The role of autophagic degradation in the heart Kazuhiko Nishida, Manabu Taneike, Kinya Otsu PII: DOI: Reference:

S0022-2828(14)00310-1 doi: 10.1016/j.yjmcc.2014.09.029 YJMCC 7911

To appear in:

Journal of Molecular and Cellular Cardiology

Received date: Revised date: Accepted date:

13 August 2014 26 September 2014 29 September 2014

Please cite this article as: Nishida Kazuhiko, Taneike Manabu, Otsu Kinya, The role of autophagic degradation in the heart, Journal of Molecular and Cellular Cardiology (2014), doi: 10.1016/j.yjmcc.2014.09.029

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ACCEPTED MANUSCRIPT Review article

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The role of autophagic degradation in the heart

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Kazuhiko Nishida, Manabu Taneike, Kinya Otsu*

Cardiovascular Division, King’s College London British Heart Foundation Centre of Excellence,

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London SE5 9NU, UK

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*Corresponding author at Cardiovascular Division, King’s College London British Heart Foundation

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Centre of Excellence, 125 Coldharbour Lane, London SE5 9NU, UK. Tel.: 020-7848-5128; Fax: 020-

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7848-5193. E-mail address: [email protected] (K. Otsu).

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Autophagy is important for the quality control of proteins and organelles. There are two kinds of autophagy, non-selective and selective autophagy. Autophagy is essential to maintain cellular homeostasis in the heart. Autophagy is a cardioprotective mechanism against external stress.

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ACCEPTED MANUSCRIPT Abstract Autophagy has evolved as a conserved process for bulk degradation and recycling of cytoplasmic components, such as long-lived proteins and organelles. Macroautophagy is the most prevalent form

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and thus referred to as autophagy. Autophagy is initially considered to be a non-selective process as an adaptive response to nutrient starvation. However, damaged mitochondria are selectively removed

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by autophagy, called mitophagy. Autophagy plays essential roles in starvation, cardiac remodeling, reverse remodeling, aging and inflammation to maintain cellular homeostasis in the heart. This review

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discusses some recent advances in understanding the basic molecular mechanisms underlying autophagosome and autolysosome formation and mitophagy and the roles of autophagy in

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cardiomyopathy. This article is part of a Special Issue entitled “Mitochondria”.

Abbreviations:

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MHC-Cre+, transgenic mice expressing Cre recombinase under the control of -myosin heavy chain

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promoter; Ambra1, activating molecule in Beclin 1-regulated autophagy; AMPK, AMP-activated protein kinase; Atg, autophagy-related; Bcl, B-cell lymphoma; Bnip3, Bcl-2/E1B 19kDa-interacting protein 3-like protein; DFCP1, double FYVE-containing protein 1; ER, endoplasmic reticulum; FoxO,

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forkhead box class O; GABARAP, -aminobutyric acid receptor-associated protein; HDAC, histone deacetylase; LC3, microtubule-associated protein 1 light chain 3; LAMP, lysosome-associated membrane protein; LVAD, left ventricular assist device support; mtDNA, mitochondrial DNA; MLC2v-Cre+, knock-in mice expressing Cre recombinase under the control of myosin light chain 2v promoter;

mTOR,

mammalian

target

of

rapamycin;

Nix,

Nip3-like

protein

X;

PE,

phosphatidylethanolamine; PINK1, PTEN-induced putative kinase protein 1; PI3K, class III phosphoinositide 3-kinase; PI3P, phosphoinositide 3-phosphate; p62/SQSTM1, sequestosome 1; ROS, reactive oxygen species; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; STX17, SNARE protein syntaxin 17; TAC, thoracic transverse aortic constriction; TF, transcription factor; TLR, Toll-like receptor; ULK, Unc-51-like kinase; Vps, vacuolar protein sorting.

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Keywords:

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Autophagy; Mitophagy; Mitochondria; Cardioprotection; Inflammation; Reverse remodeling

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1. Introduction Autophagy, literally “self-eating” in Greek, is a conserved process from yeast to mammals for bulk

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degradation and recycling of cytoplasmic components, such as long-lived proteins and organelles [13]. There are three main autophagic pathways: macroautophagy, microautophagy and chaperon-

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mediated autophagy. Macroautophagy is the most prevalent form and commonly referred to as autophagy. During autophagy, an isolation membrane (originally termed phagophore) emerges in the

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cytoplasm and forms a double membrane vesicle called an autophagosome. Autophagy-related (Atg) proteins are involved in the expansion of the isolation membrane. The cytosolic components are

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sequestered in the autophagosome. The outer membrane of the autophagosome fuses with lysosome to form autolysosome. The components and the inner membrane of autophagosome are degraded by

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lysosomal hydrolases into amino acids and lipids that are transported to the cytosol for reuse. Atg5- or

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Atg7-deficient mice die within one day of delivery due to severe nutrient deprivation until lactation starts [4, 5], suggestive of the importance of autophagy in response to starvation.

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Autophagy was initially believed to be a non-selective process. However, it has been revealed that there are selective types of autophagy, including mitochondria-specific autophagy, called mitophagy. The pathways for mitochondrial quality control are important to preserve mitochondrial homeostasis

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and to prevent cellular damage (Fig. 1) [6]. Damaged mitochondria are removed by mitophagy. Beyond quality control, mitophagy has been shown to be required for the steady-state turnover of mitochondria and for the adjustment of mitochondrion numbers to changing metabolic requirement [7]. Mitophagy is closely associated with mitochondrial fission which is a process that divides large mitochondria into smaller daughter mitochondria [8]. The smaller mitochondria can be engulfed by the autophagosome. It has been suggested that mitochondrial fragmentation precedes mitophagy for autophagosome engulfment [9]. Mitophagy can be induced under nutrient-rich conditions [7]. When autophagy is triggered during starvation, mitochondria elongate and the elongated mitochondria are spared from autophagic degradation [10]. Extensive studies have been conducted to elucidate the physiological and pathophysiological roles of autophagy in the heart using genetically modified mouse models (Table 1). Autophagy plays an 4

ACCEPTED MANUSCRIPT essential role in maintaining cellular homeostasis in starvation, cardiac remodeling, reverse remodeling, aging and inflammation (Fig. 2). This review demonstrates some recent advances in understanding the molecular mechanisms of autophagy, including mitophagy and its roles in

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cardiomyopathy.

2. Autophagy machinery

Mitochondria-associated endoplasmic reticulum (ER) membrane has been proposed as the most

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plausible candidate for the initial membrane source and/or the platform for autophagosome formation [1-3]. Autophagosome formation consists of three stages: initiation, nucleation and expansion. The

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initiation of autophagy requires the Unc-51-like kinase (ULK) complex, which contains ULK1 and/or ULK2, Atg13, focal adhesion kinase family interacting protein of 200 kDa and Atg101. The

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mammalian target of rapamycin (mTOR) complex 1 suppresses the ULK complex under nutrient-rich

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conditions. By starvation or AMP-activated protein kinase (AMPK) activation, the ULK complex is activated and translocates to ER. The autophagy-specific class III phosphoinositide 3-kinase (PI3K)

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complex, including Beclin 1, Atg14L, activating molecule in Beclin 1-regulated autophagy (Ambra1), vacuolar protein sorting (Vps)15 and Vps34, is equally important for initiating autophagosome

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formation. The ULK complex regulates the class III PI3K complex. The recruitment of Beclin 1 is also sensitive to starvation. The ER- resident soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin 17 (STX17) is activated under starved condition, binds to Atg14L, a subunit of the class III PI3K complex, and recruits the complex to the ER-mitochondria contact site [11]. The PI3K locally produces phosphoinositide 3-phosphate (PI3P), which is not a usual constituent of ER, but of autophagosome. ER acts as a scaffold for autophagosome nucleation. PI3P is essential for canonical autophagosome formation. The isolation membrane emerges from a PI3P-enriched omegashaped subdomain of ER called omegasome. Double FYVE-containing protein 1 (DFCP1) is an ERresident PI3P-binding protein. PI3P recruits DFCP1 and promotes the formation of omegasome. Thus, DFCP1 is a marker for omegasome, although its function remains unclear [12]. The PI3P-binding

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ACCEPTED MANUSCRIPT WD-repeat proteins interacting with phosphoinositides, which are Atg18 homologs, are also crucial for the maturation of omegasome and isolation membrane. Two ubiquitin-like conjugation systems, the Atg12-Atg5-Atg16L1 complex and the microtubule-

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associated protein 1 light chain 3 (LC3)-phosphatidylethanolamine (PE) conjugate, play important roles in the expansion and closure of the isolation membrane [1, 2]. Following nucleation, the Atg12-

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Atg5-Atg16L1 complex is recruited to the isolation membrane and dissociates from it upon closure of the autophagosome. The Atg12-Atg5-Atg16L1 complex is necessary for LC3-PE conjugation. The

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conjugation of PE to LC3 is involved by the sequential action of Atg4, Atg7 and Atg3. The conjugation leads to the conversion of the soluble form of LC3, LC3-I to the autophagic vesicle-

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associated form, LC3-II. Vesicles derived from Golgi, plasma membrane and endosomes contribute membrane for autophagosome expansion [1]. Coat protein complex II vesicle traffic from the ER-

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Golgi intermediate compartment are required for LC3 lipidation and autophagosome formation. Atg9

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vesicles are an important membrane source during the early steps of autophagosome formation [13]. The late stage of autophagy is referred to as the maturation or degradation stage and involves fusion

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between autophagosome and lysosome, leading to the formation of autolysosome [14]. The most important biochemical feature of lysosome is its highly acidic lumen (pH 4.5–5.0), which contains more than 50 acid hydrolases. The components and the inner membrane of autophagosome are

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degraded by the lysosomal hydrolases. The acidification of lysosome is maintained by the lysosomal membrane containing more than 20 lysosomal membrane proteins including lysosome-associated membrane proteins (LAMP)1 and 2, and more importantly, the vacuolar-type H(+)-ATPase [14]. Among the coordinated lysosomal expression and regulation network, transcription factor (TF)EB is considered to be a master regulator of lysosomal biogenesis and function and autophagy [15]. The phosphorylation of TFEB by mTOR complex 1 is sequestered in the cytosol on the lysosome surface or in combination with 14-3-3. TFEB hypophosphorylation by mTOR complex 1 suppression leads to its nuclear localization and activation of its transcriptional activity, resulting in promoting the expression of lysosome-related genes and autophagy-related genes [14]. SNAREs are the major players in controlling membrane-mediated transport events via vesicle fusion. STX17 localizes to the outer membrane of the completed autophagosome but not to the isolation membrane [16]. STX17 6

ACCEPTED MANUSCRIPT interacts with cytosolic SNARE, SNAP-29 and endosomal/lysosomal SNARE, VAMP8. They mediate the fusion between the autophagosome and lysosome, leading to the degradation of enclosed

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materials.

3. Mitophagy machinery

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Atg8 family proteins such as LC3 and the -aminobutyric acid receptor-associated protein (GABARAP) function as receptors for selective autophagy. They recognize the WXXL-like motif

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called LC3-interacting region in substrate proteins or adaptors [17]. The mechanism for selective cargo recognition is required for mitophagy. Atg32 was identified as the cargo receptor in yeast [18,

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19]. Atg32, which is an outer mitochondrial membrane protein, can interact with the isolation membrane protein Atg8 indirectly through Atg11 and directly through the WXXL-like motif [7].

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Although the mammalian Atg32 homolog has not yet been identified, selective elimination of

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damaged mitochondria has been observed. The mitochondrion-localized proteins, B-cell lymphoma (Bcl)-2/E1B 19kDa-interacting protein 3-like protein (Bnip3) and Nip3-like protein X (Nix; also

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known as Bnip3L) have been implicated in the removal of mitochondria during an autophagic response [20]. Nix functions as an autophagy receptor by binding to LC3/GABARAP proteins during reticulocyte maturation in maturing reticulocytes [21]. Bnip3 induces autophagy by interaction with

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LC3 in both mitochondria and ER [22]. It has also been reported that FUN14 domain-containing protein 1, which is localized in the outer mitochondrial membrane, mediates hypoxia-induced mitophagy [23]. PTEN-induced putative kinase protein 1 (PINK1) and the cytosolic E3 ubiquitin ligase, Parkin, are mutated in autosomal recessive Parkinson’s disease [7, 24, 25]. When mitochondria are damaged and lose mitochondrial membrane potential, PINK1 accumulates and recruits Parkin from the cytosol specifically to the damaged mitochondria. Several Parkin substrates, such as voltage-dependent anion channel 1 and mitofusins, have been identified [26, 27]. The sequestosome 1 (p62/SQSTM1) binds to ubiquitinated proteins on the mitochondria via its ubiquitin-associated domain and binds to LC3 on the isolation membrane with its WXXL-like motif, resulting in tethering mitochondria to the isolation membrane via the complex selectively [28]. Histone deacetylase (HDAC)6, as well as p62/SQSTM1, 7

ACCEPTED MANUSCRIPT is also recruited to ubiquitinated proteins on damaged mitochondria and microtubule dynein motors are required for Parkin to induce aggregation and clearance of impaired mitochondria [29]. Although the ubiquitin-binding adaptor p62/SQSTM1 binds to Parkin-ubiquitinated mitochondrial substrates,

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the requirement of p62/SQSTM1 as a mitophagy adaptor is under debate [2, 7]. Recent studies suggest that p62/SQSTM1 may be dispensable in this process [30], possibly due to redundancy with

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the related ubiquitin and LC3-binding protein neighbour of BRCA1 gene 1 [31]. The Bcl-2 family can regulate mitophagy through influencing the recruitment of Parkin to depolarized mitochondria. The

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prosurvival members of the Bcl-2 family suppress mitophagy through the inhibition of Parkin translocation to depolarized mitochondria. Conversely, BH3-only proteins accelerate Parkin

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recruitment to impaired mitochondria [32]. Ambra1 is recruited by Parkin to mitochondria in order to activate the Beclin 1 complex [33]. Parkin is expressed in many tissues [34], but some cell lines

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including HeLa cells show little or no endogenous Parkin expression [35, 36]. Thus, there may be an

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4. Autophagy in the heart

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unknown general receptor for mitophagy in mammalian cells.

Because of the high energy demand in the heart, mitochondria comprise at least 30% of the cardiomyocyte volume [37]. Rapid ablation of Atg5, in the adult heart by tamoxifen-inducible Cre-

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loxP recombination resulted in rapid left ventricular dilatation and contractile dysfunction [38]. The Atg5-deficient hearts showed the accumulation of ubiquitinated proteins, misalignment and heterogeneous size of mitochondria, and damage to the intramitochondrial structure. Thus, constitutive autophagy in the heart under baseline conditions is a homeostatic mechanism for maintaining cardiomyocyte size, global cardiac structure and function and mitochondrial morphology. In contrast, mice harboring a cardiomyocyte-specific deletion of Atg5 during cardiogenesis (Atg5f/f;MLC2v-Cre+) showed no cardiac hypertrophy or dysfunction well into adulthood, suggesting the existence of some sort of compensation for the complete loss of basal autophagy [38]. We reported the alternative pathway of autophagy, which is regulated by ULK1 and Beclin 1 [39]. The autophagosome formation depends on the small GTPase Rab9, but not Atg5/Atg7. Autophagosomes are generated in a Rab9-dependent manner by the fusion of isolation membranes with vesicles derived 8

ACCEPTED MANUSCRIPT from the trans-Golgi and late endosomes. The ULK1-dependent Atg5-independent autophagy is the dominant process of mitochondrial clearance from fetal definitive reticulocytes [40]. It is possible that the alternative autophagy may compensate the loss of conventional autophagy in the heart.

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of autophagy in cardiomyopathy in the following sections.

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However, the role of autophagy in cardiomyopathy remains to be elucidated. We will review the role

4.1. Autophagy in cardiac hypertrophy

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Cardiac hypertrophy is considered to be an initially adaptive response, counteracting the increased wall tension and helping to maintain cardiac output. However, if the heart is persistently exposed to

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increased load, cardiac hypertrophy may become maladaptive, leading to heart failure [41]. Cardiac hypertrophy is an independent risk factor for subsequent cardiac morbidity and mortality [42].

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Knockdown of Atg7 induced cardiomyocyte hypertrophy with typical characteristics in isolated rat

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neonatal cardiomyocytes [38]. HDAC inhibitors attenuated cardiac hypertrophy by suppressing autophagy [43]. The level of cardiac hypertrophy was similar between the cardiac-specific Atg5-

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deficient mice (Atg5f/f;MLC2v-Cre+) and the control mice at 2 weeks after continuous angiotensin II infusion [44] or 10 days after mild thoracic transverse aortic constriction (TAC), which did not induce cardiac dysfunction in either group [44]. These suggest that autophagy is not essential for the

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development of in vivo cardiac hypertrophy. 4.2. Autophagy in reverse remodeling Left ventricular hypertrophy is a dynamic process and not a static irreversible state [45]. Reverse remodeling such as regression of hypertrophy is a major therapeutic target for treating patients with cardiac hypertrophy. Regression of left ventricular hypertrophy has been documented after aortic valve replacement [46] or left ventricular assist device support (LVAD) [47]. Although various antihypertensive agents reduce left ventricular hypertrophy [48], the effect of anti-hypertensive treatment on cardiac hypertrophy is not satisfactory. Thus, it is necessary to identify the cellular and molecular mechanisms underlying regression of cardiac hypertrophy in order to develop novel and effective therapeutics for cardiac hypertrophy.

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ACCEPTED MANUSCRIPT Cardiac mass is determined by the balance between protein synthesis and degradation. Regression of cardiac hypertrophy could be induced by the downregulation of protein synthesis and/or the upregulation of protein degradation (Fig. 3). We infused angiotensin II by osmotic minipumps to the

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cardiac-specific Atg5-deficient mice (Atg5f/f;MLC2v-Cre+) for 2 weeks or subjected the mice to mild TAC for 10 days [44] and then removed the minipumps or released the TAC-induced pressure

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overload. The Atg5-deficient mice showed significantly less regression of left ventricular hypertrophy compared with the control mice 7 days after unloading. During the regression of cardiac hypertrophy,

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autophagy was upregulated in wild-type hearts. Hariharan et al. have reported significant regression of

cardiac hypertrophy 1 week after unloading pressure overload in wild-type mice, but not in Beclin 1

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heterozygous conventional knockout mice [49]. Autophagic activity and forkhead box class O (FoxO)1 protein expression are increased 1 week after unloading in wild-type mice. FoxO1 induces

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autophagy and is involved in mediating the regression of hypertrophy. Cao et al. have reported that

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mechanical unloading of the left ventricle by surgical transplantation of the heart leads to cardiac atrophy, increases autophagy and activates FoxO3 in wild-type mice [50]. These suggest that

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autophagy plays an important role in the regression of cardiac hypertrophy and that FoxO family proteins are critical regulators of autophagy after unloading the hypertrophic stimuli. Upregulation of autophagy mediated by FoxO family proteins may be a novel therapeutic target to reverse cardiac

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hypertrophy.

4.3. Autophagy in heart failure Although heart failure is a multifactorial clinical syndrome, the failing myocardium is viable but dysfunctional. The mechanisms underlying the development of heart failure are multiple, complex and not well understood. Mitochondrial dysfunction is observed in heart failure [51]. Damaged mitochondria generate reactive oxygen species (ROS), which damage macromolecules, namely proteins, lipids and DNA, leading to cell death (Fig. 1). Since mitochondria are the principal sites of ATP regeneration, myocardial ATP level drops in failing hearts, leading to AMPK signaling activation. Both ROS and AMPK can upregulate autophagy in failing hearts. Autophagy has been

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ACCEPTED MANUSCRIPT reported to be observed in failing myocardium [52]. Autophagy was upregulated in the failing wildtype mouse hearts 4 weeks after TAC [38]. When cardiac-specific Atg5-deficient mice (Atg5f/f;MLC2v-Cre+) were subjected to moderate TAC,

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the mice developed cardiac dysfunction and left ventricular dilatation within 1 week. The mice showed accumulation of polyubiquitinated protein, increased ER stress and promotion of apoptosis

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[38]. These results indicate that the compensatory mechanism operating at baseline may be overwhelmed and cardiomyocytes were unable to clear damaged protein. Thus, upregulation of

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autophagy in failing hearts is an adaptive response that protects cardiomyocytes. Recently, we reported that autophagy is also involved in inflammation observed in failing hearts [53].

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Mitochondria are evolutionary endosymbionts that were derived from bacteria and contain DNA similar to bacterial DNA, which represents inflammatogenic unmethylated CpG motifs. While no

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overt phenotype was observed under basal conditions, cardiac-specific ablation of DNase II, an acidic

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DNase in the lysosome, led to severe myocardial inflammation and dysfunction during pressure overload. In DNase II-deficient hearts, mitochondrial DNA (mtDNA) was accumulated in

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autolysosome. Genetically engineered Toll-like receptor (TLR)9 ablation or inhibition of TLR9 attenuated cardiac inflammation and dysfunction in DNase II-deficient hearts caused by pressure overload, suggesting that mtDNA induces inflammation mediated through TLR9 activation. TLR9-

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deficient mice subjected to pressure overload displayed attenuation of inflammatory cell infiltration and heart failure. Thus, the TLR9 signaling pathway plays a crucial role in recognizing undegraded mtDNA and modulating inflammation in stressed cardiomyocytes. The damaged mitochondria by TAC undergo an autophagic process. However, when mtDNA is not completely degraded by autophagy, TLR9 senses unmethylated CpG motifs in mtDNA and activates downstream signaling pathways which culminate in the production of proinflammatory cytokines. The secretion of the cytokines from cardiomyocytes recruits macrophages and neutrophils and then amplifies the inflammatory responses which injure the cardiomyocytes (Fig. 1). We found DNase II activity was upregulated in hypertrophic hearts but not in failing hearts after TAC. DNase II as well as TLR9 might be a therapeutic target for treating patients with heart failure.

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ACCEPTED MANUSCRIPT In contrast to the above findings, heterozygous deletion of Beclin 1, which partially reduces autophagic activity, improves cardiac function upon pressure overload [54]. In addition, robust ROS and severely damaged mitochondria can induce excessive autophagy and then autophagic cell death in

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failing hearts [55]. It is possible that excessive autophagy could be a maladaptive response [55].

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4.4. Autophagy in cardiomyopathy

Patients with Danon disease show hypertrophic and dilated cardiomyopathy, skeletal myopathy and

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mental retardation [56, 57]. LAMP2, a principal lysosomal membrane protein, is mutated in the disease. The mutation causes abnormal autophagosome accumulation because of defective fusion of

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autophagosomes with lysosomes in multiple organs, including the heart [58, 59]. A missense mutation in the B-crystallin gene (CryABR120G) triggers a severe form of desmin-

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related cardiomyopathy characterized by the accumulation of cytotoxic misfolded proteins.

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Autophagy plays an important role in protecting the heart from protein aggregation-induced cardiomyopathy such as desmin-related cardiomyopathy [60-62]. Diabetic cardiomyopathy is a heart

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muscle-specific disease that increases the risk of heart failure and mortality in diabetic patients independent of vascular pathology [63]. In diabetes, damaged mitochondria further increase ROS production through ROS-induced ROS release and induce cardiomyocyte death via leakage of pro-

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death factors (Fig. 1). The hyperglycemia and insulin resistance associated with diabetes activate the mTOR signaling pathway and then inhibit autophagy. Although cardiac autophagy is reduced, Rab9 expression is increased. The alternative autophagy dependent on Rab9 [39] may be upregulated and trigger mitophagy to protect the diabetic heart [64].

4.5. Autophagy in Aging The enlarged mitochondria characterized by swelling, loss of cristae and matrix derangement are important aspects of the age-related changes of cardiomyocytes [65]. The senescent mitochondria exhibit reduced ATP production and increased ROS generation. Since autophagy in the hearts becomes impaired during aging, the reduced autophagy may cause an accumulation of damaged mitochondria and the generation of ROS. 12

ACCEPTED MANUSCRIPT The cardiac-specific Atg5-deficient mice (Atg5f/f;MHC-Cre+) showed normal chamber size and contractile function at the age of 3 months. However, the mice began to die after the age of 6 months and exhibited a significant increase in left ventricular dimension and a decrease in the fractional

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shortening of the left ventricle at the age of 10 months, compared to control mice. The mice showed a disorganized sarcomere structure and collapsed mitochondria with decreased mitochondrial

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respiratory functions [66], indicating that inhibition of autophagy in the heart induces age-related cardiomyopathy. However, it is possible that the phenotype seen in the cardiac-specific Atg5-deficient

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mice could be time-dependent development of the phenotype and may be different from what is seen in actual aging, since the paper did not show a decrease in Atg5 expression during aging. In contrast,

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transgenic mice expressing Atg5 ubiquitously enhance autophagy and show anti-aging phenotypes including leanness, an increase in insulin sensitivity, improvement of motor function and an extended

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life span [67]. Parkin plays an important role in mitophagy [68]. The Parkin-deficient mice show

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mitochondrial dysfunction in aged hearts and a reduced life span compared with wild-type mice [69]. In contrast, the aged mouse hearts overexpressing Parkin increase mitochondrial turnover by

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autophagy, preserve mitochondrial function and attenuate proinflammatory cytokines and aging biomarkers compared with age-matched wild-type hearts [69]. These results suggest that continuous constitutive autophagy has a crucial role in maintaining cardiac structure and function during aging by

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controlling the quality of proteins and mitochondria.

5. Conclusions Autophagy has evolved as a conserved process for bulk degradation and recycling of cytoplasmic components, such as long-lived proteins and organelles. Autophagy has initially been considered to be a non-selective process as an adaptive response to nutrient starvation. However, it has been revealed that there are selective types of autophagy including mitophagy. Damaged mitochondria induce autophagy and mitophagy. Autophagy and mitophagy play an essential role in maintaining not only normal mitochondrial structure and function but also cellular homeostasis in the heart. Further

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ACCEPTED MANUSCRIPT elucidation of the mechanism of autophagy and mitophagy would have a significant impact on the treatment of heart disease.

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Financial support

This work was supported by British Heart Foundation (CH/11/3/29051, RG/11/12/29052) and King’s

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BHF Centre of Excellence (2E/08/003).

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Disclosures

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The authors declare no competing financial interests.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Incomplete degradation of damaged mitochondria by autophagy results in cardiomyocyte death and heart failure. Mitochondria are damaged by various stresses. Mitochondria segregate or exchange

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materials to repair damaged components through fission and fusion mechanism. Damaged daughter mitochondria are degraded by autophagy. ROS, harmful proteins and mtDNA leaked from undigested

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dysfunctional mitochondria directly damage cellular components. When mtDNA is not completely degraded by autophagy, the remaining mtDNA binds to TLR9 to generate proinflammatory cytokines.

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As a result, insufficient induction of autophagy results in cardiomyocyte death and heart failure.

Fig. 2. Autophagy is essential to maintain cellular homeostasis in the heart. Starvation-induced autophagy increases biosynthesis for a nutrient control. Autophagy prevents cardiac remodeling,

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inflammation or aging via quality control of proteins and mitochondria in response to pressure

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overload or long-lasting external stress during aging. Autophagy also involves in reverse remodeling

unloading.

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such as regression of cardiac hypertrophy via reduction of protein contents after hemodynamic

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Fig. 3. Cardiac mass is determined by the balance between protein synthesis and degradation including autophagy. Stresses such as hypertension and aortic stenosis induce cardiac hypertrophy (cardiac remodeling) through increased protein synthesis. Stress unloading using anti-hypertensive drugs, surgery and LVAD leads to regression of hypertrophy (reverse remodeling) by downregulation of protein synthesis and/or upregulation of protein degradation.

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Nakai et al. [38]

Nakai et al. [38] Oyabu et al. [44]

Zhu et al. [54]

Dnase II

CKO

Rapid progression of myocardial inflammation and cardiac dysfunction during TAC

Oka et al. [53]

LAMP2

conventional KO

Bcrystallin

CryABR120G CTg

Abnormal autophagosome accumulation and severely reduced heart muscle contractility from the aged mice Desmin-related cardiomyopathy characterized by the accumulation of cytotoxic misfolded proteins

Atg5

CKO

Tanaka et al. [58], Nishino et al. [59] Tannous et al. [60], Pattison et al. [61], Bhuiyan et al. [62] Oyabu et al. [44]

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Progression of cardiac dysfunction with cardiac hypertrophy 3 weeks after TAC

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Zhu et al. [54]

CTg

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Beclin 1

Aging

Reference

Beclin 1

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Reverse remodeling

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Table 1. Genetically modified mouse models to study the roles of autophagy in cardiomyopathy. Cardiac Gene Mouse model Phenotypes conditions Cardiac Atg5 CKO Progression of severe cardiac dysfunction, but hypertrophy similar cardiac hypertrophy compared with and heart control mice 1 week after TAC failure Atg5 timeRapid progression of cardiac dysfunction with dependent cardiac hypertrophy CKO Atg5 CKO Similar progression of cardiac hypertrophy between CKO mice and control mice in response to continuous angiotensin II infusion or mild TAC Beclin 1 heterozygous Less progression of cardiac dysfunction conventional compared with wild-type mice, but similar heart KO to body weight ratio 3 weeks after severe TAC

heterozygous conventional KO

Less regression of cardiac hypertrophy compared with the control mice 7 days after unloading continuous angiotensin II infusion or mild TAC Regression of cardiac hypertrophy 1 week after unloading pressure overload in wild-type mice, but not in Beclin 1 heterozygous KO mice

Hariharan al. [49]

et

Atg5

CKO

Reduced life span because of cardiac dysfunction with disorganized sarcomere structure and mitochondrial dysfunction

Taneike et al. [66]

Atg5 Parkin

ubiquitous Tg conventional KO

Extended life span with anti-aging phenotype Normal cardiac function for up to 12 months of age, but cardiac dysfunction and mitochondrial dysfunction in aged hearts and reduced life span

Parkin

CTg

Pyo et al. [67] Hoshino et al. [69], Kubli et al. [70], RodriguezNavarro et al. [71] Hoshino et al. [69]

Preserved cardiac function, preserved mitochondrial function and attenuated aging biomarkers compared with age-matched wildtype hearts Abbreviations: CKO, cardiac-specific knockout; CTg, cardiac-specific transgenic; KO, knockout; Tg, transgenic

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