Autophagy

<h1>JCI - Autophagy in cardiovascular biology</h1>

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<p>Autophagy is essential for normal maintenance, repair, and adaptation of the heart over the course of a lifetime. Evidence of autophagy in human heart disease was first reported in tissue samples from patients with dilated cardiomyopathy (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B29" target="blank">29</a>). Analysis of hearts from patients with end-stage HF indicated that cardiomyocytes died by a variety of mechanisms including necrosis, apoptosis, and autophagy, with autophagy reported as the most prominent (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B30" target="blank">30</a>).</p>

<p>Autophagy during ischemia/reperfusion. During ischemia, nutrient and oxygen supplies to the myocardium are limited, a state reminiscent of starvation. In this context autophagy can be adaptive, meeting cellular metabolic needs and eliminating damaged mitochondria, which could otherwise release damaging ROS and initiate apoptosis (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B31" target="blank">31</a>). Consistent with this, pharmacologic inhibition of autophagy in ischemia-mimicking conditions increases cardiomyocyte death, suggesting that autophagy functions as a pro-survival mechanism (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B32" target="blank">32</a>). During mild ischemic stress, activation of autophagy depends on AMPK-mediated inhibition of mTOR and is cardioprotective (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B33" target="blank">33</a>, <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B34" target="blank">34</a>). In the setting of chronic ischemia, autophagic activity can inhibit apoptosis and mitigate tissue damage (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B35" target="_blank">35</a>).</p>

<p>In most instances, an infarct-related artery recanalizes, either spontaneously or by therapeutic intervention. When oxygen and nutrients are restored to the previously ischemic tissue, cardiomyocyte autophagy is upregulated dramatically in vivo (e.g., in rat [ref. <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B36" target="blank">36</a>], rabbit [ref. <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B37" target="blank">37</a>], and swine [ref. <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B35" target="blank">35</a>]), and in cells maintained in culture (e.g., in HL-1 cells [ref. <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B38" target="blank">38</a>] and primary neonatal cardiomyocytes [refs. <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B32" target="blank">32</a>, <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B33" target="blank">33</a>]). However, detailed time-course analyses indicate that reperfusion-triggered autophagy is transient such that autophagic flux eventually declines below baseline levels (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B39" target="_blank">39</a>).</p>

<p>Reperfusion elicits a response that differs vastly from ischemia. Studies suggest that cardiac autophagy in response to reperfusion can be either adaptive or detrimental and involves beclin 1 activation independent of the AMPK/mTOR pathway (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B33" target="blank">33</a>). Indeed, whether changes in autophagic activity elicited by reperfusion are adaptive or maladaptive is the subject of debate, with experimental evidence supporting either point of view. In cultured neonatal cardiomyocytes exposed to simulated ischemia/reperfusion (I/R), chemical suppression of autophagy with 3-methyladenine enhances cell viability (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B32" target="blank">32</a>). In contrast, other studies report that autophagy is protective in simulated I/R (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B40" target="blank">40</a>, <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B41" target="blank">41</a>). Furthermore, short repetitive ischemic episodes, which elicit beneficial preconditioning effects, also induce autophagy, and when autophagy is suppressed, the protective effects of preconditioning are lost (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B35" target="blank">35</a>, <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B41" target="blank">41</a>). In HL-1 cells, I/R impairs autophagic flux at the level of both induction and processing, while enhancing autophagy protects against I/R injury (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B38" target="blank">38</a>). Moreover, autophagosomes have been detected in surviving cardiomyocytes in chronic stages of myocardial infarction, suggesting that autophagy may aid in survival. Exposure to the autophagy inhibitor bafilomycin A1 exacerbates cardiac dysfunction and remodeling (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B42" target="blank">42</a>). At present, the extent to which these discrepancies derive from differing cell types, model systems, or experimental paradigms is unclear.</p>

<p>Novel responses by beclin 1 and LAMP2 in I/R injury were recently reported (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B43" target="blank">43</a>). While beclin 1 is essential for initiation of autophagy, it can also inhibit vesicle processing late in the autophagic cascade. Cardiomyocyte autophagy was upregulated in response to I/R injury, but autophagosome clearance was defective, leading to cell death. Reperfusion increased beclin 1 levels, causing impairment of autophagosome processing and reduced levels of LAMP2, a protein critical for autophagosome-lysosome fusion. Partial beclin 1 knockdown restored autophagic processing and protected from I/R injury–induced cell death, whereas complete beclin 1 knockdown impaired autophagosome formation and increased cell death (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B43" target="blank">43</a>). Thus, beclin 1 abundance can be an important determinant of autophagic activity, either ensuring survival or triggering cell death (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B44" target="blank">44</a>). Although it is well documented that haploinsufficiency of beclin 1 protects the heart in transverse aortic constriction–induced (TAC-induced) hypertrophy (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B45" target="blank">45</a>) or I/R injury (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B33" target="blank">33</a>, <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B43" target="blank">43</a>), the actions of beclin 1 at both early and late points of the autophagic cascade complicate the interpretation of these studies.</p>

<p>With regard to human disease, analysis of tissue samples from patients with ischemic heart disease is typically confounded by co-existing HF (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B29" target="blank">29</a>). Autophagy was described in human right atrial appendages collected before cardioplegic arrest and again after reperfusion. Perioperative I/R upregulated 11 ATGs and downregulated three (of 84 examined). Elevated autophagic activity was also confirmed through observations of increased LC3-I levels and LC3-II/LC3-I ratios (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B46" target="blank">46</a>).</p>

<p>Autophagy in cardiac hypertrophy. Cardiac hypertrophy is a major risk factor for adverse cardiovascular events (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B47" target="blank">47</a>). Initial phases of hypertrophic growth of the myocardium are thought to be adaptive, serving to normalize ventricular wall stress and oxygen demand; however, this concept has never been tested. Cardiac hypertrophy involves increases in the size of individual cardiomyocytes. When it occurs in response to physiologic cues such as exercise or pregnancy, it is not associated with HF. By contrast, hypertrophy elicited by disease-related stresses such as hypertension, obesity, valvular disease, or infarction is pathological and correlates with abnormal metabolic, structural, and functional alterations, including changes in metabolic substrate utilization, disorganization of the sarcomere, alterations in Ca2+ handling, changes in contractility, cardiomyocyte loss, systolic and/or diastolic dysfunction, and electrical remodeling (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B48" target="blank">48</a>).</p>

<p>Remodeling of any tissue involves alterations in the steady-state equilibrium between protein synthesis and protein degradation. A number of studies have focused on the relationship between catabolic pathways and myocardial remodeling, with autophagy being one of the most extensively studied. The first report of autophagy in cardiac hypertrophy dates from 1983, when reduced numbers of autophagic vacuoles were noted in ventricular hypertrophy stemming from supravalvular aortic constriction, suggesting that the degradation of cytoplasmic components was inhibited (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B49" target="blank">49</a>). Cardiomyocyte-specific conditional deletion of ATG5 uncovered a key role for autophagy in the maintenance of cardiac structure and function both in the basal state and under hemodynamic stress (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B50" target="blank">50</a>). In TAC-induced pressure overload, autophagic activity increased as early as 24 hours after surgery, and load-induced pathologic remodeling was blunted in beclin 1 haploinsufficient mice (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B45" target="blank">45</a>). Conversely, cardiomyocyte-restricted overexpression of beclin 1 substantially amplified adverse remodeling (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B45" target="blank">45</a>). Subsequent studies identified protein aggregation is a proximal trigger of load-induced cardiomyocyte autophagy (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B51" target="_blank">51</a>).</p>

<p>As in the case of I/R, cardiomyocyte autophagy triggered by increased afterload appears to have both adaptive and maladaptive features. This dual nature of autophagy is a recurring theme in other organ systems and disease states (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B52" target="blank">52</a>). Indeed, we have postulated that the physiologic impact of autophagy is a continuum, and a window of optimal autophagic activation is critical to the maintenance of cellular homeostasis and function (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B53" target="blank">53</a>).</p>

<p>A shift in the balance between the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD+ and NADH) has been shown to influence autophagic flux. The prevention of downregulation of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme in the mammalian NAD+ salvage pathway (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B54" target="blank">54</a>), protects against myocardial injury by increasing NAD+ and ATP levels, inhibits apoptosis, and stimulates autophagic flux (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B55" target="blank">55</a>). NAD+ acts in part by activating sirtuin 1 (Sirt1), which then induces autophagy through nuclear localization of FoxO1 (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B56" target="blank">56</a>). In Sirt1-knockout mice, beclin 1 levels are diminished, consistent with the involvement of Sirt1 in regulation of autophagy (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B57" target="blank">57</a>). In addition, exogenous NAD+ can block phenylephrine- and angiotensin II–induced cardiac hypertrophy through activation of the Sirt3/AMPK pathway (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B58" target="_blank">58</a>). Thus, cellular events that govern NAD+ levels may emerge as therapeutic targets in cardiac remodeling.</p>

<p>In a hypertensive double-transgenic rat model harboring copies of both the human renin and angiotensinogen genes (dTGRs), caloric restriction decreased mortality, cardiomyocyte hypertrophy, vascular inflammation, cardiac damage, cardiac fibrosis, cardiomyocyte apoptosis, and transcript levels of atrial natriuretic peptide (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B59" target="blank">59</a>). These effects were independent of changes in blood pressure and were linked to increased autophagy (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B59" target="blank">59</a>).</p>

<p>The number of cellular processes regulated by microRNAs (miRNAs) has grown to include critical elements of cardiac biology, including cell size regulation, survival, action potentials, mitochondrial function, and energetics (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B60" target="blank">60</a>). Recently, Ucar et al. showed that the miRNA-212/132 family regulates both cardiac hypertrophy and autophagy by targeting FoxO3 (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B61" target="blank">61</a>), adding to the growing body of evidence for a crucial role of autophagy in the control of hypertrophy. Furthermore, because autophagy is controlled by a variety of tractable mechanisms, including histone deacetylases (HDACs), sirtuins, and miRNAs, autophagy has emerged as a promising “druggable” target in hypertrophic heart disease.</p>

<p>Autophagy in HF. The initial response of the heart to increases in afterload is hypertrophic growth (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B48" target="blank">48</a>). If the afterload stress persists, the heart becomes dilated, contractile function declines, and HF ensues (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B62" target="blank">62</a>). Indeed, this disease progression commonly occurs in patients with hypertension or ischemic heart disease (ref. <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B63" target="blank">63</a> and Figure <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#F2" target="blank">2</a>).</p>

<p>In a model of pressure overload, we found that the degree of autophagic activity correlated with the magnitude of hypertrophic growth and the rate of transition to HF (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B45" target="blank">45</a>). Consistent with these findings, transgenic mice with cardiomyocyte-restricted overexpression of beclin 1 amplified the pathologic remodeling response (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B45" target="blank">45</a>, <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B64" target="blank">64</a>). Conversely, beclin 1 haploinsufficiency halved the stress-induced autophagic response and partially rescued the HF phenotype (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B45" target="blank">45</a>). Collectively, these data suggest that autophagy can be maladaptive under conditions of severe pressure overload, a common clinical scenario.</p>

<p>It is somewhat counterintuitive that autophagy, a mechanism of protein degradation, should facilitate hypertrophic growth. However, the natural history of HF involves substantial remodeling of ventricular morphology from concentric hypertrophy to eccentric hypertrophy, a structural change that would necessitate catabolism and recycling of existing cellular structures. Because of this, autophagy is a potential target for therapeutic intervention in afterload-induced HF. To test this, we recently employed small-molecule inhibitors of HDACs, which have previously been shown to suppress pathologic cardiac remodeling (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B65" target="blank">65</a>). We hypothesized that maladaptive autophagy is HDAC dependent and that the beneficial effects of HDAC inhibitors occur due to their ability to suppress autophagy. Consistent with this model, we found that HDAC inhibition was, in fact, capable of profoundly suppressing load-induced cardiomyocyte autophagy and thereby blunted the pathologic growth response (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B64" target="blank">64</a>).</p>

<p>As discussed earlier, although excessive cardiomyocyte autophagy can be maladaptive, complete abrogation of autophagy is similarly maladaptive and can accelerate the progression to HF. For example, inactivation of Atg5 in adult heart is sufficient to trigger rapid-onset HF (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B50" target="blank">50</a>). Similarly, systemic inactivation of the gene encoding the lysosomal enzyme cysteine endopeptidase cathepsin L (Ctsl) leads to the accumulation of large dysmorphic vesicles in the cardiomyocyte cytoplasm and dilated cardiomyopathy (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B66" target="blank">66</a>), consistent with the notion that basal levels of cardiomyocyte autophagy are required for cellular proteostasis. Given this, we favor a model in which titration of cardiomyocyte autophagy within an optimal, adaptive zone is a therapeutic goal of interest (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B53" target="blank">53</a>). Importantly, HDAC inhibition suppresses, but does not eliminate, the autophagic response to stress and hence is an attractive strategy worthy of additional investigation (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B39" target="blank">39</a>, <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B64" target="_blank">64</a>).</p>

<p>Analysis of human samples has afforded additional evidence that autophagic cell death contributes to HF pathogenesis (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B30" target="blank">30</a>, <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B67" target="blank">67</a>). In patients with isolated aortic valve stenosis and varying degrees of left ventricular systolic dysfunction, cell loss, mainly due to autophagy and oncosis, was associated with the progression of left ventricular systolic dysfunction (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B5" target="blank">5</a>). More recently, analyses of biopsy samples of left ventricular myocardium from nine patients with idiopathic dilated cardiomyopathy obtained at the time of both implantation and explantation of a left ventricular assist device showed that mechanical unloading of the failing human heart was associated with decreased markers of autophagy (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B68" target="blank">68</a>). It is not known whether mechanical unloading restores autophagy to basal levels or establishes a new set point. Furthermore, as these were static endpoint studies, it is possible that initial increases in autophagic activity accompanied unloading to facilitate atrophic remodeling.</p>

<p>Mitophagy. Mitophagy is the selective sequestration of mitochondria by autophagosomes and their subsequent delivery to lysosomes for degradation. This process is important for myocardial homeostasis and stress adaptation. Destruction of damaged mitochondria by mitophagy induces cardiomyocyte preconditioning and reduces cell death during I/R (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B69" target="blank">69</a>, <a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B70" target="blank">70</a>). Oka et al. showed that impaired mitophagy leads to TLR9-mediated inflammatory responses in cardiomyocytes and induces myocarditis and dilated cardiomyopathy (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B71" target="blank">71</a>). Hoshino et al. showed that cytosolic p53 impairs autophagic degradation of damaged mitochondria, triggering mitochondrial dysfunction and HF in mice (<a href="chrome-extension://dlbfminlfioedlkcbclpmnbfjommaebc/view.html#B72" target="blank">72</a>).</p>

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