The interplay between autophagy and mitochondrial dysfunction in oxidative stress-induced cardiac aging and pathology.

YJMCC-07763; No. of pages: 9; 4C: Journal of Molecular and Cellular Cardiology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

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

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Stephanie E. Wohlgemuth a,⁎, Riccardo Calvani b, Emanuele Marzetti c a

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Article history: Received 17 September 2013 Received in revised form 8 March 2014 Accepted 10 March 2014 Available online xxxx

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Keywords: Autophagy Mitophagy Mitochondrial dynamics Quality control Hydrogen sulfide Cardioprotection

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Aging is accompanied by a progressive increase in the incidence and prevalence of cardiovascular disease (CVD). Prolonged exposure to cardiovascular risk factors, together with intrinsic age-dependent declines in cardiac functionality, increases the vulnerability of the heart to both endogenous and exogenous stressors, ultimately enhancing the susceptibility to developing CVD in late life. Both increased levels of oxidative damage and the accumulation of dysfunctional mitochondria have been observed in a wide range of cardiac diseases, which may therefore represent a common ground upon which many aspects of CVD develop. In this review, we summarize the current knowledge on the mechanisms whereby oxidative stress arising from mitochondrial dysfunction is involved in the process of cardiac aging and in the pathogenesis of CVD highly prevalent in late life (e.g., heart failure and ischemic heart disease). Special emphasis is placed on recent evidence about the role played by alterations in cellular quality control systems, in particular autophagy/mitophagy and mitochondrial dynamics (fusion and fission), and their interconnections in the context of age-related CVD. Cardioprotective interventions acting through the modulation of mitochondrial autophagy (calorie restriction, calorie restriction mimetics, and the gasotransmitter hydrogen sulfide) are also presented. This article is part of a Special Issue entitled ‘Cardiac Protein Quality Control’. © 2014 Published by Elsevier Ltd.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial ROS production and targets . . . . . . . . . . . . . . . . . . . . . . Contribution of mitochondrial dysfunction and oxidative stress to cardiac aging . . . . . Mitochondrial dysfunction and cardiac pathology . . . . . . . . . . . . . . . . . . . Cellular and mitochondrial quality control . . . . . . . . . . . . . . . . . . . . . . 5.1. Mitochondrial autophagy: the degradative effector in mitochondrial QC . . . . . 5.2. Redox regulation of macroautophagy . . . . . . . . . . . . . . . . . . . . . 5.3. Regulation of selective autophagic degradation of mitochondria . . . . . . . . . 5.4. Mitochondrial dynamics as an essential component of mitochondrial quality control 6. Autophagy in the heart and its role in cardiac aging and pathology . . . . . . . . . . . 6.1. Role of autophagy in cardiac aging . . . . . . . . . . . . . . . . . . . . . . 6.2. Role of autophagy in the pathogenesis of heart diseases . . . . . . . . . . . . . 7. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Department of Animal Sciences, University of Florida, Gainesville, FL 32611, USA Institute of Crystallography, National Research Council (CNR), Bari 70126, Italy Department of Geriatrics, Neurosciences and Orthopedics, Teaching Hospital “Agostino Gemelli”, Catholic University of the Sacred Heart School of Medicine, Rome 00168, Italy

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The interplay between autophagy and mitochondrial dysfunction in oxidative stress-induced cardiac aging and pathology

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⁎ Corresponding author at: Department of Animal Sciences, IFAS/College of Agriculture and Life Science, University of Florida, Gainesville, FL 32611, USA. Tel.: +1 352 392 7563. E-mail address: steffiw@ufl.edu (S.E. Wohlgemuth).

http://dx.doi.org/10.1016/j.yjmcc.2014.03.007 0022-2828/© 2014 Published by Elsevier Ltd.

Please cite this article as: Wohlgemuth SE, et al, The interplay between autophagy and mitochondrial dysfunction in oxidative stress-induced cardiac aging and pathology, J Mol Cell Cardiol (2014), http://dx.doi.org/10.1016/j.yjmcc.2014.03.007

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2. Mitochondrial ROS production and targets

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Mitochondria are central to cellular redox-dependent processes, as they are both the main source of ROS and critical responders to ROSmediated changes in the cellular redox state. Most cellular ROS are partially reduced forms of molecular oxygen (O2) and their derivatives, and originate from the one-electron reduction yielding superoxide anion (O•− 2 ). This incomplete reduction occurs as a result of electron leakage during normal respiration at ETC. complexes or by the enzymatic reduction of O2 [6]. Several sites have been identified within mitochondria that can produce O•− 2 [7]. Estimates indicate that 70–80% of mitochondrial O•− 2 arise from the Q cycle (ubiquinol, QH2, to ubiquinone, Q) as part of the electron transfer to cytochrome c, catalyzed by complex III [8]. Others suggest that the majority of mitochondrial O•− 2 occurs at complex I during NADH oxidation to NAD. Interestingly, only site IIIQo (at complex III) and glycerol 3-phosphate dehydrogenase can release O•− 2 into the intermembrane space (IMS). ROS released into the IMS theoretically have easier access to the cytosol, as they only need to cross the outer mitochondrial membrane (OMM), while matrix ROS have to pass across both the inner mitochondrial membrane (IMM) and the OMM [9]. Thus, site IIIQo-derived O•− 2 may possess an advantage with regard to cytosolic signaling capacity. The quantity of mROS produced is tightly regulated at several levels. Matrix O•− 2 is converted to hydrogen peroxide (H2O2) by superoxide dismutase 2 (SOD2). H2O2 can diffuse through both IMM and OMM to access the cytosol or be converted to H2O by glutathione peroxidases (glutathione peroxidase 1, Gpx1) and peroxiredoxins 3 and 5 (Prx3 and Prx5). O•− 2 released in the IMS can exit the mitochondria through voltage-dependent anion channels and be converted into H2O2 by cytosolic SOD1 [10]. The cytosol also contains glutathione peroxidases, catalase and peroxiredoxins that can reduce H2O2 to H2O [11,12]. Very recently, Gauthier et al. [13] developed a computational model to better understand the mechanisms underlying the complex balance between mROS production and scavenging in cardiac mitochondria. The model was carefully validated against a range of independent experimental data to explore how ROS production changes as a function

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The well-known free radical theory of aging, first proposed by Harman in 1956, posits that the intracellular production of reactive oxygen species (ROS) is the major determinant of lifespan [1]. The recognition of mitochondria as the main cellular sources of oxidants has prompted a refinement of the theory, usually referred to as the mitochondrial free radical theory of aging (MFRTA) [2]. According to this view, ROS generated by mitochondria would trigger a “vicious cycle” by primarily damaging mitochondrial DNA (mtDNA), which results in the synthesis of defective electron transport chain (ETC.) components and further oxidant emission. A large body of evidence exists both in support to and against the MFRTA (reviewed in [3–5]). Despite these controversies, the MFRTA has the undoubted merit of providing a theoretical framework for an enormous amount of work that has led to significant advances in the understanding of cardiovascular aging. Indeed, the role of mitochondrial oxidative stress and mitochondrial dysfunction in age-related cardiovascular pathologies is undisputed. Accumulating evidence also suggests that, in post-mitotic tissues such as the heart, mitochondrial quality control (QC) processes become inefficient in advanced age. As a result, the removal of dysfunctional mitochondria is impaired, which leads to a self-enhancing accumulation of mitochondrial damage with important implications for cardiac aging and pathology. In this review, the effects of aging on cardiac mitochondrial function are discussed, with special emphasis on recent evidence about the role played by alterations in mitochondrial ROS (mROS) production, mitochondrial QC systems and their interconnections in the context of agerelated CVD.

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of respiratory substrate, NAD/NADH redox potential, mitochondrial matrix pH, respiratory state, and inhibition of complex I or III [13]. The authors showed that ROS production was minimal at intermediate redox states, while it increased in both highly reduced/high mitochondrial membrane potential (ΔΨm) environments (e.g., high workload) and highly oxidized conditions (e.g., hypoxia) [13]. Specifically, in highly reduced environments, the increase in mROS levels was due to enhanced production, whereas in highly oxidative environments, it was largely linked to lower scavenging. Which of the two situations is more relevant to aging and CVD has yet to be established. ROS can cause posttranslational protein modifications to regulate signaling pathways. In this context, thiol groups on cysteine residues are proposed to be a major ROS target [14]. Redox modifications may influence mitochondrial function directly or indirectly by acting on mediators involved in most mitochondrial activities (reviewed in [6]). Interestingly, STAT3 appears to preserve ETC. activity by preventing ROS leakage at complex I [15]. Cardiac-specific deletion of STAT3 in mice results in the development of cardiac inflammatory fibrosis, dilated cardiomyopathy, and heart failure with advancing age [16]. Lack of STAT3 also eliminates the protective effects of ischemic preconditioning [17], while its overexpression in cardiomyocytes protects mice against doxorubicin toxicity, which is known to involve mitochondrial dysfunction [18]. The expression of a recombinant form of STAT3 that targets mitochondria and lacks the DNA-binding domain was found to protect the heart from ischemic damage by decreasing mROS production and attenuating cytochrome c release in a model of ischemia [19]. Accumulating evidence suggests that mROS are critically involved in cell homeostasis and exhibit a dual nature [12]. While very high quantities of mROS directly damage proteins, lipids and nucleic acids, lower levels function as signaling molecules to adapt to stress, and even lower amounts of mROS are required for normal cell function [12]. For instance, mROS are crucially involved in cardioprotective preconditioning pathways [20] and, notably, antioxidants render ischemic preconditioning ineffective [21]. The beneficial effects of low (physiological) mROS levels could also help explain the disappointing results of attempts to achieve cardioprotection in humans through antioxidant supplementation [22].

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3. Contribution of mitochondrial dysfunction and oxidative stress to 159 cardiac aging 160 Due to the high reliance of cardiomyocytes on mitochondrial energy metabolism, the heart is exposed to a high burden of mROS throughout the lifetime. Ultrastructural and biochemical analyses of heart tissues of old experimental animals and humans have shown evidence of elevated levels of oxidative damage to proteins, lipids and nucleic acids (reviewed in [23]). Noticeably, aberrant mitochondria, characterized by matrix derangement, loss of cristae and increased ROS generation, are encountered frequently in the myocardium of old rodents [24]. The incidence of the common 4977-bp mtDNA deletion, a typical consequence of oxidative stress, increases with age in the human heart and is estimated to be 5- to 15-fold higher in people over 40 years of age relative to younger individuals [25,26]. Moreover, levels of 8-oxo-7,8dihydro-2′deoxyguanosine in heart mtDNA, but not in nuclear DNA, are negatively correlated with longevity in different mammalian species [27]. Although correlative, these results suggest that mitochondrionmediated oxidative damage could be implicated in the process of heart aging. The mechanistic link among mtDNA mutations, mitochondrial dysfunction and cardiac aging has been provided by the characterization of mice with homozygous mutation in the exonucleaseencoding domain of mtDNA polymerase γ (PolG) [28]. PolG mice exhibit a premature aging phenotype, including cardiac enlargement, fibrosis and impaired systolic and diastolic functions [29]. Ultrastructurally, the heart of PolG mice is characterized by the accumulation of swollen and irregularly shaped mitochondria showing reduced


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5. Cellular and mitochondrial quality control

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In recent years, a great deal of research has been devoted to defining the role of QC systems in the context of heart senescence and CVD. Mitochondria possess their own QC systems that ensure the preservation of structure and function of mitochondrial proteins. Chaperones and ATP-dependent proteases are located at the OMM and IMM, as well as within the IMS and the mitochondrial matrix (reviewed in [50,51]). Recently, evidence has emerged for a mitochondrial-lysosomal degradative pathway, in which mitochondrion-derived vesicles (MDVs) transport mitochondrial cargo to the lysosome for degradation [52,53]. MDVs selectively enriched with oxidized proteins were found budding off from mitochondria in response to oxidative stress in different experimental models [52,53]. However, whether this process may function as an early response system to mitochondrial stress and damage in vivo has yet to be determined. The selective degradation of entire mitochondria through macroautophagy, or mitophagy, a term coined by Lemasters [54], may play an essential role in a number of cardiac pathologies associated with mitochondrial dysfunction. Notably, mitophagy is the only mechanism so far identified that sequesters damaged and dysfunctional mitochondria and delivers them to the lysosome for degradation [54]. Numerous studies have investigated mitochondrial QC and removal through mitophagy in a variety of disease states associated with oxidative stress. At the same time, it has become apparent that oxidative stress plays an important role in cell signaling and stress response, including the induction of selective degradation of dysfunctional mitochondria. In the following sections, we discuss about the mechanisms and regulation of macroautophagy, with specific emphasis on the interrelationship between oxidative stress and macroautophagy, the importance of mitochondrial dynamics for the process of mitophagy, the selective autophagic degradation of mitochondria, and the role that macroautophagy may play in CVD and cardiac aging.

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Increased levels of oxidative damage have been observed in a variety of cardiac diseases, including coronary artery disease, heart failure, (pathological) left ventricular hypertrophy, diabetic cardiomyopathy, and hyperkinetic arrhythmias (reviewed in [20]). As such, oxidative stress resulting from the imbalance between ROS production and scavenging is now considered to be a common denominator marking many aspects of CVD [37]. In cardiac pathological conditions, ROS (and reactive nitrogen species, RNS) are derived from several sources, such as NAD(P)H oxidase, xanthine oxidase, uncoupled nitric oxide (NO) synthase (NOS), lipoxygenase/cyclooxygenase, myeloperoxidase, and autooxidation of various substances, especially catecholamines [37]. However, dysfunctional mitochondria are thought to be the major cellular oxidant producers in the context of CVD. The relevance of mitochondrial dyshomeostasis to cardiac pathophysiology is epitomized by the role played by mitochondria in myocardial damage during ischemia/reperfusion (I/R). Mitochondrial ultrastructural and functional abnormalities are typically observed in ischemic cardiomyocytes. Under oxygen deprivation, mitochondrial oxidative phosphorylation (OXPHOS) is inhibited leading to ATP shortage. Reduced ATP levels, in turn, impair transmembrane ionic homeostasis, most importantly Ca2+ homeostasis, resulting in Ca2 + overload. The latter eventually leads to mPTP opening which causes loss of cardiomyocyte function and viability through ΔΨm dissipation and release of apoptogenic factors [38]. Simultaneously, an excessive mROS production ensues, mainly at complexes I and III of the ETC., which results in extensive oxidative damage, the severity of which is commensurate to the duration of ischemia [39]. The mechanisms by which Ca2+ promotes ROS generation are not well understood. Excess Ca2+ may enhance mROS production, in part, through the activation of the tricarboxyl acid (TCA) cycle, which results in increased NADH formation, stimulation of NOS, and activation of ROS-generating enzymes such as α-ketoglutarate dehydrogenase (reviewed in [40]). A concomitant impairment of endogenous antioxidant defenses increases the extent of oxidative stress [41]. The recovery of mitochondrial function during the reperfusion phase is largely dependent on the duration of the ischemic insult [39]. After prolonged

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O2 deprivation, extensive damage to ETC. complexes occurs, which, in conjunction with the concomitant disruption of antioxidant defenses, leads to further mROS generation and additional mitochondrial and cardiomyocyte injury during reperfusion. Notably, mPTP opening occurs mostly at the onset of reperfusion, and as a result of ROS accumulation, pH normalization and intracellular Ca2+ concentration rise [42]. Mitochondrial dysfunction and enhanced mROS generation are also involved in the pathogenesis of heart failure regardless of the etiology [43]. Indeed, mitochondria from the failing myocardium generate larger amounts of O•− 2 than those isolated from healthy hearts [44]. The increase in mROS generation was only observed when mitochondria were energized with NADH (substrate for complex I), but not with succinate (substrate for complex II). Excess O•− 2 emission, in turn, was associated with enhanced lipid peroxidation to mitochondrial membranes, decreased mtDNA copy number, reduced abundance of mitochondrial RNA transcripts, and impaired OXPHOS capacity [44]. In addition, oxidative stress impacts cardiomyocyte structure and function by activating signaling pathways involved in myocardial remodeling and failure [44–46]. Impaired mitochondrial function and increased ROS generation have been observed in the hearts of mice with type 2 diabetes mellitus [47, 48]. Interestingly, overexpression of ROS-detoxifying systems (metallothionein, catalase, and MnSOD) reverses mitochondrial dysfunction and diabetic cardiomyopathy, which suggests an important role for mROS in this condition (reviewed in [49]). In conclusion, available data indicate that mitochondrial dysfunction and mROS are likely involved in the pathogenesis of a wide spectrum of CVD. Given the important functions of ROS in physiologic conditions on the one hand, and their implication in CVD on the other, a critical research task will be to determine the “threshold” separating beneficial from detrimental ROS effects and the specific molecular targets that dictate the final outcomes of ROS actions.

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activities of ETC. complexes and higher rates of protein carbonylation [28,29]. Remarkably, the PolG heart phenotype, the cardiac mtDNA mutation load and the extent of mitochondrial protein oxidation are partially rescued by the overexpression of human catalase targeted to the mitochondrial matrix [29]. It is also noteworthy that mice with heterozygous disruption of SOD2, albeit showing a normal lifespan, exhibit elevated levels of mitochondrial oxidative damage, impaired respiration by complex I with reduced respiratory control ratio, increased susceptibility to mitochondrial permeability transition pore (mPTP) opening, and enhanced apoptotic DNA fragmentation [30,31]. An important aspect to clarify is discerning the specific alterations experienced by subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) with aging. Studies have shown that cardiac IFM isolated from old rats exhibit reduced bioenergetic efficiency and calcium (Ca2 +) retention capacity relative to organelles obtained from younger rodents [32]. The effects of aging on the redox physiology of the two cardiac mitochondrial populations are still disputed. While data by Suh et al. [33] indicate that old IFM, but not SSM produce higher rates of oxidants, others have found either the opposite [34] or no differences [35]. These contrasting findings could originate from the different methods used to quantify mROS production as well as from differential procedural manipulations during isolation [36]. In conclusion, studies in rodent models and observations in humans have made a strong case in support to mitochondrial dysfunction as a contributing factor to cardiac aging. In this context, the buildup of oxidative damage to cardiomyocyte constituents originating from the lifelong exposure to mROS seems to explain many of the alterations observed in mitochondrial function and heart structure/performance during aging.

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The process of macroautophagy can mechanistically be broken down into five steps: induction and nucleation, elongation/expansion (phagophore formation), closure and maturation of the isolation membrane (autophagosome formation), autophagosome-lysosome fusion (autolysosome formation), and lysosomal degradation. The origin of the sequestering double-membrane is debated, with the ER, Golgi apparatus, and mitochondria having been proposed as possible sources [57]. Each of the steps from sequestration to degradation is regulated by autophagy-related (Atg) proteins, at least 35 of which have been identified. As an essential QC system, macroautophagy is under tight regulation and responsive to a variety of stress signals. For a detailed description of the autophagic machinery and its regulation, the reader is referred to comprehensive, specialized reviews (for instance, [58]). It is generally accepted that ROS trigger the induction of macroautophagy [59–63]. Cysteine residues, which are susceptible to oxidative modification [14,64], are especially abundant in a number of autophagy-regulating proteins, which may be important in the redox regulation of this pathway [65,66]. In this regard, Scherz-Shouval et al. [67] proposed that ROS formation (likely H2O2) is essential for the induction of macroautophagy during starvation through oxidation of the cysteine residue 81 near the Atg4 catalytic site. Chen et al. [59] investigated the relative roles of O•− 2 and H2O2 in autophagy regulation in HeLa cells. Both glucose/glutamine/pyruvate/serum and amino acid deprivation induced •− O•− 2 and O2 plus H2O2, respectively, accompanied by increased autophagy and cytoprotection. The authors further determined that O•− 2 was the major signaling molecule in starvation-induced macroautophagy. In contrast, Dutta et al. [68] found that antimycin A-induced O•− 2 production by cardiac mitochondria, although leading to ΔΨm dissipation and oxidative damage to mtDNA, failed to induce macroautophagy or mitophagy. These contrasting findings might reflect cell type-specific redox regulation of autophagy. Surprisingly, despite the important functions of NO in cardiovascular physiology and its reactive nature, the effect of this signaling molecule on autophagy in the heart has not been explored in great detail. Rabkin and Klassen [69] analyzed gene expression in neonatal mouse cardiomyocytes exposed to a NO-donor and found that, while expression of apoptosis-related genes was induced, that of autophagyregulatory genes (Atg5l, Beclin-1 and Apgl2l) was not. On the other hand, NO has been implicated in autophagy induction in a model of cardiac oxidative stress stimulated by LPS (bacterial endotoxin lipopolysaccharide) [70]. Specifically, autophagy was induced in HL-1 cardiomyocytes (neonatal rat cardiomyocytes) and in the heart of mice challenged with LPS, and this effect was attenuated by NOS inhibition. Furthermore, autophagy was stimulated by direct exposure to NO and H2O2, and suppressed by the simultaneous administration of the

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5.3. Regulation of selective autophagic degradation of mitochondria

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The recognition of mitochondria as a major source of and target for ROS and inducers of cell death [23] has stimulated intensive research into the dissection of selective degradation of mitochondria and its triggers [71]. Mild and short-term oxidative stress, induced by rotenone or H2O2, in HeLa cells and mouse embryonic fibroblasts (MEFs) stimulated selective mitophagy rather than non-selective autophagy [60]. In addition, acute bursts of mROS production induced by a mitochondriontargeted photosensitizer followed by mitochondrial depolarization activate Parkin-dependent mitophagy [63]. Increased mROS formation can be associated with mPTP opening [38,72] and loss of ΔΨm [13], both of which have been implicated in the autophagic removal of mitochondria [73]. In isolated rat hepatocytes, nutrient deprivation caused depolarization of mitochondria and their subsequent colocalization with autophagic vacuoles [75]. Inhibition or ablation of cyclophilin D, an integral component of the mPTP [38], preserved ΔΨm and prevented starvation-induced autophagy in rat hepatocytes [75], heart-derived HL-1 cells, neonatal rat cardiomyocytes and adult cardiac cells [74]. Loss of ΔΨm in HEK293 cells treated with the mitochondrial uncoupler carbonyl cyanide mchlorophenylhydrazone (CCCP) was followed by selective degradation of mitochondria [76]. However, it is worth noting that the causative involvement of mPTP opening and ΔΨm dissipation as triggers for mitophagy has been challenged by the demonstration that polarized mitochondria were sequestered by autophagosomes in rat hepatocytes following anoxia/reoxygenation [77]. These conflicting results may suggest the presence of different upstream sensors that trigger mitophagy depending on the type and severity of the applied stress. Recently, several downstream regulatory proteins associated with mitophagy have been identified, shedding light on the selective nature of the autophagic removal of mitochondria. The phosphatase and tensin homolog-induced putative kinase 1 (PINK1)/Parkin pathway plays an integral role in this context (reviewed in [78]). In healthy mitochondria, PINK1, a serine/threonine kinase with a mitochondrial targeting sequence, is imported into the mitochondria and directed to the IMM, where it is cleaved by several proteases and ultimately degraded [79]. ΔΨm dissipation, on the other hand, leads to stabilization of PINK1 at the OMM, where it promotes the recruitment of the cytosolic E3 ubiquitin ligase cytosolic Parkin and its activation [80]. In turn, activated Parkin mediates the engulfment of dysfunctional mitochondria by autophagosomes and their selective elimination [76]. Chen and Dorn [81] recently proposed a mechanism for Parkinrecruitment in cardiomyocytes by which the OMM guanosine triphosphatase mitofusin 2 (Mfn2) was activated by PINK1 and subsequently mediated Parkin recruitment from the cytosol to depolarized mitochondria. Interestingly, a conflicting report demonstrated Parkin recruitment to depolarized mitochondria even in the absence of Mfn2 in cultured fibroblasts [76], which could indicate the presence of alternative mechanisms for Parkin translocation. Once recruited, Parkin activity is induced, a process that is likely also PINK1-dependent [82]. Parkin ubiquitinates mitochondrial proteins, including its receptor Mfn2, and thereby targets depolarized mitochondria for degradation. It is still unclear how ubiquitination of OMM proteins promotes the translocation of the autophagic isolation membrane to the tagged mitochondrion, and different models have been proposed. Adaptor proteins such as sequestosome/p62 (SQSTM1/p62) may recognize the ubiquitin-tag and facilitate the subsequent autophagic sequestration through its binding domain for MAP-LC3 [71,83].

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Macroautophagy is a broad term for processes by which intracellular constituents, including proteins, protein aggregates and organelles, are sequestered within a double-membrane structure (autophagosome), delivered to the lysosome, and degraded upon fusion of the autophagosome with the lysosome (auto(phago)lysosome) [55]. The degradation products, building blocks of macromolecules, are subsequently recycled, used for energy production, or secreted. Besides degrading intracellular material in a random, non-selective manner, macroautophagy has also been shown to selectively target microorganisms (xenophagy), and cellular organelles such as peroxisomes (pexophagy), endoplasmic reticulum (ER, reticulophagy or ERphagy), ribosomes (ribophagy), lipid droplets (lipophagy), protein aggregates (aggrephagy), and mitochondria (mitophagy) [56]. In this capacity, mitophagy becomes an essential cellular QC mechanism through which damaged mitochondria are removed before they cause further harm, for instance by emitting ROS.

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antioxidant N-acetyl-cysteine and the NO-donor or H2O2, respectively [70]. Given the incomplete understanding of redox regulation of macroautophagy, further studies are necessary to explore the role of ROS- and RNS-induced autophagy in the context of cardiac aging and CVD, to possibly identify additional targets for intervention.

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6.1. Role of autophagy in cardiac aging

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The post-mitotic nature of cardiomyocytes makes these cells especially prone to the accumulation of macromolecular and organellar oxidative damage over the lifetime [24,108]. In their “Mitochondrial– Lysosomal Axis Theory of Aging”, Brunk and Terman [109] postulated that enlarged mitochondria are a hallmark of aging and are inaccessible to autophagic removal. An age-related decline in autophagic activity may indeed exacerbate the accumulation of harmful cellular “garbage” [110,111]. Lipofuscin, an undegradable polymeric substance, also called the “age-pigment”, is primarily composed of cross-linked protein residues and lipid degradation products [112]. Terman and Brunk [112] portrayed lipofuscin-loaded lysosomes as a sink for newly synthesized lysosomal enzymes, thereby interfering with efficient lysosomal autophagy. An age-related impairment of autophagy has been reported in a variety of mammalian tissues such as the heart [113,114], liver [115], and nervous system [116,117]. The reduced expression of autophagyregulating proteins (Atg9, LC3-II and LAMP-1) observed in the heart of old rats suggests that both autophagosome maturation and processing are impaired in old age [114]. The relevance of autophagy to cardiac aging is supported by the observation that the expression of autophagyregulating proteins declines in parallel with cardiac function in mice [118]. Conversely, the induction of autophagy via rapamycin improves cardiomyocyte function in aged mice [118]. Cardiac-specific inhibition of autophagy through Atg5 knockout in mice results in a shorter lifespan and early development of left ventricular hypertrophy, reduced fractional shortening, and ultrastructural disorganization of sarcomeres [113]. In addition, Atg5-deficient mice are characterized by impairment of mitochondrial respiratory function, appearance of mitochondrial ultrastructure aberrations and accumulation of oxidatively modified mitochondrial proteins early in life [113]. Calorie restriction (CR) is one of the most robust anti-aging and cardioprotective interventions [119], which may, at least partially, act through inducing autophagy [114,120–123]. Indeed, in aged rats that underwent lifelong 40% CR, the increase in cardiac autophagic activity was associated with improved left ventricular diastolic function [124]. A similar CR regimen increased the cardiac autophagic activity in mature mice which was accompanied by reduced left ventricular mass and wall thickness, and improved cardiomyocyte contractile function [125]. Given the questionable feasibility of long-term CR in humans and the uncertainty of its efficacy, especially in the elderly population, the field of CR mimetics has become a topic of increasing scientific interest. As a general definition, CR mimetics are agents or interventions that are capable of reproducing the effects of CR without requiring significant food intake reduction. Since the identification of the first compound (2-deoxy-D-glucose) by Lane and colleagues in 1998 [126], the list of (putative) CR mimetics has grown dramatically [23], although evidence of efficacy and safety is insufficient for many of them. Resveratrol is a CR mimetic that has attracted considerable attention, in part due to its cardioprotective abilities [127]. One salient feature of this compound

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Mitochondria are dynamic organelles that move within the cell and undergo dynamic morphological changes, which are important for the regulation of intracellular energy availability, Ca++ homeostasis, metabolite and ROS/RNS production, and apoptotic signaling [94,95]. For a detailed review of mitochondrial dynamics, the reader is referred to detailed reviews (for example, [96]). In cardiomyocytes, mitochondria have a half-life of 10–25 days [97] and undergo dynamic events both in normal conditions and during disturbance of mitochondrial homeostasis [95]. The molecular machinery that enables these changes in the healthy and diseased heart have recently been reviewed [95,98,99]. Mitochondrial dynamics is also centrally involved in the regulation mitochondrial autophagy [100]. In a seminal study, Twig et al. [101] showed that, in MEFs, mitochondrial segregation through fission and selective fusion is essential for mitochondrial QC and is required for the autophagic removal of dysfunctional and damaged mitochondria. By labeling mitochondria with a photoactivatable dye, individual mitochondria and their fate through fission and fusion events were tracked. In most cases, one of the daughter mitochondria after a fission event maintained normal ΔΨ m . The other daughter mitochondrion was depolarized and exited the fusion–fission cycle, concomitant with decreased fusion capabilities and increased probability of being sequestered for subsequent autophagy [101]. These findings have been corroborated by another study in which overexpression of the fission protein Fis1 and the use of different Fis1 mutants in MEFs

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The role of autophagy in health and disease is multifaceted. Basal levels of autophagy are required for the physiological turnover of proteins and organelles [103,104]. Under conditions of cellular stress, such as starvation, hypoxia or oxidative stress, autophagy is activated and promotes cell survival [105]. Yet, if the insult is too severe, excessive autophagy may lead to impairment of function and ultimately cell death [105]. Given these vital functions, intensive research has been devoted to define the role of autophagy in cardiac aging and diseases [106,107].

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revealed that mitochondrial fission, in conjunction with mitochon- 505 drial dysfunction, triggered mitophagy [102]. 506

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Alternatively, the proteasomal degradation of ubiquitinated OMM proteins or the ubiquitinated proteins themselves may facilitate mitophagy (reviewed in [71]). How do mitochondria tagged for degradation induce the formation of the isolation (autophagosome) membrane for their ultimate sequestration and degradation? Fimia et al. [84] identified a novel player in autophagy regulation, Ambra (activating molecule in Beclin-1-regulated autophagy), and demonstrated in vitro and in vivo in mouse nervous system that Ambra is a binding partner of Beclin-1 and is required for autophagy. In a recent study, Van Humbeeck et al. [85] provided evidence that Ambra is also essential for mitochondrial clearance. The authors found that Ambra is recruited to depolarized mitochondria, where it interacts with Parkin and activates the class III phosphatidylinositol 3-kinase complex, thereby inducing phagophore formation [85]. Dorn et al. [86] found that the mitochondrial protein NIX (also known as BNIP3-like or BNIP3L), a BH3-only Bcl-2 family protein, is essential for mitophagy in erythrocytes [87]. CCCP-induced mitochondrial depolarization in MEFs and HeLa cells stimulated autophagy, Parkin translocation to mitochondria, OMM protein ubiquitination, and p62 recruitment. In contrast, Parkin, ubiquitin and p62 were not required for the stimulation of the autophagy machinery [86]. Interestingly, NIX not only controls Parkin translocation to mitochondria [86], but also possesses a LC3/GABARAP-interacting region that recruits autophagosomal membranes to mitochondria via LC3-binding [88]. The pro-apoptotic BH3-only protein BNIP3 (Bcl-2/adenovirus E1B 19 kDa interacting protein 3) is a mediator and independently implicated in cell death and regulation of mitophagy (reviewed in [89]). Myocardial oxidative stress following I/R led to increased BNIP3 activity and stimulation of cell death, suggesting a redox-sensing role of BNIP3 [90]. However, BNIP3 may play a dual role in the myocardium. Indeed, BNIP3 is proposed to be an important regulator of cardiomyocyte mitochondrial turnover via mitophagy, which may act protectively by inducing the removal of damaged organelles [89]. The tagging of mitochondria for degradation through mitophagy by BNIP3 occurs independent of mPTP opening, ROS production and increase in cytosolic Ca2+, but requires the recruitment of Parkin, the interaction between BNIP3 and LC3, and the involvement of mitochondrial fission [91–93].

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The dual nature of autophagy is especially evident in the context of various CVD conditions. For instance, in a model of pressure overload induced by aortic banding, the induction of autophagy accompanied the 581 onset of pathological left ventricular remodeling, which eventually 582 progressed into heart failure [131]. Down-regulation of the autophagic 583 flux by 50% through heterozygous disruption of Beclin-1 reduced 584 heart remodeling, while cardiac Beclin-1 overexpression exacerbated 585 hypertrophy and disease progression [131]. Similarly, moderate pres586 sure overload through transverse aortic constriction (TAC) provoked 587 cardiac hypertrophy in mice through autophagy-dependent mecha588 nisms [132]. Both TAC-induced autophagy and cardiac hypertrophy 589 were attenuated by the inhibition of histone deacetylases. Taken togeth590 er, these findings seem to point to a maladaptive function of autophagy 591 in the setting of pressure overload. In contrast, Oyabu et al. [133] iden592 tified a protective role of autophagy against angiotensin II-induced car593 diac hypertrophy, which was abolished in autophagy-deficient mice. A 594 cardioprotective role of autophagy was also shown in mice harboring 595 a cardiomyocyte-specific deletion of the autophagy-regulator Atg5 596 [134]. In adult mice, temporally controlled cardiac-specific deficiency 597 Q10 of Atg5 caused cardiac hypertrophy, left ventricular dilation and con598 tractile dysfunction, associated with sarcomere disorganization and mi599 tochondrial misalignment and aggregation. In contrast, mice with Atg5 600 deficiency early in cardiogenesis did not show such cardiac phenotype 601 under baseline conditions, but developed heart dysfunction and left 602 ventricular dilation within a week of pressure overload [134]. 603 It is worth noting that the level of autophagy was modulated to 604 different degrees in these studies. The difference between complete 605 autophagic depletion (Atg5 f/f ; MLC2v-Cre + ), reduced autophagy 606 (Beclin-1+/−), and extra-autophagic capacity (Beclin-1 overexpres607 sion) could help explain the varying effects on pathology progression 608 [135]. The fact that Atg5-deficient mice did not exhibit a cardiac pheno609 type under undisturbed conditions might point towards the existence 610 of compensatory mechanisms, which, however, may become inade611 quate under stress conditions. A partially reduced autophagic capacity, 612 on the other hand, might be barely sufficient to maintain contractile 613 function, without allowing for hypertrophic growth. Finally, an exces614 sive induction of autophagy through Beclin-1 overexpression could en615 hance hypertrophic growth and systolic dysfunction. 616 Myocardial I/R injury offers another prominent example of the two617 faced character of autophagy [136]. Early studies showed that upregula618 tion of autophagy during I/R correlated with functional recovery in 619 isolated rabbit hearts, while protracted ischemia was associated with 620 impaired autophagy and subsequent myocardial damage (reviewed in 621 [137]). In chronic ischemia, autophagy was stimulated in porcine myo622 cardium concomitant with a decline in apoptosis [138]. Similarly, au623 tophagy was upregulated in heart-derived H9c2 myoblasts subjected 624 to mild ischemia, which was associated with increased ATP concentra625 tion, preservation of ΔΨm, and significant delay of cell death [139]. Mod626 erate and severe ischemia, on the other hand, resulted in apoptosis and 627 necrosis and absence of autophagy, suggesting that the degree of au628 tophagy induction depends on the severity of the insult [139]. In 629 cardiomyocyte-derived HL-1 cells, the autophagic flux was drastically 630 reduced in response to simulated I/R (sI/R) [140]. Pharmacological or

7. Conclusions and future perspectives

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The optimal function and turnover of mitochondria appear to be critically involved in cardiac aging and CVD. The cellular processes of mitochondrial QC offer new targets for the development of therapeutics aimed at preventing and treating cardiac pathologies as well as attenuating the deterioration of heart function with aging [148]. The cardioprotective effects of H2S and the interaction of this gasotransmitter with autophagy highlight the intricate network of cellular stress response pathways, which can act both synergistically and independently from each other. Therefore, the development of cardioprotective strategies targeting autophagy and H2S signaling requires a thorough understanding of multiple factors, including the pathological context of insult, the extent of autophagy activation in a given pathophysiological setting, and the cytoprotective pathways elicited by H2S. This knowledge is necessary to exploit the cardioprotective properties of autophagy, while avoiding the detrimental effects of its defective or excessive activation.

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genetic suppression of autophagy sensitized cardiac cells to apoptotic cell death after sI/R, while activation of autophagy using rapamycin or Beclin-1 overexpression was cardioprotective [140]. These findings are in keeping with the observation that rapamycin protects Langendorffperfused rat hearts against I/R injury [141]. Matsui et al. [142] described different roles for autophagy induction during ischemia and subsequent reperfusion in vivo. Mild ischemia induced autophagy through AMPK-dependent inhibition of mTOR, featuring an adaptive role for autophagy similar to that occurring under nutrient deprivation. During subsequent reperfusion, autophagy induction was mediated by Beclin-1 upregulation, independent of AMPK. Myocardial injury occurring during the reperfusion phase was attenuated in Beclin-1+/− mice, which therefore suggests a maladaptive role for autophagy during reperfusion [142]. Studies exploring a novel therapeutic approach for the management of myocardial I/R injury have provided additional information on the complex role of autophagy in this setting (reviewed in [143]). The administration of the gasotransmitter hydrogen sulfide (H2S) at the time of reperfusion limits infarct size and preserves left ventricular function in an in vivo mouse model of I/R [144]. Interestingly, in pigs, infarct size and left ventricular function were both improved when H2S infusion started before the ischemic insult and continued throughout the reperfusion phase [145]. H2S infusion reduced Beclin-1 expression, suggesting a suppressive effect of the gasotransmitter on autophagy [145]. The protective effect of H2S preconditioning during I/R injury has recently been demonstrated in hepatocytes both in vitro and in vivo, in which cytoprotection elicited by H2S was concomitant with inhibition of autophagy through AKT activation [146]. Taken together, H2S may exert its cytoprotective actions through a multitude of cellular pathways, including, but likely not limited to attenuation of mitochondrial respiration and mROS production, antioxidant actions and autophagy modulation [147].

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resides in its ability to activate sirtuins [127], which have been associated with the lifespan extending effects of CR. Sirtuins stimulate autophagy through deacetylation of autophagic proteins such as Atg5 and Atg7 [128]. Resveratrol has shown to extend the lifespan of C. elegans [129], and to protect the rat myocardium from I/R injury in an autophagy-dependent manner [130]. A thorough characterization of the cardiac autophagic response to CR mimetics such as resveratrol may open a new venue for the development of interventions to attenuate the detrimental effects of aging on heart structure and function.

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Acknowledgments

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This work was partly supported by the Centro Study Achille e Linda Lorenzon (E.M.). We apologize to the authors whose excellent work could not be cited because of the vast literature on the subject and space limitations. We sincerely thank the anonymous reviewers for valuable input and criticism.

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Mitochondrial dysfunction in cardiac aging.

Mitochondrial dysfunction and oxidative stress in aging and cancer.

The Interplay between NLRs and Autophagy in Immunity and Inflammation.

mTOR signaling in aging and neurodegeneration: At the crossroad between metabolism dysfunction and impairment of autophagy.

The interplay between autophagy and apoptosis in the diabetic heart.

Mitochondrial oxidative stress in aging and healthspan.

Autophagy coupling interplay: can improve cellular repair and aging?

Mitochondrial autophagy promotes healthy aging.

Obesity, diabetes mellitus, atherosclerosis and chronic periodontitis: a shared pathology via oxidative stress and mitochondrial dysfunction?

Crosstalk and Interplay between the Ubiquitin-Proteasome System and Autophagy.

This old heart: Cardiac aging and autophagy.

Mercury Exposure and Endothelial Dysfunction: An Interplay Between Nitric Oxide and Oxidative Stress.

New insights into the role of mitochondrial dynamics and autophagy during oxidative stress and aging in the heart.

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence.

Neurodegeneration, mitochondrial dysfunction, and oxidative stress.

The interplay between BDNF and oxidative stress in chronic schizophrenia.

Calcium signaling alterations, oxidative stress, and autophagy in aging.

Interplay between apoptosis and autophagy in colorectal cancer.

The Intricate Interplay between Mechanisms Underlying Aging and Cancer.

Role of Mitochondrial Oxidative Stress in Glucose Tolerance, Insulin Resistance, and Cardiac Diastolic Dysfunction.

Mitochondrial aldehyde dehydrogenase 2 accentuates aging-induced cardiac remodeling and contractile dysfunction: role of AMPK, Sirt1, and mitochondrial function.

Reperfusion Injury.

Glomerular disease: autophagy failure and mitochondrial dysfunction in FSGS.

Association between heat stress and oxidative stress in poultry; mitochondrial dysfunction and dietary interventions with phytochemicals.