Regulation of Mitochondrial Dynamics and Cell Fate Rimpy Dhingra, Phd; Lorrie A

Advance Publication by-J-STAGE Circulation Journal REVIEW Official Journal of the Japanese Circulation Society http://www.j-circ.or.jp Regulation of Mitochondrial Dynamics and Cell Fate Rimpy Dhingra, PhD; Lorrie A. Kirshenbaum, PhD

Though the mitochondrion was initially identified as a key organelle essentially required for energy production and oxidative metabolism, there is considerable evidence that mitochondria are intimately involved in regulating vital cellular processes, such as programmed cell death, proliferation and autophagy. Discovery of mitochondrial “shaping proteins” (Dynamin-related protein (Drp), mitofusins (Mfn) etc.) has revealed that mitochondria are highly dynamic organelles continually changing morphology by fission and fusion processes. Several human pathologies, including cancer, Parkinson’s disease, Alzheimer’s disease and cardiovascular diseases, have been linked to abnormalities in proteins that govern mitochondrial fission or fusion respectively. Notably, in the context of the heart, defects in mitochondrial dynamics resulting in too many fused and/or fragmented mitochondria have been associated with impaired cardiac development, autophagy, and contractile dysfunction. Understanding the mechanisms that govern mitochondrial fission/ fusion is paramount in developing new treatment strategies for human diseases in which defects in fission or fusion is the primary underlying defect. Here, we provide a comprehensive overview of the cellular targets and molecular signaling pathways that govern mitochondrial dynamics under normal and disease conditions.

Key Words: Cell death; Drp1; Fission; Fusion; Mitochondrion

ollowing the discovery of the mitochondrion in the mid- eases.4 In the context of the adult heart, the functional loss of 1800 s, there has been considerable interest over the cardiac myocytes by mitochondrial-driven programmed cell F past 100 years of biological research to understand death (apoptosis or necrosis) is postulated as an underlying how this remarkable organelle controls vital cellular processes cause of ventricular remodeling and heart failure.7 More recent such as energy metabolism, proliferation and cell death. Al- studies have implicated mitochondrial fission/fusion events in though mitochondria were first established as the “power autophagy/mitophagy during ischemic injury.8,9 In this review, house” of the cell, it was not until the mid-1970 s that they we will discuss the importance of mitochondrial dynamics and were appreciated for having cellular functions beyond their fusion/fission processes under normal and stress conditions original ascribed role in energy production.1 The concept that and the effect on cardiac function in particular. mitochondria contain the necessary signaling factors for initi- ating and executing programmed cell death was considered a major paradigm shift regarding their importance in regulating Mitochondrial Dynamics cell fate. Indeed, several lines of investigation have proposed Mitochondria are highly dynamic and plastic structures that are the mitochondrion as a central gateway for integrating signals continually changing morphology.10 Multiple proteins located for apoptosis, necrosis and autophagy.2 on the outer (OMM) and inner (IMM) mitochondrial mem- Though mitochondria were initially viewed as static organ- branes have been linked to mitochondrial fission/fusion. Fission elles, it is now well appreciated that they are dynamic struc- proteins include the cytosolic GTPase Dynamin-related pro- tures that move freely throughout the cell via dynamin motor tein-1 (Drp1),11 Fission-1 (Fis1),12,13 Mitochondrial fission factor GTPases.3–5 The discovery of the mitochondrial “shaping” (Mff),14 TBC1D1515 and others, and the mitochondrial fusion proteins, the “mitofusins”, further revolutionized our under- proteins include the large GTPase mitofusins (Mfn1 and Mfn2)16 standing of mitochondrial plasticity in normal and disease localized on the OMM, as well as optic atrophy 1 (OPA1)17 lo- states (reviewed by McBride and Scorrano4, Westermann6). cated on the IMM. The coordinated dynamic activation of these These proteins enable mitochondria to rapidly change mor- proteins during cell division and cell proliferation is responsible phology and complex networks within the cell through fusion for fusion and/or fission of the OMM and IMM. and fission processes. In fact, defects in mitochondrial fission/ fusion events have been identified in a number of human pa- Significance of Mitochondrial Dynamics thologies. It is not surprising that inborn genetic errors result- In addition to maintaining mitochondrial morphology, mito- ing in abnormal mitochondrial dynamics have been linked to chondrial fission/fusion events are essential for normal embry- cancer, cardiovascular disease, and neurodegenerative dis- onic growth and development. Mice germ-lines deleted for

Received February 26, 2014; accepted February 26, 2014; released online March 20, 2014 Department of Physiology, Pharmacology & Therapeutics (R.D., L.A.K.), Faculty of Medicine (L.A.K.), The Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, University of Manitoba, Winnipeg, Manitoba, Canada Mailing address: Lorrie A. Kirshenbaum, PhD, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Rm 3016, 351 Taché Avenue, Winnipeg, Manitoba R2H 2A6, Canada. E-mail: [email protected] ISSN-1346-9843 doi: 10.1253/circj.CJ-14-0240 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Advance Publication by-J-STAGE DHINGRA R et al.

Figure 1. Regulation of mitochondrial fission. In response to fission signals, cytosolic Drp1 is recruited to mitochondria where it oligomerizes into a ring-like structure around defined sites on the mitochondrial outer membrane. Through its GTPase activity, Drp1 promotes scission of the mitochondrial membrane that progresses and results in mitochondrial fission. Mitochondrial fission may be part of the cell cycle or it may be consequent to apoptosis or autophagy depending upon the signals. Drp1 activation and inhibition is regulated by multiple signaling molecules as on the left-hand side.

either of the mitochondrial fission or fusion genes are embry- inactivate its GTPases activity and fission.22,23 onically lethal, highlighting the physiological importance of In the context of the heart, myocardial ischemia-reperfusion mitochondrial dynamics in growth and development. For ex- (IR) injury remains a major cause of morbidity and mortality ample, mice lacking Drp1 have developmental abnormalities and has recently been linked to mitochondrial abnormalities in the forebrain and die at embryonic day 12.5.18 Mfn2-defec- arising from inappropriate mitochondrial fission.24,25 In this tive embryos display abnormal placental trophoblasts, with regard, HL-1 cells exposed to simulated IR displayed frag- mice lacking either of the Mfn1 or Mfn2 alleles dying mid- mented mitochondria coupled with permeability transition pore gestation.16 The link between mutations in Mfn2 and Charcot- opening and cell death that was dependent upon Drp1.25 Nota- Marie-Tooth disease further underscores the significance of bly, overexpression of, or a GTPase defective mutant of Drp1 mitochondrial fusion at the genetic level.5 This is exemplified (Drp1K38A), or mDivi, a pharmacological inhibitor of Drp1, by studies demonstrating that germ-line deletion of both Opa-1 suppressed mitochondrial fission and IR injury in HL-1 cells.26 alleles was embryonically lethal while the heterozygous Opa-1 The concept that mitochondrial fission events mediated by mice were born viable but later developed age-related retinal Drp1 are detrimental to cardiac function was further substanti- and neuronal defects.19 Hence, despite the proteins’ structural ated by in vivo studies demonstrating that the incidence of similarities, these findings demonstrate their distinct and mitochondrial fragmentation and contractile dysfunction in unique biological properties in mitochondrial fission/fusion for mouse hearts during IR was suppressed by mDivi. Taken to- normal embryonic development. gether these findings support the notion that excessive or inappropriate mitochondrial fission during IR is detrimental.11,25 Moreover, these finding are also supported by a recent study Mitochondrial Fission demonstrating that mDivi suppressed mitochondrial fission, Drp1 is considered to be the central regulatory protein for mito- loss of mitochondrial cytochrome C and caspase activation in chondrial fission. It is localized in the cytoplasm and translocates a renal model of IR and cisplatin-induced nephrotoxicity.27 to the OMM in response to specific fission signals. Phyloge- Further inhibiting fission with a newly discovered fission in- netically, Drp1 structurally resembles the yeast ortholog Dnm1, hibitor, P110, not only inhibited mitochondrial fission but which contains a highly conserved N-terminal GTPase domain, improved mitochondrial bioenergetics and heart function after a coiled coil central domain essential for oligomerization, and ischemic injury.24 Collectively, these findings demonstrate that a C-terminal domain.20 In response to fission signals, Drp1 inappropriate mitochondrial fission mediated by Drp1 is detri- oligomerizes into ring-like structures that encompass defined mental to cardiac function under physiologically relevant con- sites on the mitochondrial OMM. Through its GTPase activity, ditions. However, Drp1-induced fission may not be univer- Drp1 promotes scission of the mitochondrial OMM lipid bi- sally detrimental, given a recent report demonstrating that lack layer, resulting in mitochondrial fission21 (Figure 1). Drp1 is of Drp1-mediated fission promoted premature cellular senes- highly regulated by post-translational modifications, including cence.28 In that study, the ubiquitin E3 ligase MARCH5 inter- SUMOylation, or phosphorylation that can either activate or acted with Drp1, Fis1 and Mfn2.28 Interestingly, a highly inter- Advance Publication by-J-STAGE Regulation of Mitochondrial Dynamics

Table. Regulation of Mitochondrial Dynamics Regulator/ Gene Mechanism Reference receptor Drp1 miR-499 miR-499 prevents fission and dephosphorylation of Drp1 by inactivating calcineurin Wang et al, 201140 Drp1 Bax/Bak Bax/Bak conjugates SUMO1 to Drp1, increasing mitochondrial-associated Drp1, Wasiak et al, 200722 mitochondrial fission and apoptosis Drp1 Bax/Bak Bax/Bak induces the cytosolic release of DDP/TIMM8a complexes, increasing Arnoult et al, 200533 mitochondrial-associated Drp1 and fission Drp1 PGAM5 PGAM5 recruits and dephosphorylates Drp1 at ser 637 and increase its GTPase Wang et al, 201238 activity, fission and necrosis Drp1 CAMP, CAMP-dependent kinase PKA phosphorylates Drp1 at ser 656 inactivating its Cribbs and Strack, Calcineurin GTPase activity; Calcineurin dephosphorylates Drp1 at ser 656, activating Drp1 200723 and mitochondrial fission Drp1 Calcineurin Dephosphorylates Drp1 at ser 637 and translocates it to mitochondria, increases Cereghetti et al, 200839 fragmentation and cell death Drp1 SOCS6 SOCS6 forms complex with PGAM5 and Drp1, dephosphorylation of Drp1 Lin et al, 201361 increases fission and death Drp1 Mid49, Mid51 Recruit and sequester Drp1 to mitochondria; inhibit its fission activity and activate Palmer et al, 201346; fusion Zhao et al, 201147 Drp1 Mid49 or Mid51 Receptor for Drp1 and activate fission Loson et al, 201345 Drp1 Mff-1 Mff-1 serves as receptor for Drp1; activates fission and cell death Gandre-Babbe and van der Bliek, 200814; Otera et al, 201044 Drp1 Bnip3 Bnip3 influences Drp1 mitochondrial targeting, resulting in fission and autophagy Lee et al, 201126 Drp1 AKAP121 AKAP121 inactivates Drp1 via PKA-dependent phosphorylation at ser 637, reduces Kim et al, 201142 the interaction of Drp1 with Fis1 and mitochondrial fragmentation and apoptosis Drp1 Aurora A, cyclin Drp1 translocates to mitochondria mediated by GTPase RALA and Aurora A or Kashatus et al, 201129 B-CDK1, RALA RALBP1, phosphorylation at ser 616 by cyclin B-CDK1 promotes mitochondrial and RALBP1 fission during mitosis Drp1 MARCH5 MARCH5 Conjugates ubiquitin to Drp1 and increases fission Park et al, 201028 Opa1, Mfn1, PGC-1α/β Pgc-1α/β–/– mice display a reduction in Mfn1 and Mfn2, structurally abnormal Martin et al, 201357 Fis1, Drp1 mitochondria and lethal cardiomyopathy during early developmental stage Opa1 ROMO1 ROMO1 prevents dissolution of mitochondrial cristae, mitochondrial fission and Norton et al, 201453 cell death by activating Opa1 Opa1 Bnip3 Bnip3 interacts with Opa1 and disrupts Opa1 complex, inhibiting fusion Landes et al, 201051 Mfn1 PGC-1α PGC-1α and coactivator ERRα transcriptionally regulate Mfn1 Martin, 201357 Mfn2 PGC-1β PGC-1β coactivator ERRα transcriptionally activate Mfn2 Liesa et al, 200856 TBC1D15 Fis1 TBC1D15 binds to its adaptor Fis1 on mitochondria to promote fission; plays role Onoue et al, 201315 in mitochondrial morphology

connected mitochondrial network coincident with the senescence ies have implicated certain Bcl-2 family members in the regu- marker β-galactosidase was observed in HeLa cells deficient lation of mitochondrial dynamics,30 which prompts yet an- for MARCH5.28 Defects in mitochondrial network dynamics other question as to whether mitochondrial fission/fusion and and senescence in MARCH5-depleted cells were rescued by cell death are parallel events or part of the same signaling axis? either inactivating Mfn1 or by ectopic expression of Drp1. The apoptotic gene, Bax, has largely been implicated in regu- These findings highlight the involvement of mitochondrial lation of mitochondrial fission through a mechanism involving fission in either preventing or delaying cellular senescence.28 Drp1.22 The exact mechanism by which Bax regulates fission Further, Drp1-mediated mitochondrial fission is also crucial is poorly understood, but it is predicated on 2 critical events for normal cell division,29 adding to the complexity and bal- involving oligomerization of Bax and Bak proteins in the ance of mitochondrial dynamics. OMM.22 The first step involves translocation of cytoplasmic Drp1 to the OMM while the second requires Drp1 activation. Regulation of Mitochondrial Fission The first step is initiated by stress signals that involve activa- Despite the emerging evidence for a critical balance between tion of mitochondrial Bax and post-translational modification mitochondrial fission and fusion for cell viability, the cellular of Drp1 (discussed below). The second critical step requires regulators and effectors of this vital process are diverse and the association of Drp1 with adaptor proteins on the surface of raise several important questions (Table). For example, how the OMM, presumably where scission takes place.22 In this does the balance between mitochondrial fission and fusion regard, SUMOylation of Drp1 increases its GTPase activity favor 1 pathway over the other during stress signals? Another resulting in mitochondrial fission.31,32 Recently, Bax/Bak have question surrounding these possibilities is whether mitochon- been shown to regulate Drp1 activity and mitochondrial fission drial fission/fusion in normal cells is regulated differently from by influencing SUMOylation of Drp1.32 Notably, the mito- that in cells undergoing apoptosis or necrosis? It remains un- chondrial-anchored protein ligase (MAPL) was identified as a clear whether distinct biological signals initiate mitochondrial mitochondrial SUMO E3 ligase.32 MAPL conjugates SUMO fission under physiological and/or pathological conditions? to Drp1 and stimulates mitochondrial fission.32 However, it Regulation of Drp1 by Bcl-2 Family Proteins Recent stud- remains unclear whether MAPL is required for SUMOylation Advance Publication by-J-STAGE DHINGRA R et al. of Drp1 by Bax/Bak proteins.22 In this regard, the oligomeriza- response to necrotic signals, receptor-interacting serine-threo- tion of Bax/Bak during apoptosis Permeabilization of the OMM nine kinases (Rip1 and Rip3) form protein complexes with the and induces release of IMM proteins, including cytochrome c mixed lineage kinase domain-like protein (MLKL) and mito- and DDP/TIMM8a.33 Interestingly, in that same study, DDP/ chondrial phosphatase phosphoglycerate mutase family mem- TIMM8a was shown to interact with the C-terminus of Drp1. ber 5 (PGAM5). During necrosis induction, PGAM5 recruits Notably, the DDP-Drp1 complexes were not found in cells and dephosphorylates Drp1 at serine 637, which triggers for- under non-apoptotic conditions, suggesting that formation of mation of Drp1 dimers linked by disulfide bridges. Presum- DDP-Drp1 complexes is required for mitochondrial targeting ably, dephosphorylation and dimerization of Drp1 increases its of Drp1 and fission during apoptosis.33 The mechanism by GTPase activity resulting in mitochondrial fission.38 Impor- which DDP influences mitochondrial retention of Drp1 and tantly, knock-down of PGAM attenuated both fission and cell execution of fission is unclear, but one explanation purports death, suggesting that PGAM5 is a critical regulator of necro- that DDP may serve as a chaperone for targeting Drp1 to mi- sis through its actions on mitochondrial fission.38 tochondria.33 An alternative explanation holds that DDP may Regulation of Drp1 by Calcineurin and Cyclic AMP-Dependent influence the oligomerization of Drp1 or its affinity for other Kinase, PKA As mentioned previously, post-translational proteins, which results in its mitochondrial localization.33 Based modifications of Drp1 are critical for its activation and/or in- on these studies, several models have proposed that activation activation. Drp1 and its metazoan ortholog have a conserved of Drp1 during cell stress mediates mitochondrial fission and phosphorylation site at serine 656, which is targeted by cyclic apoptotic cell death. However, the notion that mitochondrial AMP-dependent protein kinase and is dephosphorylated by fragmentation always results in cell death has been recently Calcineurin (CN).23 Dephosphorylation by CN activates Drp1 challenged by Parone et al who demonstrated that Bax/Bak- and promotes mitochondrial fission and cell death. However, mediated apoptosis is independent of mitochondrial fission.34 phosphorylation of Drp1 at ser 656 inactivated Drp1 and its In this regard, evidence for Bax-induced mitochondrial fis- ability to promote mitochondrial fission.23 Interestingly, in yet sion is based on a Drp1-dependent model involving permeabil- another study, CN-mediated dephosphorylation of ser 637 of ity changes to the OMM. However, it is equally plausible that Drp1 was found to be critical for mitochondrial fission.39 In re- Bax/Bak oligomers can promote mitochondrial fission by in- sponse to mitochondrial depolarization and increased cytosolic hibiting the proteins required for mitochondrial fusion, namely Ca2+, activation of CN dephosphorylates Drp1 at ser 637, which the mitofusins.35 This view is based on a recent report demon- facilitates mitochondrial translocation of Drp1 and fission.39 strating the association of Bax/Bak with Mfn1 and Mfn2 via This link between CN and Drp1 is extended by a recent study its BH3 domain.35 It was shown that under apoptotic condi- demonstrating the targeted inhibition of CNα and CNβ isoforms tions the association of Bak with Mfn2 apparently decreases by miR499 prevented dephosphorylation of Drp1, mitochon- but its association with Mfn1 increases. The Bak association drial fission and cell death.40 Collectively, these findings high- with Mfn2 is important for regulation of fission, given that a light the importance of site-specific Drp1 phosphorylation to BH3 domain mutant of Bak that retained its ability to associate promote fission in a cell- and context-specific manner. with Mfn2 was unable to trigger mitochondrial fragmenta- Moreover, the A kinase anchor proteins (AKAP) have been tion.35 These findings suggest that the dissociation of Bak from linked to PKA for regulation of mitochondrial dynamics Mfn2 is an important step in mitochondrial fission events.35 through Drp1 inhibition and cardiac cell survival during hy- Given that Bax/Bak can activate Drp1 raises the question as to poxia.41 In particular, AKAP121 was shown to inactivate Drp1 whether Bax can simultaneously influence the activity of mi- via PKA-dependent phosphorylation of Drp1 at ser 637, there- tochondrial fission and/or fusion proteins or whether fission or by reducing interaction of Drp1 with Fis1. Interestingly, a re- fusion events are regulated independently by a subset of pro- cent study demonstrated the ubiquitin ligase SIAH2 as a critical teins that are recruited by Bax/Bak complexes? For example, regulator of AKPA121 and Drp1-dependent fission. Notably, how does the inhibition of fission produce the elongated phe- Kim et al demonstrated that AKAP121 was ubiquitinated and notype of fused mitochondria or conversely how does inhibi- subsequently degraded in a manner dependent upon SIAH2.42 tion of fusion produce the fragmented “fizzed” phenotype de- Hence, regulation of AKAP121 by SIAH2 is a key nodal point scribed by Brooks et al in 2007.35 Further, the same group in Drp1 activation and mitochondrial fission. Further, mice showed that mitochondrial fragmentation was induced during germ-lines deleted for SIAH2 had smaller infarcts and reduced IR injury in the heart and was inhibited by Bcl-2 but not by incidence of cell death, which highlights the importance of the caspase inhibition, further linking the importance of Bcl-2 SIAH2-AKAP121 axis in the regulation of Drp1 and mito- family proteins to regulation of mitochondrial fission.27 chondrial fission.42 Recently, BH3-domain protein (Bnip3) has been linked to Regulation of Drp1 by Mitotic Kinases Aurora A and Cyclin mitochondrial fission during IR injury in the heart. Bnip3 is a B-CDK1 Coordinated fission and fusion of mitochondria is a hypoxia-inducible death gene that provokes mitochondrial per- critical step in normal cell division and proliferation. A recent turbations and cell death of cardiac myocytes during hypoxic/ study by Kashatus et al29 provided new insight into the regula- ischemic injury.36,37 Bnip3 can reportedly promote mitochon- tion of mitochondrial fission events during cell division. Nota- drial fission by a mechanism involving mitochondrial Drp1.26 bly, during cell division, the equal and uniform distribution of Inhibition of Drp1 with mDivi, dominant negative Drp1, mu- the mitochondria to each daughter cell is dependent upon mi- tant Drp1K38E or Mfn1 prevented Bnip3-induced mitochon- tochondrial fission. In this regard, 2 mitotic kinases have been drial fission and autophagy.26 It is currently unknown whether postulated to be involved in this process, notably the Aurora A Bnip3 impinges on other mitochondrial fission or fusion pro- kinase and cyclin B-CDK1.29 Aurora A phosphorylates the teins to induce cell death or autophagy respectively. Ras-like GTPase, RALA.29 RALA, together with its effector Regulation of Drp1 and Mitochondrial Fission by TNFα protein RALBP1, facilitate Drp1 recruitment to mitochondria.29 Receptor Signaling Pathway Recent studies have demon- Phosphorylation of Drp1 by cyclin B-CDK1 at ser 616 activates strated the involvement of the tumor necrosis factor (TNF) re- Drp1 GTPase activity at the OMM and mitochondrial fission ceptor family in mitochondrial fission and programmed necro- during mitosis. Disruption or inhibition of RALA or RALBP1 sis by a mechanism involving dephosphorylation of Drp1.38 In results in a loss of mitochondrial fission and uneven distribu- Advance Publication by-J-STAGE Regulation of Mitochondrial Dynamics tion of mitochondria in each of the newly generated daughter involvement of Mff as a critical component of the mitochon- cells.29 These findings demonstrate the importance of RALA drial fission machinery.44 and RALB1 proteins for Drp1-mediated fission and uniform Despite the considerable focus on Drp1 and Fis1 in the regu- mitochondrial distribution for normal cell division. lation of mitochondrial fission events, several new lines of investigation have identified MiD49 or MiD51 as adaptors for Drp1 Recruitment of Drp1 to Mitochondria on OMM for fission that is independent of Fis1 or Mff.45 Interest- As discussed in the previous section, mitochondrial fission ingly, MiD49 and MiD51 were shown to inhibit fission by se- requires the translocation of Drp1 to specific sites on the OMM. questering Drp1 at the level of the mitochondria, presumably These receptors or docking proteins on the OMM are critical enabling unopposed fusion of the mitochondria to occur via for mitochondrial recruitment of Drp1 and for efficient execu- unrestricted actions of Mfn1 and Mfn2.46 The inhibitory ef- tion of fission. For that reason, it is important to understand how fects of MiD51, also known as mitochondrial elongation factor Drp1 associates on the OMM. Multiple genes, including Fis1 (MIEF1) in vertebrates, on fission is further substantiated by and Mff, serve as docking sites for Drp1 on the OMM (Table).4 another group,47 who demonstrated that MIEF1 recruited Drp1 MiD49 and MiD51 are newly identified additions to this grow- to the OMM, inhibiting fusion in a manner independent of ing list of Drp1 adaptor proteins. Fis1 is an OMM protein that Fis1. Moreover, MIEF1 was shown to increase mitochondrial has been implicated as a critical receptor for Drp1 for mitochon- targeting of both Drp1 and the Drp1 GTPase defective mutant, drial fission. Although Fis1 expression produces fragmented Drp1K38A, indicating that recruitment of Drp1 to mitochondria mitochondria, silencing Fis1 results in an elongated, non- by MIEF is not dependent upon its GTPase activity.47 As a step functional mitochondrial network.12 Furthermore, depletion of toward understanding how MIEF-1 inhibits Drp1, the same Fis1 impaired normal cell cycle progression, resulting in G2/M group showed that MIEF1 reduced the GTPase activity of Drp1 arrest.43 Concordant with Fis1’s role in mitochondrial OMM independent of ser 637 phosphorylation.47 However, it is unclear fission, reperfusion injury in the heart induced mitochondrial from that study how mitochondrial MIEF1 increases the asso- defects, including fission, impaired bioenergetics, and decline ciation of Drp1 on the OMM; in other words, it is unknown how in cardiac function, that were completely reversed by the fis- MIEF1 signals the recruitment of cytosolic Drp1 to the OMM.47 sion inhibitor P110.24 Though Fis1 was initially identified as the primary OMM receptor for Drp1, recent advances in the field have suggested that Fis 1 may not be indispensable for mito- Mitochondrial Fusion chondrial fission. This notion is founded on studies in which Mitochondrial dynamics, as discussed, is extremely organized Fis1–/– mice exhibited minimal mitochondrial defects with no and balanced in the normal cell; disruption of this balance re- apparent defects in mitochondrial fission,44 which contradicts an sults in morphological changes and abnormal function of mi- earlier report by Yoon et al.12 Recently, Fis-1 has been shown tochondria, resulting in cell death. Mitochondrial fusion in- to be critical for mitochondrial turn-over and disposal.3 volves the Mfn1 and Mfn2 proteins that localize to the OMM Although Fis-1 has been shown to be dispensable for the and facilitate the fusion of the OMM of one mitochondrion actions of Drp1, Fis1 been identified as indispensable for the with another (reviewed by Chen et al16, Figure 2). OPA1, an activity of TBC1D15, a recently identified protein linked to IMM protein, is bound to the outer surface of the IMM where mitochondrial morphology. Interestingly, Fis1 can reportedly it regulates fusion of the IMM.17 Although the genes related to interact with and serve as a docking site for TBC1D15.15 When mitochondrial fusion are fairly well defined, the processes by expressed alone in cells, TBC1D15 was found to be primarily which the OMM and IMM fuse is not well understood. A hint cytosolic but when co-expressed with Fis1, it was predomi- to the processes involved in mitochondrial fusion comes from nantly mitochondrial. Moreover, the mitochondrial morphol- a recent study demonstrating some similarity between mito- ogy in cells depleted for TBC1D15 was different from that chondrial fusion and exocytosis of secretory granules medi- seen following Drp1 inhibition, which exhibited less branched ated by SNARE proteins.48 Notably, a new member of the and highly elongated mitochondria in the perinuclear region.15 phospholipase D super family, MitoPLD, has been identified These findings demonstrate that TBC1D15 has distinct non- as playing a key role in mitochondrial fusion.48,49 MitoPLD overlapping properties with Drp1. Importantly, mitochondrial localizes on the OMM in a manner dependent on mitofusins, targeting of TBC1D15 was independent of either Drp1 or Mff. where it promotes trans-mitochondrial membrane adherence Taken together, these studies reveal the involvement of yet between adjacent mitochondria and hydrolyses cardiolipin to another cytosolic protein, TBC1D15, in the regulation of mi- generate phosphatidic acid as a signaling molecule.48,49 tochondrial morphology.15 It remains to be established whether TBC1D15 influences mitochondrial fission events in the Regulation of Mitochondrial Fusion hearts during stress conditions such as IR or following myo- Opa1 is the human homolog of yeast Mgm1, and plays a cen- cardial infarction. tral role in regulating mitochondrial IMM morphology. Muta- Recently, Mff was discovered as a critical component of the tions in Opa1 have been associated with dominant optic atro- mitochondrial fission machinery.14 Interestingly, knock-down phy where degeneration of optic nerves results in blindness. of Mff apparently releases Drp1 from the OMM, resulting in a Mutations or knock-down of Opa1 promote mitochondrial fused mitochondrial state.14 Conversely, forced expression of IMM swelling and rupture, ultimately triggering the fragment- Mff increases mitochondrial recruitment of Drp1 and thus fis- ed mitochondrial phenotype.50 Importantly, loss of Opa1 by sion. Notably, the N-terminus of Mff was shown to interact small interfering RNA resulted in disorganized mitochondrial with Drp1, further supporting a role of Mff as a Drp1 adaptor cristae, loss of membrane potential, cytochrome c release, and on the OMM. Interestingly, mitochondrial fission induced by mitochondrial fragmentation.50 Notably, Bcl-2 averted apoptosis the mitochondrial proton ionophore, CCCP, was attenuated in induced by loss of Opa-1.17 In this regard, Opa1 is believed to cells following knock-down of Drp1 or Mff.14 Similarly, knock- sequester cytochrome c, hence altering Opa-1 activity that may down of Opa-1 resulted in extensive mitochondrial fragmenta- induce release of cytochrome c from mitochondria and trigger tion, cytochrome c release and apoptosis, which was suppressed the intrinsic cell death pathway.17 Bnip3, which is also linked by inhibition of Mff or Drp1.14 These studies substantiate the to mitochondrial fission, can presumably interact and disrupt Advance Publication by-J-STAGE DHINGRA R et al.

Figure 2. Regulation of mitochondrial fusion. GTPase activity of mitofusins (Mfn1, Mfn2) promotes mitochondrial adherence and promotes fusion of the outer mitochondrial membranes of adjacent mitochondria. Opa1 regulates the fusion of the inner mitochondrial membrane. Mitochondrial fusion is important for normal cell cycle and metabolism and is regulated by multiple signaling molecules as shown on the left-hand side.

Opa-1 complexes on the IMM, resulting in mitochondrial thermore, resistance of Bax/Bak double knockout hearts to fragmentation and cell death.51,52 It is unclear whether fission necrotic injury was lost when mitochondrial fission was inhib- induced by Bnip3 is therefore related to inhibition of Opa1 or ited with mDivi-1.30 Likewise, Mfn2–/– cells also displayed activation of Drp1 or both.52 Recently, Norton et al53 identified fragmented mitochondria and were resistant to necrotic injury, Reactive oxygen species modulator 1 (ROMO1) to be critical but inhibition of mitochondrial fission sensitized these cells to for normal cristae formation and mitochondrial fusion. ROMO1 necrotic cell death.30 Although that study proposed that fused is upregulated during oxidative stress and believed to be im- mitochondria sensitize cells to necrotic injury, the conclusions portant for maintaining mitochondrial cristae through its ef- were based on alterations in mitochondrial morphology. Mfn2 fects on Opa1 oligomerization.53 Inhibition of ROMO1 was has many other roles in the cell beyond its ascribed role in shown to disrupt IMM integrity and cristal structure, findings mitochondrial fusion, including the regulation of glucose me- that highlight the importance of ROMO-1 as a protective factor tabolism.55 Importantly, mitofusin knockout cells do not ex- during oxidative stress.53 hibit the normal morphological phenotype of an organized As discussed, MIEF1 promotes mitochondrial fusion.47 Mfn2 mitochondrial network typically seen in normal cells. Because and MIEF1 both induce mitochondrial fusion, but their inde- Bak can reportedly interact with Mfn2, which is important for pendent expression produces a different mitochondrial pheno- the normal fused state,35 this suggests that mitochondrial dy- type. Mfn2 expression results in perinuclear aggregation of in- namics in absence of either Bax/Bak or mitofusin proteins dividual small mitochondria, whereas MIEF1 overexpression may be regulated differently from that seen in normal cells. results in clusters of mitochondrial aggregates. Moreover, Mfn2 knock-down resulted in mitochondrial fragmentation, which Transcriptional Regulation of could be rescued by MIEF1 expression, suggesting that either Mfn2 is not required for MIEF1-induced fission or MIEF1 is a Mitochondrial Dynamics downstream effector of Mfn2. Further, knock-down of endog- Mitochondrial dynamics are critical for energy production and enous MIEF1 in HeLa cells produced fragmented mitochondria homoeostasis, but very little is known about the transcrip- that were sensitized to staurosporine-induced apoptosis.47 These tional regulation of the genes involved in this dynamic process. studies further link the fragmented mitochondrial phenotype to Peroxisome proliferator activated receptor gamma coactiva- cell death. In addition to mitofusins, G proteins (Gαq and Gα11) tor’s (PGC-1) role in the transcriptional regulation of mito- have been recently found on the OMM and IMM, respectively, chondrial biogenesis and metabolism genes has been evident and implicated in mitochondrial fission/fusion events.54 This for a number of years; recently, PGC-1 was linked to regula- view is based on a recent study demonstrating that inhibition of tion of genes associated with mitochondrial dynamics.56 Inter- Gαq/11 resulted in decreased mitochondrial fusions, coupled estingly, small mitochondria, consistent with the fragmented with reduced respiratory capacity, and ATP production.54 phenotype and reduced levels of Mfn2, were observed in Though most studies purport that mitochondrial fusion is PGC-1β knockout mice. Importantly, PGC-1β expression re- cytoprotective, a study by Whelan et al30 implicated Bax-in- stored the elongated mitochondrial phenotype, together with duced necrotic cell death in mitochondrial fusion.30 In their increased Mfn2 promoter activity.56 The same group further report, Bax/Bak double knockout had a protective effect, re- linked PGC1 to regulation of mitochondrial fission/fusion sulting in mice with smaller infarcts and less cell death. Im- events during early heart development. In particular, PGC- portantly, the reconstitution of Bax alone in Bax/Bak double 1α/β double knockout mice had structurally abnormal heart knockout MEFs resulted in a dramatic switch from the frag- mitochondria and developed severe cardiomyopathy.57 Fur- mented mitochondrial phenotype to the fused mitochondrial thermore, mitochondrial fission proteins (Drp1 and Fis1) and phenotype, with a corresponding increase in cell death. Fur- fusion proteins (Mfn1 and Opa-1) were downregulated in the Advance Publication by-J-STAGE Regulation of Mitochondrial Dynamics hearts of PGC-1α/β–/– mice.57 Notably, conditional knockout tion of normal cell physiology. Though several discoveries of PGC1 in the adult heart had no apparent effect on mito- have contributed to our basic understanding of mitochondrial chondrial fission/fusion or contractile function, suggesting that fission and fusion events under normal conditions, there re- PGC1 may play a greater role in mitochondrial dynamics dur- mains a paucity of information regarding how defects in mi- ing early heart development or in the transitional phase from tochondrial fission/fusion influence cardiac function during IR neonatal to adult when the demand for mitochondrial energy and heart failure. For that matter, the role of mitochondrial by aerobic respiration is increased.57 fission/fusion events in mitochondrial quality control and autophagy in the heart remains equally unresolved. Given the Mitochondrial Dynamics and Metabolism importance of mitochondria in energy metabolism, cardiac There is new emerging evidence that mitochondrial dynamics hypertrophy and cell survival, modulating the cellular factors are directly driven by metabolism. Recently, insulin was linked and signaling pathways that govern mitochondrial fission/fusion to mitochondrial dynamics, particularly mitochondrial fusion. in the heart may prove to be salient in the design of novel Consistent with defective insulin signaling in diabetic individu- treatment strategies for cardiac pathologies arising from de- als is concomitant impairment of mitochondrial function and fects in mitochondrial dynamics. expression of mitochondria fusion proteins Opa-1 and Mfn1/ Mfn2.58 Further, insulin treatment of cardiomyocytes and skel- Acknowledgements etal muscle cells increased the expression of Opa-1, and mito- We are grateful to Dr H. Weisman for critical comments on the manu- chondrial fusion. Notably, the ability of insulin to influence script, Ms. F. Aguilar for technical assistance and Kairee Ryplanski for metabolism was impaired in cells deficient for Mfn2 or Opa-1. secretarial assistance. Importantly, insulin-stimulated mitochondrial fusion and metabolism has been linked to the Akt-mTOR–NFκB pathway.58 Sources of Funding This work was supported by operating grants to L.A.K. from the CIHR; Mitochondrial Dynamics and Autophagy L.A.K. is a Canada Research Chair in Molecular Cardiology. In addition to regulating mitochondrial dynamics, mitochondrial shaping proteins have been linked to autophagy. Notably, Disclosures as discussed earlier, the death gene Bnip3 can trigger mitochon- None. drial fission, which is an important signal for autophagy and mitochondrial clearance. Bnip3 activates autophagy through a References mechanism involving the translocation of Drp1 and Parkin to 1. McBride HM, Neuspiel M, Wasiak S. Mitochondria: More than just mitochondria. Inhibition of Drp1 prevents Bnip3-induced au- a powerhouse. Curr Biol 2006; 16: R551 – R560. tophagy; likewise, expression of dominant negative mutant 2. Mughal W, Dhingra R, Kirshenbaum LA. 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