Autophagy in cardiomyopathies

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Autophagy in cardiomyopathies Antonia T.L. Zecha,b,1, Sonia R. Singhc,1, Saskia Schlossareka,b,1, Lucie Carriera,b, a b c

⁎,1

Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg, Hamburg, Germany German Centre for Cardiovascular Research, Partner Site Hamburg/Kiel/Lübeck, Hamburg, Germany Division of Molecular Cardiovascular Biology, The Heart Institute, Cincinnati Children's Hospital, Cincinnati, OH, United States of America

ARTICLE INFO

ABSTRACT

Keywords: Autophagy Autophagosome Lysosome Heart Cardiomyopathy

Autophagy (greek auto: self; phagein: eating) is a highly conserved process within eukaryotes that degrades longlived proteins and organelles within lysosomes. Its accurate and constant operation in basal conditions ensures cellular homeostasis by degrading damaged cellular components and thereby acting not only as a quality control but as well as an energy supplier. An increasing body of evidence indicates a major role of autophagy in the regulation of cardiac homeostasis and function. In this review, we describe the different forms of mammalian autophagy, their regulations and monitoring with a specific emphasis on the heart. Furthermore, we address the role of autophagy in several forms of cardiomyopathy and the options for therapy.

1. Introduction The concept of “dynamic state of body components” has been introduced more than 70 years ago by Rudolf Schoenheimer, who used for the first time isotopic tracer technique in metabolic research. His experiments demonstrated that the body structural proteins are in a dynamic state of synthesis and degradation [1]. Protein homeostasis is maintained by molecular chaperones that refold misfolded or mutant proteins and by one of the two major proteolytic systems, which are the ubiquitin-proteasome system (UPS) and autophagy. The UPS selectively marks proteins with ubiquitin moieties via a cascade of ubiquitin enzymes and efficiently degrades them one-by-one in the 20S proteasome core [2]. However, the UPS-mediated degradation could not explain how the cell eliminates larger protein aggregates and organelles. In the mid-1950's, the Belgian researcher Christian de Duve discovered the lysosomes [3] and was then awarded a share of the 1974 Nobel Prize in Physiology or Medicine for elucidating “the structural and functional organization of the cell”. Further observations revealed a new intracellular vesicle transporting cargo for degradation towards the lysosome and de Duve coined the term ‘autophagy’ to describe this transport. The essential role of autophagy in physiology and medicine was recognized after Yoshinori Ohsumi's research in the 1990's with the identification of many autophagy-related genes (Atg) involved in the yeast autophagy process [4]. For his discoveries, he was awarded the 2016 Nobel Prize in Physiology or Medicine. Autophagy regulates

several physiological processes that are critical for preservation of cellular function. This is particularly important in the heart because post-mitotic cardiomyocytes cannot regenerate and are therefore dependant on accurate functioning protein quality control machinery. 2. Different forms of autophagy in the heart There are three major forms of autophagy. Macroautophagy (hereafter called autophagy) involves the formation of an autophagosome, enclosing cellular proteins, debris or organelles for delivery to the lysosome for degradation (Fig. 1). Microautophagy, on the contrary, does not require autophagosome formation but direct engulfment of cellular debris/components by the lysosome. Chaperone-mediated autophagy (CMA) degrades proteins that are translocated into the lysosome via a chaperone complex (Fig. 1). Since there is not a lot known about microautophagy in the heart, we will not go into further detail of microautophagy. 2.1. Autophagy Autophagy starts with the formation of a double-membraned vesicle called autophagosome, thereby engulfing cytoplasmic proteins or organelles, followed by the fusion of the autophagosome with the lysosome to form the auto(phago)lysosome, in which the cargo is degraded by lysosomal enzymes. Autophagy can be either selective or non-

Corresponding author at: Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. E-mail address: [email protected] (L. Carrier). 1 All authors contributed equally to this review. ⁎

https://doi.org/10.1016/j.bbamcr.2019.01.013 Received 23 November 2018; Received in revised form 24 January 2019; Accepted 28 January 2019 0167-4889/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Antonia T.L. Zech, et al., BBA - Molecular Cell Research, https://doi.org/10.1016/j.bbamcr.2019.01.013

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mTOR complex 1

AMPK

Macroautophagy

ULK complex

Chaperone-mediated autophagy (CMA)

Protein aggregate Misfolded/unfolded protein

Beclin-1/Vps34 complex

KFERQ

Lysosome

LC3-I Atg7 Atg3 + PE Atg12-Atg5-Atg16L LC3-II

Hsc70 complex

Phagophore

Mitophagy

Autophagosome

Mito

Proteins

Glycophagy

Ubiquitin

Ferritin

Selective autophagy

Glycogen

Protease Lysophagy Ferritinophagy

Mitochondrion

Fig. 1. Autophagy mechanisms identified in the heart. The best studied regulators of macroautophagy in the heart are the mTOR complex 1 (negative regulator) and AMPK (positive regulator). If macroautophagy becomes activated, the phagophore structure is initiated by the ULK complex and Beclin-1/Vps34 complex. LC3-I is lipidated to LC3-II in multiple steps, involving the ubiquitin-like conjugation systems Atg7, Atg3 and Atg12-Atg5-Atg16L and is then incorporated into the phagophore membrane. Cellular waste such as excess or damaged proteins or protein aggregates can be engulfed by the phagophore either by bulk autophagy or through shuttle proteins such as p62 or NBR1 that can bind LC3-II. The membrane is then elongated and develops into an autophagosome, which is transported through the cell and eventually fuses with a lyosome to form an autolysosome, in which its content is degraded. Described forms of selective autophagy in the heart are mitophagy, glycophagy, lysophagy and ferritinophagy. Each of them is recognized by selective receptor proteins (described in the text). In chaperone-mediated autophagy (CMA), the Hsc70 complex recognizes a KFERQ-like sequence on a target protein and binds LAMP-2A on the lysosomal membrane, which then forms a multimeric complex that translocates the target protein for degradation into the lysosome.

selective. Non-selective autophagy comprises of bulk degradation of cytoplasmic content, whereas selective autophagy specifically targets damaged or superfluous cargo for degradation (Fig. 1; see Section 2.2).

and VPS34). Phosphorylation of Beclin-1 by ULK1/2 activates the VPS complex that results in the formation of phosphatidylinositol-3-phosphate (PI3P), which in turn triggers membrane elongation that develops into an autophagosome [10–13]. Autophagosome elongation and maturation is guided by two ubiquitin-like conjugation systems, ATG7-ATG3 or ATG7-ATG10 and ATG12-ATG5-ATG16L [9,14]. The E1-like activating enzyme ATG7 and the E2-like conjugating enzyme ATG10 activate and conjugate ATG12 to the lysine residue of ATG5. Then, an E3-like ligase complex is formed together with ATG16L. This ATG12-ATG5-ATG16L complex associates with the phagophore membrane. In parallel, microtubule-associated protein 1 light chain 3 (LC3), γ-aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16) are cleaved by the cysteine protease ATG4, revealing a Cterminal glycine [15–17]. Cleaved LC3 (LC3-I) is first activated by ATG7 that conjugates a phosphatidylethanolamine (PE) group and then transferred to ATG3 and incorporated into the autophagosomal membrane, resulting in LC3-II [18]. LC3-II found on the outer membrane of the autophagosome can be recycled by ATG4 that cleaves off the PE [16], whereas LC3-II found inside of the autophagosome is degraded with the cargo. LC3-II is important for cargo recognition by recruiting the shuttle proteins p62/SQSTM1 and neighbour of BRCA1 (NBR1), autophagosome biogenesis (elongation and membrane closure) and

2.1.1. Autophagy process Discovered first by genetic screens in yeast [4], over 30 autophagyrelated genes (ATG; Table 1) have been identified in mammalian cells, which encode proteins regulating the different steps of autophagy process. This process involves the nucleation, elongation and maturation of autophagosomes, followed by their fusion with the lysosomes to form auto(phago)lysosomes. Autophagosome formation starts with the generation of an isolation membrane called phagophore, commonly originated from endoplasmic reticulum (ER), but also from other types of membranes (e.g. sarcolemma, Golgi or mitochondria; [5–8]). This step is facilitated by the ULK macromolecular complex, composed of ATG13, unc-51-like autophagy-activating kinase 1 or 2 (ULK1/2), the focal adhesion kinase family interacting protein of 200 kDa (FIP200), and ATG101 [9]. Upon ULK complex activation, ATG9 is stimulated to recruit vesicles to the phagophore assembly site to begin autophagosome nucleation. Subsequently, the ULK complex activates another macromolecular complex implicated in the initiation step of autophagosome formation, composed of Beclin-1, ATG14L, vacuolar protein sorting 15 and 34 (VPS15 2

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Table 1 Major mammalian proteins involved in mammalian autophagy. Gene

Protein

Function Part of ULK-ATG13-ATG101-FIP200 complex - autophagy initiation

RB1CC1 WIPI1 WIPI2 WDR45B WDR45 ATG101 VPS15 VPS34

ULK1 ULK2 ATG2A ATG2B ATG3 ATG4A ATG4B ATG4C ATG4D ATG5 Beclin-1 ATG7 LC3A LC3B LC3C GABARAP GABARAPL1 GATE-16 GABARAPL3 ATG9A ATG9B ATG10 ATG12 ATG13 ATG14L ATG16L1 ATG16L2 FIP200 WIPI1 WIPI2 WIPI3 WIPI4 ATG101 VPS15 VPS34

Selected shuttling proteins BAG3 HDAC6 NBR1 SQSTM1 PRKN PINK1

BAG3 HDAC6 NBR1 p62 Parkin PINK1

Selective autophagy Selective autophagy Selective autophagy Selective autophagy Mitophagy Mitophagy

Selected regulators PRKAA1 FOXO1 MTOR SIRT1 TFEB

AMPK FoxO1 mTOR SIRT1 TFEB

Positive regulator (kinase) Positive regulator (binds to ATG7) Negative regulator (kinase) – part of mTORC1 complex (mTOR-RAPTOR-PRAS40-DEPTOR) Positive regulator (acetylation) Positive regulator (transcription factor)

Selected fusion proteins FYCO1 PLEKHM2 RAB7A RAB7B STX17 SNAP29 VAMP8

FYCO1 PLEKHM2 Rab7a Rab7b Syntaxin 17 SNAP29 VAMP8

Autophagosome-lysosome fusion Autophagosome-lysosome fusion Autophagosome-lysosome fusion

LAMP-1 LAMP-2A LAMP-2B LAMP-2C Cathepsin D Cathepsin A

Membrane protein Membrane proteins

Autophagy key machinery ULK1 ULK2 ATG2 ATG3 ATG4

ATG5 BECN1 ATG7 MAP1LC3A MAP1LC3B MAP1LC3C GABARAP GABARAPL1 GABARAPL2 GABARAPL3 ATG9 ATG10 ATG12 ATG13 ATG14 ATG16

Selected lysosomal proteins LAMP1 LAMP2 CTSD CTSA

Part of ATG2-WIPI complex - ATG9 recruitment and autophagosome expansion Part of ATG7-ATG3 complex - LC3 lipidation LC3 activation (cleavage) and delipidation

Part of ATG12-ATG5-ATG16L complex - LC3 lipidation Part of Beclin-1-VPS34- complex - autophagy initiation Part of ATG7-ATG3 complex - LC3 lipidation Proteins that can be lipidated and incorporated into autophagosomal membrane - important for autophagosome formation and maturation, shuttling and fusion with lysosomes

Membrane carrier, phagophore formation Involved in ATG12 conjugation to ATG5 Part of ATG12-ATG5-ATG16L complex - LC3 lipidation Part of ULK-ATG13-ATG101-FIP200 complex - autophagy initiation Autophagy initiation Part of ATG12-ATG5-ATG16L complex - LC3 lipidation Part of ULK-ATG13-ATG101-FIP200 complex - autophagy initiation Part of ATG2-WIPI complex - ATG9 recruitment and autophagosome expansion

Part of ULK-ATG13-ATG101-FIP200 complex - autophagy initiation Part of Beclin-1-VPS complex - autophagy initiation Part of Beclin-1-VPS complex - autophagy initiation

Autophagosome-lysosome fusion, STX17-SNAP29-VAMP8 complex

Lysosomal proteases

Gene names and references are given in the text.

autophagosome-lysosome fusion. However, the precise mode of action needs to be further elucidated [9]. The last step of autophagy involves the fusion of the autophagosome with a lysosome. Autophagosomes form within the cytoplasm and are then transported along microtubules in a dynein-dependant manner to

the perinuclear region. Similarly, lysosomes are transported along microtubules to the perinuclear region but in a pH-dependant manner [19]. Autophagosomes and lysosomes can fuse in two different forms: a complete fusion resulting in a hybrid organelle, called the autophagolysosome, or a kiss-and-run fusion, where only content is 3

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unidirectionally transferred from the autophagosome to the lysosome while both keeping their own vesicle's integrity. This transient membrane content exchange is independent of lysosomal acidification and seems to be characterized by multiple ‘kissing’ events [20]. The actual fusion event itself is thought to be conducted by Rab GTPases (e.g. RAB7), membrane-tethering complexes and soluble N-ethylmaleimidesensitive-factor attachment receptor (SNARE) proteins [21]. Lysosomeassociated membrane protein-2 (LAMP-2) seems to be a crucial factor for autophagosome-lysosome fusion, since its absence leads to an accumulation of autophagic vacuoles [22]. This might be due to the fact that LAMP-2 is necessary for the proper incorporation of syntaxin-17 (Qs-SNARE) into the autophagosomal membrane in order to interact with VAMP8 (R-SNARE) on lysosomes, and thus to enable the fusion [23].

cells. Under nutrient-rich conditions, mTORC1 is activated and localized on peripheral lysosomes, whereas under starvation, lysosomes move to the perinuclear region and mTORC1 is inactivated [19]. Lysosomes can directly modulate mTORC1 by sensing the level of amino acids within its lumen via the vacuolar-type H+-ATPase (V-ATPase) [33]. On the other hand, mTORC1 phosphorylates and inhibits the nuclear transport of transcription factor EB (TFEB), which regulates transcription of ATG genes and lysosomal biogenesis [34]. Furthermore, active mTORC1 negatively regulates lysosomal activity by inhibiting the ATP-sensitive Na+-channel [35]. However, if starvation persists for too long, mTORC1 is reactivated and enables autophagic lysosomal reformation [36]. Another level of autophagy regulation constitutes of post-translational modifications, such as phosphorylation, ubiquitination and acetylation [37]. As mentioned above, key events of autophagosome formation are controlled by phosphorylation. Furthermore, recent evidence indicates the presence of phosphorylation sites for cAMP-dependent protein kinase (PKA) [38] and protein kinase C [39] in the Nterminal region of LC3. PKA-mediated LC3 phosphorylation prevents LC3 incorporation into the autophagosome [38]. Another essential post-translational modification regulating autophagy is protein ubiquitination, which is performed by a series of ubiquitin enzymes composed of E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase. Protein ubiquitination serves as a signal for degradation of protein aggregates and organelles, therefore playing an important role in selective autophagy. Cargo receptors such as p62/ SQTM1 and NBR1 recognize ubiquitinated proteins/protein aggregates and shuttle them to the forming autophagosome, thus enabling degradation within autolysosomes [37]. Finally, acetylation, which consists in the addition of an acetyl group onto lysine or N-termini of proteins, plays a role in the regulation of autophagy. The best known candidate regulating autophagy is the histone de-acetylase 6 (HDAC6), which, by interacting with acetylated tubulin, mediates the retrograde transport of protein aggregates for autophagy degradation [40]. In addition, the acetyltransferase p300 can acetylate several ATG proteins and inhibit autophagy.

2.1.2. Autophagy regulation in the heart Several signaling pathways regulate autophagy in the heart [24]. Under normal conditions, autophagy is exerted at low basal levels but is highly activated in response to stress to keep cellular homeostasis and ensure energy supply. The most studied regulators of autophagy in the heart are the serine/threonine kinase, mammalian/mechanistic target of rapamycin (mTOR) and the AMP-activated protein kinase (AMPK; Fig. 1). mTOR is activated by nutrients and growth factors and inhibited during starvation. In conjunction with other proteins, mTOR forms either the mTORC1 or mTORC2 protein complex, which both play essential roles within the heart, but only mTORC1 regulates autophagy [24]. The mTORC1 consists essentially of mTOR itself, regulatory-associated protein of mTOR (RAPTOR), mammalian lethal with SEC13 protein 8 (mLST8), proline-rich AKT substrate of 40 kDa (PRAS40), and DEP domain-containing mTOR-interacting protein (DEPTOR). The activity of mTORC1 can be inhibited by rapamycin that forms a complex with FK506-binding protein of 12 kDa (FKBP12) in mammalian cells, stabilizing the RAPTOR-mTOR association and inhibits the kinase activity of mTOR [25]. Under nutrient-rich conditions, mTOR phosphorylates both ATG13 and ULK1/2, inhibiting the ULK complex activity [26]. When mTOR is inhibited, ULK1/2 autophosphorylates and subsequently phosphorylates ATG13 and FIP200, initiating phagophore formation [26]. Therefore, the ULK-ATG13-FIP200 complex is a direct target of mTOR and acts as an integrator of the autophagy signals downstream of mTORC1. A major pathway controlling mTORC1 is the phosphatidylinositol 3-kinase (PI3K) pathway. Binding of growth factors or insulin to cell membrane receptors activates PI3K and thus protein kinase B/AKT that phosphorylates and inactivates tuberous sclerosis protein 1 or 2 (TSC1/2), leading to mTORC1 activation and thus autophagy inactivation. Energy suppliers directly inhibit AMPK, leading to inhibition of TSC1/2 and autophagy inhibition. AMPK also inhibits c-jun N-terminal kinase (JNK), which under starvation activates autophagy by multi-site phosphorylation (T69, S70, S87) of B-cell lymphoma 2 (Bcl-2) and thus promoting its dissociation from Beclin-1 [27]. In case of low levels of nutrients (low ATP levels), AMPK is activated and leads to TSC1/2 activation, mTORC1 inhibition, and phagophore initiation. During energy stress, mTORC1 can also be inactivated by glycogen synthase kinase 3 beta (GSK3β), which in turn also activates TSC1/2. Under glucose deprivation, it was shown that hexokinase-II, normally important for glycolysis, inhibits mTORC1 and thus promotes autophagy. During oxidative stress, mTOR is oxidized at Cys1483, promoting autophagy [28]. Furthermore, a growing body of evidence indicates that microRNAs can regulate autophagy in the heart. Some decreased autophagy, such as miR-212 and miR132, which target the forkhead box protein O3 (FoxO3) that regulates transcription of ATG genes [29–31]. Others increased autophagy by targeting mTOR, such as miR-99a [32]. There is also a strong interconnection of lysosomes/lysosomal organization and mTORC1 activity, to date solely shown in non-cardiac

2.1.3. Autophagy monitoring in the heart Several approaches to monitor autophagy have been described in detail [41], and can be used to evaluate cardiac autophagy in the heart or in isolated cardiomyocytes. Autophagy can be monitored by evaluating the steady-state levels of autophagy-related proteins or by determining the autophagic activity/flux. The steady-state levels of autophagic markers (e.g. LC3-II) can give a hint on whether the autophagy pathway is dysregulated or not. However, increased levels of LC3-II indicate either enhanced autophagosome formation or impaired clearance. Therefore, treatment with a autophagy modulator (e.g. bafilomycin A1 or chloroquine) is required to determine the autophagic flux. Moreover, to properly validate autophagy, a combination of several assays is recommended. Visualization of double-membrane autophagosomes, single-membrane lysosomes or autolysosomes can be performed by transmission electron microscopy (TEM) in the heart [22,42–50]. Labelling is not required, but highly skilled personnel are and quantification is difficult. The main markers of autophagy (LC3-II, p62) have been widely quantified in heart or cardiomyocyte protein extract by Western blot [42,45,46,51,52] and can also be visualized and/or quantified by immunofluorescence, immunohistochemistry or flow cytometry. To date, the best method to assess autophagic activity is the evaluation of the autophagic flux. Here, one profits of the lipidation of LC3I, generating LC3-II. LC3-I is cytosolic, whereas LC3-II is found on the membrane of autophagosomes. After applying a lysosomal inhibitor, such as bafilomycin A1, chloroquine, leupeptin, pepstatin A or E64d, that prevents autophagosome-lysosome fusion or degradation within autolysosomes, there is an increase in LC3-II. When compared to the basal levels of LC3-II, an assessment of the rate of autophagosome 4

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Table 2 Different forms of autophagy studied in the heart and their characteristics. Type

Marker

Adaptor

Cargo

Regulating pathways

References

Autophagy (macroautophagy)

LC3-II, p62

p62, NBR1

K63-linked polyubiquitnated proteins

[9,14,28]

Chaperone-mediated autophagy

LAMP-2A

Ferritinophagy Glycophagy Lysophagy Mitophagy

NCOA4, LC3 GABARAPL1 LC3-II, p62 p62

Hsc70, LAMP-2A NCOA4 STBD1 p62 p62, Bnip3, Bnip3-like protein

KFERQ-like motif proteins Ferritin Glycogen Ubiquitinated lysosomes Mitochondria/ubiquitinated mitochondrial proteins

mTOR, PKA, Insulin/PKB, AMPK, GSK3β Unknown Unknown cAMP/PKA, AKT/mTOR, Ca2+ Unknown Parkin/PINK1

[41,75] [63,72] [76–78] [63,139]

[9,80,81,84]

association of the adaptor proteins with LC3-II thus enables the uptake of the mitochondria into the autophagosome (for reviews, see [63,64]). A recent study proposes a microautophagy-like-fashion pathway, where mitochondrial components are selectively packaged into small mitochondrial-derived vesicles (70–150 nm), and transported to the lysosome for degradation of their content. This pathway is supposed to keep mitochondrial homeostasis within the heart and to bridge the time interval upon stress until mitophagy takes over [65]. Mitochondrial turnover can be visualized with MitoTimer since it is tagged with an inner mitochondrial membrane signal and further mutated to change its fluorescence from GFP to DsRed within 48 h under oxidizing conditions. Recently, a conditional MitoTimer mouse model has been published [66,67]. In order to follow mitophagy, the Mito-Qc or Mt-Keima approach can be used. Mito-Qc consists of the mitochondrial fission protein 1 (FIS1) and a tandem mCherry-GFP tag. Under normal conditions the mitochondrial network is visible in red and green fluorescence, while during mitophagy mitochondria are delivered to the lysosomes where mCherry fluorescence remains stable, but GFP fluorescence is quenched due to the low pH within the lysosomes [68]. The Mt-Keima protein works in a similar way. It is tagged to a mitochondrial protein (e.g. COX VIII) and exhibits a pH-dependent excitation, while being resistant to lysosomal proteases that allows imaging of mitophagy as well as mitophagic flux [69,70]. Further, mitophagy can be specifically induced by the uncoupling agents carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and 2.4-dinitrophenol [63]. However, the uncoupling approaches are difficult to use for pathophysiological analyses in vivo, because mitochondria in tissue never completely loose mitochondrial membrane potential, which is a prerequisite for this methods. In order to inhibit mitophagy, a common autophagosome-lysosome fusion inhibitor such as bafilomycin A1 or knockout experiments (e.g. siRNAs) could be applied. Generally, whether mitophagy is impaired or not can be investigated by determining the levels of mitochondrial proteins (e.g. COX4I1), the amount of mitochondrial mass (e.g. Mitotracker, TEM, amount of mitochondrial DNA), the amount of LC3 puncta, the co-localization of LC3 (e.g. GFPRFP-LC3) and mitochondria (e.g. MitoTracker), the co-localization of autophagosomes and mitochondria (e.g. by TEM), and the co-localization of lysosomes (e.g. LysoTracker, LAMP-2) and mitochondria (e.g. MitoTracker, FIS1) and by the binding of LC3-II to mitochondria [41,71].

formation/turnover can be made. Autophagic flux has been evaluated in several models of cardiac hypertrophy either in cardiomyocytes or in vivo [44–46,53]. LC3 reporter constructs (e.g. GFP-LC3, mRFP-GFP-LC3, mTagRFPmWasabi-LC3) can be used to quantify autophagosome number (amount of LC3-positive puncta) after gene transfer in cardiomyocytes [44,53,54]. A tandem reporter construct is the better choice, since one can make use of the fact that the GFP/mWasabi fluorescence is quenched within lysosomes due to the low pH, whereas mRFP/mTagRFP maintains its fluorescence. Therefore, early autophagosomes are visible as yellow dots, whereas late autophagosomes/autolysosomes appear as red dots. Several in vivo models have been generated in order to study autophagy and its flux (after autophagy modulator treatment or starvation) in GFP-LC3 mice, cardiomyocyte-specific mCherry-LC3 mice, or cardiomyocyte-specific mRFP-GFP-LC3 mice [55–58]. 2.2. Selective forms of autophagy in the heart There is an increasing body of evidence indicating that autophagy can be a highly selective process that specifically degrades its targets, for instance mitophagy for mitochondria, glycophagy for glycogen, and lysophagy for lysosomes (Table 2, Fig. 1). In this review, we focused on selective forms of autophagy, for which research in the heart has been done. Other forms of selective autophagy very likely occur in the heart, but have not been investigated yet. 2.2.1. Mitophagy The term mitophagy describes the specific degradation of dysfunctional or long-lived mitochondria within lysosomes as part of the quality control system and nutrient provision within a cell. Especially in the heart, mitophagy is of great importance due to the high density of mitochondria (great need for energy), as well as its low regenerative potential. Mitophagy is induced by high levels of reactive oxygen species (ROS), a byproduct of ATP generation, as well as low levels of ATP, off-balanced mitochondrial membrane potential and mitochondrial DNA. Mitochondrial DNA is known to induce an immune response due to its strong resemblance to bacterial DNA. The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway is the best studied signaling that regulates mitophagy. In healthy mitochondria, the serine/threonineprotein kinase PINK1 is degraded within mitochondria. However, if the membrane potential is depolarized (damaged mitochondria), PINK1 cannot be taken up and remains on the outer membrane of the mitochondrion and is activated through auto-phosphorylation, hence recruiting the E3 ubiquitin ligase Parkin. PINK1 phosphorylates ubiquitin on damaged mitochondria and Parkin, which both lead to Parkin activation [59]. After being dissociated from the mitochondrial network (fission machinery), mitochondria can be recruited to the autophagosome after ubiquitination of outer membrane proteins and LC3 receptors such as NIP3-like protein X (NIX) [60], BCL2 interacting protein 3 (BNIP3) [61] or FUN14-domain containing protein 1 (FUNDC1) [62], by the E3-ubiquitin ligase Parkin. At the autophagosome, the

2.2.2. Glycophagy Another type of selective autophagy is glycophagy, where glycogen is selectively degraded by the autophagic machinery in order to maintain glucose homeostasis within the cell/heart. Therefore, glycogen is recruited by the adaptor starch-binding domain-containing protein 1 (STBD1) via its carbohydrate-binding domain to the autophagosome. Here, STBD1 associates with γ-aminobutyric acid receptorassociated protein-like 1 (GABARAPL1) that in turn facilitates the uptake of glycogen into the autophagosome and its subsequent degradation within the lysosomes. An interaction between STBD1 and LC3 5

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could not be determined (for review, see [63]). Glycophagy seems to be of particular importance in the heart, especially under stress, in diabetes (see below) and in the glycogenstorage Pompe disease, which is caused by the deficiency of the lysosomal α-glucosidase leading to glycogen accumulation in the lysosome and cardiac hypertrophy. Furthermore, glycophagy is transiently upregulated in the hearts of neonates and after prolonged starvation to ensure energy supply. Glycophagy seems to act in concert with the conventional breakdown of glycogen via the glycogen phosphorylase, regulated by PKA. On the other hand, glycophagy can provide larger amounts of glucose to the cell in need of energy and degrades misformed glycogen. Generally, glycogenesis is induced by the AKT/GSK3β signaling. Glycophagy can be induced by PKA signaling, inducing increased glucose uptake into the cell. Another regulating mechanism constitutes the AKT/mTOR signaling. Under nutrient-rich conditions (insulin), mTOR shuts down glycophagy by inhibiting protein phosphatase 2A (PP2A) and therefore reduces the synthesis of α-glucosidase. Furthermore, glycophagy can be enhanced by Ca2+ influx into the lysosomes as a consequence of cAMP-induced activation of the lysosomal membrane Ca2+ pump. Hence, the degradation of glycogen via the lysosomal α-glucosidase is elevated. There are not too many publications related to glycophagy monitoring. However, general autophagy monitoring assays can be applied, such as immunoblot (e.g. STDB1, GABARAPL1), TEM (glycogen-particles, autophagosomes) and immunohistochemistry (pPeriodic AcidSchiff (PAS)-glycogen). Furthermore, a glycogen assay, where the amount of cardiac glycogen is measured after amyloglucosidase digestion has been used [72].

LLOMe (L-Leucyl-L-leucine methyl ester)), autophagy modulators (e.g. bafilomycin A1), and silica and monosodium urate [76]. 2.3. Chaperone-mediated autophagy (CMA) 2.3.1. CMA process CMA is a selective form of autophagy that only degrades proteins carrying a KFERQ-like motif within lysosomes (Fig. 1) [79]. Bioinformatical analysis identified a KFERQ-like motif in about 30% of all cytosolic proteins, but at the moment few CMA substrates have been validated. The cytosolic chaperone heat shock cognate protein of 70 kDa (Hsc70) recognizes the target protein with its KFERQ-like motif and builds a complex with modulatory co-chaperones (Bag1, Hip, Hop, Hsp40) that associates with LAMP-2A on the lysosomal membrane. LAMP-2A binds to the cargo as a monomer with its cytosolic tail. However, LAMP-2A needs to form a 700 kDa multimeric translocation complex that is stabilized by luminal Hsc90. After being unfolded, the target protein is translocated into the lumen of the lysosome with the help of the lysosomal Hsc70 [80]. Then, lysosomal hydrolases degrade the target within the lysosomal lumen. LAMP-2A recognizes its target not by the KFERQ-like motif, but by a specific domain with four positive charged amino acids. The binding motif of the substrate has not been identified yet [80,81]. 2.3.2. CMA regulation in the heart So far, there is not much known about the signaling that regulates CMA. Basal activity as well as stress-induced upregulation of CMA (e.g. oxidative stress, exposure to toxics, prolonged starvation) has been reported [82,83]. This indicates its role as part of the protein quality machinery and amino acid supplier. For instance, long periods of starvation can induce CMA with a peak activity at 24 h, as shown by higher levels of LAMP-2A and Hsc70. Further, posttranslational modifications are known to induce CMA for certain proteins (for review, see [81]). CMA itself is mainly regulated by LAMP-2A, since its levels determine CMA activity. Therefore, LAMP-2A is tightly regulated on transcriptional level, and by its degradation at the lysosomal membrane via cathepsin A, and by its continuous assembly and disassembly by membrane-associated Hsc70 [80,84]. It appears that LAMP-2A levels are first increased by the blockage of its degradation and then by recruiting LAMP-2A of the luminal resident pool found in lysosomes. Further, the LAMP-2A translocation complex can be stabilized by the association of glial fibrillary acidic protein (GFAP). In the presence of cytosolic GTP, the elongation factor 1 alpha (EF1α) enables the dissociation of GFAP from the translocation complex, leading to the selfassembly of GFAP and therefore is neutralizing GFAPs stabilizing function on the translocation complex [9,80]. To date, not much is known about CMA in the heart. One study reported that CMA removes oxidized ryanodine receptor type 2 in cultured neonatal rat cardiomyocytes [85]. However, most studies were performed in liver, kidney, lungs and spleen.

2.2.3. Ferritinophagy Ferritinophagy describes the selective degradation of the chelating protein complex ferritin by macroautophagy. Ferritin is guided by the nuclear receptor coactivator 4 (NCOA4) towards the autophagosome for its subsequent degradation within the lysosome (for review, see [41]). Although the role of ferritinophagy in the heart has yet to be established, it is likely that it plays an important role since it is known that patients with iron overload present with cardiomyopathy [73]. Furthermore, high levels of serum ferritin have been associated with a higher risk of new-onset heart failure, at least in apparently healthy women [74]. To visualize ferritinophagy, iron can be detected within autolysosomes, either by TEM or by co-localization studies using calcein-AM as iron tag [41]. Furthermore, general autophagy modulators (e.g. rapamycin, bafilomycin A1, torin 1) have been used to evaluate the ferritinophagic flux in senescent cells. Moreover, endogenous levels of autophagy marker were evaluated by immunoblotting (e.g. p62; [75]). 2.2.4. Lysophagy Lysosomes are not solely a part of the autophagic machinery. They can also be targeted by autophagy as in regulating their own fate as part of the cell quality control. Thus, damaged or long-lived lysosomes are coated with galectin-3 (Gal3) protein and subsequently ubiquitinated (for review, see [76]). Next, ubiquitinated lysosomes are engulfed by autophagosomes and degraded within functioning lysosomes after fusion. It was shown that p62 and LC3 are involved in this process [77]. Lysophagy is of particular interest in the context of Danon disease, an Xchromosomal inherited disease associated with cardiomyopathy, myopathy and mild retardation (see Section 3.3). The role of lysophagy in the heart has to be established. Several approaches can be pursued to monitor lysophagy, including live cell imaging with GFP/RFP-tagged proteins (localization/amount of EGFP-Ubiquitin, EGFP-LC3B, LAMP-1-RFP, TagRFP-p62, GFP-Gal3), immunoblotting of LC3 turnover, and live cell imaging of LC3 turnover (RFP-EGFP-LC3) or lysophagy (RFP-GFP-Gal3; [77,78]). However, there is a need to pharmacologically challenge lysosomes in order to solely evaluate lysophagy, such as with lysosomotropic drugs (e.g.

2.3.3. CMA monitoring The most obvious way to monitor CMA is to validate the CMA key player LAMP-2A and Hsc70 by immunoblotting (basal levels), immunofluorescence and co-localization studies (LysoTracker, co-immunoprecipitation, pulldown/affinity isolation, immunogold EM; for review see [86]). In this regard, the best would be to determine LAMP2A and Hsc70 levels in purified/extracted lysosomes, since CMA solely takes place in a subset of lysosomes (for isolation of lysosomal fractions see [87]). Further, it is feasible to evaluate CMA dynamics and activity by measuring the translocation and degradation of CMA substrates [88] or by using fluorescent CMA reporters, such as the KFERQ-PS-CFP2 reporter [89]. LAMP-2A knockout mouse models are also suitable tools to monitor CMA, but to date there is only a liver-specific, but not a heart-specific mouse model available [90]. To fully evaluate the activity of CMA, autophagy modulation should be performed (e.g. bafilomycin 6

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Table 3 Autophagy alterations in mouse models of cardiomyopathy. Type of cardiomyopathy DRM

DCM

Mouse model

Autophagy alteration

References

Intermediates or late stage autophagosomes ↓

[97]

Autophagy-related transcripts ↓ ATG10, ATG5 and Beclin-1 ↓

[45]

Cardiomyocytes transfected with AdCryABR120G

LC3-II ↓ Autophagic flux ↓

[98]

LmnaH222P/H222P

AKT-mTORC1 pathway ↑

[104]

CryAB

R120G

LC3-II ↓, p62 and Beclin-1 ↑ Autophagic flux ↓ Lmna−/−

mTORC1 signaling ↑ Beclin-1, Atg7, p62, LC3-II, and LAMP-2A ↑

[105]

HCM

Homozygous Mybpc3-targeted knock-in

LC3-II, p62 and Beclin-1 ↑ AKT-mTORC1 pathway ↑ Autophagic flux ↓

[46,52]

Diabetic CM (type 1 diabetes)

STZ

LC3-II, p62 and cathepsin D ↑ AMPK activity ↑ Autophagosomes and lysosomes ↑

[43]

LC3-II ↓ Autophagic flux ↓ AMPK activity ↓

[44]

OVE26

LC3-II ↓ AMPK activity ↓ Autophagosomes ↓

[51]

db/db

LC3-II and p62 ↑, cathepsin D ↓ AMPK activity ↓ Mature autophagosomes and lysosomes ↓

[43]

LC3-II and Beclin-1 ↑, p62 ↓ Autophagic flux ↑

[120]

LC3-II/-I ratio and Beclin-1 ↑ Autophagosomes ↑

[121]

Autophagic vacuoles ↑

[22,49,50]

Diabetic CM (type 2 diabetes)

Danon disease

LAMP-2-deficient

Abbreviations used are: CM, cardiomyopathy; DCM, dilated cardiomyopathy; DRM, desmin-related cardiomyopathy; HCM, hypertrophic cardiomyopathy.

A1, 6-aminonicotinamide, geldanamycin). However, it has to be considered that there are no selective chemical inhibitors or activators of CMA. Thus, genetic manipulation of LAMP-2A is to date the best approach to challenge CMA activity, whereby the two other isoforms of LAMP-2 should not be targeted.

associated with a defect in autophagy. 3.1.1. Autophagy in desmin-related cardiomyopathy Although relatively rare, desmin-related cardiomyopathy (DRM) is the only cardiomyopathy for which an intracellular accumulation of protein aggregates has been shown. Therefore, it was also the first cardiomyopathy for which the concept of activating autophagy as a potential therapy was adopted, because an analogy to neurodegenerative diseases with intracellular protein accumulation was drawn, for which autophagy activation seemed to be a promising therapy. Although desmin mutations account for most of the DRM cases in humans, a R120G mutation in the desmin chaperone αB-crystallin is the best studied. Ultrastructural analysis revealed a lack of any structures that could be identified as either intermediates or late stages in the autophagic pathway in the CryABR120G mice (Table 3; [97]). Reduced LC3-II levels under basal and lysosome-inhibited conditions were observed after adenoviral-mediated gene transfer of CryABR120G in cardiomyocytes, suggesting reduced autophagic function (Table 3; [98]). Supporting this, levels of autophagy-related transcripts and of autophagy key proteins, such as ATG10, ATG5 and Beclin-1 were lower in CryABR120G than in control mice (Table 3; [45,97]). Before the therapeutic potential of treating cardiomyopathy by activating autophagy was tested in the CryABR120G mice, it was found that autophagy is an adaptive mechanism in DRM, and that blunting

3. Autophagy in cardiomyopathies Although cardiomyocytes have a low level of autophagic activity under physiological conditions, autophagy becomes important in situation of cardiac stress, such as starvation or high metabolic demand, where it acts as a protective mechanism against accumulation of toxic protein aggregates [91–93]. In the last decade, a large body of evidence indicated alterations of autophagy in a wide range of cardiac diseases, including cardiomyopathies (Table 3; [9]). 3.1. Autophagy in inherited cardiomyopathy A significant portion of cardiomyopathies is caused by genetic aberrations and the genetic causes of these inherited cardiomyopathies have been increasingly discovered over the past two decades [94–96]. The role of autophagy in inherited cardiomyopathy may be particularly important because mutant/misfolded protein is the cause of the disease. Only a few forms of inherited cardiomyopathies are known to be 7

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number of autophagosomes and autolysosomes in CryABR120G mice. Another study targeted the DRM mice with drinking water containing 0.33 mg/mL (final dose ~50 mg/kg/d) of suberoylanilide hydroxamic acid (SAHA), an FDA-approved class IIb HDAC activity inhibitor, inducing tubulin hyperacetylation and activation of autophagy (Table 4; [99]). Treatment of 3.5-month-old DRM mice with SAHA for 7 weeks reduced protein aggregate content and prevented cardiac dysfunction. The authors confirmed activation of autophagic flux through SAHA in cardiomyocytes expressing CryABR120G. Moreover, downregulation of

autophagy through hemizygous Beclin-1 knockout accelerated cardiomyopathy progression and decreased cardiac function in 9-month-old CryABR120G mice (Table 4; [91]). The first attempt to treat CryABR120G mice by autophagy activation included overexpression of ATG7, which is crucial for the elongation of autophagy, as explained in Section 2.1.1. CryABR120G mice overexpressing Atg7 and/or exercising revealed reduced protein aggregation, partial prevention of cardiac hypertrophy, fibrosis and dysfunction as well as increased survival (Table 4; [45]). Atg7 overexpression enhanced the autophagic flux and increased the Table 4 Targeting autophagy in mouse models of cardiomyopathy. Type of cardiomyopathy DRM

Subtype/mutation (mouse model)

Autophagy targeted by

Effect on cardiac phenotype

Effect on autophagy

References

CryABR120G CryABR120G

Beclin-1 hemizygous KO Atg7 overexpression and exercise

Not determined - presumably autophagy ↓ Autophagic flux ↑

[91] [45]

CryABR120G

Suberoylanilide hydroxamic acid (SAHA) COP9 signalosome downregulation (CSN8neoflox/−) UBC9 overexpression

Function ↓ Protein aggregation ↓ Hypertrophy ↓ Fibrosis ↓ Function ↑ Survival ↑ Protein aggregation ↓ Function ↑ Survival ↓

Autophagic flux ↑ (in vitro) LC3-II ↓ p62 ↑ Autophagic flux ↑

[99]

mTORC1 signaling ↓ LC3-II ↑ p62 ↓ AKT signaling ↓ (in vitro) Autophagic flux ↑ Autophagic flux ↑

[104]

OVE26 mice: LC3-II ↑ Beclin-1 ↑ AMPK phosphorylation and activity ↑ STZ mice: AMPK activity ↑ Autophagic flux ↓ Rab9 ↑ ROS ↓ Autophagic flux ↓ ROS ↓ LC3-II ↑ AMPK activity ↑

[51]

LC3-II ↓ Autophagosomes ↓ Lysosomes ↓ LC3-II ↑ SIRT1 ↑ p62 ↓ Autophagosomes ↓ Autolysosomes ↓

[43]

CryABR120G CryABR120G

Protein aggregation ↓ Hypertrophy ↓ Fibrosis ↓ Function ↑ Survival ↑ Dilation ↓ Function ↑

DCM

LMNA CM LmnaH222P/H222P

Temsirolimus

HCM

LEOPARD syndrome Ptpn11Y279C/+ Mybpc3 Mybpc3

Rapamycin

Hypertrophy ↓

Rapamycin Caloric restriction

OVE26/STZ

Metformin

Lung weight ↓ Hypertrophy ↓ Lung weight ↓ Function ↑ OVE26 mice: Function ↑ Heart failure ↓ STZ mice: Survival ↑

STZ/OVE26

BECN1+/−

Function ↑

Hypomorphic Atg16l1

Function ↑

Metformin

Cardiomyocyte apoptosis ↓ Fibrosis ↓ Function ↑ Atrophy ↓

Diabetic CM (type 1 diabetes)

STZ

Chloroquine Fenofibrate

Atrophy ↓ Fibrosis ↓ Function ↑ Function (diastolic) ↑ Fibrosis ↓ Apoptosis Fibrosis ↓ Function ↑

Chloroquine Diabetic CM (type 2 diabetes)

db/db

Desacyl ghrelin

Chloroquine

Hypertrophy ↑ Function ↓

Resveratrol

Function ↑

LC3-II/LC3-I ↑ AMPK phosphorylation ↑ Beclin-1 ↑ FoxO3 ↑ p62 ↑ Cathepsin D ↑ LC3-II ↓ Autophagosomes ↓ mTORC1 signaling ↑ p62 ↓ Cathepsin D ↑ LC3-II ↑ Autophagosomes ↑ mTORC1 signaling ↓

[100] [101]

[113] [46] [46]

[44] [44] [122]

[124] [123] [126]

[43]

[43]

(continued on next page) 8

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Table 4 (continued) Type of cardiomyopathy Age-related CM

Subtype/mutation (mouse model)

Autophagy targeted by

Effect on cardiac phenotype

Effect on autophagy

References

N/A

Atg5 conditional KO

LC3-II ↓ p62 ↑

[133]

N/A

Endothelin-1 KO

Preserved Beclin-1, Atg5 and Atg7 protein levels. LC3-II/LC3-I ↑ p62 ↓

[134]

N/A

Rapamycin

Dilation ↑ Hypertrophy ↑ Lung weight ↑ Function ↓ Survival ↓ Life span ↑ LV mass ↓ Cardiomyocyte cell size ↓ Fibrosis ↓ Function ↑ LV hypertrophy ↓ Function (diastolic) ↑

[135]

N/A

Caloric restriction

N/A

Spermidine

p62 ↓ Beclin-1 ↓ LC3-II - no significant differences No significant differences in p62, Beclin-1 or LC3-II protein levels Autophagic flux ↑ Autophagosomes ↑ Autolysosomes ↑ Cross with Atg5−/− abolishes beneficial effects

LV hypertrophy ↓ Function (diastolic) ↑ Life span ↑ LV hypertrophy ↓ Function (diastolic) ↑ Heart failure ↓

[135] [58]

Abbreviations used are: CM, cardiomyopathy; DCM, dilated cardiomyopathy; DRM, desmin-related cardiomyopathy; HCM, hypertrophic cardiomyopathy.

the COP9 signalosome by hypomorphic mutation of a component of the complex (CSN8; ~80% reduction) decreased survival of CryABR120G mice (Table 4; [100]). The COP9 signalosome is known for its CUL/ cullin deneddylation activity and has been found to positively regulate UPS activity and autophagy function. Lower LC3-II and higher p62 protein levels were found in the DRM mouse model after COP9 downregulation. Furthermore, overexpression of the SUMO-conjugating enzyme UBC9 reduced protein aggregation in cardiomyocytes, partially prevented cardiac hypertrophy, fibrosis and dysfunction, and increased survival in CryABR120G mice (Table 4; [101]). UBC9 overexpression markedly increased the number of autolysosomes determined by RFP-GFP-LC3 in CryABR120G hearts.

activation of autophagy affects DCM. It has been shown that intraperitoneal administration of 5 mg/kg/d of temsirolimus, a derivative of the mTOR inhibitor rapamycin (sirolimus), in 14-week-old LmnaH222P/H222P mice for two weeks reduced cardiac dilation and increased cardiac function (Table 4; [104]). The authors demonstrated decreased mTORC1 signaling, increased LC3-II protein and decreased p62 protein levels after temsirolimus treatment. 3.1.3. Autophagy in hypertrophic cardiomyopathy Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy, mainly associated with mutations in genes encoding sarcomeric proteins [107]. Mutations in MYBPC3, encoding cardiac myosin-binding protein C and in MYH7, encoding β-myosin heavy chain account for a majority of HCM [108]. In septal myectomies of HCM patients carrying mutations in MYBPC3 or MYH7, an accumulation of early and late autophagic vacuoles was observed by TEM, which was associated with higher protein levels of LC3-II and Beclin-1 than in donor samples [42]. LC3-II levels were also higher in septal myectomies from HCM patients carrying MYBPC3 mutations than in non-failing samples, while levels of Beclin-1 and p62 did not differ between the two groups [46]. However, several genes encoding proteins involved in the autophagy pathway were upregulated in human HCM, including p62 and Beclin-1. Since elevated LC3-II levels could result from enhanced autophagosome formation or impaired clearance (see Section 2.1.3), a conclusion whether autophagy is activated or impaired in HCM patients cannot be made yet. On the other hand, in a HCM mouse model carrying a homozygous Mybpc3 mutation, elevated levels of LC3-II, p62 and Beclin-1, and activation of the AKT/mTORC1 pathway were associated with impaired autophagic flux (Table 3; [46,52]). Rapamycin and caloric restriction, the probably most widely used activators of autophagy in research, have been described to improve cardiomyopathy in mice. Rapamycin is an FDA-approved mTOR catalytic site inhibitor. Since it inhibits the catalytic site of mTOR, it inhibits not only mTORC1, but also mTORC2, which regulates cell survival and organization [109], and may affect pathways other than autophagy. Caloric restriction (reduced calorie intake without malnutrition) activates autophagy by Sirtuin-1 activation [110], but has also other effects, such as reduction of oxidative stress, changing mitochondrial bioenergetics and cellular membrane compositions [111]. A 9-week treatment with either 2.24 mg/kg/d rapamycin by chow or 40% caloric

3.1.2. Autophagy in dilated cardiomyopathy Dilated cardiomyopathy (DCM) is familial in about 32–70% of cases [102]. Most frequently mutated genes encode titin (TTN), component of the sarcomere and Lamin A/C (LMNA), component of the nuclear lamina, representing 25% and 6% of all genotyped cases, respectively [103]. In a mouse model of DCM carrying an Lmna mutation, a hyperactivated AKT-mTOR pathway combined with defective autophagy was described (Table 3; [104]). While LC3-II levels were lower, levels of p62 and Beclin-1 were higher in LmnaH222P/H222P than in control mice. In addition, autophagic flux was impaired in LmnaH222P/H222P mice. In line with the results obtained in mice, virtually no LC3-II, but higher p62 levels were detected in left ventricular tissue from patients with LMNA mutations [104]. In cardiac muscle of Lmna−/− mice, which serve as a model of DCM caused by reduced lamin A protein levels, enhanced mTORC1 signaling was detected as well (Table 3; [105]). Furthermore, levels of Beclin-1, ATG7, p62, and LC3-II were higher in the hearts of Lmna−/− than wild-type mice. Interestingly, LAMP-2A levels were higher in Lmna−/− than in control mice, suggesting activated CMA, a process that can take place when autophagy is inhibited (see Section 2.3). Similarly, impaired autophagy was reported in patients with a severe recessive DCM and left ventricular noncompaction carrying a mutation in the PLEKHM2 gene, encoding pleckstrin homology domain-containing family M member 2 [106]. Primary fibroblasts from these patients exhibited abnormal subcellular distribution of endosomes marked by RAB5, RAB7 and RAB9, abnormal lysosomes localization and impaired autophagic flux. Although all these studies have suggested a link between autophagy activity and DCM disease progression, very few studies have explored if 9

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restriction ameliorated the cardiomyopathic phenotype in 20-week-old HCM mice (Table 4; [46]). At the beginning of the treatments, cardiac hypertrophy and dysfunction were already established. Rapamycin treatment reduced lung weight, an indicator for heart failure, in HCM mice. Caloric restriction reduced cardiac hypertrophy, dysfunction and heart failure in these mice. Both treatments restored the autophagic flux. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2 regulating the MAPK/ERK signaling pathway, can cause a rare genetic syndrome called LEOPARD syndrome that may also include the development of HCM [112]. One group treated 8-week-old mice (prior to onset of hypertrophy) or 12-week-old mice (after hypertrophy was established) with Ptpn11Y279C/+ mutation by intraperitoneal injection with 2 mg/kg rapamycin for 4 weeks daily or for 4 weeks daily with additional weekly injection for another 4 weeks (Table 4; [113]). All treated mice exhibited a reduction in cardiac hypertrophy. The authors did not show a direct effect on autophagy in the treated mice, but found that phosphorylation levels of AKT, GSK3α and TSC2, which are upstream of mTORC1 signaling, and phosphorylation of p70S6K, which is downstream of mTORC1 signaling were decreased in vitro.

Overexpression of Mir30c resulted in lower Beclin-1 level, inhibited Beclin-1-activated autophagy and improved cardiac function and structure in the diabetic mice. Support for enhanced autophagy in type 2 diabetes was also derived from human data showing higher LC3-II and Beclin-1 levels, lower p62 levels and higher number of autophagosomes in right atrial biopsies from diabetes type 2 patients with ischemic heart disease [120]. The data situation regarding the treatment of diabetic cardiomyopathy is similarly inconsistent, since several studies reported that activation of autophagy ameliorates diabetic cardiomyopathy, while others found autophagy inhibition to be beneficial (Table 4). One of the first studies performed in type 1 diabetic OVE26 and STZ mice evaluated the effect of metformin, the number 1 drug to treat diabetes type 2, which reduces through different mechanisms the release and production of liver glucose [51]. Treatment with 200 mg/kg/d metformin in drinking water for 4 months reduced lung weight, indicating prevention of heart failure and prevented cardiac dysfunction in 6-monthold OVE26 mice. Metformin increased LC3-II and Beclin-1 protein levels, enhanced AMPK activity and reduced mTORC1 signaling in OVE26 mice. Metformin also increased AMPK activity and survival in STZ mice [51]. In agreement with this, treatment with 200 mg/kg/d metformin in drinking water for 4 months enhanced autophagy and protected against cardiomyocyte apoptosis in STZ mice [122]. Another study found that although STZ mice exhibited impaired autophagy, STZ treatment of heterozygous Beclin-1 knockout (BECN1+/−) mice, which further decreased autophagy, prevented development of cardiac dysfunction, suggesting that decreased autophagy is an adaptive rather than a pathological mechanism in diabetes [44]. Similarly, cardiac dysfunction was prevented in OVE26 mice crossed with BECN1+/− mice and in Atg16L1-deficient STZ mice [44]. In line with this, Beclin-1 overexpression restored autophagic flux but worsened cardiac function in STZ mice. Pharmacological inhibition of autophagy by administration of chloroquine (10 mg/kg/d) for 14 days increased fibrosis and worsened cardiac function in STZ mice [43]. This was associated with lower LC3-II, but higher p62 protein levels, and with reduced autophagosome, but increased lysosome number [43]. Conversely, a higher dose of chloroquine (60 mg/kg/d) markedly improved diastolic dysfunction and fibrosis, increased LC3-II, p62 and Beclin-1 protein levels, and normalized the increased number of autophagosomes and autolysosomes in STZ mice [123].Treatment of STZ mice with fenofibrate (100 mg/kg every other day), a PPARα agonist that reduces oxidative stress and is FDA-approved for treatment of high cholesterol for up to 6 months normalized cardiac function, reduced cardiac fibrosis, and was accompanied by higher LC3-II and Sirtuin-1, but lower p62 protein levels [124]. Pharmacological inhibition of autophagy with chloroquine (10 mg/ kg/d) in type 2 diabetic db/db mice for 14 days worsened cardiac function and fibrosis, such as in type 1 diabetic mouse models. Moreover, chloroquine reduced LC3-II protein levels and the number of autophagosomes, whereas it increased p62 and cathepsin D protein levels, the number of lysosomes and mTORC1 signaling [43]. In contrast, activation of autophagy by a 14-day administration of 50 mg/kg/ d resveratrol, which is thought to act like a caloric restriction mimetic, improved cardiac function and suppressed fibrosis. Furthermore, resveratrol treatment reduced p62 level, the number of autophagosomes/ lysosomes, mTORC1 signaling, and, conversely, increased cathepsin D and LC3-II protein levels in db/db mice. Yet, another study tested desacyl ghrelin, a hormone with similar and distinct effects as the autophagy activator ghrelin [125], which was administered twice daily (100 μg/kg) for 10 consecutive days in db/db mice [126]. Desacyl ghrelin treatment restored cardiac function and reduced the fibrotic area in db/db mice. On the molecular level, desacyl ghrelin treatment increased LC3-II/LC3-I ratio, AMPK phosphorylation, and Beclin-1 and FoxO3 protein levels. Based on these conflicting literature reports, the role of autophagy in diabetic cardiomyopathy is overall still not clear, and especially

3.2. Autophagy in diabetic cardiomyopathy Diabetic cardiomyopathy is characterized by myocardial fibrosis and diastolic dysfunction that develops in many diabetic patients in the absence of coronary artery disease and hypertension [114]. Diabetesassociated cardiac metabolic derangements such as diminished glucose uptake and utilization, increased fatty acid oxidation and accumulation of glycogen and lipid droplets are supposed to trigger oxidative stress and autophagy dysfunction [115]. As mentioned in Section 2.1.2, insulin signaling activates the PI3K-AKT/mTORC1 pathway and thus inhibits autophagy. One would thus hypothesize that insulin deficiency (present in diabetes type 1) or insulin resistance (present in diabetes type 2) would lead to enhanced autophagy. However, insulin deficiency is usually accompanied by hyperglycemia that has been reported to inhibit autophagy in cardiomyocytes [116]. The most frequently used mouse model of type 1 diabetes is induced by intraperitoneal administration of streptozotocin (STZ), which is toxic to insulin-producing β-cells (Table 3; [117]). One study suggested enhanced autophagy in STZ mice indicated by higher protein levels of LC3-II, p62 and cathepsin D, increased AMPK activation, and accumulation of both autophagosomes and lysosomes [43]. In contrast, another study reported markedly reduced LC3-II levels, impaired autophagic flux and AMPK signaling pathway in STZ-treated mice, suggesting rather diminished autophagy (Table 3; [44]). In a genetic, and therefore more reproducible mouse model of type 1 diabetes, which overexpresses calmodulin in pancreatic β-cells (OVE26 mice), leading to deficient production of insulin [118], autophagy was also suppressed (Table 3; [51]). Lower LC3-II levels, number of autophagosomes and AMPK activity were observed in hearts of OVE26 mice than of control mice. With regard to type 2 diabetes, controversial data exist as well. Higher LC3-II and p62 protein levels, but lower cathepsin D level combined with suppressed AMPK activity were reported in hearts from db/db mice (Table 3; [43]), which serve as a type 2 diabetic mouse model and own a leptin receptor defect [119]. EM revealed autophagosomes, but no mature autolysosomes and rarely lysosomes in db/db mice, suggesting altogether reduced autophagy. In contrast, another study reported enhanced autophagy in db/db mice as evidenced by higher LC3-II and Beclin-1 levels, lower p62 levels and most importantly increased autophagic flux (Table 3; [120]). More recently, higher LC3-II/LC3-I ratio, higher Beclin-1 levels and higher number of autophagosomes were observed in hearts of db/db mice, which let the authors conclude as an induction of autophagy (Table 3; [121]). Interestingly, this study revealed a downregulation of the microRNA Mir30c in the db/db mice and that Beclin-1 is a direct target of Mir30c. 10

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whether autophagic adaptions are different between type 1 and 2 diabetes needs to be further investigated.

higher ubiquitin and p62 protein levels and lower LC3-II protein levels than in control mice. Cardiomyocyte-specific knockout of endothelin-1, a vasoconstrictor secreted by the endothelium, decreased left ventricular mass, cardiomyocyte cell size and fibrosis, preserved cardiac function and extended life span from a median survival of 25.2 months to 30.3 months (Table 4; [134]). On molecular level, endothelin-1 knockout protected from an age-dependent decline in Beclin-1, Atg5 and Atg7 protein levels and displayed a higher LC3-II/LC3-I ratio and lower p62 protein levels in mice, suggesting higher autophagic activity. Treatment of 26-month-old mice with 2.24 mg/kg/d rapamycin by chow or 40% caloric restriction for 10 weeks reversed age-induced left ventricular hypertrophy and prevented diastolic dysfunction in mice (Table 4; [135]). However, the authors only found decreased p62 and Beclin-1 protein levels, but no difference in LC3-II protein levels after rapamycin treatment, and no significant differences in any of the proteins after caloric restriction. Life-long drinking water supplementation with spermidine from 4 months of age increased life span in female mice by about 10% from ~800 days to ~880 days (Table 4; [58]). Latein-life spermidine treatment from 18 months of age still significantly increased life span by about 10%. Late-in-life spermidine treatment reversed age-induced left ventricular hypertrophy, improved diastolic function and prevented an increase in lung weight indicating pulmonary congestion and heart failure in mice. Spermidine treatment increased autophagic flux, increased number of autophagosomes and autolysosomes measured by mRFP-GFP-LC3 and induced mitophagy assessed by Mt-Keima. Cardiomyocyte-specific knockout of Atg5 abolished spermidine's beneficial effect on cardiac morphology and function.

3.3. Autophagy in Danon disease Danon disease is a rare, but severe X-linked disorder characterized by a profound HCM combined with skeletal myopathy, mental retardation and visual problems [47,127,128]. It was first described by Danon et al. in 1981 and due to excessive amounts of glycogen granules in muscle biopsies originally considered to be a lysosomal storage disease [129]. In 2000, Nishino et al. identified genetic defects in the LAMP2 gene in ten unrelated male Danon disease patients, including one of the patients from the original case report [48,129]. Most of the LAMP2 mutations led to a complete or nearly complete deficiency of LAMP-2 protein in skeletal and cardiac muscles of Danon patients [47,48]. As a major lysosomal membrane protein, LAMP-2 is involved in lysosome biogenesis, maturation and function [130], and, as mentioned in Section 2.1.1, is required for the maturation of autophagosomes by being a crucial factor for autophagosome-lysosome fusion. Alternative splicing in the last exon 9 gives rise to three different isoforms LAMP-2A, LAMP-2B and LAMP-2C, which differ in their transmembrane and cytoplasmic domains [47]. One mutation in exon 9B, which affects only the LAMP-2B isoform, is associated with a complete set of Danon disease symptoms, suggesting that Danon disease is largely due to defects of the LAMP-2B isoform [48]. This is supported by the observation that LAMP-2B is more abundantly expressed than LAMP-2A in tissues most affected in Danon disease, such as striated muscles and brain [131]. Due to its important role, one would expect that a lack of LAMP-2 leads to a disturbance of autophagy in Danon disease. Indeed, the disease is characterized by accumulation of autophagic vacuoles in skeletal and cardiac muscle [47,48]. The recent development of Danonspecific induced pluripotent stem cell-derived cardiomyocytes (iPSCCMs) allowed a deeper look in autophagy disturbance. Danon iPSC-CMs showed significantly more early autophagic vacuoles and displayed a nearly complete absence of mature autophagic vacuoles, as analysed with the tandem mRFP-GFP-LC3B reporter [53,54]. Furthermore, autophagic flux was impaired in Danon iPSC-CMs [53]. LAMP-2-deficient mice exhibit increased mortality, cardiac hypertrophy and reduced cardiac contractile function (Table 3; [22,49,50]). Similar to Danon patients, accumulation of autophagic vacuoles has been observed in the heart and skeletal muscle of LAMP-2-deficient mice [22,49,50]. In addition, excessive accumulation of autophagic vacuoles has been found in liver, pancreas and kidney [22,49]. Overall, the findings in LAMP-2deficient mice are more severe than in human patients. However, due to the severe cardiac phenotype, alterations in non-muscular tissues have not yet been studied extensively in human patients. Deficiency of the LAMP-2 protein reported in Danon patients together with Danon disease-associated features manifested in LAMP-2-deficient mice support LAMP-2 deficiency as the primary cause of Danon disease [22,48]. In addition, progression of Danon disease in humans has become better characterized clinically and treatment guidelines for Danon disease manifestations have been proposed [128]. However, the underlying pathological molecular mechanism is still not fully clear and needs further investigations in both animal and human cellular models (iPSCCMs) of Danon disease.

4. Conclusion and perspectives Although much progress has been made in elucidating autophagy in the heart, the ability to confirm whether autophagy is enhanced or inhibited in cardiomyopathy needs further improvement. Especially data from humans are difficult to obtain and interpret, as there is often high variability between patients, and determination of autophagic activity requires modulation with compounds or reporters, which is not feasible in patients directly. It will be of great interest to identify autophagy biomarkers and develop minimally- or non-invasive methods that are sensitive enough to uncover small but significant changes in autophagy. A non-invasive method could be the positron emission tomography/computed tomography that has been used to detect preamyloid oligomers in mouse hearts [136]. The generation of patientspecific iPSC-CMs [102] offers the possibility to evaluate whether autophagy is activated or inhibited and could be used to decide whether modulation of autophagy has a therapeutic benefit in the particular case. Other studies are required to decipher the importance of CMA and microautophagy in cardiomyocytes and the role of autophagy in other cardiac cells than cardiomyocytes in physiological conditions and cardiomyopathy. In addition, the elucidation of the specific molecular mechanisms modulating cardiac autophagy or through which autophagy is reactivated merits further investigation or models. Since most of the findings obtained in animal models suggest impairment of autophagy in several forms of cardiomyopathy, activation of autophagy might be an attractive option to treat cardiomyopathy in patients. Currently used activators of autophagy such as mTOR inhibitors affect cellular processes besides autophagy such as protein synthesis. Therefore, more specific activators of autophagy which target the pathway more downstream should be screened for and autophagy activation should not be excessive to minimize side effects. Another very interesting, but challenging area of research is to understand the interconnections of autophagy and other protein degradation pathways such as the UPS and the unfolded protein response in cardiomyopathy. For example, inhibition of both autophagy and UPS was found in a mouse model of HCM [46,52], loss-of-function mutation in the E3 ubiquitin ligase atrogin-1 resulted in autophagy impairment and cardiomyopathy

3.4. Autophagy in age-related cardiomyopathy Many studies have investigated the connection between autophagy and age-related cardiomyopathy, since activation of autophagy has overall been associated with an increase in life span [132]. Atg5flox/flox mice crossed with cardiomyocyte-specific Cre mice, resulting in Atg5 deficiency and reduced autophagy in cardiomyocytes, developed cardiac dilation and hypertrophy, had increased lung weight and markedly decreased cardiac function at 10 months of age and nearly all mice died around 300 days of age (Table 4; [133]). Biochemical analyses revealed 11

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in humans and mice [137,138], and the COP9 signalosome positively regulated both UPS and autophagy function in mice [100]. Thus, it will be important to look at several protein degradation pathways in parallel in the future to understand the underlying pathological mechanisms and tailor appropriate therapy.

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Transparency document

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The Transparency document associated with this article can be found, in online version.

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Acknowledgements

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The work of the authors related to this topic is supported by the DZHK (German Centre for Cardiovascular Research), the Federal Ministry of Education and Research (BMBF), the Deutsche Herzstiftung, the Helmut und Charlotte Kassau Stiftung, the Cincinnati Children's Hospital Medical Center Arnold W. Strauss award.

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Conflict of interest

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None. [26]

Author contributions

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All authors contributed equally to the writing of this manuscript.

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