contents - dpg 2021 book of abstracts

Contents DPG 2021 | Abstract Book

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DPG 2021 Book of Abstracts


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Table of Contents Table of Contents ················································································································· 2

Schedule Thursday, 30 September 2021 ··················································································· 4

Schedule Friday, 01 October 2021 ··························································································· 5

Schedule Saturday, 02 October 2021 ······················································································· 6

Plenary Lectures ·················································································································· 8

Plenary Lecture 1: Steven A. Siegelbaum ············································································ 8

Du Bois-Reymond Award: Andreas Ritzau-Jost ································································· 9

Teaching Lecture: Tobias Moser ······················································································ 10

Plenary Lecture 2: Susan M. Bailey ·················································································· 12

Plenary Lecture 3: Nikolaus Rawejwsky ············································································ 13

Plenary Lecture 4: Mariska J. Vansteensel ········································································· 14

Symposia 30 September 2021 ······························································································ 15

S 01 | Claudins - classical & non-classical ·········································································· 15

S 02 | The enlightened heart ··························································································· 21

S 03 | Linking respiration with brain function ······································································· 26

S 04 | Translational Kidney Physiology ·············································································· 33

S 05 | Microproteins in cardiovascular pathophysiology ························································ 37

S 06 | Structure & Function of Presynaptic Plasticity····························································· 43

Symposia 1 October 2021 ···································································································· 50

S 07 | When the immiscible function as one: Interaction of lipids and membrane-spanning

proteins in biological membranes ····················································································· 50

S 09 | Young Physiologists ····························································································· 56

S 10 | Microcirculation in Inflammation and Development ····················································· 61

Symposia 2 October 2021 ···································································································· 68

S 11 | Acid-base transport and metabolism: from sea life to human disease ······························ 68

S 12 | Low Oxygen - Sensing and Fighting Hypoxia ····························································· 75

Workshops 1 October 2021 ·································································································· 80

W 01 | 3Rs in Physiology ································································································ 80

W 02 | Bioinformatics - NGS data analysis and modelling of cellular networks ··························· 83

Oral Sessions 30 September 2021 ························································································· 89

OS 01 | Spotlight on Signal Transduction ··········································································· 89

OS 02 | Renal Homeostasis and Disease ·········································································· 99

OS 03 | Pulmonary Physiology and Hypoxia: Take a Breath ················································ 108

OS 04 | Inflammation: Feel the Burn ··············································································· 117


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Oral Sessions 01 October 2021 ··························································································· 126

OS 05 | Channels - Plug & Play ····················································································· 126

OS 06 | Cellular Neurophysiology: from Molecular Mechanisms to System Functions ··············· 136

OS 07 | The multicellular heart ······················································································· 146

Oral Sessions 02 October 2021 ··························································································· 156

OS 08 | Cardiovascular Systems - Mechanics to Metabolism ··············································· 156

OS 09 | Systems Neurophysiology: from Network Oscillations to Behavior ······························ 166

Poster Session A | 30 September 2021 ················································································· 178

A 01 | Neural Mechanisms of Behaviour ·········································································· 178

A 02 | Cellular Neurophysiology ····················································································· 190

A 03 | Novel Approaches to Channels and Receptors ························································· 200

A 04 | Voltage-gated Ion Channels ················································································· 214

A 05 | Transporter ······································································································· 228

A 06 | Lung Physiology and Hypoxia I ············································································· 242

A 07 | Inflammation ····································································································· 253

A 08 | Hormones ········································································································· 264

A 09 | RENAL I ··········································································································· 281

A 10 | Cardiomyocytes ································································································· 294

A 11 | Cardiac Physiology I ··························································································· 307

A 12 | Endothelial Cell Physiology I ················································································· 319

Poster Session B | 01 October 2021 ····················································································· 330

B 01 | Network Physiology ···························································································· 330

B 02 | Neuronal Pathophysiology ··················································································· 344

B 03 | Sensory Physiology ···························································································· 362

B 04 | Neuromodulation and Plasticity ············································································· 374

B 05 | Ligand-gated Ion Channels and Receptors ······························································ 388

B 06 | Ion Channel Regulation ······················································································· 402

B 07 | Lung Physiology and Hypoxia II ············································································ 416

B 08 | Blood ··············································································································· 427

B 09 | RENAL II ·········································································································· 439

B 10 | Vascular Physiology ··························································································· 452

B 11 | Endothelial Cell Physiology II ················································································ 466

B 12 | Cardiac Physiology II ·························································································· 480

Chair Index ······················································································································ 492

Author Index ···················································································································· 494

Keyword Index ················································································································· 509

Schedule Thursday, 30 September, 2021

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Schedule Thursday, 30 September 2021

Schedule Friday, 01 October, 2021

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Schedule Friday, 01 October 2021

Schedule Saturday, 02 October, 2021

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Schedule Saturday, 02 October 2021

Plenary Lectures DPG 2021 | Abstract Book

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30 September 2021 11:10 AM – 12:10 PM

Audimax

Opening

Welcome Message by the Congress President

J. Roeper (Frankfurt/Main, Germany)

Welcome Message by the Medical Director, University Clinic Frankfurt

J. Graf (Frankfurt/Main, Germany)

Welcome Message by the Vice President, Goethe University Frankfurt

B. Brüne (Frankfurt/Main, Germany)

Welcome Message by the Austrian Physiological Society

M. J. Fischer (Vienna, Austria)

Welcome Message by the LS2 Physiology Switzerland

D. Hoogewijs (Fribourg, Switzerland)

Introduction Francis W. Hoeber

J. Roeper (Frankfurt/Main, Germany)

Remembering Rudolf Höber - The Charming Professor

F. W. Hoeber (Philadelphia, USA)


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30 September 2021 1:00 PM – 2:30 PM

Audimax

Plenary Lecture 1: Steven A. Siegelbaum Chair

Jochen Roeper (Frankfurt/Main)

Neural circuits underlying social memory and its dysfunction in

neuropsychiatric disease

Steven A. Siegelbaum

Department of Neuroscience and Zuckerman Mind Brain Behavior Institute, Vagelos College of Physicians and

Surgeons, Columbia University, New York, USA

Our memories of family, friends and acquaintances plays a central role in forming our autobiographical identity, and

allows us to engage in productive and meaningful social relationships. Impairments in social memory profoundly

reduces the quality of life of individuals suffering from various neuropsychiatric disorders. Since the studies of Brenda

Milner of patient H.M., the hippocampus has been known to play a crucial role in the establishment of social memory.

However, the hippocampus is a complex structure with multiple subregions. To date, the neural circuits mediating

social memory and how circuit dysfunction results in abnormal social behavior is unknown.

Our laboratory focuses on the role of the hippocampal CA2 subregion, which undergoes alterations in individuals

with schizophrenia and bipolar disorder. Although CA2 was first identified by Lorenté de Nò in 1934, its small size

and location, interspersed between its better studied CA1 and CA3 neighbors, has limited our knowledge of CA2

function. Based on the finding that CA2 expresses a set of unique genes, we and others have developed lines of

transgenic mice that express Cre recombinase relatively selectively in CA2 principal neurons. This enables us to

label CA2 and manipulate its activity using injections of Cre-dependent viral vectors.

We find that selective silencing of CA2 greatly impairs social memory, but has little impact on other forms of

hippocampal dependent memory. In vivo electrophysiological recordings and calcium imaging experiments

demonstrate that CA2 neuron firing is enhanced during social interactions or during exposures to social odors (urine)

in a manner that enables CA2 activity to distinguish between a novel versus familiar animal, and to associate a social

stimulus with reward. Studies of a genetic mouse model of the human 22q11.2 microdeletion, which confers a 30-

fold increased risk of schizophrenia, reveal an impairment in social memory associated with abnormal CA2 neuron

activity, due in part to upregulation of the TREK-1 potassium current. Importantly, pharmacological or genetic

inhibition of TREK-1 is able to rescue both social memory and CA2 neuron social coding.


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Thus, our studies reveal a unique hippocampal subcircuit tuned to social memory, how abnormalities in this circuitry

may contribute to impairments of social memory associated with neuropsychiatric disease, and how targeting this

circuitry may provide a novel therapeutic approach for disease treatment.

Coronal section of a mouse brain Coronal section of a mouse brain labeled with green fluorescent protein in

the CA2 region of the hippocampus, an area critical for social memory.

Du Bois-Reymond Award Laudatio

Stefan Hallermann (Leipzig, Germany)

Laureate

Andreas Ritzau-Jost

University of Leipzig, Carl-Ludwig-Institute for Physiology, Faculty of Medicine, Leipzig, Germany


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1 October 2021 8:00 AM – 9:00 AM

Audimax

Teaching Lecture: Tobias Moser Chair

Rudolf Schubert (Augsburg)

The peripheral auditory system

Tobias Moser1,2

1 University Medical Center Göttingen, Institute for Auditory Neuroscience & InnerEarLab, Göttingen, Germany 2 German Primate Center & Max-Planck-Institutes for Biophysical Chemistry & Experimental Medicine, Göttingen,

Germany

The physiology of peripheral auditory system remains a highly active area of research. The physiology of the cochlea

and the auditory nerve, in particular, has been fueled by molecular approaches including human and mouse

genetics. The cochlea is part of the peripheral nervous system and houses hair cells as well as their postsynaptic

partners, the spiral ganglion neurons (SGNs), which convey the information to the auditory brainstem of the central

nervous system. While outer hair cells employ electromotility to shape active micromechanics of the cochlea, inner

hair cells are the “true” sensory cells that indefatigably transmit information at high rates and temporal preciscion.

The fan-like hair cell-neuron-array of the cochlea takes a spiral shape to sample the frequency-sorted vibrations

along the basilar membrane, comparable to a circular staircase in which each step represents a given frequency

(Fig. 1). This so-called tonotopic organization is then maintained along the entire auditory pathway reflecting hard-

wired labeled lines for the different frequencies. The central nervous system in turn provides inhibitory feedback to

hair cells and SGNs via olivocochlear efferent fibers.


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FIGURE 1. The peripheral auditory system (from Kleinlogel et al.,

Phys Rev 2020)


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1 October 2021 9:00 AM – 10:00 AM

Audimax

Plenary Lecture 2: Susan M. Bailey Chair

Jürgen Graf (Frankfurt/Main)

TWINS & TELOMERES – in Space!

Susan M. Bailey

Colorado State University, Department of Environmental & Radiological Health Sciences, Fort Collins, USA

The ends of human chromosomes are capped by telomeres, tandem arrays of highly conserved repetitive sequence

bound by a plethora of proteins that protect chromosomal termini from inappropriate degradation and loss. Telomeres

also preserve genome stability by preventing natural chromosomal ends from being recognized as broken DNA

(double-strand breaks; DSBs) and triggering inappropriate DNA damage responses (DDRs). It has long been

recognized that telomere length erodes with cellular division, and thus with aging. More recently, oxidative stress,

infection, and inflammation have been shown to also contribute to telomere shortening, as have a host of lifestyle

factors, including stress (e.g., nutritional, physical, psychological) and environmental exposures (e.g., air pollution,

UV and ionizing radiations). Therefore, telomere length dynamics (changes over time) represent a key integrating

component of the cumulative effects of genetic, environmental, and lifestyle factors, and thereby provide an

informative biomarker, and even determinant, of health status, aging, and age-related pathologies, including

dementias, cardiovascular disease (CVD), and cancer.

We speculated that assessing telomere length would be particularly relevant for astronauts on longduration missions,

because an individual’s genetic susceptibilities, exposures to galactic cosmic radiation, and distinctive “out-of-this-

world” stressors experienced, would all be captured as changes over time. We assessed telomere length and

intimately related DDRs in twin astronauts Scott and Mark Kelly, and ten unrelated astronauts before, during, and

after one-year or 6-month missions aboard the International Space Station. Most striking was our observation of

significantly longer telomeres during spaceflight, irrespective of mission duration or means of measurement.

Telomere length shortened rapidly after return to Earth for all crewmembers, and overall,

astronauts had many more short telomeres after spaceflight than they did before. Consistent with chronic exposure

to the space radiation environment, signatures of persistent DDRs were also detected during spaceflight, which

included mitochondrial and oxidative stress, as well as telomeric and chromosomal DNA damage. Together, findings

provide potential mechanistic insight, and reveal important differences in individual responses.

Acknowledgment

Funding from NASA is gratefully acknowledged (NNX14AB02G, NNX14AH51G, 80NSSC19K0434).


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1 October 2021 1:45 PM – 2:45 PM

Audimax

Plenary Lecture 3: Nikolaus Rawejwsky Chair

Ralf P. Brandes (Frankfurt/Main)

Cell-based Medicine

Nikolaus Rajewsky

Berlin, Germany

I will present how groundbreaking new single-cell sequencing technologies make it possible to not only detect

pathogenesis in single cells much earlier but also to track and target human cells during disease

progression. Combining single-cell technologies with machine learning and experimentally accessible human-

derived diseases systems (for example organoids) form the basis of “Cell Based Medicine” and are pursued by newly

formed European and national networks. I will also present unpublished science from my lab, including some

surprises about RNA.


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2 October 2021 9:30 AM – 10:30 AM

Audimax

Plenary Lecture 4: Mariska J. Vansteensel Chair

Andreas Draguhn (Heidelberg)

Communicating with the brain: from concept to real-world

implementation

Mariska J. Vansteensel, Erik J. Aarnoutse, Sacha Leinders, Mariana P. Branco, Anouck Schippers,

Zachary V. Freudenburg, Nick F. Ramsey

UMC Utrecht Brain Center, Department of Neurology and Neurosurgery, University Medical Center Utrecht,

Utrecht, Netherlands

The past decades have witnessed the emergence and fast growth of the Brain-Computer Interface (BCI) research

field. BCIs record neural signals from the brain and convert certain features thereof into control signals for a computer.

Although BCIs conceptually may serve different types of target populations, much of the current BCI research and

development is focused on resolving the communication problems of people with severe motor- and speech-

impairment due to for example amyotrophic lateral sclerosis (ALS). BCI-control is typically based on well-described

neurophysiological phenomena, but at the same time benefits greatly from recent advances in neural signal analysis.

Here, I will zoom in on one of these phenomena, the sensorimotor rhythms, and discuss the current state of the art

of using consciously and voluntarily induced changes in these signals for BCI purposes. In the second part of my

talk, I will discuss real-world implementation of BCIs. I will touch upon the potential value BCIs may have for the

quality of life of people with severe motor impairment, as well as on the importance of a user-centered design of

BCIs, where the target population of assistive technology is involved in all stages of research and development. I will

present findings of a seminal study we currently conduct at UMC Utrecht in the Netherlands, in which we aim to

validate a fully implantable, electrocorticography-based BCI for home-use by people with severe paralysis. Using

findings obtained with three severely paralyzed participants of this study (two individuals with late-stage ALS and one

individual with a brain-stem stroke), I will illustrate that fully implantable BCIs can offer a reliable signal to control

communication software, but also that real-world implementation of BCIs is associated with significant challenges,

including 24/7 functioning of the system and interindividual differences in the neural signals for BCI control. I will

conclude by discussing some of the hurdles that need to be taken before the future that is envisioned by many BCI

researchers, in which implantable BCIs are widely and clinically accessible for those who need them, could become

reality.

Symposia DPG 2021 | Abstract Book

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Symposia 30 September 2021

9:00 AM – 11:00 AM

Lecture Hall 1

S 01 | Claudins - classical & non-classical Chair

Markus Bleich (Kiel)

Rozemarijn van der Veen (Berlin)


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S 01-01

Claudin properties and function

Dorothee Günzel, Luca Meoli, Selma Dib, Ahmad Mouna, Jörg Piontek

Charité - Universitätsmedizin Berlin, Clinical Physiology/Nutritional Medicine, Medical Department, Division of

Gastroenterology, Infectiology, Rheumatology, Berlin, Germany

In freeze-fracture electron micrographs, tight junctions appear as complex network-like structures within the lateral

membrane near the apical pole of epithelial cells. For a long time, tight junctions were assigned just one task – to

form a tight barrier against unwanted transport of solutes, toxins and pathogens. Leaky junctions, although known to

ensure efficient paracellular solute transport, were assumed to simply be less intact, exhibiting less complex networks

or defective strands (strand breaks).

During the late 1990s, claudins were discovered as the major constituents of tight junction strands – able to assemble

into tight junction-like strands and networks when overexpressed in non-epithelial cell lines [1]. Some of these

claudins were identified to form paracellular ion channels, conveying anion, cation and/or water permeability to the

paracellular pathway. Among them are the two claudin-10 isoforms, claudin-10a and -10b that differ only in their N-

terminal ~80 amino acids, encoded by two alternative exons 1 of the CLDN10 gene.

Intriguingly, claudin-10a acts as a paracellular anion channel, claudin-10b as a paracellular cation channel but neither

facilitates the paracellular movement of water [2, 3]. Different mouse models and the recent discovery of patients

suffering from mutations in the CLDN10 gene [4] allow new insights into the complexity of tight junction physiology.

The disease caused by CLDN10 deficiency was dubbed HELIX syndrome by Hadj-Rabia et al. [5], an acronym of

the main symptoms of the patients: hypo- (or even an-)hydrosis and thus an inability to adequately regulate body

temperature at high ambient temperatures, electrolyte imbalance (hypokalemia, hypermagnesemia, polyuria),

lacrimal gland dysfunction, ichthyosis and xerostomia.

Consistently, Claudin-10a and -10b are expressed in the kidney, Claudin-10a in the proximal tubule, Claudin-10b

predominantly in the thick ascending limb of Henle’s loop. Both Claudin-10 isoforms allow the kidney to save energy

by facilitating passive, paracellular reabsorption of Cl- and Na+, respectively. In sweat, saliva and lacrimal glands, in

contrast, only claudin-10b is expressed. Here, it serves to move Na+ in secretory direction, i.e., against the preferred

direction of the (basolateral) Na+/K+ ATPase.With, to date, only 35 known HELIX-syndrome patients world-wide, we

are only beginning to understand the complex physiological role of paracellular ion channels.

Acknowledgment

Financial support by DFG GU 447/14-2 and DFG GRK 2318"TJ-Train" is gratefully acknowledged.

References [1] Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S 1998, Claudin-1 and -2: novel integral membrane

proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 141:1539-1550 [2] Günzel D, Stuiver M, Kausalya PJ, Haisch L, Krug SM, Rosenthal R, Meij IC, Hunziker W, Fromm M, Müller D

2009, Claudin-10 exists in six alternatively spliced isoforms that exhibit distinct localization and function. J Cell Sci. 122:1507-1517

[3] Rosenthal R, Milatz S, Krug SM, Oelrich B, Schulzke JD, Amasheh S, Günzel D, Fromm M 2010, Claudin-2, a component of the tight junction, forms a paracellular water channel. J Cell Sci. 123:1913-1921

[4] Klar J, Piontek J, Milatz S, Tariq M, Jameel M, Breiderhoff T, Schuster J, Fatima A, Asif M, Sher M, Mäbert K, Fromm A, Baig SM, Günzel D, Dahl N 2017, Altered paracellular cation permeability due to a rare CLDN10B variant causes anhidrosis and kidney damage. PLoS Genet. 13: e1006897

[5] Hadj-Rabia S, Brideau G, Al-Sarraj Y, Maroun RC, Figueres ML, Leclerc-Mercier S, Olinger E, Baron S, Chaussain C, Nochy D, Taha RZ, Knebelmann B, Joshi V, Curmi PA, Kambouris M, Vargas-Poussou R, Bodemer C, Devuyst O, Houillier P, El-Shanti H 2018, Multiplex epithelium dysfunction due to CLDN10 mutation: the HELIX syndrome. Genet Med. 20:190-201


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S 01-02

Nanoscale segregation of channel and barrier claudins enables

paracellular ion flux.

Hannes P. Gonschior1, Christopher Schmied2, Nina Himmerkus3, Jörg Piontek4, Dorothee Günzel4,

Markus Bleich3, Mikio Furuse5,6, Volker Haucke1,7, Martin Lehmann1

1 Leibniz Institute for Molecular Pharmacology (FMP), Molecular Pharmacology and Cell Biology, Berlin, Germany 2 Fondazione Human Technopole, Image Analysis Facility, Milan, Italy 3 Christian-Albrechts-University Kiel, Institute of Physiology, Kiel, Germany 4 Charité – Universitätsmedizin Berlin, Institute of Clinical Physiology/Nutritional Medicine, Medical Department,

Division of Gastroenterology, Infectiology, Rheumatology, Berlin, Germany 5 National Institute for Physiological Sciences, Division of Cell Structure, Okazaki, Japan 6 School of Life Science, SOKENDAI (Graduate University for Advanced Studies), Department of Physiological

Sciences, Okazaki, Japan 7 Free University of Berlin, Faculty of Biology, Chemistry, Pharmacy, Berlin, Germany

The paracellular passage of ions and small molecules across epithelia is controlled by tight junctions (TJ), complex

meshworks of claudin polymers that form semipermeable barriers between neighboring cells. Depending on the

claudin composition the ~10 nm thick strands can act as a paracellular seal against molecules larger than 200 Da or

as size and charge selective ion/water channels that play a fundamental role in tissue-specific ion homeostasis and

nutrient uptake. A crucial, yet unsolved question is how TJ meshworks, composed of single or multiple claudins, are

organized at the nanoscale to integrate paracellular barrier and channel functions.

Here we combine super-resolution Stimulated Emission Depletion (STED) microscopy in living and fixed cells and

different murine tissues, multivariate classification of STED images and Fluorescence Resonance Energy Transfer

to reveal the nanoscale organization of TJs formed by claudins. By visualizing single claudin strands in complex TJ

meshworks with lower than 60 nm resolution and optimized fixation and labelling conditions we could show that only

a subset of the mammalian claudins can assemble into characteristic homotypic TJ-like meshworks, whereas TJs

formed by multiple claudins display five novel nanoscale organization principles of intermixing, integration, induction,

segregation, and exclusion of strand assemblies. Importantly, channel-forming claudin-10a, claudin-10b and claudin-

15, are spatially segregated from barrier- and other channel-forming claudins via determinants mainly encoded in

their extracellular domains, while the claudin association with ZO1 adaptor proteins, channel activity and plasma

membrane located cholesterol is dispensable for the segregation. Based on that, we hypothesize that segregation of

barrier- and channel-forming claudins is a key mechanism for proper claudin channel function that is compromised

in certain patients with claudin missense mutations (e.g., Cldn10bN48K). Electrophysiological analysis of single

claudins and segregating claudin pairs in genome-engineered claudin-free epithelial cells suggests that nanoscale

segregation of distinct channel-forming claudins enables integration of barrier function and specific paracellular ion

flux across TJs via an “ion maze” as model of paracellular ion transport through complex TJ meshworks that has

wide implications for cell physiology and our understanding of tissue barrier function.


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S 01-03

Molecular architecture and assembly of the tight junction backbone

Jörg Piontek1, Caroline Hempel1, Jonas Protze2, Rita Rosenthal1, Anja Fromm1, Gerd Krause2,

Michael Fromm1, Dorothee Günzel1

1 Charité - Universitätsmedizin Berlin, Institute of Clinical Physiology/Nutritional Medicine, Medical Department,

Division of Gastroenterology, Infectiology, Rheumatology, Berlin, Germany 2 Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany

The tight junction (TJ) regulates paracellular permeability size and charge selectively. Claudins form the backbone

of TJ strands whereas other proteins regulate TJ dynamics. Models for the molecular architecture of TJ strands and

paracellular channels have been proposed but have to be further tested and refined. We analyzed the strand and

channel architecture by cellular reconstitution of strands, structure-guided mutagenesis, in silico oligomer modeling

and molecular dynamics simulations. Prototypic channel-forming (Cldn10b, Cldn15) and barrier-forming (Cldn3)

claudins were analyzed. Förster resonance energy transfer (FRET) assays indicated multistep claudin

polymerization, starting with cis-oligomerization specific to the claudin subtype, followed by trans-interaction-

triggered cis-polymerization. Alternative protomer interfaces were modeled in silico and tested by cysteine-mediated

crosslinking, confocal- and freeze fracture EM-based analysis of strand formation. The results indicated that

protomers in Cldn10b and Cldn3 strands form similar antiparallel double rows, as has been suggested for Cldn15.

Mutually stabilizing -hydrophilic and hydrophobic - cis- and trans-interfaces were identified that contained novel key

residues of extracellular segments ECS1 and ECS2. Hydrophobic clustering of the ECS1 β1β2 loops together with

ECS2-ECS2 trans-interaction is suggested to be the driving force for conjunction of tetrameric building blocks into

claudin polymers. Classic claudins, such as Cldn1 to-10 and -15, sharing critical sequence motifs are indicated to

have this polymerization mechanism in common. However, in the paracellular center of tetramers, either electrostatic

repulsion leads to formation of pores (Cldn10b, Cldn15) or electrostatic attraction to barriers (Cldn3). The models

were further supported by electrophysical analysis of Cldn10b and Cldn15 mutants expressed in MDCK-C7 cells.

Combining in vitro data and in silico modeling, this study improves mechanistic understanding of paracellular

permeability regulation by elucidating claudin assembly.


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S 01-04

Blood-nerve-barrier, claudins and drug delivery

Heike Rittner

University Hospital Würzburg, Dept Anesthesiology, Würzburg, Germany

Peripheral nerves are guarded by two barriers. The blood-nerve barrier (BNB) and myelin barrier regulate the ingress

and egress of substances to axon fibers thereby maintaining a proper neural microenvironment and

preserving optimal nerve function. The BNB consists of the perineurium and endoneurial vessels; the myelin barrier

is formed by Schwann cells at the paranode, the Schmidt-Lanterman incisures and the mesaxon. Tight and

adherence junction proteins and cytoplasmic accessory proteins form the cell-cell contacts of the BNB and myelin

barrier. The central part of the pain pathway is likewise shielded: the dorsal root ganglion, the spinal cord and the

brain all have barriers of different permeability. The leakiest one is the blood-dorsal root ganglion barrier, the tightest

one the myelin barrier.

Breakdown of peripheral and central barriers and its accompanying tight junction protein downregulation are common

features of nerve injury. Main causes of nerve injury include traumatic, metabolic and toxic damage. All of these

neuropathies have different barrier damaging patterns. For example, perineurial claudin-1 is affected in traumatic

nerve damage and diabetic polyneuropathy, but the kinetics are completely different. In patients with neuropathy of

various origins, tight junction proteins are also downregulated. Whether this is a preceeding event facilitating nerve

damage or a by-product of injury and inflammation is not clear at this moment. However, nerve edema is a very early

sign of neuropathy. So, nerve barrier sealing could reduce further damage and relief pain.

On the other side – barriers prevent the delivery of drugs. This is of interest in regional anesthesia or local treatment

of damaged nerves. Specific pathways and novel molecules have been identified like claudin peptidomimetics or

tissue plasminogen to transiently open the barrier. This is a safe procedure for delivery of analgesics – as long as

there is no inflammation in the nerve.

In summary, understanding the details of barrier function in the central and peripheral nervous system can lead to

the design of novel drugs and appraoches to treat neuroapathic pain and understand pain resolution.

Acknowledgment

Supported by the DFG (KFO5001 ResolvePAIN), the EU (ncRNAPAIN), the IZKF Würzburg and the Else-Kröner

Foundation.


of 516

S 01-05

Role of claudin-10b in the basolateral infoldings of the thick ascending

limb

Catarina Quintanova

Christian-Albrechts-University of Kiel, Institute of Physiology, Kiel, Germany

The thick ascending limb (TAL) of the loop of Henle is re-absorbing around 30% of the filtered NaCl while also being

water tight, effectively separating salt and water which contributes to the cortico-medullary osmolality gradient and

the kidney’s ability to concentrate urine. In addition, it is involved in the reabsorption of the cations K+, Ca2+ and Mg2+.

Ion transport occurs transcellularly, basic transport mechanisms involve co-transport of Na+, K+ and Cl-. Nevertheless,

the lumen positive potential generated by the transcellular transport drives paracellular cation reabsorption. In the

TAL, tight junctions (TJs) show a mosaic expression of either claudin-10b or claudin-3/-16/-19 and the tricellular

junction as a switching point between TJ properties. Claudin-10b TJ confers primarily paracellular Na+ permeability.

Absence of Claudin-10b leads to a re-arrangement of this TJ architecture and the take-over of the other claudins,

increasing divalent cation- and decreasing Na+ permeability. In addition, however, claudin-10b is also present outside

of the TJ in basolateral membrane of the TAL, where its function is not yet understood. Immunofluorescence and

high-resolution microscopy showed a dotted pattern of claudin-10 in the infoldings of the basolateral membrane which

almost reach the apical membrane. This extra-junctional expression co-localized with other membrane proteins, such

as Na+-K+-ATPase and Barttin. We investigated freshly isolated single murine TAL segments of C57BL/6 and kidney

specific (Ksp-Cre) Claudin-10 knockout mice. Although the loss of claudin-10b in the TJ increased the transepithelial

resistance, the equivalent short-circuit current as measure of transcellular electrogenic ion transport was un-altered

between genotypes. Also, the Na+-K+-ATPase inhibitor ouabain showed similar inhibition effectiveness. We

consequently, measured the relative speed of ouabain inhibition and this was increased after loosening of

intermembrane protein contacts under Ca2+-free conditions or in the absence of claudin-10. We also tested the

diffusion of fluorescein dye into the infoldings which could be trapped within the infoldings in the WT situation and

less in Claudin-10 knockout TAL. In our experiments, claudin-10b restricts the diffusion of ouabain or fluorescein

along the infoldings, suggesting a possible role in the stabilization of the infolded space by formation of a complex

between neighboring invaginated membranes.


of 516

Symposia 30 September 2021 9:00 AM – 11:00 AM

Lecture Hall 3

S 02 | The enlightened heart Chair

Tobias Brügmann (Göttingen)

Dörthe Katschinski (Göttingen)


of 516

S 02-01

Spot on! 10 years of cardiac optogenetics to investigate and treat

arrhythmia

Philipp Sasse

University Bonn, Institute of Physiology I, Bonn, Germany

Expressing the light-sensitive ion channel Channelrhodopsin-2 can be used to pace mouse and human

cardiomyocytes in the culture dish as well as a hearts of transgenic mice. Furthermore we have proven that

optogenetic methods are also very effective in terminating ventricular tachycardia and atrial fibrillation in mouse

hearts and that optogenetics could eventually represent an alternative approach to painful electrical shocks. With the

aim of translational applications, we have developed a gene transfer strategy by injection of adeno-associated virus

for Channelrhodopsin-2 expression, which enabled reliable optogenetic pacing, defibrillation and cardioversion of

wild-type mouse hearts. Using cardiomyocyte-specific expression of the light-inducible proton pump ArchT, we were

able to demonstrate that not only constant depolarization but also hyperpolarization can terminate ventricular

arrhythmia and identified a new mechanism of increased electrical sink to stop the arrhythmic wavefront.

Side effects on cardiac ion channels causing lethal arrhythmias are one reason for drug withdrawals from the market.

To screen for such effects in vitro we have developed scalable technologies combining field potential measurements

with optogenetic stimulation. This enabled reliable pacing of human pluripotent-cell derived cardiomyocytes by light

flashes at physiologically relevant heart rates and the automated frequency-dependent analysis of drug effects on

cardiac ion channel function.

For stimulation of intracellular Gs and Gi signaling cascades with light we have employed the specific light-sensitive

G-protein coupled receptors Jellyfish-Opsin and Coneopsin in cardiomyocytes. For instance, optical stimulation of

transgenic hearts expressing the Gs-coupled Jellyfish-Opsin elevated cAMP levels and accelerated the heart rate

similarly than pharmacological stimulation, but with much faster on- and off-kinetics and very high spatial precision.

In summary, we successfully applied several optogenetic methods for depolarization, hyperpolarization as well as

stimulation of Gs and Gi signaling cascades in cardiomyocytes in vitro and the mouse heart in vivo. These

technologies have been used to develop systems for drug screening as well as for better mechanistic understanding

and eventually also for treating lethal cardiac arrhythmia.

Acknowledgment

Supporte by Deutsche Forschungsgemeinschaft (German Research Foundation, 313904155/SA1785/7-1,

315402240/SA1785/8-2; 380524518/SA1785/9-1, 214362475/GRK1873/2)


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S 02-02

Thermogenetics: neurons and beyond

Vsevolod Belousov1,2,4

1 Federal Center of Brain Research and Neurotechnologies, Federal Medicval Biological Agency, Moscow, Russia 2 Pirogov Russian National Research Medical University, Center for Precision Genome Editing and Genetic

Technologies for Biomedicine, Moscow, Russia 3 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia 4 Universitätsmedizin Göttingen, Institute of Cardiovascular Physiology, Göttingen, Germany

Thermogenetics (thermal+genetics) was originally developed as a set of tools enabling control over neuronal activity

using heat-sensitive ion channels of TRP family. Advantages of thermogenetics include high conductance and fast

kinetics of TRP channels, high tissue permeability and no phototoxicity of the IR light that we use to bring millisecond-

scale heat pulses to the cells. Our studies demonstrate that thermogenetics can be used to activate neurons with

spatio-temporal precision of optogenetics. But the main advantage of thermogenetics is that TRP channels mainly

conduct calcium ions. Calcium is one of the most evolutionary ancient signaling modalities controlling metabolism,

signal transduction, and cell fate decisions in all domains of life. All major cellular functions such as cell cycle,

migration, secretion, contraction, death and many others are Ca2+-dependent. Ca2+ is a universal trigger of function

for most types of terminally differentiated cells. Here we present GECCO, the system for thermogenetic Ca2+

manipulation based on snake TRP channel optically controlled by infrared laser. GECCO is functional in animal and

plant cells and allows studying how cells decode different profiles of Ca2+ signals. Using GECCO we were able to

shape insulin release from β-cells, to identify drugs that potentiate Ca2+-induced insulin release, and to study

propagation of Ca2+ waves in plants.

Acknowledgment

The study was supported by grant 075-15-2019-1789 from the Ministry of Science and Higher Education of the

Russian Federation


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S 02-03

Control of hippocampal function and locomotion by the medial septum

Falko Fuhrmann1, Petra Mocellin2, Stefan Remy2,1

1 German Center for Neurodegenerative Diseases (DZNE), Neuroimmunology and Imaging, Bonn, Germany 2 Leibniz Institute for Neurobiology, Cellular Neuroscience, Magdeburg, Germany

Memory guided locomotion accompanies a brain-state transition that is associated with increased firing rates of CA1

pyramidal neurons and the occurrence of theta oscillations, which both correlate with locomotion velocity. We have

revealed a septo-hippocampal circuit mediated by glutamatergic neurons that links locomotor activity to hippocampal

oscillations and neuronal firing rates. Optogenetic activation of glutamatergic septal neurons in the theta frequency

band led to an entrainment of stimulus-frequency locked LFP oscillations in CA1 and increased activity of CA1

pyramidal neurons. Following the induction of hippocampal theta oscillations by a rhythmic stimulation of

glutamatergic septal neurons, locomotion was initiated within several hundreds of milliseconds (Fuhrmann et al.,

2015). Thus medial septal glutamatergic neurons not only provide entorhinal cortex (Justus et al., 2017) and

hippocampus with speed-correlated inputs, at the same time these neurons control the initiation and the speed of

locomotion.

Until now the pathways by which medial septal glutamatergic neurons control locomotion remained obscure. We now

show a direct glutamatergic medial septal pathway to ventral tegmental area (VTA) that initiates locomotion and

modulates movement onset and speed.


of 516

S 02-04

Direct optogenetic stimulation of smooth muscle cells to control

gastric contractility.

Markus Vogt1, Benjamin Schulz2, Ahmed Wagdi1, Jan Lebert3,1, Gijsbert J. C. van Belle1,

Jan Christoph3,1, Tobias Bruegmann1, Robert Patejdl2

1 University Medical Center Göttingen, Institute of Cardiovascular Physiology, Göttingen, Germany 2 University of Rostock, Oscar Langendorff Institute for Physiology, Rostock, Germany 3 University of California, Cardiovascular Research Institute, San Francisco, USA

Objective

Antral peristalsis is a prerequisite for sufficient gastric emptying. In gastroparesis, dysfunction of interstitial cells of

Cajal (ICC) and enteric neurons causes a loss of peristalsis and in return symptoms like nausea and vomiting.

Available treatment approaches, including gastric electrical stimulation, are often non-satisfying and fail to restore

gastric emptying. In this study, we investigate the use of direct optogenetic stimulation of smooth muscle cells (SMC)

via the light-gated non-selective cation channel Channelrhodopsin2 (ChR2) to control gastric motor function.

Methods

We used a transgenic mouse model expressing ChR2 fused with eYFP under the control of the chicken-β-actin

promoter. Light-induced currents were characterized via patch clamp experiments using isolated SMC. Ca2+ signals

and isometric force were analyzed in antral smooth muscle strips upon illumination with blue light (460 nm).

Intraluminal pressure and gastric propulsion were quantified in intact stomachs upon light stimulation as well as global

depolarization by 60mM K+ and pharmacological stimulation with 10 µM carbachol. Furthermore, optogenetic

stimulation was investigated in a gastroparesis model induced by neuronal- and ICC-specific damage based on

methylene blue photo-toxicity.

Results

In the tunica muscularis, membrane-bound eYFP signals were restricted to SMC (36±3%, n=5) in which light induced

depolarizing currents with the known ChR2 properties. These resulted in contractions of antral smooth muscle strips

that showed higher amplitudes than those triggered by supramaximal electrical field stimulation and comparable to

those evoked by global depolarization. This can be explained by the spread of light-induced Ca2+ transients from

ChR2 positive SMC to neighboring negative SMC via gap junctions. In isolated stomachs, panoramic illumination led

to intragastric pressure increases of 239±46% (n=6) in amplitude compared to electric field stimulation. Gastric

propulsion could only be induced in ChR2 expressing but not control wild type stomachs. In the gastroparesis model,

electric field stimulation responses were abolished, whereas light stimulation still efficiently generated pressure

waves.

Conclusions

In this study, we show that gastric contractility can be controlled by direct optogenetic stimulation of SMC with high

temporal and spatial resolution. In the future, this novel approach may be used to restore motility in patients suffering

from gastroparesis.


of 516

Symposia 30 September 2021 9:00 AM – 11:00 AM

Audimax

S 03 | Linking respiration with brain function Chair

Andreas Draguhn (Heidelberg)

Jonas-Frederic Sauer (Freiburg)


of 516

S 03-01

Breathing modulates cortico-hippocampal dynamics during offline

states

Anton Sirota1, Nikolas Karalis1,2

1 Ludwig Maximillian University Munchen, Cognition and Neural Plasticity, Faculty of Medicine, Planegg, Germany 2 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

Network dynamics have been proposed as a mechanistic substrate for the information transfer across cortical and

hippocampal circuits. During sleep and offline states, synchronous reactivation across these regions underlies the

consolidation of memories. However, little is known about the mechanisms that synchronize and coordinate these

processes across widespread brain regions. Here we address the hypothesis that breathing acts as an oscillatory

pacemaker, persistently coupling distributed brain circuit dynamics. Using large-scale recordings from seven cortical

and subcortical brain regions in quiescent and sleeping mice, we identified a novel global mechanism, termed

respiratory corollary discharge, that co-modulates neural activity across these circuits. Analysis of inter-regional

population activity and optogenetic perturbations revealed that breathing rhythm couples hippocampal sharp-wave

ripples and cortical DOWN/UP state transitions by jointly modulating excitability in these circuits. These results

highlight breathing, a perennial brain rhythm, as an oscillatory scaffold for the functional coordination of the limbic

circuit, supporting the segregation and integration of information flow across neuronal networks during offline states.


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S 03-02

Relations between respiration-induced and brain-endogenous network

oscillations

Andreas Draguhn1, Maximilian Hammer1, Jurij Brankack1, Yevgenij Yanovsky1, Adriano B. Tort2

1 Heidelberg University, Physiology and Pathophysiology, Heidelberg, Germany 2 Federal University of Rio Grande do Norte, Brain Institute, Natal, Brazil

Nasal respiration generates a state-dependent rhythmic feedback signal to the brain. During past years, collective

evidence from different groups has shown that large parts of the mammalian brain follow this self-generated sensory

cue with coherent, synchronous network oscillations. In this way, respiration-related rhythms (RR) can synchronize

neuronal activity across major parts of the brain and, hence, may be an important temporal scaffold for cognitive

processes.

We have studied RR in the mouse brain where respiration covers frequencies of ~1-11 Hz which largely overlap with

theta- (Θ) oscillations at ~ 4 – 10 Hz. Our previous results revealed several important properties of RR which clearly

distinguish them from Q-oscillations. RR is not directly generated by breathing-inducing rhythmogenic networks in

the brainstem but strictly depends on sensory feedback from nasal air flow. Similar to theta-oscillations, RR

modulates the amplitude of superimposed gamma- (γ) oscillations, creating a higher-order oscillation pattern (cross-

frequency coupling, CFC) which may support complex computations in cortical networks. The co-occurrence of both

slow rhythms (Θ and RR) in many states and brain regions and their similarities with respect to frequency and

gamma-entrainment prompted us to study their relationship in more detail.

First, we asked whether there is a causal relation between Θ, γ and RR. Recordings from the parietal cortex of mice

during REM sleep revealed that the frequencies of all three oscillations are strongly correlated. Cross-correlograms

and analysis of Granger causality showed that changes in theta frequency precede and cause subsequent changes

in RR frequency. Breathing or RR, in turn, Granger-causes changes in g frequency. Together, respiration and the

brain-endogenous Θ- and γ-oscillations are causally related with directionalities of Q affecting RR and RR affecting

γ, respectively.

Second, we tested whether RR affects Θ-g cross-frequency coupling. Indeed, the strength of Θ-g CFC depends

strongly on respiration frequency, expressing an inverted V-shaped relation: coupling is maximal at a respiration

frequency of 4-6 Hz while lower or higher respiration frequencies go along with lower coupling strength between Θ

and g.These causal relations between respiration-modulated brain activity and endogenously generated theta- and

gamma-oscillations may contribute to the impact of respiratory feedback signals on cortical information processing.

Acknowledgment

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, DR 326/15-1) and by the Alexander von

Humboldt-Foundation (grant to A.B.L. Tort).

References [1] Tort ABL, Hammer M, Zhang J, Brankack J, Draguhn A (2021) Temporal relations between cortical network

oscillations and breathing frequency during REM sleep. J Neurosci 41:5229-5242. [2] Tort ABL, Brankačk J, Draguhn A (2018) Respiration-Entrained Brain Rhythms Are Global but Often Overlooked.

Trends Neurosci 41:186-197. [3] Yanovsky Y, Ciatipis M, Draguhn A, Tort AB, Brankack J (2014) Slow oscillations in the mouse hippocampus

entrained by nasal respiration. J Neurosci 34:5949-5964.


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S 03-03

State-dependent entrainment of the prefrontal microcircuitry by

rhythmic breathing

Jonas-Frederic Sauer

University of Freiburg, Institute of Physiology I, Freiburg, Germany

Respiration is an essential rhythm of life. Recently, a series of studies has demonstrated that respiration-driven brain

rhythms are observed in a number of higher-order brain regions, including medial prefrontal cortex (mPFC, 1), barrel

cortex (2), and hippocampus (3), suggesting that the potential functions of respiration-paced brain rhythms might

extend beyond the processing of smells.

An emerging body of observations suggests that respiration entrainment of sensory and higher-oder brain areas

occurs at highest strength during immobility, suggesting that respiration rhythms might aid cortical processing during

immobile phases. However, from the perspective of emotional valence, immobility comes in quite distinct flavours:

For instance, mice display epochs of spontaneous immobility (i.e. quiet wakefulness in their homecage), a

behavioural state that is not associated with a specific emotional status. In contrast, immobility is also expressed

during negative emotional states. During tail suspension (TS) the animals are forced in an inescapable situation, to

which they stereotypically respond with alternating epochs of passive immobility and active escape attempts. If the

efficient respiration entrainment of neuronal circuits merely depends on the immobile state of the animal, it would

follow that similar patterns of network entrainment by the ongoing breathing rhythm should be observed under distinct

behavioural conditions of immobility. As an alternative hypothesis, respiration-paced activities might contribute

differentially to information processing during these distinct emotional states. Here, we tested these hypotheses in

awake mice. We focused on the medial prefrontal cortex (mPFC), which plays an important role in the control of

active vs. passive responses during TS. We find distinct behavioural state-dependent patterns of prefrontal network

entrainment by respiration. During TS, the preferred phase of prefrontal neurons shifts to earlier in the respiration

cycle. Furthermore, we observed an enhanced recruitment of layer 2/3 vs. layer 5 pyramidal neurons by respiration.

These findings suggest a physiological role of respiration-paced activities in emotional processing.

Acknowledgment

This work was supported by the German Research Foundation (SA 3609/1-1).

References [1] Biskamp, J, Bartos, M, Sauer, JF 2017, ‘Organization of prefrontal network activity by respiration-related

oscillations’,Sci Rep, doi: 10.1038/srep45508 [2] Ito, J, Roy, S, Liu, Y, Cao, Y, Fletcher, M, Lu, L, Boughter, JD, Grün, S, Heck, DH 2014, ‘Whisker barrel

cortex delta oscillations and gamma power in the awake mouse are linked to respiration ’, Nat Commun, doi: 10.1038/ncomms4572

[3] Nguyen Chi, V, Müller, C, Wolfenstetter, T, Yanovski, Y, Draguhn, A, Tort, ABL, Brankack, J 2016, ‘Hippocampal Respiration-Driven Rhythm Distinct from Theta Oscillations in Awake Mice', J Neurosci doi: 10.1523/JNEUROSCI.2848-15.2016


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S 03-04

New insights into the relationship between respiration, neural

oscillations and behaviour from human MEG studies

Daniel Kluger, Joachim Gross

University of Muenster, Institute for Biomagnetism and Biosignalanalysis, Muenster, Germany

The dynamically changing state of the human body (part of which is reflected in physiological signals such as

heartbeat, respiration and pupil dilation) influences brain activity, is in turn controlled by the brain, affects cognition

and is altered in disease. Interestingly, rhythms are a prominent feature of body and brain functions. Respiration is

inherently rhythmic. Similarly, rhythmic fluctuations in brain activity (brain oscillations) are functionally related to

cognition. Surprisingly, the cognitive and translational neuroscience literature has largely ignored the highly relevant

question to what extent changes in (oscillatory) brain

activity (between cognitive tasks or patient groups) can be attributed to changes in physiological parameters,

how rhythms in the brain reciprocally interact with rhythms in the body (such as respiration) and how both affect

behaviour. In three studies we have investigated the relationships between respiration, brain activity and behaviour

using magnetoencephalography (MEG).

First, we comprehensively mapped brain oscillations in the resting human brain that are cyclically modulated by

respiration. We observed distinct networks of brain areas with characteristic modulation pattern that extent from low

(delta) to high (gamma) frequency bands.

Second, we studied motor control using isometric contraction and observed that cortico-muscular synchronisation in

the beta band was modulated by depth of respiration and also by its phase.

Third, we presented participants with visual stimuli that were individually adjusted in contrast to lead to a defined

detection accuracy. Interestingly, we observed, that detection depends on respiratory phase and that this effect is

likely mediated by parietal alpha oscillations - a known proxy for cortical excitability.

Our findings indicate that respiration is related to brain activity and behaviour in complex ways that warrant further

investigation.

Finally, we discuss our findings in the context of models of interoceptive inference.

Acknowledgment

We acknowledge funding from the IZKF Münster.


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Network of respiration- modulated brain

oscillations (RMBOs). a, Cortical distribution of neural nodes where

oscillations were significantly modulated by

respiration. b, Side view of the entire RMBO

network (including subcortical sites) projected

on a glass brain. Nodes with similar modulation

spectra were identified with hierarchical

clustering and are coloured accordingly. c, RMBO

sites (colour-coded and numbered as in b)

closely corresponded to canonical networks of

respiratory control (RCN) and resting-state

activity.

References [1] Kluger, D.S., & Gross, J. (2020). Depth and phase of respiration modulate cortico-muscular communication.

Neuroimage, 222, 117272. [2] Kluger, D.S., Gross, J., (2021) Respiration modulates oscillatory neural network activity at rest.biorxiv [3] Kluger, D.S., Balestrieri, E., Busch, N.A., Gross, J., (2021) Respiration aligns perception with neural

excitability.biorxiv


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S 03-05

Cardiac and gastric electrical activity impact brain dynamics and

cognition in humans

Catherine Tallon-Baudry

Ecole Normale Supérieure, Inserm, Cognitive and Computational Neuroscience Lab, Paris, France

Both the heart and gastro-intestinal tract intrinsically generate their own electrical activity and are anatomically

coupled with the brain. As a result, they can be viewed as oscillators coupled to the brain. I will present how cardiac

and gastric electrical activity contribute to shaping human brain dynamics at rest, and how and why the neural

monitoring of visceral inputs might be relevant for human cognition beyond physiological regulations.


of 516

Symposia 30 September 2021 3:00 PM – 5:00 PM

Lecture Hall 1

S 04 | Translational Kidney Physiology Chair

Josef Pfeilschifter (Frankfurt/Main)

Frank Schweda (Regensburg)


of 516

S 04-01

Small leucine-rich proteoglycans in renal inflammation: Two sides of

the coin.

Liliana Schaefer

Goethe University, Frankfurt, Institute of Pharmacology and Toxicology, Frankfurt am Main, Germany

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal promoting macrophage recruitment via TLR2/4-

and CD14 co-receptors. We have now discovered that soluble biglycan triggered macrophage autophagy by

promoting autophagosome formation in primary macrophages along with increased flux of autophagic markers

Beclin-1, LC3-II, and p62. Mechanistically, biglycan evoked autophagy by acting as a novel, high-affinity ligand for

CD44, a receptor involved in adhesion, migration, lymphocyte activation, homing, and angiogenesis. The biglycan-

triggered pro-autophagic signal required TLR4/CD44 interaction. Moreover, we found a marked increase in the

number of autophagic macrophages in various organs from mice stably overexpressing soluble biglycan. Notably,

transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) caused

enhanced M1 macrophage recruitment into the kidneys of either Cd44+/+ or Cd44-/- mice but not in Cd14-/- mice. The

interaction between biglycan and CD44 increased M1 autophagy and resulted in elevated number of renal M2

macrophages and reduced tubular damage during IRI regeneration. On the other hand, the biglycan-induced M2

polarization did not depend on the CD14 co-receptor. In addition, soluble biglycan evoked autophagy in human

peripheral blood macrophages. Thus, we have discovered CD44 as a signaling co-receptor for biglycan, an

interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages, and directly involved

in preventing tubular damage in renal IRI. We hypothesize that interfering with the interaction between biglycan and

its TLR co-receptors could represent a promising therapeutic intervention to curtail renal inflammation and damage.

Acknowledgment

This work was supported by the German Research Council: SFB 1039, project B02, SFB 1177, 259130777, project

E02, SCHA 1082/6-1, and the Cardio-Pulmonary Institute (CPI), EXC 2026, Project ID: 390649896.


of 516

S 04-02

Stay flexible: The endocrine plasticity of the renal stroma

Katharina Broeker

University of Regensburg, Institute of Physiology, Regensburg, Germany

Renal stromal cells fulfill central endocrine functions – the regulation of blood pressure and extracellular volume

mediated by renin as well as the maintenance of blood oxygen levels mediated by erythropoietin (EPO). The functions

of EPO and renin converge in controlling the blood composition. EPO stimulates the erythropoiesis and thus regulates

red blood cell mass. Renin is involved in regulating the extracellular volume and consequently influences plasma

volume.

The protease renin is produced, stored and released by juxtaglomerular cells located in the media layer of afferent

arterioles at the entrance to the glomerulus. The hormone EPO on the other hand is expressed by few

tubulointerstitial cells located along the cortico-medullary border.

With their different localization and function, renin and EPO expressing cells are usually thought of as different cells.

However, they share some similarities. Regulation of EPO and renin producing cells occurs mainly through

recruitment of additional cells rather than by a gradual regulation on a single cell level. Increasing tissue hypoxia

leads to a recruitment of EPO expressing cells expanding from the cortico-medullary border throughout the

interstitium of the kidney cortex and the outer stripe of the outer medulla. Hypotension or salt deficiency lead to a

recruitment of renin producing cells along the afferent arterioles and expanding into the perivascular and

periglomerular interstitium of the cortex causing renin cell hyperplasia. Moreover, EPO and renin producing cells

descend from the same cell population, FoxD1+ stromal progenitor cells.

Emerging evidence points to an even closer relationship between these two cell types than had been originally

thought. Targeted gene deletion of the ubiquitin-ligase von-Hippel-Lindau causing a chronic stabilization of hypoxia-

inducible transcription factor-2, which is the main regulator of the EPO expression, led to an endocrine shift in

classical juxtaglomerular renin producing cells. These cells stopped to produce renin and instead started to express

EPO. Additionally, our group recently found that some interstitial cells belonging to the pool of potential EPO

producing cells actively express renin.

In this regard, this talk will focus on new aspects of the plasticity of stromal erythropoietin and renin producing cells

gained through our research and highlight new insights into the endocrine versatility of the renal interstitium.


of 516

S 04-03

Deciphering hereditary kidney disease: From clinical syndrome to

tubular physiology and back

Karl P. Schlingmann

Münster, Germany

S 04-04

Beyond the airways: Role of CFTR in the renal tubular system

Peder Berg1, Samuel L. Svendsen1, Mads V. Sørensen1, Casper K. Larsen1, Jesper F. Andersen1, Søren

J. Fangel3, Majbritt Jeppesen3, Ines Cabrita2, Karl Kunzelmann2, Rainer Schreiber2, Jens Leipziger1

1 Aarhus University, Biomedicine, Physiology, Aarhus, Denmark 2 Regensburg University, Physiology, Regensburg, Germany 3 Aarhus University Hospital, Infectious Medicine, Aarhus, Denmark

Patients with CF have an increased risk of developing electrolyte disturbances, including metabolic alkalosis. CFTR

is expressed in our kidneys, but its function has remained elusive. As the kidneys are vital to maintain acid-base and

electrolyte homeostasis, we hypothesized the metabolic alkalosis to be a direct consequence of dysfunctional CFTR

in the kidney. With the use of cell cultures, animal models, and the participation of CF patients, we investigated the

role of CFTR in renal acid-base handling. We found that in the collecting ducts, CFTR co-localizes apically with the

base secreting Cl-/HCO3--exchanger, pendrin. Loss of CFTR (in mice) or dysfunction of CFTR (in patients) causes a

markedly reduced capacity of renal base excretion. This is explained by a substantially reduced function of the base-

secreting beta-intercalated cells in the renal collecting duct. In patients, treatment with CF modulator drugs partially

normalized this phenotype. These findings motivated us to develop an in vivo CF urine test to quantify CFTR function

for validation of treatment efficacy. We now pursue this question in a larger clinical study. Preliminary data show for

CF patients, that the reduced and variable ability to acutely increase renal HCO3- excretion correlates with the

remaining lung function.


of 516


Lecture Hall 3

S 05 | Microproteins in cardiovascular

pathophysiology Chair

Ingrid Fleming (Frankfurt/Main)


of 516

S 05-01

Dissecting the role of a novel cardiac-expressed mitochondrial

microprotein

Cat Makarewich

Cincinnati Children's Hospital, Molecular Cardiovascular Biology, Cincinnati, USA

Introduction: Emerging evidence suggests that many RNA molecules currently annotated as noncoding contain short

open reading frames that code for functional small proteins called microproteins. Microproteins play critical roles in a

diverse range of essential biological processes. To identify novel cardiac-expressed microproteins, we used a

comparative genomics approach and identified mitolamban (Mtlbn) as a highly conserved 47-amino acid

transmembrane protein that is abundantly expressed in the heart.

Methods and Results: Subcellular localization studies showed that Mtlbn localized specifically to the inner

mitochondrial membrane. Immunoprecipitations and mass spectrometry analysis indicated that Mtlbn interacted with

subunits of complex III of the electron transport chain (ETC) and we observed the presence of Mtlbn in complex III

respiratory supercomplexes (SCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). Cardiac-specific

Mtlbn overexpressing transgenic (TG) mice and Mtlbn gene-deleted (knockout, KO) mice were generated to dissect

the molecular function of this protein in the heart. Mtlbn TG mice developed cardiomyopathy and died prematurely

with histological, biochemical and ultrastructural pathologic features. Metabolomic analysis indicated that hearts from

TG mice had signs of increased oxidative stress and mitochondrial dysfunction. While Mtlbn KO mice had normal

cardiac function and histological appearance, BN-PAGE analysis of purified mitochondria from KO hearts showed

altered complex III composition. Functional assessment of purified heart mitochondria from Mtlbn KO mice revealed

a reduction in complex III activity and metabolomic analysis of KO heart tissue indicated an altered metabolite profile

consistent with deficiencies in complex III activity.

Conclusions: Mtlbn is a novel heart-enriched microprotein that localizes to the inner mitochondrial membrane where

it interacts with complex III of the ETC. Our data indicate that Mtlbn serves as a critical regulator of mitochondrial

ETC activity through a direct role in affecting complex III function.


of 516

S 05-02

Novel Microproteins in the Human Heart

Norbert Hubner1,2,3

1 Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany 2 Charite-Universitätsmedizin, Berlin, Germany 3 DZHK - Partner site, Berlin, Germany

The human proteome is expected to contain many microproteins translated from presumed long noncoding RNAs

(lncRNAs), which had remained undetected because of their small size (< 100 amino acids). However, physiological

roles have been uncovered only for selected, highly conserved human microproteins and a genome-wide catalogue

of microproteins produced in human heart tissue is missing.

Integrating de novo transcriptome assemblies with ribosome profiling data of 80 human hearts, we detect active

translation of 169 previously undetected microproteins encoded by lncRNAs, 17 of which are exclusively expressed

in the heart based on GTEx expression data. Conservation analysis across 120 mammalian species reveals that

over 90% of microproteins show limited sequence conservation and are restricted to primate species. Despite their

low level of conservation, we confirm microprotein production in vivo (up to 60%) using human heart shotgun mass-

spectrometry data and a highly sensitive targeted mass-spectrometry approach on five human heart samples.

Interestingly, we find strong and significant expression co-regulation of many microproteins (93 out of 169) with

mitochondrial genes and processes. Using immunofluorescence microscopy and MS-based interactome studies, we

confirm a mitochondrial localization for dozens of these microproteins. Moreover, several microproteins show not

only organ-, but cell type-specific expression patterns as revealed by our single cell and single nuclei RNA

sequencing data of human heart tissue. We conclude that many presumed lncRNAs produce evolutionarily young,

small proteins in the human heart in vivo, a fraction of which may be involved in cardiac energy metabolism and heart

disease.


of 516

S 05-03

Function of non-coding and micropeptide-containing RNA transcripts

in vascular repair and regeneration

Andrew Baker

University of Edinburgh, Edinburgh, UK

The remodelling of the vascular system in response to injury is important in the development of a number of vascular

pathologies with unmet clinical need. My laboratory has been interested in the understand of molecular events that

are relevant to pathological remodelling and how to intervene with such pathways and mechanisms to develop

innovative therapies. To this end, we have studies protein coding genes, miRNA (miR-21, miR-143, miR-145 and

miR-214 in particular) as well as long non-coding RNA. In the area of lncRNA, we have identified several important

transcripts (SMILR, CARMN, miR-503HG in particular) that have important functional roles in the development of

vascular pathologies. For example, SMILR controls vascular smooth muscle cell proliferation and via binding to

CENPF. Further, both CARMN and miR-503HG are complex genomic loci with co-location with important miRNAs.

These lncRNAs are important in the control of cell identity and their downregulation in disease leads to vascular

damage. Finally, we have shown that a transcript linc00961/SPAAR is upregulated during endothelial cell

development and has important consequences in the control of angiogenesis. The transcript and the peptide have

important fucntions in angiogenesis and appear to oppose one another. The homologue in mouse was mutated to

assess the importance of the locus in vascular and cardiac damage. When taken together, it is clear that the non-

coding genome has critical impact upon vascular homeostasis and disease, with important functional contributions

to acute ansd chronic vascular remodelling. Therapies that target such molecular mechanisms are potentially

interesting and might lead to the development of new, advanced therapies to prevent pathological remodelling of the

vasculature.

Acknowledgment

British Heart Foundation and the European Research Council


of 516

S 05-04

Proteogenomic discovery of human endothelial smORF-encoded

microproteins.

Mauro Siragusa1,2, Johannes Graumann2,3, Carsten Künne4, Stefan Günther4, Sylvia Jeratsch2,3,

Xiaozhu Zhou1,2, Haaglim Cho5, Stefan Offermanns2,5, Ingrid Fleming1,2

1 Goethe University, Institute for Vascular Signalling, Centre for Molecular Medicine, Frankfurt am Main, Germany 2 German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main and Bad

Nauheim, Germany 3 Max Planck Institute for Heart and Lung Research, Biomolecular Mass Spectrometry, Bad Nauheim, Germany 4 Max Planck Institute for Heart and Lung Research, Bioinformatics and Deep Sequencing Platform, Bad Nauheim,

Germany 5 Max Planck Institute for Heart and Lung Research, Department of Pharmacology, Bad Nauheim, Germany

Background and Aim: Microproteins (miPs) are short peptides encoded by small open reading frames (smORFs -

shorter than 100 codons) that have been implicated in the regulation of biological processes ranging from cell growth

to cellular signalling and metabolism. Endothelial cells, as the inner lining of blood vessels, are situated in a strategic

position to respond to cues derived from the circulating blood as well as from the tissues. The aim of this study was

to identify and characterize endothelial smORF-encoded miPs.

Methods and Results: We developed a novel proteogenomic strategy consisting of RiboTag and bulk next

generation RNA-seq (NGS) as well as mass spectrometry-based proteomics to identify endothelial smORF-encoded

miPs. This yielded ~2.2k miPs encoded by previously non-annotated smORFs in primary human endothelial cells.

NGS of ribosome associated RNAs from endothelial cell-specific RiboTag mice combined with proteomic analyses

revealed the organotypic expression of murine endothelial miPs under homeostatic conditions: 907 in the thoracic

aorta, 271 in the aortic arch, ~1k in the heart and ~3k in the lung. Approximately 2.4k endothelial miPs were detected

in at least two organs. Endothelial cell activation by interleukin-1β was associated with profound alterations in

endothelial miP expression in vitro (1.6k differentially expressed miPs). Similarly, ~1.2k endothelial miPs were

differentially expressed in response to inflammation and endothelial dysfunction in mice (AAV-PCSK9 injection

combined with partial carotid artery ligation). Endothelial FLAG-tagged miPs (FLAG immunofluorescence) displayed

a diverse cellular localization, including the cytosol, plasma membrane, the endoplasmic reticulum or the nucleus.

Functional studies with selected miPs revealed their involvement in the regulation of proliferation, cell growth and the

response to oxidative stress- as well as cytotoxic drug-induced cell death. The study of selected miP interactomes

by mass spectrometry-based proteomics as well as transcriptomes by NGS upon overexpression of selected miPs

was performed to functionally characterize these novel molecular players.

Conclusions: Proteogenomic studies enabled the discovery of previously non-annotated endothelial smORFs and

their encoded miPs under homeostatic conditions. The profound alterations in the expression of miPs in response to

endothelial cell activation suggest a potential involvement in the development of cardiovascular disease.


of 516

S 05-05

Expanding the proteome with non-canonical open reading frames

Jin Chen1,2

1 University of Texas Southwestern Medical Center, Laboratory of Functional Genomics and Translational Control,

Cecil H. and Ida Green Center, Dallas, USA 2 University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas, USA

Recent studies have identified important cellular functions played by microproteins translated from unannotated open

reading frames (ORFs). However, the prevalence and functions of translated non-canonical ORFs and their encoded

proteins remain largely uncharacterized. Here, we exploit ribosome profiling and a systematic CRISPR-based

screening strategy to identify hundreds of non-canonical ORFs that are essential for cellular growth and whose

disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization

of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds

of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded

in upstream ORFs, which form stable complexes with the main, canonical protein encoded on the same messenger

RNA, thereby revealing examples of functional bicistronic operons in mammals. Together, our results point to a family

of functional human microproteins, encoded on supposedly “non-coding” regions, that play critical and diverse cellular

roles.


of 516


Audimax

S 06 | Structure & Function of Presynaptic

Plasticity Chair

Jens Eilers (Leipzig)

Stefan Hallermann (Leipzig)


of 516

S 06-01

Structural and functional vesicle pool engrams in hippocampal mossy

fiber presynaptic terminals

David Vandael1,2, Yuji Okamoto1, Silvia Jamrichova1, Peipeng Lin1, Carolina Borges-Merjane1,

Olena Kim1, Peter Jonas1

1 IST (Institute of Science and Technology) Austria, Cellular Neuroscience, Klosterneuburg, Austria 2 Netherlands Institute for Neuroscience, Royal Netherlands Academy for Arts and Sciences (KNAW), Amsterdam,

Netherlands

Post-tetanic potentiation (PTP) is an attractive candidate mechanism for hippocampus-dependent short-term

memory. Although PTP has a uniquely large magnitude at hippocampal mossy fiber–CA3 pyramidal neuron

synapses, both the physiological significance and the underlying mechanisms are unclear. To address these

questions, we combined subcellular patch-clamp recording in acute hippocampal slices with functional, “flash and

freeze’’, electron microscopy. To determine the mechanisms underlying PTP, we made paired recordings from mossy

fiber terminals and postsynaptic CA3 pyramidal neurons. Presynaptic mossy fiber terminals were stimulated in the

tight-seal cell-attached configuration, and unitary excitatory postsynaptic currents (EPSCs) were recorded in the

postsynaptic CA3 pyramidal neuron under voltage-clamp conditions. PTP was induced by high-frequency trains of

up to 100 action potentials (APs). Mossy fiber PTP showed a remarkably low induction threshold, with ~10 APs being

sufficient to induce PTP. Isolated presynaptic high-frequency stimulation induced larger PTP than simultaneous pre-

and postsynaptic stimulation, suggesting anti-associative properties of PTP. Cumulative release analysis revealed

that PTP was mainly generated by enlargement of the readily releasable pool of synaptic vesicles. To address

whether changes in functional pool size were associated with structural correlates, we performed functional, “flash

and freeze”, electron microscopy experiments. Channelrhodopsin (H134R) was expressed in dentate gyrus granule

cells, using Prox1-CreERT2 mice crossed with Ai32 reporter mice. Application of 1, 5, or 100 stimuli reduced the

number of docked vesicles in an activity-dependent manner, consistent with a depletion of the docked vesicle pool

and a full fusion mechanism of transmitter release at hippocampal mossy fiber terminals. Stimulation of granule cells

with 100 stimuli, followed by a 20-s recovery interval, revealed an increase in the number of docked vesicles beyond

the control value, indicating that overfilling of the docked vesicle pool contributes to PTP. Thus, PTP is associated

with the formation of structural changes in the presynaptic terminal. These ‘‘pool engrams” may form the basis of

hippocampus-dependent short-term memory.

Acknowledgment

This project received funding from the European Research Council (ERC) under the European Union Horizon 2020

Research and Innovation Program (grant agreement 692692 to P.J.) and the Fond zur Förderung der

Wissenschaftlichen Forschung (Z 312-B27, Wittgenstein award to P.J. and V 739- B27 to C.B.-M.).


of 516

S 06-02

Freeze-Frame Shots of Synapses in Action: Correlating Ultrastructure

and Function at the Nanoscale.

Cordelia Imig

University of Copenhagen, Department of Neuroscience, Copenhagen, Denmark

Synapses in the brain can exhibit strikingly different functional properties including the probability of neurotransmitter

release and short-term plasticity characteristics. Electron microscopy (EM) is a powerful technique to resolve the

organisation of synaptic vesicle pools at individual active zone neurotransmitter release sites with nanometer

precision. However, to link ultrastructural observations with defined synaptic activity states that operate on the

millisecond to second timescale, fast sample cryo-fixation approaches in temporal register with controlled synaptic

stimulation are required. The capture of activity-dependent exo- and endocytic events in distinct and functionally

specialized synapses embedded in a tissue-context has remained particularly challenging. In this talk, I will discuss

a methodological approach to apply optogenetic stimulation-coupled cryofixation (“flash-and-freeze) for EM to enable

the study of synaptic ultrastructure in cultured mouse brain tissue. I will highlight the power, but also the challenges

of this technology, for the study of structurally and functionally highly complex synapse types, such as hippocampal

mossy fiber synapses.


of 516

S 06-03

Disease-related variations in the UNC13A gene cause presynaptic

dysfunction

Aisha Ahmad1, Judith. J. Jans2, Holger Taschenberger1, Nils Brose1, Anita Rauch3, Reza Asadollahi3,

Noa Lipstein4,1

1 Max Planck Institute of Experimental Medicine, Department of Molecular Neurobiology, Göttingen, Germany 2 University Medical Center Utrecht, Department of Genetics, Utrecht, Netherlands 3 University of Zurich, Institute of Medical Genetics, Schlieren, Switzerland 4 Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Department of Molecular Physiology and Cell Biology,

Berlin, Germany

Intact communication between neurons occurs at synapses and is absolutely essential for information processing in

neuronal networks. A massive body of work draws a link between genetic variations in synaptic proteins and complex

brain disorders, but the mechanisms by which synaptic transmission is altered are not well-understood. At the

presynaptic compartment, hundreds of proteins act together to determine the strength, timing, and plasticity of

neurotransmitter release, thus shaping the properties of synaptic transmission. Of those, Munc13 proteins are key

regulators of neurotransmitter release, as they mediate the priming step that renders synaptic vesicles fusion-

competent. Here, we describe a novel congenital brain disorder of the synaptic vesicle priming step, characterized

by autism-spectrum disorder, a dyskinetic movement disorder, and intellectual disability. We identified an array of

disease-related variations in all Munc13-1 protein domains, and carried an electrophysiological characterization that

identified both loss- and gain of function mechanisms leading to synaptic transmission dysfunction. Our study

underscores the critical importance of fine-tuned presynaptic control in normal brain function, and adds the neuronal

Munc13 proteins and the synaptic vesicle priming process to the known etiological mechanisms of psychiatric and

neurological disease.


of 516

S 06-04

Homeostatic control of presynaptic function at a cerebellar synapse

Igor Delvendahl, Martin Müller

University of Zurich, Department of Molecular Life Sciences, Zurich, Switzerland

To allow robust behavior, synapses in the central nervous system require functional stability. Homeostatic

mechanisms can stabilize synaptic transmission and neural function across different timescales. Synaptic

homeostasis can be achieved by adaptations of postsynaptic or presynaptic function. Prolonged neural activity

perturbations cause adaptations in postsynaptic receptors abundance, termed synaptic scaling, and this form of

postsynaptic homeostasis has been intensively studied. There is also evidence for presynaptic modifications that

homeostatically stabilize neural function, but our understanding of presynaptic homeostatic plasticity in the central

nervous system is still limited.

Using whole-cell electrophysiology in combination with genetics and pharmacology, we studied homeostatic

modulation of presynaptic function at mossy fiber synapses in the mouse cerebellum. Within 20 minutes after

application of a sub-saturating concentration of a glutamate receptor antagonist, we observed homeostatic increases

in quantal content that maintain synaptic strength. This stabilization of synaptic function is driven by an increase in

the number of release-ready vesicles and fully reversible on the minute time scale. In a genetic model with reduced

AMPA receptor levels, enhanced neurotransmitter release can be sustained over months to stabilize synaptic

transmission strength. Presynaptic recordings directly demonstrate increased neurotransmitter release upon

impaired receptor function, which is also evident from increased non-AMPA-receptor-mediated

transmission. However, complete loss of an AMPA receptor subunit cannot be fully compensated by presynaptic

release modulation and leads to deficits in cerebellum-dependent associative learning.

Together, our data suggest that functional receptor perturbation can cause homeostatic release modulation at a

cerebellar synapse. Presynaptic homeostatic plasticity may contribute to ensure stable neural function on rapid and

prolonged timescales.


of 516

S 06-05

Differential presynaptic structural-functional homeostatic plasticity of

excitatory and inhibitory transmission

Andreas Ritzau-Jost1, Felix Gsell1, Nadine Ehmann1,2, Torsten Bullmann1, Lars Schmidl3,

Sebastian Maaß1, Jens Eilers1, Christian Geis3, Robert J. Kittel1,2, Stefan Hallermann1

1 University of Leipzig, Carl-Ludwig-Institute for Physiology, Faculty of Medicine, Leipzig, Germany 2 Leipzig University, Department of Animal Physiology, Institute of Biology, Leipzig, Germany 3 University Hospital Jena, Hans-Berger Department of Neurology, Jena, Germany

Neuronal networks function via an intricate interplay of excitation and inhibition that needs to be balanced to ensure

stable network function. Chronic disturbance of neuronal activity leads to homeostatic plasticity of excitatory and

inhibitory inputs. Yet, the presynaptic mechanisms that contribute to homeostatic plasticity as well as their potential

differential effects at excitatory and inhibitory synapses stay elusive.Here, we investigate excitatory and inhibitory

synapses in mature cultured neocortical neurons, following long-term activity deprivation via TTX (48hrs).

Interestingly, while inhibitory inputs initially demonstrate stronger synaptic short-term depression as compared to

excitatory synapses, TTX application exerts inverse effects leading to a strengthening of excitatory and a weakening

of inhibitory synapses. Homeostatic adaptation can be mediated by changes in the number of release-ready vesicles,

with an increase at excitatory and a decrease at inhibitory synapses. Focusing on MUNC13-1, a core constituent of

the synaptic vesicle release machinery, we investigate if the differential expression of short-term synaptic plasticity

at individual synaptic populations is accompanied by structural variations. To this end, we employ super-resolution

microscopy to correlate structure with function in order to gain a better understanding of the mechanistic coupling of

molecular composition and plasticity-induced changes in synaptic signaling.


of 516

S 06-06

Control of presynapse assembly and function by autophagy and the

endo-lysosomal system

Volker Haucke

FMP Berlin, Molecular Pharmacology & Cell Biology, Berlin, Germany

Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre-

and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity

of the membrane compartments that supply presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is

unknown. I will discuss recent advances in our understanding of how key components of the presynaptic machinery

for neurotransmitter release are transported and assembled focussing on our recent studies in Drosophila larvae and

in human iPS-derived neurons. These studies have revealed an unexpected function for a lysosome-related organelle

as the basic building block for presynaptic biogenesis.

In the second part of my lecture, I will focus on our recent studies on the role of neuronal autophagy in synaptic

transmission. In spite of its importance for neuronal health, the physiological substrates of neuronal autophagy in the

absence of proteotoxic challenge have remained largely elusive. We have used knockout mice conditionally lacking

the essential autophagy protein ATG5 and quantitative proteomics to demonstrate that loss of neuronal autophagy

causes the selective accumulation of tubular endoplasmic reticulum (ER) in axons, resulting in increased excitatory

neurotransmission and compromised postnatal viability in vivo. The gain in excitatory neurotransmission is shown to

be a consequence of elevated calcium release from ER stores via ryanodine receptors accumulated in axons and at

presynaptic sites. We propose a model in which neuronal autophagy controls axonal ER calcium stores to regulate

neurotransmission in healthy neurons and in the brain.


of 516

Symposia 1 October 2021

10:30 AM – 12:30 PM

Lecture Hall 1

S 07 | When the immiscible function as one:

Interaction of lipids and membrane-spanning

proteins in biological membranes Chair

Wing-Kee Lee (Bielefeld)

Erhard Wischmeyer (Bielefeld)


of 516

S 07-01

The versatile regulation of K2P channels by polyanionic lipids of the

phosphoinositide (PIP2) and fatty acid metabolism (LC-CoA)

Elena Riel1, Björn C. Jürs2, Sönke Cordeiro1, Marianne Musinszki1, Marcus Schewe1,

Thomas Baukrowitz1

1 University of Kiel, Physiology, Kiel, Germany 2 University of Applied Sciences and Medical University, MSH Medical School Hamburg, Hamburg, Germany

Work of the past three decades provided tremendous insight into the regulation of K+ channels - in particular Kir

channels - by polyanionic lipids of the phosphoinositide (e.g. PIP2) and fatty acid metabolism (e.g. Oleoyl-CoA).

However, comparable little is known regarding the phosphoinositide regulation in the K2P channel family and the

effects of long-chain fatty acids CoA esters (LC-CoA, e.g. Oleoyl-CoA) are so far unexplored.

By screening most mammalian K2P channels (12 in total), we report strong polyanionic lipid effects (activation and

inhibition) for all tested K2P channels. In most cases the effects of PIP2 and Oleoyl-CoA were similar causing either

activation or inhibition depending on the respective subgroup. Activation was observed for members of the TREK,

TALK and THIK subfamily with the strongest effect of PIP2 seen for TRAAK (40 fold) and of oleoyl-CoA for TALK-2

(110 fold). In contrast, inhibition was observed for members of the TASK and TRESK subfamilies ranging up to 80

%. In TASK-2 channels our results indicated an activatory as well as an inhibitory PIP2 site with different affinities.

Finally, we provided evidence that PIP2 inhibition in TASK-1 and TASK-3 channels is mediated by closure of the

recently identified lower X-gate as critical mutations (i.e. L244A, R245A) within the gate prevent PIP2 induced

inhibition. Our results disclosed K2P channels as a family of ion channels highly sensitive to polyanionic lipids (PIP2

and LC-CoA), extended our knowledge on the mechanisms of lipid regulation and implicate the metabolisms of these

lipids as possible effector pathways to regulate K2P channel activity.


of 516

S07-02

Ceramide-dependent regulation of mitochondrial function and systemic

metabolism

Jens C. Brüning

Max Planck Institute for Metabolism Research, Neuronal Control of Metabolism, Cologne, North Rhine-Westphalia,

Germany

Ceramide accumulation in non-adipose tissues has been identified as a negative regulator of glucose

tolerance and lipid metabolism. Ceramides are formed by the N-acylation of a sphingoid long chain base

and are central to sphingolipid metabolism. Individual (dihydro-) ceramide synthases (CerS)s generate

ceramides of different acyl-chain lengths (C14:0-C30:0). Only recently through the generation and

characterization of different CerS knockout mice, the specific role of distinct CerSs and their ceramide

products has begun to be elucidated. We have demonstrated that in obesity specifically CerS6-

dependently generated C16:0 ceramides are responsible for the inhibition of β-oxidation in liver and brown

adipose tissue. Strikingly, lack of CerS5, which also catalyzes the formation of C16:0 ceramides, fails to

protect from obesity or obesity-associated insulin resistance. Through the identification of distinct

proteins which interact selective with C16:0 ceramides generated by CerS6, but not with CerS5, we reveal

a novel differential role for the molecular basis, how the same ceramide class exerts distinct biological

functions, dependent on the CerS isoform it was formed by. In contrast, CerS1-dependently generated

C18:0 ceramides, and not CerS6-dependently formed C16:0 ceramides represent critical mediators of

obesity-associated insulin resistance in skeletal muscle. Collectively our experiments validate specific

CerS-proteins rather than inhibition of global ceramide synthesis as novel targets for the treatment of

obesity and obesity-associated insulin resistance.


of 516

S 07-03

Principles of cellular lipid delivery unraveled by rapid protein

perturbation

Elina Ikonen1,2

1 University of Helsinki, Faculty of Medicine, Stem Cells and Metabolism Research Program, Dept. of Anatomy,

Helsinki, Finland 2 Minerva Foundation Institute for Medical Research, Helsinki, Finland

Cell organization and membrane-related functions depend on the correct compartmentalization of lipids. Our

research aims at uncovering key mechanisms that govern the trafficking and storage of major lipid species in human

cells and elucidating disturbances in these processes that can lead to human lipid storage diseases. Lysosomes and

lipid droplets represent major cellular lipid storage sites that dynamically exchange lipids with other membrane

compartments, with the co-operation of lipid transfer proteins and membrane transport.

We are investigating machineries that are responsible for exporting lipoprotein-derived cholesterol from endo-

lysosomal compartments to the plasma membrane and from there to the endoplasmic reticulum and lipid droplets for

storage. We are also studying how lipid droplets are generated from the endoplasmic reticulum and how their

functional contacts with the mother organelle are maintained. We are taking advantage of a system recently

developed by us for rapid inducible degradation of endogenous proteins (Li et al., Nat Methods 16: 866-869, 2019),

in combination with live cell imaging, correlative light electron microscopy, and lipid analyses. This strategy is powerful

for studying rapidly adapting processes, such as lipid transport and metabolism, that typically harness compensatory

routes.


of 516

S 07-04

Sphingolipids and ABCB1 in cancer multidrug resistance

Wing-Kee Lee1,2

1 Bielefeld University, Physiology & Pathophysiology of Cells and Membranes, Bielefeld, Germany 2 Witten/Herdecke University, Physiology, Pathophysiology & Toxicology, Witten, Germany

The cancer multidrug resistance (MDR) phenotype encompasses a myriad of molecular, genetic and cellular

alterations resulting from progressive oncogenic transformation and selection. Drug efflux transporters, in particular

the MDR P-glycoprotein ABCB1, play an important role in MDR but cannot confer the complete phenotype alone

indicating parallel alterations are prerequisite. Sphingolipids are essential constituents of lipid raft domains and

directly participate in functionalization of transmembrane proteins, including providing an optimal lipid

microenvironment for multidrug transporters, and are also perturbed in cancer. We postulate that increased

sphingomyelin content, which develops early in some cancers, recruits, and functionalizes plasma membrane ABCB1

to confer a state of partial MDR, which is completed by increased glucosylceramide synthase activity and the

redistribution of ABCB1 to intracellular vesicles. Moreover, concomitant upregulation of ABCB1 and the proapoptotic

sphingolipid, ceramide, which is in turn extruded by ABCB1, culminates in increased drug resistance in renal cancer

cells. In addition, the balance of differing ceramide species switches in MDR, favoring ceramides with longer fatty

acid chain lengths, which may be crucial for the accommodation of drug transporters into the membrane bilayer.

Recent data concerning the multifaceted roles of sphingolipid metabolizing enzymes and sphingolipid-dependence

of normal and mislocalized ABCB1 expression and activity in the context of MDR and tumor cell survival will be

presented and discussed.

Acknowledgment

The presented research was funded by the Max Kade Foundation, Westermann Westdorp Foundation, Intramural

Research Program at Witten/Herdecke University, Erasmus Plus and the German Academic Exchange Service (DAAD).

Model for sphingolipids and ABCB1

The detoxifying drug transporter ABCB1 is

regulated by multiple factors governing its

expression and functionalization. Increased

total ABCB1 activity results in transport and

mislocalization of cell death promoting

molecules, such as ceramide and chemotherapeutic

drugs, leading to enhanced tumor cell survival.


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S 07-05

Regulation of cell functions by ceramide-beta-1-integrin interactions

Erich Gulbins

University of Duisburg-Essen, Dept. of Molecular Biology, Essen, Germany

The molecular mechanisms that lead to the increased susceptibility of cystic fibrosis (CF) patients to bacterial

infections are unknown. We demonstrate that β1-integrins are ectopically expressed on the luminal surface of the

plasma membrane of upper airway epithelial cells of mice and humans. Ectopic expression of β1-integrins is

mediated by increased ceramide concentrations and formation of ceramide platforms that interact with and trap β1-

integrins on the luminal pole of bronchial epithelial cell membranes. Ectopically expressed β1-integrins in CF cells

down-regulate the expression of acid ceramidase in epithelial cells via regulation of IRF-8. The IRF-8 mediated

suppression of acid ceramidase expression results in further accumulation of ceramide and reduction of surface

sphingosine levels. Interrupting this vicious cycle by inhalation of anti-β1-integrin antibodies or arginine-glycine-

aspartate (RGD) peptides, which bind to and trigger internalization of surface β1-integrins, or by genetic or

pharmacological correction of ceramide levels or by manipulation of IRF-8 expression normalizes β1-integrin

distribution and sphingosine levels in the lung. This sphingosine kills invading pathogens and prevents pulmonary P.

aeruginosainfections in CF mice.

Acknowledgment

The study was supported by the DFG.


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Symposia 1 October 2021 10:30 AM – 12:30 PM

Audimax

S 09 | Young Physiologists Chair

Gustavo Chaves (Nuremberg)

Dominik Lenz (Marburg)

Andreas Ritzau-Jost (Leipzig)

Dear Physiologists,

Welcome to the Young Physiologist Symposium! The German Physiological Society fosters early-career

physiologists and we, the Young Physiologists, are proud to again be part of this anniversary meeting’s

schedule. The symposium is organized by early-career scientists for early-career scientists, and it covers

both, up-to-date science as well as science entertainment. The scientific speakers of this symposium won

the ‘best talk’ awards during this year’s 10th Young Physiologists Meeting (March 2021),and hence will

present their current research to a broad audience:

Jan Clusmann (Aachen) works on uncovering signal pathways involved in necroptosis in glioblastomata.

Bettina Kolen’s work (Jülich) focuses on the characterization of the vesicular glutamate transporters by

electrophysiological patch-clamp techniques. Finally, Yoshiyuki Henning (Essen) investigates the

pathophysiology of age-related macular degeneration with respect to oxidative stress and hypoxia.

Furthermore, to amalgamate science and entertainment, this symposium features a special guest: Janina

Isabell Otto (https://janinaexplainsitall.de/)! As a biologist and science journalist, she presents scientific

topics with charm and humor. Be curious about an entertaining talk of the multiple award-winning science

communicator!

As does the field of physiology, this symposium covers a broad range of topics and offers up-to-date

insights into diverse research areas. We highly encourage you to engage in an active discussion following

the presentations and to get to know the Young Physiologists.

Have a pleasant stay in Frankfurt!

Best regards,

Your Young Physiologists

https://www.physiologische-gesellschaft.de/junge-physiologen/


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S 09-01

Hypoxia-inducible factor-2α exacerbates oxidative damage in a cell

culture model of age-related macular degeneration

Yoshiyuki Henning, Darius Molitor, Joachim Fandrey, Ursula S. Blind

University of Duisburg-Essen, Faculty of Medicine, Institute of Physiology, Essen, Germany

Background

Oxidative stress and hypoxia in the retinal pigment epithelium (RPE) have long been considered major risk factors

in the pathophysiology of age-related macular degeneration (AMD), a major cause of blinding among elderly people.

Dry AMD is the most common type of AMD; however, no convincing treatment is available to date. Thus, to promote

prevention and to develop novel treatment strategies against dry AMD, it is necessary to identify the

pathophysiological pathways in which oxidative stress and hypoxia are involved. In the present study, we focused on

hypoxia-inducible factors (HIF) 1α and 2α, the α-subunits of HIF-1 and HIF-2, key regulators of cellular adaptation to

hypoxic conditions. For this purpose, we developed a dry AMD cell culture model to identify the role of HIFs in AMD

pathophysiology.

Methods

We treated human RPE cells with sodium iodate (SI), an oxidative stress agent, together with DMOG, a prolyl

hydroxylase (PHD) inhibitor which increases HIF-α levels, under 3 % O2 in a hypoxic chamber to simulate AMD-

related conditions. Treatment effects were analyzed using cell viability assays, FACS, western blot, and quantitative

real-time PCR. Furthermore, siRNA knockdown was conducted and several inhibitors of cell signaling pathways were

used.

Results

Cell viability of ARPE-19 cells was significantly decreased in cells treated with SI/DMOG compared to SI or DMOG

alone, suggesting that HIF accumulation exacerbates oxidative damage. Necroptosis was identified as the main cell

death mechanism using FACS analyses and inhibitors of the necroptosis and apoptosis pathways. Moreover,

knockdown of HIF-1α and HIF-2α using siRNA revealed that HIF-2α is responsible for exacerbation of oxidative

damage. We discuss our observations in terms of the mTOR signaling pathway and autophagy inhibition, since

blocking of mTOR improved cell viability.

Conclusions

Taken together, our data suggest that selectively blocking HIF-2α could be a potential treatment strategy to protect

the aging RPE against extensive oxidative damage, which ultimately protects against AMD.


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S 09-02

Vesicular glutamate transporters are anion channels with larger

diameter pores

Bettina Kolen, Daniel Kortzak, Christoph Fahlke

Forschungszentrum Jülich, Institute of Biological Information Processing (IBI-1), Jülich, Germany

Vesicular glutamate transporters (VGLUTs) mediate the uptake of the excitatory neurotransmitter glutamate into

synaptic vesicles and are thus crucially involved in the strength of excitatory synaptic transmission. They are not only

of high physiological importance, but also functionally unique: VGLUTs are thought to be secondary active glutamate

transporters, but can also function as anion channels or as Na+-coupled phosphate transporters [1]. Neither the

mechanism nor the function are completely understood to date.

To characterize VGLUTs using electrophysiological patch clamp techniques, we used a VGLUT1 with mutations in

targeting signals in the amino- and carboxy-terminal regions and heterologously expressed VGLUT1PM as a GFP

fusion protein in the plasma membrane of HEK293T cells. We examined the function of the anion channel using a

combination of whole cell patch clamp, noise analysis and fluorescence intensity measurements.

We found that VGLUT1PM forms a strongly inward rectifying pH- and Cl--dependent chloride channel with a lyotropic

anion selectivity and a single channel amplitude of ~24 fA with Cl- as permeant anion. Next to Cl-, VGLUT1PM is also

permeable to several large anions such as glutamate, gluconate or aspartate. Glutamate differs from other large

anions in a much more positive reversal potential. Thus, VGLUT1 functions as a highly selective glutamate

transporter or channel with a large pore within synaptic vesicles. A point mutation, H120A, which is thought to affect

the transport of glutamate [2], showed altered channel gating in our measurements with markedly increased

macroscopic current amplitudes due to a twofold higher single channel amplitude. In addition, we observed changes

in anion selectivity and reduced glutamate permeability.Our results demonstrate that VGLUT1 can function as an

anion channel with a large pore and provide new insights into the different transport functions of vesicular glutamate

transporters.

Excitatory synaptic transmission at a

glutamatergic synapse.

Ca2+ influx triggers the fusion of synaptic

vesicles with the presynaptic membrane,

releasing glutamate into the synaptic cleft

where it can bind to postsynaptic receptors.

EAATs terminate the signal by transport of

glutamate into neighboring glial cells where it

is metabolized to Glutamine (Gln) and

transported back to the neurons. Within neurons,

glutamate can be synthesized from glucose or

resynthesized from Gln by PAG. Synaptic vesicles

are recycled and refilled with glutamate by

VGLUTs, driven by the electrochemical proton

gradient that is generated by the V-ATPase.

References [1] Preobraschenski, J. et al. (2018), Cell Reports, 23, 535-545. [2] Juge, N. et al. (2006), JBC, 281,39499-39506.


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S 09-03

Acid-sensing ion channels induce necroptosis in glioblastoma stem

cell lines

Jan Clusmann, David Corredor, Daniel Cortes, Nina Boersch, Yuemin Tian, Stefan Gründer

RWTH Aachen, Institute of Physiology, Aachen, Germany

Introduction and hypothesis

Necroptosis is a form of regulated cell death, inducing membrane rupture and immunogenic cell death upon

extracellular signaling, most commonly via TNF-a. Eliciting necroptosis has become a research focus in cancer

treatment as tumor cells are frequently resistant to apoptosis.

Recently, it was proposed that acid sensing ion channel 1a (ASIC1a) might induce necroptosis. ASICs are neuronal

proton-gated sodium channels, with ASIC1a being predominantly expressed in the central nervous system (CNS).

ASIC1a is, among other conditions, responsible for acid induced cell death in mouse models of ischemic stroke.

Pharmacological blockade of ASIC, but also interference with the necroptosis pathway attenuate the acid induced

cell death.

We have previously shown that cancer stem cells from glioblastoma, a highly aggressive brain tumor, express

functional ASIC1. We hypothesized that ASIC activation might decrease viability and induces necroptosis.

Results

In a tumorsphere formation assay, acidic pH (6.6) induced cell death with necrotic phenotype, decreasing viability by

about 1/3. This could be prevented by Nec-1, a necroptosis inhibitor. ASIC1 antagonist PcTx1 similarly prevented

cell death at acidic pH, the ASIC1 agonist MitTx1 induced cell death even at neutral pH. Interestingly, a fragment of

only 20 amino acids of the ASIC1a c-terminal sequence was sufficient to strongly induce cell death, even in absence

of functional ASIC1a. Western blotting revealed abundance and increased rate of phosphorylation events of several

proteins involved in the necroptosis signaling cascade at acidic pH. Electron microscopy confirmed increased

abundance of necrotic phenotypes at acidic pH.

Conclusion

Our study promotes ASIC1a as a death receptor. Induction of cell death via ASIC1a could be prevented by Nec-1,

suggesting involvement in the necroptosis pathway. As tumors often grow in an acidic microenvironment, this

pathway might shape tumor evolution and might be a potential target in tumor therapy.

References [1] Wang, Y. Z., Wang, J. J., Huang, Y., Liu, F., Zeng, W. Z., Li, Y., . . . Xu, T. L. (2015). Tissue acidosis

induces neuronal necroptosis via ASIC1a channel independent of its ionic conduction. Elife, 4. doi:10.7554/eLife.05682

[2] Tian, Y., Bresenitz, P., Reska, A., El Moussaoui, L., Beier, C. P., & Grunder, S. (2017). Glioblastoma cancer stem cell lines express functional acid sensing ion channels ASIC1a and ASIC3. Sci Rep, 7(1), 13674. doi:10.1038/s41598-017-13666-9

[3] Wang, J.-J., Liu, F., Yang, F., Wang, Y.-Z., Qi, X., Li, Y., . . . Xu, T.-L. (2020). Disruption of auto-inhibition underlies conformational signaling of ASIC1a to induce neuronal necroptosis. Nat Commun, 11(1), 475. doi:10.1038/s41467-019-13873-0

[4] Krysko, O., Aaes, T. L., Kagan, V. E., D'Herde, K., Bachert, C., Leybaert, L., . . . Krysko, D. V. (2017). Necroptotic cell death in anti-cancer therapy. Immunol Rev, 280(1), 207-219. doi:10.1111/imr.12583

[5] Stupp, R., Mason, W. P., van den Bent, M. J., Weller, M., Fisher, B., Taphoorn, M. J., . . . Mirimanoff, R. O. (2005). Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med, 352(10), 987-996. doi:10.1056/NEJMoa043330


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S 09-04

Sex Smells – Science Slam Talk

Janina I. Otto

Science Journalist, Hannover, Germany

The Young Physiologist invited Science Slam artist Janina Otto to give an entertaining Science Slam Talk. She will

be presenting snippets from her current science slam shows about pheromones and sweat. The Science Slam is a

very popular science communication format that was invented in Darmstadt, Germany. The rules are similar to a

Poetry Slam. Participants perform on stage and the audience gets to decide who wins. The format was successfully

exported to different countries around the world. Janina Otto performs on science slam events since 2015 in Germany

but also international (Mexico, Russia, Switzerland). She was awarded with a first price several times.

We are all sweating on an everyday basis even though we usually deny that fact. Usually, we do not enjoy our

sweating very much. Nevertheless, we all do it all the time. Isn’t that strange – we are humans that like to control

nearly everything from our calorie intake to rocket science, but actually we can not control our own sweating. It seems

like this is a very important function to our body. Is that possibly linked to pheromones? Is there such a thing as

human pheromones at all? If not, why does the story about human pheromones persist so stubbornly in our minds?

Studies on the subject raise questions: Does androstenone make you irresistible? Do strippers really earn more tips

when ovulating? Are women's tears perhaps total lust killers for men for entirely different reasons then you think?

These studies could be misleading us, or is there some truth to the pheromone myth?


of 516


Lecture Hall 3

S 10 | Microcirculation in Inflammation and

Development Chair

Catherine Robin (Rotterdam)

Markus Sperandio (Munich)


of 516

S 10-01

Regulation of the hematopoietic stem cell birth during embryonic

development

Catherine Robin

Hubrecht Institute & Royal Netherlands Academy of Arts and Sciences (KNAW), Regenerative Medicine Center,

University Medical Center Utrecht, Utrecht, Netherlands

The journey of hematopoietic stem cells (HSCs) starts during embryonic development where they first emerge in the

main arteries of vertebrate embryos, such as the aorta. HSCs arise from hemogenic endothelial (HE) cells via an

endothelial-to-hematopoietic transition (EHT) and the formation of intra-aortic hematopoietic clusters (IAHCs). The

molecular events controlling endothelial specification, EHT and IAHC formation, as it occurs in vivo inside the aorta,

are very complex and still poorly understood. The defined location of IAHCs/HSCs within the aortic niche also

regulates their fate, behaviour, and molecular identity via a complex extrinsic regulation. We made use of genome-

wide RNA sequencing and tomography sequencing to explore the molecular characteristics and key components of

IAHCs and precursors and the complexity of the aortic microenvironment landscape. Through interspecies

comparative RNA sequencing analyses of zebrafish, chicken, mouse, and human embryos, we identified novel

conserved processes that control HSC generation and mapped interactions between the niche and IAHCs. Among

the various factors interacting to spatiotemporally regulate HSC generation in vivo, we functionally identified the

niche-secreted ligand ADM and its IAHCs expressed receptor RAMP2, as well as SVEP1. Understanding the different

cellular and molecular aspects that regulate HSC birth in vivowill pave the way for improved HSC production in vitro

and clinical cell therapy.


of 516

S 10-02

Macrophages at the neuro-vascular interface and spinal cord

regeneration

Ferdinand le Noble

Karlsruhe Institute of Technology (KIT), Department of Cell and Developmental Biology, Karlsruhe, Germany

In man, spinal cord injury induced destruction of neuronal tissues associates with disrupted motor- and sensory

neuron signal processing, accounting for debilitating conditions. Repairing the damaged neuronal tissue, and

restoring neuronal connectivity and function is considered of high medical relevance for treating affected patients.

Unfortunately, in man neurons regenerate poorly and which molecular signaling pathways should be targeted to

restore regenerative capacity is an outstanding question in the field. In contrast to man, zebrafish show a remarkable

regenerative capacity of both the central and peripheral nervous system. In the spinal cord, regeneration involves a

complex interplay between neuron-derived cytokines like Vegf, blood vessels, oxygen radicals and the local

inflammatory milieu controlling the behavior of macrophages and neutrophils. It is postulated that macrophages

promote spinal cord regeneration by controlling post-injury inflammatory responses. However, the mechanistic basis

of how macrophages regulate regeneration is poorly understood. To address this we generated a series of (tissue

specific) mutants for neuron derived cytokines and their receptors, NADPH oxidases, as well as transgenics with a

loss or gain of macrophage scenarios and investigated repair processes upon spinal cord injury. We uncovered a

hitherto unknown role for precise titration of neuronal Vegf dynamics, essential for controlling robust spinal cord

regeneration in spatio-temporal manner.


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S 10-03

Macrophage diversity in cardiovascular inflammation

Christian Schulz

Ludwig Maximilian University, Cardiology, Munich, Germany

Cardiovascular macrophages are heterogenous and have different developmental origins. However, the association

of macrophage ontogeny with their phenotypes and functions in adulthood has been unclear. By combining immune

phenotyping with fate mapping analysis we addressed their diversity in homeostasis and inflammation. In-depth

understanding of macrophage subsets may lead to the development of targeted therapies to reduce adverse

remodeling and guide tissue healing.


of 516

S 10-04

Time of day regulation of adaptive immunity

Christoph Scheiermann

University of Geneva, Department of Pathology and Immunology (PATIM), Centre Médical Universitaire (CMU),

Genève 4, Switzerland

Migration of leukocytes from the skin to lymph nodes via afferent lymphatic vessels is pivotal for adaptive immune

responses. Circadian rhythms have emerged as important regulators of leukocyte trafficking to lymph nodes via the

blood. I will discuss that dendritic cells (DCs) have a circadian migration pattern into lymphatic vessels, which peaks

during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of

chemokines and of adhesion molecules in both mice and humans. Chrono-pharmacological targeting of the involved

factors abrogates circadian migration of DCs. I will show data demonstrating that cell-intrinsic circadian oscillations

in skin lymphatic endothelial cells and DCs co-govern these rhythms, as their genetic disruption in either cell type

ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin

lymphatics, a process that is essential for many adaptive immune responses, and relevant for vaccination and

immunotherapies.


of 516

S 10-05

MASTer the defence: Unraveling skin mast cell functions in neutrophil

recruitment.

Jan Dudeck

Otto-von-Guericke-Universität Magdeburg, Institut für Molekulare und Klinische Immunologie, Magdeburg,

Germany

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular

mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for

neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal

crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being

dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional

degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently,

intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC

degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential.

Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent

an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms,

or intentionally pushing immune defense.

MC: mast cell, MCG: mast cell granule


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S 10-06

Regulation of neutrophil recruitment during fetal ontogeny

Ina Rohwedder

Munich, Germany


of 516

Symposia 2 October 2021

10:45 AM – 12:45 PM

Lecture Hall 1

S 11 | Acid-base transport and metabolism: from

sea life to human disease Chair

Jens Leipziger (Aarhus)

Carsten Wagner (Zurich)


of 516

S 11-01

pH regulatory systems in calcifying cells of the sea urchin larva

Marian Y. - A. Hu

Christian-Albrechts-Universität zu Kiel, Institute of Physiology, Kiel, Germany

To generate CaCO3 skeletons and shells calcifying organisms need to acquire carbonate ions that largely derive

from the hydration of metabolic CO2. In this process protons are liberated that need to be removed from the

calcification front to promote calcification and to defend cellular acid-base balance. Thus, biological calcification and

pH regulation are intrinsically linked processes that remain relatively unexplored.

The sea urchin embryo develops an elaborate calcitic endoskeleton produced by the primary mesenchyme cells

(PMCs) that exocytose amorphous calcium carbonate. Using a wide range of techniques ranging from life cell

imaging and intracellular pH recordings over protein-biochemical methods to molecular technologies our work

identified a set of PMC specific ion transporters and channels that mediate the provision of HCO3- and removal of

protons liberated by the calcification process. During skeleton regeneration pHi and intracellular HCO3- levels are

increased accompanied by a change in proton transport pathways. During active mineralization, PMCs switch from

an Na+/H+-exchanger- based proton secretion mechanism to an involvement of an Otopetrin proton channel to

remove protons from the calcification front.

Using the sea urchin larva as a model organism we are able to resolve fundamental mechanisms of acid-base

transport in calcifying systems including so far unknown mechanisms to eliminate protons from the calcification front.

Energy efficient mechanisms of intracellular pH regulation to generate CaCO3 from a metabolic waste product –

carbon dioxide- demonstrates the elegance of nature in utilizing resources in the most sustainable manner. In this

way, insights into the cellular calcification mechanisms may help to develop novel approaches for biology-inspired

technologies to sequestrate carbon in times of rapid climate change.


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S 11-02

NBCn1 increases NH4+ reabsorption across renal thick ascending

limbs, the capacity for urinary NH4+ excretion, and early recovery from

metabolic acidosis

Jeppe Olsen, Samuel Svendsen, Peder Berg, Vibeke Dam, Mads Sorensen, Vladimir Matchkov,

Jens Leipziger, Ebbe Boedtkjer Aarhus University, Department of Biomedicine, Aarhus, Denmark

Background and Question: Recovery from metabolic acidosis necessitates increased renal net acid excretion

through urinary elimination of NH4+. Renal thick ascending limbs (TAL) contribute to a medullary shortcut whereby

NH4+ originating from proximal tubules is ultimately secreted in collecting ducts. NH4

+ transfer across TAL requires a

basolateral exit pathway for H+ in order to avoid intracellular accumulation. The electroneutral Na+,HCO3–-

cotransporter NBCn1 (Slc4a7) is expressed in basolateral membranes of TAL but we have lacked direct evidence

that NBCn1 contributes to acid-base handling in TAL, urinary net acid excretion, and systemic acid-base

homeostasis.

Methods: We induced metabolic acidosis in wild type and NBCn1 knockout mice, performed fluorescence-based

intracellular pH recordings, and measured NH4+ transport across isolated perfused TAL. We evaluated NBCn1

expression by quantitative RT-PCR and immunoblotting, tissue-[NH4+] in renal biopsies, NH4

+ excretion and titratable

acid in spot urine, and arterial blood gasses in normoventilated mice.

Results: Basolateral Na+,HCO3–-cotransport activity was similar in isolated perfused TAL from wild type and NBCn1

knockout mice under control conditions. During metabolic acidosis, basolateral Na+,HCO3–-cotransport activity

increased 4-fold in TAL from wild type mice but remained unchanged in TAL from NBCn1 knockout mice.

Correspondingly, NBCn1 protein expression in wild type mice increased 10-fold in inner stripe of renal outer medulla

during metabolic acidosis. During systemicacid-loading, knockout of NBCn1 inhibited the net NH4+ reabsorption

across TAL by ~60%, abolished the renal cortico-medullary NH4+ gradient, reduced the capacity for urinary NH4

+

excretion by ~50%, and delayed recovery of arterial blood pH and standard-[HCO3–] from their initial

decline.Conclusions: This study identifies NBCn1 as crucial for acid-base handling in TAL and for early renal

compensation of systemic acid-base disturbances. During metabolic acidosis, NBCn1 is required for the upregulated

basolateral HCO3– uptake and transepithelial NH4

+ reabsorption in TAL, renal medullary NH4+ accumulation, urinary

NH4+ excretion, and early recovery of arterial blood pH and standard-[HCO3

–]. We propose that NBCn1 facilitates

urinary net acid excretion by neutralizing intracellular H+ released during NH4+ reabsorption across TAL.

Acknowledgment

This work was supported by the Independent Research Fund Denmark (grants no. 4004-00137B and 10-094816 to

Ebbe Boedtkjer).

References [1] Olsen JSM, Svendsen S, Berg P, Dam VS, Sorensen MV, Matchkov VV, Leipziger J, Boedtkjer E. NBCn1 increases

NH4+ reabsorption across thick ascending limbs, the capacity for urinary NH4+ excretion, and early recovery

from metabolic acidosis. J Am Soc Nephrol 2021;32:852-865.


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S 11-03

Slc4a9 (Ae4) is essential for the appropriate response of renal ß-

intercalated cells to base loading

Helga Vitzthum1, Samuel L. Svendsen2, Peder Berg2, Christian A. Hübner3, Carsten A. Wagner4, Jens

Leipziger2, Catherine Meyer-Schwesinger1, Heimo Ehmke1

1 University Medical Center Hamburg-Eppendorf, Institute of Cellular and Integrative Physiology, Hamburg,

Germany 2 Aarhus University, Department of Biomedicine, Physiology, Health, Aarhus, Denmark 3 University Hospital Jena, Institute of Human Genetics, Jena, Germany 4 University of Zürich, Institute of Physiology, Zürich, Switzerland

Background: Renal acid (H+) and base (HCO3-) excretion rapidly change upon systemic acid-base imbalances. How

the kidney senses and reacts to acid-base imbalances is not yet fully understood. One critical mechanism to defeat

metabolic alkalosis is the urinary excretion of HCO3- from distal tubular β-Intercalated cells (β-ICs) via the HCO3

-/Cl-

exchanger pendrin (Slc26a4). Besides pendrin, β-ICs markedly express the basolateral transporter Slc4a9 (Ae4),

whose function is unknown. Here we analyzed whether the Slc4a9 (Ae4) is involved in the regulation of pendrin-

dependent HCO3- secretion.

Methods: The subcellular localization of Slc4a9 (Ae4) was visualized across species. Slc4a9-knockout (Slc4a9-/-)

and wild-type (Slc4a9+/+) mice received a normal or salt deficient diet for up to 7 days combined with an oral alkali

loading. The acid-base status, and renal abundance, subcellular distribution and activity of pendrin were analyzed.

Results: Slc4a9 (Ae4), was exclusively expressed at the basolateral aspect of ß-ICs throughout species. Slc4a9+/+

mice elicited a proper response to alkali loading, characterized by an increase of pendrin mRNA and protein

abundance, a shift of pendrin to the apical membrane, an enhanced pendrin activity in ß-ICs, and an elevated urinary

HCO3- excretion. In contrast, Slc4a9-/- littermates failed to initiate any activation of pendrin and did not increase urinary

HCO3- excretion, culminating in severe metabolic alkalosis under salt restricted conditions.

Conclusion: The basolateral transporter Slc4a9 (Ae4) is essential for the regulated urinary HCO3- excretion via

pendrin. An insufficient function of Slc4a9 (Ae4) precludes the proper reaction of ß-ICs to alkalosis and thereby

facilitates the development of life threatening acid-base disorders.


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S 11-04

Secretin: A homeostatic HCO3- hormone

Peder Berg1, Jesper F. Andersen1, Tobias Jensen1, Mads V. Sørensen1, Tobias Wang2, Hans Malte2,

Billy K. Chow3, Jens Leipziger1

1 Aarhus University, Department of Biomedicine, Physiology, Aarhus, Denmark 2 Aarhus University, Department of Biology, Zoophysiology, Aarhus, Denmark 3 University of Hong Kong, School of Biological Sciences, Hong Kong, Hong Kong

The secretin receptor is functionally expressed on the basolateral side of beta intercalated cells (β-IC) of the kidney

collecting duct. Secretin stimulates a marked urine alkalization by activating β-ICs. In mice lacking pendrin or

CFTR and in human CF patients the secretin effect on urine alkalization is absent. Here we study the secretin

receptor KO mouse (SCTR-KO) and find complete absence of acute secretin-induced urine HCO3- excretion in

bladder catheterized, anesthetized mice. In metabolic cages, net base excretion (NBE) was measured after applying

varying doses of acute NaHCO3 gavage loads. SCTR-KO mice were unable to increase NBE when administered a

moderate load were WT mice were fully responsive. These results indicate that KO mice cannot stimulate acute renal

HCO3- excretion and thus develop metabolic alkalosis. Subsequently, we studied the respiratory consequences of

acute oral base loading in WT and SCTR-KO, CFTR KO and pendrin KO mice. In WT mice, oral base loading induced

a dose-dependent metabolic alkalosis, fast urinary removal of base but no perturbation of respiration when subjected

to a moderate base-load. In contrast, SCTR KO, CFTR KO and pendrin KO mice that lack the ability to excrete

excess base into the urine, developed a marked and transient (~2h) depression of respiration when subjected to the

same base-load. The swift renal response to eliminate excess base appears a necessary physiological function to

avoid respiratory depression. The alkaline tide, i.e. the transient urinary alkalization after each meal is suggested to

be caused by fast secretin activation of the β-ICs. It may have evolved for proactive avoidance of hypoventilation in

the post-prandial state. Secretin plasma levels rise during metabolic alkalosis and we suggest secretin to be a

genuine, previously undiscovered homeostatic HCO3- hormone.


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S 11-05

New mechanistic insights how alkali therapy slows-down the

progression of chronic kidney disease

Eva M. Pastor Arroyo1,2, Nima Yassini1,3, Elif Sakiri3, Giancarlo Russo4, Soline Bourgeois1,2, Nilufar

Mohebbi5, Kerstin Amann6, Nicole Joller3, Carsten A. Wagner1,2, Pedro Henrique Imenez Silva1,2

1 University of Zurich, Institute of Physiology, Zurich, Switzerland 2 National Center of Competence in Research, NCCR Kidney.CH, Zurich, Switzerland 3 University of Zurich, Institute of Experimental Immunology, Zurich, Switzerland 4 University of Zurich, Functional Genomics Center Zürich, Zurich, Switzerland 5 University of Zurich, Division of Nephrology, Zurich, Switzerland 6 University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Institute of Pathology, Erlangen,

Germany

Chronic kidney disease (CKD) affects between 10 to 15 % of the population worldwide and halting its progression is

still challenging. Patients with deteriorating renal function retain acid. In early CKD stages, serum bicarbonate is

frequently kept within normal ranges, while acids accumulate in the interstitial and intracellular spaces. This condition

has been termed eubicarbonatemic metabolic acidosis, possibly a preclinical form of acidosis. In later stages, serum

bicarbonate is frequently reduced, causing overt metabolic acidosis. Therefore, metabolic acidosis is a common

consequence of CKD, but also functions as a driver of CKD progression. The correction of metabolic acidosis can

interrupt this vicious cycle and has been shown as an effective method to slow-down the progression of CKD. Such

correction is normally achieved with oral supplementation of alkalinizing salts (alkali therapy), like sodium bicarbonate

or sodium citrate. However, the mechanisms how alkali therapy protect renal function are not well understood, and

currently proposed hypotheses may apply only to specific etiologies or animal models.

We recently identified in a murine crystal nephropathy model that alkali therapy protects kidney function, preserves

renal alpha-klotho levels, reduces T helper cell and inflammatory monocyte abundance in kidneys, while it stimulates

multiple metabolic pathways, such as lipid, cholesterol, and iron homeostasis. Similar metabolic pathways are also

altered in kidney biopsies from kidney transplant patients with metabolic acidosis. Alkali therapy in these patients

mainly restored the expression of genes associated to bicarbonate transport and energy metabolism. However, alkali

therapy does not seem to provide its benefits via inhibition of renin-angiotensin-aldosterone system and endothelin-

1 as expected from experiments with nephrectomized rats. We expand the view how alkali therapy interacts with

mechanisms leading to poorer kidney function in CKD, which may yield fruitful insights how to generate better and

more specific therapies.


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S 11-06

Endothelial cell-mediated vascular responses to CO2 contribute to

normal brain function

Jan Wenzel

University of Lübeck, Institute for Experimental and Clinical Pharmacology and Toxicology, Lübeck, Germany

Endothelial cells form the inner layer of vessels and act as an interface between blood and the surrounding tissue.

They are able to respond to blood components including gaseous molecules like CO2. Increased CO2 induces a huge

increase of perfusion in the brain which might be necessary to remove it from this highly vulnerable tissue.

We describe a GPR4-Gαq/11-mediated mechanism by which endothelial cells sense the acidic environment that is

induced by increased CO2 and found this mechanism to be essential for CO2-induced blood flow changes in the

brain. Mice that lack this signaling pathway perform differently in CO2-induced behavior like fear responses,

respiration, and CO2-induced arousal from sleep, indicating a crucial role of the pH sensing machinery in the

vasculature for brain function.


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Audimax

S 12 | Low Oxygen - Sensing and Fighting

Hypoxia Chair

Joachim Fandrey (Duisburg)

Sandra Winning (Duisburg)


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S 12-01

Determinants of the transcriptional response to hypoxia

Johannes Schödel

University Hospital Erlangen-Nürnberg, Department of Nephrology and Hypertension, Erlangen, Germany

The discovery of the oxygen sensing mechanisms and hypoxia-inducible transcription factors (HIF) that are critically

involved in regulating red blood cell production (‘erythropoiesis’) has spawned a major global research field. In this

respect, increasing erythropoietin (EPO) levels through the application of HIF activators has been evaluated in late

stage clinical trials to correct anaemia in patients with chronic kidney disease. Conversely, overstimulation of the HIF

system is observed in tubular cells in most cases of kidney cancer and so HIF inhibitors are currently being evaluated

in that setting.

These differential effects prompted us to develop a research program for identifying mechanisms and modifiers of

HIF transcriptional activity in different cell types.

Some of the major findings of our work are: a) HIF acts as an activator of transcription; b) the repertoire of HIF target

genes is in the order of a few hundred per cell type; c) the overlap of direct HIF target genes between different cell

types is about 30-40%. These observations contrast with a high frequency of potential HIF responsive elements in

the genome (>one million). Therefore, additional mechanisms must be involved in directing HIF to its target loci.

Since DNA interaction is a prerequisite for HIF’s main function to orchestrate and re-program transcription, we applied

a variety of genome-wide and locus specific assays to identify genetic and epigenetic mechanisms, which interfere

with these interactions possibly contributing to an altered hypoxic response and renal disease

progression.Addressing these issues is of critical importance given that theHIF activators are already licensed for the

treatment of renal anemia in some countries.

Acknowledgment

Our work is funded by the DFG (387509280–SFB 1350 C5).


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S 12-02

On the function of androglobin: linking oxygen physiology and

ciliogenesis

Anna Keppner, Miguel Correia, Darko Maric, Teng Wei Koay, Ilaria Orlando, David Hoogewijs

University of Fribourg, Department of endocrinology, metabolism and cardiovascular system, Fribourg, Switzerland

The author has objected to a publication of the abstract.

S 12-03

Inflammatory hypoxia: how HIFs shape leukocyte functions

Sandra Winning, Evelyn L. Kerber, Veronika Bäcker, Katharina Flück, Stephan Settelmeier

University of Duisburg-Essen, Physiology, Essen, Germany

Oxygen shortage stabilizes the hypoxia inducible factor (HIF) α subunits and active HIFs adapt the cellular gene

expression towards a metabolic profile enabling oxygen independent ATP generation. During the last years, it has

become evident that inflammatory stimuli can also increase cellular HIF levels in the presence or absence of oxygen.

Herein, the roles of either HIF-1 or HIF-2 in different cells of the innate immune system were elucidated in animal

models ranging from acute inflammation or skeletal muscle trauma to chronic inflammation combined with tumor

growth. The data reveal that the consequences of HIF activation differed with respect to i) the cell type it occurred,

ii) the HIF orthologue that was addressed, and iii) the milieu the activation took place in. In the context of

pharmacological HIF stabilization, this underlines the need to improve our understanding of cell specific functions of

HIFs to avoid unintended consequences.


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S 12-04

The role of hypoxic signaling in Innate Lymphoid Cells for gut

homeostasis

Christian Stockmann

University of Zurich, Institute of Anatomy, Zurich, Switzerland

Gut innate lymphoid cells (ILCs) show remarkable plasticity, yet, microenvironmental factors that drive plasticity are

incompletely understood. The balance between NKp46+ Interleukin (IL-)22-producing, group 3 ILCs (ILC3s) and

Interferon (IFN-)γ-producing group ILCs (ILC1s) contributes to epithelial barrier integrity and gut homeostasis. The

gut mucosa is characterized by physiological hypoxia and cellular adaptation to low oxygen is mediated by Hypoxia-

inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not

understood. We found that mice lacking the HIF-1α isoform in NKp46+ ILCs (HIF-1α KO) show a decrease in IFN-γ-

expressing, T-box expressed in T cells (T-bet)+, NKp46+ ILC1s and a concomitant increase in IL-22-expressing,

retinoic-acid-receptor-related orphan receptor γ (RORγt)+, NKp46+ ILC3 in the gut mucosa. Single-cell RNA

sequencing revealed HIF-1α as a driver of ILC plasticity, where HIF-1α promotes the ILC1 phenotype by direct

upregulation of T-bet. Consequently, loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the

expression of IL-22-inducible pro-homeostatic genes and confers protection against intestinal damage. Consistently,

constitutive activation of the HIF pathway in NKp46+ cells decreased the expression of IL-22 and aggravates intestinal

damage. Taken together, our results suggest that HIF-1α drives ILC plasticity in the gut that mediates intestinal

homeostasis.


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S 12-05

Harnessing Hypoxia Responses for Anemia Therapy: The HIF System

in Erythropoiesis and Beyond.

Volker H. Haase1,2

1 Vanderbilt University School of Medicine, Department of Medicine and Department of Molecular Physiology and

Biophysics, Nashville, USA 2 Uppsala University, Department of Medical Cell Biology, Section of Integrative Physiology, Uppsala, Sweden

The hypoxia-inducible factor (HIF) pathway plays a central role in the regulation of physiologic hypoxia responses,

including erythropoiesis, angiogenesis and anaerobic metabolism. HIFs are heterodimeric transcription factors that

consist of an oxygen-regulated α-subunit (HIF-1α, HIF-2α or HIF-3α, respectively) and a constitutively expressed β-

subunit. HIF activity is controlled by prolyl hydroxylase domain (PHD) dioxygenases, which utilize molecular oxygen

and 2-oxoglutarate for the hydroxylation of HIF-α subunits, initiating their proteasomal degradation. Under normoxia,

HIF-α subunits, which are continuously synthesized by cells, are hydroxylated, whereas under hypoxic conditions,

HIF-α hydroxylation is attenuated, resulting in the formation of HIF transcription factors and activation of HIF

transcriptional responses.

Small molecule inhibitors of HIF-PHDs (HIF-PHIs) are a new class of orally administered drugs that activate HIF

signaling and are efficacious in correcting and maintaining hemoglobin levels in patients with chronic kidney disease.

HIF-PHIs promote erythropoiesis by a) stimulating the production of endogeneous erythropoietin in the kidney and

liver and b) by enhancing iron uptake, transport and mobilization. Although efficacious in stimulating erythropoiesis,

multiple cardiovascular and other safety concerns have been raised. This presentation will discuss mechanisms of

action of HIF-PHIs and non-erythropoietic effects of systemic HIF activation.

Acknowledgment

VHH holds the Krick-Brooks Chair in Nephrology at Vanderbilt University. Studies presented here were supported

by the National Institutes of Health, the Swedish Reaerch Council, VINNOVA and Uppsala University.

Oral Sessions DPG 2021 | Abstract Book

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Workshops 1 October 2021

3:00 PM – 5:00 PM

Lecture Hall 3

W 01 | 3Rs in Physiology Chair

Maike Windbergs (Frankfurt/Main)


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W 01-01

Human in vitro models in health and disease

Maike Windbergs

Goethe University, Institute of Pharmaceutical Technology and Buchmann Institute for Molecular Life Sciences,

Frankfurt am Main, Germany

In basic research as well as for preclinical testing of novel medicines, predictive models are necessary. Animal

models exist for many diseases. However, for some pathophysiological conditions of the human body, there is no

animal model existing or predictive readouts are missing. Furthermore, species differences between the animal

model and the human body frequently do not allow for direct translation of the data. Many approaches have been

made to develop and establish valid alternative to animal testing according to the 3R-principle (reduce, replace,

refine). Apart from ethical concerns related to animal testing, advanced human cell and tissue models offer the

perspective to faster translate new therapeutic modalities from the bench into the clinic.

We investigate in vitro models of the outer epithelia (skin, lung and intestine) as well as non-cellular barriers like

mucus. For closely mimicking the situation in the human body, our in vitro models are based on human cells isolated

from excised human tissue from clinical cooperation partners, either in primary culture or as cell lines. The focus of

our research is on complex 3D tissue models incorporating different cell types which are either cultivated under

submers conditions or are lifted towards the air-liquid-interface. Such models can be inflamed or infected to

investigate inflammatory (e.g. inflammatory bowel diseases) and infectious diseases (e.g. wound infections). Further,

as alternative for non-physiological hydrogels for cell cultivation, fibrous scaffolds based on biocompatible polymers

are developed in our projects closely mimicking the native human extracellular matrix.


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W 01-02

Computer models in (neuro)biology & 3Rs

Peter Jedlicka

JLU Giessen, Giessen, Germany

Qualitative, cartoon-based understanding of physiological processes is not sufficient and needs to be taken to a

deeper, quantitative (computational) level. Two types of computational models, namely statistical & mechanistic

models, can be used to predict subcellular, cellular and supracellular phenomena in silico. This talk will mainly focus

on mechanistic models. A simulation of a large number of parameter combinations may lead to a reduction in the

number of necessary experiments. However, experimental data from in vitro and in vivo experiments are necessary

to adjust the parameters of computer models. Relatively recently established, so called population-based

compartmental modeling will be presented as a promising tool to predict and partially replace

pharmacological/genetical perturbations. Examples from cardiac and neuronal physiology will be described to show

how population-based computational modeling enables studies of the functional impact of intercellular ion channel

variability (Britton et al. PNAS 2013, Schneider et al. bioRxiv 2021). In addition, morphological modeling (Cuntz et al.

PLoS Comput Biol 2010) will be mentioned as a useful complementary approach to traditional compartmental

modeling of electrophysiological data in neurobiology. In combination, morphological and compartmental modeling

facilitates generalization of computational predictions to any morphology and supports the search for universal

principles valid across different species and cell types (Beining et al. eLife 2017; Cuntz et al. biorxiv 2019). In line

with the principles of the 3Rs (Replacement, Reduction and Refinement), computational models are excellent tools

to support sharing of data and resources by using available experimental datasets and by distributing the source

code of new models in online databases.

Acknowledgment

The work has been supported by BMBF (01GQ1203A, 031L0229), Deutsche Forschungsgemeinschaft (JE 528/6-

1; 528/10-1), University Medical Center Giessen and Marburg (UKGM), and von Behring Röntgen Foundation.

References [1] Beining M, Mongiat LA, Schwarzacher SW, Cuntz H*, Jedlicka P* (2017) T2N as a new tool for robust

electrophysiological modeling demonstrated for mature and adult-born dentate granule cells. eLife 6:e26517. https://doi.org/10.7554/eLife.26517

[2] Britton OJ, Bueno-Orovio A, Van Ammel K, Rong Lu H, Towart R, Gallacher DJ, Rodriguez B (2013) Experimentally calibrated population of models predicts and explains intersubject variability in cardiac cellular electrophysiology. Proc Natl Acad Sci U S A 110:E2098-105; https://doi.org/10.1073/pnas.1304382110

[3] Cuntz H, Forstner F, Borst A, Häusser M (2010) One rule to grow them all: a general theory of neuronal branching and its practical application. PLoS Comput Biol. 6:e1000877; https://doi.org/10.1371/journal.pcbi.1000877

[4] Cuntz H, Bird AD, Beining M, Schneider M, Mediavilla L, Hoffmann FZ, Deller T*, Jedlicka P* (2019) A general principle of dendritic constancy – a neuron’s size and shape invariant excitability bioRxiv 787911; https://doi.org/10.1101/787911

[5] Schneider M, Gidon A, Triesch J, Jedlicka P*, Cuntz H* (2021) Biological complexity facilitates tuning of the neuronal parameter space bioRxiv 2021.05.04.442120; https://doi.org/10.1101/2021.05.04.442120


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Workshops 1 October 2021 3:00 PM – 5:00 PM

Audimax

W 02 | Bioinformatics - NGS data analysis and

modelling of cellular networks Chair

Ralf P. Brandes (Frankfurt/Main)

Daniela Wenzel (Bochum)


of 516

W 02-01

NGS data and analysis workflows

Stefan Günther

Max-Planck-Institute for Heart and Lung Research, Head of NGS core, Bad Nauheim, Germany

During the last decade, Next-generation sequencing (NGS) technologies have had a dramatic impact on the field of

basic research by constantly lowering costs combined with increasing capacity and as such have revolutionized the

study of molecular biology and genomics with excellent resolution and accuracy. These new sequencing methods

provide a range of utility-based applications, which allow for a more comprehensive analysis of all possible samples,

such as tissues, organs, biopsies, whole organisms or cultured cells. The aim of the workshop is to point out the

current possibilities of NGS for frequent questions within exemplary projects. The first part will introduce the

opportunities for bulk approaches, especially RNA sequencing. RNAseq provides insight into the transcriptome of

cells with far higher coverage and greater resolution of the dynamic range compared to previous Sanger sequencing-

and microarray-based methods. Beyond quantifying transcripts and gene expression, the data generated by RNAseq

facilitates the discovery of novel transcripts and therefore identification of alternatively spliced genes and

quantification of allele-specific expression as well as investigation of RNA subtypes like pre-mRNA, microRNA and

long ncRNA. The final part focuses on the ever-increasing need for analyses with single cell resolution for many

upcoming projects. In numerous cases, the molecular analysis of whole tissues or organs is limited due to complexity

and cellular composition of the sample. The identification of small subpopulations or the detection of specific changes

within a small cell population is usually challenging because of massive overrepresentation of signals from bulk cells.

To overcome these problems, many techniques and tools were developed during last years to obtain transcriptomics

data from hundreds to several thousands of cells from different model organisms, organs and tissues, which enables

also a broader community to apply such approaches to their research.


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W 02-02

Galaxy, an open web platform for computational analysis – examples

from epigenetic research

Ralf Gilsbach

Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt a. M., Germany

Epigenetic mechanisms include DNA methylation and histone modifications as well as spatial genome organization.

Sequencing based-methods allow analyzing these different epigenetic layers with high resolution. Analysis of the

resulting sequencing data requires dedicated computer infrastructure and operation of a variety of computational

tools.

The Galaxy platform is a web-based platform, which is accessible for wet lab researchers without programming

experience and allows big data analysis. The European Galaxy cluster (usegalaxy.eu) offers free access to a high

performance cluster with more than 2500 integrated tools. The Galaxy training network provides preconfigured

workflows, training materials and regular courses to enable wet lab researchers performing bioinformatics analysis.

In this workshop, I will give an introduction into the Galaxy platform and will demonstrate how we use it for our

epigenetic research.


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W 02-03

Analysis of transcription factor-guided regulatory networks

Marcel H. Schulz

Goethe University Frankfurt, Institute for Cardiovascular Regeneration, Frankfurt, Germany

Transcription factors (TFs) are important regulatory proteins that bind the DNA to regulate gene expression,

chromatin looping and other cellular processes. The precise coordination of transcription factor binding is important

in development, cellular differentiation and response to stress. Mis-regulation of TF binding or TF activity often results

in the predisposition to or the occurrence of disease.

Due to the complexity of TF-gene regulatory interactions and their dynamics in disease or differentiation processes,

these interactions are often described as regulatory networks.

In this talk, I will highlight recent advances for the prediction of TF-regulatory networks from different types of data,

including epigenetics and chromatin conformation capture data, such as Hi-C, or single cell data. I will illustrate how

detailed regulatory networks can be constructed in a cell-type or disease-specific manner and used for the planning

of experimental validation to verify involvement of TFs in differentiation and disease.


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W 02-04

Semi‐quantitative modeling of the TNFR1‐induced signaling pathway

Ina Koch

Goethe-University Frankfurt am Main, Molecular Bioinformatics / Institute of Computer Science, Frankfurt am Main,

Germany

Question

New experimental data enables for a holistic view on physiological processes. Thus, mathematical modeling gained

in importance. The combination of data of different quality and quantity has become a big challenge. Often, kinetic

data is not available in sufficient amount, but qualitative data. This led to applications of mathematical formalisms for

semi‐quantitative analysis, such as Petri Nets (PNs).

Methods

PNs are directed, labeled, bipartite graphs. They consist of two types of nodes, describing the passive part (places:

biochemical entities) and the active part (transitions: biochemical reactions) and directed arcs only between nodes

of different type. Movable objects (tokens) realize system’s dynamics through movement, following specific rules.

Invariant properties give basic pathways in the dynamic behavior, defining functional subnetworks. In silico knockout

analysis can verify the model and generate new hypotheses. The “token game” simulates a signal flow in

asynchronous or synchronous way.

Results: We explored the TNFR1‐induced signaling pathway for the molecular switch behavior between cell’s survival

through the NF‐κB pathway, apoptosis, and necroptosis.

We developed a PN model in terms of detailed molecular reactions for the molecular players, protein complexes,

post‐translational modifications, and cross talk. The model comprises 130 reactions and 118 biochemical entities,

and 299 arcs. 279 manatee invariants represent the combinatorial diversity of complete functional pathways,

describing signal flows from receptor activation to the cellular responses.

120 pathways steered the cell to survival, whereas 58 and 35 pathways led to apoptosis and necroptosis,

respectively. For 65 pathways, the triggered response was not deterministic, leading to multiple possible outcomes.

We investigated the detailed knockout behavior and identified important checkpoints in terms of ubiquitination within

complex I and the gene expression dependent on NF‐κB.

The in silico knockout analysis explained several scenarios and mimicked the effects of Smac mimetic and

cycloheximide.

Conclusions

The PN comprises the current knowledge of the TNFR1‐induced signaling pathway.

Without knowing any kinetic data, system’s dynamics could be analyzed and simulated.

Acknowledgment

I would like to thank Dr. Leonie Amstein, Dr. Jörg Ackermann, Prof. Simone Fulda and Prof. Ivan Dikic for great

workk, discussions, and support.


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The TNFR1‐induced signal transduction pathway

The Petri net of the TNFR1-induced signaling pathway The Petri net of the processes of the TNFR1-induced signaling

pathway. The colors highlight the manatee invariant-induced

subnetwork. The manatee invariant is formed by linear

combination of three transition invariants each highlighted in

a different color, in blue, red, and green.

References [1] Amstein et al., 2017, BMC Sys Biol 11(1):72 [2] Amstein, 2018, PhD thesis


of 516

Oral Sessions 30 September 2021

9:00 AM – 11:00 AM

Lecture Hall 2

OS 01 | Spotlight on Signal Transduction Chair

Johannes Oberwinkler (Marburg)

Mauro Siragusa (Frankfurt/Main);


of 516

OS 01-01

Neuropsin (OPN5) for selective control of Gq signaling with light

Ahmed Wagdi1, Daniela Malan2, Janosch Beauchamp1, Udhaya Bhaskar Sathya Narayanan1, Markus

Vogt1, Philipp Sasse2, Robert Patejdl3, Tobias Bruegmann1

1 University Medical Center of Gottingen (UMG), Institute for Cardiovascular Physiology, Goettingen, Germany 2 University of Bonn, Institute of Physiology, Bonn, Germany 3 University of Rostock, Oscar-Langendorff Institute, Rostock, Germany

Question: GPCRs and Gq proteins are vital for cellular behavior, especially cardiac and smooth muscle cells. Yet

very little details are known about the kinetics of the signal transduction from the extracellular space into the cytosol

because conventional methods lack spatiotemporal resolution that optogenetics can provide. However, there is no

known optogenetic tool specific for Gq proteins. Herein, we prove that Neuropsin (OPN5) can be used to selectively

control Gq signaling in cardiac and smooth muscle cells.

Methods: We characterized activation of OPN5 in HEK293 cells by measuring IP1 accumulation, Ca2+ imaging, and

patch-clamp analysis of GIRK currents. We generated a transgenic mouse expressing OPN5/eYFP driven by the

chicken-β-actin promotor and analyzed the expression pattern and rate by using tissue clearing/light-sheet

microscopy and single cell dissociation. The effect of OPN5 activation on contractility in ventricular myocytes was

measured with a custom-made software for online contractility analysis. In Langendorff-perfused hearts, the sinus

node region was illuminated with UV light and a cardiac electrogram was recorded to analyze the heart rate. In

addition, we performed isometric force measurements in smooth muscle strips from small intestine, bladder and

uterus.

Results: We prove the selective activation of Gq proteins by UV light induced (385 nm) IP3 production, Ca2+ transients

and inhibition of GIRK channels in HEK293 cells. In the transgenic animal model, OPN5/eYFP expression was found

in ~70% of the cardiomyocytes targeted to the plasma membrane. UV light induced a significant increase in

contraction velocity in ventricular myocytes, and this effect was abolished by blocking Gq proteins. Importantly, in the

right atrium HCN4 positive sinus nodal cells expressed OPN5/eYFP and illumination of this region resulted in an

average heart rate increase of 5.9±0.97% (n= 20), which was significantly higher than control hearts of negative

siblings and CD1 wild type mice (n≥15, p≤ 0.02). In the bladder, uterus and small intestine, OPN5/eYFP expression

was confined to the smooth muscle layers and UV light induced force generation which was neither observed after

Gq protein block nor in organs from CD1 wild type mice.

Conclusion: We prove that OPN5 is the first optogenetic receptor which selectively activates Gq proteins and show

its use in cardiomyocytes, intact adult hearts as well as smooth muscle tissue.


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OS 01-02

Development and Applications of Optogenetic Tools for Manipulation

of Cyclic Nucleotides.

Shiqiang Gao, Shang Yang, Jing Yu-Strzelczyk, Manfred Heckmann, Georg Nagel

University Würzburg, Physiological Institute, Department of Neurophysiology, Würzburg, Germany

Optogenetic manipulation of cells or living organisms became successful in neuroscience, especially with the

introduction of the light-gated ion channel Channelrhodopsin-2 (ChR2) as an easily applicable tool. The optogenetic

toolbox was enriched with the application of earlier discovered and characterized photoreceptors, engineered

photoreceptors, or newly discovered photoreceptors from nature. The applications were expanded from

neuroscience to other fields, like cardiovascular system etc. Very recently, a Channelrhodopsin was clinically applied

for a partial recovery of visual function in a blind patient.

Manipulation by light is attractive for an important second messenger in cells, cAMP. It was first achieved by

expressing the photoactivated adenylyl cyclase from the unicellular alga Euglena gracilis (EuPAC) in oocytes of

Xenopus laevis or in the brain of Drosophila melanogaster flies, where it yielded light-induced behavior changes. The

discovery of a small photoactivated adenylyl cyclase from the bacterium Beggiatoa (bPAC) has greatly advanced

the light-manipulation of intracellular cAMP in individual, genetically targeted cells. Due to its small size, low dark

activity, and high light activity, bPAC is a versatile tool for manipulating and studying cAMP-mediated processes.

Here, we will present our recent work on optimization of bPAC, characterization of new PACs for tight cAMP

regulation and their application in neuronal systems [1].

Furthermore, we characterized rhodopsins with enzymatic activity – first Cyclase Opsin or Cyclop [2], a light-

activated guanylyl cyclase – and concluded that it contains 8 transmembrane helices. We also characterized other

new rhodopsins with 8 transmembrane helices: RhoPDE [3], a light-regulated phosphodiesterase and 2c-

Cyclop [4,5], a light-inhibited guanylyl cyclase. We will summarize these recent advances and give an outlook on

the perspectives of light-manipulating cyclic nucleotide concentrations.

References [1] Zhang S, Lutas A, Yang S, Gao S, Diaz A, Fluhr H, Nagel G, Andermann M. 2021. Hypothalamic dopamine neurons

control the motivation to mate via persistent cAMP-PKA signaling. Nature in press. [2] Gao S, Nagpal J, Schneider M, Kozjak-Pavlovic V, Nagel G, Gottschalk A. 2015. Optogenetic manipulation of

cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp. Nat Commun 6:8046. [2] Tian Y, Gao S, Yang S, Nagel G. 2018. A novel rhodopsin phosphodiesterase from Salpingoeca rosetta shows

light-enhanced substrate affinity. Biochem J 455: 359-65. [3] Tian Y, Gao S, von der Heyde EL, Hallmann A, Nagel G. 2018. Two-component cyclase opsins of green algae are

ATP-dependent and light-inhibited guanylyl cyclases. BMC Biology 16:144. [4] Tian Y, Nagel G, Gao S. 2021. An engineered membrane-bound guanylyl cyclase with light-switchable activity.

BMC Biology 19, 54


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OS 01-03

A role for n3 fatty acids in the modulation of LPS-induced fever or

hypothermia: combined lipidomics in a multimodal pathway analysis of

brain lipid mediators

Fabian J. Pflieger1, Vannuruswamy Garikapati2, Dhaka R. Bhandari2, Janne Bredehöft1, Verena Peek1,

Elke Roeb3, Martin Roderfeld3, Jessica Hernandez1, Sabine Schulz2, Carsten Culmsee4, Sophie Layé5,

Konstantin Mayer6, Joachim Roth1, Bernhard Spengler2, Christoph Rummel1

1 Justus Liebig University Giessen, Institute of Veterinary Physiology and Biochemistry, Giessen, Germany 2 Justus Liebig University Giessen, Institute of Inorganic and Analytical Chemistry, Giessen, Germany 3 Justus Liebig University Giessen, Department of Gastroenterology, Giessen, Germany 4 Philipps University of Marburg, Institute of Pharmacology and Clinical Pharmacy, Marburg, Germany 5 Institut National de la Recherche Agronomique, Université de Bordeaux, UMR 1286, NutriNeuro: Laboratoire

Nutrition et Neurobiologie Intégrée, Bordeaux, France 6 Justus Liebig University Giessen, Department of Internal Medicine, Giessen, Germany

Question: Infection and inflammation are accompanied by an inflammatory response in the brain and by brain

controlled sickness symptoms including fever. During systemic inflammation, these responses are mainly triggered

by circulating mediators, which are detected by circumventricular organs like the vascular organ lamina terminalis

(OVLT), a critical brain structure for fever induction. We aimed to investigate the role of n3 fatty acids (FA) on brain

inflammation, immune-to-brain communication and fever during lipopolysaccharide-induced (LPS, low or high dose)

systemic inflammation in wild type and fat1 mice, which produce n3 FAs endogenously.

Methods: Multimodal analyses, combining telemetric recording of body core temperature with expression of

inflammatory marker proteins and enzymes involved in lipid metabolism, targeted LC-MS/MS, untargeted lipidomics

and matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) revealed new

spatiotemporal insights into dynamics and roles of lipid mediators in the hypothalamus, preoptic area, and the OVLT

for brain inflammation and immune-to-brain communication.

Results: The high LPS-dose induced short lasting fever in wild type (WT) mice, which was absent in fat1 mice

followed by hypothermia in both genotypes. This response was accompanied by significantly lower signs of

inflammation like circulating IL-6 levels or hypothalamic expression of components of the signal transducer and

activator of transcription-3 and nuclear factor-kB signaling of fat-1 mice after LPS-stimulation compared to WT

counterparts. Out of 1000 lipids included, a remarkable increase of the potentially anti-inflammatory

endocannabinoids monoacylglycerol MG(20:4) and MG(22:6) in fat-1 vs. WT mice 24 h after LPS treatment illustrated

changes of endocannabinoid signaling due to n3-FAs enrichment. Novel inflammatory lipid targets were revealed,

such as, acylcarnitines [CAR(16:0)], linoleic acid metabolites as well as ω-3 endocannabinoids, all of which can

modulate inflammation but remain to be further investigated as to their role in brain inflammation and sickness

responses.

Conclusion: Overall, n3-FAs enrichment promoted resolution of inflammation by pro-resolving, anti-inflammatory

mechanisms involving hypothalamic resolvin D1 dampening fever post-LPS-stimulation.

References [1] Bredehöft, J, Bhandari, D, Pflieger, JF, Schulz, S, Kang, JX, Laye, S, Roth, J, Gerstberger, R, Mayer, K,

Spengler, B and Rummel, C 2019 Visualizing and profiling lipids in the OVLT of fat-1 and wild type mouse brains during LPS-induced systemic inflammation using AP-SMALDI MSI, ACS Chemical Neuroscience, 10, 4394-4406


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OS 01-04

Adipose tissue derived medicinal signalling cells attenuate

inflammatory activation of glial cells in primary cultures of the rat

spinal dorsal horn in a co-cultivation model

Stephan Leisengang1, Laura Heilen2, Franz Nürnberger1, Daniela Ott1, Stefan Arnhold2, Joachim Roth1

1 Justus Liebig University Giessen, Institute of Veterinary Physiology and Biochemistry, Giessen, Germany 2 Justus Liebig University Giessen, Institute of Veterinary Anatomy, Histology and Embryology, Giessen, Germany

Question: Neuroinflammatory processes within the spinal dorsal horn can lead to symptoms of inflammatory and/or

neuropathic pain. In recent years involvement of spinal glial cells (e.g. microglia, astrocytes) has been highlighted in

this context (glia-neuron-interaction). Therefore, inhibition of glial activation is a promising target to alleviate

pathological pain states. Medicinal signalling cells, also termed mesenchymal stem/stromal cells, e.g. obtained from

adipose tissue (AdMSCs) are discussed to exert beneficial effects in animal models of neuropathic pain. However,

the underlying cellular mechanisms are still poorly understood. We therefore aimed to establish a model of co-

cultivation of primary cell cultures of the superficial dorsal horn (SDH) with AdMSCs to investigate effects of AdMSCs

on inflammatory activation of spinal glial cells and neurons.

Methods: We recently established a neuro-glial primary culture of the SDH to characterize cellular mechanisms upon

inflammatory stimulation with lipopolysaccharide (LPS) on spinal dorsal horn neurons and glial cells [1]. To

investigate effects of AdMSCs we now established a co-cultivation model of both cell cultures (Figure 1). After 24

hours of co-cultivation cells were stimulated with LPS for 2 hours. Supernatants were collected for measurements of

cytokine release (TNFα, IL-6). SDH cells and AdMSCs were used for immunocytochemical detection of transcription

factor activation and RT-qPCR experiments.

Results: LPS-induced increases of released TNFα and IL-6 were attenuated when SDH cells were cultured in

presence of AdMSCs. RT-qPCR experiments revealed a reduced TNFα/IL-10 ratio in SDH cells co-cultivated with

AdMSCs. While LPS treatment resulted in enhanced nuclear NFκB staining intensities in microglial cells, this

increased nuclear translocation was significantly blunted in presence of AdMSCs. Furthermore, AdMSCs showed

increased LPS-induced expression of IL-10 in RT-qPCR in both cultivation groups, while expression of COX-2, TGF-

β, and TSG-6 was not altered.

Conclusions: We show anti-inflammatory capacities of AdMSCs on LPS-exposed SDH primary cultures (Figure 2).

Released anti-inflammatory mediators by AdMSCs, e.g. IL-10, might attenuate microglial activation (via NFκB) and

suppress the release of pro-inflammatory cytokines (TNFα, IL-6) by SDH primary cells. In vivo treatment with

AdMSCs might thus serve as a tool to reduce spinal neuroinflammation and symptoms of inflammatory / neuropathic

pain.

Figure 2: Anti-inflammatory effects of AdMSCs on

spinal glial activation


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Inflammatory stimulation (LPS) of SDH primary

cells resulted in increased nuclear

translocation of NFκB in microglial cells and

enhanced production of pro-inflammatory

cytokines (IL-6, TNFα). Increased nuclear

translocation of STAT3 in astrocytes and

elevated neuronal responses upon stimulation

with glutamate were detectable. In presence of

AdMSCs LPS-induced nuclear translocation of NFκB

and cytokine production were significantly

attenuated. AdMSCs express putative anti-

inflammatory mediators (e.g. IL-10). We suggest

beneficial effects of AdMSCs on spinal

neuroinflammatory processes.

Figure 1: Co-cultivation model of SDH primary

cell cultures with AdMSCs Primary sensory neurons with their cell bodies

located in the dorsal root ganglia (DRG) can

detect noxious stimuli. Within the superficial

dorsal horn (SDH) information is transmitted on

secondary neurons and forwarded to the

respective central nervous structures. This

transmission can be modulated by glial elements

in states of inflammatory or neuropathic pain

(central sensitization). SDH primary cultures

are a useful tool to investigate effects of

inflammatory stimulation on a cellular level.

AdMSCs were obtained from abdominal fat and

cultured in fine-pored inserts for co-

cultivation.

References [1] Leisengang, S, Nürnberger, F, Ott, D, Murgott, J, Gerstberger, R, Rummel, C, Roth, J, 2020, 'Primary

culture of the rat spinal dorsal horn: a tool to investigate effects of inflammatory stimulation on the afferent somatosensory system', Pflügers Archiv - European Journal of Physiology, 472(12), 1769-1782


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OS 01-05

Gene and miRNA signatures characterizing human iPSC-derived

sensory neuron development

Maximilian Zeidler, Clemens L. Schöpf, Kai Kummer, Theodora Kalpachidou, Michaela Kress

Medical University of Innsbruck, Department of Physiology, Innsbruck, Austria

The process of pain sensing is performed by specialized sensory neurons called nociceptors that acutely or

chronically can alter their function as a consequence of neurological disorders, such as neuropathic pain or migraine.

Understanding the regulatory pathways critically implicated in nociceptor function is crucial for the detection of

disease onsets as well as optimal treatment windows. However, studies investigating nociceptive neurons are mainly

performed in rodents and are hampered by interspecies differences or even post-mortem alterations when human

dorsal root ganglia (DRG) are used as a model system.

To overcome this translational paresis, we established a human induced pluripotent stem cell (iPSC)-derived

nociceptor (iDN) culture with similar characteristics to mouse dorsal root ganglia (DRG), as indicated by

immunofluorescence staining of canonical nociceptor-specific marker and functional analysis. Since iDNs can be

used to reconstitute major developmental processes, we explored gene and miRNA signatures throughout iDN

differentiation using paired long and small RNA sequencing in three iPSC clones, with the aim to identify hub

genes/microRNAs (miRNA) and miRNA::mRNA interactions significantly regulating iDN differentiation/maturation.

Transcriptomic signatures of six timepoints (Day 0, 5, 9, 16, 26, 36) were retrieved and correlation analysis of mRNAs

as well as miRNAs expression signatures performed. Several highly correlated gene and miRNA modules emerged

using the weighted gene correlation network analysis, and stage-specific hub genes/miRNA were identified.

Multiple well described rodent sensory neuron developmental markers, such as SOX10, NEUROG1, PRDM12 and

MED13, were conserved. We further identified highly abundantly expressed neuropeptide related genes, such as

somatostatin, galanin, CGRP at D16 with putative functions in phenotype identity regulations and revealed that iDNs

resembled peptidergic sensory neurons. Since gene expression is tightly regulated post-transcriptionally by miRNAs,

we performed complex bioinformatic analyses integrating miRNA::mRNA predictions, validations as well

transcriptomic signatures. miRNA target-spaces were highly enriched for neuron related gene pathways, such as

synapse development, neurite outgrowth and ion channel regulation. Finally, we developed the online publicly

available NOCICEPTRA tool to explore mRNA and miRNA signatures, disease onset and miRNA target-spaces

throughout iDN development.

References [1] Schoepf CL*, Zeidler M*, Spiecker L, Kern G, Lechner J, Kummer KK, Kress M. Selected Ionotropic Receptors

and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors. Brain Sci. 2020;10(6).

[2] Zeidler M, Kummer KK, Schoepf CL, Kalpachidou T, Kern G, Cader MZ, et al. NOCICEPTRA: Gene and microRNA signatures and their trajectories characterizing human iPSC-derived nociceptor maturation. bioRxiv. 2021:2021.06.07.447056.


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OS 01-06

Chemotherapy induces cachexia by modulating epigenetic signaling

pathways in skeletal muscles

Mamta Amrute-Nayak1, Gloria Pegoli2, Tim Holler1, Alfredo J. Lopez-Davila1, Chiara Lanzuolo2, Arnab

Nayak1

1 MHH, Institute of Molecular and Cell Physiology, Hannover, Germany 2 Institute of Biomedical Technologies, National Research Council, Milan, Italy, Institute of Biomedical

Technologies, National Research Council, Milan, Italy, Milan, Italy

Mobility is an essential feature of the animal kingdom that determines the survival and biological success of animals.

Skeletal muscles are specialized tissues -comprising roughly 40% of human body mass- generate force to drive

movements through ATP‐dependent activity of the ´sarcomere´, the fundamental contractile unit of striated muscle.

Compromised force generation ability because of the disordered sarcomere is one of the classical hallmarks of

myopathies, such as in cachexia. Cachexia is a debilitating wasting syndrome associated with significant loss of

muscle mass in people suffering from various diseases, including cancer. Chemotherapy is the first line of treatment

for cancer patients. Notably, chemotherapeutic treatment itself is shown to induce muscle loss. Thus, in addition to

cancer-, chemotherapy-induced cachexia aggravates pre-existing conditions in patients, leading to profound loss of

muscle mass and function. Molecular mechanisms underlying chemotherapy-induced cachexia, however, remained

poorly understood. Using mouse satellite stem cell-derived primary muscle cells and mouse C2C12 progenitor cell-

derived differentiated myotubes, we found that specific classes of chemotherapeutic drugs disrupt sarcomere

organization and thereby the contractile ability of skeletal muscle cells. The sarcomere disorganization results from

loss of primary force generator molecular motor protein myosin (MyHC-II) upon drug treatment. We unraveled that

drugs impede chromatin targeting of SETD7 histone methyltransferase and perturbs association and synergetic

function of SETD7 with p300 histone acetyltransferase. The compromised transcriptional activity of these epigenetic

regulators reduced histone acetylation and further transcriptional changes leading to declined MyHC-II transcription.

Importantly, the drugs destabilize the SUMO isopeptidase enzyme, SENP3. Our studies revealed that the reduction

in SENP3 protein level is the nucleation event to deregulation of SETD7-p300 function. Collectively, we resolved a

unique epigenetic mechanism targeted by distinct chemotherapeutic drugs, leading to chemotherapy-induced

cachexia. We envisage a pharmacological intervention into SUMO isopeptidases activity as a potential tool to

alleviate conditions associated with chemotherapy-induced cachexia in striated muscle.


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OS 01-07

Mammalian Sterile-20 Like Kinase1 controls neutrophil homeostasis

through regulation of G-CSF receptor signaling

Sergi Masgrau Alsina1, Dae-Sik Lim2, Markus Sperandio1

1 LMU, Walter Brendel Center for Experimental Medicine, Munich, Germany 2 KAIST, Department of Biological Sciences, Daejeon, South Korea

Question: Neutrophils only spend 10-12 hours in the blood vasculature until they get cleared from the circulation.

Therefore, continuous release of neutrophils from the bone marrow (BM) is indispensable for proper immune function

of the organism. Key factor in regulating neutrophil homeostasis is the granulocyte colony-stimulating factor (G-CSF),

which does not only promote their development but also favours their release into the circulation. In mature

neutrophils, G-CSF interacts with the G-CSF receptor leading to activation of the JAK-Stat signaling pathway, which

controls the expression of the chemokine receptors CXCR2 and CXCR4. CXCR2 and CXCR4 are involved in

modulating neutrophil release from the BM compartment. Mammalian sterile 20-like kinase 1 (Mst1) has been shown

to be relevant in immune cell trafficking. Recently, increased numbers of neutrophils have been described in the BM

of Mst1-deficient mice suggesting a role of Mst1 in regulating neutrophil homeostasis. Accordingly, we aimed to study

the role of Mst1 in neutrophil homeostasis including G-CSF receptor signaling and neutrophil mobilization.

Methods & Results:In vivo intravenous injection of G-CSF into Mst1 knockout mice failed to induce an increase of

the circulating neutrophil pool 4 hours after the stimulation when compared to WT mice. Furthermore, using two-

photon microscopy in vivo imaging of the BM niche and systemic G-CSF stimulation, we were able to observe

defective neutrophil migratory behaviour and decreased intravasation into the BM microvasculature in the absence

of Mst1. Elucidating the molecular mechanisms of a putative regulation of G-CSF signaling by Mst1, we investigated

the expression and phosphorylation of G-CSF receptor and downstream signaling molecules using both flow

cytometry and western blot analysis. Interestingly, we detected diminished phosphorylation of Stat3 after in vitro G-

CSF stimulation in Mst1 knockout compared to WT neutrophils. In addition, up-regulation of surface CXCR2 (as

consequence of Stat3 transcriptional activity) was diminished in Mst1 knockout neutrophils elucidating that Mst1 was

affecting the G-CSF receptor signaling cascade through Stat3-dependent transcriptional mechanisms.Conclusion:

We identify Mst1 as a key player in G-CSF receptor signaling in neutrophils and show that Mst1 is critical for the

proper mobilisation and release of mature neutrophils from the bone marrow into the blood circulation.

Acknowledgment

Supported by DFG SP621/5-1 to M.S.


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OS 01-08

Zyxin protects from hypertension-induced cardiac dysfunction

Jaafar Al-Hasani1, Carla Sens-Albert1, Subhajit Ghosh1, Felix Trogisch2, Taslima Nahar1, Prisca Friede1,

Jan-Christian Reil3, Markus Hecker1

1 Heidelberg University, Department of Cardiovascular Physiology, Heidelberg, Germany 2 European Center for Angioscience, Heidelberg University, Division of Cardiovascular Physiology, Mannheim,

Germany 3 University Hospital Schleswig-Holstein, Medical Clinic II, University Heart Center Lübeck, Lübeck, Germany

Heart failure is one of the major causes of morbidity and death worldwide. It follows a remodelling process in the

heart, often caused by hypertension. In this process, cardiomyocyte hypertrophy and death as well as cardiac fibrosis

are often observed, ultimately leading to ventricular dysfunction.

Zyxin is a known mechanotransducer in vascular cells that may promote cardiomyocyte survival.

Here, we used zyxin knockout (KO) mice in an in vivo model for hypertension to analyze the role of zyxin in cardiac

function during experimental hypertension.

Hypertensive zyxin KO mice revealed a lower rise in systolic blood pressure in telemetry recordings while diastolic

blood pressure showed a normal increase.

Using echocardiography, a systolic dysfunction was identified, and isolated working heart measurements pointed to

a decrease in systolic elastance.

Hypertensive zyxin KO mice revealed increased apoptosis in heart sections along with enhanced fibrosis and an

upregulation of active focal adhesion kinase and integrins α5 and β1.

Microvascular endothelial cells were identified as possible contributors to the expression of, e.g. collagen 1α2 and

TGF-β in the cardiac tissue, which eventually stimulate myofibroblast activation, hence resulting in the excessive

scar tissue formation observed in the hearts of hypertensive zyxin KO mice.

Zyxin KO mice displayed the aforementioned cardiac phenotype, chiefly characterized by increased apoptosis and

excessive fibrosis causing cardiac dysfunction, only after induction of hypertension, no such effects were seen under

baseline conditions, i.e. at normal blood pressure.

Zyxin may thus be required for the maintenance of cardiac function in spite of hypertension.


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Oral Sessions 30 September 2021 9:00 AM – 11:00 AM

Lecture Hall 4

OS 02 | Renal Homeostasis and Disease Chair

Ioana Alesutan (Linz)

Christian Hinze (Berlin)


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OS 02-01

Study of the function of androglobin in renal ciliogenesis and its

involvement in polycystic kidney disease

Miguel C. R. M. Correia1,3, Alex Odermatt2, Darko Maric1,3, David Hoogewijs1,3, Anna Keppner1,3

1 University of Fribourg, Department of endocrinology, metabolism and cardiovascular system, Fribourg,

Switzerland 2 University of Basel, Department of pharmaceutical sciences, Basel, Switzerland 3 University of Zürich, National Center of Competence in Research Kidney Control of Homeostasis (NCCR

Kidney.CH), Zürich, Switzerland



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OS 02-02

Upregulation of putrescine degrading enzyme AOC1 is a common

mechanism after kidney injury

Tobias Sieckmann1, Neslihan Ögel1, Michael Fähling1, Muhammad I. Ashraf2, Gunnar Schley3, Emilia

Vigolo4, Felix Boivin4, Martin Reichel5, Felix Knauf5, Christian Rosenberger5, Felix Aigner2, Kai Schmidt-

Ott4, Holger Scholz1, Karin M. Kirschner1

1 Charité - Universitätsmedizin Berlin, Institut für Vegetative Physiologie, Berlin, Germany 2 Charité - Universitätsmedizin Berlin, Chirurgische Klinik, Berlin, Germany 3 Universitätsklinikum Erlangen, Nephrologie und Hypertensiologie, Erlangen, Germany 4 Max Delbrück Center for Molecular Medicine, Berlin, Germany 5 Charité - Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Nephrologie und Internistische

Intensivmedizin, Berlin, Germany

Question: The polyamines putrescine, spermidine and spermine are organic polycations that regulate many cell

functions including proliferation and differentiation. It has been shown, that genes involved in polyamine homeostasis

are dysregulated after kidney ischemia reperfusion injury. Here we examined the hypothesis that different forms of

acute and chronic kidney injury lead to similar changes in the expression patterns of the polyamine system.

Methods: Expression of genes involved in polyamine homeostasis were analyzed by RT-qPCR and RNAScope in

different models of acute and chronic kidney injury. These are: renal ischemia followed by 6 hours to 21 days of

reperfusion; kidneys after 24 hours to 7 days transplantation; rhabdomyolysis-induced kidney injury at 24 h; daily

subcutaneous injections of cyclosporin A for 6 weeks; three weeks of high oxalate diet; hypertensive nephropathy

caused by administration of Angiotensin II for 4 weeks; five weeks after two kidney, one clip renovascular

hypertension; Streptozotocin-induced diabetes mellitus after 6 weeks. The observed changes of the putrescine

degrading enzyme Aoc1 were further examined in mouse models of hypoxia (ICA induced hypoxia, tubulus specific

VHL Knockout and systemic hypoxia). Murine embryonic kidney explants were exposed to different noxae common

in kidney injury as a screening for stimuli of Aoc1 expression. Changes of Aoc1 expression were confirmed under

hyperosmotic conditions in kidney cell lines M15 and 209/MDCT as well as in primary proximal tubules. Using reporter

gene and RNA-stability assays we investigate the mechanism behind increased Aoc1 expression.

Results: We identified that the catabolic enzymes (Aoc1 and Sat1) were upregulated, and the anabolic enzymes

(Odc1, Sms) were downregulated after kidney injury. The increase of AOC1 was localized to the injured and

regenerating proximal tubules. Hypoxia and hyperosmolarity were identified in vitro as stimuli that confer an increase

in Aoc1 expression, which is based on RNA-stabilization as well as transcriptional activation. However, hypoxia did

not lead to an upregulation of Aoc1 expression in vivo.

Conclusions: Acute and chronic kidney injury models show a common pattern of dysregulation of the polyamine

system. The most striking change is the upregulation of the putrescine degrading enzyme Aoc1 in proximal tubules.

Using hyperosmolarity as a stimulus we provide first insights into the regulation of Aoc1 under harmful conditions.

Acknowledgment

This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-

ID 394046635 – SFB 1365 Renoprotection. T.S. received support from the Wilhelm Sander-Stiftung (grant no.

2018.015.1).


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OS 02-03

Role of arginase-II in hypoxia-induced renal epithelial damage and

activation of fibrotic signalling cascade

Xiujie Liang, Duilio M. Potenza, Zhilong Ren, Andrea Brenna, Xiu-Fen Ming, Zhihong Yang

University of Fribourg, Department of Endocrinology, Metabolism and Cardiovascular System - Cardiovascular and

Aging Research, Fribourg, Switzerland

Background: The ureohydrolase, type-II arginase (Arg-II), is a mitochondrial enzyme metabolizing L-arginine into

urea and L-ornithine and is strongly upregulated by hypoxia in various cell types. Arg-II is highly expressed in renal

proximal tubular epithelial cells (PTC) and is recently implicated in kidney aging. Most recent studies report

contradictory results on the role of Arg-II in renal injury under ischemia or hypoxia condition. The aim of our study is

to investigate the function of Arg-II in renal epithelial cell damage/function under hypoxia condition.

Methods and Results: Human HK-2 renal epithelial cell line was cultured under hypoxia condition for 12 to 48 hours.

Moreover, ex vivo experiments with kidneys from wild-type (WT) and genetic Arg-II deficient mice (Arg-II-/-) at age of

22 months were also studied. Our results showed that hypoxia (1% over 48 hours) induces Arg-II expression in HK2

cells, which is dependent on both hypoxia-inducible factors HIF1α and HIF2α. Treatment of the cells with

dimethyloxaloylglycine (DMOG) to enhance HIFs elevates Arg-II. Interestingly, hypoxia or DMOG upregulates

transforming growth factor β1 (TGFβ1) levels and collagen Iα1 expression, which is prevented by Arg-II silencing.

Moreover, stimulation of HK2 cells with TGFβ1 enhances collagen Iα1 and fibronectin expression, which is not

affected by Arg-II silencing. Ex vivo experiments showed enhanced epithelial Arg-II expression and damage markers,

i.e., KIM1 and NGAL, in the WT mouse kidneys under hypoxia condition, which is reduced in the Arg-II-/-

mice.Conclusion: The results demonstrate that hypoxia activates renal HIF-Arg-II-TGFβ1 cascade, participating in

hypoxia-associated renal fibrosis.


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OS 02-04

Inhibition of transforming growth factor β1 signaling in renal interstitial

cells of mice attenuates profibrotic genes and preserves erythropoietin

production in experimental kidney fibrosis

Michaela A. A. Fuchs1, Katharina A. - E. Broeker1, Julia Schrankl1, Nicolai Burzlaff3, Carsten Willam2,

Charlotte Wagner1, Armin Kurtz1

1 University of Regensburg, Institute of Physiology, Regensburg, Germany 2 Friedrich-Alexander-University Erlangen-Nürnberg, Department of Nephrology and Hypertension, Erlangen,

Germany 3 Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Chemistry and Pharmacy, Erlangen, Germany

Renal fibrosis with tissue scaring and a pronounced loss of function is the outcome of many chronic kidney diseases.

Renal anemia caused by the loss of renal erythropoietin production is another major complication for patients with

renal failure due to fibrosis. The progression of renal fibrosis is characterized by the development of matrix producing

myofibroblasts, originating from resident and immigrating cells. Myofibroblast formation and extracellular matrix

production during kidney damage are triggered by various cytokines. Among these, transforming growth factor β1

(TGFβ1) is considered a central trigger for renal fibrosis. We could detect a strongly upregulated expression of TGFβ1

and TGFβ receptor 2 (TGFβ-R2) mRNAs in renal interstitial cells in experimental fibrosis.

Our project aimed to investigate the contribution of TGFβ1 signaling in resident renal interstitial cells to renal fibrosis

in two models of experimental kidney fibrosis, namely adenine induced nephropathy and unilateral ureter occlusion.

For this purpose we generated a mouse model with the targeted inducible deletion of the TGFβ-R2 in interstitial cells

expressing the fibroblast marker PDGFR-β. Loss of TGFβ-R2 function led to a disruption of TGFβ1 signaling in these

myofibroblast progenitor cells.

In mice without functioning TGFβ-R2, expression of profibrotic genes was attenuated up to 50%. In addition, deletion

of TGFβ-R2 prevented the decline of erythropoietin production in ureter ligated kidneys. Notably, fibrosis associated

expression of alpha smooth muscle actin as a classical myofibroblast marker and overall deposits of extracellular

matrix were not altered in mice with a targeted deletion of TGFβ-R2.Our findings suggest an enhancing effect of

TGFβ signaling in resident interstitial cells that contributes to profibrotic gene expression as well as the

downregulation of erythropoietin production, but not the development of myofibroblasts in two models of renal fibrosis.

References [1] Fuchs, MAA, Broeker, KAE, Schrankl, J, Burzlaff, N, Willam, C, Wagner, C, Kurtz, A, 2021, ‘Inhibition of

transforming growth factor β1 signaling in resident interstitial cells attenuates profibrotic gene expression and preserves erythropoietin production during experimental kidney fibrosis in mice’ Kidney International, Volume 100,Pages 122-137,


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OS 02-05

Novel Epo regulating transcription factors

Darko Maric1,9, Anna Keppner1,9, Maarten Chantillon1,2, Miguel Carvalho Correia1,9, Laura Yerly1,

Isabelle A. Scerri1, Thérèse Sidibé1, Laurent Monney1, Ilaria M. C. Orlando1, Tanguy Corre3,4,9, Muriel

Bochud4,9, Alex Odermatt5,9, Roland H. Wenger6,9, Betty Gardie7, Holger Cario8, David Hoogewijs1,9

1 University of Fribourg, Department of Endocrinology, Metabolism and Cardiovascular System, Faculty of Science

and Medicine, Fribourg, Switzerland 2 University of Duisburg-Essen, Institute of Physiology, Essen, Germany 3 University of Lausanne, Department of computational biology, Lausanne, Switzerland 4 Lausanne University Hospital (CHUV), Institute of Social and Preventive Medicine (IUMSP), Lausanne,

Switzerland 5 University of Basel, Department of Pharmaceutical Sciences, Basel, Switzerland 6 University of Zürich, Institute of Physiology, Zürich, Switzerland 7 University of Nantes and University of Angers, Centre de Recherche en Cancérologie et Immunologie Nantes-

Angers, Nantes, France 8 University Medical Center Ulm, Department of Pediatrics and Adolescent Medicine, Ulm, Germany 9 University of Zürich, National Center of Competence in Research Kidney Control of Homeostasis (NCCR

Kidney.CH), Zürich, Switzerland



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OS 02-06

Blocking FGF23 function with modified C-terminal FGF23 as a novel

therapy for X-linked hypophosphatemia

Rocio Fuente1,2, Nicole Gehring1, Laura Alonso-Duran2, Carla Bettoni1, Fernando Santos2, Carsten

Wagner1, Isabel Rubio-Aliaga1

1 University of Zurich, Institute of Physiology, University of Zurich, Zurich, Switzerland 2 University of Oviedo, Department of Medicine, University of Oviedo, Oviedo, Spain

Questions: Fibroblast growth factor 23 (FGF23) is a key player in phosphate (Pi) homeostasis. It exerts its endocrine

function on the kidney by increasing Pi excretion and suppression of calcitriol levels. However, stabilizing mutations

or pathological conditions can increase FGF23 in plasma with detrimental consequences, such as

hypophosphatemia, low calcitriol levels with concomitant bone alterations (rickets and osteomalacia) and joint-

muscle pain. X-Linked Hypophosphatemia (XLH) is the most prevalent inherited FGF23-mediated hypophosphatemic

disease with severe bone deformities. Novel therapies in XLH focus on neutralizing FGF23 function using antibodies

neutralizing FGF23, yet this therapy is very costly, requires frequent infusions, and not all patients are responsive.

Based on the observation that the naturally occurring C-terminal FGF23 fragment exerts an inhibitory action on the

active FGF23 hormone, we tested the effect of two differently modified C-terminal FGF23 fragments on growth and

growth plate (GP) histology in an XLH mouse model, the PhexC733R mouse line (PHEX).

Methods: Stably transfected HEK293-αKlotho cells were used for testing the effectiveness of the two peptides to

antagonize FGF23 signaling, cFGF23 with N-terminal acetylation (Nmod) and cFGF23 with C-terminal amidation

(Cmod). PHEX weaned male mice were i.p injected with either 1mg/kg/day Nmod, Cmod or saline solution as control.

Wildtype male mice injected with saline solution were used as control. After 7 days of treatment, nose-tail and tibia

length, plasma FGF23 and phosphate levels and GP histomorphometry were analyzed.

Results: The administration of Cmod and Nmod FGF23 peptides were effective in reducing FGF23 signaling in vitro.

PHEX mice manifest the clinical symptoms of XLH: low Pi, high FGF23 levels and rickets. Results in PHEX mice

demonstrated that administration of Cmod and Nmod peptides reduced FGF23 levels in plasma, increased

longitudinal growth rate, increased body weight and tibia length. Yet, hypophosphatemia persisted. Cmod and Nmod

treatment in PHEX mice enhanced the appearance of growth cartilage and corrected the height of the hypertrophic

zone despite the hypophosphatemia stage.

Conclusions: Our data indicate that treatment with the Nmod and Cmod peptides partially reversed the pathological

symptoms of XLH. The corrections of the profound GP morphological abnormalities make them a promising therapy

to reduce the risk of bone deformities in XLH patients


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OS 02-07

Two-pore channel protein TPC1 is a determining factor for the

adaptation of proximal tubular phosphate reabsorption.

Armin Just1, Robert T. Mallmann2, Sonja Grossmann2, Norbert Klugbauer2

1 Albert-Ludwigs-Universität, Medizinische Fakultät, Physiologisches Institut, Freiburg, Germany 2 Albert-Ludwigs-Universität, Medizinische Fakultät, Institut für Experimentelle und Klinische Pharmakologie und

Toxikologie, Freiburg, Germany

Background: Two-pore channels (TPCs) constitute a small family of cation channels expressed in endo-lysosomal

compartments. TPCs have been characterized as critical elements controlling Ca2+-mediated vesicular membrane

fusion and fission. TPCs regulate endo-lysosomal trafficking and thereby control surface expression of membrane

proteins via endocytosis, recycling, and degradation. Here, we investigate the location of TPC protein 1 (TPC1) in

the kidney and its role in the dynamic regulation of tubular transport.

Methods: Immunohistochemistry and colocalization analysis were used to identify TPC1 expression and association

with tubular segments along the nephron and confocal microscopy for subcellular localization in mice. The functional

role of TPC1 in the regulation of tubular reabsorption was studied in anesthetized littermate TPC1-knockout (TPC1-

ko) and wildtype mice (wt) subjected to dynamic challenges induced by bolus injection of PTH or transitions from

baseline to alkaline to acidic load via consecutive infusion of NaCl, Na2CO3, and NH4Cl.

Results: TPC1 expression was identified in proximal, but not distal tubules with subcellular localization predominantly

below the apical membrane. The PTH-induced rise in phosphate excretion reached its peak excretion rate later and

with exaggerated peak level in TPC1-ko compared to wt-mice, suggesting impaired transition back towards higher

phosphate reabsorption with dissipation of the PTH-bolus. PTH also induced a rise in volume excretion with slower

time course than phosphate, but without differences between genotypes, suggesting that other sodium transport

mechanisms were largely unaffected by TPC1. In the acid-base transition experiment, Na2CO3 induced a strong rise

in phosphate excretion, identical between genotypes. However, the return of phosphate excretion after switching to

NH4Cl in TPC1-ko temporarily lagged behind that in wt mice. Na2CO3 also increased volume and reduced ammonium

excretion rates, and subsequent NH4Cl normalized volume and elevated ammonium excretion, but without

differences between genotypes.

Conclusions: TPC1 is expressed subapically in the proximal tubule and plays an important role in the dynamic

regulation of proximal tubular phosphate reabsorption, but apparently not other sodium or ammonium transport. This

pertains to transition towards enhanced, but not reduced phosphate reabsorption, compatible with altered trafficking

of NaPiIIa/c-transporters.

Acknowledgment

This work was supported by the Deutsche Forschungsgemeinschaft DFG (KL 1119/6-1)


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OS 02-08

Renal depletion of Ip6k1/Ip6k2 in mice reduces the expression of NaPi-

IIa and NaPi-IIc disturbing phosphate homeostasis

Betül Haykir1,2, Eva M. Pastor-Arroyo1,2, Udo Schnitzbauer1,2, Isabel Prucker4, Danye Qui4, Dorothea

Fiedler5, Adolfo Saiardi3, Henning J. Jessen4, Nati Hernando1,2, Carsten A. Wagner1,2

1 University of Zurich, Physiology Insitute, Zurich, Switzerland 2 National Center of Competence in Research NCCR Kidney.CH, Zurich, Switzerland 3 University College London, London, UK 4 University of Freiburg, Freiburg, Germany 5 Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany

Phosphate (Pi) is an essential mineral. Hyperphosphatemia leads to ectopic mineralization, increasing the risk of

cardiovascular and renal disease, while hypophosphatemia may cause rickets/osteomalacia and skeletal myopathy.

Pi homeostasis is chiefly regulated in the kidney by way of modulating the expression of the Na+-dependent Pi

cotransporters, NaPi-IIa and NaPi-IIc, at the apical membrane of proximal tubules. In non-mammalian pro- and

eukaryotes, inositol pyrophosphates such as IP7 and IP8, are small signaling molecules involved in many cellular

events including regulation of Pi. 5-IP7 is generated by inositol hexakisphosphate kinases (IP6Ks). In this study, we

investigated the role in Pi homeostasis of renal Ip6k1/2, the kinases more abundantly expressed in kidney. We

showed that of the three human paralogues of IP6Ks subtypes 1 and 2 are expressed in the kidney and renal cell

lines, whereas IP6K3 is absent. The genetic deletion of Ip6k1 and Ip6k2 in Opossum Kidney (OK) cells, a cellular

model for renal proximal epithelia, reduces 5-IP7 and Pi transport along with the expression of NaPi-IIa and NaPi-IIc.

The physiological relevance was demonstrated in vivo using mice with kidney-specific ablation of both kinases in a

doxycycline-inducible Pax8-cre recombinase system: Ip6k1/2 deficient mice exhibited highly reduced renal levels of

5-IP7 and 1,5-IP8, were hypophosphatemic, had very low expression of renal NaPi-IIa/c and negligible plasma levels

of the phosphaturic hormone fibroblast growth factor 23. Transcriptome analysis showed perturbations principally in

transport processes as well as in cell adhesion and cytoskeletal components. Thus, the results indicate that the

Ip6k1/2 are critical for renal control of Pi homeostasis and are likely involved in other renal epithelial cell processes.


of 516

Oral Sessions 30 September 2021 3:00 PM – 5:00 PM

Lecture Hall 2

OS 03 | Pulmonary Physiology and Hypoxia:

Take a Breath Chair

Andrea Olschewski (Graz)

Holger Scholz (Berlin)


of 516

OS 03-01

NFAT5/TonEBP controls metabolic adaption of pulmonary artery

smooth muscle cells in the hypoxic lung

Thomas Korff

Heidelberg University, Department of Cardiovascular Physiology, Institute of Physiology and Pathophysiology,

Heidelberg, Germany

Chronic hypoxia increases the tone of vascular smooth muscle cells (VSMCs) of pulmonary arteries and augments

their muscularization. While these adaptive responses require an adjustment of the VSMC transcriptome, critical

regulatory elements are not well defined in this context. In this study, we explored the functional role of the stress

responsive transcription factor nuclear factor of activated T-cells 5 (NFAT5) in the hypoxic lung.

Exposure to hypoxia induced a rapid nuclear translocation of NFAT5 in cultured murine VSMCs. SMC-specific

ablation of Nfat5 (Nfat5(SMC-/-)) increased the artery tone-dependent systolic pressure in the right ventricle (RVSP) of

the mouse heart and impairs its function upon exposure to hypoxia. Analyses of the lung transcriptome revealed a

robust increase in the expression of genes attributed to metabolism and mitochondrial respiration. Loss of Nfat5 in

cultured hypoxia-exposed pulmonary artery VSMCs decreases the expression of the glycolysis-related genes lactate

dehydrogenase A (Ldha) while stimulating the expression of multiple mitochondrium-associated genes including

those encoding cytochrome oxidases. Inhibition of LDHA activity as well as loss of Nfat5 stimulated the mitochondrial

production of reactive oxygen species (ROS), which may enhance VSMC constriction. Scavenging of ROS

normalized the RVSP values in hypoxia-exposed Nfat5(SMC-/-) mice.

In summary, our findings suggest a crucial role for NFAT5 in adjusting the transcriptome of hypoxia-exposed

pulmonary artery VSMCs to support an adequate glycolysis-centered metabolism. Loss of Nfat5 impairs this

response thereby fueling the mitochondrial respiration and ROS production amplifying the hypoxia-mediated

constriction of pulmonary arteries.

Acknowledgment

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) under project number 394046768-SFB

1366-A1, A6, B5, C4, Z2. The authors would like to acknowledge the excellent technical assistance of Maria

Harlacher.


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OS 03-02

Adaptation of the oxygen sensing system during normal lung

development

Karin Kirschner2, Herrmann Stehr1, Stefanie Endesfelder1, Johanna Penzlin1, Charlotte Jacobi1,

Miriam Sieg3, Jochen Kruppa4, Christof Dame1, Lina Sciesielski1

1 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Department of Neonatology, Berlin, Germany 2 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Institute for Translational Physiology, Berlin, Germany 3 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Institute of Biometry and Clinical Epidemiology, Berlin, Germany 4 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Institute of Medical Informatics, Berlin, Germany

Question: After birth, arterial oxygen pressure abruptly rises. In premature birth, this change is considered a trigger

for lung injury (in particular for bronchopulmonary dysplasia), especially if additional mechanical ventilation is

required. One would expect that the adaptation to the high oxygen levels of air-breathing life required adaptation of

the oxygen sensing system and its targets, the oxygen-regulated genes (ORGs). Herein, we describe the expression

patterns of the oxygen sensing system (hypoxia inducible factors Hif1/2/3a, prolyl hydroxylases Phd2/3) and typical

ORGs like vascular endothelial growth factor A (Vegfa), neurotrophin receptor TrkB (Trkb), glucose transporter type

1 (Glut1), carbonic anhydrase 9 (Ca9) and erythropoietin(Epo) during lung development, birth and postnatal life.

Furthermore, we explore the adaptation of the oxygen sensing system to different experimental oxygenation settings.

Methods: mRNA expression levels in the murine lung were measured by RT-qPCR from the embryonic (E12)

throughout the adult stage. To localize specific mRNAs in tissue specimens, the in situ hybridization technique

RNAscope® was used. Corresponding Hif1a protein stabilization was quantified by immunoblotting. Effects of

different oxygenation settings in lungs were studied in embryonic explant cultures exposed to 21% vs. 1% O2, early

postnatal mice exposed to 21% vs. 80% O2 and adult mice exposed to 21% vs. 8% O2 systemic hypoxia or the

hypoxia mimetic Roxadustat, respectively.

Results: Hif1α protein disappeared after birth with initiation of lung ventilation. Among the analyzed ORGs, only

Phd3 showed a significant drop in expression which correlated with the loss of Hif1a protein right after birth. This is

remarkable as we verified that embryonic lungs are able to react to hypoxia by increased ORG expression. In

contrast, hyperoxic exposure of early postnatal pubs could not decrease expression of these ORGs. Systemic

hypoxia or Roxadustat treatment only significantly increased the low-expressed transcripts Trkb and Hif3a.

Conclusion: The adult lung as best ventilated organ appears to be relatively inert to systemic oxygen changes as

from the tested ORGs, only Trkb and Hif3a were significantly induced. This capacity seemed to be required during

gestation, with the same ORGs still being oxygen-sensitive in embryonic but no longer in early postnatal or adult

stages.

Acknowledgment

The technical expertise of Nicole Dinse and Ulrike Neumann is gratefully acknowledged. This work was supported

by a German Research Foundation (DFG) grant to LS (SC132/3-1) and KK (KI1441/4-1).


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OS 03-03

A miRNA derived from the 17/92 cluster is a novel player in

programming pulmonary hypertension in response to gestational

hypoxia

Yishi Qin1, Katharina Hochkogler1,2, Luisa Lehmann1,2, Mathieu Klop1,2, Zsuzsanna Mayer3,

Stefan Holdenrieder3, Andreas Petry1, Agnes Görlach1,2,4

1 German Heart Center Munich at the Technical University Munich, Experimental and Molecular Pediatric

Cardiology, Munich, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Munich, Munich, Germany 3 German Heart Center Munich at the Technical University Munich, Institute of Laboratory Medicine, Munich,

Germany 4 Technical University of Munich, School of Medicine, Munich, Germany

Background: Hypoxia is a relatively common prenatal stress, which is not only associated with high altitude living,

but also with smoking, sleep apnea, adiposity, maternal anemia or placental insufficiency. Fetal hypoxia often leads

to IUGR (intrauterine growth restriction) and has been associated with programming of cardiovascular and metabolic

diseases in adulthood including pulmonary hypertension. While underlying pathways are not resolved in detail,

epigenetic alterations might play an important role. Hypoxia and other redox alterations can affect epigenetic

pathways including the profile of non-coding miRNAs. However, their role in cardiovascular disease programming in

response to fetal hypoxia is not well understood.

Purposes:We aimed to characterize the role of gestational hypoxia on fetal miRNA profiles and their impact on

programming cardiovascular diseases in adults.

Methods: C57BL/6J mice were exposed to fetal hypoxia for 1 day from E10.5 - E11.5. Embryos and adult offspring

were analysed by functional, immunohistochemical and molecular methods including miRNA profiling. Functional

analyses were performed in vitro in embryoid bodies and in vivo by administration of either a miRNA mimic or inhibitor.

Results: Transient gestational hypoxia for 1 day was sufficient to induce IUGR. Adult offspring derived from transient

hypoxic pregnancies not only showed reduced weight, but also developed pulmonary hypertension characterized by

pulmonary vascular remodelling, increased right ventricular pressure and right ventricular hypertrophy. Some

miRNAs derived from the 17/92 miRNA cluster were differentially regulated in embryos and in the heart of adult

offspring. In vitro analyses using embryoid bodies validated a role of some of these miRNAs in the response to

hypoxia and in cardiac development. Treatment with a miRNA inhibitor was sufficient to induce pulmonary

hypertension in adult mice whereas treating offspring derived from gestational hypoxia pregnancies with a miRNA

mimic prevented programming of pulmonary hypertension in the adult.

Conclusions: In this study we could demonstrate the hypoxic sensitivity of miRNAs derived from the 17/92

cluster. Dysregulation of a single miRNA from this cluster is sufficient to promote fetal programming of pulmonary

hypertension, thus opening novel avenues for preventing and treating this potentially devastating disease.


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OS 03-04

The endocannabinoid anandamide is an airway relaxant in health and

disease

Annika Simon1, Alexander Seidinger1, Michaela Matthey1, Laura Bindila3, Daniela Wenzel1,2

1 Ruhr-University Bochum, Medical Faculty/Institute of Physiology/Department of Systems Physiology, Bochum,

Germany 2 Rheinische Friedrich-Wilhelms-Universität Bonn, Medical Faculty/Institute of Physiology 1, Bonn, Germany 3 Johannes Gutenberg-University Mainz, University Medical Center/Institute of Physiological Chemistry/ Clinical

Lipidomics Unit, Mainz, Germany

Asthma and COPD are among the most common chronic diseases affecting children and adults worldwide. The main

treatment strategies for these airway diseases are induction of bronchodilation and suppression of inflammation.

Because we could recently show that the endocannabinoid anandamide (AEA) modulates pulmonary vascular tone

we now analyzed the effect of AEA on airways.

We examined the impact of AEA on airway tone of mouse tracheas by isometric force measurements and small

intrapulmonary airways were analyzed in precision cut lung slices (PCLS). To verify FAAH expression in airways and

different cell types we used qPCR and immunostainings. Endocannabinoid levels were quantified using liquid

chromatography-multiple reaction monitoring measurements (LC-MRM) in lung homogenates of healthy and

asthmatic mice. PGE2 and cAMP concentrations in tissue homogenates or supernatant of AEA-treated tracheas or

human airway cells were measured by ELISA. Finally, we assayed the AEA effect on airway mechanics using the

Flexivent system in healthy and OVA-sensitized mice.

AEA (10 µM) induced a very strong airway relaxation after serotonin (5-HT) pre-constriction in tracheal rings

(109±10.9%, n=5) and PCLS (83.5±19.7%), which was unaltered in Cnr1/Cnr2-/- mice (100.5±9.3% n=9). In tracheas

from FAAH-/- mice or WT mice pre-treated with the FAAH inhibitor URB597 airway relaxation by AEA was strongly

attenuated (FAAH-/-: 18.2±8,3% n=8, URB: 11.1±21.7% n=6). The important role of FAAH-dependent metabolites

for bronchorelaxation was confirmed by the lack of effect found after Met-AEA stimulation (16.5±26.2 n=5). The

cyclooxygenase inhibitor indomethacin and the combination of EP2 and EP4 inhibitors reduced AEA-dependent

airway relaxation. Similarly, AEA stimulation of tracheas, human tracheal epithelial (hTEPC) and airway smooth

muscle cells (hASMC) increased PGE2 and cAMP levels. Interestingly, in the lungs of asthmatic mice AEA levels as

well as AEA synthesis enzymes were reduced compared to healthy controls. In vivo application of AEA prevented

the increase of airway resistance in response to 5-HT compared to solvent in healthy and asthmatic mice.Our ex and

in vivo results highlight AEA as a strong airway relaxant in health and OVA-dependent asthma. The effect is mediated

by FAAH-dependent metabolites and PGE2 acting via EP2/EP4 receptors in trachea, hTEPC and hASMC. Thus,

modulation of the AEA/FAAH axis may a therapeutic concept for airway relaxation.


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OS 03-05

Embryonic alveolar macrophage progenitors are critically reliant on

epithelial GM-CSF

Samantha Sherman1, Julia Gschwend1, Frederike Ridder2, Xiaogang Feng1, Hong-Erh Liang3,

Richard Locksley3,4,5, Burkhard Becher2, Christoph Schneider1

1 University of Zürich, Institute of Physiology, Zürich, Switzerland 2 University of Zürich, Institute of Experimental Immunology, Zürich, Switzerland 3 University of California San Francisco, Department of Medicine, San Francisco, USA 4 University of California San Francisco, Department of Microbiology & Immunology, San Francisco, USA 5 University of California San Francisco, Howard Hughes Medical Institute, San Francisco, USA

Fetal monocytes that seed embryonic tissues during organogenesis differentiate into specialized tissue-resident

macrophages upon exposure to critical niche-specific factors. In the lung, alveolar macrophages (AMs) are known to

rely heavily on GM-CSF (encoded by Csf2) for their survival. Postnatally, we have unequivocally demonstrated that

AM survival is critically reliant not on hematopoietic GM-CSF but on epithelial GM-CSF derived from alveolar epithelial

type 2 cells (AT2s). It is not known, however, if differentiating fetal monocytes also require epithelial GM-CSF for AM

fate specification. To answer this question, we have developed novel transgenic Csf2 reporter mice that were used

to not only profile pulmonary GM-CSF production, but also to selectively delete Csf2 expression from populations of

interest. Flow cytometry analysis of Csf2-reporter kinetics suggests that pulmonary GM-CSF production is first

induced between E16.5 and E18.5. At E17.5, constitutive deletion of hematopoietic GM-CSF did not impact the lung

fetal monocyte population. Depletion of epithelial GM-CSF, however, revealed a severe perturbation in the

developing fetal monocytes, with significant declines in both fetal monocyte numbers and proliferation. The

aforementioned postnatal AM-AT2 relationship thus begins during embryogenesis, where nascent AT2s timely

induce GM-CSF expression to support the proliferation and differentiation of fetal monocytes towards an AM fate.

Acknowledgment

We thank Brigitte Herzog for technical expertise; M. Kopf for helpful discussion and support with mouse rederivation;

J. Rock, H. Chapman, and S. Tugues for providing mice; C. Wagner for providing the anti-NaPi-IIb; and members of

the Schneider laboratory for helpful discussions. This work was supported by grants from the UCSF Diabetes

Research Center, US National Institutes of Health (NIH), the Howard Hughes Medical Institute, and the Sandler

Asthma Basic Research (SABRe) Center at the UCSF to R.M.L, the Swiss National Science Foundation

(310030_188450) to B.B., and the Peter Hans Hofschneider Professorship for Molecular Medicine to C.S.


of 516

OS 03-06

Adiponectin counteracts mechanosensitive myofibroblast activation in

an in-vitro model of pulmonary fibrosis

Julia K. Nemeth, Annika Schundner, Manfred Frick

Ulm University, Institute of General Physiology, Ulm, Germany

Idiopathic pulmonary fibrosis (IPF) a is fatal respiratory disease with limited therapeutic options. The major hallmarks

of IPF are excessive fibroblast activation and deposition of extracellular matrix (ECM). This results in fibrotic tissue

remodeling and dramatically stiffens lung tissue. Increased matrix stiffness further promotes fibroblast activation,

matrix synthesis and contraction, establishing a feedback loop that amplifies this pathological process. The detailed

molecular mechanisms of stiffness-dependent fibroblast activation and mechanisms to reverse matrix stiffening are

yet elusive and hinder effective treatment of IPF.

We performed an unbiased, large scale RNAseq screen to identify mechanosensitive signalling pathways and

regulators of fibroblast activation in primary lung fibroblasts. Primary fibroblasts were cultured on substrates of

physiological (5 kPa PDMS)) and pro-fibrotic (plastic) stiffness for up to 14 days and samples were collected on day

0, 2, 4, 7 and 14. We identified adiponectin signaling as target in cells cultivated on matrices with pro-fibrotic substrate

stiffness. RT-qPCR, western blot and immunofluorescence studies further confirmed that adiponectin inhibits

mechanically induced expression of myofibroblast (acta2) and ECM (col1a) marker genes in primary fibroblasts.

Results from selective inhibition (APN400) or siRNA-mediated knock-down of adiponectin receptors revealed that

adiponectin signalling depends on non-canonical binding to cadherin 13. Subsequent high-throughput

pharmacological screens using a novel fibroblast reporter cell line (10-4ABFP) generated by CRISPR/cas9 technology [1] identified the p38-MAPK signaling pathway as essential mediator of mechanosensitive fibroblast activation and

ECM production. This was further confirmed by qPCR and sandwich ELISA.

In summary, our data suggest that adiponectin supresses excessive fibroblast activation and ECM deposition.

Targeting adiponectin-signaling therefore potentially slows down fibrotic tissue remodeling and constitutes a

promising therapeutic target for treatment of IPF.

References [1] Nemeth J. et al. A Novel Fibroblast Reporter Cell Line for in vitro Studies of Pulmonary Fibrosis. Front

Physiol. 2020 Oct 9;11:567675.


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OS 03-07

HIF in HHT - Hypoxia inducible factors in hereditary hemorrhagic

telangiectasia

Anna Wrobeln1, Freya Dröge2, Jonah Bosserhoff1, Joachim Fandrey1

1 University of Duisburg-Essen, Institute of Physiology, Essen, Germany 2 University of Duisburg-Essen/University Hospital Essen, Department of Otorhinolaryngology/Head and Neck

Surgery, Essen, Germany

Patients with hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Olser-Weber syndrome, suffer from

recurrent hemorrhages that originate in abnormal vascular structures. The inherited, rare disease is characterized by

multisystemic arteriovenous malformations predominantly in intestine, lung and brain.[1] The mutation affects the

transforming growth factor-β (TGF-β) pathway, that regulates angiogenesis, but also functions as a regulatory

cytokine in the immune response.[2] Additionally to the arteriovenous malformations, these patients suffer from an

impaired immune response.[3] The mechanism underlying the loss of function in HHT-immune cells is still unknown.

In general, it is well known that TGF-β interacts with hypoxia inducible factors (HIF).[4] Like TGF-β HIFs are involved

in inflammatory and angiogenic processes.[5] Although HIFs plays a central role in both, angiogenesis and immune

response, no one has yet addressed the consequences of the mutations on HIF in HHT-patients.

Within this study, for the first time, HIFs and their up- and downstream-regulated genes were analyzed in whole blood

samples from HHT-Patients as well as in organs from HHT simulating knockout-mice. Surprisingly, the analysis of

whole blood from HHT-Patients revealed a significant lower expression of HIF-1α mRNA compared to the healthy

controls. Genes upstream from HIF-1α in the inflammation response (e.g. NFκB) are unchanged, whereas HIF-1α

target genes are partly significant downregulated (e.g. IL6). Isolated Peripheral blood mononuclear cells (PBMCs)

from HHT patients fail to stabilize HIF-1α protein under hypoxic conditions (1% O2) after 4 hours of incubation, but

showed an increased protein stabilization after 24 hours under these conditions compared to healthy control PBMCs.

Reflecting human samples, organs of HHT-mice showed decreased HIF-1α in intestine, lung and brain on RNA and

protein level compared to wildtype siblings.Taken together, these results underline the relevance of HIFs in HHT.

Because the knowledge of diseases and the associated molecular mechanisms that downregulate HIF-1α in vivo

almost does not exist, further studies have to clarify these interesting findings. Moreover, investigations may identify

HIF-stabilization as a possible therapeutic target in HHT patients.

References [1] Faughnan, M.; Palda, V.; Garcia-Tsao, G.; Geisthoff, U.; McDonald, J.; Proctor, D.; Spears, J.; Brown, D.;

Buscarini, E.; Chesnutt, M.J.J.o.m.g. International guidelines for the diagnosis and management of hereditary haemorrhagic telangiectasia. 2011, 48, 73-87.

[2] Bernabéu, C.; Blanco, F.J.; Langa, C.; Garrido-Martin, E.M.; Botella, L.M. Involvement of the TGF-β superfamily signalling pathway in hereditary haemorrhagic telangiectasia. Journal of Applied Biomedicine 2010, 8, 169-177.

[3] Cirulli, A.; Loria, M.P.; Dambra, P.; Di Serio, F.; Ventura, M.T.; Amati, L.; Jirillo, E.; Sabba, C. Patients with hereditary hemorrhagic telangectasia (HHT) exhibit a deficit of polymorphonuclear cell and monocyte oxidative burst and phagocytosis: a possible correlation with altered adaptive immune responsiveness in HHT. Current pharmaceutical design 2006, 12, 1209-1215.

[4] Basu, R.K.; Hubchak, S.; Hayashida, T.; Runyan, C.E.; Schumacker, P.T.; Schnaper, H.W. Interdependence of HIF-1α and TGF-β/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression. American Journal of Physiology-Renal Physiology 2011, 300, F898-F905.

[5] Frede, S.; Berchner‐Pfannschmidt, U.; Fandrey, J. Regulation of hypoxia‐inducible factors during inflammation. Methods in enzymology 2007, 435, 403-419.


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OS 03-08

The oxygen sensor factor inhibiting HIF (FIH) catalyzes the formation of

a likely covalent protein complex with the NF-κB inhibitorβ (IκBβ)

Yulia Volkova, Roland H. Wenger, Carsten C. Scholz

University of Zürich, Institute of Physiology, Zürich, Switzerland

Oxygen sensing allows mammalian cells to adapt to oxygen depletion (hypoxia) and is thus central for their survival.

The cellular oxygen sensors prolyl-4-hydroxylase domain (PHD) proteins 1-3 and the asparagine hydroxylase factor

inhibiting HIF (FIH) confer hypoxia sensitivity to the hypoxia-inducible factor (HIF) transcriptional activator. However,

the phenotype of FIH knockout mice could not be exclusively explained by the regulation of HIF activity, indicating a

role of FIH outside of the HIF pathway. In addition, FIH also regulates IL-1β–induced NF-κB activity independently of

HIF, but the underlying molecular mechanisms are unclear. Several proteins have previously been suggested as

alternative substrates for FIH, especially proteins with ankyrin repeat domains (ARDs), but the functional relevance

of ARD hydroxylation remains to be elucidated. We previously reported that the deubiquitinase (DUB) ovarian tumor

domain-containing ubiquitin aldehyde binding 1 (OTUB1) protein (which does not contain ARDs) is a substrate for

FIH-mediated asparagine hydroxylation. Surprisingly, a subsequent study showed that FIH and OTUB1 also formed

an oxygen-dependent, extremely stable, protein-protein complex catalyzed by FIH activity, which regulated OTUB1

enzymatic function. A mass spectrometry-based analysis identified 12 additional putative stable protein complexes

formed by FIH, including IκBβ, a negative regulator of pro-inflammatory signaling. This indicated that FIH- and

oxygen-dependent stable (likely covalent) protein oligomers (“oxomers”) commonly occur in cells. Here, we confirmed

the putative FIH-IκBβ oxomer by immunoblotting. The formation of FIH-IκBβ was prevented by hypoxia, hydroxylase

inhibitors and inactivation of FIH by point mutation, clearly demonstrating that the oxomer bond was catalyzed by

FIH. Interestingly, IκBα (the most closely related protein to IκBβ; IκBα has previously been reported to be hydroxylated

by FIH) did not form an oxomer with FIH, demonstrating a FIH selectivity towards IκBβ for oxomer formation. To

identify the substrate amino acid residue for FIH within IκBβ, each asparagine was mutated to alanine, but this did

not interfere with oxomer formation (in contrast to OTUB1 where the hydroxylated asparagine 22 is essential for

oxomer formation). In summary, FIH forms a previously unknown oxomer specifically with IκBβ, which will now be

analysed for its potential impact on the regulation of NF-κB activity.


of 516

Oral Sessions 30 September 2021 3:00 PM – 5:00 PM

Lecture Hall 4

OS 04 | Inflammation: Feel the Burn Chair

Anna Keppner (Fribourg)

Flavia Rezende (Frankfurt/Main)


of 516

OS 04-01

The voltage-gated potassium channel KV1.3 regulates neutrophil

recruitment during inflammation

Roland Immler1, Wiebke Nadolni2, Annika Bertsch1, Vasilios Morikis3, Sergi Masgrau-Alsina1,

Ina Rohwedder1, Tobias Schroll1, Anna Yevtushenko1, Oliver Soehnlein4, Markus Moser5,

Thomas Gudermann2, Eytan R. Barnea6, Markus Rehberg7, Scott I. Simon3, Susanna Zierler2,8,

Monika Pruenster1, Markus Sperandio1

1 Ludwig-Maximilians-Universität München, Institute of Cardiovascular Physiology and Pathophysiology, Planegg-

Martinsried, Germany 2 Ludwig-Maximilians-Universität München, Walther-Straub Institute of Pharmacology and Toxicology, Munich,

Germany 3 University of California, Department of Biomedical Engineering, Davies, USA 4 Westfälische Wilhelms-Universität Münster, Center for Molecular Biology of Inflammation (ZMBE), Münster,

Germany 5 Technical University Munich, Institute of Experimental Hematology, Munich, Germany 6 BioIncept LLC, New York, USA 7 Helmholtz Zentrum München, Institute of Lung Biology and Disease, Helmholtz, Neuherberg, Germany 8 Johannes Kepler University Linz, Institute of Pharmacology, Linz, Austria

Question: Neutrophil functions strongly rely on changes in intracellular calcium concentrations and concomitant

calcium signaling. In lymphocytes, sustained Ca2+ influx into the cell requires a compensatory efflux of potassium via

the voltage-gated potassium channel KV1.3 to maintain membrane potential. Although, voltage-gated potassium

currents over the plasma membrane have been described in neutrophils as well, no distinct ion channel could be

attributed to this observation so far. Here, we addressed the question whether KV1.3 is involved in Ca2+ signaling of

neutrophils thereby affecting neutrophil function during acute inflammatory processes.

Methods and Results: Using in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally

expressed in human neutrophils regulating sustained store operated Ca2+ entry (SOCE) through membrane potential

stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1)

impaired intracellular Ca2+ signaling, thereby preventing cellular spreading, adhesion strengthening and appropriate

crawling under flow conditions in vitro. In contrast, interfering with KV1.3 activity did not affect β2 integrin inside-out

signaling. Intravital microscopy revealed that pharmacological blockade or genetic deletion of KV1.3 in mice

decreased intravascular neutrophil adhesion in a shear rate dependent fashion in a mouse model of TNF-α-induced

microvascular inflammation. Furthermore, we identified KV1.3 as critical regulator of neutrophil extravasation into the

inflamed peritoneal cavity. Finally, we show that KV1.3 deficient neutrophils exhibit impaired phagocytosis of E.coli

particles.

Conclusion: Taken together, our findings do not only provide evidence for a role of KV1.3 in sustained calcium

signaling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat

inflammatory disorders characterized by overwhelming neutrophil infiltration.

Acknowledgment

Supported by Deutsche Forschungsgemeinschaft CRC914 projects A01 (M.M.), B01 (M.S), B08 (O.S), and B11N

(M.P) and TRR-152 projects P14 (S.Z.) and P15 (T.G.).


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OS 04-02

MRP8/14: governing Ca2+ to support neutrophil migration

Matteo Napoli1, Roland Immler1, Ina Rohwedder1, Thomas Vogl2, Johannes Roth2, Barbara Walzog1,

Markus Sperandio1, Monika Pruenster1

1 Ludwig-Maximilans University, Cardiovascular Physiology and Pathophysiology, Walter-Brendel-Centre of

Experimental Medicine, Biomedical Center, Munich, Germany 2 University of Muenster, Institute of Immunology, Cells in Motion Interfaculty Centre, Munster, Germany

Myeloid related protein 8/14 (MRP8/14) is a Ca2+ binding protein abundantly expressed in neutrophils, where it makes

up roughly 40% of the cytosolic protein content. In inflammatory scenarios, the actively secreted MRP8/14 acts as a

danger associated molecular pattern (DAMP) regulating β2 integrin activation through a TLR4-dependent

mechanism. Nonetheless, the intracellular function of the heterodimer remains elusive.

Here, we explored whether MRP8/14 might have a role in neutrophil Ca2+ signalling during leukocyte recruitment.

Indeed, functional Ca2+ signalling is indispensable for successful post-arrest modification steps mediating cytoskeletal

rearrangement and firm adhesion under flow conditions.

First, we investigated subcellular Ca2+ activity using Ca2+ reporter wildtype (WT) and Mrp14-/- mice (functional

MRP8/14 double deficient animals) measuring Ca2+/Lymphocyte function-associated antigen 1 (LFA-1) intensities

and LFA-1 clustering during neutrophil crawling under flow by confocal microscopy. In addition, we analysed overall

Ca2+ profiles by flow cytometry and sophisticated imaging analysis in WT and MRP8/14 deficient neutrophils. Finally,

we studied MRP8/14 dependent neutrophil spreading, polarization and crawling in flow chambers coated with E-

selectin, ICAM-1 and CXCL1 and investigated leukocyte adhesion and extravasation in vivo, in WT and Mrp14-/- mice

by intravital microscopy.

In this study, we show that Mrp14-/- neutrophils form less LFA-1 clusters and display reduced Ca2+ activity within the

latter, compared to WT cells. In agreement, Ca2+ flux upon chemokine stimulation had a faster signal decay in the

absence of MRP8/14. In line with our findings on Ca2+ activity, Mrp14-/- neutrophils exhibited defective spreading,

polarization and crawling. Finally, Mrp14-/-mice unveiled impaired leukocyte adhesion and reduced extravasation in

postcapillary venules of TNF-α stimulated cremaster muscle, relative to WT mice.Based on our findings we propose

a role for cytosolic MRP8/14 as Ca2+ orchestrator in neutrophils, required for cytoskeletal rearrangements and

therefore indispensable for firm leukocyte adhesion and postarrest modifications under flow.

Acknowledgment

Supported by Deutsche Forschungsgemeinschaft CRC914 projects B11 (M.P.) and Z03 (M.S., B.W.).


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OS 04-03

A20 acts as a negative regulator of neutrophil recruitment during

sterile inflammation

Lou M. Wackerbarth1, Ina Rohwedder1, Marc Schmidt-Supprian3, Matthias Gunzer2, Averil Ma4,

Markus Sperandio1

1 LMU, Institute of Cardiovascular Physiology and Pathophysiology, Munich, Germany 2 University of Duisburg-Essen, Institute for Experimental Immunology and Imaging, Duisburg-Essen, Germany 3 TU, Department of Hematology and Medical Oncology, Munich, Germany 4 University of California San Francisco, Department of Medicine, San Francisco, USA

Question: The ubiquitin modifying enzyme A20 (tumor necrosis factor-alpha-induced protein 3, Tnfaip3) belongs to

the NF-κB family of transcription factors, which are ubiquitously expressed and regulate numerous targets particularly

in immune cells. A20 acts as a potent negative feedback inhibitor of the canonical NF-κB signaling pathway and

Tnfaip3-/- mice suffer from multi-organ inflammation and premature death. Likewise, various point mutations in the

human A20 gene are associated to auto-inflammatory diseases. In our present study, we aimed to investigate the

effect of A20 deficiency on the leukocyte adhesion cascade by analyzing A20 depleted neutrophils in an in vivo

mouse model of sterile inflammation.

Methods & Results: Since the A20 constitutive knockout is prematurely lethal in mice, we bred Tnfaip3fl/fl to Ly6g-

tdTomatoCre mice (Tnfaip3fl/flLy6gCre), to obtain animals with a highly specific deletion of A20 in mature neutrophils.

A20 depletion in our model was verified by a strong decrease in A20 mRNA and protein expression in neutrophils

derived from Tnfaip3fl/flLy6gCre mice, although no complete knockout was observed. Using intravital microscopy of

TNF-α-stimulated cremaster muscle venules, we found higher numbers of rolling neutrophils in vessels of

Tnfaip3fl/flLy6gCre mice after normalization to the white blood cell count, compared to Ly6gCre control mice.

Interestingly, neutrophil rolling velocity was not altered between the groups. Subsequently, we analyzed neutrophil

adhesion and found a significant increase in absolute numbers of adherent neutrophils/mm2 in Tnfaip3fl/flLy6gCre

mice in contrast to the control group. Finally, we stained the exteriorized and fixed cremaster muscle tissue with

Giemsa to visualize extravasated neutrophils and detected a 30% increase of extravasated neutrophils in

Tnfaip3fl/flLy6gCre mice compared to Ly6gCre mice. In a second set of in vivo experiments, the cremaster muscle

model was performed within 45 minutes after surgical preparation of the tissue without any additional stimulation.

Interestingly, we observed decreased rolling velocities along with increased adhesion of A20 deficient neutrophils

compared to Ly6gCre control mice.

Conclusion: Taken together, our results show that loss of A20 in neutrophils leads to a hyperinflammatory phenotype

with increased rolling and adhesion of neutrophils to inflamed microvessels in vivo, identifying A20 as a key negative

regulator of neutrophil function in mice.

Acknowledgment

Supported by Deutsche Forschungsgemeinschaft CRC914 project B01 (M.S.).


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OS 04-04

Insulin-like growth factor 1, but not insulin, polarizes neutrophils to an

anti-inflammatory phenotype

Rianne Nederlof1, Sophia Reidel1, Patrick Petzsch2, Karl Köhrer2, Stefanie Gödecke1, Axel Gödecke1

1 Universitätsklinikum Düsseldorf, Institüt für Herz-und Kreislaufphysiologie, Düsseldorf, Germany 2 Heinrich-Heine-Universität Düsseldorf, Biologisch-Medizinisches Forschungszentrum, Genomics &

Transcriptomics Laboratory, Düsseldorf, Germany

Neutrophils play an important role in inflammation. They have long been thought to be only pro-inflammatory.

However, recent evidence shows that neutrophils, like macrophages, can have a pro-inflammatory ‘N1’ or anti-

inflammatory ‘N2’ phenotype. Previously we have shown that insulin-like growth factor 1 (IGF1) treatment can cause

a M2 polarization in macrophages. In this study, we studied the effect of IGF1 and insulin, which has a high homology

to IGF1, on neutrophil phenotype.

Neutrophils were isolated from the bone marrow of mice using Percoll-gradient centrifugation. Neutrophils were left

untreated, or treated 4 hours with (ng/ml) IL4 (20), LPS/IFNγ (10/2), IGF1 (10) or a physiological (10) or high (100)

dose of insulin. We could show, using western blot, that both the IGF1 and insulin receptor were present on

neutrophils. As expected, treatment with IL4 upregulated anti-inflammatory ‘N2’ marker Arg1, Retnla and Chil3, and

LPS/IFNγ upregulated pro-inflammatory ‘N1’ marker TNFα, IL12a and Nos2. Treatment with IGF1 also upregulated

N2 marker. Surprisingly, insulin, even though it is known to activate the same pathways as IGF1, had no effect on

the expression of N2 genes. RNA sequencing analysis showed that IGF1 treatment caused changes in 280 genes

when compared to control, whereas insulin treatment only altered a few genes. To look at the dependence of

pathways known to be activated by IGF1 and insulin, neutrophil polarization was studied in Akt1 or Akt2 Tie2-Cre

mice, which have a knock out in endothelial and hematopoietic cells. qPCR analysis showed the absence of Akt 1 or

Akt2, in Akt1 and Akt2 Tie2-Cre mice, respectively, without affecting the other Akt isoform. Absence of these specific

Akt isoforms had no effect on IL4 or IGF1 induced N2 polarization. Also in these mice, insulin had no effect on

neutrophil phenotype. In addition, we could show that both IGF1 and insulin, but not IL4 and LPS/IFNγ caused

phosphorylation of Erk, another downstream target of the IGF1 and insulin receptor. In contrast, phosphorylation of

STAT6, which has been associated with M2 polarization in macrophages, was only observed in IL4 and IGF1 treated

neutrophils.

Like macrophages, also neutrophils can be polarized to a pro- and anti-inflammatory phenotype. Even though they

have a high homology, IGF1, but not insulin, causes polarization to an anti-inflammatory N2 phenotype. Polarization

by IGF1 is caused by non-canonical pathway activation.


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OS 04-05

Impact of the acidic inflammatory milieu on neutrophil chemotaxis

Julia Schröder, Leonie Oster, Stina Becker, Albrecht Schwab, Karolina Najder-Nalepa

Westfälische Wilhelms-University, Institute of Physiology II, Münster, Germany

Neutrophil granulocytes are the first and robust responders to the chemotactic molecules released from an inflamed

tissue. Sites of inflammation, in turn, are usually acidic and rich in inflammatory mediators. The aim of this study was

to elucidate the role of the changing microenvirontment in neutrophil chemotaxis.

To this end, we combined live cell imaging chemotaxis assays with measurements of the intracellular pH (pHi) in

varied extracellular pH (pHe). Observational studies were complemented by measurements of the activated Cdc42

Rho GTPase in acidic pH.

Our data show that pHi of neutrophils dose-dependently adapts to a given extracellular pH. Neutrophil chemotaxis

towards C5a has an optimum at pHi~7.1. Consequently, a shallow pHe gradient resembling that encountered by

neutrophils during extravasation from a blood vessel (pH~7.4) into the interstitium (pH~7.2) favors chemotaxis of

stimulated neutrophils. Lowering pHe belowpH6.8, predominantly affects neutrophil chemotaxis, while the velocity is

largely maintained. Inhibition of the Na+/H+-exchanger 1 (NHE1) with cariporide drastically attenuates neutrophil

chemotaxis at the optimal pHi.The abundance of the active GTP-bound form of Cdc42 is strongly reduced at pHe6.5.

In conclusion, we propose that neutrophil chemotaxis towards C5a is pH dependent, which can possibly be explained

through altered Cdc42 activation. Moreover, neutrophil chemotaxis requires the activity of the Na+/H+ exchanger

NHE, showing the importance of the proton homeostasis in neutrophil function.


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OS 04-06

How do human leukocytes respond to inflammatory hypoxia?

Tina Schönberger1, Bastian Tebbe2, Tina Hörbelt-Grünheidt3, Marie Jakobs3, Oliver Witzke4, Manfred

Schedlowski3, Joachim Fandrey1

1 University of Duisburg-Essen, Institute of Physiology, Essen, Germany 2 University Hospital Essen, Department of Nephrology, Essen, Germany 3 University Hospital Essen, Institute of Medical Psychology and Behavioral Immunobiology, Essen, Germany 4 University Hospital Essen, Department of Infectious Diseases, Essen, Germany

Leukocytes, which migrate from the bloodstream into inflamed tissue, need to adapt to different oxygen conditions

and hypoxia in short time. Thereby the hypoxia-inducible factors (HIF) are important transcription factors for effective

immune response and leukocyte function. In life-threatening diseases like sepsis, the host immune response to

infection is deregulated. Prior studies showed that leukocytes display decreased HIF levels in septic patients[1].

We seek to gain a more in-depth understanding of the cellular adaption processes of circulating leukocytes

challenged with inflammation and hypoxia. Therefore, we are using an experimental human endotoxemia model to

study the immune response in acute infection in healthy male subjects. Volunteers receive an intravenous injection

of 0.4 ng/kg lipopolysaccharide (LPS) as inflammatory stimulus prior or after exposure to hypoxia (10.5% normobaric

oxygen) for four hours. LPS-induced effects are assessed by flow cytometry and qRT-PCR. Further, we study LPS

treated Peripheral Blood Mononuclear Cells (PBMC) and the cell line THP-1 under normoxic (21% O2) and hypoxic

(1% O2) conditions in combination with LPS treatment, regarding HIF protein accumulation, gene expression and

surface marker expression. We want to investigate, how this setting affects HIF in humans with respect to the

inflammatory immune response.

Our recent findings show differently altered gene expression patterns of HIF and target genes in distinct immune cell

subsets (neutrophils, monocytes, T cells) upon in vivo treatment with LPS and hypoxia. Further, we observed an

increased inflammatory response to LPS after a hypoxic phase in vitro in THP-1 cells.

In conclusion, cellular adaption processes of circulating leukocytes during inflammation and hypoxia are

interdigitated. Unveiling this interplay could give rise to new treatment options for septic patients.

References [1] Schäfer ST, Frede S, Winning S, Bick A, Roshangar P, Fandrey J, Peters J, Adamzik M. Hypoxia-inducible

factor and target gene expression are decreased in patients with sepsis: prospective observational clinical and cellular studies. Anesthesiology. 2013 Jun;118(6):1426-36. doi: 10.1097/ALN.0b013e31828baa67. PMID: 23449494.


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OS 04-07

Intestinal organoids as tools to study long-term effects of cytokines in

vitro

Jasmin Ballout, Martin Diener

Justus Liebig University Giessen, Institute for Veterinary Physiology and Biochemistry, Giessen, Germany

Intestinal organoids, also known as enteroids, are three-dimensional “mini-guts” growing from intestinal stem cells

(ISC) [1]. ISCs are located in the crypt base of the intestine. Their daughter cells differentiate during the migration to

the top of the villi into distinct cell types, e.g. enterocytes, Paneth cells or goblet cells. Thus, intestinal organoids show

similar physiological functions as the native epithelium, i.e. electrolyte and nutrient transport, epithelial barrier integrity

and a high regenerative capacity [3]. Hence, ISC-derived organoids can be used e.g. for drug screening, to study

gastrointestinal diseases or host-microbe interactions and especially long-term studies can be performed easily [2].

Our study focused on the (long-term) effects of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) on

epithelial functions of murine intestinal organoids. TNFα treatment (100 ng/ml) induced “organoid swelling”, i.e. the

shape of the enteroids moved from their typical polygonal, arm-building form to a more rounded appearance with an

enlarged lumen filled with desquamated cells. The latter was caused by a higher apoptosis rate, measured as

enhanced number of caspase-3-positive cells in the lumen of TNFα treated organoids. The swelling after TNFα

pretreatment might be caused by an enhancement of Cl‒ secretion in intestinal organoids. This was supported by the

observation that TNFα pretreatment enhanced the sensitivity of the enteroids to Ca2+-dependent secretagogues in

imaging experiments with the Ca2+-sensitive dye fura-2. Depending to the period of cytokine pretreatment (1, 3 or 5

days), the maximal increase in the fura-2 ratio (peak) evoked by the acetylcholine derivate carbachol as well as the

number of responding cells to carbachol were significantly higher compared to untreated controls. Which parts of the

signaling mechanism are upregulated by TNFα is currently under investigation. At first glance, the TNFα treatment

(100 ng/ml) did not change epithelial barrier properties to a more “leaky” enteroid, because expression of the tight

junction marker zonula occludens 1 (ZO-1) did not differ between the TNFα treated and control group. Further

experiments have to be performed to characterize the effects of TNFα on intestinal organoid function more detailed.

Acknowledgment

The diligent technical assistance of Mrs. Brigitta Buß, Bärbel Schmidt and Alice Stockinger is a pleasure to

acknowledge.

References [1] Almeqdadi, Mohammad; Mana, Miyeko D.; Roper, Jatin; Yilmaz, Ömer H. (2019): Gut organoids: mini-tissues in

culture to study intestinal physiology and disease. In: American journal of physiology. Cell physiology 317 (3), C405-C419. DOI: 10.1152/ajpcell.00300.2017.

[2] Holloway, Emily M.; Capeling, Meghan M.; Spence, Jason R. (2019): Biologically inspired approaches to enhance human organoid complexity. In: Development (Cambridge, England) 146 (8). DOI: 10.1242/dev.166173.

[3] Zietek, Tamara; Rath, Eva; Haller, Dirk; Daniel, Hannelore (2015): Intestinal organoids for assessing nutrient transport, sensing and incretin secretion. In: Scientific reports 5, S. 16831. DOI: 10.1038/srep16831.


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OS 04-08

Phosphate Intake Aggravates Rise in Plasma Fibroblast Growth Factor

23 and Interleukin-6 in Murine Folic Acid-Induced Acute Kidney Injury

Ahmad Kamal Hamid1,2, Maria L. Muscalu1,2, Eva M. Pastor-Arroyo1,2, Carsten A. Wagner1,2,

Daniela Egli-Spichtig1,2

1 University of Zurich, Institute of Physiology, Zurich, Switzerland 2 Centre of Competence in Research NCCR Kidney.CH, Zurich, Switzerland

Fibroblast growth factor 23 (FGF23) is a bone-derived phosphaturic hormone that represses parathyroid hormone

(PTH) and calcitriol levels. Plasma FGF23 rises dramatically in many inflammatory diseases and independently

predicts renal, cardiovascular, and all-cause mortality. Studies have also demonstrated a link between plasma

phosphate (Pi) and renal disease severity and plasma Pi has been shown to independently exacerbate inflammatory

state, but its contribution to FGF23 elevation in acute kidney injury (AKI) remains understudied. Hence, the

comparative effects of normal (NP) and low (LP) dietary Pi in the folic acid (FA)-induced AKI mouse model has been

investigated in the context of FGF23 and sterile inflammation. Mice were fed either a 0.6% (NP) or <0.1% (LP) Pi diet

for 5 days prior to inducing FA-AKI with a single intraperitoneal FA dose of 250 mg/kg and sacrificed 24 h later.

Whereas plasma urea and creatinine were similarly elevated in the FA groups fed either diet, the LP group exhibited

a trend for an ameliorated rise in urinary neutrophil gelatinase-associated lipocalin (p=0.054), a kidney injury marker.

In FA-AKI, the LP diet curbed the rise in plasma intact (430.6 vs. 3381.7 pg/mL) and total (973.2 vs. 4126.2 pg/mL)

FGF23 compared to the NP diet, prevented the rise in plasma PTH(1–84) and calcitriol, and downregulated renal

Cyp27b1 (encoding 1α-hydroxylase). The LP diet also prevented the AKI-induced upregulation of Fgf23 in bone but

not spleen and thymus, and reduced the elevation of plasma interleukin-6 (IL-6) and upregulation of renal Il6.

Therefore, low Pi intake reduces the FA-AKI-induced rise in plasma FGF23 and IL-6 and prevents osseous Fgf23

upregulation and rise in plasma PTH(1–84) and calcitriol. These results highlight the importance of dietary Pi

restriction in patients with renal disease and lay the foundation for elucidating the mechanism underpinning the

association of FGF23 and plasma Pi withrenal disease severity and mortality.


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Oral Sessions 01 October 2021

10:30 AM – 12:30 PM

Lecture Hall 2

OS 05 | Channels - Plug & Play Chair

Angelika Lampert (Aachen)

Jochen Schwenk (Freiburg)


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OS 05-01

The molecular movement of prestin underlying outer hair cell

electromotility

Dominik Lenz1, Makoto Kuwabara1, Julia Hartmann1, Piersilvio Longo2, Britt Marie Huckschlag1,

Thomas Berger1, Annalisa Questino1, Jan-Philipp Machtens2, Dominik Oliver1

1 Philipps University, Institute for Physiology and Pathophysiology, Marburg, Germany 2 Forschungszentrum Jülich, Institute of Biological Information Processing (IBI-1), Jülich, Germany

Sensitive hearing relies on an active mechanical process termed the cochlear amplifier, mediated by cochlear outer

hair cells. Amplification results from ultrafast cellular length changes driven by membrane potential changes, a

process known as electromotility. At the molecular level, electromotility is mediated by the membrane protein prestin

(SLC26A5), a member of the SLC26 family of anion transporters. The molecular mechanism, i.e. the conformational

dynamics that generate the mechanical output, has remained unknown.

Recently, experimental structures of mammalian and bacterial homologs revealed dimeric organization of this protein

family, with each monomer organized into two major helix bundles: the core domain containing the substrate binding

site and the gate domain positioned at the dimer interface. Here, we report on a combined structural and functional

approach to elucidate the structural dynamics that generate electromotility.

Using extensive molecular dynamics simulations based on the recent cryo-EM structure of inward-facing Slc26a9

combined with homology modeling of prestin, we developed and refined an ensemble of structures that represent

various putative functional states of prestin. We first performed a cysteine accessibility scan along the core-gate

interface. Accessibility of individual amino acid positions suggested a major reorientation between core and gate

domains that exposes positions close to the central binding site towards the extracellular solution. Secondly, we

directly measured voltage dependent movement of the core domain by voltage clamp fluorometry both from

electromotile mammalian prestin and from the transport-active zebrafish SLC26A5. Fluorescence changes of an

environmentally sensitive dye attached to the core domain displayed the same voltage dependence as electromotility,

indicating that conformational rearrangement of the core domain is directly associated with prestin’s mechanical

activity. Moreover, core domain movement was modulated by transport substrate, indicating involvement in anion

transport. Third, cross-bridging of pairs of cysteines engineered into the core/gate domain interface suggested a

translational movement of the core domain relative to the gate domain.

Together these findings show for the first time that electromotility and transport by SLC26A5/prestin result from an

elevator-like movement.

Acknowledgment

Supported by DFG Research Group FOR 5046 (OL 240/8-1 to D.O. and MA 7525/2-1 to J.-P. M.). The authors

gratefully acknowledge the computing time granted through JARA on the supercomputer JURECA at

Forschungszentrum Jülich.


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OS 05-02

Impact of selenomethionine mis-incorporation on voltage-gated

sodium channels

Rama A. Hussein, Marwa Ahmed, Nikita Kuldyushev, Roland Schönherr, Stefan H. Heinemann

Friedrich Schiller University Jena and Jena University Hospital, Department of Biophysics, Center of Molecular

Biomedicine, Jena, Germany

Intake of selenomethionine (SeMet) results in integration of this amino acid instead of methionine (Met) in proteins.

Although the redox properties of SeMet and Met are markedly different, little is known about impacts of SeMet mis-

incorporation on electrical excitability. Given the importance of a Met residue in the inactivation motif of voltage-gated

sodium channels (NaV) [1], we postulated that SeMet incorporation at that position might have consequences for NaV

channel inactivation. To study SeMet mis-incorporation in channel proteins, we developed experimental protocols to

quantitatively monitor the extent of SeMet incorporation with ratiometric fluorescence imaging. The underlying

principle is a GFP-based methionine oxidation sensor with one Met residue, whose oxidation yields a calibrated

readout. With this method, we determined SeMet supplementation levels that results in certain percentages of SeMet

incorporation in newly translated protein.

HEK293T cells were transfected with DNA coding for rat NaV1.4 in SeMet media, and NaV currents were measured

with whole-cell patch clamp. Most strikingly, even with close to 100% SeMet incorporation, i.e. with almost all 66 Met

residues in the protein replaced with SeMet, NaV1.4 channels exhibited near normal activation and inactivation

properties. However, while 300-s exposure to 100 µM of the Met-specific oxidant chloramine T had almost no impact

on rNaV1.4Met channels (control), rNaV1.4SeMet channels responded with an almost complete loss of inactivation and

around 60% reduction of the peak current amplitude at -20 mV. Both effects on rNaV1.4SeMet were readily reversible

by dithiothreitol, in agreementwith the reported non-enzymatic reversibility of SeMet oxidation [2], while no such

reversibility was observed for rNaV1.4Met. Similar results were obtained for cardiac NaV1.5 and neuronal NaV1.2 and

NaV1.7. Partial substitution of SeMet in the culture media resulted in a graded response; oxidation-dependent

impairment of inactivation was detected for as low as 5% SeMet incorporation. Furthermore, functional SeMet

incorporation was even measured in TTX-sensitive NaV channels of murine dorsal root ganglia neurons after

overnight culture in SeMet media.SeMet incorporation in NaV channel proteins coinciding with oxidative insults may

therefore affect electrical excitability of neurons and muscle cells to result in hyperexcitability pathologies, similar to

congenital NaV channel gain-of-function mutations.

Acknowledgment

Support by the Research Training Groups RTG 1715 (RH) and RTG 2155 (MA, NK) financed by the German

Research Foundation (DFG).

References [1] Kassmann, M, Hansel, A, Leipold, E, Birkenbeil, J, Lu, SQ, Hoshi, T, & Heinemann, SH 2008. 'Oxidation of

multiple methionine residues impairs rapid sodium channel inactivation'. Pflugers Archiv : European journal of physiology,456(6), 1085-1095, https://doi.org/10.1007/s00424-008-0477-6: Springer

[2] Krause, RJ, and Elfarra, AA 2009, 'Reduction of L-methionine selenoxide to seleno-L-methionine by endogenous thiols, ascorbic acid, or methimazole.' Biochemical Pharmacology 77(1), 134-140, https://doi.org/10.1016/j.bcp.2008.09.022: Elsevier


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OS 05-03

Localization of Na+ channel clusters in narrowed perinexi of gap

junctional plaques modulate cardiac action potential transmission via

ephaptic coupling: a modeling study

Ena Ivanovic, Jan P. Kucera

University of Bern, Department of Physiology, Bern, Switzerland

Background: Classically, cardiac action potential propagation relies on current flow through gap junctions. However,

it has been proposed that when gap junctional coupling is reduced, propagation can be supported via ephaptic

coupling, a mechanism mediated by negative electric potentials occurring in narrow intercellular clefts of intercalated

discs (IDs) [1, 2]. Recent studies showed that Na+ channels form clusters near gap junctional plaques in nanodomains

called perinexi, where the ID cleft is even narrower [2-4]. We previously showed in a computational model that Na+

channel clustering in the ID potentiates ephaptic coupling [5]. However, the physiological relevance of Na+ channel

clusters being located in perinexi has never been examined.

Methods: We developed a high-resolution three-dimensional finite element model of two longitudinally abutting

cardiomyocytes. In the ID, we positioned a central Na+ channel cluster and a gap junctional plaque, which was located

either close to the Na+ channel cluster or remotely (Fig. 1). The Na+ channel cluster was thus located either inside or

outside the perinexus. The pre-junctional cell was excited by a depolarizing current pulse, and potentials were

recorded in the ID cleft and in the post-junctional cell, for various combinations of ID cleft widths inside and outside

the perinexus.

Results: When the Na+ channel cluster was located in the perinexus of a closely positioned gap junctional plaque

(top row in Fig. 1), varying perinexal width greatly modulated action potential transmission from one cell to the other.

This modulation occurred via the interplay of the Na+ currents and the extracellular potential in the cleft. In contrast,

when the Na+ channel cluster was located remotely from the gap junctional plaque and its perinexus (bottom row in

Fig.1), this modulation by perinexus width largely disappeared. Moreover, when the Na+ channel cluster was located

in the perinexus, more combinations of bulk cleft width and perinexus width permitted action potential transmission

(Fig. 2), and the maximal bulk cleft width leading to the excitation of the post-junctional cell increased.

Conclusion: These resultsindicate that the localization of Na+ channel clusters in the perinexi of gap junctional

plaques is crucial for action potential transmission via ephaptic coupling. Furthermore, perinexal width greatly

modulates ephaptic coupling. These findings are relevant for a comprehensive understanding of cardiac excitation.

Acknowledgment

This study was supported by the Swiss National Science Foundation (grant n° 310030-184707 to JPK).


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Figure 1 Effects of the relative position of a Na+

channel cluster and a gap junctional plaque with

its perinexus on ephaptic coupling.

(A) Schematic of the ID, with two different

locations of the gap junctional plaque (green)

and its perinexus (gray) relative to the Na+

channel cluster (red). (B) Intracellular

potentials Vi (left), Na+ currents INa through

the ID membranes (middle) and minimal

extracellular potential Ve (right) for zero gap

junctional coupling, a bulk cleft width of 60

nm, and varying perinexal widths (solid curves:

pre-junctional cell; dotted curves: post-

junctional cell; see legend).

Figure 2 Modulation of ephaptic coupling by bulk ID cleft

width, perinexus width and the relative position

of a Na+ channel cluster and a gap junctional

plaque with its perinexus on ephaptic coupling.

Effects observed on the post-junctional cell for

different combinations of bulk cleft width and

perinexus width, for the two locations of the

gap junctional plaque (green) and its perinexus

(gray) relative to the Na+ channel cluster (red)

shown in Figure 1.

References [1] Sperelakis, N, Mann, JE 1977, ‘Evaluation of electric field changes in the cleft between excitable cells’,

J Theor Biol, 64, 71-96 [2] Veeraraghavan, R, Hoeker, GS, Alvarez-Laviada, A, Hoagland, D, Wan, X, King, DR, Sanchez-Alonso, J, Chen,

C, Jourdan, J, Isom, LL, Deschenes, I, Smyth, JW, Gorelik, J, Poelzing, S, Gourdie, RG 2018, ‘The adhesion function of the sodium channel beta subunit (beta1) contributes to cardiac action potential propagation’, eLife, 7, e37610

[3] Veeraraghavan, R, Lin, J, Hoeker, GS, Keener, JP, Gourdie, RG, Poelzing, S 2015, ‘Sodium channels in the Cx43 gap junction perinexus may constitute a cardiac ephapse: an experimental and modeling study’, Pflugers Arch, 467, 2093-2105

[4] Veeraraghavan, R, Gourdie, RG 2016, ‘Stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization’, Mol Biol Cell, 27, 3583-3590

[5] Hichri, E, Abriel, H, Kucera, JP 2018, ‘Distribution of cardiac sodium channels in clusters potentiates ephaptic interactions in the intercalated disc’, J Physiol, 596, 563-589


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OS 05-04

Cav1.3 channels are linear amplifiers of firing activity in DA SN

neurons

Josef Shin1, Lora Kovacheva1, Dominique Thomas2, Strahinja Stojanovic1, Carlos Paladini4, Gerd

Geisslinger2,3, Jörg Striessnig5, Jochen Roeper1

1 Goethe University, Institute of Neurophysiology, Neuroscience Center, Frankfurt am Main, Germany 2 Pharmazentrum Frankfurt / ZAFES, Institute of clinical pharmacology, Frankfurt am Main, Germany 3 Fraunhofer Institute of Molecular Biology and Applied Ecology IME, Branch for Translational Medicine and

Pharmacology TMP, Frankfurt am Main, Germany 4 University of Texas at San Antonio, UTSA Neuroscience Institute, San Antonio, Germany 5 University of Innsbruck, Department of Pharmacology and Toxicology/ Center for Molecular Biosciences,

Innsbruck, Germany

Studying the role of Cav1.3 channels in dopamine neurons of the substantia nigra pars compacta (DA SN) and its

potential relevance in the context of neuroprotection in Parkinson disease has been complicated due to the lack of

selective pharmacological targeting (1,2,3). We combined on-cell, whole-cell and perforated patch-clamp recordings

of labelled and identified DA SN neurons in acute slice preparations of 8 – 12 weeks old C57BL6/N wildtype and

Cav1.2 DHP-/- knockin mice. In the latter, only Cav1.3 channels are sensitive to dihydropyridines (DHP) like

isradipine, while the DHP binding site of Cav1.2 has been rendered non-functional by mutagenesis. We demonstrate

that in vitro autonomous pacing is amplified by Cav1.3 channels with a linear gain of about 30% of the intrinsic

baseline frequency. The Cav1.3 gain was unaffected by somatodendritic D2 autoreceptors or the presence of DHP-

sensitive Cav1.2 channels in wildtype mice, suggesting a universal role of Cav1.3 channels as a rate-dependent

linear amplifier in DA SN neurons. To characterize Cav1.3 channel function across the entire dynamic firing range of

DA SN neurons observed in vivo (ca. 1-50 Hz), we utilized the dynamic-clamp technique for evoking these

frequencies via in silico NMDAR conductances. By using 300 nM isradipine, we demonstrated that Cav1.3 channels

linearly amplified firing frequencies across the entire range selectively in lateral DA SN neurons. We also evaluated

clinically relevant (3 – 10 nM) concentrations of isradipine in vitro in a similar fashion. These low nanomolar

concentrations partially reduced lateral DA SN firing across the dynamic range by about 25%, consistent with

incomplete Cav1.3 channel inhibition. We then tested whether in vivo firing of lateral DA SN neurons was selectively

reduced by 25% upon systemic application of isradipine (3 mg kg-1) as predicted by our in vitro experiments. Our in

vivo recordings confirmed these predictions for lateral DA SN neurons and also demonstrated that medial DA SN

neurons were not affected in vivo by clinically relevant concentrations of israpipine. Our study demonstrated that

Cav1.3 channels function as linear amplifiers of autonomous pacemaking and selectively boost firing frequency in

vivo across the entire dynamic firing range of lateral DA SN neurons. We also show that targeting the most vulnerable

DA neurons in the lateral SN with DHPs in the clinical concentration range is feasible and might be applicable in

Parkinson Disease.

References [1] Chan, C.S. et al. 2007, 'Rejuvenation' protects neurons in mouse models of Parkinson's disease. Nature 447,

1081-1086 [2] Guzman, J. N. et al., 2018, Systemic isradipine treatment diminishes calcium-dependent mitochondrial

oxidant stress. J. Clin. Invest. 128, 2266-2280 [3] Ortner, N. J. et al. 2017, Lower affinity of isradipine for L-type Ca2+ channels during substantia nigra

dopamine neuron-like activity: implications for neuroprotection in Parkinson's disease. J. Neurosci. 2946-16.


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OS 05-05

miR-221/222 influence calcium handling in cardiomyocytes by targeting

L-type Ca2+ channels

Maria Knyrim1, Stephanie Binas1, Udo Kloeckner1, Sindy Rabe1, Sigrid Mildenberger1, Katja Quarch1,

Nicole Strätz1, Danny Misiak2, Claudia Grossmann1, Michael Gekle1, Barbara Schreier1

1 Martin Luther University Halle-Wittenberg, Julius-Bernstein-Institute of Physiology, Halle (Saale), Germany 2 Martin Luther University Halle-Wittenberg, Institute of Molecular Medicine, Halle (Saale), Germany

While it is established that miR-221/222 are associated with cardiac remodelling and disease, the precise role needs

to be evaluated. Our group previously demonstrated that upregulated miR-221/222 expression in hearts from

epidermal growth factor receptor knockout (EGFR KO) mice was correlated to downregulated cardiac ion channel

genes. Among the regulated ion channel genes were subunits of the L-type Ca2+ channel (LTCC; Cacna1c as pore-

forming subunit, Cacnb2 and Cacna2d1 as auxiliary subunits) which are predicted targets of both miRs [1].

Additionally, Cacna1c, Cacnb2 and Cacna2d1 mRNA expression was decreased in the hearts of mice with cardiac

hypertrophy due to angiotensin II-infusion. These mice also showed increased miR-221/222 levels [1]. The aim of

this study was to analyse the possible role of miR-221/222 in electrical remodelling in cardiomyocytes.

Dual luciferase reporter assays revealed Cacna1c as a direct target of miR-221/222. In order to determine a possible

impact of miR-221/222-dependent regulation on ion channel function, LTCC current density was analysed by whole

cell patch clamp recording. HL-1 cells transfected with miR-221 or -222 mimics showed a reduction in LTCC current

density while the voltage-dependence of activation was not altered. To investigate if this regulation has an impact on

Ca2+ homeostasis, ratiometric fluorescence microscopy was performed in HL-1 cells and calcium and contractility

measurements were performed in neonatal cardiomyocytes (neoCMs). Transient transfection of HL-1 cells with miR-

221/222 mimics led to slower depolarization-dependent Ca2+ entry and increased proportion of non-responding cells.

Angiotensin II-induced Ca2+ release from the sarcoplasmic reticulum was not affected by miR-221/222. Furthermore,

response to β-adrenergic stimulation (isoprenaline) was altered in neoCMs. In miR-222-transfected neoCMs the

isoprenaline-induced positive inotropic effect on the intracellular Ca2+ transient was lost and the positive chronotropic

effect on spontaneous beating activity was strongly reduced. This could lead to a reduced contractility and systolic

dysfunction of the whole heart.

This study adds a new role of miR-221/222 in cardiac electrical remodelling by showing the impact on β-adrenergic

regulation of L-type Ca2+ channel function, cardiomyocyte calcium handling and contractility.

References [1] Binas S, Knyrim M, Hupfeld J, Kloeckner U, Rabe S, Mildenberger S, et al. miR-221 and -222 target CACNA1C

and KCNJ5 leading to altered cardiac ion channel expression and current density. Cell Mol Life Sci [Internet]. 2020;77(5):903–18. Available from: https://doi.org/10.1007/s00018-019-03217-y


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OS 05-06

TRPM3-mediated calcium transients intertwined with dynamic

mitochondrial activation: possible mechanisms of NGF-induced latent

sensitization in chronic nonspecific low back pain

Dan Wang1, Qi Gao2, Ina Schäfer1, Handan Mörz1, Ulrich Hoheisel1, Karl Rohr2, Wolfgang Greffrath1,

Rolf D. Treede1

1 Heidelberg University, Mannheim Centre for Translational Neuroscience, Mannheim, Germany 2 Heidelberg University and DKFZ, Biomedical Computer Vision Group, BioQuant, IPMB, Heidelberg, Germany

Transient receptor potential ion channel melastatin 3 (TRPM3) is an important nociceptive sensor in the peripheral

nervous system and modulates long-term potentiation (LTP) related to memory processes as a downstream effector

of neurosteroids (primarily pregnenolone sulfate, PregS) in the hippocampus. However, the function of TRPM3 in

synaptic plasticity involving pain sensitization is still less well explored. Based on the prevalent distribution of TRPM3

on dorsal root ganglia neurons (DRGs) at the presynaptic level, we now directly address TRPM3-associated

mechanisms of nerve growth factor (NGF)-induced latent sensitization in nonspecific low back pain (LBP), focusing

on presynaptic plasticity. By live-cell imaging approaches combined with our newly developed software tools-

mitoflash tracker, we specifically target TRPM3-mediated calcium transients in DRGs exposed to NGF treatment and

mitoflash pulsations in HEK cells upon PregS challenge, showing that NGF preincubation accelerated calcium influx

through TRPM3 by 37% (*p<0.05) and slowed intracellular calcium clearance by 31% (*p<0.05) without significantly

upregulating TRPM3 expression in DRGs; acute NGF treatment attenuated the tachyphylaxis to repeated application

of PregS from 46% to 12% (*p=0.001), prolonged the rise time of calcium transients from 2.3 to 2.7 min (*p=0.046),

and slowed intracellular calcium clearance by 50% (*p<0.05); compared with depolarization by high potassium

solution, PregS caused more mitochondrial calcium loading in DRGs (41% vs. 20%, *p=0.045), and induced a steady-

state mitochondrial calcium elevation that can be imitated by HEK cells. In HEK cells, a basal ROS increase was

accompanied by a steep rise in the incidence of mitoflash pulsations by a factor of 4.7 (*p<0.001). The present study

for the first time demonstrates that NGF-induced latent sensitization is partly ascribed to the potentiation of NGF on

TRPM3-mediated calcium transients of DRGs in response to PregS, providing evidence for the involvement of

TRPM3 in presynaptic modulation of neuroplasticity in nonspecific LBP, and preliminarily disclosed increased

pulsatile mitoflashes downstream of PregS-induced TRPM3 activation, proposing that the frequency response of

mitoflash pulsations could be one of the earliest events initiating synaptic plasticity implicated in pain sensitization

and be a new perspective toward the investigation of chronic nonspecific LBP.

Acknowledgment

We thank Professor Heping Cheng for the mt-cp-YFP plasmid donation.

References [1] Baron R, Binder A, Attal N, Casale R, Dickenson AH & Treede RD (2016) Neuropathic low back pain in clinical

practice. Eur J Pain (United Kingdom) 20: 861–873 doi:10.1002/ejp.838 [2] Hoheisel U, Reuter R, De Freitas MF, Treede RD & Mense S (2013) Injection of nerve growth factor into a low

back muscle induces long-lasting latent hypersensitivity in rat dorsal horn neurons. Pain 154: 1953–1960 [3] Fu ZX, Tan X, Fang H, Lau PM, Wang X, Cheng H & Bi GQ (2017) Dendritic mitoflash as a putative signal for

stabilizing long-term synaptic plasticity. Nat Commun 8: 1–11 [4] Mulier M, Ranst N Van, Corthout N, Munck S, Berghe P Vanden, Vriens J, Voets T & Moilanen LJ (2020)

Upregulation of TRPM3 drives hyperexcitability in nociceptors innervating inflamed tissue. Elife: 1–27 [5] Flatters SJL (2015) The contribution of mitochondria to sensory processing and pain. Prog Mol Biol Transl

Sci 131: 119–146


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OS 05-07

Lipids directly control permeability and ion selectivity of TMEM16 lipid

scramblases

Andrei Kostritskii1,2,3, Jan-Philipp Machtens1,2,3

1 Forschungszentrum Jülich, Institute of Biological Information Processing (IBI-1), Molekular- und Zellphysiologie,

Jülich, Germany 2 RWTH Aachen University, Institute of Clinical Pharmacology, Aachen, Germany 3 Forschungszentrum Jülich, JARA-HPC, Jülich, Germany

Ca2+-activated lipid scramblases of the TMEM16 protein family (also known as anoctamins) are transmembrane

proteins, which mediate the bidirectional transport of lipids between the leaflets of cellular membranes and

additionally operate as ion channels. Ionic currents mediated by TMEM16 lipid scramblases are implicated in various

physiological processes, including apoptosis, immune response, and cell-volume regulation [1]. Despite recent

progress in their structural characterization, the molecular mechanisms underlying ion conduction of these dual-

function proteins remain poorly understood [2]. In particular, little is known about the determinants of ion selectivity

in TMEM16 lipid scramblases, which are variously reported to conduct anion-, cation-, or non-selective currents.

Here, we used extensive atomistic molecular dynamics (MD) simulations, combined with the recently developed

Computational Electrophysiology approach, to obtain high-resolution insights into the dynamics of ion conduction in

TMEM16 lipid scramblases [3]. Our simulations of fungal nhTMEM16 and human TMEM16K revealed that ions are

mainly conducted through a structured but yet dynamic proteolipidic pore, which is partly formed by lipid headgroups.

Lining the ion permeation pathway, the lipid headgroups directly interact with permeating ions. Interestingly, these

interactions depend on the polarity of the applied voltage, which affects orientation of the lipid headgroups. Moreover,

we discovered that the lipid headgroups flank the neck region of the pore and could control its permeability state. We

also found that ion selectivity of TMEM16 lipid scramblases depends not only on positioning and orientation of

charged residues in the pore, but also on membrane lipid composition. In particular, the ion selectivity prominently

changes when anionic lipids are present in the membrane. Our analysis demonstrated that such change is mainly

caused by an increase in membrane surface charge at the entrances to the pore. In summary, we identified the

molecular mechanisms of ion conduction in TMEM16 lipid scramblases and discovered the direct effects of

membrane lipids on the ion permeability and selectivity of these dual-function proteins. Furthermore, our results

showcase how state-of-the-art molecular simulations can provide insights into the modulatory role of the cell

membrane in the physiological function of membrane proteins at unprecedented resolution.

Acknowledgment

The authors gratefully acknowledge the computing time granted through JARA on the supercomputer JURECA at

Forschungszentrum Jülich. References [1] Kunzelmann, K., Nilius, B., Owsianik G., Schreiber R., Ousingsawat J., Sirianant L., Wanitchakool P.,

Bevers E.M., Heemskerk J.W.M. 2014, ‘Molecular functions of anoctamin 6 (TMEM16F): a chloride channel, cation channel, or phospholipid scramblase?’, Pflügers Archiv - European Journal of Physiology, 466,407–414

[2] Kalienkova V., Mosina V.C., Paulino C. 2021, 'The Groovy TMEM16 Family: Molecular Mechanisms of Lipid Scrambling and Ion Conduction.', Journal of Molecular Biology, 166941

[3] Kostritskii A.Y., Machtens J.-P. 2021 'Molecular mechanisms of ion conduction and ion selectivity in TMEM16 lipid scramblases', Nature Communications, 12,2826


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OS 05-08

Paneth cell expansion and granule secretion are modulated by

TMEM16A and TMEM16F.

Rainer Schreiber, Jiraporn Ousingsawat, Karl Kunzelmann

Universität Regensburg, Institut für Physiologie, Regensburg, Germany

Introduction: Paneth cells form part of the innate immune response. Expansion of Paneth cell during intestinal

metaplasia, in inflammatory bowel disease and in response to mucosal damage has been reported. The mechanisms

that regulate the secretion in response to stimuli are poorly understood. Paneth cells immediately release granules

upon exposure of the apical surface to LPS or by basolateral muscarinic stimulation. We reported a contribution of

the Ca2+ activated Cl- channel TMEM16A and the phospholipid scramblase TMEM16F to cellular exocytosis. Both

TMEM16A and TMEM16F enhance receptor-mediated Ca2+ increase in the apical sub-membranous compartment,

thereby supporting the exocytic machinery of goblet cells in airways and intestine. In the absence of TMEM16A and

TMEM16F, intracellular Ca2+ levels in the apical pole of goblet cells were found to be attenuated, and thus basal and

ATP-activated mucus release is compromised. In the present study we therefore analyzed the role of TMEM16A and

TMEM16F for Paneth cell expansion in vivo and examined their contribution to agonist-induced release of intracellular

granules.

Methods: intestinal knockout mice, histology, organoids, video imaging of granule secretion.

Results: In crypts from jejunum and ileum of 6 months and 1 year old mice, knockout of intestinal TMEM16F

increased the number of Paneth cells, size of Paneth cells, number of intracellular granules and lysozyme content.

The results indicated hyper- and metaplasia of Paneth cells in the absence of TMEM16F. Moreover, intestinal crypts

were isolated from jejunum and cultured to form intestinal organoids. Basolateral application of ATP induced a

transient apical release of intracellular granules, whereas muscarinic stimulation by carbachol (CCH) caused

complete granular depletion. Apical ATP-induced transient secretion of granules was sensitive to the TMEM16A

blockers CaCC-AO1 and Ani9 and was significantly reduced by knockdown of TMEM16A. The CCH-induced granule

depletion was not impaired by inhibition of TMEM16A, but was reduced in organoids from intestinal TMEM16F

knockout mice.

Conclusion: These results provide evidence that TMEM16A and TMEM16F are required for fusion of Paneth cell

granules with the apical membrane and exocytosis. Absence of TMEM16F causes deregulation of size and number

of Paneth cells in vivo. While TMEM16F contributes to muscarinic secretion, TMEM16A is required for ATP-induced

exocytosis of granules.

Acknowledgment

Supported by DFG project number 387509280, SFB 1350 (project A3).


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Oral Sessions 01 October 2021 3:00 PM – 5:00 PM

Lecture Hall 1

OS 06 | Cellular Neurophysiology: from

Molecular Mechanisms to System Functions Chair

Kristina Lippmann (Leipzig)

Jonas-Frederic Sauer (Freiburg)


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OS 06-01

Directed and acyclic synaptic topology in the human layer 2/3 temporal

cortex

Yangfan Peng1, Antje Bjelde1, Franz X. Mittermaier1, Henrike Planert1, Sabine Grosser2,

Pawel Fidzinski3, Henrik Alle1, Imre Vida2, Jörg R. P. Geiger1

1 Charité-Universtitätsmedizin Berlin, Institut für Neurophysiologie, Berlin, Germany 2 Charité-Universtitätsmedizin Berlin, Institut für integrative Neuroanatomie, Berlin, Germany 3 Berlin Institute of Health at Charité-Universtitätsmedizin Berlin, NeuroCure Clinical Research Center, Berlin,

Germany

Recurrent excitatory synapses dominate the output of pyramidal neurons, but their function for the local computation

remains unclear. While high and non-random reciprocity among pyramidal neurons has been described in juvenile

rodent cortices, the exact connectivity rules governing these connections is still unknown. In humans, the L2/3 of the

cortex is greatly expanded compared to other mammals, and the functional architecture of local excitatory

connectivity has not been studied yet. We performed multi-neuron patch-clamp recordings of up to 10 pyramidal

neurons simultaneously in human temporal cortical tissue obtained from 22 epilepsy patients. We recorded from

more than 1000 pyramidal neurons and detected more than 1000 monosynaptic excitatory connections in around

9000 tested connections. Higher-order network motif analysis and reconstruction of anatomical cell positions

identified specific connectivity rules: The synaptic connectivity among pyramidal neurons within clusters is

heterogenous across local clusters with the network architecture being acyclic and anatomically directed. These

findings may have important theoretical implications for local information flow in the expanded human L2/3 cortex.

Taken together, the newly identified functional properties combined with anatomical determinants of synaptic

connectivity highlight potential species-specific differences of neocortical network architecture.

Acknowledgment

Current funding and address of Y. Peng: DFG Walter-Benjamin Fellow at MRC Brain Network Dynamics Unit,

University of Oxford, UK.


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OS 06-02

Presynaptic ATP concentration decreases during physiological activity

at a central synapse

Lukas Kunstmann, Isabelle Straub, Jens Eilers, Johannes Hirrlinger, Stefan Hallermann

Universität Leipzig, Carl Ludwig Institut für Physiologie, Leipzig, Germany

Brain tissue is highly energy demanding. The synapses of the brain require a lot of energy to maintain

neurotransmitter release from the presynaptic terminals. Studies on cultured neurons suggest that ATP production

can compensate the presynaptic ATP consumption. However, presynaptic ATP dynamics in intact brain tissue is still

poorly understood. Here, we perform high-resolution measurements of presynaptic ATP levels at cerebellar mossy

fiber to granule cell synapses in acute brain slices to investigate the presynaptic ATP dynamics under physiological

conditions. Cerebellar mossy fibers convey sensory information using a broad range of firing frequencies between 1

and 1000 Hz including sustained firing at a few 100 Hz in vivo, and are thus ideally suited to study presynaptic ATP

consumption at different physiological activities. We simultaneously measured the presynaptic ATP levels with

the Förster resonance energy transfer (FRET) based ATP-sensor ATeam1.03(YEMK) and excitatory postsynaptic

currents (EPSC) at granule cells during electrical stimulation of single mossy fiber axons. The basal FRET ratio did

not correlate with the depth of the cerebellar mossy fiber boutons in the slice. Synaptic transmission at a frequency

of 100 Hz for 5 and 10 s decreased the FRET ratio by 4 ± 0.6 % and 7.7 ± 1.7 % (n = 6 and 9), respectively.

Furthermore, synaptic transmission at a lower frequency at 20 Hz for 30 s, resembling a weak physiological activity

at this synapse, decreased the FRET ratio by 3.7 ± 0.9 % (n = 7). Our data thus indicates that the presynaptic ATP

concentration decreases during physiological neuronal activity at a central synapse.


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OS 06-03

Chloride transport-independent function of Cl-/H+ exchangers and its

role in neuronal excitability

Juan David Sierra Marquez1, Antje Willuweit2, Angelika Lampert3, Christoph Fahlke1, Raul E. Guzman1

1 Forschungszentrum Jülich, Institute of Biological Information Processing, Molecular and Cellular Physiology (IBI

1), Jülich, Germany 2 Forschungszentrum Jülich, Institute of Neuroscience and Medicine, Medical Imaging Physics (INM-4), Jülich,

Germany 3 RWTH Aachen University, Institute of Physiology, Medical Faculty, Aachen, Germany

The chloride transport activity, the main function of intracellular Cl-/H+ exchangers, is evolutionary conserved and

regulates a variety of multiple physiological and cellular functions. It supports the acidification of intracellular

organelles by regulating luminal chloride homeostasis [1, 2]. ClC-3 is highly expressed in the central nervous (CNS)

and the peripheral nervous system (PNS). Clcn3‐/‐ mouse model shows severe neurodegeneration in the CNS [3] and

exhibits a behavioral phenotype of hyperalgesia [4], highlighting the physiological relevance of this transporter within

both systems. Here we studied the role of ClC-3 in the regulation of neuronal excitability in nociceptive neurons and

microglia homeostasis using Clcn3‐/‐ mouse model. Dorsal root ganglion (DRG) neurons from mutant mice show

enhanced neuronal excitability due to altered Na+ and K+ current densities. Ablation of ClC-3 does not affect the

viability of the DRG nor of the dorsal horn neurons. They show an increased expression of CC-chemokine ligand 2

(CCL2), accompanied by the detection of reactive microglia within the spinal cord, suggesting a central sensitization

(Fig 1). DRGs from Clcn3E281Q/Clcn4‐/‐ mice, which are virtually devoid of any Cl-/H+‐mediated transport, show no

signs of altered neuronal excitability. Action potential properties and K+ current densities were similar to the control

condition, suggesting that the transport mechanism of ClC-3 is not involved in the regulation of neuronal excitability

in sensory cells. This work unveils a novel function for ClC‐3 that independent of its chloride transport activity and is

going beyond to simply contribute to the acidification and osmotic balance of endosomes.

Acknowledgment

We thank Dr. Karlijn van Aerde and Dr. Dirk Feldmeyer to provide the Igor-based macro to analyze the action potential

data, Dr. Ute Becherer to provide the protocol used for the extraction and culturing of DRG neurons. We are grateful

to the staff of our animal facility and to Nicola Kornadt-Beck for invaluable support in all aspects of animal work. This

work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) (GU 2042/2-1) to REG.


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Figure 1. Changes in the pain signaling pathway in the Clcn3-/- mouse model

References [1] Guzman, RE, Miranda-Laferte, E, Franzen, A, Fahlke, C 2015, ‘Neuronal ClC-3 splice variants differ in

subcellular localizations, but mediate identical transport functions’, Journal of Biological Chemistry, 290(43), 25851-62

[2] Bose, S, He, H, Stauber, T 2021, ‘Neurodegeneration Upon Dysfunction of Endosomal/Lysosomal CLC Chloride Transporters’, Frontiers in Cell and Developmental Biology, 9, 639231

[3] Weinert, S, Gimber, N, Deuschel, D, Stuhlmann, T, Puchkov, D, Farsi, Z, Ludwig, C, Novarino G, López-Cayuqueo, K, Planells-Cases, R, Jentsch, T 2020 ‘Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration’ The EMBO Journal, 39(9), e103358

[4] Pang, R-P, Xie, M-X, Yang, J, Shen, K-F, Chen, X, Su, Y-X, Yang, C, Tao, J, Liang, S-J, Zhou, J-G, Zhu, H-Q, Wie, X-H, Li, Y-Y, Qin, Z-H, Liu, X-G 2016, ‘Downregulation of ClC-3 in dorsal root ganglia neurons contributes to mechanical hypersensitivity following peripheral nerve injury’, Neuropharmacology, 110, 181-9


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OS 06-04

Auxiliary subunits control function and distribution of AMPA receptor

complexes in hippocampal NG2 glia during development

Gerald Seifert, Stefan Hardt, Stefan Passlick, Aline Timmermann, Dario Tascio, Ronald Jabs,

Christian Steinhäuser

University of Bonn, Medical Faculty, Institute of Cellular Neurosciences, Bonn, Germany

NG2 glial cells are equipped with functional AMPA and GABAA receptors and receive direct synaptic input from

glutamatergic and GABAergic neurons. We combined functional and molecular techniques to analyse properties and

expression of auxiliary subunits of AMPA receptors, so-called transmembrane AMPA receptor regulatory proteins

(TARPs) and cornichons (CNIHs) in NG2 cells of the juvenile and adult hippocampus.

To identify Ca2+ permeable AMPA receptors, polyamine derivatives (Naspm and IEM-1460) were exposed

intracellularly as well as applied by bath application. Strong blockage of AMPA receptor currents by polyamines was

observed in NG2 glia from adult mice, indicating developmental upregulation of Ca2+ permeable AMPA receptors.

Molecular analysis revealed frequent expression of auxiliary subunits TARPs γ4, γ7, γ8, and CNIH-2 in NG2 glia, but

all of the aforementioned subunits were downregulated during development. The profound downregulation of TARP

γ8 during development encouraged us to apply a specific TARP γ8 antagonist, JNJ55511118. Application of JNJ

blocked receptor currents that were insensitive to blockers of Ca2+ permeable receptors, indicating that TARP γ8 is

mainly associated with Ca2+ impermeable AMPA receptors. Additionally, the high blocking efficiency of extracellularly

applied polyamine derivatives in NG2 glia of adult mice indicates expression of Ca2+ permeable and Ca2+

impermeable AMPA receptors. Moreover, postsynaptic receptor currents on NG2 glia were sensitive to Naspm and

IEM-1460, indicating expression of Ca2+ permeable AMPA receptors on postsynaptic glial membranes.

Ca2+ permeable AMPA receptors through somatic AMPA receptors may regulate proliferation and differentiation of

NG2 glia. Pharmacological characterization and deletion of AMPA receptors and/or TARP/CNIH subunits may help

to decipher specific roles of AMPA receptor/TARP complexes in neural signaling.

Acknowledgment

Supported by DFG (SE 774/6, STE 552/5).


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OS 06-05

5-HT4 receptor interacts with adhesion molecule L1 to modulate

morphogenic signaling in neurons

Daria Guseva1, Simon Bennet Sonnenberg2, Jonah Rauer2, Christoph Göhr2, Nataliya Gorinski2, Sophie

Kristin Schade2, Dalia Abdel Galil2, Vladimir Naumenko3, André Zeug2, Stephan C. Bischoff1, Evgeni

Ponimaskin2

1 University of Hohenheim, Department of Nutritional Medicine, Stuttgart, Germany 2 Hannover Medical School, Department of Cellular Neurophysiology, Hannover, Germany 3 Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, Novosibirks,

Germany

Morphological remodeling of dendritic spines is critically involved in memory formation and depends on adhesion

molecules. Serotonin receptors are also implicated in this remodeling, though the underlying mechanisms remain

enigmatic. Here, we uncovered a signaling pathway involving the adhesion molecule L1 and serotonin receptor 5-

HT4 (5-HT4R). Using FRET imaging, we demonstrated a physical interaction between 5-HT4R and L1 and found that

5-HT4R/L1 hetero-dimerization facilitates mitogen-activated protein kinase activation in a Gs-dependent manner. We

also found that 5-HT4R/L1-mediated signaling is involved in G13-dependent modulation of cofilin activity. In

hippocampal neurons in vitro, the 5-HT4R/L1 pathway triggers maturation of dendritic spines. Thus, the 5-HT4R/L1

signaling module represents a previously unknown molecular pathway regulating synaptic remodeling.

Acknowledgment

This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) through

DFG Grant GU 1521/4-1


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OS 06-06

Cyclin-dependent kinase 5 (CDK5) regulates the phase shift of the

circadian clock at early night.

Andrea Brenna1,3, Micaela Borsa2, Gabriella Saro3, Iwona Olejniczak3, Jurgen Ripperger3,

Dominique Glauser3, Xiu-Fen Ming1, Zhihong Yang1, Antoine Adamantidis2, Urs Albrecht3

1 University of Fribourg, Cardiovascular and Aging Research, Department of Endocrinology, Metabolism, and

Cardiovascular System, Faculty of Science and Medicine, Fribourg, Switzerland 2 Inselspital University Hospital Bern, Zentrum für Experimentelle Neurologie, Department of Neurology, Bern,

Switzerland 3 University of Fribourg, Department of Biology, Faculty of Science and Medicine, Fribourg, Switzerland

The circadian rhythms are self-sustained physiological pathways that oscillate over 24 hours and respond to defined

stimuli such as blue light. Their resetting ensures that livings' biological pathways are aligned with the light: dark

cycles based on the earth's rotation. Administration of blue light, at the early or late night, can reset these rhythms [1]. The first case generated a phase delay of circadian responses (the rhythm is slowed down). In the second one, a

phase advance is promoted (the rhythm is sped up). Molecularly, light primes the phosphorylation of the

transcriptional factor CREB at serine 133 (Ser-133) via Ca2+/CamKinases cascade. This event promotes gene

expression and modulates the circadian phase shift associated with resetting. However, the spatial and temporal

dynamics associated with the kinases cascade are still unknown. Using an in vivo approach (mice), we show that

Cyclin-Dependent Kinase 5 (CDK5), a proline-directed serine-threonine kinase belonging to the Cdc2/Cdk1 family,

regulates the cascade. CDK5 promotes neurogenesis, neuronal migration, axon guidance, and regulation of the

circadian clock [2,3]. CDK5 dysfunction leads to neurodegenerative diseases. Here we demonstrate, performing a

stereotaxic injection of adeno associate viruses (AAVs), that silencing Cdk5 in the suprachiasmatic nuclei (SCN) of

the hypothalamus abolishes the resetting of the circadian clock at early night. This phenotype correlates to a lack of

light-dependent CREB phosphorylation. Interestingly, in Cdk5 silenced mice, CREB is already phosphorylated in the

dark, but the level remains steady after the illuminating pulse. This event is associated with the aberration of the

CamKinase cascade. In wt scramble mice, the light pulse evokes Calcium (Ca2+) entrance in the SCN neurons via

Cav3.1 channels. Ca2+ is transferred from Cav3.1 to CamKIV through calmodulin (Cam), promoting CamKIV

phosphorylation and activation, which leads to CREB phosphorylation. In Cdk5 silenced mice, CamKIV is already

phosphorylated without any light pulse, which correlates to a perinuclear localization of Cam already in the dark,

mirroring the steady phosphorylation level of CREB. These events correlate to the dysregulation of Ca2+ influx, which

is decoupled from the light pulse in silenced mice, as observed via in vivo calcium imaging. Altogether, we define

CDK5 as an upstream regulator of the Ca2+/CamKinase cascade that mediates the circadian resetting at early night.

References [1] Tsuyoshi, H, Yoshitaka. 2004 ‘Resetting mechanism of central and peripheral circadian clocks in mammals’,

Zoolog Sci, Apr;21(4):359-68. doi: 10.2108/zsj.21.359. publishers

[2] Kawauchi, T. 2014 ‘Cdk5 regulates multiple cellular events in neural development, function and disease’, Development, Growth & Differentiation 56:335–348.

[3] Brenna, A, Olejniczak, I,A, Chavan, R, Ripperger, J,A, Langmesser, S, Cameroni, C, Hu, Z, De Virgilio, C, Dengjel, J, Albrecht, U. 2019 ‘Cyclin-dependent kinase 5 (CDK5) regulates the circadian clock’, Elife 2019 Nov 5;8:e50925. doi: 10.7554/eLife.50925.


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OS 06-07

gp130-induced TRPA1 upregulation in uninjured but not injured

neurons in a murine model for neuropathic pain

Theodora Kalpachidou1, Philipp Malsch1, Yanmei Qi1, Norbert Mair1, Stephan Geley2, Serena Quarta1,

Kai K. Kummer1, Michaela Kress1

1 Medical University of Innsbruck, Institute of Physiology, Innsbruck, Austria, Austria 2 Medical University of Innsbruck, Institute of Pathophysiology, Innsbruck, Austria, Austria

Upon a peripheral nerve injury, primary nociceptive afferents exhibit pronounced alterations that can result in the

development of neuropathic pain. Although the transient receptor potential ankyrin 1 ion channel (TRPA1) is an

emerging target for the treatment of pain, its mechanistic relevance in neuropathic pain pathogenesis is not fully

understood. We employed a transgenic mouse model with a conditional depletion of the interleukin-6 signal

transducer gp130 specifically in Nav1.8 expressing sensory neurons (SNS-gp130-/-) and subjected it to the spared

nerve injury (SNI) model for neuropathic pain. Control mice subjected to SNI developed a severe mechanical

hypersensitivity, evident by reduced mechanical thresholds in the von Frey behavioral test in vivo, as well as

increased mechanosensitivity of unmyelinated primary afferents in ex vivo skin-nerve recordings. In contrast, SNS-

gp130-/- mice did not develop any signs of mechanical hypersensitivity at the treated paw, and this was accompanied

by low levels of Trpa1 mRNA in sensory neurons, which could be partially restored by adenoviral re-expression of

gp130 in vitro. Importantly, microfluorimetric calcium measurements revealed that uninjured but not injured neurons

derived from SNI treated control mice developed increased responsiveness to the TRPA1 agonist cinnamon

aldehyde. In contrast, neurons from SNI treated SNS-gp130-/- mice were significantly less responsive. Our study

demonstrates increased TRPA1 responsiveness after peripheral nerve injury specifically in uninjured but not

injured neurons and this depended on the IL-6 signal transducer gp130. We provide novel mechanistic insight into

TRPA1 regulation following nerve injury and highlight the significance of TRPA1 as an important target in neuropathic

pain disorders.

Acknowledgment

This work was supported by the Austrian Research Funding Agency FWF (P 28611 to MK) and the graduate

program Signal Processing In Neurons (SPIN-DK W1206-B18 to MK).


of 516

OS 06-08

Reduction of waiting impulsivity by Gi-cascade activation in layer 5

pyramidal neurons of the anterior cingulate cortex in mice

Bastiaan van der Veen1, Sampath Kapanaiah1, Kasyoka Kilonzo1, Peter Steele-Perkins1,

Stefanie Schulz1, Martin Jendryka1, Birgit Liss1,3, Anton Pekcec2, Wiebke Nissen2, Dennis Kätzel1

1 University of Ulm, Institute for Applied Physiology, Ulm, Germany 2 Boehringer Ingelheim Pharma GmbH & Co, Discovery Research, Biberach an der Riss, Germany 3 University of Oxford, New College, Oxford, UK

Introduction: Lack of proper impulse control has been found in many psychiatric disorders, such as ADHD,

substance abuse and bipolar disorder. However, currently used medication in these disorders is often unspecific or

not very effective. The medial prefrontal cortex has been implicated in various forms of impulsivity, e.g. waiting

impulsivity, stopping impulsivity, delayed and probabilistic discounting. Sub-regions of the prefrontal cortex (PFC)

have been implicated in impulse control, however the role of its distinct subregions is not fully clear. Therefore, we

sought to clarify the specific role of those sub-regions in waiting impulsivity in behaving mice and find a possible new

drug-target to reduce waiting impulsivity.

Methods: A chemogenetic approach was used to selectively activate or inhibit G-protein-cascades on excitatory

cells by Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), specifically expressed in

excitatory cells in distinct prefrontal sub-regions of mice. The mice were trained and tested on the 5-choice-serial-

reaction-time task (5CSRTT). During testing, DREADDs were activated by application of CNO in a latin-square

design, counterbalancing for targeted sub-region and DREADD vector. Separately, differentially expressed GPCRs

in identified target cells were determined using Cytosplore to find a suitable drug-target, which was afterwards

validated pharmacologically.

Results: In the 5CSRTT, we found that activating Gi-DREADDs in excitatory cells of the ACC decreases premature

responding when impulse control was challenged. Triggering Gi-signalling in layer 5 pyramidal neurons (L5PNs) were

sufficient to elicit the anti-impulsive effect. Grm2 was the most differentially expressed Gi-coupled GPCR gene in

L5PNs among 402 surveyed GPCR-encoding genes. Pharmacological activation of mGlu2 receptors (encoded by

Grm2) elicited a similar anti-impulsive effect as the chemogenetic modulation.

Conclusion: Decreasing activity of layer-5 excitatory neurons in the ACC can improve impulse control when

challenged. A similar reduction of waiting impulsivity was found using drugs that activate mGluR2, suggesting

mGluR2 agonists as a possible pharmacological intervention to ameliorate waiting impulsivity.

Acknowledgment

Bosiljka Tasic, Zizhen Yao, Hongkui Zeng provided the ACC gene expression dataset, Thomas Akam developed

the pyControl used for the 5-choice operant box system, Janet R. Nicholson provided crucial advice.


of 516

Oral Sessions 01 October 2021 3:00 PM – 5:00 PM

Lecture Hall 2

OS 07 | The multicellular heart Chair

Petra Kleinbongard (Essen)

Alexander P. Schwoerer (Hamburg)


of 516

OS 07-01

Overexpression of human BAG3P209L in mice causes restrictive

cardiomyopathy due to sarcomere disruption and protein aggregate

formation

Kenichi Kimura1, Astrid Ooms1, Kathrin Graf-Riesen1, Maithreyan Kuppusamy2, Andreas Unger3,

Julia Schuld4, Jan Daerr4, Achim Lother5, Caroline Geisen1, Lutz Hein5, Satoru Takahashi6, Guang Li7,

Wilhelm Röll8, Wilhelm Bloch9, Peter F. van der Ven4, Wolfgang A. Linke3, Sean M. Wu7,

Pitter F. Huesgen2,10, Jörg Höhfeld4, Dieter O. Fürst4, Bernd K. Fleischmann1, Michael Hesse1

1 University of Bonn, Institute of Physiology I, Medical Faculty, Bonn, Germany 2 Forschungszentrum Jülich, Central Institute for Engineering, Electronics and Analytics, ZEA-3, Jülich, Germany 3 University of Münster, Institute of Physiology II, Münster, Germany 4 University of Bonn, Department of Molecular Cell Biology, Institute for Cell Biology, Bonn, Germany 5 University of Freiburg, Institute of Experimental and Clinical Pharmacology and Toxicology, Division II, Faculty of

Medicine, Freiburg, Germany 6 University of Tsukuba, Department of Anatomy and Embryology, Faculty of Medicine, Tsukuba, Japan 7 Stanford University School of Medicine, Stanford Cardiovascular Institute, Stanford, USA 8 University of Bonn, Department of Cardiac Surgery, Bonn, Germany 9 German Sport University Cologne, Department of Molecular and Cellular Sport Medicine, Cologne, Germany 10 University of Cologne, CECAD, Medical Faculty, and University Hospital, and Institute for Biochemistry, Faculty

of Mathematics and Natural Sciences, Cologne, Germany

The co-chaperone BAG3 (Bcl-2 associated athanogene 3) is strongly expressed in cross-striated muscles and plays

a key role in the turnover of muscle-proteins as a member of the CASA (chaperone-assisted selected autophagy)

complex. An amino acid exchange (P209L) in the human BAG3 gene, caused by a single base mutation, gives rise

to a severe dominant childhood muscular dystrophy, restrictive cardiomyopathy, and respiratory insufficiency. To get

deeper insights into the pathophysiological mechanisms of the disease, we generated a transgenic mouse model of

the human mutation BAG3P209L, in which a fusion protein consisting of the human BAG3P209L and the green fluorescent

protein eGFP can be conditionally overexpressed. Expression of human BAG3P209L-eGFP in mice caused Z-disc

disintegration and formation of protein aggregates leading to a severe phenotype between the second and fourth

week of life, including decreased body weight, skeletal muscle weakness, and heart failure, as observed in patients.

Echocardiography revealed that the BAG3P209L-mice suffer from restrictive cardiomyopathy and Sirius-red-staining of

heart tissue showed extensive fibrosis. RNA-Seq and proteomic analysis revealed changes in the protein quality

control system and increased autophagy in hearts from hBAG3P209L-eGFP overexpressing mice. Also, the mutation

renders hBAG3P209L less soluble in vivo and induces protein aggregation but does not abrogate hBAG3 binding

properties. Thus, we have established a mouse model recapitulating the human disease and found that the disease

mechanism is due to accumulation of hBAG3P209L and mouse BAG3, causing sequestering of components of the

protein quality control system and the autophagy machinery leading to the reduction of the stability and functional

organization of sarcomeres. We have also tested a gene therapy approach utilizing expression of shRNA against

hBAG3 in cardiac muscle via AAV and demonstrate that this halted and even partially reversed major phenotypic

features of the disease.

Acknowledgment


of 516

This work was supported by the German Research Foundation (FOR1228 to D.O.F. and FOR1352 to B.K.F., D.O.F.,

and J.H., FOR2743 to J.H., P.F.H., D.O.F., M.H.) and the Seventh Framework Program for Research and

Technological Development of the EU (MUZIC; D.O.F.). B.K.F., A.L. and L.H. are members of CRC1425, funded by

the German Research Foundation. The Galaxy server that was used for some calculations is in part funded by

Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992/1 2012) and German Federal Ministry

of Education and Research (BMBF grants 031 A538A/A538C RBC, 031L0101B/031L0101C de.NBI-epi, 031L0106

de.STAIR (de.NBI)).


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OS 07-02

The human endothelial-specific lncRNA PCAT19 limits angiogenic

function

James A. Oo1,2,5, Katalin Pálfi1,2,5, Timothy Warwick1,2,5, Frederike Boos1,2,5, Ilka Wittig3,1,5,

Stefan Guenther4, Matthias Leisegang1,2,5, Ralf Brandes1,2,5

1 Goethe University, Institute for Cardiovascular Physiology, Frankfurt am Main, Germany 2 Partner site RheinMain, German Center of Cardiovascular Research (DZHK), Frankfurt am Main, Germany 3 Goethe-University, Functional Proteomics, SFB 815 Core Unit, Frankfurt am Main, Germany 4 ECCPS Bioinformatics and Sequencing Facility, Max-Planck-Institute for Heart and Lung Research, Bad

Nauheim, Germany 5 Cardio-Pulmonary Institute, Cardio-Pulmonary Institute, Frankfurt am Main, Germany

Background: Long noncoding RNAs (lncRNA) provide a new layer of gene expression control and are crucial for

vascular development and cardiovascular disease. We set out to identify novel endothelial lncRNAs which could be

exploited to treat vascular disease.

Methods and Results: A search for differentially expressed and cell-specific lncRNAs in RNA-seq datasets identified

“prostate cancer associated transcript 19” - PCAT19 as a top candidate. PCAT19 is restricted to human endothelial

cells and is induced by cellular confluence as observed in cultured endothelial cells. Knockdown of PCAT19

increased endothelial proliferation and migration, while overexpression had the opposite effect. Importantly, this also

applied to angiogenic capacity, wherein depletion of PCAT19 increased endothelial spheroid outgrowth potential.

RNA-seq after knockdown of the lncRNA suggested that PCAT19 affects genes associated with cell-cycle

progression, cellular senescence, atherosclerosis and the PI3K-Akt pathway. PCAT19 pulldown experiments

followed by mass spectrometry identified multiple DNA damage response proteins as interaction partners of PCAT19.

Assays to investigate DNA double-strand breaks, including TUNEL and Comet assays, revealed that depletion of

PCAT19 results in substantial levels of DNA damage.Conclusions: LncRNA PCAT19 is induced by endothelial cell-

cell contacts to decrease cell-cycle progression, endothelial proliferation and growth. Inhibition of PCAT19 releases

this proliferative break and thereby increases the endothelial angiogenic capacity.


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OS 07-03

Gq protein inhibitor FR900359 – A therapeutic option for pulmonary

hypertension?

Alexander Seidinger1, Gabriele M. König2, Evi Kostenis2, Bernd K. Fleischmann3, Daniela Wenzel1,3

1 Ruhr-University Bochum, Systems Physiology, Bochum, Germany 2 University of Bonn, Pharmaceutical Biology, Bonn, Germany 3 University of Bonn, Physiology I, Bonn, Germany

Gq proteins are prominent key regulators in the pulmonary vasculature and determine tone regulation in health and

disease. Because currently applied drugs for pulmonary hypertension (PH) mainly target single G protein-coupled

receptors or single enzymes we wanted to test the specific pan-Gq inhibitor FR900359 (FR) on pulmonary arterial

tone regulation in mouse ex and in vivo as well as on human pulmonary artery smooth muscle cell (hPASMC) growth

in vitro and compare the effects with clinically used PH drugs.

The effect of FR on arterial tone ex vivo was examined in isometric force measurements of pulmonary arteries (PAs)

in a wire-myograph, in precision-cut lung slices and in the isolated perfused lung (IPL) model of mouse. Vasodilatory

effects and the impact of FR on hPASMC growth were compared with those of the endothelin receptor antagonist

bosentan, the prostacyclin analog iloprost and the phophodiesterase inhibitor sildenafil. The acute effect of FR on

pulmonary hemodynamics in vivo was determined after intratracheal application of FR and subsequent serotonin

injection by catheter measurements. The chronic effect of FR on PH was examined by a prevention and a treatment

study in a hypoxia-induced PH model.

We found a strong vasodilatory effect of FR (10 µM) on PAs in isometric force measurements (74 ± 2.8 %, n=7) that

was superior to relaxation by equal concentrations of bosentan (8.8 ± 2.9 %, n=5), iloprost (27.5 ± 3.9 %, n=7) and

sildenafil (34.6 ± 3.5 %, n=6), all p<0.001 vs FR. A strong vasorelaxation by FR has also been found in lung slices

(93.0 ± 1.2 %, n=7) and in the IPL model (98.9 ± 6.4 %, n=6). In a cell growth assay FR (1 µM), similar to bosentan

and iloprost but unlike sildenafil, reduced hPASMC numbers to 76.8 ± 2.1 % (n=3, p<0.01) compared to vehicle

control. Right ventricular (RV) catheter measurements demonstrated that FR acutely reduces a serotonin-induced

blood pressure increase by 65 % compared to solvent controls in vivo. Finally, FR prevented and reversed RV systolic

pressure increase, vessel remodeling and RV hypertrophy in the PH mouse model.

Thus, we show that the Gq inhibitor FR is a strong pulmonary vasorelaxant in mice ex and in vivo and limits hPASMC

growth in vitro. The efficacy of FR appears superior to clinically used PH drugs, as it can reduce both vascular tone

and hPASMC growth. Finally, we can show that FR prevents and reverses pathological changes in a PH mouse

model suggesting FR as a promising therapeutic option.


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OS 07-04

Unbiased screen of the cardiac proteome in a rat model of heart failure

with preserved ejection fraction (HFpEF)

Franziska Koser1, Mahmoud Abdellatif2, Clara Türk3, Marcus Krüger3, Simon Sedej2, Wolfgang Linke1

1 University Hospital Münster, Institute of Physiology II, Münster, Germany 2 Medical University of Graz, Division of Cardiology, Graz, Austria 3 University of Cologne, Institute for Genetics, Cologne, Germany

Background: Despite high public-health importance, the pathomechanism of HFpEF remains elusive and specific

therapy is lacking. A key feature in HFpEF patients is diastolic dysfunction, caused in part by altered stiffness of the

cardiomyocyte protein titin. Good patient-mimicking animal models of HFpEF are sparse but useful for elucidating

the impact of age, gender and comorbidities on HFpEF progression. The Zucker diabetic fatty/Spontaneous

hypertensive heart failure F1 hybrid (ZSF1) rat is an established animal model of HFpEF induced by metabolic

syndrome, the most prevalent cause and comorbidity of HFpEF.

Objective: To test whether previously suggested pathomechanistic pathways of HFpEF are altered in hearts of ZSF1

obese rats, and to gain new insights in the HFpEF pathomechanism using unbiased proteomic screens.

Methods & Results: Eight-week-old female and male ZSF1 obese and lean rats were fed with a Purina diet up to

the onset of the HFpEF phenotype in the ZSF1 obese rats at around 20 weeks of age.

Unbiased proteomic screens revealed the substantial reprogramming of the acetylome, but not the proteome or

phosphoproteome, in ZSF1 obese vs. lean rats highlighting obesity as a crucial comorbidity in the HFpEF

pathogenesis. Proposed features of the HFpEF pathomechanism could only be partially confirmed in ZSF1 obese

rats by immunoblot-based quantitation. Neither eNos activity, nor iNos expression were altered, contradicting the

presence of oxidative/nitrosative stress. In contrast, low-grade meta-inflammation was confirmed by increased CD68

and P-selectin expression, indicating macrophage infiltration and microvascular endothelial inflammation,

respectively. Moreover, PKG activity and PKG site-specific titin phosphorylation were decreased, potentially

increasing titin-based cardiomyocyte stiffness. Beside titin hyperacetylation, which may also contribute to the

increased cardiomyocyte stiffness in HFpEF, we propose disturbed Ca2+-handling as an additional contributor to

diastolic dysfunction in HFpEF patients. Hyperphosphorylation of RyR2 and hyperacetylation of SERCA2a in hearts

of ZSF1 obese rats could lead to SR-Ca2+-leakage via RyR2 and reduced SR Ca2+-reuptake via decreased SERCA2a

activity.

In conclusion, our findings support suggestions that both mechanical and metabolic stress are disease causing

elements of HFpEF. We confirm the ZSF1 obese rat is an appropriate model for HFpEF, and reveal new insights in

the HFpEF pathomechanism.


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OS 07-05

Impact of ubiquitin E3 ligase Siah-2 on cardiomyocytes and fibroblasts

in a model of overload-induced RV failure

Nicole Molenda1, Simone Kraut2, Ling Li1, Daniela Haag2, Klaus-Dieter Schlüter1, Norbert Weissmann2,

Susanne Rohrbach1

1 Justus Liebig University, Institute of physiology, Giessen, Germany 2 Justus Liebig University, Excellence Cluster Cardio-Pulmonary System, Giessen, Germany

Question: The ubiquitin E3 ligase Siah-2 (seven in absentia homolog 2) is one of the proteins regulating the hypoxic

response. Siah-2-mediated ubiquitination leads to destabilization of hypoxia regulators such as prolyl hydroxylase

(PHD)1 and PHD3. Here, we investigated the role of Siah-2 in a model of right ventricular hypertrophy (RVH) induced

by pulmonary artery banding (PAB) in mice.

Methods: Adult Siah-2 KO or WT mice were analyzed 3 weeks after Sham or PAB surgery. Cardiac remodeling was

characterized by echocardiography, hemodynamic measurements, histology and blood analyses. Isolated

cardiomyocytes or fibroblasts were utilized in functional studies.

Results: We observed an increased RV expression and phosphorylation (Ser28) of Siah-2 in mice after PAB which

could also be confirmed in tissue from patients suffering from RV failure.

Echocardiographic analyses demonstrated a better preservation of RV function (TAPSE, cardiac output) in Siah-2

KO compared to WT mice after RV overload. This funtional improvement could be confirmed by serum brain

natriuretic peptide (BNP). Siah-2 KO mice were also largely protected from RVH compared to WT mice. Accordingly,

Siah-2 deficient RV cardiomyocytes displayed a decreased hypertrophy (cell size) and reduced mRNA expression of

load-induced genes such as BNP or beta-myosin heavy chain. However, the contractile response of isolated

cardiomyocytes was not altered compared to WT cells after PAB.

While WT mice showed strongly increased collagen content and collagen mRNA/protein expression in the RV as

well as increased circulating TGF-beta 1 after PAB, this was significantly reduced in Siah-2 KO animals. Loss of

Siah-2 induced reduced migration and proliferation as well as SMAD2/3 phosphorylation in isolated fibroblasts in the

absence and presence of TGF-beta 1. The reduced fibrosis in Siah2-deficient RVs appears in part to be due to an

increased anti-fibrotic Apelin signaling and its crosstalk with attenuated TGF-ß1 signaling pathways. Accordingly,

activation of the Apelin receptor (APJ) lead to a more pronounced reduction in collagen synthesis, fibroblast migration

and myofibroblast transdifferentiation in Siah2-deficient compared to WT fibroblasts.

Conclusions: Siah-2 acts as a negative cardiac fibrosis and hypertrophy regulator in response to RV overload

induced by PAB. Understanding the precise role of Siah-2 may provide novel therapeutic targets directed against the

development of cor pulmonale.


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OS 07-06

Arg-II deficiency protects against cardiac aging

Duilio M. Potenza1, Guillaume Ajalbert1, Marie-Noelle Giraud2, Stephane Cook2, Xiu-Fen Ming1,

Zhihong Yang1

1 University of Fribourg, Department of Endocrinology, Metabolism and Cardiovascular System - Cardiovascular

and Aging Research, Fribourg, Switzerland 2 University of Fribourg, Department of Endocrinology, Metabolism and Cardiovascular System - Cardiology,

Fribourg, Switzerland

During aging, alterations of the cardiac structure and function enhance the susceptibility to heart failure. The need to

identify new strategy for preventing the age-associated heart functional decline is becoming urgent as the elderly

population continues to grow. In such context, increasing attention has been given to the mitochondrial enzyme

arginase II (Arg-II) which plays an important role in promoting oxidative stress and cellular senescence. In specific,

Arg-II expression was reported to increase in aging heart. Moreover, Arg-II deficiency was shown to extend lifespan

of mouse models.

This project was designed to investigate the role of Arg-II in the aging heart and the consequences of Arg-II ablation

in terms of heart fibrosis and function. In this view, experiments were conducted using young (3-4 months) and old

(20-22 months) wild type (WT) and Arg-II-/- mice. Cardiac function was assessed in vivo by echocardiography, and

ex vivo using the Langendorff apparatus. Upon global ischemia, reperfusion injury was assessed by measuring

hemodynamic function and infarct size.

Arg-II but not Arg-I expression was significantly enhanced in aging heart of WT mice. Ablation of Arg-II gene

significantly ameliorates the heart performance as shown by preserved ejection fraction in old Arg-II-/- mice when

compared to age-matched WT animals. Isolated Arg-II-/- heart showed a significantly faster and better hemodynamic

recovery during reperfusion. Moreover, triphenyl-tetrazolium chloride staining shows higher infarct area in old WT

when compared with age-matched Arg-II-/- mice. The preserved heart function in aged Arg-II-/- mice was accompanied

with reduced expression of cytokines/chemokines, e.g. IL1-β and IL6, and MCP1, decreased macrophage infiltration,

and an attenuated tissue fibrosis.

In summary, our study suggests that enhanced Arg-II expression favors cardiac inflammation and fibrosis, leading to

cardiac functional decline in aging. Targeting Arg-II may be a promising therapeutic strategy to treat age-associated

cardiac dysfunction.


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OS 07-07

Optogenetic manipulation of the heterocellular heart

Marbely Calderón Fernández1, Ana Simon Chica1,2, Eike Moritz Wülfers1, Teo Puig Walz1,

Callum Zgierski-Johnston1, Franziska Schneider-Warme1, Peter Kohl1

1 University Heart Center Freiburg • Bad Krozingen, Institute for Experimental Cardiovascular Medicine, 79110,

Germany 2 Spanish National Cardiovascular Research Center, Carlos III (CNIC), Myocardial Pathophysiology Area, 28029,

Spain

The heart is composed of cardiomyocytes (CM) and non-myocytes (NM), the latter including interstitial fibroblasts

(FB) and resident macrophages (MΦ). NM have long been suspected to electrically couple to CM in native

myocardium. We are interested in how FB and MΦ electrotonically couple to CM and may alter electrical activity

during myocardial remodeling in response to injury.

We used Cre-loxP recombination to selectively express fluorescent reporters and optogenetic actuators in NM

populations of murine hearts. We optically cleared hearts using X-CLARITY and imaged them with super-resolution

confocal microscopy. This allowed us to reconstruct 3D models of FB and MΦ in situ. We also characterized the

electrophysiological properties of resident cardiac MΦ, using RNA sequencing, single-cell patch-clamp, showing the

functional expression of Cx43 and different voltage-gated K+ channels. Based on our structural and functional data,

we developed a computational model describing cardiac MΦ electrophysiology, which we then used to quantitatively

assess CM-MΦ coupling in silico. In on-going experiments, we utilize Channelrhodopsin-2 (ChR2) and a fluorescent

voltage reporter (VSFP2.3) to study heterocellular coupling between CM, FB and MΦ, with a focus on NM effects on

cardiac activity in post-injury hearts.

In healthy cleared ventricles, we found that FB networks consist of elongated, thin strands of interconnected cells,

which appear to wrap around CM with finger-like nano-protrusions. Volume and surface area of 3D FB does not differ

statistically between atrial and ventricular walls (e.g. average surface area in right atrial and left ventricular

myocardium are 2,130±280 μm2 and 1,770±190 µm2 [N=3 hearts], respectively). MΦ appear as solitary cells in intact

ventricular myocardium with an average surface area of 1,160±80 µm2 (N=3 hearts). In isolated and cultured cells,

we found that passive electrophysiological properties of MΦ such as capacitance and membrane resistance scale

with surface area. In silico models of CM-MΦ coupling illustrate that those parameters are directly linked to the

electrical load of MΦ coupled to CM.

FB and MΦ exhibit surprisingly similar cell dimensions in intact tissue, although they differ in their distribution and

their electrophysiology. Upon injury, these cell populations undergo changes leading to larger NM numbers in the

scar and scar border, altering the balance of NM and CM, and thus altering their impact on cardiac physiology.

Acknowledgment

We thank Stefanie Perez Feliz for expert technical support. We acknowledge the imaging support of the SCI-MED

facility at the Institute for Experimental Cardiovascular Medicine (IEKM).


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OS 07-08

Impact of left and right heart failure on skeletal muscle in a novel two-

stage model in rats

Fabienne Knapp1, Bernd Niemann2, Ling Li1, Michael Kracht3, Rainer Schulz1, Susanne Rohrbach1

1 Justus-Liebig-University, Institute of Physiology, Giessen, Germany 2 UKGM, Department of Cardiac and Vascular Surgery, Giessen, Germany 3 Justus-Liebig-University, Rudolf Buchheim Institute of Pharmacology, Giessen, Germany

Background: Exercise intolerance, a hall mark of patients with right (RV) and left ventricular (LV) failure, is

associated with poor quality of life and increased mortality. Patients suffering from heart failure show a massive

impairment of their skeletal muscles, which contributes to increased morbidity. Animal models which result in a

gradual increase in afterload resemble closely the human situation and enable the investigation of the impact of RV

and LV failure on peripheral organs such as skeletal muscles. RV and LV failure may mediate different effects, but

this was never compared systematically.

Methods: Pulmonary artery banding (PAB) or aortic banding (AOB) was performed in weanling rats with a non-

constricting clip, to study the transition from compensated cardiac hypertrophy (7 weeks post surgery) to heart failure

(22 weeks after PAB, 26 weeks after AOB). Cardiac function was characterized by echocardiography (Vevo2100).

Skeletal muscle abnormalities associated with heart failure were comparatively analyzed in both models by

histological and mitochondrial analyzes or qPCR/Western Blot respectively.

Results: AOB and PAB animals at the compensated stage show a similar hypertrophy in the respective ventricle but

maintained LV or RV function. Whereas animals at the decompensated stage show massively reduced LV or RV

function. Two clearly distinguishable stages of left and right heart disease and a comparable severity of the respective

heart failure were reached.

Peripheral skeletal muscles (gastrocnemius and soleus muscle) showed a reduced weight at the stage of cardiac

decompensation as well as mitochondrial dysfunction and altered respiratory chain gene expression, enhanced

proteasome activation and increased atrophy markers like the E3 ubiquitin ligases Atrogin-1 and MuRF-1. The

diaphragm did not demonstrate any differences in most parameters. Alterations in skeletal muscle were more

pronounced in the AOB model.

An increase in the plasma levels of systemic mediators such as IL-6, TNF-α and angiotensin II was observed in both

models, but significantly more distinct in the AOB model as well.

Conclusions: LV and RV failure seem to affect peripheral organs differently, suggesting a major impact of impaired

systemic circulation. Mitochondrial dysfunction and upregulation of myostatin were identified as the earliest signs of

skeletal muscle impairment in heart failure.


of 516

Oral Sessions 02 October 2021

10:45 AM – 12:45 PM

Lecture Hall 2

OS 08 | Cardiovascular Systems - Mechanics to

Metabolism Chair

Tobias Brügmann (Goettingen)

Nazha Hamdani (Bochum)


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OS 08-01

Transcriptional bursting underlies unequal allelic expression and force

generation among cardiomyocytes in Hypertrophic Cardiomyopathy

patients

Valentin Burkart1, Kathrin Kowalski1, Julia Beck1, David Aldag-Niebling1, Tim Holler1, Ante Radocaj1,

Britta Keyser2, Jolanda van der Velden3, Cristobal dos Remedios4, Sean Lal4, Denise Hilfiker-Kleiner5,

Theresia Kraft1, Judith Montag1

1 Hannover Medical School, Molecular and Cell Physiology, Hannover, Germany 2 Hannover Medical School, Department of Human Genetics, Hannover, Germany 3 VU University Medical Centre, Department of Physiology, Amsterdam, Netherlands 4 University of Sydney, Department of Cardiology, Sydney, Australia 5 Hannover Medical School, Clinic of Cardiology and Angiology, Hannover, Germany

Evidence accumulates that alleles of most genes are transcribed independently from each other in stochastic bursts.

This leads to unequal ratios of mRNA from both alleles among individual cells within a tissue. In patients with

heterozygous mutations, this heterogeneity among cells may cause functional imbalance and trigger disease

phenotype.

Hypertrophic cardiomyopathy (HCM) is mostly caused by heterozygous mutations in sarcomeric genes. The most

commonly affected genes are myosin binding protein C (cMyBP-C, MYBPC3), β-myosin heavy chain (β-MyHC,

MYH7), cardiac troponin T (cTnT, TNNT2) and cardiac troponin I (cTnI, TNNI3). Mutations alter force generation of

cardiomyocytes; however, alterations can be highly variable, e.g. increasing or decreasing calcium sensitivity. Thus,

a common pathomechanism remains elusive.

Here we asked whether transcription in bursts could provide a potential mechanism underlying HCM-development in

patients with mutations in different HCM-genes and with different primary alterations in force generation. Using single

molecule RNA-fluorescence in situ hybridization (smRNA-FISH) on tissue sections from donor controls and HCM-

patients with MYH7-mutation R723G, TNNI3-mutation R145W and MYBPC3 truncation mutation c.927-2G>A, we

show that burst-like transcription is a common transcriptional mechanism of the three genes. We additionally show

that substantial allelic imbalance from cell to cell is associated with this burst-like transcription, as revealed by allele-

specific single cell RT-PCR analysis for MYH7 and TNNI3. In the cMyBP-Ctrunc patient, we detected a highly variable

distribution of wildtype cMyBP-C from cell to cell presumably associated with burst-like transcription. Furthermore,

we showed a large variability among individual cardiomyocytes from HCM-patients with mutations in β-MyHC,cMyBP-

C and cTnI in calcium-dependent force generation. The variability was detected for both, calcium-sensitizing and

calcium-desensitizing mutations.

In summary, we provide evidence that three commonly affected genes in HCM are transcribed in bursts. The random

expression of both alleles may lead to highly variable fractions of mutant vs. wildtype at mRNA and protein level from

cell to cell. Due to mutation-induced alterations in contractile function this could underlie contractile imbalance

between neighboring cardiomyocytes and thereby contribute to development of hypertrophy, fibrosis and

cardiomyocyte disarray in HCM patients.


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OS 08-02

Micromechanical investigations of myofibrils carrying the HCM

mutation G716R reveal slower relaxation

Anja Brunkhorst, Birgit Piep, Tim Holler, Theresia Kraft, Bogdan Iorga

Hannover Medical School, Deparment of Molecular and Cell Physiology, Hannover, Germany

Aim: Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death. Most of the known

mutations causing HCM occur in sarcomeric proteins, e.g. in the gene for myosin heavy chain (MYH7). One highly

malignant mutation within the MYH7 is Gly716Arg (G716R), located in the converter domain of the β-myosin heavy

chain (β‐MyHC). To understand the underlying mechanism that leads to the disease, the potential primary functional

effects caused by this mutation were investigated at subcellular level with isolated myofibrils.

Method: Due to their small size, myofibrils allow determination of the contractile force with high temporal resolution.

We measured steady‐state and kinetic force related parameters of human ventricular myofibrils (hvMFs) isolated

from adult donor heart (wild-type β-MyHC) and from a patient heart carrying the mutation G716R in β-MyHC.

Results: At saturating calcium concentration [Ca2+] donor and patient hvMFs develop similar maximum force (donor:

46 kPa, patient: 60 kPa). The rate constants of Ca2+ induced force development (kACT) and force redevelopment

induced by a rapid slack‐restretch maneuver (kTR) were also similar (donor: kACT=0.59±0.21 s-1, kTR=0.59±0.16 s-1;

patient: kACT=0.49±0.13 s-1, kTR=0.53±0.12 s-1). At submaximal [Ca2+], to simulate physiological conditions,

myofibrillar force response to calcium was different, but after PKA treatment, to normalize the possible differences in

the PKA‐mediated phosphorylation of the sarcomeric proteins, the response was not significantly different (donor:

pCa50=5.53±0.11, patient: pCa50=5.54±0.09; p=0.67). After PKA treatment, relaxation of the patient hvMFs showed

significantly slower kinetics (donor: τREL=555 ms, patient: τREL=695 ms, p=0.04), which may contribute to a

“sarcomeric” diastolic dysfunction. Therefore, we relaxed initially fully activated hvMFs at low, submaximal [Ca2+], to

mimic an incomplete Ca2+ removal, which often has been reported in intact HCM cardiomyocytes. Preliminary results

indicate that relaxing mutant hvMFs seem to act more sensitive to the calcium level change than wild-type hvMFs.

Conclusion: Slower relaxation kinetics could be affected directly or indirectly (via thin filaments) by the mutant

myosin. Further, in depth investigations, especially regarding the impact of myosin on the process of myofibril

relaxation, are under way.


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OS 08-03

Optogenetics enables Ca2+-included Ca2+ release from the intracellular

stores by subcellular targeting of the ryanodine receptor

Wanchana Jangsangthong1, Alexander Gottschalk2, Stephan Lehnart3, Martina Krüger4, Philipp Sasse1

1 University of Bonn, Institute of Physiology I, Medical Faculty, Bonn, Germany 2 Johann Wolfgang Goethe-University Frankfurt, Institute of Biochemistry, Frankfurt, Germany 3 University Medical Center Göttingen, Cellular Biophysics and Translational Cardiology, Heart Research Center

Göttingen, Department of Cardiology & Pneumology, Göttingen, Germany 4 Heinrich-Heine-University Düsseldorf, Department of Cardiovascular Physiology, Medical Faculty, Düsseldorf,

Germany 5 University of Bonn, Institute of Physiology I, Medical Faculty, Bonn, Germany

Introduction: In cardiomyocytes, ryanodine receptor type 2 (RyR2) gates Ca2+ release from sarco/endoplasmic

reticulum (SR/ER). Pathological Ca2+ leak through RyR2 plays a crucial role for lethal arrhythmias. For therapeutic

applications, screening of RyR2-modulators is required but a cellular model is lacking because of the challenge to

physiologically trigger RyR2 opening by cytosolic Ca2+ elevation.

Methods: We developed a new light-gated Ca2+-permeable Channelrhodopsin-2 variant (CatChUP) and targeted it

close to RyR2 to obtain light-induced Ca2+ release (LICR) from the SR/ER. In the first attempt, CatChUP-EYFP was

C-terminally fused to the RyR2-interacting protein Junctin (CatChUP-EYFP-Junctin). As a second attempt CatChUP-

EYFP was fused to the N-terminus of human RyR2 (OptoRyR2) with a 157 Å long natural linker to span the distance

from the SR/ER membrane.

Results/Discussion: CatChUP-EYFP-Junctin was expressed in the intracellular compartments of cardiomyocytes.

Ca2+ imaging showed LICR in HL-1 cardiomyocytes, which was confirmed by light-induced frequency acceleration in

ES-cell derived cardiomyocytes. Unfortunately, patch clamp analysis showed residual photocurrents suggesting that

CatChUP-EYFP-Junctin was not exclusively targeted to the SR/ER but also expressed at the plasma membrane,

which prevents the use for actual LICR. In contrast, expression of OptoRyR2 in HEK293 cells was restricted to the

SR/ER membrane and did not result in photocurrents. Western blots confirmed the expected size of OptoRyR2 of

~640 kDa compared to RyR2 of ~560 kDa. Ca2+ imaging showed Ca2+ release by activating the RyR2 with Caffeine

in OptoRyR2-expressing HEK293 cells but not in WT-HEK293 cells suggesting intact RyR2 function. Furthermore,

Illumination of cell with light led to a biphasic LICR in OptoRyR2-expressing HEK293 cells with an initial slow rise

followed by a second, much faster Ca2+ release. Importantly, application of the RyR-blocker Tetracaine abolished the

second fast component without affecting the initial phase.

Conclusion: Opening of CatChUP within OptoRyR2 by light leads to an initial Ca2+ release from the SR/ER until a

cytosolic Ca2+ threshold level is reached for triggering RyR2 opening with a second faster Ca2+ release event.

Because Ca2+ is the physiological trigger to open RyR2, OptoRyR2 in HEK293 cells will be a valuable tool to screen

for therapeutic and toxic effects of RyR2-modulators.

Acknowledgment

Funding: Deutsche Forschungsgemeinschaft (German Research Foundation, 313904155/SA1785/7-1,

315402240/SA1785/8-2; 380524518/SA1785/9-1)


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OS 08-04

Bridging the gap: the role of Connexin-43 in modeling arrhythmogenic

diseases with iPSC-cardiomyocytes

Nina D. Ullrich1,2, Xijian Ke1, Markus Hecker1,2

1 Heidelberg University, Institute of Physiology and Pathophysiology, Heidelberg, Germany 2 German Center for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim, Heidelberg, Germany

Arrhythmogenic diseases comprise a family of diseases that feature myocardial abnormalities and ventricular

arrhythmias. As one common pathological characteristic, electrical signal transmission across the heart is affected

leading to life threatening heart rhythm abnormalities and conduction disorders. Arrhythmogenesis is related to the

displacement of gap junctions-forming protein Connexin-43 (Cx43) from intercalated discs to lateral membranes, but

the underlying mechanisms of the pathophysiological remodeling processes remain elusive. Recent advances in the

development of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) suggests to use these cells

specifically for disease modeling. Our previous research demonstrated that iPSC-CMs express lower levels of Cx43

compared with native primary CMs, leading to similar electrical properties and conduction deficiencies as seen in

arrhythmogenic diseases.

In this project, we used iPSC-CMs as a model for arrhythmogenic diseases and tested the hypothesis that Cx43

expression is modulated by chronic cell stress. The aim of this study was to identify key molecular players that are

involved in the regulation of Cx43 expression at the sarcolemma and to restore robust electrical signal propagation

across engineered cardiac tissue.

Combining chronic tachypacing and direct microcontact printing techniques, iPSC-CMs were electrically stimulated

during long-term culture. Using the whole-cell voltage and current clamp techniques and confocal Ca2+ imaging,

electrical properties and Ca2+ signal propagation were investigated in 2D-printed cell strands. Our data demonstrate

that Cx43 expression is significantly decreased after tachypacing. Moreover, we have identified the micro-RNA miR-

1 as a major regulator of Cx43 expression. Blocking miR-1 using the oligonucleotide anti-miR-1 not only restored

Cx43 expression at the membrane, but also significantly increased the voltage-dependent sodium current, which is

responsible for the action potential upstroke velocity and cell excitability. Furthermore, inhibition of miR-1 significantly

enhanced Ca2+ wave propagation across iPSC-CMs and synchronized electrical field potentials as assessed by multi-

electrode arrays.In conclusion, our results point towards miR-1 as suppressor of Cx43 expression under conditions

of severe chronic stress. Therefore, miR-1 may represent a novel target for pharmacological treatment of

arrhythmogenic remodeling processes involving Cx43.

Acknowledgment

This project was supported by a research scholarship to XK.


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OS 08-05

Ischemic preconditioning enhances rapid modulation of titin-based

cardiomyocyte stiffness after ischemia-reperfusion in pig hearts

Celerina Jahns1, Sebastian Kötter1, Lale Azer-Güney1, Sabine Bongardt1, Andreas Skyschally2,

Gerd Heusch2, Petra Kleinbongard2, Martina Krüger1

1 Heinrich Heine-Universität Düsseldorf, Medical Faculty, Institute of Cardiovascular Physiology, Düsseldorf,

Germany 2 University of Essen, West German Heart and Vascular Center, Medical School, Institute for Pathophysiology,

Essen, Germany

Titin largely defines cardiomyocyte passive tension and plays an important role for the structural integrity of the

sarcomeres. We previously demonstrated that myocardial ischemia/reperfusion (I/R) in mice caused rapid PKCα-

dependent phosphorylation of titin, which increased cardiomyocyte passive stiffness and contributed to remodeling

of non-ischemic (remote) myocardium. We speculate that increased myocyte stiffness improves stability of the

remote myocardium and is beneficial to maintain myocardial function until scar formation is completed. Here, we

aimed to translate our findings to the Göttingen mini pigs, and to test how cardioprotection by ischemic

preconditioning affects titin modification and cardiomyocyte stiffness in left ventricular biopsies of remote

myocardium. Pigs underwent 60 min myocardial ischemia by LAD occlusion and 10-120 min reperfusion, without or

with ischemic preconditioning by 2 cycles of 3-min coronary occlusion and 2 min of reperfusion 15 min before

sustained I/R (n=5+5). To identify involved signaling pathways, we further tested a third group, which underwent

vagotomy prior to I/R (n=5). Biopsies were taken from non-ischemic left ventricular tissue before and after 55 min

ischemia, at the end of ischemic preconditioning and after 10-120 min reperfusion. Titin phosphorylation was

analyzed using Western blot, passive tension was determined by stepwise stretching of isolated cardiomyocytes.

Ischemia significantly increased phosphorylation of PKCα to 130% and of titin S11878 to 160%, and increased

passive tension at sarcomere lengths of 2-2.4 µm. Ischemic preconditioning caused similar increases of PKCα

phosphorylation to 170% and of titin S11878 phosphorylation to 150%, which persisted during the following ischemia.

Preconditioned hearts showed an additional increase in relative titin S11878 phosphorylation to 200% in the

reperfusion phase and cardiomyocyte passive tension remained significantly elevated throughout the protocol.

Importantly, vagotomy prevented the ischemia-induced alterations of titin and titin-based cardiomyocyte passive

stiffness. We conclude that I/R-induced changes of titin-based cardiomyocyte properties are relevant processes in

both small and large animal models. Our data further suggest that even short ischemic events initiate rapid signal

transduction from ischemic to non-ischemic tissue. Finally, first results indicate that titin-based cardiomyocyte

stiffening after I/R depends on vagal signaling.


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OS 08-06

Cystathionine gamma lyase delays endothelial senescence

Sofia-Iris Bibli1,2, Jiong Hu1,2, Matthias Leisegang1,2, Mauro Siragusa1,2, Janina Wittig1,2,

Maria-Kyriaki Drekolia1,2, Ilka Wittig1, Oliver Mueller2,3, Ralf Brandes1,2, Andreas Weigert1,

Andreas Papapetropoulos4, Fragiska Sigala4, Ingrid Fleming1,2

1 Goethe University, Frankfurt am Main, Germany 2 German Center for Cardiovascular Research, Frankfurt am Main, Germany 3 University of Kiel, Kiel, Germany 4 National and Kapodestrian University of Athens, Athens, Greece

Question: Several mechanisms have been proposed to contribute to age-related changes in the vasculature and in

endothelial cell function. However, there is a real lack of information on the aged human endothelium – largely

because of the scarcity of access to native human endothelial cells from well phenotyped collectives. This study set

out to unravel novel metabolic-related mechanisms that contribute to vascular endothelial senescence.

Methods and Results: To compare young and aged native endothelial cells, mesenteric arteries were obtained

(during non-cardiovascular disease-related interventions), from young (20±3.4 years of age) and aged (80±2.3 years

of age) human donors. Endothelial cells were isolated and FACS sorted (CD144+ aSMA- cells) within 45 minutes,

and frozen after a short recovery period (20 minutes) in Hanks Buffer supplemented with 5% orthologous serum.

Endothelial cells from aged donors demonstrated a significant reduction (~16%) in telomere length and a ~54%

increase in the expression of senescence-associated b-galactosidase activity. Metabolomic analysis revealed major

reduction in the capacity of the aged endothelium to catabolize cysteine, an effect that was accompanied by a

decrease in the expression of cystathionine g-lyase (CSE). Loss of CSE resulted in increased expression and nuclear

translocation of p53, which was acetylated on lysine120 and activated, as indicated by enriched transcription of its

direct target gene p21. p53 activation impacted on endothelial cell metabolic responses by inhibiting the transcription

of the glucose transporter Glut1. Glut 1 downregulation in the aged endothelium had marked consequences on

energy production, which in turn affected the assembly of the telomerase complex, leading to endothelial senescence

and impaired vascular repair. Mice lacking endothelial cell CSE exhibited premature lethality coupled with a loss in

endothelial regenerative capacity in a mouse model of aortic regeneration and enhanced senescence. The effects

were again attributed to the CSE dependent activation of the p53, which reduced telomerase activity in an energy

dependent manner.

Conclusions: In native human and murine arterial endothelial cells cystathionine gamma lyase (CSE) was identified

as a novel regulator of endothelial senescence. CSE represents a novel link between altered endothelial cell

metabolism and telomere attrition as well as endothelial cell reparative responses.


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OS 08-07

Compartment-specific production of reactive oxygen species (ROS)

affects the metabolism and function of the heart

Ana Maria Vergel Leon1, Jia Guo1, Aline Jatho1, Gijsbert J. van Belle1, Cristina M. Furdui2,

Vsevolod V. Belousov1,3,4, Dörthe M. Katschinski1, Maithily S. Nanadikar1

1 University Medical Center Goettingen, Institute for Cardiovascular Physiology, Goettingen, Germany 2 Wake Forest University Health Sciences, Section on Molecular Medicine, Department of Internal Medicine,

Winston-Salem, USA 3 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Department of Metabolism and Redox Biology,

Moscow, Russia 4 Pirogov Russian National Research Medical University, Center for Precision Genome Editing and Genetic

Technologies for Biomedicine, Moscow, Russia 5 Federal Center for Cerebrovascular Pathology and Stroke, Moscow, Russia

A cohesive relationship between ROS and cardiac dysfunction has been studied for many years. An imbalance in

ROS to the antioxidant system is often referred to as one important cause for cardiac failure. However, the precise

mechanism and especially specific ROS targets are not known. Hydrogen peroxide (H2O2), an important ROS, has

the capacity to reversibly modify protein cysteine thiols (-SH) to sulfenic acid (-SOH), which can further be oxidized

to sulfinic (-SO2H) and sulfonic (-SO3H) acids. These kinds of “redox switches” can further have adverse effects on

the protein function and thus their downstream pathways. In this study, we generated and employed a mouse model

with a genetically encoded cardiomyocyte-specific and nucleus-targeted DAO-HyPer biosensor. D-amino acid

oxidase (DAO) catalyzes the production of H2O2 endogenously in presence of its substrate D-alanine. HyPer is a

ratiometric biosensor that can monitor the production of H2O2. Thus endogenous, controllable and compartment-

specific production of H2O2 can be achieved by utilizing this mouse model. In the in vivo studies, upon treating the

DAO-HyPer mice with D-alanine in drinking water the mice developed a cardiac dysfunction phenotype as compared

to their control group treated with L-alanine. The fractional area shortening (FAS) and the ejection fraction (EF) were

significantly decreased after just 7 days of D-alanine treatment, with both parameters going from moderate to

severely impaired after 21 days. Surprisingly, just in 2 days, this phenotype was reversed after removing D-alanine

out of the drinking water. Redox proteomics analysis performed with cardiac samples of the D-alanine treated DAO-

HyPer mice revealed that the most redox modified candidates are proteins located in the mitochondria. Isocitrate

dehydrogenase 3γ (IDH3γ), a part of the TCA cycle is one of the top candidates. Additional biochemical and functional

analysis we performed points towards a switchable change in cellular metabolism.


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OS 08-08

Maintenance on mitochondrial complexes ensure bioenergetic function

in differentiated cells

Ilka Wittig1, Juliana Heidler1, Heiko Giese2, Ralf P. Brandes3

1 Goethe University, Functional Proteomics, Institute for Cardiovascular Physiology, Frankfurt, Germany 2 Goethe University, Molecular Bioinformatics, Frankfurt, Germany 3 Goethe University, Institute for Cardiovascular Physiology, Frankfurt, Germany

The assembly sequence of mitochondrial complexes was intensively investigated in proliferating cells. These studies

mostly reflect the de-novo assembly and give only limited information on the protein complex dynamics in

differentiated cells and tissues. The state of protein complexes in postmitotic tissues might rather be a balance

between biosynthesis and degradation. It is an important question whether protein complexes are always assembled

de-novo or if remodeling and repair mechanisms maintain mitochondrial function. The combination of blue native

electrophoresis with quantitative mass spectrometry identifies even scarce sub-complexes, assembly intermediates,

and complex remodeling [1]. In this study, we combined complexome profiling and pulse stable isotope labeling of

amino acids in cell culture (pSILAC) [2] to study turnover and half-life of single proteins within protein complexes in

differentiated post-mitotic C2C12-myotubes. The results represent a comprehensive data collection of dynamics in

all stable mitochondrial protein complexes. Complete replacement of all complexes of the oxidative phosphorylation

system (OXPHOS) requires about a month. We detected higher turnover rates between interface subunits of dimeric

and oligomeric ATP synthase and supercomplexes suggesting more dynamics between as within complexes. We

explored the maintenance plan of complex I (Fig.1). Freshly translated proteins assemble into spare parts and replace

old modules within complex I. Application of the developed method to patients with a mitochondrial disorder

discovered a novel factor involved in quality control and repair of complex I [3]. In detail, we identified subunits of

complex I with higher turnover rates in the parts of electron transport modules, suggesting quality control and service

mechanisms within assembled complexes guarantee for full bioenergetic function in postmitotic tissues

Acknowledgment

This research was funded by Deutsche Forschungsgemeinschaft (DFG) grant SFB815/Z1 and German Federal

Ministry of Education and Research (BMBF, Bonn, Germany) grant to the German Network for Mitochondrial

Disorders (mitoNET, 01GM1906D).


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Fig. 1 Turnover rates and repair of complex I as heatmap within the mouse complex I

structure [4].

References [1] Heide, H.; Bleier, L.; Steger, M.; Ackermann, J.; Dröse, S.; Schwamb, B.; Zörnig, M.; Reichert, A.S.; Koch,

I.; Wittig, I.; et al. Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex. Cell Metab. 2012, 16, 538–549, doi:10.1016/j.cmet.2012.08.009.

[2] Schwanhäusser, B.; Gossen, M.; Dittmar, G.; Selbach, M. Global analysis of cellular protein translation by pulsed SILAC. Proteomics 2009, 9, 205–209, doi:10.1002/pmic.200800275.

[3] Stenton SL, Sheremet NL, Catarino CB, Andreeva NA, Assouline Z, Barboni P, Barel O, Berutti R, Bychkov I, Caporali L, Capristo M, Carbonelli M, Cascavilla ML, Charbel Issa P, Freisinger P, Gerber S, Ghezzi D, Graf E, Heidler J, Hempel M, Heon E, Itkis YS, Javasky E, Kaplan J, Kopajtich R, Kornblum C, Kovacs-Nagy R, Krylova TD, Kunz WS, La Morgia C, Lamperti C, Ludwig C, Malacarne PF, Maresca A, Mayr JA, Meisterknecht J, Nevinitsyna TA, Palombo F, Pode-Shakked B, Shmelkova MS, Strom TM, Tagliavini F, Tzadok M, van der Ven AT, Vignal-Clermont C, Wagner M, Zakharova EY, Zhorzholadze NV, Rozet JM, Carelli V, Tsygankova PG, Klopstock T, Wittig I, Prokisch H. Impaired complex I repair causes recessive Leber's hereditary optic neuropathy. J. Clin. Invest. 2021, 131, doi:10.1172/JCI138267.

[4] Agip, A.-N.A.; Blaza, J.N.; Bridges, H.R.; Viscomi, C.; Rawson, S.; Muench, S.P.; Hirst, J. Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states. Nat. Struct. Mol. Biol. 2018, 25, 548–556, doi:10.1038/s41594-018-0073-1.


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Oral Sessions 02 October 2021 10:45 AM – 12:45 PM

Lecture Hall 3

OS 09 | Systems Neurophysiology: from

Network Oscillations to Behavior Chair

Julia Schiemann (Homburg)

Torfi Sigurdsson (Frankfurt/Main)


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OS 09-01

Single-trial decoding of working memory-based choices from inter-

regional connectivity in mice and humans

Daniel Strahnen, Sampath K. Kapanaiah, Dennis Kätzel

Ulm University, Applied Physiology, Ulm, Germany

Working memory (WM), the capacity to briefly and intentionally maintain mental items, is key to goal-directed

behaviour and impaired in various psychiatric disorders. While several brain regions and connections have been

correlatively associated with WM performance, the degree of the species- and task-specificity of such findings

remains unclear. Here, we investigate WM correlates in three task paradigms each in mice and humans with

simultaneous multi-site electrophysiological recordings, whereby an already published dataset was used for the

analysis of WM correlates in humans (1). By decoding individual choices from several hundred functional connectivity

measures using a common model across subjects, we unveiled task-phase-specific WM-related connectivity from

unbiased analysis of predictor weights. Only a few common connectivity patterns emerged across tasks. In rodents,

these were thalamus-prefrontal cortex delta- and beta-frequency connectivity during memory encoding and

maintenance, respectively, and hippocampal-prefrontal delta- and theta-range coupling during retrieval. In humans,

task-independent WM correlates were exclusively in the gamma-band. Mostly, however, the predictive connectivity

patterns were unexpectedly specific to each task and always widely distributed across brain regions, calling for a

refined conceptualization of WM in translational psychiatry.

Acknowledgment

We thank Elizabeth Johnson (UC Berkeley and Wayne State University) for providing the human dataset, Benjamin

F. Grewe (Institute of Neuroinformatics, Zürich) for advice on machine learning, Thomas Akam (University of Oxford)

for assistance with development of operant boxes, and Birgit Liss (Ulm University), David Bannerman (University of

Oxford) and Alexei Bygrave (Johns Hopkins University) for general support and/or advice. This work was funded by

the Else-Kroener-Fresenius/German-Scholars-Organization Programme for excellent medical scientists from abroad

(GSO/EKFS 12; to D.K.), the Juniorprofessorship programme of Baden-Württemberg (to D.K.), the DFG (KA 4594/2-

1; to D.K.), and the Brain and Behaviour Research Foundation (NARSAD Young Investigator Award 22616 to D.K.).

References [1] Johnson, E. L. et al. Dynamic frontotemporal systems process space and time in working memory. PLOS

Biol.16, e2004274 (2018).


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OS 09-02

Optimized awake-rat-fMRI habituation strategy leads to consistent

BOLD brain activation

Gabriele Russo1,2, Xavier Helluy1,3, Mehdi Behroozi3, Denise Manahan-Vaughan1

1 Ruhr University Bochum, Department of Neurophysiology, Medical Faculty, Bochum, Germany 2 Ruhr University Bochum, International Graduate School of Neuroscience, Bochum, Germany 3 Ruhr University Bochum, Department of Biopsychology, Faculty of Psychology, Institute of Cognitive

Neuroscience, Bochum, Germany

Questions: Functional Magnetic Resonance Imaging (fMRI) is a non-invasive indirect indicator of neuronal activity

[1] that is widely used to study brain functions under a broad range of applications [2]. Over the last few years, fMRI

has been established in awake rodents, but one limitation facing researchers is that acute stress caused by the

experimental conditions (physical restraint and loud noise) is likely to introduce biases and confound data

interpretation: stress not only impacts the neurobiological function of the brain [3], but also increases the likelihood

of head motion during imaging. Therefore, we asked ourselves whether a gradual habituation to the MRI environment

may serve to improve stress levels and preserve data integrity.

Methods: In this study we assessed fecal corticosterone metabolites (FCM) levels [4] in adult rats during gradual

habituation to fMRI in the awake state. After each habituation event, stress levels were determined by conducting an

ELISA analysis of the corticosterone concentration of fecal boli. Each sample was analysed in triplicate. After the

conclusion of the habituation procedure, in order to assess the sensitivity and stability of our setup, the animals were

presented with an auditory task expected to produce a basic activation pattern [5].

Results: Although FCM levels became elevated after the exposure to a dark environment and to the head fixation

device, incremental exposure to the various scanner conditions (physical restraint, head fixation, noise) resulted in a

return of FCM levels to pre-handling levels. Moreover, the activated structures that emerged from the auditory fMRI

group analysis are in agreement with the ones characterizing the central auditory pathway

Conclusions: In view of the above, we can conclude that effective habitation to the awake fMRI conditions can be

achieved by gradual habituation to the experimental procedures over a period of several weeks of training. In addition,

the benefits of a gradual habituation procedure extend to the motion and breathing parameters and to the robust

BOLD pattern that we were able to detect. On the other side, in the absence of a gradual habituation, experimental

data are likely to be confounded by the influence of stress-related elevations in FCM.

Acknowledgment

This work was supported by a grant from the German Research Foundation (Deutsche Forschungsgemeinschaft,

DFG) to Denise Manahan-Vaughan (SFB 874/B3, project number: 122679504). We gratefully acknowledge Jens

Colitti-Klausnitzer Beate Krenzek technical assistance. We thank Nadine Kollosch for animal care.


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Awake rat fMRI under low-stress level conditions - graphical

abstract

References [1] Ogawa S, Tank DW, Menon R, Ellermann JM, Kim SG, Merkle H, et al. Intrinsic signal changes accompanying

sensory stimulation: functional brain mapping with magnetic resonance imaging. Proc Natl Acad Sci. 1992 Jul 1;89(13):5951–5

[2] Angenstein F. The role of ongoing neuronal activity for baseline and stimulus-induced BOLD signals in the rat hippocampus. NeuroImage. 2019 Nov 15;202:116082

[3] Diamond DM, Bennett MC, Fleshner M, Rose GM. Inverted-U relationship between the level of peripheral corticosterone and the magnitude of hippocampal primed burst potentiation. Hippocampus. 1992;2(4):421–30

[4] Cinque C, Zinni M, Zuena AR, Giuli C, Alemà SG, Catalani A, et al. Faecal corticosterone metabolite assessment in socially housed male and female Wistar rats. Endocr Connect. 2018 Feb;7(2):250–7

[5] Cheung MM, Lau C, Zhou IY, Chan KC, Cheng JS, Zhang JW, et al. BOLD fMRI investigation of the rat auditory pathway and tonotopic organization. NeuroImage. 2012 Apr;60(2):1205–11


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OS 09-03

Changed cholinergic signaling to the medial prefrontal cortex in a

mouse model of neuropathic pain

Kai K. Kummer1, Miodrag Mitric1, Theodora Kalpachidou1, Marie-Luise Edenhofer1, Anna Seewald2,

Francesco Ferraguti2, Michaela Kress1

1 Medical University of Innsbruck, Institute of Physiology, Innsbruck, Austria 2 Medical University of Innsbruck, Institute of Pharmacology, Innsbruck, Austria

Chronic neuropathic pain constitutes a major public health issue, but the underlying disease mechanisms are only

partially understood. Although the involvement of the medial prefrontal cortex (mPFC) in the chronification of pain is

well established, the role of cholinergic signaling to the mPFC for the processing of painful stimuli has so far not been

assessed.

We therefore investigated cholinergic synaptic transmission in acute mPFC slices from spared nerve injure (SNI) and

control mice by multielectrode array (MEA) or patch-clamp recordings using pharmacological or endogenous

optogenetic cholinergic stimulation. Furthermore, we investigated the functional and morphological properties of

basal forebrain (BF) cholinergic neurons, which are the main source of acetylcholine in the mPFC.

MEA recordings demonstrated reduced mPFC network activity in response to pan-cholinergic and M1 receptor

activation 7d after SNI, which could also be observed in patch-clamp recordings of prelimbic (PrL) but not infralimbic

(IL) layer 5 pyramidal neurons. Also, cholinergic neurons in the BF showed more depolarized membrane potentials

together with a decrease in inhibitory postsynaptic current frequency, as well as an increased dendritic length and

complexity after SNI.

These findings suggest that in a mouse model of neuropathic pain cholinergic signaling is altered in the PrL, and that

these changes are based on SNI induced disinhibition and subsequent hyperexcitability of BF cholinergic neurons.


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OS 09-04

Increased activity of EAAT1 anion channels impairs hippocampal

neuronal inhibition and causes epilepsy in episodic ataxia 6

Yulia Kolobkova1,2, Miriam Engels1, Sabrina Behuet3, Sebastian Bludau3, Christoph Fahlke1,

Peter Kovermann1

1 Forschungszentrum Jülich GmbH, Institut for Biological Information Processing (IBI-1), Molecular and Cellular

Physiology, Jülich, Germany 2 Medical School Hamburg, Medical Faculty (University), Department Human Medicine, Hamburg, Germany 3 Forschungszentrum Jülich GmbH, IInstitute of Neuroscience and Medicine (INM-1), Structural and Functional

Organisation of the Brain, Jülich, Germany

Question: Episodic ataxia type 6 is an inherited neurological condition characterized by combined ataxia and

epilepsy. A severe case of episodic ataxia type 6 is caused by the P290R mutation in glial EAAT1 glutamate

transporter [1]. EAAT1 has a dual function as a glutamate transporter and anion channel [2, 3], and P290R was

shown to impair glutamate transport and to enhance anion channel activity by EAAT1 [4]. We generated a knock-in

mouse model (Slc1a3P290R/+) and showed that ataxia in mutant mice is caused by Bergmann glia apoptosis triggered

by augmented Cl- efflux, and subsequent cerebellar degeneration [5]. However, cerebellar degeneration cannot

account for the severe epileptic phenotype of Slc1a3P290R/+mice. Therefore, the P290R-induced cellular

pathomechanisms that lead to epileptic seizures have remained elusive.

Methods: Glutamatergic and GABAergic synaptic transmission was studied in acute brain slices of

Slc1a3P290R/+ mice, before and after epilepsy onset. We used fluorescence lifetime imaging microscopy with a

chloride-sensitive dye to determine intracellular chloride concentrations in hippocampal glial cells. Protein expression

was examined by immunohistochemistry and western blot analysis.

Results: Tonic GABAergic transmission was decreased in dentate gyrus granule cells from Slc1a3P290R/+ mice before

seizure onset (P20) whereas phasic GABAergic and glutamatergic transmission were unaltered in juvenile animals

of the same age. Expression of the GABAAR δ subunit was unaltered in Slc1a3P290R/+mice, indicating that

extrasynaptic GABA concentrations are decreased in juvenile mutant animals. EAAT1 is highly expressed in

hippocampal radial glia-like cells (RGLs) that modulate neurotransmission in dentate gyrus. We found intracellular

chloride concentrations to be reduced in Slc1a3P290R/+ RGLs, indicating increased driving forces for GABA uptake via

Cl--dependent GABA transporters.

Conclusions: Our study shows how increased EAAT1 chloride channel activity can cause epilepsy by modifying

synaptic GABA concentrations and suggests that EAAT1 anion channels may functionally link glutamatergic and

GABAergic synaptic transmission.

Acknowledgment

We are grateful to Christoph Aretzweiler, Marcel Böttcher, and Tanja Mertens, for technical assistance with the care

and genotyping of animals. We acknowledge the help of Nikola Kornadt-Beck with the legal aspects of animal

breeding.

References [1] Jen JC, Wan J, Palos TP, et al., 2005, 'Mutation in the glutamate transporter EAAT1 causes episodic ataxia,

hemiplegia, and seizures', Neurology, 65:529-534. [2] Fahlke C, Kortzak D, Machtens JP, 2016, 'Molecular physiology of EAAT anion channels', Pflügers Archiv,

468:491-502. [3] Machtens JP, Kortzak D, Lansche C, et al., 2015, 'Mechanisms of anion conduction by coupled glutamate

transporters', Cell, 160:542-53. [4] Winter N, Kovermann P, Fahlke C, 2012, 'A point mutation associated with episodic ataxia 6 increases

glutamate transporter anion current', Brain, 135:3416-3425.


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[5] Kovermann P, Untiet V, Kolobkova Y, et al, 2020, 'Increased glutamate transporter-associated anion currents cause glial apoptosis in episodic ataxia 6', Brain Communications 2020, 2: fcaa022.


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OS 09-05

Influence of Isoluminance on Gamma Band Synchrony in Area V1

Björn Mattes1,2,4, Gareth Bland1, Johanna Klon-Lipok1,3, Wolf Singer1,2,3, Katharine Shapcott1,2

1 Ernst Strüngmann Institute (ESI) for Neuroscience in Cooperation with Max Planck Society, Singer Lab, Frankfurt,

Germany 2 Frankfurt Institute for Advanced Studies, Frankfurt, Germany 3 Max Planck Institute for Brain Research, Frankfurt, Germany 4 Technical University of Darmstadt, Institute of Psychology, Darmstadt, Germany

Isoluminant stimuli are hard to process perceptually and have been shown to reduce gamma band activity. Previous

studies compared a limited amount of stimuli that had either luminance contrast or isoluminant properties and thus

limit the generalisability of results. To thoroughly investigate the cortical response at isoluminance we used grating

stimuli to explore the complete red-green luminance space and separately recorded perceptual and V1 neural

responses. This was done by first quantifying the perceptual luminance with a flicker task of a chromatically opponent

red-green disc stimulus randomly increasing or decreasing red and green brightness separately. The explored

luminance space spanned across the entire brightness levels available. From this we constructed 41 red-green

luminance gratings and measured the neural responses in two awake monkeys. This gave us neural activity in a 3

dimensional space, red luminance, green luminance and perceptual luminance. The strength of gamma band activity

depended on the perceptual luminance contrast, while its frequency was modulated by all three measured

dimensions. At isoluminance, the gamma band activity was strongly reduced while peak and sustained spiking activity

where mostly reduced. Some channels, however, also showed increased neuronal activity in response to

isoluminance. By controlling for the strong effect of perceptual luminance, we also derived the residual responses to

red and green luminance. The modulation of the mean activity of V1 spiking responses was much more

heterogeneous on individual channels than for the gamma, with a majority of channels increasing mean activity in

response to green and decreasing in response to red while also the opposite effect was reliably observed in other

channels. We also observed strong locking of spike times to the gamma band activity at high perceptual luminance

contrasts. The channels that did not show a dependence on the perceptual luminance manipulation had a far lower

phase locking and lower spike rates. We showed that population oscillations in the gamma range covered the

perceptual luminance space while population mean spiking activity seemed tied to the low level dimensions of red or

green luminance. However, the timing of these spiking responses followed the perceptual luminance dimension. This

argues for the importance of timing of population activity in perception in early visual area V1.


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OS 09-06

Activity-Dependent Plasticity of Gamma-Oscillations Mediated by

Calcium-Permeable AMPA-Receptors and Metabotropic Activation of

Parvalbumin-Interneurons

Michael D. Hadler1, Henrik Alle1, Jörg R. P. Geiger1,2

1 Charité - Universitätsmedizin Berlin, Institute of Neurophysiologiy, Berlin, Germany 2 Charité - Universitätsmedizin Berlin, NeuroCure Cluster of Excellence, Berlin, Germany

Synaptic recruitment of parvalbumin-positive interneurons (PVIs) is required to generate gamma oscillations (γ, 30-

80 Hz), a network rhythm linked to cognition in health and aberrant behavior in neuropsychiatric disorders. However,

little is known of how PVI synaptic inputs and network γ-activity interact during development. Investigating how known

mechanisms of PVI synaptic plasticity affect subsequent γ-activity may link these phenomena.

We prepared acute hippocampal slices of adolescent mice and performed local field potential recordings in CA3

and/or CA1 and transiently induced low frequency γ-activity (30-40 Hz) by bath-application of kainate. After one hour

of washout, a return to baseline activity and an additional resting period, an identical second induction of γ-activity

resulted in a roughly doubled amplitude of peak power, which lasts up to three hours. In simultaneous CA3-CA1

recordings, we found a similarly pronounced increase of phase-locked low frequency γ-activity in CA1. In isolated

"CA1-Mini" slices we also observed an increase of power in high frequency γ-activity (50-60 Hz) during the second

induction period. Reminiscent of synaptic plasticity, we call this form of activity-dependent network plasticity "γ-

potentiation".

By bath-applying pharmacological antagonists to our paradigm and recording in CA3, we prevented γ-potentiation

by blockade of calcium-permeable AMPA-receptors and either protein kinase A or C. However, blockade of NMDA-

or GluK1-receptors, L- or T-Type calcium channels did not affect γ-potentiation. Blockade of either mGluR1 or

mGluR5 reduced the magnitude of γ-potentiation by 50%, blocking both prevented γ-potentiation altogether. In mice

lacking mGluR5 specifically in PVIs (PV-mG5-/-), γ-potentiation was similarly attenuated by 50% compared to wild-

type littermates, with no additional effect of pharmacological blockade of mGluR5. Interestingly, in both wild-type and

mutant animals, the mGluR5 positive allosteric modulator VU-0409551 increased the magnitude of γ-potentiation,

resulting in a complete rescue in the mutant.

Our data demonstrate an activity-dependent mechanism of network plasticity in the hippocampus strongly

reminiscent of PVI synaptic plasticity in its pharmacological profile [1, 2], directly linked to PVIs in the PV-mG5-/-

animal model [3]. Investigating how metabotropic activation of PVIs affects subsequent γ-activity may inform future

therapeutic strategies for neuropsychiatric disease.

Acknowledgment

This study was funded by the Deutsche Forschungsgemeinschaft (FOR 2143, Cluster of Excellence NeuroCure

EXC 257, Research Unit Interneuron Synaptic Plasticity).


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Figure 1: γ-Potentiation in Mouse Hippocampal

CA3 A Methods. Left, Brain slices were cut and

recordings obtained from CA3. Centre, Kainate

(KA) induced network activity in the γ-frequency

range. Right, We recorded from up to 8 slices

and applied pharmacological agents.

B Left Exemplary voltage trace and spectrogram.

Red marks a power increase of the respective

frequencies. Right, Close-up traces of time

points 1 - 4 with power spectra. C Time-power plot of control experiments. Insets

"γ1" and "γ2" denote the intervals used for

analysis. Power values were normalized to the

value obtained during "γ1".

D γ-Potentiation lasts up to three hours.

γ-Potentiation Is Modulated by mGluR5-Activation

on PVIs A PV-mG5 animal model. mGluR5 can be

pharmacologically blocked by MPEP or positively

allosterically modulated by VU-0409551 (VU-0409,

VU).

B Above, Time-power plot of all experiments in

wild-type and mutant animals with respective

blockade of mGluR5 with MPEP. Below, Boxplots

comparing differences in initial peak γ-power

and subsequent potentiation. C Above, Time-power plot of all experiments in

wild-type and mutant animals with respective

positive allosteric modulation of mGluR5 with

VU-0409551. Below, Boxplots comparing

differences in initial peak γ-power and

subsequent potentiation.

References [1] Hainmüller, T., et al. 2104. 'Joint CP-AMPA and group I mGlu receptor activation is required for synaptic

plasticity in dentate gyrus fast-spiking interneurons.' PNAS September 9, 2014. 111(36) 13211-13216 [2] Zarnadze, S. et al., 2016. 'Cell-specific synaptic plasticity induced by network oscillations.' eLife

2016;5;e14912 [3] Barnes, S.A., et al. 2015. 'Disruption of mGluR5 in parvalbumin-positive interneurons induces core features

of neurodevelopmental disorders.' Mol. Psychiatry volume 20, pages 1161-1172.


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OS 09-07

KATP channel mutation in fast-spiking GABAergic interneurons disrupts

hippocampal network activity

Marie-Elisabeth Burkart, Josephine Kurzke, Jens-Karl Eilers, Kristina Lippmann

Leipzig University, Carl-Ludwig-Institute for Physiology, Leipzig, Germany

ATP-sensitive potassium channels (KATP channels) essentially mediate cell metabolism and electrical activity by

coupling intracellular ATP levels to K+ membrane conductance. Gain-of-function mutations of KATP channels result in

developmental delay and epilepsy with neonatal diabetes, i.e., DEND syndrome. While the role of KATP channels in

beta cells and the cause of neonatal diabetes in this syndrome is well understood, the pathophysiology of the

neurological symptoms remains to be elucidated. We hypothesized that KATP channels are key players in

parvalbumin+ interneurons (PV-INs), which play a pivotal role in generating both hippocampal sharp-wave ripples

(SWRs) and gamma frequency oscillations (30-100 Hz) and whose dysfunction is associated with epilepsy and

cognitive comorbidities. Therefore, we asked whether constitutively open KATP channels in PV-INs may be

responsible for the neurological symptoms of the DEND syndrome.

We investigated this question by using in vitro and in vivo local field potential (LFP) recordings as well as patch-

clamp recordings of PV-INs in the hippocampal area CA1 of adult mice.

We found that opening KATP channels pharmacologically in all neurons reduces both the frequency of in

vitro hippocampal SWRs and gamma oscillations. To specifically investigate the role of KATP channels in PV-INs, we

expressed the most common Kir6.2 gain-of-function mutation V59M heterozygously in PV-INs (PV-V59M) and found

that SWRs and gamma oscillations were severely impaired in PV-V59M mice similar to our pharmacological findings.

Additionally, we found a reduction in power of hippocampal gamma oscillations during wakefulness in in vivo LFP

recordings of PV-V59M mice. Both kinds of LFP recordings also indicated an increased susceptibility towards

epileptiform activity. To study underlying cellular properties, we performed patch-clamp recordings of PV-INs in

hippocampal slices showing no major alterations in passive membrane and active firing properties in PV-V59M mice.

However, the power of intrinsic membrane oscillations in the gamma range was significantly decreased and fewer

cells revealed gamma as their peak frequency.Thus, our findings provide evidence that the KATP channel mutation

V59M expressed only in PV-INs can account for altered network oscillations potentially underlying neurological

symptoms of the DEND syndrome and highlight the crucial role of PV-INs in oscillatory activity.


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OS 09-08

The neural architecture of sleep regulation and sensory gates –

Insights from Drosophila

Davide Raccuglia, Raquel Suárez-Grimalt, Cedric Brodersen, Jörg Geiger, David Owald

Universitätsmedizin Berlin, Institute of Neurophysiology, Berlin, Germany

Sleep disruption is a hallmark of insomnia, severely diminishes sleep quality and is linked to depression and deficits

in attention and learning. To maintain sleep, neural networks in our brains create selective sensory “gates” that block

sensory information while we sleep. Since sleep is a phenomenon widely conserved throughout the animal kingdom,

it is likely that all animals share similar strategies to filter sensory information. However, the fundamental

neurophysiological principles and neural interactions behind the creation of selective sensory gates for sleep

regulation are essentially unknown.

During sleep in mammals, reptiles and even in Zebrafish, neural networks undergo large-scale synchronizations of

slow-wave oscillatory activity. Interestingly, cognitive impairments in sleep deprived but awake mammals have been

linked to local synchronization of slow-wave oscillations (SWO) in specific neural networks, indicating a role for SWO

in shutting down sensory processing in specific neural networks and signaling the need for sleep. Using genetically

encoded voltage indicators we have recently discovered that even in the fruit fly Drosophila melanogaster electrical

SWO play an important role in the regulation of sleep drive and sensory gating. We found that sleep drive mediating

R5 neurons synchronize their electrical patterns to generate network-specific SWO that facilitate consolidated sleep

phases. Our findings therefore suggest that SWO might be an evolutionary optimized strategy to block sensory

information and provide consolidated sleep. However, the underlying neural interactions and neurophysiological

principles that could explain how SWO create selective sensory gates remain unclear.

In Drosophila, the less complex brain, unprecedented genetic accessibility and the sleep-related physiological

analogies of the neural networks involved in sleep offer a unique opportunity to study and understand the neural

architecture of sleep regulation. In my talk I will focus on a specific recurrent circuitry in Drosophila, demonstrating

how network-interactions provide the neurophysiological framework to construct sensory gates that facilitate sleep

quality.

Poster Session A DPG 2021 | Abstract Book

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Poster Session A | 30 September 2021

5:00 PM – 7:00 PM

Foyers

A 01 | Neural Mechanisms of Behaviour Chair

Timo Kirschstein (Rostock)

Birgit Liss (Ulm)


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A 01-01

Dissecting the roles of neurotensin and dopamine neuron

subpopulations in driving innate behaviours

Hanna E. van den Munkhof, Francisco J. de los Santos, Tatiana Korotkova

University of Cologne, Institute of Vegetative Physiology, Medical Faculty, Cologne, Germany

Innate motivated behaviours such as feeding, social interaction and exploration are crucial for survival and

reproduction. Evolutionarily conserved brain areas including the lateral hypothalamus (LH), the lateral septum (LS)

and the ventral tegmental area (VTA) are part of the circuitry controlling motivation, yet it remains unclear how exactly

genetically identified subpopulations within this circuitry direct specific reward-related behaviours. Two different

subpopulations in the LH and LS produce the neuromodulator neurotensin, while the VTA contains a large population

of dopamine neurons. Both neurotensin and dopamine have been associated with motivated behaviour and

locomotion.

First, we separately manipulated the activity of neurotensin neurons in the LH and LS using chemogenetics

(DREADDs), while mice engaged in a range of motivated behaviours such as feeding and social interaction. We

applied MoSeq, an unsupervised machine learning algorithm developed for depth images, to automatically classify

mouse behaviour on a subsecond scale, thus enabling unbiased identification of behavioural syllable sequences with

sub-second precision.

Second, we recorded activity of individual VTA dopamine neurons using calcium imaging, while mice freely explored

an arena with various rewards including a running wheel, to assess if neuronal responses depend on reward type.

We found that activation of neurotensin neurons in LH versus LS altered social interaction and food and water intake

in an opposite direction. Subsecond analysis of behaviour using MoSeq revealed changes in multiple behavioural

syllables related to motivation. For example, activation of LH neurotensin neurons increased expression of syllables

involved in water seeking, while decreasing those involved in social exploration. VTA dopamine neurons responded

differently depending on reward type. Neurons that were excited in the food zone showed decreased activity in the

running wheel, and vice versa. These results suggest that neurotensin and dopamine neurons selectively regulate

innate behaviours, with differences between neurotensin brain regions and even between individual VTA dopamine

neurons.

Acknowledgment

We thankfully acknowledge support from the ERC Consolidator Grant (HypFeedNet, TK), the Max Planck Society

(TK) and the DFG (431549029, TK; 233886668/GRK1960, FS).


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A 01-02

Prefrontal pyramidal neurons are critical for all phases of working

memory

Pascal Vogel1, Johannes Hahn1,2, Sevil Duvarci1, Torfi Sigurdsson1

1 Goethe University Frankfurt, Institute of Neurophysiology, Neuroscience Center, Frankfurt, Germany 2 Max Planck Institute for Brain Research, IMPRS for Neural Circuits, Frankfurt, Germany

The prefrontal cortex (PFC) has long been known to be essential for working memory (WM), yet exactly how it

contributes to this fundamental cognitive process remains incompletely understood. The PFC has primarily been

viewed as being responsible for the maintenance of information over a delay, but it is unclear whether it also plays a

more general role during WM. To address this, we performed task phase-specific optogenetic silencing of pyramidal

neurons in the medial PFC (mPFC) of mice performing a spatial delayed non-match-to-sample (DNMS) WM task on

a T-maze. To decompose the working memory process into distinguishable memory stages, each trial of this task

consisted of three phases (sample, delay, and choice phase), designed to reflect the encoding, maintenance, and

retrieval/usage of spatial information. We validated our optogenetic silencing strategy using extracellular recordings

in the mPFC, confirming selective inhibition of the vast majority of recorded putative pyramidal neurons in a robust

and temporally precise manner. Silencing during task performance revealed that the mPFC was required not only

during the delay phase but also during the sample and choice phase of the task, suggesting a global contribution of

mPFC pyramidal neurons to the entire working memory process that is not restricted to individual memory stages.

To gain further insights into how WM is supported by mPFC pyramidal neurons, we recorded their activity during all

phases of the task using calcium imaging with head-mounted miniscopes. Consistent with our optogenetic results,

mPFC pyramidal neurons were active during all task phases. Linear modeling using multiple task variables revealed

that they were most strongly influenced by the animals’ position and running direction, indicating a fundamental role

in spatial navigation. Spatial activity patterns were also influenced by other task variables including previously visited

goal arms, suggesting a role in encoding and maintaining to-be-remembered information. Taken together, these

results delineate the functional contribution of mPFC pyramidal neurons to WM, extending their role beyond the

maintenance of information.

Acknowledgment

Pascal Vogel and Johannes Hahn contributed equally


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A 01-03

Regulation of social behaviors by the lateral septum

Francisco J. de los Santos Bernal1, Robson Scheffer Teixeira1, Letizia Moscato2,

Hanna van den Munkhof1, Hae-Na Choi1, Tatiana Korotkova1

1 Institut for Vegetative Physiology, Faculty of Medicine, University of Cologne, Cologne, Germany 2 Max Planck Institute for Biological Cybernetics, Sensory and Sensorimotor Systems, Tubingen, Germany

Social behaviors, either conflictive or cooperative, are crucial for the survival and reproduction. Neural circuit

mechanisms underlying the regulation of various social behaviors remain largely elusive. Further, little is known about

how the brain computes choices when competing stimuli for mutually exclusive basic behaviors, e.g. social interaction

vs. feeding, occur simultaneously. Aggression and feeding-related behaviors are regulated by the lateral septum

(LS), which is connected with hypothalamic areas as well as with the prefrontal cortex and the hippocampus. We

have recently shown that somatostatin-expressing (Sst) neurons in the LS promote food-seeking (Carus-Cadavieco

et al., Nature 2017). Here we investigated functions of two cell populations in the LS, Sst-, and neurotensin-

expressing (NT) cells, in social interactions, ranging from stressful to rewarding, as well as in feeding-related

behaviors. Combining optogenetics, chemogenetics and calcium imaging in behaving mice, we found differential

neuronal activity and neuronal network dynamics in the LS selectively changes during different stages of social

behaviors. Optogenetic activation of NT cells resulted in increased social interaction, accompanied by a decreased

dominant behavior to conspecifics. At the same time, opto- or chemogenetic activation of NT cells in the LS

decreased food intake, also in the absence of conspecifics. Subsecond analysis of behavior using MoSeq, an

unsupervised machine learning algorithm, upon chemogenetic activation of NT cells, revealed changes of multiple

behavioral modules on a subsecond scale. Conversely, optogenetic activation of Sst cells in LS promoted food

approach and decreased social interactions. Analysis of spatial coding revealed that a subset of Sst cells could be

classified as place cells while exploring an open field, and Sst cells exhibited a more precise spatial coding than NT

cells in the presence of different stimuli. Taken together, our results suggest that Sst- and NT-expressing

subpopulations in LS complementary regulate multiple aspects of innate behaviors.

Acknowledgment

Acknowledgements: We gratefully acknowledge support by the ERC Consolidator Grant (FeedHypNet, to TK), the

DFG (233886668/ GRK1960, to FS) and the RTG (233886668/ GRK1960).


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A 01-04

Region-specific neuronal encoding of the hippocampus in novel object

learningRegion-specific neuronal encoding of the hippocampus in

novel object learning

Thu-Huong Hoang1,2, Juliane Böge1, Denise Manahan-Vaughan1

1 Ruhr University Bochum, Neurophysiology, Bochum, Germany 2 Ruhr University Bochum, International Graduate School of Neuroscience, Bochum, Germany

Long-term depression (LTD) and long-term potentiation (LTP) are key mechanisms underlying learning and memory.

Different forms of spatial learning triggers nuclear immediate-early gene (IEG) expression in the hippocampus that

is input-specific and also subcompartment-specific. It was previously shown that distinct components of spatial

learning are tightly associated with the differentiated expression of LTP and LTD.

Question: It is still unknown which hippocampal subcompartment processes object memory when the nature of the

object in space is different. In the present study, we explored to what extent nuclear IEG expression as a response

to patterned afferent stimulation of the hippocampus that leads to long-term plasticity. In freely behaving rodents, by

means of in vivo electrophysiological recording, changes in basal synaptic transmission were obtained in the

hippocampal subregions when animals were engaged in different aspects of novel spatial learning, or received sole

afferent stimulation to induce synaptic plasticity.

Method: By means of fluorescence in situ hybridization to detect nuclear expression of Homer1a, we mapped the

activity of the hippocampal subregions during the expression of synaptic plasticity triggered by afferent patterned

stimulation. We also examined IEG expression that is triggered during exploration of a novel holeboard or novel

spatial constellations of landmark features of the environment in conjunction with afferent stimulation. This approach

corresponds to the facilitation of persistent LTP or LTD through spatial learning.

Results: We found that LTP or LTD that are induced by afferent stimulation of the perforant path results in nuclear

IEG expression across all hippocampal subregions. By contrast learning-facilitation of LTP and LTD results in highly

localized, subcompartment specific IEG expression.

Conclusions: Our observations are consistent with a precise role of the hippocampal subfields in the encoding of

spatial content information. Furthermore, our data provide evidence for the segregation of information processing in

the hippocampus that supports the parallel map theory and the two-stream hypothesis.

Acknowledgment

We gratefully thank Ute Neubacher, Beate Krenzek and Jens Collitti-Klausnitzer for their great technical support. We

thank Nadine Kollosch for animal care.

This work was funded by the Deutsche Forschungsgemeinschaft (SFB 874/B3, project number: 122679504 to DMV).


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A 01-05

Leptin receptor-expressing neurons of the lateral hypothalamus favour

exploration over consumption

Rebecca Figge1,2, Anne Petzold1,2, Tatiana Korotkova1,2

1 University of Cologne, Institute for Vegetative Physiology, Cologne, Germany 2 Max Planck Institute for Metabolism Research, Neuronal Circuits and Behavior, Cologne, Germany

The cell population of leptin receptor (LepRb) expressing cells detects the anorectic hormone leptin and regulates

feeding behaviour. In the lateral hypothalamus (LH) this cell population is involved in the regulation of motivated

behaviours. Using optogenetics, we specifically stimulated LepRb cells in LH (LepRbLH) and investigated its effects

on foraging and exploratory behaviour. The identification of their distinct functionality helps to understand underlying

neuronal mechanisms in health and pathology.

LepRbLH-activation reduced food intake despite acute hunger. Additional Ca2+-imaging experiments indicated an

inhibition of LepRbLH whenever mice were located in the food zone. We could further demonstrate that the activation

of LepRbLH also reduced water intake despite acute thirst. When given the choice LepRbLH-stimulated male mice

preferred the exploration of a female to consummatory stimuli despite food or water deprivation.

Moreover, we found that mice displayed a changed state-dependent locomotion upon LepRbLH-activation. While

control mice showed a reduced locomotion after consecutive food deprivation, this reduction was not observed upon

LepRbLH-activation. In an elevated plus maze activation of LepRLH cells led to an increased exploration of open arms,

suggesting reduction of anxiety.

These results suggest that LepRbLH regulate multiple innate behaviours, promoting social interaction at the cost of

foraging behaviour.

Acknowledgment

We gratefully acknowledge support from the ERC Consolidator Grant (HypFeedNet, to Tatiana Korotkova), the

DFG (CRC1451, to Tatiana Korotkova) and the RTG-NCA (GRK1960, to Rebecca Figge).


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A 01-06

Enhancement of impulse control and sustained attention by Gq-protein

signaling in parvalbumin interneurons of the anterior cingulate cortex

Martin Jendryka1, Uwe Lewin1, Bastiaan van der Veen1, Sampath Kapanaiah1, Thomas Akam2, Birgit

Liss1, Anton Pekcec3, Wiebke Nissen3, Dennis Kätzel1

1 University Ulm, Institute of Applied Physiology, Ulm, Germany 2 University of Oxford, Department of Experimental Psychology, Oxford, Germany 3 Boehringer Ingelheim Pharma GmbH & Co. KG, Div. Research, Biberach, Germany

Impairment of prefrontal parvalbumin-positive interneurons has been linked to several psychiatric disorders and may

underlie some of the cognitive dysfunctions they present. In mice, optogenetic stimulation of prefrontal PV-

interneurons in the low gamma-range (30-60 Hz) has been found to improve some, rather specific, aspects of

cognitive control, including cognitive flexibility and attentional task engagement, whereas stimulation at lower

frequency was mostly ineffective or even detrimental. Hence, it remains unclear whether pharmacological

augmentation of prefrontal PV-cell function is a potential treatment strategy and, if so, which cognitive functions could

benefit from it. We therefore tested whether stimulation of Gq-protein-coupled receptors (GqPCRs) in PV interneurons

of the anterior cingulate cortex (ACC) could improve attention and impulse control. When activating the

chemogenetic GqPCR hM3Dq in ACC PV-cells, we found a selective enhancement of sustained attention and a

reduction of motor impulsivity assessed in the 5-choice-serial-reaction-time task (5-CSRTT). Both effects were driven

by a selective reduction of active erroneous (incorrect and premature) responses but were largely independent from

each other within individual animals. This cognition-enhancing effect was mainly apparent when cognitive control

was challenged by an extended waiting time or by systemic antagonism of GluN2B-containing NMDA-receptors.

Stimulation of the Gi-protein-coupled receptor hM4Di in ACC PV-cells did not cause any modulation of impulse control

or attention. Further, no change in these cognitive domains was seen if hM3Dq was activated in somatostatin-positive

interneurons of the ACC. These results suggest that the activation of GqPCRs expressed in PV-cells of the ACC may

be a viable strategy to enhance cognitive control.


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A 01-07

Contributions of the dorsal and ventral hippocampus to spatial working

memory and firing patterns in the medial prefrontal cortex

Susanne S. Babl1,2, Torfi Sigurdsson1

1 Goethe University Frankfurt, Institute of Neurophysiology, Frankfurt am Main, Germany 2 Max Planck Institute for Brain Research, IMPRS for Neural Circuits, Frankfurt am Main, Germany

Spatial working memory (SWM), the ability to remember a spatial cue over a short period of time (Dudchenko, 2004),

is a critical process for guiding behavioral actions. An extensive body of literature has repeatedly confirmed the joint

involvement of medial prefrontal cortex (mPFC) and hippocampus (HPC) in the successful execution of SWM (Kesner

et al., 1996; Floresco et al., 1997; Yoon et al., 2008). Recent studies suggest that both the dorsal and the ventral

hippocampal poles interact with the mPFC during SWM at the level of single cells and local field potentials

(Sigurdsson et al., 2010; Spellman et al., 2015; Jadhav et al., 2016; Maharjan et al., 2018). But to date it is not clear,

in which SWM phases the two hippocampal poles are involved and which task-relevant information they transmit to

the mPFC.

To address these questions, we infused mice with the viral construct AAV5-CamKII-ArchT-GFP in dorsal HPC

(dHPC) or ventral HPC (vHPC) and trained them to perform a delayed non-match-to-sample task in a T-maze. We

inhibited pyramidal neurons in either dHPC or vHPC optogenetically during the different phases of SWM, namely

encoding, maintenance or retrieval, while simultaneously recording neuronal activity in the mPFC.

In many aspects of SWM we found contrasting roles of the two hippocampal poles. dHPC inhibition during goal

encoding and retrieval, but not during maintenance, strongly impaired behavioral performance. The vHPC on the

other hand was only critically involved in encoding, but not in maintenance or retrieval. In the mPFC, neuronal firing

rates were modulated in various ways by both dHPC and vHPC inhibition, showing either excitation or inhibition.

However, only vHPC inhibition, but not dHPC inhibition, decreased goal-selective firing of mPFC neurons during the

encoding phase. In contrast, mPFC firing patterns relating to the linearized position between start and goal of the

maze were modified by dHPC inhibition. These findings highlight complementary roles of the dorsal and ventral

hippocampal poles in signaling spatial information to the mPFC during SWM.

Inhibition of dHPC or vHPC during SWM and

recording of neuronal firing rates in the mPFC

A, Schematic of SWM task in the T-maze

consisting of three phases: Encoding,

maintenance and retrieval. B+C, Expression of

inhibitory opsin ArchT in dHPC (B) or vHPC (C)

and recording of neuronal firing rates in mPFC

and local field potential in dHPC (B) or vHPC

(C) during optogenetic inhibition.


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A 01-08

Complementary coding of sexual drive, hunger and thirst by the lateral

hypothalamus

Anne Petzold1,2, Hanna van den Munkhof1,2, Rebecca Figge1,2, Tatiana Korotkova1,2

1 University of Cologne, Institute for Vegetative Physiology, Cologne, Germany 2 Max Planck Institute for Metabolism Research, Cologne, Germany

Several, at times competing, needs have to be coordinated and prioritised to ensure survival and reproduction. Little

is known about the representation and orchestration of multiple innate behaviours. Here, we report two

complementary neural populations of the lateral hypothalamus (LH) that cooperate to enable the appropriate

prioritisation of competing needs. Using a combination of deep-brain calcium imaging in freely moving animals and

cell-type specific activity manipulations, we show that leptin receptor-expressing (LepR) neurons track satiation to

promote the prioritisation of sexual exploration over feeding. Conversely, neurotensin-expressing (Nts) neurons track

food consumption to promote congruous water intake and discount social exploration. We further uncover that both

populations regulate distinct consummatory phases. These complementary populations may provide a fundamental

process to coordinate the pursuit of orthogonal needs according to state and opportunity.

Acknowledgment

We thankfully acknowledge support from the ERC Consolidator Grant (HypFeedNet, to TK), the DFG (CRC1451,

to TK) and the RTG-NCA (GRK1960, to RF).


of 516

A 01-09

Dopamine circuits underlying fear extinction learning

Ximena I. Salinas Hernández, Sevil Duvarci

Johann Wolfgang Goethe University Frankfurt, Institute of Neurophysiology, Frankfurt am Main, Germany

Extinction of conditioned fear is a learning process, during which the repeated presentations of a stimulus that no

longer predicts an aversive outcome lead to a gradual decrease in fear responses. Deficits in this form of safety

learning are a hallmark of anxiety disordersand thus understanding the neural basis of fear extinction has clinical

significance. In order to initiate extinction learning, the absence of the expected aversive outcome (unconditioned

stimulus, US) must be detected and signaled to the brain regions mediating fear extinction. However, the neuronal

substrates and mechanisms underlying this prediction error signal have remained largely elusive. We have recently

shown that a subset of VTA dopamine (DA) neurons is activated by the omission of the aversive US during fear

extinction, specifically during the beginning of extinction when the US omission is unexpected. Furthermore,

temporally specific optogenetic inhibition of DA neurons at the time of the US omission prevents extinction,

demonstrating that this signal is necessary for fear extinction learning. Conversely, enhancing this DA signal using

temporally-specific optogenetic excitation was sufficient to accelerate extinction learning. Together, our findings

demonstrated that a subset of VTA DA neurons signal a prediction error signal to drive fear extinction learning.

However, how and through which neural circuits this DA signal initiates extinction learning is still unknown. To address

this question, we are currently investigating the neural circuitry mediating this DA prediction error signal during fear

extinction using a combination of calcium recordings and optogenetic techniques.

Acknowledgment

We thank Jasmine Sonntag for technical support.


of 516

A 01-10

Dendritic integration of dopamine signals in dopaminergic neurons of

the fly

Michael-Marcel Heim, David Owald

Charité CCO Berlin Mitte, Neurophysiology, Berlin, Germany

Dopaminergic signaling is thought to be at the basis of goal directed behavior and instrumental for specific forms of

learning. Amongst these is associative learning which is found in vertebrates but also invertebrates. In the Drosophila

brain dopaminergic neurons innervate a higher-order processing center called the mushroom bodies. Here,

dopaminergic signaling alters the synaptic weights between intrinsic Kenyon cells that represent sensory information

and efferent cells called mushroom body output neurons. Individual dopaminergic neurons transport specific sensory

information encoding valence signals. However, it is less clear how competing inputs to the dopaminergic neuron

network are integrated. With this work, we present evidence for an inhibitory signaling pathway at the level of

dopaminergic neurons innervating the fly mushroom bodies. Moreover, we provide first evidence that this signaling

pathway is located at the dendritic sites of these neurons and resembles mammalian dopamine receptor 2 (D2) auto-

receptor signaling.


of 516

A 01-11

Classical “oral” vs. multi station settings for mock examinations in

physiology:students' perceptions and test quality

Robert Patejdl, Denise Franz, Marco Heerdegen, Timo Kirschstein, Falko Lange, Steffen Müller,

Valentin Neubert, Thomas K. Noack, Gesine Reichart, Rüdiger Köhling

University of Rostock, Oscar-Langendorff-Institute of Physiology, Rostock, Germany

Background & Aims: Oral examinations have a long-standing tradition in medicine, including the basic sciences. It

has repeatedly been reported that students prepare more intense for oral than written examinations. Furthermore,

oral examinations allow to assess subject-specific communication skills and are perceived as a more valid

preparation for later professional challenges than written exams. Nevertheless, over the past decades, classical

“orals” have been reduced and replaced by written exams due to doubts concerning the limited reliability and

objectivity of oral examinations. As an altervative the so-called objective structured clinical (or practical) examinations

(OSCE or OSPE, respectively) have been established for the assessment of students' skills. Only scarce data exists

regarding the implementation of OSPE-like assessments in physiology at German universities.

Methods: Students were assessed in both, an OSPE with seven stations and a classical oral examination covering

mostly neurophysiological issues. The order of both was set randomly. Following the test, the results of assessments

were analyzed with repect to difficulty and reliability. Furthermore, students were asked to participate in an electronic

survey to collect their personal perceptions of the examination.

Results: Thirty-one students particitpated, 22 completed the evaluation-survey. Cronbach's alpha was 0.79 for the

OSPE. The majority of students (91%) perceived the examinations to be aligned with the contents of previous lessons

and practical classes in physiology. The degree of difficulty was perceived to be rather similar between the stations

by 61% of participants. Ninety-five percent of participants stated that they received important feedback regarding

their individual performance during the OSPE. Although 69% stated that the OSPE had been more challenging and

straining, 77% stated that they had received more valuable feedback in the OSPE compared to the classic oral

exmination.

Discussion: The developed OSPE that was perceived as a valuable instrument to measure of students' performance

level in physiology. Its formal parameters of test quality were high and will be compared to classic orals in a second

sample by July 2021. More data is needed to judge the specific contribution of OSPEs for assessing knowledge and

understanding in physiology.


of 516

Poster Session A | 30 September 2021 5:00 PM – 7:00 PM

Foyers

A 02 | Cellular Neurophysiology Chair

Volkmar Leßmann (Magdeburg)

Jakob von Engelhardt (Mainz)


of 516

A 02-01

An in vivo patch-clamp study of K-ATP Channels in dopamine midbrain

neurons

Richard Egger, Jochen Roeper

Goethe University Frankfurt, Institute of Neurophysiology, Frankfurt, Germany

We have recently established deep in vivo patch-clamp recordings of dopamine (DA) midbrain neurons in mice

(Otomo et al. 2020). We now apply this approach to study the functional role of ATP-sensitive potassium (K-ATP)

channels. We have previously demonstrated that the expression of K-ATP channels in DA neurons in the medial

substantia nigra (SN) are required for in vivo burst firing and in turn for novelty-induced exploratory behavior

(Schiemann et al. 2012). However, the moment-to-moment contribution of K-ATP channels to ongoing in vivo activity

of DA neurons has not yet been explored. To address this question, we added 100 µM of the K-ATP channel blocker

tolbutamide to the internal pipette solution to inhibit these channels selectively in the target cell in vivo. Previous

control experiments comparing electrical in vivo activity in on-cell and whole-cell mode demonstrated stable firing

pattern and gave no evidence that in vivo whole-cell patching per sealtered intrinsic K-ATP channel activity (Otomo

et al. 2020). When dialyzing tolbutamide into DA SN neurons, we observed distinct effects. In n = 11/22 cells, the

firing frequency rapidly increased, which was accompanied by a net membrane depolarization. Some of these cells

(n = 7 of 11) went into depolarization block, from which they were rescued by injection of negative currents. In

contrast, DA neurons in the ventral tegmental area (VTA, n = 11) did not alter their in vivo firing activity in response

to tolbutamide dialysis. This preliminary data set suggests that in a least a subset of DA SN neuron K-ATP channels

are continuously open and shape ongoing electrical activity. Similar to previously reported for pancreatic alpha-cells

(Göpel et al. 2000) open K-ATP channels in some DA neurons might be necessary for action potential firing.

Acknowledgment

Jochen Roeper - Guidance and Supervision

Beatrice Fischer - Lab Management

Jasmine Sonntag - Histology

Niklas Hammer - Support

Strahinja Stojanovic - Support

References [1] Otomo et al. (2020) Nature Communications 11:6286 [2] Schiemann et al. (2012) Nature Neuroscience 15:1272-80 [3] Göpel et al. (2000) J. Physiol 528:509-20.


of 516

A 02-02

Functional heterogeneity of cerebellar granule cells

Philipp O'Neill, Martin Müller, Igor Delvendahl

University of Zurich, Department of Molecular Life Sciences, Zurich, Switzerland

The human brain contains approximately 50 billion cerebellar granule cells (GCs), accounting for over half of the total

number of neurons. Classic theories and computational models of cerebellar function have typically treated GCs as

a homogeneous population. However, recent evidence points towards differences in electrical properties and

morphology between GCs, as well as heterogeneity of excitatory synapses between mossy fibres (MFs) and GCs.

Heterogeneity has been shown to improve robustness of neural networks and learning in computational models.

Given that the cerebellum is involved in motor learning, the question arises of how diversity in membrane properties

and synaptic input influences the function of GCs.

Here we perform whole-cell patch-clamp recordings in acute cerebellar brain slices from mice. By measuring cellular

responses to current injections, spontaneous synaptic activity, and evoked synaptic transmission, we extract an

extensive set of cellular and synaptic properties across many individual GCs. We quantify the diversity of the

electrophysiological parameter space using bioinformatics approaches and address the functional role of

heterogeneity within the GC population by computational network modelling. We observe large differences in

variation and distributions of the different electrophysiological parameters, with the majority of measured properties

having skewed, non-normal distributions. Basic cellular characteristics such as resting membrane potential or action

potential shape exhibit a rather narrow distribution, whereas the excitability of GCs differs strongly between cells. In

general, MF-GC synaptic parameters are relatively variable between GCs, in particular the amplitude of excitatory

postsynaptic currents and the number of presynaptic release-ready vesicles.

Together, our data indicate that GC properties are non-uniform and that several cellular and synaptic characteristics

have heavy-tailed distributions, creating a considerable degree of functional diversity within the large GC population.

This approach enables us to systematically characterize differences between GCs and to elucidate functional aspects

of heterogeneity on the population level.


of 516

A 02-03

Machine learning based image analysis for automated quantification of

neuronal subtypes, and of protein levels in subcellular compartments

Shoumik Roy1, Gautami Amarnath1, Max Häusler1, Sonja Müller1, Johanna Wiemer1, Dominik Wuttke2,

Alina Glebova1, Helene Hollman1, Julia Benkert1, Nicole Wiederspohn1, Christina Pötschke1, Moritz

Münchmeyer2,3, Rosanna Parlato1, Johanna Duda1, Birgit Liss1,4

1 Ulm University, Institute of Applied Physiology, Ulm, Germany 2 Wolution GmbH & Co. KG, Munich, Germany 3 University of Wisconsin-Madison, Department of Physics, Madison, USA 4 University of Oxford, Linacre College & New College, Oxford, UK

Quantitative image analysis represents an important tool in physiological research, but manual analysis is time-

consuming and prone to bias. Here, we optimized machine learning based automated analysis (MLA) of

immunostained mouse midbrain sections, to quantify neuron numbers in defined areas, and signal intensities in

subcellular compartments. We use MLA to classify subtypes of dopaminergic midbrain neurons (DAN) in the

Substantia nigra (SN) and the Ventral tegmental area (VTA), and to address mechanisms of differential vulnerability

of DAN subtypes towards activity- and Ca2+-associated metabolic stress.

We used tyrosine-hydroxylase (TH) and dopamine transporter (DAT) immunostaining as markers for DAN and their

cytoplasm, the ion channel Kv4.3 as DAN plasma-membrane marker, and DAPI (4',6-diamidino-2-phenylindole)

staining to define the nucleus. We used the Wolution platform, customized algorithms, and supervised training for

recognition of TH-positive cells, subcellular compartment discrimination and fluorescence signal quantification. We

compared automated with manual image analysis. For automated image acquisition, we used AperioVersa 8 (Leica)

and Stedycon (Abberior Instruments) set-ups.

We found that our MLA approach is well-suited for unbiased and fast quantification of cell numbers and signal

intensities in subcellular compartments, and we are currently extending it to quantify viral transfection rates. Our

approach allows analysis of large neuron numbers, and hence facilitates the identification of small populations of

neuronal subtypes, based on differential protein expression. Accordingly, we detected a novel subpopulation of TH-

positive SN neurons (~10%) showing no DAT protein expression, mainly located in the lateral SN.Moreover, we

analyzed the expression of the neuronal Ca2+ sensor NCS-1 and of class II histone deacetylases (HDACs 4,5,7,9),

as these proteins undergo subcellular compartmental shuttling in a Ca2+ dependent manner, and are involved in

defining differential DAN pathophysiology. NCS-1 and all class II HDACs are expressed at mRNA and protein levels

in DAN, with HDAC7 and HDAC9 being differentially expressed in SN and VTA. Moreover, NCS-1 and HDAC4 were

mainly located at the plasma membrane and in the cytoplasm, while HDAC7 was located predominantly in the

nucleus. We are currently further analyzing the DAT-negative TH-positive SN neuron population, and the relative

redistribution of HDACs in response to metabolic stressors.


of 516

A 02-04

Projection-specific hierarchical organisation of somato-dendritic

dopamine 2 receptor inhibition among midbrain dopamine neurons

Niklas Hammer, Jochen Roeper

Goethe University Frankfurt, Institute of Neurophysiology, Frankfurt am Main, Germany

Most midbrain dopamine (DA) neurons are inhibited by activation of somato-dendritic D2-autoreceptors (D2R) (1).

The presence of synaptic D2R signalling between pre- and postsynaptic DA neurons in midbrain slices was

demonstrated in vitro (2). In addition, spontaneous D2R mediated IPSCs were recorded in DA neurons showing

functional dendro-dendritic vesicular DA release between midbrain DA neurons (3). Previous studies from our group

reported different D2R and GIRK2 expression levels in DA neurons projecting to distinct target areas (4), but the

functional contribution of synaptic and extrasynaptic D2R signaling within and across these DA subpopulations is

currently unknown. Distinct somato-dendritic D2R-signaling might also contribute to the different functional in vivo

properties we recently identified (5).

Therefore, we recorded electrically evoked, D2R-mediated slow inhibitory postsynaptic currents (eIPSCs) in

retrogradely identified DA neurons in vitro. These sulpiride-sensitive sIPSCs displayed kinetics and current peak

amplitude ranges in agreement with previous studies (2). However, we observed significant differences in peak D2R-

sIPSC amplitudes between DA neurons projecting to ventral (i.e. lateral shell of the nucleus accumbens (lNAcc),

medial shell of the nucleus accumbens (mNAcc)) and dorsal striatum (e.g. dorsomedial striatum (DMS), dorsolateral

striatum (DLS)). Mean eIPSC amplitudes of lNAcc-projecting DA neurons displayed at least two-fold larger currents

compared to DA neurons projecting to other compartments of the striatum (lNAcc: 27.1 ± 2.5 pA (n = 21); mNAcc

=12.2 ± 3.1 pA (n = 9); DMS = 16.3 ± 1.4 pA (n = 23); DLS =12.5 ± 1.3pA (n=18).

To investigate the interaction between defined DA subpopulations, we developed a projection-specific viral approach

to optogenetically excite a projection-selective pool of presynaptic DA neurons while recording a single postsynaptic

DA neuron with retrogradely defined axonal projections. Optogenetically stimulating presynaptic DA neurons

projecting to DLS while recording a DA neuron projecting to lNAcc, we recorded robust optically-evoked, sulpiride-

sensitive D2-IPSC (oIPSC) (lNAccDLS: 14.9 ± 3.7 pA (n = 13). In contrast, when reversing pre- and postsynaptic DA

neurons, little to no oIPSCs were recorded in DLS projecting DA neurons (DLSlNAcc: 3.6 ± 1.4 pA (n = 9). These

results suggest a lateral-to-medial hierarchical organisation of D2R-inhibition among distinct DA subpopulation in the

midbrain.

References [1] Lacey, M.G., Mercuri, N.B., North, R.A., 1987, ‘Dopamine acts on D2 receptors to increase potassium

conductance in neurons of the rat substantia nigra zona compacta’, The Journal of Physiology, 392, 397-416 [2] Beckstead, M. J., Grandy, D. K., Wickman, K., Williams, J. T. 2004, ‘Vesicular Dopamine Release Elicits an

Inhibitory Postsynaptic Current in Midbrain Dopamine Neurons’, Neuron, 42, 939-946 [3] Gantz, S. C., Bunzow, J. R., Williams, J. T., 2013, ‘Spontaneous inhibitory synaptic currents mediated by a

G protein-coupled receptor’, Neuron, 78, 807-812 [4] Lammel, S., Hetzel, A., Häckel, O., Jones, I., Liss, B., Roeper, J., 2008, ‘Unique Properties of

Mesoprefrontal Neurons within a Dual Mesocorticolimbic Dopamine System’, Neuron, 57, 760-773 [5] Farassat, N., Costa, K. M., Stojanovic, S., Albert, S., Kovacheva, L., Shin, J., Egger, R., Somayaji,

M., Duvarci, S., Schneider, G., Roeper, J. 2019, ‘In vivo functional diversity of midbrain dopamine neurons within identified axonal projections’, eLife, 8, 1-27


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A 02-05

Intraindividual Physiomic Heterogeneity of Layer 2/3 Pyramidal

Neurons in the Human Temporal Cortex

Henrike Planert1, Franz X. Mittermaier1, Sabine Grosser2, Pawel Fidzinski3, Ulf C. Schneider1, Henrik

Alle5, Imre Vida2, Jörg Geiger1, Yangfan Peng1

1 Charité Universitätsmedizin Berlin, Institute for Neurophysiology, Berlin, Germany 2 Charité Universitätsmedizin Berlin, Institute for integrative Neuroanatomy, Berlin, Germany 3 Charité Universitätsmedizin Berlin, Berlin Institute of Health / Neurocure Clinical Research Center, Berlin,

Germany 4 University of Oxford, MRC Brain Network Dynamics Unit, Oxford, Germany 5 Charité Universitätsmedizin Berlin, Department of Neurosurgery, Berlin, Germany

Neurons exhibit great heterogeneity in their physiological, anatomical and molecular properties, even within

established cell types. However, it is as yet unclear whether this within-type heterogeneity arises from pooling across

different individuals or represents a canonical principle.

We set out to explore the extent and principles of physiological heterogeneity by performing patch-clamp recording

of more than 1000 layer 2/3 pyramidal neurons from the human temporal cortex, as well as post-hoc anatomical

staining and reconstruction. In acute resected tissue from 22 patients undergoing temporal pole surgery, up to ten

neurons were recorded simultaneously. We analyzed cellular and synaptic electrophysiological parameters from up

to 88 pyramidal neurons and 177 monosynaptic connections per individual patient.

Intraindividual variability of single physiological properties was severalfold larger than variability between patients.

Hierarchical clustering of the high-dimensional parameter space revealed distinct clusters of functionally similar

neurons. While intraindividual synaptic properties were highly diverse, we found functional cell subtype-specific

differences in synaptic properties.

Our results suggest a generic organizational principle of the human cortical microcircuit that is shared among

individuals. Taken together, we identified distinct functional subtypes within the human layer 2/3 pyramidal neuron

population that may relate to previously described transcriptomic heterogeneity. This has important implications for

the computational capacity and highlights potential species-specific differences of the cortical microcircuit.

Acknowledgment

Current funding and address of Y. Peng: DFG Walter-Benjamin Fellow at MRC Brain Network Dynamics Unit,

University of Oxford, UK.


of 516

A 02-06

Optogenetic interrogation of dendritic integration in pacemaking

neurons

Lior Tiroshi, Osnat Oz, Lior Matityahu, Joshua A. Goldberg

The Hebrew University of Jerusalem, Department of Medical Neurobiology, Jerusalem, Israel

Pacemaker neurons discharge autonomously irrespective of synaptic inputs. For pacemakers, the question of

whether a weak synaptic input is excitatory or inhibitory is replaced by the question of whether it advances or delays

the next action potential (AP). The instantaneous phase response curve (PRC) of a pacemaker, describes how a

weak depolarizing synaptic input perturbs the pacemaker’s period (e.g., advances or delays the next AP), as a

function of when the depolarizing input arrives along the period of the pacemaker’s cycle.

PRCs have proven useful in theoretical and experimental neuroscience as a tool to succinctly characterize the

composite effect of membrane nonlinearities on synaptic integration. Thus, in the context of synaptic integration

measuring the pacemaker’s PRC can replace the need for a complete biophysical characterization of its ionic

conductances. However, this formalism does not account for: a) synaptic inputs that arrive at dendritic locations that

are electronically distant from the axosomatic pacemaker; or for b) dendritic nonlinear ionic conductances that can

transform the way synaptic inputs are integrated and how they affect the axosomatic pacemaker.

To address these shortcomings, we previously generalized the PRC formalism and defined a dendritic PRC (dPRC)

to account for distal dendritic inputs and dendritic nonlinearities. I will present how we have begun – using

optogenetics – to implement this formalism in order to measure dPRCs and the impact of dendrites on the response

properties of pacemaking neurons. We have found that dendrites of substantia nigra pars reticulata (SNr) projection

neurons are essentially linear, and introduce a ~15 millisecond delay to the latency of the their population response

to a sudden increase in synaptic input, as captured by their peristimulus time histogram (PSTH). This latency

demonstrates the dendritic delays can commensurate with synaptic delays, providing an alternative interpretation to

the structure of PSTHs. I will also present unpublished data demonstrating how dendrites of striatal cholinergic

interneurons that exhibit strong restorative and amplifying nonlinearities arising from hyperpolarization-activated

cyclic nucleotide-gated (HCN) and persistent voltage-activated Na+ channels, respectively, impact their dendritic

processing. I will show that our analysis can also indicate where along the dendrite (e.g., proximally or distally) these

two nonlinearities exert their effect.

Acknowledgment

This work was funded by a European Research Council (ERC) Consolidator Grant (no. 646886) to J.A.G.

References [1] Goldberg JA, Deister CA and Wilson CJ (2007). Response properties and synchronization of rhythmically

firing dendritic neurons. Journal of Neurophysiology, 97(1): 208-19.

[2] Tiroshi L and Goldberg JA (2019). Population dynamics and entrainment of basal ganglia pacemakers are shaped by their dendritic arbors. PLoS Computational Biology 15(2):e1006782 (2019).


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A 02-07

Glutamate fluorescent nanosensors: engineering and characterization

Kateryna Ryndia, Claudia Alleva, Christoph Fahlke

Forschungszentrum Jülich, Institute of Biological Information Processes (IBI-1), Jülich, Germany

L-Glutamate (Glu) is the predominant excitatory neurotransmitter and an intermediate of primary metabolism in the

vertebrate central nervous system. It plays an important role for normal brain function and contributes to the

pathophysiology of various neuropsychiatric illnesses [1, 2, 3].

Glutamate is released from presynaptic nerve terminals via exocytosis of synaptic vesicles. Vesicular glutamate

transporters accumulate glutamate in synaptic vesicles harnessing the electrochemical proton gradient generated by

primary active H+-ATPases. The strength of glutamatergic synapses critically depends on glutamate concentrations

in synaptic vesicles.

Based on its ubiquitous nature and prominent role, various genetically encoded nanosensors were developed to

detect local concentration changes in the synaptic and perisynaptic space, but sensors that report on glutamate

accumulation in synaptic vesicles are missing. Since synaptic vesicles exhibit an acidic lumen and since glutamate

accumulation is associated with changes of cation and anion concentrations, such a sensor needs to be pH-, cation-

and anion-independent. Moreover, low affinity is required to report on high mM concentrations in synaptic vesicles.

In this work, molecular dynamics simulations (MD), protein biochemistry and fluorescence spectroscopy were

conducted to characterize and to improve a glutamate sensor that is based on the GluBP domain of a bacterial

periplasmic glutamate binding protein (ybeJ). We studied ligand binding and interaction with Na+ / K+ ions through

atomistic molecular dynamics simulations. MD revealed hot-spots of interaction between these ions and the binding

domain, suggesting a potential role of ions in the binding process or in the following conformational change. In

combination with the fast switching alchemical transformations we identified mutations that are involved not only in

K+/Na+ binding, but also decrease the affinity to glutamate. In order to study glutamate binding and to verify the

prediction of our simulations by fluorescence spectroscopy, we are currently expressing WT and mutant GluBP in E.

coli and purifying it by affinity chromatography, as well as transfecting living tsA201 cells.

We expect our studies will pave the way to engineer a new glutamate sensor with decreased Na+ / K+ sensitivity,

lower glutamate affinity and reduced pH dependence that will allow to study intravesicular glutamate concentrations

in the near future.

References [1] Vandenberg, RJ, Ryan, RM 2013, Mechanisms of Glutamate Transport . Physiol Rev, 93(4), 1621-57, doi:

10.1152/physrev.00007.2013. PMID: 24137018. [2] Alleva, C, Machtens, JP, Kortzak, D et al. 2021, Molecular Basis of Coupled Transport and Anion Conduction

in Excitatory Amino Acid Transporters, Neurochem Res, https://doi.org/10.1007/s11064-021-03252-x. [3] Meldrum, BS 2000, Glutamate as a neurotransmitter in the brain: review of physiology and pathology. J Nutr.

130(4S Suppl), 1007S-15S, doi: 10.1093/jn/130.4.1007S, PMID: 10736372. [4] Okumoto, S, Looger, LL, Micheva, KD, Reimer, RJ, Smith, SJ, Frommer, WB 2005, Detection of glutamate

release from neurons by genetically encoded surface-displayed FRET nanosensors, Proc Nat Acad Sci USA, 102(24), 8740-5, doi: 10.1073/pnas.0503274102, PMID: 15939876, PMCID: PMC1143584.

[5] Coates, C, Kerruth, S, Helassa, N, Török, K 2020, Kinetic Mechanisms of Fast Glutamate Sensing by Fluorescent Protein Probes. Biophysical Journal, 118, 117-127, https://doi.org/10.1016/j.bpj.2019.11.006.


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A 02-08

Sodium channel inactivation and maximal firing rates in dopamine

neuron subpopulations

Tabea Ziouziou1, Christopher Knowlton2, Niklas Hammer1, Carmen Canavier2, Jochen Roeper1

1 Goethe University, Institute for Neurophysiology, Neuroscience Center, Frankfurt, Germany 2 Louisiana State University Health Sciences Center, Department of Cell Biology and Anatomy, School of Medicine,

New Orleans, USA

Based on electrophysiological properties, dopamine (DA) midbrain neurons can be segregated into two main

subpopulations: conventional DA neurons projecting to dorsal striatum or the lateral shell of nucleus accumbens and

atypical DA neurons projecting – among others - to medial shell and core regions of nucleus accumbens or the medial

prefrontal cortex. While some of their distinct biophysical properties (Lammel et al. 2008) and different firing pattern

in vivo (Farassat et al. 2019) have already been described, their differences regarding maximal firing rates and their

distinct onset of depolarization block have not yet been studied in detail.

By systematically exploring the parameter space of a computational single-compartment model of DA neurons, we

identified slow inactivation of voltage-gated sodium channels as a candidate mechanism that might differentiate

between conventional and atypical DA neurons. In particular, the model suggested that conventional DA neurons

enter depolarization at lower firing rates because of enhanced slow inactivation of voltage-gated channels and that

depolarization block could be overcome by injection of additional depolarizing current. This prediction was tested

with projection-specific in vitro patch-clamp experiments in identified and retrogradely labeled DA neurons. We

confirmed that lateral shell-projecting, conventional DA neurons entered depolarization block at lower frequencies

(17.0 ± 3.3 Hz; n = 7, N = 3) compared to the medial shell-projecting, atypical DA neurons (31.9 ± 4.6 Hz; n = 13, N

= 3). In addition, only lateral shell-projecting, conventional DA neurons triggered an action potential (amplitude 37.5

± 10.2 mV, peak slope 15.1 ± 4.8 V/s, threshold for spike generation = 5 V/s; n = 7, N = 3) induced by additional

depolarizing current injection during depolarization block, while atypical DA neurons showed a significantly slower

and smaller depolarizing responses (amplitude 17.7 ± 3.8 mV, peak slope 4.5 ± 1.7 V/s, threshold for spike generation

= 5 V/s; n = 13, N = 3). To further test the predictions of the computational models, we currently study gating properties

of voltage-gated sodium channels in defined conventional and atypical DA neurons.

Acknowledgment

This work was supported by NIH R01 DA041705 to CCC and JR.

References [1] Lammel S., Hetzel A., Häckel O., Jones I., Liss B., Roeper J. 2008, 'Unique properties of mesoprefrontal

neurons within a dual mesocorticolimbic dopamine system', Neuron; 57: 760–773.

[2] Farassat N., Costa K.M., Stojanovic S., Albert S., Kovacheva L., Shin J.,Egger R., Somayaji M., Duvarci S., Schneider G., Roeper J. 2019, 'In vivo functional diversity of midbrain dopamine neurons within identified axonal projections', eLife; 8: e48408.


of 516

A 02-09

Distinct contribution of postsynaptic Ca2+ sources and Ca2+-permeable

AMPA receptors to timing-dependent LTP induced by two distinct low

repeat STDP paradigms at SC-CA1 synapses

Babak Khodaie1,3, Efrain Cepeda-Prado1, Elke Edelmann1,2,3, Volkmar Leßmann1,2,3

1 Otto-von-Guericke University, Institute of Physiology, Magdeburg, Germany 2 Center for Behavioral Brain Sciences (CBBS), Magdeburg, Germany 3 OVGU ESF-funded International Graduate School ABINEP, Magdeburg, Germany

STDP is a cellular model for learning that employs low frequency repeats of coincident action potential (AP) firing in

pre- and postsynaptic neurons to induce timing-dependent LTP (t-LTP). Recent evidence suggested that t-LTP at

SC-CA1 synapses is equally robust as high-frequency stimulation induced conventional LTP. While conventional SC-

CA1 LTP depends on postsynaptic Ca2+ rise through NMDA receptors and L-type VGCCs, the contribution of intra-

and extracellular Ca2+ sources to t-LTP is not well understood.

We set out to decipher the origin of postsynaptic Ca2+ that triggers induction of SC-CA1 t-LTP elicited by

physiologically relevant low repeat STDP stimulation. We performed whole cell patch clamp recordings in acute

hippocampal slices from 4 weeks old male mice and used either a canonical (1EPSP/1AP) or a burst (1EPSP/4APs)

STDP protocol, repeated 6 times at 0.5 Hz, to induce t-LTP in CA1 pyramidal cells. The importance of postsynaptic

Ca2+ signaling for t-LTP was initially tested with application of the Ca2+ chelator BAPTA (10 mM) via the patch pipette,

which blocked t-LTP induced by both protocols. Bath application of APV (50 µM) and Nifedipine (25 μM) were used

to test the role of NMDAR and L-type VGCCs. Contribution of mGluR signaling was tested by co-application of the

mGluR1 antagonist YM298198 (1 μM), and the mGluR5 antagonist MPEP (10 μM). The role of GluA2-lacking

calcium-permeable AMPARs (cp-AMPARs) was tested by bath applied inhibitors NASPM (100 µM) or IEM-1460 (100

µM). To test ER Ca2+stores, we bath applied 2-APB (100 µM) to inhibit IP3 receptors, and intracellular ryanodine

(100 µM) to block ryanodine receptors (RyRs).

Our results showed that t-LTP induction by the 2 protocols (6x 1:1 and 6x 1:4) recruited diverse induction

mechanisms. Both protocols relied on activity of cp-AMPARs, while only the canonical protocol needed Ca2+ entry

via NMDARs and L-type VGCCs. Metabotropic mGluR1 and mGluR5 contributed to t-LTP induction with the burst

protocol; the canonical protocol induced t-LTP did not involve group I mGluRs. However, activation of IP3 and RyR

receptor sensitive internal Ca2+ stores was necessary to allow t-LTP with any of the 2 protocols.

Thus, while our study reveals distinct mechanisms for the initial postsynaptic Ca2+ rise required for the 2 low repeat

t-LTP protocols, both paradigms critically rely on sustained Ca2+ release from the ER and Ca2+ influx through cp-

AMPARs during test stimulation after the induction paradigm.

Acknowledgment Funding: OVGU ESF-founded International graduate school ABINEP.


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Foyers

A 03 | Novel Approaches to Channels and

Receptors Chair

Rüdiger Köhling (Rostock)

Philipp Sasse (Bonn)


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A 03-01

Observing Rapid Phosphatidylinositol 3,4-bisphosphate Kinetics

during Activation of Voltage Sensitive Phosphatase in Mammalian

Cells using Total Internal Reflection Microscopy at high Frame Rates

Christian R. Halaszovich, Vijay Renigunta, Dominik Oliver

Philipps-Universität Marburg, Institut für Physiologie, Marburg, Germany

Voltage sensitive phosphatases (VSPs) are PI(4,5)P2/PI(3,4,5)P3-5- and PI(3,4)P2/PI(3,4,5)P3-3-phosphatases

consisting of a voltage sensor domain (VSD) and a catalytic domain (CD) (Halaszovich 2009; Kurokawa 2012). The

VSD senses membrane voltage and controls the CD. Thus, membrane voltage controls VSP activity. The effect of

VSP on phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) is paradox, as it is produced by the 5-phosphatase activity

as well as degrade by the 3-phosphatase activity. Grimm & Isacoff (2016) reported that this leads to a transient

increase in PI(3,4)P2 immediately after activation of Ciona intestinalis (Ci) VSP in Xenopus leavis oocytes. Detection

of phosphatidylinositol (PI) species in living cells is usually performed using either diffusible probes that translocate

from the membrane into the cytosol or vice versa, or using Förster resonance energy transfer (FRET) based probes,

the rely on changes in proximity of a FRET donor / acceptor pair. Membrane anchored FRET based probes are

virtually independent of diffusion. Grimm & Isacoff needed to use a membrane anchored FRET-based PI(3,4)P2

probe since diffusible probes proved to be too slow to detect these fast PI(3,4)P2 transients.

PI probes that translocate from the plasma membrane into the cytosol are notoriously slow when used in oocytes.

Presumably this is due to large diffusion distances. These probes show rapid effects in mammalian cells, where the

diffusion distances are small compared with oocytes. We decided to test if such a probe can detect PI(3,4)P2

transients caused by VSP activation.

We expressed Ci-VSP together with the canonical PI(3,4)P2 probe TAPP1-PH-GFP in Chinese hamster ovary (CHO)

cells. Cells were depolarized using the whole-cell patch clamp technique to activate the VSP. Changes in membrane

binding of the probe were visualized and quantified using Total Internal Reflection (TIRF) microscopy at a frame rate

of 50 fps.

As expected, we were able to detect PI(3,4)P2 transients at sufficiently high activity of the VSP, i.e. at activation

voltages ≥ 40 mV.

We conclude that the limitations of translocation based PI probes as to their speed, while being significant in Xenopus

laevis oocytes, are of no concern in mammalian cells.

References [1] Grimm SS, Isacoff EY. Allosteric substrate switching in a voltage-sensing lipid phosphatase. Nat Chem Biol.

2016 Apr;12(4):261-7. [2] Halaszovich CR, Schreiber DN, Oliver D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-

bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5'-phosphatase. J Biol Chem. 2009 Jan 23;284(4):2106-13.

[3] Kurokawa T, Takasuga S, Sakata S, Yamaguchi S, Horie S, Homma KJ, Sasaki T, Okamura Y. 3' Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP). Proc Natl Acad Sci U S A. 2012 Jun 19;109(25):10089-94.


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A 03-02

Mapping the interaction surface between CaVß and actin filaments

using cross-linking mass spectrometry and computational biology

Francisco J. Castilla Porras1, Mercedes Alfonso-Prieto2,3, Beatrix Santiago-Schübel4, Patricia Hidalgo1

1 Forschungszentrum Jülich, IBI-1, Jülich, Germany 2 Forschungszentrum Jülich, INM-9, Jülich, Germany 3 Forschungszentrum Jülich, IAS-5, Jülich, Germany 4 Forschungszentrum Jülich, ZEA-3, Jülich, Germany

Voltage-gated calcium channels (CaVs) are the major pathway of extracellular calcium entry into excitable cells

controlling a variety of Ca2+-induced signals, including cardiac contraction, hormone secretion, synaptic transmission

and gene expression. The core complex of these channels is formed by an α1 pore-forming subunit containing the

ion conduction pathway (CaVα1) and an accessory β-subunit (CaVβ) [1-2]. CaVβ associates directly with actin

filaments (F-actin); this association increases CaVs insertion at the cell surface in cardiac cells, whereas in neurons

augments the size of the readily-releasable pool of synaptic vesicles [3-4]. However, the lack of information on the

CaVβ/F-actin interaction surface hampers our understanding of how it regulates the aforementioned functions.

Here, we use chemical cross-linking combined with tandem mass spectrometry (XL-MS) to determine distance

constraints in the CaVß/F-actin assembly, which in turn are used to guide docking modelling of the protein-protein

complex. In silico alanine scanning is used to identify hot-spots residues, which are then experimentally validated by

in vitro binding assays. Lastly, this work will set the basis for in vivo experiments whereby mutants CaVβ proteins will

be expressed in primary cultured cells and the functional consequence of disrupting the CaVβ/F-actin association for

synaptic transmission will be assessed using cellular electrophysiology.

Altogether, our studies will provide a model of the CaVß/F-actin interaction surface and pave the way for further

analysis of its physiological and pathological roles.

References [1] Chen, Y. H., Li, M. H., Zhang, Y., He, L. L., Yamada, Y., Fitzmaurice, A., ... & Yang, J. (2004).

Structural basis of the α 1–β subunit interaction of voltage-gated Ca 2+ channels. Nature, 429(6992), 675-680.

[2] Stölting, G., de Oliveira, R. C., Guzman, R. E., Miranda-Laferte, E., Conrad, R., Jordan, N., ... & Hidalgo, P. (2015). Direct interaction of CaVβ with actin up-regulates L-type calcium currents in HL-1 cardiomyocytes. Journal of Biological Chemistry, 290(8), 4561-4572.

[3] Conrad, R., Stölting, G., Hendriks, J., Ruello, G., Kortzak, D., Jordan, N., ... & Hidalgo, P. (2018). Rapid turnover of the cardiac L-Type CaV1. 2 channel by endocytic recycling regulates its cell surface availability. Iscience, 7, 1-15.

[4] Guzman, G. A., Guzman, R. E., Jordan, N., & Hidalgo, P. (2019). A tripartite interaction among the calcium channel α1-and β-subunits and F-actin increases the readily releasable pool of vesicles and its recovery after depletion. Frontiers in cellular neuroscience, 13, 125.


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A 03-03

Inference of kinetic schemes for ion channels by a Bayesian network

based algorithm

Jan Münch, Ralf Schmauder, Klaus Benndorf

FSU, UKJ Institut für Physiologie 2, Jena, Germany

Inferring the complex functional dynamics of ion channels from ensemble currents is a daunting task due to limited

information in the data, leading often to a poorly determined inference of kinetic schemes. We present results of a

Bayesian network analysis method for posterior estimation of the rates of a linear stochastic network such as ion

channel data. The Bayesian network is an analytical stochastic approximation of the underlying master equation

which enables a precise modeling of this class of stochastic processes with better accuracy and precision than

traditional deterministic rate equations.

To establish the Bayesian network we mathematically generalized an existing Kalman Filter framework (Moffat 2007)

to enable analysis of signals with state-dependent open-channel noise and Poisson- distributed fluorescence data.

We benchmark this generalization against the two gold standard algorithms. We show that over-simplification by rate

equations, which ignores autocorrelation of the intrinsic noise, leads to unreliable credibilty volumes or, in the

maximum likelihood framework, to unreliable confidence volumes.

Our full Bayesian treatment of macroscopic data such as patch-clamp (PC) or patch-clamp fluorometric data has

many favorable modeling properties compared to traditional maximum-likelihood techniques. PC data can be very

weakly informative on rate parameters of a complex Markov process, leading sometimes to complete parameter

unidentifiability. This problem is often completely undetected in maximum-likelihood approaches while the Bayesian

posterior immediately reveals that data information vs. model complexity conflict. We show that often this parameter

unidentifiability origiates from inexpertly chosen default uniform priors on the rate matrix. To further alleviate

parameter identifiability we exemplify the benefits of performing information fusion on arbitrary prior information such

as theoretical concepts like diffusion limited binding on a single rate. The sentence is not clear. Doing so leads to a

concentration of the posterior distribution of all other rates Is this clear? .

While ignoring autocorrelation of the intrinsic noise leads additionally to a break down of model identification by

information criteria, our algorithm enables a robust identification of the true model by continuous model expansion,

the Widely Applicable Information Criterion (WAIC), and variance-based model selection.

Acknowledgment We thank Prof Michael Habek and Prof. Frank Noe for their helpful discussions

References [1] Moffatt L. Estimation of ion channel kinetics from fluctuations of macroscopic currents. Biophys J. 2007

Jul 1;93(1):74-91. doi: 10.1529/biophysj.106.101212. Epub 2007 Apr 6. PMID: 17416622; PMCID: PMC1914441. [2] Milescu LS, Akk G, Sachs F. Maximum likelihood estimation of ion channel kinetics from macroscopic

currents. Biophys J. 2005;88(4):2494-2515. doi:10.1529/biophysj.104.053256


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A 03-04

Gold nanoparticles functionalized with adrenaline stimulate β1- but not

β2-receptors

Rebecca Claßen1, Annabelle Mattern2, Annemarie Wolf3, Ervice Pouokam1, Klaus-Dieter Schlüter3,

Mathias S. Wickleder2, Martin Diener1

1 Justus-Liebig-University Giessen, Institute of Veterinary Physiology and Biochemistry, Giessen, Germany 2 University of Cologne, Institute of Inorganic Chemistry, Cologne, Germany 3 Justus-Liebig-University Giessen, Institute of Physiology, Giessen, Germany

Nanomedicine has gained high attention in the last years due to the wide range of possible applications of

nanostructures. These nanostructures can be functionalized by binding a high number of ligands on their surface and

thereby they can be used for specific drug targeting [1]. Due to the plenty of ligands stabilized on a single

nanostructure, they can show multivalent interactions at the receptor side. In this project, the two catecholamines

adrenaline and noradrenaline were bound to the surface of spherical gold nanoparticles. Catecholamines in general

induce different biological effects in the organism via stimulation of adrenergic receptors. In our study, we wanted to

find out whether adrenaline and noradrenaline functionalized nanoparticles can stimulate adrenergic receptors of

different biological tissues.

As the properties of nanoparticles influence their effects [2], adrenaline and noradrenaline functionalized gold

nanoparticles were synthesized with diameters between 8 to 14 nm. Their influence on rat intestinal β-receptors was

tested in Ussing chamber experiments. Adrenaline functionalized nanoparticles (9 nm) led to a decrease of short-

circuit current caused by induction of potassium secretion. It is known that native adrenaline stimulates potassium

secretion by binding to β1- and β2-receptors [3]. To find out with which receptor subtype the nanoparticles interact,

we performed isometric tension measurements with bronchial and tracheal segments from rats whose contractility is

highly dependent on stimulation of β2-receptors. While native adrenaline led to a concentration-dependent relaxation

after interaction with β2-receptors, neither the adrenaline nor the noradrenaline functionalized nanoparticles (8 to 14

nm) led to a relaxation of airway smooth muscle. In contrast, adrenaline functionalized nanoparticles (9 nm) were

able to raise the contractility of cardiomyocytes which highly express β1-receptors. Additionally, a selective β1-

receptor-blocker could not totally inhibit the nanoparticles' action as it did in the positive control. This might be

explained by multivalent interactions of the functionalized nanoparticles and the β1-receptors resulting in a stronger

receptor binding.

These results suggest that adrenaline functionalized nanoparticles stimulate β1-adrenergic receptors in a multivalent

way while not stimulating β2-receptors.

References [1] Patra, Jayanta K.; Das, Gitishree; Fraceto, Leonardo F.; Campos, Estefania V. R.; Del Rodriguez-Torres,

Maria P.; Acosta-Torres, Laura S.; Diaz-Torres, Luis A.; Grillo, Renato; Swamy, Mallappa K. S.; Sharma, Shivesh; Habtemariam, Solomon; Shin, Han-Seung; 2018, 'Nano based drug delivery systems: recent developments and future prospects', Journal of Nanobiotechnology, 16, 71

[2] Albanese, Alexandre; Tang, Peter S.; Chan, Warren C. W.; 2012, 'The effect of nanoparticle size, shape and surface chemistry on biological systems', Annual review of biomedical engineering, 14, 1-16

[3] Zhang, Jin; Halm, Susan T.; Halm, Dan R.; 2009, 'Adrenergic activation of electrogenic K+ secretion in guinea pig distal colonic epithelium: involvement of beta1- and beta2-adrenergic receptors', American journal of physiology. Gastrointestinal and liver physiology, 297, 269-77


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A 03-05

The versatile supportive roles of MARVEL proteins in tight junctions,

unveiled with super-resolution microscopy

Rozemarijn van der Veen1, Hannes Gonschior1, Thomas Stellwag1, Vini Natalia1, Carolin Dunker1,

Volker Haucke1,2, Martin Lehmann1

1 Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Department Molecular Pharmacology and Cell Biology,

Berlin, Germany 2 Freie Universität Berlin, Faculty of Biology, Chemistry, Pharmacy, Berlin, Germany

Tight junctions (TJs) connect neighboring cells in epithelia and endothelia and are important for tissue

compartmentalization, as they not only establish a paracellular barrier, but also prevent apical and basolateral lipids

from mixing. TJs have a tissue-specific composition of transmembrane and cytosolic proteins. In correspondence

with this, pathogenic mutations in TJ proteins often also have tissue-specific effects. While cytosolic TJ proteins

mediate cytoskeletal interaction and signaling, transmembrane proteins, in particular members of the claudin family,

form of a fibril-based meshwork and are the major determinants of the TJ barrier. Members of another family of

transmembrane TJ proteins, the tetra-spanning MARVEL (MAL and related proteins for vesicle trafficking and

membrane link) proteins, cannot form meshworks by themselves. Studies suggest that these MARVEL proteins have

a supportive role in the TJ, but the molecular details remain very poorly understood.

In this study, we used super-resolution stimulated emission depletion microscopy, with a lateral resolution of ~50 nm,

to image TJ strands (of 10nm thickness) and meshwork architecture. With this newly achieved level of detail, we

show that MARVEL proteins differentially support the structure and the dynamics of meshworks created by other TJ

proteins. Functional consequences of these findings were studied by performing ion flux and transepithelial

resistance measurements on epithelial monolayers. For this, we used a unique epithelial cell line that lacks the major

TJ proteins and thus allows for their individual reintroduction and examination. Overall, this project demonstrates the

complexity of TJ support by the MARVEL protein family, showing distinct effects on the structure and function of

meshworks created by different proteins. These findings can be a basis for future studies that advance our

understanding of tissue-specific phenotypes in TJ-related disorders.


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A 03-06

Using photo-physical properties for optimized fluorescent ligands and

binding assays

Ralf Schmauder, Andrea Schweinitz, Christian Sattler, Klaus Benndorf

Jena University Hospital, Institute of Physiology II, Jena, Germany

Most real-time studies on receptor ligand–interactions require fluorescently labeled ligands. At moderate

concentrations (nM-µM), however, background from unbound fluorescent ligands is not negligible. Distinguishing free

from bound fluorescent ligands requires either an (inert) reference dye to counterstain the solution or ligands with

distinguishable properties between free and bound state, e.g. ligands only fluorescing in the bound state.

However, such ligands with state-dependent brightness are usually based on either quenching interaction between

ligand and fluorophore moiety or stabilizing interactions between fluorophore moiety and receptor. Unfortunately,

these interactions are additional coupled reactions in the system, potentially producing systematic errors if not

considered.

For fluorescent ligands based on cyanine fluorophores as Dy547 and Dy547P1, we show that the ligand binding

alters the photo-physics, presumably the cis-trans isomerisation of the fluorophore, leading to increased fluorescent

lifetimes and molecular brightness.

Quenching interactions between ligand- and fluorophore moiety were excluded by quenching analysis (Stern-Volmer

Plots) before synthesizing the fluorescent ligands. Fluorescence correlation measurements confirm the altered photo

physics for protein conjugated fluorophores. Lifetime measurements showed an increased fluorescence lifetime by

more than factor five upon binding. As example, binding of fluorescently labeled cGMP to CNG channels and binding

of fluorescently labeled ATP to P2X channels are shown. The altered fluorescence lifetime allows for direct detection

of labeled, bound ligands by FLIM microscopy or lifetime-gated detection in the presence of unbound free fluorescent

ligands or fluorophores.

We also show, with anisotropy imaging combined with patch-clamp fluorometry that the increased lifetime leads to a

reduced anisotropy of fluorescence from bound ligands, raising caution in interpreting anisotropy-based binding

assays using cyanine-fluorophores.


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A 03-07

Light-controlled artificial action potentials in Xenopus laevis oocytes

Christian vom Dahl1, Emanuel Müller1, Florian Walther1, Dominic Feind1, Eugena Sylaj1,

Annabelle Spinti1, Georg Nagel2, Christian Sattler1, Thomas Zimmer1

1 University Hospital Jena, Institute of Physiology II, Jena, Germany 2 Julius-Maximilians-University, Institute of Physiology–Neurophysiology, Biocentre, Würzburg, Germany

Voltage-gated sodium (Nav) channels respond to membrane depolarization by opening of the pore, leading to a

massive sodium influx and thereby mediating the fast upstroke of action potentials (AP) in excitable tissue. The small

initial membrane depolarization to a threshold potential, required for Nav channel activation, is normally mediated by

ionotropic receptors reacting to external stimuli like mechanical stretch, temperature changes, and various ligands.

In our experiments, we used an optogenetic approach to generate AP in non-excitable cells. We first coupled dimers

of Channelrhodopsin-2 (ChR2), a light-gated ion channel from the green alga Chlamydomonas reinhardtii, directly to

the C- or N-terminus of the human cardiac voltage-gated Na+ channel (Nav1.5). Expression of the fusion proteins in

Xenopus laevis oocytes and electrophysiological recordings by the two-microelectrode voltage clamp technique

resulted in robust Nav whole-cell currents and in typical light-induced ChR2 photocurrents. Using two of the C-terminal

fusion proteins, we were able to elicit AP by short 50 ms light pulses. In these two constructs, the electrophysiological

properties of the Na+ channel were not significantly different from wildtype Nav1.5. In a second set of experiments,

we linked ChR2 to the auxiliary β1 subunit of Nav channels. As shown by heterologous expression in Xenopus

oocytes, the β1-ChR2 fusion kept its ability to modify the kinetics of different Nav channels. Furthermore, the resulting

photocurrents were sufficient to elicit AP. As expected, co-expression of different voltage-gated potassium channels

significantly shortened AP duration. In contrast, incorporation of LQT3 mutations in Nav1.5 led to significant AP

prolongation. Ongoing research aims to transfer the new optogenetic tools to HEK293 and human stem cells.

Acknowledgment We thank K. Schoknecht, G. Ditze, G. Sammler, P. Hachenburg, C. Ranke, and S. Bernhardt for excellent technical

assistance.


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A 03-08

Establishing minimal viral proteins as model system to study

selectivity filter gating in potassium channels

Nils Drexler1, Ulf-Peter Hansen2, Indra Schroeder1

1 University Hospital Jena, Friedrich Schiller University Jena, Physiologie II, Jena, Germany 2 Christian-Albrechts-Universität zu Kiel, Department of Structural Biology, Kiel, Germany

Question

Potassium (K+) channels play a critical role in many physiological processes. Their adaptation to physiological needs

is achieved by the regulation of gating, i.e. switching between conductive and nonconductive conformational states.

One of the major gates in K+ channels is the selectivity filter (SF), for example in hERG1 and K2P2 channels . The

SF is strongly conserved within the K+ channel family3. SF gating is regulated by the ion occupancy in the filter K+

binding sites2 as well as a hydrogen (H) bond network anchoring the SF to the rest of the protein via the pore helix.

This has been shown i.e. for the C-type inactivation in Kv channels3.

We have recently quantified the role of the ion occupancy in a minimal viral model channel (Kcv), consisting of only

82 amino acids (AA) per monomer4. In this project, we focus on the H-bond network in the same model system. The

poster reports on the first phase of the project, an alanine scan to identify the critical players.

Methods

Single-point alanine mutations were introduced via mutagenesis PCR. The channel proteins were expressed in vitro

and reconstituted into planar lipid bilayers for recording of single-channel currents5. Gating beyond the temporal

resolution of the experimental setup was analyzed with extended beta distributions5.

Results and Conclusion

The mutated channels presented a wide variety of phenotypes:

• Non-functional channels

• Channels with lowered conductance

• Changes in open probability

• Strongly reduced protein stability

Two aromatic AA in the pore helix turned out to be critical for channel function, the alanine mutations were non-

functional. By conservative mutations at these positions, channel function could be partially rescued. These two

aromatic AA are also important for C-type inactivation in Kv channels3.

The mutation at the outer pore mouth resulting in quickly degrading channel proteins was previously identified as

being important for C-type inactivation in other K+ channels1,3.

Confirming our previous results4, the mutants displayed a robust correlation between single-channel conductance

(as a measure of K+ binding site occupation) and SF gating.

In summary, Kcv channels are an ideal model system to study the molecular mechanisms underlying SF gating in K+

channels.

Acknowledgment The work has been supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation),

research grant HA 712/14-3 to U.P.H. and Heisenberg Fellowship SCHR 1467/4-1 and FOR 2518 (DynIon, SCHR

1467/6-1) to I. S.

References


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[1] Köpfer, D. A. et al. A molecular switch driving inactivation in the cardiac K+ channel hERG. PLoS One7, e41023 (2012).

[2] Schewe, M. et al. A non-canonical voltage-sensing mechanism controls gating in K2P K+ channels. Cell164, 937–949 (2016).

[3] Cordero-Morales, J. F., Jogini, V., Chakrapani, S. & Perozo, E. A multipoint hydrogen-bond network underlying KcsA C-type inactivation. Biophys. J.100, 2387–2393 (2011).

[4] Rauh, O., Hansen, U.-P., Scheub, D. D., Thiel, G. & Schroeder, I. Site-specific ion occupation in the selectivity filter causes voltage-dependent gating in a viral K+ channel. Sci. Rep.8, 10406 (2018).

[5] Schroeder, I. How to resolve microsecond current fluctuations in single ion channels: The power of beta distributions. Channels9, 262–280 (2015).


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A 03-09

Competitive Inhibitors of Membrane Associated Cyclic Nucleotide

Binding Proteins for TIRFM Approaches

Stefan Kuschke, Ralf Schmauder, Klaus Benndorf

Jena University Hospital, Institute of Physiology II, Jena, Germany

Our aim is to observe and evaluate single binding events of cyclic nucleotides to various proteins in supported

membranes patches. In particular, we investigate whether there is specific binding cooperativity in hyperpolarization-

activated cyclic nucleotide-gated channels of the mouse (mHCN2 channels). Towards this, we employed a total

internal reflection microscopy (TIRFM) approach that utilizes fluorescence-labelled cAMP derivatives (fcAMP). This

allowed us to measure binding and unbinding of fcAMP to the mHCN2 channel as well as to other proteins in real

time.

Due to overexpression of ion channels in conventional electrophysiological measurements the endogenous

background is often ignored. However, at the single molecule or native expression level it is no longer negligible

since cAMP or respectively fcAMP will also bind to other binding sites such as membrane bound cyclic nucleotide

binding proteins. To address this problem we tried to quantitatively compete or block possible cAMP binding proteins

such as protein kinase A (PKA), phosphodiesterase (PDE) and exchange protein directly activated by cAMP (EPAC).

Therefore, a mixture of different inhibitors was characterized and applied with the highest reduction of binding to

endogenous proteins achieved by adding 100 µM Piclamilast, 100 µM ht31 Protein kinase A Anchoring inhibitor

peptide, and 65 µM 8-pCPT-2‘-O-Me-cAMP to the fcAMP solution. Upon application of inhibitors individually, we

quantified the portion of specific binding to membrane bound proteins and the overall background signal.

Using patch-clamp measurements, we could show that the inhibitors have no effect on the functionality of mHCN2

ion channels or their modulation by cAMP, making them a convenient tool to suppress unwanted cAMP binding sites.

By repeating the experiments with a differently labeled fcAMP derivative, we could show that the type of dye attached

to cAMP has a surprisingly high impact on the binding to cAMP binding proteins. The presented mixture of inhibitors

allowed us to follow binding and unbinding events to single molecules with a significantly reduced background. As

an additional result we obtain information on the density of endogenous membrane-located cAMP binding proteins.


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A 03-10

Cystic Fibrosis: A non-invasive method to measure CFTR-function and

mRNA-restoration of defect CFTR

Vanessa Mete1,2, Anna Katharina Kolonko1, Lea Schnüttgen1, Jörg Große-Onnebrink2, Heymut Omran2,

Michael Weber1

1 University of Muenster, Institute of Animal Physiology, Muenster, Germany 2 University Hospital Muenster, Department of General Paediatrics, Muenster, Germany

Cystic fibrosis (CF), caused by the malfunction of the chloride channel cystic fibrosis transmembrane conductance

regulator (CFTR), is the most common genetic disorder in the Caucasian population. The lack of CFTR function leads

to an imbalance in homeostasis of ion and water transport in secretory epithelia. In the lung, this prohibits mucociliary

clearance leading to the common clinical picture. In this study, a state-of-the-art Ussing chamber named Multi

Transepithelial Current Clamp (MTECC) was tested for its suitability to measure changes in conductivity via CFTR

in brushings of primary nasal epithelial cells. Cell material of 10 CF patients, 2 heterozygous individuals and 8 healthy

donors was cultured under air-liquid interface conditions. CFTR activity was evaluated after cAMP application.

Findings show that CF cells could be distinguished from healthy controls (ΔcAMP 0.83 ± 0.21 > -0.06 ± 0.32 mS/cm²;

p≤0.0001). Furthermore, these MTECC results were compared to sweat chloride test results, which is the primary

method to diagnose CF, and gene statues of the patients, showing that they are relatable to clinical findings. To

establish whether the method is also applicable to test treatment responses, CF cells were transfected with wtCFTR-

mRNA. Therefore, chitosan-based nanocapsules were used to introduce the nucleic acid to the cells and indeed

CFTR activity was improved for more than 48 h after transfection (p≤0.0001). MTECC may provide additional data

for future studies and allow theratyping when multiple treatment options are available.


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A 03-11

Targeting nicotinic acetylcholine receptors for precisely monitoring

activities at mouse neuromuscular junctions

Jing Yu-Strzelczyk, Shiqiang Gao, Shang Yang, Manfred Heckmann

Wuerzburg University, Department of Neurophysiology, Institute of Physiology, Wuerzburg, Germany

At the neuromuscular junction (NMJ), nicotinic acetylcholine receptors (nAChRs) are the primary receptors in muscle

for motor nerve-muscle communication. There the nAChRs respond to the neurotransmitter acetylcholine and control

muscle contractions. Mutations of the muscular nAChRs lead to diseases like congenital myasthenic syndromes

(CMSs) and multiple pterygium syndromes etc.

The muscle-type nAChR is a ligand-gated cation channel permeable to Ca2+, Na+ and K+, thus its activity can be

monitored by genetically encoded calcium indicators (GECI) like GCaMP. The postjuncational membrane of NMJ is

packaged with nAChRs at a concentration of as high as 10 000 receptors/µm2 in a clustered pattern. Post synaptic

responses are induced by presynaptic release with spatio-temporal dynamics. To look deeper into this with improved

resolution and kinetics, we fused GCaMP directly with nAChR to monitor this activity.

Two up to date versions of fast GCaMPs, jGCaMP7f or 8f were fused to the cytosolic loop region of the nAChR α

subunit. The nAChR α-jGCaMP (nAChR α-jGCaMP together with β, γ and δ subunits) transfected HEK cells showed

nice plasma membrane-targeted expression and light up upon acetylcholine application in fluorescence microscopy.

In HEK cells and Xenopus oocytes, electrophysiology studies employing outside-out excised patch clamp technique

combining with fast drug application method indicated that the engineered nAChR-jGCaMP behaved identical to the

wild type nAChR. The engineered nAChR-jGCaMP fusion will facilitate in vitro studies such as drug screening. As a

follow up, we will knock in the jGCaMP8f to the corresponding genomic location in the mouse and fuse jGCaMP8f

with endogenous nAChR for further in vivo investigations.


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Figure 1. Illustration of nAChR-GCaMP

jGCamp7fs or 8fs are linked to the cytosal of

nAChR alpa subunit . Upon AChR binding, Calcium

ions influx through nAChR and active the

jGCaMPs.

Figure 2. Electrophysiology and fluorescence

imaging of nAChR-jGCaMP (A) Diagram of outside-out patch clamp from

Xenopus oocyte. (B) Superimposed current traces

of outside-out patch from nAChR (black) and

nAChR-jGCaMP7f (red) expressing Xenopus

oocytes induced by 1ms 1mM ACh pulses. (C)

Diagram of Fluorescence macroscopy for jGCaMPs.

D, Images of nAChR-jGCaMP8f transfected HEK

cells before and after 10 µM ACh Application,

and after wasch off.

References [1] Dudel J, Heckmann M (2002) Quantal endplate currents from newborn to adult mice and the switch from

embryonic to adult channel type. Neurosci Lett 326:13-16,Elsevier. [2] LDana, H., Sun, Y., Mohar, B. et al. (2019) High-performance calcium sensors for imaging activity in

neuronal populations and microcompartments. Nat Methods 16, 649–657, Nature Publisher Group. [3] Lastname, GN, LastnaZhang, Yan; Rózsa, Márton; Bushey, Daniel; Zheng, Jihong; Reep, Daniel; Broussard,

Gerard Joey; et al. (2020): jGCaMP8 Fast Genetically Encoded Calcium Indicators. Janelia Research Campus. Online resource.

[4] Nagwaney S, Harlow ML, Jung JH, Szule JA, Ress D, Xu J, Marshall RM, McMahan UJ (2009) Macromolecular connections of active zone material to docked synaptic vesicles and presynaptic membrane at neuromuscular junctions of mouse. J Comp Neurol. 513, 457–68, Wiley.


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Foyers

A 04 | Voltage-gated Ion Channels Chair

Thomas Baukrowitz (Kiel)



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A 04-01

Nav1.7/N1245S and Nav1.7/E1139K: gating characteristics of voltage-

gated sodium channel Nav1.7 variants with unclear disease relevance

Kim Le Cann1, Jannis Meents1, Jannis Körner1,2, Vishal Sudha Bhagavath Eswaran1, Petra Hautvast1,

Ingo Kurth3, Angelika Lampert1

1 RWTH Aachen University Hospital, Institut für Physiologie, Aachen, Germany 2 RWTH Aachen University Hospital, Department of Anaesthesiology, Aachen, Germany 3 RWTH Aachen University Hospital, Institute of Human Genetic, Aachen, Germany

The voltage-gated sodium channel 1.7 (Nav1.7) is expressed widely in peripheral sensory neurons and is involved

in pain signaling. Loss-of-function mutations lead to insensitivity to pain while gain-of-function mutations are linked

to severe neuropathic pain such as IEM (inherited erythromelalgia). Recently, variants of SCN9a with unclear disease

relevance were identified in patients, such as p.N1245S described in a 50-year-old woman with IEM (Haehner et al.

2018), or p.E1139K identified in a 39-year-old woman suffering from severe pain.

Question: What is the biophysical impact of both Nav1.7/N1245S and Nav1.7/E1139K variants, located respectively

in domain III and between domains II and III?

Methods: Whole-cell voltage-clamp was performed in HEK cells heterologous expressing, the WT Nav1.7 or one

variant form co-transfected with GFP. 3D computer simulation was used to identify the molecular interaction partners

of Nav1.7/N1245S variant, based on the cryogenic electron microscopy structure of hNav1.7 (Huaizong Shen,

Dongliang Liu, Kun Wu 2019).

Results: Both variants revealed no change in voltage dependence of activation and steady-state fast inactivation.

The kinetics of fast inactivation and deactivation were also similar. p.N1245S showed an enhanced steady-state slow

inactivation. Homology modeling experiments revealed one additional potential interaction partner specific to the

p.N1245S variant.

Conclusion: These patch-clamp and 3D computer simulation experiments suggest that both p.N1245S and

p.E1139K variants may not be responsible for the symptoms of these patients. It is also possible that a heterologous

expression system is unable to reveal the potential variants’ biophysical changes. Other affected genes may be

necessary or that modulatory proteins needed for these variants to show their effect. Our findings stress that genetic

variants identified in patients need to be investigated in their context.

References [1] Haehner, A et al. 2018. “Mutation in Na v 1 . 7 Causes High Olfactory Sensitivity.” European Journal of

Pain 22: 1767–73. [2] Huaizong Shen, Dongliang Liu, Kun Wu, Jianlin Lei and Nieeng Yan. 2019. “Structures of Human Na Channel in

Complex with Auxiliary Subunits and Animal Toxins.” Science 363(March): 1303–8.


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A 04-02

High-throughput electrophysiology reveals temperature dependence of

sodium channel activation

Sophia Kriegeskorte1, Raya Bott1, Ralf Hausmann2, Angelika Lampert1

1 University Hospital, RWTH Aachen University, Institute of Physiology, Aachen, Germany 2 University Hospital, RWTH Aachen University, Institute of clinical Pharmacology, Aachen, Germany

Pain syndromes such as small fiber neuropathy (SFN) can be linked to mutations in voltage-gated sodium

channels (Navs). Several Nav mutations are described, and many were shown by manual patch-clamping to display

gain-of-function characteristics. Within the framework of the Sodium Channel Network Aachen (SCNAachen) we aim

to study Nav gating and its pathophysiological role in neuronal excitability. In the project presented here we

performed a comparative study of the activation and fast inactivation properties of Nav1.7, Nav1.3, Nav1.6 and

Nav1.5 stably expressed in HEK cell lines on the SyncroPatch 384i, a high-throughput electrophysiology robot. We

specifically focused on the temperature dependence and revealed a shift of Nav1.3 activation to more depolarized

potentials at increasing temperatures, while the other subtypes were less affected. Our recordings stress the

importance to consider temperature as a regulator for channel gating, as it may have a significant impact on cellular

excitability and its pathophysiology, which may differ from what is expected.

Acknowledgment Supported by a grant from the Interdisciplinary Centre for Clinical Research within the faculty of Medicine at the

RWTH Aachen University (IZKF TN1-1/IA 532001). AL and RH are the winners of

the 2020 SynchroPatch 384i award.


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A 04-03

Modulation of K+channel N(β)-type inactivation by reactive sulfur

species

Kefan Yang1, Ina Coburger1, Toshinori Hoshi2, Roland Schönherr1, Stefan H. Heinemann1

1 Friedrich Schiller University Jena and Jena University Hospital, Center of Molecular Biomedicine, Department of

Biophysics, Jena, Germany 2 University of Pennsylvania, Department of Physiology, Philadelphia, USA

A-type voltage-gated K+ (Kv) channels open on membrane depolarization and undergo subsequent rapid inactivation,

which is mediated by their N-terminal “ball” domains of select pore-forming α subunits (e.g., Kv1.4, Kv3.4) or of

cytoplasmic ancillary β subunits (e.g., Kvβ1.1), with importance in fine-tuning cellular excitability. Reactive sulfur

species (RSS) from the hydrogen sulfide donor NaHS and the polysulfide donor Na2S4 regulate the inactivation of

Kv1.4 and Kv3.4 channels via targeting cysteine residues in their ball domains [1]. Since members of the Kvβ family

often harbor cysteines in their ball domains, we investigated if and how NaHS/Na2S4 and the cysteine-specific

modifier DTNP (2,2′-dithiobis[5-nitropyridine]) affect N(β)-type inactivation.

Non-inactivating rat Kv1.1 and human Kv1.5 channels were coexpressed with different human Kvβ subunits in

HEK293T cells, and currents were measured before and after extracellular application of NaHS/Na2S4 or DTNP in

the whole-cell patch-clamp configuration at 19-21 °C.

N(β)-type inactivation conferred by Kvβ1.1 (C7), Kvβ1.2 (C8/C28) and Kvβ3.1 (C7/C22), containing one or two

cysteines, was sensitive to RSS and DTNP because of the named cysteines. While the inactivation of Kv1.1+Kvβ1.1-

C7S was insensitive to DTNP, RSS readily removed inactivation. Inactivation removal was partially recovered by the

reducing reagent dithiothreitol. Additionally, co-immunoprecipitation showed the interaction of Kv1.1 and Kvβ1.1-C7S

is unaffected by Na2S4. Both types of experiments suggest that RSS do not result in dissociating the β subunit from

the channel complex. In contrast, RSS failed to disrupt inactivation in Kv1.5+Kvβ1.1-C7S, pointing toward the

involvement of polysulfide targets in the pore-forming α subunit. In fact, a mutagenesis study revealed that C35/C36

in the Kv1.1 N terminus confer this particular RSS sensitivity: when mutated to alanine, the fast inactivation of Kv1.1-

C35A:C36A+Kvβ1.1-C7S was insensitive to RSS.In summary, RSS impair N(β)-type inactivation of Kv channels

when cysteines are available in the Kvβ N termini, thus broadening the physiological scenarios under which RSS

may modulate cellular electrical excitability. A double-cysteine motif in the N terminus of Kv1.1 appears to be an

additional RSS target and constitutes a unique example of an RSS-specific functional impact that cannot be mimicked

by DTNP-mediated cysteine modification.

References [1] Yang, K., Coburger, I., Langner, J.M. et al. Modulation of K+ channel N-type inactivation by sulfhydration

through hydrogen sulfide and polysulfides. Pflugers Arch - Eur J Physiol 471, 557–571 (2019)


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A 04-04

Biophysical characterisation and interplay of voltage-gated Ca2+

currents and A-type K+ currents in substantia nigra dopaminergic

neurons

Aisylu Gaifullina1, Gabriele Sarigu1, Nicole Wiederspohn1, Christina Poetschke1, Wolfgang Graier3,

Birgit Liss1,2

1 Ulm University, Institute of Applied Physiology, Ulm, Germany 2 University of Oxford, Linacre College & New College, Oxford, UK 3 Medical University of Graz, Gottfried Schatz Research Center, Molecular Biology & Biochemistry, Graz, Austria

Dopaminergic neurons within the Substantia nigra (SN DAN) display an autonomous pacemaker-activity, even in full

synaptic isolation, that is accompanied by oscillatory increases in free cytosolic Ca2+- and metabolic stress-levels.

The characterisation of voltage-gated Ca2+ channels (Cav) and of Ca2+ sensitive, voltage-gated A-type K+ (Kv4)

channels in SN DAN, and their functional interplay, is of particular interest as they both modulate the pacemaker-

activity of SN DAN, and both have been linked to impacting the vulnerability of these neurons to degeneration in

Parkinson’s disease (PD). However, a comprehensive dissection of the contribution of distinct Cav subtypes to bulk

Cav currents, and their impact on A-type K+ currents in health and PD-paradigms is missing. We are characterising

Cav and Kv4 currents, and activity-related Ca2+ dynamics in SN DAN in mouse brain slices, using whole-cell patch

clamp techniques in combination with Ca2+ imaging, specific pharmacology, as well as transgenic mice.

We found that all Cav subtypes contribute to somatodendritic bulk Cav currents in SN DAN, but their relative

contributions varied at different membrane potentials. More precisely, low voltage-activated Cav currents were mainly

blocked by the T-type Cav inhibitor Z941 (10 µM), with a maximum at around -70 to -60 mV. The L-type Cav blocker

isradipine (1 μM), as well as the R-type Cav blocker SNX-482 (100 nM), each inhibited ~35% of high voltage-activated

Ca2+ currents, with a maximum at about -40 mV (SNX-482 data were complemented by Cav2.3 KO mouse analysis).

And the P/Q/N-Cav selective blocker ω-conotoxin-MVIIC (1 µM) inhibited ~ 25% of bulk Cav currents, with a

maximum around -10 mV. Our Ca2+ imaging data indicate that the mean activity-related oscillatory Ca2+ signals in

SN DAN increase over time (by ~ 40%). This effect is likely caused by elevated metabolic stress, as it was not

observed in the presence of glutathione (10 mM). We also addressed the main Ca2+ source for stimulation of A-type

channels in SN DAN, that are built by Kv4.3/Kv4.2 α-, and KChip3/KChip4 β-subunits. A-type K+ currents were ~

50% reduced when intracellular Ca2+ was chelated by 10 mM BAPTA, while inhibition of the ER Ca2+ ATPase SERCA

increased A-type currents by ~50%.We are currently analysing how different Cav and other Ca2+ sources modify A-

type K+ currents as well as activity related Ca2+ oscillations in SN DAN, under control conditions and in PD-paradigms.


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A 04-05

Seizures, behavioral deficits and adverse drug responses in two new

genetic mouse models of HCN1 epileptic encephalopathy

Andrea Merseburg1,2,3, Jacquelin Kasemir1,2, Eric W. Buss5, Felix Leroy5, Hans Bock5, Alessandro

Porro6, Anastasia Barnett5, Simon E. Tröder4, Birgit Engeland1,2, Malte Stockebrand1,2, Anna Moroni6,

Steven A. Siegelbaum5, Dirk Isbrandt1,2,3, Bina Santoro5

1 German Center for Neurodegenerative Diseases (DZNE), Experimental Neurophysiology, Bonn, Germany 2 University of Cologne, Institute for Molecular and Behavioral Neuroscience, Cologne, Germany 3 University of Cologne, Center for Molecular Medicine Cologne, Cologne, Germany 4 University of Cologne, In vivo Research Facility, Faculty of Medicine and University Hospital Cologne, Cologne,

Germany 5 Columbia University, Department of Neuroscience, The Kavli Institute for Brain Science, Mortimer B. Zuckerman

Mind Brain Behavior Institute, New York, USA 6 University of Milan, Department of Biosciences, Milan, Italy

De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases

of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here,

we examine two knock-in mouse models replicating de novo mutations in the HCN1 (hyperpolarization-activated

cyclic nucleotide-gated non-selective cation channel 1) voltage-gated channel gene, p.G391D and p.M153I

(Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drug resistant neonatal- and childhood-onset epilepsy,

respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures.

Hcn1G380D/+ animals had an overall more severe phenotype, with pronounced alterations in the levels and distribution

of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical

reports from HCN1 patients, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin

resulted in the paradoxical induction of seizures in both lines, consistent with an effect to further impair inhibitory

neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists,

indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our

results underscore the need to tailor effective therapies for specific channel gene variants, and how strongly validated

animal models may provide an invaluable tool towards reaching this objective.


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A 04-06

α-Adrenoreceptor-Blocker Phentolamine Mesylate Inhibits Human

Sodium Channels Nav1.5 and Nav1.7

Idil Toklucu1, Julia Stingl3, Angelika Lampert1, Jannis Körner1,2

1 University Hospital, RWTH Aachen University, Institute of Physiology, Aachen, Germany 2 Medical Faculty, University Hospital, RWTH Aachen University, Department of Anesthesiology, Aachen, Germany 3 University Hospital, RWTH Aachen University, Institute of Clinical Pharmacology, Aachen, Germany

Purpose: Phentolamine mesylate (PM) is an α-adrenoreceptor antagonist, inducing vasodilatation at its injection

site, thus leading to faster systemic clearance of locally injected drugs. It is e.g. injected as a reversal agent after

treatment with soft-tissue local anesthesia and vasoconstrictors to avoid unwanted numbness. Previous studies

suggested that PM may also interact with voltage-gated sodium channels (VGSC), like local anesthetics do, and thus

may directly lead to additional side effects like cardiac arrhythmia. Here, we investigate whether PM affects the

VGSCs Nav1.7, which is primarily expressed in peripheral nociceptors and is one of the direct targets of local

anesthetics and Nav1.5, which is primarily expressed in cardiomyocytes.

Methods: Heterologously expressed hNav1.7 in HEK293 cells were investigated with whole cell patch-clamp

electrophysiology. hNav1.5 in HEK293 cells and hNav1.7 in CHO cells were investigated with the automated planar

patch-clamp platform Syncropatch 384i (Nanion Technologies). PM in concentrations of 16, 32, 64, 128, 256 and

512 μM, a vehicle control or 100 μM mexiletine HCl were applied during the recordings. The concentration–response

relationships were obtained. Steady-state fast inactivation and use-dependent block of Nav1.7 and Nav1.5 were also

characterized in the presence of these compounds.

Results: PM inhibited HEKNav1.7 with an IC50 of 72.10 μM, CHONav1.7 with an IC50 of 57.92 μM and HEKNav1.5

more potently with an IC50 of 18.08 μM. PM induced a hyperpolarization of the voltage-dependence of steady-state

fast inactivation both in HEKNav1.5 and in CHONav1.7. Moreover, it led to a use-dependent block of HEKNav1.5;

but not of CHONav1.7.

Conclusions: Our findings suggest that the effect of PM on VGSC can conflict with its current indication as an

antidote for local anesthetics. Mexiletine e.g., blocks VGSC in a low micromolar range, comparable to PM. Thus,

interactions by PM may reinforce the effect of local anesthetics as well as competing for the same binding site on the

channel protein. Effects of PM on different channel gating modalities and in combination with local anesthetics will

be investigated in future studies.

Acknowledgment Supported by a grant from the Interdisciplinary Centre for Clinical Research within the Faculty of Medicine at the

RWTH Aachen University (IZKF TN1-1/IA 532001), and the Syncropatch Award by Nanion.


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Phentolamine inhibits VGSC Nav1.5 more potently

than Nav1.7. Dose-Response curves of PM in HEKNav1.7

(obtained by manual patch-clamp), CHONav1.7 and

HEKNav1.5 (obtained with automated patch-clamp

setup SyncroPatch). The mean peak inward

currents after perfusion were measured and

normalized to the mean of peak currents before

the PM application for comparison.


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A 04-07

Modulation of KV10.1 channel gating kinetics by locally released Fe2+

Gemini Chandra1, Philipp Rühl1, Matthias Westerhausen2, Christoph Drees3, Jacob Piehler3, Stefan H.

Heinemann1

1 Friedrich Schiller University Jena and Jena University Hospital, Department of Biophysics, Center of Molecular

Biomedicine, Jena, Germany 2 Friedrich Schiller University Jena, Institute of Inorganic and Analytical Chemistry, Jena, Germany 3 Osnabrück University, Department of Biology/Chemistry and Center of Cellular Nanoanalytics, Osnabrück,

Germany

Iron metabolism plays crucial roles in cellular functions and its dysregulation causes disorders such as anaemia or

iron overload. The main limitation in studying the physiological impact of Fe2+ is its oxidation under ambient conditions.

Therefore, suitable methods for the controlled supply of Fe2+ to monitor its impact in biological systems are required.

Here we examined dicarbonyl-bis(cysteamine)iron (II) (CORM-S1) as a light-triggered source of Fe2+ [1] in

combination with frequency upconverting nanoparticles (UCNP) for the local modulation of voltage-gated ether à go-

go K+ channels (KV10.1).

KV10.1 channels were expressed in HEK293T cells and channel activation kinetics was measured in whole-cell

patch-clamp experiments. Extracellular solutions with free Fe2+ or Fe3+ ions were made from 0.1 N HCl stock solutions

of FeCl2 and FeCl3, respectively. While application of 100 µM Fe3+ did not alter activation kinetics, 100 µM Fe2+

slowed down activation kinetics at 40 mV from about 30 to 200 ms, similarly to about 1 mM Mg2+ [2]. CORM-S1 at

80 µM, applied to the extracellular solution, itself had no impact on channel kinetics, but illumination of the cell and

its surrounding with 470 nm LED light (1 s, 310 µW, 20x objective) readily released Fe2+ and slowed down KV10.1

activation equivalent to about 80 µM Fe2+. Diffusion of CORM-S1 and Fe2+ into and out of the focus volume

determined recovery time and maximal repetition interval.

Since illumination of the CORM-S1-containing bath solution decomposes all CORM-S1 in the light path of the wide-

field microscope, we also examined the option of local UV/VIS light generation by membrane-targeted UCNPs,

excited with 976-nm infrared (IR) laser light. Biofunctionalized, Tm3+-based UCNPs were coupled to the plasma

membrane by an extracellular nanobody fused to the transmembrane helix of the platelet-derived growth factor

receptor. In 80 µM CORM-S1, an IR-mediated slowing of KV10.1 channel activation equivalent to at least 5 µM Fe2+

in bulk solution was achieved. This impact of locally generated Fe2+ immediately vanished after stop of irradiation,

indicating the local confinement of Fe2+ release.

In summary, extracellular Fe2+ markedly slows down activation gating of KV10.1 channels. CORM-S1 is a suitable

compound for the quick release of Fe2+ when illuminated with 470-nm light. By combining CORM-S1 and UCNPs,

infrared light-triggered, spatially confined manipulation of biological systems with Fe2+ is achieved.

Acknowledgment Support by the German Academic Exchange Service (GC) and the German Research Foundation (SHH,

HE2993/16-1, 18-1).

References [1] Gessner, G., P. Rühl, M. Westerhausen, T. Hoshi, and S. H. Heinemann. 2020. 'Fe2+-mediated activation of BKCa

channels by rapid photolysis of CORM-S1 releasing CO and Fe2+', ACS Chem Biol, 15: 2098-2106. [2] Terlau, H., J. Ludwig, R. Steffan, O. Pongs, W. Stühmer, and S. H. Heinemann. 1996. 'Extracellular Mg2+

regulates activation of rat eag potassium channel', Pflügers Arch, 432: 301-312. Edition, Page, Place of publication: publishers


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A 04-08

Mechanism of Kv channel inhibition: binding of cellular lipids to side-

pockets induces allosteric selectivity filter inactivation

Marcus Schewe1, Aytug K. Kiper2, Stefanie Marzian2, Wojciech Kopec3, Mauricio Bedoya4, David

Ramirez4, Elena B. Riel1, Annemarie Köhler1, Susanne Rinne2, Bert L. de Groot3, Wendy Gonzalez4,5,

Thomas Baukrowitz1, Niels Decher2

1 Kiel University, Institute of Physiology, Kiel, Germany 2 University of Marburg, Institute of Physiology and Pathophysiology, Marburg, Germany 3 Max Planck Institute for Biophysical Chemistry, Computational Biomolecular Dynamics Group, Göttingen,

Germany 4 Universidad de Talca, Centro de Bioinformatica y Simulacion Molecular, Talca, Chile 5 Universidad de Talca, Millennium Nucleus of Ion Channels-Associated Diseases (MiNICAD), Talca, Chile

Most voltage-gated K+ channels (Kv channels) are strongly affected by polyunsaturated fatty acids (PUFAs) and

several effector mechanisms have been reported including channel activation via voltage sensor modulation, open

channel block via a central cavity site as well as induction of inactivation at the selectivity filter (SF inactivation) via

an allosteric mechanism. The latter inactivation process in Kv channels is essential for the regulation of cellular

excitability, however, the molecular mechanism underlying its modulation by PUFAs is still elusive.Noteworthy,

cooperative inhibition of Kv channels by the coumarin derivative Psora-4 requires drug binding to the central cavity

and a newly identified binding site in the side-pockets of the channel protein.Here we report that PUFAs and Psora‐4

share a similar co-operative and Rb+-sensitive inactivation-like mechanism which we identified by scanning

mutagenesis, pore blocker and cysteine modification analysis, permeant ion experiments, ligand docking and

computational electrophysiology approaches. Thus, we demonstrate that PUFAs do not act by a pure pore plugging

mechanism but that a substantial contribution to channel inhibition occurs via a SF inactivation mechanism initiated

by binding of lipids within the hydrophobic side-pockets. We further highlight the transduction pathway (interaction

cascade) up the S6 segment directly ending in the SF gate, that finally undergoes a collapse by a two-stage process.

An asymmetrical constriction is formed between the second glycines (G374) in the SF following consecutively flipping

of aspartate residues (D375) behind the SF. Strikingly, we show that Rb+ as permeant ion directly stabilize the SF

gate and subsequently antagonizes this mechanism resulting in slowed PUFA induced inactivation. These findings

demonstrate that lipid induced inhibition can arise by different mechanisms in one Kv channel but clearly underscore

the side-pockets as a universal hub for lipid/hormone/drug binding that regulate Kv channel activity by an allosteric

SF inactivation mechanism.

Acknowledgment We thank all members of our labs for technical assistance and helpful comments. This work was supported by a

grant of the Studienstiftung des deutschen Volkes to S.M., by the Deutsche Forschungsgemeinschaft (DFG) grant

DE1482‐4/1 to N.D. and Fondecyt Grant 1140624 to W.G. The DFG supported M.S., W.K., B.L. de G. and T.B. as

part of the Research Unit FOR2518, DynIon.


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A 04-09

The role of protein flexibility and an Eyring barrier in the selectivity

filter gating of a K+ channel

Oliver Rauh1, Jennifer Opper1, Maximilian Sturm1, Nils Drexler3, Deborah D. Scheub1, Ulf-Peter Hansen2,

Gerhard Thiel1, Indra Schroeder3,1

1 Technische Universität Darmstadt, Plant Membrane Biophysics, Darmstadt, Germany 2 Christian-Albrechts-Universität zu Kiel, Department of Structural Biology, Kiel, Germany 3 University Hospital Jena, Friedrich Schiller University Jena, Physiology II, Jena, Germany

Question: In potassium (K+) channels, the selectivity filter (SF) region discriminates between different ion species

and also functions as a physiological gate, for example in hERG1 and K2P2 channels. One main regulator of SF

gating is a hydrogen bond network anchoring the SF to the pore helix3. One critical player in this network is an amino

acid at the transition between the SF and the pore. In contrast to the SF itself, it is not conserved, and various

mutations are tolerated, but they change SF gating, for example C-type inactivation in KcsA and Kv channels.

We have recently confirmed, that the same position also controls SF gating in a minimal viral model channel (KcvNTS),

which consists of only 82 amino acids (AA) per monomer4. Here, we isolate the energetic contribution of single

mutations at this position in concatemeric constructs of KcvNTS.

Methods: The channel proteins were expressed in vitro and reconstituted into planar lipid bilayers for recording of

single-channel currents. The SF gating is faster than the nominal temporal resolution of the experimental setup was

therefore analyzed with extended beta distributions5.

Results and Conclusion: KcvNTS channel shows voltage-depending gating with sub-millisecond kinetics in the SF.

The voltage sensitivity arises from the voltage-dependent ion occupation of the individual K+ binding sites the SF,

especially the S0 binding site. The signal from this sensor is mediated to the gate via changes in protein flexibility.

The mutation S42T modifies the above-mentioned hydrogen bond network and consequently greatly stabilizes the

open state of the SF while leaving the stability of the closed state unaffected. The introduction of zero to four S42T

mutations leads to a steady parallel shift of the voltage dependency of the rate constant of channel closure. This

indicates that ion occupation in the SF and its effect on flexibility is not affected by the mutation. Instead, the Eyring

barrier mediating the effect of flexibility on the gate is increased by about 0.4 kcal/mole per mutated subunit.

In summary, the detailed kinetic analysis of concatemeric channels allows to separate the effects of mutations on

different elements of the gating mechanism, in this case: the occupancy of a modulator binding site and the energy

barriers dividing two conformational states of the protein.

Acknowledgment The work has been supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation),

research grant HA 712/14-3 to U.P.H. and Heisenberg Fellowship SCHR 1467/4-1 and FOR 2518 (DynIon, SCHR

1467/6-1) to I. S., and the European Research Council (NoMAGIC) under Grant Agreement No 695078 to G. T.

References [1] Köpfer, D. A. et al. A molecular switch driving inactivation in the cardiac K+ channel hERG. PLoS One7,

e41023 (2012). [2] Schewe, M. et al. A non-canonical voltage-sensing mechanism controls gating in K2P K+ channels. Cell164,

937–949 (2016). [3] Cordero-Morales, J. F., Jogini, V., Chakrapani, S. & Perozo, E. A multipoint hydrogen-bond network

underlying KcsA C-type inactivation. Biophys. J.100, 2387–2393 (2011). [4] Rauh, O., Hansen, U.-P., Scheub, D. D., Thiel, G. & Schroeder, I. Site-specific ion occupation in the

selectivity filter causes voltage-dependent gating in a viral K+ channel. Sci. Rep.8, 10406 (2018). [5] Schroeder, I. How to resolve microsecond current fluctuations in single ion channels: The power of beta

distributions. Channels9, 262–280 (2015).


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A 04-10

Rodents Have Two Functional Epithelial Sodium Channels Isoforms

Sean M. Gettings1,2, Stephan Maxeiner3, Maria Tzika1, Matthew Cobain1, Irina Ruf4, Fritz Benseler5, Nils

Brose5, Gabriela Krasteva-Christ3, Greetje Vande Velde2, Matthias Schönberger2, Mike Althaus6

1 Newcastle University, School of Natural and Environmental Sciences, Newcastle upon Tyne, UK 2 KU Leuven, Biomedical Imaging, Department of Imaging and Pathology, Faculty of Medicine, Leuven, Belgium 3 Saarland University School of Medicine, Institute for Anatomy and Cell Biology, Homburg, Germany 4 Senckenberg Research Institute and Natural History Museum Frankfurt, Division of Messel Research and

Mammalogy, Frankfurt am Main, Germany 5 Max-Planck-Institute of Experimental Medicine, Department of Molecular Neurobiology, Göttingen, Germany 6 Institute for Functional Gene Analytics, Department of Natural Sciences, Bonn-Rhein-Sieg University of Applied

Sciences, Rheinbach, Germany

Introduction: The epithelial sodium channel (ENaC) plays a key role in sodium homeostasis in tetrapod vertebrates.

Four ENaC subunits (α, β, γ and δ) form heterotrimeric αβγ- or δβγ-ENaCs. While the physiology of αβγ-ENaCs is

well understood, the function of δβγ-ENaC is unknown. The SCNN1D gene coding for δ-ENaC is a pseudogene in

mice/rats, limiting research into δ-ENaC physiology. We investigated whether δ-ENaC is generally absent across

rodents or limited to specific rodent suborders.

Methods: The presence of potentially functional SCNN1D genes was analysed in all currently available sequenced

rodent genomes. Transmembrane currents generated by human and guinea pig αβγ- and δβγ-ENaCs expressed in

Xenopus oocytes were measured with two-electrode voltage-clamp and cell-attached patch-clamp techniques.

Sodium self-inhibition (SSI) and activation by extracellular protease (chymotrypsin, 2 mg/ml), two mechanisms

controlling ENaC activity, were characterised as previously described (Wichmann et al. 2019).

Results: While SCNN1D was lost in five rodent lineages, including Muridae (mice/rats), SCNN1D is present in

species from 21 of the 35 rodent families. Fusion of exons 11 and 12 of the SCNN1D creates a ‘super-exon’ in

Hystricognathi, a suborder containing the Caviidae family (guinea pigs). The 'super-exon' causes intron DNA

sequences to be translated into a structurally flexible part of the δ-ENaC extracellular domain. Despite the presence

of the 'super-exon', whole-cell and single-cell electrophysiology revealed that guinea pig δβγ-ENaCs generate robust

amiloride-sensitive currents. Amiloride sensitive currents were significantly larger than those generated by guinea

pig αβγ-ENaCs and comparable to human ENaCs. Unlike the human isoforms, the single channel conductance of

guinea pig αβγ- and δβγ-ENaC were not significantly different. In both species, the magnitude of SSI was greater in

αβγ-ENaCs as compared to δβγ-ENaCs. Extracellular chymotrypsin provoked a strong activation in human and

guinea pig αβγ-ENaCs compared to a small activation of human δβγ-ENaCs and lack of effect on guinea pig δβγ-

ENaCs.

Conclusion: δ-ENaC is not generally absent from rodents but was independently lost in five lineages. Guinea pigs

have two functional αβγ- and δβγ-ENaC isoforms with similar biophysical and regulatory features to human

orthologues. Guinea pigs represent a commercially available rodent model for studying mammalian δ-ENaC

physiology.

Acknowledgment


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Stephan Maxeiner is a recipient of funding by intramural seed grants (HOMFOR 2017-2019) by the Medical School

of Saarland University. Maria Tzika was supported through an undergraduate summer studentship by The

Physiological Society. Gabriela Krasteva-Christ receives funding from the DFG (SFB-TR152/P22) and Greetje

Vande Velde is supported by a G0H9818N Odysseus grant from the Research Foundation Flanders (FWO).

References [1] Wichmann L et al. (2019), ‘An extracellular acidic cleft confers profound H+-sensitivity to epithelial

sodium channels containing the δ-subunit in Xenopus laevis’, J Biol Chem 294(33):12507-12520.


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Foyers

A 05 | Transporter Chair

Markus Bleich (Kiel)

Carsten Wagner (Zurich)


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A 05-01

Functional consequences of mutations in human EAAT2 associated to

epileptic encephalopathy

Peter Kovermann1, Yulia Kolobkova1,2, Christoph Fahlke1

1 Forschungszentrum Jülich GmbH, Institute for Biological Information Processing, Molekular- und Zellphysiologie

(IBI-1), Jülich, Germany 2 Medical school Hamburg, Hamburg, Germany

Question: Mutations in SLC1A2 encoding the glutamate transporter EAAT2 are associated with severe forms of

epileptic encephalopathy in heterozygous patients [1-3]. EAAT2 is expressed in glial cells and in presynaptic nerve

terminals and functions as secondary-active glutamate transporter and as anion channel [4,5]. How the disease-

associated mutations affect EAAT2 function and how such alterations cause epilepsy is insufficiently understood.

Methods: We here compare the functional consequences of the three currently known mutations associated with

epileptic encephalopathy, predicting the amino acid exchanges p.Gly82Arg, p.Leu85Pro and p.Pro289Arg in human

EAAT2, by heterologous expression in mammalian cells, biochemistry, confocal imaging and whole-cell patch clamp

recordings of EAAT2 transport and anion currents.

Results: We observed reduced glutamate transport and enhanced chloride currents for all tested mutations. Two of

the mutations exchange amino acid residues that contribute to formation of the EAAT anion pore and enlarge the

pore diameter sufficiently to permit passage of glutamate. p.Gly82Arg and p.Leu85Pro EAAT2 thus function as

glutamate efflux pathways. The mutation p.Pro289Arg solely increases anion currents despite a lower membrane

expression and decreases glutamate uptake.

Conclusions: Glutamate efflux and reduced glial glutamate uptake will augment ambient glutamate and cause

glutamate excitotoxicity and neuronal hyperexcitability in affected patients.

Acknowledgment We would like to thank Dr. M. Hediger, Universität Bern, for providing the expression construct for WT hEAAT2 and

Arne Franzen and Petra Thelen for excellent technical assistance.


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Mutations in human EAAT2 associated with

epileptic encephalopathy (A) The positions of the epileptic

encephalopathy-associated mutations p.Gly82Arg

(NM_4171.3: c.244G>A), p.Leu85Pro (NM_4171.3:

c.254T>C), and p.Pro289Arg (NM_4171.3: c.866C>G)

in the membrane topology of hEAAT2

(trimerization/transport domains:

green/magenta). Multiple alignments of human

EAAT isoforms show a strict conservation of G82,

L85, and P289. (B) A homology modelling derived

structure of hEAAT2 onto the open channel

structure from the archeal aspartate transporter

Gltph indicates that the disease-associated

residues are in close proximity to the pore

region of hEAAT2.

References [1] Guella I, McKenzie MB, Evans DM, et al. De Novo Mutations in YWHAG Cause Early-Onset Epilepsy. American

journal of human genetics. Aug 03 2017;101(2):300-310. doi:10.1016/j.ajhg.2017.07.004 [2] Epi KC, Epilepsy Phenome/Genome P, Allen AS, et al. De Novo Mutations in SLC1A2 and CACNA1A Are Important

Causes of Epileptic Encephalopathies. American journal of human genetics. Aug 4 2016;99(2):287-98. doi:10.1016/j.ajhg.2016.06.003

[3] Epi KC, Epilepsy Phenome/Genome P, Allen AS, et al. De novo mutations in epileptic encephalopathies. Nature. Sep 12 2013;501(7466):217-21. doi:10.1038/nature12439

[4] Pines G, Danbolt NC, Bjoras M, et al. Cloning and expression of a rat brain L-glutamate transporter. Nature. Dec 3 1992;360(6403):464-7. doi:10.1038/360464a0

[5] Chaudhry FA, Lehre KP, van Lookeren Campagne M, Ottersen OP, Danbolt NC, Storm-Mathisen J. Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. Neuron. Sep 1995;15(3):711-20.


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A 05-03

Inhibition of the Na+/H+-exchanger NHE1 reduces fibrosis in pancreatic

cancer

Zoltan Pethö1, Karolina Najder1, Stephanie Beel2, Maria Wolters3, Klavs Grantins3, Eva Wardelmann3,

Andrea Oeckinghaus2, Albrecht Schwab1

1 University Münster, Institute of Physiology II, Münster, Germany 2 University Münster, Institute of Tumor Biology, Münster, Germany 3 University Münster, Gerhard-Domagk-Institute of Pathology, Münster, Germany

Pancreatic ductal adenocarcinoma (PDAC) is accompanied by marked tumor fibrosis, caused primarily by pancreatic

stellate cells (PSCs). Moreover, impaired ductal HCO3- secretion in transformed tumor ducts leads to a relative

alkalinization of the interstitial pH, which we hypothesize to activate PSCs and ultimately induce fibrosis. In this study,

we aim to test this theory in vivo in genetically induced murine pancreatic cancer, and thus, confirm key underlying

mechanisms in ex vivo PDAC-derived primary murine PSCs.

We conducted our experiments with adult tumor-bearing KPfC mice having pancreas-specific KRAS and p53

mutations. For treatment, we applied the Na+/H+-exchanger NHE1 inhibitor cariporide (6 mg/kg/d) or vehicle i.p. for

four weeks, with or without additional chemotherapy in the form of gemcitabine (100 mg/kg/d). After treatment, we

isolated the pancreata, and conducted histological analysis, immunohistochemistry and Western Blot experiments.

Collagen deposition was determined with sirius red staining. Moreover, we isolated primary PSCs for further in vitro

experiments such as pH-measurements, immunocytochemistry and cell migration assays.

Upon applying the Na+/H+-exchanger NHE1-inhibitor cariporide in vivo as a supportive therapy of the

chemotherapeutic agent gemcitabine, we found that tumor fibrosis decreases compared to vehicle therapy. In primary

PDAC-derived PSCs we found that PSCs from the double-treated group are less activated and migrate more slowly

than PSCs from the vehicle-treated group, but do not functionally down-regulate NHE1. In conclusion, we propose

cariporide as a feasible ancillary therapy for pancreatic cancer.

Acknowledgment IZKF Münster (Schw2/020/18), Marie Skłodowska-Curie Innovative Training Network (ITN) Grant Agreement

number: 813834 - pHioniC - H2020-MSCA-ITN-2018, DFG Chembion GRK2515


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A 05-04

Analysing the transport mode of SLC26A9

Britt Marie Huckschlag1, Piersilvio Longo2, Jan-Philipp Machtens2, Dominik Lenz1, Dominik Oliver1

1 Philipps University, Institute for Physiology and Pathophysiology, Marburg, Germany 2 Forschungszentrum Jülich, Institute of Biological Information Processing (IBI-1), Jülich, Germany

The SLC26 transporters are a family of multifunctional anion exchangers encoded by 10 genes. They are expressed

throughout the body and play an important role in electrolyte transport and homeostasis. The family member

SLC26A9 is for example involved in gastric acid regulation and chloride transport in airway epithelia. While most

SLC26 isoforms work as coupled transporters, SLC26A9 appears to transport in a channel-like, uncoupled mode.

The underlying mechanism, however, remains unclear, and evidence supporting either a bona-fide channel or rather

an uncoupled alternating access mechanism has been published.

Based on a recently published cryo-EM structure (Walter et al, eLife 2019), we performed MD simulations that

revealed distinct inward- and outward-facing states, arguing directly for an alternate access transport mediated by

an elevator-like movement of the so-called core domain. To probe for the predicted outward-open state

experimentally, we applied a cysteine accessibility approach. Individual cysteines were introduced by site-directed

mutagenesis and accessibility to MTS reagents applied from the extracellular side was assessed by effects on

transport activity. Transport currents were measured with whole-cell voltage-clamp in CHO cells transfected with the

various SLC26A9 cysteine mutants.

In agreement with the MD simulations, positions in transmembrane domains TM1 and TM3 predicted to be accessible

only in the hypothetical outward-facing state were sensitive to external MTS reagents, as shown by decreased

transport currents. These results are in agreement with an uncoupled alternating access transport mechanism by

SLC26A9, rather than with a static channel pore. Equivalent positions in TM8 will be examined in the future.

Acknowledgment Supported by DFG Research Group FOR 5046 (OL 240/8-1 to D.O. and MA 7525/2-1 to J.-P. M.). The authors




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A 05-05

Cryptosporidium parvum infection modulates intestinal glucose uptake

in calves

Franziska Dengler1, Cora Delling2, Harald Hammon3, Wendy Liermann3, Christiane Helm4, Reiner

Ulrich4, Lisa Bachmann3

1 University of Veterinary Medicine Vienna, Department of Biomedical Sciences, Wien, Austria 2 Leipzig University, Institute of Parasitology, Leipzig, Germany 3 Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany 4 Leipzig University, Institute for Veterinary Pathology, Leipzig, Germany

Question: Beyond posing an immense economic problem in calf rearing, infection with C. parvum is a life-threatening

zoonosis causing massive diarrhea especially in children and immune-deficient people. So far, there is no fully

effective therapy available, which is at least partly due to lacking understanding of the pathophysiology. Previous

studies in cell culture models indicated an interference of the infection with intestinal glucose uptake and metabolism.

Therefore, we aimed to investigate the effects of infection with C. parvum in neonatal calves on the transepithelial

transport and systemic glucose metabolism in vivo.

Methods: Neonatal calves were infected with 2x107C. parvum oocysts (N=5) while the control group (N=4) was

administered H2O orally. The feeding regime was identical in all animals. Clinical parameters and fecal shedding of

C. parvum oocysts were monitored for one week. Blood samples were taken before infection, on day 4 post infection

(p.i.) and 7 p.i. On day 7 p.i. the calves were sacrificed, and the isolated distal jejunum epithelium was mounted in

Ussing chambers. Electrophysiological parameters were measured and the electrogenic glucose uptake via Na+-

linked transport (SGLT1) was evaluated.

Results: Experimentally infected calves started shedding C. parvum oocysts on day 3±1 p.i. Plasma glucose levels

of the infected calves were lower compared to the control group on days 4 p.i. (p=0.078) and on day 7 p.i. (p=0.002;

t-test), indicating metabolic distress in the infected animals. In line with this, plasma urea concentrations tended to

be higher in infected calves on day 7 p.i. (p = 0.053; t-test).

Ussing chamber experiments revealed a significant increase of phlorizin-sensitive electrogenic transport of glucose

across the infected epithelia compared to the control group after 2 hours of incubation (p<0.05; paired t-test). Tissue

conductance was similar in all groups.

Conclusions: Our results indicate an upregulation of SGLT1 in C. parvum infected jejunum epithelium but

simultaneously lower systemic blood glucose levels and enhanced amino acid degradation in the infected animals.

This might indicate an increased metabolic demand due to the infection and/or a nutritional competition of the

parasite, diminishing the glucose supply of the host. In contrast to the prevailing view that glucose uptake is disturbed

in C. parvum infection, the enterocytes seem to attempt to compensate and increase their transport capacities for

glucose uptake.


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A 05-06

Cysteine cross-bridging reveals a translational movement in prestin

(SLC26A5)

Dominik Lenz1, Piersilvio Longo2, Jan-Philipp Machtens2, Dominik Oliver1

1 Philipps University Marburg, Institute for Physiology and Pathophysiology, Marburg, Germany 2 Forschungszentrum Jülich, Institute of Biological Information Processing (IBI-1), Jülich, Germany

Sensitive hearing relies on an active mechanical process termed the cochlear amplifier, mediated by cochlear outer

hair cells. Amplification results from ultrafast cellular length changes driven by membrane potential changes, a

process known as electromotility. At the molecular level, electromotility is mediated by the membrane protein prestin

(SLC26A5), a member of the SLC26 family of anion transporters. The molecular mechanism, i.e. the conformational

dynamics that generate the mechanical output, has remained unknown. Recently, experimental structures of

mammalian and bacterial homologs revealed dimeric organization of this protein family, with each monomer

organized into two major helix bundles: the core domain containing the substrate binding site and the gate domain

positioned at the dimer interface.

Here, we report on a combined structural and functional approach to elucidate the structural dynamics that generate

electromotility. Using extensive molecular dynamics simulations based on the recent cryo-EM structure of inward-

facing Slc26a9 combined with homology modeling of prestin, we developed and refined an ensemble of structures

that represent various putative functional states of prestin. We mutated pairs of cysteines into positions in the

core/gate domain interface predicted to be in close proximity in the inward-facing state. By inducing either Cd-linkage

or disulfide formation we were able to reduce electromotility, indicating an enhanced rigidity and validating the MD

simulated model.

These findings support the notion of an elevator-like movement of the core domain perpendicular to the membrane

plane resulting in a change of prestin’s circumference, thereby modulating overall membrane area and cell length.

Studies on positions in close proximity only in the modeled outward-facing state will be conducted in the future.

Acknowledgment Supported by DFG Research Group FOR5046 (OL240/8-1 to D.O. and MA 7525/2-1 to J.-P. M.). The authors




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A 05-07

Markov state modelling of VGLUT anion channels

Bart Borghans, Daniel Kortzak, Christoph Fahlke

Forschungszentrum Jülich, Institute of Biological Information Processing (IBI-1), Molecular and Cellular Physiology,

Jülich, Germany

Vesicular glutamate transporters (VGLUTs) accumulate glutamate in synaptic vesicles. They also function as anion

channels, and neither the molecular basis nor the cellular roles of this dual function is sufficiently understood.

VGLUTs can be targeted to the plasma membrane of HEK293T cells by mutating N- and C-terminal retention signals

(VGLUTPM) and studied with whole cell patch clamp [1]. VGLUT1PM anion channels are closed at basic external pH

and open upon hyperpolarising voltage steps at acidic pH. To kinetically describe this behaviour, we studied VGLUT1

current responses to fast changes in pH and chloride concentration (≤1 ms solution exchange) and voltage. Fig.1

depicts representative current responses to pH jumps from 8.5 to 5 at different holding potentials, illustrating slow

activation upon external acidification and fast deactivation upon returning to alkaline external pH. Activation and

deactivation time constants are only minimally voltage dependent. Noise analysis [3] revealed very short channel

open times (around 100 µs) that increased upon membrane hyperpolarization, but were not pH-dependent. External

[Cl-] shifted the voltage dependence of activation of VGLUTPM anion channels to more positive potentials. To describe

this behaviour we designed a Markov state model [2] with two 2 apo-protonation-open pathways to account for

biexponential activation in absence of cytoplasmic chloride, as well as one chloride-activated pathway (Fig. 2). Only

opening steps were assumed to change with voltage, whereas transitions between closed states were voltage-

independent. Initial values for all state interactions were mutated using a script using the DEAP evolutionary algorithm

to optimise sum-of-squares between experimental results and the model’s output of cumulative open states. This

model can accurately simulate VGLUT1PM anion currents with regard to the pH dependence of relative steady state

currents, its chloride concentration and its voltage dependence, as well as time courses upon pH and [Cl-] changes.

We are planning to use such models to also describe VGLUT glutamate currents to quantitatively describe the

different transport modes of vesicular glutamate transporters.

Figure 1: Representative current responses of HEK293T

cells expressing VGLUT1PM to pH switches from 8.5

to 5 and back (blue) and predictions of the

kinetic model (black).


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Figure 2: Kinetic scheme to describe VGLUT1 anion

currents. Only interactions between closed and

open states are voltage dependent.

References [1] Eriksen, J, et al. 2016, ‘Protons regulate vesicular glutamate transporters through an allosteric

mechanism’, Neuron, 90, 768-780. [2] Zifarelli, G, et al. 2021, ‘The joy of Markov models—channel gating and transport cycling made easy’, The

Biophysicist, 2, 70–107. [3] Alekov, A, et al. 2009, ‘Channel-like slippage modes in the human anion/proton exchanger ClC-4’, J Gen

Physiol, 133, 485-96.


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A 05-08

Glutamate transporters contribution to retinal ganglion cell

susceptibility to neurodegeneration

Isabella Boccuni1, Claus Bruehl1, Carlos Bas Orth2, Richard Fairless3, Andreas Draguhn1

1 Heidelberg University, Institute for Physiology and Pathophysiology, Heidelberg, Germany 2 Heidelberg University, Institute of Anatomy and Cell Biology, Heidelberg, Germany 3 University Clinics Heidelberg, Department of Neurology, Heidelberg, Germany

Glutamate transporters (GluTs) ensure a fast, spatiotemporally defined synaptic transmission by glutamate

clearance. At the same time, they allow for a low, but relevant ambient glutamate concentration in the extrasynaptic

space. Glutamate can exert protective or toxic effects on neurons, depending on its concentration and on the

localization of NMDA-type glutamate receptors (NMDARs)1. Extrasynaptic NMDARs are prone to mediate neurotoxic

effect, and their tonic activation by ambient glutamate has been shown to be a major mechanism of

neurodegeneration in various brain regions.

We analysed the regulation of ambient glutamate levels around rat retinal ganglion cells (RGCs) which express a

consistent pool of extrasynaptic NMDARs. This may be relevant in early stages of multiple sclerosis where RGCs

show signs of glutamate excitotoxicity prior to the typical axonal demyelination2. It is therefore important to unravel

the regulation of glutamate in the retinal network and the reaction of RGCs to stimulation of synaptic and extrasynaptic

glutamate receptors.

We used whole-mount preparations of the rat retina to study the function of αRGCs, a major class of RGCs, which

have been shown to display subtype-specific vulnerabilities to degeneration3. Hence, we first performed whole-cell

patch clamp recordings to classify the αRGCs according to their ON/OFF light responses.

Ganglion cells showed strong spontaneous postsynaptic activity which, upon block of GABA- and glycine-receptors

revealed multiple phasic excitatory postsynaptic currents. We asked whether there is an additional, glutamate-

mediated tonic current component in healthy ganglion cells. Perfusion of the retina with TBOA, an EAAT-1 and -2

transporters blocker, induced a tonic inward current which was blocked by application of the NMDAR antagonist MK-

801. By enhancing NMDARs activity, RGCs showed an inward shift of the tonic current suggesting the physiological

presence of ambient glutamate in absence of glutamate stimulation.

These data indicate that efficient glutamate uptake provides a “modulatory cap” around glutamatergic synapses at

RGCs, avoiding strong activation of extrasynaptic NMDAR. Subsequent experiments on rats exposed to retina

glutamate excitotoxicity and at diverse stages of experimental autoimmune optic neuritis (AON, a model of multiple

sclerosis) will reveal whether this protective mechanism is disturbed, and whether it can be reconstituted by virus-

induced over-expression of GluTs.

Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft (FOR 2289 “CALCIUM HOMEOSTASIS IN

NEUROINFLAMMATION AND DEGENERATION”, project 3).

References 1 Hardingham, G. E. & Bading, H., 2010, Nature Reviews Neuroscience vol. 11 682–696, Synaptic versus

extrasynaptic NMDA receptor signalling: Implications for neurodegenerative disorders. 2 Fairless, R. et al., 2012, J. Neurosci.32, 5585–5597, Preclinical retinal neurodegeneration in a model of

multiple sclerosis. 3 Mayer, C. et al., 2018, Neuroscience393, 258–272, Selective Vulnerability of αOFF Retinal Ganglion Cells

during Onset of Autoimmune Optic Neuritis.


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A 05-09

Pancreatic Stellate Cells Functionally Express the Na+/Ca2+-Exchanger

Thorsten Loeck, Micol Rugi, Karolina Najder-Nalepa, Zoltan Pethö, Albrecht Schwab

University of Münster, Insitute of Physiology 2, Münster, Germany

Question: The pancreatic ductal adenocarcinoma (PDAC) is a malignant disease with high mortality. 90 % of the

tumor tissue consists of fibrotic stroma which is produced by pancreatic stellate cells (PSCs). PSCs are responsible

for the extracellular matrix turnover. These cells get highly activated in PDAC. The properties of the microenvironment

in PDAC are characterized among others by increased stiffness and pressure as well as by hypoxia and acidity.

Intracellular Ca2+ ([Ca2+]i) affects many cellular processes including PSC activation in cancer. Ca2+ regulation

involves the activity of the Na+/Ca2+-exchanger (NCX) and other proteins of the "Ca2+ signalling toolkit". The NCX

transports 1 Ca2+ against 3 Na+ over the cell membrane in a forward or reverse mode. The regulation of transport

mode is set by the transmembranous gradients of the Ca2+ and Na+ concentrations as well as by the membrane

potential. This work is aimed at showing the functional expression of the NCX in PSCs, and the transmembranous

gradients as well as the membrane potential of PSCs to predict the driving force of the NCX in PSCs.

Methods: The expression of the NCX was shown by (q)PCR and Western blot. Ionic imaging experiments were done

for functional analysis of the NCX. For this purpose, [Ca2+]i, [Na+]i and membrane potential were determined using

fluorescent dyes.

Results: The NCX was detected by (q)PCR on the gene level and by Western blot on the protein level in PSCs. Our

results showed that NCX1 is the main isoform. In activated PSCs the most prominent splice variant is NCX1.3. The

functionality of the NCX was shown by inducing a driving force switch and meanwhile detecting a change of [Ca2+]i

in PSCs.

To forecast the driving forces of the NCX, the resting values for [Ca2+]i (90.8±7.6 nM), [Na+]i (7.4±0.4 mM) and the

membrane potential (-41±2 mV) were measured. These values predict that the NCX operates in the forward mode

under resting conditions. Piezo1 is highly expressed on PSCs. It mediates at least in part the mechanosensitivity of

PSCs. Its activation with Yoda1 leads to an increase of the intracellular Na+ and Ca2+ concentrations and causes a

depolarization of the cell membrane potential. This elicits a switch of the NCX transport mode in PSCs.

Conclusion: This study analyzed for the first time the expression of the NCX in PSCs. Moreover, determining

transmembranous Na+, Ca2+ and potential gradients are important to understand the [Ca2+]i regulation in PDAC

through the NCX.

Acknowledgment This project was supported by the Deutsche Forschungsgemeinschaft (SCHW 407/22-1; GRK 2515/1, Chembion).


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A 05-10

Exploring the transport cycle of a bacterial homolog of vesicular

glutamate transporters

Nataliia Dmitrieva1, Claudia Alleva1,7, Mercedes Alfonso-Prieto2,3, Paolo Carloni2,4,5, Christoph Fahlke1

1 Forschungszentrum Jülich, Institute of Biological Information Processing (IBI-1), Molekular- und Zellphysiologie,

Jülich, Germany 2 Forschungszentrum Jülich, Institute for Advanced Simulation (IAS-5) and Institute of Neuroscience and Medicine

(INM-9), Computational Biomedicine, Jülich, Germany 3 Heinrich Heine University Düsseldorf, Cécile and Oskar Vogt Institute for Brain Research, University Hospital

Düsseldorf, Medical Faculty, Düsseldorf, Germany 4 Forschungszentrum Jülich, JARA-HPC, Jülich, Germany 5 RWTH Aachen University, Department of Physics, Aachen, Germany 6 Forschungszentrum Jülich, JARA Institute Molecular Neuroscience and Neuroimaging (INM-11), Jülich, Germany 7 Stockholm University, Department of Biochemistry and Biophysics and Science for Life Laboratory, Stockholm,

Sweden

The solute carrier 17 (SLC17) family encompasses transporters that harness electrochemical proton gradients to

accumulate glutamate, aspartate or nucleotides in intracellular compartments. Members of the family facilitate

transport with distinct stoichiometry, however, the underlying transport mechanisms remain insufficiently understood.

The recent determination of the structure of a bacterial homologous protein, the D-galactonate transporter (DgoT)

from E. coli in two different conformations, has opened the way to use computational approaches to study substrate

transport by SLC17 transporters [1]. DgoT shows high sequence and structural similarity with mammalian vesicular

glutamate transporters (VGLUTs) and sialin, but differs in substrate selectivity and proton coupling: it mediates

electrogenic transport, with at least 2 H+ co-transported with one molecule of D-galactonate. Four charged residues

located in membrane-spanning region (D46, E133, R47 and R126) have been suggested to be involved in proton

transfer and substrate binding.

We study DgoT transport mechanisms combining computational and experimental approaches. As typical major

facilitator superfamily (MFS) member, the protein uses an alternate access mechanism [2] to perform its function.

Major structural changes are coupled with local rearrangements of transmembrane (TM) helices TM1 and TM7 (Fig,

1A) that gate access to the substrate-binding site from outside, similarly to those in other MFS transporters [3]. Using

all-atom molecular dynamics simulations, we demonstrated that protonation of key residues D46 and E133 leads to

movement of the gating helices in DgoT, resulting in full opening and allowing substrate access only in the double

protonated state (Fig. 1A-B, 2). Substrate binding then induces the closure of the periplasmic gate (Fig. 1C, 2). These

computational studies are complemented by experiments. We demonstrated functional activity of DgoT

overexpressed in E. coli by monitoring changes in pH of cells suspension after addition of galactonate, but not of its

epimer gluconate. Mutation of either of four charged residues in transmembrane region abolishes transport.

Moreover, purified DgoT shows selective binding of galactonate measured by nano differential scanning fluorimetry

and stopped-flow techniques.

Future work will elucidate possible similarities and differences of the DgoT tansport mechanisms with mammalian

SLC17 members, such as VGLUT or sialin.


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Fig. 1

Fig. 2

References [1] Leano JB, Batarni S, Eriksen J, Juge N, Pak JE, Kimura-Someya T, Robles-Colmenares Y, Moriyama Y, Stroud

RM, Edwards RH 2019, 'Structures suggest a mechanism for energy coupling by a family of organic anion transporters.' PLOS Biology 17(5): e3000260. https://doi.org/10.1371/journal.pbio.3000260

[2] Quistgaard, E, Löw, C, Guettou, F, Nordlund P 2016, 'Understanding transport by the major facilitator superfamily (MFS): structures pave the way.' Nat Rev Mol Cell Biol17, 123–132. https://doi.org/10.1038/nrm.2015.25

[3] Qureshi AA, Suades A, Matsuoka R, Brock J, McComas SE, Nji E, Orellana L, Claesson M, Delemotte L, Drew D. 2020, 'The molecular basis for sugar import in malaria parasites.' Nature578, 321–325. https://doi.org/10.1038


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Foyers

A 06 | Lung Physiology and Hypoxia I Chair

Wolfgang Kübler (Berlin)

Eric Metzen (Essen)


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A 06-01

The impact of roxadustat on glucose metabolism and ischemic

tolerance of brain cells

Sarah Madai, Hugo Marti, Reiner Kunze

Heidelberg University, Institute of Physiology and Pathophysiology, Heidelberg, Germany

In many organs including the central nervous system (CNS) hypoxia-inducible factors (HIF) are important cellular

regulators for the adaptation to ischemia. Although, the HIF pathway is known to change the metabolic flux from

oxidative phosphorylation (OXPHOS) to anaerobic glycolysis in certain organs, the role of HIF for the glucose

metabolism of the CNS remains unclear. Thus, we aimed to investigate whether activation of the HIF pathway in

brain-resident cells by roxadustat, a second-generation prolyl-4-hydroxylase (PHD) inhibitor, leads to metabolic shift

from OXPHOS to glycolysis. Moreover, we analyze if potential HIF-mediated metabolic reprogramming affects the

ischemic tolerance of brain cells in vitro.

All experiments were performed with primary cortical neurons and astrocytes derived from newborn C57BL/6 mice.

Real-time reverse transcription-PCR and western blotting were applied to determine the expression of enzymes of

the glucose metabolism. A Seahorse Extracellular Flux Analyzer was used to assess the bioenergetic state of cells

by measuring surrogates for glycolytic activity (extracellular acidification rate) and mitochondrial respiration (oxygen

consumption rate). Lactate concentrations in the cell culture supernatants were quantified by an enzymatic assay.

LDH release assay and dead-cell protease activity assay were applied to determine the viability of cells exposed to

oxygen-glucose deprivation (OGD).

The expression of HIF-targeted glucose transporter and glycolytic enzymes was increased upon roxadustat treatment

in both neurons and astrocytes. Up-regulation of pyruvate dehydrogenase kinase 1 (PDK1) caused inhibition of the

pyruvate dehydrogenase (PDH) complex, which enhanced conversion of pyruvate to lactate. Accordingly,

extracellular lactate levels of roxadustat-treated cells were returned to baseline upon co-stimulation with

dichloroacetate, a PDK1 inhibitor. Metabolic flux analyses indicated an increased glycolytic activity, whereas

mitochondrial respiration was lowered in roxadustat-treated cells. Roxadustat pre-treatment improved the viability of

astrocytes, but not neurons, upon OGD stress.Conclusively, our current results imply a metabolic switch from

oxidative phosphorylation to glycolysis via PDK1-mediated inhibition of PDH in neurons and astrocytes upon

pharmacological activation of HIF. Future studies will clarify whether improved ischemic tolerance of roxadustat-

treated astrocytes is due to HIF-induced metabolic reprogramming.


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The role of the Hypoxia-inducible Factor 2α in focal cerebral ischemia

Vincent von Oepen, Joachim Fandrey, Tristan Leu

University of Duisburg-Essen, Institute of Physiology, Essen, Germany

Stroke is one of the major reasons for death and disability. Insufficient blood flow in stroke interferes with proper brain

oxygenation causing areas of hypoxia. Under hypoxic conditions hypoxia-inducible factors (HIFs) accumulate and

function as transcription factors. To determine the role of HIFs in stroke, particularly HIF-2α, regarding the regulation

of neurotrophic, growth or apoptotic factors and infarct size, we use an in vivo model with transient middle cerebral

artery occlusion (tMCAO). Within this model, we provoke an ischemic stroke in wild type and Hif-2α-/-mice with a

conditional knockout of Hif-2α in neuronal cells. In addition, we created a human in vitro model with SY5Y cells, a

neuroblastoma cell line, in order to be able to transfer the in vivo insights of the mouse to human cells.

On both, Hif-2α deficient and wild type mice, tMCAO was performed for two hours and protein and RNA samples

were generated of ischemic and control brains. To simulate stroke in SY5Y cell culture, oxygen-glucose-deprivation

(OGD) experiments were performed under almost anoxic conditions (0.2% oxygen) and without glucose. RNA and

protein samples were hourly taken for up to 4 hours to analyse different neurotrophic factors, well-known HIF target

genes, genes related to apoptosis and different growth factors. First results show a significant increased expression

of Nrg1 already after 1 h OGD. NRG1 (Neuregulin-1) is important for synaptic plasticity and well known to be

neuroprotective in stroke by reducing the infarct size and decreasing the post-ischemic inflammatory response of the

brain1. With the drug Roxadustat we can induce HIF accumulation and expression of the HIF target gene Nrg1 which

might help to integrate new built neurons in the existing network after ischemic damage.The role of HIF-2α in stroke

is still not well understood and controversially discussed. With our investigations, we try to better understand the

impact of HIF-2α on neuronal survival and regeneration during and after ischemia. This may help to provide new

therapeutically approaches for a better outcome of stroke.

References [1] Zhenfeng Xu, Gregory D. Ford, DaJoie R. Croslan, Ju Jiang, Alicia Gates, Robert Allen, and Byron D.

Ford, Neuroprotection by neuregulin-1 following focal stroke is associated with the attenuation of ischemia-induced pro-inflammatory and stress gene expression, Neurobiology of diseases, Volume 19, Issue 3, August 2005, Pages 461-470


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The role of lactate dehydrogenase in pathology of ischemic stroke

Hanna Weisbecker, Gesine Reichart, Xiati Guli, Timo Kirschstein, Falko Lange, Rüdiger Köhling

Rostock University Medical Center, Oscar Langendorff Institute of Physiology, Rostock, Germany

As stroke is the most common cause of dependency worldwide, the main purpose of our project is to improve

therapeutic approaches in the treatment of ischemic stroke. Our approach is based on an anti-oxidative strategy

induced by the inhibition of lactate dehydrogenase (LDH). The focus of our investigations primarily is on the

penumbra, the hypoperfused area around the infarct core, which displays potentially reversible impairment and is

therefore of great therapeutic interest. Reactive oxygen species (ROS) are presumed to play a major role in the

ischemic cascade of pathological changes in the penumbra. Our project aims to reduce ROS and thereby penumbra

size and neurological damage in the affected area.

Question: Can inhibition of LDH improve outcome after ischemic stroke?

The idea behind inhibiting LDH is to slow down or even stop astrocyte-neuron-lactate-shuttling which is the transport

of glucose-derived lactate from astrocytes into neurons. In neurons the lactate will be used for oxidative energy

production which is unfavorable under stroke- (oxygen deprivation) and also reperfusion- (increased ROS generation)

conditions.

Methods: We established an in vivo tMCAO (transient middle cerebral artery occlusion) mouse model and an ex

vivo model of oxygen-glucose deprivation (OGD) in acute brain slices. In these models, the effects of LDH inhibition

using histological, protein biochemical and electrophysiological analyses were investigated.

Results: The treatment of in vivo tMCAO ischemic stroke with LDH-blocker stiripentol showed a reduction in total

infarct size which was attributed to the penumbra part. Basal electrophysiological characterization of LDH-blocker

effects showed a reduction in synaptic transmission compared to control slices. Changing energy source from

glucose to ketone bodies disposed this difference. In the ex vivo strokemodel of OGD, network excitability was

significantly reduced emulating the functional impairment of the in vivo stroke. However, neither LDH inhibition with

oxamate nor the introduction of ketone bodies reversed this deficit.

Conclusions:LDH inhibition in vivo may be beneficial in terms of reducing stroke size, but inhibition of the regional

astrocyte-neuron-lactate shuttle per se is not sufficient to explain the in vivo effect.


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A 06-04

S-nitrosoalbumin linked perfluorocarbon based artificial oxygen

carriers in ischemia reperfusion injury

Tobias Glück1, Michael Kirsch2, Katja B. Ferenz1,3

1 University of Duisburg-Essen, Institute of Physiology, 45147, Germany 2 University of Duisburg-Essen, Institute of Physiological Chemistry, 45147, Germany 3 University of Duisburg-Essen, CeNIDE (Center for Nanointegration, Duisburg-Essen), 47057, Germany

O2 supply byalbumin-derived artificial oxygen carriers with a perfluorodecalin (PFD) core (A-AOC)[1],[2] combined with

transcriptional reprogramming via hypoxia-inducible factors (HIF) may improve outcome of ischemia reperfusion

injury in transplantation medicine. As nitric oxide (NO) inhibits HIF-degrading enzymes (prolyl-hydroxylases, PHD)

under normoxia[3] this study aimed to combine O2 & NO supply by synthesizing NO-releasing A-AOCs (NO-A-AOCs).

NO-A-AOCs were synthesized by high pressure homogenization of PFD+S-nitrosoalbumin (S-NO-BSA). To obtain

S-NO-BSA, reduced albumin was incubated with S-nitrosoglutathione (GSNO). NO-release from either S-NO-BSA

or NO-A-AOCs (initiated by glutathione (GSH) or copper) was measured with an amperometric NO-electrode

connected to an OROBOROS-Oxygraph-2k; alkylated S-NO-BSA served as negative control. Human kidney cells

(HK-2) were chosen as an in vitro model to test both biocompatibility (CellTiter-Glo® luminescent cell viability assay)

and effects of NO-A-AOCs on HIF activity (Western blot). GSNO was used as S-nitrosated model compound to inhibit

PHDs under normoxia.

In HK-2 cells the presence of GSNO/GSH stabilized HIF-1a but not HIF-2a up to 6h. Successful nitrosation of S-NO-

BSA was confirmed by NO-release in the low µM range in presence of either GSH or copper but they failed by using

alkylated S-NO-BSA (<100nM). However, NO-release was decreased after processing S-NO-BSA to NO-A-AOCs.

In conclusion, GSNO released enough NO to stabilize HIF-1a in HK-2 cells. While pure S-NO-BSA released high

amounts of NO, S-NO bonds appeared to be fragile to high pressure homogenization resulting in decreased but still

sufficient NO release from NO-A-AOCs.

References [1] Wrobeln A, Laudien J, Groß-Heitfeld C, Linders J, Mayer C, Wilde B, Knoll T, Naglav D, Kirsch M, Ferenz KB.

Albumin-derived perfluorocarbon-based artificial oxygen carriers: A physico-chemical characterization and first in vivo evaluation of biocompatibility, Eur J Pharm Biopharm, 2017 Jun, 115:52-64

[2] Wrobeln A, Jägers J, Quinting T, et al. Albumin-derived perfluorocarbon-based artificial oxygen carriers can avoid hypoxic tissue damage in massive hemodilution, Sci Rep, 2020,10(1),11950

[3] Metzen E, Zhou J, Jelkmann W, Fandrey J, Brüne B, Nitric oxide impairs normoxic degradation of HIF-1alpha by inhibition of prolyl hydroxylases, Mol Biol Cell, 2003 Aug, 14(8), 3470-81


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A 06-05

Cardiac dysfunction after 72 h of normobaric hypoxia in rats

Annekathrin Hoschke1, Elias Neubert1, Beate Raßler1, Aida Salameh2, this project is a collaboration of

the University of Leipzig, Carl Ludwig Institute for Physiology and the Heart Center, Department of

Pediatric Cardiology

1 University of Leipzig, Carl-Ludwig-Institut of Physiologie, Leipzig, Germany 2 Heart Centre, Department of Pediatric Cardiology, Leipzig, Germany

Background: Previous results showed that normobaric hypoxia (10% O2 in N2) depressed left ventricular (LV)

inotropic function. Acute hypoxia (6 h) decreased LV systolic pressure (LVSP) to 68%, LV contractility even to 55%

of normoxic values. In the lung, first signs of pulmonary edema (PE) occurred.

Question: In the present study, we investigated hemodynamic responses and markers of myocardial injury after

prolonged (72 h) hypoxia.

Methods: Rats (n = 32) were exposed over 72h either to normal room air (N) or to normobaric hypoxia in a chamber

with 10% O2 in nitrogen (H). The animals received infusion with 0.9% NaCl (0.1 ml/h). At the end of the experiment,

heart catheterization was performed for examination of hemodynamic function. Finally, lung and heart tissue were

obtained for histological and immunohistochemical analyses.

Results: After 72h of hypoxia, left ventricular (LV) function had recovered compared to acute hypoxia but was still

significantly below normoxic values. LVSP, LV contractility and cardiac index (CI) were 80-86% of normoxic values.

In contrast, right ventricular (RV) systolic pressure and contractility were even mildly elevated (111 and 107% of

normoxic values, respectively) but this difference was not significant. Hypoxia induced a more than three-fold

increase of apoptosis-inducing factor in myocardial cells. Nitrotyrosin, a marker of oxidative stress, was in the hearts

of hypoxic rats about 150% of normoxic values. Hypoxic rats presented a significantly elevated hematocrit (48.8 %

compared to 43.5 % in normoxic animals). In addition, we observed that hypoxic rats had poor food intake (only 43%

of normoxic animals), and this was accompanied by a significant loss in body weight (12% compared to baseline).

Conclusions: These results indicate that after 72 h of hypoxia, hypoxic LV depression recovers partially but not

completely. LV function might be compromised by increased hematocrit. We assume that energy metabolism is

impaired under hypoxic conditions. Moreover, increased oxidative stress and apoptosis of myocardial cells might

contribute importantly to LV dysfunction. Further analyses are necessary to elucidate the mechanisms of hypoxia-

induced cardiac dysfunction.

(Authors AH and EN contributed equally to this work.)


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A 06-06

Delayed hypoxic response in immune cells of patients with hereditary

hemorrhagic telangiectasia

Jonah Bosserhoff1, Joachim Fandrey1, Freya Dröge2, Anna Wrobeln1

1 University of Duisburg Essen, Institute of Physiology, Essen, Germany 2 University Hospital Essen, Department of Otorhinolaryngology, Head and Neck Surgery, Essen, Germany

Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber disease is a genetic disorder,

which manifests in epitaxis and mucocutaneous telangiectasisa. The underlying pathology is an endothelial dysplasia

due to mutations in gene sequences that belong to the TGF-β- (transforming growth factor beta) and BMP-pathway

(bone morphogenetic gene). TGF-β is a major player in the immune response, consequently, besides the obvious

arteriovenous malformations, HHT-patients suffer from an impaired immune response which has already been shown

in several publications1. It was shown that the ability of mononuclear cells to migrate is decreased in HHT. Hypoxia

inducible factors (HIF) are known to interact with TGF-β2. Although, both HIF and TGF-β- play a significant role in

inflammatory processes3, there is no data on the HIF expression in HHT-patients.

We investigated for the first time how the expression of several genes in PBMCs (peripheral blood mononuclear

cells) isolated from blood samples of HHT-patients and healthy controls behave under hypoxic (1% O2) and normoxic

(20% O2) conditions. For protein and RNA analysis, the PBMCs were incubated for 4 and 24 hours and the samples

were quantified via Western Blotting or RT-PCR. After 4 hours of hypoxic incubation, HHT-PBMCs failed to stabilize

HIF-1α protein, whereas healthy controls showed the expected hypoxic increase compared to the normoxic samples.

After 24 hours of hypoxic incubation HIF-1α protein was increased in HHT-PBMCs, while in healthy control PBMCs

HIF1-α protein decreased already. HIF-1α, HIF-2α and the related target genes, as well as TGF-β and the genes

corresponding to the pathway were quantified. PBMCs from HHT-patients showed a massive up regulation of HIF-

1α-mRNA after 24 hours under hypoxic conditions compared to the PBMCs from healthy controls.

The results underline that HIF plays a relevant role in the immune response of HHT-patients. In future work we will

focus on the mechanisms of delayed HIF-1α up regulation under hypoxic conditions in HHT-patients.

If the delayed response of HIFs influences the symptoms of the patients, treatment with HIF-stabilizing drugs could

be a therapeutic option restoring the physiological function of the cells.

References [1] Cirulli A, Loria MP, Dambra P, Di Serio F, Ventura MT, Amati L, Jirillo E, Sabbà C. Patients with

Hereditary Hemorrhagic Telangectasia (HHT) exhibit a deficit of polymorphonuclear cell and monocyte oxidative burst and phagocytosis: a possible correlation with altered adaptive immune responsiveness in HHT. Curr Pharm Des. 2006;12(10):1209-15

[2] Lei R, Li J, Liu F, Li W, Zhang S, Wang Y, Chu X, Xu J. HIF-1α promotes the keloid development through the activation of TGF-β/Smad and TLR4/MyD88/NF-κB pathways. Cell Cycle. 2019 Dec;18(23):3239-3250

[3] Palazon A, Goldrath AW, Nizet V, Johnson RS. HIF transcription factors, inflammation, and immunity. Immunity. 2014 Oct 16;41(4):518-28


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A 06-07

The Hippo pathway coactivator Yes-Associated Protein 1 modulates

p22phox expression under severe hypoxic conditions

Xinpei Chen1, Philipp Mutzel1, Kilian Hierdeis1, Johannes Trefz2, Gabryela Huning Kuhnen2, Martin

Schönfelder2, Henning Wackerhage2, Agnes Görlach1,3,4, Andreas Petry1

1 German Heart Center Munich, Experimental and Molecular Cardiology, Munich, Germany 2 Technical University of Munich, Department of Sport and Health Sciences, Munich, Germany 3 DZHK (German Centre for Cardiovascular Research), partner site Munich, Munich, Germany 4 Technical University of Munich, School of Medicine, Munich, Germany

Background: The Hippo pathway is an evolutionarily conserved regulator of organ size and tumorigenesis that

negatively regulates cell growth and survival. YAP (gene YAP1) is the main effector downstream of the Hippo key

components Mst1/2 and Lats1/2. Activation of Lats1/2 induces YAP phosphorylation and nuclear exit/ proteolytic

degradation, thereby negatively regulating YAP activity. YAP itself regulates the transcriptional activity of TEAD, but

also of other transcription factors like p73 or RUNX2 in the nucleus, thereby controlling growth and death in many

cell types. In the adult heart, YAP has been shown to contribute to the regenerative response to myocardial infarction,

a condition characterized by severe hypoxia/ischemia and increased load of reactive oxygen species (ROS). While

the multicomponent family of NADPH oxidases contributes to ROS generation in the compromised heart, it is not

known whether there is a link between YAP and NADPH oxidases under these conditions.

Aim: We investigated whether YAP is sensitive to severe hypoxia and interferes with NADPH oxidases in

cardiomyoblasts.

Results: Exposure of H9C2 cardiomyoblasts to 0.1% oxygen resulted in increased levels of YAP mRNA and protein

within 4 hours as well as in nuclear translocation and enhanced activity of YAP. Silencing of YAP increased the levels

of apoptotic markers and reduced cell cycle progression under severe hypoxia. Interestingly, YAP silencing also

decreased ROS generation under basal and hypoxia/reoxygenation conditions, while overexpression of a

constitutively active YAP mutant increased ROS generation and cell cycle progression. To test the involvement of

NADPH oxidases in this response, H9C2 cells were depleted of p22phox, the common subunit of most NADPH

oxidases. Depletion of p22phox prevented YAP-induced ROS generation and cell cycle progression. Moreover, YAP

increased p22phox mRNA and protein levels under normoxia and hypoxia. Reporter gene assays, EMSA (electro-

mobility-shift-assay) and ChIP analyses showed that TEAD and YAP bind to the p22phox promoter in H9C2 cells.

Conclusion: These data identify YAP as a transcriptional coactivator of p22phox under severe hypoxic conditions

in cardiomyoblasts showing a novel link between the Hippo pathway and NADPH oxidases in the heart.


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A 06-08

A Microfluidic System for Simultaneous Raman Spectroscopy, Patch-

Clamp Electrophysiology and Live-Cell Imaging to Study Key Cellular

Events of Single Living Cells in Response to Acute Hypoxia

Kerstin Ramser2, Joel Wahl2, Anders G. Andersson2, Simone Kraut1, Natascha Sommer1, Norbert

Weissmann1, Fenja Knoepp1

1 Justus-Liebig-University Giessen, Excellence Cluster Cardio-Pulmonary Institute (CPI), University of Giessen and

Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany 2 Luleå University of Technology, Department of Engineering Sciences and Mathematics, Luleå, Sweden

Question: The ability to sense changes in oxygen availability is fundamentally important for the survival of all aerobic

organisms. However, cellular oxygen sensing mechanisms and pathologies remain incompletely understood, and

studies of acute oxygen sensing in particular have produced inconsistent results. Current methods cannot

simultaneously measure the key cellular events in acute hypoxia (i.e. changes in redox state, electrophysiological

properties and mechanical responses) at controlled partial pressures of oxygen (pO2). The lack of such a

comprehensive method essentially contributes to the discrepancies in the field.

Methods: We developed an airtight microfluidic system that combines i) Raman spectroscopy, ii) patch-clamp

electrophysiology and iii) live-cell imaging under precisely controlled pO2. Merging these modalities allows label-free

and simultaneous observation of O2-dependent alterations in multiple cellular redox couples of pulmonary arterial

smooth muscle cells (PASMCs), cellular membrane potential and contraction.

Results: The pO2 was continuously recorded via an optical O2 sensor that was placed inside the microfluidic system.

Concomitant with the evenly decreasing pO2, an immediate cellular membrane depolarization was observed in patch-

clamp recordings, which was fully reversible upon return to normoxia. Simultaneously recorded Raman spectra

revealed that hypoxia induced an increase in NADH and cytochrome c (Fe2+) while a decrease in NAD+ and

cytochrome c (Fe3+) was observed. The opposite behavior was detected during reoxygenation. While showing the

changes in redox state and membrane potential, the PASMCs immediately contracted upon hypoxic exposure.

However, in contrast to the reversibility of redox state and membrane potential, the hypoxia-induced contraction

continued (albeit at a decreased rate) upon return to normoxic conditions.

Conclusions: Although our multimodal setup has here been used to measure PASMCs, it is applicable and

transferrable to any other cell type. The results conclusively demonstrate that our multimodal approach allowed – for

the first time – simultaneous measurement of the cellular processes involved in acute O2 sensing. Thus, our technique

has the unique ability to provide an unambiguous picture of the key cellular events underlying various physiologic

and pathologic conditions.

Acknowledgment This study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) –

Project numbers 452531259 and 268555672 (CRC 1213, project A06) as well as the Swedish Research Council

(grant 2016-04220).


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A 06-09

Actin dynamics in hypoxia- from the cytoplasm to the nucleus

Maxime Olmos1, Gijsbert J. van Belle1, Sabine Krull1, Argyris Papantonis2, Felipe Opazo3, Dörthe M.

Katschinski1, Anke Zieseniss1

1 Georg-August University, University Medical Center, Institute of Cardiovascular Physiology, Göttingen, Germany 2 Georg-August University, University Medical Center, Institute of Pathology, Göttingen, Germany 3 Georg-August University, University Medical Center, Department of Neuro- and Sensory Physiology, Göttingen,

Germany

The hypoxia-inducible factors (HIFs) are critically important for the transcriptional response to low oxygen availability

(hypoxia). Hypoxia plays a central role in many physiological (e.g. development and adaption to altitude) and

pathophysiological processes (e.g. ischemic diseases, wound healing and cancer). Activation of the HIF-pathway is

the classical way of cells to adapt to hypoxia. Up to date hundreds of genes have been described to be regulated in

a HIF-dependent manner. However, in the overall adaptive transcriptional response to hypoxia, transcriptional

repression is an equally important process. Still, the underlying mechanisms are much less understood. Transcription

can be repressed through several mechanisms: inhibition of the transcriptional machinery, inhibition of transcriptional

activators or chromatin remodeling that changes the chromatin accessibility.

Actin is a central component of the eukaryotic cytoskeleton and is involved in a wide variety of cellular processes.

Besides having a well-established role as part of the cytoskeleton, actin is also present in the nucleus where it is

involved in fundamental nuclear processes. Nuclear actin is part of the chromatin remodeling complex, is involved in

histone-modification, has a role in transcriptional regulation by all three RNA polymerases and regulates transcription

factor activity.The various nuclear functions of actin make nuclear actin a prime candidate to participate in

transcriptional changes in hypoxia. Hypoxia changes cytoplasmic actin dynamics making it likely that nuclear actin

dynamics are affected by hypoxia, too. Indeed, using several methods we observed the rapid and reversible, HIF-

independent reduction of the nuclear actin pool in hypoxia (1% O2) in several murine and human cell lines. This

reduction was paralleled by a decrease in RNA polymerase II (RNApol2) phosphorylation and RNApol2 clusters as

seen by immunofluorescence and STED microscopy. Additionally, RNApol2 binding was analyzed using ChIP-seq

experiments. First evaluation using SICER peak calling show little overall differences in RNApol2 coverage

comparing normoxia and early hypoxia. Nonetheless, in acute hypoxia ~150 enriched regions comprising well-

described hypoxia regulated genes, and even more (i.e.~ 350) repressed regions, were identified.

Further investigation of nuclear actin dynamics in hypoxia might help to shed light on the adaptive gene expression

in hypoxia.


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A 06-10

Transcriptome profiling of HIF1a and HIF2a CRISPR/Cas9 knock-out

cell lines reveal distinct response pathways to hypoxia

Simon Kelterborn, Katharina Krueger, Lorenzo Catanese, Karin Kirschner, Holger Scholz

Charite – Universitatsmedizin Berlin, Institut für Vegetative Physiologie, Berlin, Germany

Neuroblastoma is the second most common childhood cancer and develops in sympathetic ganglion cells. Tumor

metastasis and a clinical progression highly correlate with oxygen deficiency in tumor tissue. Cells evolved several

pathways to respond and adapt to low oxygen conditions which are controlled by several hypoxia-inducible factors

(HIFs). To study which factor induces malignant tumor progression in neuroblastoma, we individually inactivated

HIF1a, HIF2a or HIF1b in neuroblastoma-derived Kelly cells and performed whole genome transcriptome sequencing

(RNA-Seq).

In unmodified Kelly cells that were held under hypoxic conditions for 24 h, 3448 genes were significantly up- or

downregulated (padj. < 0.05 & -1 > log2F > 1). In the absence of HIF1a or HIF2a, the number of genes that are

differentially expressed compared to control hypoxic conditions are 263 and 1005, respectively, indicating that in

Kelly cells, HIF2a plays the major role in hypoxia signaling.

Under the top hits of genes that are not differentially expressed anymore under hypoxia when HIF1a is inactivated

are carbonic anhydrase 9 (CA9), BCL2 interacting protein 3 (BNIP3), pyruvate dehydrogenase kinase (PDK1) or

6−phosphofructo−2−kinase (PFKFB3) - all implicated in regulation of apoptosis or misregulated metabolism in cancer

cells.

In the absence of HIF2a, many transcription factors are found in the top hits as e.g. SRY−box transcription factor 7

(SOX7), paired box 7 (PAX7) and Wilms tumor protein 1 (WT1), the latter an established marker for malignant

neuroblastoma. Additionally, many of the top targets are involved in GTP-signaling e.g. Rho-related BTB domain-

containing protein 2 (RHOBTB2), Rac/Cdc42 Guanine Nucleotide Exchange Factor 6 (ARHGEF6) or FYVE, RhoGEF

And PH Domain Containing 5 (FGD5).

This data set in combination with the ongoing experiments about the hypoxic response in the absence of HIF1b is a

rich source to unravel the different response pathways to hypoxia in neuroblastoma cells.


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Foyers

A 07 | Inflammation Chair

Joachim Fandrey (Duisburg)

Agnes Görlach (Munich)


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A 07-01

The lncRNA MANTIS is required for the pro-inflammatory response of

monocytes

Corina Ratiu, Beatrice Pflüger-Müller, Matthias Leisegang, Ralf Brandes

Goethe Universität Frankfurt, Kardiovaskuläre Physiologie, Frankfurt, Germany

Objective: Long non-coding RNAs (lncRNAs) are novel, important molecules regulating cellular gene expression.

Human monocytes are important inflammatory cells which contribute to arteriosclerosis development und

inflammatory processes. We studied monocytic lncRNA expression to identify potential novel anti-inflammatory

mechanisms.

Results: RNA sequencing of human monocytes and the monocytic cell line THP1 revealed several highly expressed

lncRNAs, among them MANTIS. Interestingly, MANTIS expression massively decreased during monocyte

differentiation pointing to a specific function of the lncRNA in monocytes. Given that monocytes sense danger

associated molecules like TLR-ligands, we speculated that MANTIS might be required for the inflammatory response

of monocytes. To study this aspect, THP-1 cells were treated with TLR ligands (Lipopolysaccharide -LPS, Flagellin,

Lipoteichoic acid) with and without CRISPR/Cas9-mediated deletion of MANTIS. Importantly, TLR-mediated

proinflammatory gene expression was massively attenuated after knockout of MANTIS. Luciferase reportergen

assays demonstrated that deletion of MANTIS suppresses NF-kB but not AP-1 and STAT1 promoter activity.

MANTIS, however, did not affect NFkB nuclear recruitment. In contrast, TLR-mediated recruitment of the NF-kB

subunit p65 to the transcriptional start site of inflammatory genes was significantly reduced after MTS knockout as

demonstrated by chromatin-immunoprecipitation (ChIP).

Conclusion: The lncRNA Mantis is involved in NF-kB signaling and therefore impacts expression of inflammation-

related genes. MANTIS is a novel attractive target for anti-inflammatory therapy.


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A 07-02

The purinergic P2Y6 receptor is crucially involved in innate immune

activation in an in vitro model of acute respiratory distress syndrome

Michael Fauler, Eva Wirsching, Manfred Frick


ALI/ARDS (Acute Lung Injury/Acute Respiratory Distress Syndrome) is a frequent cause of respiratory failure in

critically ill patients suffering from various illnesses, including Covid-19, or following direct or indirect lung trauma.

ALI/ARDS aggravates the disease course in the need for intensive care. Despite intense basic and clinical research,

mortality remains high. ARDS is caused by an exuberant activation of the innate immune response. This leads to the

breakdown of the alveolar air-blood-barrier and formation of an intra-alveolar exudative oedema. In previous work,

we have developed an in vitro model of the alveolar barrier, composed of rat primary alveolar epithelial cells and

alveolar macrophages. Applying this model, we were able to identify a threshold-like elicitation. However, cellular

and molecular mechanisms of this activation process are not well understood. In this study, we investigated the role

of the purinergic system.

Rat primary alveolar cells were cultivated on cell culture inserts for three to four days. Alveolar macrophages (CD68+)

were isolated from broncho-alveolar lavage fluid and seeded on top of the epithelial layer. This co-culture was

maintained for another two to three days to reach transepithelial electrical resistances above 600 W×cm2.

Macrophages were subsequently activated to gain a M1-polarized state by lipopolysaccharide (LPS) and Interferon-

γ. Transepithelial electrical resistance drastically decreased towards about 100 W×cm2 and was accompanied by an

increase in permeability towards high-molecular dextrans. Increase in permeability depended on macrophage

density. The non-specific purinergic antagonist suramin avoided this barrier break-down. Therefore, we screened for

effects of specific agonists and inhibitors for various P2Y and P2X7 receptors. We identified and confirmed P2Y6 as

the critical receptor involved in the macrophage-initiated activation process, since blockage of P2Y6 completely

abolished barrier break-down in a concentration-dependent manner.

In summary, we identified the purinergic system as indispensably involved in the elicitation of an innate immune

response in an in vitro model of the alveolar epithelium. Furthermore, using pharmacological tools, we demonstrate

that purinergic signals are mediated by P2Y6 receptors.


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A 07-03

A20 and the non-canonical NF-κB pathway as key regulators of

neutrophil function in fetal ontogeny

Ina Rohwedder1, Lou M. Wackerbarth1, Kristina Heinig1, Annamaria Ballweg1, Claudia Nussbaum2,

Johannes Altstätter1, Myriam Ripphahn1, Melanie Salvermoser1, Tobias Straub3, Matthias Gunzer4,

Marc Schmidt-Supprian5, Christian Schulz6, Averil Ma7, Barbara Walzog1, Matthias Heinig8,

Markus Sperandio1

1 Ludwig-Maximilians Universität, Institute for Cardiovascular Physiology and Pathophysiology, Planegg, Germany 2 Ludwig-Maximilians Universität, Dr. von-Haunersches-Kinderspital, Munich, Germany 3 Ludwig-Maximilians Universität, Core Facility Bioinformatics, Planegg, Germany 4 University of Duisburg-Essen, Institute for Experimental Immunology and Imaging, Essen, Germany 5 TU Munich, Department of Hematology and Medical Oncology, Munich, Germany 6 Ludwig-Maximilians Universität, Medizinische Klinik und Poliklinik I, Munich, Germany 7 University of California, Department of Medicine, San Francisco, USA 8 Helmholtz Zentrum, Institute of Computational Biology, Muncih, Germany

Question: Premature infants are at high risk to develop neonatal sepsis shortly after birth. While this has been linked

to their immature and functionally limited innate immune response, it is still elusive how this maturation is regulated.

Therefore, we aimed to investigate the molecular mechanisms behind this ontogenetic maturation process.

Methods & Results: In a transcriptomic analysis of human fetal and adult neutrophils, we found 128 differentially

regulated genes, with a prominent focus on genes being involved in the RelB-regulated non-canonical NFκB signaling

pathway. We detected higher levels of p52 and pronounced nuclear RelB localization in fetal neutrophils compared

to adult cells, indicating a higher baseline activity of non-canonical NFκB signaling. This pathway also acts as a

negative regulator of the classical NFκB signaling cascade. Accordingly, we observed down-regulation of the

canonical, inflammatory NFκB pathway by reduced phosphorylation of IκB upon stimulation. Also in vivo, intrauterine

LPS stimulation led to impaired induction of neutrophil adhesion in yolk sac vessels of E14.5 compared to E17.5

mouse fetuses. In our transcriptomic analysis, we found upregulation of the TNFAIP3 transcript encoding for the

ubiquitin modifying enzyme A20, a well characterized negative regulator of canonical NFκB signaling. To clarify the

role of A20 in neutrophil recruitment, we generated A20-overexpressing Hoxb8 cells and analyzed their adhesion to

E-selectin/ICAM1 and CXCL1 as an inflammatory substrate under flow. As expected, we observed reduced adhesion

of A20-overexpressing Hoxb8 cells compared to control cells in our flow chambers, indicating that high levels of A20

impair the inflammatory response in neutrophils.

Conclusion: Taken together, our results identify A20 and the non-canonical NFκB pathway as key regulators of

neutrophil function in mice and human fetuses, which eventually lead to the development of new therapeutic

approaches in the treatment of newborn infants suffering from severe infections.

Acknowledgment Supported by Deutsche Forschungsgemeinschaft CRC914 projects A02 (B.W.), A10 (C.S), and B01 (M.S).


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A 07-04

The role of the secreted modular calcium binding protein 1 in the

resolution of inflammation

Fredy Delgado Lagos1,2, Urun Ukan1,2, Andreas Weigert3, Beate Fisslthaler1,2, Mauro Siragusa1,2,

Ingrid Fleming1,2

1 Goethe-Universität, Institute for Vascular Signalling, Frankfurt am Main, Germany 2 German Centre for Cardiovascular Research, Partner Site Rhine-Main, Frankfurt am Main, Germany 3 Goethe-Universität, Institute of Biochemistry I, Frankfurt am Main, Germany

Question: The resolution of inflammation is an essential process for the restoration of tissue homeostasis after injury

or infection. Macrophages play an essential role in both parts of this response and while more than 80% of

macrophages exhibit an inflammatory phenotype (M1) in the early stages of inflammation, their repolarization to the

M2c phenotype is a prerequisite for resolution. Transforming growth factor β (TGF-β) released by apoptotic

neutrophils drives the latter process and given that secreted modular calcium binding protein (SMOC1) has been

reported to modulate TGF-β signalling, the aim of this study was to determine whether SMOC1 contributes to

macrophage polarization.

Methods and Results: SMOC1 was highly expressed in murine and human M1 macrophages, although its

expression was not necessary for polarization (iNOS, TNF-α and IL-1β showed no significant differences). Analysis

of the peritoneal lavage from mice with zymosan-induced peritonitis revealed that Ly6C+ monocytes and TNF-α levels

were elevated in SMOC1-deficient (SMOC1+/-)mice during the resolution of the inflammation. When TGF-β signalling

was assessed, SMOC1 deficiency tipped TGF-β signalling towards ALK1 and induced the upregulation of Id3. This

shift was associated with a dysregulated TGF-β-dependent miR146/miR155 inflammatory axis, which is crucial for

the modulation of the immune response. Rab7, a target of miR-146, was downregulated in M2c macrophages from

SMOC1+/- mice and in phagocytosis assays these cells demonstrated an increase in uptake, but poor material

digestion that eventually resulted in cell rupture. Proteomic analysis of M2c macrophages from SMOC1 deficient

mice revealed that key proteins that participate in the lysosomal degradation (VATG1 and MPRI) were

downregulated.

Conclusions: Altogether these results demonstrate that SMOC1 deficiency leads to an improper debris clearance

during the inflammatory process, causing a delay in the resolution of the inflammation.


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A 07-05

Insulin-like growth factor 1 induces an anti-inflammatory neutrophil

phenotype by non-canonical signal transduction

Sophia Reidel1, Rianne Nederlof1, Patrick Petzsch2, Karl Köhrer2, Katharina Voigt1, Axel Gödecke1

1 Universitätsklinikum Düsseldorf, Institut für Herz- & Kreislaufphysiologie, Düsseldorf, Germany 2 Universitätsklinikum Düsseldorf, BMFZ - Genomics & Transcriptomics Laboratory, Düsseldorf, Germany

Neutrophils were long thought to play a detrimental role in ischaemic heart disease, but recent data showed that

neutrophils also play an important role in cardiac remodelling and can exhibit an anti-inflammatory N2-phenotype or

a pro-inflammatory N1 phenotype [2,3;4,5]. Previous experiments showed that short-term treatment with insulin-like

growth factor (IGF1) preserves cardiac function after myocardial infarction (MI). This effect was mediated by myeloid

cells (neutrophils, macrophages) [1]. Therefore, it was the aim to analyse to what extent and how IGF1 modulates

neutrophil function.

Murine bone marrow neutrophils were isolated by density gradient centrifugation and were left untreated or polarized

for 4 hours in vitro with IL-4 (20 ng/ml) or IGF1 (10 ng/ml).

Transcript expression analysis and qRT-PCR were performed to analyse to what extent transcriptional changes were

triggered by growth factor and cytokine induced polarization after 4 hours. These analyses revealed that IL-4, and

interestingly also IGF-1, induces a reparative N2-like neutrophil phenotype, characterised by the induction of

Arg1,Retnlα and Chi3l3. As expected, RNAseq analysis showed, that IL-4 treatment upregulated genes involved in

the IL-4 pathway. Surprisingly, IGF1 treatment induced almost the same pathways in neutrophils as IL-4. Therefore,

the canonical IL-4 signaling pathway Jak-Stat was analysed. This revealed that IL-4, and surprisingly also IGF- 1,

induced phosphorylation of Stat6. Additionally, Jak1/2/3/Tyk2 was inhibited with pharmacological inhibitors

(InSolution Jak Inhibitor I, Ruxolitinib, BMS-911543). Inhibition of all Jaks prevented IL-4 induced upregulation of N2

marker genes. Also IGF1 induced polarization was prevented by Jak inhibition. Specific inhibition of Jak2 showed

that IL-4- and IGF1-induced polarization is most likely dependent on Jak2 activation, as N2 marker gene expression

was reduced. Western blot analysis as well revealed that Stat6 phosphorylation was reduced after Jak2 inhibition.

In conclusion, these findings indicate that IGF1 is able to polarize neutrophils to a N2-like phenotype most likely via

the non-canonical Jak2-Stat6 axis, comparable to that induced by the classical M2/N2 polarizer IL-4.

References [1] Heinen, A., Nederlof, R., Panjwani, P., Spychala, A., Tschaidse, T., Reffelt, H., Boy, J., Raupach, A.,

Godecke, S., Petzsch, P., Kohrer, K., Grandoch, M., Petz, A., Fischer, J. W., Alter, C., Vasilevska, J., Lang, P., and Godecke, A. (2019) IGF1 Treatment Improves Cardiac Remodeling after Infarction by Targeting Myeloid Cells. Mol Ther27, 46-58

[2] Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D. S., Weinrauch, Y., and Zychlinsky, A. (2004) Neutrophil extracellular traps kill bacteria. Science303, 1532-1535

[3] Kolaczkowska, E., and Kubes, P. (2013) Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol13, 159-175

[4] Ma, Y. G., Yabluchanskiy, A., Iyer, R. P., Cannon, P. L., Flynn, E. R., Jung, M. R., Henry, J., Cates, C. A., Deleon-Pennell, K. Y., and Lindsey, M. L. (2016) Temporal neutrophil polarization following myocardial infarction. Cardiovasc Res110, 51-61

[5] Fridlender, Z. G., Sun, J., Kim, S., Kapoor, V., Cheng, G. J., Ling, L. N., Worthen, G. S., and Albelda, S. M. (2009) Polarization of Tumor-Associated Neutrophil Phenotype by TGF-beta: "N1" versus "N2" TAN. Cancer Cell16, 183-194


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A 07-06

The soluble epoxide hydrolase plays a key role in modulating skin

inflammation

Zumer Naeem1, Timo Frömel1, Sven Zukunft1, Arnaud Huard2, Andreas Weigert2, Bruce Hammock3,

Ingrid Fleming1

1 IVS, Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main,

Germany, frankfurt, Germany 2 Institute of Biochemistry I, Goethe-University Frankfurt, Frankfurt am Main 60590, Germany, Frankfurt, Germany 3 Department of Entomology and Nematology and Comprehensive Cancer Center, University of California, Davis,

CA, USA, Davis, USA

Cytochrome P450 (CYP) enzymes are membrane bound, heme containing terminal oxidases that metabolize

endogenous poly-unsaturated fatty acids (PUFAs) to their corresponding epoxides, generating bioactive lipid

mediators that have been attributed anti-inflammatory properties. These bioactive epoxides are further metabolized

by the soluble epoxide hydrolase (sEH) to corresponding less active diols.

PUFA metabolism is highly active in the skin but little is known about the role of the CYP-sEH axis in this organ. LC-

MS/MS based free fatty acid analyses revealed that dorsal skin from wild-type mice is enriched in sEH-derived PUFA

diols, that were lacking in skin from sEH-/- mice. Phenotypically, the proliferation of basal keratinocytes was greater

in sEH-deficient mice, which also demonstrated thicker differentiated spinous and corneocyte layers than wild-type

mice. In the latter animals, the topical application of a sEH inhibitor induced a similar hyperkeratosis phenotype.

Although the inhibition of the sEH is generally associated with anti-inflammatory effects, sEH deletion made skin

more prone to inflammation triggered by depilation. Indeed, hair removal resulted in pronounced skin irritation and

the infiltration of immune cells including neutrophils, Langerhans cells, monocytes and macrophages within 24 hours

of depilation of sEH-/- mice. sEH-/- mice were also more sensitive to Imiquimod-induced psoriasis and developed

thicker psoriasis plaques compared to the control group. This phenomenon was also associated with increased

neutrophil infiltration and was coincident with an increase in 12,13-epoxyoctadecenoic acid (EpOME) and leukotrine

B4 (LTB4). Both 12,13-EpOME and LTB4 have been reported to attract and activate neutrophils in the skin of burn

victims. Ex vivo studies revealed that 12,13-EpOME significantly increased neutrophil adhesion and neutrophil

elastase activity as well as neutrophil trans-endothelial migration.In summary, the sEH is highly active in murine skin

and its deletion alters the abundance of fatty acids in different skin layers which has consequences on keratinocyte

differentiation in the epidermis. The deletion of the sEH resulted in an exaggerated immune response during atopic

dermatitis suggesting that this may be an important side effect of sEH inhibitor therapy.

References [1] Kendall AC, Pilkington SM, Massey KA, Sassano G, Rhodes LE, Nicolaou A. Distribution of bioactive lipid

mediators in human skin. J Invest Dematol. 2015;135:1510–1520. doi: 10.1038/jid.2015.41 [2] Oyoshi MK, He R, Li Y, Mondal S, Yoon J, Afshar R, Chen M, Lee DM, Luo HR, Luster AD, Cho JS, Miller LS,

Larson A, Murphy GF, Geha RS. Leukotriene B4-driven neutrophil recruitment to the skin is essential for allergic skin inflammation. Immunity. 2012;37:747–758. doi: 10.1016/j.immuni.2012.06.018

[3] Hu J, Dziumbla S, Lin J, Bibli S-I, Zukunft S, Mos J de, Awwad K, Frömel T, Jungmann A, Devraj K, Cheng Z, Wang L, Fauser S, Eberhart CG, Sodhi A, Hammock BD, Liebner S, Müller OJ, Glaubitz C, Hammes H-P, Popp R, Fleming I.

[4] Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy. Nature. 2017;552:248–252. doi: 10.1038/nature25013


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A 07-07

Do Albumin-derived perfluorocarbon-based artificial oxygen carriers

activate immune cells?

Linda M. Tchuendem1, Katja B. Ferenz1,2, Shah Bahrullah Shah1

1 University of-Duisburg-Essen, University Hospital Essen, Institute of Physiology, Essen, Germany 2 CeNIDE, University of Duisburg-Essen, Duisburg, Germany

Although albumin-derived perfluorocarbon-based artificial oxygen carriers (A-AOCs) are well established in our

research group1, interactions with immune cells have not been investigated so far. To explore whether A-AOCs

activate immune cells, we used the human monocytic cell line Tohoku Hospital Pediatrics-1 (THP-1), a common

model for primary human macrophages (PHM) after differentiation with phorbol12-myristate13-acetate (PMA).

To determine the optimal PMA concentration resulting in PHM, THP-1 cells (1x106cells/ml) were incubated with either

25 or 10ng/ml PMA for 72h, followed by 24h resting without PMA. The acquisition of M0 macrophage-like phenotype

(e.g. cell adhesion, spreading) was assessed microscopically & confirmed at the molecular level by quantifying the

expression of monocyte (CD32) and macrophage (CD14) surface markers using flow cytometry (1x106 cells/ml); non-

differentiated THP-1 served as control. Cytotoxicity of A-AOCs was checked with lactate dehydrogenase (LDH)

assay. PHM (1x104cells/ml/well, 10ng/ml PMA for 72h+24h resting without PMA) were treated with medium +/-17-

2% of A-AOCs for 4h. Influence of A-AOCs on cell adhesion was analyzed by using western blot checking for

intracellular adhesion molecule-1 (ICAM-1) expression. PHM, treated with medium, LPS (1µg/ml) or 10-2% of A-

AOCs were exposed to 1% O2 for 24h in a hypoxia station. To investigate the uptake of A-AOC by PHM (5x105

cells/ml) were labelled with TRITC-Concanavalin A (100µg/ml) for 30min. Subsequently, cells were incubated with

4% of FITC-labelled-A-AOCs (1mg/ml) for 30min and 2h & visualized at 580 + 519nm with a fluorescence

microscope.

Both PMA concentrations tested resulted in the M0 phenotype, which (for 10ng/ml PMA) was confirmed by higher

CD14 and lower CD32 expression as compared to non-differentiated THP-1 cells. LDH assay revealed toxicity only

for 17% A-AOCs & positive control. In addition, hypoxia increased ICAM-1 expression (associated with pro-

inflammatory conditions) in LPS-treated control cells but not in A-AOCs-treated cells. Furthermore, fluorescent

microcopy showed uptake of A-AOCs by PHM already after 30min, which further increased after 2h of incubation.

Taken together, our results demonstrate that 10ng/ml PMA are sufficient to ensure stable differentiation of THP-1

cells into PHM. Moreover, our A-AOCs (up to 10%) are well tolerated by PHM. Furthermore, PHM, as part of the

innate immune response, do recognize and engulf A-AOCs.

References [1] Wrobeln A, 2017, Eur J Pharm Biopharm. 115, 52-64.


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A 07-08

Differential microRNA expression during monocyte-macrophage

differentiation in THP-1 cells

Gerhild Euler1, Oezden Tastan1, Rainer Schulz1, Mariana Parahuleva2

1 Justus Liebig Universität, Physiologisches Institut, Gießen, Germany 2 UKGM, Innere Medizin, Kardiologie/Angiologie, Marburg, Germany

In atherosclerosis, macrophages lodge in the intima and subintima of arteries, and contribute to the formation of

obstructive atherosclerotic plaques that are prone to rupture, leading to thrombosis, myocardial infarction (MI) or

stroke. Just recently, we identified eight monocytic microRNAs (miRs) that are expressed differentially in human

advanced coronary atherosclerotic plaques or acute myocardial infarction. Since the differentiation of monocytes to

macrophages is a prerequisite for lodging of macrophages in the atherosclerotic plaque, we determined in this study,

if those miRs are also differentially expressed during monocyte-macrophage differentiation.

The monocytic human cell line, THP1, was used in the study. THP1 cells were grown in RPMI 1640 medium up to

80 % confluence. To induce macrophage differentiation, cells were stimulated with PMA (phorbol-12-myristate-13-

acetate, 5 ng/ml) for one, two and three days. mRNA expression of CD14, a marker of macrophage differentiation,

started to rise within one day and was upregulated 31-times compared to non-stimulated cells after three days (n=4,

p<0.05). Under the same conditions, miR expressions were determined. Under the eight miRs that were differentially

expressed in atherosclerotic plaques or monocytes of MI patients, three miRs (miR-21, -99a, -223) did not change

their expression in PMA stimulated THP1 cells. Upregulation under PMA stimulation for three days was identified for

miR-1 (6.3-times vs. control), miR-22 (3.4-times vs. control), and miR-143 (2.7-times vs. control) (n=10, p<0.05,

respectively). In contrast, let-7f and miR-92a were downregulated within the first day of PMA stimulation (0.4-times

and 0.5-times vs. control, n=10, p<0.05, respectively), and turned to control levels after three days.

In conclusion, differential miR expressions can be found during monocyte-macrophage differentiation in THP1 cells.

These resemble in parts the differential miR expressions in human atherosclerotic plaque samples. Therefore, those

miRs may influence monocyte-macrophage differentiation, and may become interesting therapeutic targets in the

treatment of atherosclerosis.


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A 07-09

Soluble epoxide hydrolase is required in mediating inflammatory

resolution

Xiaoming Li, Sebastian Kempf, Jiong Hu, Ingrid Fleming

Goethe University, The Institute for Vascular Signalling, Frankfurt am Main, Germany

Background: Polyunsaturated fatty acids (PUFAs) play essential roles in mediating inflammation and its resolution.

PUFA metabolites generated by the cytochrome P450 (CYP) - soluble epoxide hydrolase (sEH) axis are known to

regulate macrophage activation/polarization but little is known about their role in the resolution of inflammation.

Methods: Monocytes were isolated from murine bone marrow or human peripheral blood and differentiated to naïve

macrophages (M0). Thereafter cells were polarized using LPS and IFNγ (M1), IL-4 (M2a), or TGFβ1 (M2c).

Expression profile analysis was performed by RNA sequencing, RT-qPCR and Western blotting. Phagocytosis of

zymosan and oxo-LDL was also assessed.

Results: The expression of sEH was comparable in M0, M1 and M2a macrophages but markedly elevated in M2c

polarized cells. The increase in sEH expression elicited by TGF-β relied on the TGFβ receptor ALK5 and the

phosphorylation of SMAD2, which was able to bind to the sEH promoter. In macrophages lacking sEH, M2c

polarization was incomplete and characterized by lower levels of pro-resolving phagocytosis associated receptors

(Tlr2 and Mrc1), as well as higher levels of the pro-inflammatory markers; Nlrp3, IL-1β and TNFα. Fitting with the

failure to upregulate phagocytosis associated receptors, the phagocytosis of zymosan and ox-LDL were less efficient

in M2c macrophages from sEH-/- mice, which also demonstrated a retarded resolution of inflammation (zymosan-

induced peritonitis) in vivo. At the molecular level, the failure to upregulate Tlr2 and Mrc1 expression as well as the

maintained expression of NLRP3 in sEH-deficient macrophages were accounted by the decreased expression of

PPARγ.Conclusions: Taken together, our data indicates that sEH expression is required for the effective M2c

polarization of macrophages and thus the resolution of inflammation.


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A 07-10

An in vitro model for ALI/ARDS

Eva Wirsching, Michael Fauler, Manfred Frick


The alveolar-capillary barrier is composed of epithelial and endothelial cells interacting across a thin basal

membrane. Damage of the alveolar-capillary barrier plays a key role in the patho-physiology of various lung diseases,

including acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).

To investigate the molecular mechanisms of barrier damage in ALI/ARDS, we established a fully humanized in vitro

model of the alveolar-capillary barrier. We co-cultured alveolar epithelial cells (hAELVi), in-vitro differentiated

macrophages, neutrophilic granulocytes and, for some experiments, microvascular endothelial cells as well as

primary alveolar type II cells on opposite sides of porous cell culture inserts.

Major functional characteristics of the barrier in vivo are a tight epithelium and its immune responsiveness. We

measured transcellular electrical resistance (TER) and permselectivity of fluorescently labelled dextrans to determine

epithelial integrity. Under air-liquid-interphase conditions, the epithelial layer established a high TER and a stable

barrier which was not affected by addition of pro-inflammatory stimuli. In contrast, thrombin and proinflammatory

mediator TNFα, significantly, but reversibly, reduced the TER of the endothelial layer and increased expression of

inflammatory markers ICAM-1 and E-selectin, respectively. Neutrophils (PMN) and macrophages, which contribute

and aggravate barrier damage in vivo, significantly exacerbated barrier break-down in response to lipopolysaccharide

(LPS) and interferon-γ treatment.

In summary, these results confirm validity of the model for investigating molecular mechanism of ALI/ARDS in vitro.


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Foyers

A 08 | Hormones Chair

Michael Föller (Stuttgart)

Ralf Mrowka (Jena)


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A 08-01

pH-dependence of activation and interconnectivity of pancreatic beta

cells within islets in acute tissue slices

Sandra Postic1, Wen-Hao Tsai4, Srdjan Sarikas1, Marko Gosak2,3, Shi-Bing Yang4, Marjan Slak

Rupnik1,2

1 Medical University of Vienna, Physiology/Center for Physiology and Pharmacology, Vienna, Austria 2 University of Maribor, Faculty of Medicine, Maribor, Slovenia 3 University of Maribor, Faculty of Natural Sciences and Mathematics, Maribor, Slovenia 4 Academia Sinica, Institute of Biomedical Sciences, Taipei, Taiwan

pH in bodily fluids is highly regulated and deviations interfere with the physiological function. Doubling of the H+

concentration (pH 7.4 to 7.1) has been reported to enhance insulin secretion at low stimulatory glucose concentration

in pancreatic beta cells. Insulin is a key hormone in regulation of metabolic homeostasis. Beta cells function as a

collective within a functional units called islets of Langerhans. Islets are surrounded by lobes of acinar cells and

ductal cells, the two cell types that are capable to directly influence the pH of their microenvironment. In this study,

we used the pancreatic tissue slice approach, which gave us the opportunity to simultaneously study all before

mentioned cells in their native environment, control pH in the extracellular medium, and perform high temporally and

spatially resolved functional calcium imaging. We developed an analysis pipeline that allows us to automatically

detect ROIs corresponding to individual cells, as well as annotate cytosolic Ca2+ changes as individual events of

different time scales, ranging from sub-second to hundred second events. We used a triple protocol, with three

sequential stimulations with physiological stimulatory glucose (8 mM), where the middle section was used to test the

effect, while sections 1 and 3 provided control conditions before and after. We observed that doubling of H+

concentration increased the frequency of the Ca2+ events and the halfwidth length of these events. In addition, we

determined that switch to pH 7.1 enabled beta cells to activate already at substimulatory glucose concentration.

Finally, the beta cell network properties were changed as well, showing reduced node degree and average clustering

efficiency at pH 7.1, indicating reduced network connectivity, forming more waves that incorporated fewer beta cells.

Insulin release measurements confirmed that the higher activity of beta cells measured as cytosolic Ca2+ oscillations

was accompanied also with higher insulin release.

Acknowledgment Project was done thanks to Austrian Science Fund/Fonds zur Forderung der Wissenschaftlichen Forschung

bilateral grant I 3562 I


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pH influences the activity of beta cells

A) Immunofluorescence image of the recorded

slice with labeled ROIs B)Time course of the

[Ca2+]c changes of cells indicated in the panel

A, exposed to a triple 8 mM glucose stimulation

protocol. C) Frequency histograms of the event

halfwidth durations. D) Onset time of the [Ca2+]c

events at all measured time scales. Note that

the events prolonged and increased in frequency.

E) Expanded time traces from a representative

ROI indicating a slow event from a transient

phase (*) and a plateau phase (**) as indicated

in panel F. F)Time trace of the average of all

ROIs in the presented islet (n=196).

pH-dependent spatiotemporal organization of

intercellular calcium waves in islets

A,C) Space-time graphs and B,D) raster plots of

binarized calcium activity of all cells in the

representative islet in the plateau phase

under 8 mM glucose and pH: A,B) 7.4 and C,D)

7.1. The colors of dots signify different

calcium waves and purple dots in the x-y plane

in panels A and C denote the coordinates of

individual beta cells. E) The box-plots in panel

signify the distribution of relative wave sizes

in different pH levels pooled from 3 different

islets for each pH level.

References [1] Hegyi, P., J. Maleth, V. Venglovecz and Z. Rakonczay, Jr. 2011, "Pancreatic ductal bicarbonate secretion:

challenge of the acinar Acid load.", Front Physiol, 2: 36 [2] Marciniak, A., C. M. Cohrs, V. Tsata, J. A. Chouinard, C. Selck, J. Stertmann, S. Reichelt, T. Rose, F.

Ehehalt, J. Weitz, M. Solimena, M. Slak Rupnik and S. Speier 2014, "Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology.", Nat Protoc, 9(12): 2809-2822.

[3] Pace, C. S., J. T. Tarvin and J. S. Smith 1983, "Stimulus-secretion coupling in beta-cells: modulation by pH.", Am J Physiol, 244(1): E3-18.

[4] Postić, S., S. Sarikas, J. Pfabe, V. Pohorec, L. K. Bombek, N. Sluga, M. S. Klemen, J. Dolenšek, D. Korošak, A. Stožer, C. Evans-Molina, J. D. Johnson and M. S. Rupnik 2021, "Intracellular Ca2+ channels initiate physiological glucose signaling in beta cells examined in situ.", bioRxiv, 2021.2004.2014.439796.


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A 08-02

The impact of cAMP and the role of Epac2A on the collective activity of

pancreatic beta cell networks

Maša Skelin Klemen1, Jurij Dolenšek1, Lidija Križančić Bombek1, Viljem Pohorec1, Marko Gosak1,2,

Marjan Slak Rupnik3,1, Andraž Stožer1

1 Faculty of Medicine, University of Maribor, Institute of Physiology, Maribor, Slovenia 2 Faculty of Natural sciences and Mathematics, University of Maribor, Department of Physics, Maribor, Slovenia 3 Medical University of Vienna, Center for Physiology and Pharmacology, Vienna, Austria

The pancreatic islets of Langerhans are multicellular micro-organs within which communication among a variety of

cells with unique functions must occur to ensure a proper control of metabolic homeostasis [1]. The insulin-secreting

beta cells are the most abundant cell type within the islets and exhibit a strong intercellular connectivity, which

provides the required means for coordinated responses of beta cell populations. The latter is not only a necessity for

a well-regulated secretion of insulin but also one of the underlying factors, which contribute to the pathogenesis of

diabetes [2]. Furthermore, beta cell signaling and cell-to-cell communication in islets have also been reported to be

modulated by cAMP signaling, but the findings about the effects of PKA-dependent and PKA-independent

mechanism involving Epac2A are rather diverse [3]. In our study we addressed this issue and performed functional

multicellular calcium imaging in beta cells in mouse pancreas tissue slices after stimulation with glucose and forskolin

in wild-type and Epac2A knock-out mice. To quantify the multicellular activity patterns, we performed network

analyses [4]. Our results reveal that beta cells from Epac2A knock-out mice displayed a slightly higher active time in

response to glucose compared with wild-type littermates, and increased cAMP increased the active time via a large

increase in oscillation frequency and small decrease in oscillation duration in both Epac2A knock-out and wild-type

mice. Functional network properties during stimulation with glucose did not differ in Epac2A knock-out mice, but the

presence of Epac2A was crucial for the protective effect of increased cAMP in preventing a decline in beta cell

functional connectivity with time. Moreover, islets from Epac2A knock-out mice are characterized by a faster response

to stimulatory glucose, whereas increased cAMP prolonged beta cell activity during deactivation in an Epac2A-

independent manner [5]. Our findings provide a deeper insight into the involvement of cAMP signaling in intra- as

well as intercellular activity in beta cells, which is vital for the development of novel incretin-based pharmacological

management strategies of diabetes [3].

Acknowledgment We thank Rudi Mlakar for his excellent technical assistance. We also thank Professor Susumo Seino, Kobe

University Graduate School of Medicine, Kobe, Japan and Professor Martina Schmidt, University of Groningen,

Groningen, the Netherlands, for providing Epac2A KO mice to establish our colony.

This research was funded by the Slovenian research agency, grant numbers P3-0396, I0-0029, N3-0048, N3-0133,

and J3-9289.

References [1] Dolensek, J, Rupnik, MS, Stozer, A. Structural similarities and differences between the human and the mouse

pancreas. Islets 2015, 7, e1024405. [2] Head, WS, Orseth ML, Nunemaker, CS, Satin, LS, Piston, DW, Benninger, RK. Connexin-36 gap junctions

regulate in vivo first- and second-phase insulin secretion dynamics and glucose tolerance in the conscious mouse. Diabetes 2012, 61, 1700.

[3] Stožer, A, Leitgeb, EP, Pohorec, V, Dolenšek, J, Bombek, LK, Gosak, M, Klemen, MS. The Role of cAMP in Beta Cell Stimulus-Secretion and Inter-cellular Coupling. Cells 2021 (accepted).

[4] Gosak, M, Markovič, R, Dolenšek, J, Rupnik, MS, Marhl, M, Stožer, A, Perc, M. Network science of biological systems at different scales: A review. Physics of Life Reviews 2018, 24, 118.


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[5] Klemen, MS, Dolenšek, J, Bombek, LK, Pohorec, V, Gosak, M, Rupnik, MS, Stožer, A. The Effect of cAMP and the Role of Epac2A During Activation, Activity, and Deactivation of Beta Cell Networks. Preprints, 2021, 10.20944/preprints202105.0064.v1.


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A 08-03

C-type Natriuretic Peptide (CNP) prevents the activation of lung

fibroblasts to myofibroblasts

Anna-Lena Friedrich1, Eva Lessmann1, Lisa Krebes1, Clemens Ruppert2, Andreas Günther2, Michaela

Kuhn1, Swati Dabral1

1 University of Würzburg, Physiologische Institut, Physiologie I, Wuerzburg, Germany 2 University of Giessen, University of Giessen and Marburg Lung Center, Giessen, Germany

Rationale: Idiopathic Pulmonary Fibrosis (IPF) is a progressive parenchymal lung disease with limited therapeutic

options. Pathologically altered lung (myo-) fibroblasts exhibiting increased proliferation, migration and collagen

production, drive IPF development and progression. Fibrogenic factors such as Platelet derived growth factor-BB

(PDGF-BB) contribute to these pathological alterations. Endogenous counter-regulating factors are barely known.

Published studies have described a protective role of exogenously administered C-type Natriuretic Peptide (CNP) in

pathological tissue remodeling, e.g. in heart and liver fibrosis. CNP and its cyclic GMP producing guanylyl cyclase B

(GC-B) receptor are expressed in the lung, but it is unknown whether CNP can attenuate lung fibrosis. To address

this question, we performed studies in primary cultured lung fibroblasts.

Results: To examine the effects of the CNP/GC-B pathway on PDGF-BB - induced collagen production, proliferation

and migration in vitro, lung fibroblasts were cultured from control and GC-B knockout mice. Lung fibroblasts from

patients with IPF and controls were obtained from the UGMLC Biobank. CNP (10 nM and 100 nM) markedly and

similarly increased cGMP levels in both, murine and human lung fibroblasts as measured by radioimmunoassay,

demonstrating GC-B/cGMP signalling. CNP reduced PDGF-BB-induced proliferation and migration of lung fibroblasts

as analyzed by BrdU incorporation assay and scratch assay, respectively. CNP strongly decreased PDGF-BB-

induced collagen 1/3 expression as measured by immunocytochemistry and immunoblotting. It is known that the

profibrotic actions of PDGF-BB are partly mediated by phosphorylation and nuclear export of Forkhead Box O3

(FoxO3), a transcription factor downregulated in IPF. CNP prevented PDGF-BB elicited FoxO3 phosphorylation and

nuclear exclusion. CNP signalling and functions were abolished in GC-B-deficient lung fibroblasts. We are

investigating whether the protective actions of CNP are preserved in IPF fibroblasts.

Conclusions: Taken together the results show that CNP moderates the PDGF-BB-induced activation and

differentiation of human and murine lung fibroblasts to myofibroblasts. This effect is mediated by GC-B/cGMP

signalling and possibly by FoxO3 regulation/stabilization. To follow up the pathophysiological of these findings, we

are generating mice with fibroblast-restricted GC-B deletion for studies in the model of bleomycin-induced pulmonary

fibrosis.

Acknowledgment This study was entirely financed by the Deutsche Forschungsgemeinschaft (DFG KU 1037/6-1).


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A 08-04

The endocannabinoid Anandamide mediates anti-inflammatory effect

by activating NR4A nuclear receptors

Beatrice Pflüger-Müller, Tom Teichmann, Ralf Brandes

Goethe Universität Frankfurt, Kardiovaskuläre Physiologie, Frankfurt, Germany

Objective: Endocannabinoids are important lipid mediators which excert a multitude of different functions in the body.

In particular the endocannabinoid anadamide (AEA) is considered an important signalling molecule in the

cardiovascular system. We set-out to determine the function of AEA in vascular smooth muscle cells.

Results: RNA-Seq analysis of human aortic smooth muscle cells (HAoSMCs) treated with and without AEA revealed

disparten effects on gene expression, suggesting that AEA induces pro-inflammatory gene expression but also has

an important anti-inflammatory component. The anti-inflammatory effect of AEA was unique as it was not mediated

by any other endocannabinoid nor by endocannabinoid receptors. To identify the potential receptor mediating this

effect, the RNAseq data were reanalyzed: AEA massively induced the nuclear receptor family NR4A. Indeed,

knockdown of NR4A blocked the AEA-mediated anti-inflammatory effect and prevented parts of the AEA-induced

gene expression. Artificial NR4A agonists (CsnB, C-DIM12 and PGA1) mimic parts of the AEA response, albeit at

much higher concentrations. Further experiments revealed, that binding of AEA to NR4A recruits the nuclear

corepressor NCoR1 to mediate gene inhibition. To demonstrate direct interaction of NR4A with AEA, microscale

thermophoresis was performed, which indeed demonstrated a specific binding with exceedingly high efficacy.

Conclusions: By activating NR4A AEA elicits a novel and specific anti-inflammatory response in smooth muscle

cells. AEA therefore represents a lead structure for a novel class of anti-inflammatory drugs.


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A 08-05

Using multilayer network analysis to unveil the nature of intercellular

signals in the pancreatic islets of Langerhans

Marko Šterk1,2, Jurij Dolenšek1,2, Maša Skelin Klemen2, Lidija Križančić Bombek2, Andraž Stožer2,

Marko Gosak1,2

1 University of Maribor, Faculty of Natural Sciences and Mathematics, Maribor, Slovenia 2 University of Maribor, Faculty of Medicine, Maribor, Slovenia

The pancreas is a complex organ that is home to micro-organs called islets of Langerhans which contain different

cell types, among which the insulin-producing beta cells are the most abundant ones. They are electrically excitable

and highly heterogeneous [1]. Despite this inherent heterogeneity, electrical coupling via gap junctions facilitates

their well-coordinated behavior and coherent Ca2+ concentration oscillations when stimulated with glucose. Advanced

network analyses based on thresholded pairwise correlations of Ca2+ signals have proven to be a valuable tool to

quantify intercellular communication patterns [2,3]. It has been postulated that so-called hub cells, i.e. cells with many

functional connections, are importantly involved in the coordination of collective cellular activity and these features

were suggested to be linked to an efficient spreading of intercellular Ca2+ waves [2]. The later were found to be

triggered within spatially organized and metabolically highly active beta cell subpopulations, which act as pacemakers

[4]. However, if these two types of cells overlap, remains unclear. In this study we aimed to address these issues by

combining multicellular Ca2+ imaging in pancreatic tissue slices with novel multilayer network approaches. By this

means we compared the roles of cells in each Ca2+ wave (wave-based network layer) with their role in the classic

correlation-based functional network. Our results show that cells harboring many connections in the correlation-based

network were more active and their oscillations also appeared to be more stable and regular. When comparing the

roles of individual cells in the correlation- and wave-based networks, we observed a significant correlation between

the fractions of connections in both types of networks. Furthermore, highly connected cells tended to exhibit shorter

activation time delays with respect to their neighbors, which indicates that they are better signal conductors. As such,

they probably importantly contribute to the synchronized islet activity and facilitate efficient signal propagation through

the cellular network. However, we did not observe a clear correlation between the pacemaker properties of cells and

their number of functional connections. Therefore, our results do not support the hypothesis that hub cells act as

wave initiators, which is one of the hottest and controversial topics in the islet community [5].

References [1] R. K. P. Benninger and D. J. Hodson, 2018. New Understanding of β-Cell Heterogeneity In Situ Islet

Function. Diabetes 67(4), 537-547. [2] N. R. Johnston et al., 2016. Beta Cell Hubs Dictate Pancreatic Islet Responses to Glucose. Cell Metabolism

24(3), 389-401. [3] M. Gosak et al., 2018. Network science of biological systems at different scales: A review. Physics of Life

Reviews 24, 118-135. [4] M. J. Westacott, 2017. Spatially Organized β-Cell Subpopulations Control Electrical Dynamics across Islets

of Langerhans. Biophysical Journal 113(5), P1093-1108. [5] L. S. Satin et al., 2020. “Take Me To Your Leader”: An Electrophysiological Appraisal of the Role of Hub

Cells in Pancreatic Islets. Diabetes 65(5), 830-836.


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A 08-06

C-type natriuretic peptide (CNP)/cGMP signalling attenuates

hyperproliferation and metabolic switch of lung pericytes from patients

with pulmonary hypertension

Minhee Noh1, Lisa Krebes1, Jan Dudek2, Christoph Maack2, Vinicio de Jesus Perez3, Michaela Kuhn1,2,

Swati Dabral1

1 University of Wuerzburg, Institute of Physiology, Wuerzburg, Germany 2 University Hospital Wuerzburg, Comprehensive Heart Failure Center (CHFC), Wuerzburg, Germany 3 Stanford University, Divisions of Pulmonary and Critical Care Medicine and Stanford Cardiovascular Institute,

Palo Alto, USA

Rationale: Pericytes are mural cells within the basement membrane of capillaries, providing structural support and

controlling vascular tone. In the lungs, pericytes contribute to the integrity of the alveolar-capillary interface and

coordinate vascular repair in response to injury. Loss of proper pericyte-endothelial communication and a

hyperproliferative/contractile phenotype contribute to vascular pathologies such as Pulmonary Hypertension (PH) [1].

In this situation, pericytes switch their metabolism to higher glycolysis rates to match the increased energy demands.

We aim to study the effect of C-type natriuretic peptide (CNP), signalling through the cGMP-producing guanylyl

cyclase B (GC-B) receptor, on this proliferative and metabolic switch.

Methods and Results:Cultured human pericytes from controls and PH lung specimens were kindly provided by Dr.

Perez [1]. CNP (10-100 nM) markedly and similarly increased cGMP levels in control and PH pericytes,

demonstrating GC-B/cGMP signalling. Upon platelet derived growth factor-BB (PDGF-BB) stimulation, PH pericytes

exhibited higher proliferation coupled with enhanced rate of glycolysis compared to controls (BrdU incorporation and

seahorse analyses). Notably, such PDGF-BB effects were significantly attenuated by CNP pre-treatment. To dissect

the mechanisms, the expression levels of various components of the glycolytic pathway (Glucose Transporter 1

(GLUT1), Hexokinase 2 and Lactate Dehydrogenase) as well as of their well-known regulator hypoxia-inducible factor

1 alpha (HIF1α) were analysed by subcellular fractionation followed by immunoblotting. PDGF-BB markedly

upregulated membrane GLUT1 and nuclear HIF1α levels. Notably these changes were significantly prevented by

CNP in both control and PH pericytes.

Conclusions and Outlook: Cultured human pericytes from PH lung specimens exhibit greater baseline proliferative

ability and rate of glycolysis as well as increased responses to PDGF-BB as compared to controls. Notably, CNP

attenuates these changes. Such protective effects of CNP may be partly mediated by inhibition of HIF1α-dependent

GLUT1 induction. To further characterize these findings and their relevance in vivo, we are studying metabolomics

and glucose uptake of cultured pericytes as well as hypoxia-driven PH in mice with pericyte-restricted GC-B deletion

[2]. Understanding the effects and signalling pathways of CNP in pericytes may unravel novel targets for therapies

of PH.

Acknowledgment Supported by the Deutsche Forschungsgemeinschaft (DFG KU 1037/7-1)

References [1] Yuan K, et al., 2016, 'Increased Pyruvate Dehydrogenase Kinase 4 Expression in Lung Pericytes Is Associated

with Reduced Endothelial-Pericyte Interactions and Small Vessel Loss in Pulmonary Arterial Hypertension.', Am J Pathol. 186, 2500-14


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[2] Špiranec K, et al., 2018, 'Endothelial C-Type Natriuretic Peptide Acts on Pericytes to Regulate Microcirculatory Flow and Blood Pressure.', Circulation, 138, 494-508


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A 08-07

Keeping it low: Thyroid hormones are upstream regulators of

physiological adaptations to the subterranean habitat

Patricia Gerhardt1, Alexandra Heinrich1, Sabine Begall2, Yoshiyuki Henning1

1 University of Duisburg-Essen, Institute of Physiology, Essen, Germany 2 University of Duisburg-Essen, Department of General Zoology, Essen, Germany

Around 250 rodent species worldwide live in underground burrow systems. These subterranean animals face harsh

environmental conditions, because underground burrow systems are characterized by food shortage, lack of water,

high risk of overheating, hypoxia, and hypercapnia. To adapt to these harsh environmental conditions, many

subterranean rodents have evolved a low body core temperature (Tb) and a low resting metabolic rate (RMR), but

the underlying molecular mechanisms are unknown thus far. Previous studies on the Ansell’s mole-rat (Fukomys

anselli), an African subterranean rodent species, have shown peculiarities in this species’ thyroid hormone (TH)

system. Most intriguingly, while the transcriptionally-active hormone triiodothyronine (T3) was circulating in rodent-

typical concentrations, serum concentrations of the prohormone thyroxine (T4) were far below the mammalian range,

resulting in a hitherto unknown T3/T4 ratio. Since THs are crucially involved in thermoregulation and metabolic

regulation, we hypothesized that this unique TH phenotype is an ecophysiological adaptation to maintain low Tb and

RMR to cope with harsh subterranean burrow conditions. To test this hypothesis, we treated Ansell’s mole-rats with

T4 or vehicle solution (n=5 per group) for 4 weeks using implantable osmotic pumps. Furthermore, we implanted

temperature loggers to assess Tb and measured RMR via indirect calorimetry. At the end of the treatment period,

several organs were collected to analyze treatment effects on molecular level. ELISA, histology and qPCR analyses

revealed systemic effects of upregulated TH concentrations, confirming successful treatment. Furthermore, T4-

treated animals had significantly higher Tb compared to the control group. Since T4-treated animals had higher RMR,

while we found no signs of increased non-shivering thermogenesis, it is most likely that increased Tb was a

consequence of upregulated RMR. In line with upregulated RMR, animals in the T4 group exhibited body weight loss

of 13.8 ± 5.0 g (initial mean body weight: 115.5 ± 15.1 g). Based on these findings, we conclude that there is a strong

selective pressure on low RMR and Tb in this subterranean rodent species, which is kept in a steady state by

upstream regulation through THs. Our study highlights the need to further promote research on thyroid hormone

biology in different animal species to understand how these versatile hormones affect animal biology.

Acknowledgment The authors thank J.C. Becker for providing access to the Qubit Fluorometer for cDNA measurements. We

especially thank J. Fandrey for fruitful discussions and providing lab space at the Institute of Physiology, University

of Duisburg-Essen.


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A 08-08

Impact of ryanodine receptors on calcium-signaling of beta-cells

Johannes U. Pfabe, Sandra Postic, Srdjan Sarikas, Marjan Slak Rupnik

Medical University of Vienna, Institute of Physiology, Vienna, Austria

Question: Calcium is an important second messenger for insulin release. Likewise, ryanodine receptors (RyR) play

an important role in many physiological functions. They act as transporters of Ca2+ from the endoplasmic reticulum

to the cytosol and can, thus, influence insulin release. The current consensus model of Ca2+-signalling in β-cells does

not assign a major role for intracellular Ca2+ release. The question is whether RyR can be connected to Ca2+

oscillations and how this can add a missing layer to the model describing the physiological activity of β-cells.

Methods: Pancreas of C57BL/6J mice got injected with 1.9% low-melting point agarose into the bile duct to embed

the tissue and extracted. We used slices with a thickness of 140 µm and stained them with Calbryte 520 (Kd = 1.2

µM) as a fluorescence marker to measure calcium concentrations in the cytosol. Islets within the slices were imaged

under a confocal laser scanning microscope with a frequency of 20-30 Hz. Data was analysed within the analysis

pipeline, which recognizes individual cells as ROIs and can detect individual Ca2+ events at all time scales by

sequential filtering. For event detection, we use at least a z-score of 3 corresponding to a p-value of p<0.01.

Results: Stimulation of islets with 8 mM glucose shows a reproducible and robust response in form of calcium

oscillations. Like previous findings, we can distinguish between an initial transient phase and a plateau phase.

We could detect cytosolic Ca2+ oscillations in 3 major time domains following stimulation with 8 mM glucose. It was

possible to distinguish between ultra-fast (<1 second), fast (2-8 seconds), and slow events (10-100 seconds). When

we tried to block the oscillations by adding high concentration of ryanodine (100 µM) the ultra-fast and fast

components were not detectable any longer. Adding low-concentration of ryanodine (100 nM) at substimulatory 6

mM glucose, we could trigger Ca2+ oscillations in the ultra-fast and fast time domains.

Conclusions: Our experiments show that we could record Ca2+-oscillations as ultra-fast, fast, and slow events.

Blocking RyR by high ryanodine eliminated calcium events in the sub-second range, however, slow events remained.

In contrast, stimulatory ryanodine concentration was sufficient to stimulate Ca2+-oscillations in the ultra-fast and fast

time domain.

This shows that RyR are a sufficient and necessary component for calcium signalling in β-cells.

Acknowledgment Project “Beta-cells in diet-induced diabetes and remission” (I 3787-B21) funded by FWF.

FWF leading agency: Institute of Physiology, Medical University of Vienna

ARRS-FWF Partner: Institute of Physiology, Faculty of Medicine, University of Maribor


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Islet activity in sub-stimulatory (6 mM) and

stimulatory (8 mM) glucose A) A light microscopy image of the investigated

islet.

B) Representative traces of the ROIs inside the

pancreatic islet. #1-3 indicate legs of the

experiment. C) Histogram showing number of events in

correlation to their halfwidth for each leg. D) Plot of the halfwidth of the events, which

occured during the experiment time. E) Calcium oscillations during transient (*) and

plateau (**) phase. F) Sum over the whole islet.

Blocking RyRs removes the ultra-fast and fast

events A) A light microscopy image of the investigated

islet.

B) Representative traces of the ROIs inside the

pancreatic islet. #1-3 indicate legs of the

experiment. C) Histogram showing number of events in

correlation to their halfwidth for each leg.

Arrow: Indicates loss of ultra-fast events. D) Plot of the halfwidth of the events, which

occured during the experiment time. E) Calcium oscillations during transient (*) and

plateau (**) phase during the different steps of

the experiment. The plateau phase disappears

when ryanodine gets applied. F) Sum over the whole islet.


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A 08-09

T3 reduces transcriptional activity of MYBPC3 and TNNI3

Kathrin Kowalski1, Jan N. Riesselmann1, Natalie Weber1, Birgit Piep1, Annika Franke2, Ulrich Martin2,

Robert Zweigerdt2, Theresia Kraft1

1 Hannover Medical School, Molecular and Cell Physiology, Hannover, Germany 2 Hannover Medical School, Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG),

Hannover, Germany

The hormone triiodothyronine (T3) binds to a receptor that subsequently acts as transcription factor for several

different genes. For sarcomeric proteins it is known that T3 induces switch from β-myosin heavy chain (MyHC) to α-

MyHC and regulates transition from N2BA-titin to N2B-titin. T3 is used to mature human pluripotent stem cell-derived

cardiomyocytes (hPSC-CMs) by inducing cardiac (ventricular) properties (e.g. activation of mTOR pathway,

expression of SERCA2a and MLC2v). In this study, we aim to elucidate the effect of T3-treatment on the

transcriptional activity of the sarcomeric genes MYBPC3 and TNNI3.

We analyzed hPSC-CMs treated for seven days with T3 and 14 days after stopping treatment compared to untreated

controls. Transcriptional activity was analyzed by quantification of active transcription sites (aTS) per nucleus/cell

using mRNA fluorescence in situ hybridization. High transcriptional activity is indicated by low numbers of cells

without aTS.

Untreated hPSC-CMs showed high levels of MYBPC3 transcription, only 2% of nuclei showed no aTS. This

transcriptional activity remained unchanged under T3-treatment and 14 days after stopping treatment and was similar

to untreated controls. For TNNI3 we observed a substantial reduction in transcriptional activity from 26.5% nuclei

without aTS to 55.5% upon T3-treatment up to 76.4% at day 14 after stopping. To analyze effects of T3 on both genes

in vivo, we analyzed aTS in isolated ventricular cardiomyocytes from thyroidectomized (Thyrect)-rats (3-4 weeks after

operation, n=3) vs. Sham-operated rats (n=3). Thyrect-rats do not produce T3 and serve as an in vivo model for lack

of T3. In accordance with our findings in hPSC-CMs, TNNI3 showed an increased transcriptional activity upon T3-

withdrawal due to extraction of thyroid gland. In addition, MYBPC3 transcription also increased in Thyrect-rats,

indicating an influence of T3 on MYBPC3 transcription.In summary, we show that T3-addition reduces TNNI3

transcriptional activity in hPSC-CMs and lack of T3 in the Thyrect-rat model increases the transcription of MYBPC3

and TNNI3. Our findings indicate that T3-treatment can be detrimental for expression of particular cardiomyocyte-

specific genes and may thus negatively influence hPSC-CM maturation with regard to these genes.


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A 08-10

Dual mode of action of acetylcholine on cytosolic calcium oscillations

in pancreatic beta and acinar cells in situ

Nastja Sluga1, Sandra Postić2, Srdjan Sarikas2, Ya-Chi Huang2, Andraž Strožer1, Marjan Slak Rupnik1,2,3

1 Faculty of Medicine, University in Maribor, Institute of Physiology, Maribor, Slovenia 2 Medical University of Vienna, Center for Physiology and Pharmacology, Vienna, Australia 3 Alma Mater Europaea, European Center Maribor, Maribor, Slovenia

Questions: Acetylcholine (ACh) released by postganglionic fiber through the activation of the muscarinic receptors

directly triggers insulin release from islet beta cells [1] and digestive enzyme secretion in acinar cells [2]. We aimed

to determine whether ACh in physiological concentration can modulate beta and acinar cells responses to

physiological stimulatory glucose concentration in pancreas tissue slices. Furthermore, we questioned whether

measured responses to ACh in physiological concentration can be used to differentiate between exocrine acinar and

endocrine beta cells.

Methods: Pancreas from C57BL/6J mice were isolated and stained with Ca2+ indicator Calbryte 520. Next,

[Ca2+]c imaging on the upright confocal system was performed, while 6 or 8 mM glucose-containing extracellular

solutions were perifused to the pancreas slice [3,4]. ACh was used at supraphysiological (25 µM) or the physiological

(50 nM) concentrations. Analyses were performed as described before by Postić et al [5].

Results: Glucose and ACh in the physiological and supraphysiological range stimulate [Ca2+]coscillations in both

beta and acinar cells. Stimulation with 50 nM ACh in beta cells produced an elevation of the frequency of

the [Ca2+]cduring the plateau phase. A prominent effect of physiological ACh stimulation is complete synchronization

of the activation onsets between different beta cells in an islet. In acinar cells, the addition of 50 nM ACh triggered

[Ca2+]c oscillations, which were better synchronized compared to glucose stimulation alone. Acinar cells responded

to ACh in the physiological range sooner than beta cells. At supraphysiological ACh concentration, the frequency of

[Ca2+]c oscillations in individual beta cells was increased, however, the synchronization of the events on the plateau

phase between beta cells in an islet was completely lost. In acinar cells, supraphysiological ACh concentration

evoked a distinct [Ca2+]c transient, while further oscillations were inhibited.

Conclusions: Fresh tissue slices are the method of choice to simultaneously assess different endocrine and

exocrine cell types in the pancreas. Here we evaluated different functional features upon stimulation with ACh in beta

and acinar cells.

Acknowledgment MSR receives grants by the Austrian Science Fund/Fonds zur Förderung der Wissenschaftlichen Forschung

(bilateral grants I3562-B27 and I4319-B30). MSR and AS further received financial support from the Slovenian

Research Agency (research core funding programs P3-0396 and I0-0029, as well as projects N3-0048, N3-0133

and J3-9289). Open Access Funding by the Austrian Science Fund (FWF).


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Muscarinic activation with ACh and IP3 signaling

pathway in beta and acinar cell

(a) Acinar and beta cells express muscarinic

receptors (MXRs), activation of which results in

1,4,5-inositol trisphosphate (IP3) and

diacylglycerol (DAG) production.

IP3 specifically binds to IP3-receptor on the ER

and triggers Ca2+ mobilization, leading to an

oscillatory rise in [Ca2+]c. Additionally,

ryanodine receptors (RYR) can be activated. (b)

Color-coded time courses for beta (light brown)

and acinar cells (blue) simultaneously

stimulated with 8 mM glucose in combination with

a physiological (50 nM) or a supraphysiological

(25 μM) ACh concentration.

Time scales and heterogeneity of responses of

cytosolic Ca2+ oscillations

(a) Location of several beta, acinar and non-

active or non-beta cells in pancreas tissue

slices. (b) Time course of the [Ca2+]c changes in

a beta, acinar and non-active or non-beta cells

shown in pancreas tissue slice in (a). Frequency

histogram of the event halfwidth durations for

active beta cells is presented on (c) and (d),

onset time of the [Ca2+]c events at measured time

scales for active beta cells. (e) Frequency

histogram of the event halfwidth durations in

acinar cell and (f), onset time of the [Ca2+]c events at measured time scales for functional

regions of acinar cells.

References [1] Van Der Zee, E.A.; Buwalda, B.; Strubbe, J.H.; Strosberg, A.D.; Luiten, P.G.M. Immunocytochemical

localization of muscarinic acetylcholine receptors in the rat endocrine pancreas. Cell Tissue Res. 1992, 269, 99–106.

[2] Gautam, D.; Han, S.J.; Heard, T.S.; Cui, Y.; Miller, G.; Bloodworth, L.; Jürgen, W. Cholinergic stimulation of amylase secretion from pancreatic acinar cells studied with muscarinic acetylcholine receptor mutant mice. J. Pharmacol. Exp. Ther. 2005, 313, 995–1002.

[3] Speier, S.; Rupnik, M. A novel approach to in situ characterization of pancreatic β-cells. Pflüg. Arch. 2003, 446, 553–558.

[4] Stožer A, Dolenšek J, Rupnik MS. Glucose-Stimulated Calcium Dynamics in Islets of Langerhans in Acute Mouse Pancreas Tissue Slices. PLoS ONE. 2013;8(1):e54638.

[5] Postić, S.; Sarikas, S.; Pfabe, J.; Pohorec, V.; Bombek, L.K.; Sluga, N.; Klemen, M.S.; Dolenšek, J.; Korošak, D.; Stožer, A.; et al. Intracellular Ca2+ channels initiate physiological glucose signaling in beta cells examined in situ. BioRxiv 2021. Available online: https://www.biorxiv.org/content/10.1101/2021.04.14.439796v2 (accessed on 14 May 2021).


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A 08-11

NoxO1 and Erbin- a cooperation to control EGF-signaling ?

Maureen Hebchen1, Tim Schader1, Niklas Müller1, Christina Reschke1, Johannes Graumann2, Katrin

Schröder1

1 Goethe University, Institute for Cardiovascular Physiology, Frankfurt, Germany 2 Max Planck Institute for Heart and Lung Research, Biomolecular Mass Spectrometry, Bad Nauheim, Germany

Question: The NADPH organizer NoxO1, a subunit of the ROS-producing Nox1 complex, is involved in

angiogenesis, proliferation, and controls differentiation of colon epithelial cells [1,2]. Based on its higher expression

compared to the other Nox1 subunits we assume NoxO1 to exert additional functions, presumably by interacting with

other proteins than Nox1.

In a proximity-dependent biotin labeling approach (BioID), Erbin (ErbB2-interacting protein) [3] was identified and

validated as an interaction partner of NoxO1. Interestingly, neither the effects on cellular processes nor the

mechanisms behind are known so far. As Erbin is a negative regulator of epidermal growth factor (EGF)-signaling

[4], we speculate that NoxO1 impacts on EGF-signaling as well.

Methods and Results: Overexpression of NoxO1 in HEK293 cells results in a reduced EGF-dependent

phosphorylation of Akt and Erk1,2.

Proximity Ligation Assays showed NoxO1’s localization close to EGF receptor under resting conditions. Importantly,

upon treatment of the cells with EGF, NoxO1 was not internalized together with the EGF receptor.

Immunofluorescence uncovered an increased early endosome formation in NoxO1 overexpressing cells, which was

prevented by Erbin knockdown. Moreover, NoxO1 decreased total EGF receptor protein expression without changing

EGF receptor mRNA level or ErbB2 / EGF receptor phosphorylation.

Interestingly, siRNA based Erbin knockdown lowered EGF-induced superoxide generation by Nox1 / NoxA1 / NoxO1

in HEK cells.

Conclusion: We identified NoxO1 as a novel interaction partner of Erbin, which delays EGF-signaling. NoxO1

promotes EGF receptor internalization and potentially facilitates receptor degradation. We conclude that NoxO1 and

Erbin cooperatively regulate EGF-signal transduction and EGF receptor turnover.

References [1] Moll, Franziska; Walter, Maria; Rezende, Flávia; Helfinger, Valeska; Vasconez, Estefania; Oliveira, Tiago

de et al. (2018): NoxO1 Controls Proliferation of Colon Epithelial Cells. In: Frontiers in Immunology 9:973 [2] Schader, Tim; Reschke, Christina; Spaeth, Manuela; Wienstroer, Susanne; Wong, Szeka; Schröder, Katrin

(2020): NoxO1 Knockout Promotes Longevity in Mice. In: Antioxidants 9(3):226 [3] Borg, J. P.; Marchetto, S.; Le Bivic, A.; Ollendorff, V.; Jaulin-Bastard, F.; Saito, H. et al. (2000):

ERBIN: a basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor. In: Nature cell biology 2(7):407-414

[4] Santoni, Marie-Josée; Kashyap, Rudra; Camoin, Luc; Borg, Jean-Paul (2020): The Scribble family in cancer: twentieth anniversary. In: Oncogene 39 (47):7019–7033


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Foyers

A 09 | RENAL I Chair

Armin Kurtz (Regensburg)

Jakob Völkl (Linz)


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A 09-01

Chronic high phosphate diet is detrimental for bone health and alters

energy balance without major renal alterations

Isabel Rubio-Aliaga1, Marko Ugrica1, Soline Bourgeois1, Arezoo Daryadel1, Nati Hernando1, Pieter

Pieter Giebtsze2, Hannelore Daniel2, Carsten A. Wagner1

1 University of Zurich, Physiology, Zurich, Switzerland 2 Technical University of Munich, Nutritional Physiology, Munich, Germany

Question: Current hypotheses postulate that a higher consumption of processed foods in the last decades increase

phosphate levels in plasma. In chronic kidney disease patients, increased phosphate plasma levels are associated

with increased mortality and cardiovascular risk and an accelerated loss of kidney. Whether increased plasma

phosphate levels may be also detrimental for renal function and overall health in the healthy population is under

debate.

Method: We fed healthy mature adult mice ( 6 months old) with a standard phosphate (0.6 % w/w) and a high

phosphate diet (1.2 % w/w), mimicking the reported increased phosphate consumption in the overall population. After

12 months feeding, we assessed kidney function, energy metabolism and bone health.

Results: Animals fed a high phosphate diet showed apparently unaltered glomerular filtration rate, but lower blood

urea and higher urea clearance. Urinary ammonium excretion was markedly increased, suggesting an increased acid

load. Blood and urinary pH were more acidic in animals fed a high phosphate diet, which may be responsible for the

higher bone resorption observed and the decreased total and cortical bone mineral density. Moreover, a chronic high

phosphate intake led to hypoglycaemia. Amino acid profiling in blood showed that feeding a high phosphate diet led

to hypoglycinemia and decreased blood levels of 1-methylhistidine, a muscle turnover marker. Whereas mice under

a standard phosphate diet increased their weight over the 12 months feeding trial, mice under a high phosphate diet

did not gain weight despite similar food intake levels. Finally, indirect calorimetry experiments indicated decreased

carbohydrate utilization in mice fed a high phosphate diet when compared to mice fed a standard phosphate diet.

Conclusion: Chronic phosphate intake does not affect kidney function but alters energy balance and is detrimental

for bone health probably due to a chronic increased acid load.


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A 09-02

Osmo-Sensitivity of the adrenal gland: An intrinsic pathway to adapt

aldosterone secretion to salt intake

Allein Plain, Laura Knödl, Ines Tegtmeier, Sascha Bandulik, Richard Warth

University of Regensburg, Department of Physiology/Medical Cell Biology, Regensburg, Germany

Question: Aldosterone is a key regulator of salt balance and long-term control of blood pressure. The regulation of

aldosterone secretion by plasma K+ and angiotensin-II is well known. However, precise adjustment to salt intake

must be made to avoid excessive aldosterone secretion, which is associated with hypertension.

It is difficult to study the adaptation of aldosterone secretion to salt intake because cell models are not suitable and

living animals have a high degree of complexity. Therefore, we established a method to perfuse isolated mouse

adrenal glands and studied the effect of osmolality/plasma Na+ on the secretion of aldosterone.

Methods: Mouse adrenal glands were perfused ex-vivo, and aldosterone secretion was stimulated with K+ and

angiotensin-II at different osmolalities. Aldosterone in the perfusate was determined via ELISA. Quality controls of

the preparations were performed by histochemical staining with propidium iodide and Hoe33342, and by hypoxia-

inducible factor (HIF) staining.

Results:Ex-vivo isolated perfused mouse adrenal glands were sensitive to stimulation with K+ and angiotensin-II.

Osmolality/plasma Na+ modulated K+-induced secretion of aldosterone at the lower range of physiological K+

concentration (2-4 mmol/l). At high K+ concentrations in the perfusate (6 and 8 mmol/l), aldosterone secretion was

high, independent of osmolality. On the other hand, angiotensin-II-induced aldosterone secretion was strongly

dependent on osmolality over a wide range of concentrations.Conclusion: For the first time, mouse adrenal glands

were successfully perfused. This method provides unique opportunities to study the intrinsic regulation of aldosterone

in native adrenal glands. In particular, angiotensin-II-induced secretion, which links aldosterone secretion via renin

to blood pressure and stress levels, was strongly modulated by plasma Na+. This regulation ensures that aldosterone

secretion is suppressed under conditions in which the body is not deficient in Na+. These data establish the mouse

adrenal gland as an intrinsically osmo-sensitive organ. Future studies are needed to test if defects of osmo-sensitivity

are associated with forms of hyperaldosteronism and hypertension in humans.


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A 09-03

Functional relevance of renin produced by renal interstitial cells

Claudia Lehrmann, Anna Federlein, Robert Götz, Katharina Krieger, Rosmarie Heydn,

Katharina Broeker, Philipp Tauber, Frank Schweda

University of Regensburg, Physiology, 93053, Germany

Question: In addition to its physiological role, the renin-angiotensin system contributes significantly to the

progression of cardiovascular and renal diseases. Renin is expressed not only in juxtaglomerular cells but also in

interstitial cells of the kidney. The functional role of interstitial renin is completely unclear and was investigated in this

study.

Methods: Because renin producing interstitial cells express PDGF-receptor β (PDGFRβ), a PDGFRβ–Cre mouse

line was used to induce cell-specific deletion of renin in interstitial cells (PDGFRβ-Cre/Ren KO). Plasma renin and

prorenin concentrations, renal function and histology, and renin expression in interstitial cells were determined in

wildtype and PDGFRβ-Cre/Ren KO mice under control conditions and in the adenine nephropathy model.

Results: Whereas the number of renin expressing interstitial cells was rather low under control conditions, it

increased markedly in wildtype mice subjected to adenine nephropathy (renin mRNA in situ hybridization). As

expected, renin mRNA was not detected in interstitial cells of PDGFRβ-Cre/Ren KO mice under control or disease

conditions. Adenine nephropathy was associated with suppression of plasma renin concentration in both

genotypes. In contrast, plasma concentration of enzymatically inactive prorenin, which is associated with

exacerbated progression of renal disease, was increased 3-fold in adenine-fed wildtype mice compared with

healthy controls. In PDGFRβ-Cre/Ren KO, plasma prorenin concentration was reduced to 50% of wildtype mice

under control conditions and remained at this low level in adenine nephropathy. Glomerular filtration rate (GFR)

was not different between wildtype and KO mice under control conditions and decreased in both genotypes in

adenine nephropathy. However, the attenuation of GFR was significantly less in KO (-60% vs. control) than in WT

(-75% vs. control). Consistent with the improvement in renal injury, the increase in mRNA expression of fibronectin

and F4/80 in response to adenine was significantly attenuated in KO compared with WT mice.Conclusion: Renin

expression in PDGFRβ-positive interstitial cells is markedly stimulated in adenine nephropathy and contributes to

circulating prorenin levels. Deletion of renin in PDGFRβ-positive cells blunts the increase in plasma prorenin

concentration and ameliorates the reduction in GFR and renal injury in adenine nephropathy, indicating a role for

interstitial renin as a disease-promoting factor.

Acknowledgment A. Federlein, R. Götz, K. Krieger, R. Heydn, K. Broeker, P. Tauber, F. Schweda


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A 09-04

Mice with reduced renal angiotensin I-converting enzyme are protected

against aristolochic acid nephropathy

Annett Juretzko1, Antje Steinbach1, Jeannine Witte1, Uwe Lendeckel2, Rainer Rettig1

1 University of Greifswald, Institute of Physiology, Greifswald, Germany 2 University of Greifswald, Institute of Clinical Chemistry and Laboratory Medicine, Greifswald, Germany

Question: As it is known, that the renal renin-angiotensin system (RAS) is involved in the development of chronic

kidney disease, we investigated whether mice (C57BL/6J-tm(ACE3/3) = ACE-/-) with reduced renal angiotensin I-

converting enzyme (ACE) are protected against aristolochic acid nephropathy (AAN). To elucidate potential

molecular mechanisms, we assessed renal abundances of major components of the renal RAS.

Methods: AAN was induced using aristolochic acid I (AAI, 3 mg/kg body weight, i.p., every three days for six weeks

followed by six weeks without treatment). At the end of the protocol, the glomerular filtration rate (GFR) was

determined using inulin clearance and the kidneys were removed. Renal protein abundances of renin,

angiotensinogen (AGT), ACE2 and Mas receptor (MasR) were determined in ACE-/- and C57BL/6J control mice by

Western blot analyses. Abundances of specific protein species were determined relative to total protein abundance

and normalized to controls. Renal ACE activity was determined using a colorimetric assay and renal angiotensin

(Ang) (1-7) concentration was determined by an ELISA.

Results: ACE-/- mice had an 88 % lower renal ACE activity than controls. GFR was similar in vehicle-treated mice of

both strains. AAI decreased GFR in controls but not in ACE-/- mice. Furthermore, AAI decreased renal ACE activity

in controls but not in ACE-/- mice. Vehicle-treated ACE-/- mice had significantly higher renal ACE2 and MasR protein

abundances than controls. AAI decreased renal ACE2 protein abundance in both strains. Furthermore, AAI increased

renal MasR protein abundance, although the latter effect did not reach statistical significance in ACE-/- mice. Renal

Ang(1-7) concentration was similar in vehicle-treated mice of both strains. AAI increased renal Ang(1-7) concentration

in ACE-/- mice, but not in controls.

Conclusions: Mice with reduced renal ACE are protected against AAN. Our data suggest that in the face of renal

ACE deficiency, AAI may activate the ACE2/Ang(1-7)/MasR axis, which in turn may deploy its reno-protective effects.

Acknowledgment We thank Dr. R. A. Gonzalez-Villalobos, Dept. of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles,

CA, USA for providing the C57BL/6J-tm(ACE3/3) mice.


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A 09-05

Empagliflozin reduces hyperfiltration after uninephrectomy (UNx), but

does not protect from renal injury in a UNx/DOCA/salt mouse model

Philipp Tauber1, Frederick Sinha1, Raffaela Berger2, Wolfram Gronwald2, Katja Dettmer-Wild2,

Frank Schweda1

1 University of Regensburg, Institue of Physiology, Regensburg, Germany 2 University of Regensburg, Institute of Functional Genomics, Regensburg, Germany

The inhibition of renal glucose reabsorption by SGLT2 has proven its therapeutic efficacy in patients with diabetic

nephropathy. Clinical studies have shown that the beneficial effects of SGLT2 inhibitors on kidney function are also

relevant in non-diabetic patients with chronic kidney disease (CKD). However, the diabetes-independent

mechanisms of renal protection by SGLT2 inhibitors are still a matter of debate. A critical factor of nephroprotection

might be the reduction of glomerular hyperfiltration by SGLT2 inhibitors. In this study, we investigated the renal

benefits of the SGLT2 inhibitor Empagliflozin (EMPA) in mouse models with non-diabetic hyperfiltration and

progressing kidney disease in order to unravel the mechanisms of nephroprotection by SGLT2 inhibition.

The effect of EMPA administration was investigated in four different mouse models with varying degrees of glomerular

damage caused by non-diabetic hyperfiltration, arterial hypertenison and hypervolemia. Wildtype mice (WT) or mice

with genetic predisposition for glomerular injury were subjected to unilateral nephrectomy (UNx) and a combination

of deoxycorticosterone acetate (DOCA) treatment and/or high NaCl diet. Renal function (GFR, albuminuria, urine

volume) was monitored for 4-6 weeks before mice were sacrificed and kidneys used for histological examination.

EMPA did not affect baseline GFR in WT animals, but decreased hyperfiltration after UNx by 24 %. In all our kidney

disease models single kidney GFR was raised by 35-112 % after UNx depending on baseline GFR of the

corresponding model. But, in contrast to WT animals, EMPA had no effect on hyperfiltration in our kidney disease

models, regardless of the severity of renal impairment caused by DOCA/salt treatment. In addition, all EMPA-treated

animals showed the same progression of albuminuria, renal fibrosis and hypervolemia as H2O control animals. EMPA

treatment induced glucosuria, osmotic diuresis and in some disease models kidney hypertrophy, possibly due to a

compensatory increase in tubular reabsorption activity of distal tubule segments.

Taken together, our data proved that EMPA reduces UNx-induced hyperfiltration in untreated WT animals. However,

the effect on hyperfiltration was not observed in UNx/DOCA/salt mouse models and EMPA did not protect from

progression of kidney damage. Therefore, the UNx/DOCA/salt mouse model seems not suitable for investigations of

nephroprotective mechanisms of SGLT2 inhibitors.

Acknowledgment We thank the Robert Götz, Katharina Krieger and Rosmarie Heydn (Institute of Physiology, University of

Regensburg, Regensburg, Germany for their excellent technical assistance.


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A 09-06

Relationship between GFR, intact PTH, oxidized PTH, non-oxidized PTH

as well as FGF23 in patients with CKD

Steffen Rausch1, Shufei Zeng2, Uwe Querfeld3, Martina Feger1, Bernhard K. Krämer2, Michael Föller1,

Berthold Hocher2

1 University of Hohenheim, Department of Physiology, Stuttgart, Germany 2 University of Heidelberg, Univ. Med. Centre Mannheim, Fifth Dep. of Medicine

(Nephrology/Endocrinology/Rheumatology, Heidelberg, Germany 3 Charité-Universitätsmedizin Berlin, Department of Nephrology, Berlin, Germany

Question: Fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) are regulators of renal phosphate

excretion and vitamin D metabolism. In chronic kidney disease (CKD), circulating FGF23 and PTH concentrations

progressively increase as renal function declines. Oxidation of PTH at 2 methionine residues (positions 8 and 18)

causes a loss of function. The impact of n-oxPTH and oxPTH on FGF23 synthesis, however, and how n-oxPTH and

oxPTH concentrations are affected by CKD, is yet unknown.

Methods: The effects of oxidized and non-oxidized PTH 1-34 on Fgf23 gene expression were analyzed in UMR106

osteoblast-like cells. Furthermore, we investigated the relationship between n-oxPTH and oxPTH, respectively, with

FGF23 in two independent patients’ cohorts (620 children with CKD and 600 kidney transplant recipients).

Results: While n-oxPTH stimulated Fgf23 mRNA synthesis in vitro, oxidation of PTH in particular at Met8 led to a

markedly weaker stimulation of Fgf23 expression. The effect was even more pronounced when both Met8 and Met18

were oxidized. In both clinical cohorts, n-oxPTH – but not oxPTH – was significantly associated with FGF23

concentrations, independent of known confounding factors. Moreover, with progressive deterioration of kidney

function, intact PTH (iPTH) and oxPTH increased substantially, whereas n-oxPTH increased only moderately.

Conclusions: Mainly non-oxidized PTH, not oxidized PTH, stimulates Fgf23 gene expression. The increase in PTH

with decreasing GFR is mainly due to an increase in oxPTH in more advanced stages of CKD.


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A 09-07

Renal Nox4 contributes to systemic redox homeostasis by controlling

cysteine and folate metabolism

Flávia Rezende1,2, Melina Lopez1,2, Timothy Warwick1,2, Niklas Müller1,2, Pedro Felipe Malacarne1,2,

Maria P. Pacheco3, Tamara J. R. Bintener3, Thomas Sauter3, Katrin Schröder1,2, Luciana Hannibal4,

Ralf Brandes1,2

1 Goethe-University, Institute for Cardiovascular Physiology, Frankfurt am Main, Germany 2 German Center of Cardiovascular Research, Rhein Main, Germany 3 Université du Luxembourg, Life Sciences Research Unit, Luxembourg, Germany 4 University Medical Center Freiburg, Centre for Paediatric and Adolescent Medicine, Freiburg, Germany

The NADPH oxidase Nox4 produces H2O2 and is highly expressed in the kidney. Its expression is reduced in diabetic

renal disease, suggesting its role in normal renal function. To uncover the physiological role of Nox4 in the kidney,

we first defined its localization. As demonstrated by in situ hybridization (RNAscope) combined with

immunofluorescence, Nox4 is selectively expressed in the proximal tubule. As this part of the kidney mediates mass

reabsorption and secretion, WT (wild type) and Nox4-/- (tamoxifen-inducible, global Nox4 knockout mice) were put

on a sugar/fat diet, with low sodium (130mg/kg chow), low micro nutrients and no protein. Urine samples (day 0, 3,

14), renal cortex and plasma (day 14) were analyzed by global untargeted LC/MS for metabolites. Metabolic

reconstruction using Fastacore showed a significant downregulation on extracellular and mitochondrial transport;

metabolism of nucleotides, inositol phosphate and folate in Nox4-/- mice. Moreover, metabolites of histidine

catabolism were significantly upregulated in Nox4-/-. Systematic differences in cysteine, cystine and

homocysteine were observed in plasma and renal cortex of Nox4-/- as compared to WT animals. The results using

metabolomics indicate that the physiological function of Nox4 in the kidney is to control reabsorption and metabolism

of amino acids and vitamins of the complex B which are important for redox homeostasis.


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A 09-08

The chloride channel CFTR is not required for cyst growth in an

ADPKD mouse model

Khaoula Talbi1, Inês Cabrita1, Andre Kraus2, Sascha Hofmann1, Kathrin Skoczynski2,

Karl Kunzelmann1, Bjoern Buchholz2, Rainer Schreiber1

1 University of Regensburg, Physiological Institute, Regensburg, Germany 2 Friedrich-Alexander-University, Department of Nephrology and Hypertension, Erlangen, Germany

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic disease caused by

mutations in either PKD1 or PKD2 genes. ADPKD ischaracterized by cyst development induced by enhanced cell

proliferation and electrolyte secretion into the cyst lumen, which leads to progressive loss of renal function. The

cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) is believed to be the main channel

responsible for chloride secretion. However, while the role of CFTR for cyst formation in vivo remained to be

demonstrated, the calcium-activated Cl- channel TMEM16A was shown recently to be essential for cyst formation in

vivo in a mouse model for ADPKD.

Methods: Single knockout of Pkd1 (Pkd1-/-) and double knockout of Pkd1 and Cftr (Pkd1-/-/Cftr-/- ) were induced in

male mice by intraperitoneal injection of tamoxifen. Noninduced mice served as control. We performed histological

analysis to determine the cystic index and Ki-67 staining to examine cell proliferation. Expression of TMEM16A and

CFTR were demonstrated by immunohistochemistry , and whole cell Cl- currents were examined by patch-clamp and

iodide-induced quenching of yellow fluorescent protein (YFP).

Results: Knockout of Pkd1 induced renal cysts and cell proliferation, which was not attenuated by additional

knockout of Cftr. Similarly, enhanced cell proliferation was still observed in Pkd1-/- /Cftr-/- kidneys. Upregulation of

both TMEM16A and CFTR was detected by immunostaining in Pkd1-/- mice, and upregulation of TMEM16A-

expression was still detectable in Pkd1-/- CFTR-/- mice. Patch clamp analysis indicated a minor contribution of cAMP-

activated CFTR currents to whole cell Cl- currents in primary renal epithelial cells from Pkd1-/- mice, while Ca2+

activated TMEM16A currents dominated. Results from patch clamp experiments were confirmed by YFP-

fluorescence assay.

Conclusion: CFTR chloride currents are not required to develop a cystic phenotype in an ADPKD mouse model.

Enhanced cell proliferation and Cl- secretion leading to cyst formation is caused primarily by the upregulation of the

calcium-activated Cl- channel TMEM16A.

Acknowledgment We are grateful to Prof. Dr. Dorien J.M. Peters (Department of Human Genetics, Leiden University Medical Center,

Leiden, The Netherlands) for providing us with the ADPKD mouse model. The mice with a floxed CFTR allele were

kindly provided by Dr. Mitchell Drumm and Dr. Craig Hodges (Case Western Reserve University, 10900 Euclid

Avenue, Cleveland, Ohio 44106-7219).


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A 09-09

Cellular crosstalk protects renal cells against acidosis-induced

inflammatory and fibrotic alterations.

Marie-Christin Schulz, Gerald Schwerdt, Michael Gekle

Martin-Luther-Universität, Julius Bernstein Institute of physiology, Halle (Saale), Germany

Pathogeneses of CKD often comprises inflammation, fibrosis and epithelial-to-mesenchymal transition (EMT).

Inflammation and fibrosis are often accompanied by interstitial acidosis. Moreover, altered cellular crosstalk can be

involved in the development of these pathomechanisms. Aim of this study was to assess the influence of extracellular

acidosis on proximal tubule cells and renal fibroblasts in dependence of cellular crosstalk.

Proximal tubule cells (NRK-52E) and renal fibroblasts (NRK-49F) were used in mono and co-culture. Cells were

treated with acidic media for 48h. Protein expression of inflammatory (TNF, COX-2), epithelial (E-cadherin),

mesenchymal (vimentin) and fibrotic (collagen I, fibronectin) proteins was measured by western blot. Secreted

collagen I and fibronectin was quantified by ELISA. Subcellular distribution of E-cadherin was assessed by

immunofluorescence and epithelial barrier function by FITC-dextran diffusion. mRNA-expression was measured by

qPCR and activity of matrix metalloproteases (MMPs) by an fluorescence assay.

Inflammation: Acidosis led to an increase of COX-2 protein and mRNA expression in NRK-52E as well as TNF protein

but not mRNA expression in NRK-49F in monoculture. In co-culture acidosis led to a decrease of COX-2 and TNF

protein in both cell lines. The COX-2 mRNA was increased in NRK-52E as well as the TNF mRNA in NRK-49F.

EMT: Acidosis led to an increase of vimentin protein and mRNA in both cell lines. In NRK-52E the E-cadherin

expression was unchanged but subcellular E-cadherin showed a disturbed distribution. The cellular barrier function

was decreased. Under co-culture conditions in both cell lines the acidosis-induced impact on vimentin protein

vanished whereas the vimentin mRNA was increased. In NRK-52E E-cadherin protein expression was unchanged.

Fibrosis: Acidosis led to an increased secretion and mRNA expression of collagen I and fibronectin in NRK-52E in

monoculture. In NRK-49F the secretion of collagen I was increased and fibronectin was unchanged. In co-culture the

total collagen I secretion was unchanged and fibronectin secretion was decreased. In NRK-52E increase of collagen

I and fibronectin mRNA was measured. Regarding MMP-activity the only observed acidosis-induced alteration was

a decrease NRK-52E cells, in co-culture.

1 These data provide evidence for acidosis-induced inflammatory and fibrotic response as well as EMT.

2 Cellular crosstalk is protective against acidosis-induced alterations.


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A 09-10

Example for Integrative Pathophysiology: Radiogenic Urinary Bladder

(UB)

Michael C. Michailov1, Eva Neu1, Thomas Plattner1, Tatjana Senn1, Hartwig W. Bauer1,2, Alfons

Hofstetter1,3, Gero Hohlbrugger1,4, Helmut Madersbacher1,5, Ernst R. Weissenbacher1,6

1 Institute Umweltmedizin c/o ICSD/IAS e.V. POB 340316, 80100 Muenchen, Germany (Int. Council Sci.

Develop./Int. Acad. Sci. Berlin-Bratislava-Innsbruck-Muenchen-NewDelhi-Paris-Sofia-Vienna), Muenchen,

Germany 2 FU Berlin & Univ. München, Med. Fac., Berlin, Germany 3 Univ. München, Klinikum Großhadern (Ex-Dir.), München, Germany 4 Univ. Innsbruck, Med. Fac. & Univ. Vienna, Innsbruck, Austria 5 Med. Univ. Innsbruck (Klinikum Innsbruck, Ex-Dir.), Innsbruck, Austria 6 Univ. München, Med. Fac. & Practice Premium Med., München, Germany

INTRODUCTION:

Vesicalpathophysiology is essential for whole organisms – cystitis can cause pyelonephritis followed by renal

hypertension. Model for multidimensional&holistic, i.e. functional&morphological investigations of radiocystitis is

given.

METHODS:

UB-motor/electrical activities: X-/gamma-radiation=XR, microscopy (ref).

RESULTS:

I. Patients (Cystotonometry, n=150). Increased vesical pressure, prolonged micturition, decreased urothelial

potentials is observed 1-12months after oncological radiotherapy (Gamma-/X-rays70Gy).

II. Pathophysiology. XR induce tonic contractions in detrusor preparations: Human=H (>1Gy, surgical

tissue)/guinea-pig=GP/rat (>10-50Gy), increase of spontaneous phasic/SPC=4.04 ±0.75/min, decrease of tonic

contractions of trigonum/STC=0.28± 0.15/min: Negative chronotropic effect after irradiation (>10Gy) is observed.

SPC/STC appear also in UB in-toto/in-vitro et in-vivo (n=420,p<0.01). Detrusor myocytes (intracellular-rec,

n=120,p<0.01): After stretch (3-80mN) appear electrical spike-transformation (63.29±4.96/min) into burst-plateaus

(1.54±1.18/min) augmentation rate of spikes-rise (RR: 0.41±0.19/3.27±0.76V/sec) & -fall (RF

0.43±0.28/2.32±0.58V/sec resp), caused by mechanosensitive ionic channels. No information about radiogenic

effects: Research could clarify pathogenesis of radiogenic incontinence.

III. Pathomorphology. After local XR of UB in-vivo/rats (2-6 weeks) appear histologically hyperemia, lymphocyte

infiltration, interstitial fibrosis. Ultrastructure (electron microscopy) demonstrate essential differences between H-

&GP-detrusor, eg average normal myocyte-separation H:500nm, GP:100-150nm. XR-200Gy increase cytoplasmic-

vacuoles near nuclei, augmented damage-susceptibility.

CONCLUSION:

Similar models for an integrative pathophysiology (I.-IV.) about immediate/acute/chronic radiogenic effects on

different level (patient to cellular-molecular) can permit better analysis of pathogenesis, eg radiocystitis supporting

more effective therapy-prophylaxis of bladder hypertonia, incontinence,etc.

REFERENCES:


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IARR-2019-Manchester (IntCongrRadRes) Abs-Book Po.no 116,117,124,192. IARR-2015-Kyoto,

AB:p201,255,825.

DPG-2019-Ulm, Acta Physiol. 227,S719, p108-109.

FEPS-2018-London-Europhysiol. AB:p334P-337P.

IUPS-2017-Rio-de-J. (physiol.), AB.No 997,999,1001,1003. IUPS-2009-Kyoto JPhysiolSci 59/S1:p168.

SIU-2016-B.Aires, J.Urol 34/S1,126&223. SIU-2007-Paris, J.Urol 70/S3A:232-3. SIU-2004-Honolulu BrJUrol

94:24-5/258-9/305.


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A 09-11

The impact of RAAS-modulating drugs on the metastatic behavior of

melanoma cells

Yannic Becker, Christian Stock

Hannover Medical School, Gastroenterology, Hepatology and Endocrinology, Hannover, Germany

Angiotensin II (ATII) receptor blockers (ARBs) and inhibitors of the angiotensin converting enzyme (ACE) are applied

to lower blood pressure in hypertensive patients. However, a number of clinical studies cannot rule out a possible

relationship between ARB therapy and an increased risk of cancer. Recent studies show that ARBs affect melanoma

cell behavior, possibly by interfering with the regulation of the Na+/H+ exchanger 1 (NHE1). Whether or not the ACE

as a potential ATII receptor and signal transducer is also involved is a moot question.

The expression of ACE and the angiotensin receptor types 1 and 2 (AT1R, AT2R) in human melanoma (MV3) cells

was verified by qRT-PCR. Employing BCECF and the NH4Cl/acid pre-pulse technique, inhibition of ACE, AT1R and

AT2R by Lisinopril, Losartan and EMA, respectively, was found to decrease NHE1 activity. Time-lapse video

microscopy revealed that MV3 cells seeded on a confluent Huh7 (liver cancer) cell layer became significantly more

invasive in response to each one of the drugs as shown by both the number of invasive cells and the time of invasion.

The drugs did not affect the integrity of the epithelial Huh7 cell layer, measured as transepithelial electrical resistance.

In contrast, Losartan alone decreased MV3 cell invasion of a reconstituted collagen I matrix, stimulated by Huh7-

conditioned medium. This effect could be antagonized by simultaneous application of Lisinopril. EMA, on the other

hand, counteracted the anti-proliferative effect of Lisinopril. None of the drugs affected MV3 cell adhesion to a

collagen I matrix.

In summary, AT1R, AT2R and ACE inhibitors decreased NHE1 activity, however, increased MV3 cell invasion of an

epithelial Huh7 cell layer without weakening its integrity. While AT1R and ACE inhibition did not cause an additive

effect on MV3 cell invasion of the Huh7 cell layer, ACE inhibition by Lisinopril counteracted the inhibitory effect of

Losartan on stimulated MV3 cell invasion of a collagen I matrix. We conclude that in addition to AT1R, also AT2R and

ACE modulate human melanoma cell invasion.


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Foyers

A 10 | Cardiomyocytes Chair

Katharina Bottermann (Düsseldorf)

Wolfgang Linke (Münster)


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A 10-01

Effects of a phospholipase A2 homologue from the venom of the snake

Bothrops asper on the cell membrane and sarcomeric proteins of rat

cardiac muscle cells

Alfredo J. López Dávila1, Natalie Weber1,2, Faramarz Matinmehr1, Theresia Kraft1, Julián Fernández3,

José María Gutiérrez3, Bruno Lomonte3

1 Medizinische Hochschule Hannover, Institut für Molekular- und Zellphysiologie, Hannover, Germany 2 Medizinische Hochschule Hannover, Institut für translationale und therapeutische Strategien, Hannover, Germany 3 Universidad de Costa Rica, Instituto Clodomiro Picado, San José, Costa Rica

Venom from Bothrops asper snake contains high concentrations of Myotoxin-II (Mt-II), a Lys-49 phospholipase A2

homologue lacking catalytic activity. Mt-II is highly toxic for skeletal muscle cells but displays variable toxicity to other

cell types. In skeletal muscle Mt-II triggers spontaneous intracellular calcium transients, disruption of the cell

membrane, hypercontraction and cell collapse leading to muscle necrosis. Recent reports have strongly suggested

that Mt-II enters into the cytosol of skeletal muscle cells, after interacting with proteins of the cell membrane. However,

intracellular cytotoxic effects have not been described for Mt-II so far.

We used cardiac muscle cells as a model for studying the cytotoxicity of Mt-II on the cell membrane and intracellular

proteins from a molecular and biophysical perspective. In intact rat cardiomyocytes Mt-II induced a) disruption of the

cell membrane, b) delayed calcium transients after electrical stimuli, c) increased intracellular calcium concentration

and d) irreversible hypercontraction. On the contrary, in demembranated rat cardiomyocytes, Mt-II did not modify

isometric force generation, nor rate constants, calcium sensitivity and cooperativity of force development. Rat cardiac

muscle cells are a suitable model to study the cytotoxic effects of Mt-II on striated muscle. All detected effects harm

the cell membrane and the excitation contraction coupling, inclusive the calcium handling. Mt-II does not target

sarcomeric proteins and possible cytotoxic effects of its internalization remain unknown.

References [1] Massimino ML, Simonato M, Spolaore B et al. (2018) Cell surface nucleolin interacts with and internalizes

Bothrops asper Lys49 phospholipase A2 and mediates its toxic activity. Scientific reports 8(1):10619. doi: 10.1038/s41598-018-28846-4.

[2] Gutiérrez JM, Escalante T, Hernández R et al. (2018) Why is Skeletal Muscle Regeneration Impaired after Myonecrosis Induced by Viperid Snake Venoms? Toxins 10(5). doi: 10.3390/toxins10050182

[3] Vargas-Valerio S, Robleto J, Chaves-Araya S et al. (2021) Localization of Myotoxin I and Myotoxin II from the venom of Bothrops asper in a murine model. Toxicon : official journal of the International Society on Toxinology 197:48–54. doi: 10.1016/j.toxicon.2021.04.006


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A 10-02

Differences in myosin light chain composition influence the sliding

velocity of actin filaments on otherwise identical slow skeletal and

venticular myosin molecules

Jennifer Reitmeier, Maral Mohebbi, Tianbang Wang, Petra Uta, Theresia Kraft, Mamta Amrute- Nayak,

Tim Scholz

Hannover Medical School, Molecular and Cell Physiology, Hannover, Germany

In vitro motility assays are a powerful method for studying the function of tissue purified or expressed motor proteins

such as myosins. The actin sliding in vitro motility assay on surface-bound myosin molecules is generally used to

compare different myosin isoforms and constructs or to study disease-associated mutations of e.g. human ventricular

myosin. Due to sample and experimental limitations, fibers from slow skeletal muscles such as M. soleus are used

as a model system to study slow myosin function in the cellular environment as both slow skeletal and ventricular

muscle express the same myosin heavy chain.

However, we found that gliding of actin filaments on tissue purified ventricular myosin is significantly faster than on

slow skeletal muscle myosin. This difference in gliding speed is independent of the assay chamber surface, and it

also persists when tissue purified native thin filaments from M. soleus or ventricular tissue were used instead of

unregulated actin filaments. We also found that the source of native thin filaments exerts a wide influence on the

sliding velocity. While cardiac thin filaments accelerate sliding, slow skeletal muscle thin filaments decrease the

sliding velocity both on cardiac and slow skeletal myosin molecules. As the faster actin gliding on ventricular myosin

could not be explained by contaminations with faster atrial myosin, we tested for differences in myosin light chain

composition. Unlike ventricular myosin molecules, M. soleus myosin contains the essential myosin light chain MyLC-

1sa in addition to the essential light chain MyLC-1sb/v, even on the single muscle fiber level. A higher relative content

of MyLC-1sa correlated to significant slower actin gliding.

From our results we conclude that the presence of the essential myosin light chain MyLC-1sa slows down actomyosin

interactions, and, therefore, an influence of myosin and native filaments sources should be considered, e.g. when

disease-associated mutations in cardiac myosin are studied.

Acknowledgment The authors would like to thank the Fritz-Thyssen-Stiftung for a grant to MAN and the Young Faculty programm 2.0

Hannover Medical School for financial support to TS.


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A 10-03

Muscular dystrophy in mice overexpressing myofibrillar myopathy

causing hBAG3P209L

Kathrin Graf-Riesen1, Stefan Arnhold2, Bernd K. Fleischmann1, Michael Hesse1

1 University of Bonn, Medical Faculty, Institute of Physiology I, Bonn, Germany 2 University of Gießen, Institute of Veterinary Anatomy, Histology and Embryology, Gießen, Germany

The co-chaperone BAG3 (Bcl-2 associated athanogene 3) is strongly expressed in cross-striated muscles and plays

a key role in the turnover of muscle-proteins as a member of the CASA (chaperone-assisted selected autophagy)

complex. An amino acid exchange (P209L) in the human BAG3 gene, caused by a single base mutation, gives rise

to a severe dominant childhood muscular dystrophy, restrictive cardiomyopathy, and respiratory insufficiency.

To get deeper insights into the pathophysiological mechanisms of the disease, we generated a transgenic mouse

model of the human mutation BAG3P209L, in which a fusion protein consisting of the human BAG3P209L and the green

fluorescent protein eGFP can be conditionally overexpressed. Ubiquitous overexpression of BAG3P209L-eGFP leads

to a severe phenotype starting a few weeks after birth, including decreased body weight, skeletal muscle weakness,

and heart failure.

Examination of skeletal muscles of transgenic mice revealed a robust expression of the BAG3P209L-eGFP transgene.

The histological structure of the skeletal muscle of CAG-BAG3P209L-mice was impaired compared to littermate

controls. Particularly striking was the increased number of centralized nuclei, which is a sign of muscle regeneration,

while the number of satellite cells in soleus and EDL muscles was similar to controls. Immunofluorescence stainings

for alpha-actinin and desmin showed protein aggregate formation and Z-disc disruption in CAG-BAG3P209L-mice

Similar to the hearts of CAG-BAG3P209L-mice there was also a significant amount of fibrosis in skeletal muscle.

Interestingly, we noticed a switch in skeletal muscle fiber types from fast-twitch to slow-twitch types as has been

described for other muscular dystrophies. Additionally, MHC-IIb fibers had a smaller diameter in the EDL muscle of

CAG-BAG3P209L-mice. For functional assessment, we performed myograph measurements and found a striking

decrease of force development in CAG-BAG3P209L-mice. First experiments with a motion tracker system hinted that

CAG-BAG3P209L-mice are less active than their littermate controls.

In summary, we could show that CAG-BAG3P209L-mice display symptoms of severe muscular dystrophy as described

in patients suffering from BAG3P209L myofibrillar myopathy.

Acknowledgment K. Graf-Riesen is supported by the DFG (FOR 2743)


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A 10-04

Measuring metabolic fluxes and mitochondrial function in intact

cardiac tissue slices

Andre Heinen1, Katharina Bottermann2, Stefanie Gödecke1, Axel Gödecke1

1 Medizinische Fakultät und Universitätsklinikum Düsseldorf, Heinrich-Heine-Universität, Institut für Herz- und

Kreislaufphysiologie, Düsseldorf, Germany 2 Medizinische Fakultät und Universitätsklinikum Düsseldorf, Heinrich-Heine-Universität, Institut für Pharmakologie

und Klinische Pharmakologie, Düsseldorf, Germany

Question: Analysis of substrate metabolism in different cell lines using extracellular flux analyser has become a

standard tool in multiple research areas. It would be of interest to use this technique also in cardiac tissue as changes

in cardiomyocyte metabolism are a hallmark of many cardiac diseases including diabetic cardiomyopathy and heart

failure. However, the results from metabolic analysis of isolated cardiomyocytes might be affected by the isolation

and cultivation procedure that are required for cell selection and attachment to the cell culture plates. Therefore, we

aimed to develop a Seahorse XFe 24 flux analyser-based method for the metabolic and mitochondrial determination

of intact cardiac tissue slices.

Methods: Mouse cardiac tissue was embedded in low melting agarose, and sliced in tangential orientation starting

from the epicardium using a vibratome. Tissue pieces of these slices were punched out to yield samples of

comparable size, and were transferred to “islet capture plates”. Glucose (Glc), glutamine (Glu) and pyruvate (P)

enriched medium was used, and oxygen consumption rates (OCR) were measured at baseline, after FCCP-

stimulation and after electron transport chain inhibition by rotenone and antimycin A (Rot/AA). As fatty acids are an

important substrate in cardiomyocytes, additional experiments were performed using a substrate combination of Glc,

Glu and palmitate. Long-chain fatty acid (LCFA) metabolism was inhibited by etomoxir, and Glc/Glu metabolism by

UK5099/BPTES.

Results: Using Glc, Glu and P as exogenous substrates, FCCP increased OCR by 163±72% compared to baseline

(figure 1A, n=7). 86% of the maximal OCR were due to mitochondrial electron chain activity. Cardiomyocytes seem

to be resistant to Oligomycin as OCR did not decrease as expected. In experiments with Glc, Glu and palmitate as

substrate, inhibition of LCFA- and Glc/Glu metabolism reduced FCCP-stimulated OCR by 52±7 pmol/min (figure 1B,

n=3). The dependency on LCFA-oxidation was 52±13% of all pathway inhibition (Glc+Glu+LCFA) indicating that both

LCFA and Glc/Glu are metabolised.Conclusions: Cardiac metabolism and mitochondrial function can be measured

in intact cardiac tissue slices using extracellular flux analyser. Importantly, this method offers a promising approach

to investigate cardiac metabolism and substrate preferences, as not only Glc/Glu but also LCFA are metabolised.

Figure 1


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A 10-05

Subcellular control of cAMP microdomain signaling using targeted

optogenetics

Berivan Mansuroglu, Philipp Sasse

University of Bonn, Institute of Physiology I, Medical Faculty, Bonn, Germany

In cardiomyocytes cAMP/PKA-dependent phosphorylation of L-type Ca2+ channels (LTCC) and Ryanodine receptors

2 (RyR2) increases contractile force but their relative contribution is difficult to predict and due to the close vicinity of

both proteins, it is unclear, if cAMP/PKA microdomains are functionally separated or cross-talking. To investigate

functional differences from selective phosphorylation we aim to generate LTCC and RyR2 cAMP microdomains by

light using subcellular targeting of the photo-activated adenylate cyclase from Turneriella parva (TpPAC)1. TpPAC

was targeted together with EYFP or mCitrine to the RyR2 by fusion with FKBP12.6, due to its high affinity to RyR22

and to the LTCC by fusing with a nanobody (nb.F3), which was shown to bind the LTCC ß4 subunit3. Non-targeted

TpPAC-EYFP served as control for global cytosolic cAMP elevation. Intact light-dependent cAMP generation was

analyzed in HEK293 cells co-expressing the TpPAC constructs and the cAMP-sensitive luminescence-based

GloSensor, in which blue light elevated cAMP levels in a light dose-dependent manner.

For functional analysis, constructs were expressed in neonatal mouse cardiomyocytes by nucleofection. Non-

targeted TpPAC showed EYFP distribution in α-actinin positive cardiomyocytes without signs of aggregation

indicating homogeneous cytosolic expression. In contrast, TpPAC-mCitrine-FKBP12.6 localized around the nuclear

envelope indicating RyR2/sarcoplasmic reticulum targeting and nb.F3-TpPAC-EYFP showed mixed results ranging

from intracellular aggregations, uniform expression and some cell membrane targeting. Initial functional

characterization was performed by analyzing light-dependent changes of spontaneous beating. Brief blue light

flashes resulted in transient acceleration of beating frequency in a light dose-dependent manner to a maximum

increase of +72% for non-targeted, +61% for RyR2 targeted and +27% for LTCC-targeted TpPAC.

In conclusion, fusion constructs for subcellular targeted optogenetics enables to generate localized cAMP levels. In

future, combination with patch clamp and Ca2+ imaging will allow to determine the relative contribution of LTCC and

RyR2 phosphorylation to inotropy and potential pathologies from non-balanced phosphorylation.

Acknowledgment Deutsche Forschungsgemeinschaft (German Research Foundation, 313904155/SA1785/7-1,

315402240/SA1785/8-2; 380524518/SA1785/9-1, 214362475/GRK1873/2)

References [1] Penzkofer A., Tanwar M., Veetil S.K., Kateriya S. Photo-dynamics of photoactivated adenylyl cyclase TpPAC

from the spirochete [2] Despa, S., Shui, B., Bossuyt, J. B. C., Lang, D., Kotlikoff, M I., Bers, D. M. Junctional Cleft [Ca2+]i

Measurements Using Novel Cleft-Targeted Ca2+ Sensors. Circ Res. 115:339-347, doi: 10.1161/CIRCRESAHA.115.303582, 2014.

[3] Morgenstern T., J., Park, J., Fan, Q., R., Colecraft, H., M. A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVβ subunits. eLife 8:e49253, doi: 10.7554/eLife.49253, 2019.


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A 10-06

Frequency modulation of cardiomyocytes by optogenetic stimulation

of Gi signaling using long wavelength coneopsin.

Daniela Malan1, Milan Cokić1, Tobias Brügmann2,3, Philipp Sasse1

1 University of Bonn, Physiology I, Bonn, Germany 2 University Medical Center Göttingen, Institute for Cardiovascular Physiology, Göttingen, Germany 3 German Center for Cardiovascular Research (DZHK), Göttingen, Germany

G-protein-coupled receptors play a pivotal role in regulating cardiac function. To study these receptors and

downstream signaling with high spatial and temporal precision in cardiomyocytes, optogenetic tools to activate Gs

with blue light have been reported before. The aim of this study was to show optogenetic activation of the important

counteracting Gi signaling cascade in cardiomyocytes using the red light-activated long wavelength-sensitive cone

opsin (LWO).

Transgenic mouse embryonic stem cells expressing the human LWO in fusion with EYFP under the control of the

CAG promoter were generated and two independent lines were differentiated into cardiomyocytes within embryoid

bodies. α-actinin positive cardiomyocytes showed LWO-EYFP expression at the plasma membrane and illumination

with 625 nm led to a nearly complete block of the beating activity in both cell lines (block of activity: 98.5 ± 1.5 %,

n=11 and 84.1 ± 6.1 %, n=11). Application of identical light pulses to control cardiomyocytes expressing only GFP

did not affect beating. Baseline beating frequency in the dark was similar between LWO and control clones,

suggesting that LWO has no dark activity in cardiomyocytes.

By using various calibrated light intensities, we determined a light sensitivity for 10 s pulses with a half maximal

effective light intensity of 1.2 µW/mm2 and a maximum effect at 100 µW/mm2. Compared to pharmacological

activation of the Gi-coupled M2 receptors with Carbachol (CCh), optogenetic reduction of beating rate by LWO was

similar in magnitude but was much faster in peak activation (LWO 0.82 ± 0.10 s; CCh 33.43 ± 7.85 s) and 50%

recovery after stimulation (LWO 0.80 ± 0.33 s; CCh 99.57 ± 21.86 s, all values n=7, p<0.01). LWO did specifically

activate the Gi signaling pathway because incubation with pertussis toxin to block Gi proteins completely abolished

light effects. The reduction of frequency was mainly through activating GIRK channels by Gi-β/γ subunits because

the specific blocker Tertiapin reduced the light effect by ~80%.

Thus, LWO is a suitable optogenetic tool to specifically activate the Gi signaling pathway in cardiomyocytes with red

light and with much higher temporal resolution than pharmacologic stimulation. The red light sensitivity of LWO will

allow combination with the blue-light sensitive optogenetic Gs coupled receptor Jellyfish Opsin to study interaction of

sympathetic and parasympathetic stimulation in cardiomyocytes in vitro and in vivo.

Acknowledgment Funding: Deutsche Forschungsgemeinschaft (German Research Foundation, 313904155/SA1785/7-1,

380524518/SA1785/9-1, 214362475/GRK1873/2)


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A 10-07

Functional Analysis Of Radiation Effects On Organs At Risk Following

Microbeam Irradiation

Bernd Frerker1, Elisabeth Schültke1, Rüdiger Köhling2,3, Falko Lange2,3, Kathrin Porath2,

Paula Stöhlmacher2, Stefan Fiedler4, Julian P. Moosmann5, Fabian Wilde5, Mitzi Klein6, Michael Lerch7,

Timo Kirschstein2,3, Guido Hildebrandt1

1 Rostock University Medical Center, Department of Radiation Oncology, Rostock, Germany 2 Rostock University Medical Center, Oscar Langendorff Institute of Physiology, Rostock, Germany 3 University of Rostock, Center of Transdisciplinary Neurosciences, Rostock, Germany 4 European Molecular Biology Laboratory (EMBL), Fiedler Team, Hamburg, Germany 5 Helmholtz-Zentrum Hereon, Outstation at Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany 6 Australian Nuclear Science and Technology Organisation (ANSTO), Australian Synchrotron, Melbourne, Australia 7 University of Wollongong, Centre for Medical Radiation Physics (CMRP), Wollongong, Australia

Background: The heart and the oesophagus are frequently exposed to radiation during the treatment of thoracic

tumours. They either can be in the planning target volume (PTV, high dose region), or they are exposed to lower

doses as organs at risk (OAR). Consequently, there is a risk to develop acute or late side effects.

Oesophagus: The radiation induced oesophagitis is one of the most common and dose-limiting acute toxicities during

the treatment of thoracic tumours. Affected patients complain dysphagia due to mucosal damage, but mucosal

damage is not a mandatory finding. Alternatively, the dysphagia may be due to radiation induced motility disorder.

Clinical trials found an impaired oesophageal transit during radiation therapy, but the results are controversially

discussed. Preclinical data supporting this assumption is rare.

Heart: Like the oesophagus, the heart may suffer from high doses of ionizing radiation during irradiation of the thorax.

This may lead to pericarditis, aortic stenosis or insufficiency, disorders of the conduction system and coronary heart

disease.

Methods: In our study, we investigated the effects of microbeam irradiation (MBI), an experimental approach on the

base of spatial dose fractionation of X-ray beams in the micrometer range. Synchrotron generated X-ray beams are

collimated into planar and quasi-parallel microbeams resulting in narrow, typically 25-50µm wide peak-dose regions,

which are separated by 200-400 µm wide valley-dose regions. Preclinical data showed that good tumour control can

be achieved with MBI while sparing normal tissue. Both organs were excised from adult Wistar rats. To test several

doses of MBI on the heart, the ex vivo perfusion model of the Langendorff setup was employed and cardiac

parameters like heart rate (via ECG), aortic and left ventricular pressure were determined. The oesophagus was

tended in the organ bath and carbachol-induced contractility was recorded.

Results:Oesophagus: No harmful effects of MBI on the contractility of the smooth muscles were found, but the

intracellular mechanisms of contraction were impaired. Heart: After irradiation with clinically relevant doses of MBI,

no effects on cardiac parameters were found in the Langendorff setup, whereas in control experiments with MBI

doses up to 4k Gy, arrhythmia and loss of contractility of the left ventricle were present.

References [1] Murro D, Jakate S. Radiation esophagitis. Archives of pathology & laboratory medicine 2015;139:827–30; doi:

10.5858/arpa.2014-0111-RS. [2] Bradley J, Movsas B. Radiation esophagitis: Predictive factors and preventive strategies. Seminars in

radiation oncology 2004;14:280–6; doi: 10.1016/j.semradonc.2004.06.003.


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[3] Sasso G, Rambaldi P, Sasso FS, Cuccurullo V, Murino P, et al. Scintigraphic evaluation of oesophageal transit during radiotherapy to the mediastinum. BMC gastroenterology 2008;8:51; doi: 10.1186/1471-230X-8-51.

[4] Türkölmez S, Atasever T, Akmansu M. Effects of radiation therapy on oesophageal transit in patients with inner quadrant breast tumour. Nuclear medicine communications 2005;26:721–6; doi: 10.1097/01.mnm.0000171785.03403.6f.

[5] Grotzer MA, Schültke E, Bräuer-Krisch E, Laissue JA. Microbeam radiation therapy: Clinical perspectives. Physica medica : PM : an international journal devoted to the applications of physics to medicine and biology : official journal of the Italian Association of Biomedical Physics (AIFB) 2015;31:564–7; doi: 10.1016/j.ejmp.2015.02.011.


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A 10-08

Optogenetic stimulation of Gs signaling increases ventricular

arrhythmia triggering and maintenance

Vanessa Dusend, Philipp Sasse

University of Bonn, Institute of Physiology I, Bonn, Germany

β-adrenergic activation is a major risk factor for ventricular tachycardia (VT) after myocardial infarction and in patients

with catecholaminergic polymorphic ventricular tachycardia (CPVT). In CPVT β-adrenergic Gs activation promotes

the VT trigger by amplifying calcium leak from the sarcoplasmic reticulum during diastole causing delayed

afterdepolarizations. However, traditional pharmacological interventions do neither allow regional distinguishing nor

temporal precise investigation of β-adrenergic effects. In order to investigate these effects on VT trigger and

morphology, we established optogenetic Gs-stimulation by generating a mouse line expressing Jellyfish opsin

(JellyOp), a light-activated Gs-coupled GPCR in cardiomyocytes. This line was crossed with a calsequestrin (Casq2)

knock-out mouse as a model for CPVT (JellyOp x Casq2-/-).

Ventricular illumination in explanted Langendorff-perfused JellyOp x Casq2-/- hearts induced premature ventricular

contractions (PVC) or triggered VT, but only when heart rate was pharmacologically reduced with the muscarinergic

agonist carbachol (n=5). To investigate regional PVC occurrence, we illuminated the endocardium of the left ventricle

using a light catheter calibrated to match epicardial illumination intensity and area. Endocardial illumination induced

PVC in ventricular-paced JellyOp x Casq2-/- hearts with more than 10-fold higher light-sensitivity than epicardial

illumination (N=5). These findings indicate an increased propensity for PVC generation particularly in the

endocardium upon selective Gs-activation in the ventricle, facilitated by low heart rate.

To analyze VT prevalence we performed S1S2 programmed electrical stimulation protocols and found that

illumination of the right ventricle of JellyOp mice increased the likelihood for VT induction and maintenance (N=3).

Moreover, we electrically induced stable running monomorphic VT in JellyOp mice after pharmacologically shortening

action potentials with pinacidil. Interestingly, ventricular illumination led to a decrease of VT frequency from 20.2±0.2

Hz to 18.6±0.2 Hz (n=27, N=5) which in some (N=2) led to degradation of monomorphic VT into high frequency

ventricular fibrillation (VF).

In conclusion, optogenetic methods allow regional discrimination of adrenalin effects on ventricular arrhythmia

showing that Gs activation not only promotes triggering of VT by PVC generation but also enhances VT inducibility,

maintenance and the risk of degradation into VF.

Acknowledgment Funded by the Deutsche Forschungsgemeinschaft (German Research Foundation, 313904155/SA1785/7-1,

380524518/SA1785/9-1, 214362475/GRK1873/2)


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A 10-09

Imaging-Based Assay for Screening of Cell Cycle Modifying

Substances in Postnatal Cardiomyocytes

Cora Becker1, Carmen C. Garcia2, Patricia Freitag1, Dennis Schade2, Bernd K. Fleischmann1,

Michael Hesse1

1 University of Bonn, Medical Faculty, Bonn, Germany 2 University of Kiel, Department of Pharmaceutical & Medicinal Chemistry, Kiel, Germany

Cardiovascular diseases are the main cause of death in the industrial world. Especially large myocardial infarctions

and the resulting left ventricular dysfunction are chronic diseases with a poor prognosis. Due to limited endogenous

regeneration of the adult heart, we are exploring strategies to re-initiate the cell cycle in cardiomyocytes (CMs) as a

potential treatment strategy. We hypothesize that controlled induction of CM proliferation will ultimately result in an

improvement of contractile function of the heart. We are using CMs from double transgenic αMHC-H2B-

mCherry/CAG-eGFP-anillin mice as a read-out system, as this enables the identification of CM nuclei and of cell

cycle variations, namely acytokinetic mitosis (mitosis without cytokinesis), resulting in binucleated CMs, and

endoreplication (mitosis without cytokinesis and karyokinesis), leading to polyploid CMs. Using this system, we are

screening for pro-proliferative substances (e.g. Pan-kinase inhibitors) in P1 and P6 postnatal CMs of αMHC-H2B-

mCherry/CAG-eGFP-anillin mice in order to identify substances inducing authentic cell division. We have identified

several screening hits, however we found that these induced preferentially an increase in cell cycle activity leading

mainly to binucleation or polyploidy, but not cytokinesis. This demonstrates that none of the compounds tested so

far induce authentic cell division in mice. Because of strong species dependent effects, we are currently working on

the establishment of an αMHC-H2B-mCherry/CAG-eGFP-Anillin double transgenic hiPS cell line with the aim of

screening pro-proliferative substances in human iPS cell-derived cardiac organoids.


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A 10-10

Ultrastructural Dynamics of Cardiomyocyte Contraction

Joachim Greiner1,2, Thomas Kok1,2, David Kaltenbacher1,2, Peter Kohl1,2, Eva Rog-Zielinska1,2

1 University Heart Center Freiburg·Bad Krozingen, Institute for Experimental Cardiovascular Medicine, Freiburg,

Germany 2 University of Freiburg, Faculty of Medicine, Freiburg, Germany

The structure and function are tightly interlinked in cardiomyocytes. However, the ultrastructural dynamics during the

cardiac action potential, including mechanical organelle deformation, are poorly understood. Dynamics of contracting

cardiomyocytes are conventionally investigated using light microscopy, a modality severely limited in terms of its

ability to resolve individual organelles. An imaging method that can truly provide structural data beyond the light

diffraction limit is electron microscopy – however, conventional electron-based approaches are unable to provide

precise temporal resolution.

Here, we use action potential-synchronised high pressure-freezing to assess the ultrastructural dynamics during

cardiomyocyte contraction with dual-axis electron tomography, resulting in a spatial resolution of (1.2 nm)3 and

millisecond temporal resolution. In brief, cardiomyocytes were isolated from precision-cut left-ventricular rabbit tissue,

high pressure-frozen at prescribed intervals post-electrical stimulation, freeze-substituted, heavy metal-stained,

resin-embedded, and cut into 300 nm sections. Then, the cardiomyocytes were imaged using dual-axis electron

tomography using a 300 kV transmission electron microscope. The resulting image volumes were reconstructed and

segmented utilising fully convolutional neural networks into 3D organelle models. When necessary, reconstructions

were refined within a custom-developed software. This workflow allowed for a time-efficient segmentation of

organelles, such as the sarcoplasmic reticulum, the transverse-axial tubular system, caveolae, and mitochondria

during several phases of contraction.

Our proof-of-principle study resolves the structural dynamics of contracting cardiomyocytes in a nanoscopic, three-

dimensional, and millisecond-accurate manner. Precise understanding of cardiac ultrastructure and its mechanical

modulation, ultimately to be achieved in human cardiomyocytes under both physiological and pathophysiological

conditions, is key in advancing our current comprehension and development of state-of-the-art diagnostic and

interventional approaches towards cardiac diseases.

Acknowledgment JG, TK, DK, PK, and ERZ are members of the Collaborative Research Centre SFB1425 of the German Research

Foundation (DFG #422681845).


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A 10-11

Effects of long-term immobilisation on endomysium of the soleus

muscle in humans

Georgina K. Thot1, Carolin Berwanger1, Edwin Mulder1, Jessica K. Lee1, Yannick Lichterfeld1,

Bergita Ganse2,3, Ifigeneia Giakoumaki2, Hans Degens2,4, Ibrahim Duran5, Eckhard Schönau5,6,

Christoph S. Clemen1,7, Bent Brachvogel6, Jörn Rittweger1,6

1 German Aerospace Center (DLR), Institute of Aerospace Medicine, Cologne, Germany 2 Manchester Metropolitan University, Research Centre for Musculoskeletal Science & Sports Medicine,

Department of Life Sciences, Manchester, UK 3 Saarland University, Innovative Implant Development, Department of Surgery, Homburg, Germany 4 Lithuanian Sports University, Institute of Sport Science and Innovations, Kaunas, Lithuania 5 Cologne University Hospital and Medical Faculty, Center of Prevention and Rehabilitation, Cologne, Germany 6 University of Cologne, Department of Pediatrics and Adolescent Medicine, Cologne, Germany 7 University of Cologne, Institute of Vegetative Physiology, Medical Faculty, Cologne, Germany

Question: It is well known that immobilized muscles atrophy. While muscle fibres have been extensively examined,

less is known about the intramuscular connective tissue and its smallest component, the endomysium. Animal data

from Williams and Goldspink suggest an increase in endomysium content with immobilisation [1], but there have so

far been no studies on humans. We therefore hypothesized that the proportional amount of endomysium in the soleus

muscle in humans increases during immobilisation.

Methods: Muscle biopsies of the soleus muscle were extracted from 21 healthy volunteers participating in the 60-

day 6° head-down tilt bed rest study AGBRESA, a collaborative project between NASA, ESA and DLR. The objective

of the AGBRESA was to test the effectiveness of short-duration continuous and intermittent centrifugation, i.e.,

artificial gravity, as a countermeasure to microgravity-induced adaptations. Biopsies were taken at baseline (BDC)

and during day 6 and day 55 of head-down tilt bed rest (HDT6 and HDT55, respectively). Muscle biopsies were snap

frozen in liquid nitrogen, sectioned on a cryostat and stained using immunofluorescence-labelled laminin γ-1. Laminin

γ-1 is an integral component of the basement membrane and can be used to determine the endomysium area. The

endomysium-to-fibre-area ratio (EFAr, in %) was assessed as a measure related to stiffness, and the endomysium-

to-fibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be

attributed to muscle fibre shrinkage.

Results: Muscle fibres had atrophied (p=0.0031) by 16.6% (SD 28.2%) at HDT55. ENAr increased on day 55 of bed

rest (p<0.001), whereas no significant effect of bed rest was found regarding EFNr (p=0.62). We found no significant

effects of either the continuous or intermittent centrifugation countermeasure on muscle fibre atrophy, ENAr or EFNr.

Conclusions: These results demonstrate that a disuse-induced increase in EFAr is explicable by muscle fibre

atrophy, as there was no increase in EFNr. Assuming there is no fibre loss during 55 days of bed rest, this indicates

that the absolute amount of connective tissue in the endomysium remained unchanged. However, the relative

increase of endomysium per fibre is likely to affect muscle stiffness and thus function.

Acknowledgment Special thanks to the entire team of the DLR Institute of Aerospace Medicine in Cologne for the great operational

support and to the participants of the AGBRESA study.

References [1] Williams, P. E.; Goldspink, G. 1984, 'Connective tissue changes in immobilised muscle'. Journal of Anatomy,

138(2), 343–350, Great Britain


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Foyers

A 11 | Cardiac Physiology I Chair

Jörg Heineke (Heidelberg)

Susanne Rohrbach (Gießen)


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A 11-01

Cardiac stiffness is tuned through unfolded domain oxidation (UnDOx)

and in-register aggregation in the distal I-band region of titin.

Christine M. Loescher1, Martin Breitkreuz2, Yong Li1, Alexander Nickel3, Andreas Unger1,

Alexander Dietl4, Andreas Schmidt5, Belal Mohamed6, Sebastian Kötter7, Joachim Schmitt8,

Marcus Krüger5,9, Martina Krüger7, Karl Toischer6, Christoph Maack3, Lars Leichert10, Nazaha Hamdani2,

Wolfgang Linke1

1 University of Münster, Institute of Physiology II, Münster, Germany 2 Ruhr University Bochum, Institute of Physiology, Bochum, Germany 3 University Clinic Würzburg, Comprehensive Heart Failure Center Würzburg, Würzburg, Germany 4 University Hospital Regensburg, Department of Internal Medicine II, Regensburg, Germany 5 University of Cologne, Institute for Genetics, Cologne, Germany 6 University Medicine Göttingen, Department of cardiology and Pneumology, Göttingen, Germany 7 Heinrich Heine University, Department of Cardiovascular Physiology, Düsseldorf, Germany 8 Heinrich Heine University, Department of Pharmacology and Clinical Pharmacology, Düsseldorf, Germany 9 University of Cologne, Center for Molecular Medicine and Excellence Cluster 'Cellular Responses in Aging-

Associated Diseases' (CECAD), Cologne, Germany 10 Ruhr University Bochum, Institute for Biochemistry and Pathobiochemistry, Bochum, Germany

Question: Titin stiffness, a main determinant of myocardial stiffness, is thought to be modulated by oxidative stress.

In vitro, titin oxidation is promoted by unfolding of titin immunoglobulin-like (Ig) domains, which in turn cannot properly

refold after oxidation. However, it is unknown whether titin oxidation occurs in vivo and if the required unfolding can

be detected through the differential oxidation of sarcomeric I-band (extensible) and A-band (inextensible) titin.

Further, both an increase and decrease in passive tension have been suggested as the effect of titin oxidation,

making it hard to predict how oxidative stress alters titin-based passive tension.

Methods & Results: Titin oxidation was studied in ex vivo perfused (Langendorff) mouse hearts treated with 0.1 mM

H2O2 and in vivo in the aorto-caval shunt mouse heart, a chronic volume overload model developing oxidative stress.

Titin oxidation was quantifiedby isotope-coded affinity tag labelling of heart tissue followed by mass spectrometry.

Oxidative stress significantly increased the oxidized (GSSG) to reduced (GSH) glutathione ratio. Hundreds of

cysteines in titin became more oxidized under oxidative stress. I-band and A-band titin sites showed similar increases

in oxidation level under ex-vivo non-stretch conditions. However, in preload-stressed shunt hearts, I-band titin was

more oxidized than A-band titin. In isolated skinned human cardiomyocytes, we determined the mechanical effects

of titin Ig domain unfolding (through stretch) and oxidation. Oxidation was induced with 2 mM GSH/0.5 mM diamide

(S-glutathionylation) or 35 µM protein disulfide isomerase (disulfide bonding) at sarcomere length 1.7 or 2.3 µm for

30 min. When oxidized under stretched conditions, passive force decreased after S-glutathionylation but increased

after disulfide bonding. Finally, recombinantly expressed Ig domains from the distal I-band titin region were thermally

unfolded then S-glutathionylated, which consistently resulted in increased aggregation.Conclusion: Titin oxidation

occurs in vivo and elastic titin becomes more oxidized than A-band titin following stretch through unfolded domain

oxidation (UnDOx; 1). UnDOx modulates passive stiffness through oxidation type-specific modification of titin and

promotes aggregation of unfolded titin domains. We propose a mechanism whereby UnDOx enables the controlled

homotypic interactions within the distal titin spring region to modulate passive stiffness of the heart.

References [1] 1) Loescher CM, Breitkreuz M, Li Y, Nickel A, Unger A, Dietl A, Schmidt A, Mohamed BA, Kötter S, Schmitt

JP, Krüger Markus, Krüger Martina, Toischer K, Maack C, Leichert LI, Hamdani N, Linke WA. Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx).Proc Natl Acad Sci USA 117: 24545-56, 2020.


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A 11-02

Titin-based skeletal muscle function is impaired in a mouse model with

diet-induced obesity and after cardiac ischemia/reperfusion

Benedikt Wolframm1, David Monteiro Barbosa1, Zhaoping Ding2, Katja Wegener3, Maria Grandoch3,

Martina Krüger1

1 Heinrich Heine-University Düsseldorf, Medical Faculty, Institute of Cardiovascular Physiology, Düsseldorf,

Germany 2 Heinrich Heine-University Düsseldorf, Medical Faculty, Institute of Molecular Cardiology, Düsseldorf, Germany 3 Heinrich Heine-University Düsseldorf, Medical Faculty, Institute of Pharmacology and Clinical Pharmacology,

Düsseldorf, Germany

Question: Patients with type 2 diabetes often suffer from reduced muscular strength and atrophy of the skeletal

muscles. The muscular filament protein titin is an important determinant of the passive elastic properties of striated

muscle. We have previously demonstrated that diabetes as well as cardiac infarction mediate increased cardiac titin-

based passive stiffness (PT) by phosphorylation of the titin PEVK region at S11878 and S12022. Here, we tested if

cardiac ischemia and reperfusion affects titin and skeletal muscle function in a mouse model of diet-induced obesity.

Material & Methods: Eight-week-old C57BL/6- mice were fed a high-fat and high-sucrose diet (HFD) for 10 weeks.

The animals then underwent surgical cardiac ischaemia for 45 minutes, followed by reperfusion for 21 days. Psoas

and extensor digitorum longus (EDL) skeletal muscles were excised and used for analyses. SDS-PAGE was used

to separate proteins according to their molecular weight. Titin phosphorylation was determined using Western

blotting. Active and passive forces of skinned skeletal muscle fibre bundles were measured using the MYOSCOPE

system (Myotronic). Fibre bundles were permeabilized, stretched step-wise and passive tension was recorded.

Maximum force development was determined by incubating the fibres in activating solution with pCa 4.5.

Results: In psoas and EDL of control animals, I/R reduced titin-based passive tension and significantly impaired

maximum force development at pCa4.5. HFD had a similar impact on active and passive forces. In psoas muscle of

HFD-fed mice, reduced force development correlated with hypo-phosphorylation of titin at serine 12022. In HFD-fed

mice I/R affected muscle function in a muscle-type specific manner. In EDL muscle from HFD-fed mice, I/R led to

reduced passive tension and reduced active force development. In psoas muscle, I/R increased active and passive

tension in the HFD group.

Conclusion: Taken together, both HFD and cardiac I/R lead to a dynamic adjustment of titin-based passive stiffness.

We hypothesize that the partly opposite effects of I/R on active and passive force development in psoas and EDL of

HFD are caused by altered fibre type and MHC isoform composition. We observed a close correlation of passive

tension changes with maximum force generation, again highlighting the role of titin in modulating striated muscle

function.


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A 11-03

Cardiac Titin ubiquitination and degradation by autophagy and the

ubiquitin-proteasome-system

Erik Müller, Senem Salcan, Sabine Bongardt, David Monteiro Barbosa, Martina Krüger,

Sebastian Kötter

Medical Faculty Heinrich-Heine University, Cardiovascular Physiology, Düsseldorf, Germany



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A 11-04

Quantifying the contribution of titin and actin filaments to

cardiomyocyte passive stiffness

Johanna K. Freundt, Christine M. Loescher, Wolfgang A. Linke

WWU, Institute of Physiology II, Münster, Germany

Question: Pathological changes to cardiac stiffness impair the function of the diseased heart, notably in heart failure

(HF) with preserved ejection fraction. The giant sarcomere protein titin bears passive load in cardiomyocytes (CMs),

and increased titin-based stiffness occurs in HF. Other cytoskeletal structures, such as actin filaments, also contribute

to total CM stiffness. Actin and titin interact in the sarcomeric I-band. Here, we aimed to quantify the contribution of

titin and actin filaments to CM passive stiffness, using a new genetic mouse model that allows the specific and acute

cleavage of the titin springs.

Methods & Results: In the mouse model, a tobacco etch virus (TEV) protease-recognition site and a HaloTag were

cloned into elastic titin (HaloTag-TEV knock-in (KI)). This cassette allows for specific in-situ cleavage of titin during

mechanical measurements of permeabilized mouse CMs and visualization of successful cleavage by tracking the

fluorescence signal of cells incubated with fluorophore-conjugated HaloLigand (which binds covalently to HaloTag),

using confocal microscopy or protein gel electrophoresis. Recombinant TEV protease caused the rapid, specific, and

complete cleavage of cardiac titin in homozygous KI heart samples, and 50% cleavage in heterozygous KI. In CMs

stained with HaloLigand-Alexa488, HaloTag-TEV titin was equally distributed within individual cells and throughout

all cells. Single CMs isolated from homozygous KI hearts were stretched and the resulting force recorded before/after

cleavage of titin using TEV protease. The specific titin cleavage resulted in a 61±5% (n=10) reduction in elastic force.

Actin filaments were severed using a Ca2+-independent gelsolin fragment. This treatment reduced the elastic force

of single CMs by 30±6% (n=7). Cleavage of titin first and actin second decreased the total force by 67±4% (n=10)

while cleavage of actin first and titin second reduced the total force by 63±7% (n=7).Conclusions: The HaloTag-

TEV mouse allows, for the first time, the direct and reliable quantitation of the titin contribution to CM stiffness. Our

findings show that the intact titin springs are responsible for the majority of the elastic forces of the mouse CM. Actin

filaments contribute much less to CM elastic force than titin. The order of cleavage of these cytoskeletal structures is

important, suggesting the presence of a cellular tensegrity architecture.


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A 11-05

Insulin-like Growth Factor 1 Improves Cardiac Function after Acute

Myocardial Infarction in a Prediabetic Mouse Model

André Spychala, André Heinen, Alexandra Zimmerhofer, Rianne Nederlof, Olesia Omelchenko,

Axel Gödecke

Universitätsklinik Düsseldorf, Institut für Herz- und Kreislaufphysiologie, Düsseldorf, Germany

Background: T2DM is a major comorbidity worsening the outcome after acute myocardial infarction (MI). Insulin-

like growth factor 1 (IGF1) is an anabolic hormone similar to insulin in molecular structure and signaling. IGF1 controls

proliferation, differentiation and metabolism of cells. In previous studies we have shown that short-term IGF1

treatment after MI improved cardiac function and reduced scar size in mouse hearts. Here we investigate whether

IGF1 maintains cardiac function after MI in prediabetes.

Methods: Mice were fed standard chow or high fat/high sucrose diet (HFHSD) for 10 weeks to induce obesity and

prediabetes. Phenotyping was performed by measurements of blood glucose, plasma insulin, glucose (GTT) and

insulin tolerance tests (ITT). A catheter in the left ventricle carried out direct pressure volume measurements. Mice

were subjected to 45 min LAD coronary artery occlusion and 1-week reperfusion. IGF1 or vehicle were given over 3

days using osmotic mini pumps. Heart function was analyzed echocardiographically (EC) before and one week after

MI including strain analysis. In addition, metabolic profiling was carried out by expression analysis for both the area

at risk and the remote area. Cardiac macrophage population after MI was determined by FACS.

Results: HFHSD led to increased blood glucose levels with up to 200 mg/dl and elevated insulin levels with 1.3 ng/L

in comparison to 0.4 ng/l in standard chow mice. GTT revealed glucose intolerance. However, exogenous insulin

was able to lower blood glucose in the ITT, indicating prediabetes but no T2DM. Cardiac pressure volume

measurements revealed no changes between both groups. EC indicated that cardiac function after MI under

prediabetic conditions worsened to the same extent as in the chow group (EF=~35%). Treatment of IGF1 after MI

preserved cardiac function not only in the chow group but also in the prediabetes group, with higher EF (chow:45.5%;

IGF1:48.2%). Expression of glucose marker gens were downregulated in HFHSD mice after MI, whereas fat marker

genes were higher expressed in HFHSD mice. However, treatment of IGF1 did not alter this expression profile. FACS

analysis revealed no effect of IGF1 on macrophage populations in the ventricle of HFHSD mice 3 days after MI.

Conclusion: Despite a similar signal transduction mediated by IGF1 and insulin, IGF1 was able to preserve cardiac

function after MI in a prediabetic mouse model. However, the underlying mechanism has to be further elucidated.


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A 11-06

Pathomechanisms of human cardiomyopathy due to a titin truncation

studied in hiPSC-derived cardiomyocytes

Anna Hucke1, Andrey Fomin2,3, Lukas Cyganek4,5,2, Marion von Frieling-Salewski1, Malte Tiburcy5,3,

Wolfram H. Zimmermann3,5,6, Wolfgang Linke1,2,3

1 University of Münster, Institute of Physiology II, Münster, Germany 2 University Medical Center, Clinic for Cardiology and Pneumology, Göttingen, Germany 3 German Centre for Cardiovascular Research, partner site Göttingen, Göttingen, Germany 4 University Medical Center, Stem Cell Unit, Göttingen, Germany 5 University Medical Center, Institute of Pharmacology and Toxicology, Göttingen, Germany 6 University of Göttingen, Cluster of Excellence, Göttingen, Germany

A heterozygous truncation in TTN, which codes for the muscle protein titin, is the most common genetic cause of

dilated cardiomyopathy (DCM). The pathogenicity of a TTN truncating variant (TTNtv) is highest for mutations in the

constitutively expressed A-band region of titin. Here, we study how the presence of a TTNtv and the pharmacological

modulation of intracellular protein quality control (PQC) pathways affect titin protein expression and contractility of

cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC), with the aim to explore the

pathomechanisms of TTNtv-DCM.

Human iPSC-CMs with a TTNtv were generated from a DCM patient with a truncation in A-band TTN-exon 327 and

from a healthy hiPSC-CM line, in which the truncation was induced in M-band TTN-exon 359 using CRISPR/Cas9

gene editing. Wildtype (WT-)hiPSC-CMs were used as controls. Cells were matured for 1-3 months in 2D-culture.

Additionally, ring-shaped engineered heart muscles (EHMs) were cultured from 1-month-old hiPSC-CMs and

matured for an additional month. PQC pathways were modulated using the following drugs: autophagy-activator

rapamycin, autophagy-inhibitor bafilomycin-A, and proteasome inhibitors MG132 or bortezomib; DMSO was used as

the vehicle-only control. Titin protein expression was analyzed by loose gel electrophoresis and western blot.

In both the 2D TTNtv-hiPSC-CMs and TTNtv-EHMs, we observed lower wildtype (wt-)titin protein content and stable

expression of truncated (tr-)titin proteins compared to control cells. The reduced wt-titin expression in TTNtv-EHM

was associated with contractile deficiency compared to WT-EHM, as it causes sarcomere insufficiency. The wt-titin

protein content could be restored by incubating proteasome inhibitors for 5-6 days, which also ameliorated the

contractile deficit. Tr-titin protein content increased upon proteasome-inhibition, while modulation of autophagy had

no significant effects. Genetic correction of the A-band-TTNtv in EHMs using CRISPR/Cas9 eliminated the tr-titin

proteins and raised the wt-titin proteins to levels found in WT-controls, while contractility was fully rescued. Our

studies of patient-derived and CRISPR/Cas9-edited hiPSC-CMs and EHMs suggest that pathomechanisms of TTNtv-

DCM include the accumulation of tr-titin proteins worsened by proteasome-inactivation, and the loss of wt-titin protein

in TTNtv-CMs (titin haploinsufficiency). Rescue of wt-titin protein content is a promising potential treatment.


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A 11-07

Long-term cultivation of human atrial myocardium as a tool to

investigate atrial fibrillation

Maximilian Klumm1,2, Dominik Fiegle1, Victoria Baron1, Michael Weyand2, Tilmann Volk1, Christian

Heim2, Thomas Seidel1

1 Friedrich-Alexander-Universität Erlangen-Nürnberg, Institut für Zelluläre und Molekulare Physiologie, Erlangen,

Germany 2 Friedrich-Alexander-Universität Erlangen-Nürnberg, Herzchirurgische Klinik, Erlangen, Germany

Question: Cardiac tissue culture is an emerging research tool in cardiac physiology. While several groups have

demonstrated the feasibility of long-term culture of human or animal ventricular tissue slices, it is still unclear if long-

term culture of human atrial myocardium is possible as well. It would permit the verification and extension of findings

from clinical studies and animal models. Furthermore, it would allow for screening of drugs and therapeutic

interventions related to atrial fibrillation. The goal of this study is to develop protocols for long-term cultivation of

beating human atrial myocardium and to mimic atrial fibrillation in vitro.

Methods: After written informed consent, atrial tissue was donated by patients with sinus rhythm undergoing open

heart surgery with cardiopulmonary bypass. Trabeculae (pectinate muscles) were prepared from the right-atrial

appendage and installed into cultivation chambers at 37° C. Diastolic preload was set to 1.5 mN. After an adaptation

period of 2 days with 0.5 Hz pacing, stimulation frequency was set to 1 Hz or 6 Hz and maintained for several days.

Contractile force was monitored continuously. Beta-adrenergic response (isoprenaline), stimulation threshold,

contractile refractory period and maximum captured frequency were assessed periodically. After cultivation, tissues

were used for MTT viability assays and structural investigation by confocal microscopy.

Results: All 24 cultured trabeculae obtained from 6 patient samples survived 12 days in culture, as indicated by MTT

viability assays, tissue microstructure and functional responses to electrical stimulation and isoprenaline. In one

experiment, trabeculae paced at 1 Hz were constantly beating for more than 3 weeks. In 8 trabeculae from two patient

samples, we compared functional parameters after 5 days of either 1 Hz (n=4) or 6 Hz pacing (n=4). Mean contractile

refractory period was 175±28 ms and 140±21 ms, maximum captured frequency was 6.3±0.7 Hz and 7.0±1.4 Hz,

and stimulation threshold was 58±4 mA and 34±1 mA, respectively. Confocal microscopy suggested changes in

cellular connexin-43 distribution after high frequency pacing.

Conclusions: We demonstrate a workflow that allows for the long-term cultivation of human atrial myocardium and

its functional assessment. We expect that this method will provide novel insights into the physiology and

pathophysiology of human atrial myocardium, particularly in the setting of atrial fibrillation.


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A 11-08

Analysis of peripheral adipose tissue lipolysis role in cardiac

remodeling after myocardial infarction

Heba Zabri, Luzhou Wang, Anne Petz, Dominik Semmler, Jens W. Fischer, Katharina Bottermann

Heinrich-Heine-University Duesseldorf, Medical Faculty University Hospital Duesseldorf, Department of

Pharmakology and Clinical Pharmakology, Duesseldorf, Germany

The sympathetic nervous system (SNS) is compensatory activated during and after myocardial infarction (MI).

Catecholamines released by the SNS activate β3-adrenergic receptors in peripheral adipose tissue (AT) and thereby

stimulate lipolysis. We hypothesize that increased levels of circulating free fatty acids (FFAs) lead to cardiac

metabolic disturbances during and after MI and thereby add to cardiac dysfunction. Inhibiting peripheral AT lipolysis

might therefore be beneficial for cardiac remodeling and may serve as a new therapeutic way to improve cardiac

function after MI.

To inhibit lipolysis the small molecule inhibitor of adiposetriglyceride lipase (ATGL), the first and rate limiting step in

the cascade of lipolysis, was used.

The effect of Atglistatin (Atgli) on lipolysis was tested ex vivo in gonadal white adipose tissue (gWAT) explants with

or without stimulation of lipolysis via Isoproterenol (Iso). Non-esterified fatty acid (NEFA) levels were measured to

assess lipolysis. Iso successfully increased NEFA levels. Atglistatin treatment significantly suppressed both, basal

NEFA levels without Iso stimulation as well as after Iso stimulation. In vivo a mouse model of 1 h ischemia and 28 d

of reperfusion (rep) was used. Atgli was administered from day 1 to 28 of rep via food (2mg Atglistatin/5g chow) to

12 weeks old C57Bl/6J male mice. Food intake was comparable between the groups. Efficiency of Atgli treatment

was shown by a decrease in body weight (BW) (Veh: 27,9±2,6g, Atgli: 24,7±2,5g) and gWAT/BW ratio (Veh:

11,32±2,64mg/g, Atgli: 7,99±0,93mg/g) as described before by Schweiger et al.

Cardiac blood was analyzed via flow cytometry and revealed slight changes in circulating immune cell composition

by Atgli treatment. While the abundance of neutrophils and monocytes was not affected, the fraction of T- and B-

cells seemed to be shifted with an increase in T-cells (Veh: 24,4±5%, Atgli: 31,5±3,2%) and a decrease in B-cells

(Veh: 47,4±4,8%, Atgli: 42,2±3%). Cardiac systolic pump function, measured weekly by echocardiography, was

unchanged between Atgli and vehicle treated mice. However, left ventricular mass (LV Mass: Veh 132,8±27,4mg,

Atgli 103,7±25,7mg) and left ventricular anterior and posterior wall thickness (LVAW,d: Veh 0,827±0,19mm, Atgli

0,65±0,22mm, LVPW,d: Veh 0,771±0,2mm, Atgli 0,66±0,17mm) were smaller in Atgli treated animals indicating an

influence of adipose tissue lipolysis on cardiac remodeling after myocardial infarction.


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A 11-09

Characterization of changes in peripheral white adipose tissue after

myocardial infarction

Luzhou Wang, Heba Zabri, Dominik Semmler, Jens W. Fischer, Katharina Bottermann

Heinrich-Heine-University Düsseldorf, Medical Faculty and University Hospital Düsseldorf, Department of

Pharmacology and Clinical Pharmacology, Düsseldorf, Germany

The loss in cardiac pump function during and after myocardial infarction (MI) leads to a compensatory increase in

systemic β-adrenergic stimulation, which is one of the strongest stimuli for white adipose tissue (WAT) lipolysis. To

investigate the influence of increased lipolysis during and after MI on different WAT depots, visceral (gondal WAT,

gWAT) and subcutaneous (inguinal WAT, iWAT) AT were analyzed histologically and for gene expression levels at

different time points after MI.

C57Bl/6J mice underwent 1-hour ischemia and 24 hours, 7 or 28 days of reperfusion (rep). Tail blood was collected

at baseline and after 30 minutes of reperfusion to measure non-esterified fatty acid (NEFA) levels. Mice which

underwent ischemia, showed a significant increase in circulating NEFA levels compared to baseline and sham

operated mice (Sham: 0,532 ± 0,11mmol/l, Ischemia: 0,742 ± 0,15 mmol/l).

Analysis of adipocyte size by H&E-staining revealed smaller adipocytes in iWAT of ischemic mice already after 24h

of reperfusion, which was even more pronounced after 7d rep. gWAT of ischemic mice showed a small trend towards

smaller adipocytes after 7d rep. Due to a higher number of nuclei visible in the H&E staining we speculated that iWAT

was infiltrated after MI. Subsequent MAC2 staining for macrophages revealed a higher number of macrophages and

crown like structures in iWAT of ischemic mice after 7d rep (Sham: 17,98 ± 14,15/mm2, Ischemia: 37,79 ±

12,22/mm2). Additional analysis of TNF-α gene expression using qPCR showed a significant increase of TNF-α

expression in both gWAT and iWAT after 7d rep.

As AT is not only a connective tissue storing energy, but also is considered as an important endocrine organ,

expression level of several adipokines was measured. The anti-inflammatory and cardioprotective adipokine

adiponectin was downregulated in iWAT already after 24h rep. Surprisingly, also the pro-inflammatory adipokine

resistin was downregulated in both gWAT and iWAT after 24h rep.

Taken together, the iWAT depot seems to be more prone to MI induced changes, characterized by smaller

adipocytes, macrophage infiltration and reduced adipokine expression.


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A 11-10

No functional TRPA1 in cardiomyocytes

Clara Hoebart1, Natalia Rojas-Galvan1, Cosmin I. Ciotu1, Ibrahim Aykac2, Lukas Reissig3,

Wolfgang Wenninger3, Attila Kiss2, Bruno Podesser2, Michael J. Fischer1, Stefan Heber1

1 Medical University of Vienna, Center for Physiology and Pharmacology, Vienna, Austria 2 Medical University of Vienna, Center for Biomedical Research, Vienna, Austria 3 Medical University of Vienna, Division of Anatomy, Vienna, Austria

Question: Our main objective was to study the role of transient receptor potential channel subtype ankyrin 1 (TRPA1)

in the heart, specifically during myocardial ischemia. We started by detecting the receptor in cardiomyocytes. As

reference, the related ion channel TRPV1 was investigated.

Methods: Microfluorimetry was used to measure intracellular Ca2+ ions in primary mouse cardiomyocytes and the

cardiac cell lines H9c2 and HL-1, while they were stimulated with TRPA1 and TRPV1 agonists or paced electrically.

Additionally, TRPA1 and TRPV1 mRNA levels were quantified by qPCR in mouse and human hearts, primary

cardiomyocytes and cardiac cell lines. Dorsal root ganglia served as positive controls for both methods.

Results: All tested TRPA1 agonists, including the established allyl isothiocyanate (AITC) and the more potent PF-

4840154 and JT010, failed to elicit a TRPA1-mediated response in native primary mouse cardiomyocytes and the

cardiac cell lines H9c2 or change the spontaneous activity of HL-1 cells. Moreover, TRPA1 was at best marginally

expressed in primary mouse cardiomyocytes and cardiac cell lines. Similarly, the TRPV1 agonist capsaicin was not

able to elicit a response in primary mouse cardiomyocytes or cardiac cell lines H9c2 and HL-1. In human pluripotent

stem cells differentiated to cardiomyocytes and in human heart samples TRPA1 mRNA levels were substantially

lower than in dorsal root ganglia.

Conclusion: There is no evidence for the expression of functional TRPA1 in cardiomyocytes. However, this does not

exclude a role of the channel in the physiology or pathophysiology of the heart.


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Foyers

A 12 | Endothelial Cell Physiology I Chair

Markus Hecker (Heidelberg)

Daniela Wenzel (Bochum)


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A 12-01

The nuclear Corepressor NCoR1 limits endothelial angiogenic function

Tom Teichmann, Beatrice Pflüger-Müller, Ralf Brandes

Goethe University Frankfurt am Main, Cardiovascular Physiology, Frankfurt, Germany

Objective: Angiogenesis is the process of new blood vessel formation, which is essential during development and

tissue repair during vascular diseases, such as angiopathy or arteriosclerosis. The endothelial angiogenic function is

initiated upon physiological deficiencies resulting in phenotypic changes of endothelial cells (ECs). We hypothesized

that the endothelial phenotype can be manipulated to switch from a resting to a pro-angiogenic state by altering

global gene expression. In order to study this aspect, we characterized the function of nuclear repressor complexes

in human umbilical vein endothelial cells (HUVEC) as well as in mouse aortic tissue from different knockout mice.

Results: Screening of HUVECs revealed a particular high expression of the Corepressors NCoR1, SMRT and

CoREST. Knockdown of these genes by siRNA demonstrated that exclusively the loss of NCoR1, but not SMRT or

CoREST increases the endothelial cell angiogenic capacity as determined by spheroid out-growth assay. Contrary,

proliferation and migration of NCoR1 deficient endothelial cells was decreased. To determine the underlying

mechanism, RNAseq was performed. Loss of NCoR1 significantly upregulates the expression 1200 genes including

the majority of genes involved in the tip cell formation of angiogenic sprouts such as ESM1, FLT4 and NOTCH4.

Accordingly, KEGG pathways analysis of the Top 50 up- or downregulated genes indicates a positive regulation of

angiogenesis. Furthermore, a compensatory sprouting assay using spheroids built out of non-target control- and

NCoR1 deficient cells was performed. There we could show that more than 70% of the NCoR1 deficient cells take

over the tip cell role within the same spheroid.

Conclusion: NCoR1 represents an interesting target in endothelial cells allowing the positive modulation of

pathophysiological angiogenesis related processes such as diabetes or atherosclerosis.


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A 12-02

Endothelin receptor B controls the production of fibroblast growth

factor 23

Martina Feger1, Franz Ewendt2, Matthias Menzel3, Berthold Hocher4, Michael Föller1

1 University of Hohenheim, Department of Physiology, Stuttgart, Germany 2 Martin Luther University Halle-Wittenberg, Institute of Agricultural and Nutritional Sciences, Halle (Saale),

Germany 3 Fraunhofer Institute for Microstructure of Materials and Systems (IMWS), Halle (Saale), Germany 4 University of Heidelberg, University Medical Centre Mannheim, Fifth Department of Medicine

(Nephrology/Endocrinology/Rheumatology), Heidelberg, Germany

Question: Endothelin-1 (ET-1) is a member of the endothelin family of peptide hormones first discovered as

endothelium-derived mediators regulating vascular tone. ET-1 also regulates the proliferation and differentiation of

bone cells that synthesize fibroblast growth factor 23 (FGF23). FGF23 is a hormone controlling renal phosphate and

vitamin D metabolism. Here, we studied the role of ET-1 and endothelin receptor B (ETB) for FGF23 production.

Methods:Fgf23 gene expression was studied in IDG-SW3 bone cells by quantitative RT-PCR. ETB-expressing

(etb+/+) and rescued ETB-deficient mice (etb-/-) were studied in metabolic cages. Their serum FGF23, PTH, and

1,25(OH)2D3 concentrations were determined by ELISA, serum and urinary phosphate and Ca2+ by photometric

methods.

Results: ET-1 and ETB agonist sarafotoxin 6c suppressed Fgf23 mRNA in IDG-SW3 cells. Serum C-terminal and

intact FGF23 as well as bone Fgf23 mRNA levels were significantly higher in etb-/- mice than in etb+/+ mice. Renal

phosphate excretion was significantly higher in etb-/- mice despite lower phosphate levels. In addition, etb-/- animals

exhibited calciuria and a significantly higher serum 1,25(OH)2D3 concentration compared to etb+/+ mice.

Conclusions: ETB-dependent ET-1 signaling is a potent suppressor of FGF23 formation. This effect is likely to be

of clinical relevance given the use of endothelin receptor antagonists in various diseases.


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A 12-03

HIF1α-AS1 is an anti-angiogenic DNA:DNA:RNA triplex forming long

non-coding RNA in endothelial cells

Matthias S. Leisegang1,6, Jasleen Kaur Bains2, Sandra Seredinski1,6, Soni S. Pullamsetti4,

Ralf Gilsbach1,6, Ilka Wittig5,6, Ivan G. Costa3, Harald Schwalbe2, Ralf P. Brandes1,6

1 Goethe University, Institute for Cardiovascular Physiology, Frankfurt am Main, Germany 2 Goethe University, Institute for Organic Chemistry and Chemical Biology, Frankfurt am Main, Germany 3 RWTH Aachen, Institute for Computational Genomics, Aachen, Germany 4 Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany 5 Goethe University, Functional Proteomics, Frankfurt am Main, Germany 6 German Center of Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany

Objective: Long-noncoding RNAs (lncRNAs) are potent regulators of the vascular system. The lncRNA HIF1α-AS1

is located on the antisense strand of the important Hypoxia-inducible factor 1α gene and is induced in aortic

aneurysms. The function and the mode of action of the lncRNA are, however, unknown and were identified here.

Results: HIF1α-AS1 was reduced in endothelial cells isolated from glioblastoma and from lungs of patients with

pulmonary arterial hypertension. In contrast, reoxygenation after hypoxia induced HIF1α-AS1. Functionally,

knockdown of HIF1α-AS1 in endothelial cells increased their angiogenic capacity. HIF1α-AS1 localized to condensed

nuclear regions and associated with double stranded DNA. Triplex-Sequencing, triplex domain finding and RNA

immunoprecipitation indicated that HIF1α-AS1 forms DNA:DNA:RNA triplexes on specific repressive sites of the

genome, among them the HIF1α and EPH receptor A2 (EPHA2) genes. Exchange of the triplex forming region of

HIF1a-AS1 with other known triplex forming regions by CRISPR Arcitect abolished its effects on gene expression.

Protein interaction studies revealed that HIF1α-AS1 interacts with the human silencing hub (HUSH) complex, which

contains the epigenetic repressor MPP8. HIF1α-AS1 recruits the HUSH complex to sites of triplex formation to pro-

angiogenic genes like EPHA2.Conclusions: These results suggest that HIF1α-AS1 forms DNA:DNA:RNA triple

helices in endothelial cells to recruit epigenetic silencer complexes. This mechanism is operative in vascular tissue

and controls the expression of the highly important genes HIF1α and ephrin receptor A2. Through this mechanism,

HIF1α-AS1 limits the aniogenic response.


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A 12-04

Pericyte actions of atrial natriuretic peptide (ANP) improve

angiogenesis

Rebecca Bosch1, Katarina Špiranec Spes1, Katharina Völker1, Lisa Krebes1, Berin Upcin2, Süleyman

Ergün2, Franziska Werner1, Michaela Kuhn1

1 University of Würzburg, Institute of Physiology I, Würzburg, Germany 2 University of Würzburg, Institute of Anatomy and Cell Biology, Würzburg, Germany

Background. Angiogenesis, the sprouting of new blood vessels from preexisting capillaries, is a key process for the

repair of the microvascular damage and rescue of ischemic tissue. This complex process is triggered by the crosstalk

between microcirculatory pericytes and endothelial cells, which maintain an intimate mutual communication through

direct interaction as well as via paracrine factors. This communication is essential for the control of endothelial cell

proliferation and differentiation, microvascular stability and the barrier function. This project elucidates the role of

atrial natriuretic peptide (ANP) in this interaction.

Methods and Results. In vitro:ANP, via its cyclic GMP-forming guanylyl cyclase-A (GC-A) receptor, stimulated the

proliferation and vitality of cultured murine brain and lung pericytes. Ex vivo: ANP enhanced the sprouting of

capillaries from cultured murine aortic rings in a concentration dependent manner compared to basal conditions.

These results indicate a direct influence of ANP on the formation of complete new vessel structures. The participation

of pericytes, which originate from the vasa vasorum of the aorta, was confirmed by immunohistochemistry. In vivo:

To study the role of pericyte ANP/GC-A/cGMP signalling in reparative neoangiogenesis we subjected mice with

pericyte-restricted deletion of GC-A and control littermates [1] to hind limb ischemia by surgical femoral artery

excision. Serial non-invasive laser Doppler perfusion imaging showed that during the first 2 weeks after surgery

(phase of arteriogenesis), the perfusion in the ischemic relative to the non-ischemic hind limb was similar in both

genotypes. However, significant impairment of blood flow recovery was observed between days 21 and 35 in KO

mice (phase of neoangiogenesis). Histology demonstrated that capillary densities in the gastrocnemic muscles of the

ischemic legs were significantly lower in pericyte GC-A KO mice compared to controls.

Conclusions. Together these results indicate that pericyte ANP/GC-A/cGMP signalling may improve the pericyte-

endothelial crosstalk and neo-angiogenesis after ischemia. The characterization of these effects and of participating

molecular pathways may unravel novel targets for therapies improving microcirculatory reperfusion.

Acknowledgment This study is supported by the Deutsche Forschungsgemeinschaft (DFG KU 1037/13-1). Present address of KŠS:

Fidelta Biotechnology, Zagreb, Croatia.

References [1] Špiranec Spes K, Chen W, Krebes L, Völker K, Abeßer M, Eder Negrin P, Cellini A, Nickel A, Nikolaev VO,

Hofmann F, Schuh K, Schweda F, Kuhn M, ‘Heart-Microcirculation Connection: Effects of ANP (Atrial Natriuretic Peptide) on Pericytes Participate in the Acute and Chronic Regulation of Arterial Blood Pressure’, Hypertension, 2020;76,1637-1648


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A 12-05

Quantifying the collective motion of individual endothelial cells

responding to regional injury

Anselm Hohlstamm1, Mats L. Moskopp1,2, Andreas Deussen1, Peter Dieterich1

1 TU Dresden, Institut für Physiologie, Medizinische Fakultät Carl Gustav Carus, Dresden, Germany 2 Vivantes Klinikum im Friedrichshain, Charité Academic Teaching Hospital, Klinik für Neurochirurgie, Berlin,

Germany

Question: Confluent dynamics of endothelial cells is an important precondition for vessel integrity. In addition, it

plays an important role in vascular repair mechanisms. To ensure this function, cells show complex movements

adapting to external influences. Collective cell migration can especially emerge under conditions of injury. Thus, we

aim to classify and quantify the dynamics of individual vascular cells in a confluent monolayer which is exposed to

local injury.

Methods: Human umbilical vein endothelial cells (HUVECs) were seeded and cell nuclei were stained with the

fluorescent dye Hoechst 33342 (dilution 1:10.000). A well-defined gap of 500 µm was created by placing inserts into

the microscope slides before seeding cells. HUVECs were observed for 24 hours via time-lapse microscopy

(sampling interval = 10 min). An automated segmentation and tracking algorithm allowed to follow the paths of several

10.000 individual cells per experiment. These paths were analysed regarding their individual movements in

relationship to their neighbours and with respect to the overall wound closure.

Results: Cells performed continuous activemovements and proliferated lively. Despite of this non-equilibrium

behaviour, the cell layer remained confluent. This dynamic is characterized by an exponential (non-Gaussian) velocity

distribution of the cells and a slowly decreasing mean squared velocity over time. In addition, the spatial velocity

correlation function showed an exponential decay over lengths of up to 60 µm carrying forces of interactions beyond

the next nearest neighbour cells. It is noteworthy, that the cell-cell interaction showed a repulsive, exponentially

decaying force between cells. The analysis of the mean squared displacement (msd) indicates a stronger cell

localisation with increasing cell density, which is quantified by a transition to a sub-diffusive behaviour. In comparison,

the msd near the edge in the wound healing assay was enhanced by a factor of five and showed a more persistent

dynamics. This observation is further supported by the enlarged velocity autocorrelation time of up to four hours.

Conclusion: By examining migration we quantified the dynamics of confluent cells with different functions of

correlations and potentials of cell-cell interactions. This enables us to quantify the complex cell movements and to

understand the changes that are triggered by wounds or external pharmaceutical stimuli in a quantitative way.


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A 12-06

Nuclear endothelial nitric oxide synthase interacts with RNA-binding

proteins to modulate endothelial gene expression.

Xiaozhu Zhou1,2, Ilka Wittig2,3, Mauro Siragusa1,2, Ingrid Fleming1,2

1 Goethe University, Institute for Vascular Signalling, Centre for Molecular Medicine, Frankfurt am Main, Germany 2 German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany 3 Goethe University, Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Frankfurt am Main, Germany

Background and Aim: Endothelial nitric oxide (NO) synthase (eNOS) is responsible for the production of NO, a

gasotransmitter that regulates vascular tone and endothelial homeostasis. The effect of NO can be attributed to its

interaction with heme-containing proteins, including soluble guanylyl cyclase, or to the reaction of nitrogen oxide with

cysteines, a process referred to as S-nitrosation. Although eNOS expression was thought to be restricted to the cell

membrane and Golgi apparatus, a nuclear localization in endothelial cells has also been reported. This study

assessed the mechanisms and consequences of eNOS nuclear translocation in endothelial cells.

Methods and Results: Stimulation of primary human endothelial cells with vascular endothelial growth factor (VEGF

– 50 ng/ml, 10 minutes) induced eNOS nuclear translocation. Proteomic studies demonstrated the VEGF-dependent

association of nuclear eNOS with 81 proteins, most of which have been reported to be S-nitrosated. One such protein

was Double-Stranded RNA-Specific Adenosine Deaminase, an enzyme involved in the hydrolytic deamination of

adenosine to inosine in double-stranded RNA (dsRNA), leading to decreased dsRNA stability. The shRNA-mediated

knockdown of eNOS in primary endothelial cells was associated with an increase in the amount of dsRNA

(immunofluorescence). Conversely, the expression of eNOS-wild type or eNOS-Y657F (gain-of-function mutant) in

HEK cells led to a lower dsRNA content compared to cells expressing the loss-of-function mutant eNOS-Y657D.

Nuclear eNOS was also associated with core components of paraspeckles, specialized nuclear compartments

involved in the regulation of gene expression. Indeed, the shRNA-mediated knockdown of eNOS in primary human

endothelial cells profoundly affected the expression of >4k genes.

Conclusions: These results demonstrate that eNOS and NO signalling modulate nuclear processes that are

essential for the regulation of endothelial gene expression.


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A 12-07

Impact of the mechanosensitive ion channel Piezo1 in vascular

inflammation

Benedikt Fels, Katja Schweim, Franziska Unrein, Kristina Kusche-Vihrog

University of Lübeck, Institute of Physiology, Lübeck, Germany

Vascular tone needs to be continuously monitored and regulated by endothelial cells (ECs) to ensure sufficient blood

flow. ECs have a sophisticated system of sensors and transducers to cope with this continuously altering mechanical

input and to maintain their function. EC surface is highly flexible and can adjust their mechanical properties, alternate

between ‘soft’ and ‘stiff’ conditions. These nanomechanical properties correlate with a proper endothelial function

and we hypothesize that the mechanosensitive ion channel Piezo1 is an important player in this context.

To test this hypothesis, we analyzed the effects of physiological relevant shear stress on primary human ECs

(HUVECs), using the Ibidi pump system and AFM-based nanoindentation for the quantification of the cell cortex

mechanics. Cortical actin was stained with Phalloidin-TRITC and Piezo1 expression/membrane abundance was

analyzed with qPCR and/or via immunofluorescence staining. To elucidate the impact of Piezo1 in vascular

inflammation, we quantify the monocyte adhesion on ECs in dependency of Piezo1 activation.

Application of chronic laminar shear stress (8 dyn/cm2) significantly enhances the presence of polymerized cortical

actin and increased cortex stiffness by +19 ± 0.05 % compared to non-flow control conditions (N=3, n=35-39). Within

stiff ECs, the mRNA expression of Piezo1 was increased by 1.5 ± 0.15 fold compared to the control group (N=5,

n=3). This was confirmed by Piezo1 staining’s and increased membrane abundance of +44 ± 2 % in stiff ECs

compared to control (N=6, n=13). We next stimulate ECs with 5 µM Yoda1, a Piezo1 channel agonist leading to -24

± 1.5 % reduced cortical stiffness in the ECs compared to the solvent control group (N=6, n=145). Stimulation with

Yoda-1 thereby did not change Piezo1 mRNA expression nor number of Piezo1 channels within the membrane

indicating increased open probability. In contrast, Piezo1 mediated changes in cortical stiffness lead to decreased

number of adherent monocytes on the EC surface.

It can be concluded, that Piezo1 is involved in the regulation of endothelial nanomechanics and function. Changes

in Piezo1 channels function impact on immune cell adhesion, an important step within the inflammatory cascade.

Since dysfunctional mechanosignaling is linked to vascular pathologies, Piezo1 channels could serve as both

predictors and anti-inflammatory pharmaceutical targets.


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A 12-08

Suppressor of cytokine signaling protein 3-deficiency in myeloid cells

regulates angiogenesis via enhanced apoptotic endothelial cell

engulfment

Behnaz A. Hosseini1, Irina Korovina2, David Sprott1, Andreas Deussen1, Anne Klotzsche - von Ameln1

1 Faculty of Medicine, Technische Universität Dresden, Institute of Physiology, Dresden, Germany 2 Faculty of Medicine, Technische Universität Dresden, OncoRay, National Center for Radiation Research in

Oncology, Dresden, Germany

Mononuclear phagocytes, such as macrophages and microglia, are key regulators of organ homeostasis including

vascularization processes.

In the current study, we investigated the role of the suppressor of cytokine signaling protein 3 (SOCS3) in myeloid

cells as a regulator of mononuclear phagocyte function and their interaction with endothelial cells in the context of

sprouting angiogenesis.

As compared to SOCS3-sufficient counterparts, we found that SOCS3-deficient microglia and macrophages

displayed an increased phagocytic activity towards primary apoptotic endothelial cells. This phenomenon was

associated with an enhanced expression of the opsonin growth arrest-specific 6 (Gas6), a major pro-phagocytic

molecule.

Furthermore, we found that myeloid SOCS3-deficiency significantly reduced angiogenesis in an ex-vivo mouse aortic

ring assay, which could be reversed by the inhibition of the Gas6 receptor Mer. Together, SOCS3 in myeloid cells

regulates the Gas6/Mer-dependent phagocytosis of endothelial cells and thereby angiogenesis-related processes.

Our findings provide novel insights into the complex crosstalk between mononuclear phagocytes and endothelial

cells and may therefore contribute to a more comprehensive understanding of angiogenetic processes.


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A 12-09

Hypoxia-inducible factor-2α exacerbates oxidative damage in a cell

culture model of age-related macular degeneration

Yoshiyuki Henning, Ursula Blind, Darius Molitor, Joachim Fandrey

University of Duisburg-Essen, Faculty of Medicine, Institute of Physiology, Essen, Germany

Background: Oxidative stress and hypoxia in the retinal pigment epithelium (RPE) have long been considered major

risk factors in the pathophysiology of age-related macular degeneration (AMD), a major cause of blinding among

elderly people. Dry AMD is the most common type of AMD; however, no convincing treatment is available to date.

Thus, to promote prevention and to develop novel treatment strategies against dry AMD, it is necessary to identify

the pathophysiological pathways in which oxidative stress and hypoxia are involved. In the present study, we focused

on hypoxia-inducible factors (HIF) 1α and 2α, the α-subunits of HIF-1 and HIF-2, key regulators of cellular adaptation

to hypoxic conditions. For this purpose, we developed a dry AMD cell culture model to identify the role of HIFs in

AMD pathophysiology.

Methods: We treated human RPE cells with sodium iodate (SI), an oxidative stress agent, together with DMOG, a

prolyl hydroxylase (PHD) inhibitor which increases HIF-α levels, under 3 % O2 in a hypoxic chamber to simulate

AMD-related conditions. Treatment effects were analyzed using cell viability assays, FACS, Western blot, and

quantitative real-time PCR. Furthermore, siRNA knockdown was conducted and several inhibitors of cell signaling

pathways were used.

Results: Cell viability of ARPE-19 cells was significantly decreased in cells treated with SI/DMOG compared to SI or

DMOG alone, suggesting that HIF accumulation exacerbates oxidative damage. Necroptosis was identified as the

main cell death mechanism using FACS analyses and inhibitors of the necroptosis and apoptosis pathways.

Moreover, knockdown of HIF-1α and HIF-2α using siRNA revealed that HIF-2α is responsible for exacerbation of

oxidative damage. We discuss our observations in terms of the mTOR signaling pathway and autophagy inhibition,

since blocking of mTOR improved cell viability.

Conclusions: Taken together, our data suggest that selectively blocking HIF-2α could be a potential treatment

strategy to protect the aging RPE against extensive oxidative damage, which ultimately protects against AMD.


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A 12-10

Transcriptome profiling of two different H9c2 cell lines overexpressing

the cytosolic renin isoform named renin-b

Janine Golchert1, Doreen Staar1, Jonathan Bennewitz1, Heike Wanka1, Sabine Ameling2, Uwe Völker2,

Jörg Peters1

1 University Medicine Greifswald, Institute of Physiology, Greifswald, Germany 2 University Medicine Greifswald, Interfaculty Institute for Genetics and Functional Genomics, Department of

Functional Genomics, Greifswald, Germany

Question: Besides the classical secretory renin-a the presence of another cytosolic, non-secretory isoform named

renin-b has been described. Interestingly, in contrast to renin-a, cardioprotective effects have been demonstrated for

renin-b under ischemia-related conditions in H9c2 cardiomyoblasts. Using gene expression profiling we investigated

renin‐b effects on signaling pathways that may be involved in cardioprotection.

Methods: In the present study we used H9c2 cell lines overexpressing either the full-length renin-b transcript

including its 5’UTR located in intron A of the renin gene [exon(1a-9)renin] or a shortened renin-b transcript lacking

the 5’UTR [exon(2-9)renin]. Isolated total RNA samples of these two cell lines as well as of control cells expressing

only the empty pIRES vector were subjected to microarray-based transcriptome analysis (n = 3 or 4 per group).

Finally, pathway analysis of genes exhibiting significantly different mRNA levels was carried out using the Ingenuity

Pathway Analysis (IPA) software. Additionally, selected key regulatory proteins involved in these pathways were

analyzed regarding their phosphorylation state using western blot analysis.

Results: The transcriptome analysis revealed altogether 3645 and 4059 significantly different expressed transcripts

for exon(1a-9)renin and exon(2-9)renin overexpressing cells compared to the control group, respectively (pFDR < 0.05,

fold change ≥ |1.5|). Of these, 2762 transcripts were altered in both groups. The results of the in silico pathway

analysis indicated that renin-b overexpression is associated with numerous pathways. These data suggest an

involvement of the mTOR, Akt, and ERK signaling. Moreover, analyses on protein level showed significantly

increased phosphorylation of mTOR and Akt kinases in renin-b overexpressing cells (p < 0.05, fold change > 1.5),

which may also point towards an activation of these pathways.

Conclusions: In conclusion, we have identified several potential candidate pathways that might be involved in

mediating cardioprotective effects. Among these, mTOR and Akt are of particular interest since they play an essential

role in signal transduction of cell growth, proliferation, and metabolism.

Poster Session B DPG 2021 | Abstract Book

of 516

Poster Session B | 01 October 2021

5:00 PM – 7:00 PM

Foyers

B 01 | Network Physiology Chair

Josef Bischofberger (Basel)

Simon Rumpel (Mainz)


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B 01-01

Investigating putative pacemaker currents of R5 neurons in a reduced

complexity in vivoDrosophila sleep model

Anatoli Ender1,2, Davide Raccuglia1, David Owald1,2

1 Charité - Universitätsmedizin, Neurophysiology, Berlin, Germany 2 NeuroCure, Charité - Universitätsmedizin, Berlin, Germany

Sleep behavior is highly conserved across the animal kingdom and can be observed in vertebrates as well as in

invertebrates. In Drosophila melanogaster, a model system highly accessible with genetics, sleep manifests in

sustained periods of quiescence and an increased arousal threshold. Recently, we discovered slow wave oscillations

that can be associated with sleep need in fruit flies [1]. These oscillations can be measured in the R5 network of the

ellipsoid body, a set of 10 cells per hemisphere, which encodes sleep drive in Drosophila melanogaster. The power

of the oscillations increases with sleep need and exhibits diurnal variations. These finding allows us to address

fundamental principles of sleep regulating systems in a model brain of reduced numerical complexity. Indeed, we

find that at a single cell level R5 neurons switch from a spontaneous irregular firing activity to a more regular bursting

behavior depending on the animal’s sleep pressure. Periodic activity patterns of cells are often generated by intrinsic

membrane currents. In the present study we aim to identify putative pacemaker currents in the R5 system of the

model organism Drosophila melanogaster.

We use in vivo patch clamp recordings to characterize the neuronal intrinsic properties and the mechanisms

underlying the rhythmic firing pattern of R5 neurons. Furthermore, we utilized receptor specific RNAi lines and

pharmacological approaches to identify the receptors which are critical for the local spontaneous oscillations. We

isolated tetrodotoxin resistant currents which might play a role in the generation of the rhythmic activity. Eventually,

we aim to understand the role of the identified receptors on the sleep pattern of the animals.

References [1] Raccuglia D, Huang S, Ender A, Heim MM, Laber D, Suárez-Grimalt R, Liotta A, Sigrist SJ, Geiger JRP, Owald

D. 'Network-Specific Synchronization of Electrical Slow-Wave Oscillations Regulates Sleep Drive in Drosophila.' Current biology : CB vol. 29,21 (2019): 3611-3621.e3.


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B 01-02

Dysregulation of the cortico-hippocampal network activity in the

neonatal SCN2A mutant mice

Nima Mojtahedi, Yury Kovalchuk, Daria Savitska, Olga Garaschuk

Eberhard Karls Universität Tübingen, Institute of Physiology, Department of Neurophysiology, Tübingen, Germany

Pathogenic variants of SCN2A, encoding the voltage-gated Na+ channel NaV1.2, are often associated with epileptic

encephalopathy, autism and intellectual disability. Whether these disorders reflect cell-intrinsic or network-driven

dysfunctions remains, however, unclear. Here we analyzed the effect of a missense mutation in SCN2A

(p.Ala263Val), originally found in a patient with neonatal-onset seizures, by comparing large-scale cortico-

hippocampal activity in wild type (WT) and heterozygous SCN2AA263V mice.

To do so, large-scale Ca2+ imaging was conducted in acute horizontal slices of 7 WT and 9 SCN2A 3-day-old mice,

labeled with Oregon Green BAPTA-1. Principal component analysis of region-specific mean fluorescence traces of

WT and SCN2A mice revealed clear differences in the overall activity pattern. Analyses of the amplitude, frequency,

duration and inter-event intervals of both Ca2+ transients and bursts, revealed no difference in the event frequency

as well as the inter-event or intra-burst intervals. However, event amplitude and network burstiness increased

substantially in SCN2A mice, with significantly higher burst width, frequency and fraction compared to WT mice. At

high temporal resolution, each Ca2+ signal represented a wave of activity, originating in a given pacemaker region.

In WT mice, 61% of pacemakers were of cortical origin whereas 64% of pacemakers in SCN2A mice were generated

in the hippocampus. The mean instantaneous wave velocities did not differ between the two groups. The empirical

distributions of areas, covered by individual waves, showed significant differences within some ranges, with a higher

fraction of waves propagating over the larger areas in SCN2A mice. Hidden Markov Model with two (basal and active)

hidden states, revealed a subpopulation of very long (> 30 s in duration) Ca2+ signals, present exclusively in SCN2A

mice. The latter were accompanied by seizure-like electrical activity, often covered an entire cortico-hippocampal

formation and were mostly triggered either in the hippocampus or the entorhinal/temporal cortex.

Together, the data suggest that the presence of the p.Ala263Val mutation in NaV1.2 modifies endogenous cortico-

hippocampal activity by significantly enhancing network burstiness, shifting the pacemaking activity towards the

hippocampus and increasing the size of propagating waves. These changes support the generation of yet infrequent

seizure-like activity in neonatal SCN2AA263V mice.


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B 01-03

NDNF interneuron-mediated GABAergic inhibition in the mouse dentate

gyrus

Katharina Behr, Josef Bischofberger

University of Basel, Institute of Physiology, Department of Biomedicine, Basel, Switzerland

Neurogliaform (NGF) cells are present in both superficial and deep layers of the neocortex as well as in the

hippocampal network where they comprise about 10% of the inhibitory interneuron population.

While it is known that Neuron-derived neurotrophic factor (NDNF)-positive NGF cells mediate important GABAergic

feed-forward inhibition in layer 1 of the neocortex (Abs et al. 2018), the role of this interneuron subtype within the

hippocampal circuitry remains largely unknown.

To study the function of NDNF interneurons in the hippocampus, we generated NDNF-Cre transgenic mice

expressing floxed YFP-channelrhodopsin (ChR2). Immunohistochemical analysis revealed most abundant

expression in the outer molecular layer (OML) of the DG. To investigate the functional properties of the GABAergic

synapses formed by these interneurons, we used whole-cell patch-clamp recordings to assess postsynaptic currents

in DG GCs in 2- to 3-month-old adult mice. The NDNF interneurons were stimulated using short laser pulses (1 to 2

ms) with an intensity ranging from 1.5 to 10 mW. Optogenetic stimulation of NDNF interneurons activated GABAA

receptor-mediated postsynaptic currents with slow decay time courses in the range of 67 to 104 ms. This decay

kinetic was substantially slower than the decay time constants of synaptic currents that are mediated by either

perisomatic basket cell synapses (23.4 ± 2.2 ms), or GABAergic synaptic currents evoked by extracellular stimulation

in the inner molecular layer (48.36 ± 4.4 ms), most likely mediated by cholecystokinin (CCK)-positive hilar

commissural associational pathway (HICAP) cells.Taken together, NDNF-positive interneurons mediate slow

dendritic inhibition in DG GCs.

Acknowledgment This work was supported by the Swiss National Science Foundation.


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B 01-04

Microglial THIK-1 K+ channels regulate neuronal function and network

activity in the hippocampus

Michael Surala, Ecem Tütüncü, Zoltan Gerevich, Christian Madry

Charité - Universitätsmedizin, Institut für Neurophysiologie, Berlin, Germany

Microglia are the innate immune cells of the central nervous system. Their activity is crucial for establishing complex

brain functions in the developing and adult brain. Under healthy conditions, THIK-1 is the dominant K+ channel in

microglia, and sets the cells’ membrane potential due to its constitutive and ATP-gated activity. We previously showed

that reduction of THIK-1 activity leads to a reduction in microglial ramification, surveillance and cytokine release.

Given the importance of these functions for microglia-neuron interactions, we here investigated the role of THIK-1 for

neuronal function at the cellular and network level in acute hippocampal brain slices from one-month-old mice with

genetically deleted THIK-1 K+ channels. Our data show an increase in the power of kainate-induced gamma band

oscillatory network activity (30-90 Hz), a hallmark of hippocampal network function important for information

processing which greatly depends on GABAergic interneuron function. The increase in gamma power in mice lacking

THIK-1 was most prominent in CA1 and was absent in miniature slices comprising the CA1 region only, which

suggests the involvement of CA3-CA1 feed-forward inhibition. THIK-1-induced modulation of gamma activity was

also reflected by changes in synaptic transmission in the CA3-CA1 network involving inhibitory and excitatory

neurons. Specifically, a decreased frequency, but not amplitude, of miniature excitatory postsynaptic currents

(mEPSCs) occurred in interneurons receiving input from the Schaffer collaterals and targeting dendrites of pyramidal

cells in THIK-1 KO mice. In contrast, neither mEPSC frequency nor amplitude was altered in CA1 pyramidal cells.

However, the latter exhibited an increased frequency of spontaneous EPSCs and increased EPSC amplitudes upon

electrical stimulation of CA3 Schaffer collaterals in THIK-1 KO mice. These findings suggest a reduced GABAergic

inhibition from interneurons targeting the dendrites of pyramidal cells, due to a reduced number of presynaptic

excitatory contacts on these interneurons, which may arise from impaired microglial dynamics affecting synaptic

pruning and/or deficits in cytokine levels in the absence of THIK-1. Collectively, our data show that THIK-1 is a key

regulator of microglia-neuron communication.

Acknowledgment Supported by the DFG, CRC/TRR167 NeuroMac.


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B 01-05

Place fields of single spikes in hippocampus involve Kcnq3 channel-

dependent entrainment of complex spike bursts

Changwan Chen1,2, Xiaojie Gao3,4, Franziska Bender3,4, Heun Soh5, Mahsa Altafi6,

Sebastian Schütze3,7, Matthias Heidenreich3,7, Maria Gorbati3,4, Michaela-Anca Corbu1,

Marta Carus-Cadavieco3,4, Tatiana Korotkova1,3,4, Anastasios V. Tzingounis5, Thomas J. Jentsch3,4,7,

Alexey Ponomarenko3,4,6

1 Max Planck Institute for Metabolism Research, Cologne, Germany 2 Institut für Vegetative Physiologie, University of Cologne, Cologne, Germany 3 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany 4 NeuroCure Cluster of Excellence, Charité Universitätsmedizin, Berlin, Germany 5 University of Connecticut, Storrs, CT, USA 6 Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen,

Germany 7 Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany

Hippocampal pyramidal cells encode an animal’s location by single action potentials and complex spike bursts. These

elementary signals are believed to play distinct roles in memory consolidation. The timing of single spikes and bursts

is determined by intrinsic excitability and theta oscillations (5-10 Hz). Yet contributions of these dynamics to place

fields remains elusive due to the lack of methods for specific modification of burst discharge. Here, we recorded the

discharge of pyramidal cells from the hippocampal CA1 area in freely behaving Kcnq3-/- and wild-type mice using

movable silicon probes to study network and circuit mechanisms of spatial representations by bursts and single action

potentials. Ex vivo, the M-current was reduced in pyramidal cells lacking Kcnq3-containing M-type K+ channels. We

found that pyramidal cells in vivo spontaneously fired more bursts, which consisted of a larger number of spikes, in

Kcnq3-/- than in wild-type mice. Average interspike intervals within bursts were progressively shorter with increasing

burst length in Kcnq3-/-. In the knock-out, the timing of complex spike bursts was altered during spatial navigation -

the theta rhythmic modulation of burst firing at the population level was attenuated during spatial navigation, but not

during immobility-related theta oscillations. On the other hand, pyramidal cells in the mutant featured an intact intrinsic

pause of single spikes firing after a burst. This intact intrinsic property and theta-unlocked burst times led to a temporal

offset between intrinsic and oscillatory excitability. We found that place fields of single spikes in different

environments had reduced relative size in Kcnq3-/- mice compared to control animals. Finally, we examined theta

entrainment of pyramidal cell bursts by optogenetically stimulating various types of projections from the medial

septum. This manipulations revealed that neither theta rhythmic medial septal GABA-ergic nor cholinergic inputs

alone, but rather their joint activity, is necessary and sufficient for theta rhythmic entrainment of bursts during spatial

navigation. Our results suggest that altered spatial representations by bursts and single spikes may contribute to

deficits underlying cognitive disabilities associated with KCNQ3-mutations in humans.

Acknowledgment This work was supported by a SAW grant of the Leibniz Gemeinschaft (TJJ) and Deutsche

Forschungsgemeinschaft (DFG; Exc 257 NeuroCure, TJJ, TK and AP; SPP1665, 1799/1-2, Heisenberg

Programme, 1799/3-1, AP).


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B 01-06

Inhibitory control of the nucleus reuniens by the medial prefrontal

cortex.

Gilda Baccini1, Johanne G. de Mooij-van Maalsen2,3, Alina Mogk1, Felix Kohlhaas1, Peer Wulff1

1 Christian Albrechts Universität zu Kiel, Institute of Physiology, Kiel, Germany 2 Vrije Universiteit Amsterdam, Faculty of Science, Molecular and Cellular Neurobiology, Amsterdam, Netherlands 3 Vrije Universiteit Amsterdam, Amsterdam Neuroscience - Mood, Anxiety, Psychosis, Stress & Sleep, Amsterdam,

Netherlands

The integration of executive and mnemonic functions is required for adaptive behaviour. In rodents as well as in

humans such integration is thought to depend on a circuit connecting the medial prefrontal cortex (mPFC) and the

hippocampus (HC). Whereas, the HC sends direct projections to the mPFC, no direct projections from mPFC to HC

exist. The nucleus reuniens (Re), a midline-thalamic region, forms a prominent bi-directional link between mPFC and

HC. The excitatory connections within this higher-order cortico-thalamic-cortical circuit have been extensively

described and have received increasing attention in the past few years. However, little is known about the inhibitory

control of this system. We have here used a combination of retrograde and anterograde viral tracing methods to

explore the existence of inhibitory cortical control of the RE. We found that different long-range GABAergic projections

account for about 5% of all projections from mPFC to RE.

Acknowledgment Felix Kohlhaas was supported by a stipend of the Medical Faculty, Kiel University, Alina Mogk was supported by

DFG grant GRK2154, further support came from DFG grant FOR2143


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B 01-07

Respiration paces prefrontal neuronal activity during intense threat

Shani Folschweiller, Jonas F. Sauer

Medical Faculty, Albert-Ludwigs-University Freiburg, Institute of Physiology I, Freiburg, Germany

Respiration, subdivided in inspiration and expiration, has been shown to affect human emotion recognition via nasal

airflow [1]. Furthermore, recent studies performed on rodents revealed that respiration plays a key role in higher order

cognitive processes, namely by entraining the medial prefrontal cortex (mPFC) neuronal activity during freezing

induced by Pavlovian auditory conditioning [2] . We recently found that the mPFC activity is reliably entrained by

respiration during despair-like behavior in a tail suspension test (TST) [3], suggesting that respiration-related rhythms

(RR) might aid prefrontal processing during threat.

To address this hypothesis, we performed local field potential and single unit recordings in the mPFC of mice while

monitoring the respiration during different behavioral states.

We found that respiration paces the activity of a majority of mPFC neurons during immobility, whether this immobility

was emotionally neutral, as in the home cage, or linked to threat during TST. Nonetheless, we observed that neurons

fire preferentially during inspiration when the mice where in their home cage, and switched toward firing more during

the transition from expiration to inspiration when the mice were subjected to TST stress. Furthermore, immobility

during the TST induced a robust increase in the percentage of cells coupled to the respiration, but solely in the

superficial layers 2/3, when compared to neutral immobility. This localized change in RR entrainment suggests that

a different macro-circuit of the mPFC is recruited by the respiration during intense threat. The respiration frequency

and amplitude is strongly modulated by cognitive states, and unlike other sensorial afferences, projections from the

olfactory bulb by-pass the thalamus to directly project to brain regions involved in emotions and cognition. These

characteristics, along with increasing electrophysiological evidences, indicate that the RR role in the brain extends

beyond olfactory processing.

Acknowledgment We thank the German Research Foundation (DFG) for supporting this research and Prof. Marlene Bartos, director

of the Physiology Department I.

References [1] Zelano, C, Jiang, H, Zhou, G, Arora, N, Schuele, S, Rosenow, J, Gottfried, JA 2016, ‘Nasal Respiration

Entrains Human Limbic Oscillations and Modulates Cognitive Function’, Journal of Neuroscience, 36 (49) 12448-12467, Washington, DC: Society for Neuroscience

[2] Moberly, AH, Schreck, M, Bhattarai, JP, Zweifel, LS, Luo, W, Ma, M 2018, ‘Olfactory inputs modulate respiration-related rhythmic activity in the prefrontal cortex and freezing behavior’, Nature Communications, 9:1528, New York, NY: Springer Nature

[3] Biskamp, J, Bartos, M, Sauer, JF 2017, ‘Organization of prefrontal network activity by respiration-related oscillations’, Scientific reports, 7:45508, New York, NY: Springer Nature


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B 01-08

The brain state-specific neuronal activity in the CA1 region of the

hippocampus over the course of Slow-Wave and Rapid Eye Movement

Sleep

Diego Marco Pagano1, Nima Mojtahedi1, Yury Kovalchuk1, Niels Niethard2, Olga Garaschuk1

1 University of Tübingen, Department of Neurophysiology, Institute of Physiology, Tübingen, Germany 2 University of Tübingen, Institute of Medical Psychology and Behavioral Neurobiology, Tübingen, Germany

Sleep shapes the cortical and subcortical network activity contributing to global homeostatic downscaling as well as

upscaling of selected neural subnetworks, thus consolidating newly encoded and relevant information. According to

the active systems consolidation theory, the intense crosstalk between the hippocampus and the neocortex during

sleep is responsible for the integration of newly encoded information into preexisting long-term memories. Sleep

spindles, waxing and waning oscillations occurring during the slow-wave sleep (SWS), provide an optimal time

window for synaptic plasticity and the information transfer from the hippocampus to the cortex.

Here, we used high resolution in vivo two-photon calcium imaging in combination with EEG and EMG recordings to

assess the temporal dynamics of the CA1 pyramidal cell activity across SWS and rapid eye movement (REM) sleep

and to identify cell subpopulations, whose activity was modulated differently within and across sleep stages. First,

we measured the effect of the brain state transitions on the properties of CA1 pyramidal cells. During sleep, the

number of active cells progressively decreased during SWS prior to REM and then decreased even more profoundly

over the course of REM sleep. In individual cells, including the cells active during sleep spindles, the mean frequency

of spontaneous calcium transients also decreased across the REM episodes. This reduction in activity persisted in

the subsequent episodes of SWS and wakefulness. However, we identified opposing dynamics dependent on

whether cells were selectively active during sleep spindles or slow oscillations (SO). Whereas the cells active during

sleep spindles increased their activity, the activity was decreasing in SO-active cells, thus supporting the idea that

SOs might play an important role in homeostatic downscaling during sleep.

Overall, our results suggest that sleep selectively modulates the activity of different subgroups of cells in the CA1

region of the hippocampus: SWS enables either upregulation or downregulation through spindle-related and SO-

related processes, respectively, whereas REM sleep mediates general downscaling of the neural network.

References [1] Tononi, G., & Cirelli, C. (2014). Sleep and the Price of Plasticity: From Synaptic and Cellular Homeostasis

to Memory Consolidation and Integration. Neuron, 81(1), 12–34. [2] Klinzing, J. G., Niethard, N., & Born, J. (2019). Mechanisms of systems memory consolidation during sleep.

Nature Neuroscience, 22, 1598–1610 [3] Fernandez, L., & Lüthi, A. (2020). Sleep Spindles: Mechanisms and Functions. Physiological reviews, 100(2),

805–868.


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B 01-09

Fast spiking interneurons as potential switch between alpha and

gamma-band oscillatory activity in the human neocortex

Ecem Tütüncü1, Florian Wildner1, Zoltan Gerevich1, Pawel Fidzinski2,3,4, Ulf C. Schneider5,

Ulrich-Wilhelm Thomale6, Angela M. Kaindl7,8,9, Jörg R. P. Geiger1

1 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Institute of Neurophysiology, Berlin, Germany 2 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Clinical and Experimental Epileptology, Department of Neurology, Berlin, Germany 3 Epilepsy-Center Berlin-Brandenburg, Institute for Diagnostics of Epilepsy, Berlin, Germany 4 Berlin Institute of Health at Charité, NeuroCure Clinical Research Center, Berlin, Germany 5 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Department of Neurosurgery, Berlin, Germany 6 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Department of Neurosurgery, Division Pediatric Neurosurgery, Berlin, Germany 7 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Department of Pediatric Neurology, Berlin, Germany 8 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Center for Chronically Sick Children (SPZ), Berlin, Germany 9 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu

Berlin, Institute of Cell Biology and Neurobiology, Berlin, Germany

Synchronized network activity is implicated in neuronal information processing and various cognitive functions.

Dynamic switching between alpha and gamma-band oscillatory activity has been described as a neurophysiological

correlate of attentional modulation in humans. In the rodent brain, fast-spiking interneurons are thought to play an

essential role in the generation of gamma-band oscillations. Yet little is known about the microcircuitry underlying

human neocortical gamma oscillations and the role fast-spiking interneurons might play.

Using neurosurgical resection material from 7 patients aged between 3 weeks and 43 years, we set out to investigate

human neocortical gamma oscillations in acute brain slices while recording local field potentials (LFPs) or performing

whole-cell patch-clamp recordings of fast-spiking interneurons. To visually identify the latter, we used acute

fluorescence labeling with the dye H2-DCFDA, which is largely selective for fast-spiking interneurons (Gotti et al.

2021).

Kainic acid induced oscillatory activity in the gamma frequency band in layer 2/3 and layer 5 in acute brain slices of

temporal and frontal cortical areas. We found, like in rodents, excitatory synaptic transmission onto fast-spiking

interneurons during whole-cell patch-clamp recordings to be largely mediated by calcium-permeable (CP) AMPA

receptors, since evoked postsynaptic currents were substantially blocked by 1-naphthylacetyl-spermine (Naspm), a

selective blocker of CP-AMPA receptors. Furthermore, during LFP-recordings Naspm reversibly decreased the

frequency of oscillatory activity from a gamma to an alpha range.Thus, our preliminary findings suggest that human

neocortical gamma oscillations are highly dependent on fast-spiking interneuron excitation mediated by CP-AMPA

receptors. This property of the human microcircuitry seems to be preserved irrespective of cortical region, layer, and

developmental stage. Fast-spiking interneurons might represent the key regulatory elements in dynamic switching

between oscillatory frequencies underlying cognitive processing. Further investigations will be needed to corroborate

this essential function of human fast-spiking interneurons.

References [1] Gotti, G.C., Kikhia, M., Wuntke, V., Hasam-Henderson, L.A., Wu, B., Geiger, J. R. P., Kovács, R., 2021, ‘In

situ labeling of non-accommodating interneurons based on metabolic rates’, Redox Biology, 38, 101798 (2021)


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of 516

B 01-10

Slow wave oscillations and circuit interactions underlying sleep

regulation and sensory gating in the fruit fly Drosophila melanogaster

Raquel Suárez-Grimalt1,2,3, Davide Raccuglia1,3, David Owald1,2,3

1 Charité – Universitätsmedizin Berlin, Institute of Neurophysiology, Berlin, Germany 2 Charité – Universitätsmedizin Berlin, Einstein Center for Neurosciences Berlin, Berlin, Germany 3 Freie Universität Berlin, Belin, Germany

Keeping the right balance between sleep and wakefulness is of critical importance for survival and fitness. In

mammals, electrical slow-wave activity (SWA) is correlated to the upkeep of this balance while implicated in filtering

external sensory stimuli during sleep. We have recently discovered that the fruit fly Drosophila melanogaster show

SWA within the sleep-regulating R5 network mediating sleep drive and the maintenance of consolidated sleep [1].

As the neurophysiological principles of mediating sleep drive seem to be evolutionarily conserved, we aim at using

Drosophila, a model organism with a less complex brain and high genetical accessibility, to understand how SWA

and neuronal interactions can block sensory information during sleep. Here, we use all-optical electrophysiology to

investigate how individual network components of the sleep-regulating and sensory filtering circuitry interact to create

network oscillations. We also aim at linking our neurophysiological results to the regulation of sleep behaviour and

locomotion using a combination of optogenetic tools and behavioural assays.

References [1] Raccuglia et al., 2019, Current Biology 29, 1–11


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B 01-11

Hippocampal output processing in layer VI of the medial entorhinal

cortex

Janis Winterstein, Märt Rannap, Andreas Draguhn, Alexei V. Egorov


The entorhinal cortex (EC) constitutes a major interface between the hippocampus and telencephalic structures.

Multimodal sensory information enters the hippocampal formation via neurons located in superficial layers (II and III)

of the EC. In turn, the deep layers (V and VI) receive a substantial part of the hippocampal output. Recently, we

investigated the processing of hippocampal output signals within layer V (Va and Vb) of the medial EC (mEC).

However, it is still largely unknown how hippocampal activity patterns propagate to and are processed in layer VI

(LVI).

Here, we performed whole-cell patch-clamp recordings from identified mEC LVI neurons combined with local field

potential recordings from the hippocampal CA1 area (or the subiculum) in acute horizontal mouse brain slices.

Location and morphology of recorded neurons were confirmed by labeling of the biocytin-filled neurons and

immunostaining for the transcription factor Ctip2 (a marker for layer Vb neurons).

Morphologically, layer VI was mainly found to comprise horizontal pyramidal neurons exhibiting a main dendrite

running parallel to the cell layer, or neurons with multipolar organization. The axons of LVI neurons travelled towards

the subiculum and frequently also to superficial layers of the mEC, indicating far-reaching innervation. In the vast

majority of LVI glutamatergic neurons electrophysiological recordings revealed a characteristic delayed firing pattern

with only minor firing adaptation in response to depolarizing current steps and a lag of hyperpolarization-activated

sag potential. Furthermore, LVI neurons were able to generate muscarinic acetylcholine receptor-dependent

persistent activity, as previously described for LV neurons.To study the propagation of hippocampal output signals

to mEC LVI, we injected a ChR2-expressing AAV into the ventral hippocampus (CA1/subiculum) and activated the

axons of infected neurons by illuminating LVI with blue light. Most LVI neurons showed strong synaptic responses to

the light pulses with a median delay time of 2.32 ms, suggesting monosynaptic input from the ventral hippocampus.

Correspondingly, a naturally occurring hippocampal activity pattern, the sharp wave-ripple complex (SPW-R),

propagated efficiently to LVI neurons, causing well discernible compound postsynaptic responses. These findings

suggest a critical involvement of mEC LVI in hippocampal-neocortical signal propagation.

Acknowledgment Supported by the DFG Grant No. 430282670 (EG134/2-1)


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B 01-12

Multi-Electrode Array Analysis Identifies Complex Dopamine

Responses and Glucose Sensing Properties of Substantia Nigra

Neurons in Mouse Brain Slices

Nadja Mannal1, Katharina Kleiner1, Christina Poetschke1, Birgit Liss1,2

1 Ulm University, Institute of Applied Physiology, Ulm, Germany 2 University of Oxford, Linacre College & New College, Oxford, UK

Dopaminergic midbrain neurons (DAN) are important for a variety of brain functions like movement control, cognition,

motivation, and glucose homeostasis. Hence, identifying mechanisms that control the activity of DAN are of particular

interest. They display intrinsic pacemaker activities, but only in DAN within the Substantia nigra (SN) are these

accompanied by intracellular Ca2+ oscillations. Ca2+ promotes transmitter-release and ATP production, but

constitutes a stressful burden that underpins their heightened vulnerability to neurodegenerative stressors.

Accordingly, SN DAN are particularly affected by additional metabolic stress, and their progressive degeneration

causes the main motor-symptoms of Parkinson’s disease (PD). Given the high vulnerability of SN DAN, their activity

is regulated by complex mechanisms, notably by dopamine itself, via inhibitory dopamine D2-autoreceptor responses

that can undergo prominent desensitization. However, we lack a detailed mechanistic understanding.

We studied SN DAN activity and its control by dopamine and glucose with extracellular multi-electrode array (MEA)

recordings from mouse midbrain slices. Our optimized MEA- and spike sorting-protocols allowed high throughput

and long recording times. According to individual dopamine-responses, we identified two distinct SN cell-types, in

similar abundancy: dopamine-inhibited, and also dopamine-excited neurons. Dopamine-excited SN neurons were

either silent in the absence of dopamine, or they displayed pacemaker-activity, similar to that of dopamine-inhibited

neurons. Spontaneous firing-rates of DA inhibited SN neurons were significantly higher at high glucose-levels

(∼20%). Moreover, transient glucose-deprivation (1 mM) induced a fast and fully reversible pacemaker frequency

reduction. To directly address and quantify glucose sensing properties of SN DAN, we continuously monitored their

electrical activity, while altering extracellular glucose concentrations stepwise from 0.5 mM up to 25 mM. SN DA

neurons were excited by glucose, with EC50 values ranging from 0.35 to 2.3 mM.

In conclusion, we identified a novel subtype of dopamine-excited SN neurons, and a complex, joint regulation of

dopamine-inhibited neurons by dopamine and glucose, within the range of physiological brain glucose-levels. We

are currently addressing the molecular mechanisms of dopamine-excited SN neurons, as well as of glucose sensing

in dopamine-inhibited SN neurons.


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Poster Session B | 01 October 2021 5:00 PM – 7:00 PM

Foyers

B 02 | Neuronal Pathophysiology Chair

Dirk Isbrandt (Köln)

Hans-Georg Schaible (Jena)


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B 02-01

Neutropenia enhances the brain and peripheral inflammatory response

to LPS-induced hypothermic severe systemic inflammation.

Jessica Hernandez1, Fabian Pflieger1, Jenny Schneiders1, Karsten Krüger2, Thomas Reichel2,

Christoph Rummel1

1 Justus-Liebig Universität, Institut für Veterinär-Physiologie und -Biochemie, Giessen, Germany 2 Justus-Liebig Universität, Institut für Sportwissenschaft, Giessen, Germany

Introduction: The innate immune system plays a pivotal role in shaping acute inflammatory responses through

immune-to-brain signaling. This includes central nervous system-induced sickness responses, such as fever,

lethargy, and adipsia, which are known to occur during systemic inflammation. Previous studies have shown that

leukopenia can alter sickness responses leading to a prolonged fever response. Moreover, neutropenic fever is a

severe clinical status of unknown origin.

Hypothesis: We aimed to investigate the effects of neutropenia on the sickness response and immune-to-brain

signaling during acute systemic inflammation in mice.

Methods: To induce neutropenia and systemic inflammation, mice received an intraperitoneal injection of anti-

polymorphonuclear serum (PMN) followed by a high dose intraperitoneal injection of lipopolysaccharide (LPS, 2.5

mg/kg) 24 or 48 hours later. Brains, peripheral tissue, and serum were collected at 4h or 24h after LPS-stimulation

for detection of peripheral/brain inflammatory markers. To investigate the physiological significance of neutropenia,

we continuously recorded locomotor activity, core body temperature, food, and water intake using a telemetric system

over the course of the experiment.

Results: PMN reduced circulating neutrophils by approximately 20% compared to control mice that received NRS in

dose response experiments. Initial experiments revealed that neutropenia inhibited recruitment of neutrophil

granulocytes (NG) to the brain, brain nuclear factor interleukin-6-activation, and was associated with inhibited LPS-

induced expression of the anti-inflammatory cytokine interleukin 10 and NG specific chemokine CXCL1 (48h). PMN-

pretreatment exacerbated LPS-induced hypothermia compared to NRS controls (24h), while adipsia, anorexia, and

loss in body weight were not affected by neutropenia (4h and 24h). Further analyses revealed that LPS-induced

increase in corticosterone serum levels were higher (24h) and levels of circulating cytokines were enhanced (4h and

24h) in PMN mice when compared to NRS counterparts.

Conclusion: Together, our ongoing experiments suggest an anti-inflammatory role of NG with neutropenia

exacerbating sickness and immune responses during systemic inflammation.


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B 02-02

Glutamate receptor expression in human brain tumors and perampanel

action in rodent glioma

Richard Gade1, Franziska Zessin1, Falko Lange1,2, Katrin Porath1, Jens Hartung1, Thomas Freiman3,

Christian Henker3, Rüdiger Köhling1,2, Timo Kirschstein1,2

1 Rostock University Medical Center, Oscar-Langendorff-Institute of Physiology, Rostock, Germany 2 University of Rostock, Center for Transdisciplinary Neurosciences Rostock, Rostock, Germany 3 Rostock University Medical Center, Department of Neurosurgery, Rostock, Germany

Seizures are a common comorbidity of glioblastoma. Both, glioblastoma and tumor-associated epilepsy share several

pathological mechanisms, which drive tumor progression and generation of seizures. On key player is the

neurotransmitter glutamate. In glioma, extracellular glutamate levels were found to be elevated up to 100-times higher

than in unaffected brains. On the one hand, this may contribute to an increased glioma cell growth and, on the other

hand, may lead to epileptic discharges and excitotoxicity, which in turn may facilitate tumor bulk expansion. Several

glutamate receptors like mGluR3 are known to be upregulated in glioblastoma. In contrast to glioblastoma, there are

hardly any data for brain metastasis.

In our study, we analysed the expression of glutamate receptors in tissues of glioblastoma and brain metastasis

obtained from surgical resections. Additionally, clinical data like the presence of seizures were recorded. We were

able to identify a panel of genes that are expressed differently between the two cohorts. As expected, mGluR3 was

found to be higher expressed in glioblastoma than in samples of brain metastasis, but there are also differences in

the group of the AMPA receptors.

Therefore we further asked, if an AMPA receptor antagonist may not only attenuate an epileptic phenotype, but also

may affect tumor progression. As shown by our group in vitro, AMPA receptor antagonist perampanel acts in an

antiproliferative manner and additionally may attenuate glutamate levels in human glioblastoma cells. To further

investigate the role of AMPA receptors in glioma and tumor-associated epilepsy, we employed F98 glioma cells as

an orthotopic tumor model in Fischer rats. Perampanel was tested in a monotherapy setting and also in combination

with standard radiochemotherapy (RCT). Epileptiform activity was recorded with video EEG monitoring in vivo and

in electrophysiological analysis of brain slices bearing F98 glioma in vitro. With respect to F98 gliomas, the tumor

size was estimated and expression of AMPA receptors was analysed. In EEG recordings, we could demonstrate that

perampanel abolished a tumor-associated epileptogenic phenotype. Furthermore, electrophysiological recordings

suggested neuroprotective effects by perampanel in combination with RCT. With respect to the tumor disease, a

highly reduced glioma size after RCT was determined whereas additional perampanel substitution had no further

effect.


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B 02-03

Short-term pallidal deep brain stimulation alters synaptic

communication of striatal medium spiny neurons in the animal model

of the dystonic dtsz mutant hamster

Denise Franz1, Marco Heerdegen1, Julia Hörnschemeyer1, Nadja Engel1, Valentin Neubert1, Anika

Lüttig2, Stefanie Perl2, Franz V. Plocksties3, Angelika Richter2, Rüdiger Köhling1

1 University Medical Center Rostock, Oscar Langendorff Institute of Physiology, Rostock, Germany 2 University of Leipzig, Institute of Pharmacology, Pharmacy and Toxicology/ Veterinary Faculty, Leipzig, Germany 3 University Rostock, Institute of Applied Microelectronics and Computer Engineering, Rostock, Germany

Question: Deep brain stimulation of the globus pallidus internus (GPi) indicated the most relevant therapeutic option

for patients with severe dystonias. However, the mechanisms of the deep brain stimulation (DBS) of the GPi are far

from understood. Dystonia, a hyperkinetic movement disorder, is thought to result from disequilibrium of the direct

and indirect pathway originates from disturbance in the striatum. In the dtsz mutant hamster, a model of inherited

generalized, paroxysmal dystonia, it has been already shown that the number of parvalbumin-positive gabaergic

interneurons declined, which leads to uncontrolled projections via the medium spiny neurons (MSN). We

hypothesized that DBS via backfiring, or indirectly via thalamic and cortical coupling, modifies striatal network

function.

Methods: The dtsz mutant hamsters were bilaterally implanted with stimulation electrodes targeting the

entopeduncular nucleus (EPN; equivalent of the human GPi). DBS (130 Hz, rectangular pulse of 50 μA and 60 μs)

and sham-DBS were performed in vivo in unanaesthetized animals. Acute brain slices of both groups were

immediately prepared after the 3 h DBS as well as from untreated dtsz mutant hamsters (native group) to assess the

effects of electrode implantation. With whole-cell patch clamp recordings, we investigated the spontaneous cortico-

striatal synaptic activity and the characteristics of the D1- (direct pathway) and D2- (indirect pathway) MSN, which

are capable of being differentiated by adding the D2-agonist sumanirole.

Results: In view of spontaneous release activity from cortical projections, our study indicated a strong dampening

effect on the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSC) of the stim-group in

contrast to sham-group. The specific cellular parameters of D1- and D2-MSN, which include resting membrane

potential, input resistance, membrane capacity, rheobase, and firing properties of action potentials, did not differ

between native-, sham-, and stim- group.

Conclusions: To conclude, while EPN-DBS obviously dampens spontaneous presynaptic glutamate release at

cortico-striatal synapses, there is no alteration on MSN properties. Future studies will consider the effects of long-

term stimulation on synaptic plasticity by a novel implantable stimulation system which enables DBS over weeks.

Acknowledgment This study is supported by the German Research Foundation (DFG) within the Collaborative Research Centre

(SFB 1270/1 ELAINE 299150580).

References


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[1] {cke_protected}{C}%3C!%2D%2DStartFragment%2D%2D%3EHeerdegen M, Zwar M, Franz D, Hörnschemeyer J, Neubert V, Plocksties F, Niemann C, Timmermann D, Bahls C, van Rienen U, Paap M, Perl S, Lüttig A, Richter A, Köhling R. Mechanisms of pallidal deep brain stimulation: Alteration of cortico-striatal synaptic communication in a dystonia animal model. Neurobiol Dis. 2021 Jul;154:105341. doi: 10.1016/j.nbd.2021.105341. Epub 2021 Mar 19. PMID: 33753292. {cke_protected}{C}%3C!%2D%2DEndFragment%2D%2D%3E

[2] Paap M, Perl S, Lüttig A, Plocksties F, Niemann C, Timmermann D, Bahls C, van Rienen U, Franz D, Zwar M, Rohde M, Köhling R, Richter A. Deep brain stimulation by optimized stimulators in a phenotypic model of dystonia: Effects of different frequencies. Neurobiol Dis. 2021 Jan;147:105163. doi: 10.1016/j.nbd.2020.105163. Epub 2020 Nov 6. PMID: 33166698.


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B 02-04

Electrophysiological profile of dopaminergic neurons as a potential

marker for vulnerability in Parkinson disease mouse model

Josefa Zaldivar-Diez1,2, Lora Kovacheva1, Joseph Shin1, Jose A. Obeso2,3, Ledia F. Hernández2,3,

Jochen Roeper1

1 Goethe University, Institute of Neurophysiology, Frankkurt Am Main, Germany 2 HM-CINAC, HM Puerta del Sur / Fundación de Investigación HM Hospitales, Madrid, Spain 3 Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED), Madrid, Spain

Parkinson disease (PD) is the second most common neurodegenerative disorder affecting 5 million people

worldwide and expected to increase up to 10 million by 20301. Clinically, PD is characterized by the presence of

resting tremor, bradykinesia, rigidity and postural instability2. These cardinal signs are thought to arise as a

consequence of the early and prominent loss of ventro-lateral dopamine neurons of the substantia nigra pars

compacta (DA SNpc) and the associated dopamine depletion within related striatal projection areas3.

DA SN neurons are considered a major target in PD compared to more resistant neighboring DA neurons in the

ventral tegmental area (VTA). However, even within the SN, there are regional differences in vulnerability4. In

humans, DA SNpc neurons in the ventro-lateral tier show an earlier and higher loss compared to those in the dorso-

medial tier. This differential pattern in primates corresponds to higher vulnerability of lateral compared to medial DA

SNpc neurons in rodents.

In the current project, we aim to establish a mild 6-OHDA lesion mouse model where about 50% of the most

vulnerable lateral DA SNpc neurons survive. We will then study the in vivo and in vitro electrophysiology features of

surviving DA lateral SNpc neurons in order to identify functional impairments or adaptations that might modulate their

high innate vulnerability compared to the more medial ones.

References [1] Dorsey, ER; Constantinescu, R; Thompson, JP; Biglan, KM; Holloway, RG; Kieburtz, K; Marshall, FJ; Ravina,

BM; Schifitto, G; Siderowf, A; Tanner, CM. Projected number of people with Parkinson disease in the most populous nations, 2005 through 2030. Neurology., 2007, 68 (5), 384-386.

[2] Kalia LV, Lang AE. Parkinson disease in 2015: Evolving basic, pathological and clinical concepts in PD. Nature Reviews Neuroly., 2016, 12 (2), 65-66.

[3] Redgrave P, Rodriguez M, Smith Y, Rodriguez-Oroz MC, Lehericy S, Bergman H, Agid Y, DeLong MR, Obeso JA. Goal-directed and habitual control in the basal ganglia: implications for Parkinson's disease. Nature Reviews Neuroscience.,2010, 11 (11), 760-772.

[4] Kordower JH, Olanow CW, Dodiya HB, Chu Y, Beach TG, Adler CH, Halliday GM, Bartus RT. Disease duration and the integrity of the nigrostriatal system in Parkinson’s disease. Brain., 2013, 136, 2419–2431.


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B 02-05

Galvanotactic migration of patient-derived brain tumor cells

Falko Lange1,2, Daria S. E. Abady1, Katrin Porath1, Anne Einsle1, Michael Linnebacher3,

Rüdiger Köhling1,2, Timo Kirschstein1,2

1 Rostock University Medical Center, Oscar-Langendorff-Institute of Physiology, Rostock, Germany 2 University of Rostock, Center for Transdisciplinary Neurosciences Rostock, Rostock, Germany 3 Rostock University Medical Center, Molecular Oncology and Immunotherapy, Department of General, Visceral,

Vascular and Transplantation Surgery, Rostock, Germany

High-grade gliomas have one of the worst survival prognoses of all common human tumor diseases. Even after

tumour resection and adjuvant radiochemotherapy, there often remain only a few months of survival after the

diagnosis, since relapses almost always occur. Several pathophysiological mechanisms were identified, that

contribute to this highly limited outcome, with cell migration being one of the hallmarks. The molecular mechanism

by which glioma cells migrate and invade into the healthy brain is complex and poorly understood so far. One stimulus

of migration can be electrical fields. The migration of cells along a direct current electrical field referred to as

galvanotaxis and can be in the direction of either the positive or the negative electrode. Interestingly, in previous

studies based on permanent cell lines, an increased cellular motility to both the cathode and the anode were

documented.

In the present study, we employed patient-derived low-passage glioblastoma cells and cells of brain metastasis to

investigate the galvanotactic migration in vitro. Therefore, we cultivated these cells in direct current chambers and

analysed migratory behaviour under different electric field strengths.

Under control conditions without direct current, all employed cell lines migrated during the observation period, with

the glioblastoma cells showing an overall higher velocity than cells derived from metastasis. Under direct current

conditions, the glioblastoma cells showed mainly anodal migration behaviour. Remarkably, the brain metastasis cells

presented no change in response to the electric field stimulation.

To gain more insights into possible mechanism, we exposed the cells to small molecule kinase inhibitors that may

interfere in central signalling pathways that are associated with migration. Proliferation of the cells and induction of

apoptosis were determined. Based on these data, selected concentration of the kinase inhibitors were co-

administered to the direct current studies and again migratory behaviour was estimated.

Our results suggest that glioblastoma cells are able to actively migrate along electrical direct current fields. In vitro,

the migration can be in part restricted by kinase inhibitors.


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B 02-06

Seizure-associated potassium signaling in the endothelial-pericytic

syncytium of the ex vivo neurovascular unit

Mirja grote Lambers1, Henrike Planert1, Agustin Liotta1, Christian Madry1, Jörg R. P. Geiger1,2,

Richard Kovács1

1 Charité – University Medicine Berlin, Institute of Neurophysiology, Berlin, Germany 2 NeuroCure, Cluster of Excellence, Charité – Universitätsmedizin Berlin, Berlin, Germany

Involvement of the neurovascular unit (NVU) in epileptogenesis has gained attention recently, however, the

mechanisms of postictal neurovascular uncoupling and hypoperfusion are still not known in every detail.

[1,2] Epileptic seizures come along with large increases in extracellular potassium concentration which is

redistributed to blood flow where potassium acts as a strong vasoactive signal. [3,4,5]

Here we studied seizure-associated potassium signaling at pericyte/astrocyte/EC interface with special emphasis on

electrical coupling and voltage-dependent Ca-channels (VDCCs) and their possible contribution to hyper- or postictal

hypoperfusion.

Whole cell patch clamp recordings were obtained from visually identified astrocytic endfeet, pericytes and ECs,

whereas seizure-associated changes in extracellular potassium concentration were recorded with ion-sensitive

electrodes. GAP junctional communication was revealed by dye coupling while VDCC or metabotrop receptor-

mediated intracellular Ca-signaling was monitored by Oregon green-BAPTA 1 or rhod-2 fluorescence. The missing

vasotonus was mimicked by the application of the thromboxane analogue U46619 and epileptiform activity was

induced by the voltage dependent potassium channel inhibitor 4-aminopyridine.

Endothelial-pericytic syncytium remains functionally connected in OHSCs and reacts to neuronal activity by

potassium-mediated changes in the membrane potential. Pericytes along the capillaries differed with regard to

morphology, electrophysiological properties and coupling strength. Pericytes depolarized during seizures, constricted

upon U46619 and presented metabotrop receptor mediated Ca-signaling. However, membrane potential changes in

thin strand pericytes were virtually indistinguishable from astrocytic endfeet and ECs, whereas in a subset of pericytes

we observed inwardly rectifying and voltage dependent potassium currents. The contribution of VDCCs to Ca-

signaling could be revealed in the presence of the L-type VDCC agonist Bay-K-8644.

In conclusion, the GAP-junctional coupling among thin strand pericytes and ECs enabled the quick retrograde spread

of voltage changes, whereas putative pre-capillary mesh or ensheathing pericytes were adapted to transduce a

capillary-generated voltage into a change in intracellular calcium concentration. On the other hand, our results did

not support the hypothesis that postictal neurovascular uncoupling observed in OHSCs would be mediated by

VDCCs.

Acknowledgment The study was supported by the DFG grant: AL/NB 408355133 for MgL and AL, and the Sonnenfeld Foundation to

RK. The authors are deeply indebted to Ms Andrea Wilke for her technical assistance.


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Fig. 1 Brain pericytes are coupled via

endothelium creating a syncytium up to 450μm

distance

a. Whole cell patch clamp recording of a

targeted pericyte using an Alexa488 dye-filled

pipette. b. Dye diffuses via gap junctions in

both directions of the vessel revealing a gap

junctional coupled network of pericytes through

ECs. Coupling strength was quantified measuring

fluorescence intensity of the cells c. Double

patch recordings of a pericyte couple in a

NeuroTracer labelled capillary showing a

directionality in coupling (n=2). Despite the

directionality of the coupling, the spontaneous

depolarizations (arrow) were identical in both

pericytes (traces drifted 2mV to enhance

visibility).

Fig. 2 4AP (100µM) induced seizure like activity

leads to changes in potassium reversal potential

a. Recordings with ion-sensitive electrodes

reveal a 18.5 ± 2.42mV drift during seizure-like

activity equivalent to an extracellular

potassium accumulation of 7.156 ± 0.68mM (n=9).

Whole cell patch clamp recordings of pericytes

and astrocytes present with similar pattern of

recurrent membrane depolarizations. b. Two types

of pericytes could be distinguished by showing

either an ohmic profile of the IV-curve or

rectification and possible activation of voltage

dependent potassium currents. c. Astrocytes show

a stereotypic ohmic current to voltage

relationship and no coupling to pericytes.

References [1] Farrell JS, Gaxiola-Valdez I et al. 2016, ‘Postictal behavioural impairments are due to a severe prolonged

hypoperfusion/hypoxia event that is COX-2 dependent.’ eLife, 2016;5:e19352, doi: 10.7554/eLife.19352 [2] Prager O, Kamintsky L et al. 2019, ‘Seizure-induced microvascular injury is associated with impaired

neurovascular coupling and blood-brain barrier dysfunction.’ Epilepsia 60(2):322-336, doi: 10.1111/epi.14631

[3] Kovács R, Heinemann U et al. 2012, ‘Mechanisms underlying blood-brain barrier dysfunction in brain pathology and epileptogenesis: role of astroglia.’ Epilepsia, 53, 6:53-9. doi: 10.1111/j.1528-1167.2012.03703.x

[4] Filosa JA 2010 ‘Vascular tone and neurovascular coupling: considerations toward an improved in vitro model.’ Front Neuroenergetics, 2:16, doi: 10.3389/fnene.2010.00016

[5] Bellot-Saez A, Kékesi O et al. 2017, ‘Astrocytic modulation of neuronal excitability through K+ spatial buffering.’ Neurosci Biobehav Rev., 77:87-97, doi: 10.1016/j.neubiorev.2017.03.002


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B 02-07

The role of the ubiquitin-like protein ubiquilin-1 on epileptic activity in

mice in vitro.

Tabea Kürten, Natascha Ihbe, Thomas Mittmann

University Medical Center of the Johannes Gutenberg University, Institute of Physiology, Mainz, Germany

Posttraumatic epilepsies (PTEs) constitute a major public health concern and a large proportion of acquired

epilepsies. However, their prevention remains a matter of intense research, as no effective anti-epileptogenic

treatment preventing late seizure-onset has been implemented in clinical practice yet [1]. The present study provides

new insights on the role of the GABAA-receptor stabilizing protein ubiquilin-1, its regulation and modulation under in

vitro epilepsy conditions in mice. The ubiquitin-like protein ubiquilin-1 forms a functional link between the

ubiquitination machinery and the proteasome [2]. It accumulates at inhibitory synapses, prevents early GABAA-

receptor degradation and increases the receptor cell surface levels [3; 4]. Diminished ubiquilin-1 expression levels

have been reported in samples from temporal lobe epilepsy (TLE)-patients and in a rat model of epilepsy [3]. In our

study, we used an in vitro mouse model of epilepsy to study hippocampal and cortical ubiquilin-1 expression levels.

Epileptiform events were evoked pharmacologically in acute slices by application of picrotoxin (50 μM) and kainic

acid (500 nM). Extracellular recordings were conducted in the hippocampal CA1 region and monitored by

Multielectrode Array (MEA) recordings. Interestingly, Western blot quantification revealed a significant decrease in

the ubiquilin-1 expression in cortical and hippocampal lysates in a time window of 1-7 hours after onset of epileptiform

activity. To achieve a potential rescue of ubiquilin-1 expression in our in vitro epilepsy model, we included the non-

selective monoamine oxidase inhibitor nialamide (NM). Western blot quantification confirmed our hypothesis that via

NM-administration (10 μM), the previously observed reduction in ubiquilin-1 expression could be fully reversed. To

disclose potential alterations in excitability, we further conducted MEA-recordings and compared the picrotoxin-

dependent dose-response relationships. Here, the NM-application significantly mitigated epileptiform activity with

regard to the number of discharges and the mean peak amplitude. Our data indicate aside from an increased

ubiquilin-1 expression a potential neuroprotective effect of NM involving the monoamine transmitter system. Future

investigations on the role of specific monoamine transmitter systems might be a useful strategy to

regain physiological excitatory-inhibitory (E/I) balance in posttraumatic epileptogenesis.

References [1] Lucke-Wold, B. P., Nguyen, L., Turner, R. C., Logsdon, A. F., Chen, Y. W., Smith, K. E., Huber, J. D.,

Matsumoto, R., Rosen, C.L., Tucker, E. S., Richter, E. 2015. Traumatic brain injury and epilepsy: Underlying mechanisms leading to seizure. Seizure, 33, 13-23.

[2] Ko, H.S., Uehara, T., Tsurama, K., Nomura, Y. 2004. Ubiquilin interacts with ubiquitylated proteins and proteasome through its ubiquitin-associated and ubiquitin-like domains. FEBS Lett, 566, 110-4.

[3] Zhang, Y., Li, Z., Gu, J., Zhang, Y., Wang, W., Shen, H., Chen, G., Wang, X. 2015. Plic-1, a new target in repressing epileptic seizure by regulation of GABAAR function in patients and a rat model of epilepsy. Clin Sci (Lond), 129, 1207-23.

[4] Bedford, F. K., Kittler, J. T., Muller, E., Thomas, P., Uren, J. M., Merlo, D., Wisden, W., Triller, A., Smart, T.G., Moss, S.J. 2001. GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1. Nat Neurosci, 4, 908-16.


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B 02-08

RNAscope analysis of A53T α-synuclein transgenic mice and post

mortem human brains identifies altered expression of Cav and Kv4

channels in neurons displaying differential vulnerability to

degeneration in Parkinson’s disease.

Nicole Wiederspohn1, Desirée Spaich1, Christina Pötschke1, Jochen Roeper2, Joshua Goldberg3,

Thomas Arzberger4,5, Johanna Duda1, Birgit Liss1,6

1 Ulm University, Institute of Applied Physiology, Ulm, Germany 2 Goethe University Frankfurt, Institute of Neurophysiology, Frankfurt, Germany 3 The Hebrew University of Jerusalem, Department of Medical Neurobiology, Jerusalem, Israel 4 Ludwig-Maximilians-University Munich, Department of Psychiatry and Psychotherapy, Munich, Germany 5 Ludwig-Maximilians-University Munich, Center for Neuropathology and Prion Research, Munich, Germany 6 University of Oxford, New College, Oxford, UK

The two major neuropathological hallmarks of Parkinson’s disease (PD) are differential neuronal vulnerability and

formation of toxic α-synuclein aggregates, so-called Lewy bodies (LB). While dopaminergic (DA) neurons within the

Substantia nigra (SN) exhibit the highest cell loss during PD and its animal models, cholinergic neurons within the

dorsal motor nucleus of the vagus (DMV) show extensive LB-formation and a similar pacemaker-activity, but are less

affected by degeneration in PD. The cause for this differential neuronal vulnerability is still unclear, but cell-type

specific ion channel activity, activity-related metabolic stress, and Ca2+ homeostasis are important factors.

Recent studies analysed mice overexpressing mutant human α-synuclein (A53T, PARK1), causing a familial form of

PD. SN DA neurons of A53T mice displayed elevated metabolic stress as well as redox-impaired A-type K+ channel

(Kv4) function, while in DMV neurons metabolic stress levels were even lower, compared to wildtype (WT). Here, we

addressed differential expression of voltage gated Ca2+ channels (Cav) and/or of Ca2+ and voltage gated Kv4

channels as underlying mechanism. We quantified expression of Cav and Kv4 channel α and β subunits in mouse

and in human neurons (from post mortem brains) via RNAscope in situ hybridization, combined with a custom-

designed algorithm (Wolution) for automated cell recognition and signal quantification.

We found that already in WT, selectively Cav3.1 mRNA levels were significantly higher in vulnerable SN DA

compared to more resistant DMV neurons. In A53T-mice, Cav3.1 levels were further reduced in DMV, while they

were further elevated in SN DA neurons. A-type channel α (Kv4.3/Kv4.2) and β (KChip3/4) subunits were also

differentially expressed in in DA and DMV neurons. Moreover, in remaining human SN DA neurons from PD patients,

mRNA-levels for Cav3.1, as well as for Kv4.3 and KChip3 were significantly higher, compared to those of age-

matched unaffected controls.

We propose that elevated Cav3.1 levels in SN DA neurons constitute a compensatory response to their progressive

loss in PD, to stimulate Ca2+ dependent ATP-production and dopamine release in the remaining neurons, but it also

elevates metabolic stress. The latter could be countered by higher functional expression of inhibitory A-type K+

channels, in a neuroprotective feedback loop. We currently further analyse this hypothesis at the functional level.


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B 02-09

Hippocampal alterations and behavioural deficits in a mouse model of

Fabry disease

Jeiny Luna-Choconta, Kai K. Kummer, Theodora Kalpachidou, Michaela Kress

Medical University of Innsbruck, Institute of Physiology, Innsbruck, Austria

Accumulation of glycosphingolipids in internal organs and the nervous systems is a main characteristic of Fabry

disease (FD). This lysosomal storage disorder is caused by a defective x-chromosomal α-galactosidase A (αGal)

gene, leading to dysfunctions in the metabolism of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3).

FD patients develop neurological symptoms, such as cognitive deficits, with older age that may be caused by Gb3

accumulating in diverse regions of the central nervous system. We therefore explored Gb3 accumulation and

behaviour during the progression of FD in a murine transgenic α-Gal A-/0 (Gla-KO) model of FD.

To determine the effect of Gb3 accumulations in brains of FD mice, we performed immunohistochemistry using anti-

Cd77 for Gb3, anti-Cd31 for blood vessels (endothelial cells), and anti-Tmem119 for microglial morphology.

Behavioural differences were assessed in Gla-KO and wild type (wt) mice using the open field test for general motor

behaviour and anxiety, the Rotarod test for sensorimotor coordination, as well as the Barnes maze for spatial memory

assessment.

We found Gb3 accumulation in the hippocampal dentate gyrus, a brain area that is involved in the spontaneous

exploration of novel environments. Gla-KO mice showed decreased thickness of blood vessels and alteration of

microglia morphology characterized by a smaller number of intersections of microglial processes. Overall, Gla-KO

mice showed deficits in exploratory behaviour and latency to complete the task in the Barnes maze test. No

alterations were observed in the Rotarod test.

To summarize, Gb3 accumulation in the hippocampal dentate gyrus of Gla-KO mice was associated with a deficit in

exploration of new environments but not sensorimotor coordination, suggesting that region-specific structural

alterations might be causally involved in cognitive deficits in FD. These findings might link Gb3 accumulation to

neuroimmune and vascular changes as a potential cause of dementia developing in FD patients at older ages.

Acknowledgment The project has received funding from the European Union’s Horizon 2020 research and innovation programme

under the Marie Skłodowska-Curie Grant Agreement No 764860 “TOBeATPAIN”.


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B 02-10

Neurodegeneration and obsessive-compulsive disorder in SPRED2-

deficient mice is associated with brain ventricle enlargement and

ultrasonic vocalization changes

Denis Hepbasli1, Sina Gredy1, Melanie Ullrich2, Martin Pauli1, Kai Schuh1

1 University of Wuerzburg, Institute of Physiology, Würzburg, Germany 2 University Clinic Wuerzburg, Center for Rare Diseases, Würzburg, Germany

Neurodegenerative diseases are characterized by progressive neuronal cell death. Common consequences are

movement coordination problems (ataxia), mental disabilities (dementia), and compulsive behaviors. To date, the

underlying mechanisms remain quite unclear. Here we show that the deficiency of SPRED2, a protein ubiquitously

expressed in brain and a potent suppressor of Ras/ERK-MAPK cascades [1], causes neurodegeneration in

combination with a changed ultrasonic vocalizations (USV) usage in mice.

SPRED2-KO mice show excessive self-grooming leading to severe facial lesions despite normal skin sensitivity.

Obsessive-compulsive grooming behavior is accompanied by an increased release of stress hormones from the

hypothalamic-pituitary-adrenal axis and by reduced anxiety behavior as detected by behavioral tests [2]. Prompted

by increased brain ventricles detected by exemplary MRIs and by augmented levels of Tau and P-Tau in brains of

SPRED2-KOs, one objective of this study was to verify if OCD in SPRED2-KOs is accompanied by

neurodegeneration. To proof our hypothesis, we quantified the ventricle system volume in brains of young and old

WT and SPRED2-KOs by cutting PFA-fixed brains in 100 µm slices, measuring ventricle areas in each slice, and by

reconstruction of 3D models with Imaris. This quantification of all ventricle volumes revealed a significant increase of

the ventricle system of aged SPRED2-KOs (Figure 1). To determine related differences in USV usage, we analyzed

call rate, call subtype profile, and acoustic parameters (i.e. duration, bandwidth, and mean peak frequency) in young

and old SPRED2-KOs. We recorded USVs of male mice, while interacting with females, analyzed the calls of both

with the deep-learning DeepSqueak Software, trained to recognize and categorize the emitted USVs [3]. Thereby,

we were able to group these calls into 10 different call groups (Figure 2, a-j), and found significant differences in the

pure number of calls (Figure 2k) and multiple variations in which those calls were used by the young and old WT and

SPRED2-KO mice.

Our findings confirmed neurodegeneration in SPRED2-KOs by an increase of the ventricle system volume and

provided the first classification of WT vs. SPRED2-KO USVs. This indicates that SPRED2 is involved in the

pathogenies of neurodegenerative disorders and in developmental processes required for USV learning.


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Subdivision of the 10 different call types and

total number of calls Figure 1: a-j: Examples of calls, generated by

DeepSqueak. a: Short. b: Flat. c: Up. d: Down.

e: U. f: Inverted U. g: Step Down. h: Step Up.

i: Complex. j: Harmonic. k: Total number of

Calls: Two-way ANOVA demonstrating the averaged

sum of calls per five-minute recording of young

and old animals from WT and KO (genotype p <

0.05; age p < 0.05).


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Exemplary 3D models of mouse brains Figure 2: WT in the left column and KO in the

right column. A/B: view at -45°/145°/0° angle of

young animals. C/D: caudal view of

young animals. E/F: view at -45°/145°/0° angle

of old animals. G/H: caudal view of old animals.

Highlighted in purple is the ventricle system,

which showed a significant genotype-dependent

enlargement in older animals.

References [1] Ullrich, M., et al., Identification of SPRED2 (Sprouty-related Protein with EVH1 Domain 2) as a Negative

Regulator of the Hypothalamic-Pituitary-Adrenal Axis. J Biol Chem, 2011. 286(11): p. 9477-88. [2] Ullrich, M., et al., OCD-like behavior is caused by dysfunction of thalamo-amygdala circuits and

upregulated TrkB/ERK-MAPK signaling as a result of SPRED2 deficiency. Mol Psychiatry, 2018. 23(2): p. 444-458.

[3] Coffey, K.R., R.G. Marx, and J.F. Neumaier, DeepSqueak: a deep learning-based system for detection and analysis of ultrasonic vocalizations. Neuropsychopharmacology, 2019. 44(5): p. 859-868.


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B 02-11

Microstructure of ingestive behaviour of patients one year after Roux-

en-Y gastric bypass

Michele Serra1, Daniela Alceste1, Ivana Raguz1, Daniel Gero1, Jeannette Widmer1,

Andreas Thalheimer1, Steinert E. Robert1, Alan C. Spector2, Marco Bueter1

1 University Hospital Zurich, Department of Surgery and Transplantation, Zürich, Switzerland 2 Florida State University, Department of Psychology, Tallahassee, USA

Bariatric surgery (BS) is a surgical procedure that supports improvement and remission of many obesity-related

comorbidities, and sustained weight loss (WL). However, a subset of patients undergoing surgery fails to achieve

adequate WL. Extensive research increased the understanding of the wide range of mechanisms underlying the

effectiveness of BS, where altered satiety and hunger are considered important factors for WL after RYGB [1].

Recently, the drinkometer, was introduced as a successful tool for the study of ingestive microstructure in humans

[2]. This allows to investigate in humans the results obtained in animal studies [3] but also to investigate the relation

between the measured behaviour and what the patients report. From two previous studies [4,5], we know that, after

RYGB, the ingestive microstructure undergoes progressive changes, however, one year after surgery, the behaviour

appears to be stable in the short term. We aimed to identify one-year postoperative microstructural parameters of

ingestive behaviour explaining the variation in WL after RYGB. Secondly, we aimed to investigate if the reported pre-

and post-ingestive sensations of one-year-postoperative RYGB patients are in relation with their microstructure of

ingestive behaviour.

One-hundred-seventeen one-year-postoperative RYGB female patients who received a RYGB were assessed for

eligibility, and 50 were enrolled. After an overnight fasting, patients were asked to consume 800 ml of a high-sugar,

high-fat liquid meal. Their ingestive behaviour was measured with the drinkometer. Self-reported pre- and post-

ingestive sensations were measured with a 100-mm horizontal visual analogue scale (VAS). All microstructural

parameters of the ingestive behaviour were analysed in combination using regression to explain the variation in WL

12 months after Roux-en-Y gastric bypass.

The reported sensations do not correlate with the measured ingestive behaviour. Some microstructural parameters,

such as suck rate and suck duration, appear to explain the postoperative WL. Also, different patterns of behaviour

were found.

These results suggest that microstructure of ingestive behaviour measured with the drinkometer may help determine

predictors for less successful WL after bariatric surgery. If verified in larger cohorts, this may form the basis for

individualised pre- and postoperative support to optimise WL outcome.


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Macro- and microstructural parameters of

ingestive behavior A: Exemplary drinkometer recordings. The two

panels on the left and the two panels on the

right show the recorded ingestive behaviour of

two different Roux-en-Y gastric bypass (RYGB)

patients with a 14-day interval. B: Graphical

representation of ingestive behavior and its

macro- and microstructural components. C:

Protocol for the recording of self-reported pre-

and post-ingestive sensations with 100-mm

horizontal visual analogue scale (VAS).

References [1] Miras, AD, le Roux, CW 2013, ‘Mechanisms underlying weight loss after bariatric surgery’, Nat rev

Gastroenterol Hepatol, 10(10), 575-584 [2] Gero, D, File, B, Justiz, J, Steinert, RE, Frick, L, Spector, AC, Bueter, M 2019, ‘Drinking microstructure

in humans: A proof of concept study of a novel drinkometer in healthy adults’, Appetite, 133, 47-60 [3] Gero, D, Bálint, F, Alceste, D, Frick, LD, Serra, M, Ismaeil, A, Steinert, RE, Specter, AC, Bueter, M 2021

‘Microstructural changes in human ingestive behavior after Rox-en-Y gastric bypass during liquid meals’, JCI Insight (accepted)

[4] Alceste, D, Serra, M, Raguz, I, Gero, D, Thalheimer, A, Widmer, J, File, B, Ismaeil, AEM, Steinert, R, Spector, A, Bueter, M 2021, ‘Lack of correlation between microstructure of ingestive behavior and body weight loss in patients one year after Roux-en-Y gastric bypass’ (not published)

[5] Johnson AW 2018, ‘Characterizing ingestive behavior through licking microstructure: Underlying neurobiology and its use in the study of obesity in animal models’, Int J Dev Neuroscience


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B 02-12

Long-term in vivo electrophysiological recordings of dopamine

function in the Df(16)A+/- mouse model of the 22q11.2 deletion

syndrome

Solmaz Bikas, Anastasia Diamantopoulou, Jochen Roeper

Goethe University, Institute of Neurophysiology, Frankfurt, Germany

Dopamine (DA) dysregulation has been long hypothesized to underlie core schizophrenia deficits, with updated

clinical imaging data reappraising the role of dopamine in this severe chronic mental health disorder that affects

approximately 1% of the general population. Furthermore, abnormalities in DA function are crucial for a wide range

of disorders including mental disorders such as schizophrenia (SCZ) or neurodegenerative disorders like Parkinson

disease (PD). Striatal DA elevation along with mesocortical reduced DA release describe the pattern of DA

dysfunction in SCZ, while initial DA depletion in the posterior putamen is seen in PD. To study DA dysregulation in

relevance to human disease, here we make use of the Df(16)A+/- mouse model, a validated animal model of the

22q11.2 Deletion Syndrome (DS), which represents the highest genetic risk factor for schizophrenia, and recently

associated with increased risk of early onset PD. We established in vivo extracellular recordings of pharmacologically

identified DA midbrain neurons in awake freely moving Df(16)A+/- mice and wild type littermates during novelty-

induced hyperactivity in open field and in response to unexpected sensory stimuli. In addition, we used the genetically

encoded DA sensor dLight to monitor extracellular striatal DA dynamics with fiber photometry in the same paradigms.

Analysis of the first data set on striatal DA dynamics revealed elevated DA release in the dorsolateral striatum of

Df(16)A+/- mice, compared to wild-types, upon presentation of sensory (visual) stimuli. We are currently aiming to

identify genotype-related differences in electrical activity of DA neurons and striatal DA signaling.


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Foyers

B 03 | Sensory Physiology Chair

Michael Fischer (Wien)

Stefan Gründer (Aachen)


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B 03-01

Are spontaneous spikes of the mitral cells of the rodent olfactory bulb

modulated by intrinsic theta oscillations?

Luna Jammal Salameh, Alice Stephan, Veronica Egger

University of Regensburg, Neuroscience, Regensburg, Germany

Both spontaneous and odor-evoked network activity in the rodent olfactory bulb (OB) are patterned by respiration,

resulting in oscillations within the theta regime. Previously, we observed theta rhythms also in local field potential

recordings (LFP) in a semi-intact nose-brain preparation of the juvenile rat that is uncoupled from respiration (Perez

et al. 2015). Thus, we hypothesize that the respiratory theta rhythm taps into an intrinsic theta resonance of the

bulbar network.

Here we investigate the properties of this intrinsic theta rhythm in our preparation. First, we observed harmonics of

theta, in line with recent reports from hippocampal LFP (Peroti et al., 2019). With regard to stability, we observed that

both the theta oscillation frequency and its power persisted for at least 60 min (average frequency 2.1 ± 0.3 Hz, n =

10 preparations).

Then we tested if different frequencies of oscillations appear in different recording areas within the same OB. We

found that the same oscillation frequencies persist in all locations checked, suggesting that the frequency is a

character of the OB and not of the locus (n=14).

Since we recorded multi-unit activity from putative mitral cells within the LFP, we asked if this spontaneous spiking

activity could be correlated with the ongoing theta rhythm. We developed an algorithm to filter and isolate the spikes

and to determine their phase relative to the LFP theta, and applied circular statistical tests (Rayleigh, Kuiper) to check

for preferred phases of firing. In all recordings from the 6 preparations analyzed so far, we always observed

significant correlations between spike phase distributions and LFP theta within subsets of 1 minute recording

intervals. These correlations did not persist throughout the entire recordings but were observed within an average

fraction of 0.4 ± 0.1 of the total number of recorded intervals. We also tested for the robustness of these results by

using various spike detection thresholds and analysis intervals.

This finding serves as an additional control that the observed intrinsic theta rhythm is not a recording artefact. If that

was actually the case, mitral cells should fire randomly with respect to the recorded theta rhythm. . Moreover, our

results align well with in vivo recordings showing a correlation between mitral cell spontaneous activity and breathing

(Fukunaga et al 2012) and prove that the preparation allows to investigate rhythmic bulbar activity at the single cell

level.

Acknowledgment •Funding: DFG

•Collaboration: Mathias Dutschmann, Florey Institute, Melbourne


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B 03-02

Lipopolysaccharide (LPS) evokes a state of tolerance in neuro-glial

primary cultures of the rat afferent somatosensory system

Franz Nürnberger, Stephan Leisengang, Daniela Ott, Jolanta Murgott, Rüdiger Gerstberger,

Christoph Rummel, Joachim Roth

Justus-Liebig-University, Institute of Veterinary Physiology and -Biochemistry, Giessen, Germany

Question: Bacterial lipopolysaccharide (LPS) may contribute to the manifestation of inflammatory pain within

structures of the afferent somatosensory system. LPS can induce a state of refractoriness to its own effects termed

LPS-tolerance. We employed primary neuro-glial cultures from rat dorsal root ganglia (DRG) and the superficial

dorsal horn (SDH) of the spinal cord, mainly including the substantia gelatinosa to establish and characterize a model

of LPS-tolerance within these structures.

Methods: Tolerance was inducedby pre-treatment of both cultures with 1 µg/ml LPS for 18 h, followed by a short-

term stimulation with a higher LPS-dose (10 µg/ml for 2 h). Cultures treated with solvent were used as controls. Cells

from DRG or SDH were investigated by means of RT-PCR (expression of inflammatory genes) and

immunocytochemistry (translocation of inflammatory transcription factors into nuclei of cells from both cultures).

Supernatants from both cultures were assayed for tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) by highly

sensitive bioassays.

Results: At the mRNA-level, pre-treatment with 1 µg/ml LPS caused reduced expression of TNF-a and enhanced

IL-10/TNF-a expression ratios in both cultures upon subsequent stimulation with 10 µg/ml LPS, i.e. LPS-tolerance.

SDH cultures further showed reduced release of TNF-a into the supernatants and attenuated TNF-a-

immunoreactivity in microglial cells. In the state of LPS-tolerance, macrophages from DRG and microglial cells from

SDH showed reduced LPS-induced nuclear translocation of the inflammatory transcription factors NFkB and NF-IL6.

Nuclear immunoreactivity of the IL-6-activated transcription factor STAT3 was further reduced in neurons from DRG

and astrocytes from SDH in LPS-tolerant cultures.

Conclusion: A state of LPS-tolerance can be induced in primary cultures from the afferent somatosensory system,

which is characterized by a down-regulation of pro-inflammatory mediators. Thus, this model can be applied to study

the effects of LPS-tolerance at the cellular level, for example possible modifications of neuronal reactivity patterns

upon inflammatory stimulation.


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Influence of LPS-tolerance on DRG primary

cultures In immunocytochemistry experiments the nuclear

translocation of the transcription factors NFκB

and NF-IL6 in macrophages was reduced. In

addition, an elevated IL-10/TNF-α ratio was

shown in RT-PCR. The LPS-induced increase of

inflammatory cytokines TNF-α and IL-6 was not

reduced in the state of LPS-tolerance. Further

immunocytochemistry experiments showed reduced

nuclear immunoreactivity of the transcription

factor STAT3 in neurons, however the LPS-induced

increase of nuclear NF-IL6 was not reduced in

LPS-tolerance. LPS stimulation had no effect on

the TRPV1 expression in DRG primary cells.

Influence of LPS-tolerance on SDH primary

cultures TNF-α expression as well as TNF-α

immunoreactivity in microglial cells was

reduced. In further immunocytochemistry

experiments the nuclear translocation of the

transcription factors NFκB and NF-IL6 was

reduced in microglial cells. In addition, an

elevated IL-10/TNF-α ratio was shown by RT-PCR.

Further experiments showed reduced nuclear

immunoreactivity of the transcription factor

STAT3 in astrocytes and highly sensitive

bioassays revealed a reduced release of TNF-α

and IL-6 into supernatants of SDH primary

cultures in the state of LPS-tolerance.


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B 03-03

Cytokine induced regulation of TRPV1 in iPSC-derived nociceptors

Clemens L. Schöpf1, Maximilian Zeidler1, David Zimmermann1, Georg Kern1, Andrea Gollner1,

Wilfried Posch2, Michaela Kress1

1 Medical University of Innsbruck, Institute of Physiology, Innsbruck, Austria 2 Medical University of Innsbruck, Division of Hygiene and Medical Microbiology, Innsbruck, Austria

We recently characterized iPSC-derived nociceptors (iDNs) and compared the expression of selected ionotropic

receptors and voltage gated calcium channels side-by-side to mouse DRG neurons. The nociceptor-specific, heat

sensitive TRPV1 channel was robustly expressed in differentiated iDNs thus providing an interesting target to study

potential effects of neuro-immune interactions since receptors for proinflammatory (TNFa, IL-6, IFNg) and anti-

inflammatory (TGFb, IL-4) cytokines were concomitantly upregulated during iDN development. First data indicated a

strong effect of activated peripheral blood lymphocytes (PBLs) on the cell survival of co-cultured iDNs when

compared to non-activated PBLs. This together with the expression of relevant cytokine receptors prompted us to

study the interaction of pro and anti-inflammatory cytokines and its effects on TRPV1 expression and function in

iDNs.

We found a robust upregulation of TRPV1 by TNFa and IL-6 in iDN subcellular structures including the soma, axons,

and synaptic boutons, whereas TGFb or IL-4 short-term application (30min) showed no significant effects when

compared to untreated controls. Long term treatments with IL-4 and TGFb for a period of 24h caused cell protective

effects manifesting in a high number of axons as well as synaptic boutons, while long term adjustment of IL-6 and

TNFa caused a highly reduced number of iDN connections. Finally, the role of glia cells in this setting was investigated

by including IFNg treatments in iDNs cultured with or without Ara-C. Interestingly, we found strong effects of IFNg

only in iDNs without Ara-C treatment including a substantial number of active glia-like cells. Thus, we speculated that

the nociceptive effect of IFNg was due to non-neuronal cell activation whereas TNFa and IL-6 directly targeted TRPV1

expression in iDNs. For in depth functional assessments, calcium imaging and whole-cell patch clamp experiments

are currently performed to assess the regulatory role of cytokine stimulation on TRPV1 responsiveness in iDNs.

These data will further underpin the functional compliance of human iDNs as an alternative animal free model system

to study molecular mechanisms of pathological nociceptor function and pain.

References [1] Schoepf, C. L., Zeidler, M., Spiecker, L., Kern, G., Lechner, J., Kummer, K. K., & Kress, M. (2020).

Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors. Brain Sci, 10(6). doi:10.3390/brainsci10060344

[2] Zeidler, M., Kummer, K. K., Schöpf, C. L., Kalpachidou, T., Kern, G., Cader, M. Z., & Kress, M. (2021). NOCICEPTRA: Gene and microRNA signatures and their trajectories characterizing human iPSC-derived nociceptor maturation. bioRxiv, 2021.2006.2007.447056. doi:10.1101/2021.06.07.447056


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Comparison of two protocols for a more rapid differentiation of

induced pluripotent stem cells to peripheral sensory neurons

Anil Kalia1, Corinna Rösseler1, Vincent Truong2, Patrick Walsh2, Angelika Lampert1

1 RWTH Aachen University, Institute of Physiology, Aachen, Germany 2 Anatomic Incorporated, Minneapolis, USA

Peripheral sensory neurons play an important role in the transmission of signals for the sensing of temperature,

touch, pressure and noxious stimuli mediated by various ion channels and receptors expressed at the peripheral

nerve endings. Clinically, peripheral sensory neuropathies result in complete loss of pain to severe painful

neuropathic symptoms. Human sensory neurons in a dish provide an important tool to study the pathophysiological

mechanisms of neurological disorders. Directed differentiation of induced pluripotent stem cells (iPSCs) to sensory

neurons involves the step-wise inhibition and activation of selected signaling pathways important for embryogenesis

to direct the generation of specific cell types. Time required to obtain mature and homogenous neuronal population

for disease modelling remains a key challenge to study human nociception in vitro.

In this study, we are comparing a standard protocol for manufacturing sensory neurons from human iPSCs

(Chambers et al., 2012)1 to a novel protocol through a 24-hour primal ectoderm intermediate (Walsh et al., 2020)2

developed by Anatomic. iPSCs (Fig. a) derived from blood cells and fibroblasts of two healthy subjects were used in

the study. iPSCs obtained were then differentiated into sensory neurons (Fig. b) using both protocols. Peripheral

neuronal identity was confirmed with immunostaining using the markers Peripherin and Tuj1. Whole cell patch clamp

was used to characterize the electrophysiological phenotype of neurons. Tetrodotoxin (TTX) was used to confirm the

presence of TTX resistant sodium ion channels (Nav1.8) in nociceptors.

We found that Anatomic protocol results in differentiation of iPSCs to immature neurons in 7 days as compared to

10 days in Chambers protocol. Anatomic protocol results in highly dense and homogenous neuronal culture which

was not observed with Chambers protocol. Current clamp recordings indicate TTX resistant sodium currents in

neurons at Day 14 and mature action potentials by Day 28 of maturation with both the protocols. We also confirmed

the identity of TTXr currents in neurons with selective Nav1.8 blocker (A-887826).

Differentiation of iPSCs to sensory neurons serve as a platform to study individual patient’s phenotype in a dish. This

study will help us to further exploit optimized generation of iPSC derived nociceptors for disease modelling and

identification of personalized treatment to address unmet medical need for peripheral neuropathies.

Acknowledgment "Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - 368482240/GRK2416"


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a. iPSCs iPSCs at Day 0 of differentiation.

b. Sensory neurons Sensory neurons at Day 14 of Maturation.

References [1] Chambers SM, Qi Y, Mica Y, Lee G, Zhang XJ, Niu L, Bilsland J, Cao L, Stevens E, Whiting P, Shi SH, Studer

L., 2012, ‘Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors’, Nature Biotechnology, 30, 715-20, PMID: 22750882

[2] Walsh P, Truong V, Nayak S, Saldías Montivero M, Low WC, Parr AM, Dutton JR., 2020, ‘Accelerated differentiation of human pluripotent stem cells into neural lineages via an early intermediate ectoderm population’, Stem Cells, 38(11):1400-1408, PMID: 32745311


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B 03-05

Mechanisms underlying elimination of adult-born neurons in the

mouse olfactory bulb

Yury Kovalchuk, Xin Su, Nima Mojtahedi, Olga Garaschuk

Institut of Physiology, Department of Neurophysiology, Tuebingen, Germany

Adult neurogenesis in the rodent olfactory system provides the brains of grown-ups with an additional level of

plasticity and cognitive flexibility, which is important for odor-dependent learning and odor pattern separation. Adult-

born neuronal progenitors are generated in the subventricular zone, wherefrom they migrate through the rostral

migratory stream into the olfactory bulb and differentiate into inhibitory interneurons. Approximately 90% of adult-

born cells become granule cells, whereas the others become juxtaglomerular neurons (JGNs).

Adult-born cells, arriving daily into the olfactory bulb, either integrate into the neural circuitry or get eliminated. It

remains, however, unclear whether there are any fundamental morphological or functional differences between the

surviving and the subsequently eliminated cells. Using in vivo two-photon imaging, we monitored longitudinally the

dendritic morphogenesis, odor-evoked responsiveness, endogenous Ca2+ signaling, and survival/death of adult-born

juxtaglomerular neurons. We found that maturation of adult-born JGN is accompanied by a significant reduction in

dendritic complexity, with surviving and subsequently eliminated cells showing similar degrees of dendritic complexity

and dendritic remodeling. Moreover, ~63% of subsequently eliminated adult-born JGNs acquired odor-

responsiveness before death, with amplitudes and time courses of odor-evoked responses similar to those recorded

in the surviving cells. We observed, however, a significant long-lasting enhancement of the endogenous Ca2+

signaling in subsequently eliminated JGNs, visible already 6 days before death.

Taken together, our data identify the ongoing endogenous activity as a key player influencing the fate of adult-born

JGNs. Such ongoing activity can integrate both the cell-intrinsic firing and the influence of the steady-state odor

environment reaching either a pro- or an anti-apoptotic level. Because some odorants increase and others decrease

the levels of endogenous activity in JGNs, this model reconciles the seemingly opposing findings showing that odor

enrichment either decreases or increases survival of adult-born JGNs.


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B 03-06

Role of parvalbumin-positive GABAergic neurons in the basal forebrain

in a neuropathic pain mouse model

Marie-Luise Edenhofer, Michaela Kress, Kai K. Kummer

Medical University of Innsbruck, Institute of Physiology, Innsbruck, Austria

Chronic neuropathic pain is a serious public health problem that affects ~5 % of the European population and exerts

major impacts on the patients’ quality of life. However, the central mechanisms underlying the chronification of pain

are only partially understood. Basal forebrain (BF) cholinergic inputs to the medial prefrontal cortex (mPFC), a brain

region associated with emotional and cognitive aspects of pain, are changed in respective animal models and one

neuron type that might be of special importance for this regulation are parvalbumin-positive (PV) GABAergic

interneurons. These neurons produce widespread inhibition of large brain areas due to their high firing frequencies

and synchronized activity and play an important role in maintaining the balance of excitation and inhibition (E/I). In

order to elucidate the participation of these neurons in pain-associated changes of BF cholinergic neurons we

investigated the synaptic strength of local BF PV GABAergic neurons.

We performed whole cell patch clamp recordings from PV neurons in acute BF slices from spared nerve injure (SNI)

and sham-operated mice one week after injury. Enhanced yellow fluorescent protein (eYFP) was selectively

expressed in PV neurons via stereotactic injection of a floxed viral vector into the BF of PV-Cre mice.

BF PV neurons exhibited reduced excitability and lower firing rates in response to ramp-shaped depolarizing current

injections after SNI. The threshold current to reach a depolarization block was increased in SNI mice compared to

sham controls. Consistently, the input-frequency (I-F) linear slope was reduced in the SNI group following a

polynomial fitting of the I-F curve. Notably, we found three different cluster of PV neurons showing either high input

resistance with HCN channel currents or low input resistance with or without HCN channel currents.

Our findings suggest that alterations in cholinergic synaptic transmission in chronic pain were caused by reduced

inhibitory inputs mediated by local BF GABAergic PV neurons. The responsible changes in input regions to the BF

are currently investigated.


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B 03-07

Assessing the role of µ-Opioid Polymorphism A118G on human iPSC-

derived peripheral ==> central synapses.

Franziska Kahnt1, Katrin Schrenk-Siemens2, Claudio Acuna1

1 Chica and Heinz Schaller Foundation, Heidelberg University Hospital, Institute of Anatomy and Cell Biology,

Heidelberg, Germany 2 University of Heidelberg, Department of Pharmacology, Heidelberg, Germany

Opioids are one of the most commonly used forms of pain treatment. However, response to medication varies from

person to person, making it hard to efficiently treat patients suffering from pain. It is not yet fully understood what

causes these differences, but one approach suggests genetic factors such as polymorphisms as the origin of the

great individuality of pain sensation and treatment response. The A118G polymorphism in µ Opioid-Receptor 1

(MOR1) is associated with higher pain sensitivity and reduced activity of the μ-opioid system, but the cellular

mechanisms mediating this remain largely unknown. In this project, we take advantage of recent advances in stem

cell technologies to assess the role of the A118G polymorphism at the cellular level in peripheral to central human

synapses derived from induced-pluripotent stem cells (iPSCs).

In order to re-constitute functional peripheral-to-central human neural pathways,we generated induced glutamatergic

neurons as well as nociceptor-like neurons from iPSCs carrying the A118G polymorphisms using forced expression

of NGN2 or NGN1, respectively, and then co-cultured them in vitro for 4-6 weeks. To induce spike-mediated

transmitter release from nociceptors onto central glutamatergic neurons, we selectively expressed channelrhodopsin

in presynaptic nociceptors and recorded light-evoked synaptic transmission using patch clamp electrophysiology

from postsynaptic glutamatergic neurons. We then assessed the impact of the MOR1 agonist [D-Ala2, N-MePhe4,

Gly-ol5]-enkephalin (DAMGO) on peripheral-to-central synaptic transmission in iPSC lines carrying the two MOR1

variants. This project is expected to ultimately provide critical insight into how genetic variants contribute to population

variability in pain sensitivity and responsiveness to medical treatment of pain with opioids.

Acknowledgment This work is supported by SFB1158, the Heidelberg Pain Consortium.


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B 03-08

Electrophysiological stimulation of the olfactory bulb triggers

information encoding in olfactory and non-olfactory structures

Christina Strauch1, Thu-Huong Hoang1, Frank Angenstein2,3,4, Denise Manahan-Vaughan1

1 Ruhr-University Bochum, Medical Faculty, Department of Neurophysiology, Bochum, Germany 2 Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Functional Neuroimaging Group, Magdeburg,

Germany 3 Leibnitz Insitute for Neurobiology, Magdeburg, Germany 4 Otto-von Guericke University, Medical Faculty, Magdeburg, Germany

Question: Olfactory information is projected from the olfactory bulb (OB), the first processing station in the central

nervous system, via the lateral olfactory tract to the regions of the olfactory cortex, including the anterior olfactory

nucleus (AON) and piriform cortex. Several brain areas are known to be closely linked to odor information processing

e.g. the orbitofrontal cortex, amygdala, hippocampus or specific thalamic nuclei, but little is known about the extent

to which other regions of the brain engage in olfactory information processing. Our aim was to examine brain activity

resulting from OB stimulation to induce long-term potentiation (LTP) in the anterior piriform cortex (aPC), or during

OB stimulation that does not change synaptic strength in the aPC.

Methods: We used functional magnetic resonance imaging (fMRI) in adult rats to examine blood oxygen level

dependent (BOLD) responses in the brain triggered by patterned electrophysiological stimulation of the OB. For this,

animals were implanted with a simulating electrode into the OB and recording electrode in the aPC. Two afferent

protocols were used: one which induced LTP and one which did not change basal synaptic transmission in the aPC.

Significant BOLD effects were verified using fluorescence in-situ hybridization to detect nuclear immediate early gene

(IEG) expression triggered by OB stimulation.

Results: During both types of afferent stimulation, BOLD responses increased in several brain areas, including AON,

piriform cortex, entorhinal cortex, nucleus accumbens, prelimbic-infralimbic cortex, dorsal medial prefrontal cortex

and basolateral amygdala. The afferent protocol that induced LTP in the aPC resulted in a significant increase in

BOLD responses in the AON, piriform cortex and infralimbic-prelimbic cortex compared to the afferent protocol that

has no effect on synaptic strength in the aPC. Moreover, nuclear IEG expression in these regions was enhanced

after afferent stimulation resulting in LTP in the aPC.

Conclusion: Stimulation of the OB, resulting in encoding in the form of LTP in the aPC, activates a whole network

of brain regions. Besides regions of the olfactory system including AON and piriform cortex, non-olfactory regions in

particular the infralimbic and prelimbic cortices, are involved in processing and encoding of olfactory information. This

suggests that valence evaluation, is a key component of olfactory information encoding and storage.

Acknowledgment This work was funded by a German Research Foundation grant to DMV (SFB874/B1; project no.: 122679504)


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B 03-09

A novel approach to build an in vitro innervated skin model with

directional neurite guidance

Srijeeta Nag Biswas1, Susan Babu2, Andreas Kist3, Corinna Rösseler1, Laura De Laporte2,

Angelika Lampert1

1 Uniklinik RWTH Aachen University, Institute of Physiology, Aachen, Germany 2 DWI – Leibniz-Institut für Interaktive Materialien, Aachen, Germany 3 Friedrich-Alexander-University Erlangen-Nürnberg, Department for Artificial Intelligence in Biomedical

Engineering, Erlangen, Germany 4 Uniklinik RWTH Aachen University, Institute of Physiology, Aachen, Germany 5 DWI – Leibniz-Institut für Interaktive Materialien, Aachen, Germany 6 Uniklinik RWTH Aachen University, Institute of Physiology, Aachen, Germany

Cutaneous free nerve endings are responsible for transducing noxious stimuli from the surface of the skin to the

central nervous system, which can potentially be perceived as pain. Cutaneous innervation has become an important

factor in skin pathophysiology, as the interaction between sensory neurons and skin cells modulates and

regulates their morphology and their function. Cutaneous nerve fiber degeneration has been observed in patients

suffering from small fiber neuropathy. However, it is difficult to investigate these fine nerve endings in vivo using

routine diagnostics.

Several studies have focused on replicating the interaction between sensory neurons and keratinocytes, by

developing an innervated skin model in vitro. Most of these studies relied on the effects produced by neuroactive

compounds, secreted in part by either sensory neurons or keratinocytes. On the other hand, nerve regeneration

studies using different methods, such as various topography cues, nanofiber/tube scaffold or laminin have shown

directional guidance of neurite outgrowth.

We aim to develop an in vitro 3D innervate skin model with directional guidance elements for the growing neurites,

where the incorporated guidance elements provide both topography cues and a cell adhesive environment.

We use murine dorsal root ganglion (DRG), seeded inside an anisotrophic PEG (polyethylene glycol) based hydrogel

modified with fibronectin. The bulk of the hydrogel matrix, termed Anisogel, contains several rod shaped microgels

dispersed throughout. Before gelation, the microgels are aligned in the preferred direction using an external magnetic

field. The whole DRG is cultured for a period of 7 days. The neurite growth was assessed by immunostaining with

TUJ1, the microgel stained with Rhodamine, and imaged with confocal microscopy. Neurite outgrowth was quantified

using Vaa3D, an open-source visualisation and analysis software. Growing direction was quantified using custom

written software in Python.

We observed that aligned microgels influenced the growing direction of the neurite endings, in the preferred vertical

direction within 7 days of culture in vitro.

We achieved directional neuronal outgrowth that forms the base of the potential skin model. Currently, we are working

towards a co-culture model of keratinocytes with mouse DRG neurons. This would provide a physiologically relevant

model, that can be expanded to include induced pluripotent stem cell-derived nociceptors from pain patients.


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Foyers

B 04 | Neuromodulation and Plasticity Chair

Marlene Bartos (Freiburg)

Olga Garaschuk (Tübingen)


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B 04-01

Preferential frequency-dependent induction of synaptic depression by

the lateral perforant path and of synaptic potentiation by the medial

perforant path inputs to the dentate gyrus

Hardy Hagena, Jens Colitti-Klausnitzer, Valentyna Dubovyk, Denise Manahan-Vaughan

Ruhr-University Bochum, Department of Neurophysiology, Bochum, Germany

The encoding of spatial representations is enabled by synaptic plasticity. The entorhinal cortex sends information to

the hippocampus via the lateral (LPP) and medial perforant (MPP) paths that transfer egocentric item-related and

allocentric spatial information, respectively. To what extent LPP and MPP information-relay results indifferent

homosynaptic synaptic plasticity responses is unclear. We examined the frequency dependency (at 1, 5, 10, 50, 100,

200 Hz) of long-term potentiation (LTP) and long-term depression (LTD) at MPP and LPP synapses in the dentate

gyrus (DG) of freely behaving adult rats.

Methods: Seven- to eight-week old male Wistar rats underwent chronic implantation of a monopolar recording

electrode in the dentate gyrus and a bipolar stimulation in the MPP or LPP. Immunohistochemistry was performed to

examine the expression of GluN1, GluN2A and GluN2B subunits of the NMDA receptor.

Results: We show that whereas the MPP–DG synapses exhibit a predisposition toward the expression of LTP, LPP–

DG synapses prefer to express synaptic depression. The divergence of synaptic plasticity responses is most

prominent at afferent frequencies of 5, 100, Hz and 200 Hz. Priming with 10 or 50 Hz significantly modified the

subsequent plasticity response in a frequency-dependent manner, but failed to change the preferred direction of

change in synaptic strength of MPP and LPP synapses. Evaluation of the expression of GluN1, GluN2A, or GluN2B

subunits of the NMDA receptor revealed equivalent expression in the outer and middle thirds of the molecular layer

where LPP and MPP inputs convene, respectively, thus excluding NMDA receptors as a substrate for the frequency-

dependent differences in bidirectional plasticity.

Conclusion: These findings demonstrate that the LPP and MPP inputs to the DG enable differentiated and distinct

forms of synaptic plasticity in response to the same afferent frequencies and thus may support the functional

differentiation of allocentric and item information provided to the DG by the MPP and LPP, respectively. We propose

that allocentric spatial information, conveyed by the MPP is encoded through hippocampal LTP in a designated

synaptic network. This network is refined and optimized to include egocentric contextual information through LTD

triggered by LPP inputs.

Acknowledgment This work was funded by a Deutsche Forschungsgemeinschaft grant (SFB 1280/A04, project number: 316803389)

to DMV.


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B 04-02

The E3 ligase Thin controls homeostatic plasticity through

neurotransmitter release repression

Martin Baccino-Calace, Katharina Schmidt, Martin Müller

Universtiy of Zurich, Department of Molecular Life Sciences, Zurich, Switzerland

Synaptic proteins and synaptic transmission are under homeostatic control, but the relationship between these two

processes remains enigmatic. Here, we systematically investigated the role of E3 ligases, key regulators of protein

degradation-mediated proteostasis, in presynaptic homeostatic plasticity (PHP). An electrophysiology-based genetic

screen of 157 E3 ligase-encoding genes at the Drosophila neuromuscular junction identified thin, an ortholog of

human tripartite motif-containing 32 (TRIM32), a gene implicated in several neural disorders, including Autism

Spectrum Disorder and schizophrenia. We demonstrate that thin functions presynaptically during rapid and sustained

PHP. Presynaptic thin negatively regulates neurotransmitter release under baseline conditions by limiting the number

of release-ready vesicles, independent of gross morphological defects. We provide genetic evidence that thin controls

release through dysbindin, a schizophrenia-susceptibility gene required for PHP. Thin and Dysbindin localize in close

proximity within presynaptic boutons, and Thin degrades Dysbindin in vitro. Thus, the E3 ligase Thin links protein

degradation-dependent proteostasis of Dysbindin to homeostatic regulation of neurotransmitter release.

Acknowledgment We are grateful to members of the Müller lab for helpful discussions and critical comments on the manuscript. We

thank Dr. Damian Szklarczyk for help with STRING-based protein-protein interaction analysis used for prioritization

of E3 ligases. This research was funded a Swiss National Science Foundation Assistant Professor grant (PP00P3–

15), and an European Research Council Starting grant (SynDegrade-679881) to MM.


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B 04-03

Differential presynaptic homeostatic plasticity of excitatory and

inhibitory transmission

Andreas Ritzau-Jost1, Felix Gsell1, Nadine Ehmann1,2, Torsten Bullmann1, Sebastian Maas1,

Jens Eilers1, Robert J. Kittel1,2, Stefan Hallermann1

1 Carl-Ludwig-Institute for Physiology, Faculty of Medicine, University of Leipzig, Leipzig, Germany 2 Department of Animal Physiology, Institute of Biology, University of Leipzig, Leipzig, Germany

Activity in neuronal circuits arises from the interplay of excitatory and inhibitory synaptic signaling, which is delicately

balanced to maintain stable network activity. Long-term perturbation of network activity induces homeostatic plasticity

of excitatory and inhibitory transmission. However, the presynaptic mechanisms of homeostatic plasticity and

potential differences between excitatory and inhibitory connections remain poorly understood. To determine the

presynaptic parameters, we measure short-term plasticity of excitatory and inhibitory inputs at mature cultured

neocortical neurons, while pharmacologically minimizing the saturation of postsynaptic receptors. These inputs

demonstrate fundamentally different short-term plasticity, with stronger depression due to higher vesicular release

probability at inhibitory compared with excitatory synapses. Homeostatic plasticity induced by 48 hours of activity

deprivation with Tetrodotoxin (TTX) has an opposing effect on excitatory and inhibitory transmission, leading to a

strengthening of excitatory and a weakening of inhibitory synapses. This differential effect is mediated by pre- and

postsynaptic changes including an increase in the number of release-ready vesicles at excitatory synapses and their

decrease at inhibitory synapses. Thus, our data reveal differential but concordant presynaptic plasticity of excitatory

and inhibitory synapses to stabilize the activity in neuronal networks.


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B 04-05

Serotonin modulates the synaptic input of somatostatin-expressing

interneurons in the mouse cingulate cortex

Nathalie Schmitz, Therese Riedemann

Ludwig-Maximilians-Universität München, Physiological Genomics, Planegg-Martinsried, Germany

Serotonin is a potent modulator of neuronal activity in the central nervous system. It is synthesized by serotonergic

neurons of the nucleus raphé in the brainstem and released in response to reward but also in the context of fear and

aggression. The biological effects of serotonin are mediated via serotonin (5-HT) receptors of which there are 7

families, comprising in total 14 different 5-HT receptor subtypes. Except for the 5-HT3 receptor, which is a ligand-

gated cation channel, all 5-HT receptors are metabotropic receptors. In the medial prefrontal cortex, the receptor

families 5-HT1, 5-HT2 and 5-HT3 are most abundantly expressed. The effects of serotonin on the synaptic input of

somatostatin-positive interneurons (SOM+ INs) are currently not known. With the help of immunocytochemistry, we

initially determined that the serotonin receptor subtype 5-HT1A is expressed in around 60% of SOM+ INs and in around

50% of parvalbumin-positive (PV+) INs. In addition, we could show that axons and, to a lesser extent, dendrites of

SOM+ INs are directly innervated by serotonergic fibers. Using whole-cell patch-clamp recordings, we next focused

on the question of whether serotonin affects the spontaneous synaptic input of SOM+ INs or pyramidal cells. Bath

application of 10 µM serotonin had no effect on excitatory synaptic currents recorded in SOM+ INs or pyramidal cells.

In contrast, serotonin induced a robust and reversible increase in the frequency of spontaneous inhibitory currents

(sIPSCs) in both cell types. Interestingly, inhibition of the 5-HT1A receptor by WAY10135 [1 µM] was unable to block

the serotonin-induced increase in sIPSC frequency in either cell type suggesting that this effect was not mediated

via the 5-HT1A receptor. Ionotropic 5-HT3A receptors are mainly expressed on a class of GABAergic interneurons,

namely the class of 5-HT3A receptor+ INs that are mainly present in cortical layers I-III. Indeed, we could show that

pressure application of 50 µM serotonin onto the soma of LI interneurons induced a strong inward current in these

cells. Therefore, we next asked whether inhibition of the 5-HT3 receptor by Y25130 [1 µM] had any impact on the

serotonin-mediated increase in sIPSC frequency of SOM+ INs and pyramidal cells. Interestingly, 5-HT3A receptor

inhibition efficiently blocked the serotonin-induced increase in sIPSC frequency in either cell type suggesting that 5-

HT3A receptor+ INs preferentially innervate SOM+ INs and pyramidal cells.


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B 04-06

Optogenetically controlled activity pattern determines survival rate of

developing neocortical neurons

Anne Sinning, I. Emeline W. F. Sang, Jonas Schroer, Lisa Halbhuber, Davide Warm, Jenq-Wei Yang,

Heiko J. Luhmann, Werner Kilb

University Medical Center Mainz, Johannes Gutenberg University, Institute of Physiology, Mainz, Germany

A substantial proportion of neurons undergoes programmed cell death during early development. This process is

attenuated by increased levels of neuronal activity and enhanced by suppression of activity. To uncover whether the

mere level of activity or also the temporal structure of electrical activity affects neuronal death rates, we

optogenetically controlled spontaneous activity of synaptically-isolated neurons in developing cortical cultures. Our

results demonstrate that action potential firing of primary cortical neurons promotes neuronal survival throughout

development. Chronic patterned optogenetic stimulation allowed to effectively modulate the firing pattern of single

neurons in the absence of synaptic inputs, while maintaining stable overall activity levels. Replacing the burst firing

pattern with non-physiological, single pulse pattern significantly increased cell death rates as compared to

physiological burst stimulation. Furthermore, physiological burst stimulation led to an elevated peak in intracellular

calcium and an increase in the expression level of classical activity-dependent targets, but also decreased Bax/BCL-

2 expression ratio and reduced caspase 3/7 activity. In summary, these results demonstrate at the single-cell level

that the temporal pattern of action potentials is critical for neuronal survival versus cell death fate during cortical

development, beside the pro-survival effect of action potential firing per se.

Acknowledgment This research was funded by grants of the Deutsche Forschungsgemeinschaft to H.J.L. and A.S. (CRC 1080).

References [1] Wong Fong Sang IE, Schroer J, Halbhuber L, Warm D, Yang JW, Luhmann HJ, Kilb W, Sinning A. (2021)

Optogenetically Controlled Activity Pattern Determines Survival Rate of Developing Neocortical Neurons Int J Mol Sci. 22(12):6575. doi.org/10.3390/ijms22126575

[2] Blanquie O, Kilb W, Sinning A, Luhmann HJ (2017) Homeostatic interplay between electrical activity and neuronal apoptosis in the developing neocortex. Neurosci. 358:190-200


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B 04-07

Role of electrical activity in the regulation of Reelin expression in

developing neocortical neurons

Kira Engeroff, Davide Warm, Oriane Blanquie

University Medical Center of the Johannes Gutenberg University, Institute for Physiology, Mainz, Germany

Reelin is a large extracellular matrix protein secreted during early development by Cajal-Retzius neurons, a pioneer

and transient neuronal population. The main function of Reelin is to control neuronal migration and ensure the proper

lamination of cortical layers. Defects in Reelin expression or secretion leads to dramatic anatomical and

neurodevelopmental disorders: mice genetically knocked-out for Reelin or for its downstream protein Dab1 display

inverted cortical layers and are used as model for schizophrenia. Although Cajal-Retzius neurons disappear at the

end of cortical development, these neurons are well integrated in the developing network and are electrically active.

However, whether the activity displayed by Cajal-Retzius neurons play a role in Reelin expression and/or secretion

is not known. In this project, we use electrophysiology and molecular biology in dissociated cultures from immature

cortical neurons to investigate the role of electrical activity in the regulation of Reelin expression and secretion. This

project will be fundamental to better understand the mechanisms underlying the proper formation of the neocortex

and might reveal how pathological immature activity patterns, e.g. epileptic activity, can lead to dramatic

neurodevelopmental disorders.

Acknowledgment The current project was funded by the by the Deutsche Forschung Gemeinshaft (BL 1633/1-1) and by the intramural

research funding of the Johannes Gutenberg-Universität Mainz. We thank Prof. HJ Luhmann for his material and

scientific support.

References [1] Causeret, F., Moreau, M. X., Pierani, A. & Blanquie, O 2021 "The multiple facets of Cajal-Retzius neurons"

Development148. Blanquie, O., Liebmann, L., Hübner, C. A., Luhmann, H. J. & Sinning, A 2016 "NKCC1-Mediated GABAergic Signaling Promotes Postnatal Cell Death in Neocortical Cajal–Retzius Cells", Cereb. Cortex27, bhw004. D’arcangelo G, Miao GG, Chent S-C, Soares HD, Morgan JI, Curran T. n.d 1999 "A protein related to extracellular matrix proteins deleted in the mouse mutant reeler" Neuron 24(2):471-9. Ringstedt T, Ibáñez CF, Linnarsson S, Wagner J, Arenas E, Ernfors P, Lendahl U, Kokaia Z. 1998. BDNF regulates reelin expression and Cajal-Retzius cell development in the cerebral cortex. Neuron. 21:305–315


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B 04-08

Vesicle super-priming mediates LTP at neocortical layer 5 pyramidal

neurons

Iron Weichard, Stefan Hallermann, Jana Nerlich

Leipzig University, Carl-Ludwig-Institute for Physiology, Faculty of Medicine, Leipzig, Germany

Long-term potentiation (LTP) is a potential mechanism for experience-dependent learning and memory. LTP can be

expressed both pre- and postsynaptically. The mechanisms of presynaptic transmitter release and its regulation

during plasticity remain poorly understood. Particularly, the relation of LTP with super-priming has not been studied

carefully. To address this question we focused on LTP at synapses at layer 5 pyramidal neurons of the

somatosensory cortex, which have been shown to exhibit presynaptic LTP referred to as redistribution of the synaptic

efficacy. We performed whole cell recordings from layer 5 pyramidal neurons in acute cortical slices with extracellular

stimulation of local excitatory inputs and measured paired-pulse ratio, short-term plasticity during high-frequency

transmission, and recovery from depression before and after the induction of spike timing-dependent LTP. After LTP,

the EPSC amplitude was increased by 63 ± 13 % in 21 responder cells and by 31 ± 9 % in all 39 investigated cells

(merge of responder and non-responder cells; Wilcoxon signed-rank test p<0.001 and p<0.001, respectively). LTP

decreased the paired pulse ratio and increased the depression during high-frequency transmission. The number of

release-ready vesicles estimated by back-extrapolation techniques was higher after LTP. Interestingly, the recovery

from synaptic depression was slower and contained a second slow component after LTP. Synaptic potentiation

induced with the adenylyl cyclase activator forskolin mimicked features of electrically induced LTP. Quantal analysis

with four short-term plasticity models indicated a substantial increase in the number of super-primed vesicles during

spike timing-dependent and forskolin-induced LTP. Super-primed vesicle pools exhibit both a higher release

probability and slower recovery from depression, explaining the observed changes in release probability and recovery

from depression during LTP. Our data therefore indicate that cAMP-dependent super-priming of vesicles mediates

presynaptic LTP at layer 5 pyramidal neurons.


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B 04-09

Dopamine D1/D5 receptor regulation of hippocampal long-term

depression that is triggered by locus coeruleus activation in vivo

Natalia Babushkina1,2, Hardy Hagena1,2, Denise Manahan-Vaughan1,2

1 Ruhr-University Bochum, Department of Neurophysiology, Bochum, Germany 2 Ruhr-University Bochum, International Graduate School of Neuroscience, Bochum, Germany

Question: Synaptic plasticity in the forms of long-term potentiation (LTP) and long-term depression (LTD) is a cellular

mechanism that supports hippocampus-dependent memory formation. The effectiveness of encoding and storage

information is influenced by neuromodulation related to novelty acquisition. Noradrenaline (NA) and dopamine (DA)

are two neuromodulators that are released in response to novel experience. The main source of NA in the brain is

the locus coeruleus (LC). Changes in LC activity that is triggered by novel experience leads to NA release in the

hippocampus, particularly in the dentate gyrus (DG). Furthermore, patterned stimulation of the LC causes LTD in the

CA1 and the DG in vivo that depends on β-adrenergic receptor activation. In recent years it has emerged that the LC

is capable of co-release DA, in addition to NA, in the hippocampus. In the current study we employ in vivo

electrophysiology to examine the role of dopamine D1/D5 receptors in DG synaptic plasticity that is induced by

patterned activation of the LC.

Methods: Adult male rats underwent implantation of a recording electrode into the DG, stimulation electrodes in the

medial perforant path and the LC, and a guide cannula into the lateral cerebral ventricle to permit pharmacological

treatment. Following recovery from surgery, we examined the effect of dopamine D1/5-ligand treatment on synaptic

plasticity evoked in the DG by LC stimulation.

Results: Whereas D1/D5-receptor agonist application has no effect on LC-mediated LTD, D1/D5-receptor

antagonism modulates synaptic plasticity evoked by the LC in the DG of freely behaving rats.

Conclusions: Our results suggest a graded contribution of D1/D5 receptors to LC-mediated LTD in the

hippocampus.

This work was funded by the Deutsche Forschungsgemeinschaft (SFB 874/B3, project number: 122679504).

Acknowledgment We thank Christina Strauch and Juliane Böge for technical assistance.


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B 04-10

Dynorphin neuropeptides decrease the apparent proton affinity of

ASIC1a activation and unbind during channel opening.

Audrey M. Ortega-Ramirez1, Lilia Leisle1, Michael Margreiter2,3, Giulia Rossetti2,4,5, Stefan Gründer1

1 RWTH Aachen University, Institute of Physiology, Aachen, Germany 2 Forschungszentrum Jülich, Computational Biomedicine – Institute for Advanced Simulation/Institute of

Neuroscience and Medicine and Jülich Supercomputing Centre, Jülich, Germany 3 RWTH Aachen University, Institute of Organic Chemistry, Aachen, Germany 4 Forschungszentrum Jülich, Jülich Supercomputing Center (JSC), Jülich, Germany 5 RWTH Aachen University, Institute of Neurology, Aachen, Germany

Prolonged acidification of tissue pH is a component in several neurological disorders, for example in ischemic stroke.

Acid-sensing ion channel 1a (ASIC1a) responds directly to acidic extracellular pH, and its activity mediates

neurotoxicity during sustained acidosis. The opioid peptide Big Dynorphin (Big Dyn) shows an overlapping expression

pattern with ASIC1a in the central nervous system, is a potent endogenous modulator of ASIC1a and has also been

implicated in neuronal damage. In fact, Big Dyn binds to the acidic pocket of ASIC1a and shifts the steady-state

desensitization to more acidic pH values, thereby enhancing ASIC1a activity and acidosis-induced neuronal death.

Targeting this interaction for therapeutic purposes would aid the development of neuroprotective drugs. Based on a

molecular binding model for a dynorphin peptide and ASIC1a, which has been confirmed in functional experiments

and identified specific interaction pairs and forces, we used photo-crosslinking to attach Big Dyn to ASIC1a

covalently. This resulted in a dramatically decreased proton sensitivity of activation, yet the channels were still able

to open. Our data indicate that Big Dyn must dissociate from the acidic pocket during the transition from closed to

open states. Together, we provide an experimentally confirmed binding model for a dynorphin peptide and ASIC1a

with novel mechanistic insight.

Acknowledgment This work has been supported by the Deutsche Forschungsgemeinschaft (GR1771/8-1). The HEK293 ASIC1a

knock-out cell line was a generous gift from Stephan A. Pless (University of Copenhagen). The plasmid for

incorporation of p-azido-L-phenylalanine in mammalian expression systems (pIRE4-Azi) has been generously

provided by Irene Coin (University of Leipzig).

References [1] Yoder, N., Yoshioka, C., Gouaux, E., 2018, "Gating mechanisms of acid-sensing ion channels", Nature 555,

397–401, https://doi.org/10.1038/nature25782 [2] Sherwood, T.W., Askwith, C.C., 2009, "Dynorphin opioid peptides enhance acid-sensing ion channel 1a

activity and acidosis-induced neuronal death", J Neurosci 29, 14371–14380, https://doi.org/10.1523/JNEUROSCI.2186-09.2009

[3] Borg, C.B., Braun, N., Heusser, S.A., Bay, Y., Weis, D., Galleano, I., Lund, C., Tian, W., Haugaard-Kedström, L.M., Bennett, E.P., Lynagh, T., Strømgaard, K., Andersen, J., Pless, S.A., 2020, "Mechanism and site of action of big dynorphin on ASIC1a", Proc Natl Acad Sci U S A 117, 7447–7454, https://doi.org/10.1073/pnas.1919323117

[4] Gründer, S., Pusch, M, 2015, "Biophysical properties of acid-sensing ion channels (ASICs)", Neuropharmacology, 94, 9-18, https://doi.org/10.1016/j.neuropharm.2014.12.016


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B 04-11

Dopamine D2-like receptor family modulates rat spatial memory and

hippocampal synaptic plasticity in the CA1 region

Violeta M. Caragea1,2, Denise Manahan-Vaughan1,2

1 Ruhr-University Bochum, Department of Neurophysiology, Medical Faculty, Bochum, Germany 2 Ruhr-University Bochum, International Graduate School for Neuroscience, Bochum, Germany

Questions: Dopamine (DA) is known as a key modulator of cognition and of hippocampal information processing.

DA exerts its influence through two receptor families: the D1-like receptor family (including D1 and D2 receptors) and

the D2-like receptor family (including D2, D3, and D4 receptors). A crucial difference between these families is that

they are differently coupled to adenylyl cyclase (AC). As such, while the D1-like receptors are positively coupled to

AC, the D2-like receptors are negatively coupled to the enzyme. The D1-like receptor family is well recognized as a

strong modulator of hippocampal synaptic plasticity at diverse synaptic populations within the trisynaptic circuit. Still,

much less is known yet about the D2–like receptor family. Therefore, in our study, we asked what would be the effect

on in vivo synaptic plasticity at Schaffer collaterals (SC) – Cornu Ammonis (CA1) synapses under D2-like receptor

blockade. Moreover, we also investigated the effects of D2-like receptor antagonism on both episodic and semantic-

like memory in rats.

Methods: Adult Long-Evans rats underwent implantation of a stimulating electrode in the SC and a recording

electrode in the stratum radiatum of the CA1 region of the hippocampus. A cannula was implanted in the ipsilateral

cerebral ventricle. D2-like receptor-mediated modulation of evoked short-term potentiation, long-term potentiation,

and short-term depression was examined in vivo. Moreover, object and spatial memory were tested under the same

pharmacological treatment.

Results: Intracerebral treatment with a D2-like receptor antagonist, at a dose that did not influence basal synaptic

transmission, significantly altered both synaptic potentiation and depression in the CA1 region, at all tested durations

of synaptic plasticity. Moreover, the antagonist also modulated memory in rats. While in an episodic-like memory

task, D2-like receptor antagonism led to a lack of discrimination of recently displayed objects, in a semantic-like

memory task, the antagonist abolished discrimination both in simple and in complex configurations designed to test

cumulative memory formation.

Conclusions: In contrast to effects reported for other hippocampal areas, dopamine D2-like receptors contribute to

a greater degree to the regulation of synaptic plasticity in the hippocampal CA1 region in vivo. Blocking D2-like

receptors has also an impact on both episodic and semantic-like memory.

Acknowledgment This work was funded by the Deutsche Forschungsgemeinschaft (SFB 874/B10, project number: 122679504 to

DMV).

References [1] Hagena, H, Manahan-Vaughan, D (2016), 'Dopamine D1/D5, But not D2/D3, Receptor Dependency of Synaptic

Plasticity at Hippocampal Mossy Fiber Synapses that Is Enabled by Patterned Afferent Stimulation, or Spatial Learning', Frontiers in Synaptic Neuroscience, 8, 31

[2] Hansen, N, Manahan-Vaughan, D (2014), 'Dopamine D1/D5 receptors mediate informational saliency that promotes persistent hippocampal long-term plasticity', Cerebral Cortex, 24(4), 845–858

[3] Jay, TM (2003), 'Dopamine: a potential substrate for synaptic plasticity and memory mechanisms', Progress in Neurobiology, 69(6), 375–390

[4] Lisman, JE, & Grace, AA (2005), 'The hippocampal-VTA loop: Controlling the entry of information into long-term memory', Neuron, 46(5), 703–713


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[5] Manahan-Vaughan, D, Braunewell, KH (1999), 'Novelty acquisition is associated with induction of hippocampal long-term depression', Proceedings of the National Academy of Sciences of the United States of America, 96(15), 8739–8744


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B 04-12

Neuronal excitability in rat cerebral cortex is controlled by the

neuropeptide Galanin

Fatima Gimeno-Ferrer1, Annett Eitner1, Reinhard Bauer3, Alfred Lehmenkuhler2, Hans-Georg Schaible1,

Frank Richter1

1 University Hospital Jena, Institute of Physiology I, Jena, Germany 2 Center for Medical Education, Düsseldorf, Germany 3 University Hospital Jena, Center for Molecular Biomedicine, Jena, Germany

The neuropeptide Galanin plays a role in metabolism, sleep control and stress resilience. In hippocampus, Galanin

controls the release of neurotransmitters, and exerts neuroprotective effects. To test whether Galanin is able to

interact with neuronal activity in cerebral cortex, we recorded ongoing brain activity and induced cortical spreading

depolarization (CSD) before and after application of Galanin.

In spontaneously breathing anesthetized adult rats (sodium thiopentone, 100 mg/kg, i.p.) the direct current (DC-)

electrocorticogram was recorded with arrays of glass microelectrodes in two areas (treated with galanin and

untreated) and different depths. We CSD induced by microinjection of 1 M KCl. CSD-related DC potential shifts,

changes in extracellular potassium concentration and changes in regional cerebral blood flow were continuously

monitored. Galanin at concentrations of 10-6, 10-7, 10-8, 10-9, and 10-10 M was applied for 3 h and then washed away

with artificial cerebrospinal fluid. To test for specificity of the galanin effects, we pretreated one area of the cortex

with a Galanin receptor2 antagonist (GalR2 antagonist M871) at 3 nM for 2 hours followed by Galanin at 10-7 M for

another 2 hours.

Galanin at concentrations of 10-6, 10-7, and 10-8 M increased the threshold for elicitation of CSD, reduced amplitudes

of CSD within 4 hours down to 40 % of controls (best effect with Galanin at 10-7 M) and significantly slowed

propagation velocity of CSD (largest effect at 10-8 M 2.97 to 2.38 mm/min). In some rats CSD remained restricted to

the untreated area. Galanin at 10-9 and 10-10 M had no significant effects on CSD. In the washout phase after the

effective concentrations of galanin ictal discharging or repetitive seizure activity were observed in 25 % of the rats.

The pretreatment with the GalR2 antagonist alone decreased CSD amplitudes in the first hour of application, and

then amplitudes recovered. When Galanin at 10-7 M was applied subsequently, no changes in CSD and no

pathological discharging activity were observed.

We conclude that galanin is an important controller of cortical neuronal activity. Local topical application of Galanin

reduced and slowed CSD, i.e. it reduced neuronal excitability in cerebral cortex. Vice versa, washing out of a Galanin

pretreatment induced neuronal hyperexcitablity. Therefore Galanin in cerebral cortex could be an interesting target

for treating pathological brain states accompanied by neuronal hyperexcitablity.

Acknowledgment This project has received funding from the European Union’s Horizon 2020 research and innovation programme

under the Marie Skłodowska-Curie Grant Agreement No 764860.


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B 04-13

Pharmacokinetic profile of tetrahydrocannabinol and cannabidiol in

mice following oral cannabis extracts administration

Cristiana Dumbraveanu1,2, Kai K. Kummer1, Katharina Strommer2, Astrid Neumann2, Michaela Kress1

1 Medical University of Innsbruck, Institute of Physiology, Innsbruck, Austria 2 Bionorica research GmbH, Innsbruck, Austria

Introduction:Cannabis sativa contains over 100 bioactive substances, predominantly the cannabinoids delta-9-

tetrahydrocannabinol (THC) and cannabidiol (CBD) that are generally accepted therapeutics in nausea, epileptic

disorders and pain. Other constituents of the cannabis plant remain insufficiently explored and there is insufficient

understanding of potential additive, synergetic or antagonistic interactions or effects on absorption and bioavailability

of THC or CBD. Therefore, we aimed to explore and compare the pharmacokinetics of THC and CBD in two sets of

plant extracts, rich in cannabinoids and cannabinoid-depleted, with semisynthetic (THC) or isolated (CBD)

cannabinoids.

Methods: The pharmacokinetic profile of medical marihuana extract rich in cannabinoids (THC+), cannabinoid-

depleted extract (THC-), THC at corresponding concentration, and CBD was investigated in C575BL/6J mice.

Formulations were prepared in sesame oil and administered by oral gavage as a single dose. Concentrations of THC

and its metabolites, 11-Hydroxy-THC (OH-THC) and 11-nor-9-carboxy-THC (THC-COOH), as well as CBD were

evaluated in plasma, brain, and spinal cord up to 6 hours after application using mass spectrometry.

Results: THC or CBD from all formulations administered were detectable in plasma with Cmax at 2 hours. THC+

yielded a faster rise and significantly higher THC levels already at 60 min in plasma and brain as compared to THC.

As expected, THC was hardly detectable in plasma or nervous tissue after administration of cannabinoid-depleted

extract (THC-). For THC+ the maximum concentration was reached faster than for THC in brain, but not spinal cord

suggesting faster resorption of THC when applied as extract. This was supported by faster and increased peaks of

THC metabolites in plasma and brain for THC+. Regarding THC metabolites, highest concentration levels in brain

were reached for the psychoactive metabolite OH-THC, while in plasma COOH-THC showed the highest

concentrations. CBD single compound administration showed two times higher bioavailability in brain and spinal

cord, in comparison to THC as a single compound.

Conclusion: Our results support enhanced bioavailability of THC from cannabinoid-rich medical marihuana extracts

compared to THC single compound. Importantly, the presence of CBD and THC in the spinal cord after oral

application is reported for the first time.

Acknowledgment This work is part of the TOBeATPAIN project and has received funding from the European Union’s Horizon 2020

research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No 764860.


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Foyers

B 05 | Ligand-gated Ion Channels and Receptors Chair

Bernd Fakler (Freiburg)



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B 05-01

Novel tools for studying the function of heteromeric kainate and AMPA

receptor complexes

Andreas Reiner

Ruhr-Universität Bochum, Cellular Neurobiology, Faculty of Biology and Biotechnology, Bochum, Germany

AMPA and kainate receptors belong to the family of ionotropic glutamate receptors (iGluRs), which are tetrameric,

glutamate-gated ion channels that generate excitatory postsynaptic currents and have important modulatory

functions in the brain [1]. Both, AMPA and kainate receptors can form functional homomeric channels, but recent

work continues to highlight the role of heteromeric assemblies for CNS function. Furthermore, new structural data

give insight into the subunit assembly within these receptor complexes. However, the functional contributions of

individual subunits to channel gating, i.e. to glutamate-induced activation, desensitization and recovery remain poorly

understood.

In order to investigate the role of individual subunits, we extended previous work on the chemo-optogenetic control

of iGluRs. This approach relies on labeling genetically-modified receptor subunits with photoswitchable tethered

ligands (PTLs) that allow to reversibly turn on- and off ligand binding with short pulses of light of specific wavelengths

[2]. Using MAG-type (maleimide-azobenzene-glutamate) photoswitches, which undergo light-induced trans-to-cis

isomerization, and previous screens that identified photoswitchable GluK2 subunits, we now extended this approach

to other kainate and also AMPA receptor subunits. Using different attachment sites for photoswitch tethering, we

established receptor variants that can be either activated (photo-agonism) or inhibited with light (orthosteric photo-

antagonism). These tools offer ultimate specificity for liganding of individual subunits and allow to study iGluR gating

with high spatio-temporal resolution in complex environments. We now used these tools on different heteromeric

receptor populations. These experiments, in combination with genetic engineering of ligand binding sites and

pharmacological tools, uncovered how partial occupancy states can result in partial desensitization and how recovery

from desensitization may depend on receptor occupancy. This has also important consequences, for instance when

subunit-specific agonists and antagonists are used on heteromeric receptor populations (see e.g. [3]). Current work

also addresses the assembly propensities of different heteromers and their expression in different cell types. Using

photoswitchable iGluRs we will continue to study heteromeric receptor function in different neuronal cell types, where

additional modulatory factors are present.

Acknowledgment This work was in part supported by the NRW-Rückkehrprogramm and the Deutsche Forschungsgemeinschaft

(DFG RE 3101/3-1).

References [1] Reiner, A, Levitz, J, 2018, ‘Glutamatergic signaling in the central nervous system: Ionotropic and

metabotropic receptors in concert’, Neuron, 98, 1080-1098. [2] Reiner, A, 2018, ‘New light on neurotransmitter-gated receptors: Optical approaches for controlling

physiological function', Neuroforum, 24, A159. [3] Pollok, S, Reiner, A, 2020, ‘Subunit-selective iGluR antagonists can potentiate heteromeric receptor

responses by blocking desensitization’, Proc. Natl. Acad. Sci. USA, 117, 25851-25858.


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B 05-02

Forced subunit assembly in homo- and heterotrimeric P2X receptors

Milica Gusic, Christopher Unzeitig, Klaus Benndorf, Christian Sattler

Friedrich Schiller Universität, Physiologie, Jena, Germany

ATP is not only an energy source, it is also a signaling molecule and a player in the purinergic system. P2X receptors

(P2XRs) belong to this system and are permeable for cations like sodium, potassium and calcium. They are

expressed in various tissues and found in neuronal and immune cells. Thus, the function of the receptors is also

diverse, ranging from synaptic transmission, to pain and inflammation. In mammals, seven distinct subunits are

described which can assemble into homo- and heteromeric trimers. The trimeric structure was postulated by

biochemical crosslinking and blue native PAGE experiments and, later on, was proven by the first crystal structure

of the zebrafish P2X4R.

In this functional study, we used the approach of subunit concatenation to define number and position of the P2XR

subunits in the trimeric complex. Two sets of concatamers were constructed and functionally characterized using the

patch-clamp technique in the whole-cell or outside-out configuration. The first line follows the question how P2X2

and P2X3 subunits are arranged in a heteromeric complex of P2X2/3 receptors. The second line asks for the impact

of the number of functional binding sites on the gating of homomeric P2X7 receptors.

Conclusions from our results are: 1. Subunit concatenation allows for the expression of defined P2X receptors. 2.

P2X2/3 receptors can assemble in the stoichiometries (P2X2)2(P2X3)1 and (P2X2)1(P2X3)2, but the construct P2X2-

P2X3-P2X3 matches mostly the results from the co-expression of P2X2 and P2X3 subunits. 3. Occupation of a single

binding site suffices for P2X7 receptor activation, whereas the occupation of all three binding sites maximally

stabilizes the open state.


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B 05-03

Expression and composition of NMDARs varies between brain regions

and is altered in Alzheimer’s disease

Michaela Back, Eric Jacobi, Mariel Braunbeck, Sonia Ruggieri, Jakob von Engelhardt

University Medical Center of the Johannes Gutenberg University Mainz, Institute of Pathophysiology, 55128,

Germany

N-methyl-D-aspartate receptors (NMDARs) are involved in the pathogenesis of various neurodegenerative diseases,

which often manifest in specific brain areas. Alzheimer’s disease (AD), for instance, affects neurons in the

hippocampus and neocortex. The composition and distribution of synaptic and extrasynaptic NMDARs varies in

different brain regions. Thus, it is of interest whether a specific NMDAR subunit composition in a brain area explains

its vulnerability to distinct neurodegenerative diseases. We therefore studied the expression and subunit composition

of NMDARs in brain regions typically involved in neurodegenerative processes with WT and with AD model mice.

We report here the different amplitudes and kinetics of synaptic and extrasynaptic NMDAR-mediated currents of CA1

pyramidal cells (PCs), dentate gyrus granule cells (GCs), PCs of the somatosensory cortex and medium spiny

neurons of the striatum. The data suggest that number and composition of NMDARs differ between brain regions.

Differently to often described in literature, we find no evidence for preferential expression of GluN2B-containing

NMDARs at extrasynaptic sites, but that the majority of extrasynaptic NMDARs contain the GluN2A subunit

(diheteromeric GluN1/GluN2A and triheteromeric GluN1/GluN2A/GluN2B NMDARS) in all brain areas studied.

NMDARs are involved in mediating amyloid beta (Aβ) toxicity, and NMDAR expression can be affected by Aβ in AD

model mice. It was shown that the neocortex is involved earlier in AD pathology than the hippocampus. We tested

whether the region-specific susceptibility to Aβ results from a differential expression of NMDARs. Indeed, we find

that NMDAR expression is different in AD mice in the hippocampus compared to the neocortex [1,2]. NMDAR currents

are decreased in synaptic and extrasynaptic sites in the hippocampus of AD mice, but not in the neocortex. In the

neocortex, however, NMDAR subunit composition at extrasynaptic sites is altered, indicating a subunit switch from

GluN1/GluN2A to triheteromeric GluN1/GluN2A/GluN2B NMDARs. Furthermore, deletion of NMDAR subunits

protects PCs in the somatosensory cortex, but not GCs in the dentate gyrus from Aβ-mediated spine loss.

In conclusion we find differences in the expression and composition of NMDARs in various brain regions and in AD

model mice. These differences in subcellular location and subunit expression might account for the susceptibility of

different brain regions to neurodegenerative processes.

Acknowledgment We thank Barabara Biesalski for technical help.

References [1] Müller et al., NMDA receptors mediate synaptic depression, but not spine loss in the dentate gyrus of adult

amyloid Beta (Aβ) overexpressing mice, Acta Neuropathologica Communications (2018) 6:110 https://doi.org/10.1186/s40478-018-0611-4

[2] Back, M.K.; Ruggieri, S.; Jacobi, E.; von Engelhardt, J. Amyloid Beta-Mediated Changes in Synaptic Function and Spine Number of Neocortical Neurons Depend on NMDA Receptors. Int. J. Mol. Sci. 2021, 22, 6298. https://doi.org/ 10.3390/ijms22126298


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B 05-04

Somatostatin specifically modulates GABABbut not GABAAreceptor-

mediated synaptic transmission

Therese Riedemann, Bernd Sutor

Ludwig-Maximilians-Universität München, Physiological Genomics, Planegg-Martinsried, Germany

Inhibitory modulation of glutamatergic information processing is a prerequisite for proper network function. Among

the many groups of interneurons, somatostatin-expressing interneurons (SOM+ INs) seem to play an important role

in the maintenance of physiological brain activity. Imbalances between the excitation to inhibition ratio can manifest

as neurological diseases such as recurrent seizures. In addition, mood disorders haven been linked to SOM+ INs,

especially in the cingulate cortex. We have recently investigated the neurochemical, morphological and

electrophysiological variety of SOM+ INs in the cingulate cortex (Riedemann et al., 2016; 2018; Riedemann and

Sutor, 2019). To gain further insight into the function of SOM+ INs, we analyzed the role of SOM on pyramidal cell

and GABAergic interneuron excitability. We found that SOM exerts long-lasting effects on pyramidal cell excitability

by inducing a potassium conductance and membrane potential hyperpolarization. In contrast, SOM induces only a

moderate reduction of cell excitability in GABAergic INs. In agreement with these findings, we find that SOM -by

acting pre- and a postsynaptically- depresses evoked and spontaneous glutamatergic synaptic transmission. In

addition, we show that SOM specifically modulates GABABR-mediated synaptic transmission but has little effect on

GABAAR-mediated transmission.


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B 05-05

NMDA receptor inhibition fosters the collective pancreatic beta-cell

activity.

Lidija Križančić Bombek1, Marko Šterk1,2, Maša Skelin Klemen1, Marjan Slak Rupnik1,3,4, Marko

Marhl1,2,5, Andraž Stožer1, Marko Gosak1,2

1 University of Maribor, Faculty of Medicine, Institute of Physiology, Maribor, Slovenia 2 University of Maribor, Faculty of Natural Sciences and Mathematics, Maribor, Slovenia 3 Medical University of Vienna, Center for Physiology and Pharmacolog, Vienna, Austria 4 Alma Mater Europaea, ECM, Maribor, Slovenia 5 University of Maribor, Faculty of Education, Maribor, Slovenia

NMDA receptors promote repolarization in pancreatic beta cells and thereby reduce glucose-stimulated insulin

secretion. Therefore, NMDA receptors are a potential therapeutic target for diabetes [1]. While the mechanism of

NMDA receptor inhibition in beta cells is rather well understood at the molecular level, its possible effects on the

collective cellular activity have not been addressed to date, even though proper insulin secretion patterns result from

well-synchronized beta-cell behavior. The latter is enabled by strong intercellular connectivity, which governs

propagating calcium waves across the islets and makes the heterogeneous beta-cell population work in synchrony

[2]. Since a disrupted collective activity is an important and possibly early contributor to impaired insulin secretion

and glucose intolerance, it is of utmost importance to understand the possible effects of NMDA receptor inhibition on

beta-cell functional connectivity.

To address this issue, we combined two highly sophisticated research methods, namely the confocal functional

multicellular calcium imaging in mouse tissue slices and the multilayer network science approach [3, 4].

Our results revealed that NMDA receptor inhibition increases, synchronizes, and stabilizes beta cell activity (Fig.1)

without affecting the velocity or size of calcium waves. To explore intercellular interactions more precisely, we made

use of the multilayer network formalism by regarding each calcium wave as an individual network layer, with weighted

directed connections portraying the intercellular propagation. NMDA receptor inhibition stabilized both the role of

wave initiators and the course of waves. The findings obtained with the experimental antagonist of NMDA receptors,

MK-801, were additionally validated with dextrorphan, the active metabolite of the approved drug dextromethorphan,

as well as with experiments on NMDA receptor KO mice.

In sum, our results provide additional and new evidence for a possible role of NMDA receptor inhibition in the

treatment of type 2 diabetes and introduce the multilayer network paradigm as a general strategy to examine the

effects of drugs on connectivity in multicellular systems.

Acknowledgment We thank Maruša Rošer, Rudi Mlakar, and Maša Čater for their excellent technical assistance.


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Quantitative and network measures of beta-cell

activity. Temporal evolution of the average oscillation

frequency (red), average oscillation duration

(black), and average coactivity (blue) of beta-

cells in glucose only protocol G (A) and in

glucose with MK-801 protocol MK (B). Light grey

curves = average Ca2+ signal. Panels (C-H) =

relative change in oscillation frequency,

oscillation duration, relative active time,

inter-oscillation interval variability, average

coactivity, and node degree in the functional

beta-cell networks from interval 1 to interval 2

for all recordings in protocols G (cyan) and MK

(violet).

References [1] Marquard, J, Otter S, et al. 2015, Characterization of pancreatic NMDA receptors as possible drug targets

for diabetes treatment. Nat Med, 21:363–76. [2] Benninger, RKP, Head, WS, et.al. 2011, Gap junctions and other mechanisms of cell-cell communication

regulate basal insulin secretion in the pancreatic islet. J Physiol, 589(22):5453–66. [3] Stožer, A, Gosak, M, et al. 2013, Functional Connectivity in Islets of Langerhans from Mouse Pancreas

Tissue Slices. Shvartsman S, editor. PLoS Comput Biol, 9(2):e1002923. [4] Gosak, M, Markovič, R, et al. 2018, Network science of biological systems at different scales: A review,

Physics of Life Reviews. Elsevier B.V., Vol.24, p.118–35.


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B 05-06

Unravelling the intricate cooperativity of subunit gating in P2X2 ion

channels

Christian Sattler1, Thomas Eick1, Sabine Hummert1, Christopher Unzeitig1, Ralf Schmauder1,

Andrea Schweinitz1, Maik Otte1, Eckhard Schulz2, Frank Schwede3, Klaus Benndorf1

1 Friedrich Schiller Universität, Physiology, Jena, Germany 2 Fachhochschule Schmalkaden, Elektrotechnik, Schmalkaden, Germany 3 BIOLOG, Bremen, Germany

Ionotropic purinergic receptors (P2XRs) are expressed in several cell types and they are involved in diverse

physiological and pathophysiological processes like pain, inflammation or synaptic transmission. Binding of

extracellular ATP at the interphases of two neighbored subunits induces a conformational change of the pore forming

transmembrane domains TM2, resulting in a nonspecific cation flux. A central role of the b-14 sheet, which connects

the binding pocket with the pore, and in particular the amino acid H319 has been suggested for transmission of the

signal within one subunit.

Here we performed global fit strategies to kinetically unravel the activation of the three subunits in P2X2 receptors.

The strategy is based on four complex and intimately coupled kinetic schemes, sharing the majority but not all

parameters, which were fitted to multiple current data sets recorded from wild-type receptors and mutated receptors,

sensitized by the mutation H319K, with either ATP or its fluorescent derivative 2-[DY-547P1]-AET-ATP (fATP) as

ligand. The extended global fit allowed us to determine 26 rate constants for wild-type P2X2 channels and to gain

new insights into the gating mechanism.

Conclusions from our results are: 1. The steep concentration-activation relationship typical for wild-type P2X2

channels is caused by a subunit flip reaction with strong positive cooperativity that overbalances a pronounced

negative cooperativity for the three ATP binding steps. 2. The net probability fluxes in the kinetic scheme are

characterized by a marked hysteresis in the activation-deactivation cycle for the third binding step. 3. The facilitated

opening in the H319K mutant is caused by a significantly enhanced flipping reaction when only one ligand is bound.


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B 05-07

Modulation of gamma oscillations by purinergic P2X4 channels via

dendritic calcium influx into fast spiking interneurons

Florian Wildner, Tim S. Neuhäusel, Alexander Klemz, Marina Hager, Richard Kovács,

Jörg R. P. Geiger, Zoltan Gerevich

Charité — Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin

and Berlin Institute of Health, Institute of Neurophysiology, 10117 Berlin, Germany

Synaptic excitation on fast spiking interneurons is thought to play a crucial role in the modulation of the power of

gamma oscillations. Changes in the postsynaptic calcium concentration in fast-spiking interneurons is a key regulator

of synaptic strength. Ionotropic P2X4 receptors (P2X4R), activated by extracellular ATP and known to have a high

calcium permeability, were suggested to be an effective calcium source able to alter synaptic plasticity. Here, we

investigated if the activation of P2X4Rs alters excitatory synaptic currents in fast spiking interneurons in the CA3

area of the hippocampus and accordingly the power of gamma oscillations.

Using rat acute hippocampal slices, we show that activating P2X channels inhibits gamma oscillations induced by

cholinergic agents. This inhibition was occluded when slices were preincubated with naphthyl-spermine, a blocker of

calcium permeable AMPA receptors which are thought to largely mediate excitation onto fast-spiking interneurons.

Thus, P2XR mediated inhibition of gamma oscillations may act by regulation of calcium permeable AMPA receptors

in fast-spiking interneurons. Consistently, immunofluorescence stainings indicate that P2X4Rs are expressed on the

proximal dendrites of CA3 parvalbumin positive interneurons, an area where synapses from mossy and associational

fibers are located.Calcium imaging from CA3 fast spiking interneurons revealed that activation of P2XRs increases

dendritic calcium concentration. Excitatory postsynaptic currents (EPSCs) recorded from CA3 fast spiking

interneurons were evoked by electrical tract stimulation of associational fibers in stratum oriens and showed a main

component of calcium permeable AMPA receptors. Activation of P2X4Rs reversibly inhibited these EPSCs in a

calcium dependent manner.The results indicate that P2X4Rs are a possible source of dendritic calcium in CA3 fast

spiking interneurons and are able to modulate excitatory synaptic transmission and the state of hippocampal gamma

network activity.


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B 05-08

CKAMP44 unsilences and modulates function of relay neuron

synapses during dLGN development

Florian Hetsch, Danni Wang, Xufeng Chen, Jiong Zhang, Muhammad Aslam, Marcel Kegel,

Jakob von Engelhardt

University Medical Center of the Johannes Gutenberg University Mainz, Institute of Pathophysiology, Mainz,

Germany

Relay neurons of the dLGN receive input from retinal ganglion cells via retinogeniculate synapse. These connections

undergo pruning in the first two weeks after eye opening. The remaining connections are strengthened several-fold

by the insertion of AMPA receptors (AMPARs) into weak or silent synapses. We found that the AMPAR auxiliary

subunit CKAMP44 is expressed throughout development in the dLGN. Genetic deletion of CKAMP44 resulted in a

decrease in synaptic strength and an increase in the number of silent synapses of young animals. In addition,

CKAMP44 modulates synaptic properties by rendering short-term depression in retinogeniculate synapses stronger.

This effect is explained by the influence of CKAMP44 on AMPAR gating properties, in particular by the slow recovery

from desensitization of AMPARs that interact with CKAMP44. The reduction in short-term depression in relay neurons

of P9-11 CKAMP44 knockout mice led to a fascilitation of depolarization-induced L-type Ca²+-channel-driven plateau

potentials. The development of dLGN anatomy was not affected in CKAMP44 knockout mice as evidenced by

unaltered relay neuron morphology and input segregation at different developmental stages. These results show that

CKAMP44 promotes maturation and modulates function of retinogeniculate synapses during development of the

visual system.


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B 05-09

Dimerization and kinetics within groups of metabotropic glutamate

receptors

Taulant Kukaj, Christian Sattler, Thomas Zimmer, Ralf Schmauder, Klaus Benndorf

Friedrich Schiller University Jena, Institute of Physiology II, Jena University Hospital, Jena, Germany

Metabotropic Glutamate Receptors (mGluRs) are part of family C of G-protein coupled receptors. They are eight

members and that are classified into three groups based on sequence homology, pharmacology and downstream

signaling. mGluRs are expressed widely in the CNS. Members of group I, II, and III are located in postsynaptic sites,

presynaptic sites, and both pre- and postsynaptic sites, respectively.

mGluRs differ from other GPCRs by having a large extracellular domain, which is composed of a ligand-binding

domain (LBD) and a cysteine rich domain (CRD). The ligand-binding domain is responsible for dimerization, which

is required for receptor function. Previously it has been shown that mGluRs exist only as obligatory homodimers, but

recent findings suggest that mGluRs can also form heterodimers. Evidence for heterodimerization in neurons has

been presented so far for mGluR1/5, mGluR2/3, mGluR2/4, and mGluR2/7. Further studies are needed to explore

heterodimerization within mGluRs.

Herein, we used FRET to investigate dimerization of members of group I and II. In each subunit, we placed either

CFP (donor) or YFP (acceptor) in the second i2-loop. On one hand dimerization was determined by acceptor photo

bleaching and on the other kinetics was studied by combining a fast piezo-driven solution exchange system in small

outside-out membrane patches from Xenopus oocytes using a confocal microscope.

We observed that activation kinetics in members of group I are significantly faster compared to members of group II

and III, whereas deactivation varies even within members of each group. Furthermore, we show heterodimerization

and kinetics between members within each group, and among members of group I and II, and II and III, but not

between group I and III. In all heterodimers providing FRET signals, the slower subunit in the homodimer dominates

the kinetics, in both activation and deactivation. In heterodimers between groups, at least one subunit from members

of group II must be present.


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B 05-10

Mapping the ligand binding site on a peptide-gated ion channel

Michèle Bachmann1, Audrey Ortega-Ramírez1, Lilia Leisle1, Axel Schmidt2, Stefan Gründer1

1 RWTH Aachen University, Institute of Physiology, Aachen, Germany 2 University of Bonn, Institute of Human Genetics, Bonn, Germany

The ligand-gated cation channels from the freshwater polyp Hydra magnipapillata, termed Hydra Na+ channels

(HyNaCs), belong to the DEG/ENaC gene family. They are activated by endogenous neuropeptides RFamide I and

II and contribute to tentacle contraction of Hydra. In total, 11 different HyNaC isoforms have been characterised,

most of which form heterotrimeric channels eliciting inward currents upon ligand binding. Yet, the ligand binding site

remains undefined. Here, we aim to map the binding site of RFamide II on the heterotrimer comprising HyNaC2,

HyNaC3 and HyNaC5. First, to improve the expression levels of Hydra proteins in HEK293 cells we performed codon

optimisation of the genes. Western Blot analysis of whole protein lysates showed dramatically increased protein

levels. Second, to map the ligand binding site we successfully incorporated the photo-crosslinking unnatural amino

acid 4-azido-L-phenylalanine (AziF) at 20 different positions in the extracellular domain of HyNaC subunits. The site

selection was based on in silico molecular docking results of the ligand to a homology model, guided by functional

analysis of substitution mutants. Upon UV irradiation, AziF forms covalent bonds with molecules at distances

characteristic for specific interaction, thus providing details on the interaction interface. We aimed to detect the

wildtype peptide within the crosslinked product using a monoclonal antibody. Because this produced no specific

binding, we are currently affinity-purifying the antibody. Photocrosslinking allowed us, however, to successfully

determine the subunit order within the heterotrimer as 2-5-3 (and not 2-3-5). In addition, we measured individual

alanine substitutions of the seven amino acids of RFamide II to determine the importance of individual positions for

apparent affinity. Mapping of the peptide binding site on the HyNaC2/5/3 heterotrimer will help elucidate the

evolutionary conservation of the ligand binding site in DEG/ENaC channels.


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B 05-11

Juvenile epilepsy mediated by SCN2A mutation p.C258R

Alina Köppel1,2, Mahdi Jamili1, Enrico Leipold1,3, Ulrich Brandl2, Stefan H. Heinemann1

1 Friedrich Schiller University Jena and Jena University Hospital, Center of Molecular Biomedicine, Department of

Biophysics, Jena, Germany 2 Jena University Hospital, Clinics for Neuropediatrics, Jena, Germany 3 University of Lübeck, Department of Anesthesiology and Intensive Care, Lübeck University Hospital, Center for

Brain, Behavior and Metabolism (CBBM), Lübeck, Germany

Mutations in SCN2A, the gene encoding the α subunit of NaV1.2 voltage-gated sodium channel, are associated with

a wide spectrum of mild and severe epileptic disorders and other neurological diseases [1]. Finding an appropriate

therapy often remains difficult, especially in patients with intractable epilepsies caused by de novo mutations. Here

we report on an index patient with a severe early-childhood epileptic encephalopathy, mental retardation, and

movement disorder. Genetic analysis revealed a heterozygous de novo mutation in SCN2A, p.C258R, replacing an

arginine for a completely conserved cysteine in S5 of domain I. The functional impact was assessed in whole-cell

recordings of transiently transfected HEK293T cells, and the actions of antiepileptic drugs that were part of the

therapeutic strategy were determined.

Depending on the membrane voltage and pulse paradigm, mutant C258R displayed a mixed gain- and loss-of-

function phenotype. Peak current density was reduced by about a factor of 2.8 with a minor contribution of reduced

single-channel conductance. Most markedly, kinetics of fast inactivation was slowed down above -50 mV and

accelerated below. Voltage dependence of activation was only marginally affected (half-activation voltage of C258R

-50.2 ± 0.5 mV vs -46.6 ± 0.5 mV for wild-type (WT) with no effect on steepness. Voltage dependence of steady-

state inactivation, both with 0.5 and 10 s conditioning, was slightly left-shifted and was significantly shallower for the

mutant: 10.3 ± 0.2 mV vs 5.8 ± 0.1 mV, n > 50, P < 0.001), thus resulting in less current at holding voltages (-80 to -

110 mV). Trains of depolarizing pulses (20 to 83 Hz) resulted in stronger cumulative inactivation of C258R. Under

steady-state conditions (-120 mV holding, step to -20 mV), 100 µM lamotrigine and phenytoin blocked the mutant

more strongly than the WT (20-30% vs 10%), while topiramate was ineffective. Lamotrigine and phenytoin resulted

in progressive use-dependent block; for lamotrigine this block was more pronounced for the WT than for C258R.

Topiramate induced use-dependent block that immediately saturated after the second pulse in a train.Mutation

C258R in NaV1.2 channels, although displaying slower inactivation mostly results in a mild loss-of-function

phenotype. The mutation furthermore has a differential impact on the action of typically administered antiepileptic

drugs.

References [1] Ogiwara, I., Ito, K., Sawaishi, Y., Osaka, H., Mazaki, E., Inoue, I., ... & Yamakawa, K. (2009). 'De novo

mutations of voltage-gated sodium channel αII gene SCN2A in intractable epilepsies'. Neurology, 73(13), 1046-1053.


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B 05-12

Dopaminergic modulation of spike timing-dependent plasticity along

the dorso-ventral gradient of the mouse hippocampal CA1 region

Babak Khodaie1,3, Elke Edelmann1,2,3, Volkmar Leßmann1,2,3

1 Otto-von-Guericke University, Institute of Physiology, Magdeburg, Germany 2 Center for Behavioral Brain Sciences (CBBS), Magdeburg, Germany 3 OVGU ESF-funded International Graduate School ABINEP, Magdeburg, Germany

In recent years, a growing number of studies focused on the functional diversity of CA1 pyramidal cells (CA1 PCs)

along the longitudinal axis of the hippocampus. Moreover, heterogeneous expression of neurotransmitter and

neuromodulator receptors along this longitudinal hippocampal axis were reported, suggesting a gradient of both D1-

and D2-like receptor densities between dorsal and ventral poles of this axis. However, few studies investigated the

contribution of this receptor diversities to activity-dependent plasticity measured at single postsynaptic CA1 PCs

along the hippocampal axis.

Our spike timing dependent plasticity (STDP) protocols, consisting of either a canonical (1EPSP/1AP) or a burst

(1EPSP/4AP) pairing protocol, repeated just 6 times at 0.5 Hz, were used to induce t-LTP in slices taken from dorsal

(DH), intermediate (IH) or ventral (VH) hippocampus. To investigate dopamine receptor dependence of t-LTP, either

D1 receptor (D1R; 10 μM SCH23990) or D2R (10 μM Sulpiride) antagonists were bath applied alone or co-applied

to test the modulatory role of dopamine during t-LTP induction along the dorso-ventral axis. T-LTP induced by the 6x

1:4 protocol in VH was blocked by either D1 or D2R antagonism, but remained unaffected in DH. In contrast, t-LTP

induced by the 6x 1:1 protocol required co-application of D1- and D2R antagonists in DH and VH, whereas in IH t-

LTP induced by both protocols depended on D1 and/or D2R.

Our study revealed a gradient- and STDP paradigm-dependent dopaminergic modulation of t-LTP along the

longitudinal axis of the hippocampus, which possibly depends on the gradient in D1 and D2R expression levels

(VH>IH>DH).

Acknowledgment Funding: OVGU ESF-founded International graduate school ABINEP.


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Foyers

B 06 | Ion Channel Regulation Chair

Klaus Benndorf (Jena)

Angelika Lampert (Aachen)


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B 06-01

Heterodimerization of ‘silent’ subunits within the Two-Pore-Domain

Potassium Channel Family

Merten Prüser, Marianne Musinszki, Thomas Baukrowitz

CAU Kiel, Institute of Physiology, Kiel, Germany

Two-pore domain potassium (K2P) channels are a ubiquitously expressed family of channels that have a crucial role

in the maintenance of a cell’s resting membrane potential and, thus, the regulation of cellular excitability. Unlike other

groups of potassium channels, they are assembled as dimers and consist of two subunits, with each subunit made

up of four transmembrane segments (TM1-TM4) and two pore forming extracellular loops (P1 and P2). The P1 and

P2 loops of each subunit form a pseudotetrameric selectivity filter (SF) that contains a highly conserved amino acid

sequence.

In recent years an increasing number of intra- and intergroup K2P-channel heterodimers have been reported and it

appears that some of the heterodimers discovered have regulatory and pharmacological properties that are very

different from those of their corresponding homodimers. Interestingly, the identified heterodimers include some of the

‘silent’ family members, which do not seem to form functional homodimeric channels at the plasma membrane (i.e.

the TWIK subfamily and TASK-5 channels). Thus, heterodimerization with ‘silent’ subunits could constitute a

regulatory mechanism which increases potassium channel diversity at low cost for the cell. We systematically

investigated intergroup K2P channel heterodimerisation with ‘silent’ subunits of the TWIK-subfamily. To this end, we

developed and tested dominant negative mutants of TWIK-1/2 channels and at least one member of all other K2P

subfamilies by substituting singular amino acids at/near the SF with amino acids whose residues have

different/opposite charges (i.e. G -> E, T -> K). Coexpression of native channels and their corresponding mutants in

Xenopus laevis oocytes yielded concentration-dependent inhibition of the wild-type current for e.g. TREK-1/TREK-1

G159E, TREK-1/TREK-1 T156K, and TWIK-1/TWIK-1 T117K in two-electrode voltage clamp (TEVC) studies. We

then coexpressed the dominant-negative mutants with members of all K2P subfamilies to detect unidentified

heterodimerization partners of ‘silent’ subunits. Our results suggest that members of the TWIK subfamily can

assemble with TRESK, a channel involved in migraine and pain perception, which comprises a promising

pharmacological target.

In future, we aim to complete our comprehensive screen to reveal unknown heterodimers within K2P family and to

characterize biophysical, regulatory and pharmacological properties of the identified heterodimers.


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B 06-02

Identification of proton-gated DEG/ENaC ion channels in Nematostella

vectensis

Katharina B. Foreman1, José M. Aguilar-Camacho2, Yehu Moran2, Stefan Gründer1

1 RWTH Aachen University, Department of Physiology, Aachen, Germany 2 The University of Jerusalem, Department of Ecology, Evolution and Behavior, Alexander Silberman Institute of

Life Sciences, Faculty of Natural Sciences, Jerusalem, Israel

Ion channels from the degenerin/ epithelial sodium channel (DEG/ENaC) gene super family are very diverse in

function and activating stimuli. Nematostella vectensis is a model organism of the phylum Cnidaria, which is sister to

all bilaterian organisms and, therefore represents a valuable model to gain more insight into the evolution of the

nervous system. We cloned several DEG/ENaCs from N. vectensis, which we named Nematostella Na+ channels

(NeNaCs). Currently, we are functionally characterizing them in Xenopus oocytes with the Two-Electrode Voltage

Clamp system (TEVC). We expressed several combinations of NeNaCs in oocytes and exposed them

to diverse stimuli, including numerous neuropeptides. In addition, to activate these channels independent of a

specific stimulus, we introduced the “DEG mutation” which leads to constitutive activation. Here we present the

results on NeNaC2 and NeNaC14, two homomeric ion channels activated by protons in an acidic pH range. We

characterized location, activation and inhibition mechanisms for each. We anticipate that the study of NeNaCs will

reveal new insights into the evolution of DEG/ENaC ion channels.


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B 06-03

The role of KCa3.1 channels in overcoming erlotinib chemo-resistance

in non-small cell lung cancer cells

Luca Matteo Todesca, Etmar Bulk, Albrecht Schwab

Westfälische Wilhelms-Universität Münster, Institute of Phisiology II, Münster, Germany

Question: KCa3.1 channels play an important pathophysiological role in non-small cell lung cancer (NSCLC). They

promote aggressiveness by modulating processes such as migration, proliferation and tumor cell extravasation. EGF

receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as erlotinib are commonly used to treat NSCLC but almost all

patients initially responding to erlotinib develop acquired resistance. The aim of this project is to study how the

blockage of KCa3.1 channels with senicapoc helps to overcome the resistance to erlotinib. We put a particular

emphasis on apoptosis and integrin signaling.

Methods: We started by comparing microarray data sets of erlotinib-sensitive and -resistant NSCLC cells to evaluate

the differentially expressed genes and their correlation with other genes. In order to evaluate integrin signaling we

studied cell-matrix adhesion with single-cell force microscopy using an atomic force microscope. Apoptosis was

quantified with caspase-3 staining assays and annexin-V staining by fluorescent imaging.

Results: Microarray data analysis with the R software showed a differential expression of the KCa3.1 channel in the

erlotinib-resistant cells. This differential expression is highly correlated to that of 31 genes which are part of 10

different signaling pathways including integrin signaling and apoptosis. Adhesion experiments revealed an increased

adhesion force of NSCLC cells to the ECM-like matrix while applying the KCa3.1 blocker senicapoc or a combination

of erlotinib and senicapoc. This is most likely due to an increased expression of β1-integrin as shown by Western

blot. To distinguish between plasma membrane and intracellular (mitochondrial) KCa3.1 channels, we inhibited KCa3.1

channels with the non-permeant peptide Maurotoxin. It does not increase cell adhesion and β1-integrin expression,

which is consistent with a role of mitochondrial KCa3.1 channels. Caspase-3 and Annexin-V staining assays revealed

that senicapoc treatment also induces apoptosis also in erlotinib-resistant cells. Induction of apoptosis is comparable

to that induced by doxorubicin which was used as positive control.

Conclusions: Taken together, our results suggest that KCa3.1 channels inhibition with senicapoc in NSCLC resistant

cells can help to overcome erlotinib resistance by an increase of cell adhesion to the ECM matrix and inducing at the

same time apoptosis due to a caspase-3 activation mechanism.

Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft (GRK 2515/1, Chembion).


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B 06-04

CLCA1 regulates airway mucus production and ion secretion through

TMEM16A

Raquel Centeio, Jiraporn Ousingsawat, Khaoula Talbi, Rainer Schreiber, Karl Kunzelmann

University of Regensburg, Department of Physiology, Regensburg, Germany

Background: Transmembrane protein 16A (TMEM16A), a Ca2+-activated chloride channel, and Ca2+-activated

chloride channel regulator 1 (CLCA1), have both been independently associated with inflammatory airway disease,

goblet cell metaplasia, mucus production/secretion and cytokine/chemokine secretion. CLCA1 is a secreted

metalloproteinase containing a von Willebrand factor type A (vWFA) domain, known to mediate protein-protein

interactions and extracellular signaling. Recently, a mechanism was established through which direct binding of

CLCA1’s vWFA stabilizes TMEM16A at the cell plasma membrane in heterologous-expressing cells, thereby

increasing Ca2+-activated chloride currents (CaCC). Whether such a mechanism is existing under conditions of

endogenous expression and in vivo and is responsible for mediating the downstream effects that are common to

both proteins, remains unclear.

Methods/Results: Secreted N-terminal CLCA1 containing vWFA (N-CLCA1) was produced by CLCA1-expressing

HEK293T cells. N-CLCA1-containing media was applied to Calu-3 human airway epithelial cells expressing

TMEM16A endogenously. N-CLCA1 increased PM-expression of TMEM16A as demonstrated by

immunocytochemistry, while whole-cell patch-clamp detected enhanced CaCC. Human BCi-NS1 airway epithelial

cells grown on permeable supports were stimulated with N-CLCA1. Alcian blue staining of acidic mucins showed

enhanced production of mucus that was inhibited by siRNA-knockdown of TMEM16A. These findings were

reproduced in vivo, by directly applying N-CLCA1-medium to mouse airways via intratracheal instillation.

Immunostaining of TMEM16A in mouse airways showed upregulation of PM-expression. Moreover, mucus

production and secretion were both upregulated, when quantified by alcian blue staining. N-CLCA1-induced mucus

secretion was even further augmented in airways of asthmatic mice sensitized by ovalbumin, which is known to

strongly enhance expression of TMEM16A. Notably, CaCC appeared not to be enhanced, as equivalent short circuit

currents measured in mouse tracheas in open-circuit Ussing chambers were not increased.

Conclusions/Future perspectives: The present data suggest a limited contribution of TMEM16A/CLCA1 to ion

secretion in mouse airways, but demonstrate a clear role of CLCA1 for airway mucus secretion, which requires

TMEM16A. Thus, CLCA1 has a clear prosecretory role and may also shape the innate immune response, by

stimulating cytokine/chemokine secretion with the help of TMEM16A.

Acknowledgment The technical assistance by Patricia Seeberger is greatly appreciated. Funding was supported by UK CF Trust

SRC013, DFG KU756/14-1, DFG project number 387509280, SFB 1350 (project A3) and Gilead Stiftung.


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B 06-05

Defective X-Gating of TASK-1 in a Novel Channelopathy Associated

with Sleep Apnea

Janina Sörmann1,2, Marcus Schewe3, Peter Proks1,2, Thibault Jouen-Tachoire1, Shanlin Rao4,

Thomas Müller5, Thomas Baukrowitz3, Matthew E. Hurles6, Caroline F. Wright7, Stephen J. Tucker1,2

1 University of Oxford, Clarendon Laboratory, Department of Physics, Oxford, UK 2 University of Oxford, Kavli Institute for Nanoscience Discovery, Oxford, UK 3 Univeristy of Kiel, Department of Physiology, Kiel, Germany 4 Unversity od Oxford, Department of Biochemistry, Oxford, UK 5 Bayer AG, Research & Development, Pharmaceuticals, Wuppertal, Germany 6 Wellcome Sanger Institute, Wellcome Genome Campus, Human Genetics Programme, Cambridge, UK 7 University of Exeter Medical School, Institute of Biomedical and Clinical Science, Exeter, UK

A recent study identified 28 novel genes with a high burden of de novo mutations associated with developmental

disorders [1]. One of these genes (KCNK3)encodes TASK-1, a member of the Two-Pore Domain (K2P) family of K+

channels. Nine probands with a total of six unique mutations have now been identified, each heterozygous for a

single de novo missense mutation in KCNK3 and shared a common phenotype which includes developmental delay

and also sleep apnea requiring nocturnal oxygen. TASK-1 is expressed in many tissues associated with sleep apnea

and has previously been implicated in many of the pathways which control breathing, chemosensitivity and oxygen

sensing [2,3]. However, mutations in KCNK3 are currently associated with a different clinical disorder, Pulmonary

Arterial Hypertension [4], and so its link with sleep apnea remains incompletely understood. The recent crystal

structure of TASK-1 revealed a lower ‘X-gate’ created by intersection of the two M4 helices [5] and which has

previously been implicated in the control of TASK-1 activity. Interestingly, all six disease mutations cluster adjacent

to the X-gate in TASK-1. We found these mutant TASK-1 channels result in a markedly higher basal activity and

single channel recordings demonstrated up to a 10-fold increase in open probability. Molecular dynamics simulations

of both WT and mutant structures of TASK-1 also revealed these mutations promote opening of the X-gate. Finally,

we observed that these mutations render TASK-1 insensitive to GqPCR-mediated inhibition that normally

characterizes wild type channels, but retain their sensitivity to inhibition by BAY1000493 [5], a close analogue of a

drug in clinical trials for the treatment of sleep apnea which exhibits highly efficacious block in the nanomolar range.

These findings demonstrate a clear role for TASK-1 in sleep apnea and identify possible therapeutic strategies for

this new channelopathy as well as for sleep apnea in general.

Acknowledgment This work was supported by the BBSRC (UK) and the DFG (Germany).

References [1] Kaplanis, J., et al., (2020) Evidence for 28 genetic disorders discovered by combining healthcare and

research data.Nature, 586: 757-762. [2] Trapp, S., et al., (2008) A Role for TASK-1 (KCNK3) Channels in the Chemosensory Control of Breathing.

Journal of Neuroscience, 28: 8844-8850. [3] López-Barneo, J., et al., (2016) Oxygen sensing by the carotid body: mechanisms and role in adaptation to

hypoxia.American Journal of Physiology, 310: C629-C642. [4] Bohnen, M.S., et al., The Impact of Heterozygous KCNK3 Mutations Associated With Pulmonary Arterial

Hypertension on Channel Function and Pharmacological Recovery. Journal of the American Heart Association, 2017. 6(9).

[5] [5] Rödström, K., et al., (2020) A lower X-gate in TASK channels traps inhibitors within the vestibule.Nature, 582: 443-447.


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B 06-06

Differential effect of Diltiazem on rod and cone CNG channels: A viable

solution for retinitis pigmentosa patients?

Valerie Popp1, Kathrin Groeneveld1,2, Christian Melle2, Frank Schwede3, Vasilica Nache1

1 University Hospital Jena, Institute of Physiology II, Jena, Germany 2 University Hospital Jena, Biomolecular Photonics Group, Jena, Germany 3 Biolog Life Science Institute GmbH & Co. KG, Bremen, Germany 4 University Hospital Jena, Institute of Physiology II, Jena, Germany

Retinitis pigmentosa (RP) is a group of hereditary diseases where a primary degeneration of rod photoreceptors is

followed by a secondary loss of cones, leading first to severe impairment of vision and finally to blindness. So far,

there is no efficient cure for this condition. A common symptom for many RP forms is the dysregulation of cGMP

homeostasis, which induces an increased activation of cyclic nucleotide-gated (CNG) channels and excessive Ca2+

influx. CNG channels play an essential role within visual transduction, being responsible for the “dark current” in the

absence of light. The treatment strategy to inhibit selectively the targets for the high cGMP was already considered,

but so far with no conclusive outcome1. The studies with Diltiazem, a well-known anti-hypertensive drug, produced

also contradictory results2.

By combining electrophysiology and confocal patch-clamp fluorometry, we assessed the effect of D- and L-cis-

Diltiazem on rod and cone CNG channels, heterologously expressed in Xenopus oocytes. We show that under

pathologically high cGMP-conditions, Diltiazem had a differential effect on retinal CNG channels, with a stronger

inhibition of rod- vs. cone- CNG channels and the effect of L- exceeding that of D-cis-Diltiazem. Specifically, we

observed ~92% inhibition of rod CNG channel activity with 100 µM L-cis-Diltiazem and only ~36% with D-cis-

Diltiazem at a similar concentration. At physiological cGMP, neither D- nor L-cis-Diltiazem showed an appreciable

inhibitory effect. Both D- and L-cis-Diltiazem acted on retinal CNG channels in a voltage- and cGMP-dependent

manner. Interestingly, Diltiazem can assert its inhibitory effect only after cGMP-induced channel activation, most

probably by obstructing its conductive pore. Although we observed no influence of Diltiazem on ligand binding and

channel activation, the deactivation kinetic was delayed and slowed down by a factor of ~2. In conclusion, our results

indicate that only L-cis-Diltiazem has the desired traits for an effective RP treatment: (1) it can inhibit the rod CNG

channels only under RP-like cGMP conditions and has no effect on these channels in healthy cells, (2) it changes

the gating kinetics of the CNG channels only moderately and (3) it shows a cGMP-dependent rod vs. cone selectivity.

Further studies on established animal models for retinal degenerative diseases would be necessary to verify the

effect and the therapeutical potential of L-cis-Diltiazem.

Acknowledgment This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, TRR 166

ReceptorLight project B01 and Project 437036164 to VN).

References [1] Vighi, E., Trifunovic, D., Veiga-Crespo, P., Rentsch, A., Hoffmann, D., Sahaboglu, A., et al., 2018,

Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration. Proc Natl Acad Sci U S A., 115(13): E2997-E3006.

[2] Barabas, P., Cutler, P.C., Krizaj, D., 2010, Do calcium channel blockers rescue dying photoreceptors in the Pde6b ( rd1 ) mouse? Adv Exp Med Biol. 664: 491-9.


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B 06-07

The Two-Pore-Domain Potassium Channel TWIK-2 is inhibited by

Members of the Phosphoinositide-Phospholipase C Signaling Pathway

Marianne Musinszki, Sönke Cordeiro, Thomas Baukrowitz

Christian-Albrechts University, Institute of Physiology, Kiel, Germany

Two-Pore Domain Potassium (K2P) channels are important regulators of cellular excitability and involved in diverse

physiological and pathophysiological processes including e.g. sleep, secretion, anaesthesia, and pain. They are

modulated by diverse intra- and extracellular stimuli like stretch, temperature and pH and respond to cellular signaling

pathways via cellular signaling lipids and phosphorylation. K2P channels comprise 15 members in 6 subfamilies,

most of which have been investigated in the past decade with the TREK and TASK families well studied. However,

some K2P channels are referred to as ‚silent’ since they show little or no current in standard expression systems, i.e.

the TWIK subfamily and TASK-5 channels. Therefore, they are poorly characterized and their physiological role and

regulation is not clear.

The TWIK-2 channel is expressed e.g. in vascular smooth muscle, vascular and airway endothel and spleen. It was

suggested to reside in intracellular compartments or act as regulatory subunit when forming heterodimers with other

K2P channels1. TWIK-2 channel currents were recorded in lysosomes2, but the channel was also proposed as

potassium efflux channel in inflammasome activation in macrophages3.

Here we report that TWIK-2 channels are regulated by members of the PLC signaling pathway in transiently

transfected HEK293 cells. To overcome the limitation of small currents at the plasma membrane, we removed a

known retention motif and substituted extracellular potassium for rubidium, which yielded larger currents facilitating

characteritzation of the channel.

We found that the membrane-permeable diacylglycerol (DAG) analogon DiC8 inhibited TWIK-2 currents in whole-

cell patch clamp recordings by 50%. DAG is a product of phospholipase C (PLC)-mediated hydrolysis of membrane

phosphoinositides, i.e. phosphatidylinositol-4,5-bisphosphate (PIP2). Consistently, direct pharmacological activation

of PLC with m3mfbs reduced TWIK-2 currents by almost 90%. Thus, we aim to understand the regulation mechanism

by cellular lipids and their metabolites and to reveal receptor pathways converging on the respective lipid species.

References [1] Chavez, R.A., Gray, A.T., Zhao, B.B., Kindler, C.H., Mazurek, M.J., Mehta, Y., Forsayeth, J.R., Yost, C.S.,

1999. TWIK-2, a New Weak Inward Rectifying Member of the Tandem Pore Domain Potassium Channel Family. The Journal of Biological Chemistry 274, 7887–7892. https://doi.org/10.1074/jbc.274.12.7887

[2] Bobak, N., Feliciangeli, S., Chen, C.-C., Ben Soussia, I., Bittner, S., Pagnotta, S., Ruck, T., Biel, M., Wahl-Schott, C., Grimm, C., Meuth, S.G., Lesage, F., 2017. Recombinant tandem of pore-domains in a Weakly Inward rectifying K+ channel 2 (TWIK2) forms active lysosomal channels. Scientific Reports 7, 649. https://doi.org/10.1038/s41598-017-00640-8

[3] Di, A., Xiong, S., Ye, Z., Malireddi, R.K.S., Kometani, S., Zhong, M., Mittal, M., Hong, Z., Kanneganti, T.-D., Rehman, J., Malik, A.B., 2018. The TWIK2 Potassium Efflux Channel in Macrophages Mediates NLRP3 Inflammasome-Induced Inflammation. Immunity 49, 56-65.e4. https://doi.org/10.1016/j.immuni.2018.04.032


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B 06-08

Stimulatory effects of butyrate on TRPV3 and TRPV4 with implications

for Ca2+ uptake in ruminants

Franziska Liebe1, Hendrik Liebe1, Stefan Mergler2, Gerhard Sponder1, Friederike Stumpff1

1 Freie Universität Berlin, Institute of Veterinary Physiology, Berlin, Germany 2 Charité - Universitätsmedizin Berlin, Experimental Ophthalmology, Berlin, Germany

Microbial fermentation of plant material within the rumen releases large quantities of short-chain fatty acids (SCFA),

which stimulate the transport of Ca2+ across the rumen in vivo and in vitro [4,3]and induce a rise in short-circuit

current. Although typical Ca2+ channels (TRPV5, TRPV6) are not expressed by the rumen, TRPV3 has emerged as

a possible candidate [1,2]. Furthermore, qPCR data suggest expression of TRPV4. We confirm protein expression

of the bovine form of TRPV4 (bTRPV4) by the ruminal epithelium, which could be detected on the level of the protein

in Immunoblots, but also via immunofluorescence staining in native ruminal epithelium and model epithelia grown in

cell culture. Apart from cytosolic expression, faint staining of the apical membranes was observed. We further

investigated effects of Na-butyrate (30 mmol∙L‐1) on bTRPV3 and bTRPV4 via patch-clamp measurements and

calcium fluorescence imaging. The effects of Na-butyrate on whole-cell currents tended to be small and variable in

both bTRPV4 and non-expressing control cells. Conversely, in cells overexpressing bTRPV3, a strong stimulation

on Na+ currents was observed with highest effect at pH 6.4 (p < 0.001 versus bTRPV4 and control). Furthermore,

current kinetics were affected with activation of current at positive potentials and pronounced tail-currents upon

repolarization. In calcium fluorescence imaging at pH 6.4, initial cytosolic calcium concentration [Ca2+]i was similar in

all three cell-types (~ 30 nmol·L-1). In both bTRPV3 and bTRPV4 HEK-293 cells, [Ca2+]i rose to a peak-value within

one minute after application of NaBu 6.4, subsequently dropping to a lower plateau of 170 ± 18 (bTRPV3) and 135

± 7 nmol·L‐1 (bTRPV4) (p < 0.001 versus baseline). In control cells, a significantly smaller increase (p < 0.001) in

[Ca2+]i was observed that remained constant at 101 ± 8 nmol·L-1. We conclude that both bTRPV3 and bTRPV4 may

contribute to the stimulatory effects of SCFA on the absorption of cations across the rumen.

Acknowledgment The support of the Deutsche Forschungsgemeinschaft (STU 258/7-1) is gratefully acknowledged.

References [1] Liebe F, Liebe H, Kaessmeyer S, Sponder G, Stumpff F (2020) Pflugers Archiv : European journal of

physiology 472:693-710. [2] Schrapers KT, Sponder G, Liebe F, Liebe H, Stumpff F (2018). PloS one 13:e0193519. [3] Schröder B, Vossing S, Breves G (1999) J Comp Physiol (B) 169:487-494 [4] Wilkens MR, Muscher-Banse AS (2020) Animal 14:s29-s43. doi:10.1017/s1751731119003197


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B 06-09

Potential role of K2P2.1 channels in the mechano- and pH-sensitivity of

pancreatic stellate cells

Micol Rugi, Zoltan Phetö, Thorsten Loeck, Verena Hofschröer, Albrecht Schwab

University of Münster, Institut für Physiologie II, Münster, Germany

Question: An extensive desmoplastic reaction and a unique pH landscape are two of the main features of the

pancreatic ductal adenocarcinoma (PDAC). Desmoplasia is the result of the uncontrolled activation of pancreatic

stellate cells (PSCs). Mechanosensitive ion channels are involved in the activation of these cells. Among them, K2P2.1

channels are known to be sensitive to pH. Little is known about the role of K2P2.1 channels in PSCs and PDAC so

far. Our project seeks to solve this question by studying the interplay between the pH and the mechano-modulation

of K2P2.1 in PSCs.

Methods: The expression of K2P2.1 channels was ascertained by RT-qPCR and immunofluorescence assays both

in murine PSCs and human PS1 cells. Furthermore, the expression of K2P2.1 was also studied by

immunohistochemistry in human PDAC samples. As a surrogate of the PSC activation we investigated their migratory

activity. We tested different conditions, such as different pH, pressure and Spadin (K2P2.1 inhibitor) on PSCs from

wildtype mice and K2P2.1 knock-out mice. Pressure (+ 100 mmHg above ambient pressure) was applied in a self-

made pressure chamber. The assays were evaluated with live-cell imaging.

Results: Both RT-qPCR and immunofluorescence assays confirmed K2P2.1 expression in both murine and human

PSCs. Immunohistochemistry staining pointed out a weaker staining of the channels in the tumor tissue, while the

pancreatic islet are strongly positive for it. A strong involvement of the K2P2.1 in the mechano-sensing of the murine

PSCs was detected during migration. In particular K2P2.1 knockout PSCs showed a weaker reaction after the

preincubation with pressure, while wild type PSCs showed the tendency to increase their speed in pHe 7.4 and

decrease it in pHe 6.6. The acute application of pressure causes PSCs to decrease their velocity. Normal speed was

recovered after the removal of pressure. In physiological and acidic conditions both wild type and K2P2.1 knockout

PSCs showed no significant differences in their speed. After the treatment with Spadin wild type cells showed the

tendency to decrease their area.

Discussion: Murine K2P2.1 channels are known to be weakly affected by extracellular acidification. Our hypothesis

takes into account that under pressure and in an acidic environment the role of K2P2.1 activation by pressure may

counterbalance the depolarizing effect of Ca2+ entry via other mechano-sensitive ion channels and thereby allow their

response to external stimulation.

Acknowledgment This work was supported by European Marie Skłodowska-Curie Innovative Training Network (ITN) pH and Ion

Transport in Pancreatic Cancer–pHioniC (Grant Agreement number: 813834; H2020-MSCA-ITN-2018). AS thanks

the support from the Deutsche Forschungsgemeinschaft (DFG; SCHW 407/22-1 and GRK 2515/1, Chembion).


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B 06-10

Regulation and gating mechanism of the two-pore domain potassium

THIK-1 channel (KCNK 13)

Bisher Eymsh, Elena Riel, Sönke Cordeiro, Thomas Baukrowitz

Kiel University, Insitiute of physiology, Kiel, Germany

The two-pore domain potassium channels (K2P) are the most recently discovered subgroup of potassium channels

consisting of 15 members. Unlike other K+ channels K2P channel subunits are composed of 4 transmembrane

domains with 2 pore forming domains. Accordingly, K2P channels are composed of only two subunits. In previous

studies we and others have shown that K2P channels are differentially gated. K2P channels are modified by a variety

of stimuli including physical (temperature, tension), chemical (pH) and intracellular signaling (phosphorylation). While

TREK channels are gated at the selectivity filter only, other K2P channels possess an additional gate at the

intracellular entrance to the central cavity (X-gate in TASK-1 channels). THIK-1 channels (Two-pore Domain

Halothane Inhibited K+) are only known to be slightly activated by arachidonic acid and inhibited by volatile

anesthetics like halothane. They are widely expressed in the CNS but their physiological functions and gating

mechanisms are unknown. In this study we investigated the possible intracellular gate of the THIK-1 channel. In

addition, we investigated stimuli leading to gating of the channels.

Pharmacological screening of THIK-1 channels revealed a very strong activation by membrane phosphoinositide and

long-chain fatty acid Coenzyme A esters (up to 30-fold activation). This activation was abolished after cleavage of

the C-terminus at a previously described caspase-8 cleavage site. By introduction of cysteins at different positions in

the gating path of THIK-1 channels we investigated the accessibility of these amino acids in then open confirmation

(PIP2 activated) and in the closed confirmation (unstimulated). This was done by irreversible modification by different

MTS reagents in patch clamp measurements of macro-patches from Xenopus oocytes in the inside-out configuration.

This configuration was used to allow the application of modifying MTS reagents from the inside. In addition, we

identified the PIP2 binding site in the C-terminus of THIK-1 channels.

In conclusion, in PIP2 we identified the first clear activator of THIK-1 channels. The here identified binding site in the

C-terminus couples the PIP2 dependence to the caspase-8 dependent apoptosis pathway. In addition, we identified

the structures in the channel relevant for gating mechanisms.


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B 06-11

Pharmacological inhibition of KCa3.1 channels leads to increased ROS

production in non-small lung cancer cells.

Etmar Bulk, Luca M. Todesca, Marius Rieke, Albrecht Schwab

University of Muenster, Institute of Physiology II, Muenster, Germany

The generation of reactive oxygene species (ROS) is a constant process during the aerobic metabolism of eucaryotic

cells. At low levels, ROS are important for several cellular functions such as cell signaling and calcium homeostasis.

However, high levels of ROS can result in oxidative cell damage since the physiological antioxidant defence system

of a cell is limited. The production of ROS has been correlated with several human diseases including cancer. The

main producers of ROS are mitochondria.

Here, we investigated whether the inhibition of KCa3.1 channels could have an effect in the generation of ROS in non-

small cell lung cancer (NSCLC) cells. By applying Western blotting, we detected KCa3.1 expression levels in

mitochondrial fractions of different lung cancer cells. This result was confirmed via immunofluorescence. KCa3.1

channels are colocalized with mitochondria in non-small cell lung cancer cells. Using the fluorescent dye Rhodamine

123, we analyzed the mitochondrial membrane potential of NSCLC cells. The KCa3.1 channel inhibitor TRAM-34

decreases Rhodamine 123 fluorescence intensity by 15 % corresponding to a hyperpolarization of the mitochondrial

membrane potential. In a final experiment, we examined the impact of the KCa3.1 channel inhibitors TRAM-34 and

senicapoc on the ROS production of lung cancer cells. Using the mitochondrial superoxide indicator MitoSOX™ Red

we detected an increase of ROS production in NSCLC cells following the application of the KCa3.1 inhibitors TRAM-

34 or senicapoc.

In summary, our results reveal the presence and a functional role of KCa3.1 channels in mitochondria of NSCLC cells.

Their pharmacological inhibition induces the generation of ROS.


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B 06-12

A truly promiscuous channel: D-menthol, L-menthol, and the

conductance of TRPV3 to NH4+

Hendrik Liebe, Franziska Liebe, Gisela Manz, Gerhard Sponder, Friederike Stumpff

Freie Universität Berlin, Institute for Veterinary Physiology, Berlin, Germany

Keratinocytes within stratified squamous epithelia such as the skin or the rumen abundantly express TRPV3 channels

with functions that are poorly understood. In the ruminant forestomach, a role in the transport of Ca2+ and NH4+ has

emerged [3,1]. In human skin, TRPV3 participates in the keratinisation process, with gain of function mutations in

humans leading to severe hyperkeratosis [2]. Given the importance of NH4+ for protein metabolism, we wished to find

out if the human homologue hTRPV3 conducts NH4+. Furthermore, while stimulatory effects of L-menthol on TRPV3-

mediated Ca2+ influx are well documented, effects of D-menthol have not been studied.

Accordingly, HEK-293 cells were transiently transfected with a HA-Strep-hTRPV3 construct subcloned into a pIRES2-

AcGFP1 vector, while controls were transfected using the empty vector. Cells were investigated with the whole-cell

configuration of the patch clamp technique. After filling with a Na-Gluconate pipette solution, cells were consecutively

superfused with NaCl or NH4Cl Ringer. Significant rises in inward current and the reversal potential in overexpressing

cells and controls were observed, which suggests that permeability to NH4+ exceeded that to Na+. Simultaneous

stimulation of outward currents carried by Na+ were also observed and may reflect effects of swelling, pH, or activation

of current by a permeant ion. In cells expressing hTRPV3, but not in controls, both inward and outward currents could

be stimulated by either D-menthol or L-menthol. While the effects of a second application of menthol were always

significantly larger than that of the first, no difference between the two enantiomers emerged. The stimulatory effects

of D-menthol on TRPV3 were confirmed via Ca2+ imaging. We conclude that chirality plays no role in the effects of

menthol on TRPV3. Furthermore, like the bovine homologue, the human TRPV3 channel conducts NH4+. In addition

to the well-documented role of the TRPV3 channel in Ca2+ signalling in the human skin, a further function of this non-

selective channel may thus be to supply NH4+ for the synthesis of glutamine and ultimately, involucrin, loricrin, and

filaggrin as building blocks of the corneocyte envelope.

Acknowledgment The financial support of the Sonnenfeld Stiftung is gratefully acknowledged.

References [1] Liebe F, Liebe H, Kaessmeyer S, Sponder G, Stumpff F (2020) Pflügers Archiv 472:693-710. [2] Nilius B, Biro T (2013) Exp Dermatol 22:447-452. [3] Schrapers KT, Sponder G, Liebe F, Liebe H, Stumpff F (2018) PLoS One 13:e0193519.


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B 06-13

Characterisation of a new combined SCN5A and GPD1L variant

associated with Brugada Syndrome

Francesca Semino1,2, Claus Bruehl1, Fabrice Darche2, Michael Koenen2, Patrick Schweizer2,

Andreas Draguhn1

1 Heidelberg University, Institute of Physiology and Pathophysiology, Heidelberg, Germany 2 Medical University Hospital Heidelberg, Department of Cardiology, Heidelberg, Germany

Brugada syndrome (BrS) is a cardiac disease leading to typical ECG changes in the right precordial leads associated

with a high risk for polymorphic ventricular tachycardia and sudden cardiac death. Although the genetic influence in

BrS pathophysiology is known, the causal relation between genetic variants and Brugada phenotype is still largely

unclear. SCN5A loss-of-function mutations are the most common cause of BrS. The SCN5A gene encodes for the

voltage-gated sodium channel Nav1.5 underlying the fast sodium influx in the early phase of the myocardial action

potential. Recently variants in other genes that modulate the cardiac sodium current have been identified in BrS

patients, including the GPD1L gene encoding the glycerol-3-phosphate dehydrogenase-1-like protein. Although its

function is not fully understood, a GPD1L mediated cascade with subsequent activation of kinases and

phosphorylation of the Nav1.5 channel is assumed to alter the sodium current.

In this study two previously undescribed mutations (SCN5A-G1661R and GPD1L-A306del) were identified in a

German family. While an individual presenting only the GPD1L-A306del variant was phenotypically healthy, two

individuals with only the SCN5A-G1661R mutation were diagnosed with BrS. A fourth individual presenting both

variants showed the typical BrS ECG and also suffers from conduction disease. To establish the underlying

pathophysiology, Na+-currents were measured in transiently transfected HEK293 cells, using the whole-cell-voltage

clamp technique. For the transfection different combinations of plasmids encoding either the wildtype or the mutated

SCN5A and GPD1L were used. Transient sodium current as well as the characteristic parameters for activation,

inactivation and recovery properties of the channels were detected.

While homozygote expression of SCN5A-G1661R mutation failed to produce current, the heterozygote constellation

showed a reduction in current amplitude to around 50% without changes of the biophysical properties of Na+-currents,

suggesting haploinsufficiency as the leading pathomechanism. On the other hand, preliminary data on the GPD1L-

A306del variant indicate a more complex pathophysiology, that alters the biophysical properties of the sodium

channel and may have additional impact on the sodium current. The latter observation has to be clarified and is

currently under investigation. Together this study supports the role of the altered Nav1.5 in the genesis and

maintenance of BrS.


of 516


Foyers

B 07 | Lung Physiology and Hypoxia II Chair

Paul Dietl (Ulm)

David Hoogewijs (Fribourg)


of 516

B 07-01

Without You I´m Nothing: Hypoxia-inducible Factor 2α is Essential for

Proper Brain Function

Timm Schreiber1,2, Theresa Quinting1, Tristan Leu1, Kira Kleszka1, Joachim Fandrey1

1 University Duisburg-Essen, Department of Physiology, Essen, Germany 2 University Witten/Herdecke, Department of Physiology, Pathophysiology and Toxicology and Center for

Biomedical Education and Research (ZBAF), Witten, Germany

Sufficient tissue oxygenation is required for regular brain function; thus, oxygen supply must be tightly regulated to

avoid hypoxia and irreversible cell damage. If hypoxia occurs the hypoxia-inducible factor (HIF) will accumulate and

coordinate adaptation of cells. However, even under atmospheric O2 conditions we found stabilised HIF-2α in brains

of adult mice. Hence, we are interested in the role of HIF-2α in brain function, with a special focus on plasticity of the

brain. Mice with a neuro-specific knockout of HIF-2α showed a reduction of pyramidal neurons in the retrosplenial

cortex. Accordingly, behavioural studies showed disturbed cognitive functions. In search of the underlying

mechanisms, we found deficits in migration in HIF-2a deficient neuronal stem cells (NSCs), cultured as neurospheres,

due to altered expression patterns of genes highly associated with neuronal patterning. Interestingly, we found that

synaptogenesis is highly oxygen dependent, and loss of HIF-2α leads to a remarkable decrease in synaptic markers.

To further elucidate the role of HIF-2α in synapse-associated processes, we will combine environmental enrichment

to enhance synaptic plasticity with RNA expression analysis and electrophysiological measurements. Hypoxia is also

one of the key components in neuropathological conditions such as stroke. To elucidate the influence of HIF-2α

during regeneration after ischemic stroke, we challenged neurospheres with oxygen/glucose-deprivation (OGD).

After OGD, NSCs migrated significantly less, with no differences considering genotypes. Additionally, mRNA

analyses showed a strong effect of HIF-2α on expression of genes involved in neurogenesis. Combined, we showed

that HIF-2a is essential for proper brain function.


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B 07-02

Effects of pharmacological Gq protein inhibition on airway

hyperresponsiveness and airway remodeling in chronic asthma

Jennifer M. Dietrich1,2, Annika Simon4, Michaela Matthey4, Gabriele König3, Reynold A. Panettieri5,

Bernd K. Fleischmann1, Daniela Wenzel1,4

1 University of Bonn, Medical Faculty, Institute of Physiology I, Bonn, Germany 2 University of Bonn, Research Training Group 1873, Bonn, Germany 3 University of Bonn, Institute of Pharmaceutical Biology, Bonn, Germany 4 Ruhr University of Bochum, Department of Systems Physiology, Bochum, Germany 5 Rutgers, Department of Medicine, Rutgers Institute for Translational Medicine and Science, New Brunswick, USA

Gq proteins play a key role in the regulation of airway tone in the lung and are hence targets for the treatment of lung

diseases, e.g. asthma. The pathophysiology of asthma is characterized by airway hyperresponsiveness,

inflammation and remodeling. Although airway remodeling contributes to the decline of lung function, current

therapeutic strategies are not able to prevent this process. We therefore wondered, whether the pan Gq-inhibitor FR

900359 (FR) could attenuate airway hyperresponsiveness and airway remodeling.

The effect of FR (5 µg/d) was assessed in a Balb/c mouse model of chronic ovalbumin (OVA)-induced asthma. Airway

hyperresponsiveness was determined with a FlexiVent system and lungs were processed for histology. In addition,

effects of FR (1µM) on mucus secretion were also investigated by ELISA in precision cut lung slices (PCLS) of human

asthmatics harvested post-mortem. To examine in vitro effects of FR on goblet cells, human bronchial epithelial cells

were grown in an air liquid interface (ALI) culture. The impact of FR (10 µM) on goblet cell differentiation and mucus

secretion was tested after stimulation with Gq-coupled receptor agonists Carbachol (CCh, 100 µM), ATP (100 µM)

and thrombin (1 U/ml).

Intratracheal application of FR reduced airway hyperresponsiveness in mice with chronic OVA-induced asthma

(3.9±0.4 cmH2Os/mL n=9 (FR) vs 9.2±1.9 cmH2Os/mL n=8 (vehicle), p<0.001). Histological analysis of lung sections

from OVA mice revealed that FR treatment reduced collagen deposition (6.2±0.3 µm2/µm perimeter basal membrane

(pbm), n=11 (FR) vs 7.4±0.4 µm2/µm pbm (vehicle)) around the airways in asthmatic lungs and strongly attenuated

goblet cell metaplasia (0.03±0.0 PAS+ cells/µm pbm, n=11) compared to vehicle controls (0.06±0.01 PAS+ cells/µm

pbm (vehicle), n=11, p<0.05). FR was able to reduce Muc5AC secretion of PCLS (0.66±0.08 mg/mL (FR) vs.

1.03±0.02 mg/mL (vehicle), n=6 p<0.01) as well as of bronchial epithelial cells grown in ALI culture upon stimulation

with Gq receptor agonists e.g. CCh: 0.47±0.06 OD450nm, n=4 (FR) vs 2.12±0.3, n=3 (vehicle), p<0.05.

These results show that the pan-Gq inhibitor prevents airway hyperresponsiveness and airway remodeling in a

chronic asthma mouse model and inhibits mucus secretion. Our data demonstrate that blocking of Gq signaling is a

promising strategy for the treatment of airway hyperresponsiveness and remodeling.


of 516

B 07-03

Calcium-mediated proteolysis of cilia-associated androglobin

Teng Wei Koay, David Hoogewijs

University of Fribourg, Section of Medicine, Department of Endocrinology, Metabolism and Cardiovascular System,

Fribourg, Switzerland



of 516

B 07-04

Analysis of growth and radiation sensitivity of HIF-1α deficient cultured

murine hepatocytes

Akram Hamidi, Alexandra Wolf, Rositsa Dueva, Melani Kaufmann, Kirsten Göpelt, Eric Metzen


Hypoxia-inducible factor (HIF) is the main cellular transcription factor to regulate adaptation to hypoxia. HIF-1 is

composed of a constitutively expressed β-subunit and an α-subunit which is degraded in the presence of oxygen.

HIF-1 supports tumor growth by adaptation of cell metabolism and stimulation of angiogenesis. HIF-1 expression in

tumor cells is known to mediate resistance to ionizing radiation (IR) in vitro. The aim of this study was to establish

cultured murine hepatocyte derived cells (mHDC) as an in vitro model. In mHDC, we analyzed the role of HIF-1 in

the regulation of apoptosis induction and in oxidative metabolism. We have generated genetically modified mice that

bear a loxP flanked exon 2 of HIF-1α and tamoxifen-inducible Cre expression. We have established transformed

hepatocyte cultures from these mice, which are HIF-1 deficient after ex vivo tamoxifen treatment as shown by

Western blotting (WB). To investigate the effect of HIF-1α KO on apoptosis, WB was performed using PARP-1

antibodies. To confirm these results, pro-apoptotic caspase-3 activity was analysed. We also investigated the impact

of HIF-1α deletion on cellular respiration. To this end, we measured the oxygen consumption rate (OCR) and

extracellular acidification rate (ECAR). To investigate the effect of HIF-1α KO on IR-induced DNA damage, the

kinetics of γH2AX induction were monitored by WB and immunofluorescent staining. Assessment of mRNA levels of

HIF-1α target genes showed that all target genes analyzed were downregulated in KO cells in hypoxia. Elevated

apoptosis levels were demonstrated after irradiation of HIF-1α depleted mHDC. Neither of the HIF-deficient cell

systems was able to reduce oxygen consumption in response to hypoxia as control cells did, which indicates that

HIF-1 is necessary and sufficient for reducing mitochondrial oxygen consumption in hypoxia. Furthermore, HIF-1α

depletion in mHDC led to an increase of DNA damage after IR. The amount of IR-induced DNA damage decreased

after the first 6 hours showing that the surviving cells completed DNA repair. However, the significant increase of

DNA damage and the increase of apoptosis in the first 6 hours after IR pointed to a possible alteration of DNA repair

after HIF-1α depletion. Taken together, the KO of HIF-1α combined with irradiation in mHDC increased apoptosis

under hypoxic conditions and led to an increase of DNA damage after IR.

References [1] Moeller BJ, Cao Y, Li CY, Dewhirst MW. Cancer Cell 2004; 5:429-441 [2] Moeller BJ, Dreher MR, Rabbani ZN, Schroeder T, Cao Y, Li CY, Dewhirst MW.. Cancer cell 2005; 8:99-110


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B 07-05

Prolyl 4-hydroxylase domain 1-3 (PHD1-3) enzymatic activity leads to

oxygen-dependent likely covalent protein interactions in cells

Agnieszka E. Jucht1, Alexander von Kriegsheim2, Roland H. Wenger1, Carsten C. Scholz1

1 University of Zurich, Institute of Physiology, Zurich, Switzerland 2 University of Edinburgh, Institute of Genetics and Cancer, Edinburgh, UK

Limited oxygen availability (hypoxia) occurs in a range of (patho-)physiological conditions, including development,

exercise, ischemia, anaemia, stroke and inflammation. Cellular oxygen sensing is a crucial mechanism to sense

changes in local oxygen availability and to survive. The four cellular oxygen sensors prolyl-4-hydroxylase domain

(PHD) proteins 1-3 and the asparagine hydroxylase factor inhibiting HIF (FIH) confer oxygen sensitivity to the HIF

transcription factor. Under normoxic conditions, PHD1-3 hydroxylate two proline residues in HIF-α subunits, leading

to HIF-α proteasomal degradation. In hypoxia, the oxygen sensors are no longer active, HIF-α dimerises with the β

subunit, and enhances the transcription of hundreds of genes involved in hypoxia adaptation. Pharmacologic HIF

hydroxylase inhibitors (HIs) were recently approved for the treatment of renal anaemia in China and Japan. Preclinical

studies indicate that such inhibitors are also beneficial for the treatment of other hypoxia-associated diseases,

including ischemia, stroke and inflammation. But the underlying molecular mechanisms of the protective effect of the

HIs is largely unknown. The relevance of PHDs and FIH for oxygen (patho-)physiology independent of HIF is currently

unclear, but such regulations may contribute to the HI protective effect. We recently found that FIH forms an oxygen-

dependent stable (likely covalent) protein oligomer with the deubiquitinase OTUB1 (FIH-OTUB1). FIH-OTUB1

oxomer formation regulates OTUB1 enzymatic activity, representing a novel mode of oxygen-dependent cellular

signalling. It was previously reported that the prolyl-3-hydroxylase OGFOD1 forms a denaturing-conditions-resistant

protein complex with its substrate RPS23, indicating that such complex formation is not exclusive for the asparagine

hydroxylase FIH. Thus, we analysed if PHD1-3 also form such complexes. PHD1-3 were ectopically expressed in

the presence or absence of hypoxia. We observed several high molecular weight immunoblot signals that were

sensitive to PHD inhibition. Moreover, the high molecular weight signals were detected after immunoprecipitation of

denatured samples, indicating covalent interactions. Mass spectrometry analyses of native and denatured

interactomes of PHD1-3 identified several putative target proteins for stable protein complex formation. These results

provide evidence that PHD activity also targets proteins outside the HIF pathway.


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B 07-06

Regulation of PITX2 and ABCB1 in oncogenic multidrug resistance by

tumorigenic HIF2 in VHL-deficient renal cancer cells

Calista J. L. Ow1,2, Timm Schreiber1,3, Frank Thévenod1, Wing-Kee Lee1,4

1 Witten/ Herdecke University, Institute for Physiology, Pathophysiology and Toxicology, Witten, Germany 2 University of Manchester, Faculty of Biology, Medicine and Health, Manchester, UK 3 University of Duisburg-Essen, Institute for Physiology, Essen, Germany 4 Bielefeld University, Physiology and Pathophysiology of Cells and Membranes, Medical School OWL, Bielefeld,

Germany

The tumour suppressor von Hippel-Lindau (VHL) is involved in ubiquitination and subsequent proteasomal

degradation of hypoxia-inducible factors (HIFs). Under hypoxic conditions, HIFα- subunits are stabilized, leading to

dimerization with HIFβ and transactivation of genes for metabolism, survival and angiogenesis. Human renal cancer

cells A498 are VHL-deficient and harbor stabilized HIF2, whereas Caki-1 cells are VHL-intact and HIF1-dominant.

Previous studies demonstrated increased paired-like homeodomain transcription factor 2 (PITX2) and its target gene,

the ABCB1 drug transporter, in A498 cells. The relationship between VHL, HIF, PITX2 and ABCB1 remains unclear.

Transient transfection of A498 cells with HIF-1α or HIF-2α siRNA (25nM, 72h, >85% protein knockdown) attenuated

PITX2 protein by up to 75% by immunoblotting and PITX2 promoter activity by ~40%. Immunoprecipitation studies

did not propose direct HIF-PITX2 interaction. HIF-regulated PITX2 expression was proportional to ABCB1

expression, which was decreased at both mRNA (~20%) and protein (~70%) levels after HIF-1α or HIF-2α

knockdown. Furthermore, HIF/PITX2 target gene CCND1 was reduced by >60%, but PITX2 target LCN2 increased

>3-fold. HIF-2α siRNA was more effective than HIF-1α siRNA in altering PITX2 and its target genes. Conversely, in

Caki-1 cells, HIF siRNA augmented PITX2 and ABCB1 mRNA and protein by ~1.5- fold and ~2-fold, respectively,

and elevated CCND1 and LCN2 ~1.2-fold. These findings suggest PITX2 and ABCB1 are positively regulated by

tumorigenic HIF-2 in drug-resistant A498 cells whereas they are suppressed by HIF1 in more drug-sensitive Caki-1

cells. These data may provide better understanding of membrane transporters and possible upstream causes of

multidrug resistance in different renal cancers.

Acknowledgment C. J. L. Ow was supported by an Erasmus+ Traineeship.


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B 07-07

Oxomers - a new type of hypoxia sensing/signalling?

Carsten C. Scholz

University of Zurich, Institute of Physiology, Zürich, Switzerland

Cellular oxygen sensing is of vital importance for cells and tissues in order to adapt when cellular oxygen demand

exceeds its supply (hypoxia). The major cellular oxygen sensors are prolyl-4-hydroxylase domain (PHD) proteins 1-

3 and factor inhibiting HIF (FIH). PHDs and FIH hydroxylate hypoxia-inducible factor (HIF) α subunits, which reduce

HIF-α half-life (PHDs) or modulate HIF transactivation activity (FIH), respectively. The relevance of PHDs and FIH

for oxygen (patho-)physiology independent of HIF is currently unclear. We recently found that FIH forms an “oxomer”

– defined as an oxygen-dependent stable (likely covalent) protein oligomer – with the deubiquitinase OTUB1 (FIH-

OTUB1). FIH-OTUB1 oxomer formation was more sensitive to hypoxia than HIF-α hydroxylation and regulated

OTUB1 enzymatic activity. Interestingly, conditional Otub1 deletion in mice phenocopied Hif1an (the gene encoding

FIH) knockout (KO) mice, showing a metabolic and a respiratory phenotype. The phenotype of Hif1an KO mice

showed no clear overlap with classical functions of HIF. Thus, OTUB1 is currently the most likely physiological

relevant target protein of FIH. A mass spectrometry-based screen identified 12 additional putative FIH-dependent

oxomers, of which we now validated two. Moreover, it has previously been reported that the prolyl-3-hydroxylase

OGFOD1 forms a denaturing conditions-resistant protein complex with its substrate RPS23, depending on OGFOD1

enzymatic activity. This indicated that oxomer formation might not be restricted to the asparagine hydroxylase FIH.

Using immunoblotting and mass spectrometry, we analysed the potential of PHD1-3 to form oxomers and identified

several complexes and potential target proteins. PHD1-3 oxomer formation was prevented in hypoxia and by

treatment with the hydroxylase inhibitor desferrioxamine, demonstrating that oxomer formation was dependent on

PHD1-3 catalytic activity. In summary, oxomer formation is not restricted to a single enzyme or substrate, but all

major cellular oxygen sensors catalyse oxomer formation. We therefore hypothesise that oxomer formation

represents a novel mode of hypoxia sensing and signalling.


of 516

B 07-08

HIF-1 and p53 – friends or foes in the inflammatory colorectal

carcinoma?

Stefanie Saoub, Joachim Fandrey, Anna Wrobeln


The colorectal carcinoma belongs to the most common cancers in developed countries. The prevalence for chronic

inflammatory colorectal disease constitutes approximately 0.5 - 0.7 % in Germany1. These patients show a 2- to 6-

fold higher risk of developing colorectal carcinomas (CRC)2. CRCs have been associated with mutations in the

tumour suppressor p53 and often develop hypoxic areas. p53 plays a key role for cell cycle arrest and apoptosis and

is mutated in approximately half of human cancers3. Interestingly, invasive and metastasizing tumour growth strongly

depends on hypoxia-inducible factors (HIF)4. Several studies have now provided evidence for crosstalk between HIF-

1α and p53, although the exact interaction between HIF-1α and p53 remains controversial5.

In this study, we investigate the interaction between HIF-1α and p53 in human colorectal carcinoma cells (HCT116).

By performing quantitative PCRs and Western Blotting, we compared the transcriptional and the translational activity

of both HIF-1α and p53 as well as their specific target genes (HIF-1α, HIF-2α, p53, Actin Beta, MDM2, BAX, P21,

NOXA, VEGF). We incubated the cells either under normoxic (20 % O2) or hypoxic (1 % or 0.1 % O2) conditions for

4 or 24 hours. Our first results indicate that the p53 protein accumulates under severe hypoxia (0.1 % O2) after 24

hours of incubation. This result is supported by the observation that the p53 targets BAX and P21 are exclusively

upregulated after 24 hours of hypoxia at 0.1 % O2. In contrast, HIF-1α expression is downregulated after 4 hours of

hypoxia at 0.1 % O2 but also after 24 hours of hypoxia at 1 % as well as at 0.1 %. HIF-1α protein is increased after 4

hours of hypoxia at 1 % and 0.1 % O2. After 24 hours of hypoxia at 1 % O2 HIF-1α protein returns to normoxic levels

while the protein remains high after 24 hours at 0.1 % O2.This is also reflected by expression of the HIF-target gene

VEGF.

These results indicate that in HCT116 cells HIF-1α and p53 might interact under severe hypoxic conditions (0.1 %

O2) which mimic the tumour microenvironment of colorectal tumours. Further analysis of the interaction between HIF-

1α and p53 in HCT116 cells under inflammatory conditions as in the tumour microenvironment will contribute to better

understanding the molecular processes involved in the development of inflammatory colorectal carcinoma.

References [1] Hoffmann, J. C., Klump, B., Kroesen, A., Siegmund, B.: Chronisch-entzündliche

Darmerkrankungen, 3. Auflage, Springer, Berlin, Heidelberg 2020. [2] Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet. 2001 Feb

17;357(9255):539-45 [3] Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997 Feb 7;88(3):323-31. [4] Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003 Oct;3(10):721-32. [5] Amelio I, Melino G. The p53 family and the hypoxia-inducible factors (HIFs): determinants of

cancer progression. Trends Biochem Sci. 2015 Aug;40(8):425-34.


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B 07-09

The connection between the hypoxia-inducible factor 2 and primary

cilia in neuronal regeneration

Tristan Leu, Jannik Denda, Joachim Fandrey


Communication between cells plays an important role in development and regeneration. If this is disturbed, it leads

to serious effects not only on individual cells, but on the organism as a whole. A possible cause for such a disrupted

communication is an oxygen deficiency. Ischaemia with oxygen deficiency in the brain is often due to stroke. This

usually results in permanent damage to neuronal cells and thus entire brain areas due to limited regeneration. An

essential player in cell communication are primary cilia. These apical cell surface organelles play a crucial role in

signal transduction in mostly all eukaryotic cells and thus influence cell growth, migration and differentiation also in

the central nervous system. If the development of primary cilia is disturbed, thereby affecting a structural change or

function of the cilia, it can cause various diseases dealing with functional and developmental disturbances of the

brain. Recent studies have shown that the transcription factor HIF-2α is located in and at the primary cilium [1]. The

main function of HIF-2 is to regulate the oxygen saturation in the cells, but we recently showed that HIF-2 is also

involved in differentiation of new neurons after ischemia in vitro [2]. However, the function of HIF-2α in relation to

primary cilia is unknown.In this study, we investigate the role of HIF-2 on signal transmission of primary cilia under

normoxic and hypoxic conditions in wild type and Hif-2α-knockout cells. For this purpose, we determined the

localization of the HIF-2α protein at and inside primary cilia by immunhistochemistry and verified the signalling

pathways involved in ciliary communication by qPCR analyses of specific target genes. We now report a connection

between HIF-2α in cilia and the mTor, Wnt, Notch and Hippo signalling pathway. Considering the important role of

primary cilia in cell communication and thus also in cell regeneration, the aim of this work is to determine the possible

functions of HIF-2 in primary cilia and the effect of HIF-2 on known signalling pathways to identify a role of cilia and

HIF-2 in neuronal regeneration.

References [1] Wann AK, Thompson CL, Chapple JP, Knight MM (2013) Interleukin-1beta sequesters hypoxia inducible factor

2alpha to the primary cilium. Cilia 2:17. doi:10.1186/2046-2530-2-17 [2] Leu T, Fandrey J, Schreiber T (2021) (H)IF applicable: Promotion of neurogenesis by induced HIF 2 signaling

after ischemia. Pflugers Arch. (in revision)


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B 07-10

The oxygen sensor prolyl-4-hydroxylase 2 (PHD2) negatively impacts

spheroid growth of MCF-7 cells and epithelial to mesenchymal

transition

Friederike K. Kosyna, Johanna-Theres Borutta, Mareike Otto, Reinhard Depping

University of Luebeck, Institute of Physiology University of Lübeck and Center for Structural and Cell Biology in

Medicine, Lübeck, Germany

Hypoxia evolves due to an imbalance between oxygen availability and consumption and is a characteristic feature of

solid tumors. Therefore, hypoxia-regulated pathways play a major role in the regulation of cancer cell metabolism.

The heterodimeric transcription factor hypoxia-inducible factor (HIF) consists of an oxygen-labile α-subunit and a

constitutive β-subunit and is a master regulator of the cellular adaptation to hypoxia. HIF specific target genes are

involved in the activation of numerous cellular processes including cell survival, glycolytic energy metabolism,

erythropoiesis, vascular remodeling and angiogenesis. The prolyl-4-hydroxylase PHD2 appears to be the main HIF-

1α regulator implying PHD2 may have a regulatory role in the pathogenesis of cancer. However, the role of PHD2

expression in tumor cells is highly diverse and cell type specific. In tumor research, three-dimensional (3D) tumor

spheroid culture models display in vivo microenvironmental properties of cultured tumor cells ex vivo. 3D tumor

spheroids grow with zones of cellular heterogeneity providing gradients of nutrients and oxygen.

In this study, we set out to further evaluate the relationship between PHD2 expression and tumor development in

two-dimensional (2D) and 3D cell culture models of MCF-7 breast cancer cells. Using the CRISPR/CAS9 approach,

we generated a stable PHD2 knockout MCF-7 cell line. Cell viability and cytotoxicity assays revealed that knockout

of PHD2 negatively impacts cellular proliferation of MCF-7 cells without affecting cell viability. By HIF-dependent

reporter gene studies and qPCR analysis we demonstrated that PHD2 knockout inhibits the HIF-dependent hypoxia

response pathway. PHD2 knockout cells formed tight spheroids that were larger than MCF7 wild-type spheroids.

Finally, we showed that the epithelial to mesenchymal transition (EMT) is affected by PHD2 knockout. Taken

together, we provide further insights into the functional consequences of an inhibition of PHD2 expression in MCF-7

breast cancer cells demonstrating the critical role of PHD2 in the regulation of tumor growth.


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Foyers

B 08 | Blood Chair

Dörthe Katschinski (Göttingen)

Kristina Kusche-Vihrog (Lübeck)


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B 08-01

Genetic suppressor element (Gse) 1 in megakaryopoiesis and platelet

production

Timo Frömel1,4, Zumer Naeem1,4, Weijia Yu3, Rüdiger Popp1,4, Anastasia Kyselova1,4, Bernhard Brüne2,

Clara Türk10,9, Stefan Günther6, Andreas Weigert2, Mario Looso7, Marcus Krüger10,9, Michael Rieger3,8,5,

Ingrid Fleming1,4

1 Goethe University, Institute for Vascular Signalling, Frankfurt, Germany 2 Goethe University, Institute for Biochemistry I, Frankfurt, Germany 3 Goethe University, Hematology/Oncology, Frankfurt, Germany 4 German Centre for Cardiovascular Research (DZHK) partner site RheinMain, Frankfurt, Germany 5 Frankfurt Cancer Institute, Frankfurt, Germany 6 Max-Planck-Institute for Heart and Lung Research, Bioinformatics and Deep Sequencing Platform, Bad Nauheim,

Germany 7 Max-Planck-Institute for Heart and Lung Research, Bioinformatics Core Unit (BCU), Bad Nauheim, Germany 8 German Cancer Consortium (DKTK) and German Cancer Research Center, Heidelberg, Germany 9 University of Cologne, Center for Molecular Medicine Cologne (CMMC), Cologne, Germany 10 University of Cologne, CECAD Research Centre Institute for Genetics, Cologne, Germany

Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, plays an important role in the

regulation of physiological and pathophysiological hematopoiesis, especially megakaryopoiesis. LSD1 is known to

have a role in leukemia and myeloid differentiation as well as in hematopoietic progenitor and stem cell function.

Functionally, LSD1 demethylates both Lys-4 (H3K4me) as well as Lys-9 (H3K9me) of histone H3 meaning that

depending on the cellular context it can act as either a coactivator or corepressor. The demethylation of H3K4me is

facilitated by the formation of a complex consisting of Rcor1, Rcor2, Rcor3, HDAC1 and HDAC2. Here, we describe

an additional partner in this repressive complex i.e. genetic suppressor element (GSE) 1; a potential intrinsically

disordered protein. Using co-immunoprecipitation approach GSE1 was shown to interact with LSD1 and to map the

site of interaction using a peptide array. Given the role of LSD1 in megakaryopoiesis, a potential link between GSE1

and the differentiation and maturation of megakaryocytes was assessed using a PF4-Cre x Gse1fl/fl mouse model.

The megakaryocyte specific knockout of GSE1 resulted in a significant increased number of vWF/CD41 positive

megakaryocytes in the spleen as well as the bone marrow, whereas the platelet numbers in the peripheral blood

were decreased. Moreover in-vitro experiment revealed a decreased proplatelet formation as well as nuclear

abnormalities in the megakaryocytes derived from Gse1 deficient animals. Downstream signalling analysis using

RNA-Seq and proteomics revealed a modulation of several Gfi1b/LSD1 downstream targets, affecting for example

cytoskeleton remodelling. Taken together these data indicate that GSE1 and its downstream products seems to be

important in the physiological megakaryopoiesis.


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B 08-02

Impact and long-term compatibility of albumin-derived

perfluorocarbon-based artificial oxygen carriers on regenerative

capacity of small animals models, after high blood loss

Shah Bahrullah Shah1,2, Katja Bettina Ferenz1

1 Universität klinikum Essen (University of Duisburg-Essen), Institute of physiology, Essen, Germany 2 Erwin L. Hahn Institute for Magnetic resonance imaging ( University of Duisburg-Essen), Essen, Germany

Albumin-derived perfluorocarbon-based artificial oxygen carriers (A-AOCs) mimic red blood cells carrying oxygen to

the organs. These A-AOCs can be an alternative to allogenic blood by solving the problems related to e.g. donor

availability, effort of screening for infections, transportation or storage & immune-modulation. Because A-AOCs

already showed promising results in isolated organs and short-term in vivo studies in rats[1-3], this study investigated

their long-term compatibility up to two weeks in a clinically relevant setting after moderate normovolemic

hemodilution.

For normovolemic hemodilution, rats were anesthetized & the hematocrit level was reduced stepwise from the normal

45.5% to 18% in 5 dilution steps by using either A-AOCs in plasma-like carrier solution or pure plasma-like solution

(control group) for volume replacement, respectively. Reaching 18% hematocrit level, animals survived for either 0,

7 or 14 days. Blood samples from the animals were collected with each dilution stept at regular intervals during follow-

up and analyzed for blood gases, acid base status, blood count and met-hemoglobin formation. At the end of the

observation period, blood & organs were collected to measure tissue alterations (e.g. hematoxylin-eosin staining,

periodic acid–Schiff staining) and bio-distribution of A-AOCs.

All animals treated with A-AOCs survived throughout the designated survival period according to their group. The

hematocrit level of 18% already doubled within 7-days in both, control and A-AOCs-treated animals. Similarly, the

reticulocyte count in these groups already increased as an early sign of hematocrit recovery. The met-hemoglobin

accumulation, lactate and potassium levels as well as blood glucose remained in the normal physiological range

during hemodilution and follow-up in all groups (0, 7 & 14 days). As expected, A-AOCs accumulated in the spleen,

whereas all other organs remained unaffected by A-AOC treatment.

For the first time, this study demonstrated long-term compatibility of A-AOCs in a clinically relevant animal setting. A-

AOCs were well tolerated and did not affect erythropoiesis in animals recovering from moderate anemia caused by

normovolemic hemodilution up to 2 weeks. To track bio-distribution and organ retention of A-AOCs and thus confirm

histological results, combined 19F/1H MRI using a specialized rat bird-cage just started in cooperation with the E. L.

Hahn Institute, University of Duisburg-Essen.

References [1] A. Wrobeln et al., “Albumin-derived perfluorocarbon-based artificial oxygen carriers: A physico-chemical

characterization and first in vivo evaluation of biocompatibility,” Eur. J. Pharm. Biopharm., vol. 115, pp. 52–64, 2017, doi: 10.1016/j.ejpb.2017.02.015.

[2] A. Wrobeln et al., “Functionality of albumin-derived perfluorocarbon-based artificial oxygen carriers in the Langendorff-heart,” Artif. Cells, Nanomedicine Biotechnol., 2017, doi: 10.1080/21691401.2017.1284858.

[3] A. Wrobeln et al., “Albumin-derived perfluorocarbon-based artificial oxygen carriers can avoid hypoxic tissue damage in massive hemodilution,” Sci. Rep., vol. 10, no. 1, 2020, doi: 10.1038/s41598-020-68701-z.


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B 08-03

Antioxidative properties of the phytoalexin trans-Resveratrol

experience a boost through interactions with albumin-derived artificial

oxygen

Ozan Karaman1,2, Michael Kirsch2, Katja B. Ferenz1,3

1 University Hospital Essen, Insitute of Physiology, Essen, Germany 2 University Hospital Essen, Institute of Biochemistry, Essen, Germany 3 University of Duisburg-Essen, Center for Nanointegration Duisburg-Essen, Duisburg, Germany

Trans-resveratrol (tRES) exhibits a remarkable anti-oxidative and –inflammatory capacity. Unfortunately, low

solubility in aqueous solutions and high metabolization rate in vivo impair the therapeutic potential of tRES in

biomedical setups. Local hyperoxygenation leads to the formation of reactive oxygen species (ROS) and subsequent

harmful oxidative processes are described as a typical side effect of artificial oxygen carriers (AOCs). To (I) increase

tRES stability and to (II) counteract negative effects of local hyperoxygenation potentially caused by AOCs, tRES

was coupled to albumin-derived AOCs (A-AOCs) comprised of an bovine serum albumin (BSA) shell encapsulating

a perfluorodecalin core [1] forming tRES-loaded A-AOCs (RESLOCs).

Potential interactions (binding and release profile) between tRES and the BSA shell of the A-AOCs were examined

by mixing 1000 µL of 17 vol% A-AOCs with an aqueous 0.05 mg/mL tRES solution at 20°C shielded from light.

Subsequently, free tRES was determined photometrically at 304 nm.

To demonstrate that tRES still effectively acts as an antioxidative compound when bound to A-AOCs, radical

scavenging capacity was tested in two different approaches of an ABTS•+ decolorization assay, one being based on

an enzyme cascade comprised of glucoseoxidase and horseradishperoxidase while the other inspired by Re et al.

[2] utilized potassium persulfate for radical generation. RESLOCs were exposed to ABTS•+ containing solutions at

20°C. Different time points (up to 120 minutes) were utilized to detect remaining ABTS•+ in presence of RESLOCs

photometrically at 734 nm.

A-AOCs albumin shell successfully interacts and binds tRES. Aditionally, partial release of bond tRES from the A-

AOCs surface could be observed. Utilization of RESLOCs in both approaches of the ABTS•+ decolorization assay

resulted in a drastic and significant extinction of ABTS•+ absorption, even when the radical was present in high excess.

When compared to aqueous solutions of free t-RES, free albumin or a combination of both, the radical scavenging

effect of RESLOCs exceeded these controls by more than a hundredfold.

Conducted experiments delivered first insights about the anti-oxidative potential of tRES as additive in A-AOCs. The

observations unequivocally showed that the formed RESLOCs considerable increase anti-oxidative properties of

tRES.

References [1] Wrobeln, A., Laudien, J., Groß-Heitfeld, C., Linders, J., Mayer, C., Wilde, B., ... & Ferenz, K. B. (2017).

Albumin-derived perfluorocarbon-based artificial oxygen carriers: A physico-chemical characterization and first in vivo evaluation of biocompatibility. European journal of pharmaceutics and biopharmaceutics, 115, 52-64

[2] Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free radical biology and medicine, 26(9-10), 1231-1237.


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B 08-04

The role of carbon flux from cysteine in the regulation of stemness.

Janina Wittig1,2, Linda Kessler3, Maria Kyriaki Drekolia1,2, Ilka Wittig4,2, Sven Zukunft1,2, Jiong Hu1,2,

Sebastian Kempf1,2, Daqiang Pan1, Stefan Günther5, Mario Looso6, Matthias Leisegang7,2,

Dominik Fuhrmann8, Andreas Weigert8, Wolfram-Hubertus Zimmermann9,10, Gergana Dobreva3,

Sofia-Iris Bibli1,2, Ingrid Fleming1,2

1 Goethe University, Institute for Vascular Signalling, Centre for Molecular Medicine, Frankfurt, Germany 2 German Center of Cardiovascular Research (DZHK), Partner Site Rhein-Main, Frankfurt, Germany 3 Medical Faculty Mannheim, Heidelberg University, Anatomy and Developmental Biology, Mannheim, Germany 4 Goethe University, Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Frankfurt, Germany 5 Max Planck Institute for Heart and Lung Research, Deep Sequencing Platform, Bad Nauheim, Germany 6 Max Planck Institute for Heart and Lung Research, IT Service Group, Bad Nauheim, Germany 7 Goethe University, Institute for Cardiovascular Physiology, Centre for Physiology, Frankfurt, Germany 8 Goethe University, Institute of Biochemistry I, Faculty of Medicine, Frankfurt, Germany 9 University Medical Center, Georg-August-University, Institute of Pharmacology, Heart Research Center,

Göttingen, Germany 10 German Center of Cardiovascular Research (DZHK), Partner Site Göttingen, Göttingen, Germany

Question: Exit from pluripotency has been linked with metabolic reprogramming, but the role of metabolism in the

preservation of stemness has not been addressed. The aim of this study was to use multi-omic approaches to

investigate how metabolic alterations regulate stemness and exit from pluripotency.

Methods and Results: Targeted metabolomics in human induced pluripotent stem cells (iPSCs) and after

differentiation into all three germ layers revealed that pluripotency is characterized by enhanced catabolism of the

cysteine metabolites. Unbiased 13C-metabolic and -proteomic flux analyses were performed for cysteine in iPSCs

and after differentiation into all three germ layers. This reveled that carbons from cysteine preferentially acetylate

histone 2B lysines to preserve chromatin accessibility for pluripotency-related transcription. To assess the role of

cysteine metabolism in the preservation of pluripotency, iPSCs were cultured in media either containing only cysteine

as source of energy (TgCys) or depleted of cysteine (ΔCys). ΔCys medium reduced proliferation and led to ferroptotic

cell death, a process characterized by loss of the reduced glutathione equivalents. Ferrostatin, an inhibitor of

ferroptosis, improved survival of the ΔCys-treated iPSCs but failed to preserve pluripotency. Rather it was driving the

differentiation of iPSCs towards the mesodermal lineage as indicated by increased transcription (RNA seqencing)

and translation (FACS analysis) of eomesodermin and homeobox protein MIXL1. In contrast, the TgCys approach

maintained pluripotency even in the absence of other key metabolites such as glucose, glutamine, pyruvate and fatty

acids. RNA sequencing revealed that stemness-related transcripts NANOG and octamer-binding transcription factor

4 were preserved in TgCys-treated iPSCs. Also, Assay for Transposase-Accessible Chromatin sequencing revealed

that in the TgCys condition chromosome regions responsible for the preservation of pluripotency remained

accessible. In mice the simultaneous genetic deletion of two enzymes responsible for cysteine metabolic flux to

acetyl-coA (i.e. cystathionine-γ-lyase and -β-synthase) led to developmental failure in murine embryos.Conclusion:

These observations suggest that cysteine metabolism maintains stemness through the inhibition of ferroptosis and

the preservation of histone marks responsible for pluripotency-related chromatin accessibility and transcription.


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B 08-05

CCR3-dependent eosinophil recruitment is regulated by

sialyltransferase ST3Gal-IV

Wang Liu1, Ina Rohwedder1, Omar E. Bounkari2, Caspar Ohnmacht3, David Frommhold4,

Jürgen Bernhagen2, Jamey D. Marth5, Markus Sperandio1

1 Ludwig-Maximilians-Universität München, Institute of Cardiovascular Physiology and Pathophysiology, Walter

Brendel Center of Experimental Medicine, Biomedical Center(BMC), München, Germany 2 Ludwig-Maximilians-Universität München, Institute for Stroke and Dementia Research (ISD), München, Germany 3 Helmholtz Center München, Institute of Allergy Research, München, Germany 4 Kinderklinik Memmingen, München, Germany 5 University of California, Center of Nanomedicine, Sanford Burnham Medical Research Institute, Santa Barbara,

USA

Questions: Eosinophil recruitment is a classical hallmark of many allergic and helminthic diseases. Recruitment of

eosinophils is highly dependent on binding of chemokine CCL11 (Eotaxin1) to its receptor CCR3, which subsequently

triggers α4β1 integrin activation and adhesion to the endothelial lining. Our group could previously identify the α2,3

sialyltransferase ST3Gal-IV to be essential for CXCR2-triggered firm neutrophil arrest, but so far the role of α2,3-

sialyation in the CCL11/CCR3 axis has not been investigated.

Methods & Results: Using St3gal4-deficient mice, we set out to investigate ST3Gal-IV-dependent eosinophil

recruitment in the mouse cremaster muscle 4h after intrascrotal injection of CCL11. We analyzed eosinophil

extravasation by whole mount Giemsa staining and found reduced numbers of extravasated eosinophils in St3gal4-

deficient mice. To study the functionality of the CCR3 receptor in the absence of ST3Gal-IV, we analyzed CCL11

binding to CCR3 and observed reduced CCL11 binding capacity of St3gal4-deficient eosinophils. Consequentially

also the internalization of CCR3 following CCL11 ligation, an important step to fully activate eosinophil effector

functions, was diminished in these cells. To verify that absence of ST3Gal-IV is indeed resulting in reduced sialyation

of CCR3, we finally performed lectin-pulldown assays. While in wildtype cells, we were able to pull down CCR3

together with WGA (wheat germ agglutinin lectin from Triticum vulgare), this was strongly reduced in St3gal4-deficient

eosinophils.

Conclusions: Taken together, our results demonstrate an important role of ST3Gal-IV and thus α2,3-sialyation in

CCR3-induced eosinophil recruitment.

Acknowledgment Supported by Mizutani Foundation (Grant Ref. Nr. 090063).


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B 08-06

A hierarchical regulatory network analysis of the vitamin D induced

transcriptome reveals novel regulators and complete VDR dependency

in monocytes

Timothy Warwick1,3, Marcel H. Schulz2,3, Stefan Günther4, Ralf Gilsbach1,3, Carsten Carlberg5,

Antonio Neme6,5, Ralf P. Brandes1,3, Sabine Seuter1,3,5

1 Goethe Universität, Institute for Cardiovascular Physiology, Frankfurt am Main, Germany 2 Goethe Universität, Institute for Cardiovascular Regeneration, Frankfurt am Main, Germany 3 German Center for Cardiovascular Research (DZHK), Partner sire Rhein-Main, Frankfurt am Main, Germany 4 Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany 5 University of Eastern Finland, Institute of Biomedicine, Kuopio, Finland 6 National Autonomous University of Mexico, Institute for Applied Mathematics, Sierra Papacal Merida, Mexico

Question: Can the complex transcriptional response of monocytic cells to 1α,25-dihydroxyvitamin D3

(1,25D) treatmentbe elucidated by predicting gene targets of transcription factors (TFs) downstream of the vitamin D

receptor, and are downstream factors important to the modulation of cardiovascular disease risk by vitamin D?

Background: 1,25D deficiency has been associated with adverse cardiovascular outcomes in patients, and as such

has been researched heavily in the context of cardiovascular disease, including atherosclerosis. Monocytes are

heavily involved in atheroma progression, and treatment of monocytic cells with 1,25D provokes a widespread and

extended transcriptomic response, which could subsequently modulate the development of cardiovascular disease.

1,25D mediates its transcriptomic effects via its corresponding nuclear receptor, vitamin D receptor (VDR). A network

of TFs downstream of VDR must also act to alter gene expression of 1,25D-target genes. However, it is unclear

which TFs are involved, which genes they regulate, and whether they are important to cardiovascular disease.

Methods: The TEPIC workflow was used to leverage RNA sequencing, FAIRE sequencing, and VDR ChIP-

sequencing data to predict TF binding at differentially expressed genes following 1,25D treatment. TFs predicted to

be bound by VDR were assigned to differentially expressed genes, and a TF network centred on VDR was created

and validated using sequencing data.

Results: VDR target genes were identified in THP-1 cells at both early (2.5h, 4h) and late (24h) time points after

1,25D treatment. Among these were 47 TFs whose expression was upregulated following 1,25D treatment. These

TFs were subset into early and late targets of VDR, and gene targets for each TF were identified by TF binding site

prediction. A TF network was constructed and the gene targets of CEBPA and ETS1 after 1,25D treatment were

validated using ChIP sequencing data. Pathways related to modulation of inflammation were significantly

overrepresented among the gene targets of ETS1, which included many inflammatory cytokines and chemokines,

indicating the importance of downstream TFs in modulating inflammation in the cardiovascular system. Genomic

knockout of VDR completely ablated the transcriptional response to 1,25D in THP-1 cells.

Conclusion: There exists a complex network of TFs downstream of VDR which are required to facilitate the

transcriptional response of THP-1 cells to 1,25D treatment.


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B 08-07

Effect of secreted modular calcium binding protein 1in soluble epoxide

hydrolase on macrophage polarization

Urun Ukan1,2, Xiaoming Li1,2, Fredy Delgado Lagos1,2, Ingrid Fleming1,2

1 Goethe Universität, Institut für Vascular Signalling, Frankfurt am Main, Germany 2 German Centre for Cardiovascular Research, Partner Site Rhine-Main, Frankfurt am Main, Germany

Question: Monocyte-derived macrophages are plastic cells and can modulate the inflammatory process via

polarization to classical or alternative phenotypes. TGF-β plays a critical role in the process of inflammation

resolution, and drives macrophage polarization to alternatively activated M2c phenotype. Polyunsaturated fatty acids

play a role in macrophage activation and this study set out to assess how the expression of the soluble epoxide

hydrolase (sEH) pathway was affected by macrophage polarization.

Methods and Results: The sEH was expressed in human and murine macrophages and unaffected by polarization

(with LPS and IFN-γ) to the classically activated (M1) phenotype or to the alternatively activated (M2a) phenotype

with IL-4. However, the repolarization of M1 macrophages to the pro-resolving M2c phenotype with TGF-β1 resulted

in a clear increase in sEH expression. The TGF-β-induced increase in sEH expression was preceded by the

phosphorylation of SMAD2 and was attenuated by the ALK5 antagonist; SD208. The activity of ALK5 can be

modulated by the binding of secreted modular calcium binding protein 1 (SMOC1), and in SMOC1-defient (SMOC1+/-

mice) TGF-β increase the expression of sEH (mRNA and protein). M2c macrophages from SMOC1+/- mice also

demonstrated a deficient M2c polarization that was associated with increased phagocytosis of zymosan particles.

This phenotype could be rescued by the addition of recombinant SMOC1 prior to the addition of TGF-β1. Failure to

upregulate sEH expression also resulted in a failure in M2c polarization and was linked with an altered fatty acid

profile.

Conclusions: In conclusion, TGF-β1 can regulate the expression of sEH via an ALK5 and SMOC1-dependent

mechanism.


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B 08-08

Characterization of glucose-crosslinked albumin‐derived artificial

oxygen carriers

Jan-Eric Sydow1, Jürgen Linders2,3, Christian Mayer2,3, Katja B. Ferenz1,3

1 University Hospital Essen, University of Duisburg-Essen, Institute of Physiology, Essen, Germany 2 University of Duisburg-Essen, Institute of Physical Chemistry, Essen, Germany 3 University of Duisburg-Essen, CeNIDE, Essen, Germany

Further development of alternatives for blood supplies is needed. Perfluorodecalin (PFD)-based nanocapsules with

an albumin shell showed relevant oxygen transport capacity.[1-3] In this study, the albumin shell of these PFD-based

oxygen carriers was modified with glucose to enhance dispersion stability in aqueous, crystalloid carrier solution

resulting in glucose-crosslinked albumin‐derived artificial oxygen carriers.

These oxygen carriers were synthesized via ultra-sonication of human serum albumin and PFD, followed by

introducing glucose through UV radiation and heating overnight according to the Maillard reaction. Their

hydrodynamic radius was measured by tracking their molecular motion utilizing dark-field microscopy. Atomic force

microscopy was used to investigate their surface properties and dimensions. All measurements were performed

directly after synthesis up to a period of one year.

Results showed spherically-shaped oxygen carriers that remained stable in shape and size (average radius of 100

nm) directly after synthesis up to a period of more than two weeks, while growing only slightly to an average radius

of 125 nm after one year of storage (Figure 1). Atomic force microscopy emphasized these results, revealing oxygen

carriers of similar shape and radius.

In conclusion, crosslinking the albumin shell with glucose resulted in durable and stable glucose-crosslinked

albumin‐derived artificial oxygen carriers with improved dispersion stability in crystalloid carrier solution as compared

to non-crosslinked oxygen carriers. Future experiments investigating oxygen transport capacity and blood cell

interactions will complement knowledge and highlight therapeutic relevance of this novel artificial oxygen carrier.

Fig. 1: Size distribution histograms Size distribution histograms of synthesized

oxygen carriers; dark‐field microscopy; (A) one

day after synthesis; (B) one year after

synthesis.

References [1] Wrobeln A, et. al. 2017, ‘Albumin-derived perfluorocarbon-based artificial oxygen carriers: A physico-

chemical characterization and first in vivo evaluation of biocompatibility’, Eur J Pharm Biopharm, 115, 52-64, Elsevier B. V.

[2] Wrobeln A, et. al. 2017, ‘Functionality of albumin-derived perfluorocarbonbased artificial oxygen carriers in the Langendorffheart’, Artif Cells Nanomed Biotechnol, 45(4), 723-730, Taylor & Francis.


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[3] Jägers J, et. al. 2021, ‘Perfluorocarbon-based oxygen carriers: from physics to physiology’, Pflügers Arch Eur J Physiol, 473, 139-50, Springer.


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B 08-09

Human hemoglobin and hematocrit values at varying altitude: evidence

for a precise oxygen sensing system

Max Gassmann

University of Zurich, Veterinary Physiology, Zurich, Switzerland

It is well accepted that living at and travelling to high altitude is a physiological challenge. While lowlanders have to

acclimatize to reduced oxygen supply (and cold) by increasing red blood cell production, highlanders living in different

regions of the world have developed various adaptive mechanisms to cope with these harsh conditions. Indeed, in

contrast to South American Highlanders (Quechua and Aymaras), Tibetans only moderately elevate their hematocrit

and hemoglobin level at high altitude. We recently asked the question if an elevation lower than 1500 m above sea

level shows an impact on Hb levels in men. To answer this question, we analyzed blood from 70,000 Swiss men

aged 18-22 years and observed a significant increase of Hb values for every 300 meters of augmented altitude. Our

data provides convincing evidence that even altitudes below 1500 m must be considered when defining normal

hemoglobin and hematocrit values. Accordingly, the WHO is currently re-discussing their suggested correction

factors used to adjust hemoglobin at altitude that represent the basis to define anemia. We also speculate on why

such a precise oxygen sensing system has evolved.

References [1] Staub K., Häuseler M., Bender N., Morozova I., Eppenberger P., Panczak R., Zwahlen M., Schaer D.J.,

Maggiorini M., Ulrich S., Gassmann N.N., Muckenthaler M.U., Rühli F.J., Gassmann M. (2020). Hemoglobin concentration of young men at residential altitudes between 200 and 2000m mirrors Switzerland’s topography. Blood Feb 10. Comment by: Beall C.M., Blood: Hemoglobin, altitude, and sensitive Swiss men. Blood (2020) 135(13): 984-985. doi: 10.1182/blood.2020005251.

[2] Gassmann M., Mairbäurl H., Livshits L., Seide S., Hackbusch M., Malczyk M., Kraut S., Gassmann N.N., Weissmann N., Muckenthaler M.U. (2019). The increase in hemoglobin concentration with altitude varies among human populations. Ann N Y Acad Sci Aug;1450(1):204-220.

[3] Gassmann M., Cowburn A., Gu H., Li J., Rodriguez M., Babicheva A., Jain P.P., Xiong M., Gassmann N.N., Yuan J.X., Wilkins M.R., Zhao L. (2021). Hypoxia-induced pulmonary hypertension-Utilizing experiments of nature. Br J Pharmacol. 2020 May 28. doi: 10.1111/bph.15144. 2021 Jan;178(1): 121-131.

[4] Muckenthaler M.U., Mairbäurl H., Gassmann M. (2020). Iron metabolism in high-altitude residents. J Appl Physiol (1985) 2020 Oct 1;129(4):920-925.

[5] Gassmann M. and Muckenthaler M.U. (2015). Adaptation of iron requirement to hypoxic conditions at high altitude. J Appl Phys; Dec 15; 119(12): 1432-1440.


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B 08-10

The role of the secreted modular calcium binding protein 1 in platelet

function

Fredy Delgado Lagos1,2, Anastasia Kyselova1,2, Voahanginirina Randriamboavonjy1,2,

Beate Fisslthaler1,2, Mauro Siragusa1,2, Ingrid Fleming1,2

1 Goethe-Universität, Institute for Vascular Signalling, Frankfurt am Main, Germany 2 German Centre for Cardiovascular Research, Partner Site Rhine-Main, Frankfurt am Main, Germany

Question: Secreted modular calcium binding protein 1 (SMOC1) is a matricellular protein essential for angiogenesis

that acts as an ALK5 antagonist in the TGF-β signalling. Although SMOC1 expression is known to be regulated by

platelet enriched microRNA-223 (miR-223), nothing is known about the expression or function of SMOC1 in platelets.

Here we report that SMOC1 is expressed in platelets from healthy humans and mice and that its expression is

increased platelets from diabetic patients, when miR-223 levels are decreased.

Methods and Results: To elucidate the function of SMOC1, functional assays were performed using platelets from

wild-type and SMOC1+/- mice (which express no detectable SMOC1 protein). In the absence of SMOC1, platelets

demonstrated an attenuated response to thrombin (Ca2+ increase, aggregation and β3 integrin phosphorylation),

while responses to other platelet agonists were unaffected. Although SMOC1 has been associated with TGF-β

signalling, no evidence was found that this pathway participated in the platelet dysfunction. Mutagenesis analysis

revealed that the Kazal domain of SMOC1 interacted with thrombin, enhancing its activity. These effects were

prevented by pre-incubation with a SMOC1 monoclonal antibody. Platelets from diabetic individuals and miR223-

deficient mice, which expressed higher levels of SMOC1, demonstrated hyper-responsiveness to thrombin that were

also reversed by antibodies directed against SMOC1.

Conclusions: In conclusion, SMOC1 has been identified as a novel thrombin-enhancing protein that potentiate its

activity and as a consequence regulates platelet function. The increased expression of SMOC1 in platelets from

patients with diabetes could partly account for platelet hyperreactivity making strategies that target SMOC1, or its

interaction with thrombin, attractive therapeutic approaches to normalize platelet function.


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Foyers

B 09 | RENAL II Chair

Pontus Persson (Berlin)

Markus Ritter (Salzburg)


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B 09-01

3D structure analysis of cleared human renal organoids derived from

induced pluripotent stem cells by laser scanning microscopy

Kathrin Groeneveld4, Jasmin Dilz1, Isabel Auge1, Ralf Schmauder2, Andreas Kurtz3, Ralf Mrowka1,4

1 Friedrich-Schiller-Universität, UKJ Exp. Nephrologie, Jena, Germany 2 Friedrich-Schiller-Universität, UKJ Physiologie II, Jena, Germany 3 Charité, Universitätsmedizin, Berlin, Germany 4 Friedrich-Schiller-Universität, Thimedop, Jena, Germany

Question: Our aim is to establish a human iPS model system to enable translational structural studies of kidney

tissue to study development, diseased stages, injury and toxicity effects.

Methods: The generation of renal organoids from iPS cells was based on a method developed and published by

Przepiorski et al from the Davidson lab in 2018. Renal typical gene expression was determined by qPCR. Histology

stainings were performed on fixed and non-fixed samples. We followed an adjusted clearing protocol by Klingberg et

al (2016) from the Gunzer lab. The organoids were first dehydrated during several washing steps with increasing

alcohol solutions (EtOh and 1-Propanol). For the final clearing step, organoids were transferred into ethyl-3-

phenylprp-2-enoate (ethyl cinnamate, ECi) or into CytoVista™ 3D Cell Culture Clearing Reagent (thermofisher).

Laser-scanning micrographs of cleared organoids (one-photon excitation) were compared to micrographs of native

organoids from both one- and two-photon excitation.

Results/Conclusion: We did successfully establish protocols to derive and clear renal organoids from induced

pluripotent stem cells, which show identifying features of renal structures in their optical appearance (such as tubular

structures) as well as renal typical gene expression. The clearing procedure led to an increased depth of light

penetration and to an increased acuity of the laser scans beneath the organoid surfaces. The depth of the optical

resolution did depend on the used excitation wavelength.

We predict that a standardized production and clearing of renal organoids will strongly enhance the possibilities for

structural studies in the field of physiology and nephrology.

Acknowledgment The project on which these results are based was funded by the Federal Ministry of Education and Research,

funding number 01EK1612B (µ-iPS Profiler).


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B 09-02

Peroxisomal proliferator-activated receptor gamma (PPARγ) in the

regulation of FGF23 production

Lisa Wolf, Michael Föller

University of Hohenheim, Department of Physiology, Stuttgart, Germany

Question: Fibroblast growth factor 23 (FGF23) is an endocrine and paracrine factor mainly synthesized in bone. It

is a pivotal regulator of renal phosphate and vitamin D metabolism along with αKlotho, a transmembrane protein

expressed in the kidney that serves as a co-receptor for FGF23. The FGF23 plasma concentration correlates with

progression and coutcome in chronic kidney disease and further cardiovascular disorders whereas αKlotho is a

powerful anti-aging factor. Insulin controls the synthesis of FGF23. PPARγ enconded by Pparg is a transcription

factor implicated in adipocyte function and insulin sensitivity. Glitazones are PPARγ agonists and were used as drugs

in the treatment of diabetes. Here, we studied the role of PPARγ for FGF23 production

Methods: Fgf23 gene expression was examined in UMR106 osteoblast-like cells by qRT-PCR. Pparg expression

was down-regulated by siRNA-mediated gene silencing.

Results: UMR106 cells expressed Pparg. PPARγ agonists piogliatazone and ciglitazone significantly enhanced

Fgf23 gene expression in a dose-dependent manner in UMR106 cells. SiRNA specific for Pparg, but not non-sense

siRNA abrogated the pioglitazone effect on Fgf23 in UMR106 cells. Moreover, the stimulatory effect of cigliatzone on

Fgf23 was signifiantly attenuated in the presence of PPARγ antagonist SR-202.

Conclusions: Glitazones are powerful stimulators of Fgf23 gene expression, an effect dependent on PPARγ. The

observed effect is likely to influence the cardiovascular safety of this class of antidiabetics.


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B 09-03

Role of renal acetylcholinesterase in the regulation of kidney function

Sabrina J. Habel, Philipp Tauber, Robert Götz, Anna Federlein, Katharina Krieger, Frank Schweda

Universität Regensburg, Biologie und Vorklinische Medizin, Physiologie II, Regensburg, Germany

Question: Proper kidney function requires communication between cells of different renal compartments such as

tubules and interstitial cells. The underlying intrarenal signaling cascades are only partially understood. Therefore,

this study aimed at identifying yet unrecognized communication signals of collecting ducts (CD).Since CDs are

centrally involved in urine concentration, mice received either water ad libitum or were subjected to water deprivation

for 24 hours (wd). CDs were isolated by microdissection and gene expression was determined using mRNA

microarrays (Affymetrix). Results were confirmed in independent experiments at the mRNA and protein level using

real time PCR (qPCR), in situ hybridization (ISH) and immunofluorescence staining. For functional analysis, renal

perfusion and urine flow was determined in the isolated perfused mouse kidney model (IPMK).

Results: Besides other mRNAs coding for signaling molecules or relevant enzymes, collecting ducts expressed

acetylcholinesterase (AChE) at high abundance already under control conditions (c) and AChE was markedly

upregulated by water deprivation (wd). qPCR confirmed upregulation of AChE in renal cortex, outer medulla and

isolated CDs while no regulation occurred in the inner medulla and in glomeruli. ISH showed clear and selective

AChE mRNA expression in CDs and glomeruli and confirmed upregulation of AChE in CDs. In contrast to wd, CDs

of mice with high urinary flow (loop diuretics) had low AChE mRNA expression levels. AChE protein was located

around CDs in the outer medulla while in the inner medulla it was distributed over the entire interstitium

(immunofluorescence). Since AChE inactivates acetylcholine (ACh) it might be involved in the regulation of ACh

signaling in the kidney. ACh concentration-dependently stimulated renal perfusion and urine flow in IPMKs. Inhibition

of AChE (donepezil hydrochloride) augmented the effects of ACh on kidney function, suggesting a functional role of

AChE in the kidney.Conclusion: AChE expression increases in cortical and outer medullary CDs in response to

water deprivation. AChE protein is secreted from collecting duct cells and distributes over the entire interstitium,

where it might degrade ACh and thereby regulate ACh signaling in the kidney. This hypothesis is underlined by the

fact that ACh stimulates renal perfusion and urine flow and these effects are augmented by pharmacological inhibition

of AChE.

Increased AChE mRNA expression in mouse kidney

tissue by water deprivation


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ISH (A) Outer medulla (green: AQP2, collecting

ducts, red: megalin, proximal tubule, pink:

NKCC2, loop of henle) and qPCR (B) results of

AChE mRNA expression.


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B 09-04

Mapping leaks in live epithelia

Jan F. Richter1, Ralf Schmauder2

1 Jena University Hospital, Institute of Anatomy II, Jena, Germany 2 Jena University Hospital, Institute of Physiology II, Jena, Germany

Compartimentalization of the body, as well as separation from the outside are maintained by multifactorial barriers.

An essential part are epithelial sheets that limit exchange of solutes, thereby regulating passage of smaller solutes

like ions. At the same time, epithelia are normally much less permeable for larger solutes. Understanding the

mechanisms of transepithelial passage of the larger, potentially antigenic solutes, is particularly important for

discrimination of physiological and pathological macromolecule permeation.

At present, the “leak pathway” across epithelial barriers cannot be evaluated in sufficient detail. To overcome this

limitation, we employed FRET-based live cell imaging of macromolecular tracers to achieve real-time monitoring of

barrier function at single-cell level. Using epithelial cells on transwells, we are able to detect when, where and for

how long such barrier defects to macromolecules occur. Basal macromolecule passage through these epithelial

sheets occurred within minutes and at only a few locations where cell junctions reorganized. Commonly used global

measures to define the leak pathway thus appear to average contributions from leaks that may actually originate

from only a few hotspots rather than reflecting the majority of cell junctions.

This novel approach is expected to facilitate understanding of basal barrier function in general and will allow to

delineate initial and causative biological processes in epithelial barrier pathologies.


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B 09-05

Acute adaptation to oral phosphate in the absence of Parathyroid

hormone (PTH)

Arezoo Daryadel1,3, Betül Haykir1, Milica Bugarski2,3, Carla Bettoni1, Udo Schnitzbauer1,

Nati Hernando1, Andrew Hall2,3, Carsten A. Wagner1,3

1 University of Zürich, Institute of Physiology, Zürich, Switzerland 2 University of Zürich, Institute of Anatomy, Zürich, Switzerland 3 National Center of Competence in Research Kidney.CH, Zürich, Switzerland

Background: Dietary intake of inorganic phosphate (Pi) is a major regulator of renal Pi reabsorption. Ingestion of

high dietary Pi induces a rapid adaptive phosphaturia through downregulation of NaPi-IIa and NaPi-IIc Pi transporters

in the brush border membrane of proximal tubules. These changes are accompanied by modifications in the levels

of several hormones such as parathyroid hormone (PTH), FGF-23, 1,25-(OH)2 vitamin D3, insulin and dopamine,

which mediate to some extent the effect of Pi on the kidney. To further clarify the role of PTH and other Pi-sensitive

mechanisms, we delivered an oral Pi bolus to WT and Pth KO mice and investigated the short term response of the

kidney after 1, 4 and 12 hours. Additionally, the direct effect of Pi and PTH on NaPi-IIa and –IIc expression was

investigated in ex vivo kidney slices.

Results: Oral Pi administration increased plasma Pi and decreased plasma calcium in all animals after 1 h but all

values normalized within the next hours except for calcium in Pth KO mice. Pi decreased NaPi-IIa and NaPi-IIc

abundance in WT animals within 1 hour, an effect reduced in Pth KO mice. We measured PTH, PTHrP, dopamine

and FGF23. PTH increased after 1 hr in WT animals but normalized after 4 hrs. PTHrP was elevated in Pth KO mice

after 4 hrs and FGF23 was higher in Pi delivered WT and Pth KO mice after 4 hrs. Blocking FGF23 signaling partially

blunted the effect of Pi in WT but not in Pth KO mice. In vitro incubation of kidney slices with high Pi did not induce

NaPi-IIa and –IIc degradation.

Conclusion: Our data demonstrate that 1) PTH is involved in the rapid downregulation of renal Pi transporters, 2)

that Pi has no direct effects in kidney slices, 3) FGF23 signaling contributes in WT mice to Pi transporter

downregulation, 4) signals other than PTH, FGF23, and dopamine must contribute to renal adaption. The role of

PTHrp remains to be established.


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B 09-06

Localization of Angiotensin II Type 1 receptor gene expression in

rodent and human kidneys

Julia Schrankl1, Michaela A. A. Fuchs1, Katharina A. - E. Broeker1, Christoph Daniel2, Armin Kurtz1,

Charlotte Wagner1

1 University of Regensburg, Institute of Physiology, Regensburg, Germany 2 Friedrich-Alexander University Erlangen Nürnberg, Department of Nephropathology, Erlangen, Germany

The kidneys are an important target for angiotensin II (ANG II). In the adult kidneys the effects of ANG II are mediated

mainly by ANG II type 1 (AT1) receptors. AT1 receptor expression has been reported for a variety of different cell

types within the kidneys, suggesting a broad spectrum of actions for ANG II. Since there have been heterogeneous

results in the literature regarding the intrarenal distribution of AT1 receptors, this study aimed to obtain a

comprehensive overview about the localization of AT1 receptor expression in mouse, rat and human kidneys. Using

the cell specific and high-resolution RNAscope technique, we performed colocalization studies with various cell

markers to specifically discriminate between different segments of the tubular and vascular system. Overall we found

a similar pattern of AT1 mRNA expression in mouse, rat and human kidneys. AT1 receptors were detected in

mesangial cells and renin-producing cells. In addition, AT1 mRNA was found in interstitial cells of the cortex and outer

medulla. In rodents, late afferent and early efferent arterioles expressed AT1 receptor mRNA, but larger vessels of

the investigated species showed no AT1 expression. Tubular expression of AT1 mRNA was species-dependent with

a strong expression in proximal tubules of mice while expression was undetectable in human tubular cells. These

findings suggest that the (juxta)glomerular area and the tubulointerstitium are conserved expression sites for AT1

receptors across species and might present the main target sites for ANG II in adult human and rodent kidneys.


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B 09-07

Development of a High Throughput Metabolomics Assay to Analyze

Intact Isolated Nephron Compartments

Johannes Jägers, Markus Rinschen

University Aarhus, Biomedicine, Aarhus, Denmark

The metabolome is the intra- and intercellular entity of small organic molecules and their reaction network. The vast

abundance of organic substances interacts with the body and modulate the biology of the cell on genomic,

transcriptomic, and proteomic level. Artificial setups such as cell culture are known to have an altered metabolism

and/or are lacking polarity that alters the membrane transport. Animal models allow for investigations of healthy and

diseased tissue but are time consuming and do not allow for direct interrogation of metabolism.

Here, we use labeled metabolites (15N or 13C) to investigate both transport and acute metabolic changes in isolated

nephron segments. We combine fast tissue extraction of selected intact proximal tubules and glomeruli in high purity

and a large-scale experimental setup of acute metabolic response with cutting-edge UPHLC-triplequadrupole based

MS-technology for targeted metabolomics in a multiplexing format. Metabolites were resolved at the single tubule

and glomerular level. Fractions enriched for proximal tubules allowed for metabolic analysis in the timescale of

minutes and hours. The extracted tubules or glomeruli were incubated and treated in 96 well plates, and experiments

analyzing metabolic activity were carried out in timescales of minutes to hours. Amino acid uptake, metabolism and

gluconeogenesis, and its modulation by different inhibitors was monitored in dependence of time, concentration, and

competitive substances.

Conclusion: A high-throughput method to quantify glomerular and tubular metabolism and metabolome was

developed.

References [1] Rinschen, M.M., Ivanisevic, J., Giera, M. et al. Identification of bioactive metabolites using activity

metabolomics. Nat Rev Mol Cell Biol 20, 353–367 (2019)


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B 09-09

The B1 H+-ATPase (Atp6v1b1) subunit is required for non-type A

intercalated cell function during alkalosis

Soline Bourgeois1, Jana Kovacikova1, Milica Bugarski2, Carla Bettoni1, Andrew Hall2, Carsten A.

Wagner1

1 University of Zurich, Institut of Physiology, Zurich, Switzerland 2 University of Zurich, Institut of Anatomy, Zurich, Switzerland

Non-type A intercalated cells (IC) in the connecting tubule (CNT) and cortical collecting duct (CCD) express the

luminal Cl-/HCO3- exchanger pendrin and apical and/or basolateral vacuolar H+-ATPases containing the B1 subunit

isoform. One of the main functions of non-type A ICs is the excretion of bicarbonate during metabolic alkalosis.

Mutations in the B1 subunit (ATP6V1B1) in man cause distal renal tubular acidosis due to its importance in acid

secretion by type A ICs. However, the function of the B1 isoform in non-type A ICs has remained elusive.

Induction of metabolic alkalosis by 0.28 M NaHCO3 in drinking water and one i.p. injection of 2mg

deoxycorticosterone resulted in a more pronounced alkalosis in ATP6v1b1-/- mice with increased blood bicarbonate,

hypokalemia, and hypochloremia despite a reduced lung minute volume. Determination of the relative abundance of

the different subtypes of cells in the collecting duct system revealed a remodelling of CNT and CCD in the cortex

with an increase of type A ICs and a compensatory increase of non-type A ICs in collecting ducts of the outer

medullary in ATP6v1b1-/-kidneys. Furthermore, total pendrin expression and pendrin activity in non-type A cells of ex

vivo microperfused CCD were reduced in ATP6v1b1-/- mice. Basolateral H+-ATPase activity in pendrin expressing

cells was strongly reduced, even though the expression of the B2 isoform was increased at the basolateral side of

pendrin expressing cells. Moreover, the E1 and A H+-ATPase subunits of the V0 domain did not fully associate with

the a4 H+-ATPase subunit of V1 domain at the basolateral pole of ATP6v1b1-/- non-type A ICs indicating impaired

assembly of V0 and V1 H+-ATPase domains. Finally, cellular signalling involving an increase in cAMP production can

stimulate pendrin activity. Indeed, in the basolateral presence of isoproterenol, activating β2-adrenergic receptors

and stimulating cAMP production, pendrin activity was increased in non-type A ICs of ex vivo microperfused CCDs

from ATP6v1b1+/+ mice but not in non-type A ICs from ATP6v1b1-/- mice.

Thus, this study demonstrates for the first time that 1- the B1 subunit is required for the assembly of a complete and

functional basolateral vacuolar H+-ATPases complexes in intercalated cells. Moreover, the B1 H+-ATPase subunit is

critical for normal non-type A ICs function and protects against alkalosis.


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B 09-10

Claudin-10b in the basolateral infoldings of the thick ascending limb

Catarina Quintanova1, Nina Himmerkus1, Samuel L. Svendsen2, Otto von Schwerdtner1,

Cosima Merkel1, Lennart Pinckert1, Kerim Mutig3, Tilman Breiderhoff4, Dominik Müller4,

Dorothee Günzel5, Markus Bleich1

1 Christian-Albrechts-University of Kiel, Institute of Physiology, Kiel, Germany 2 Aarhus University, Department of Biomedicine/Physiology, Aarhus, Denmark 3 Charité-Universitätsmedizin Berlin, Department of Anatomy, Berlin, Germany 4 Charité-Universitätsmedizin, Department of Pediatric Nephrology, Berlin, Germany 5 Charité-Universitätsmedizin, Institute of Clinical Physiology, Berlin, Germany

Question: The thick ascending limb (TAL) of the loop of Henle is the essential segment for salt homeostasis and

urinary concentration. Basic transcellular transport mechanisms involve co-transport of sodium (Na+), potassium (K+)

and chloride (Cl-) and paracellular cation permeability. In the TAL, tight junctions (TJs) showed a mosaic expression

of either claudin-10b or claudin-3/-16/-19. TJ dominated by claudin-10b confer mainly paracellular Na+ permeability.

Claudin-10b is also present outside of the TJ in basolateral membrane infoldings.

Methods: Freshly isolated single murine TAL segments of C57Bl6 and kidney specific (Ksp-Cre) Claudin-10

knockout mice were investigated for localization, protein expression and function by fluorescence microscopy and

electrophysiology measurements.

Results: Claudin-10 immunofluorescence revealed TJ staining and extra-junctional expression in a dotted pattern

that could be followed from the basement membrane into the apical membrane. This extra-junctional expression co-

localized with other membrane proteins, such as Na+-K+-ATPase and Barttin. To study a possible role of claudin-10

in the accessibility of the infoldings we measured the relative speed of ouabain inhibition. Speed of inhibition was

increased after loosening of intermembrane protein contacts under Ca2+-free conditions or in the absence of claudin-

10. Fluorescein dye could be trapped within the infoldings in the WT situation but much less so in Claudin-10 knockout

TAL.

Conclusions: Claudin-10 shows extra-junctional expression and co-localization with ion transport proteins in the

basolateral infoldings of TAL. As the presence of claudin-10 impedes free diffusion of ouabain or fluorescein along

the infolded space, we suggest a possible role of claudin-10 in the control of the infolded space by complex formation

between neighboring invaginated membranes.


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B 09-11

TNFa induces epithelial barrier perturbation by a self-enhancing effect

on TNFR-1 in porcine jejunal epithelial cells

Linda Droessler1, Valeria Cornelius1, Alexander Markov2, Salah Amasheh1

1 Freie Universität Berlin, Institut für Veterinär-Physiologie, Berlin, Germany 2 St. Petersburg State University, Department of General Physiology, St. Petersburg, Russia

Introduction: The pro-inflammatory cytokine Tumor necrosis factor alpha (TNFα) has been described to increase

the intestinal permeability by affecting tight junctions (TJ). In our study, the non-transformed porcine jejunal epithelial

cell line IPEC-J2 was employed to analyze these effects in detail [1].

Methods: IPEC-J2 cells were grown on cell culture inserts until confluency. Recombinant human TNFα (1000 U/mL)

was added to the basolateral compartment of the permeable supports, and transepithelial electrical resistance

(TEER) was recorded for 48 hours. For signaling analyses, the specific Myosin light chain kinase blocker ML-7 was

employed. Subsequently, tight junction proteins and the TNFα receptor (TNFR) were further analyzed by

immunohistochemistry and immunoblotting. Statistical analysis was performed using one-way ANOVA for normally

distributed data, Kruskal-Wallis test for not normally distributed data, and Dunnett’s test for correction of multiple

testing. In signaling experiments, Tukey Kramer method was used to ensure pairwise comparisons.

Results: An incubation with TNFα led to a decrease in TEER after 48 hours, while controls remained unchanged.

Western blot analysis of TJ proteins revealed a marked decrease of claudin-1, claudin-3 and occludin, after

incubation with TNFα. Furthermore, an incubation with the cytokine induced a dose-dependent increase of the

expression of its own specific receptor TNFR-1. Confocal microscopy confirmed all observations. Blocker

experiments revealed that ML-7 prevented the TNFα-induced decrease of resistance and claudin expression as well

as the increase of TNFR-1.

Conclusion: TNFα induced an increase of TNFR-1 and a decrease of claudin-1, -3, and occludin in IPEC-J2,

resulting in a barrier perturbation of monolayers mediated via MLCK. This mechanism contributes to explain the

exponential nature of TNFα effects on epithelial integrity in inflammatory processes.

Acknowledgment This work was supported by a grant of the German Research Foundation, DFG grant no. AM141/11-2.

References [1] Schierack P, Nordhoff M, Pollmann M, Weyrauch KD, Amasheh S, Lodemann U, Jores J, Tachu B, Kleta S,

Blikslager A, Tedin K, Wieler LH 2006, Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine, Histochem Cell Biol, 125, 293-305.


of 516

B 09-12

Enhancing and disrupting motility: Methylene blue as an experimental

tool for studying regular and altered murine gastric function

Robert Patejdl, Benjamin Schulz

University of Rostock, Oscar-Langendorff-Institute of Physiology, Rostock, Germany

Question: Alterations of gastric smooth muscle function are the hallmark of gastroparesis. Instead of an actual

impairment of smooth muscle cells (SMC), the most frequent causes of gastric dysmotility are lesions and functional

impairments of the motility-controlling networks formed by interstitial cells Cajal (ICC) and enteric neurons within the

stomach wall. While methylene blue has been shown to be a photosensitizer the enables selective lesioning of

neurons and ICC in intestinal tissues of various species, its selective uptake in murine stomach smooth has not yet

been demonstrated. Furthermore, the motility-enhancing effects of methylene blue have not yet been systematically

studied in any gastric tissue.

Methods: Longitudinal and circumferential strips of smooth muscle from the gastric antrum and corpus of CD1-mice

were excised and transferred to organ baths for isometric force measurements. Whole stomachs were explanted and

catheterized for intraluminal pressure measurements. Following reference activations using high-K+, carabachol

(CCh) and electric field stimulation, methylene blue (MB) was added and illumination or time control measurements

with repeated activations were conducted.

Results: A specific pattern of MB uptake into ICC and neurons was observed in both isolated stomachs and muscle

strips. In each, contractions were significantly enhanced by MB (organs: mean increase 6.1±1.7cmH2O, n=7 strips:

0.8±0.3cmH2O n=12, p for all <0.01). In parallel, the frequency of contractions declined. The MB-triggered responses

were unaltered by preincubation with atropine or tetrodotoxine but were diminished in the presence of perhexilline.

Following MB-washout, responses to high potassium and carbachol were preserved in both illuminated and in control

preparations. Following illumination with red light (650nm, 0.3mW/mm²), spontaneous activity was reduced and

desynchronized and responses to electric field stimulation were abolished.

Conclusions:The results of this study indicate that methylene blue can be used as an experimental tool for inducing

acute damage to enteric neurons and ICC in murine gastric tissues. Furthermore, we found surprisingly strong

stimulatory effects of methylene blue on the motor patterns of the isolated murine stomach. Although the exact

underlying mechanisms of MB deserve further study, it seems a promising tool to study pharmacological, physical or

genetic approaches to modify gastric smooth muscle function.


of 516


Foyers

B 10 | Vascular Physiology Chair

Martina Krüger (Düsseldorf)

Markus Sperandio (Munich)


of 516

B 10-01

Cytochrome P450 reductase maintains vessel function by regulating

eNOS activity and the metabolic fate of arachidonic acid

Pedro Malacarne1,2, Corina Ratiu1, Niklas Müller1,2, Anna Gajos-Draus1,3, Melina Lopez1,2,

Timothy Warwick1, Beatrice Pflüger-Müller1,2, Mauro Siragusa4,2, Wolf-Hagen Schunck5,

Ingrid Fleming4,2, Ralf P. Brandes1,2, Flavia Rezende1,2

1 Institute for Cardiovascular Physiology, Goethe-University, Frankfurt am Main, Germany 2 German Center of Cardiovascular Research, Partner Site Rhein Main, Frankfurt am Mian, Germany 3 National Science Center, Poland, Poland, Poland 4 Institute for Vascular Signaling, Goethe-University, Frankfurt am Main, Germany 5 Max-Delbrück-Centrum für Molekulare Medizin, MDC, Berlin, Germany

Cytochrome P450 reductase (POR) provides electrons for the catalytic activity of the cytochrome P450

monoxyenases. One function of the latter is to metabolize polyunsatturated lipids like arachidonic acid into

vasodilatory epoxyeicosatrienoic acid (EETs). The relevance of POR is the vascular system largely unknown. To

study this, we generated an endothelial-specific, tamoxifen-inducible knockout mouse of POR (ecPOR-/-). In an in

vivo Angiotensin II infusion model, acute deletion of POR increased the blood pressure as measured by telemetry

and tail cuff (137.4 ± 15.9 mmHg in WT; 152.1 ± 7.154 mmHg in ecPOR-/-). Under basal condition ecPOR-/- already

exhibited endothelial dysfunction in the aorta and mesenteric artery [Aortic LogEC50 acetylcholine-dependent

relaxation CTR: -7.6M ecPOR-/- -7.2M]. This effect was linked to lower plasma nitric oxide levels (CTR: 236.8 ±77.4;

ecPOR-/- 182.8 ±34.1 nmol/L), attenuated eNOS activity (Heavy arginine/citruline assay) and reduced eNOS

phosphorylation of Serine 1177. Furthermore, CYP450-dependent EET production was lower in ecPOR-/- mice

alongside with an increased production of COX-derived vasoconstriction prostanoids, such as thromboxane and

prostaglandins. In line with this, aortic expression of genes of eicosanoid production was increased (RNAseq).

Importantly, the cycloxyogenase COX inhibitor Naproxen selectively reduced the AngII-induced hypertersion in

ecPOR-/- mice (telemetry). In conclusion: endothelial POR regulates eNOS activity and impacts on arachidonic acid

metabolism. In the absence of POR, EET production is reduced and arachidonic acid is shunt to prostaglandins and

thromboxanes. Thus, loss of POR induces vascular dysfunction and hypertension.


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B 10-02

A circulation model simulating low venous blood flow rate to study

hemodynamical effects in the venous system

Ali Aghajafari1, Sina Moztarzadeh2, Foivos L. Mouzakis2, Mohammad Esmaeil Barbati1,

Houman Jalaie1, Khosrow Mottaghy2

1 University Hospital RWTH Aachen, Clinic for Vascular Surgery, Aachen, Germany 2 University Hospital RWTH Aachen, Institute of Physiology, Aachen, Germany

QUESTION

To mimic the hemodynamic of the venous system is a rather considerable challenge due to demands on flexibility

and compliance of the vessels, its low blood pressure, flow behavior, and its “elliptical” cross-section. This study aims

to realize and develop a venous circulation model suitable for studies of pathological behavior of some venous

diseases such as deep vein thrombosis (DVT) and May-Thurner Syndrome (MTS) to investigate the eligibility of

various venous stents. However, so far, extensive research has been conducted on coronary and arterial stents. This

model is realized here to investigate the hemodynamic of a venous system at different flow conditions.

METHODS

An in-vitro fluid circulation model is developed applying special thin-wall silicon tubes, silicon and PVC elastic tubes

possessing circular cross-sections. Special sensors for very low-pressure monitoring and applying pump systems for

peristaltic or alternatively continues flow perfusion of the system. Several stents of different manufactures were

examined. As fluids are used water, glycerin, and animal blood to compare the effect of viscosity and shear stress

but also blood trauma.

RESULTS

The blood flow rates in the “venous” tube segments range from 250 – 1000 ml/min. The pressure drop was across

the length of the tube 0.01 – 1.2 cmH2O and was in fair agreement with the values calculated by the Hagen-Poiseuille

equation. A metal wire string was used as a stent model to study the variations of the parameters and the results

showed clear changes in the pressure drop as it was increased up to 4 times higher. The produced hemolysis in the

case of using animal blood was as low as 8.9 mg/dl.

CONCLUSIONS

The results demonstrate clearly that this circulation model is suitable for an extensive comparative study of different

pathological venous conditions and the application of stents. Furthermore, it is planned to extend the circulation

model by including elastic tubes to simulate the iliac vein compression.


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B 10-03

Adenosine signaling in the pulmonary vasculature

Jana Lewandowski1, Alexander Seidinger1, Daniela Wenzel1,2

1 Ruhr-University Bochum, Systems Physiology, Bochum, Germany 2 University of Bonn, Physiology I, Bonn, Germany

Adenosine receptors are G protein-coupled receptors that are widely expressed in the cardiovascular system. In

blood vessels, adenosine has been shown to induce vasorelaxation or vasoconstriction depending on the tissue type

and the receptor activated. In the pulmonary vasculature A2B receptors (A2BR) appear to be of special importance

for pathophysiological states, as they are upregulated in humans with pulmonary hypertension (PH). However, the

effect of A2BR activation on the tone of pulmonary arteries (PAs) is still unclear.

We therefore want to investigate the effects of adenosine as well as receptor specific agonists and antagonists on

vascular tone regulation in the lung. First, adenosine, A2BR agonists and antagonists will be tested in isometric force

measurements of large mouse PAs. The underlying signaling pathway will be investigated by pharmacological

compounds and transgenic mouse models. Then, A2BR activation will be analyzed in smaller PAs using precision-

cut lung slices and the isolated perfused lung model of mouse. The effects of adenosine, A2BR agonists and

antagonists will be further tested in cell growth assays using primary cell cultures of human and mouse smooth

muscle cells. Finally, the role of A2BR will be specified in healthy mice and a mouse model of PH in vivo.

In isometric force measurements of mouse PAs, adenosine preincubation resulted in a right shift of serotonin (5-HT)

dose-response curves compared to controls (EC50con: -6.97 (n=7), EC50Ade: -6.54 (n=7), p<0.001). Preincubation

with the A2BR specific antagonist PSB-603 abrogated the effect of adenosine (EC50con: -6.64 (n=4), EC50PSB+Ade: -

6.68 (n=3), p>0.05). Similar to adenosine, the A2BR specific agonist BAY 60‐6583 induced a right-shift of 5-HT dose

response curves (EC50con: -6.61 (n=5), EC50BAY: -6.14 (n=6), p<0.01) and again, preincubation with PSB-603

inhibited the effect (EC50con: -6.42 (n=3), EC50PSB+BAY: -6.39 (n=4), p>0.05).

First results indicate that adenosine and A2BR agonists induce vasorelaxation of large pulmonary vessels via A2B

receptors. Therefore, activation of A2B could be an interesting therapeutic approach in states with elevated

pulmonary blood pressure.


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B 10-04

Endothelial cysteinolysis in age-related cardiac hypertrophy

Maria Kyriaki Drekolia1,2, Ilka Wittig2,3, Baktybek Kojonazarov4, Janina Wittig1,2, Stefan Günther5,

Carolin Mogler6, Sofia-Iris Bibli1,2, Ingrid Fleming1,2

1 Goethe University, Institute for Vascular Signalling, Centre for Molecular Medicine, Frankfurt am Main, Germany 2 German Center of Cardiovascular Research (DZHK), Partner Site Rhein-Main, Frankfurt am Main, Germany 3 Goethe University, Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Frankfurt am Main, Germany 4 Justus-Liebig-University Gießen, Gießen, Germany 5 Max Planck Institute for Heart and Lung Research, Department I Cardiac Development and Remodelling, Bad

Nauheim, Germany 6 Technical University of Munich, Institute of Pathology, Munich, Germany

Question: Cysteine is metabolized in endothelial cells by the enzyme cystathionine gamma lyase (CSE) which also

generates the H2S, a potential angiocrine factor. H2S generation and cysteine catabolism regulate endothelial cell

activation and mechanosensing. This project set out to characterize the effects of reduced endothelial cell cysteine

catabolism (inducible endothelial cell-specific CSE knockout mice/ CSEiΔEC mice) and to identify potential CSE-

associated angiocrine effects on cardiomyocytes.

Methods and results: Analysis of CSE activity andH2S levels (HPLC-MS/MS) in endothelial cells from 1, 3, 6 and

18 month old mice revealed a gradual decrease in H2S production. This decrease was associated with the enhanced

phosphorylation of CSE on Ser377, which inhibits its activity. To determine whether the deletion of endothelial cell

CSE impacts on cardiac function, CSEiΔEC and wild type littermates were studied. Cardiac echocardiography and

μCT analyses revealed an age dependent systolic and diastolic dysfunction, which was enhanced in 18 month old

CSEiΔEC mice - based on left ventricle posterior wall thickness. In addition, pulsed-wave Doppler recording showed

severe right ventricular dysfunction, with significantly shorter pulmonary acceleration time and tricuspid annular plane

systolic excursion measures. Histopathological characterization of hearts confirmed severe hypertrophy without signs

of inflammation in hearts from CSEiΔEC mice compared to their wild type littermates. Mechanistically, endothelial CSE

deletion impacted on the cardiomyocyte transcriptional program. RNA sequencing of cardiomyocytes isolated from

18 month old wild type and CSEiΔEC mice, revealed changes in hypertrophic (i.e. myosin heavy chain 6 and 7) and

metabolic related transcripts (i.e. carbonic anhydrase 3, fatty acid synthase). Validation of the most upregulated

transcripts confirmed these observations, while treatment with the sulfur donor (sodium polysulthionate) reversed

this effect. The most altered gene was cytochrome P450 2E1 (CYP2E1), and its upregulation could be confirmed at

the protein level.

Conclusions: Age-related alterations in the endothelial cysteine catabolism impact on cardiac function and

cardiomyocyte transcriptional program. Studies are ongoing to investigate the link between CSE and CYP2E1 as

well as the impact of cardiomyocyte CYP2E1 on heart function.


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B 10-05

The baroreceptor reflex brought to life outside the classroom – a non-

supervised e-learning based laboratory class on the Active Standing

Test

Tobias Heinrich, Isabel Wageringel, Heimo Ehmke, Alexander P. Schwoerer

University Medical Centre Hamburg-Eppendorf, Department of Cellular and Integrative Physiology, Hamburg,

Germany

Background: In-classroom laboratory classes with hands-on experiences traditionally form a fundamental part of

teaching physiology. In the context of limited resources or a pandemic situation, non-supervised e-learning based

laboratory classes may be a valuable extension or replacement. The baroreceptor reflex is a fundamental aspect of

cardiovascular physiology. It is regularly visualized and experienced by students during a voluntary provocation of

orthostatic stress using the Active Standing Test (AST).

Aims: The current study aimed to transform the established AST into an e-learning class with predefined

characteristics: it should be asynchronous, be feasible for conduction by the students (self-experience) without the

need of medical and technical equipment or supervision. Furthermore, it should be applicable to different curricula,

provide textbook-conform and easy interpretable data with low interindividual variances, and motivate students to

reflect the experimental results.

Methods: To this end, the AST was simplified and embedded into a framework of digital material allowing

independent student performance. Implementation was realised in 1st and 2nd year curricula of human medicine,

dental medicine, midwifery and pharmacy. The frameworks and interactive elements were specifically adapted to the

characteristics of the different curricula.

Results: Here, we provide detailed experimental data of 217 students who participated in the class within 1 year.

Consistent with texbook knowledge, students reported a highly reproducible ~30% increase of heart rate during

standing. The evaluation documented high levels of feasibility and satisfaction within all careers.

Conclusion: This study demonstrates that a fundamental process of cardiovascular physiology can be successfully

addressed in a non-supervised e-learning based laboratory class. The high quality of individual results allows a broad

application within different health-care related curricula. Due to its feasibility and its modular design, it may also be

highly useful in virtually all pre- and post-graduate settings worldwide that address cardiovascular physiology.


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B 10-06

Quantification of the extracellular adenosine metabolism on cells from

patients with Arterial Calcification due to Deficiency of CD73

Jule Richter, Peter Dieterich, Andreas Deußen, Melanie Martin

Faculty of Medicine Carl Gustav Carus, TU Dresden, Institute for Physiology, Dresden, Germany

Loss-of function mutations in the CD73 encoding gene result in a rare autosomal recessive disease, which is known

as Arterial Calcification due to Deficiency of CD73(ACDC) or Calcification of Joints and Arteries (CAJA) (NEJM 2011;

364:432-442). CD73 is an ectonucleotidase that hydrolyses extracellular AMP to adenosine. Despite the general

expression of this enzyme in many tissues and organs, patients with ACDC show strong but very local ectopic

calcifications. These mainly affect the small joint capsules of the hands and feet and the arteries of the extremities

causing joint pain and limb ischemia. This phenotype raises the question of how the CD73 deficiency may be

compensated, e.g. by upregulation of other enzymatic activities like the tissue non-specific alkaline phosphatase

(TNAP). To answer this question, we measured the activities of ectonucleotidases involved in the extracellular

adenosine metabolism. In cell culture experiments with cells from patients with ACDC and healthy controls we

assayed the activity of AMP hydrolysing enzymes by adding etheno(ɛ)-AMP in different initial concentrations (1 – 50

µM) dissolved in a buffer with an extracellular fluid-like composition. Supernatant was collected at defined intervals

and analysed by high performance liquid chromatography with fluorescence detection. This enabled us to quantify

the enzymatic activities from time-concentration curves. Control cells almost completely degraded ɛAMP to

ɛAdenosine within 2 hours. By contrast, patient cells barely showed ɛAMP degradation over this time period. When

incubating control cells together with ɛAMP and an inhibitor of CD73 we obtained time-concentration curves similar

to those from patient cells. In conclusion, the quantification of enzymatic activity in cell culture experiments confirmed

the non-functional CD73 in cells from patients with ACDC. Furthermore, we found no comparable, alternative catalytic

activity in patient cells under our experimental conditions. This suggests that extracellular adenosine production from

AMP predominantly rests on the activity of CD73 without an upregulation of other AMP-hydrolysing enzymatic

activities such as TNAP.


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B 10-07

Endothelial actions of C-type natriuretic peptide prevent systolic

hypertension, aortic stiffness and atherosclerosis in female mice

Franziska Werner1, Lydia Schröder1, Sarah Schäfer2, Melanie Rösch2, Katharina Völker1, Lisa Krebes1,

Marco Abeßer1, Kai Schuh1, Hideo A. Baba3, Frank Schweda4, Alma Zernecke2, Michaela Kuhn1

1 University of Würzburg, Institute of Physiology, Würzburg, Germany 2 University Hospital Würzburg, Institute of Experimental Biomedicine, Würzburg, Germany 3 University Hospital Essen, University of Duisburg-Essen, Institute of Pathology, Essen, Germany 4 University of Regensburg, Institute of Physiology, Regensburg, Germany

Background: C-Type Natriuretic Peptide (CNP) participates in the paracrine communication of endothelial cells with

neighbours like pericytes and perivascular resident mast cells (1,2). Thereby CNP regulates microcirculatory flow

and blood pressure and stabilizes the vascular barrier. In addition, its´ cyclic GMP-producing guanylyl cyclase-B (GC-

B) receptor is expressed in endothelial cells themselves. To elucidate the role of endothelial CNP signalling in

vascular homeostasis, we generated a novel genetic mouse model with endothelial deletion of GC-B (EC GC-B KO

mice).

Methods and results: Telemetric and tail cuff recordings revealed that female KO mice have mildly but significantly

increased systolic blood pressure already at young ages. Doppler Ultrasound demonstrated increased aortic pulse

wave velocity, an indication of stiffness. Indeed, stainings of aortic sections showed that the total, media and

adventitial thickness and the number of elastin breaks were increased, whereas the collagen/elastin ratio was

decreased in such KO mice as compared to controls. Concomitantly the expression levels of the endothelial adhesion

proteins E-Selectin and VCAM-1 were increased in the former. Notably, we never observed such genotype-

dependent changes in male KO mice. Aortic stiffness and hypertension are well known risk factors for atherosclerosis.

To delineate a possible protective endothelial role of CNP in this disease, we intercrossed the EC GC-B KO and

control mice with low density lipoprotein receptor deficient (LDLR-/-) mice. Feeding a western type diet (21% fat) for

10 weeks did not impact arterial blood pressure. Despite, the double KO females had greater plaque areas and

heights in their aortic roots (aldehyde fuchsin stainings). This was accompanied by enhanced macrophage infiltration

(immunostainings with Mac-2). Moreover, the number and area of necrotic cores within the plaques were larger,

suggesting unstable plaques. Remarkably such genotype-dependent changes again only ocurred in female mice.

Conclusions: The endothelial CNP/GC-B/cGMP pathway protects against aortic stiffness and atherosclerosis. Our

own and published observations indicate sex differences in vascular cGMP signalling, CNP outweighing in females

and nitric oxide in males. Consistently LCZ696, a drug inhibiting neprilysin-mediated CNP degradation, reduced the

risk of hospitalization more in women than in men with HFpEF (3), supporting the human relevance of our

experimental findings.

Acknowledgment Supported by the Deutsche Forschungsgemeinschaft (DFG KU 1037/8-1) and by the Universitätsbund Würzburg

(AZ 21-25).

References (1) Špiranec K, Chen W, Werner F, Nikolaev VO, Naruke T, Koch F, Werner A, Eder-Negrin P, Diéguez-Hurtado R,

Adams RH, Baba HA, Schmidt H, Schuh K, Skryabin BV, Movahedi K, Schweda F, Kuhn M 2018 'Endothelial C-Type Natriuretic Peptide Acts on Pericytes to Regulate Microcirculatory Flow and Blood Pressure', Circulation, 138, 494-508


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(2) Chen W, Werner F, Illerhaus A, Knopp T, Völker K, Potapenko T, Hofmann U, Frantz S, Baba HA, Rösch M, Zernecke A, Karbach S, Wenzel P, Kuhn M 2020 'Stabilization of Perivascular Mast Cells by Endothelial CNP (C-Type Natriuretic Peptide)', Arterioscler Thromb Vasc Biol, 40, 682-696

(3) McMurray JJV, Jackson AM, Lam CSP, Redfield MM, Anand IS, Ge J, Lefkowitz MP, Maggioni AP, Martinez F, Packer M, Pfeffer MA, Pieske B, Rizkala AR, Sabarwal SV, Shah AM, Shah SJ, Shi VC, van Veldhuisen DJ, Zannad F, Zile MR, Cikes M, Goncalvesova E, Katova T, Kosztin A, Lelonek M, Sweitzer N, Vardeny O, Claggett B, Jhund PS, Solomon SD 2020, 'Effects of Sacubitril-Valsartan Versus Valsartan in Women Compared With Men With Heart Failure and Preserved Ejection Fraction: Insights From PARAGON-HF', Circulation, 141, 338-351


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B 10-08

Interaction with αvβ3-integrin mediates the cardiovascular protective

effects of Developmental Endothelial Locus-1 after established

angiotensin II-induced hypertension.

Michael Amponsah-Offeh1, George Hajishengalis2, Triantafyllos Chavakis3, Andreas Deußen1,

Irakli Kopaliani1

1 Medical Faculty, TU Dresden, Institute of Physiology, Dresden, Germany 2 University of Pennsylvania, Penn Dental Medicine, Philadelphia, USA 3 University Clinic Dresden, Institute of Clinical Chemistry, Dresden, Germany

Introduction: Hypertension remains a global health problem as it poses a major risk for development of CVDs.

Target organ damage through remodeling is a dominant manifestation of hypertension and inflammation has been

shown to mediate the target organ damage during hypertension. Hence, novel anti-inflammatory strategies are

needed for better management of hypertension-related organ damage.

Aim: We investigated the therapeutic role Del-1 and its mechanisms of action in hypertension related organ damage.

Methods: Hypertension was induced by subcutaneous infusion of ANGII in WT mice for 2 weeks. Systolic blood

pressure (SBP) was measured with tail cuff method. Starting from the 6th until the 12th day, mice were treated by daily

injection of soluble recombinant Del-1 or its point mutant Del-1-RGE which cannot bind to αvβ3-integrin. Endothelial

function was assessed ex vivo with Mulvany-Myography, inflammatory cells were assessed with flow cytometry.

Aortic and cardiac remodelling parameters were quantified by histology.

Results: Compared with vehicle, all ANGII-infused mice had elevated SBP consistent with hypertension after 6 days.

Further elevation of SBP after day 6 was observed only in non-treated (ANGII+Fc) or mutant treated ANGII-infused

mice (ANGII+Del-1-RGE-Fc). Increase in SBP after day 6 was abrogated in ANGII-infused mice treated with Del-1

(ANGII+Del-1-Fc). This was associated with better maintained endothelium dependent aortic relaxation and the

decreased aortic wall stiffness in ANGII+Del-1-Fc mice. In comparison to ANGII+Del-1-RGE-Fc or ANGII+Fc mice,

aortic and cardiac accumulation of CD45+ leukocytes, CD45+/IL-17A+ and TCRβ+T cells were markedly lower in the

ANGII+Del-1-Fc mice. Additionally, ANGII induced reduction in anti-inflammatory regulatory T-cells

(CD25+FOXP3+Tregs) was prevented in the Del-1 treated mice. Degradation of elastin was hindered in ANGII+Del-

1-Fc mice and increases of adventitial collagen deposition and medial thickness were reduced. Compared

with ANGII+Del-1-RGE-Fc or ANGII+Fc mice, the hearts of ANGII+Del-1-Fc mice had significantly decreased

interstitial and pericoronary collagen as well as a smaller cardiomyocyte cross-sectional area and an increased

lumen-to-wall ratio.

Conclusion: We demonstrate the potential efficacy of Del-1 to protect from adverse cardiovascular remodeling and

damage after ANGII-induced hypertension which is mainly dependent on its binding to αvβ3-integrin and related anti-

inflammatory actions.

Acknowledgment Funding by the German Research foundation (DFG) and National Institute of Health (NIH)


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B 10-09

Role of the NADPH oxidase 4 in restenosis development after vascular

injury in mice

Giulia K. Buchmann1,2, Christoph Schürmann1,2, Manuela Spaeth1,2, Wesley Abplanalp3,

Lukas Tombor3, David John3, Timothy Warwick1,2, Flavia Rezende1,2, Andreas Weigert4, Ajay M. Shah5,

Martin-Leo Hansmann6, Norbert Weissmann7, Stefanie Dimmeler3, Katrin Schröder1,2, Ralf P. Brandes1,2

1 Goethe-University Frankfurt am Main, Institute for Cardiovascular Physiology, Frankfurt, Germany 2 Goethe-University Frankfurt am Main, German Center for Cardiovascular Research (DZHK), Frankfurt, Germany 3 Goethe University Frankfurt am Main, Institute of Cardiovascular Regeneration, Frankfurt, Germany 4 Goethe University Frankfurt am Main, Faculty of Medicine, Institute of Biochemistry I, Frankfurt, Germany 5 King’s College London, School of Cardiovascular Medicine & Sciences, London, UK 6 University Hospital Frankfurt, Department of Pathology, Frankfurt, Germany 7 Universities of Giessen and Marburg Lung Center (UGMLC), Excellence Cluster Cardio-Pulmonary Institute (CPI),

Giessen, Germany

Background: Oxidative stress is known to be a risk factor for development of cardiovascular diseases. NADPH

oxidases are important producers of reactive oxygen species. The H2O2 producing NADPH oxidase Nox4 is

described to play a protective role the vasculature and is also known to regulate differentiation of smooth muscle

cells (SMC). On this basis, the role of Nox4 for restenosis development was determined in the mouse carotid artery

injury model.

Methods and Results: Wire injury, a common model to study restenosis in mice, was used to induce vascular injury

in C57BL/6 and tamoxifen inducible global Nox4 knockout mice. Neointima area measurements after injury did not

reveal differences after loss of Nox4. Time-resolved single-cell RNA-sequencing (scRNAseq) and massive-analysis-

of-cDNA-ends (MACE)-RNAseq of selectively neointima obtained by laser capture microdissection was performed

to understand this finding. Single cell RNA sequencing of C57BL/6 mice at different time points after injury

recapitulated the wound healing process and revealed high expression of Nox4 in quiescent SMCs, but low

expression in proliferative, neointima forming SMCs. In accordance with this, neointimal gene expression of control

and Nox4 knockout samples was not altered in inflammatory phases after injury.

Conclusions: Nox4 is differently expressed upon carotid injury; its expression is lost in neointima forming,

proliferative SMCs. Therefore, the NADPH oxidase 4 plays no role in the development of restenosis in mice.


of 516

B 10-10

NADPH oxidase 4 and endothelium-derived hyperpolarization factor

can maintain vascular function in small resistance arteries

Patrick Diaba-Nuhoho, Coy Brunssen, Henning Morawietz, Heike Brendel

University Hospital Carl Gustav Carus Dresden, Technische Universität Dresden, Division of Vascular Endothelium

and Microcirculation, Department of Medicine III, Dresden, Germany

Purpose: Endothelial dysfunction precedes the onset of cardiovascular and vascular complications. NOX4-

generated H2O2 might play a role in vasodilation of small resistance arteries in our mice models.

Methods: Vascular function of 2nd and 3rd branches of isolated mesenteric artery segments from 10-week-old WT

and Nox4-/- mice was analysed using a wire myograph.

Results: PE-induced contraction by calcium-release from intracellular compartments was similar in WT and Nox4-/-

mice. Endothelium dependent vasorelaxation induced by ACh was also similar in WT and Nox4-/- mice. However,

application of catalase on vessel segments decreased ACh-induced vasorelaxation only in WT but not Nox4-/- mice.

Blocking the large conductance calcium-dependent potassium channels (BK channels) with paxilline induced

endothelial dysfunction in WT mice but not Nox4-/- mice. In both WT and Nox4-/- mice cyclooxygenase-2 (COX-2)

inhibitor diclofenac showed no change in endothelial function. The highest impact on endothelium-dependent

vasorelaxation had NOS inhibitor L-NAME. Incubation with L-NAME induced endothelial dysfunction in both mice

strains. In addition, endothelial dysfunction could be further impaired by application of paxilline to L-NAME-blocked

mesenteric segments of WT and Nox4-/- mice. Both WT mice and Nox4-/- mice showed significant endothelial

dysfunction when blocked with paxilline and diclofenac. However, smooth muscle function was unchanged between

groups.

Conclusion: In both WT and Nox4-/- mice, nitric oxide is the main vasodilator and can be regulated by endothelium-

derived hyperpolarisation factors (EDHF). The presence of NOX4 in the mesenteric artery of WT mice mediates

vasodilatory and compensatory mechanisms when normal physiological responses are inhibited.

Acknowledgment This work was supported by the Government of Ghana (Ministry of Education) and the German Government

(Deutscher Akademischer Austauschdienst German Academic Exchange Service (DAAD) (91642764) to P.D.N.

We acknowledge the Medical Faculty of the University Hospital, Carl Gustav Carus, TU Dresden for institutional

support.


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B 10-11

An interdisciplinary course on peripheral vascular physiology –

oscillatory pulse wave diagnostics and venous occlusion

plethysmography

Tobias Heinrich1, Robert Bähring1, Axel Larena-Avellaneda2, Jürgen Querengässer3, Olaf Solbrig3,

Heimo Ehmke1, Alexander P. Schwoerer1

1 University Medical Centre Hamburg-Eppendorf, Department of Cellular and Integrative Physiology, Hamburg,

Germany 2 Asklepios Hospital Altona, Department of Vascular and Endovascular Surgery, Hamburg, Germany 3 medis. Medizinische Messtechnik GmbH, Ilmenau, Germany

Introduction: Integrative laboratory classes on peripheral vascular physiology are challenging to design. Ideally,

they should concomitantly address aspects of vascular physiology, pathophysiology and the resulting diseases.

However, the basic science and clinical practice approaches to vascular function differ fundamentally in methodology

and aims. This divergence hampers the successful implementation of a translational laboratory class, especially

when high feasibility is required to allow students’ performance. Therefore, most established classes focus on either

physiological or clinical methodology leaving a translational gap between basic science and clinical practice.

Aims: Here, we describe an integrative laboratory class on leg perfusion. We provide experimental results of the

students, document learning success and students’ satisfaction.

Methods: The activity was integrated in the 2nd and 3rd year of an integrative medical curriculum. It was immediately

preceded by an advanced physiology seminar on vascular properties and a lecture on vascular medicine.

Results: Using oscillography and impedance venous occlusion plethysmography, the course addressed key aspects

of the arterial and venous vascular system: 1) arterial pulse wave properties, 2) regional differences in systolic blood

pressure, 3) reactive hyperemia, 4) venous capacity and venous outflow. All experiments were well feasible and

provided textbook-conform data with high relevance for vascular physiology and vascular medicine. Students

documented a positive perception and a substantial improvement of knowledge.

Conclusions: Our integrative laboratory class successfully bridged the translational gap between vascular

physiology and vascular medicine. It was highly feasible, provided excellent experimental data and was positively

perceived by students.


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B 10-12

Sex-specific differences of endothelial progesterone and estrogen

receptor stimulation in mouse aorta

Basant Elsaid1,2, Birgit Zatschler1, Irakli Kopaliani1, Andreas Deussen1

1 Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Department of Physiology, Dresden,

Germany 2 Faculty of Medicine, Ain Shams University, Department of Physiology, Cairo, Egypt

Female blood vessels constrict less and relax more than male vessels to adrenergic stimulation, but the exact

mechanisms governing these differences are not well understood.

We investigated the effects of female sex hormones and their specific receptor agonists on vascular relaxation.

Isolated aorta from male and female wild type mice preconstricted with phenylephrine were exposed to 17 β estradiol,

progesterone and specific estrogen receptors agonists (ERα, ERβ and GPER) in a Mulvany Myograph. Vessel

relaxation was determined for concentrations ranging from 10-8 to 10-5 M. To assess the role of endothelial signaling

the endothelium was mechanically removed.

Female aorta relaxation in response to estrogen showed no significant difference as compared to male aorta (12 ± 3

vs. 8 ± 2 %). After removal of endothelium relaxations of 4% in male and 5% in female remained (p=0.65 and 0.18

vs. intact endothelium in male and female respectively). Progesterone showed similar relaxations in female and male

aorta with endothelium (11 ± 1 and 11 ± 2 %). After removal of the endothelium, this relaxant effect decreased to 2%

in male (p≤0.02), whereas in female vessels 54% of the relaxation response was maintained (p=0.4).

The relaxant effect of ERα agonist did not differ between female and male aorta in the presence of endothelium (22

± 4 and 22 ± 4 %). Also, ERβ agonist showed a similar relaxation in female and male aorta (14 ± 3 and 14 ± 4 %).

After removal of endothelium relaxation toward ERα agonist was blunted by73% and 65% in female and male

respectively, (p≤0.02). When endothelial cells were removed ERβ agonist showed a 70% decrease in relaxation with

a trend toward significance in female (p= 0.1), whereas only a 27% decrease in relaxation was noted in male aorta

(p=0.8). In presence of endothelium GPER agonist (10-6 to 10-4 M) relaxed male and female blood vessels to a similar

extent (12 ± 2 vs. 10 ± 3 %). After removal of endothelium this relaxant effect was completely abolished in male

(p<0.05), but not significantly diminished in female aorta (p=0.8).

Estrogen and progesterone cause relaxations in male and female aorta. The endothelium is involved in mediating

this sex hormone dependent relaxation. Between male and female aorta differences exist with respect to

progesterone as well as ERβ- and GPER-induced relaxations via the endothelium.


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Foyers

B 11 | Endothelial Cell Physiology II Chair

Ingrid Fleming (Frankfurt/Main)

Axel Gödecke (Düsseldorf)


of 516

B 11-01

The NADPH Oxidase Nox4 Promotes Endothelial Differentiation

Fabian Hahner1, Tim Warwick1, Giulia Buchmann1, Ralf P. Brandes1, Ralf Gilsbach1, Arne Hansen2,

Wesley Abplanalp3, Katrin Schröder1

1 Goethe University Hospital, Institute for Cardiovascular Physiology, Frankfurt, Germany 2 University Medical Center Hamburg-Eppendorf, Department of Experimental Pharmacology and Toxicology,

Hamburg, Germany 3 Goethe University Hospital, Institute for Cardiovascular Regeneration, Frankfurt, Germany

Nox4 is the only constitutively active NADPH oxidase. It directly produces H2O2 and contributes to cell homeostasis

and forces differentiation. In the vasculature, Nox4 is mainly expressed in endothelial cells. We therefore, hypothesize

that Nox4 contributes to endothelial differentiation.

In the process of endothelial differentiation from iPSCs, Nox4 expression increased and knockout of Nox4 prolonged

the abundance of pluripotency markers such as Oct4 and Nanog. Simultaneously expression of endothelial markers

such as Vegfr2 and Pecam1 was delayed in differentiating Nox4-depleted iPSCs. Accordingly, iPSC-ECs ability to

form tubes and sprouts upon treatment with VEGF, was dependent on Nox4. In an in vivo matrigel plug assay, Nox4-

/- iPSC-ECs integrated less into the newly formed vascular network.

As an underlying mechanism, we identified that triple methylation of histone 3 (H3K27me3) was increased in cells

lacking Nox4 due to a lower nuclear abundance of the demethylase JmjD3 in Nox4 deficient iPSC-ECs. We found

JmjD3 to be redox sensitive and specifically oxidized by Nox4. Indeed, rescuing the absence of Nox4 by treatment

of the cells with H2O2 increased the expression of endothelial genes such as CD31 in iPSCs.

We conclude that Nox4 mediated oxidation of JmjD3 enables the demethylase to demethylate H3K27me3, which

forced endothelial marker expression and differentiation.


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B 11-02

In vitro mechanical stimulation of an endothelial and a cardiac cell line

using a cell stretching device (IsoStretcher) and visualization of the

stretch induced response via calcium imaging

Ulrike Schöler1,2, Anna-Lena Merten1,2, Yang Guo3,4,5, Boris Martinac3,5, Sebastian Schürmann1,2,

Oliver Friedrich1,2,3

1 Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute of Medical Biotechnology, Erlangen, Germany 2 Friedrich-Alexander-Universität Erlangen-Nürnberg, School in Advanced Optical Technologies, Erlangen,

Germany 3 Victor Chang Cardiac Research Institute, Molecular Cardiology and Biophysics Division, Darlinghurst, Australia 4 Victor Chang Cardiac Research Institute, Cardiac Physiology and Transplantation Division, Darlinghurst, Australia 5 University of New South Wales, Faculty of Medicine, Kensington, Australia

Cells of the cardiovascular system are constantly exposed to varying mechanical forces caused by the blood flow.

Certain cardiovascular diseases may alter those forces. Mechanosensitive ion channels sense external mechanical

stimuli and transduce them into intracellular electrochemical signals. Interest in understanding this

mechanotransduction at both the cellular and subcellular level is steadily increasing. The IsoStretcher, a cell

stretching device developed at our institute, can be used to apply radial stretch to cells attached to a transparent

custom-made silicone elastomer polydimethylsiloxane (PDMS) chamber (Fig. 1). The IsoStretcher can be mounted

on most fluorescence microscopes and allows for life cell imaging during stretch.

Cells of the human umbilical vein endothelial cell line, EA.hy926, and the mouse atrial cardiomyocyte cell line, HL-1

(which shows spontaneous calcium fluctuations), were seeded onto PDMS chambers coated with extracellular matrix

proteins. After adhesion to the elastomer substrate, cells were stained with the calcium indicator Fluo-4-AM (HL-1)

or Cal-520-AM (EA.hy926). After recording the baseline fluorescence intensity of the non-stretched cells, the

chambers were stretched to 15 % (HL-1) or ~20 % (EA.hy926) radial stretch. The stretch was maintained and the

calcium signal within the stretched cells was recorded (Fig. 2). Since the field-of-view inevitably moves upon stretch,

we developed an ImageJ image analysis routine which allows the automated allocation of the cells before and during

stretch via feature extraction followed by application of the ImageJ plugin bUnwarpJ [1]. This provides the possibility

to evaluate the calcium signal within the same cell in the non-stretched and stretched state.

From previous preliminary data, we found that stretch might affect baseline intensity and peak frequency in HL-1

cells [2]. A more detailed analysis using the automated routine gives deeper insight into the stretch induced response

as the automated procedure enables the analysis of a large number of cells. Preliminary results show a weak

transient increase in fluorescence intensity (integrated density) in EA.hy926 cells upon stretch.

In conclusion, the IsoStretcher is a valuable device to directly visualize mechanotransduction in vitro upon controlled

isotropic stretch. Both model cell lines for the cardiovascular system, EA.hy926 and HL‐1 cells, respond to stretch,

with the response being different depending on the cell type.


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Acknowledgment This work was supported by the German Science Foundation (DFG) projects 383071714 (FR 2993/23-1), the

Bavarian Equal Opportunities Sponsorship – Realisierung von Chancengleichheit von Frauen in Forschung und

Lehre (FFL) – Realization Equal Opportunities for Women in Research and Teaching, the Erlangen Graduate

School in Advanced Optical Technologies (SAOT) by the German Research Foundation (DFG) in the framework of

the German excellence initiative, and mobility grants from the German Academic Exchange Service (DAAD) and

Universities Australia (UA) scheme (#57389224).

Fig. 1: The IsoStretcher and the custom-made

silicone elastomer chamber

A: The IsoStretcher, a device to apply isotropic

stretch to living cells attached to an elastic

PDMS chamber. Optimized for microscopic studies,

it can easily be mounted on the microscope stage

of most commercial microscopes. B: Schematic

drawing of a cell (green) attached to a silicone

elastomer chamber (not to scale). The chamber is

mounted onto six pins (red circles) which are

driven by a stepper motor and move in radial

direction depicted by the red arrows. Movement

of the pins applies stretch to the chamber and

thus to the attached cell.

Fig. 2: In vitro stretch stimulation and calcium

imaging of model cells of the cardiovascular

system

A: Exemplary curves of two HL-1 cells stained

with the calcium indicator dye Fluo-4-AM

subjected to 15 % radial stretch after 2

minutes. B: Exemplary curves of two EA.hy926

cells stained with the calcium indicator dye

Cal-520-AM subjected to 19 % radial stretch

after 3 minutes. While the HL-1 cells show a

clear increase in fluorescence intensity and a

change in peak frequency of the calcium

fluctuations, the EA.hy926 cells show a short

transient increase in fluorescence followed by a

decrease of the signal.

References [1] Arganda-Carreras, I, Sorzano, COS, Marabini, R, Carazo, JM, Ortiz-de-Solórzano, C, Kybic, J. 2006,

'Consistent and Elastic Registration of Histological Sections using Vector-Spline Regularization', Lecture Notes in Computer Science, CVAMIA: Computer Vision Approaches to Medical Image Analysis, 4241, 85-95, Springer Berlin / Heidelberg

[2] Guo Y, Merten AL, Schöler U, Yu ZY, Cvetkovska J, Fatkin D, Feneley MP, Martinac B, Friedrich O. 2021, 'In vitro cell stretching technology (IsoStretcher) as an approach to unravel Piezo1-mediated cardiac mechanotransduction', Prog Biophys Mol Biol, 159, 22-33, https://doi.org/10.1016/j.pbiomolbio.2020.07.003


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B 11-03

Redox stimuli differentially module metabolic and transcriptomic

response by endothelial cells

Niklas Mueller, Kurt Noack, Timothy Warwick, Katrin Schröder, Ralf P. Brandes, Flávia Rezende

Goethe University Frankfurt, Cardiovascular Physiology, Frankfurt am Main HESSEN, Germany

Endothelial cells (EC) have a dynamic metabolism to attend their biosynthetic and bioenergetic demand as well as

to cope with environmental cues. The vascular milieu is rich in oxygen, and therefore, EC have a complex and

integrated metabolic and genetic program to sense and respond to oxidative stimuli. However, how this is

orchestrated is completely unknown. In this study we compared how EC integrate a redox and an energetic response

to diverse stimuli in a timely manner. For this, we treated HUVEC (human umbilical vein endothelial cells) with either

menadione (5µM) or H2O2 (10 and 300µM) but also developed a lentiviral chemogenetic system with DAO (D-amino

acid oxidase which converts D-amino acids into imino acids and produces H2O2 as byproduct ) to gradually release

H2O2 intracellularly. Cells were treated for 3, 10, 30, 90, 270 or 900 minutes and subjected to untargeted

metabolomics (LC/MS) and the later four time points to RNAseq. H2O2 at low concentration as well as D-alanine (D-

Ala) stimulation led to no changes in the metabolic response and just mild gene regulation. Instead, D-Ala treated

HUVEC show a gradual oxidation of peroxiredoxins to their dimers without overoxidation (SO3). These findings

suggest a very high reducing redox capacity of EC towards intracellular reactive oxygen species and large regulation

through peroxiredoxins.

Interestingly, H2O2 (300µM)and menadione induced the strongest energetic and redox responses. The first increased

Cys-GSH and GSSG (oxidized glutathione) ≥ 15 fold after ten minutes which was followed by an increase in S-lactoyl-

GSH and accompanied by a reduction in GSH (reduced glutathione). In contrast, menadione led to an acute and

very transient formation of only S-lactoyl-GSH after ten minutes. Regarding tri-carboxilic acid cycle, menadione

decreased isocitrate and likely re-wired glutamine to maintain α-ketoglutarate levels. Conversely, α-ketoglutarate was

largely reduced in H2O2 treated HUVEC. Gene expression changes induced by H2O2 (300µM) peaked at 270 minutes

with cell cycle and p53 signalling as the most down and upregulated, respectively. In conclusion, our study shows

that intracellular production of H2O2 by DAO expressing cells leads to minor metabolic and genetic responses,

whereas exogenous H2O2 and menadione induced a timely and pinpointed metabolic response in EC.


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B 11-04

Model based quantification of the extracellular nucleotide degradation

in smooth muscle cells, endothelial cells and whole aortic rings of rats

Arvid J. S. Pietsch, Peter Dieterich, Melanie Martin, Andreas Deußen

TU Dresden / Universitätsklinikum Dresden, Institut für Physiologie, Dresden, Germany

Question: Several diseases of ectopic calcification of vessels are associated with disturbances of components of

the extracellular nucleotide cascade. In view of this observation, it is the aim of this study to quantify the hydrolysis

of AMP to Adenosine for smooth muscle cells and endothelial cells of rats and compare the results with the

metabolism in ex vivo whole aortic rings of rats.

Methods: Etheno-AMP (eAMP) was given to cells and aortic rings in several initial concentration (1 - 50 µM). The

time course of the decay of eAMP to etheno Adenosine (eAdo) was quantified by HPLC over 3 hours with a time

interval of 15 min. Data were analyzed with models for enzymatically driven degradation. Model parameters and

model selection was performed by Bayesian data analysis.

Results: Bayesian data analysis showed that a simple Michaelis-Menten-Kinetics was suited best to capture the

measured biological system. As a result, we could estimate effective Michaelis-Menten constants Km and Vmax for

different experimental conditions: We obtained Km = 70 ± 10 µM and a very small Vmax = 0.023 ± 0.005 nmol/min for

rat aortic endothelial cells. By contrast, rat aortic smooth muscle cells showed a lower Km= 31 ± 17µM and higher

Vmax = 0.5 ± 0.2 nmol/min for eAMP degradation. The analysis of complete aorta rings of rats gave an increased Km

= 70 ± 10 µM and Vmax = 0.4 ± 0.1 nmol/min. The removal of the endothelium did not change Km = 76 ±13 µM

significantly, but increased Vmax = 0.6 ± 0.1 nmol/min slightly. To relate cell and aortic ring experiments, we assumed

that surfaces of the cell culture and the rat aortic ring represent the amount of enzyme. Taking into account about 20

layers of smooth muscle cells for the aorta ring, this scaling gave very similar Vmax values per cell area for both types

of experiments. The differences of Km -values between isolated smooth muscle cells and whole aorta might arise due

to diffusion limitation between layers of smooth muscle cells in the aortic rings resulting in an enhanced apparent Km

value for the tissue. Removal of the endothelium has very little effect on the degradation of AMP in rat aorta.

Conclusion: In rat aorta endothelial cells contribute very little to overall extracellular AMP degradation to adenosine.

AMP degradation is very active, however, in aortic smooth muscle. Further experiments must elucidate the

contribution of CD73 (ecto-5´-nucleotidase) and alkaline phosphatase in this process.


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B 11-05

The role of polyunsaturated fatty acids (PUFAs)in endothelial cell

specification

Giorgia Ciliberti1, Rushendhiran Kesavan1, Rüdiger Popp1, Timo Fromel1, Andreas Weigert2,

Ingrid Fleming1

1 Goethe University Frankfurt, Institute for Vascular Signaling, Center for Molecular Medicine, Frankfurt am Main,

Germany 2 Goethe University Frankfurt, Institute of Patho-Biochemistry, Frankfurt am Main, Germany

Polyunsaturated fatty acids (PUFAs) are important membrane components as well as precursors of signaling

molecule and have been shown to play an important role in the modulations of vascular relaxation, cardiac

performance, inflammation and angiogenesis. PUFAs are classified in ω-3 and ω-6 fatty acids which can be

converted into epoxides by cytochrome P450 (CYP) enzymes and further processed to vicinal diols by the soluble

epoxide hydrolase (sEH).

This study was designed to characterize the ability of PUFA-derived epoxides and diols to promote angiogenesis and

lymph-angiogenesis. In isolated aortic rings from 8-10 weeks old mice, a stronger proliferative response was

observed in sEH-/- animals compared to wild-type. Instead, no significant difference in lymphatic endothelial cell

(LEC) sprouting was observed in thoracic duct explants. A decreased lymphatic vasculature area was however

detected in ears of 10 weeks old sEH-/- mice. As our observations indicated that immature endothelial cells may

more responsive to epoxides and diols, specific epoxide and diol pairs were added to spheroid-based co-cultures of

breast cancer (PyMT) cells and mouse embryonic stem cells (mESCs). RT-qPCR and FACS analyses revealed that

ω-6 epoxides and ω-3 diols promoted the development of blood-endothelial cells as evidenced by the markers

EPHB4, VEGFR2/1 and PECAM1. An increase in LECs expressing the markers Prox-1, COUP-TFII and VEGFR3,

was detected in spheroids treated with ω-3 epoxides or ω-6 diols treatment.

In conclusion, our data suggest a differential effect of PUFA-derived CYP- and sEH– metabolites on endothelial cell

specification and vascular development. While ω-6 epoxides/ω-3 diols favor blood endothelial cell identity and

proliferation, -3 epoxides/ -6 diols promote lymphatic cell commitment of vascular progenitors and contribute to

lymphatic morphogenesis.


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B 11-06

Cytochrome P450 reductase prevents cardiac hypertrophy through

endothelial cell crosstalk

Melina Lopez1,2, Pedro F. Malacarne1,2, Deepak P. Ramanujam3, Jiong Hu2,4, Ingrid Fleming2,4,

Stefan Engelhardt3, Ralf Brandes1,2, Flávia Rezende1,2

1 Goethe-University, Institute for Cardiovascular Physiology, Frankfurt am Main, Germany 2 German Center of Cardiovascular Research (DZHK), Partner Site Rhein Main, Frankfurt am Main, Germany 3 Technical University, Institute of Pharmacology and Toxicology, Münich, Germany 4 Goethe-University, Institute for Vascular Signaling, Frankfurt am Main, Germany

The cytochrome P450 reductase (POR) transfers electrons and thus drives the function of all cytochrome P450-

dependent enzymes (CYP450). In the vascular system, the CYP450-POR system has been linked to vascular

epoxyeicosatrienoic acids (EET) production and endothelial function. The importance of the CYP450 system in the

heart is complexe and cell-specific. The aim of the present study was to determine the role of endothelial POR in the

mouse heart, using a tissue-specific deletion approach. RNAseq revealed that cardiac endothelial cells expresse

numerous CYPs, which have been linked to different functions: CYP2 family members produce EETs, whereas CYP4

family generated omega fatty acids. Cardiac hypertrophy induced by transverse aortic constriction greatly increased

endothelial CYP4b1 and POR expression. Endothelial-specific, tamoxifen-inducible POR knockout mice (ecPOR-/-)

show endothelial dysfunction and increased blood pressure in AngII-induced hypertonie. Strikingly, these mice also

exhibit heart hypertrophy, an increase in cardiomyocyte area, a reduced ejection fraction and an increased left

ventricle size during systole (echocardiography) as compared to WT animals. Arotic RNAseq revealed a significant

increase in genes annotated to electron transport chain and thermogenesis (Cox8b, Cox7a1) and hypertrophic

cardiomyopathy (lamin and tropomyosin) in response to POR deletion. Collectively, our data suggests that

endothelial cells support adjacent cells with fatty acids which are the major energetic source of cardiomyocytes. In

the absence of POR this endothelial function is compromised leading to negative cardiac metabolic remodeling.


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B 11-07

The role of sEH related lipid mediatiors on Pericyte-Endothelial Cell

Junctions of the diabetic heart

Sebastian Kempf1,2, Xiaoming Li1,2, Stephan Klatt1,2, Jiong Hu1,2, Ingrid Fleming1,2

1 Goethe University, Institute for Vascular Signalling, Frankfurt am Main, Germany 2 Partner Site Rhein-Main, German Center of Cardiovascular Research (DZHK), Frankfurt am Main, Germany

Polyunsaturated fatty acid (PUFA) metabolites generated by the sequential action of cytochrome P450 (CYP) and

soluble epoxide hydrolase (sEH) enzymes are important modulators of vascular function and integrity. Given the

reported role of the sEH in regulating vascular stability in the retina, this study aimed to explore the effects of the

PUFA metabolites generated by CYP/sEH on pericyte-endothelial cell junctions and to characterize the effects of

sEH on vessel density during the development of diabetic cardiomyopathy.

A comparison of wild-type mice and animals with type1 diabetes (Ins2Akita mice), revealed higher cardiomyocyte

expression of the sEH in the diabetic group. The diabetic mice also displayed characteristics of heart failure with

preserved ejection fraction (HFpEF) at the age of 6 months, and developed heart failure with reduced ejection fraction

(HFrEF) by 12 months. Immunohistochemistry revealed a significant increase in left ventricular capillary density in

12-month-old Ins2Akita mice versus their non-diabetic littermates. However, pericyte coverage was markedly

reduced in the diabetic mice. Consistent with the change in sEH expression, fatty acid profiling of the left ventricle

reveled increased levels of sEH-derived PUFA diols, e.g. 12,13-dihydroxy-octadecenoic acid (12,13-DiHOME) and

14,15-dihydroxyeicosatrienoic acid (14,15-DHET) in the diabetic group. In vitro studies with 12,13-DiHOME revealed

that it targeted endothelial cell-endothelial cell interactions to disrupt barrier function and increase permeability.

Similarly, 12,13-DiHOME disrupted pericyte–endothelial cell junctions, and induce on pericyte migration.Taken

together our data indicate that the increase in sEH expression in cardiomyocytes of the diabetic heart increases the

generation of 12,13-DiHOME, which disrupts endothelial cell-pericyte junctions and contributes to a decrease in

vascular pericyte coverage. Studies are ongoing to determine the consequences of sEH inhibition on pericyte

coverage and vascular density in the diabetic heart.

Acknowledgment Deutsche Forschungsgemeinschaft SFB 834/B13


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B 11-08

RNA:DNA:DNA triple helix structures are important for human

endothelial cells undergoing endothelial-to-mesenchymal transition

Sandra Seredinski1, Timothy Warwick1, Stefan Guenther2, Alessandro Bonetti3,4, Ralf Gilsbach1,

Matthias S. Leisegang1, Ralf P. Brandes1

1 Goethe University, Institute for Cardiovascular Physiology, Frankfurt, Germany 2 Max-Planck-Institute for Heart and Lung Research, ECCPS Bioinformatics and Sequencing Facility, Bad

Nauheim, Germany 3 AstraZeneca, Translational Genomics, Discovery Science, Mölndal, Sweden 4 Yokohama City University, RIKEN Yokohama Institute, Yokohama, Japan

Objective: Formation of RNA:DNA:DNA triple helix structures are known for more than 50 years. However, little is

known about their function in living cells. Long non-coding RNAs (lncRNAs) are involved in gene regulation

mechanisms and for some of them triplex formation has been shown. We propose that lncRNA-mediated

RNA:DNA:DNA triplices are important for endothelial physiology and gene expression regulation. Therefore we

investigate genome-wide triplex-mediated gene regulation by characterizing the first endothelial triplexome in human

endothelial cells undergoing endothelial-to-mesenchymal transition (EndMT).

Results: Thousands of triplex-associated RNAs were enriched after immunoprecipitation with anti-dsDNA antibody

in a triplex-sequencing approach. RNA candidates forming these triplices varied strongly between native and EndMT-

undergoing endothelial cells. Surprisingly, lncRNAs as well as mRNAs were involved. In silico prediction of triplex-

forming RNAs and potential triplex target sites revealed a huge number of potential triplex-regulated genes. Using

an alternative approach, where RNA and DNA interacting complexes were ligated and sequenced (RADICL-seq),

we could confirm many of the predicted RNA-DNA interactions. Nuclear RNA-Seq as well as an assay for

transposase accessible chromatin (ATAC-Seq) yielded that specific lncRNA candidates performing triplex formation

could potentially alter gene expression of their target genes and play a role in EndMT.

Conclusion: The data indicate that triplices strongly contribute to gene regulation. The triplexomes are changing in

the course of EndMT. This suggests that RNA:DNA:DNA triplices play an important role in physiological processes

as well as related diseases.


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B 11-09

The role of endothelial NFAT5 in the context of cerebral ischemia

Paul Wacker, Reiner Kunze, Thomas Korff


Local cerebral hypoperfusion causes ischemic stroke leading to a critical shortage of oxygen, glucose and further

nutrients in the affected brain area. Ischemic stroke is associated with a loss of blood-brain barrier integrity

predominantly due to structural breakdown of interendothelial junctions. Moreover, adaptive angiogenic responses

in vascular endothelial cells contribute to brain tissue repair, as well as functional recovery. The nuclear factor of

activated T cells 5 (NFAT5), also known as a tonicity-responsive enhancer-binding protein (TonEBP), is a

transcription factor which is induced by different stress-related stimuli such as hypoxia, osmotic stress and pro-

inflammatory cytokines. This project aims to investigate the impact of endothelial cell-specific knockout of NFAT5 on

the outcome from ischemic stroke in mice.

Endothelial cell-specific NFAT5 knockout mice (NFAT5(EC)-/-) and NFAT5fl/fl littermates were subjected to either

transient middle cerebral artery occlusion (MCAO) or sham surgery followed by 1 or 28 d of reperfusion. Sensorimotor

function was determined by neurological deficit scoring and Rotarod motor performance test. Cresyl violet staining

was used to quantify infarct lesion size and brain swelling. Moreover, we conducted immunofluorescent analyses to

estimate apoptosis, neuronal degeneration and angiogenic processes.

Upon acute stroke, NFAT5(EC)-/- mice showed an increased infarct size and worsened neurological impairment,

whereas total numbers of apoptotic cells and degenerated neurons were not different in comparison to NFAT5fl/fl

littermates. During the post-acute stage of stroke, NFAT5(EC)-/- mice exhibited more pronounced sensorimotor deficits

and enhanced brain atrophy as compared to NFAT5fl/fl mice. Four weeks upon onset of cerebral ischemia, the density

of total and lectin-perfused microvessels as well as the number of proliferating endothelial cells across the infarct

lesion were comparable between NFAT5(EC)-/- and NFAT5fl/fl mice, which, however, argues against a substantial role

of endothelial NFAT5 for adaptive post-ischemic angiogenesis.

Based on these initial findings, we hypothesize that NFAT5 acts as a stress-responsive transcription factor in

endothelial cells during ischemic stroke. Further studies will be conducted to identify NFAT5 target genes that are

involved in the endothelial cell response to ischemic stress.


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B 11-10

Zinc inhibits high glucose-induced osteogenic transdifferentiation and

calcification of vascular smooth muscle cells via NF-kB suppression

Ioana Alesutan1, Laura Henze2, Misael Estepa2, Burkert Pieske2,5, Florian Lang4, Kai U. Eckardt3,

Jakob Voelkl1,3,5

1 Johannes Kepler University, Institute for Physiology and Pathophysiology, Linz, Austria 2 Charité – Universitätsmedizin, Department of Internal Medicine and Cardiology, Berlin, Germany 3 Charité – Universitätsmedizin, Department of Nephrology and Medical Intensive Care, Berlin, Germany 4 Eberhard Karls University Tübingen, Department of Vegetative and Clinical Physiology, Tübingen, Germany 5 DZHK, Berlin, Germany

Question: Medial vascular calcification is accelerated and associated with high mortality in diabetes mellitus patients.

Reduced serum zinc levels are frequently observed in these patients and are correlated with serum calcification

propensity, showing an increased risk for calcification. Vascular calcification is an active process involving osteogenic

transdifferentiation of vascular smooth muscle cells (VSMCs). The present study investigated the possible direct

effects of zinc supplementation on osteogenic transdifferentiation and calcification of VSMCs during hyperglycemic

conditions.

Methods: Experiments were performed in primary human aortic VSMCs exposed to excessive glucose

concentrations without and with additional zinc supplementation.

Results: Zinc treatment suppressed high glucose-induced expression of osteogenic markers core-binding factor

alpha-1 (CBFA1) and tissue-nonspecific alkaline phosphatase (ALPL) in VSMCs. Moreover, zinc blunted the

enhanced VSMC calcification induced by high glucose during pro-calcifying conditions. High glucose exposure

activated NF-kB in VSMCs, effects inhibited in the presence of zinc. Zinc treatment increased the expression of TNF

alpha-induced protein 3 (TNFAIP3), a NF-kB pathway suppressor, while knockdown of TNFAIP3 abolished the

inhibitory effects of zinc on high glucose-induced osteogenic markers expression in VSMCs. In addition, the effects

of zinc on TNFAIP3 as well as osteogenic markers expression during exposure to excessive glucose levels were

abolished by silencing of the zinc-sensing receptor G protein-coupled receptor 39 (GPR39).

Conclusions: Zinc inhibits osteogenic transdifferentiation and calcification of VSMCs triggered by exposure to high

glucose concentrations, effects involving GPR39-dependent up-regulation of TNFAIP3 and NF-kB suppression.

Thus, zinc supplementation may reduce the progression of vascular calcification during hyperglycemic conditions.


of 516

B 11-11

Effect of two renin isoforms on cardiomyoblast survival under

starvation conditions

Heike Wanka1, Philipp Lutze2, Alexander Albers1, Janine Golchert1, Doreen Staar1, Jörg Peters1

1 University Medicine Greifswald, Institute of Physiology, Greifswald, Germany 2 Leibnitz Institute for Farm Animal Biology, Institute of Muscle Biology & Growth, Dummerstorf, Germany

Question: Renin transcripts encoding a cytosolic renin isoform (renin-b) have been discovered that may in contrast

to the commonly known secretory renin (renin-a) exert protective effects. Here, we compared the effect of renin-b

and renin-a overexpression in H9c2 cardiomyoblasts on apoptosis and necrosis as well as on potential mechanisms

involved in cell death processes.

Methods: To mimic ischemic conditions, cardiac H9c2 cells transfected with an empty pIRES vector or an vector

containing renin-a or renin-b were exposed to glucose starvation or combined oxygen-glucose starvation (OGD) for

24 h. Then apoptosis rate (CaspACE+ cells, Annexin V+ cells), necrosis rate (ratio LDH release to LDH content), ROS

accumulation (MitoSOX+ cells) and mitochondrial membran potential ∆Ψm (JC1 Ratio red/green FLI) as well as ATP

content were determined.

Results: Under glucose starvation or OGD, control pIRES cells exhibited markedly increased necrotic and apoptotic

cell death accompanied by enhanced ROS accumulation, loss of mitochondrial membrane potential and decreased

ATP levels. In renin-a cells, the effects of starvation were similar or even exaggerated, while renin-b cells were

markedly protected from OGD-induced damaget as demonstrated in the table.

Conclusion: From these data we conclude that renin-b overexpression mediates cardioprotective effects under

glucose depletion and OGD. Since glucose depletion or OGD markedly increased the expression of renin-b in non-

transfected H9c2 cells and renin-a overexpression appeared to be harmful, our data suggest that an endogenous

system exist based on the expression of renin-b that protects cells from glucose depletion or OGD induced cell death.

Effects of glucose starvation or OGD on H9c2

cells overexpressing renin-a or renin-b.. Effects of renin-b or renin-a overexpression in

H9c2 cardiomyoblasts on apoptosis and necroasis

as well as on potential mechanisms involved in

the induction of cell death (ROS accumulation,

breakdown of mitochondrial membrane potential,

ATP depletion). Cardiac cells were exposed to

glucose starvation and oxygen-glucose depletion

for 24 h.


of 516

B 11-12

Governing of the endothelial cell phenotype by the highly endothelial-

specific long non-coding RNA LINC00607

Frederike M. Boos1,2,5, James A. Oo1,2,5, Ilka Wittig2,3, Stefan Günther4, Matthias S. Leisegang1,2,5,

Ralf P. Brandes1,2,5

1 Goethe University, Institute for Cardiovascular Physiology, Vascular Research Centre, Frankfurt am Main,

Germany 2 German Center of Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany 3 Goethe University, Functional Proteomics, Frankfurt am Main, Germany 4 Max-Planck-Institute for Heart and Lung Research, ECCPS Bioinformatics and Sequencing Facility, Bad

Nauheim, Germany 5 Goethe University, Cardio-Pulmonary Institute (CPI), Frankfurt am Main, Germany

Background: Long non-coding RNAs (lncRNAs) are described as molecular switches in cellular differentiation,

movement and in the reprogramming of cell states by altering gene expression patterns. However, in endothelial

cells their role is not well understood. We identified LINC00607 as a novel and functionally important endothelial-

enriched lncRNA governing the endothelial phenotype.

Results: Among the ten highest expressed lncRNAs in endothelial cells is LINC00607, as shown by deep-endothelial

RNA-seq and FANTOM5 search. Silencing of LINC00607 with siRNAs or LentiCRISPR-knockout in human

endothelial cells increased cellular proliferation and inhibited angiogenic sprouting in response to VEGF-A. Anti-

sense oligo pulldown of the predominantly nuclear lncRNA followed by Mass spectrometry revealed the chromatin

remodeler SMARCA3 and the DNA-binding protein SSBP1 as protein interaction partners. Chromatin remodeling is

a dynamic epigenetic process leading to modifications in chromatin architecture. Thereby altering the access of the

transcriptional machinery to the chromatin. CRISPR-mediated endothelial cell knockout of LINC00607 followed by

RNA-Seq revealed important phenotypic cues for LINC00607: Genes related to endothelial function including FLT1,

Cadherins, PECAM1 and VWF, were strongly downregulated, whereas genes involved in TGFß- and NOTCH-

signaling pathways were upregulated upon loss of LINC00607. The results indicate that LINC00607 might function

as a guide for SMARCA3 and SSBP1 them to specific target sites to maintain endothelial function.

Conclusions: LINC00607 is a highly expressed endothelial-specific lncRNA important for vascular phenotype

control. The lncRNA appears to have a scaffolding function within the SMARCA3 chromatin-remodeling complex,

facilitating and leading efficient key endothelial gene transcription to control the endothelial phenotype.


of 516


Foyers

B 12 | Cardiac Physiology II Chair

Andre Heinen (Düsseldorf)

Zhihong Yang (Fribourg)


of 516

B 12-01

Cardiac p38 MAPKα is an Essential Mediator of Metabolic Signaling for

Adapting to pressure overload

Lisa Kalfhues1, Anne Hemmers1, Katharina Bottermann1, Vici Oenarto1, Chantel Galang1, Thurl Harris2,

Axel Gödecke1

1 Heinrich-Heine University Düsseldorf, Institute of Cardiovascular Physiology, Düsseldorf, Germany 2 University of Virginia, Department of Pharmacology, Charlottesville, USA

Question: Cardiac metabolic remodeling is one of the early changes driving the progression of heart failure and is

present even before overt cardiac dysfunction. We previously showed that the tamoxifen inducible deletion of cardiac

p38 MAPKα (KO) leads to a pronounced left ventricular dilation with a strongly impaired heart function only after 2

days of angiotensin (ANGII) treatment (EF [%]: KO 34 ±7; Ctrl 56 ±11). Additionally, these hearts are insulin resistant

and developing a metabolic dysfunction causing impaired energy supply.

Methods and Results: After 2 days of ANGII exposure KO mice exhibit enhanced cardiac AKT phosphorylation.

Activated AKT reduced activating AMPKT172 and increased inhibitory AMPKS485 phosphorylation shifting AMPK to the

inactive form which might contribute to the reduced energy supply in KO hearts. The increased AKT phosphorylation

could be explained by strongly increased plasma insulin levels ([µg/L]: KO 3.4 ±2.5; Ctrl 0.3 ±0.1), whereas blood

glucose levels remained unaltered ([mg/dl]: KO 95 ±21; Ctrl 86 ±15). Interestingly, in AKT1 and AKT2 KO hearts the

insulin mediated AMPK inhibition was not found underlining the importance of AKT in AMPK inhibition.

The increased insulin levels in KO hearts suggests a crosstalk between the stressed heart and pancreatic β-cells.

Western Blot analysis revealed a significantly reduced insulin amount (to app. 33 %) in β-cells, however, transcript

expression analyses exhibit no changes in Ins1 and Ins2 levels. These results pointing to a potential excessive insulin

secretion from pancreatic islets.

To investigate a longer-term effect on these hearts, mice were treated with ANGII for 3 and 7 days, respectively. First

results showed that heart function was strongly diminished after 3 days of ANGII exposure (EF [%]: KO 35 ±14; Ctrl

58 ±12) and interestingly largely recovered within 12 days (EF [%]: KO 45 ±6; Ctrl 55 ±4). 7 days of ANGII treatment

led to a sustained impaired cardiac performance in KO animals (EF [%]: KO 42 ±15; Ctrl 73 ±6), whereby recovery

experiments are still ongoing.

Conclusion: Cardiomyocyte specific p38 MAPKα KO mice develop hyperinsulinemia, cardiac insulin resistance and

altered metabolic signaling. These alterations are hallmarks of a diabetic cardiomyopathy, which promotes cardiac

failure under pressure overload. Thus, cardiac p38 MAPKα is a key regulator for metabolic signaling and potentially

for the interorgan communication between the failing heart and the pancreas.


of 516

B 12-02

Identification and functional epigenetic modulation of regulatory

elements in cardiac myocytes

Patrick Laurette1,2, Martin Schwaderer3, Núria Díaz i Pedrosa1,2, Rebecca Bednarz1,2, Ralf Gilsbach1,2

1 Institute of Cardiovascular Physiology, Goethe University Frankfurt, Frankfurt am Main, Germany 2 German Center of Cardiovascular Research, Partner site RheinMain, Frankfurt am Main, Germany 3 Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg, Germany

Background: Epigenome studies in cardiac myocytes revealed dynamic establishment of active regulatory sites

during development and disease (Gilsbach et al., Nat. commun. 2018). The aim of this project was to identify target

promoters of distal regulatory sites associated with Gata4 and assess their functional relevance.

Methods: We performed Hi-ChIP, Gata4-ChIP, and epigenomic mapping experiments to annotate Gata4-positive

regulatory elements and their interacting target genes in murine cardiac myocytes (HL-1 cells). We then combined

pooled CRISPR interference of Gata4-bound enhancers with single-cell transcriptome sequencing to dissect the

contribution of hundreds of these enhancers to the expression of interacting target genes simultaneously. ChIP-seq,

RNA-seq and Western Blot analysis were carried out to confirm the functional consequences of selected promoter

and enhancer perturbations.

Results: Our ChIP experiments characterized the epigenome of HL1 cells and revealed that Gata4 binding is highly

enriched on strong enhancers. Gata4 Hi-ChIP showed that these enhancers are associated with a significant number

of putative target gene promoters. The pooled CRISPRi assay allowed us to screen the functionality of these

enhancer-gene interactions with high throughput. Among those, we identified a strong Gata4-bound enhancer on

chromosome 18, which physically interacts with the 320kb distant promoter of Gata6, a key cardiac transcription

factor implicated in pathological cardiac hypertrophy.

CRISPRi mediated perturbation of this enhancer induced a reduction in Gata6 gene expression, protein levels and

genome-wide binding as well as more than 600 genes significantly associated with cardiovascular developmental

and stress processes.

Conclusion: This study unraveled enhancer-promoter interactions in cardiac myocytes and showed that functional

(epi)genetic-modulation of distal regulatory elements allows steering of gene expression programs. Furthermore, this

project underlines the importance of lineage-specific cis-regulatory elements at the crossroads of cardiac

transcription factor networks in the regulation of the cardiac myocytes transcriptome.


of 516

B 12-03

Store-operated calcium entry increases nuclear calcium concentration

in rat ventricular myocytes

Julia Hermes, Jens Kockskämper

University of Marburg, Institute of Pharmacology and Clinical Pharmacy, Marburg, Germany

Question: Store-operated calcium (Ca) entry (SOCE) via TRPC and Orai/Stim proteins may contribute to cardiac

remodeling during hypertrophy and heart failure. Transcription is regulated, in part, via Ca-dependent mechanisms.

It is unknown, however, if and how SOCE may alter intranuclear Ca concentration in cardiomyocytes. Hence, we

developed a protocol to elicit SOCE in adult cardiomyocytes and characterized its impact on nuclear Ca regulation.

Methods: Isolated rat ventricular myocytes were loaded with Fluo-4/AM (8μM, 20-30min) and electrically-stimulated

at 1Hz. Cytoplasmic and nuclear Ca transients (CaTs) were imaged simultaneously using linescan confocal

microscopy. Afterwards, CaTs were eliminated by zero Ca solution containing thapsigargin (0.5μM), verapamil (5μM)

and KB-R7943 (10μM) to block sarcoplasmic reticulum (SR)-Ca-ATPase, L-type Ca channels and Na-Ca exchange,

respectively. Next, the SR was depleted of remaining Ca by two bolus applications of caffeine. Finally, Ca (2mM)

was added to elicit SOCE.

Results: Using this protocol, re-addition of Ca (2mM) elicited a slow Ca increase in the cytoplasm and nucleus, i.e.

SOCE. In a total of 39 cells, SOCE amounted to 0.64±0.07 ΔF/Frest in cytoplasm vs 0.70±0.08 ΔF/Frest in nucleus

(P<0.01) or to 23±3% in cytoplasm vs 41±6% in nucleus (P<0.001) when normalized to the CaT in the very same

cell. There was a linear correlation between the amplitude of SOCE in the nucleus and cytoplasm with a slope of

1.13 (r=9607, P<0.001). Gadolinium (1mM, n=45) greatly reduced SOCE both in cytoplasm (to 0.18±0.03 ΔF/Frest)

and nucleus (to 0.19±0.03 ΔF/Frest) (both P<0.001 vs ctrl). S66 (1μM, n=26) or BTP-2 (3μM, n=36), inhibitors of

Orai/Stim, reduced SOCE by 40% and 45% in cytoplasm and by 40% and 51% in nucleus, respectively. SKF96365

(5μM, n=28), an inhibitor of TRPC channels, reduced SOCE by 50% in cytoplasm and by 57% in nucleus. Despite

large reduction of electrically-stimulated CaTs, SOCE persisted in detubulated ventricular myocytes (n=84)

amounting to 0.56±0.04 ΔF/Frest in cytoplasm and 0.58±0.04 ΔF/Frest in nucleus.

Conclusion: Following block of Ca influx and SR function, SOCE can be observed upon re-addition of Ca in Ca-

depleted adult ventricular myocytes. SOCE is mediated by Orai/Stim- and TRPC-dependent Ca influx. Nuclear SOCE

(1) is comparably large as and (2) appears to depend on cytoplasmic SOCE and (3) does not require T-tubules. The

results suggest that SOCE may increase nuclear Ca and alter transcription.


of 516

B 12-04

Decelerated calcium handling in SPRED2-deficient cardiomyocytes

Hannes Häbich1, Melanie Ullrich1, Marco Abeßer1, Michael Kohlhaas2, Alexander Nickel2, Christoph

Maack2, Sina Gredy1, Kai Schuh1

1 University of Würzburg, Institute für Vegetative Phyiology, Würzburg, Germany 2 University Hospital Würzburg, Comprehensive Heart Failure Center, Würzburg, Germany

SPRED proteins are known inhibitors of the MAPK-signaling pathway, a major regulator of cell proliferation and

differentiation. SPRED2-/- mice show multiple signs of cardiac arrhythmias, hypertrophy and heart failure. It has also

been shown that the absence of SPRED2 leads to impaired autophagy. This results in an intracellular accumulation

of vacuolar structures and non-functional mitochondria. Immunohistological staining revealed accumulation of

autophagosomal and lysosomal markers, which was confirmed quantitatively at the protein level.

To identify the connection between affected signaling pathways and arrhythmias, we examined expression and

function of potentially involved proteins in isolated cardiomyocytes. We used Western Blots to quantify proteins and

their phosphorylation status. Membrane potentials and intracellular calcium was measured with the dyes di-8-

ANEPPS and Indo in an IonOptix system. Mitochondrial function was analyzed with an Oroboros FluoRespirometer.

We found that expression of CaV1.2 (n(WT)=17, n(KO)=16, p=0.0097) and SERCA2a (n(WT)=10, n(KO)=8,

p=0.0029) was reduced in SPRED2-/- cardiomyocytes. The expression of Nav1.5, NCX, CaMK and P-CaMK was not

significantly changed. At 1 and 2 Hz stimulation frequencies, the action potential rise was faster, i.e. about 25% of

the time to peak of WT cardiomyocytes (1Hz: n(WT)=67, n(KO)=68, p=0,0085) and the action potential maximum

amplitude was 13% higher than in WT (n(WT)=67, n(KO)=68, p=0.0033). The repolarization time to 90% of the resting

membrane potential at 1 Hz stimulation frequency was prolonged by 33% (n(WT)=67, n(KO)=68, p=0.0064) and the

SPRED2-/- sarcomeres were approx. 1.5% longer at the systole (n(WT)=73, n(KO)=62, p=0.0070). Diastolic calcium

content was 8.5% lower in SPRED2-/- mice (n(WT)=73, n(KO)=62, p=0.0004). Functional mitochondrial analysis

showed no significant difference.From these data we concluded that the ion channels CaV1.2 and SERCA2a as well

as the intracellular calcium are reduced in cardiomyocytes of SPRED2-/- mice. Reduced intracellular calcium leads

to weaker pre-tensioning of the sarcomeres, which corresponds to the measured difference in length and may also

trigger the arrhythmias. Further, we concluded that the altered expression of proteins and the consequences for

cardiomyocyte function are a consequence of SPRED2-dependent MAPK pathway dysregulation.

Acknowledgment We thank Alice Schaaf, Michelle Gulentz and Annette Berbner for excellent technical assistance.

KS is supported by the DFG (SCHU1600/6-1).

References [1] Ullrich, M., 2019, ‘SPRED2 deficiency elicits cardiac arrhythmias and premature death via impaired

autophagy’, Journal of Molecular and Cellular Cardiology, 129:13-26, PubMed, https://pubmed.ncbi.nlm.nih.gov/30771306/


of 516

B 12-05

Decoding of promoter-enhancer interactions in human cardiac

myocytes

Rebecca Bednarz1,2, Patrick Laurette1,2, Katrin Streckfuß-Bömeke3,4, Ralf Gilsbach1,2

1 Goethe University, Institute of Cardiovascular Physiology, Frankfurt am Main, Germany 2 DZHK Rhein Main, Frankfurt am Main, Germany 3 University Medicine Göttingen, Cardiology and Pneumology, Göttingen, Germany 4 University of Würzburg, Institute of Pharmacology and Toxicology, Würzburg, Germany



of 516

B 12-06

Analysis of signaling defects in BAG3P209L- myofibrillar myopathy

Kerstin Klemm1, Luke M. Judge2,3, Bruce R. Conklin4,3, Bernd K. Fleischmann1, Michael Hesse1

1 University of Bonn, Medical Faculty, Institute of Physiology I, Bonn, Germany 2 University of California, Department of Pediatrics, San Francisco, USA 3 Gladstone Institutes, San Francisco, USA 4 University of California, Department of Medical Genetics and Molecular Pharmacology, San Francisco, USA

Patients with an amino acid exchange (P209L) in the BAG3 gene suffer from severe myofibrillar myopathy and/or

restrictive cardiomyopathy. The average life expectancy is only about 20 years. BAG3 plays a key role in the turnover

of muscle-proteins as a member of the CASA (chaperone-assisted selected autophagy) complex. Our goal is to gain

a mechanistic understanding of BAG3-associated muscle diseases.

To this end, we generated a human induced pluripotent stem cell (iPSC) line harboring the BAG3P209L mutation as

well as a BAG3 -/- from a well-characterized iPSC line by gene editing. Furthermore, we analysed a patient-specific

iPSC line derived from a patient with BAG3P209L-myofibrillar myopathy. All iPSC lines were successfully differentiated

into twitching skeletal muscle cells using an established differentiation protocol and also phenotypically analysed.

Interestingly, both the deletion of the BAG3 gene and the BAG3P209L mutation appear to result in morphological

abnormalities, such as the formation of thinner skeletal myotubes and an increase in apoptosis compared with

myotubes derived from control iPSCs. Initial stainings for structural components and members of the protein quality

control system also suggest differences. We are currently investigating the mechanisms underlying the disrupted

skeletal myocyte phenotype in the disease cell lines.

Acknowledgment K. Klemm is supported by the GRK 1873 - funded by the German Research Foundation (DFG).


of 516

B 12-07

Promoter-enhancer interactions in cardiac fibroblasts

Lisa M. Weiss1,2, Rebecca Bednarz1,2, Ralf Gilsbach1,2

1 Goethe University, Institute for Cardiovascular Physiology, Frankfurt, Germany 2 Partner site RheinMain, German Center of Cardiovascular Research (DZHK), Frankfurt, Germany

Question/Background

The heart is a heterocellular organ. Main cardiac cell types include cardiac myocytes, endothelial cells and fibroblasts

(FB). These different cell types express cell type-specific as well as common genes. Cell type-specific gene

expression is controlled by regulatory elements including distal transcription factor binding sites, which enhance gene

expression (enhancers). Enhancers interact with their target gene promoters via long-range chromatin interactions.

These chromatin interactions occur within topologically associated domains (TAD). However, the functional spatial

organization of cardiac FB is yet to be unraveled.

Methods

We used a transgenic approach to lineage trace FB nuclei, specifically a Tcf21-Cre and an inducible nuclear GFP-

reporter. This approach allowed fluorescent-activated sorting of FB nuclei. These nuclei were used for chromatin

interaction analysis using Hi-C and chromatin accessibility analysis (ATAC). With sorted FB we performed gene

expression analysis (RNA-seq). ATAC data was used to annotate enhancers and chromatin interaction analysis was

applied to link enhancers to their target genes. These data were compared to data obtained for non-FB.

Result

Chromatin interaction analysis of FB revealed specific promoter enhancer interactions and higher order chromatin

organization (TADs). Specifically, higher chromatin analysis showed that Postn, which is involved in the activation of

FB to myofibroblasts, is located in a 400kb TAD in FB. A comparison of FB and non-FB ATAC data shows that this

TAD contains FB-specific enhancers. Chromatin interaction data show a significantly interaction (p<0.05) between

the Postn promoter and an enhancer region located 350kb downstream. Our whole genome analysis further identified

FB-specific interactions at the Col1a1 and Col1a2 loci, which orchestrated with FB gene expression.

Conclusion

Here we provide a whole genome annotation of regulatory elements and spatial genome organization in cardiac

fibroblasts. These data identify and link regulatory elements to their respective target genes in cardiac fibroblasts.


of 516

B 12-08

Mitochondrial calcium transients in rat atrial myocytes show

frequency-dependent regulation and subcellular differences

Victoria Hammer, Carsten Culmsee, Jens Kockskämper

University of Marburg, Institute of Pharmacology and Clinical Pharmacy, Marburg, Germany

Question: Mitochondrial calcium (mitoCa) is an important regulator of cardiac metabolism. Moreover, Ca uptake by

mitochondria may affect cardiac Ca signaling and arrhythmogenesis. Our aim was (1) to establish reliable

measurements of mitoCa and (2) to characterize regulation of mitoCa transients in atrial myocytes.

Methods: Atrial myocytes were isolated from 12-16 weeks old rats. MitoCa or cytoplasmic Ca (cytoCa) was

measured in cells loaded with x-Rhod-1-AM (5 μM, 30 min) or Fluo-4-AM (8 μM, 20 min), respectively. Confocal

linescan imaging was employed to visualize subcellular Ca transients (CaTs).

Results: Following CoCl2 quenching of cytoplasmic x-Rhod-1 fluorescence, atrial myocytes showed a striped pattern

of x-Rhod-1 fluorescence coinciding with mitochondria as evidenced by co-staining with MitoTracker Green. Electrical

stimulation (0.5 Hz) of atrial myocytes (n=15) after CoCl2 quenching revealed transient increases of mitoCa with an

amplitude of 0.39±0.06 ΔF/Frest, rise time of 64±7 ms and tau of decay of 92±10 ms. Treatment of atrial myocytes

with the actin blocker cytochalasin D (10 μM, 12 min) greatly reduced cell shortening. MitoCaTs in the presence of

cytochalasin D were similar to the transients in its absence. Increasing stimulation frequency (from 0.5 to 1.0 to 2.0

Hz) caused a progressive increase in diastolic mitoCa, a decrease of the amplitude of mitoCaTs and acceleration of

mitoCaT decay. CytoCaTs (Fluo-4) showed similar frequency-dependent behavior. Comparison of central vs

subsarcolemmal mitoCaTs (n=37) at 0.5 Hz stimulation revealed higher diastolic Ca (0.97±0.01 (ct) vs 0.93±0.02 (ss)

F0/Frest, p=0.02), lower systolic Ca (1.28±0.03 (ct) vs 1.43±0.06 (ss) F/Frest, p<0.01) and lower amplitude of CaTs

(0.31±0.02 (ct) vs 0.50±0.05 (ss) ΔF/Frest, p<0.01) in central vs subsarcolemmal mitochondria. Again, cytoCaTs

exhibited similar subcellular differences.

Conclusion: x-Rhod-1-AM loading followed by CoCl2 quenching enables reliable measurements of mitoCaTs in

electrically-stimulated rat atrial myocytes. MitoCaTs exhibit marked frequency-dependent alterations and subcellular

differences similar to cytoCaTs suggesting that cytoCaTs are an important determinant of mitoCaTs.


of 516

B 12-09

Membrane remodeling and maturation of Ca2+ handling in 3D-shaped

hiPSC-cardiomyocytes

Fatemeh Kermani1, Markus Hecker1,2, Nina D. Ullrich1,2

1 Heidelberg University, Institute of Physiology and Pathophysiology, Heidelberg, Germany 2 German Center for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim, Heidelberg, Germany

Human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)present an immature phenotype, which

structurally and functionally differs from adult CMs. Specifically, hiPSC-CMs do not form any transverse membrane

(t-) tubules essential for the development of efficient excitation-contraction (EC-) coupling. As a consequence, hiPSC-

CMs exhibit pronounced spontaneous Ca2+ release events indicative of immature Ca2+ handling. Several

experimental strategies are currently examined to trigger maturation processes and achieve better Ca2+ handling in

these cells. Here we test the hypothesis that combination of structural and molecular remodeling can synergistically

alter the microarchitecture with the aim to improve Ca2+ signaling and EC-coupling in these novel CMs.

After differentiation, single hiPSC-CMs were cultured in laser-written three-dimensional (3D) microscaffolds

mimicking the cuboid form of adult CMs. In parallel, the scaffolding protein Bin1, a promising candidate for the

induction of t-tubules, was expressed by AAV6-mediated gene transduction; dsRed served as indicator for positive

expression of Bin1-containing and empty control vectors. Structural changes of cell shape and microarchitecture

were examined by confocal live cell imaging and immunocytochemistry. Functional adaptations were evaluated by

measuring Ca2+ currents and Ca2+ transients using the whole-cell voltage-clamp technique in combination with laser-

scanning confocal microscopy.

Our results demonstrate that hiPSC-CMs adapt to reshaping and form cuboid cells in rectangular 3D-microscaffolds.

As a consequence, cells have established a longitudinal axis for efficient unidirectional contraction. Moreover, Bin1-

expression successfully induced sarcolemmal invaginations in reshaped hiPSC-CMs, indicating formation of an early,

yet unstructured tubular membrane network. Detailed analysis revealed a significant increase of network branches

and tubular junctions compared to control. Investigation of EC-coupling demonstrated enhanced Ca2+ handling

properties in reshaped and Bin1-expressing cells: shorter Ca2+ transients and faster Ca2+ removal after release

suggest maturation of cytosolic Ca2+ handling.In summary, our data reveal significant remodeling processes both at

the structural and functional level upon forced growth in 3D-cuboids and enhanced Bin-1 expression. We conclude

that both cell morphology and membrane organization are required to drive novel hiPSC-CMs toward robust Ca2+

handling.

Acknowledgment This project is supported by the DFG (to N.D.U.).


of 516

B 12-10

Single molecule detection of RNA modifications in cardiac cells

Núria Díaz i Pedrosa1,2, Lisa M. Weiß1,2, Maximilian Staps3, André Schneider3, Thomas Braun3,

Ralf Gilsbach1,2

1 Goethe University Frankfurt, Institute of Cardiovascular Physiology, Frankfurt, Germany 2 German Center for Cardiovascular Research, Partner site RheinMain, Frankfurt, Germany 3 Max Planck Institute for Heart and Lung Research, Department of Cardiac Development and Remodeling, Bad

Nauheim, Germany

RNA modifications are key regulators of RNA stability, thus regulating gene expression at a posttranscriptional level.

N6-Methyladenosine (m6A) is the most studied RNA modification in the cardiovascular field. It is known to decorate

essential transcripts for the cardiac linage, but the exact position of the modification remains unknown. The

establishment of m6A is performed by the Methyltransferaselike 3 (Mettl3) RNA-specific enzyme. Direct RNA

sequencing (DRS) using nanopores is a third-generation sequencing technique that allows us to identify cardiac RNA

modifications (the epitranscriptome) with base pair resolution. In this work, we combine DRS and cardiomyocyte-

specific Mettl3 KO mice to identify m6A sites. After performing DRS on ventricles from WT and Mettl3 KO mice, we

obtain a sequencing depth of over 1,5 Mio reads in average per sequencing run (WT1 2,45 Mio, WT2 1,81 Mio, KO1

1,15 Mio and KO2 1,63 Mio). These data identify more than 1.800 significantly modified RNA regions in the cardiac

transcriptome. Integration of data from cardiomyocyte-specific Mettl3 KOs reveal that more than 22% of the modified

sites detected by DRS in cardiomyocyte-specific transcripts are sensitive to ablation of Mettl3 and thus represent

m6A sites. Remarkably, we observe that transcripts losing m6A modifications are upregulated. In contrast,

expression of non-cardiomyocyte transcripts, which are not affected by the cardiomyocyte-specific deletion, are not

altered. In conclusion, direct RNA sequencing using nanopores allows us to detect RNA modifications in an unbiased

manner with base pair resolution. Integration of Mettl3 KO data identifies m6A modifications in cardiomyocyte

transcripts. Our data suggest that m6A plays a role in posttranscriptional gene expression regulation in

cardiomyocytes via promoting mRNA degradation, as transcripts losing the modification tend to have higher

expression values.


of 516

B 12-11

Pro-survival effects of renin-b in H9c2 cells under anoxia: role of

angiotensin generation

Heike Wanka1, Philipp Lutze2, Doreen Staar1, Katharina Bracke1, Janine Golchert1, Jörg Peters1

1 University Medicine Greifswald, Institute of Physiology, Greifswald, Germany 2 Leibnitz Institute for Farm Animal Biology, Institute of Muscle Biology & Growth, Dummerstorf, Germany

Question:The renin-angiotensin system is known to regulate blood pressure as well as water- and electrolyte

balance. An activated RAS is involved in the development of hypertension and hypertension-related organ damage.

However, renin transcripts have been discovered encoding a cytoplasmatic renin isoform, termed renin-b, which may

be protective.

Methods: Cardiac H9c2 cells with knock down of renin or overexpression of renin combined with anti-renin treatment

using the inhibitor CH732 were exposed to anoxia for 24 h and then analyzed regarding apoptosis rate (Caspase+

cells, Annexin V+ cells), necrosis rate (LDH release vs LDH content) as well as ATP content.

Results: Here we demonstrate that depletion of renin-b encoding transcripts by small interference RNA decreased

ATP levels and increased basal necrosis as well as apoptosis rates. Furthermore, renin-b depletion potentiated the

anoxia-induced increase of necrosis rates. Vice versa, overexpression of renin-b prevented the anoxia-induced

increase of caspase-mediated apoptosis rates. Besides, cells overexpressing renin-b exhibited even reduced

mitochondrial mediated apoptosis rates under anoxia, when compared with normoxic conditions, as indicated by

Annexin V labeling. However, whereas the protective effect of renin-b on caspase-mediated apoptosis during anoxia

was completely blocked by the renin inhibitor CH732, the effect on mitochondrial-mediated apoptosis was not

affected by CH732 at all.

Conclusions: From these data we conclude that renin-b overexpression mediates cardioprotective effects under

anoxia with respect to mitochondrial induced apoptosis angiotensin-independently, but with respect to caspase

induced apoptosis in an angiotensin-dependent manner.

Effects of renin targeting siRNA or renin

inhibitor CH732 on H9c2 cells.

Upper table demonstrates effects of renin

targeting siRNA, while lower table represents

effects of tne renin inhibitor CH732 on cardiac

H9c2 cells. Cells were exposed to basal

conditions or anoxia and analyzed regarding

apoptotic and necrotic cell death as well as ATP

content.

Chair Index DPG 2021 | Abstract Book

of 516

Chair Index

A

Alesutan, Ioana ..........................................OS 02 B

Bartos, Marlene ........................................... B 04 Baukrowitz, Thomas .................................... A 04 Benndorf, Klaus ........................................... B 06 Bischofberger, Josef .................................... B 01 Bleich, Markus .................................... S 01, A 05 Bottermann, Katharina ................................. A 10 Brandes, Ralf P. ....................................... , W 02 Brügmann, Tobias ........................... S 02, OS 08 C

Chaves, Gustavo ......................................... S 09 D

Dietl, Paul .................................................... B 07 Draguhn, Andreas ..................................... , S 03 E

Eilers, Jens .................................................. S 06 F

Fakler, Bernd ............................................... B 05 Fandrey, Joachim ............................... S 12, A 07 Fischer, Michael .......................................... B 03 Fleming, Ingrid .................................... S 05, B 11 Föller, Michael ............................................. A 08 G

Garaschuk, Olga .......................................... B 04 Gödecke, Axel ............................................. B 11 Görlach, Agnes ............................................ A 07 Graf, Jürgen ......................................................... Gründer, Stefan ........................................... B 03 H

Hallermann, Stefan .............................. PL1, S 06 Hamdani, Nazha ........................................OS 08 Hecker, Markus ........................................... A 12 Heineke, Jörg .............................................. A 11 Heinen, Andre .............................................. B 12 Hinze, Christian .........................................OS 02 Hoogewijs, David ......................................... B 07 I

Isbrandt, Dirk ............................................... B 02 K

Katschinski, Dörthe ............................. S 02, B 08

Keppner, Anna .......................................... OS 04 Kirschstein, Timo .......................................... A 01 Kleinbongard, Petra ................................... OS 07 Köhling, Rüdiger ........................................... A 03 Krüger, Martina ............................................. B 10 Kübler, Wolfgang .......................................... A 06 Kurtz, Armin .................................................. A 09 Kusche-Vihrog, Kristina ................................ B 08 L

Lampert, Angelika ........................... OS 05, B 06 Lee, Wing-Kee .............................................. S 07 Leipziger, Jens ............................................. S 11 Lenz, Dominik ............................................... S 09 Leßmann, Volkmar ....................................... A 02 Linke, Wolfgang ............................................ A 10 Lippmann, Kristina ..................................... OS 06 Liss, Birgit ..................................................... A 01 M

Metzen, Eric ................................................. A 06 Mrowka, Ralf ................................................ A 08 O

Oberwinkler, Johannes .............................. OS 01 Olschewski, Andrea ................................... OS 03 P

Persson, Pontus ........................................... B 09 Pfeilschifter, Josef ........................................ S 04 R

Rezende, Flavia ........................................ OS 04 Ritter, Markus ............................................... B 09 Ritzau-Kost, Andreas ................................... S 09 Robin, Catherine .......................................... S 10 Roeper, Jochen ................................ , A 04, B 05 Rohrbach, Susanne ...................................... A 11 Rumpel, Simon ............................................. B 01 S

Sasse Philipp ................................................ A 03 Sauer, Jonas-Frederic ...................... S 03, OS06 Schaible, Hans-Georg .................................. B 02 Schiemann, Julia ....................................... OS 09 Scholz, Holger ........................................... OS 03 Schubert, Rudolf .................................................. Schweda, Frank ........................................... S 04 Schwenk, Jochen ...................................... OS 05 Sigurdsson, Torfi ....................................... OS 09

Chair Index DPG 2021 | Abstract Book

of 516

Sperandio, Markus ............................. S 10, B 10 V

van der Veen, Rozemarijn ........................... S 01 Völkl, Jakob ................................................. A 09 von Engelhardt, Jakob ................................. A 02 W

Wagner, Carsten ................................ S 11, A 05

Wenzel, Daniela ................................ W 02, A 12 Windbergs, Maike ........................................ W 01 Winning, Sandra ........................................... S 12 Wischmeyer, Erhard ..................................... S 07 Y

Yang, Zhihong .............................................. B 12

Author Index DPG 2021 | Abstract Book

of 516

Author Index

A

Aarnoutse, Erik J. ................................................ Abady, Daria S. E. .................................. B 02-05 Abdellatif, Mahmoud ............................ OS 07-04 Abeßer, Marco .......................... B 10-07, B 12-04 Abplanalp, Wesley .................... B 10-09, B 11-01 Acuna, Claudio ....................................... B 03-07 Adamantidis, Antoine ........................... OS 06-06 Aghajafari, Ali ......................................... B 10-02 Aguilar-Camacho, José M. ..................... B 06-02 Ahmad, Aisha ......................................... S 06-03 Ahmed, Marwa .................................... OS 05-02 Aigner, Felix ......................................... OS 02-02 Ajalbert, Guillaume .............................. OS 07-06 Akam, Thomas ....................................... A 01-06 Albers, Alexander ................................... B 11-11 Albrecht, Urs ........................................ OS 06-06 Alceste, Daniela ...................................... B 02-11 Aldag-Niebling, David .......................... OS 08-01 Alesutan, Ioana ....................................... B 11-10 Alfonso-Prieto, Mercedes ......... A 03-02, A 05-10 Al-Hasani, Jaafar ................................. OS 01-08 Alle, Henrik .......... OS 06-01, OS 09-06, A 02-05 Alleva, Claudia .......................... A 02-07, A 05-10 Alonso-Duran, Laura ........................... OS 02-06 Altafi, Mahsa ........................................... B 01-05 Althaus, Mike .......................................... A 04-10 Altstätter, Johannes ................................ A 07-03 Amann, Kerstin ....................................... S 11-05 Amarnath, Gautami ................................ A 02-03 Amasheh, Salah ..................................... B 09-11 Ameling, Sabine ..................................... A 12-10 Amponsah-Offeh, Michael ...................... B 10-08 Amrute-Nayak, Mamta ........... OS 01-06, A 10-02 Andersen, Jesper F. ................. S 04-04, S 11-04 Andersson, Anders G. ............................ A 06-08 Angenstein, Frank .................................. B 03-08 Arnhold, Stefan ...................... OS 01-04, A 10-03 Arzberger, Thomas ................................. B 02-08 Asadollahi, Reza ..................................... S 06-03 Ashraf, Muhammad I. ............ OS 02-02, B 05-08 Auge, Isabel ............................................ B 09-01 Aykac, Ibrahim ........................................ A 11-10 Azer-Güney, Lale ................................. OS 08-05 B

Baba, Hideo A. ....................................... B 10-07 Babl, Susanne S. .................................... A 01-07 Babu, Susan ........................................... B 03-09

Babushkina, Natalia ............................... B 04-09 Baccini, Gilda ......................................... B 01-06 Baccino-Calace, Martin .......................... B 04-02 Bachmann, Lisa ...................................... A 05-05 Bachmann, Michèle ................................ B 05-10 Back, Michaela ....................................... B 05-03 Bäcker, Veronika .................................... S 12-03 Bähring, Robert ...................................... B 10-11 Bailey, Susan M. ........................................ PL 02 Baker, Andrew ........................................ S 05-03 Ballout, Jasmin ..................................... OS 04-07 Ballweg, Annamaria ............................... A 07-03 Bandulik, Sascha .................................... A 09-02 Barbati, Mohammad Esmaeil ................. B 10-02 Barnea, Eytan R. .................................. OS 04-01 Barnett, Anastasia .................................. A 04-05 Baron, Victoria ........................................ A 11-07 Bas Orth, Carlos ..................................... A 05-08 Bauer, Hartwig W. .................................. A 09-10 Bauer, Reinhard ..................................... B 04-12 Baukrowitz, Thomas . S 07-01, A 04-08, B 06-01,

B 06-05, B 06-07, B 06-10 Beauchamp, Janosch ........................... OS 01-01 Becher, Burkhard ................................. OS 03-05 Beck, Julia ............................................ OS 08-01 Becker, Cora .......................................... A 10-09 Becker, Stina ........................................ OS 04-05 Becker, Yannic ....................................... A 09-11 Bednarz, Rebecca ..... B 12-02, B 12-05, B 12-07 Bedoya, Mauricio .................................... A 04-08 Beel, Stephanie ...................................... A 05-03 Begall, Sabine ........................................ A 08-07 Behr, Katharina ...................................... B 01-03 Behroozi, Mehdi ................................... OS 09-02 Behuet, Sabrina .................................... OS 09-04 Belousov, Vsevolod ............... S 02-02, OS 08-07 Bender, Franziska .................................. B 01-05 Benkert, Julia .......................................... A 02-03 Benndorf, Klaus ........ A 03-03, A 03-06, A 03-09,

B 05-02, B 05-06, B 05-09 Bennewitz, Jonathan .............................. A 12-10 Benseler, Fritz ........................................ A 04-10 Berg, Peder . S 04-04, S 11-02, S 11-03, S 11-04 Berger, Raffaela ..................................... A 09-05 Berger, Thomas .................................... OS 05-01 Bernhagen, Jürgen ................................. B 08-05 Bertsch, Annika .................................... OS 04-01 Berwanger, Carolin ................................. A 10-11 Bettoni, Carla .......... OS 02-06, B 09-05, B 09-09


of 516

Bhandari, Dhaka R. ............................. OS 01-03 Bibli, Sofia-Iris ......... OS 08-06, B 08-04, B 10-04 Bikas, Solmaz ......................................... B 02-12 Binas, Stephanie ................................. OS 05-05 Bindila, Laura ....................................... OS 03-04 Bintener, Tamara J. R. ........................... A 09-07 Bischofberger, Josef ............................... B 01-03 Bischoff, Stephan C. ............................ OS 06-05 Bjelde, Antje ........................................ OS 06-01 Bland, Gareth ...................................... OS 09-05 Blanquie, Oriane ..................................... B 04-07 Bleich, Markus .......................... S 01-02, B 09-10 Blind, Ursula ............................. S 09-01, A 12-09 Bloch, Wilhelm ..................................... OS 07-01 Bludau, Sebastian ............................... OS 09-04 Boccuni, Isabella .................................... A 05-08 Bochud, Muriel ..................................... OS 02-05 Bock, Hans ............................................. A 04-05 Boedtkjer, Ebbe ...................................... S 11-02 Boersch, Nina ......................................... S 09-03 Böge, Juliane .......................................... A 01-04 Boivin, Felix ......................................... OS 02-02 Bonetti, Alessandro ................................ B 11-08 Bongardt, Sabine ................... OS 08-05, A 11-03 Boos, Frederike ..................... OS 07-02, B 11-12 Borges-Merjane, Carolina ....................... S 06-01 Borghans, Bart ........................................ A 05-07 Borsa, Micaela ..................................... OS 06-06 Borutta, Johanna-Theres ........................ B 07-10 Bosch, Rebecca ..................................... A 12-04 Bosserhoff, Jonah .................. OS 03-07, A 06-06 Bott, Raya ............................................... A 04-02 Bottermann, Katharina ............. A 10-04, A 11-08,

A 11-09, B 12-01 Bounkari, Omar E. .................................. B 08-05 Bourgeois, Soline ...... S 11-05, A 09-01, B 09-09 Brachvogel, Bent .................................... A 10-11 Bracke, Katharina ................................... B 12-11 Branco, Mariana P. .............................................. Brandes, Ralf P.OS 07-02, OS 08-06, OS 08-08,

A 07-01, A 08-04, A 09-07, A 12-01, A 12-03, B 08-06, B 10-01, B 10-09, B 11-01, B 11-03, B 11-06, B 11-08, B 11-12

Brandl, Ulrich .......................................... B 05-11 Brankack, Jurij ........................................ S 03-02 Braun, Thomas ....................................... B 12-10 Braunbeck, Mariel ................................... B 05-03 Bredehöft, Janne ................................. OS 01-03 Breiderhoff, Tilman ................................. B 09-10 Breitkreuz, Martin ................................... A 11-01 Brendel, Heike ........................................ B 10-10 Brenna, Andrea .................. OS 02-03, OS 06-06 Brodersen, Cedric ................................ OS 09-08 Broeker, Katharina ................ S 04-02, OS 02-04, A 09-03, B 09-06

Brose, Nils ................................ S 06-03, A 04-10 Bruegmann, Tobias ............... S 02-04, OS 01-01 Bruehl, Claus ............................ A 05-08, B 06-13 Brügmann, Tobias .................................. A 10-06 Brüne, Bernhard ..................................... B 08-01 Brüning, Jens .......................................... S07-02 Brunkhorst, Anja ................................... OS 08-02 Brunssen, Coy ........................................ B 10-10 Buchholz, Bjoern .................................... A 09-08 Buchmann, Giulia ..................... B 10-09, B 11-01 Bueter, Marco ......................................... B 02-11 Bugarski, Milica ........................ B 09-05, B 09-09 Bulk, Etmar ............................... B 06-03, B 06-11 Bullmann, Torsten .................... S 06-05, B 04-03 Burkart, Marie-Elisabeth ....................... OS 09-07 Burkart, Valentin ................................... OS 08-01 Burzlaff, Nicolai .................................... OS 02-04 Buss, Eric W. .......................................... A 04-05 C

Cabrita, Inês ............................. S 04-04, A 09-08 Calderón Fernández, Marbely .............. OS 07-07 Canavier, Carmen .................................. A 02-08 Caragea, Violeta M. ................................ B 04-11 Cario, Holger ........................................ OS 02-05 Carlberg, Carsten ................................... B 08-06 Carloni, Paolo ......................................... A 05-10 Carus-Cadavieco, Marta ........................ B 01-05 Carvalho Correia, Miguel ...................... OS 02-05 Castilla Porras, Francisco J. ................... A 03-02 Catanese, Lorenzo ................................. A 06-10 Centeio, Raquel ...................................... B 06-04 Cepeda-Prado, Efrain ............................. A 02-09 Chandra, Gemini .................................... A 04-07 Chantillon, Maarten .............................. OS 02-05 Chavakis, Triantafyllos ........................... B 10-08 Chen, Changwan .................................... B 01-05 Chen, Jin ................................................ S 05-05 Chen, Xinpei ........................................... A 06-07 Chen, Xufeng ......................................... B 05-08 Cho, Haaglim .......................................... S 05-04 Choi, Hae-Na .......................................... A 01-03 Chow, Billy K. ......................................... S 11-04 Christoph, Jan ........................................ S 02-04 Ciliberti, Giorgia ...................................... B 11-05 Ciotu, Cosmin I. ...................................... A 11-10 Claßen, Rebecca .................................... A 03-04 Clemen, Christoph S. ............................. A 10-11 Clusmann, Jan ....................................... S 09-03 Cobain, Matthew .................................... A 04-10 Coburger, Ina ......................................... A 04-03 Cokić, Milan ............................................ A 10-06 Colitti-Klausnitzer, Jens .......................... B 04-01 Conklin, Bruce R. ................................... B 12-06 Cook, Stephane .................................... OS 07-06


of 516

Corbu, Michaela-Anca ............................ B 01-05 Cordeiro, Sönke ......... S 07-01, B 06-07, B 06-10 Cornelius, Valeria ................................... B 09-11 Corre, Tanguy ...................................... OS 02-05 Corredor, David ...................................... S 09-03 Correia, Miguel ...................... S 12-02, OS 02-01 Cortes, Daniel ......................................... S 09-03 Costa, Ivan G. ......................................... A 12-03 Culmsee, Carsten .................. OS 01-03, B 12-08 Cyganek, Lukas ...................................... A 11-06 D

Dabral, Swati ............................ A 08-03, A 08-06 Daerr, Jan ............................................ OS 07-01 Dam, Vibeke ........................................... S 11-02 Dame, Christof ..................................... OS 03-02 Daniel, Christoph .................................... B 09-06 Daniel, Hannelore ................................... A 09-01 Darche, Fabrice ...................................... B 06-13 Daryadel, Arezoo ...................... A 09-01, B 09-05 de Groot, Bert L. ..................................... A 04-08 de Jesus Perez, Vinicio .......................... A 08-06 De Laporte, Laura ................................... B 03-09 de los Santos Bernal, Francisco J. ........ A 01-01,

A 01-03 de Mooij-van Maalsen, Johanne G. ........ B 01-06 Decher, Niels .......................................... A 04-08 Degens, Hans ......................................... A 10-11 Delgado Lagos, Fredy A 07-04, B 08-07, B 08-10 Delling, Cora ........................................... A 05-05 Delvendahl, Igor ....................... S 06-04, A 02-02 Denda, Jannik ......................................... B 07-09 Dengler, Franziska ................................. A 05-05 Depping, Reinhard .................................. B 07-10 Dettmer-Wild, Katja ................................ A 09-05 Deussen, Andreas ..... A 12-05, A 12-08, B 10-12 B 10-06, B 10-08, B 11-04 Diaba-Nuhoho, Patrick ........................... B 10-10 Diamantopoulou, Anastasia .................... B 02-12 Díaz i Pedrosa, Núria ............... B 12-02, B 12-10 Dib, Selma .............................................. S 01-01 Diener, Martin ........................ OS 04-07, A 03-04 Dieterich, Peter .......... A 12-05, B 10-06, B 11-04 Dietl, Alexander ...................................... A 11-01 Dietrich, Jennifer M. ................................ B 07-02 Dilz, Jasmin ............................................ B 09-01 Dimmeler, Stefanie ................................. B 10-09 Ding, Zhaoping ....................................... A 11-02 Dmitrieva, Nataliia .................................. A 05-10 Dobreva, Gergana .................................. B 08-04 Dolenšek, Jurij .......................... A 08-02, A 08-05 dos Remedios, Cristobal ..................... OS 08-01 Draguhn, Andreas .... S 03-02, A 05-08, B 01-11,

B 06-13 Drees, Christoph ..................................... A 04-07

Drekolia, Maria Kyriaki ......... OS 08-06, B 08-04, B 10-04

Drexler, Nils .............................. A 03-08, A 04-09 Droessler, Linda ..................................... B 09-11 Dröge, Freya ......................... OS 03-07, A 06-06 Dubovyk, Valentyna ............................... B 04-01 Duda, Johanna ......................... A 02-03, B 02-08 Dudeck, Jan ........................................... S 10-05 Dudek, Jan ............................................. A 08-06 Dueva, Rositsa ....................................... B 07-04 Dumbraveanu, Cristiana ......................... B 04-13 Dunker, Carolin ...................................... A 03-05 Duran, Ibrahim ........................................ A 10-11 Dusend, Vanessa ................................... A 10-08 Duvarci, Sevil ........................... A 01-02, A 01-09 E

Eckardt, Kai U. ....................................... B 11-10 Edelmann, Elke ........................ A 02-09, B 05-12 Edenhofer, Marie-Luise ......... OS 09-03, B 03-06 Egger, Richard ....................................... A 02-01 Egger, Veronica ...................................... B 03-01 Egli-Spichtig, Daniela ........................... OS 04-08 Egorov, Alexei V. .................................... B 01-11 Ehmann, Nadine ....................... S 06-05, B 04-03 Ehmke, Heimo ........... S 11-03, B 10-05, B 10-11 Eick, Thomas .......................................... B 05-06 Eilers, Jens ........ S 06-05, OS 06-02, OS 09-07,

B 04-03 Einsle, Anne ........................................... B 02-05 Eitner, Annett .......................................... B 04-12 Elsaid, Basant ........................................ B 10-12 Ender, Anatoli ......................................... B 01-01 Endesfelder, Stefanie ........................... OS 03-02 Engel, Nadja ........................................... B 02-03 Engeland, Birgit ...................................... A 04-05 Engelhardt, Stefan .................................. B 11-06 Engels, Miriam ...................................... OS 09-04 Engeroff, Kira ......................................... B 04-07 Ergün, Süleyman .................................... A 12-04 Estepa, Misael ........................................ B 11-10 Euler, Gerhild ......................................... A 07-08 Ewendt, Franz ........................................ A 12-02 Eymsh, Bisher ........................................ B 06-10 F

F. Hernández, Ledia ............................... B 02-04 Fähling, Michael ................................... OS 02-02 Fahlke, Christoph S 09-02, OS 06-03, OS 09-04,

A 02-07, A 05-01, A 05-07, A 05-10 Fairless, Richard .................................... A 05-08 Fandrey, Joachim S 09-01, OS 03-07, OS 04-06,

A 06-02, A 06-06, A 12-09, B 07-01, B 07-08, B 07-09

Fangel, Søren J. ..................................... S 04-04


of 516

Fauler, Michael ......................... A 07-02, A 07-10 Federlein, Anna ........................ A 09-03, B 09-03 Feger, Martina .......................... A 09-06, A 12-02 Feind, Dominic ........................................ A 03-07 Fels, Benedikt ......................................... A 12-07 Feng, Xiaogang ................................... OS 03-05 Ferenz, Katja B. ........ A 06-04, A 07-07, B 08-02,

B 08-03, B 08-08 Fernández, Julián ................................... A 10-01 Ferraguti, Francesco ........................... OS 09-03 Fidzinski, Pawel ...... OS 06-01, A 02-05, B 01-09 Fiedler, Dorothea ................................. OS 02-08 Fiedler, Stefan ........................................ A 10-07 Fiegle, Dominik ....................................... A 11-07 Figge, Rebecca ........................ A 01-05, A 01-08 Fischer, Jens W. ....................... A 11-08, A 11-09 Fischer, Michael J. .................................. A 11-10 Fisslthaler, Beate ...................... A 07-04, B 08-10 Fleischmann, Bernd K. ...... OS 07-01, OS 07-03,

A 10-03, A 10-09, B 07-02, B 12-06 Fleming, Ingrid ....... S 05-04, OS 08-06, A 07-04,

A 07-06, A 07-09, A 12-06, B 08-01, B 08-04, B 08-07, B 08-10, B 10-01, B 10-04, B 11-05, B 11-06, B 11-07

Flück, Katharina ...................................... S 12-03 Föller, Michael ........... A 09-06, A 12-02, B 09-02 Folschweiller, Shani ................................ B 01-07 Fomin, Andrey ........................................ A 11-06 Foreman, Katharina B. ........................... B 06-02 Franke, Annika ....................................... A 08-09 Franz, Denise ........................... A 01-11, B 02-03 Freiman, Thomas ................................... B 02-02 Freitag, Patricia ...................................... A 10-09 Frerker, Bernd ........................................ A 10-07 Freudenburg, Zachary V. .............................. PL4 Freundt, Johanna K. ............................... A 11-04 Frick, Manfred ......... OS 03-06, A 07-02, A 07-10 Friede, Prisca ...................................... OS 01-08 Friedrich, Anna-Lena .............................. A 08-03 Friedrich, Oliver ...................................... B 11-02 Frömel, Timo ............. A 07-06, B 08-01, B 11-05 Fromm, Anja ........................................... S 01-03 Fromm, Michael ...................................... S 01-03 Frommhold, David .................................. B 08-05 Fuchs, Michaela A. A. ............ OS 02-04, B 09-06 Fuente, Rocio ...................................... OS 02-06 Fuhrmann, Dominik ................................ B 08-04 Fuhrmann, Falko .................................... S 02-03 Furdui, Cristina M. ............................... OS 08-07 Fürst, Dieter O. .................................... OS 07-01 Furuse, Mikio .......................................... S 01-02 G

Gade, Richard ........................................ B 02-02 Gaifullina, Aisylu ..................................... A 04-04

Gajos-Draus, Anna ................................. B 10-01 Galang, Chantel ..................................... B 12-01 Galil, Dalia Abdel .................................. OS 06-05 Ganse, Bergita ....................................... A 10-11 Gao, Qi ................................................. OS 05-06 Gao, Shiqiang ........................ OS 01-02, A 03-11 Gao, Xiaojie ............................................ B 01-05 Garaschuk, Olga ....... B 01-02, B 01-08, B 03-05 Garcia, Carmen C. ................................. A 10-09 Gardie, Betty ........................................ OS 02-05 Garikapati, Vannuruswamy .................. OS 01-03 Gassmann, Max ..................................... B 08-09 Gehring, Nicole ..................................... OS 02-06 Geiger, Jörg R. P. .............. OS 06-01, OS 09-06,

OS 09-08, A 02-05, B 01-09, B 02-06, B 05-07 Geis, Christian ........................................ S 06-05 Geisen, Caroline ................................... OS 07-01 Geisslinger, Gerd ................................. OS 05-04 Gekle, Michael ....................... OS 05-05, A 09-09 Geley, Stephan ..................................... OS 06-07 Gerevich, Zoltan ........ B 01-04, B 01-09, B 05-07 Gerhardt, Patricia ................................... A 08-07 Gero, Daniel ........................................... B 02-11 Gerstberger, Rüdiger .............................. B 03-02 Gettings, Sean M. ................................... A 04-10 Ghosh, Subhajit .................................... OS 01-08 Giakoumaki, Ifigeneia ............................. A 10-11 Giese, Heiko ......................................... OS 08-08 Gilsbach, Ralf .......... W 02-02, A 12-03, B 08-06,

B 11-01, B 11-08, B 12-02, B 12-05, B 12-07, B 12-10

Gimeno-Ferrer, Fatima ........................... B 04-12 Giraud, Marie-Noelle ............................ OS 07-06 Glauser, Dominique .............................. OS 06-06 Glebova, Alina ........................................ A 02-03 Glück, Tobias ......................................... A 06-04 Gödecke, Axel ....... OS 04-04, A 07-05, A 10-04,

A 11-05, B 12-01 Gödecke, Stefanie ................. OS 04-04, A 10-04 Göhr, Christoph .................................... OS 06-05 Golchert, Janine ........ A 12-10, B 11-11, B 12-11 Goldberg, Joshua A. ................. A 02-06, B 02-08 Gollner, Andrea ...................................... B 03-03 Gonschior, Hannes P. .............. S 01-02, A 03-05 Gonzalez, Wendy ................................... A 04-08 Göpelt, Kirsten ........................................ B 07-04 Gorbati, Maria ......................................... B 01-05 Gorinski, Nataliya ................................. OS 06-05 Görlach, Agnes ...................... OS 03-03, A 06-07 Gosak, Marko .. A 08-01, A 08-02, A 08-05, B 05-

05 Gottschalk, Alexander .......................... OS 08-03 Götz, Robert ............................. A 09-03, B 09-03 Graf-Riesen, Kathrin .............. OS 07-01, A 10-03 Graier, Wolfgang .................................... A 04-04


of 516

Grandoch, Maria ..................................... A 11-02 Grantins, Klavs ....................................... A 05-03 Graumann, Johannes ............... S 05-04, A 08-11 Gredy, Sina ............................... B 02-10, B 12-04 Greffrath, Wolfgang ............................. OS 05-06 Greiner, Joachim .................................... A 10-10 Groeneveld, Kathrin .................. B 06-06, B 09-01 Gronwald, Wolfram ................................. A 09-05 Gross, Joachim ....................................... S 03-04 Große-Onnebrink, Jörg ........................... A 03-10 Grosser, Sabine ..................... OS 06-01, A 02-05 Grossmann, Claudia ............................ OS 05-05 Grossmann, Sonja ............................... OS 02-07 Grote Lambers, Mirja .............................. B 02-06 Gründer, Stefan ........ S 09-03, B 04-10, B 05-10,

B 06-02 Gschwend, Julia .................................. OS 03-05 Gsell, Felix ................................ S 06-05, B 04-03 Gudermann, Thomas ........................... OS 04-01 Guenther, Stefan ................... OS 07-02, B 11-08 Gulbins, Erich ......................................... S 07-05 Guli, Xiati ................................................ A 06-03 Günther, Andreas ................................... A 08-03 Günther, Stefan ....... S 05-04, W 02-01, B 08-01,

B 08-04, B 08-06, B 10-04, B 11-12 Günzel, Dorothee ..... S 01-01, S 01-02, S 01-03,

B 09-10 Gunzer, Matthias ................... OS 04-03, A 07-03 Guo, Jia ............................................... OS 08-07 Guo, Yang .............................................. B 11-02 Guseva, Daria ...................................... OS 06-05 Gusic, Milica ........................................... B 05-02 Gutiérrez, José María ............................. A 10-01 Guzman, Raul E. ................................. OS 06-03 H

Haag, Daniela ...................................... OS 07-05 Haase, Volker H. .................................... S 12-05 Habel, Sabrina J. .................................... B 09-03 Häbich, Hannes ...................................... B 12-04 Hadler, Michael D. ............................... OS 09-06 Hagena, Hardy ......................... B 04-01, B 04-09 Hager, Marina ......................................... B 05-07 Hahn, Johannes ..................................... A 01-02 Hahner, Fabian ....................................... B 11-01 Hajishengalis, George ............................ B 10-08 Halaszovich, Christian R. ....................... A 03-01 Halbhuber, Lisa ...................................... B 04-06 Hall, Andrew ............................. B 09-05, B 09-09 Hallermann, Stefan S 06-05, OS 06-02, B 04-03,

B 04-08 Hamdani, Nazaha ................................... A 11-01 Hamid, Ahmad Kamal .......................... OS 04-08 Hamidi, Akram ........................................ B 07-04 Hammer, Maximilian ............................... S 03-02

Hammer, Niklas ........................ A 02-08, A 02-04 Hammer, Victoria .................................... B 12-08 Hammock, Bruce .................................... A 07-06 Hammon, Harald .................................... A 05-05 Hannibal, Luciana ................................... A 09-07 Hansen, Arne ......................................... B 11-01 Hansen, Ulf-Peter ..................... A 03-08, A 04-09 Hansmann, Martin-Leo ........................... B 10-09 Hardt, Stefan ........................................ OS 06-04 Harris, Thurl ............................................ B 12-01 Hartmann, Julia .................................... OS 05-01 Hartung, Jens ......................................... B 02-02 Haucke, Volker .......... S 01-02, S 06-06, A 03-05 Häusler, Max .......................................... A 02-03 Hausmann, Ralf ...................................... A 04-02 Hautvast, Petra ....................................... A 04-01 Haykir, Betül .......................... OS 02-08, B 09-05 Hebchen, Maureen ................................. A 08-11 Heber, Stefan ......................................... A 11-10 Hecker, Markus .... OS 01-08, OS 08-04, B 12-09 Heckmann, Manfred .............. OS 01-02, A 03-11 Heerdegen, Marco .................... A 01-11, B 02-03 Heidenreich, Matthias ............................. B 01-05 Heidler, Juliana ..................................... OS 08-08 Heilen, Laura ........................................ OS 01-04 Heim, Christian ....................................... A 11-07 Heim, Michael-Marcel ............................. A 01-10 Hein, Lutz ............................................. OS 07-01 Heinemann, Stefan H. .......... OS 05-02, A 04-03,

A 04-07, B 05-11 Heinen, André .......................... A 10-04, A 11-05 Heinig, Kristina ....................................... A 07-03 Heinig, Matthias ...................................... A 07-03 Heinrich, Alexandra ................................ A 08-07 Heinrich, Tobias ....................... B 10-05, B 10-11 Helluy, Xavier ....................................... OS 09-02 Helm, Christiane ..................................... A 05-05 Hemmers, Anne ..................................... B 12-01 Hempel, Caroline .................................... S 01-03 Henker, Christian .................................... B 02-02 Henning, Yoshiyuki .... S 09-01, A 08-07, A 12-09 Henze, Laura .......................................... B 11-10 Hepbasli, Denis ...................................... B 02-10 Hermes, Julia ......................................... B 12-03 Hernandez, Jessica ............... OS 01-03, B 02-01 Hernando, Nati ....... OS 02-08, A 09-01, B 09-05 Hesse, Michael ...... OS 07-01, A 10-03, A 10-09,

B 12-06 Hetsch, Florian ....................................... B 05-08 Heusch, Gerd ....................................... OS 08-05 Heydn, Rosmarie .................................... A 09-03 Hidalgo, Patricia ..................................... A 03-02 Hierdeis, Kilian ....................................... A 06-07 Hildebrandt, Guido ................................. A 10-07 Hilfiker-Kleiner, Denise ......................... OS 08-01


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Himmerkus, Nina ...................... S 01-02, B 09-10 Hirrlinger, Johannes ............................ OS 06-02 Hoang, Thu-Huong ................... A 01-04, B 03-08 Hocher, Berthold ....................... A 09-06, A 12-02 Hochkogler, Katharina ......................... OS 03-03 Hoebart, Clara ........................................ A 11-10 Hoeber, Francis W. ................................ Opening Hofmann, Sascha ................................... A 09-08 Hofschröer, Verena ................................ B 06-09 Hofstetter, Alfons .................................... A 09-10 Hoheisel, Ulrich ................................... OS 05-06 Höhfeld, Jörg ....................................... OS 07-01 Hohlbrugger, Gero .................................. A 09-10 Hohlstamm, Anselm ............................... A 12-05 Holdenrieder, Stefan ............................ OS 03-03 Holler, Tim ......... OS 01-06, OS 08-01, OS 08-02 Hollman, Helene ..................................... A 02-03 Hoogewijs, David S 12-02, OS 02-01, OS 02-05,

B 07-03 Hörbelt-Grünheidt, Tina ....................... OS 04-06 Hörnschemeyer, Julia ............................. B 02-03 Hoschke, Annekathrin ............................ A 06-05 Hoshi, Toshinori ...................................... A 04-03 Hosseini, Behnaz A. ............................... A 12-08 Hu, Jiong . OS 08-06, A 07-09, B 08-04, B 11-06,

B 11-07 Hu, Marian Y. - A. ................................... S 11-01 Huang, Ya-Chi ........................................ A 08-10 Huard, Arnaud ........................................ A 07-06 Hübner, Christian A. ............................... S 11-03 Hubner, Norbert ...................................... S 05-02 Hucke, Anna ........................................... A 11-06 Huckschlag, Britt Marie .......... OS 05-01, A 05-04 Huesgen, Pitter F. ................................ OS 07-01 Hummert, Sabine .................................... B 05-06 Huning Kuhnen, Gabryela ...................... A 06-07 Hurles, Matthew E. ................................. B 06-05 Hussein, Rama A. ................................ OS 05-02 I

Ihbe, Natascha ....................................... B 02-07 Ikonen, Elina ........................................... S 07-03 Imenez Silva, Pedro Henrique ................ S 11-05 Imig, Cordelia ......................................... S 06-02 Immler, Roland ................... OS 04-01, OS 04-02 Iorga, Bogdan ...................................... OS 08-02 Isbrandt, Dirk .......................................... A 04-05 Ivanovic, Ena ....................................... OS 05-03 J

Jabs, Ronald ........................................ OS 06-04 Jacobi, Charlotte .................................. OS 03-02 Jacobi, Eric ............................................. B 05-03 Jägers, Johannes ................................... B 09-07 Jahns, Celerina .................................... OS 08-05

Jakobs, Marie ....................................... OS 04-06 Jalaie, Houman ...................................... B 10-02 Jamili, Mahdi .......................................... B 05-11 Jammal Salameh, Luna .......................... B 03-01 Jamrichova, Silvia .................................. S 06-01 Jangsangthong, Wanchana .................. OS 08-03 Jans, Judith. J. ....................................... S 06-03 Jatho, Aline ........................................... OS 08-07 Jedlicka, Peter ....................................... W 01-02 Jendryka, Martin .................... OS 06-08, A 01-06 Jensen, Tobias ....................................... S 11-04 Jentsch, Thomas J. ................................ B 01-05 Jeppesen, Majbritt .................................. S 04-04 Jeratsch, Sylvia ...................................... S 05-04 Jessen, Henning J. ............................... OS 02-08 John, David ............................................ B 10-09 Joller, Nicole ........................................... S 11-05 Jonas, Peter ........................................... S 06-01 Jouen-Tachoire, Thibault ........................ B 06-05 Jucht, Agnieszka E. ................................ B 07-05 Judge, Luke M. ....................................... B 12-06 Juretzko, Annett ..................................... A 09-04 Jürs, Björn C. .......................................... S 07-01 Just, Armin ........................................... OS 02-07 K

Kahnt, Franziska .................................... B 03-07 Kaindl, Angela M. ................................... B 01-09 Kalfhues, Lisa ......................................... B 12-01 Kalia, Anil ............................................... B 03-04 Kalpachidou, Theodora ..... OS 01-05, OS 06-07,

OS 09-03, B 02-09 Kaltenbacher, David ............................... A 10-10 Kapanaiah, Sampath ......... OS 06-08, OS 09-01,

A 01-06 Karalis, Nikolas ....................................... S 03-01 Karaman, Ozan ...................................... B 08-03 Kasemir, Jacquelin ................................. A 04-05 Katschinski, Dörthe M. .......... OS 08-07, A 06-09 Kätzel, Dennis ...... OS 06-08, OS 09-01, A 01-06 Kaufmann, Melani .................................. B 07-04 Kaur Bains, Jasleen ............................... A 12-03 Ke, Xijian .............................................. OS 08-04 Kegel, Marcel ......................................... B 05-08 Kelterborn, Simon ................................... A 06-10 Kempf, Sebastian ...... A 07-09, B 08-04, B 11-07 Keppner, Anna ..... S 12-02, OS 02-01, OS 02-05 Kerber, Evelyn L. .................................... S 12-03 Kermani, Fatemeh .................................. B 12-09 Kern, Georg ............................................ B 03-03 Kesavan, Rushendhiran ......................... B 11-05 Kessler, Linda ......................................... B 08-04 Keyser, Britta ........................................ OS 08-01 Khodaie, Babak ........................ A 02-09, B 05-12 Kilb, Werner ............................................ B 04-06


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Kilonzo, Kasyoka ................................. OS 06-08 Kim, Olena .............................................. S 06-01 Kimura, Kenichi ................................... OS 07-01 Kiper, Aytug K. ........................................ A 04-08 Kirsch, Michael ......................... A 06-04, B 08-03 Kirschner, Karin ... OS 02-02, OS 03-02, A 06-10 Kirschstein, Timo ...... A 01-11, A 06-03, A 10-07,

B 02-02, B 02-05 Kiss, Attila ............................................... A 11-10 Kist, Andreas .......................................... B 03-09 Kittel, Robert J. ......................... S 06-05, B 04-03 Klatt, Stephan ......................................... B 11-07 Klein, Mitzi .............................................. A 10-07 Kleinbongard, Petra ............................. OS 08-05 Kleiner, Katharina ................................... B 01-12 Klemm, Kerstin ....................................... B 12-06 Klemz, Alexander ................................... B 05-07 Kleszka, Kira ........................................... B 07-01 Kloeckner, Udo .................................... OS 05-05 Klon-Lipok, Johanna ............................ OS 09-05 Klop, Mathieu ....................................... OS 03-03 Klotzsche - von Ameln, Anne ................. A 12-08 Klugbauer, Norbert .............................. OS 02-07 Kluger, Daniel ......................................... S 03-04 Klumm, Maximilian ................................. A 11-07 Knapp, Fabienne ................................. OS 07-08 Knauf, Felix .......................................... OS 02-02 Knödl, Laura ........................................... A 09-02 Knoepp, Fenja ........................................ A 06-08 Knowlton, Christopher ............................ A 02-08 Knyrim, Maria ...................................... OS 05-05 Koay, Teng Wei ........................ S 12-02, B 07-03 Koch, Ina ............................................... W 02-04 Kockskämper, Jens .................. B 12-03, B 12-08 Koenen, Michael ..................................... B 06-13 Kohl, Peter ............................. OS 07-07, A 10-10 Köhler, Annemarie .................................. A 04-08 Kohlhaas, Felix ....................................... B 01-06 Kohlhaas, Michael .................................. B 12-04 Köhling, Rüdiger .... A 01-11, A 06-03, A 10-07, B

02-02, B 02-03, B 02-05 Köhrer, Karl ........................... OS 04-04, A 07-05 Kojonazarov, Baktybek ........................... B 10-04 Kok, Thomas .......................................... A 10-10 Kolen, Bettina ......................................... S 09-02 Kolobkova, Yulia .................... OS 09-04, A 05-01 Kolonko, Anna Katharina ........................ A 03-10 König, Gabriele ...................... OS 07-03, B 07-02 Kopaliani, Irakli ......................... B 10-08, B 10-12 Kopec, Wojciech ..................................... A 04-08 Köppel, Alina .......................................... B 05-11 Korff, Thomas ........................ OS 03-01, B 11-09 Körner, Jannis .......................... A 04-01, A 04-06 Korotkova, Tatiana ... A 01-01, A 01-03, A 01-05,

A 01-08, B 01-05

Korovina, Irina ........................................ A 12-08 Kortzak, Daniel ......................... S 09-02, A 05-07 Koser, Franziska .................................. OS 07-04 Kostenis, Evi ......................................... OS 07-03 Kostritskii, Andrei .................................. OS 05-07 Kosyna, Friederike K. ............................. B 07-10 Kötter, Sebastian .... OS 08-05, A 11-01, A 11-03 Kovacheva, Lora ................... OS 05-04, B 02-04 Kovacikova, Jana ................................... B 09-09 Kovács, Richard ....................... B 02-06, B 05-07 Kovalchuk, Yury ........ B 01-02, B 01-08, B 03-05 Kovermann, Peter ................. OS 09-04, A 05-01 Kowalski, Kathrin ................... OS 08-01, A 08-09 Kracht, Michael ..................................... OS 07-08 Kraft, Theresia ..... OS 08-01, OS 08-02, A 08-09,

A 10-01, A 10-02 Krämer, Bernhard K. .............................. A 09-06 Krasteva-Christ, Gabriela ....................... A 04-10 Kraus, Andre .......................................... A 09-08 Krause, Gerd .......................................... S 01-03 Kraut, Simone ........................ OS 07-05, A 06-08 Krebes, Lisa A 08-03, A 08-06, A 12-04, B 10-07 Kress, Michaela OS 01-05, OS 06-07, OS 09-03,

B 02-09, B 03-03, B 03-06, B 04-13 Krieger, Katharina .................... A 09-03, B 09-03 Kriegeskorte, Sophia .............................. A 04-02 Križančić Bombek, Lidija ......... A 08-02, A 08-05,

B 05-05 Krueger, Katharina ................................. A 06-10 Krüger, Karsten ...................................... B 02-01 Krüger, Marcus ....... OS 07-04, A 11-01, B 08-01 Krüger, Martina OS 08-03, OS 08-05, A 11-01, A

11-02, A 11-03 Krull, Sabine ........................................... A 06-09 Kruppa, Jochen .................................... OS 03-02 Kucera, Jan P. ...................................... OS 05-03 Kuhn, Michaela ......... A 08-03, A 08-06, A 12-04,

B 10-07 Kukaj, Taulant ........................................ B 05-09 Kuldyushev, Nikita ................................ OS 05-02 Kummer, Kai K. OS 01-05, OS 06-07, OS 09-03,

B 02-09, B 03-06, B 04-13 Künne, Carsten ...................................... S 05-04 Kunstmann, Lukas ................................ OS 06-02 Kunze, Reiner ........................... A 06-01, B 11-09 Kunzelmann, Karl .. S 04-04, OS 05-08, A 09-08,

B 06-04 Kuppusamy, Maithreyan ....................... OS 07-01 Kürten, Tabea ......................................... B 02-07 Kurth, Ingo .............................................. A 04-01 Kurtz, Andreas ........................................ B 09-01 Kurtz, Armin ........................... OS 02-04, B 09-06 Kurzke, Josephine ................................ OS 09-07 Kusche-Vihrog, Kristina .......................... A 12-07 Kuschke, Stefan ..................................... A 03-09


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Kuwabara, Makoto ............................... OS 05-01 Kyselova, Anastasia ................. B 08-01, B 08-10 L

Lal, Sean ............................................. OS 08-01 Lampert, Angelika .. OS 06-03, A 04-01, A 04-02,

A 04-06, B 03-04, B 03-09 Lang, Florian ........................................... B 11-10 Lange, Falko ............. A 01-11, A 06-03, A 10-07,

B 02-02, B 02-05 Lanzuolo, Chiara ................................. OS 01-06 Larena-Avellaneda, Axel ........................ B 10-11 Larsen, Casper K. ................................... S 04-04 Laurette, Patrick ....................... B 12-02, B 12-05 Layé, Sophie ........................................ OS 01-03 Le Cann, Kim .......................................... A 04-01 le Noble, Ferdinand ................................ S 10-02 Lebert, Jan .............................................. S 02-04 Lee, Jessica K. ....................................... A 10-11 Lee, Wing-Kee .......................... S 07-04, B 07-06 Lehmann, Luisa ................................... OS 03-03 Lehmann, Martin ....................... S 01-02, A 03-05 Lehmenkuhler, Alfred ............................. B 04-12 Lehnart, Stephan ................................. OS 08-03 Lehrmann, Claudia ................................. A 09-03 Leichert, Lars .......................................... A 11-01 Leinders, Sacha ................................................... Leipold, Enrico ........................................ B 05-11 Leipziger, Jens ......... S 04-04, S 11-02, S 11-03,

S 11-04, A 12-03, B 11-08, B 11-12 Leisegang, Matthias S. ........................................ Leisengang, Stephan ............. OS 01-04, B 03-02 Leisle, Lilia ................................ B 04-10, B 05-10 Lendeckel, Uwe ...................................... A 09-04 Lenz, Dominik ......... OS 05-01, A 05-04, A 05-06 Lerch, Michael ........................................ A 10-07 Leroy, Felix ............................................. A 04-05 Lessmann, Eva ....................................... A 08-03 Leßmann, Volkmar ................... A 02-09, B 05-12 Leu, Tristan ................ A 06-02, B 07-01, B 07-09 Lewandowski, Jana ................................ B 10-03 Lewin, Uwe ............................................. A 01-06 Li, Guang ............................................. OS 07-01 Li, Ling ................................ OS 07-05, OS 07-08 Li, Xiaoming ............... A 07-09, B 08-07, B 11-07 Li, Yong .................................................. A 11-01 Liang, Hong-Erh .................................. OS 03-05 Liang, Xiujie ......................................... OS 02-03 Lichterfeld, Yannick ................................ A 10-11 Liebe, Franziska ....................... B 06-08, B 06-12 Liebe, Hendrik .......................... B 06-08, B 06-12 Liermann, Wendy ................................... A 05-05 Lim, Dae-Sik ........................................ OS 01-07 Lin, Peipeng ............................................ S 06-01 Linders, Jürgen ....................................... B 08-08

Linke, Wolfgang ... OS 07-04, OS 07-01, A 11-04, A 11-01, A 11-06

Linnebacher, Michael ............................. B 02-05 Liotta, Agustin ......................................... B 02-06 Lippmann, Kristina ................................ OS 09-07 Lipstein, Noa .......................................... S 06-03 Liss, Birgit OS 06-08, A 01-06, A 02-03, A 04-04,

B 01-12, B 02-08 Liu, Wang ............................................... B 08-05 Locksley, Richard ................................. OS 03-05 Loeck, Thorsten ........................ A 05-09, B 06-09 Loescher, Christine M. ............. A 11-01, A 11-04 Lomonte, Bruno ...................................... A 10-01 Longo, Piersilvio ..... OS 05-01, A 05-04, A 05-06 Looso, Mario ............................. B 08-01, B 08-04 López Dávila, Alfredo J. ........ OS 01-06, A 10-01 Lopez, Melina ............ A 09-07, B 10-01, B 11-06 Lother, Achim ....................................... OS 07-01 Luhmann, Heiko J. ................................. B 04-06 Luna-Choconta, Jeiny ............................ B 02-09 Lüttig, Anika ............................................ B 02-03 Lutze, Philipp ............................ B 11-11, B 12-11 M

Ma, Averil .............................. OS 04-03, A 07-03 Maack, Christoph ...... A 08-06, A 11-01, B 12-04 Maas, Sebastian ....................... S 06-05, B 04-03 Machtens, Jan-Philipp .... OS 05-01, OS 05-07, A

05-04, A 05-06 Madai, Sarah .......................................... A 06-01 Madersbacher, Helmut ........................... A 09-10 Madry, Christian ....................... B 01-04, B 02-06 Mair, Norbert ........................................ OS 06-07 Makarewich, Cat ..................................... S 05-01 Malacarne, Pedro ...... A 09-07, B 10-01, B 11-06 Malan, Daniela ...................... OS 01-01, A 10-06 Mallmann, Robert T. ............................. OS 02-07 Malsch, Philipp ..................................... OS 06-07 Malte, Hans ............................................ S 11-04 Manahan-Vaughan, Denise .. OS 09-02, A 01-04,

B 03-08, B 04-01, B 04-09, B 04-11 Mannal, Nadja ........................................ B 01-12 Mansuroglu, Berivan .............................. A 10-05 Manz, Gisela .......................................... B 06-12 Margreiter, Michael ................................. B 04-10 Marhl, Marko .......................................... B 05-05 Maric, Darko ......... S 12-02, OS 02-01, OS 02-05 Markov, Alexander ................................. B 09-11 Marth, Jamey D. ..................................... B 08-05 Marti, Hugo ............................................. A 06-01 Martin, Melanie ......................... B 10-06, B 11-04 Martin, Ulrich .......................................... A 08-09 Martinac, Boris ....................................... B 11-02 Marzian, Stefanie ................................... A 04-08 Masgrau Alsina, Sergi ........ OS 01-07, OS 04-01


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Matchkov, Vladimir ................................. S 11-02 Matinmehr, Faramarz ............................. A 10-01 Matityahu, Lior ........................................ A 02-06 Mattern, Annabelle ................................. A 03-04 Mattes, Björn ....................................... OS 09-05 Matthey, Michaela ................. OS 03-04, B 07-02 Maxeiner, Stephan ................................. A 04-10 Mayer, Christian ..................................... B 08-08 Mayer, Konstantin ................................ OS 01-03 Mayer, Zsuzsanna ............................... OS 03-03 Meents, Jannis ....................................... A 04-01 Melle, Christian ....................................... B 06-06 Menzel, Matthias .................................... A 12-02 Meoli, Luca ............................................. S 01-01 Mergler, Stefan ....................................... B 06-08 Merkel, Cosima ....................................... B 09-10 Merseburg, Andrea ................................. A 04-05 Merten, Anna-Lena ................................. B 11-02 Mete, Vanessa ........................................ A 03-10 Metzen, Eric ............................................ B 07-04 Meyer-Schwesinger, Catherine .............. S 11-03 Michailov, Michael C. .............................. A 09-10 Mildenberger, Sigrid ............................ OS 05-05 Ming, Xiu-Fen .... OS 02-03, OS 06-06, OS 07-06 Misiak, Danny ...................................... OS 05-05 Mitric, Miodrag ..................................... OS 09-03 Mittermaier, Franz X. ............. OS 06-01, A 02-05 Mittmann, Thomas .................................. B 02-07 Mocellin, Petra ........................................ S 02-03 Mogk, Alina ............................................. B 01-06 Mogler, Carolin ....................................... B 10-04 Mohamed, Belal ...................................... A 11-01 Mohebbi, Maral ....................................... A 10-02 Mohebbi, Nilufar ..................................... S 11-05 Mojtahedi, Nima ......... B 01-02, B 01-08, B 03-05 Molenda, Nicole ................................... OS 07-05 Molitor, Darius .......................... S 09-01, A 12-09 Monney, Laurent .................................. OS 02-05 Montag, Judith ..................................... OS 08-01 Monteiro Barbosa, David .......... A 11-02, A 11-03 Moosmann, Julian P. .............................. A 10-07 Moran, Yehu ........................................... B 06-02 Morawietz, Henning ................................ B 10-10 Morikis, Vasilios ................................... OS 04-01 Moritz Wülfers, Eike ............................. OS 07-07 Moroni, Anna .......................................... A 04-05 Mörz, Handan ...................................... OS 05-06 Moscato, Letizia ...................................... A 01-03 Moser, Markus ..................................... OS 04-01 Moser, Tobias ..................................................TL Moskopp, Mats L. ................................... A 12-05 Mottaghy, Khosrow ................................. B 10-02 Mouna, Ahmad ....................................... S 01-01 Mouzakis, Foivos L. ................................ B 10-02 Moztarzadeh, Sina .................................. B 10-02

Mrowka, Ralf .......................................... B 09-01 Mueller, Niklas ........................................ B 11-03 Mueller, Oliver ...................................... OS 08-06 Mulder, Edwin ......................................... A 10-11 Müller, Dominik ....................................... B 09-10 Müller, Emanuel ..................................... A 03-07 Müller, Erik ............................................. A 11-03 Müller, Martin ............. S 06-04, A 02-02, B 04-02 Müller, Niklas ............. A 08-11, A 09-07, B 10-01 Müller, Sonja .......................................... A 02-03 Müller, Steffen ........................................ A 01-11 Müller, Thomas ....................................... B 06-05 Münch, Jan ............................................. A 03-03 Münchmeyer, Moritz ............................... A 02-03 Murgott, Jolanta ...................................... B 03-02 Muscalu, Maria L. ................................. OS 04-08 Musinszki, Marianne .. S 07-01, B 06-01, B 06-07 Mutig, Kerim ........................................... B 09-10 Mutzel, Philipp ........................................ A 06-07 N

Nache, Vasilica ....................................... B 06-06 Nadolni, Wiebke ................................... OS 04-01 Naeem, Zumer ......................... A 07-06, B 08-01 Nag Biswas, Srijeeta .............................. B 03-09 Nagel, Georg ......................... OS 01-02, A 03-07 Nahar, Taslima ..................................... OS 01-08 Najder-Nalepa, Karolina ....... OS 04-05, A 05-03,

A 05-09 Nanadikar, Maithily S. .......................... OS 08-07 Napoli, Matteo ...................................... OS 04-02 Narayanan, Udhaya Bhaskar Sathya ... OS 01-01 Natalia, Vini ............................................ A 03-05 Naumenko, Vladimir ............................. OS 06-05 Nayak, Arnab ........................................ OS 01-06 Nederlof, Rianne .... OS 04-04, A 07-05, A 11-05 Neme, Antonio ........................................ B 08-06 Nemeth, Julia K. ................................... OS 03-06 Nerlich, Jana .......................................... B 04-08 Neu, Eva ................................................. A 09-10 Neubert, Elias ......................................... A 06-05 Neubert, Valentin ...................... A 01-11, B 02-03 Neuhäusel, Tim S. .................................. B 05-07 Neumann, Astrid ..................................... B 04-13 Nickel, Alexander ..................... A 11-01, B 12-04 Niemann, Bernd ................................... OS 07-08 Niethard, Niels ........................................ B 01-08 Nissen, Wiebke ..................... OS 06-08, A 01-06 Noack, Kurt ............................................. B 11-03 Noack, Thomas K. .................................. A 01-11 Noh, Minhee ........................................... A 08-06 Nürnberger, Franz ................. OS 01-04, B 03-02 Nussbaum, Claudia ................................ A 07-03


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O

Obeso, Jose A. ....................................... B 02-04 Odermatt, Alex .................... OS 02-01, OS 02-05 Oeckinghaus, Andrea ............................. A 05-03 Oenarto, Vici ........................................... B 12-01 Offermanns, Stefan ................................ S 05-04 Ögel, Neslihan ..................................... OS 02-02 Ohnmacht, Caspar ................................. B 08-05 Okamoto, Yuji ......................................... S 06-01 Olejniczak, Iwona ................................ OS 06-06 Oliver, Dominik ... OS 05-01, A 03-01, A 05-04, A

05-06 Olmos, Maxime ....................................... A 06-09 Olsen, Jeppe .......................................... S 11-02 Omelchenko, Olesia ............................... A 11-05 Omran, Heymut ...................................... A 03-10 O'Neill, Philipp ........................................ A 02-02 Oo, James A. ......................... OS 07-02, B 11-12 Ooms, Astrid ........................................ OS 07-01 Opazo, Felipe ......................................... A 06-09 Opper, Jennifer ....................................... A 04-09 Orlando, Ilaria ........................ S 12-02, OS 02-05 Ortega-Ramírez, Audrey .......... B 04-10, B 05-10 Oster, Leonie ....................................... OS 04-05 Ott, Daniela ............................ OS 01-04, B 03-02 Otte, Maik ............................................... B 05-06 Otto, Janina I. ......................................... S 09-04 Otto, Mareike .......................................... B 07-10 Ousingsawat, Jiraporn ........... OS 05-08, B 06-04 Ow, Calista J. L. ..................................... B 07-06 Owald, David OS 09-08, A 01-10, B 01-01, B 01-

10 Oz, Osnat ............................................... A 02-06 P

Pacheco, Maria P. .................................. A 09-07 Pagano, Diego Marco ............................. B 01-08 Paladini, Carlos ................................... OS 05-04 Pálfi, Katalin ......................................... OS 07-02 Pan, Daqiang .......................................... B 08-04 Panettieri, Reynold A. ............................. B 07-02 Papantonis, Argyris ................................ A 06-09 Papapetropoulos, Andreas .................. OS 08-06 Parahuleva, Mariana .............................. A 07-08 Parlato, Rosanna .................................... A 02-03 Passlick, Stefan ................................... OS 06-04 Pastor-Arroyo, Eva M. .......... S 11-05, OS 02-08,

OS 04-08 Patejdl, Robert .... S 02-04, OS 01-01, A 01-11, B

09-12 Pauli, Martin ............................................ B 02-10 Peek, Verena ....................................... OS 01-03 Pegoli, Gloria ....................................... OS 01-06 Pekcec, Anton ....................... OS 06-08, A 01-06

Peng, Yangfan ....................... OS 06-01, A 02-05 Penzlin, Johanna .................................. OS 03-02 Perl, Stefanie .......................................... B 02-03 Peters, Jörg ............... A 12-10, B 11-11, B 12-11 Pethö, Zoltan ............................ A 05-03, A 05-09 Petry, Andreas ....................... OS 03-03, A 06-07 Petz, Anne .............................................. A 11-08 Petzold, Anne ........................... A 01-05, A 01-08 Petzsch, Patrick ..................... OS 04-04, A 07-05 Pfabe, Johannes U. ................................ A 08-08 Pflieger, Fabian ..................... OS 01-03, B 02-01 Pflüger-Müller, Beatrice . A 07-01, A 08-04, A 12-

01, B 10-01 Phetö, Zoltan .......................................... B 06-09 Piehler, Jacob ......................................... A 04-07 Piep, Birgit ............................. OS 08-02, A 08-09 Pieske, Burkert ....................................... B 11-10 Pieter Giebtsze, Pieter ........................... A 09-01 Pietsch, Arvid J. S. ................................. B 11-04 Pinckert, Lennart .................................... B 09-10 Piontek, Jörg ............. S 01-01, S 01-02, S 01-03 Plain, Allein ............................................. A 09-02 Planert, Henrike ...... OS 06-01, A 02-05, B 02-06 Plattner, Thomas .................................... A 09-10 Plocksties, Franz V. ................................ B 02-03 Podesser, Bruno ..................................... A 11-10 Poetschke, Christina ................ A 04-04, B 01-12 Pohorec, Viljem ...................................... A 08-02 Ponimaskin, Evgeni .............................. OS 06-05 Ponomarenko, Alexey ............................ B 01-05 Popp, Rüdiger .......................... B 08-01, B 11-05 Popp, Valerie .......................................... B 06-06 Porath, Katrin ............ A 10-07, B 02-02, B 02-05 Porro, Alessandro ................................... A 04-05 Posch, Wilfried ....................................... B 03-03 Postic, Sandra ........... A 08-01, A 08-08, A 08-10 Potenza, Duilio M. .............. OS 02-03, OS 07-06 Pötschke, Christina .................. A 02-03, B 02-08 Pouokam, Ervice .................................... A 03-04 Proks, Peter ............................................ B 06-05 Protze, Jonas ......................................... S 01-03 Prucker, Isabel ..................................... OS 02-08 Pruenster, Monika .............. OS 04-01, OS 04-02 Prüser, Merten ........................................ B 06-01 Puig Walz, Teo ..................................... OS 07-07 Pullamsetti, Soni S. ................................ A 12-03 Q

Qi, Yanmei ............................................ OS 06-07 Qin, Yishi .............................................. OS 03-03 Quarch, Katja ....................................... OS 05-05 Quarta, Serena ..................................... OS 06-07 Querengässer, Jürgen ............................ B 10-11 Querfeld, Uwe ........................................ A 09-06 Questino, Annalisa ............................... OS 05-01


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Qui, Danye ........................................... OS 02-08 Quintanova, Catarina ................ S 01-05, B 09-10 Quinting, Theresa ................................... B 07-01 R

Rabe, Sindy ......................................... OS 05-05 Raccuglia, Davide ... OS 09-08, B 01-01, B 01-10 Radocaj, Ante ...................................... OS 08-01 Raguz, Ivana .......................................... B 02-11 Rajewsky, Nikolaus ...................................... PL3 Ramanujam, Deepak P. ......................... B 11-06 Ramirez, David ....................................... A 04-08 Ramser, Kerstin ...................................... A 06-08 Ramsey, Nick F. .................................................. Randriamboavonjy, Voahanginirina ........ B 08-10 Rannap, Märt .......................................... B 01-11 Rao, Shanlin ........................................... B 06-05 Raßler, Beate ......................................... A 06-05 Ratiu, Corina ............................. A 07-01, B 10-01 Rauch, Anita ........................................... S 06-03 Rauer, Jonah ....................................... OS 06-05 Rauh, Oliver ............................................ A 04-09 Rausch, Steffen ...................................... A 09-06 Rehberg, Markus ................................. OS 04-01 Reichart, Gesine ....................... A 01-11, A 06-03 Reichel, Martin ..................................... OS 02-02 Reichel, Thomas ..................................... B 02-01 Reidel, Sophia ....................... OS 04-04, A 07-05 Reil, Jan-Christian ............................... OS 01-08 Reiner, Andreas ...................................... B 05-01 Reissig, Lukas ........................................ A 11-10 Reitmeier, Jennifer ................................. A 10-02 Remy, Stefan .......................................... S 02-03 Ren, Zhilong ........................................ OS 02-03 Renigunta, Vijay ..................................... A 03-01 Reschke, Christina ................................. A 08-11 Rettig, Rainer .......................................... A 09-04 Rezende, Flávia ........ A 09-07, B 10-01, B 10-09,

B 11-03, B 11-06 Richter, Angelika .................................... B 02-03 Richter, Frank ......................................... B 04-12 Richter, Jan F. ........................................ B 09-04 Richter, Jule ............................................ B 10-06 Ridder, Frederike ................................. OS 03-05 Riedemann, Therese ................ B 04-05, B 05-04 Rieger, Michael ....................................... B 08-01 Rieke, Marius .......................................... B 06-11 Riel, Elena ................. S 07-01, A 04-08, B 06-10 Riesselmann, Jan N. .............................. A 08-09 Rinne, Susanne ...................................... A 04-08 Rinschen, Markus ................................... B 09-07 Ripperger, Jurgen ................................ OS 06-06 Ripphahn, Myriam .................................. A 07-03 Rittner, Heike .......................................... S 01-04 Rittweger, Jörn ....................................... A 10-11

Ritzau-Jost, Andreas ....... PL1, S 06-05, B 04-03 Robert, Steinert E. .................................. B 02-11 Robin, Catherine .................................... S 10-01 Roderfeld, Martin .................................. OS 01-03 Roeb, Elke ............................................ OS 01-03 Roeper, Jochen .. OS 05-04, A 02-01, A 02-04, A

02-08, B 02-04, B 02-08, B 02-12 Rog-Zielinska, Eva ................................. A 10-10 Rohr, Karl ............................................. OS 05-06 Rohrbach, Susanne ............ OS 07-05, OS 07-08 Rohwedder, Ina ... S 10-06, OS 04-01, OS 04-02,

OS 04-03, A 07-03, B 08-05 Rojas-Galvan, Natalia ............................ A 11-10 Röll, Wilhelm ........................................ OS 07-01 Rösch, Melanie ....................................... B 10-07 Rosenberger, Christian ........................ OS 02-02 Rosenthal, Rita ....................................... S 01-03 Rösseler, Corinna ..................... B 03-04, B 03-09 Rossetti, Giulia ....................................... B 04-10 Roth, Joachim ...... OS 01-03, OS 01-04, B 03-02 Roth, Johannes .................................... OS 04-02 Roy, Shoumik ......................................... A 02-03 Rubio-Aliaga, Isabel .............. OS 02-06, A 09-01 Ruf, Irina ................................................. A 04-10 Ruggieri, Sonia ....................................... B 05-03 Rugi, Micol ................................ A 05-09, B 06-09 Rühl, Philipp ........................................... A 04-07 Rummel, Christoph . OS 01-03, B 02-01, B 03-02 Ruppert, Clemens .................................. A 08-03 Russo, Gabriele .................................... OS 09-02 Russo, Giancarlo .................................... S 11-05 Ryndia, Kateryna .................................... A 02-07 S

Saiardi, Adolfo ...................................... OS 02-08 Sakiri, Elif ............................................... S 11-05 Salameh, Aida ........................................ A 06-05 Salcan, Senem ....................................... A 11-03 Salinas Hernández, Ximena I. ................ A 01-09 Salvermoser, Melanie ............................. A 07-03 Sang, I. Emeline W. F. ........................... B 04-06 Santiago-Schübel, Beatrix ...................... A 03-02 Santoro, Bina .......................................... A 04-05 Santos, Fernando ................................. OS 02-06 Saoub, Stefanie ...................................... B 07-08 Sarigu, Gabriele ..................................... A 04-04 Sarikas, Srdjan .......... A 08-01, A 08-08, A 08-10 Saro, Gabriella ..................................... OS 06-06 Sasse, Philipp ...... S 02-01, OS 01-01, OS 08-03,

A 10-05, A 10-06, A 10-08 Sattler, Christian ....... A 03-06, A 03-07, B 05-02,

B 05-06, B 05-09 Sauer, Jonas F. ........................ S 03-03, B 01-07 Sauter, Thomas ...................................... A 09-07 Savitska, Daria ....................................... B 01-02


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Scerri, Isabelle A. ................................ OS 02-05 Schade, Dennis ...................................... A 10-09 Schade, Sophie Kristin ........................ OS 06-05 Schader, Tim .......................................... A 08-11 Schaefer, Liliana ..................................... S 04-01 Schäfer, Ina ......................................... OS 05-06 Schäfer, Sarah ........................................ B 10-07 Schaible, Hans-Georg ............................ B 04-12 Schedlowski, Manfred ......................... OS 04-06 Scheffer Teixeira, Robson ...................... A 01-03 Scheiermann, Christoph ......................... S 10-04 Scheub, Deborah D. ............................... A 04-09 Schewe, Marcus ........ S 07-01, A 04-08, B 06-05 Schippers, Anouck ............................................... Schley, Gunnar .................................... OS 02-02 Schlingmann, Karl P. .............................. S 04-03 Schlüter, Klaus-Dieter ............ OS 07-05, A 03-04 Schmauder, Ralf ....... A 03-03, A 03-06, A 03-09,

B 05-06, B 05-09, B 09-01, B 09-04 Schmidl, Lars .......................................... S 06-05 Schmidt, Andreas ................................... A 11-01 Schmidt, Axel .......................................... B 05-10 Schmidt, Katharina ................................. B 04-02 Schmidt-Ott, Kai .................................. OS 02-02 Schmidt-Supprian, Marc ........ OS 04-03, A 07-03 Schmied, Christopher ............................. S 01-02 Schmitt, Joachim .................................... A 11-01 Schmitz, Nathalie .................................... B 04-05 Schneider, André .................................... B 12-10 Schneider, Christoph ........................... OS 03-05 Schneider, Ulf C. ...................... A 02-05, B 01-09 Schneiders, Jenny .................................. B 02-01 Schneider-Warme, Franziska .............. OS 07-07 Schnitzbauer, Udo ................. OS 02-08, B 09-05 Schnüttgen, Lea ..................................... A 03-10 Schödel, Johannes ................................. S 12-01 Schöler, Ulrike ........................................ B 11-02 Scholz, Carsten C. .. OS 03-08, B 07-05, B 07-07 Scholz, Holger ....................... OS 02-02, A 06-10 Scholz, Tim ............................................. A 10-02 Schönau, Eckhard .................................. A 10-11 Schönberger, Matthias ........................... A 04-10 Schönberger, Tina ............................... OS 04-06 Schönfelder, Martin ................................ A 06-07 Schönherr, Roland ................. OS 05-02, A 04-03 Schöpf, Clemens L. ............... OS 01-05, B 03-03 Schrankl, Julia ....................... OS 02-04, B 09-06 Schreiber, Rainer ... S 04-04, OS 05-08, A 09-08,

B 06-04 Schreiber, Timm ....................... B 07-01, B 07-06 Schreier, Barbara ................................ OS 05-05 Schrenk-Siemens, Katrin ........................ B 03-07 Schröder, Julia ..................................... OS 04-05 Schröder, Katrin ........ A 08-11, A 09-07, B 10-09,

B 11-01, B 11-03

Schröder, Lydia ...................................... B 10-07 Schroeder, Indra ....................... A 03-08, A 04-09 Schroer, Jonas ....................................... B 04-06 Schroll, Tobias ...................................... OS 04-01 Schuh, Kai ................. B 02-10, B 10-07, B 12-04 Schuld, Julia ......................................... OS 07-01 Schültke, Elisabeth ................................. A 10-07 Schulz, Benjamin ...................... S 02-04, B 09-12 Schulz, Christian ...................... S 10-03, A 07-03 Schulz, Eckhard ..................................... B 05-06 Schulz, Marcel H. .................... W 02-03, B 08-06 Schulz, Marie-Christin ............................ A 09-09 Schulz, Rainer ....................... OS 07-08, A 07-08 Schulz, Sabine ..................................... OS 01-03 Schulz, Stefanie ................................... OS 06-08 Schunck, Wolf-Hagen ............................. B 10-01 Schundner, Annika ............................... OS 03-06 Schürmann, Christoph ............................ B 10-09 Schürmann, Sebastian ........................... B 11-02 Schütze, Sebastian ................................ B 01-05 Schwab, Albrecht OS 04-05, A 05-03, A 05-09, B

06-03, B 06-09, B 06-11 Schwaderer, Martin ................................ B 12-02 Schwalbe, Harald ................................... A 12-03 Schweda, Frank .... A 09-03, A 09-05, B 09-03, B

10-07 Schwede, Frank ....................... B 05-06, B 06-06 Schweim, Katja ....................................... A 12-07 Schweinitz, Andrea ................... A 03-06, B 05-06 Schweizer, Patrick .................................. B 06-13 Schwerdt, Gerald .................................... A 09-09 Schwoerer, Alexander P. .......... B 10-05, B 10-11 Sciesielski, Lina .................................... OS 03-02 Sedej, Simon ........................................ OS 07-04 Seewald, Anna ..................................... OS 09-03 Seidel, Thomas ...................................... A 11-07 Seidinger, Alexander ......... OS 03-04, OS 07-03,

B 10-03 Seifert, Gerald ...................................... OS 06-04 Semino, Francesca ................................ B 06-13 Semmler, Dominik .................... A 11-08, A 11-09 Senn, Tatjana ......................................... A 09-10 Sens-Albert, Carla ................................ OS 01-08 Seredinski, Sandra ................... A 12-03, B 11-08 Serra, Michele ........................................ B 02-11 Settelmeier, Stephan .............................. S 12-03 Seuter, Sabine ........................................ B 08-06 Shah, Ajay M. ......................................... B 10-09 Shah, Shah Bahrullah .............. A 07-07, B 08-02 Shapcott, Katharine .............................. OS 09-05 Sherman, Samantha ............................ OS 03-05 Shin, Josef ............................................ OS 05-04 Shin, Joseph ........................................... B 02-04 Sidibé, Thérèse .................................... OS 02-05 Sieckmann, Tobias ............................... OS 02-02


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Sieg, Miriam ......................................... OS 03-02 Siegelbaum, Steven A. ................... PL1, A 04-05 Sierra Marquez, Juan David ................ OS 06-03 Sigala, Fragiska ................................... OS 08-06 Sigurdsson, Torfi ...................... A 01-02, A 01-07 Simon Chica, Ana ................................ OS 07-07 Simon, Annika ....................... OS 03-04, B 07-02 Simon, Scott I. ..................................... OS 04-01 Singer, Wolf ......................................... OS 09-05 Sinha, Frederick ..................................... A 09-05 Sinning, Anne ......................................... B 04-06 Siragusa, Mauro . S 05-04, OS 08-06, A 07-04, A

12-06, B 08-10, B 10-01 Sirota, Anton ........................................... S 03-01 Skelin Klemen, Maša . A 08-02, A 08-05, B 05-05 Skoczynski, Kathrin ................................ A 09-08 Skyschally, Andreas ............................ OS 08-05 Slak Rupnik, Marjan . A 08-01, A 08-02, A 08-08,

A 08-10, B 05-05 Sluga, Nastja .......................................... A 08-10 Soehnlein, Oliver ................................. OS 04-01 Soh, Heun ............................................... B 01-05 Solbrig, Olaf ............................................ B 10-11 Sommer, Natascha ................................. A 06-08 Sonnenberg, Simon Bennet ................ OS 06-05 Sørensen, Mads V. .... S 04-04, S 11-02, S 11-04 Sörmann, Janina .................................... B 06-05 Spaeth, Manuela .................................... B 10-09 Spaich, Desirée ...................................... B 02-08 Spector, Alan C. ..................................... B 02-11 Spengler, Bernhard ............................. OS 01-03 Sperandio, Markus OS 01-07, OS 04-01, OS 04-

02, OS 04-03, A 07-03, B 08-05 Spinti, Annabelle ..................................... A 03-07 Špiranec Spes, Katarina ......................... A 12-04 Sponder, Gerhard ..................... B 06-08, B 06-12 Sprott, David ........................................... A 12-08 Spychala, André ..................................... A 11-05 Staar, Doreen ............ A 12-10, B 11-11, B 12-11 Staps, Maximilian ................................... B 12-10 Steele-Perkins, Peter ........................... OS 06-08 Stehr, Herrmann .................................. OS 03-02 Steinbach, Antje ..................................... A 09-04 Steinhäuser, Christian ......................... OS 06-04 Stellwag, Thomas ................................... A 03-05 Stephan, Alice ........................................ B 03-01 Šterk, Marko ............................. A 08-05, B 05-05 Stingl, Julia ............................................. A 04-06 Stock, Christian ...................................... A 09-11 Stockebrand, Malte ................................. A 04-05 Stockmann, Christian ............................. S 12-04 Stöhlmacher, Paula ................................ A 10-07 Stojanovic, Strahinja ............................ OS 05-04 Stožer, Andraž ........... A 08-02, A 08-05, B 05-05 Strahnen, Daniel .................................. OS 09-01

Strätz, Nicole ........................................ OS 05-05 Straub, Isabelle .................................... OS 06-02 Straub, Tobias ........................................ A 07-03 Strauch, Christina ................................... B 03-08 Streckfuß-Bömeke, Katrin ...................... B 12-05 Striessnig, Jörg ..................................... OS 05-04 Strommer, Katharina .............................. B 04-13 Strožer, Andraž ...................................... A 08-10 Stumpff, Friederike ................... B 06-08, B 06-12 Sturm, Maximilian ................................... A 04-09 Su, Xin .................................................... B 03-05 Suárez-Grimalt, Raquel ......... OS 09-08, B 01-10 Sudha Bhagavath Eswaran, Vishal ........ A 04-01 Surala, Michael ....................................... B 01-04 Sutor, Bernd ........................................... B 05-04 Svendsen, Samuel L. S 04-04, S 11-02, S 11-03,

B 09-10 Sydow, Jan-Eric ..................................... B 08-08 Sylaj, Eugena ......................................... A 03-07 T

Takahashi, Satoru ................................ OS 07-01 Talbi, Khaoula .......................... A 09-08, B 06-04 Tallon-Baudry, Catherine ....................... S 03-05 Taschenberger, Holger ........................... S 06-03 Tascio, Dario ........................................ OS 06-04 Tastan, Oezden ...................................... A 07-08 Tauber, Philipp .......... A 09-03, A 09-05, B 09-03 Tchuendem, Linda M. ............................. A 07-07 Tebbe, Bastian ..................................... OS 04-06 Tegtmeier, Ines ...................................... A 09-02 Teichmann, Tom ...................... A 08-04, A 12-01 Thalheimer, Andreas .............................. B 02-11 Thévenod, Frank .................................... B 07-06 Thiel, Gerhard ........................................ A 04-09 Thomale, Ulrich-Wilhelm ........................ B 01-09 Thomas, Dominique ............................. OS 05-04 Thot, Georgina K. ................................... A 10-11 Tian, Yuemin .......................................... S 09-03 Tiburcy, Malte ......................................... A 11-06 Timmermann, Aline .............................. OS 06-04 Tiroshi, Lior ............................................. A 02-06 Todesca, Luca M. ..................... B 06-03, B 06-11 Toischer, Karl ......................................... A 11-01 Toklucu, Idil ............................................ A 04-06 Tombor, Lukas ....................................... B 10-09 Tort, aDRIANO B. ................................... S 03-02 Treede, Rolf D. ..................................... OS 05-06 Trefz, Johannes ...................................... A 06-07 Tröder, Simon E. .................................... A 04-05 Trogisch, Felix ...................................... OS 01-08 Truong, Vincent ...................................... B 03-04 Tsai, Wen-Hao ....................................... A 08-01 Tucker, Stephen J. ................................. B 06-05 Türk, Clara ............................. OS 07-04, B 08-01


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Tütüncü, Ecem ......................... B 01-04, B 01-09 Tzika, Maria ............................................ A 04-10 Tzingounis, Anastasios V. ...................... B 01-05 U

Ugrica, Marko ......................................... A 09-01 Ukan, Urun ............................... A 07-04, B 08-07 Ullrich, Melanie ......................... B 02-10, B 12-04 Ullrich, Nina D. ....................... OS 08-04, B 12-09 Ulrich, Reiner .......................................... A 05-05 Unger, Andreas ..................... OS 07-01, A 11-01 Unrein, Franziska ................................... A 12-07 Unzeitig, Christopher ................ B 05-02, B 05-06 Upcin, Berin ............................................ A 12-04 Uta, Petra ............................................... A 10-02 V

van Belle, Gijsbert J. ............. S 02-04, OS 08-07, A 06-09

van den Munkhof, Hanna ........ A 01-01, A 01-03, A 01-08

van der Veen, Bastiaan ......... OS 06-08, A 01-06 van der Veen, Rozemarijn ...................... A 03-05 van der Velden, Jolanda ...................... OS 08-01 van der Ven, Peter F. .......................... OS 07-01 Vandael, David ....................................... S 06-01 Vande Velde, Greetje ............................. A 04-10 Vansteensel, Mariska J. ............................... PL4 Vergel Leon, Ana Maria ....................... OS 08-07 Vida, Imre .............................. OS 06-01, A 02-05 Vigolo, Emilia ....................................... OS 02-02 Vitzthum, Helga ...................................... S 11-03 Voelkl, Jakob .......................................... B 11-10 Vogel, Pascal .......................................... A 01-02 Vogl, Thomas ...................................... OS 04-02 Vogt, Markus ......................... S 02-04, OS 01-01 Voigt, Katharina ...................................... A 07-05 Volk, Tilmann .......................................... A 11-07 Völker, Katharina ...................... A 12-04, B 10-07 Völker, Uwe ............................................ A 12-10 Volkova, Yulia ...................................... OS 03-08 vom Dahl, Christian ................................ A 03-07 von Engelhardt, Jakob .............. B 05-03, B 05-08 von Frieling-Salewski, Marion ................. A 11-06 von Kriegsheim, Alexander ..................... B 07-05 von Oepen, Vincent ................................ A 06-02 von Schwerdtner, Otto ............................ B 09-10 W

Wacker, Paul .......................................... B 11-09 Wackerbarth, Lou M. ............. OS 04-03, A 07-03 Wackerhage, Henning ............................ A 06-07 Wagdi, Ahmed ....................... S 02-04, OS 01-01 Wageringel, Isabel .................................. B 10-05 Wagner, Carsten ................................. OS 02-06

Wagner, Carsten A. S 11-03, S 11-05, OS 02-08, OS 04-08, A 09-01, B 09-05, B 09-09

Wagner, Charlotte ................. OS 02-04, B 09-06 Wahl, Joel ............................................... A 06-08 Walsh, Patrick ........................................ B 03-04 Walther, Florian ...................................... A 03-07 Walzog, Barbara .................... OS 04-02, A 07-03 Wang, Dan ........................................... OS 05-06 Wang, Danni ........................................... B 05-08 Wang, Luzhou .......................... A 11-08, A 11-09 Wang, Tianbang ..................................... A 10-02 Wang, Tobias ......................................... S 11-04 Wanka, Heike ............ A 12-10, B 11-11, B 12-11 Wardelmann, Eva ................................... A 05-03 Warm, Davide ........................... B 04-06, B 04-07 Warth, Richard ....................................... A 09-02 Warwick, TimothyOS 07-02, A 09-07, B 08-06, B

10-01, B 10-09, B 11-01, B 11-03, B 11-08 Weber, Michael ...................................... A 03-10 Weber, Natalie .......................... A 08-09, A 10-01 Wegener, Katja ....................................... A 11-02 Weichard, Iron ........................................ B 04-08 Weigert, Andreas OS 08-06, A 07-04, A 07-06, B

08-01, B 08-04, B 10-09, B 11-05 Weisbecker, Hanna ................................ A 06-03 Weiss, Lisa M. .......................... B 12-10, B 12-07 Weissenbacher, Ernst R. ........................ A 09-10 Weissmann, NorbertOS 07-05, A 06-08, B 10-09 Wenger, Roland H. ... OS 02-05, OS 03-08, B 07-

05 Wenninger, Wolfgang ............................. A 11-10 Wenzel, Daniela .. OS 03-04, OS 07-03, B 07-02,

B 10-03 Wenzel, Jan ............................................ S 11-06 Werner, Franziska .................... A 12-04, B 10-07 Westerhausen, Matthias ......................... A 04-07 Weyand, Michael .................................... A 11-07 Wickleder, Mathias S. ............................. A 03-04 Widmer, Jeannette ................................. B 02-11 Wiederspohn, Nicole . A 02-03, A 04-04, B 02-08 Wiemer, Johanna ................................... A 02-03 Wilde, Fabian ......................................... A 10-07 Wildner, Florian ........................ B 01-09, B 05-07 Willam, Carsten .................................... OS 02-04 Willuweit, Antje ..................................... OS 06-03 Windbergs, Maike .................................. W 01-01 Winning, Sandra ..................................... S 12-03 Winterstein, Janis ................................... B 01-11 Wirsching, Eva ......................... A 07-02, A 07-10 Witte, Jeannine ....................................... A 09-04 Wittig, IlkaOS 07-02, OS 08-06, OS 08-08, A 12-

03, A 12-06, B 08-04, B 10-04, B 11-12 Wittig, Janina .......... OS 08-06, B 08-04, B 10-04 Witzke, Oliver ....................................... OS 04-06 Wolf, Alexandra ...................................... B 07-04


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Wolf, Annemarie ..................................... A 03-04 Wolf, Lisa ................................................ B 09-02 Wolframm, Benedikt ............................... A 11-02 Wolters, Maria ........................................ A 05-03 Wright, Caroline F. .................................. B 06-05 Wrobeln, Anna ........ OS 03-07, A 06-06, B 07-08 Wu, Sean M. ........................................ OS 07-01 Wulff, Peer .............................................. B 01-06 Wuttke, Dominik ..................................... A 02-03 Y

Yang, Jenq-Wei ...................................... B 04-06 Yang, Kefan ............................................ A 04-03 Yang, Shang .......................... OS 01-02, A 03-11 Yang, Shi-Bing ........................................ A 08-01 Yang, Zhihong ... OS 02-03, OS 06-06, OS 07-06 Yanovsky, Yevgenij ................................ S 03-02 Yassini, Nima .......................................... S 11-05 Yerly, Laura ......................................... OS 02-05 Yevtushenko, Anna ............................. OS 04-01 Yu, Weijia ............................................... B 08-01 Yu-Strzelczyk, Jing ................ OS 01-02, A 03-11

Z

Zabri, Heba ............................... A 11-08, A 11-09 Zaldivar-Diez, Josefa .............................. B 02-04 Zatschler, Birgit ...................................... B 10-12 Zeidler, Maximilian ................ OS 01-05, B 03-03 Zeng, Shufei ........................................... A 09-06 Zernecke, Alma ...................................... B 10-07 Zessin, Franziska ................................... B 02-02 Zeug, André .......................................... OS 06-05 Zgierski-Johnston, Callum .................... OS 07-07 Zhang, Jiong ........................................... B 05-08 Zhou, Xiaozhu .......................... S 05-04, A 12-06 Zierler, Susanna ................................... OS 04-01 Zieseniss, Anke ...................................... A 06-09 Zimmer, Thomas ...................... A 03-07, B 05-09 Zimmerhofer, Alexandra ......................... A 11-05 Zimmermann, David ............................... B 03-03 Zimmermann, Wolfram-Hubertus .......... A 11-06,

B 08-04 Ziouziou, Tabea ...................................... A 02-08 Zukunft, Sven ........................... A 07-06, B 08-04 Zweigerdt, Robert ................................... A 08-09

Keyword Index DPG 2021 | Abstract Book

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Keyword Index

1-10

17 β estradiolol ....................................... B 10-12 3Rs ........................................................ W 01-02 β subunit ................................................. A 04-03 A

A20 ......................................................... A 07-03 A2B receptors ......................................... B 10-03 absorption ............................................... B 04-13 acetylcholine ........................................... A 08-10 Acetylcholinesterase ............................... B 09-03 Acid sensing ion channel ........................ S 09-03 Acid-base .................................. S 11-02, S 11-04 acid-base balance .................................. S 11-05 acid-base disorder .................................. S 11-03 Acid-sensing ion channels ...................... B 04-10 acinar cells .............................................. A 08-10 Active Standing Test ............................... B 10-05 activity pattern ........................................ B 04-06 acute human neocortical slices .............. B 01-09 acute oxygen sensing ............................. A 06-08 ADAR ...................................................... A 12-06 adhesion molecule L1 .......................... OS 06-05 adiponectin .......................................... OS 03-06 ADPKD ................................................... A 09-08 adrenal gland .......................................... A 09-02 Adrenergic Receptors ............................. A 03-04 adrenergic signaling ............................... A 10-08 Adult neurogenesis ................................. B 03-05 aging .................................................... OS 07-06 airway inflammation ................................ B 06-04 airway tone .......................................... OS 03-04 Aldosterone ......................................... OS 02-01 aldosterone regulation ............................ A 09-02 ALI/ARDS ............................................... A 07-10 alkali loading ........................................... S 11-03 alkali therapy .......................................... S 11-05 alkalosis .................................................. B 09-09 alpha-blocker .......................................... A 04-06 alveolar epithelial type 2 cells (AT2s) .. OS 03-05 alveolar macrophages ......................... OS 03-05 alveolar-capillary barrier ......................... A 07-10 Alzheimer's disease ................................ B 05-03 Ammonium ............................... S 11-02, B 06-12 AMPA receptor ...................... OS 06-04, B 05-08 amplifying pathway ................................. A 08-02 Androglobin ........................... OS 02-01, B 07-03 ANG II independent effects .................... B 12-11 Angiogenesis .......... OS 07-02, A 12-01, A 12-08

Angiotensin II .......................................... B 09-06 angiotensin(1-7) ..................................... A 09-04 Animal Models ........................................ B 02-12 anion conductance ................................. A 05-07 anisotropy ............................................... B 03-09 anterior cingulate cortex ......................... A 01-06 antidiabetic drugs ................................... B 05-05 apoptosis ................................................ B 04-06 Arginase 2 .......................... OS 02-03, OS 07-06 aristolochic acid nephropathy ................. A 09-04 arrhyhtmia .............................................. S 02-01 arrhythmogenic diseases ..................... OS 08-04 Arterial Calcification due to Deficiency of CD73

(ACDC) ............................................... B 10-06 artifical oxygen carrier ............................ A 06-04 Artificial Blood ......................................... B 08-03 Artificial Oxygen Carrier .......... A 07-07, B 08-02,

B 08-03, B 08-08 asthma .................................................. OS 03-04 AT1 receptors ......................................... B 09-06 atherosclerosis ......................... A 07-08, B 10-07 ATP metabolism ................................... OS 06-02 Atrial fibrillation ....................................... A 11-07 atrial myocytes ....................................... B 12-08 Atrial Natriuretic Peptide ......................... A 12-04 A-type K+ currents ................................. A 04-04 audition ................................................. OS 09-02 automated patch-clamp .......................... A 04-02 autophagy ............................................... S 04-01 B

BAG3 ........................................ A 10-03, B 12-06 baroreceptor reflex ................................. B 10-05 basal forebrain ........................................ B 03-06 Bayesian statistics .................................. A 03-03 beta cells .................................. A 08-08, A 08-10 beta cells’ NMDA receptors .................... B 05-05 Bioinformatics ........................................ W 02-03 Blood therapeutics .................................. B 08-02 brain ....................................................... S 03-05 brain endothelial cells ............................. S 11-06 brain metastases .................................... B 02-02 brain oscillations ..................................... S 03-04 brain tumor ............................................. B 02-05 breathing ................................................ S 03-01 Burst-like Transcription ......................... OS 08-01 C

cachexia ............................................... OS 01-06


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Calcification of Joints and Arteries (CAJA) . B 10-06

Calcium ...... A 05-09, A 08-08, B 06-08, B 12-04, B 12-08

calcium channels ................................. OS 05-04 calcium dynamics as multicellular networks B 05-

05 calcium imaging ........ A 01-08, A 03-11, A 08-02,

B 01-08 calcium signaling ................... OS 04-01, B 12-09 calpains .................................................. B 07-03 cAMP ...................................................... A 03-09 cancer ..................................................... S 09-03 carcinogenesis .......................... S 07-04, B 07-06 cardiac .................................................... A 11-01 cardiac fibroblasts ................................... B 12-07 cardiac function ...................................... A 06-05 Cardiac hypertrophy ............................... B 10-04 cardiac myocytes .................................... B 12-03 cardiac proteome ................................. OS 07-04 Cardiac research .................................... B 12-10 cardiac sodium channels ..................... OS 05-03 cardiomyocyte cell cycle variations......... A 10-09 cardiomyocyte signaling ...................... OS 08-05 cardiomyocytes OS 05-05, OS 08-03, A 10-01, A

10-05, A 10-06, A 10-10, B 12-04 cardiomyopathies ................................ OS 07-01 cardioprotection ...................................... B 11-11 cardiovascular disease ........................ OS 03-03 cav1.3 .................................................. OS 05-04 CaVβ/F-actin complex ............................ A 03-02 Cell cycle ............................................. OS 07-02 cell death ................................................ B 12-11 cell excitability ......................................... A 08-01 Cell stretching device ............................. B 11-02 central sensitization ............................. OS 06-03 ceramide .................................... S07-02, S 07-04 Cerebellum .......................................... OS 06-02 cerebral ischemia ................................... A 06-02 CFTR ........................................ S 11-04, A 09-08 channel ..................................... S 01-03, S 09-02 channelrhodopsin ................................... A 03-07 chemogenetics in 5-CSRTT ................... A 01-06 chemo-optogenetics ............................... B 05-01 chemotaxis .......................................... OS 04-05 Chloride .................................................. S 09-02 chloride homeostasis ........................... OS 09-04 chloride-transport activity ..................... OS 06-03 cholinergic ........................................... OS 09-03 chromatin interactions ............................ B 12-07 chronic asthma ....................................... B 07-02 chronic kidney disease ............. S 11-05, A 09-09 cilia ......................................................... B 07-09 ciliogenesis ............................................. S 12-02 CKAMP44 ............................................... B 05-08

claudin .................................................... S 01-03 Claudin segregation ............................... S 01-02 claudin-10 ............................................... S 01-01 claudin-10b ............................................. S 01-05 claudins .................................................. B 09-11 CLCA1 .................................................... B 06-04 clearing ................................................... B 09-01 CNG channels ........................................ B 06-06 cNMP .................................................... OS 01-02 CNP ........................................................ B 10-07 CO2 ........................................................ S 11-06 cochlea ................................................... A 05-06 co-culture ................................................ A 09-09 Collapsible tubes .................................... B 10-02 Collecting Duct ....................................... B 09-03 colon carcinoma ..................................... B 07-08 compartmental & morphological modeling W 01-

02 complexome profiling ........................... OS 08-08 computational electrophysiology .......... OS 05-07 Coneopsin .............................................. A 10-06 connectin ................................................ A 11-03 Connective tissue ................................... A 10-11 Connexin-43 ......................................... OS 08-04 contractile force .................................... OS 08-02 Corepressor ............................................ A 12-01 cortex ...................................................... B 05-04 cortical microcircuit ............................... OS 06-01 cortical presynaptic terminals ................. S 06-01 Cortico-striatal projections ...................... B 02-03 Crosslinking-mass spectrometry ............ A 03-02 C-type Natriuretic Peptide ........ A 08-06, A 08-03 CYP2E1 .................................................. B 10-04 cyst growth ............................................. A 09-08 cystathionine gamma lyase ... OS 08-06, B 10-04 cysteine catabolism ................................ B 08-04 Cytochrome P450 reductase .................. B 10-01 Cytokines ................................................ B 03-02 cytosolic renin ........................... B 11-11, B 12-11 D

decoding analysis ................................. OS 09-01 Deep brain stimulation ............................ B 02-03 DEG/ENaC ............................................. B 05-10 dendritic morphology .............................. B 03-05 Dentate gyrus ......................................... B 01-03 Depolarization block ............................... A 02-08 developemntal disorder .......................... B 06-05 development ........................................... B 04-06 developmental epileptic encephalopathies . A 04-

05 Developmental Physiology ..................... S 10-02 Diabetes ................................................. B 08-10 diabetes mellitus ..................................... B 11-10 diabetic cardiomyopathy ......................... B 12-01


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Dietary Pi ................................................ B 09-05 differentiation .......................................... B 11-01 Diltiazem ................................................. B 06-06 disease-relevant SNPs ........................... B 12-05 dLGN ...................................................... B 05-08 docking ................................................... A 03-02 dopamine ................ OS 05-04, A 01-01, B 04-11 Dopamine A 01-09, A 02-01, A 02-08, B 02-12, B

05-12 Dopamine D2 Autoreceptor .................... A 02-04 dopaminergic neurons ............................ B 01-12 Dorsal Root Ganglion (DRG) .................. B 03-09 Drosophila ................. B 01-01, B 01-10, B 04-02 DRS ........................................................ B 12-10 drug transporter ...................................... S 07-04 Drug-target .......................................... OS 06-08 Dystonia .................................................. B 02-03 E

E3 ligases ............................................... B 04-02 echocardiography ................................ OS 01-08 ecophysiological adaptation ................... A 08-07 Ectonucleotidases .................................. B 10-06 EGF signaling ......................................... A 08-11 e-learning ................................................ B 10-05 electron microscopy ................................ A 10-10 electrophysiological profile ..................... B 02-04 electrophysiology .................................... A 05-04 Emotion .................................................. B 01-07 ENaC ...................................................... A 04-10 Endocannabinoids ................. OS 03-04, A 08-04 Endomysium ........................................... A 10-11 endothelial cell pericyte interaction ......... B 11-07 endothelial cells ......... S 05-04, A 12-05, B 11-01 Endothelial function ................................ B 10-10 endothelial GC-B .................................... B 10-07 endothelial nanomechanics .................... A 12-07 endothelial senescence ....................... OS 08-06 endothelial-to-hematopoietic transition ... S 10-01 energy metabolism ................................. A 06-05 eNOS ...................................................... B 10-01 eosinophils .............................................. B 08-05 epigenetic signaling ............................. OS 01-06 Epigenetics ............... S 12-01, W 02-03, B 08-06 Epilepsy .... OS 09-04, B 02-06, B 04-12, B 05-11 epileptic encephalopathy ........................ A 05-01 Epithelial Sodium Channel ..................... A 04-10 Epoxyeicosatrienoic acids ...................... B 10-01 Erbin ....................................................... A 08-11 erlotinib resistance .................................. B 06-03 Erythropoietin ........ S 04-02, S 12-05, OS 02-05,

OS 02-04 Extinction ................................................ A 01-09 extracellular acidosis .............................. A 09-09 extracellular matrix .............................. OS 03-06

F

Fabry disease ......................................... B 02-09 Fast Spiking Interneurons ........ B 01-09, B 05-07 Fear conditioning .................................... A 01-09 Feeding behaviour .................................. A 01-08 ferroptosis ............................................... B 08-04 fetal hypoxia ......................................... OS 03-03 fetal immune system .............................. A 07-03 fever ..................................................... OS 01-03 FGF-23 ................................................... B 09-05 FGF23-Peptides ................................... OS 02-06 Fibroblasts ............................................ OS 07-07 Fibrosis ................................................... A 09-03 flash and freeze ...................................... S 06-01 FLIM ....................................................... A 03-06 fluorescence in situ hybridization ........... A 01-04 fluorescent ligands ................................. A 03-06 fluorescent nanosensor .......................... A 02-07 fMRI ....................................... OS 09-02, B 03-08 FRET ...................................................... B 05-09 functional interactions ............................. B 12-05 Functionalized Nanoparticles ................. A 03-04 G

GABAergic neurons ................................ B 01-06 GABAergic transmission ........................ B 01-03 gain-of-function ....................................... A 05-01 galvanotaxis ........................................... B 02-05 Gamma ................................................. OS 09-05 Gamma Oscillations ............. OS 09-06, B 01-04,

B 01-09, B 05-07 gap junctions ........................................ OS 05-03 g-csf ...................................................... OS 01-07 gene regulation ....................................... B 07-06 gene therapy ........................................ OS 07-01 glioblastoma ........................................... B 02-02 glioma ..................................................... B 02-05 Globin ..................................................... S 12-02 glucose ................................................... A 05-05 glucose sensing ...................................... B 01-12 glucose-stabilized nanoparticles ............ B 08-08 glucose tolerance ................................... S 07-02 glutamate ................................................ A 02-07 glutamate receptor ................................. B 02-02 glutamate transporter ........... OS 09-04, A 05-08,

A 05-01 Glycolysis ............................................... A 06-01 GM-CSF ............................................... OS 03-05 gp130 ................................................... OS 06-07 GPCR ..................................................... B 05-09 Gq ......................................................... OS 01-01 Gq protein inhibition ............................... B 07-02 Gq proteins ........................................... OS 07-03 Growth .................................................. OS 02-06


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Growth-Plate ........................................ OS 02-06 gut homeostasis ..................................... S 12-04 H

H9c2 cell death. ...................................... B 11-11 H-ATPase ............................................... B 09-09 HCM ...................................... OS 08-02, A 08-09 HCN Channel ........................... A 02-06, A 03-09 HCN1 ...................................................... A 04-05 Heart ....................... S 02-01, S 03-05, OS 07-06 heart disease .......................................... S 05-02 Heart failure ......... OS 01-08, OS 07-08, B 12-01 heart regeneration .................................. A 10-09 HELIX syndrome .................................... S 01-01 Hematopoietic stem cells ........................ S 10-01 Hemodilution ........................................... B 08-02 hemoglobin ............................................. B 08-09 hereditary hemorrhagic telangiectasia . OS 03-07 heterodimerisation .................................. B 06-01 heteromeric receptor assembly .............. B 05-01 HFpEF ................................................. OS 07-04 HHT ........................................................ A 06-06 Hidden Markov ....................................... A 03-03 HIF ............................. S 12-01, S 12-03, B 07-01 HIF-2a .................................................. OS 02-05 high altitude ............................................ B 08-09 high throughput analysis ......................... B 09-07 high-pressure freezing ............................ A 10-10 Hippo pathway ........................................ A 06-07 hippocampal network activity ............... OS 09-07 hippocampal synaptic plasticity .............. A 01-04 hippocampus ............ A 01-07, A 02-09, B 05-12,

B 01-08, B 01-11, B 02-09, B 04-09, B 04-11 homeostasis ............................. S 06-04, B 04-03 hPSC-CMs .............................................. A 08-09 Hyperosmolarity ................................... OS 02-02 Hypertension ......................... OS 01-08, B 10-08 Hypertrophic Cardiomyopathy ............. OS 08-01 Hypothalamus ......................................... A 01-08 Hypoxia . S 12-01, S 12-03, OS 02-03, OS 02-05,

OS 03-01, OS 03-02, OS 04-06, A 06-01, A 06-06, A 06-07, A 06-08, A 06-09, A 06-10, A 12-09, B 07-01, B 07-06, B 08-09, B 07-10

Hypoxia-inducible factor ... S 09-01, S 12-05, OS 03-07, A 06-02, B 07-08, B 07-09

I

idiopathic pulmonary fibrosis ............... OS 03-06 IGF-1 ...................................................... A 07-05 immediate early gene ............... A 01-04, B 03-08 Immobilisation ......................................... A 10-11 immune cells ........................................... A 07-07 immune system ...................................... A 06-06 Immune-to-brain communication ............ B 02-01 immunity ................................................. S 12-03

Impulsivity ............................................. OS 06-08 in vitro assay .......................................... A 10-02 in vitro epilepsy ....................................... B 02-07 in vitro model .......................................... A 07-10 in vivo patch clamp ................... A 02-01, B 01-01 Inactivation ............................................. B 05-11 Inflammation .......... S 04-01, S 12-04, OS 04-03,

OS 04-06, A 07-01, A 07-04, A 08-04, B 08-05, B 10-08

Inflammatory pain ................................. OS 01-04 inflammatory resolution .......................... A 07-09 infoldings ................................................ S 01-05 Ingestive behavior .................................. B 02-11 innate behaviour ..................................... A 01-01 Innate Immunity ...................................... A 07-02 Innate Lymphoid cells hypoxia ............... S 12-04 Innervated skin model ............................ B 03-09 Insulin ................................................... OS 04-04 Insulin-like growth factor 1 .................... OS 04-04 intact cardiac tissue slices ..................... A 10-04 integrative pathophysiology .................... A 09-10 integrins .................................................. S 07-05 intercalated disc ................................... OS 05-03 interdisciplinary integration ..................... B 10-11 interneuron ............................................. B 01-04 Interstitial Cells ....................................... A 09-03 intestinal organoids .............................. OS 04-07 intracellular trafficking ........................... OS 02-07 invariant and knockout analysis ............ W 02-04 ion channels .............. A 03-06, A 04-05, B 06-11 ionotropic glutamate receptors (iGluRs) . B 05-01 iPSC ........ OS 01-05, B 03-03, B 03-04, B 03-07,

B 09-01 iPSC-derived cardiomyocytes .............. OS 08-04 Ischemia ................................................. B 11-09 ischemia reperfusion injury ..................... A 06-04 Ischemic stroke ...................................... A 06-03 Isoluminance ........................................ OS 09-05 K

K+ channel ............................................. S 07-01 K+ channel inactivation .......................... A 04-03 K2P channel ............. S 07-01, B 06-01, B 06-05,

B 06-07, B 06-10 K2P channel gating ................................ B 06-10 K2P2.1 .................................................... B 06-09 KATP ...................................................... A 02-01 KATP channel ...................................... OS 09-07 KCa3.1 ................................................... B 06-03 keratinocyte ............................................ B 06-12 Kidney .................... S 04-01, OS 02-03, B 09-06 Kidney Injury ......................................... OS 02-02 KV1.3 .................................................... OS 04-01


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L

laboratory class ...................................... B 10-11 Lactate dehydrogenase .......................... A 06-03 Lateral Septum ....................................... A 01-03 Leukocyte recruitment ......................... OS 04-02 leukocytes ............................................ OS 04-06 ligand-gated ion channel ........................ B 05-10 light chain ............................................... A 10-02 lipid regulation ........................................ A 04-08 lipid scramblases ................................. OS 05-07 lipid-bilayer ............................... A 03-08, A 04-09 lipidomics ............................................. OS 01-03 lncRNA ................... S 05-02, OS 07-02, A 07-01 locomotor activity ................................. OS 06-06 locus coeruleus ....................................... B 04-09 long-term depression .............................. B 04-09 Low back pain ...................................... OS 05-06 LPS ...................................................... OS 01-03 LPS tolerance ......................................... B 03-02 L-type Ca2+ channel ........................... OS 05-05 Lung ........................................................ A 07-02 lung development ................................ OS 03-02 M

machine learning ................................. OS 09-01 Macrophages ......... S 10-03, OS 07-07, A 07-02,

B 08-07 maintenance ........................................ OS 08-08 Markov state model ................................ A 05-07 Mas receptor ........................................... A 09-04 mathematical modelling .......................... B 11-04 Mechanosensitive ion channels B 06-09, B 11-02 Mechanotransduction ............................. B 11-02 medial entorhinal cortex ......................... B 01-11 medial prefrontal cortex ......... OS 09-03, B 01-06 medical marihuana ................................. B 04-13 Medicinal Signalling Cells .................... OS 01-04 MEG ....................................................... S 03-04 megakaryopoiesis ................................... B 08-01 Memory ................................................... A 02-09 memory consolidation ............................. S 03-01 Metabolic acidosis .................................. S 11-02 metabolic fluxes ...................................... A 10-04 Metabolism ................................ S07-02, A 06-01 metabolomics ........................... B 09-07, B 11-03 Methionine oxidation ............................ OS 05-02 mGluR5 ............................................... OS 09-06 mGluRs ................................................... B 05-09 Microbeam Irradiation ............................. A 10-07 microdomain ........................................... A 10-05 microfluidics ............................................ A 06-08 Microglia ................................................. B 01-04 microproteins ............................ S 05-02, S 05-04 microRNA ............................................... A 07-08

migration ................................................. A 12-05 miR-221/222 ......................................... OS 05-05 miRNA .................................................. OS 03-03 mitochondria .......................... OS 08-08, B 12-08 mitoflash ............................................... OS 05-06 mixed neuro-glial cultures ...................... B 03-02 Monocytes .............................................. A 07-01 mPFC .................................... OS 06-08, A 01-02 mucus secretion ..................................... B 07-02 multicellular functional imaging .............. A 08-01 multielectrode array ................................ B 02-07 multi-electrode array (MEA) ................... B 01-12 multipatch ............................................. OS 06-01 Multivalency ............................................ A 03-04 Munc13 ................................................... S 06-03 Muscle Physiology ................................ OS 01-01 muscular dystrophy ................................ A 10-03 myocardial infarction ............................ OS 08-05 myofibrillar myopathy ............ OS 07-01, B 12-06 myofibrils .............................................. OS 08-02 Myofibroblasts ........................................ A 08-03 myosin .................................................... A 10-02 myotoxin ................................................. A 10-01 N

Na+/Ca2+-Exchanger (NCX) .................. A 05-09 nAChR .................................................... A 03-11 NADPH oxidase ....................... A 06-07, B 10-09 NADPH oxidase 4 .................................. B 10-10 Nanopore ................................................ B 12-10 NCoR1 .................................................... A 12-01 NDNF interneurons ................................ B 01-03 Necroptosis .............................. S 09-03, A 12-09 Neoangiogenesis .................................... A 12-04 nerve ...................................................... S 01-04 Network oscillations .................. S 03-02, B 01-01 Neural Development ............................... B 07-01 neurobiology ........................................... S 10-02 neuroblastoma ........................................ A 06-10 Neurodegeneration ................... B 02-10, B 05-03 Neuroinflammation ............................... OS 01-04 neuromuscular junction .......................... A 03-11 neuronal excitability .............................. OS 06-03 neuronal regeneration ............................ B 07-09 neuropathic pain ... OS 06-07, OS 09-03, B 03-06 Neuropeptide ............................ B 04-10, B 05-10 neuroprotection ...................................... B 04-12 Neurotensin .............................. A 01-01, A 01-03 neurotransmission .................................. S 06-03 neurovascular coupling .......................... B 02-06 neuro-vascular interface ......................... S 10-02 Neutropenia ............................................ B 02-01 neutrophil ............................ OS 01-07, OS 04-05 Neutrophil cytoskeletal mouldability ..... OS 04-02 neutrophil recruitment ........................... OS 04-03


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Neutrophils ......... OS 04-01, OS 04-04, A 07-03, A 07-05

NFAT5 .................................................... B 11-09 NG2 glia ............................................... OS 06-04 nitric oxide .............................................. A 06-04 NMDAR .................................................. B 05-03 nociceptor-immune interactions .............. B 03-03 noise analysis ......................................... S 09-02 Non-small cell lung cancer ....... B 06-03, B 06-11 normobaric hypoxia ................................ A 06-05 Nose-Brain preparation .......................... B 03-01 NoxO1 .................................................... A 08-11 nuclear actin ........................................... A 06-09 nuclear eNOS ......................................... A 12-06 Nuclear receptors ................................... A 08-04 nucleotides ............................................. B 11-04 nucleus ................................................... B 12-03 nucleus reuniens .................................... B 01-06 O

Olfactory bulb ......................................... B 03-01 olfactory system ...................................... B 03-08 optogenetics .......... S 02-01, OS 08-03, A 03-07,

A 10-05, A 10-06, A 10-08 Optogenetics ..... OS 01-01, OS 01-02, OS 07-07 Organ At Risk ......................................... A 10-07 oscillation .... S 03-03, B 01-07, B 01-10, B 01-11 osmosensitivily ....................................... A 09-02 oxidation ................................................. A 11-01 oxidative damage ................................... S 09-01 Oxidative Stress ..................................... B 11-03 oxygen .................................................... S 12-02 oxygen-regulated genes ...................... OS 03-02 P

P. aeruginosa ......................................... S 07-05 p38MAPK ............................................... B 12-01 p53 ......................................... OS 08-06, B 07-08 pain ........................................... S 01-04, B 03-07 pain syndromes ...................................... A 04-02 paired RNA and small RNA sequencing ..............

OS 01-05 pancreatic beta cells ............................... A 08-02 pancreatic ductal adenocarcinoma ......... A 05-03 Pancreatic Ductal Adenocarcinoma (PDAC) .......

A 05-09 Paneth cell ........................................... OS 05-08 Parameter indentifiabilty ......................... A 03-03 parasite infection .................................... A 05-05 paraspeckles .......................................... A 12-06 PARK ...................................................... B 02-08 Parkinson Disease .................................. B 02-04 parvalbumin interneurons ....................... A 01-06 parvalbumin neurons .............................. B 03-06 parvalbumin positive interneurons ....... OS 09-06

parvalbumin+ interneurons ................... OS 09-07 patch-clamp recordings .......................... A 04-06 patient ..................................................... S 01-04 PDAC ..................................................... B 06-09 pendrin ..................................... S 11-03, B 09-09 perfusion ................................................. S 11-06 Pericyte ..................... A 08-06, A 12-04, B 02-06 persistent Na current .............................. A 02-06 Petri net model ...................................... W 02-04 pH ......................................................... OS 04-05 pH homeostasis ...................................... A 05-03 phagoctosis ............................................ A 12-08 pharmacokinetics ................................... B 04-13 PHD2 ...................................................... B 07-10 Photo-crosslinking .................................. B 04-10 photoreceptors ..................................... OS 01-02 pH-sensitivity .......................................... A 08-01 Piezo1 .................................................... A 12-07 PIP2 ........................................................ S 07-01 place cells ............................................... B 01-05 plasticity .................................... B 04-03, B 04-08 Platelets .................................................. B 08-10 polarization ............................................. A 07-05 Polyamines ........................................... OS 02-02 Polycystic kidney disease ..................... OS 02-01 Polyunsaturated fatty acids .................... B 11-07 population-based models ...................... W 01-02 post-translational proteolysis .................. B 07-03 potassium channels ... A 03-08, A 04-09, B 06-07 Prefrontal cortex ....................... S 03-03, A 01-07 Presynapse ........................... OS 06-02, B 04-03 presynaptic ............................................. B 04-08 progesterone .......................................... B 10-12 promoter-enhancer interactome ............. B 12-05 protein expression .................................. B 02-07 Protein-quality-control ............................ A 11-03 proximal tubule ..................................... OS 02-07 PTH ........................................................ B 09-05 pulmonary artery .................................. OS 03-01 Pulmonary fibrosis .................................. A 08-03 Pulmonary Hypertension ...... OS 07-03, B 10-03,

A 08-06 pulmonary vascular system .................... B 10-03 pulmonary vasculature ......................... OS 07-03 Purinergic Signalling ............................... B 05-07 Q

quasi-linear membrane ........................... A 02-06 R

Radiation Effects .................................... A 10-07 radiogenic motor reactions ..................... A 09-10 Ras/ERK-MAPK ..................................... B 02-10 reactive oxygen species ......................... A 06-03 Reactive sulfur species .......................... A 04-03


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regulation ................................................ B 06-07 renal fibrosis ........................................ OS 02-04 Renal Hemodynamics ............................ S 12-05 renal interstitial cells ............................... S 04-02 renal organoids ....................................... B 09-01 Renin ........................................ S 04-02, A 09-03 Resistance arteries ................................. B 10-10 Respiration . S 03-02, S 03-03, S 03-04, B 01-07 restenosis ............................................... B 10-09 restrictive cardiomyopathy ...................... A 10-03 retinal degeneration ................................ S 09-01 Retinal ganglion cells .............................. A 05-08 retinal pigment epithelium ....................... A 12-09 Retinitis pigmentosa ............................... B 06-06 RiboTag .................................................. S 05-04 RNA sequencing ....... S 10-01, W 02-01, A 06-10 Rodents .................................................. A 04-10 ROS ........................................................ B 06-11 rumen ..................................................... B 06-08 ryanodine ................................................ A 08-08 ryanodine receptor type 2 .................... OS 08-03 RYGB ..................................................... B 02-11 S

Sarcomere organization ...................... OS 01-06 Schizophrenia ......................................... B 02-12 SCN2A .................................................... B 05-11 screening assay ...................................... A 10-09 scRNAseq .............................................. W 02-01 Secretin .................................................. S 11-04 secretion .............................................. OS 04-07 sEH ........................................... A 07-09, B 08-07 selective vulnerability .............................. B 02-04 selectivity filter .......................... A 03-08, A 04-09 Selectivity filter inactivation ..................... A 04-08 Selenomethionine ................................ OS 05-02 sensory hair cell ...................................... A 05-06 sensory neuron development .............. OS 01-05 Sensory Neurons .................................... B 03-04 serotonin ................................................. B 04-05 serotonin receptor 5-HT4 ..................... OS 06-05 SGLT1 .................................................... A 05-05 sialylation ................................................ B 08-05 sickness response .................................. B 02-01 single nephron analysis .......................... B 09-07 single-cell RNA sequencing .................... B 10-09 skeletal muscle .................................... OS 07-08 skeletal muscle cells ............................... B 12-06 sleep ......................................... S 03-01, B 01-10 Sleep apnea ........................................... B 06-05 sleep spindles ......................................... B 01-08 SMOC1 ..................................... A 07-04, B 08-10 smooth muscle cells ............................ OS 03-01 Snake venom .......................................... A 10-01 SOCS3 ................................................... A 12-08

Sodium ................................................... A 02-08 sodium channel ........................ A 04-02, A 04-06 sodium-proton-exchanger type 1 ............ A 05-03 Somatostatin ............................ A 01-03, B 05-04 Somatostatin-expressing interneurons ... B 04-05 spatial memory ....................................... B 02-09 Spatial working memory ........... A 01-02, A 01-07 Sphingosine ............................................ S 07-05 Spike timing-dependent plasticity .......... A 02-09,

B 05-12 spine formation ..................................... OS 06-05 spreading depression ............................. B 04-12 SPRED2 ................................................. B 02-10 SPRED2-deficiency ................................ B 12-04 stat3 ...................................................... OS 01-07 stem cell specification ............................ B 08-04 stem cell-derived cardiomyocytes .......... B 12-09 stem cells ............................................... B 11-01 stereotaxic surgery ............................... OS 06-06 stomach .................................................. S 03-05 store-operated calcium entry .................. B 12-03 streetlight effect .................................... OS 09-01 stress .................................................... OS 09-02 structured oral examinations physiology

reliability OSCE OSPE ........................ A 01-11 Subcellular and quantitative Ca2+ analysis .... OS

04-02 subcellular patch-clamp recording .......... S 06-01 substantia nigra dopaminergic neurons . A 04-04 substrate preferences ............................. A 10-04 subterranean rodent ............................... A 08-07 super-priming ......................................... B 04-08 Super-resolution microscopy .... S 01-02, A 03-05 Susceptibility to neurodegeneration ....... A 05-08 Synapse ................................ S 06-03, OS 06-01 Synaptic plasticity ...... S 06-04, B 04-02, B 04-11 synaptic transmission S 06-04, B 03-07, B 04-05,

B 05-04 synaptic vesicle ...................................... A 05-07 synthetic blood supplement .................... B 08-08 T

TAL ......................................................... S 01-05 Target organ damage ............................. B 10-08 TARP .................................................... OS 06-04 TEVC ...................................................... B 06-01 TGF-b ................................................... OS 02-04 TGF-β ....................................... A 07-04, B 08-07 TGFβ1 .................................................... A 07-09 theta ....................................................... S 03-02 Theta oscillations .................................... B 03-01 THIK-1 .................................................... B 06-10 thyroid hormone ..................................... A 08-07 Tight junction ............. S 01-01, S 01-02 S 01-03,

A 03-05, B 09-11


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TIRFM ..................................................... A 03-09 Titin ........................................ OS 07-04, A 11-01 TMEM16 .............................................. OS 05-07 TMEM16A .............................. OS 05-08, B 06-04 TMEM16F ............................................ OS 05-08 TNFR1-induced signaling pathway ........ W 02-04 TNFα .................................................... OS 04-07 TPC channel ........................................ OS 02-07 transcription ............................................ A 06-09 Transcription factor regulation ............... W 02-03 Transcription factors ............................... B 08-06 Transcriptomics ....................... W 02-01, B 11-03 Translational Pain Models ...................... B 03-04 transporter .............................................. A 05-04 trans-Resveratrol .................................... B 08-03 Triiodothyronine ...................................... A 08-09 TRPA1 ................................................. OS 06-07 TRPM3 ................................................ OS 05-06 TRPV1 .................................................... B 03-03 TRPV3 ...................................... B 06-08, B 06-12 t-tubules .................................................. B 12-09 Tumor necrosis factor alpha ................... B 09-11 tumor spheroid ........................................ B 07-10 turnover .................................................. A 11-03 two-photon in vivo calcium imaging ........ B 03-05 U

urinary bladder ........................................ A 09-10

V

V1 ......................................................... OS 09-05 vascular calcification ................ B 11-04, B 11-10 Vascular functions .................................. B 11-09 vascular inflammation ............................. A 12-07 vascular physiology ................................ B 10-11 Venous blood flow .................................. B 10-02 Venous stents ......................................... B 10-02 ventricular arrhythmias ........................... A 10-08 Vitamin D ................................................ B 08-06 voltage gated calcium channels ............. B 02-08 voltage gated Kv4/KChip3 A-type channel ..........

B 02-08 Voltage gated sodium ion channel ....... OS 05-02 voltage sensing ...................................... A 05-06 voltage-gated Ca2+ channels................. A 04-04 Voltage-gated K+ channels .................... A 04-08 voltage-gated sodium channel ............... A 03-07 W

Water deprivation ................................... B 09-03 Weight loss ............................................. B 02-11 wound healing ........................................ A 12-05 Z

zinc ......................................................... B 11-10

contents - dpg 2021 book of abstracts

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