Doctoral thesis at Oslo, 2018

Yangchen Dhondup

Toll-like receptor 9 signalling in

heart failure

© Yangchen Dhondup, 2018

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-156-5

All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo.

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To Passang and Lhakpa

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Table of Contents Acknowledgements ......................................................................................................................................... 4 List of papers .................................................................................................................................................. 6 Selected abbreviations ................................................................................................................................... 7 1.Introduction ................................................................................................................................................. 8 1.1 Heart failure ............................................................................................................................................... 9 1.1.1. Definition and epidemiology .............................................................................................................. 9 1.1.2 Systolic vs. diastolic HF ..................................................................................................................... 10 1.1.2.1 Diastolic HF .................................................................................................................................... 11 1.1.3 Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and regulation of Ca2+ homeostasis in HF ... 12 1.1.4 Myocardial remodelling ..................................................................................................................... 14 1.2 Inflammation in CVD/HF ........................................................................................................................ 16 1.2.1 Inflammation and cytokines .............................................................................................................. 16 1.2.2 Inflammation in clinical HF ............................................................................................................... 17 1.2.3 Pathogenic role of local and systemic inflammation in HF ............................................................... 19 1.2.4 The role of macrophages in the failing heart ..................................................................................... 21 1.3 The innate immune system ...................................................................................................................... 23 1.3.1 PRRs and PAMPS ............................................................................................................................. 23 1.3.2 TLRs ................................................................................................................................................... 24 1.3.3 DAMPs-Mediators in CVD ............................................................................................................... 26 1.3.4 TLR9 can be activated by TLR9 ........................................................................................................ 26 1.3.5 TLR9 in the heart ............................................................................................................................... 28 2. Aims of the thesis ...................................................................................................................................... 31 3. Summary of results .................................................................................................................................. 32 Paper 1 ................................................................................................................................................... 32 Paper 2 ................................................................................................................................................... 33 Paper 3 ................................................................................................................................................... 34 4. Methods ..................................................................................................................................................... 35 4.1 Establishment of SERCA2a KO model ................................................................................................ 35 4.2 Establishment of SERCA2a-TLR9KO model ...................................................................................... 37 4.3 Ethics ..................................................................................................................................................... 38 5. Methodological considerations ................................................................................................................ 39 5.1 Human study and control subjects ........................................................................................................ 39 5.2 Mouse models of HF ............................................................................................................................. 41 . 5.3 Histological scoring of inflammation ................................................................................................... 44 5.4 Immunohistochemistry and image based quantification ....................................................................... 46 5.5 Quantification of fibrosis ...................................................................................................................... 48 5.6 Echocardiography and phase contrast magnetic resonance (PC-MRI) ................................................. 49 5.7 Statistics ................................................................................................................................................ 52 6. Discussion of results ................................................................................................................................. 53 6.1 Tissue injury and release of nucleic acids ............................................................................................. 53 6.2 TLR9 activation and systemic inflammation ........................................................................................ 56 6.3 Direct vs. indirect cardiac consequences of systemic TLR9 activation ................................................ 58 6.4 Intracellular vs. extracellular mtDNA ................................................................................................... 61 6.5 Acute vs. chronic activation of TLR9 ................................................................................................... 63 7. The role of TLR9 and future perspectives ............................................................................................. 65 8. Conclusion ................................................................................................................................................. 67 9. References ................................................................................................................................................. 68 10. Appendix ................................................................................................................................................ 84

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Acknowledgements

Science is teamwork. The work that resulted in this thesis was carried out at the Research

Institute for Internal Medicine (IMF) at Oslo University Hospital, Rikshospitalet and at the

Institute for Experimental Medical Research (IEMR) at Oslo University Hospital, Ullevaal in

Oslo.

I would like to thank my supervisor, Leif Erik, for giving me the opportunity to be a part of the

“Heart failure group”. Thank you for your continuous engagement and availability during my

PhD studies.

Next, I would like to give a special thanks to my supervisor, Arne. You taught me the value of

positivity, curiosity as well as humbleness and critical thinking in science. I’m truly grateful for

your engagement in my projects, and for always being open to my ideas and suggestions as well

as for your feedback when writhing my thesis. Thank you for your friendship.

Also, I would like to thank Pål for always keeping an eye on my PhD projects, making sure our

end goals were reached at all time. Your combination of kindness, righteousness, down-to-earth

attitude and supreme knowledge is what makes you a unique role model and an inspiration to all

leaders. I would also thank my senior supervisor Lars for providing me with human tissue

samples and for your engagement in my project.

During my first year I learned that in science hard work does not necessarily equal results worth

publishing. Laboratory work was challenging and for someone who had just graduated from

medical school, my confidence and motivation quickly reached bottom. I realized that this was a

whole new academic field of learning for me, and I gained a tremendous respect for science, the

psychological aspect of it and for the work behind every scientific paper. I learned that

guidelines for clinical everyday use truly are guidelines, as opposed to universal truths that can

be applied for each individual, and that there are so many variables that may influence the

effects of medications.

When I recall my second year I realize that I spent most of my time at the animal facility at

Ullevaal, learning how to breed mice, and I ultimately overcame my fear of mice bits- which

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resulted in an interesting side-project when I developed a glove to use in laboratory work with

mice. I was thrilled of having established a novel double KO mouse model, the SERCA2a-TLR9

KO. I hope this will encourage scientists to do follow-up studies on TLR9 in the future. My third

year was mostly enticed by writing papers and keeping my motivation up.

As mentioned above, the work behind this thesis could never have been carried out without

collaborators. I would like to thank Helge for his positivity, compassion and for always

reminding me how fun medicine is! I will miss our meetings discussing histological slides, and I

wish you the best of luck in your retirement years. Moreover, I would like to thank my research

group: pharmacist, Ingrid, for her patients with me in the lab, Katrine and Azita for assisting me

with genotyping, as well as the rest of the group: Alexandra, Maria, Marina, Øystein, Jonas,

Mieke, Linn, Trine, Aurelia and lastly Thor, for helping me with the statistics. Thanks to all the

excellent co-authors, especially: Christen, Erik, Shakil, Håvard, Jan Magnus, Lily and Solveig.

Thank you, Ivar, for your availability, and for performing echocardiography on the mice. Thank

you, Geir, for keeping an overall perspective on the project and for your contribution to the

papers.

I’m truly grateful for have had the opportunity to work with such inspirational people, and I

hope that my acquired scientific knowledge will be of use in the future. I would like to thank my

family and my dear Magnus for your unconditional love and support. Also, thanks to all my

friends for your support. I learned a lot during my three years at the institute, but mostly I

learned about myself and it gave me perspective of what truly is important in life. Finally, thanks

to the patients and to all supporters of The Norwegian Health Association.

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List of papers

Paper 1

Low circulating levels of mitochondrial and high levels of nuclear DNA predict mortality in

chronic heart failure

Yangchen Dhondup, Thor Ueland, Christen Peder Dahl, Erik Tandberg Askevold, Øystein

Sandanger, Arnt Fiane, Ingrid Kristine Ohm, Ivar Sjaastad, Alexandra Vanessa Finsen, Anne

Wæhre, Lars Gullestad, Pål Aukrust, Arne Yndestad*, Leif Erik Vinge*

J Card Fail. 2016;22(10):823-8.

Paper 2

Sustained TLR9 activation promotes systemic and cardiac inflammation, and aggravates

diastolic heart failure in SERCA2a KO mice

Yangchen Dhondup, Ivar Sjaastad, Helge Scott, Øystein Sandanger, Lili Zhang, Solveig Bjærum

Haugstad, Jan Magnus Aronsen, Trine Ranheim, Sigve Dhondup Holmen, Katrine Alfsnes,

Muhammad Shakil Ahmed, Håvard Attramadal, Lars Gullestad, Pål Aukrust, Geir Christensen,

Arne Yndestad, Leif Erik Vinge.

PLoS One. 2015;10(10):e0139715. Paper 3

Toll-like receptor 9 promotes survival in SERCA2a KO heart failure mice

Yangchen Dhondup, Ivar Sjaastad, Øystein Sandanger, Jan Magnus Aronsen, Muhammad Shakil

Ahmed, Håvard Attramadal, Alexandra Vanessa Finsen, Lili Zhang, Trine Ranheim, Katrine

Alfsnes, Pål Aukrust, Geir Christensen, Arne Yndestad*, Leif Erik Vinge*

Mediators of inflamm. 2017;2017:9450439.

* Authors contributed equally to the paper

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Selected abbreviationsAbsent in melanoma Coronary artery disease Cyclic guanosine monophosphate C-type Lectin receptors Cardiomyocyte Cytosin phosphate Guanine Cardiovascular disease Danger associated molecular pattern Dendritic cells Dilated cardiomyopathy Ejection fraction Extracellular matrix Heart failure Heart failure with midrange ejection fraction Heart failure with preserved ejection fraction Heart failure with reduced ejection fraction Interferon beta Inhibitory protein of kappa B Interleukin Knock out Lipopolysaccharide Leucine rich repeats Left ventricle MerCreMer Myocyte chemoattractant peptide 1 Myocardial infarction Macrophage inflammatory protein 1 Matrix metalloproteinase Messenger RNA Mitochondrial DAMP Mitochondrial DNA Myeloid differentiation factor 88 Nuclear factor kappa B Nuclear DNA Nucleotide binding oligomerization domain New York Heart Association Pathogen associated molecular pattern Protein kinase G Pattern recognition receptor Retinoic acid inducible gene I Sarco/endoplasmic reticulum calcium ATP-ase Sarcoplasmic reticulum ST-segment elevation MI Systemic inflammatory response syndrome TIR-domain-containing adaptor-IFNß -dependent Toll-like receptor Tumor necrosis factor Toll/interleukin-1 receptor Wild type

AIM CAD cGMP CLRs CM CpG CVD DAMP DC DCM EF ECM HF HFmrEF HFpEF HFrEF IFNß IκB IL KO LPS LRR LV MCM MCP-1 MI MIP-1 MMP mRNA MTD mtDNA MyD88 NF-κB nDNA NOD NYHA PAMP PKG PRR RIGI SERCA SR STEMI SIRS TIR TLR TNF TRIF WT

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1. Introduction

Cardiovascular diseases (CVDs) are leading causes of death and disability in the world (1,2). A

great majority of these deaths are caused by arterial atherosclerosis (3,4) resulting in stroke and

ischemic heart disease, e.g. myocardial infarction (MI). Heart failure (HF) is a severe condition

caused by the heart’s inability to maintain a blood flow that meets the body’s requirement.

Though MI is the most common cause of HF (5), other frequent causes may be hypertension,

valvular diseases or cardiomyopathies. Moreover, other causes may be: congenital heart disease,

pulmonary hypertention, heart arrhythmias (e.g. atrial fibrillation), myocarditis, pericarditis and

cardiotoxic substances (e.g. alcohol) (5), as well as chronic diseases such as diabetes, HIV,

hyperthyroidism, hypothyroidism, hemochromatosis and amyloidosis (6). HF involves a

substantial risk of morbidity and mortality, and it is the most common condition for hospital

admissions in people aged >65 years, making it a major socioeconomic burden. Although there

have been great improvements in the management of this disease over the past decade, the

mortality and morbidity is still high, and the disease prevalence is continuing to rise, due to an

aging population, earlier diagnosis and increased awareness.

Since the 1990s numerous clinical and experimental studies have demonstrated that low-grade

inflammation may play a role in the progression of HF. Cardiac stress or injury involves the

release of intracellular cell debris, which initiates recruitment of inflammatory cells in an attempt

to clean and heal the affected area. Though cardiac inflammation conveys protective means

during initial tissue damage or infection, it may also be detrimental if deregulated or prolonged

and can cause maladaptive cardiac remodelling (7,8). Hence, a timely and concentrated

resolution of the inflammatory response is necessary for proper wound healing and restoration of

cardiac function. Our understanding of inflammatory mechanisms underlying HF is still

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incomplete and increased knowledge on activation of the inflammatory pathways during HF is

needed until therapy targeting inflammation can be introduced in the management of HF.

1.1 Heart failure

1.1.1. Definition and epidemiology

HF is a clinical syndrome defined by the European Society of Cardiology as an “abnormality of

cardiac structure or function, leading to failure of the heart to deliver oxygen at a rate

commensurate with the requirements of the metabolizing tissues” (9). HF is recognized by the

following typical symptoms: dyspnoea, fatigue, reduced exercise tolerance, orthopnoea,

nocturnal cough and signs; elevated jugular venous pressure, ankle oedema, tachycardia and

pulmonary crackles (5,9).

HF is a major public health issue with a prevalence of over 23 million worldwide (10) and

accounts for approximately 2% of the adult population in developed countries (5,9,10) with the

prevalence rising to ≥10% among people of 70 years of age or older (9). The prevalence of HF is

continuing to increase due to earlier diagnosis and awareness, as well as improvements in

therapy and management of other forms of CVD (10). Moreover, HF disorder involves a 5-year

mortality of 45–60% (5,11).

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1.1.2 Systolic and diastolic HF

The most common clinical parameter used to describe HF is based on measurement of left

ventricular (LV) ejection fraction (EF). Mathematically, EF is described as the stroke volume

divided by the end-diastolic volume ((EDV-ESV)/EDV) (9). HF with reduced ejection fraction

(HFrEF) is defined as ejection fraction (EF) <40% (12), also termed systolic HF, and it is the

best characterized type of HF in terms of pathophysiology and treatment (9).

Over the years, an increased awareness has been devoted to HF with preserved ejection fraction

(HFpEF) (8), defined as EF≥50% (12), also termed diastolic HF. Recently, a new term for HF

patients with EF 40-49% (12) was described as HF with midrange EF (HFmrEF). These patients

are believed to have a mild systolic dysfunction, but with characteristics of diastolic dysfunction

(12). Patients with diastolic HF accounts for at least 50% of HF cases, with only slightly lower

mortality compared with patients with systolic HF (10,13). Whereas evidence-based HF

treatment has greatly improved the prognosis of systolic HF patients over the past three decades,

the prognosis of diastolic HF patients has remained unchanged (14). This is supported by several

clinical trials, which demonstrates positive effects on systolic HF patients by using standard HF

therapy and only neutral effects on diastolic HF patients (15). However, at the current time there

is an on-going debate as to whether treatment with the aldosterone antagonist, spironolactone,

(TOPCAT study) could improve clinical outcomes in these patients (16) The main reason for

these inadequate effects is most likely the different underlying pathological mechanisms in

diastolic HF and the higher prevalence of metabolic disorders (15,17,18), as well as non-cardiac

comorbidities or causes, e.g. hypertension, diabetes, atrial fibrillation, chronic ischemic heart

disease, aging etc. in this subgroup (5,9, 10,19,20).

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1.1.2.1 Diastolic HF

Diastolic dysfunction is characterized by left ventricular (LV) stiffness. The pathogenesis is

complex and probably involves several mechanisms, ultimately leading to one common

macroscopic phenotype featured with increased LV-filling pressure. As the main theme of this

thesis is inflammation the focus in this subchapter will be on this.

Some of the main driving mechanisms behind the phenotype are alterations in the extracellular

matrix (ECM) and/or in the cardiomyocyte (CM). Studies have suggested that TGF-β induced

trans-differentiation of fibroblasts into myofibroblasts promotes increased myocardial collagen.

This, in addition to the level of inflammatory cells has been correlated with diastolic HF (21).

Moreover, the Health ABC study reported a strong association between inflammatory cytokines,

such as interleukin (IL)-6 and tumour necrosis factor (TNF), and diastolic HF (22). The

preceding inflammation with the changes in ECM ultimately leads to fibrosis. Moreover,

metabolic disorders, oxidative stress, reduced nitric oxide bioavailability and down-regulated

NO-mediated cyclic guanosine monophosphate (CAMPS) and protein kinase G (PKG)

signalling, have been linked to LV dysfunction. These factors may all contribute to the changes

in titin, a sarcomere protein, and are believed to enhance CM and LV stiffness. The consequence

of a stiffened heart is increased filling pressures to preserve normal LV end-diastolic volumes

(23). Other important driving mechanisms behind diastolic HF are impaired LV active

relaxation. Interruptions in the Ca2+-handling of cardiac cells may lead to impaired relaxation of

LV during the diastolic phase of the cardiac cycle. The change in CM Ca2+ homeostasis is a

hallmark of HF pathogenesis, and is thought to underlie both mechanical and

electrophysiological dysfunction in HF (24).

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1.1.3 Sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) and regulation of

Ca2+ homeostasis in HF

The Sarcoplasmic reticulum (SR) constitutes the main compartment for Ca2+ storage in cardiac

cells (25). Excitation-contraction coupling includes Ca2+ influx through sarcolemma L-type

channels. This involves Ca2+ induced Ca2+ release from the SR through ryanodine receptor

channels with subsequent binding of Ca2+ to myosin, triggering contraction. Ca2+ re-enters into

the SR via the SERCA pump and cellular efflux is conducted through the Na+-Ca2+ exchanger

(Figure 1)(25). In human HF, excitation-contraction coupling is impaired (24) with less

SERCA2a protein expression (25) and altered phosphorylation of phospholamban, the regulator

of SERCA activity (26). Along with increased Ca2+ leakage through ryanodine receptor

channels, this causes high cytosolic and low SR Ca2+ concentrations resulting in increased

diastolic Ca2+ content (27). In mammals, there are three genes encoding several SERCA protein

isoforms. SERCA1a and SERCA1b are expressed in adult and neonatal fast-twitch skeletal

muscles, whereas SERCA2a is selectively expressed in heart and slow-twitch skeletal muscles.

SERCA2b is expressed nearly ubiquitously, thus considered the housekeeping isoform and

SERCA3 is expressed in a limited number of non-muscle cells (28). Brody´s and Darier´s

disease are two human genetic diseases associated with mutations in the SERCA pump (28).

Though these conditions are relatively rare, gene modified SERCA2a KO mice are used

experimentally to study diastolic dysfunction (29,30). As opposed to other murine HF models,

the SERCA2a KO mice display prolonged relaxation deficit due to slowed rate of Ca2+ uptake

(31) This results in a diastolic dysfunction, with subsequent enlargement of the left atrium,

pulmonary congestion and fluid retention due to increased central venous pressure. At about 7

weeks post induction of HF, the mice start to decompensate without preceding dilatation of the

LV (29).

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Modified from Bers DM: Nature. 2002;415:198-205

Action potential

SERCA2a

Figure 1. Excitation-contraction coupling

An action potential induces Ca2+ influx through sarcolemma L-type channels. Ca2+ induced Ca2+ release from the SR

through ryanodine receptor channels with subsequent binding of Ca2+ to myosin, triggers contraction. Ca2+ re-enters into

the SR via the SERCA pump and cellular efflux is conducted through the sodium Na+-Ca2+exchanger.

1

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1.1.4 Myocardial remodelling

Myocardial remodelling may be caused by tissue loss, pressure overload (aortic stenosis) and/or

hypertension), inflammatory heart muscle disease (myocarditis), idiopathic dilated

cardiomyopathy (DCM) or volume overload (valvular regurgitation) (32). Moreover,

remodelling involves an increase in heart size, a more spherical shape and altered cardiac

function in response to cardiac injury (32,33). At a molecular/cellular level, remodelling is

characterized by cardiac myocyte growth, re-expression of foetal genes, changes in the

expression of proteins involved in excitation-contraction coupling, changes in myocyte energetic

and metabolic state, as well as necrosis, apoptosis, oxidative stress and changes in the ECM (32).

When cardiac function is disrupted, the body elicits countermeasures to maintain hemodynamic

homeostasis, e.g. fluid retention, release of neurohormones, increased sympathetic drive (34).

However, over time these mechanisms turn maladaptive and promote development of HF. To

characterize the mechanisms that turn from adaptive into maladaptive responses is one of the

major tasks in HF research.

Though the CM is an essential cell involved in the remodelling process, the interstitium,

fibroblasts, coronary vasculature (32) and inflammatory cells (35) are likewise important

contributors. Fibroblasts play a key role in ventricular remodelling (36), and are responsible for

maintaining the balance between synthesis and degradation of the ECM, which consists of

collagens, proteoglycans, glycoproteins, growth factors, cytokines and proteases. Such proteases

termed matrix metalloproteinases (MMPs) degrade collagen-especially MMP-9 in humans.

Moreover, fibroblasts have been suggested to be “sentinel cells” that sense injury and attract

inflammatory cells to a wounded area with the production of cytokines and chemokines (35).

Though tissue inflammation is considered to be beneficial for tissue healing in the initial phase,

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these changes become maladaptive if the inflammatory response is prolonged, leading to fibrosis

and myocardial dilatation eventually causing systolic HF (36).

Several studies have suggested that the neurohormones that are involved in the renin-

angiotensin-aldosterone system (RAAS) and the adrenergic system, contribute to inflammation

(37,38). Animal experiments on angiotensin II (ATII) have shown inflammatory cytokine

induced inflammation in endothelium (39). Also, AT II (40) and aldosterone (41) induced

intracellular ROS production with subsequent inflammation has been demonstrated. Moreover,

as monocytes and lymphocytes express β-adrenergic receptors, experimental studies have shown

that increased catecholamine may induce inflammatory responses (42). This may suggest that

neurohormones, induced by RAAS-and the β-adrenergic activation, perhaps represents

components that interact with inflammatory cytokines rather than them being separate systems

(43).

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1.2 Inflammation in CVD/HF

In 1990 Levine and colleagues reported elevated levels of circulating TNF in patients with

chronic HF (44), and this finding lead to a new research area: inflammation in HF. Since then,

numerous clinical studies have implied activation of inflammatory pathways both locally in the

heart and in blood as a potentially important pathological event in the initiation and progression

of the syndrome (45,46,47,48).

1.2.1 Inflammation and cytokines

Inflammation is a generic response, and therefore considered a mechanism of innate immunity,

which is vital for host defence to eliminate the initial cause of cell injury, clear out necrotic cells

and induce tissue repair (49). In response to infections, cascades of signals lead to the

recruitment of inflammatory cells to the affected area such as neutrophils and macrophages.

These cells phagocytize infectious agents and produce inflammatory mediators such as cytokines

and chemokines which are pharmacologically active low weight proteins that are secreted from a

variety of different cell types, and may promote either autocrine and/or paracrine effects. This

leads to activation of lymphocytes and elicit adaptive responses. However, in the absence of

pathogens the inflammatory response is also essential for tissue and wound repair, e.g. ischemia-

reperfusion (I/R) injury or chemical damages. This type of inflammation is termed “sterile

inflammation” indicating the absence of microorganisms (50). Though the initial phase of sterile

inflammation is beneficial, e.g. post- MI, too much and prolonged inflammation is detrimental,

causing harmful remodelling and eventually HF with severe hemodynamic stress.

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1.2.2 Inflammation in clinical HF

Several clinical studies have shown that some inflammatory markers play a significant role and

can provide prognostic information in HF patients (51,52,53). Reports show that increased

cytokines such as interleukin (IL)-1 (54), IL-6 (55) TNF and IL-1β concentrations are associated

with poorer prognosis, which may suggests that they reflect important pathogenic pathways

during HF (56,57,58). Moreover, a paper in 2014 demonstrated that inflammatory markers such

as IL-6, TNF and C-reactive protein (CRP) were associated with HF risk and could predict the

development of diastolic HF (HFpEF) (59).

In chronic HF the most important and most studied cytokine is TNF (55) and it has been

recognised as a key cytokine involved in the remodelling process (7, 60,61,62,63). However,

despite the knowledge that TNF and other cytokines are strongly associated with HF, several

clinical trials have been unsuccessful to reach primary end points in the attempt to antagonize

inflammatory mediators, e.g. TNF (58). Several studies have been conducted on the soluble TNF

receptor, etanercept on HF patients. The RENEWAL trial resulted in chronic HF hospitalization

and/or death (58,64) and the RECOVER and RENAISSANCE trial observed no increase in

mortality, however none of them showed improvement in HF either (58). Another anti-TNF

therapy against HF was the ATTACH trial, with the use of a human monoclonal antibody with

an anti-TNF murine Fab, named infliximab. High-dose infliximab resulted in increased mortality

and HF hospitalization and as a consequence of this study, high-dose infliximab became

contraindicated for HF patients (58). In addition to anti-inflammatory therapy, other approaches

to counteract inflammation in HF have been attempted, broad-based immunomodulators, e.g.

intravenous immunoglobulin (IVIg) against a total imbalanced cytokine network, rather than

only one cytokine (56,65). Also, methotrexate and immune modulation therapy and even

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autoimmune therapies have been suggested (58). At the current time, there are exciting on-going

trials on inflammation as a target for CVD.The CIRT trial (66) is assessing whether low-dose

methotrexate reduces MI, stroke or death in patients with type 2 diabetes or metabolic syndrome

who have had heart attack or stable CAD. The CANTOS trial (67) is assessing whether

inhibition of IL-1β with canakinumab can reduce MI, stroke and death in post MI patients with

increased CRP.

Of other cytokines associated with HF, is IL-6, in which increased circulating levels have been

associated with CM hypertrophy, myocardial dysfunction and myocyte atrophy (55). Other

cytokines, such as IL-8 and macrophage inflammatory protein-1 (MIP-1), have also strongly

been associated with cardiac remodelling (7,48,65,68,69,70). Of the anti-inflammatory

cytokines, IL-10 is considered the most important as it down regulates TNF, IL-6 and IL-1

synthesis (55).

Though we do not know the complete underlying mechanism, systemic inflammation is believed

to play a central role in the progression on HF (43). This is particularly relevant as many HF

patients have comorbid diseases, and may be increasingly important with a growing elderly

population. Nevertheless, it seems that no single inflammatory cytokine provides sufficient

discrimination to justify the transition to everyday clinical use as a prognosticator in HF.

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1.2.3 Pathogenic role of local and systemic inflammation in HF

Clinical data supports the hypothesis of persistent low-grade myocardial and systemic

inflammation in HF. Still, we do not fully understand the implications of this inflammatory

activation in patients with HF, i.e., whether it is beneficial, detrimental or simply that it does not

affect the progression of the clinical disorder. However, although clinical evidence is still

ambiguous, numerous experimental studies demonstrate a pathogenic role of several

inflammatory cytokines in HF.

Primarily, HF involves inflammation induced by non-infectious pathological events such as

hemodynamic overload and stress in several cell types, or tissue hypoxia and ischemia through

the production of reactive oxygen species (ROS) and Nuclear factor kappa B (NF-κB)

transcription which all lead to cytokine production (43). NF-κB is the main regulator of

transcription of inflammatory genes. As indicated initially in this chapter, the disease is

characterized with local cardiac inflammation involving the myocardium itself with both CMs

and non-CMs such as fibroblasts, smooth muscle cells, endothelial cells, e.g. TNF production

from the heart (71,72). Such local inflammation may activate immune mediators and lead to the

activation of proinflammatory cytokines (55) in an autocrine or paracrine manner (43). However,

such release from the heart may activate extra-myocardial tissue cells that contribute to this

inflammation, e.g. leukocytes, platelets and macrophages as well as peripheral organs, e.g. liver

and lungs (43), thus inducing a low-grade systemic inflammation (44,48,71,72). As there is

strong evidence of increased myocardial and circulating proinflammatory cytokines during HF,

this has been suggested to be a consequence of increased NF-κB activity (55).

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As in human HF trials, TNF has been thoroughly studied in animal HF models. TNF binds to

TNF receptors, and while type I (TNF-RI) activation has shown detrimental effects, type II

(TNF-RII) activation has shown protective effects (73). Still, most animal studies seem to

indicate beneficial effects by inhibiting TNF in general (74, 75). Reports on rats (76) and dogs

(72) have shown that infusion with TNF reversibly impairs cardiac function, and the latter lead

to systolic dysfunction (77). Moreover, several studies on transgenic mice that overexpress

cardiac TNF have been conducted and demonstrate detrimental effects (78, 79,80,81).

Among other inflammatory cytokines that have been studied in animals is IL-6, which has been

shown to induce negative inotropic effects (82,83,84) as well as hypertrophy, fibrosis, and

diastolic dysfunction (85). IL-1β is a cytokine in the IL-1 family that has gained growing

attention as previous clinical studies have shown a significant beneficial effect by inhibiting IL-

1β (55,86,87). Studies have shown that IL-1β deficient mice display cardiac dysfunction (63,87).

This suggests that IL-1β may aggravate hypertrophic remodelling (88).

As in clinical HF trials, inhibition of proinflammatory responses have been thoroughly

investigated as a therapeutic strategy experimentally. IL-10 is a major anti-inflammatory

cytokine, and IL-10 deficient mice have demonstrated increased cardiac hypertrophy, fibrosis

and cardiac dysfunction in response to isoproterenol (89). However, IL-10 injections have even

shown to significantly reduce cardiac hypertrophy, fibrosis and preserve cardiac function in

different models of cardiac hypertrophy as well (89).

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1.2.4 The role of macrophages in the failing heart

More recently the research focus has changed from cytokines to the role of inflammatory cells

per se. In healthy murine hearts, resident macrophages constitute about 6-8% of the non-CMs

(90,91). Macrophages are specialized mononuclear phagocytes (92) that ultimately derive from

the haematopoietic CD34+ stem cells in bone marrow (93). However, before entering the organ

or tissue, the precursor cells of macrophages while circulating in the blood, are monocytes.

When they finally enter the injured myocardium, they differentiate into macrophages in response

to different chemokines (92): macrophage colony stimulating factor (M-CSF)- which is

important for macrophage survival (94), TNF (95), platelet derived endothelial cell growth factor

(PD-ECGF) and transforming growth factors α and β (TGFα and β), which indirectly contribute

to fibrosis (96). Also IL-1 and insulin-like growth factor (IGF) are among the important

mediators (97).

Post MI remodelling involves three stages: inflammation, scar formation and scar remodelling

with overlapping time frames (98). The removal of dead cell debris followed by wound healing

is considered the primary role of the macrophage post-MI (98). The initial innate immune

response is characterized by an early phase and a reparative phase, the latter occurs around day

3. The early phase involves mobilisation of neutrophils and monocytes to the necrotic tissue and

the reparative phase involves macrophage phenotype transformation, followed by fibroblast

activation and collagen synthesis, which is necessary for scar formation. Both of these phases are

crucial for both mice and humans (99). Moreover, macrophages stimulate endothelial cell

induced angiogenesis, which is initiated by tissue hypoxia (98,99,100). Macrophages express

MMPs, in which MMP9 may be the most important in post- MI remodelling (100). In addition to

contributing to angiogenesis, MMPs degrade collagen during remodelling. Macrophages also

express tissue inhibitor of metalloproteinases (TIMPs), which inhibit MMPs, thus the balance

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between MMPs and TIMPs determine net LV remodelling (101). The two macrophage

activation patterns are: 1) the proinflammatory M1 macrophage activation (classical) and 2) the

anti-inflammatory M2 macrophage activation (alternative), and they display different markers.

Porcheray and colleagues demonstrated that macrophage activation occurs first through the M1

pathway, and then shifts to the M2 pathway, and this shift between pathways is reversible.

However, as both pathways are activated at varying time-point post-MI, both subgroups are most

likely to be present simultaneously (102). The role of cardiac-resident macrophages in chronic

post-MI remodelling is not yet fully understood (99). The balance between the two macrophage

activation phases as well as the interaction between collagen synthesis and degradation are

among the many factors that determine the net LV remodelling process. At this point, we do not

completely understand this interaction and how the quantitative relationships of the mediating

factors are regulated. Though inflammation is necessary for optimal wound healing in post-MI

remodelling, scenarios where too much inflammation resulting in prolonged remodelling, seems

to be detrimental.

As mentioned above, mechanical overload and oxidative stress may initiate inflammation.

However, studies show that certain components of the innate immune system are able to

recognize specific molecular patterns in pathogens. These pattern recognition receptors (PRRs)

are able to activate signalling pathways that lead to the production of cytokines and chemokines,

which attract leukocytes to the affected area and ultimately combat harmful microbes (103). One

subgroup of PRRs is the Toll-like receptors (TLRs), in which they do not only respond to

microbes but potentially also non-infectious agents with similar structures. Studies have shown

that they convey a significant role in HF, and that they contribute to both local myocardial

inflammation as well as systemic inflammation (43).

23

1.3 The innate immune system

1.3.1 PRRs and PAMPS

As discussed above, an important step in the elucidation of new inflammatory target for therapy

in HF, is to more precisely characterize the inflammatory pathways that are activated during

these complex disorders. In the present thesis we focus on the role of Toll-like receptor-9

(TLR9), a component of the innate immune system.

The innate immune system consists of germ line-encoded PRRs, and recognizes evolutionary

conserved structures termed pathogen associated molecular patterns (PAMPs). PAMPs are found

in bacteria, viruses or fungi (104,105) and include microbe-specific carbohydrates, lipids and

peptides or combinations of these components. Different PRRs recognize different agents, and

they are expressed in macrophages, dendritic cells (DC), phagocytes and B-lymphocytes

(104,105) as well as in non-immune cells such as endothelial cells, fibroblasts (106,107,108) and

CMs (109,110). Until now, five major groups of PRRs have been discovered, and they have

been classified depending on their location within the cell: cytoplasmic PRRs include Retinoic

acid inducible gene I (RIGI)-like receptors (RLRs), Nucleotide binding oligomerization domain

(NOD)-like receptors (NLRs) and Absent in melanoma (AIM)-like receptors (ALRs)

(111,112,113). Transmembrane PRRs include C-type Lectin receptors (CLRs) and the TLRs.

24

1.3.2 TLRs

Recent studies have shown that TLRs are involved in the inflammatory response during HF, and

represent a bridge between infectious and non-infectious derived inflammation in stressed,

injured and dead cells (114,115). The first TLR, “Toll”, was first identified in 1985 in a fruit fly

(Drosophila melanogaster) (116,117). Over the years, ten human and twelve murine

homologues of the Drosophila TLR have been found, of which TLR 1-9 are the best

characterized. TLRs are transmembrane signalling receptors localized either in the plasma

membrane (TLR 1, 2, 4, 5 and 6) or within endolysosomes (TLR 3,7,8 and 9). The

transmembrane TLRs recognize microbial cell wall structures such as lipopeptides (TLR2),

glycolipids (TLR4) or flagellin (TLR5) while endolysosomal TLRs recognize nucleic acids as

RNA (TLR3, TLR7, TLR8) or DNA (TLR9) (108, 118,119). They have mostly been studied in

immune cells, however TLR2, -3, -4, -5 and -9 have been identified in CMs as well

(43,120,121).

TLRs are type 1 integral membrane glycoproteins composed of a horseshoe shaped N-terminal

ectodomain consisting of leucine rich repeats (LRR) which are responsible for ligand binding.

The trans membrane section is composed of a single membrane spanning helix terminating in a

C-terminal cytoplasmic domain (122), and binding is either direct or indirectly dependent on the

presence of a co-receptor. The cytoplasmic domain is termed the Toll/interleukin-1 receptor

(TIR) domain, homologous to IL-1/IL-18 receptors. Upon activation, all TLRs form either

homo-or heterodimers in M-shaped complexes, and the two dimerized TLRs frequently share a

single ligand (122). Upon TLR activation, the TIR domain recruits the appropriate adaptor

protein, with subsequent downstream signalling through various key structures. This results in

transcription of specific genes with recruitment of leukocytes to the affected area. Downstream

25

signalling may either be 1) myeloid differentiation factor 88 (MyD88) adaptor protein-

dependent or 2) TIR-domain-containing adaptor-inducing interferon beta (IFNß)-dependent

(TRIF) (123) (Figure 2). Each TLR recognizes specific PAMPs or danger associated molecular

patterns (DAMPs). The latter is released in the absence of pathogens and induces “sterile

inflammation” (124).

Modified from Kawai T: Immunity. 2011: 637–50

Figure 2. Overview of TLR signalling pathways

Signalling pathways in inflammatory cell types such as macrophages (MP), inflammatory monocytes (iMO),

plasmacytoid dendritic cells (pDCs), conventional dendritic cells (cDCs) and lamina propria DCs (LPDCs).

Heterodimers of TLR1-2 and TLR2-6, as well as TLR4 and TLR5 are expressed on the cell surface. PAMP induced

recruitment of adaptor proteins such as MyD88, TIRAP, TRIF, and TRAM leads to NF-κB induced production of

inflammatory cytokines. In steady state, TLR3, TLR7, and TLR9 are primarily localized in the endoplasmic

reticulum (ER). Upon activation, they traffic to the endosomes mediated by the chaperon protein, UNC93B1. TLR7

and TLR9 may initiate two signalling pathways depending on cell type; In pDCs, TLR7 and TLR9 signalling may

lead to MyD88-dependent 1) NF-κB mediated signalling from the endosome or 2) IRF7 mediated signalling from the

lysosome-related organelle (LRO) after the receptors are transported from the endosome. These two pathways lead to

the induction of inflammatory cytokines and type I IFN. In cDCs and macrophages, TLR7 and TLR9 induce

inflammatory responses by activating NF-κB via MyD88, but fail to activate IRF7.

2

26

1.3.3 DAMPs-Mediators in CVD

DAMPS are endogenous molecules, largely released upon pathological stimuli, e.g. cellular

stress or damage. This results in sterile inflammation (125,126) through activation of PRRs.

Different classes of DAMPs have been suggested: (i) cell derived, e.g. crystalline uric acid

(127), heat shock proteins (128), high mobility group box 1 (HMGB1) (129), and nucleic acids

(130) (ii) derived from breakdown of ECM, e.g. hyaluronic acid (131) and fibronectin (132,133)

or (iii) plasma derived, e.g. oxidized LDL and palmitate (134). Increased circulating levels of

DAMPs have been described in several clinical studies, e.g. patients with massive lung

embolism (135), patients with cancer (136,137) or autoimmune diseases, e.g. rheumatoid

arthritis (138,139,140). Most importantly, experimental studies using mice deficient in different

PRRs strongly suggest pathogenic effects of DAMPs during sterile inflammation. Recently,

Zhang and colleagues found elevated plasma levels of mitochondrial DNA (mtDNA) in trauma

patients (141), and reports suggest that mitochondrial DAMPs consisting of N-formyl peptides

and mtDNA are able to induce inflammation, and more specifically activate TLR9 (142).

1.3.4 TLR9 can be activated by mtDNA

Hemmi and colleagues first described TLR9 in 2000 as a TLR recognizing bacterial DNA (142).

TLR9 was primarily studied in B-lymphocytes and pDCs. However, recent reports suggest that

TLR9 is also expressed in numerous other cellular entities like cardiac fibroblasts, monocytes,

neutrophil granulocytes, respiratory epithelial cells, endothelial cells, vascular smooth muscle

cells, intestinal epithelium and CMs (105, 139,143,144,145,146). During physiological resting

conditions, TLR9 resides within the ER of the cells (147, 148,149) and exists as a preformed

dimer. Upon activation, TLR9 is transported from ER to endolysosomes, with subsequent

27

conformational changes as well as cleavage of the ligand binding LRR-domain, resulting in a

functional receptor (147,150). Researchers have proposed that it is the cleavage of the C-

terminal fragment, which mediates ligand recognition (151,152) however the details behind this

are not yet fully understood yet.

TLR9 is activated by specific nucleotide sequences named unmethylated cytosine-phosphate-

guanine (CpG)-DNA-repeats. These are abundant in bacterial DNA and mtDNA, however rather

limited in nuclear mammalian DNA. The similarities of mtDNA to bacterial DNA are thought to

be a consequence of a distant common evolutionary step. According to the endosymbiotic

theory, a prokaryotic cell fused with a pre-eukaryotic cell, thus establishing the mitochondrion,

necessary for multicellular life. When mtDNA is endocytosed into the endolysosomes, it

activates the residing TLR9 leading to a cascade of downstream signalling. Still, we do not know

which part of the CpG structure is responsible for TLR9 activation. Studies have shown that

oxidation of mtDNA may be of importance to the binding process. Others propose that the

binding is independent of the CpG-motif base composition and that mtDNA phosphodiester 2’-

deoxyribose backbone is responsible for activation (153). Furthermore, there have been reports

suggesting that the preceding stages of endosomal compartmentalization of TLR9 is essential for

the receptor to discriminate between endogenous and pathogenic DNA, and subsequent ligand

binding to TLR9 (147,153,154).

28

1.3.5 TLR9 in the heart

Since the report by Hemmi and colleagues (142), only a handful of studies have investigated

TLR9’s role in the heart and even fewer studies have examined its role in HF. At this stage most

studies have been conducted on animal models, although recently there have been a few studies

on mtDNA in humans.

In 2010, Zhang and colleagues observed increased circulating mitochondrial DAMPs (MTDs),

consisting of formal peptides and mtDNA, in trauma patients with muscle injuries. Moreover,

the researchers found that MTDs from human liver, myositis and fracture haematoma attracted

polymorph nuclear neutrophils (PMN). This was also demonstrated in rat muscle and liver. As

several studies demonstrate that CpG can activate TLR9 in PMNs (155,156,157), Zhang and

colleagues wondered if mtDNA induces similar responses at clinical plasma levels. Thus, in

vitro, they incubated PMN with CpG or mtDNA, and observed co-existent low dose N-formal-

Met-Leo-Phi (fly), a synthetic peptide that simulates bacteria, lead to IL-8 release. This finding

demonstrated clinically significant activation of PMN secretion by mtDNA/TLR9 and that

activated TLR9 could elicit organ injury in a sepsis-like manner (139). Moreover, they

introduced these DAMPs as representatives of a link between trauma and systemic

inflammation, i.e. systemic inflammatory response syndrome (SIRS). In 2012, our group

compared circulating mtDNA in patients with ST-segment elevation MI (STEMI) to patients

with stable angina, after being treated with percutaneous coronary intervention (PCI)(158). We

found that 3 hours post PCI mtDNA levels were significantly increased in the STEMI group,

followed by a rapid decrease, reaching the same levels as in the control patients with stable

angina pectoris 3 days post-PCI. Also, the peak mtDNA levels were higher in STEMI patients

with trans mural MI compared to STEMI patients with non-trans mural MI. Also, in the STEMI

29

group, mtDNA levels after 3 hours correlated with maximum troponin T levels. This study

demonstrated for the first time that focal myocardial necrosis due to MI could lead to the release

of mtDNA, and that the plasma levels correlated with the degree of myocardial damage.

As mentioned initially in this chapter, most studies on TLR9 in the heart have been conducted on

various animal models. Experiments on mice have shown that 4 hours of pre-treatment with

CpG followed by pressure overload HF induced by transverse aortic constriction (TAC) 12

hours later, can attenuate cardiac hypertrophy and function (159). The study by Velten and

colleagues demonstrated that priming with CpG could attenuate the inflammatory response by

modulating cardiac gene expression as well as cellular growth and proliferation. The latter was

seen as reduced CCL2 and CCL4 and reduced macrophage activation and infiltration (159).

Moreover, CpG pre-treatment attenuated collagen deposition in TAC induced HF. The net result

was attenuated hypertrophy, remodelling and LV function. Another study observed that priming

with CpG 1 hour prior to myocardial ischemia followed by reperfusion, could attenuated

apoptosis and reduced infarct size determined by Triphenyltetrazolium chloride (TTC) staining

through PI2K/Act signalling pathway (160). A recent study from our group, investigated mice

subjected to 30 minutes of ischemia, immediately followed by injections with CpG and 24 hours

later hearts were re-perfused (146). Though TLR9 activation displayed evidence of reduced

cardiac monocyte and granulocyte infiltration, paradoxically there were increased circulating

immune cells. Moreover, TLR9 activation increased several cardiac inflammatory genes.

However, CpG induced systemic TLR9 activation upon ischemia/reperfusion (I/R) did not

influence infarct size despite several alterations in inflammatory parameters (146).

Though TLR9 activation evidently have shown salutary effects, some studies challenge this view

30

by demonstrating adverse effects. One study by Knuefermann and colleagues demonstrated that

CpG stimulation wild type (WT) mice with intact TLR9 resulted in a clear inflammatory

response by several cytokines, e.g. TNF, IL-6 and IL-1β (161). This was not seen in the TLR9

knock out (KO) mice. Moreover, isolated CMs from the CpG induced WT mice displayed

reduced contractility (161). Boehm and colleagues supported this finding by demonstrating

septic HF and increased mortality in WT mice injected with CpG i.p., followed by

pharmaceutical inhibition 30 minutes after. By comparison, these effects were not present in

TLR9 KO mice (162). Finally, in 2012 Oka and colleagues published a scientific work,

supporting the harmful effects that had been reported in previous research. They demonstrated

that mtDNA that escapes from autophagy, can activate cardiac TLR9 within lysosomes resulting

in inflammation and cardiac dysfunction. The group studied in vivo CM-specific

deoxyribonucleic (DNase)2a inhibition, i.e. DNase2a KO mice, that were subjected to TAC

which lead to increased intracellular mtDNA induced TLR9 signalling, since mtDNA was not

degraded. This involved early increase in cardiac infiltration of inflammatory cells and increased

messenger RNA (mRNA) expressions of several cytokines. Ten days after TAC, the DNase2a

KO mice developed severe HF and demonstrated increased mortality (163). Moreover, both

TLR9 depletion and pharmaceutical inhibition of TLR9 demonstrated attenuated cardiac

function. Even WT mice with intact DNase2a and TLR9 presented with less inflammation and

improved cardiac function.

The above-mentioned studies emphasize the complexity of studying TLR9 activation in hearts,

as there are many considerations to make when interpreting the ambiguous results. The research

on TLR9 in the heart is far from settled. As long as HF remains a challenge worldwide and more

studies reveal that TLR9 may play a central role in the pathogenesis, one should consider to

invest in more translational studies on TLR9 in the future.

31

2. Aims of the thesis

We hypothesized that mtDNA is released during chronic HF and may impact cardiac function by

activating TLR9. This hypothesis was investigated using differential experimental approaches

combined with analyses in clinical material from patients with HF. Our specific aims were to:

1. Analyse circulating levels of mtDNA and nuclear DNA (nDNA) in HF patients and to

investigate their correlations to clinical and biochemical parameters.

2. Determine the pathophysiological consequence of sustained systemic TLR9 stimulation in

experimental chronic HF.

3. Explore the pathophysiological consequence of attenuated TLR9-signalling in

experimental chronic HF.

32

3. Summary of results

Paper 1

Low circulating levels of mitochondrial and high levels of nuclear DNA predict mortality in

chronic heart failure

Aim: We aimed to investigate circulating levels of mtDNA and nDNA from 84 chronic HF

patients with New York Heart Association (NYHA) functional class I-IV.

Our main findings:

• High circulating levels of nDNA are associated with increased mortality.

• High circulating levels of mtDNA are associated with increased survival.

• Patients with HF have increased circulating mtDNA and nDNA compared to controls.

Conclusion: Plasma levels of mtDNA and nDNA are elevated in human HF. High levels of

nDNA are associated with mortality, whereas elevated levels of mtDNA are associated with

increased survival. This study suggests a rationale for exploring TLR9, a putative mtDNA

receptor, as a new target in treatment of human HF.

33

Paper 2

Sustained TLR9 activation promotes systemic and cardiac inflammation, and aggravates

diastolic heart failure in SERCA2a KO mice

Aim: We aimed to investigate the impact of sustained, systemic TLR9 activation on cardiac and

systemic inflammation in SERCA2a KO HF mice and the consequences on HF progression and

phenotype.

Our main findings:

• Sustained TLR9 stimulation increases cardiac monocyte/macrophage infiltration and cytokine

mRNA expression, as well as systemic lymphocyte infiltrations in lungs and liver in SERCA2a

KO mice.

• Sustained TLR9 stimulation aggravates HF and promotes premature death in SERCA2a KO

mice.

Conclusion: Sustained activation of TLR9 causes cardiac and systemic inflammation, and

deterioration of SERCA2a depletion-mediated HF.

34

Paper 3

Toll-like receptor 9 promotes survival in SERCA2a KO heart failure mice

Aim: We aimed to investigate the consequences of endogenous TLR9 signalling in SERCA2a

KO HF mice.

Our main findings:

• The absence of TLR9 promotes a significant premature death in SERA2a KO HF mice

despite no echocardiography, biochemical or histological evidence of altered HF phenotype.

Conclusion: In mice with SERCA2a depletion-mediated diastolic HF, the absence of TLR9

reduces life expectancy compared to mice with cardiac TLR9 present. Despite thorough

investigation to what may have caused the premature death, we were unsuccessful to pinpoint

what was causing the difference in HF phenotype between mice with and without TLR9. Thus,

further studies on alternative explanations need to be conducted.

35

4. Methods

4.1 Establishment of SERCA2a KO model

In paper 2 and 3 in this thesis, we used the conditional SERCA2a KO model based on the Cre-

lox P method first described in the early 1980s (164,165). Christensen and colleagues (30,166)

established the lox-P-flanked SERCA2a model in which the gene resides between two loxP sites

(170). By crossing this model with a mouse with the Cre-lox P gene, this resulted in the

MerCreMer (MCM) SERCA2a flox/flox. MCM is a fusion protein, consisting of the Cre enzyme

and two oestrogen-binding domains sensitive to tamoxifen (anti-oestrogen). MCM activation by

tamoxifen, leads to Cre-recombinase mediated excision of the SERCA2a gene (173). Cre is

controlled by a CM-specific mediated promotor; α-myosin heavy chain (α-MHC) (Figure 3).

36

Reprinted and modified with permission from Professor Ole M. Sejersted: ”Lessons from the SERCA knock-out mouse” (Sejersted O.M. M.D. PhD. Lessons from the SERCA knock-out mouse. Lecture obtained as power point presentation on 4th December 2015,IEMR.)

Figure 3. SERCA2 gene modification in CMs of adult mice

A) SERCA2 prior to gene excision is named SERCA2 flox/flox with loxP sites on both sides of the target gene. After

gene excision, i.e. SERCA2 KO, the gene is inactivated, i.e. not able to re-distribute Ca2+ into the sarcoplasmic

reticulum during diastole. This leads to increased Ca2+ concentration in the cytosol and thus a relaxation deficit.

B) MCM is a fusion protein, consisting of the Cre enzyme and two oestrogen-binding domains, sensitive to

tamoxifen. MCM activation leads to Cre redistribution into nucleus and SERCA2a gene excision. Cre is controlled

by a CM-specific mediated promotor; α-myosin heavy chain (α-MHC).

FF

KO

SERCA2a flox/flox

Exon Exon

LoxP1 LoxP2

LoxP1/2 SERCA2a KO

3A Inducible SERCA2a excision (Andersson KB 1998-2003)

SERCA2a gene modification (Christensen G 1996-1998)

MM M

3B

37

4.2 Establishment of SERCA2a-TLR9 KO model

In paper 3, we employed a three-generation breeding strategy by crossing the αMHC-MCM-

SERCA2a flox/flox model with the single TLR9 KO, giving rise to four comparable mouse lines

consisting of two HF models (SERCA2a KO and SERCA2a-TLR9 KO) and two control models:

WT and TLR9 KO (Figure 4). The breeding strategy was based on expected number of offspring

per female mouse. By crossing the conditional KO and the TLR9 KO, there were three sets of

genes that had to be merged into one animal. These genes were Cre, the floxed SERCA gene and

the deleted TLR9. The breeding strategy is illustrated in Figure 4A in which the SERCA KO

was crossed with the TLR9 KO, establishing a 50% chance of an offspring with all three genes

(heterozygous). In the second generation, displayed in Figure 4B, we crossed two heterozygous

animals, which resulted in the desired homozygous floxed SERCA, the TLR9 KO and Cre.

Normal gene

MCM

SERCA2a

TLR9

G1 4A Normal gene

MCM

SERCA2a

TLR9

G2 4B

Normal gene

MCM

SERCA2a

TLR9

G3 4C

Figure 4. Establishment of the SERCA2a-TLR9KO model

A) In Generation one (G1), a MCM-SERCA2a flox/flox was crossed with the single TLR9 KO, giving rice to mice (G2)

with MCM and heterozygote floxed SERCA2a and TLR9 KO genes. B) G2 mice were crossed C) giving rice to mice

(G3) with MCM, homozygote floxed SERCA2a and TLR9 KO genes.

38

4.3 Ethics

In paper 1, all patient and control subjects that were recruited at Oslo University Hospital

entered the study voluntarily after receiving appropriate study information and signing consent

forms. The study protocol and all human tissue sampling were approved by the Regional

Committee for Medial and Health Research Ethics and conformed to the Declaration of Helsinki.

In paper 2 and 3, all animals were cared for according to the Norwegian Animal Welfare Act,

which conforms to the National Institutes of Health guidelines (NIH publication no. 85– 23,

revised 1996). Experiments were approved by the Norwegian National Animal Research

Committee (paper 2 FOTS ID 5319; paper 3 FOTS ID 6941) and conformed to the Guide for the

Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH

Publication No. 85-23, revised 1985).

39

5. Methodological considerations

5.1 Human study and control subjects

A prerequisite in clinical studies is to have suitable patient populations and comparable healthy

control subjects. Patients with chronic HF in NYHA I-IV with stable LVEF ≤ 55 were recruited

and consisted of a mixture of HFrEF, HFmrEF and HFpEF. Though, the majority of the

population consisted of HFrEF patients (n=71), and the rest consisted of HFmrEF patients

(n=12) and one HFpEF patient. As the patient samples were assessed at our tertiary hospital;

Oslo University Hospital, Rikshospitalet, Oslo, Norway, most of them were categorized as

NYHA II and III (Distribution: NYHA I n=5; NYHA II, n= 29; NYHA III n=40, NYHA IV

n=10). This may lead to a systematic patient population bias, however by merging NYHA I/II

and III/IV, we were able to minimize this effect. The patients were carefully clinically

characterized according to patient history, routine physical examinations, echocardiography and

coronary angiography. Based on these measurements, the underlying cause of HF was classified

as coronary artery disease (CAD), DCM (genetic) or other sub groups (hypertrophic

cardiomyopathies, aortic insufficiency, unknown aetiology). NYHA classification was based on

the patient’s subjective report. To limit confounding factors and increase homogeneity, patients

with acute coronary syndromes within the last 6 months, congenital heart disease, post-radiation

affected hearts and right ventricular (RV) diseases as well as concomitant diseases, e.g.

malignancies, autoimmune disorders, or liver or kidney failure, were excluded.

Since age and gender affects immune responses (167,168), we found it crucial to include

comparable healthy controls. Seventy-two age- and gender matched healthy blood donors with

40

no prior medication requirements except contraceptives, allergy medication or medication for

hypothyroidism, served as controls. In addition, controls were selected based on case history and

clinical examination and a few selected blood samples within normal range limits (CRP,

proBNP, haemoglobin, leukocyte count, creatinine, cholesterol and metabolic tests).

Unfortunately, we did not obtain complete data of all patients and controls.

Venous blood samples were analysed for mtDNA and DNase1, however as they may be

influenced by several factors it was necessary to standardize the collection criteria, processing

and storage. To avoid contamination, samples were kept in pyrogen-free tubes with EDTA as

anticoagulant (plasma) or no addition (serum). To further avoid induction of inflammatory

responses, storage at room temperature was kept to a minimum by immediately placing samples

on ice and centrifuging them within 15 minutes at 2000g for 20 minutes (plasma) or allowed to

clot at room temperature for <1 hour before centrifugation at 1500g for 15 minutes (serum).

Similar to cytokine measurements from blood samples, DNA is affected by time and temperature

as well as the frequencies of freeze and thaw cycles prior to analysis (169) and repeated freeze

thawing will inevitably accelerate the activity of circulating DNase1. In paper 1, all blood

samples had been frozen and thawed minimum 2 times prior to the experiments, whereas control

samples had been thawed less than three times which is considered acceptable (169). However,

all samples had been stored at -80°C until assayed. For most analyses in this thesis the choice of

serum or plasma was dictated by the availability of samples.

41

5.2 Mouse models of HF

The overall goal in medical research is to increase the knowledge of human diseases and to

discover new treatment modalities. However, the majority of research projects with goals of

reaching mechanistic insight are not immediately accessible using human patients, due to ethical

considerations, and this warrants the use of experimental animal models. In our projects we have

used the mouse as an experimental animal model.

There are several genetic murine models to study HF, among others: 1) overexpression of a

specific gene 2) Gene KO mice. One example of gene overexpression models that may represent

clinical HF is muscle lim protein KO mice in which one gene encoding muscle lim, an actin-

based cytoskeletal protein that regulates myogenic differentiation, is interrupted (170). This may

represent Lamin A/C mutations resulting in DCM in humans (171). However, some murine

models may serve as models for cardiomyopathies, without mimicking human diseases. As

opposed to the monogenetic causes of HF, these models presents with a single protein of

importance in general human HF. One example may be mice with nuclear Ca2+/calmodulin

kinase II overexpression in CMs (172) or mice with disrupted Ca2+ handling within CMs. 3) In

gene KO models one may study conditional KO mice and in paper 2-3 in this thesis, we used

SERCA2a KO mice (30). As opposed to most experimental models of HF, which primarily

represents systolic HF, the complete KO of SERCA, with a short transition phase of

compensated function, eventually leads to diastolic dysfunction (30,166). As mentioned in the

introduction of this thesis (chapter 1.1.3), SERCA2 mutations have been reported in humans

(28). Moreover, several mutations in phospholamban, the protein involved in regulating SERCA,

have been reported to lead to DCMs in humans (173,174). This may indicate a clinical relevance

42

of studying such mutations in human HF. As recent studies have suggested that the pathogenesis

of systolic and diastolic HF development is somewhat different (21), the relevance of using

specific models that represent either one of these sub conditions becomes obvious. Moreover, as

previously mentioned in this thesis, the SERCA2a KO is a conditional KO restricted to CMs

allowing for cell specific studies and flexibility as for study initiation. It is however important to

emphasize that overexpression of the Cre enzyme alone has been reported to express around

35% (175) of the genes, which makes it crucial to select appropriate control groups when

designing experimental studies using MCM SERCA2a KO mice (30,166). Due to the possibility

of uncontrolled Cre induced gene regulations, we used MCM control mice for MCM SERCA2a

KO mice. In traditional gene KO models, one single gene has been inactivated, making it

possible to study the gene of interest in a given disease context.

As different as they appear, mice share a surprisingly similar biology to humans, which e.g.,

makes their immune systems comparable in several aspects (176). Mice are cost and space

effective, and due to their short life span are they appealing as an animal model of chronic

human diseases. Also, they reproduce quickly and can be genetically engineered. Thus, they can

act as a bridge between in vitro and in vivo experiments to proof of concept data. Despite the

advantages of utilizing mouse models (177), it is essential to keep in mind that mice are not furry

humans with a tail, and interpretations from mouse models must be made with caution as the

lack of the complexity of humans is inevitable (178). Among the disadvantages of using mice

are that, unlike humans, mouse hearts have adapted to function at very high heart rates. Also,

mice and humans express different cardiac proteins that may affect the pathogenesis, as mouse

ventricular CMs express fast α-MHC, which involves faster kinetics than the slow β-MHC found

in human ventricles. However, upon disease progression, fast α-MHC is down regulated and

43

slow β-MHC is up regulated at the protein level in both species during HF. The SERCA protein,

which is responsible for re-distributing Ca2+ back into the SR during diastole filling, accounts for

90-92% of Ca2+ re-distribution in rodents (179,180). In comparison, it only accounts for 76% of

the re-distribution in humans, whereas NCX accounts for the majority of the rest in both groups

(181). The use of KO mice is not un-problematic as one particular gene of interest may serve

different roles in humans and in mice(182). Another challenge in KO mice is that since the gene

most often is inactivated in all cells from birth, the body may early adapt to the physiological

changes with unknown compensating factors that may impact cardiac function -and that are not

present in human HF. In contrast, human HF is most often developed from the adult ages (apart

from congenital HF) involving a gradual detrimental development. Other considerations are that

it is important that littermates are crossed to reduce gene pool variations (183) and that

upbringing environments should be the same (temperature, food, etc.). All animals in our studies

were stationed at the same animal facility.

It seems that is no “ideal” animal model of the human cardiovascular system, and obviously with

all the limitations one should assess different animal models, both small and large, when

studying CVDs.

44

5.3 Histological scoring of inflammation

Though HF is a source of systemic inflammation, secondary organ damage, e.g. lung and liver

congestion, renal failure etc., as well as comorbidities, e.g. diabetes, hypertension, rheumatic

diseases etc., may all contribute to systemic inflammation. As HF and systemic inflammation

often co-exist, we investigated the impact of these two conditions combined in paper 2 using the

SERCA2a KO.

Among our collaborators, a trained pathologist analysed haematoxylin eosin stained slides of

hearts, livers and lungs, and established a scoring system based on pure histological

observations. He then applied this system to score organ inflammation while being blinded to

genotype and intervention. As we could not find any predefined scoring system for hearts in the

literature, we designed a novel system in which scoring of hearts was based on standard methods

used to analyse biopsies from heart transplants to evaluate the degree of rejection. Initially, we

tried to indicate the number of muscle fibres as cells per visual field with 200X magnification.

However, due to poor reproducibility we quickly had to discard this approach in favour of

another approach: scoring muscle fibres relative to nuclei as nuclear-to-cytoplasm ratio. We

decided to score gradual changes in the cytoplasm from 0, in which there was absence of nuclear

variation, to 4 in which there were many light cells with large nuclei and with nucleoli present.

Very few heart samples (n =3) displayed vacuolization or necrosis. Red cytoplasm, often glass-

like, and pyknosis (shrinkage due to condensation of chromatin) of nuclei is considered a sign of

cell death, and were rarely seen. As such, the majority of evaluating cardiac cell stress and/or

death was based on nuclear-to-cytoplasm ratio evaluation. Some of the challenges we

encountered were: How light should “light” cells be? How large nuclei and nucleoli were

necessary for a certain score? For each slide the following factors were considered: Light cells

45

are often considered as a sign of cell damage, frequently caused by swelling of mitochondria,

intracellular fluid (oedema), still they could also represent artefacts caused by poor fixation. As

for the two latter causes, we would expect more generalized changes of the muscle fibres as

opposed to our slides in which the changes were patchy. Infiltrations of lymphocytes were

scored from 0, in which there was absence of cells, to 2 in which there were several cell per

visual field. When evaluating each slide some of the challenges were: How many lymphocytes

are needed to make up an infiltrate that would result in a certain score? In example, does it have

to be more areas of infiltrates than 50% to achieve the highest score? Another relevant factor to

consider was how many visual fields are analysed per biopsy. In our study the whole biopsy was

analysed (Scott H. M.D. PhD., 2014 Personal communication).

Finally, to evaluate systemic inflammation we used scoring systems that were already described

in the literature (184,185,186). This made it much easier to extrapolate to our study for

evaluating inflammation in lungs and liver, than the previous cardiac sections. For evaluating

vascular lung inflammation, we applied the classification system for grading pulmonary allograft

rejection (184), and inflammations were scored from 1 to 2. However, as there is no current

established scoring system for the highest score 3: lymphocyte infiltration of lung intima, we

used the same criteria which would qualify for grading “vasculitis score 1” in renal graft

rejections according to the Banff classification (186) (Scott H. M.D. PhD., 2014 Personal

communication).

We chose this approach to assess an unbiased estimate on inflammation in the heart, lungs and

liver. Other approaches could have been tried, such as flow cytometrical quantification of

leukocyte infiltrates, though this would have required separate mice only for this procedure. For

46

detecting DNA fragmentations, e.g. necrotic cells, one could perhaps use Terminal

deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. However this method is

primarily used to detect apoptotic cells, and the accuracy of this method has been questioned, as

it does not distinguish between apoptosis, necrosis and autolytic cell death (187).

5.4 Immunohistochemistry and image based quantification

Immunohistochemistry (IHC) is an important and complimentary tool for visualizing the specific

location of a given protein. IHC relies on uncovering antigens for specific binding to specific

antibodies. These antigen-antibody complexes are visualized by microscopy, either by

fluorochromes in ultraviolet light or simply with a coloured histochemical reaction (used in our

studies) (188). As in all molecular assays, IHC comes with challenges in each step of the

protocol. Some of these pre-analytical factors involve quality of the tissues, the choice and

duration of fixation medium, slice thickness, the level of antigen expression/cell preservation

within the tissue- all of which impacts the end results. In our studies organs were fixated over

night in 4% formalin (the most common fixation medium) and subsequently changed into

phosphate buffered saline (PBS) the next day, prior to paraffin embedding to prevent chemical

modification or degradation of antigens. It is critical to uncover all antigens as well as avoiding

unspecific binding. For heat induced epitope retrieval, we incubated the tissue in citric acid

buffer (pH 6) at 96 degrees for 20 minutes, and used a commercially available blocking buffer

(Rodent block M; Biocare Medical, Concord, CA). Appropriate tissue block has considerable

impact on the end results. Moreover, primary antibody sensitivity and specificity is crucial for

detecting the target antigen only and all of the protein. The medium for detecting peroxidase

activity and visualization of antigen, as well as incubation time is essential for avoiding under-

47

or overexposure of the tissue. We used chromogen for immune peroxidase, resulting in clear and

saturated protein detection with acceptable background colouring.

For cell quantification in paper 2 and 3 and we chose a digital method as opposed to visual

counting. We believe this increased the sensitivity of detecting positively stained cells if done

with proper caution and discretion. In paper 2, high-resolution images of histological sections

were acquired using a Nikon Eclipse E400 microscope with 40x objective and images were

automatically stitched using Hugin Panorama Photo Stitcher 2013 (189) to form a complete

rendering of the slide. Finally, images were thresholded using three-color channels adapted to

the target stain with ImageJ (version 1.49, National Institutes of Health, Bethesda, MD). In paper

3 we used an automated slide scanner system (Axio Scan Z1, Carl Zeiss Microscopy, Munich,

Germany), which resulted in an even higher precision level, as we cannot exclude the possibility

of photo capturing overlap when conducted manually. Images were inspected using the Zen Lite

Blue software (Carl Zeiss Microscopy). Prior to measurement of the stained area, all slides were

investigated manually regardless of the scanning method (automatic or photo). To avoid

including non-cardiac tissue in the analyses, connective tissues, e.g. endothelium in vessels,

epicard and endocard, and obvious artefacts from tissue processing were excluded as well as the

right ventricle. The stained area was adjusted for the total area of the section resulting in a

relative quantification of the amount of cells stained for macrophages with MAC-2. To avoid

biased results, a blinded operator conducted the procedures. In paper 2 and 3, both the operator

and the analyst were blinded to the different experimental groups.

48

5.5 Quantifications of fibrosis

In humans a common cause of passive relaxation deficit is increased fibrosis, this being the

result of an imbalance between the synthesis and degradation of collagen, as well as qualitative

alterations of the ECM. Hydroxyproline is a major component of collagen (190) and plays a key

role in maintaining collagen stability (191). Hydroxyproline is present in only few proteins apart

from collagen, making it a sensitive and specific marker for collagen detection. The amount of

hydroxyproline was measured by drying cardiac tissue in a vacuum centrifuge, hydrolysis in 6M

HCl and quantification by applying High-pressure liquid chromatography (HPLC) (192). The

HPLC method allows for relatively precise quantification of the total collagen end product, as

compared to visual assessment by IHC, assessment of collagen mRNA or measuring protein

levels. HPLC makes it possible to quickly separate different materials based on polarity by using

a pressure pump and produces high-resolution results. Still, the method has some disadvantages

as it is quite expensive and it requires experience to manage correctly.

In paper 3, we applied picrosirius red staining as a supplement protocol for collagen detection.

This is one of the best-understood techniques of collagen histochemistry. After incubating with

Weigherts haematoxylin for visualization of nuclei, slides were stained in picrosirius red for 1

hour before wash in acidified water to prevent the loss of dye. The quantification of slides was

performed similarly as for the inflammatory cells (see section 5.1.4). Though hydroxyproline

determination is favourable for collagen quantification, picrosirius red staining provides visual

information of distribution in the heart, e.g. distribution of perivascular fibrosis versus interstitial

fibrosis. As such, both methods give valuable information about the collagen and are

complementing.

49

5.6 Echocardiography and phase contrast magnetic resonance (PC-MRI)

Human hearts are more than two orders of magnitudes larger than mouse hearts with a heart rate

that is up to ten times lower. Thus, cardiac measurements of mice require markedly higher

resolution than in humans for comparable data yield. Despite the differences in heart dimensions

and frequencies, the cardiac mechanical features of mice do resemble human hearts greatly, and

are indeed transferable to human conditions (193,194,195,196).

In paper 2 and 3, we assessed cardiac function and death as the end parameter. An important

aspect for achieving comparable data is to standardize the amount of anaesthesia during

echocardiography. In our studies, mice were anesthetized with a mixture of oxygen and

isoflurane while being carefully monitored for optimal depth of anaesthesia by observing heart

rate during echocardiography. They were initially anesthetized in a chamber with a mixture of 2-

3% isoflurane and 97% oxygen and further on a mask with a mixture of 1.75% isoflurane and

98.25% oxygen. It is crucial to standardize the anaesthesia to limit inter-variation between the

animals, to avoid statistical type I errors. This protocol for anaesthesia has been standardized at

the Institute of Experimental Medical Research, Oslo University Hospital Ullevaal by years of

extensive experiments. During the procedure, stable conditions were maintained by recording

ECG and respiration. Body temperature was monitored and stabilized by a rectal probe, either on

a heated pad with the mouse in a supine position on top during echocardiography or by hot air

during PC-MRI assessments. Note, during the initial part of extended PC-MRI there was some

body heat loss due to preparations. Moreover, the duration of the procedure is a key factor for

keeping the body state as physiologic as possible. In our studies the echocardiography and PC-

MRI did not last longer than 10 and 75 minutes per animal, respectively, and all mice recovered

from anaesthesia within 1-2 min.

50

Echocardiography is the go-to standard to evaluate global cardiac function and is frequently used

as it effective (short duration per animal) with a relatively low cost. It provides with 2D or 3D

(M-mode or B-mode) images with relatively high temporal resolution. Nevertheless, as opposed

to MRI, the reproducibility is low as it relies on geometric assumptions when calculating

volumes (197). Also, high resolution may be challenging as shadowing by the sternum in

transthoracic echocardiography limits the geometric views available for the operator (197). Such

low spatial resolution limits information on velocity and becomes dependent on the direction of

the transducer making results less reliable (198,199). For evaluating cardiac dimensions, LVEF

and LV fractional shortening (LVFS), 2D-imaging by M-mode was assessed. LVEF was

assessed to evaluate cardiac function, however there are some differences that needs to be

considered when comparing measurements in human and in mice. For calculating cardiac output

blood flow Doppler was assessed. All dimensional measurements in B-mode were controlled

with M-mode images.

MRI is considered the gold standard for assessing cardiac morphology and physiology (200,201)

as it provides high spatial resolution with reliable data of regional myocardial function. MRI

offers information about velocity, displacement and strain measurements and, as opposed to

echocardiography; it overcomes all limitations in geometry (199), though it involves a longer

acquisition time and more extensive data analysis. As opposed to Cine MRI, which is the

fundamental MRI tool for studying cavity volumes, LVEF, cardiac output and myocardial wall

thickening in animal hearts in vivo (202,203,204), we used PC-MRI or velocity-encoded MRI.

This is an even more advanced imaging technique as it integrates motion of blood and tissue into

one signal phase and trans mural velocity. Both Cine and PC-MRI may allow for information

about trans mural variations (205), but only PC-MRI allows for transmural variation of velocity

51

(206).

As studies have shown that LVEF and LVFS measured by echocardiography are rather

insensitive parameters of systolic dysfunction (207), we used PC-MRI to measure LV

longitudinal strain (fractional change in length of an object relative to its original length), which

is a far more sensitive parameter to systolic dysfunction. Longitudinal strain allows for

measurements of muscle fibre shortening in axial directions and yields favourable geometrical

control during the cardiac cycle. As PC-MRI allows for temporal and spatial control during

every stage of the cardiac cycle, it is considered superior for recording and analysing diastolic

function as well (Sjaastad I M.D. PhD., 2014 Personal communication).

As good quality of images is a prerequisite for optimal analyses, it is essential to use the same

operator to limit the inter-animal variations. Though there has been great advancement since the

first publication on PC-MRI in 2003 (208), there is still a need for optimization of temporal

resolutions. In our studies, the echocardiography operator has over ten years of experience.

Importantly, both the operator and the analyst were blinded to genotype and intervention. In his

thesis, E. Espe M.Sc., Ph.D. describes the PC-MRI protocol that has been established at the

Institute of Experimental Medical Research, Oslo University Hospital Ullevaal over several

years (209) and the MRI recording and analyses were conducted by a blinded and trained

physicist, using Matlab (The MathWorks, Natick, MA).

52

5.7 Statistics

In paper 1, we tested for normal distribution in SPSS and the resulting skewed data were

analysed by non-parametric statistics, e.g. Mann-Whitney U test with data shown as ±SEM,

Wilcoxon matched-pairs signed rank test and Spearman correlation for unpaired, paired and

correlation analysis, respectively. Kaplan-Meier analysis with log-rank test was used to analyse

all-cause mortality stratified by median mtDNA or nDNA levels. Due to limited outcome and

observations, a stepwise Cox regression was conducted and estimate hazard ratios included age,

NT-proBNP, LVEF, creatinine and NYHA class in addition to the DNA species. The factors that

remained in the regression model were NT-proBNP, mtDNA and nDNA. All analyses were

performed with GraphPad Prism version 6 or IBM SPSS version 22. Probability values are two-

tailed and P<0.05 was considered significant.

Based on low study power and unknown genotype distributions, we assumed that the data was

skewed in paper 2 and 3. Unpaired data was analysed using Graphpad Prism 6 (GraphPad, San

Diego, CA), ANOVA Kruskal-Wallis test, and subsequent Mann-Whitney non-parametric test

for comparison of two groups. Survival analyses were conducted using Log rank (Mantel Cox

test). Results are shown as mean±SEM. In paper 2, we used IBM SPSS Statistics version 22 Chi

square tests to compare the distribution of score numbers between the groups after scoring

inflammation in heart, lung and liver tissue. Probability values of P<0.05 were considered

significant.

53

6. Discussion of results

6.1 Tissue injury and release of nucleic acids

The link between endogenous release of the putative TLR9 agonist mtDNA and initiation of

systemic inflammatory activation was demonstrated in 2010, by studying multi-traumatized

patients with severe striated muscular damage (139). Based upon this initial observation and the

knowledge that the myocardium is densely populated by mitochondria, we hypothesized that MI

also could cause release of circulating mtDNA with similar putative inflammatory actions.

Indeed, in 2012 our group published a paper demonstrating that plasma mtDNA levels were

increased in STEMI patients after PCI (158). As mtDNA is released upon cellular stress and

damage as seen in, e.g. MI, we hypothesized that a prolonged state of hemodynamic stress, such

as HF, also could cause intracellular and extracellular mtDNA release upon disease progression

and that this could activate cardiac TLR9 and possibly impact cardiac function.

In paper 1 we studied circulating mtDNA and nDNA in patients with chronic HF and found that

both markers were increased. However, while high levels of nDNA were associated with high

NYHA class, high NT-proBNP levels, high troponin levels and low LVEF, this was not the case

for mtDNA. Moreover, whereas increased concentrations of nDNA were associated with

increased mortality, high levels of mtDNA were associated with increased survival. This

observation was corroborated with an even better prediction of mortality in subgroups with high

nDNA combined with low mtDNA levels. The mortality findings may appear paradoxical as

both mtDNA and nDNA were increased in HF patients. Unfortunately, for the negative

correlation between high mtDNA levels and mortality, we have no clear explanation at present.

There may be unknown beneficial effects by mtDNA, e.g. synthesis of protective mediators

54

against harmful metabolites during tissue damage. In paper 3, TLR9 activation was associated

with survival as SERCA2a-TLR9 KO mice displayed premature death. This suggests some

beneficial effects of TLR9 activation, perhaps through increased mtDNA mediated activation.

As we did not study mtDNA release on a cell-specific level, one may hypothesize that the origin

of mtDNA may be CMs. Still, we cannot exclude the possibility that mtDNA may be released

from other cell-types and organs, e.g. leukocytes. Nevertheless, we did not find increased levels

of circulating leukocytes in the HF patients as we only found a trend towards a correlation

between leukocytes and both DNA forms. Moreover, other potential sources of mtDNA may be

lungs and skeletal muscle.

We observed increased mtDNA and nDNA in HF patients, and though this may be caused by

increased release into the circulation, another possible explanation may be reduced degradation.

Three nucleases have been identified, DNase1, 2 and 3, of which DNase1 is primarily found in

the circulation and DNase2 is found intracellular. Our findings in 2012 (158), of reduced levels

of plasma mtDNA three days post revascularization could be caused by increased DNase1 levels

as this has been demonstrated in previous studies in patients with acute MI (216). Moreover, in a

prolonged inflammatory state such as HF, our findings of higher levels of both mtDNA and

nDNA in patients may be a consequence of increased nucleic acid release and/or reduced

degradation. To our surprise, measurements of circulating DNase1 was not significantly

different between HF patients and controls, which might support the hypothesis of an increased

nucleic acid release. This hypothesis is supported by studies that show increased DNase1 in the

acute period (217) as well as later during HF development (218). Unfortunately we did not

elaborate the investigation beyond measuring serum levels of DNase1 at one isolated time point.

To assess a possible time-dependant variation of nucleic acid concentrations we would have to

measure levels serially over time. Moreover, we did not assess DNAse1 activity. Nevertheless,

55

based on these findings we cannot exclude the possibility of increased DNase1 activity upon HF.

Finally, due to limited availability of tissue from HF patients and controls, we unfortunately did

not measure intracellular DNase2. Further mechanistic studies on the effects of increased

mtDNA will be crucial to shed light on this topic.

In contrast to our findings in HF, mtDNA has been suggested as a strong predictor of 15-day

mortality in massive pulmonary embolism and to be a more accurate prognostic marker than

nDNA (134). Still, the 15-day mortality in massive lung embolism could reflect acute failure of

the RV with subsequent circulatory decompensating effects, as opposed to the impact of

increased mtDNA on survival. In general, although pulmonary embolism and HF cannot be

compared as these are completely different disorders, it is likely that plasma mtDNA levels are

dynamic and not static, meaning a high level of mtDNA may serve as an accurate predictor of

mortality in an acute setting, however when reaching a more chronic state, the association may

change.

Circulating nucleic acids are currently being evaluated as biomarkers in several different

diseases (210,211,212,213,214,215). One of the criteria for biomarkers in clinical use is high

sensitivity and specificity, e.g. ≥0.9 (186). Though HF is a highly prevalent disease in the

Norwegian population, the positive predictive value would probably be too low. However,

neither mtDNA nor nDNA is organ specific as it may be released upon all damaged cells,

making them difficult to interpret for clinical everyday use. Also, one has to remember that

while release of the larger and more robust nDNA may be a marker of tissue damage and cell

death, the release of the smaller mtDNA is markedly lower and is more prone to degradation by

circulating nucleases. Instead, circulating nucleic acids may perhaps serve as a supplement to

established cardiac biomarkers such as troponin T and BNP, or to muscle damage in general

56

such as creatine kinase or creatine kinase-MB, the latter of which is primarily released from the

heart, but also from skeletal muscles.

6.2. TLR9 activation and systemic inflammation

Zhang et al demonstrated that SIRS was associated with mtDNA-mediated activation of TLR9 in

PMNs (139). Moreover, they showed that injections of MTDs into mouse peritoneum could

cause peritonitis, and that i.v. MTD injections into rats lead to systemic inflammation and severe

lung injury (139). Another experimental study demonstrated that increased circulating mtDNA

over a six-week period in hypertensive rats, caused hypertension and vascular dysfunction by

activation of TLR9 (219). As mentioned in the introduction of this thesis (chapter 1.3.5), several

studies on CpG induced cardiac TLR9 activation have been conducted, though with ambiguous

results. Some studies have shown that repeated CpG stimulation may attenuate cardiac

dysfunction during HF (159,160,161,220), whereas others and we have found that CpG

injections is detrimental to the heart (162,221).

Behrens and colleagues demonstrated that repeated TLR9 stimulation in healthy mice could

cause hemophagocytic lymphohistiocytosis disease in mice, thus suggesting a large

inflammatory potential of repeated TLR9 activation (185). HF patients may present with

concomitant diseases (lung diseases, rheumatic diseases etc.) that may contribute to the already

low-grade systemic inflammatory state. In paper 2 we assessed this topic by establishing

systemic inflammation in mice, by CpG injections. Note that we are aware of the several aspects

with this model that may not represent low-grade systemic inflammation in humans. When

comparing our study with the paper by Behrens and colleagues, both groups observed a cytokine

57

storm in the CpG B treated mice. This caused leukopenia, a common consequence of depressed

bone marrow or hemophagocytosis in bone marrow, as well as peripheral cell-destruction,

apoptosis etc. As for peripheral organs, both groups found increased inflammation in the CpG B

treated WT mice. Surprisingly we did not observe this pattern in the lungs. A possible

explanation could be that the sample size in each group was simply too low to detect any

difference. In the SERCA2a KO control mice we found no difference in circulating white blood

cells (wbc), cytokine profile or cardiac macrophage infiltration compared to WT mice. However,

the SERCA2a KO mice did display increased inflammation in liver, lungs and heart well as

collagen I and III mRNA deposition and spleen weight. The finding was supported by the

SERCA2a KO model in paper 3, in which we found increased lung weight, cardiac macrophage

infiltration and collagen II and III mRNA deposition, compared to WT mice, reflecting a HF

phenotype regardless of TLR9 depletion. We did not find any difference between SERCA2a-

TLR9 KO and SERCA2a KO mice in any of the inflammatory parameters, thus confirming that

HF induces a low-grade systemic inflammation.

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6.3 Direct vs. indirect cardiac consequences of systemic TLR9 activation

In our in vivo studies of SERCA2a KO induced HF with chronic TLR9 activation and TLR9

deficiency, we observed aggravated cardiac function and premature death. There are several

considerations to make when evaluating these effects. By injecting CpG B i.p. we created a non-

physiological systemic inflammation in the mice, which made it difficult to distinguish between

direct TLR9 activation and indirect activation, making the model “dirty” (Figure 5A). An

alternative approach would be to induce HF in a SERCA2a-TLR9 KO mouse with a conditional

CM-specific TLR9 knock in, with subsequent assessment of cardiac function (Figure 5B). A

second and more feasible alternative would be to eliminate the bone marrow of SERCA2a KO

mice with irradiation, and reconstitute with bone marrow from TLR9 KO mice with induction of

HF (Figure 5C). To further investigate if the findings by Oka and colleagues could be

generalized for HF, one approach could be to use another murine HF model, e.g. a CM-specific

SERCA2a-DNAse2a KO mouse (Figure 1D). In paper 3, we found that the SERCA2a KO mice

with TLR9 intact had increased survival as compared to SERCA2a-TLR9 KO mice, thus

supporting a beneficial effect of TLR9 activation. One may hypothesize that if the SERCA2a-

DNAse2a KO model likewise would demonstrate increased survival, this would be the opposite

result of what Oka and colleagues demonstrated as they found increased mortality in the

DNase2a KO mice with TAC induced HF. Thus, the main conditioning in this case would

perhaps be different HF aetiology i.e. TAC vs. SERCA2a KO. On the other hand, if the

SERCA2a-DNase2a KO model demonstrated increased mortality, it would corroborate with the

findings by Oka and colleagues. Interestingly, it would also impair the assumption that the CpG

induced systemic inflammation in paper 2 was the cause of premature death in the SERCA2a

KO mice. This is because the only distinguishing factor between the SERCA2a KO model,

promoting survival in paper 2, and the SERCA2a-DNase2a KO model would be conditioned by

endogenous TLR9 signalling. i.e. the SERCA2a-DNase2a KO mice would accentuate the CM

59

TLR9 signalling due to unlimited TLR9 signalling. In all the alternative models, the only impact

on cardiac function would be derived from endogenous mtDNA released from the heart. Though

such “cleaner” models would probably make it easier to distinguish between direct and indirect

cardiac effects, this would unfortunately be much more methodologically challenging and

require more resources than we were able to provide at the time. Thus, at this point the question

remains unresolved as of whether cardiac TLR9 could be activated indirectly through

extracellular mtDNA or, as Oka and colleagues (163) suggests, exclusively by intracellular

mtDNA.

Several systemic inflammatory conditions involve disturbances in the innate immune system and

may involve altered TLR9 signalling. Still, we cannot ignore that sustained TLR9 stimulation

does not necessarily represent a clinically relevant inflammatory condition. Also, the SERCA2a

KO model does not adequately represent the molecular basis for, or the clinical features of

diastolic HF. Thus, different HF models reflecting both systemic and diastolic HF, needs to be

investigated to fully understand the role of TLR9. Moreover, future studies on what role

comorbidities, e.g. systemic inflammation, play in HF, and how HF impacts systemic

inflammation, would be necessary and crucial.

60

5A

SERCA2a-DNAse2a KO5D

TLR9

SERCA2a-TLR9 KO with HF

SERCA2a KO HF

TLR9 KO BM cells

5B

5C

Figure 5. CpG B induced systemic inflammation in SERCA2a KO model

A) CpG B injections i.p. lead to unspecific TLR9 activation in peripheral organs with the release of inflammatory

cytokines into the bloodstream. This positive feedback circle made it difficult to distinguish between direct cardiac

TLR9 activation and indirect activation. Direct activation of cardiac TLR9 can be assessed by; B) induction of HF in a

SERCA2a-TLR9 KO mouse with a conditional CM-specific TLR9 knock in. C) eliminate the bone marrow of

SERCA2a KO mice with irradiation, and reconstitute with BM from TLR9 KO mice followed by subsequent induction

of HF. D) CM-specific SERCA2a-DNAse2a KO mouse to study TLR9 activation in CMs specifically.

5A

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6.4 Intracellular vs. extracellular mtDNA

Oka and colleagues observed that unlimited mtDNA mediated TLR9 activation in CMs could

induce myocarditis and HF (163). During baseline conditions, the deletion of DNAse2a did not

show any cardiac phenotype. However, ten days after induction of TAC the mice displayed HF

and premature death. Interestingly, there was no significant difference in the amount of

extracellular mtDNA between DNase2a KO mice and control mice with TAC-induced HF (163).

Thus, intracellular increased mtDNA was not reflected in systemic measurements and they

excluded the possibility of circulating mtDNA for being the cause the TLR9 mediated

inflammatory response. This must be interpreted with caution as mtDNA release can be elicited

by small interventions, e.g. harvesting blood samples, making the risk of background noise high.

As opposed to Oka and colleagues, we found increased extracellular mtDNA in humans with

HF, though unfortunately we were not able to investigate the source of this increase. In addition

to the data presented in paper 1, we also investigated TLR9 mRNA levels in cardiac tissues of

HF patients (n=18) (unpublished data). We analysed paired cardiac tissue samples, which were

obtained at two time-points; first, upon implantation of left ventricular assist device (LVAD) and

second, at the time of heart transplantation. As demonstrated in Figure 6A, we found that along

with the clinical improvement during LVAD treatment, there was a reduction in cardiac TLR9

mRNA after LVAD treatment (p=0.002). Moreover, during LVAD treatment there was a

reduction in BNP mRNA expression, which correlated with the changes in cardiac TLR9 mRNA

expression (Figure 6B; r=0.47, p<0.05). Unfortunately, due to logistic reasons we were not able

to assess plasma samples of these patients for investigation of mtDNA levels. A potential

increase in plasma mtDNA levels at the time of LVAD insertion could strengthen the possibility

of a cardiac origin of the increased mtDNA levels from paper 1. As Oka and colleagues, who

observed increased intracellular mtDNA release within CMs upon TAC, we could potentially

62

isolate CMs from these patients at the time of LVAD insertion with subsequent labelling of

mtDNA. This would substantiate the hypothesis of a CM cell-specific origin even further. As

mentioned initially in the discussion (chapter 6.1), we cannot exclude that mtDNA may originate

from other cell-types, and a cardiac origin could still indicate a non-CM origin, e.g. fibroblasts,

endothelial cells etc. As mentioned earlier, one feasible origin may be leukocytes, as we found a

trend towards a correlation between leukocytes and both DNA forms, though not significant.

Also, Zhang and colleagues (139) demonstrated mtDNA mediated TLR9 activation in

polymorph nuclear granulocytes.

6A

6B

Figure 6. Reduced myocardial expression of TLR9 mRNA with LVAD treatment.

Heart biopsies from 18 patients with HF were serially sampled at the time of LVAD insertion and at the time of heart

transplantation. (A) mRNA expression of TLR9 at the time of LVAD insertion and at the time of transplantation. (B)

Fold change in mRNA expression of TLR9 correlated to fold change in BNP mRNA expression. *P<0.05, **P<0.01 vs.

controls.

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6.5 Acute vs. chronic activation of TLR9

A recent study by Shintani and colleagues suggested an alternative non-canonical TLR9

signalling pathway in CMs and neurons, as opposed to the classic canonical pathway in immune

cells (222). They observed increased AMPK activation and cell survival, e.g. autophagy (223),

as a result of inhibiting SERCA2 by CpG B induced TLR9 activation. In paper 3, we studied

genetically modified SERCA2a-TLR9 KO mice and found that the absence of TLR9 was

associated with increased mortality whereas mice with cardiac TLR9 present promoted survival.

We investigated levels of AMPK activation in these mice, however we did not find any

differences (data not shown). Both studies demonstrated beneficial effects of TLR9 activation. In

contrast to Shintani and colleagues, who stimulated TLR9 with CpG B, we could assume that

autocrine and/or paracrine mtDNA was the activating ligand in the SERCA2a model with TLR9

present. Unfortunately we did not investigate this on a mechanistic level.

When studying the results from our group in 2012 (158), paper 1 and 3, it may seem that

circulating mtDNA is modulated dynamically from the acute setting to death. A hypothesis may

be that initial myocardial damage, e.g. MI, may cause increased mtDNA release (or reduced

degradation) into the circulation, with a nuclease mediated fast return to baseline in an attempt to

reduce harmful effects (Figure 7A). Upon prolonged mtDNA release, e.g. HF, this may elicit a

second phase of low-grade TLR9 activation, which in paper 3 was associated with survival.

These results suggest that at least some degree of mtDNA mediated TLR9 activation may be

necessary for survival.

In paper 1, low levels of mtDNA were associated with death, possibly caused by reduced TLR9

activation (Figure 7C). However, both our paper from 2012 on STEMI patients and paper 2 in

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this thesis, suggest that excessive amounts of TLR9 ligands (either mtDNA or CpG) are

associated with cardiac damage and premature death. These are only speculations. As mentioned

earlier, despite many similarities, mice and humans far from the same. We have only measured

high circulating mtDNA concentrations in HF patients, assuming that mtDNA is the endogenous

TLR9 ligand, and we have not measured TLR9 activity in these patients. Moreover, we do not

fully understand what the implications of increased mtDNA levels are during MI and whether

TLR9 even does convey modulating effects upon I/R (146). As such, it is with great humbleness

and caution that this hypothesis is presented in this thesis, as evidently the implications of having

increased or reduced extra-or intracellular mtDNA is not yet determined- nor what the impact of

TLR9 activation is on patient outcome.

Figure 7. mtDNA is modulated dynamically from the acute setting to death.

A) MI may cause increased mtDNA release into the circulation, with a nuclease mediated fast return to baseline to

reduce harmful effects. B) Upon prolonged mtDNA release this may elicit a second phase TLR9 activation. C) Both

too much and too little TLR9 stimulation may be harmful and may cause premature death.

A A A

B

C 7

B

CC C

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7. The role of TLR9 and future perspectives

Though the literature on the effects of cardiac TLR9 demonstrates opposing results, there are

several factors that need to be considered. First, one has to distinguish between CpG induced

TLR9 activation and endogenous mtDNA receptor activation, as we do not fully understand

which part of the CpG that is responsible for ligand binding and receptor activation. Thus, we do

not know to what extent mtDNA and CpGs are comparable for TLR9 activation. Second, we

need to distinguish between studies conducted on whole-heart TLR9 and studies on a particular

cell type, e.g. CM. Moreover, we need to distinguish between studies on different cell-types. As

most studies on TLR9 have been conducted on immune cells, it is not unlikely that immune cells

might be responsible for the TLR9 signalling as they convey a stronger TLR9 potency. The

paper by Shintani and colleagues is the first to compare the inflammatory TLR9 response of the

canonical pathway in immune cells vs. the non-canonical pathway in adult CMs (216,217).

Although the hypothesis of SERCA as being a central mediator of TLR9 signalling still needs to

be thoroughly investigated, the comparison between immune cells and adult CMs is novel and

intriguing. Third, paper 2 and 3 were conducted on SERCA2a KO mice, which promotes both

systolic and diastolic HF, primarily the latter. As most studies on cardiac TLR9 have been

conducted in models with systolic dysfunction, this needs to be considered when comparing the

results as the two subtypes of HF probably convey differences in the pathogenesis.

As mentioned in the introduction of this thesis, it is evident that inflammation plays a crucial

role in HF, regardless of the up-stream aetiology. Based on the results in paper 1 and 3, it may

seem that some degree of TLR9 activation is associated with survival in both mouse and human

HF. Opposing results, in paper 2 and in our pilot study on LVAD patients, suggest detrimental

66

effects of TLR9 activation with premature death in mice and higher TLR9 mRNA expression

prior to alleviation with LVAD (Unpublished data, Figure 6). Based on the results in this thesis,

a hypothesis may be that both too much (see paper 2 and LVAD pilot) and too little (see paper 1

and 3) TLR9 stimulation is harmful (Figure 7C). TLR9 activation results in activation of NF-κB,

which promotes the production and release of the proinflammatory cytokine, TNF. A large

amount of clinical trials have been conducted on TNF and likewise it seems that too much of

TNF and too little of TNF both provide adverse effects. Again, this fits with our results

indicating that a delicate balance is needed for favourable effects (43).

Evidently, the pathophysiological role of cardiac TLR9 is not resolved. The conflicting results in

the studies on cardiac TLR9 may be explained by several factors; 1) Difference in study design

2) Different mouse models of HF 3) Physiological differences in mice and humans 4)

Endogenous TLR9 activation vs. CpG mediated activation 5) TLR9 activation in MI vs. HF etc.,

however, it is safe to claim that the studies on TLR9 in HF do not settle the question as to

whether TLR9 promote detrimental or salutary effects nor if TLR9 is particularly important in

specific subtypes of HF. As the elderly population is increasing followed by a higher prevalence

of patients with HF and comorbid diseases, it is not unlikely that diastolic HF will become more

frequent and require more hospital- and socioeconomic resources. As such, more studies on

inflammation and HF, particularly diastolic HF should be considered. Larger studies on plasma

mtDNA in HF patients, with hard primary end-points would be intriguing. Moreover, studies on

TLR9 in human cardiac tissue, e.g. functional studies should be assessed and from our

knowledge, human TLR9 protein in cardiac tissue has not been successfully obtained. As such,

more mechanistic and clinical research is needed to increase our knowledge of what role

inflammation conveys in human HF and to identify potential therapeutic targets.

67

8. Conclusion

mtDNA is increased during human HF, which may serve as a potential TLR9 activating DAMP.

High circulating levels of nDNA are associated with increased mortality and high levels of

mtDNA are associated with increased survival. An even better prediction of mortality is seen in

subgroups with high nDNA combined with low mtDNA levels. Signalling through TLR9 is

necessary for survival in SERCA2a KO HF mice though not through modulation of distorted HF

specific hemodynamic or structural parameters. Finally, we found that systemic inflammation

caused by CpG B mediated TLR9 activation causes deterioration of cardiac function and

premature death in SERCA2a KO mice.

More specifically the findings presented in this thesis are:

• High circulating levels of nDNA are associated with increased mortality and high circulating

levels of mtDNA are associated with increased survival.

• Sustained TLR9 stimulation leads to premature death in SERCA2a KO mice, possibly by

aggravating diastolic HF as indicated by echocardiography and PC-MRI. Both systemic and

cardiac inflammation is increased with sustained TLR9 stimulation.

• Absence of TLR9 promotes premature death in SERCA2a KO mice, however this does not

significantly influence diastolic HF in SERCA2a KO mice, as indicated by echocardiography

nor does it influence cardiac monocytes/macrophages infiltration or cardiac collagen levels in

SERCA2a KO mice.

Our data suggests that circulating mtDNA is increased during human HF. Moreover, mtDNA

levels significantly impact survival in human HF and experimental studies demonstrate that a

delicate balance of TLR9 activation is necessary for beneficial remodelling and survival.

68

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