Mitochondrial Dynamism and Cardiac Fate

Transcription

1 Circulation Journal Official Journal of the Japanese Circulation Society Advance Publication by-j-stage REVIEW Mitochondrial Dynamism and Cardiac Fate A Personal Perspective Gerald W. Dorn II, MD Defects in mitochondrial biogenesis are well known to contribute to cardiac dysfunction. By contrast, mechanistic details of essential homeostatic mechanisms that maintain mitochondrial health in the heart are only recently being uncovered, and the pathological potential of these processes is largely hypothetical. I will review the role of mitochondrial dynamics, focusing on cyclic organelle fission and fusion, in normal and diseased hearts. Special attention is given to recent insights into the non-canonical functioning of the mitofusin 2 (Mfn2) outer mitochondrial membrane fusion protein as a regulator of sarcoplasmic-reticular calcium crosstalk and a critical determinant of mitophagic culling of damaged mitochondria. Because mitochondrial fusion in normal adult cardiomyocytes occurs so slowly and infrequently, I postulate that the major function of Mfn2 in the heart may not be to redundantly promote mitochondrial fusion with Mfn1, but to centrally orchestrate mitochondrial quality control. Key Words: Mfn2; Mitochondrial fission; Mitochondrial fusion; Mitophagy; Parkin Mitochondria are cellular powerhouses, but are also gateways to apoptosis. 1,2 Accordingly, multiple research studies over the past decade have defined context-specific molecular effectors of mitochondrial-mediated programmed cardiomyocyte death. My laboratory contributed to this area by developing the first in vivo mouse model to prove that cardiomyocyte apoptosis is sufficient to induce heart failure (HF). 3 Subsequently, 2 Bcl-2 family mitochondrial death proteins that are the genetically programmed effectors of cardiomyocyte apoptosis (Nix in cardiac hypertrophy and Bnip3 in cardiac ischemia) were identified. 4 7 These 2 transcriptionally regulated proteins localize primarily to the outer mitochondrial membranes (OMM) where they facilitate Bax/Bak-mediated OMM permeabilization, cytochrome c release, and activation of caspase-dependent apoptosis signaling. 8 Recently, added and unexpected functions of these mitochondrial death proteins were uncovered. Nix localizes (in addition to mitochondria) to endoplasmic/sarcoplasmic reticulum (ER/SR) where it regulates SR-mitochondrial calcium crosstalk that opens mitochondrial permeability transition pores (MPTPs), 9,10 inducing a non-apoptotic form of programmed cell death, designated programmed necrosis. 11,12 Nix also plays an important role in mitochondrial quality control, targeting dysfunctional mitochondria for mitophagic elimination. 13,14 Thus, pro-apoptotic mitochondrial Bcl2-family proteins are multi-taskers that also modulate programmed necrosis and mitophagy. It is at the OMM where mitochondrial and extra-mitochondrial factors interface and interact to determine mitochondrial integrity, and hence cell fate. The normal cytosolic/organelle OMM barrier maintains a protective internal milieu essential to the functioning of the mitochondrial matrix electron transport pathway enzymes that generate ATP. The OMM also protects the cell by compartmentalizing soluble pro-apoptotic mitochondrial factors, such as cytochrome c. Accordingly, the OMM barrier is stabilized by anti-apoptotic Bcl2-factors such as Bcl-xL, and destabilized by pro-apoptotic Bcl2-proteins such as Bax, Bak, Nix, and Bnip3. Loss of OMM integrity (ie, OMM permeabilization ) is the initial step in mitochondrial pathway apoptosis. If mitochondria are gateways to cell death, then these gateways are constantly changing size, shape, and location via organelle transport, fission, and fusion. This is mitochondrial dynamics. The molecular mechanisms of mitochondrial fission and fusion are understood and have recently been reviewed in depth. 15,16 The OMM is also the physical and biochemical nexus for mitochondrial fusion/fission. Mitochondrial fusion mediated by mitofusin (Mfn) 1 and Mfn2, and fission mediated by dynamin-related protein 1 (Drp1), occur with far less frequency in normal adult cardiac myocytes than in the more commonly studied fibroblasts, 17 but recent work has uncovered essential roles for Mfn-mediated mitochondrial fusion and mitochondria-sr tethering in cardiac homeostasis Conversely, pathologic mitochondrial fragmentation is observed in cardiomyocyte apoptosis and HF, 21 although it is unclear if mitochondrial fission is an important effector of, or just a collateral event in, apoptosis. 22,23 Because of the need for large amounts of ATP to fuel excitation-contraction coupling (~6 kg/day), hearts have the greatest density of mitochondria of any organ. It is axiomatic that mitochondrial health is essential to general health, 24 and past research has focused on the cardiac consequences of altered Received April 4, 2013; accepted April 5, 2013; released online April 25, 2013 Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA Mailing address: Gerald W. Dorn II, MD, Philip and Sima K Needleman Professor, Washington University Center for Pharmacogenomics, 660 S Euclid Ave, Campus Box 8220, St. Louis, MO 63110, USA. gdorn@dom.wustl.edu ISSN doi: /circj.CJ All rights are reserved to the Japanese Circulation Society. For permissions, please cj@j-circ.or.jp

2 DORN II GW Figure 1. The mitochondrial fission/fusion cycle and its key players. Fusion (blue) is mediated by OMM mitofusins (Mfn) and IMM optic atrophy 1 (Opa1). In non-cardiomyocytes, increased fusion produces mitochondrial networks with increased interorganelle connectivity. Fission (red) is mediated by recruitment of cytosolic Drp1 to the OMM and its subsequent oligomerization and constriction at fission sites. Drp1 translocation to mitochondria can be pharmacologically inhibited with mitochondrial division inhibitor (Mdivi) compounds. Mitochondrial fragmentation is essential for cell mitosis and mitophagic elimination of senescent organelles, and is increased in apoptosis. IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. mitochondrial metabolism and biogenesis. Numerous recent investigations have revealed the mitochondria to be effectors of programmed cardiomyocyte death (apoptosis or programmed necrosis) and sources of damaging reactive oxygen species (ROS). By comparison, studies of mitochondrial dynamism in the heart have lagged, possibly because a role for mitochondrial fusion in cardiac health or disease has tended either to be ignored (it is difficult to measure something you can t directly observe) or dismissed (it is easy to disregard something you can t measure). Given robust myocardial expression of Mfn1 and Mfn2, which have overlapping functions to promote mitochondrial fusion, it may also have been assumed that dysfunction or deficiency of cardiac Mfn1 would be compensated for by functionally redundant Mfn2, and vice versa. These ideas proved incorrect on 2 levels; first, unique functions of Mfn2, such as SR-mitochondrion tethering and involvement in mitophagic mitochondrial pruning (discussed later) are not shared by Mfn1 and second, the ability of one of the Mfn to promote mitochondrial fusion by interacting either homotypically with itself (ie, Mfn1-Mfn1 or Mfn2-Mfn2) or heterotypically with its counterpart (ie, Mfn1-Mfn2) creates a situation in which dominant negative mutants of either Mfn protein have the opportunity to suppress the function of both. I will provide an overview of recent advances and evolving concepts in this area. Mitochondrial Fragmentation and Fission Mitochondrial fragmentation is the term used to describe abnormally small mitochondria, such as those that are frequently observed in apoptotic cells. 25,26 Often it is assumed that the presence of mitochondrial fragments is evidence of increased mitochondrial fission. However, small mitochondria can be produced by any imbalance that favors fragmentation over the normal steady state of mitochondrial fusion and fission (ie, either by an increase in the rate of organelle fission or a decrease in the rate of organelle fusion) (Figure 1). It is notable that mitochondrial fragmentation induced by suppressing mitochondrial fusion does not appear sufficient to cause cardiomyocyte apoptosis (discussed in detail later), 19 suggesting either that the association between mitochondrial fragmentation and apoptosis is correlative (ie, true, true, and causally unrelated) 27,28 or that organelle fragmentation resulting from forced mitochondrial fission has different effects on the proclivity to cell death than fragmentation accruing from impaired organelle fusion. The predominant molecular mediator of mitochondrial fission is Drp1 (aka Dlp1), one of several evolutionarily conserved GTPases that modulate mitochondrial dynamics. Under normal conditions, Drp1 is approximately 97% cytoplasmic, but under situations leading to organelle fission it translocates to the OMM, self-assembles into oligomers at sites of future mitochondrial division, and undergoes a GTP-dependent conformational change that constricts and then fragments the organelle. 29 Depending on the cellular context, Drp1-mediated mitochondrial fission can lead to mitophagic elimination of a dysfunctional daughter organelle, partitioning of the cellular mitochondrial network in preparation for cell division, or release of mitochondrial-derived apoptogens that induce programmed cell death. The importance of Drp1-mediated mitochondrial fission to cell and tissue health is underscored by a recently described, naturally occurring de novo human Drp1 mutation that alters its ability to oligomerize at mitochondria and causes a lethal syndrome of microcephaly with abnormal retinal development. 30 Neurological phenotypes are likewise induced by experimental germline Drp1 gene ablation. 31 Because Drp1 induces the mitochondrial fission that is frequently observed prior to or during apoptosis, 32 it has been suggested that pharmacologic inhibition of Drp1 with Mdivi (Mitochondrial division inhibitor) compounds might reduce programmed cell death after tissue ischemia-reperfusion injury (eg, myocardial or cerebral infarction). 33,34 To date only a

3 Mitochondrial Dynamics in the Heart Figure 2. Sequence of events leading to mitochondrial fusion. The outer membranes (OMM) of separate mitochondria are first tethered by intermolecular interactions between Mfn1 (yellow) and/ or Mfn2 (green). The 2 OMMs then undergo Mfn GTPase-dependent fusion to form a single organelle with a common intermembrane space, but separate matrix compartments. Finally, the 2 inner membranes (IMM) undergo Opa1 (orange) GTPase-dependent fusion to complete the production of a single daughter organelle with the components of both parents. MARF, Drosophila mitofusin; Mfn, mitofusin. few studies have tested this approach, but early results appear promising: Ong et al pretreated mice with Mdivi prior to experimental cardiac ischemia and observed decreased mitochondrial fragmentation and (for low-dose, but not higher dose Mdivi) approximately 50% decrease in the size of resulting myocardial infarctions. 35 Cardiomyocyte apoptosis was not measured in the Mdivi-treated hearts, but Mdivi treatment decreased cardiomyocyte death after in vitro simulated ischemia in the same study. More recently, a similar protective effect of Mdivi was described for HF induced by surgical pressure overloading in mice, with reduced caspase activation and decreased LC3 (an autophagy marker) and p62/sqstm1 (a mitophagy chaperone) staining after Mdivi treatment. 36 These results support a pathophysiological role for Drp1 as a promoter of both cardiomyocyte apoptosis and mitophagy after ischemic injury, which seems to confirm the notions originated by Nunnari et al. 32,37 Mitochondrial fragmentation/fission is frequently observed in ischemic neurons within the penumbra of cerebral strokes, where it has been mechanistically linked to programmed neuronal death. 16,38 Accordingly, the efficacy of Drp1 inhibition with Mdivi has also been assessed in experimental stroke. Grohm et al pretreated mice with Mdivi prior to transient focal ischemia and observed a 30 50% reduction in infarct volume. 39 In vivo mechanistic studies were not performed, but the authors observed that in vitro Mdivi treatment decreased stressinduced mitochondrial fragmentation and depolarization in cultured neurons. The only other in vivo Mdivi brain ischemia study reported that low-dose (1.2 mg/kg), but not higher dose (2.4 mg/kg), intravenous Mdivi administered prior to transient cerebral ischemia in rats modestly decreased 2 markers of apoptosis: peri-infarct neuronal TUNEL positivity and cytochrome c staining (infarct size was not reported). 40 It remains unexplained why low-dose, but not higher dose, Mdivi is protective in ischemic hearts and brains. 35,40 Contrasting with the benefits that accrue from pharmacologic Drp1 inhibition with Mdivi, conditional Drp1 gene ablation in cerebellar Purkinje cells has uncovered an essential requirement for Drp1 in tissue health: 41 in the absence of functional Drp1, Purkinje cell mitochondria enlarged and developed respiratory dysfunction with an increased sensitivity to ROS that led to neuronal death. These observations seem to be consistent with a protective function for Drp1 in normal tissues, wherein mitochondrial fragmentation limits organelle injury and toxicity. Accordingly, when Drp1-mediated fission is impaired, the mitochondria are larger and more interconnected. This increased mitochondrial connectivity facilitates rapid transmission of deleterious cell signals, such as ROS and calcium waves, throughout the cell and increases the overall level of cellular damage accruing from a given pathologic stimulus. 42,43 Drp1 is therefore essential to normal mitochondrial health, but can be co-opted by apoptosis pathways after ischemic injury. Other possible considerations that may help explain the disparity between the results of pharmacologic Drp1 inhibition and genetic Drp1 ablation include that Drp1 has the potential to modify post-ischemic cell death via mechanisms distinct from its effects on mitochondrial fragmentation, or the possibility that the cytoprotective effects of systemically administered Mdivi are not entirely explained by its local inhibition of Drp1 in ischemic tissues. Evidence for and Against Mitochondrial Fusion in the Heart Mitochondrial fusion has been directly observed in almost all cell types, except cardiomyocytes. 17 In cultured fibroblasts, mitochondrial fusion is a 3-step process requiring, in order, OMM tethering, OMM fusion, and inner membrane (IMM) fusion. Organelle tethering and OMM fusion are accomplished by Mfn GTPases constitutively localized to the OMM. IMM fusion requires a different GTPase, Opa1, that also mediates cristae remodeling 44 (Figure 2). As with mitochondrial fission, the biological importance of mitochondrial fusion is revealed by the chronic neurodegenerative diseases that are induced by naturally occurring loss-of-function mutations: Charcot-Marie- Tooth syndrome is caused by Mfn2 mutations and dominant optic atrophy is caused by Opa1 mutations (see reference 15). Because heart disease is not commonly observed in these neurological syndromes, mitochondrial fusion and fission have been considered by some to be irrelevant to normal heart function. Until recently, the widely held notion was that cardiomyocytes mitochondria are essentially static organelles trapped within the highly organized subcellular architecture of this unique cell type.

4 DORN II GW When my group first entered this area of investigation, we asked the question If mitochondrial fusion and fission are so important in other cell types, why should cardiomyocytes (in which mitochondria are 30% of cell mass and mitochondrial fusion and fission proteins are abundantly expressed) be the only exception? We initially studied Drosophila, which expresses a single mitofusin (dmfn, aka MARF) in the heart tube, to determine the functional cardiac consequences of interrupting mitochondrial fusion in cardiac myocytes. Studying fruit flies also permitted us to use genetically-encoded mitochondrial-targeted green fluorescent protein to interrogate cardiomyocyte mitochondrial morphometrics in living heart tubes. 18 Cardiomyocyte-specific RNAi-mediated suppression of dmfn induced both mitochondrial fragmentation and dilated cardiomyopathy (DCM), and the mitochondrial and cardiac abnormalities provoked by dmfn/marf deficiency were rescued by cardiac-specific expression of either human Mfn1 or Mfn2. These early results proved that mitochondrial fusion occurs in invertebrate hearts (because, in its absence, we observed mitochondrial fragmentation) and demonstrated that human Mfn1 and Mfn2 can each substitute functionally to promote cardiomyocyte fusion in dmfn-deficient fruit fly hearts. To determine if an intact mitochondrial fusion apparatus is also essential to the normal functioning of mammalian hearts, we performed an analogous and more detailed experiment in mice. 19 Cardiac-specific combined ablation of Mfn1 and Mfn2 using Nkx2.5-Cre proved to be embryonic lethal. Therefore, we used tamoxifen-dependent combined Mfn1/Mfn2 ablation in cardiomyocytes (myh6-mer-cre-mer) to evaluate the consequences of disrupting mitochondrial fusion in the adult mouse heart. Combined Mfn1/Mfn2 ablation induced progressive cardiac dilation and mitochondrial fragmentation that was lethal after approximately 8 weeks. Measuring the rate of mitochondrial fragmentation under conditions of unopposed fission indicated that the in vivo mouse cardiomyocyte mitochondrial fusion/fission cycle takes approximately 15 days (compared with just a few minutes in cultured fibroblasts), providing an explanation for why mitochondrial fusion is not observed in short-term studies of isolated adult cardiac myocytes. Together, the phenotype produced by cardiac-specific suppression of Mfn/MARF in Drosophila and the data accruing from conditional combined ablation of Mfn1 and Mfn2 in mouse hearts prove that, although mitochondrial fusion occurs very slowly in cardiac myocytes, fusion proteins are evolutionarily conserved and their presence is essential for the maintenance of cardiac homeostasis. Because previous studies had linked mitochondrial fragmentation and apoptosis, 25,26 we were surprised that programmed cardiomyocyte death is not a feature of the cardiomyopathy caused by suppressing mitochondrial fusion in the fruit fly or mouse heart. Functional dissociation of mitochondrial fragmentation and cardiomyocyte apoptosis in hearts with defective mitochondrial fusion reveals that the roles of cardiac mitochondrial fusion and fission factors are more complicated than previously recognized. As indicated later, evidence is accumulating that the mitochondrial proteins known (and named) primarily for their roles in organelle fusion and fission have many other important functions in the heart and elsewhere. Heart Diseases Caused by Defective Mitochondrial Fusion Cardiac disease is not a finding of Charcot-Marie-Tooth syndrome caused by loss-of-function Mfn2 mutations, or of dominant optic atrophy caused by loss-of-function Opa1 mutations. 15 Thus, there has been relatively little interest in mitochondrial fusion factors as causal or contributory factors in human heart disease. This perspective is evolving, however, partly because of recognition that mutations within different structural domains of Mfn2 can induce organ dysfunction in a tissue-specific manner. Indeed, an increasing number of natural and experimental cardiomyopathies are being attributed to an imbalance in mitochondrial fusion/fission. The DCM of the Python mouse, created by ENU mutagenesis, is caused by a mutation in the mitochondrial fission factor Drp1/Dnm1l, 46 and a form of heritable bovine DCM is caused by a mutation in the fusion factor Opa3. 47,48 Two recent independent reports describe cardiomyopathy and mitochondrial dysfunction in mice having only one functional Opa1 allele (Opa +/ ). 49,50 Based on this accumulating information, we postulate that rare loss-of-function mutations of mitochondrial fission and fusion proteins may yet be uncovered as one of the causes of human heritable cardiomyopathy that is not explained by the more common, classical mutations of sarcomeric and other genes. Mfn2 Mutations and the Heart Our laboratory has utilized Drosophila not only to understand the biology of cardiomyocyte mitochondrial fusion/fission, but also for initial in-heart studies of mutant human fusion proteins. Thus, we use Drosophila similar to many laboratories using neonatal rat cardiac myocytes (NRCM) as a rapid and inexpensive system for characterizing cardiomyocyte-autonomous effects of factors we have predetermined to have functional biological significance in non-myocytes. Advantages of the Drosophila platform over NRCM include (1) fruit flies have a working heart tube that shortens (contracts) and remodels over time in a manner that recapitulates mammalian cardiomyopathy; (2) genetic manipulation for cardiomyocyte-specific overexpression, suppression, or replacement of endogenous fruit fly gene products with their wild-type or mutant mammalian counterparts is straightforward; (3) genetically-encoded markers have been developed for in-situ studies of mitochondrial and SR structure and cardiomyocyte calcium signaling; (4) HF symptoms can be assessed independently of cardiac contractile function using a negative geotaxis fruit fly stress test, 28 and finally, (5) results obtained in Drosophila heart tubes are rapidly translatable to in vivo mammalian hearts ,45 We recently used the Drosophila platform to assess the pathologic potential of the rare Mfn2 R400Q mutant (rs ) that was detected in 1 individual by the 1,000 Genomes Project (no clinical data available). We bioinformatically determined that Mfn2 400Q mutated the first heptad repeat (ie, HR1) region of Mfn2 and was predicted to be poorly tolerated (ie, pathologic). By comparing the effects of wild-type Mfn2 R400 and mutant Mfn2 Q400 on mitochondrial morphology in cultured mammalian cells and Drosophila heart tubes, we not only established that the mutant is defective in inducing mitochondrial fusion, but we further showed that it is a potent dominant inhibitor of mitochondrial fusion mediated by wildtype Mfn1 and/or Mfn2. 45 When expressed in Drosophila heart tubes, mutant Mfn2 400Q provoked mitochondrial fragmentation, DCM, and HF, closely recapitulating the phenotypes induced by cardiomyocyte-specific dmfn/marf deficiency. 18 Because of its dominant negative activity, we postulated that the Mfn2 400Q mutation may be sufficient to cause human heart disease in heterozygous carriers. A related human Mfn2 mutation (M393I) within the HR1 domain is also defective in

5 Mitochondrial Dynamics in the Heart promoting mitochondrial fusion, but does not exhibit dominant inhibition of normal Mfn1 or Mfn2; it caused only a mild cardiomyopathy in Drosophila. We anticipate that Mfn2 M393I is unlikely to cause human disease, except in the almost impossibly rare circumstance where it may occur as a homozygous mutation. Mfn2 in Mitochondrial-SR Calcium Crosstalk Our studies of Nix and Bnip3 in programmed cardiomyocyte death and HF had increased our awareness that both proteins induce cell death through 2 distinct mechanisms: OMM permeabilization that initiates caspase-dependent apoptosis, 4 6 and mitochondrial depolarization that leads to programmed necrosis. Mitochondrial depolarization suggested involvement of the MPTP. 9 Indeed, we found that Nix alters the in vivo sensitivity of the MPTP by localizing to the SR of the cardiomyocyte and facilitating export of calcium to adjacent mitochondria; exclusive localization of Nix to the SR (via forced expression of an SR-targeted mutant) increased the SR calcium content, whereas Nix removal from the SR (by gene ablation or expression of a mutant Nix that forced localization to mitochondria) decreased the SR calcium content. These Nix effects on SR calcium content explain its ability to induce mitochondrial depolarization: Nix-mediated increases in SR calcium enhance delivery of SR calcium to the mitochondria, which induces the mitochondrial permeability transition and results in mitochondrial depolarization. The consequence for the cardiomyocyte is non-apoptotic programmed death via programmed necrosis. 9,10,51 Transcriptionally regulated Nix (or in ischemia, Bnip3) therefore simultaneously orchestrates 2 mechanistically distinct pathways that lead to stress-dependent cardiomyocyte death: mitochondrial-localized Nix induces Bax/ Bak-dependent OMM permeabilization and caspase-dependent apoptosis, whereas SR-localized Nix induces MPTP opening, leading to mitochondrial depolarization and death by programmed necrosis. The broad relevance of these parallel death pathways was demonstrated in studies of similar design in which we showed that Nix-mediated programmed pancreatic β-cell death is a cause of murine diabetes. 52,53 The underlying assumption of a MPTP-dependent mechanism for Nix-mediated programmed necrosis is that the amount and concentration of calcium released from cardiac SR/ER is sufficient to open the MPTP. However, both mitochondrial calcium uptake and MPTP opening requires a high concentration of calcium (0.1 1 mmol/l) that is difficult to achieve in cardiomyocytes because calcium released in boluses from the SR is rapidly dissipated into the cytoplasm. Calcium diffusion combined with this requirement for high calcium concentrations (recently confirmed by the molecular identification of a mitochondrial calcium uniporter 54 ) would seem to indicate that MPTP opening can only occur under the most extreme conditions of massive calcium release, which is clearly not the case. Rizzuto and Pozzan resolved the apparent paradox of SR-mitochondrial calcium crosstalk that can open MPTP in the face of a requirement for impossibly high concentrations of calcium by postulating the existence of calcium microdomains between the ER/SR and mitochondria. 55,56 Within such microdomains, calcium diffusion would be limited, permitting mitochondrial delivery of a concentrated calcium bolus from the SR. More recently, de Brito and Scorrano proved that the OMM fusion protein, Mfn2, uniquely tethers the ER to mitochondria in fibroblasts, establishing a molecular basis for calcium microdomains. 57 Together with our collaborators, we proved the existence of this same Mfn2 tethering function Figure 3. Mitochondrion-SR tethering by Mfn2 facilitates interorganelle calcium crosstalk and regulates mitochondrial metabolism. Intermolecular interactions between mitochondrial Mfn1 or Mfn2 and SR-localized Mfn2 physically tether the 2 organelles, creating privileged calcium microdomains through which calcium released from the SR can be taken up by mitochondria without diffusing into the cytosol. Mitochondrial sensing of SR calcium release is a mechanism for increasing ATP production in anticipation of and in proportion to increased cardiac workload. Mfn, mitofusin; SR, sarcoplasmic reticulum. between cardiomyocyte mitochondria and the SR, and demonstrated that the calcium microdomains formed by Mfn2 tethers are essential for rapid mitochondrial sensing of increased SR calcium release during the early phase of increased cardiac work (Figure 3). 20 Ablating the Mfn2 mitochondria- SR tether does not adversely affect the young adult mouse heart at rest, but the resulting distortion of the normal, privileged interorganelle calcium export and uptake delays the mitochondrial ATP response to acute stress. Others have suggested that Mfn2 is essential for calcium-stimulated opening of cardiomyocyte MPTPs, presumably also by tethering mitochondria to the SR. 58 Thus, Mfn2 appears to be a centrally positioned orchestrator of both homeostatic mitochondrial fusion and mitochondrion-sr calcium crosstalk, modulating mitochondrial ATP production according to changing physiological demand and facilitating cardiomyocyte death in response to non-lethal stress by inducing programmed necrosis. Mfn2 and Mitochondrial Quality Control Cycles of mitochondrial fission and fusion can obviously determine organelle structure and interconnectivity, but extensive mitochondrial networks are not observed in adult cardiac myocytes. Instead, cardiomyocytes mitochondria exist as individual small organelles closely packed together in large groups arranged between SR myofilaments. This subcellular architecture appears optimal for continuously supplying mitochondrial ATP to the SR for calcium cycling and for the SR contractile machinery. Cardiomyocytes also differ from other cells in that a mitochondrial fission-fusion cycle occurring over several minutes in HeLa and Cos-7 cells or cultured fibroblasts 59 takes approximately 2 weeks in in vivo adult cardiomyocytes. 19 As mitochondrial networking and network remodeling does not appear to occur in normal cardiomyocytes, and because mitochondrial fusion is infrequent in these cells, we postulate that a major function of mitochondrial fission and fusion in the heart is to determine the fate of individual organelles.

6 DORN II GW Figure 4. Mitochondrial fission and fusion proteins orchestrate mitochondrial culling. Senescent or damaged mitochondria (Top) segregate their functional (green) and dysfunctional (red) components, which are then separated into different daughter organelles (Bottom) via Drp1-mediated fission. The healthy hyperpolarized daughter mitochondrion (green) is fusion-competent and re-joins the cellular mitochondrial pool. Depolarization of the smaller, damaged daughter mitochondrion (red) stabilizes PINK1 kinase, which phosphorylates Mfn2, enabling it as the OMM receptor for translocated Parkin. Mitochondrial localization of Parkin promotes its ubiquitination of multiple OMM proteins (not shown), which targets the damaged mitochondrion for autophagosomal elimination (ie, mitophagy). OMM, outer mitochondrial membrane. One of the functions of mitochondrial fission (at least in cultured cells) is to physically separate damaged mitochondrial components within a senescent parent organelle into one of the 2 daughter organelles, then segregate the resulting dysfunctional daughter mitochondrion from functionally competent organelles and specifically target it for mitophagic elimination. 60,61 By contrast, mitochondrial fusion acts as preventative maintenance, permitting relatively normal or minimally damaged organelles to exchange DNA, metabolic enzymes, and membrane phospholipids, thus correcting small functional problems and maintaining overall mitochondrial homeostasis. 62,63 It is notable that mitochondrial fission/fusion cycles are temporally linked to mitochondrial dysfunction that is most often manifested as dissipation of the IMM electrochemical gradient ( Ψm), commonly referred to as IMM depolarization. Mitochondrial depolarization also activates mitophagy. 60,64 66 Because IMM depolarization is central to both organelle fission and mitophagy, and because it typically occurs abruptly rather than as a continuous stochastic deterioration, as would be expected from gradual organelle senescence, 60 it seems likely that mitochondrial fission plays a central role in mitochondrial quality control. Consistent with this schema, overexpressing Drp1 both increases the sensitivity of mitochondria to fission and accelerates stress-induced mitophagy. 67 Mitochondrial fission generates small, depolarized mitochondria that undergo mitophagy. By contrast, mitochondrial fusion is restricted to highly polarized healthy mitochondria with good Ψm. 68,69 This is the sequestration mechanism by which dysfunctional and damaged mitochondrial components of partially depolarized mitochondria are prevented from mixing with and contaminating those of healthy mitochondria. Thus, the 2 opposing processes in mitochondrial dynamism also exert opposite effects on mitochondrial quality control. What then is the function of Mfn2, which plays central roles in both fusion of healthy mitochondria and mitophagic elimination of depolarized mitochondria? 70 The role of Mfn2 in mitophagy is widely assumed to be as a Parkin ubiquitination substrate that is eliminated with the rest of the mitochondrion during mitophagy. 71,72 However, a recent study found that Parkin-mediated ubiquitination of the yeast Mfn ortholog, fuzzy onion, can either destabilize or stabilize Mfn, depending upon which lysine is ubiquitinated. 73 Thus, we propose that ubiquitination of Mfn by Parkin (and possibly by other mitochondrial-targeted E3 ligases such as MULAN 74 and MARCH5 75 ) is a mechanism for functional crosstalk between the mitophagy and mitochondrial dynamics pathways. In the course of studying mouse hearts lacking either Mfn2 alone or both Mfn1 and Mfn2 (but not those lacking Mfn1 alone), we observed accumulation of mitochondria with abnormal cristae, and proposed that this might reflect the presence of a defect in mitochondrial pruning in Mfn2-deficient hearts. 19 Mitochondrial pruning or culling refers to the quality control mechanism whereby functionally abnormal and/ or senescent mitochondria are identified and selectively eliminated from cells via the mechanism of mitophagy. 76 Mitophagic quality control is essential to maintaining a normal mitochondrial population and to preventing cytotoxicity induced by ROS generated from damaged mitochondria. 64 In most tissues, the major pathway directing damaged mitochondria to mitopha-

7 Mitochondrial Dynamics in the Heart Figure 5. Intermolecular Mfn-Drp1 interactions regulate mitochondrial fusion. In the basal state (Left) Drp1 is cytosolic and Mfn2 is constitutively localized on the outer mitochondrial membrane (OMM). Intra- and intermolecular interactions between the Mfn2 heptad repeat (HR) domains, HR1 and HR2, suppress Mfn2-mediated transorganelle tethering by maintaining a tightly folded conformation that conceals the critical HR2 domain. If cytosolic Drp1 is recruited to the OMM, it can promote Mfn2 unfolding by interacting with the Mfn2 HR1 domain (Right). Because binding of Mfn2 HR1 and Drp1 coiled-coiled domains is mutually exclusive with intramolecular Mfn2 HR1-HR2 binding, Drp1-Mfn2 binding exposes the HR2 domain and promotes trans-interaction with the HR2 domains of Mfn2 molecules on neighboring mitochondria, thus tethering the 2 organelles. Mfn1 may interact in the same manner as Mfn2. Drp1, dynamin-like protein; Mfn, mitofusin. Mfn2 as an Orchestrator of Mitochondrial Fate The identification of PINK1-phosphorylated Mfn2 as a mitochondrial Parkin receptor emphasizes the multidimensional role of this fusion protein in mitochondrial biology. As discussed earlier, and similar to Mfn1, Opa1, and Drp1, Mfn2 is a GTPase; enzymatic activity is necessary for OMM fusion. 84 Mfn2 is also a binding partner for other mitochondrial proteins, acting as a physical hub through which their actions can be coordinated. The canonical mitochondrial tethering and fusion function of Mfn2 function requires either homotypic (Mfn2-Mfn2) or heterotypic (Mfn2-Mfn1) binding to other Mfn molecules on adjacent mitochondria via their respective C-terminal second HR domains. 85 [Heterotypic binding may be more efficient for organelle fusion than either of the posgy is the PINK1-Parkin pathway, mutations of which cause Parkinson s disease. 76 Mitochondrial damage or senescence leads to loss of the IMM electrochemical gradient, stabilizing the kinase PINK1, which in turn induces Parkin translocation. 64 Because Parkin is a E3 ubiquitin ligase, its localization to the mitochondria induces ubiquitination of OMM proteins and leads to selective mitophagic elimination of the polyubiquitinated organelle. The importance of this pathway for cardiac function is supported by reports that germline deficiency of either PINK1 or Parkin sensitizes the heart to dysfunction related mechanistically to defective mitophagy Mitochondrial enlargement in cardiac Mfn2 knockout (KO) mice, but not age-matched control or cardiac Mfn1 KO mice, 20 is the inverse of the expected consequence of deficiency in a critical mitochondrial fusion factor (ie, mitochondrial fragmentation). Nevertheless, the seemingly paradoxical finding of mitochondrial enlargement after tissue-specific Mfn2 ablation is also observed in hepatocytes 80 and neurons. 81 Collectively, these studies support an essential role for Mfn2 in culling abnormal mitochondria, independent of its effects in promoting mitochondrial fusion. To discover the mechanistic basis for impaired mitophagy in the absence of Mfn2, we examined the consequences of Mfn2 (and Mfn1) cardiac ablation on PINK1- Parkin pathway activity, 82 We observed that Parkin and Mfn2 co-immunoprecipitate in HEK293 cells and adult myocardium, and determined that their physical association depends upon PINK1-mediated phosphorylation of Mfn2 on T111 and S442. Parkin translocation, mitochondrial ubiquitination, and p62/sqstm1 (a mitophagy chaperone) activation after PINK activation by FCCP-induced mitochondrial depolarization 64 were all defective in Mfn2 null, but not Mfn1 null, murine cardiomyocytes. The consequence of PINK1-Parkin pathway interruption through Mfn2 ablation was age-dependent accu- mulation of structurally and functionally abnormal cardiomyocyte mitochondria that ultimately produced DCM. The murine cardiomyopathic phenotype provoked by deletion of Mfn2 (which, after PINK1-mediated phosphorylation, functions as the Parkin receptor specific to depolarized mitochondria) was reproduced in Drosophila by mutational ablation of Parkin itself. Thus, Mfn2 is a signaling intermediary between PINK1 and Parkin, being a substrate of PINK1 and a mitochondrial binding partner (ie, receptor) for Parkin (Figure 4). Given that Mfn ubiquitination can either promote or suppress fusion, 73 Parkin-dependent Mfn2 ubiquitination on depolarized mitochondria seems not to be a specific mechanism for eliminating this OMM fusion protein and suppressing mitochondrial fusion as some have suggested. Instead, Mfn2 appears to be one of dozens of mitochondrial proteins that undergo Parkin-mediated ubiquitination subsequent to mitochondrial depolarization. 83

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Dynaminrelated protein Drp1 is required for mitochondrial division in mamsible homotypic interactions. 86 ] Intriguingly, Mfn2 also binds to Drp1 molecules located on the same mitochondrion via its internal HR1 domain. 87 The nature of the Mfn2-Drp1 interaction suggests the following mechanism: intermolecular binding of HR1 to HR2 within a given Mfn2 molecular produces a closed structure, not unlike a closed safety pin, which is unfavorable to trans-mfn2 tethering (Figure 5). Drp1 can interrupt the intramolecular Mfn2 HR1-HR2 interaction by binding to the Mfn2 HR1 domain, inserting itself into and opening the safety pin. This open configuration favorably repositions the Mfn-Mfn binding HR2 domain, increasing the probability of a transmolecular interaction with opposing Mfn1 or Mfn2 molecules on another organelle. In this manner Mfn2-Drp1 crosstalk can facilitate mitochondrial tethering, potentially leading to increased fusion in a negative feedback loop that opposes Drp1-mediated organelle fission. Mfn2 binds to proapoptotic Bax and Bak, helping to modulate, or be modulated by, apoptosis signaling. 86,88,89 Mfn2 also binds to, or is bound by, the E3 ubiquitin ligases Parkin, 82 Huwe1, 90 and Mul1. 91 Finally, Mfn2 is a phosphoprotein, being a substrate for protein kinase A, JNK, and PINK1. 82,90,92,93 It seems clear that phosphorylated Mfn2 is more susceptible to polyubiquitination, and therefore to degradation. 82,90 Whether Mfn2 ubiquitination plays an important role in modifying mitochondrial fusion, as has been suggested, 71,91,94,95 or is simply one of many OMM protein ubiquitination events that precedes and facilitates mitophagic elimination of the organelle, 96 is not completely clear. Based on our findings, 82 we favor the idea that Mfn2 phosphorylation is critically important for recruiting ubiquitin ligases to damaged mitochondria, and that subsequent Mfn2 polyubiquitination is simply a marker for the broader actions of these ligases on multiple mitochondrial proteins. 97 Summary The work reviewed here challenges the notion that cardiac mitochondria in normal hearts are relatively static energy factories that drive excitation-contraction coupling, and under pathological conditions become the effectors of programmed cell death. Instead, these tissue-specific mitochondria are constantly reacting to intra- and extracellular conditions through dynamically regulated fusion/fission that directly affects mitochondrial metabolic function, replication, culling of dysfunctional organelles, calcium signaling, and sensitivity to programmed cardiomyocyte elimination. Mitochondrial dynamism is therefore essential to normal cardiac function at multiple mechanistic levels. 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