Autophagy as a therapeutic target for ischaemia/reperfusion injury? Concepts, controversies, and challenges

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1 Cardiovascular Research (2012) 94, doi: /cvr/cvr358 SPOTLIGHT REVIEW Autophagy as a therapeutic target for ischaemia/reperfusion injury? Concepts, controversies, and challenges Karin Przyklenk 1,2,3 *, Yi Dong 1,2, Vishnu V. Undyala 1, and Peter Whittaker 1,3 1 Cardiovascular Research Institute, Wayne State University School of Medicine, Elliman Building, Room 1107, 421 E Canfield, Detroit, MI 48201, USA; 2 Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA; and 3 Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, MI, USA Received 20 September 2011; revised 6 December 2011; accepted 27 December 2011; online publish-ahead-of-print 2 January 2012 Abstract Autophagy is the tightly orchestrated cellular housekeeping process responsible for the degradation and disposal of damaged and dysfunctional organelles and protein aggregates. In addition to its established basal role in the maintenance of normal cellular phenotype and function, there is growing interest in the concept that targeted modulation of autophagy under conditions of stress (most notably, ischaemia/reperfusion) may represent an adaptive mechanism and render the myocardium resistant to ischaemia/reperfusion injury. Our aims in this review are to: (i) provide a balanced overview of the emerging hypothesis that perturbation of autophagy may serve as a novel, intriguing, and powerful cardioprotective treatment strategy and (ii) summarize the controversies and challenges in exploiting autophagy as a therapeutic target for ischaemia/reperfusion injury Keywords Autophagy Myocardial ischaemia Myocardial reperfusion Reperfusion injury This article is part of the Spotlight Issue on: Reducing the Impact of Myocardial Ischaemia/Reperfusion Injury 1. Introduction The development of novel strategies to limit lethal myocardial ischaemia/reperfusion injury and its attendant, long-term deleterious consequences has, for four decades, been the subject of intensive investigation. 1 3 Attention to date has focused largely on the attenuation of necrosis and type I (apoptotic) programmed cell death. However, a novel, intriguing, and in many respects provocative paradigm has recently emerged: i.e. the concept that targeted modulation of autophagy in heart cells (including cardiomyocytes, fibroblasts, and mast cells) may: (i) render myocytes resistant to ischaemia/reperfusion injury and (ii) have longer term effects on post-infarction cardiac healing and remodelling. In this review, we provide a brief overview of the key molecular components and physiological roles of autophagy, summarize the data that have provided the impetus for the nascent hypothesis of autophagy as treatment, and discuss the controversies and challenges in exploiting autophagy as a therapeutic strategy for ischaemia/reperfusion injury. 2. Basic concepts 2.1 Autophagy: definition and key molecular components Autophagy (or, more precisely, macroautophagy) is the tightly regulated intracellular catabolic process that serves as the cellular quality control mechanism for the disposal of damaged and dysfunctional organelles and protein aggregates. This is achieved by isolating and delivering subcellular components to lysosomes for subsequent degradation and hydrolysis, thereby: (i) culling damaged organelles and potentially toxic protein aggregates, and (ii) salvaging and recycling amino acids and other substrates for protein synthesis and ATP generation Current understanding of the molecular machinery of autophagy 4,7 14 has been summarized in several recent, comprehensive reviews. In brief, the autophagic pathway consists of four distinct and consecutive steps: (i) initiation; (ii) formation of autophagosomes (i.e. the double-membrane structures that encircle their cargo of damaged * Corresponding author. Tel: ; fax: , kprzykle@med.wayne.edu Published on behalf of the European Society of Cardiology. All rights reserved. & The Author For permissions please journals.permissions@oup.com.

2 198 K. Przyklenk et al. cytosolic constituents); (iii) generation of autolysosomes via docking and fusion with lysosomes; and (iv) final degradation and recycling of the sequestered cargo (illustrated in Figure 1A). Among the multiple protein complexes and signalling pathways that reportedly participate in orchestrating this process, a cohort of key mediators have emerged. These include: The target of rapamycin complex (TORC), particularly TORC1, the components of which serve as the sensors for energy and nutrient balance. Under conditions of stress (including hypoxia and ischaemia), a signalling cascade is initiated involving phosphorylation of AMP-activated protein kinase (AMPK) and subsequent inhibition of mammalian target of rapamycin (mtor). Inhibition of mtor, in concert with other protein partners, provides the critical step in initiating autophagosome formation. Class I and Class III phosphatidylinositol-3 kinase (PI3-kinase) play integral and reportedly opposing roles in the autophagic process: Class I PI3-kinase inhibits the induction of autophagy via phosphorylation of Akt and activation of mtor. In contrast, Class III PI3-kinase forms a complex together with Beclin 1 and other proteins to facilitate assembly and elongation of the autophagosomal membrane. The cytosolic precursor microtubule-associated protein 1 light chain 3 (LC3) is cleaved and converted to LC3-I and then activated and converted to LC3-II. This protein, LC3-II, is incorporated in the autophagosomal membrane and, together with autophagy-related gene (Atg) products, most notably Atg5 is critical for completing the elongation and maturation of the autophagosome. Identification of these critical components of the autophagic process has had the dual benefit of yielding mechanistic insights into the mechanisms of this catabolic process, and of practical importance, providing a molecular framework that can be utilized to monitor the status of autophagic activity in cells or tissues. That is: while direct electron microscopic visualization of structurally distinctive doublemembrane autophagosomes is undoubtedly the gold standard for the detection and assessment of autophagy, fluorescent labelling of autophagosomes (achieved via tagging of LC3 with the fusion proteins green fluorescent protein, GFP, or mcherry), fluorescent detection of autophagolysosomes (achieved by administration of cell-permeable acidotropic dyes), together with standard immunoblotting for expression of Beclin 1, LC3-II, and other key proteins have all emerged as tools to determine whether autophagy has been up- or downregulated by pathological or pharmacological stimuli (summarized in Figure 1 (A) Schematic illustration of the four phases of autophagy. (B) Summary of key molecular mediators, methods of assessment and tools utilized to perturb the autophagic process. Methods to assess and modify autophagic flux are also highlighted. This summary is not exhaustive but, rather, focuses on specific components that have been monitored or utilized to gain insights into the role of autophagy in myocardial ischaemia/reperfusion injury.

3 Autophagy as a therapeutic target for ischaemia/reperfusion injury 199 Figure 1B; reviewed in Klionsky et al. 14 ). These proteins and kinases also serve as foci for the modulation of autophagic activity: i.e. pharmacological agents (rapamycin, 3-methyladenine (3-MA), wortmannin, Bafilomycin A1, and chloroquine) as well as genetic manipulation of Atg5 and Beclin 1 have been utilized to perturb the induction, assembly, docking, or degradation of autophagosomes (Figure 1B). 14 Finally, it is important to note that the aforementioned endpoints are static indices that do not reflect the dynamic nature of the autophagic process. That is: an increased abundance of autophagosomes (assessed by electron microscopy, fluorescence-tagged LC3 puncta, etc.) or augmented expression of LC3-II and Beclin 1 may be evidence of an up-regulation in autophagy or, alternatively, may be a consequence of a backlog of autophagosomes caused by defects in fusion and clearance 14,15 (termed frustrated autophagy 15 ). To distinguish between these possibilities and provide definitive proof of a perturbation in the autophagic process, concurrent assessment of autophagic flux is required. Methods that have been utilized in models of ischaemia/reperfusion (highlighted in Figure 1B) include: Comparison of LC3-II abundance in the presence and absence of Bafilomycin A1 or chloroquine (i.e. agents that inhibit formation or degradation of autophagolysosomes). Immunoblot assessment of the polyubiquitin-binding protein p62 (also called SQSTM1; sequestrome 1). This assay reflects clearance of protein aggregates, with an increased signal considered indicative of inhibition of autophagic flux. 14,15 Tandem fluorescent tagging of LC3 with GFP and monomeric red fluorescent protein for simultaneous detection of both autophagosomes (indicated by LC3 puncta labelled both red and green) and autophagolysomes (LC3 puncta labelled red). 14,17,18 Assessment of flux obviously introduces additional technical challenges and thus, not surprisingly, data on autophagic flux in ischaemic-reperfused myocardium remain limited. 2.2 Established (patho)physiological roles of autophagy Under unstressed conditions, virtually all eukaryotic cells (including cardiomyocytes) display a basal, housekeeping level of autophagy that is critical for maintenance of normal phenotype and function. This essential homeostatic role of autophagy is underscored by the fact that molecular defects or global homozygous knockout of key autophagy genes are lethal while, in the heart, cardiac-specific disruption of Atg5 is associated with sarcomere disarray, hypertrophy, dilatation, and impaired contractility Moreover, evidence obtained in multiple models (from yeast to mammals) and in multiple tissues (including heart) has established that up-regulation of autophagy is a highly conserved adaptive mechanism to promote cell survival under conditions of starvation, energy deprivation, and metabolic stress. 4 9,11,22,23 Autophagy is, however, tightly associated with apoptosis and, under certain conditions, functions as a destructive (rather than adaptive) pathway: i.e. type II (autophagic) programmed cell death Indeed, it is this pivotal life-or-death (patho)physiological role of autophagy that has prompted investigators to interrogate its involvement in the stressful circumstances of myocardial ischaemia/ reperfusion injury. 3. Controversy I: autophagy is upregulated in ischaemic-reperfused myocardium As mentioned previously, there is exhaustive evidence that autophagy is initiated under conditions of energy deficit. Given the fact that depletion of high energy phosphate stores, as well as intracellular calcium overload, generation of reactive oxygen species and opening of the mitochondrial transition pore have all been reported to trigger autophagy (reviewed in Gustafsson and Gottlieb 10 and Dong et al. 11 ), it is perhaps not surprising that up-regulation of autophagy has been described in ischaemic-reperfused cardiomyocytes. Seminal studies conducted by Matsui et al. 26 in the in vivo mouse model revealed that 20 min of coronary artery occlusion was associated with a robust increase in expression of LC3-II (assessed by immunoblotting and normalized to LC3-I) in the at-risk region of the heart vs. non-ischaemic shams. These data, implicating up-regulation of autophagy with ischaemia per se, were supplemented by documentation of increased density of punctuate staining (indicative of increased numbers of autophagosomes) in left ventricular (LV) tissue sections obtained from GFP-LC3 transgenic mice following the 20 min episode of coronary occlusion. This concept of up-regulation of autophagy with ischaemia has been corroborated in isolated cardiomyocytes, with evidence of increased numbers of autolysosomes provided by fluorescence microscopy following administration of a cell-permeable probe that purportedly accumulates exclusively in acidic vesicles. 27 Moreover, and of arguably greater interest in terms of therapeutic relevance, data obtained from isolated cells, isolated buffer-perfused hearts and in vivo models suggest that 16,17,26 29 autophagy is up-regulated following ischaemia/reperfusion and may, indeed, be amplified by reintroduction of oxygen to ischaemic myocardium. 26 However, in marked contrast to these observations, meticulous analysis of autophagy by GFP LC3 fluorescence, by immunoblotting for LC3-II expression and by the gold standard of electron microscopy (allowing direct visualization of doublemembrane autophagosomes) revealed no evidence of increased autophagy in the ischaemic territory or peri-infarct region of mice subjected to 24 h of sustained ischaemia or transient, 1 or 4 h periods of coronary artery occlusion followed by 4 h of reflow. In fact, the peri-infarct zone was characterized by a diminished (rather than increased) number of autophagosomes vs. shams. 30 Recent comprehensive experiments by Loos et al. 23 may resolve this apparent discrepancy. Using cultured H9c-2 cells, the authors report differential induction of cell death that was dependent on the severity and duration of the ischaemic insult. Mild ischaemia (achieved by using 2-deoxy-D-glucose in the ischaemic buffer) was associated with apoptosis and up-regulation of autophagy (assessed by quantifying GFP LC3 puncta), and interestingly, an accompanying increase (rather than the expected decrease) in ATP concentration. However, in marked contrast, moderate or severe ischaemia achieved by metabolic inhibition with sodium dithionate alone or together with 2-deoxy-D-glucose induced apoptotic and necrotic cell death with no evidence of autophagy. 23 Thus, although the precise conditions that constitute mild, moderate, and severe ischaemia are undoubtedly model- and species-dependent, differences in the duration of coronary occlusion (20 min vs. 1 4 h) may underlie the presence vs. absence of an up-regulation in autophagy in the in vivo mouse model.

4 200 K. Przyklenk et al. 4. Controversy II: autophagy modulates ischaemia/reperfusion injury Given the dichotomous life-or-death role of autophagy, together with reports in most (but not all) studies that autophagy is up-regulated in ischaemic-reperfused myocardium, this raises the pivotal question: is up-regulation of autophagy good or bad for the fate of the cardiomyocytes? There is general agreement that up-regulation of autophagy during 23,26,29,31 33 ischaemia is protective. Evidence in support of this concept was provided by Loos et al. 23 Data obtained in their H9-c2 model of mild ischaemia revealed that up-regulation of autophagy was associated with preservation of mitochondrial membrane potential and sarcolemmal membrane integrity and a resultant significant delay in the onset of irreversible cell injury (both necrotic and apoptotic cell death). This favourable, ischaemia-induced autophagic response is reportedly initiated by phosphorylation of AMPK, inhibition of mtor, or both (i.e. the key molecular sensors of cellular energy and nutrient depletion) 23,26,32 a concept supported by the observation of suppressed ischaemia-induced autophagosome formation in mice with genetic inhibition of AMPK achieved by cardiacspecific expression of a dominant-negative form of the kinase. 26,32 The aforementioned studies report that an overall up-regulation in the degradation and disposal of damaged organelles is associated with an increased resistance to ischaemia-induced injury. Most recently, emerging attention has focused on the selective autophagic targeting and clearance of defective mitochondria a phenomenon termed mitophagy. 34 Evidence obtained in the in vivo mouse model revealed that up-regulation of mitophagy (achieved by genetic deletion of two molecular inhibitors of mitophagy, p53 and TP53-induced glycolysis and apoptosis regulator, TIGAR) attenuated apoptotic cell death and had a favourable effect on cardiac remodelling following permanent coronary artery ligation. 35 Moreover, treatment of p53 2/2 and TIGAR 2/2 mice with chloroquine abrogated the cardioprotection conferred by knockout of p53 and TIGAR, an effect accompanied by the accumulation of damaged mitochondria in the ischaemic myocardium. 35 Taken together, these data support the paradigm that up-regulation of autophagy and, more specifically, mitophagy renders myocardium resistant to ischaemic injury. In contrast to the consensus obtained in models of ischaemia, the role of autophagy in ischaemia/reperfusion (beneficial vs. detrimental) is controversial. A substantial body of evidence suggests that up-regulation of autophagy is cardioprotective and profoundly attenuates myocardial ischaemia/reperfusion injury. 16,36 40 The first and perhaps most compelling support for this paradigm was provided by Hamacher-Brady et al. 16 Using cultured HL-1 cells subjected to simulated ischaemia/reperfusion, the investigators demonstrated that knockdown of Beclin 1 (and the expected, attendant impairment of autophagy) was accompanied by an increase in apoptotic cardiomyocyte death, whereas over-expression of Beclin 1 (and up-regulation of autophagy) was cytoprotective. Moreover, the favourable, prosurvival effect of Beclin 1 over-expression was abrogated by co-expression of dominant negative Atg5 K130R. 16 Additional insights have been gained from recent studies investigating the mechanisms by which transcription factors (in particular, members of the forkhead family [FoxO1, FoxO3] and signal transducer and activator of transcription 1, STAT1) modulate the vulnerability of cardiomyocytes to ischaemia/reperfusion injury In mice with cardiac-specific deletion of FoxO1 and FoxO3, infarct size was exacerbated vs. wild-type controls, an effect that was accompanied by a decrease in expression of autophagy-related proteins including LC3-II. 38 Conversely, cardiac deletion of STAT1 conferred resistance to ischaemia/reperfusion, an effect that correlated with increased expression of LC3-II and Beclin 1 and was reversed by pre-ischaemic administration of 3-MA. 39 Finally, up-regulation of autophagy has been reported to play a causal role in the infarct-sparing effect of the two established gold standards of cardioprotection: ischaemic preconditioning, and postconditioning. 43 This positive association is based on observations of increased autophagosome formation (as assessed by electron microscopy, mcherry-lc3 fluorescence and immunoblot analysis of LC3-II and Beclin 1) in response to brief antecedent preconditioning ischaemia, 40,41 the reported failure of preconditioning and postconditioning to limit infarct size in cohorts treated with wortmannin, 40,43 and, most notably, the finding that preconditioning-induced cardioprotection in isolated buffer-perfused hearts is attenuated by perfusion with Tat-conjugated Atg5 K130R. 41 As in models of permanent coronary occlusion, the concept of cardioprotection via up-regulation of autophagy has been refined to focus on the mitochondria: recent data have demonstrated that up-regulation of mitophagy plays a causal role in the reduction of infarct size achieved with preconditioning. 42 There is, however, robust evidence that up-regulation of autophagy in ischaemia/reperfusion (and, in particular, during reperfusion) is detrimental and exacerbates, rather than attenuates, cardiomyocyte death. In contrast to the aforementioned studies, there are reports that the standard preconditioning stimulus (repeated 10 min episodes of brief ischaemia) is not associated with up-regulation of autophagy in the in vivo pig model, 44 and the cytoprotective effect of postconditioning are accompanied by a reduction in (rather than augmentation of) Beclin 1 expression. 45 Furthermore, genetic suppression of Beclin 1, achieved by RNAi, was shown to augment (rather than impair) cell survival in cardiomyocytes subjected to simulated ischaemia/ reperfusion, 27 whereas cardiac-specific heterozygous Beclin 1 +/2 mice exhibited smaller infarct sizes and an attenuation of apoptotic cell death, following coronary artery occlusion reperfusion. 26 Subsequent studies provided insights into the cellular mechanisms by which autophagy is up-regulated (and infarct size is reportedly exacerbated) in ischaemic-reperfused myocardium: reperfusion-induced generation of reactive oxygen species and activation/dephosphorylation of the beta-isoform of glycogen synthase kinase-3 (GSK-3b) have both been implicated to play a role. 17,46 5. Dichotomy: a recurring theme Potential explanations for these discrepant outcomes, particularly among studies that employed presumably direct and selective genetic approaches to perturb autophagy, 16,26 are speculative, and include the possibilities that the smaller infarct sizes seen in Beclin 1 +/2 mice may be due to: (i) a persistent but modest, reperfusion-associated increase in autophagosome abundance in these heterozygotes or (ii) improved autophagic flux. 15,26 Nonetheless, this concept of dichotomy is clearly a recurring theme, and autophagy may be an example of the Goldilocks principal : i.e. the presence vs. absence of an up-regulation in autophagy, and consequences of this up-regulation (beneficial vs. detrimental) appear to be a matter of timing and degree conditions must be just right. In

5 Autophagy as a therapeutic target for ischaemia/reperfusion injury 201 this regard, Matsui et al. have proposed a complex, dual role of this catabolic process in mediating the fate of cardiomyocytes. First, up-regulation of autophagy under conditions of ischaemia and nutrient deprivation achieved via FoxO, AMPK, and mtor-dependent mechanisms is adaptive and contributes to cardioprotection. Second, amplification of autophagy following relief of ischaemia may be adaptive or maladaptive in a stimulus-dependent manner (and dependent in part on the severity and duration of the ischaemic insult), with robust up-regulation of Beclin 1 being the proposed molecular hallmark of excessive up-regulation of autophagy and exacerbated cardiomyocyte death. 13,26,32,47 6. Multiple targets and longer term effects? Autophagy and post-infarction remodelling Most studies conducted to date have focused on the role of autophagy in determining the acute fate of ischaemic-reperfused cardiomyocytes. However, given the evidence obtained in other models that autophagy is up-regulated under conditions of hypertension and the progression to hypertrophy and heart failure, 13,48,49 it is not surprising that the scope of investigation is being expanded to include the possible longer term consequences of autophagy in healing and remodelling. This later period (days to weeks post-infarction) represents an attractive and potentially more accessible time frame for intervention: not only is the window of opportunity considerably wider than in the acute phase of ischaemic or ischaemia/reperfusion injury but the challenges of delivering pharmacological therapies are presumably resolved because of revascularization. Among the as-yet limited number of studies that have explored this issue, there is emerging agreement that up-regulation of autophagy (and, specifically, mitophagy 35 ) is associated with a favourable attenuation of adverse post-infarction LV remodelling. 35,50,51 Specifically, in wild-type mice 51 and rats 50 subjected to coronary artery ligation, surviving cardiomyocytes at the margins of the infarct exhibited an increase in autophagosome formation (assessed by electron microscopy, expression of LC3-II) 51 and autophagic flux 50 at 1 4 weeks post-infarction. Moreover, LV dysfunction, hypertrophy, and remodelling were exacerbated by Bafilomycin A1 and attenuated by mtor inhibitors (rapamycin and everolimus), thereby implicating cause-and-effect. 50,51 This concept is supported by the observation that mice with cardiac-specific deletion of FoxO1 and FoxO3 (and an attendant decrease in expression of autophagy-related proteins) were characterized by exacerbated LV dilatation, a worsening of LV dysfunction and a two-fold increase in fibrotic area at 4 weeks following permanent coronary artery occlusion when compared with genetic controls. 38 The aforementioned studies imply that the salutary effects on LV remodelling were due to up-regulation of autophagy in surviving periinfarct cardiomyocytes. However, healing and remodelling involves multiple cell types most notably, fibroblasts and mast cells which contain the molecular machinery required for autophagy (Figure 2). The crucial role of fibroblasts in both the immediate healing phase and subsequent longer term remodelling is complex, and there are both advantages and disadvantages to intervening in these processes. That is, promoting collagen production during healing could limit infarct expansion while, in contrast, augmented collagen deposition in non-infarcted myocardium could increase myocardial stiffness and thereby impair LV function. In this regard, data obtained by Aranguiz-Urroz et al. 52 in cultured adult rat cardiac fibroblasts suggested that ß 2 -adrenergic stimulation by isoproterenol and norepinephrine initiated an up-regulation in autophagosome formation and autohpagic flux (detected by standard methods used in cardiomyocytes, including electron microscopy, GFP LC3 fluorescence and immunoblot analysis of LC3-II). This up-regulation in autophagy led Figure 2 Perturbation of autophagy in fibroblasts (A) and mast cells (B). Proposed effects on collagen deposition/degradation and healing.

6 202 K. Przyklenk et al. to an increase in collagen type I degradation and, although extracellular collagen secretion was not assessed, activation of ß 2 -adrenergic receptors promoted degradation of intracellular collagen. Interestingly, the ß 2 -adrenergic-stimulated increase in autophagic flux was blocked by propranolol, but not by atenolol, a finding consistent with a selective adrenergic receptor response. These results led the authors to speculate that such action might mitigate or even prevent myocardial fibrosis induced by adrenergic stimulation. 52 Additional insight into this issue was recently provided by Marchesi et al. 53 using a mouse model in which the gene for proprotein convertase 5/6 (an enzyme that converts precursor proteins into their biologically active forms) was selectively inactivated in endothelial cells: fibroblasts co-cultured with these endothelial cells exhibited an increase in expression of LC3-II (implying up-regulation of autophagy) and decreased collagen production. Taken together, these studies appear to reiterate the recurring theme of dichotomy: down-regulation of autophagy in fibroblasts within the developing scar may increase collagen production and hence promote healing while, in contrast, increased fibroblast autophagy in the viable myocardium may help limit increases in interstitial collagen and hence maintain cardiac function (Figure 2). There is also evidence that autophagy could hypothetically influence healing and remodelling via the actions of mast cells. Bone marrowderived mast cells from mice that lacked Atg7 exhibited an impaired degranulation response both in vitro and in vivo. 54 In addition, the authors found that LC3-II was localized in the secretory granules. Mast cells can and do release a host of mediators during degranulation such as histamine, tumour necrosis factor-a, chymase, and tryptase agents that have potentially important roles in both mediating cardiac injury and in promoting wound healing (reviewed in Frangogiannis et al. 55 ). Thus, interference in the degranulation process by inhibition of autophagy could have significant implications. Once again, the timing and the location, or both, of inhibiting degranulation are important. Mast cell degranulation to set in motion the repair process appears beneficial; however, degranulation of resident mast cells reportedly contributes to myocardial ischaemia/reperfusion injury and may exacerbate necrosis (Figure 2). On the contrary, inhibition of degranulation may impair healing but have a favourable, infarct-sparing effect. 56 Finally, there is intriguing data to suggest that collagen may also play a direct role in the autophagy process, i.e. extracellular matrix may regulate autophagy, primarily through integrin-mediated signalling. 57 Differential regulation of autophagy was demonstrated in HeLa cells plated on different collagen types, 58 with higher basal levels of autophagy observed in cells plated on type IV collagen (primarily found in basement membranes) than in cells plated on type I collagen (the primary myocardial interstitial collagen). Conversely, under conditions of nutrient starvation, up-regulation of autophagy was greater in cells plated on type I collagen than it was for the type IV collagen substrate. These data may add another level of complexity, in that cellular environment may play a contributing role in mediating the autophagic response. 7. Modulation of autophagy as treatment for ischaemia/ reperfusion injury? 7.1 The good news Given the evidence that, in many (albeit not all) models: (i) autophagy is up-regulated in ischaemic-reperfused myocardium and (ii) perturbation of autophagy can influence the short-term fate of ischaemic-reperfused cardiomyocytes and, potentially, the longer term phase of healing and remodelling, these data raise the pivotal question: is modulation of autophagy a tenable treatment strategy for ischaemia/reperfusion injury? The concept of autophagy as a treatment paradigm is supported by studies that have documented a robust association between brief antecedent preconditioning ischaemia, up-regulation of autophagy in response to the preconditioning stimulus, and cardioprotection. Indeed, Gottlieb and colleagues 41 have proposed that autophagy is essential for preconditioning-induced protection, and, moreover, have reported that up-regulation of autophagy is a common theme in the cardioprotection achieved with pre-ischaemic administration of multiple, unrelated pharmacological agents, including 2-chlorocyclopentyladenosine (CCPA: adenosine A 1 receptor agonist), diazoxide (agent that opens mitochondrial ATP-sensitive potassium channels), UTP (purinergic receptor agonist), ranolazine (sodium channel blocker), and sulfaphenazole (cytochrome P450 inhibitor). 41,59,60 Reduction of infarct size has also been demonstrated in mice pre-treated with the mtor inhibitor rapamycin 61 (thereby implicating the involvement of autophagy), in hearts from rats receiving chronic pre-treatment with low doses of polyphenols (resveratrol and a supplemented form of resveratrol: confirmed by the authors to initiate an increase in autophagosome abundance and LC3-II expression), 28,62 and following modest induction of endoplasmic reticular stress with administration of tunicamycin or thapsigargin (confirmed to up-regulate autophagy). 63 Of note, data in all of the preceding studies were obtained using cultured cells or rat and mouse models (in vivo or isolated bufferperfused hearts). Indeed, recent evidence from our group was the first to demonstrate an association between pharmacological perturbation of autophagy and cardioprotection in a large animal (porcine) model of myocardial ischaemia/reperfusion; we found that chloramphenicol succinate (CAPS: a cytochrome P450 inhibitor), administered 10 min before the onset of coronary occlusion, evoked (i) a robust, 6.2-fold increase in expression of LC3-II at 10 min after treatment (the time corresponding to the onset of ischaemia) vs. baseline (Figure 3A) and (ii) a significant, 50% reduction in myocardial infarct size when compared with placebo-controls (Figure 3B). 64 Interestingly, in addition to the up-regulation in LC3-II, the reduction of infarct size seen with CAPS pre-treatment was also associated with a 2.4-fold increase in expression of Beclin 1 (Figure 3A) an observation that is in apparent contrast to the tenet that up-regulation of Beclin 1 is a characteristic of maladaptive autophagy and a harbinger of exacerbated cardiomyocyte death. Our study was also among the first to assess whether prophylaxis is required or whether the favourable effects of an agent that perturbs autophagy persist with delayed treatment. In this regard, we found that the infarct-sparing effect of CAPS was maintained (albeit attenuated) when treatment was given at 15 min before the onset of reflow (Figure 3B) Limitations and inconsistencies The profound cardioprotection obtained with CAPS in the clinically relevant swine model is consistent with the concept that therapeutic up-regulation of autophagy may provide a novel strategy to attenuate ischaemia/reperfusion injury. However, despite the translational implications of data obtained using this model, there are two caveats that warrant consideration. First, the data do not provide evidence of cause-and-effect, and it is unlikely that causality will be confirmed

7 Autophagy as a therapeutic target for ischaemia/reperfusion injury 203 Figure 3 (A) Expression of Beclin-1 and LC3-II with CAPS pre-treatment in the swine model. Myocardial samples were obtained at baseline and at 10 min following administration of chloramphenicol succinate (CAPS). Top: original immunoblots. Bottom: immunoreactivity of Beclin-1 and LC3-II, corrected for GAPDH expression and normalized to baseline values (mean + SEM). (B) Reduction of infarct size with CAPS in the porcine model. Images of heart slices obtained from 1 control, a CAPS-pre-treated pig and 1 pig that received CAPS-delayed treatment. Heart slices were incubated in triphenyltetrazolium chloride; using this method, viable myocardium stains red, whereas necrotic tissue (denoted by arrows) is unstained and this appears pale. Reprinted with permission from Sala-Mercado et al. 64 using currently available techniques; i.e. in contrast to the isolated rat heart model, in which perfusion of Tat-conjugated Atg5 K130R was shown to attenuate sulfaphenazole-induced (and preconditioning-induced) cardioprotection, 41,59 the use of comparable molecular strategies in the in vivo pig is challenging and feasibility is limited. Second, none of the pharmacological agents that have been used including CAPS selectively target the autophagic pathway. For agents that modulate autophagy via perturbation of PI3-kinase Akt mtor signalling, concerns regarding selectivity are particularly relevant given: (i) the complex role of this pathway in autophagy 11,12,28,64 66 and (ii) evidence PI3-kinase Akt mtor are components of the well-documented, cardioprotective reperfusion injury salvage kinase pathway. 43,67,68 Novel agents that selectively target autophagy are in development (reviewed in Nemchenko et al. 12 ) and, once available, will undoubtedly assist in the resolution of these issues. Enthusiasm for autophagy as a treatment paradigm must also be balanced by the seemingly disparate reports that up-regulation of autophagy is not a universal requirement for the cardioprotection achieved with preconditioning, 44 postconditioning, 45 or conditioningmimetic agents. For example, the well-established protective effects of urocortin were attributed to an inhibition (rather than increase) in Beclin 1 expression, 27 cardioprotection achieved with rapamycin in rat myocardium was shown to be independent of rapamycin-induced up-regulation of autophagy, 69 whereas pre-ischaemic administration of D-myo-inositol-1,4,5-trisphosphate hexasodium the sodium salt of the endogenous second messenger inositol 1,4,5-trisphosphate (IP3), a negative regulator of autophagy mimicked the reduction in infarct size seen with preconditioning. 70,71 Other puzzling inconsistencies include the observations that both wortmannin and rapamycin abrogated the infarct-sparing effect of postconditioning, 43 and the cardioprotection achieved with hydrogen sulfide in the in vivo porcine model was accompanied by down-regulation of mtor together with decreases (rather than increases) in LC3-II and Beclin 1 expression, 72,73 Future studies incorporating assessment of autophagic flux may yield insights into the reasons for these seemingly discrepant observations. 8. Challenges and future directions Perturbation of autophagy has emerged as a novel, intriguing, and potentially powerful molecular approach to modulate myocardial

8 204 K. Przyklenk et al. ischaemia/reperfusion injury. There are, however, challenges that must be addressed and gaps in knowledge that must be resolved before autophagy can be considered as a tenable therapeutic target. These include, first and foremost, the recurring theme of dichotomy: i.e. the question of whether up-regulation of autophagy is good or bad is simplistic and, based on current evidence, is dependent on magnitude (the Goldilocks principle ), timing (ischaemia vs. reperfusion vs. the longer term healing phase), and, potentially, on site and cell type (cardiomyocytes vs. fibroblasts and mast cells, located within the risk region vs. remote normally perfused myocardium). The interrelationships among these multiple factors are undoubtedly complex and are, at present, incompletely defined. As a result, the precise conditions and therapeutic window in which induction of autophagy will yield cardioprotection have not been established. A second challenge is the development of pharmacological agents that specifically and selectively target the autophagic process, with the caveat that the agents do not simply augment autophagosome abundance and result in frustrated autophagy. 15 A third, potentially confounding issue is that all studies to date in which up-regulation of autophagy was associated with cardioprotection were conducted using healthy adult cohorts. Given recent evidence that the efficacy of preconditioning and postconditioning is lost in aging and diabetic models, together with the fact that autophagy is attenuated under these conditions, 79,80 it will be important to establish whether up-regulation of autophagy can evoke cardioprotection in the face of these clinically relevant co-morbidities. If these challenges can be successfully resolved, the concept of autophagy as treatment for myocardial ischaemia/reperfusion injury would unquestionably merit clinical investigation. 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