Mitochondrial dynamics: Orchestrating the journey to advanced age.

YJMCC-08071; No. of pages: 7; 4C: Journal of Molecular and Cellular Cardiology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc

Review article

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Mitochondrial dynamics: Orchestrating the journey to advanced age

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Agnieszka K. Biala, Rimpy Dhingra, Lorrie A. Kirshenbaum ⁎

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The Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Winnipeg, Manitoba R2H 2A6, Canada Department of Physiology, Faculty of Health Sciences, College of Medicine, University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada Department of Pharmacology& Therapeutics, Faculty of Health Sciences, College of Medicine, University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada

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Article history: Received 19 January 2015 Received in revised form 30 March 2015 Accepted 19 April 2015 Available online xxxx

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Keywords: ROS Oxidative stress Aging Mitochondrial dynamics Cell death

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Aging is a degenerative process that unfortunately is an inevitable part of life and risk factor for cardiovascular disease including heart failure. Among the several theories purported to explain the effects of age on cardiac dysfunction, the mitochondrion has emerged a central regulator of this process. Hence, it is not surprising that abnormalities in mitochondrial quality control including biogenesis and turnover have such detrimental effects on cardiac function. In fact mitochondria serve as a conduit for biological signals for apoptosis, necrosis and autophagy respectively. The removal of damaged mitochondria by autophagy/mitophagy is essential for mitochondrial quality control and cardiac homeostasis. Defects in mitochondrial dynamism fission/fusion events have been linked to cardiac senescence and heart failure. In this review we discuss the impact of aging on mitochondrial dynamics and senescence on cardiovascular health. This article is part of a Special Issue entitled: CV Aging. © 2015 Published by Elsevier Ltd.

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1. Introduction . . . . . . . . . . . . . . . . 2. Theories of aging and the mitochondrion . . . 3. Mitochondrial damage and aging . . . . . . 4. Molecular regulation of the aging heart . . . . 5. Aging and caloric restriction . . . . . . . . . 6. Mitochondrial dynamics in the senescent heart 7. Mitophagy in the senescent heart . . . . . . Conflict of interest . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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1. Introduction

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The search for the “Fountain of Youth” dates as far back as the 5th century BC in which the Greek historian Herodotus describes waters with magical rejuvenating powers that would reverse the effects of time on age. Indeed, throughout historical record there are countless anecdotes of expeditions commissioned by kings and aristocrats alike seeking the ambrosia that would promise youth and immortality. The concept of reversing or even halting the effects of age on the body and

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⁎ Corresponding author at: Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre Rm. 3016, 351 TachéAvenue, Winnipeg, Manitoba, Canada, R2H 2A6. Tel.: +1 204 235 3661; fax: +1 204 233 6723. E-mail address: [email protected] (L.A. Kirshenbaum).

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restoring one's youth has withstood the tests of time to even modern day society. In fact one can easily find advertisements and infomercials for a plethora of “health products” that claim to magically reverse the effects of time and age on the body. Hence, our modern day obsession with age has become a multi-billion dollar industry world-wide driven by the desire to be “forever young”. Aging leads to a progressive decline in the normal physiological function of the organism. These age-related defects include DNA strand breaks, defective cell proliferation and repair, mitochondrial dysfunction, accumulation of oxidized proteins, and lipids, organ failure that ultimately lead to senescence and death. In fact, the inability of the body to remove cells that have become genetically unstable or irreversibly injured by apoptosis or necrosis respectively is considered a central underlying mechanism for the increased prevalence of human cancers in the aging population. This perhaps is best exemplified by the

http://dx.doi.org/10.1016/j.yjmcc.2015.04.015 0022-2828/© 2015 Published by Elsevier Ltd.

Please cite this article as: Biala AK, et al, Mitochondrial dynamics: Orchestrating the journey to advanced age, J Mol Cell Cardiol (2015), http:// dx.doi.org/10.1016/j.yjmcc.2015.04.015

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Though several theories of cellular aging have been suggested, oxidative stress from increased production of reactive oxygen species

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2. Theories of aging and the mitochondrion

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(ROS) has been identified as a major contributing factor [5]. Adaptive mechanisms to overcome the potentially harmful effects of molecular oxygen occurred over millions of years ago of the evolutionary pressure and the appearance of the first land creatures that utilized atmospheric oxygen for aerobic respiration. Indeed, the evolution of multi-cellular organisms that relied on mitochondrial oxidative processes and respiration for ATP synthesis also required adaptive mechanisms to detoxify the lethal effects of mono- and divalent oxygen species [6–8]. Early studies in aerobic metabolism in eukaryotic cells revealed that molecular divalent oxygen served as a terminal electron acceptor in the mitochondrial electron transport chain during aerobic respiration reviewed in [6,9]. Here the sequential tetravalent reduction of molecular oxygen via the mitochondrial electron transport chain complexes (I–IV) to yield water and carbon dioxide is a critical mechanism for efficient ATP synthesis and prevention of reactive oxygen species (ROS). Univalent or monovalent reduction of molecular oxygen through electron leakage within the electron transport chain or other cellular sources leads to the generation of highly reactive ROS intermediates that include the superoxide anion, hydrogen peroxide, hydroxyl radical, peroxy radicals and other minor species [6, 10]. In addition to the mitochondrial electron transport chain, other cellular sources of ROS include membrane bound NADPH oxidases [11], H2O2-generating monoamine oxidases [12] and p66shc, and ROSinduced/ROS-released [13]. If left unchecked ROS intermediates can induce cellular injury including DNA strand breaks, oxidation of lipids, proteins and carbohydrates resulting in organelle dysfunction. Increased ROS production has been shown to disrupt normal cardiac function and contractile abnormalities resulting in heart failure reviewed in [14–16]. Both enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic antioxidants including water (vitamin C) and lipid soluble antioxidants (vitamins A, E) neutralize ROS and protect against oxidative stress injury [16,17]. Hence, an imbalance between excess ROS production and anti-oxidant capacity would trigger deleterious cellular defects resulting in cell death and is the fundamental tenant of the ROS theory of aging [18]. For example, increased ROS production has been reported in the aged myocardium and is believed to underlie in part the cellular defects associated with heart failure [19–21]. The mitochondrion has emerged as a convergence point for not only regulating cellular respiration but also signaling pathways linked to cell

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fact that defects in the mechanisms that govern autophagy, which is predominantly required for catabolic recycling of damaged organelles and prevention of proteo- and lipo-toxic stress, are considered a major driving force for cellular senescence with aging. In the context of the heart, cardiac senescence is a major clinical problem since age-associated defects in contractility, calcium handling, cell metabolism and mitochondrial function have been identified as a primary underlying defects associated with senescence and heart failure reviewed in [1]. In this regard, heart failure represents a major financial and socioeconomic burden to the health care system, since individuals diagnosed with heart failure require costly long-term care. In fact, the co-morbid effects of aging and frailty coupled with cardiovascular disease are expected to escalate over the next several years in the baby boomer population. Next to cancer, age-associated complications with cardiovascular disease account for more than half the reported deaths in North America. For example, cardiac senescence resulting from the accumulation of oxidized proteins, lipids and increased ROS production from dysfunctional organelles such as mitochondria contribute to vascular remodeling, and impaired contractility (Fig. 1). Hence, a better understanding of the underlying molecular mechanisms that contribute to cardiac senescence and heart failure would be of tremendous scientific and clinical importance [2]. As a physiological/pathological process aging induces distinct biochemical changes to proteins and lipids that alter the structural integrity and function of the vasculature and myocardium. In this regard, recent evidence suggests that cellular senescence is a contributing factor to atherosclerosis. Indeed, endothelial dysfunction activates pro-inflammatory cytokines resulting in inflammation, pro-coagulatory responses and thrombosis. Endothelium-dependent vasodilation is impaired during senescence largely due to increased oxidative injury and decreased nitric oxide production. Macroscopically, arteries lose their normal elastic properties and become calcified and rigid which contributes to vascular remodeling and dysfunction [3,4].

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Fig. 1. Signaling pathways involved in cardiac senescence. Figure depicts cellular senescence signaling pathways in the heart. Increased age predisposes to development of senescencerelated events resulting in endothelial cell dysfunction, fibrosis, hypertrophy, atherosclerosis, contractile dysfunction, myocardial infarction (MI) and heart failure. The hallmarks of mitochondrial senescence included mtDNA mutations, respiratory chain defects, ROS production, loss of ΔΨm, Ca2+ overload, nuclear DNA damage and telomere attrition. Perturbations in mitochondrial dynamism (fission/fusion) impair autophagy/mitophagy resulting in cellular senescence. Nicotinamide adenine dinucleotide phosphate (NADPH); p66, mono amino oxidase (MAO); reactive oxygen species (ROS); Mitofusin 1&2 (Mfn1&2), optic atrophy type 1 (Opa1); Dynamin-related Protein1 (Drp1); Mitochondrial fission 1 (Fis1), Bcl2 19 kDa interacting protein3 (Bnip3)/Bnip3-like protein (Nix/Bnip3FL); light chain protein3 (LC3).


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4. Molecular regulation of the aging heart

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Shortly, after birth cardiac myocytes exit the cell cycle and lose their ability to actively replicate. This inherent property of the post-mitotic heart has been suggested to be a key underlying factor of cellular senescence and heart failure, since damaged cardiac myocytes are not replaced by new ones. The underlying mechanism for cellular senescence in the adult heart has been linked to developmental down-regulation of key cell cycle proteins as well as to telomere attrition. Telomeres, are located at the end of chromosomal DNA, and protect DNA during replication. However, since telomere length shortens with each round of DNA replication, telomeres and telomerase activity can be used as an index of cellular senescence [43–45]. In this regard, telomerase deficient mice (Terc −/−) were reported to have shorter telomere lengths than wild type mice that were accompanied with increased senescence. In addition, elevated levels of cyclin-dependent kinase inhibitor p21CIP1WAF1 were observed in the aortae of Terc −/− mice with DNA aging marker—γH2AX—in endothelial cells, as well as cell cycle inhibitors p16INKa and p19ARF [46–50]. High levels of cell cycle inhibitors p16INK4a/Rb and p14ARF/p53 have been reported in the aged myocardium [51] as well as senescent endothelial and vascular smooth muscle cells [52–54].

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5. Aging and caloric restriction

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As stated above the mitochondrion is a major source of ROS production during cellular metabolism and has been intimately linked to senescence. Pioneering studies in worms, flies and rodents conclusively revealed that life span of the model organism was dramatically increased by simply restricting the caloric intake [55]. This was attributed to a reduction in mitochondrial ROS production and presumably oxidative injury [56]. Collectively, these studies revealed for the first time an unprecedented relationship between caloric restriction, and longevity [57]. Several lines of investigation have linked the Sirtuins (Sirt 1–7) as molecular caloric sensors [58]; [59–61]. Sirt-1 is the mammalian homolog to the yeast Sir-2 longevity factor which was demonstrated to be critical for regulating life span of yeast. Sirt-1 is an NAD+ regulated histone deacetylase activated in cells during caloric restriction or nutrient stress [58,62,63]. The discovery of Sirt-1 and its activation by caloric restriction further links cellular metabolism to vital cellular processes involved in aging including apoptosis and autophagy [58]. Several proteins known to regulate metabolism and proliferation are intimately coupled to Sirt-1 highlighting the inner connectivity of these pathways. These include the tumor suppressor p53, Forkhead Box Protein O1/3 (FOXO1/3), mitochondrial biogenesis regulator, peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC-1 α), nuclear factor κB (NF-κB) and mechanistic target of rapamycin (mTOR) as reviewed in [64]. By targeting these different signaling pathways, Sirt1 suppresses aging by impinging simultaneously on multiple cellular pathways crucial for cell longevity such as DNA repair, mitochondrial biogenesis, cell metabolism, and cell death thereby delaying aging and prolonging lifespan [62,65,66]. Pharmacological approaches to activate

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In addition to its many complex biochemical functions, the mitochondrion is uniquely distinguished from other cellular organelles because it contains its own mitochondrial DNA (mtDNA). The mtDNA is circular and located within the mitochondrial matrix. It is continually turned over through replicative and degradative processes. Notably, certain proteins of the respiratory electron transport chain are encoded by mtDNA, whereas other mitochondrial including cytochrome c and other heme constituent proteins are encoded by nuclear DNA. Disorders associated with mtDNA mutations generally fall into two classes: those linked to specific, maternally-inherited mtDNA mutations and those linked to mutations in nuclear-encoded genes that maintain normal fidelity and stability of mtDNA [33]. The incidence and frequency of mtDNA mutations increase exponentially with age and contribute to cellular senescence [34,35]. The mitochondrial theory of aging was substantiated in studies in mice expressing error-prone mitochondrial DNA polymerase γ (PolgD257A) defective for proof-reading activity. The increased mutation frequency from loss of function of DNA proof-reading activity resulted in the expression of defective respiratory chain proteins and premature aging [33,36,37]. Moreover, these mitochondrial defects resulted in pre-mature aging, dilated cardiac hypertrophy and mice dying of congestive heart failure by six months of age [38]. Through the process of mitochondrial fusion, the mtDNA of one mitochondrion can be transmitted or shared with other mitochondria within the cell. Therefore, the fusion of healthy mitochondria with mitochondria harboring mutations within their mtDNA during replication could be detrimental and profoundly influence the quality of mitochondrial pool within the cell [39–41]. Tam et al. [42] used a mathematical in silico model to predict the frequency of mtDNA mutations and accumulation over time. In this study, the authors demonstrated that lowered rates of mitochondrial fission and fusion events with age resulted in a

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predicted increase in the disproportionate distribution of mutant mtDNA to mitochondria. Presumably based on this model, the higher prevalence of mtDNA mutations with age would result in impaired mitochondrial respiration and organ failure. Based on this study, a critical role for age dependent regulation of mitochondrial fission–fusion events and mitochondrial turnover rates via mitophagy as discussed above is important for averting the accumulation of mitochondria with damaged mtDNA and contamination of the mitochondrial pool. Collectively, these findings highlight the importance of mitochondrial dynamism for ensuring normal mitochondrial homogeneity with increased age; this will be discussed in more detail below.

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death by apoptosis, necrosis and autophagy during aging [22]. The link between mitochondrial perturbations and aging has been previously associated with activation of the intrinsic mitochondrial death pathway and increased apoptotic cell death [23]. In fact, mitochondrial perturbations that have been suggested to underlie cellular aging have also been intimately linked to autophagy/mitophagy. In the most classical sense autophagy is defined as a catabolic process by which macromolecular structures, including proteins, and organelles such as mitochondria are re-cycled by an elaborate autophagosome–lysosomal system into their native biochemical constituents, carbohydrates, lipids and proteins for generating ATP during caloric restriction or nutrient stress. In this context several lines of investigation have suggested that the removal of damaged mitochondria by mitophagy is critical for maintaining mitochondrial quality control and cellular homeostasis for two important and salient reasons [24]. Selective removal of dysfunctional mitochondria by mitophagy during caloric restriction would presumably confer a survival advantage by eliminating damaged mitochondria that would otherwise trigger mitochondrial-driven cell death. Moreover, mitophagy of damaged mitochondria would ensure that only healthy mitochondria are maintained within the cell for mitochondrial biogenesis [24–28]. It must be stated however that beyond a certain threshold excessive mitophagy/autophagy is maladaptive and promotes cell death [29,30]. Previous work by our laboratory demonstrated, that the inducible protein Bnip3 triggered maladaptive autophagy in cardiac myocytes and necrotic cell death following p53 activation [30]. In the same study we demonstrated that interventions that suppressed autophagy, were equivalently sufficient for suppressing cell death—indicating that at least in this context excess autophagy induced by Bnip3 was maladaptive and promoted death [30]. Hence, a growing body of experimental evidence suggests that age associated apoptosis and autophagy may not be mutually exclusive or independently regulated events but commonly linked through several Bcl-2 family proteins including Beclin-1, Bnip3, Bim, Bnip3L, Bax, Bak and others reviewed in [31,32].

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There is a growing body of evidence that suggest that mitochondria are highly dynamic organelles constantly remodeling through fission and fusion to produce fragmented or highly organized mitochondrial networks. Mitochondrial fission and fusion are highly regulated events and critical for the normal growth, development and functioning of the cell reviewed in [78–80]. Recently, defects in mitochondrial dynamism (fission/fusion) have been linked to a variety of human pathologies and age-associated diseases [81,82]. Given the importance of the mitochondrion in the regulation of cellular metabolism it is not surprising that genetic abnormalities in mitochondria inherited or induced have such a dramatic and profound impact on human disease, especially in organs such as the heart which rely heavily on mitochondria for normal physiological function. The molecular signaling pathways by which mitochondria direct cell growth, autophagy and cell death remain to be elucidated. Mitochondrial dynamics is regulated by a number of cellular proteins related to the dynamin motor GTPases that regulate fission and fusion of mitochondrial outer and inner membranes. These include key fission proteins GTPase Drp1 (Dynamin related protein-1) [83] and Fis1 [84, 85] and mitochondrial fusion proteins Mitofusins 1&2 (Mfn1/2) [86,87] and Optic Atrophy 1 (Opa1) [88]. The physiological importance of mitochondrial fission and fusion process in growth, development and aging is supported by the reports that mice germ-line deleted for

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either of the mitochondrial fission or fusion proteins (Drp1, Mfn2 or Opa-1) were found to be embryonic lethal [86,89]. Mice with disrupted mitochondrial fusion ability developed several mutations in mtDNA, and displayed impaired respiratory function [87]. However, mice heterozygous for the inner mitochondrial membrane fusion protein Opa-1 were viable but developed age dependent retinal and nerve degeneration [90]. Recently, an essential and critical role for mitochondrial fusion has been suggested for normal function of the heart. In this report, knockdown of OPA-1 and Mitochondrial Assembly Regulatory Factor (MARF) the functional homologue of human Mitofusins) in the heart tubes of Drosophila melanogaster increased heterogeneity in mitochondria, displaying heart tube dilation and contractile impairment [91]. In another interesting report, lipid accumulation during aging was linked to decreased expression of Mfn2 and impaired autophagy. In this study, which was conducted in both young and old mice, Zhao et al. demonstrated an elevated levels of triglycerides in the liver and heart muscles of old mice, associated with a reduction in PGC-1α, mitochondrial fusion protein, Mfn2 and declined mitochondrial function [92]. Although mitochondrial fusion is essential for normal cardiac myocyte homeostasis, in cells defective for mitophagy, mitochondrial fusion can reportedly trigger dilated cardiomyopathy from the promiscuous fusion of senescent damaged mitochondria with normal healthy ones thereby contaminating the cellular pool of mitochondria [93]. Together these studies highlight the importance of mitochondrial fission and fusion for normal development and aging. This is best illustrated by a recent report by Park et al. [94] which showed the importance of mitochondrial fission in preventing onset of senescence. Here, MARCH5, a mitochondrial-associated ubiquitin ligase was shown to regulate mitochondrial fission and fusion, MARCH5 depletion in HeLa cells resulted in highly interconnected elongated mitochondrial network, increased expression of Mfn1, coupled with increased senescence associated marker β-galactosidase [94]. Interestingly, defects in mitochondrial network and senescence in MARCH5 depleted cells was reversed by either inactivating Mfn1 or inducing fission by Drp1 overexpression, suggesting the importance of mitochondrial fission events in preventing or delaying senescence [94]. It remains to be seen whether these events are equivalently operational in the vasculature or heart muscle itself with age. Another study in young and old mice, demonstrated increased mitochondrial fusion in aged muscle concordant with increased Mfn1 and Mfn2. Interestingly, Opa-1 levels which have been implicated in mitochondrial inner membrane fusion, were down-regulated in aged muscle. The increased mitochondrial fusion in aged muscle was accompanied by lower fission rates related to a decline in Fis-1 since Drp-1 levels remained unchanged [95]. Aging related decline in cardiovascular performance is also associated with thickening and increased stiffness of arteries and impaired endothelial cell function. Endothelium produces nitric oxide and other vasoactive materials that are important for maintaining cardiovascular health. Recently endothelium dysfunction during ischemia–reperfusion (I–R) has been linked to excessive mitochondrial fission from increased oxidative stress injury [96]. In this study, it was demonstrated that endothelial cells subjected to I–R underwent changes in mitochondrial morphology and exhibited phenotypic changes including mitochondrial fission that were partially suppressed by either anti-oxidants and inhibition of NO synthase or DRP1, suggesting a tentative link between endothelial cell dysfunction and mitochondrial fragmentation induced by oxidative stress [96]. There is a growing body of literature that supports the notion that active mitochondrial dynamics is critical for delaying senescence, through either mitochondrial repair process or mitophagy, however, an alternative view by Figge et al. proposed reduced mitochondrial dynamics and fission–fusion cycles would instead delay the detrimental effects of aging and senescence [97]. Although, this study was based on an in silico modeling of mitochondrial turnover and which will require further in vivo testing for validation.

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Sirt1 with resveratrol or SRT1720 were shown to protect endothelial cells from senescence through inhibition of p53–p21 pathway [67]. SRT1720 was also reported to normalize NF-κB and TNF-α levels in old mice, ameliorating endothelial dysfunction, oxidative stress injury and inflammation [68]. The metabolic link between Sirt-1 and senescence is profound. Senescent cells are typified by reduced metabolic activity and increased AMP (or ADP) to ATP ratios. This activates AMP-activated protein kinase (AMPK), a central regulator of metabolic signaling which induces growth arrest and autophagy by inhibiting mechanistic target of rapamycin (mTOR) pathway [69,70]. A recent report by the Finkel laboratory demonstrated that attenuating mTOR activity in mice expressing hypomorphic mTOR(Δ/Δ) alleles that express only 25% of normal mTORC1 and mTORC2 activity increased lifespan and decelerated aging in mice—supporting a link between metabolism, aging and life-span [71]. Moreover, a recent study by our laboratory demonstrated that mTOR inhibition with Rapamycin or hypoxia activated the Bcl-2 protein Bnip3 triggering autophagy in cardiac myocytes. Our studies demonstrated a direct functional link between nutrient sensing properties of mTOR and autophagy induced by Bnip3 [72]. Concordantly, Sirt1-mediated deacetylation of Foxo3 was shown to promote survival in a kidney model of caloric restriction, further highlighting the link between cellular senescence and metabolic signaling. Moreover, deacetylation of p53 by Sirt1 blunted p21 activation and cellular aging [73,74]. Taken together these findings collectively reveal the highly interconnected relationship between cell metabolism, growth and senescence [74]. Interestingly in contrast to Sirt-1 which is predominantly localized to the nucleus [75], Sirt-3 is reportedly localized to mitochondria and was recently demonstrated to deacetylate cyclophilin D (CypD), putative to be the regulator of the mitochondrial permeability transition pore (mPTP) [76]. In the study, deacetylation of CypD suppressed the age-dependent increase in mitochondrial swelling and cardiac dysfunction [76]. Moreover, Sirt3-deficiency was linked with increased vulnerability to ischemia–reperfusion injury, suggesting that the activation of Sirt3 may play an important role in preserving mitochondrial function through its effects on CypD during cellular stress [77]. However, it remains to be tested whether CypD inhibition by Sirt3 will protect mitochondria from mPTP in the aged myocardium, given that the study by Basso et al. demonstrated that mPTP can also occur in the absence of CypD in the liver in vitro and in vivo [2].

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This work was supported by grants to LAK from the Canadian Insti- 473 tutes of Health Research. AKB holds a post-doctoral fellowship from 474 Q6 the Manitoba Health Research Council and IMPACT-CIHR Fellowship. 475

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As stated above mitochondria are a major source of ROS which is a contributing factor to oxidative stress during aging. Proteotoxic stress, associated with senescence induced mitochondrial ROS production and oxidative stress injury results in the accumulation of oxidized proteins, contributing to cardiac dysfunction. To this date there is a growing body of evidence suggesting that autophagy and ubiquitin proteasome pathways become defective with age [98,99], resulting in accumulation of damaged mitochondria and misfolded proteins that negatively impact the overall function of the cell. It can be seen that in this context the removal of severely oxidized proteins or organelles within myocardium, by autophagy and ubiquitin proteasome pathways would be beneficial [100]. Aberrant mitochondria are removed in the process of mitophagy in order to sustain cardiovascular dynamics [26]. In fact in a recent study young versus old mice demonstrated the importance of autophagy for preventing the deleterious effects of senescence—induced cardiomyopathy [101]. In this study, a marked decline in Beclin-1 and ULK-1, along with increased levels of p62 levels indicative of impaired autophagy and cardiac dysfunction was observed in old versus younger animals. Bnip3 and related protein Nix (for Nip-like protein X) are known to induce mitophagy through the recruitment of LC3II/GABARAP/Atg8 proteins to the damaged mitochondria for removal by mitophagy [102,103]. Recently, an essential role for Nix and Bnip3 genes in mitochondrial quality control by eliminating damaged mitochondria by autophagy has been reported to be critical for preventing age induced cardiomyopathy [103,104]. In this study expression of Bnip3 and Nix was linked to adverse cardiac remodeling leading to cardiomyopathy, while mice deficient for both Nix and Bnip3 genes (Bnip3/Nix double knock-out) displayed age-dependent cardiac dysfunction and accumulation of aberrant mitochondria presumably from the lack of sufficient mitochondrial clearance—supporting the notion that autophagy/ mitophagy is essential for removing damaged organelles [105]. However, as stated earlier the exact role of Bnip3 and Nix in mitochondrial quality control and age-induced cardiomyopathy remains poorly understood. Previous work by our laboratory demonstrated that Bnip3 triggered mitochondrial perturbations resulting in mitochondrial depolarization, mPTP opening and cell death during hypoxia. Notably, the cell death induced by Bnip3 was abrogated by inhibiting autophagy with 3-MA or Atg7 knock-down [30,106]. These findings suggest that autophagy-induced by Bnip3 at least in this context was detrimental. Based on these findings as well as those of others we speculate that Bnip3 activation likely triggers the depolarization of mitochondria which then serves as a signal receptor for recruitment of GABARAP/ Atg8 and mitochondrial clearance [103,104]. We refer the reader to the article and works by Dr. Roberta Gottlieb in this issue for more in depth and detailed analysis of autophagy/mitophagy in the heart. Several reports have suggested interplay between mitochondrial dynamics and age induced mitophagy. Lee et al. described a mechanism by which Drp1 and E3-ubiquitin ligase Parkin are recruited to mitochondria in a manner dependent upon Bnip3. Moreover inhibition of Drp1 prevented Bnip3-induced autophagy and expression of dominant negative mutant Drp1K38E or Mfn1 also prevented Bnip3 induced mitochondrial fission and autophagy [104], suggesting that fission of mitochondria is a critical stage for clearance by mitophagy. Mfn2 is a key protein of mitochondrial fusion that is regulated by PTEN-Induced Putative Kinase protein 1 (PINK1)/Parkin mitophagy system [107,108]. Accordingly, this study demonstrated that Mfn2 was critical for removal of defective mitochondria by serving as a mitochondrial docking site for ubiquitin E3-ligase Parkin. Interestingly, these elegant studies demonstrated that mitochondrial associated PINK1 acts on both Mfn2 and Parkin, it phosphorylates Mfn2 thereby recruiting Parkin to mitochondria whereby Parkin then ubiquitinates Mfn2 [107,109]. Notably the absence of Mfn2 prevented Parkin translocation to mitochondria resulting in accumulation of dysfunctional mitochondria and impaired respiration. The association of age related defects in mitochondrial dynamics

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appears to be a critical nodal point in the maintenance of a viable normal regulation of myocardial function. Over the next several years it will be interesting to see whether interventions that selectively target metabolic signaling and mitochondrial dynamics will be sufficient to rejuvenate the senescent heart improving cardiovascular health and longevity and bring us a little closer to the elusive “Fountain of Youth”.

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7. Mitophagy in the senescent heart

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A journey through chemical dynamics.

Postponing parenthood to advanced age.

Mitochondrial Dynamics in Mitochondrial Diseases.

The regulation of mitochondrial dynamics.

To unite or divide: mitochondrial dynamics in the murine outer retina that preceded age related photoreceptor loss.

Astrocytes: Orchestrating synaptic plasticity?

Mitochondrial dynamics in astrocytes.

Cyclin G2 promotes hypoxia-driven local invasion of glioblastoma by orchestrating cytoskeletal dynamics.

ACF7 regulates inflammatory colitis and intestinal wound response by orchestrating tight junction dynamics.

Erratum: ACF7 regulates inflammatory colitis and intestinal wound response by orchestrating tight junction dynamics.

Journey to the center of the centrosome.

A Journey to Kitovu.

Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age.

The Journey.

Mitochondrial dynamics and the cell cycle.

The journey from proton to gamma knife.

The journey to independent nurse practitioner practice.

Orchestrating energy for shifting busyness to strategic work.

PS1dE9 mouse model of Alzheimer's disease.

The Journey to Adult Congenital Heart Disease.

The journey from stem cell to macrophage.

Creatine kinase-MB: the journey to obsolescence.

Metabolic regulation of mitochondrial dynamics.

Redox Homeostasis and Mitochondrial Dynamics.