UNIVERSITÀ DEGLI STUDI DI TORINO

Dipartimento di Biotecnologie Molecolari e Scienze per la Salute

SDSB – Struttura Didattica Speciale di Biotecnologie

Corso di Laurea in Biotecnologie

Tesi di Laurea di I livello

Mitochondrial Dynamism and Mitophagy in the Heart

Candidato: Relatore: Alessandra Murabito Prof.ssa Mara Brancaccio

a.a. 2014/2015

Index

Abstract....................................................................................................................3

1. Introduction..........................................................................................................4

1.1 Mitochondrial Fission....................................................................................4

1.2 Mitochondrial Fusion....................................................................................6

2. The Heart: an Unexpected Exception...................................................................7

3. How Mitochondrial Dynamism Affects Mitochondrial Quality Control.............9

4. PINK1-Parkin-Mediated Mitophagy Signaling Pathway...................................11

5. Mitochondrial Dynamism - Mitophagy Crosstalk in the Heart.........................18

6. Conclusions........................................................................................................22

Bibliography...........................................................................................................23

2

Abstract

Mitochondria are highly dynamic organelles undergoing fission, fusion and

mitophagy processes. In many cell types mitochondria form an interconnected

reticular network and dynamism is frequent, perhaps continuous, in such cells. An

exception though is present in the heart. Recent results in vivo cardiomyocyte-

specific genetic manipulation have revealed that mitochondrial dynamic factors

function in cardiomyocytes in different ways. In fact, in this cell-type there is a

partial absence of mitochondrial network and a slow mitochondrial turnover.

Nevertheless, the mitochondrial fusion proteins mitofusins (Mfn)1 and 2 and optic

atrophy (Opa)1, and the mitochondrial fission protein dynamin related protein

(Drp)1 are highly expressed in mammalian heart, wherein genetic ablation

provokes dramatic cardiac dysfunction. All these studies suggest that fission and

fusion have an important role in cardiac mitochondrial quality control. In this

work I will describe in more detail a major regulator of these processes, the

PTEN-induced putative kinase1-Parking mitophagy pathway.

3

1. Introduction

It has become axiomatic that mitochondria constantly undergo fission, fusion and

migration, collectively termed “mitochondrial dynamism”, within the cells.

Mitochondrial networks are constantly remodeling, with sections of the network

periodically breaking away and then re-estabilishing new interconnection

elsewhere. Relative frequency of fission and fusion has also a determinant role in

mitochondrial morphology. When fusion and fission are balanced, mitochondrial

aspect ratio (length/width) is stable. However, when the balance shifts in favor of

more fusion, mitochondrial elongation and network connectivity increase. On the

other hand, when an imbalance produces less fusion, this will lead to an

accumulation of smaller, shorter, fragmented organelles2. Mitochondrial

morphometry seems especially plastic during cell mitosis. In fact network

disassembly not only facilitates cytokinesis in growing and dividing cells, but also

helps to deliver equal proportions of the parental cell mitochondrial pool to each

daughter cell. In these cells, fission is an essential process that permits to populate

them with adequate numbers of mitochondria1-5.

1.1 Mitochondrial Fission

In order to replicate and expand the cellular mitochondrial pool, mitochondria

undergo fission. Through this process, from one mitochondrion, two daughters are

produced. Drp1 has a key role in this mitochondrial dynamic: it is a GTPase with

an amino terminal GTPase domain, a middle domain and a carboxyl terminal

GTPase effector domain (Fig 1A). This protein has to be recruited to the outer

mitochondrial membrane (OMM) to promote fission because in normal condition

is present in the cytosol. But how Drp1 is able to promote fission? After the

localization to the OMM, the GED domain (GTPase effector domain) interacts

with the middle domain of adjacent molecules to promote head-to-toe

oligomerization (Fig 1B). Following the GTPase-mediated constriction, Drp1

ligates and separates the IMM (inner mitochondrial membrane) and the OMM.

There are multiple events that stimulate Drp1 translocation form the cytosol, such

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as apoptosis, mitosis, mitophagy and mitochondrial remodeling. As a consequence

of impaired mitochondrial fission, fibroblasts derived from Drp1 deficient mice

are hyperlongated and partially resistant to mitochondrial fragmentation induced

by pharmacological uncoupling agents. It is thought that Drp1 is able to bind the

OMM via resident receptor protein such as mitochondrial fission protein factor

(Mff), since it lacks of an hydrophobic transmembrane domain. In mammals, Mff

recruits Drp1 often at sites where mitochondria make contact with the

5

Fig 1A: Schematic Drp1 molecular structure.

Fig1B: GTP-dependent constriction of oligomeric ring structures constricts and severs the mitochondrion.

endoplasmic reticulum (ER)1. Therefore, thanks to this process cells are able to

maintain an adequate pool of mitochondria and to modify their structure before

mitosis. This is called symmetrical fission and produces from one mitochondrion

two equal daughters. Mitochondrial networks subsequently have to be re-

estabilished in each daughter cell via generalized organelle fusion.

1.2 Mitochondrial Fusion

The process of fusion is more complicated then the fission one. A complication is

compartmentalization of mitochondria that are composed by a central matrix,

enclosed by the cristae/inner mitochondrial membrane (IMM), which is separated

from the outer membrane (OMM) by intermembrane space. The integrity of the

compartments has to be maintained through mitochondrial fusion which thus

occurs layer by layer. The first step involves physical tethering of two

mitochondria via trans-interaction between the carboxyl terminal domains of

OMM mitofusins of two organelles. In fact, Mfn 1 and 2 are both GTPases like

Drp1 and both isoforms have a cytosolic N terminal GTPase domain, a

hydrophobic transmembrane domain and two cytosolic hydrophobic heptad

repeated coiled-coiled domains (HR1 and HR2). Consequently, Mitofusins insert

into the OMM with the majority of the protein being exposed to the cytosol and

available to interact with cytosolic factors. This position allows them to

participate in information exchange between the mitochondrion and its host cell.

Since Mfn1 and Mfn2 are quite similar, the interaction between mitofusins of

different organelles can occur in either a homotypic or heterotypic manner, with

the heterotypic shown to be more efficient and to yield a higher rate of successful

fusion events (Fig 2). Mitofusin-mediated physical tethering of mitochondria is

GTPase-independent and fully reversible, whereas GTP hydrolysis is essential to

irreversible OMM fusion. OMM fusion maintains the structural integrity of the

IMM and matrix, thus avoiding ROS formation. After the OMM fusion, IMM

fusion can occur. A key factor in this process is Opa1, also a GTP-ase protein.

With the loss of Opa1, mitochondrial tethering and OMM fusion still occur

(through the actions of Mfn1 and Mfn2), but absence of Opa1- mediated IMM

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Fig 2: (A)Schematic description of the molecular structure of Mitofusins and their role in fusion. (B) Opa1 molecular structure.

fusion produces mitochondria that are not only structurally heterogenous, but also

exhibit generalized dissipation of the normal IMM electrochemical potential and

profoundly impaired cellular respiration. These results thus point to a major role

for Opa1 in maintaining normal crista morphology that is essential for proper

assembly and functioning of electron transport chain supercomplexes1.

2. The Heart: an Unexpected Exception

The general characterization of mitochondria as “highly dynamic” does not apply

to adult cardiomyocytes. Compared to prototypical cultured fibroblast, normal

cardiac mitochondria exhibit a smaller, rounder, so-called fragmented

morphometry and do not routinely travel within the cell. Cardiomyocyte

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mitochondria appear static and no networks are observed. It appears from

ultrastructural examination of myocardium that individual cardiomyocyte

mitochondria exist as relatively short organelles, collectively grouped together

between myofibrillar elements in lanes that extend parallel to the long axis of the

cell. Even in the perinuclear region of cardiomyocytes, where mitochondria tend

to be clustered in large numbers, filamentous organelles and interconnected

networks are not observed (Fig 3).

The explanation might be that intrinsically fragmented mitochondria confer a

biomechanical advantage to striated muscle, because interconnected organelles

would constitute an internal resistance element, detrimental to myocyte

contraction. Finally, cardiomyocyte mitochondria do not undergo fusion and

fission at a rate that can be directly measured. For this reason, the widely accepted

notion that mitochondrial fragmentation is detrimental to mitochondrial and

cellular functioning likely does not apply to adult cardiomyocytes. Indeed, even

lacking interconnected mitochondrial networks, these cells have both the greatest

mitochondrial density and the highest respiratory capacity of any mammalian cell.

The maintenance of mitochondrial fitness is clearly a physiological imperative for

this organ. This thus suggests that mitochondrial dynamism plays different roles

according to the biological context2.

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Fig 3: Murine fibroblasts and human induced pluripotent stem cells (hiPSC)-derived cells costained with anti-TOM20(green) and anti-cardiac Troponin T(cTnT,red), and mounted in DAPI. In the image of hiPSC-derived cells, the cTnT-positive cell is a cardiomyocyte with mostly fragmented mitochondria, and the cTnT-negative cell is a fibroblast with filamentous mitochondria. Mouse cardiomyocytes were isolated from an adult mouse heart expressing a mitochondrial-targeted fluorescent green marker. The picture on the left is a transmission electron microscopy image of adult mouse heart.

3. How Mitochondrial Dynamism Affects Mitochondrial QualityControl

The life cycle of mitochondria resembles the one of primordial bacteria from

which they are descendant. Mitochondria replicate, as it said before, through

symmetrical fission: a healthy parent organelle produces 2 healthy daughters that

grow by adding new components generated by mitochondrial biogenesis and by

fusing with other healthy mitochondria. But over time or as a consequence of a

stress, there are two possibilities: as long as the stress is below to a critical

threshold, the mitochondrion can go under fusion in order to mitigate the effects

of environmental damage through the exchange of proteins and lipids with other

mitochondria. If the damage is too severe for correction through biogenic or

fusion-mediated repair, the mitochondrion is removed via mitophagy. Why this

mitophagic quality control is so important? This is due to the fact that damaged

mitochondria become cytotoxic factories producing ROS. Mitochondrial ATP

synthesis depends on an electrochemical gradient generated across the inner

mitochondrial membrane through a series of redox reactions: the transfer of

electrons between complexes of the electron transport chain is coupled to

extrusion of protons (hydrogen ions) across the inner mitochondrial membrane

and into the mitochondrial intermembrane space. Reversal of this proton flow

powers the mitochondrial ATP synthase, a molecular rotor. The terminal electron

acceptor is molecular oxygen, forming superoxide anion that is normally reduced

through the sequential actions of superoxide dismutase and catalase (to form

water). When electrons prematurely leak from complexes I or III of the electron

transport chain, or when specific endogenous mitochondrial enzymes such as

Romo1 (reactive oxygen species modulator 1) are activated, damaging superoxide

radicals and hydrogen peroxide can accumulate within or escape from

mitochondria and attack critical proteins, lipids, and mitochondrial or nuclear

DNA. Because production of ROS in damaging amounts does not normally occur,

substantial ROS production is a marker of mitochondrial dysfunction. In order to

protect themselves from ROS, cells use a sophisticated surveillance and

elimination mechanism to identify and remove dysfunctional mitochondria. The

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cellular decision to remove a dysfunctional mitochondrion is necessarily

dichotomous. Either the organelle is retained or it is targeted for autophagy. If

only severely damaged mitochondria are culled by mitophagy, the cell may have

to face toxicity deriving from damaged organelles that have not achieved high

threshold of dysfunction necessary to trigger their elimination. However if the cell

lowers its threshold for mitochondrial removal, this will lead to elimination of

still functional organelles, thus depriving the cell of ATP. Nature has addressed

this quandary by integrating mitochondrial dynamism with mitophagy in the form

of asymmetrical mitochondrial fission. It has been shown that mitochondria that

are targeted for mitophagy have a relatively depolarized membrane potential

before being targeted for mitophagy. This subpopulation also rarely is involved in

fusion and fission processes as mitofusins and Opa 1 are either cleaved or

degraded. This process is induced by either inner membrane depolarization or

reduced mitochondrial ATP production. Thus, mitochondria that are expected to

be eliminated through mitophagy are characterized by being relatively depolarized

and remaining solitary. The use of a photoactivable green fluorescent protein, that

was targeted to the matrix of mitochondria and photoactivated by a laser, showed

the existence of an asymmetrical fission event. This process produces 1 healthy

daughter, with a normal hyperpolarized potential, and 1 daughter that can have

depolarized membrane potential that may recover or persist. The latter one, who

has less chances to being involved in fusion events, will become part of the

preautophagic pool (Fig 4). Evidence supports that asymmetrical fission is the

result of uneven distribution of dysfunctional mitochondrial components,

including oxidized or older proteins. This model explains the role of fusion in

mitochondria quality control as it may allow for the redistribution of damaged

components while fission and mitophagy are responsible for the elimination. The

selectivity of the fusion process for the healthy daughter mitochondrion is

therefore key to the efficiency of the quality control. Selectivity depends on the

transition of dysfunctional daughter mitochondria from fusion-competent to

permanently solitary status. This process may be mediated by coupling the

degradation or post-translational modification of fusion proteins to the

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Fig 4: Diagram of symmetrical replicative fission and asymmetrical fission leading to selective mitophagy of the impaired daughter mitochondrion.

corresponding bioenergetic function of healthy and impaired daughter

mitochondria. The mechanism by which damaged components are partitioned

from healthy counterparts before asymmetrical fission is not known but one

possibility is that misfolded and damaged proteins are passively excluded because

they are physically incompatible with the supermolecular structure of IMM

respiratory complexes. The elements that do not fit, segregate and accumulate

and, after fission, mitophagy occurs1.

4. PINK1-Parkin-Mediated Mitophagy Signaling Pathway

The word mitophagy is a contraction of mitochondria and autophagy, and it refers

to the process by which cells eat their own mitochondria. Engulfment of

mitochondria in autophagosomes and the subsequent transfer to lysosomes can

occur during generalized macroautophagy as during nutrient deprivation, or as a

highly selective process that targets dysfunctional mitochondria. The most widely

explored mechanism for homeostatic mitochondrial quality control is PINK1-

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Parkin-mediated mitophagy. One of the key proteins in this pathway is PINK1

(PTEN induced putative kinase 1). PINK1 kinase is encoded by the PINK1 gene,

which like all but 13 proteins encoded by mitochondrial DNA, is part of the

nuclear genome. For this reason, after the translocation by the ribosomal

apparatus, PINK1 has to be imported into mitochondria. In normal condition, as

fast as PINK1 is imported, it is proteolytically degraded. So healthy mitochondria

have little PINK1 protein or PINK1 kinase activity. However, if ROS,

mitochondrial depolarization, or protein misfolding occur, PINK1 senses these

inputs and initiates the appropriate response. PINK1 degradation process is

interrupted, causing its accumulation and the consequent PINK1-mediated

phosphorylation of its substrates. Conceptually, healthy mitochondria make an

effort to degrade PINK1, thus staying alive by avoiding mitophagic destruction. In

damaged mitochondria that no longer support PINK1 degradation, passive

accumulation of PINK1 triggers the mitophagic destruction of the organelles.

Unprocessed 63kDa PINK1 protein is transported across the OMM translocase

complex of the OMM, delivered to the IMM by translocase of the IMM, and

proteolytically processed in a manner typical for imported mitochondrial matrix

proteins. In healthy mitochondria, PINK1 is rapidly cleaved by presentilin-

associated rhomboid like protein in the matrix, generating a 52 kDa PINK1

fragment that escapes into the cytosol and undergoes proteosomal degradation. If

mitochondria are damaged, translocase of the IMM is not functional, protecting

the kinase from translocation. The physical association between PINK1 and the

OMM is also maintained. From this location, PINK1 phosphorylates available

substrates, such as ubiquitins, and also inhibits fusion of the damaged

mitochondrion. Among PINK1 substrates is Parkin that is another major player of

mitophagy. There are different views about the biochemical events that induce

mitochondrial Parkin localization and that interrupt mitochondrial fusion. The

importance of this mechanism in mitochondrial quality control suggests a

signaling redundancy and that multiple pathways are involved. PINK1

phosphorylates Parkin on Ser65, and Parkin polyubiquitinates mitofusins. A

former hypothesis suggested a key role of PINK1-mediated phosphorylation in

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Parkin recruitment to damaged mitochondria. However, despite PINK1 does

phosphorylate Parkin, recent studies have revealed that PINK1-phosphorylated

ubiquitin, and not PINK1- mediated phosphorylation of Parkin itself, is essential

to enable Parkin to work as an E3 ubiquitin ligase. The observation that PINK1

can phosphorylate ubiquitin complexes on OMM proteins, and not just free

ubiquitin, has suggested that nonspecific anchoring of Parkin to phospho-

ubiquitinated OMM proteins is a mechanism for its PINK1-mediated recruitment

to mitochondria. This is consistent with the idea that Parkin binding to phospho-

ubiquitin can accelerate Parkin-mediated OMM protein ubiquitination as a

positive feedback amplification loop. However, it is unlikely that the primary

event for Parkin recruitment relies on pre-existing ubiquitinated OMM proteins1.

Two candidates that may have a crucial role in the recruitment of Parkin are

mitofusins (Mfn1 and 2). These two mitochondrial outermembrane fusion proteins

are Parkin ubiquitination substrates. Because mitophagy is stimulated by Parkin-

mediated ubiquitination of mitochondrial proteins, one possibility is that Mfn1

and/or Mfn2 are mediators of the PINK1-Parkin signaling pathway. Although

ubiquitination can explain a role of mitofusins downstream Parkin, the upstream

signaling has long remained unknown. In order to evaluate if and which

mitofusins are involved in this process, human embryonic kidneys (HEK) cells

were transfected with tagged mitofusins and Parkin. Immunoprecipitation of

Parkin showed an association with Mfn2, but not Mfn1, and this co-

immunoprecipitation was enhanced in cells also overexpressing Parkin. (Fig 5

A,B). The oxidative phosphorylation inhibitor carbonyl cyanide 4-

(trifluoromethoxy) phenylhydrazone (FCCP) depolarizes mitochondria and

stimulates PINK1-mediated translocation of Parkin to mitochondria, thus targeting

damaged organelles for mitophagy. Consistent with a role for Mfn2 in this

process, FCCP treatment stimulated Parkin binding to endogenous fibroblast

Mfn2. Overexpression of PINK1 enhanced the effect of FCCP, instead its

inhibition mediated by RNA interference lowered it. Cardiomyocytes naturally

express Mfn2 and Parkin in large amount and, because of their high endogenous

expression of these factors, no transfection is needed.

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These cells were treated with FCCP and only in the Mfn1-null cells Parkin

translocation was stimulated. In Mfn2 null cardiomyocytes Parkin was not

distributed in punctae near mitochondrial-rich perinuclear region but remained

diffusely cytosolic (Fig 5C), demonstrating a requirement for endogenous Mfn2 to

promote Parkin localization to depolarized mitochondria. Parkin recruitment

appears to be a unique property of Mfn2. It is also known that Mfn2 is a Parkin

ubiquitination substrate. This might require PINK1-stimulated association of

Parkin and Mfn2 with mitochondria. A complete concordance of ubiquitination

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Fig 5: Interaction of Mfn2 with Parkin in a PINK1-dependent manner and its requirement forParkin translocation to depolarized mitochondria. (A and B) Fibroblasts were transfected with Flag-Mfn1 (A) or Mfn2 (B), PINK1, and/or hemagglutinin (HA) Parkin; immunoprecipitated (IP) with anti-Flag; and immunoblotted (IB). Right-hand gels show IB ofinput homogenates. (C) Subcellular Parkin redistribution (green) induced by mitochondrial depolarization with FCCP in wild-type, Mfn1-deficient, and Mfn2-deficient mouse cardiomyocytes. KO, knockout; DAPI, 4 ,6-diamidino- 2-phenylindole.

with PINK1-stimulated Mfn2 binding to Parkin in HEK cells and with FCCP-

stimulated binding of endogenous cardiac Mfn2 to Parkin in isolated perfuse

mouse hearts has been observed, demonstrating the importance of PINK1-Mfn2-

Parkin interactions on ubiquitination. Conversely, Mfn2 (but not Mfn1) gene

ablation in cardiomyocytes decreased mitochondrial ubiquitination stimulated by

mitochondrial depolarization, following the same pattern seen for Parkin

translocation. Moreover, the mitophagic response, measured as punctal

accumulation of the mitophagy adaptor protein p62 [also called sequestosome 1

(SQSTM1)], was impaired in FCCP-treated Mfn2- deficient cardiac myocytes.

Together, these data support a model in which Mfn2 functions as a receptor to

which cytosolic Parkin binds on depolarized mitochondria, provoking

ubiquitination of mitochondrial proteins that target the organelle for autophagic

elimination. Enhancement of Mfn2- Parkin association by PINK1 also reveals

Mfn2 as a substrate for PINK1 phosphorylation. Indeed, the use of Phos-tag gels

and the mobility shifts, indicate that Mfn2 might be phosphorylated at one or

more sites (Fig 6A). Also phosphoserine immunoreactivity of immunoprecipitated

Mfn2 is increased in cells overexpressing PINK1(Fig 6B). In order to evaluate if

PINK1-mediated phosphorylation of Mfn2 is required for Mfn2-Parkin

interaction, amino acid residues Lys 219, Asp 362 and Asp 384 were mutated in

Alanine in order to inactivate PINK1 catalytic domain. These mutants failed to

promote Mfn2-Parkin binding or to induce the characteristic change in Mfn2

mobility in Phos-tag gels (Fig 6C). To establish that Mfn2 is a substrate of PINK1

and the mitochondrial binding partner for Parkin, the phosphorylation sites of

Mfn2 were mapped and then functional consequences were examined.

Bioinformatics analysis identified three highly conserved potential Mfn2

phosphorylation sites: Thr 111 (T111), Ser 442 (S442), and Tyr 448 (Y448) .

Since PINK1 is a serine threonine kinase Mfn2 T111 and S442 were mutated to

alanine, in order to prevent their phosphorylation. Both Mfn2 mutations decreased

PINK1-stimulated Mfn2-Parkin binding, whereas simultaneous mutation of both

residues (T111A/S442A) completely abrogated PINK1- stimulated interaction

between Mfn2 and Parkin (Fig 6E).

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16

Fig 6: PINK1 phosphorylation of Mfn2 T111 and S442 dictates Parkin binding. (A) Mfn2electrophoretic mobility shifts (arrowheads) induced by PINK1 in SDS–polyacrylamide gelelectrophoresis (top) and Phos-Tag (bottom) gels. Exploded view of lanes 4 and 5 is shownto the right. (B) PINK1-mediated Mfn2 phosphorylation (arrowheads) by anti-phosphoserine IB. (C) (Left) Mfn2-Parkin co-immunoprecipitation study with functionaland kinase-defective (KD) PINK1; (right) Mfn2 Phos-Tag phosphorylation study withfunctional and KD PINK1. (D) Effects of Mfn2 T111A and S442A mutations on PINK-stimulated Mfn2-Parkin binding. WT, wild type. (E) Abrogation of PINK1-stimulated Mfn2-Parkin binding by Mfn2 T111A/S442A mutation and induction of PINK1-independentMfn2-Parkin binding by Mfn2 T111E/S442E mutation.

In agreement with these loss-of-function data, Mfn2 mutations that mimic PINK1

phosphorylation of Mfn2 (T111E/S442E) conferred PINK1-independent binding

activity to Parkin4. Collectively, these studies reveal a potential mechanism by

which Mfn2 orchestrates the PINK1-Parkin mitochondrial quality control

apparatus. In summary, stabilized PINK1 located on the OMM phosphorylates

Mfn2, transforming it into a receptor to which Parkin can bind, thereby bringing it

into physical proximity to its many mitochondrial ubiquitination substrates.

PINK1-mediated phosphorylation of the free ubiquitin activates Parkin E3

ubiquitin ligase activity, and its phosphorylation of ubiquitinated OMM proteins

amplifies mitophagy signaling (Fig 7).

A mechanistic link between PINK1, Mfn2, and Parkin can explain how the

PINK1–Parkin pathway simultaneously initiates mitophagy and shuts down

fusion, as required to preclude mitochondrial contagion. It is possible that PINK1-

mediated phosphorylation may instantaneously convert Mfn2 from a fusion

protein to a Parkin-binding protein. This mechanism of modulating mitochondrial

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Fig 7: Molecular events leading to selective mitophagy of dysfunctionalmitochondria.

fusion in mitophagy would have the advantage of being more rapid and direct

than Parkin-mediated Mfn2 ubiquitination, extraction, and proteasomal

degradation. Indeed, the importance of Parkin-mediated ubiquitination of specific

proteins, leading to their selective elimination from soon-to-be autophagocytized

organelles, is unclear. Parkin-mediated ubiquitination of Mfn1 and Mfn2 might

deplete these fusion-promoting proteins by targeting them for proteasomal

degradation, thus interrupting mitochondrial fusion and placing the organelle in

quarantine until it can be mitophagically eliminated. Parkin does not selectively

ubiquitinate profusion proteins; it also ubiquitinates the profission protein, Drp1,

which would move the fission/fusion equilibrium in the opposite way, toward

mitochondrial fusion. Indeed, Parkin promiscuously ubiquitinates ≥100 OMM

proteins, essentially painting the organelle with a coat of ubiquitin that attracts

autophagosomes. Thus, during mitophagy at least, Parkin-mediated OMM

ubiquitination does not seem to fine tune OMM protein expression of organelles

that, in any case, are shortly headed to the graveyard. Furthermore, there is little

delay between Parkin localization to mitochondria and their engulfment by

autophagosomes. Live cell microscopy of cultured cells revealed that Parkin

localization, focal protein ubiquitination, and regional mitochondrial

fragmentation with autophagosomal engulfment all occur within minutes of

mitochondrial injury. This provides little time for selective extraction and

proteasomal degradation of mitofusins1.

5. Mitochondrial Dynamism - Mitophagy Crosstalk in the Heart

To investigate the functional impact of mitochondrial quality control mechanisms

on cardiac pathophysiology, genetic ablation of Drp1 was performed in mouse

cardiomyocyte to interrupt mitochondrial fission. A combination of Drp 1 fl/fl

alleles with myh6 promoter-driven modified estrogen receptor (MER)-Cre

transgene was used to generate mice that could be grown to adulthood prior to

cardiac-specific Drp1 deletion using tamoxifen. The same was performed for

Mfn1 and Mfn2 genes (Fig 8A). No tamoxifen effects were found on cardiac

function during the 8 weeks of the studies. Conditional deletion of both Drp1 and

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Mfn1/Mfn2 induced progressive left ventricular enlargement and a decline in

ejection performance. Six to seven weeks after Drp1 deletion full-blown heart

failure was evident (Fig 8B), but the decline in ejection performance was less

severe in Mfn1/Mfn2 double knockout (DKO) hearts (Fig 8C).

Indeed, Drp1 deficiency induced ventricular wall thinning and chamber dilatation

and also cardiac mass (heart weight corrected for body weight) increased by only

12% 6 weeks after Drp1 deletion (Fig 8B). This is called dilated cardiomyopathy.

By comparison, combined Mfn1/Mfn2 deletion provoked eccentric ventricular

remodeling with ventricular wall thickening and an increase in cardiac mass 6

weeks after Mfn1/Mfn2 ablation (Fig 8C). This is eccentric hypertrophy. Drp 1

deletion, and the ensuring fission inhibition, should have an impact on

mitochondrial morphology, causing an enlargment of the organelles, while

Mitofusins deletion, and fusion inhibition, should lead to smaller and fragmented

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B C

Fig 8: (A) Schematic of tamoxifen-inducible myh6-Cre-mediated Drp1 orMfn1/Mfn2 gene deletion.(B and C) Cardiac phenotypes induced by Drp1 orMfn1/Mfn2 gene deletion.(Upper left) Four-chamber view of hearts. (Upperright) Ratio of left ventricular (LV) end-diastolic radius (r) to wall thickness (h).(Lower left) Serial M-mode echocardiographic analyses. (Lower right)Quantitative group data showing % change of LV end-diastolic dimension (EDD)and fractional shortening (FS).

mitochondria. Mitochondria observed after Drp1 ablation had a relatively normal

appearance, suggesting a link between this structure and myocardial thinning.

Because the sine qua non of dilated cardiomyopathy is cardiomyocyte loss with

replacement fibrosis, examination of Drp1- and Mfn1/Mfn2-deficient hearts for

cardiomyocyte dropout was necessary. Strikingly, Drp1 deficiency provoked a

progressive increase in myocardial fibrosis that involved the 40% of the

myocardium by 6 weeks while myocardial fibrosis was not a feature of

Mfn1/Mfn2 null hearts. Replacement fibrosis is linked to cardiomyocyte death.

Accordingly, TUNELpositive cardiomyocytes were increased in Drp1 null hearts,

but this does not reliably differentiate between apoptosis and necrosis. Evans blue

staining and visualization of focal complement complex activation in Drp1-

deficient myocardium confirmed cardiomyocyte necrosis while Mfn1/Mfn2 DKO

myocardium showed no cardiomyocyte death, consistent with no myocardial

fibrosis. Mitophagic culling of damaged mitochondria is mediated in part by

mitochondrial localization of the autophagy chaperone protein p62/SQSTM1, and

binding to autophagosomal microtubule associated protein 1 light chain 3 (LC3).

Mitochondrial p62 increased up to 9-fold in Drp1 null hearts, but was unchanged

in Mfn1/Mfn2 DKO hearts. Likewise, mitochondrial LC3-II increased in Drp1

null hearts, but decreased in Mfn1/ Mfn2 DKO hearts. Levels of fibroblast growth

factor (Fgf) 21, a marker of mitochondria insufficiency, increased more rapidly

and to a greater extent after Drp1 ablation than after Mfn1/Mfn2 deletion. Thus,

myocardial autophagosome-mitochondria interactions increased after interrupting

mitochondrial fission, but not after suppressing mitochondrial fusion. By contrast,

levels of two proteases involved in the mitochondrial unfolded protein response

were normal in Drp1 null hearts, but were increased in Mfn1/Mfn2 DKO hearts.

These results raised the possibility of reciprocal defects in mitochondrial quality

control. Because of the impossibility to directly measure mitochondrial mitophagy

in vivo, an in vitro system, wherein Drp1 of Mfn1 and Mfn2 were conditionally

manipulated using adenoviral-encoded Cre and their respective floxed genes, was

developed in cultured murine embryonic fibroblasts. Confocal fluorescence

studies of Drp1- deficient MEFs stained with MitoTracker Green showed

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mitochondrial hyperfusion with a loss of mitochondrial content within 2 days.

Even the electrochemical potential (Δψm) was not adversely impacted by

mitochondrial elongation induced by Drp1 deletion in either MEFs, or in Drp1

null hearts. Normal respiratory function and absence of increased ROS production

in isolated Drp1-deficient cardiac mitochondria further support a high fitness level

in the retained mitochondria. The same approach was used with Mfn1 and Mfn2

genes. Conditional ablation of Mfn1 and Mfn2 decreased mitochondrial aspect

ratio, reflecting the so-called fragmentation induced by unopposed fission, with no

change in mitochondrial content. Similar to Mfn1/Mfn2 DKO hearts, cell

membrane integrity was not impaired by conditional ablation of Mfn1 and Mfn2

in MEFs. Nevertheless, dissipation of mitochondrial electrochemical potential was

evident in the fragmented mitochondria of MEFs lacking both mitofusins and the

same was present in hearts with also a severe respiratory impairment and ROS

production. Taken together, the results show that conditional interruption of

mitochondrial fission or fusion has parallel effects in MEFs and mouse hearts:

mitochondrial fitness is maintained after ablating Drp1, but is severely

compromised by combined mitofusin deficiency. This is due to the fact that Drp1

ablation provokes the absence of asymmetrical fission: healty daughter

mitochondria are not generated to renew the mitochondrial pool and fully

depolarized daughter mitochondria are not generated to be culled through

mitophagy. Fusion can still occur, leading to a cell wide mitochondrial

dysfunction. It is a matter of time for the depolarized mitochondria to reach the

threshold for mitophagy activation, provoking a strong reduction in mitochondrial

content. When instead fusion is inhibited, Mfn2 absence is able to impair

mitophagy signaling independent of mitochondrial fusion, for its role in PINK1-

Parkin mediated mitophagy pathway. This prevents the normal mitochondrial

culling. Moreover, the organelle fusion renewal mechanism for reintroduction of

the healty daughter mitochondria produced by asymmetric fission cannot occur in

the absence of mitofusins (Fig 9). All these evidences show the implication of

fission and fusion proteins in quality control3.

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6. Conclusions

Molecular crosstalk between mitochondrial dynamism and mitophagy effectors

leads to a complex and effective mechanism that exclusively eliminate damaged

mitochondria, thus ensuring fitness of the overall cellular mitochondrial

collective. The dichotomous role of Mfn2 as a mitochondrial fission factor or as a

Parkin receptor suggests that mitochondrial fusion and mitophagy are mutually

exclusive, thus protecting healty mitochondria from fusion-mediated

contamination by dysfunctional organelles.

PINK1-Parkin mediated mitophagy signaling pathway, with its function in

mitochondrial fitness, has been proven to have a crucial role in the correct

functioning of the heart, but it is involved and deregulated also in other

diseases, including degenerative pathologies such as Parkison's disease,

thus uncovering multiple opportunities for therapeutic intervention1.

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Fig 9: Schematic representation of the role of Drp1 andMfn1/2 ablation in cardiac physiophatology.

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