Regulation of Autophagy by Metabolic and Stress Signaling Pathways in the Heart

Abstract

Autophagy is an essential process for the maintenance of cellular homeostasis in the heart under both normal and stress conditions. Autophagy is a key degradation pathway and acts as a quality control sensor. It protects myocytes from cytotoxic protein aggregates and dysfunctional organelles by quickly clearing them from cell. It also responds to changes in energy demand and mechanical stressors to maintain contractile function. The autophagic-lysosomal pathway responds to serum starvation to ensure that the cell maintains its metabolism and energy levels when nutrients run low. In contrast, excessive activation of autophagy is detrimental to cells and contributes to development of pathological conditions. A number of signaling pathways and proteins regulate autophagy. These include the AMPK/mTOR pathway, FoxO transcription factors, Sirt1, oxidative stress, Bcl-2 family proteins, and the E3 ubiquitin ligase Parkin. In this review, we will discuss how this diverse cast of characters regulates the important autophagic process in the myocardium.

INTRODUCTION

Autophagy is an evolutionarily conserved catabolic process that is responsible for the degradation of cytoplasmic components via the lysosomal pathway ¹. In the absence of stress, autophagy complements the function of the proteasome by degrading long-lived proteins. It also plays an important role in cellular quality control and is responsible for clearing protein aggregates and dysfunctional organelles that could become toxic to the cell. This quality control function is particularly important in post-mitotic cells such as myocytes and neurons that are not easily replaced. Autophagy increases under conditions of limited nutrients and degrades cytoplasmic material to provide the cell with amino acids and fatty acids. The breakdown of organelles and proteins ensures that the cell can maintain its metabolism and energy level when nutrients run low ². Autophagy is also upregulated by many other stressors including opening of the mitochondrial permeability transition pore (mPTP) ³, ER stress ⁴, and increased production of reactive oxygen species (ROS) ⁵.

Autophagy has important functions in the myocardium and its dysregulation has been implicated in a wide variety of cardiovascular pathologies. For instance, Danon disease is a fatal cardiomyopathy caused by a defect in the fusion between autophagosomes and lysosomes that leads to an accumulation of autophagosomes in the myocytes ^{6, 7}. Also, cardiac specific deletion of the autophagy-related gene 5 (Atg5), a critical autophagy protein, in the adult heart leads to disruption of autophagy with subsequent buildup of dysfunctional mitochondria and development of cardiac dysfunction ⁸. Similarly, deletion of Atg7 in skeletal muscle leads to accumulation of impaired mitochondria and a corresponding increase in intracellular ROS levels ⁹. Increased autophagy is commonly observed in the heart with acute and chronic ischemia, heart failure and dilated cardiomyopathy ^{5, 10–14}.

Since cardiac myocytes are terminally differentiated and possess extremely limited regenerative capacity, rapid adaptive activation of autophagy in response to metabolic or mechanical stress is critical for the maintenance of normal cardiac function. Activation of autophagy will generate intracellular nutrients and energy required to survive the stress, and will also remove damaged organelles such as leaky mitochondria that can be harmful to the cell ¹⁵. Here, we review our current knowledge of metabolic and stress signaling pathways that regulate autophagy in the myocardium.

Initiation of Autophagy

The study of autophagy in yeast has provided detailed knowledge about autophagic signaling pathways and advanced our general understanding of autophagy in the heart. At least sixteen different Atg gene products coordinate the formation of an autophagosome ¹⁶. When the cell receives a signal to initiate autophagy, an isolation membrane (also called phagophore) is formed (Figure 1). Although the origin of the membrane is still unclear, recent studies have found that sarcoplasmic/endoplasmic reticulum ¹⁷, mitochondrial outer membrane,¹⁸ and plasma membrane ¹⁹ can all serve as sources of the isolation membrane. Unc-51-like kinase (ULK1), which forms a complex with Atg13 and focal adhesion kinase-family interacting protein of 200 kD (FIP200), is a key regulator of the initiation of autophagy ^{20, 21}. The initial phagophore formation (nucleation) requires assembly of the Beclin1 (Atg6 in yeast)-vacuolar sorting protein 34 (Vps34)-Vps15 complex ²². Beclin1 is regulated by the anti-apoptotic proteins Bcl-2 and Bcl-X_L, which bind Beclin1 to inhibit activity and induction of autophagy ^23–25. Subsequent expansion of the membrane is mediated by two ubiquitin-like conjugation systems, Atg12 and Atg8 (microtubule-associated protein 1 light chain 3 (LC3) in mammals) that together promote assembly of the Atg16L complex and the processing of LC3 ^{26, 27}. The isolation membrane elongates until the edges fuse around its target(s) forming a double-membrane structure called the autophagosome. The autophagosome then moves along the microtubules to the microtubule organizing center where it fuses with a lysosome ²⁸. The contents are degraded by lysosomal digestive enzymes, and the breakdown products, including amino acids, lipids, nucleosides, and carbohydrates, are released into the cytosol where they can be used by synthetic and metabolic pathways.

Figure 1.

Schematic of the autophagic process. The plasma membrane, mitochondria, and E/SR serve as sources of the isolation membrane. The initiation of autophagy is regulated by the ULK1 protein complex. The initial phagophore formation requires Beclin1-Vps34-Vps15 complex and expansion of the membrane is mediated by two ubiquitin-like conjugation systems, Atg12 and LC3. The membrane elongates until the edges fuse around its target forming the autophagosome. The autophagosome then fuses with the lysosome and the content is degraded.

Autophagy and Energetic Balance

Induction of autophagy is critical for the maintenance of cardiac function under starved conditions ^{2, 29}, and inhibition of autophagy results in reduced ATP levels and development of severe cardiac dysfunction ². Moreover, autophagy plays an important role in salvaging myocytes during acute myocardial infarction (MI) by preserving amino acid and ATP levels. Autophagy was rapidly enhanced in the region bordering the infarction ^30–32, and inhibiting autophagy prior to coronary ligation resulted in increased injury. In contrast, starvation of mice for 24 h prior to MI enhanced autophagy after occlusion of the left descending coronary artery and reduced infarct size compared to normally fed mice ³⁰. Furthermore, amino acid and ATP levels were significantly reduced 4 h post-infarction in controls, but hearts of mice subjected to starvation prior to MI had preserved amino acid and ATP levels. Inhibition of autophagy abolished the protective effect of starvation ³⁰. Clearly, this suggests that activation of autophagy during MI preserves ATP levels and maintains an adequate amino acid pool, which can be used for energy production and protein synthesis that required for the proper response to the ischemic insult.

The serine/threonine kinase mammalian target of rapamycin (mTOR) i s an important negative regulator of autophagy in mammalian cells ^{33, 34} and integrates intracellular signals such as growth factors, amino acids, glucose, and energy status ³⁵. In the heart, mTORC1 plays an important role in regulating cellular homeostasis, and cardiac specific deletion of mTOR leads to development of a fatal dilated cardiomyopathy ³⁶. mTOR exists in a multiprotein complex known as mTOR complex 1 (mTORC1) which includes Raptor, proline-rich Akt substrate of 40-kDa (PRAS40), and MTOR associated protein LST8 homolog (Mlst8) ³⁷. It has been reported that mTORC1 suppresses autophagy by binding and phosphorylating the autophagy-initiating kinase ULK1 ³⁸. When inactivated, mTORC1 dissociates from ULK1, freeing it to initiate formation of the isolation membrane. Also, perfusion of isolated hearts with rapamycin, an inhibitor of mTOR and activator of autophagy, prior to I/R reduced infarct size suggesting that mTOR is an important regulator of essential cellular functions during ischemia ^{39, 40}.

The mTOR pathway is regulated by the 5’-AMP-activated protein kinase (AMPK) (Figure 1). AMPK is an intracellular energy sensor that is activated in response to changes in the AMP/ATP ratio. Activation of AMPK results in adaptive changes in growth and metabolism under conditions of low energy. AMPK stimulates autophagy by inhibiting mTOR through phosphorylation and activation of the tuberous sclerosis complex 2 (TSC2) ⁴¹ and Raptor ⁴². TSC2 exists in a complex with TSC1 in cells, and the TSC1/2 complex inhibits the mTOR activator Rheb ⁴³. TSC2 acts as a GTPase activating protein towards Rheb and by stimulating the conversion of active Rheb-GTP into the inactive Rheb-GDP, the TSC1/2 complex inhibits mTOR ⁴⁴. Raptor is an essential mTOR binding partner and is responsible for recruiting substrates to the mTORC1 complex ^{45, 46}. Recently, it was reported that AMPK can directly activate autophagy by directly activating ULK1. Kim et al. discovered that under glucose starvation, AMPK promotes autophagy by directly activating ULK1 through phosphorylation of Ser 317 and Ser 777 ³⁸. In contrast, during nutrient sufficiency, high mTOR activity prevents ULK1 activation by phosphorylating ULK1 Ser757 and thus disrupting the interaction between ULK1 and AMPK. When nutrients are limited, as seen in myocardial ischemia, AMPK acts as a checkpoint by inhibiting cellular growth and inducing autophagy in cardiac myocytes. The AMPK-mTOR pathway is an important regulator of autophagy in response to glucose deprivation in neonatal myocytes as inhibition of AMPK reduced autophagy and increased cell death in these cells ¹³. Also, transgenic mice with cardiac specific expression of a dominant negative AMPK had attenuated induction of autophagy in response to ischemia in vivo ¹³. This suggests that AMPK-induced autophagy regulation via inhibition of mTOR plays an important role in the adaptation to ischemia.

Recently, glycogen synthase kinase-3 (GSK-3) was identified to be a regulator of the mTOR pathway in cardiac cells. GSK-3 is an important signaling molecule and is involved in regulating gene transcription, protein translation, and apoptosis, as well as hexose metabolism ⁴⁷. GSK-3β is known to function downstream of phosphatidylinositol 3-kinase (PI3K) and Akt ⁴⁸, and, in the heart, inhibition of GSK-3β has been reported to be cardioprotective ^{14, 48–50}. Similar to AMPK, GSK-3β was found to inhibit mTOR signaling and activate autophagy via phosphorylation of TSC2 ^51,52. Moreover, Zhai et al. discovered that GSK-3β is a critical upstream regulator of mTOR during both ischemia and reperfusion in the heart ¹⁴.

Regulation of Autophagy by FoxO Transcription Factors and the NAD-dependent deacetylase SIRT1

Autophagy is also regulated by the FoxO (Forkhead box-containing protein, O subfamily) transcription factors ⁵³. FoxO1 and FoxO3 are highly expressed in the heart ⁵⁴ and regulate autophagy by activating transcription of the Atg genes ⁵⁵. It was recently reported that glucose deprivation of cardiac myocytes induced translocation of FoxO1 and FoxO3 to the nucleus where they activated transcription of genes involved in autophagy ^55,56. In addition, transgenic mice overexpressing FoxO3 in the heart showed increased levels of autophagy which correlated with the development of cardiac atrophy ⁵⁷. In contrast, genetic deletion of FoxO3 resulted in development of cardiac hypertrophy ⁵⁸. Both FoxO1 and FoxO3 have been reported to induce expression of the BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) ^{59, 60} which is a potent inducer of autophagy.

Acetylation/deacetylation status of certain proteins is also linked to regulation of autophagy. The Sirtuin 1 (Sirt1) is a NAD-dependent deacetylase which is upregulated in response to starvation or caloric restriction ^{61, 62}. Interestingly, Lee et al. found that starvation-induced autophagy was reduced in Sirt1−/− MEFs and that Sirt1 regulates autophagy via deacetylation of Atg5, Atg7 and Atg8 ⁶³. Another study recently reported that deacetylation of FoxO1 by Sirt1 was an essential step for autophagy in glucose-deprived cardiac myocytes as FoxO1 mutants that cannot be deacetylated by Sirt1 inhibited induction of autophagy ⁵⁶.

Induction of Autophagy by Stress Signals

Many studies have reported that autophagy is upregulated in the heart in response to ischemia/reperfusion and hemodynamic stress ^{5, 10, 13}. Multiple signaling pathways are activated in these hearts, and many of them are likely to contribute to the activation of autophagy. For instance, increased oxidative stress activates autophagy in cells, including cardiac myocytes ^{5, 64}. Production of ROS played an important role in inducing autophagy during I/R in vivo, and the presence of an anti-oxidant significantly reduced the level of autophagy ⁵. Interestingly, mitochondria are a major source of ROS, which promotes activation of autophagy in cells. For instance, inhibition of electron transport chain complexes I or II induced ROS production and autophagy in cells, whereas the presence of a ROS scavenger decreased autophagy ⁶⁵. Increased ROS could affect autophagy by directly acting on the autophagy proteins. Hydrogen peroxide was reported to oxidize and subsequently inactivate Atg4 during starvation. Atg4 is responsible for recycling of LC3 by cleaving phosphatidylethanolamine (PE) from PEconjugated LC3 and inhibition of Atg4 leads to accumulation of LC3-PE (LC3-II) and increased autophagosome formation ⁶⁶. In addition, oxidative stress might indirectly activate autophagy by causing damage to organelles such as mitochondria that induces mitochondrial autophagy.

The mitochondrial permeability transition pore (mPTP) causes permeabilization of the inner mitochondrial membrane resulting in swelling of the inner membrane with subsequent rupture of the outer membrane ⁶⁷. Opening of the mPTP have been reported to activate autophagy in mammalian cells ^68–70. In contrast, starvation-induced autophagy was significantly reduced in the presence of cyclosporine A, an inhibitor of the mPTP ³. Cyclophilin D (CypD) is an essential component of the mPTP, and CypD-deficient mice are resistant to mPTP opening ⁷¹. This study found that fasting failed to induce autophagy in CypD-deficient mice while, transgenic mice overexpressing cypD in the heart showed enhanced levels of autophagy under normal and fasting conditions compared to wild type mice ³. This study suggests that the mPTP plays an important role in starvation-induced autophagy. In contrast, Quinsay et al. found that the BH3-only protein Bnip3 induced autophagy independent of the mPTP in cardiac myocytes ⁷². Although the mPTP plays a role in the induction of autophagy, it is unclear if loss of mitochondrial membrane potential due to pore opening alone is responsible for the induction of autophagy or if factor(s) released from the mitochondria activate the autophagic pathway.

The endoplasmic reticulum (ER) is important in regulating cytosolic Ca²⁺ homeostasis and proper folding of newly synthesized proteins ⁷³. Defects in ER function lead to activation of the ER stress pathway and activation of autophagy ⁴ via down-regulation of mTOR signaling ⁷⁴, monocyte chemotactic protein-1 induced protein (MCPIP) ⁷⁵, and JNK/p38 and activating transcription factor 4 (ATF4)-dependent activation ⁷⁶. Interestingly, treatment of hearts with low doses of tunicamycin or thapsigargin, two different inducers of ER stress, resulted in induction of autophagy and reductions in apoptosis and infarct size ⁷⁷. This suggests that ER stress-mediated autophagy can serve a protective role in the heart.

Mitochondrial Autophagy in the Myocardium

For a long time, autophagy was considered to be a non-selective process in which the autophagosomes randomly sequestered material in the cytosol for degradation. However, many studies have now demonstrated that autophagy can be selective and specifically remove damaged organelles and toxic protein aggregates (Figure 2). Bnip3 and Bnip3-like (Bnip3L/Nix) belong to the BH3-only proteins of the Bcl-2 family that are primarily localized to mitochondria in cells. Both Bnip3 and Nix are potent inducers of mitochondrial autophagy ^78–80. Sandoval et al. found that Nix was required for mitophagy in erythroid cells ⁸¹. Interestingly, Nix was not required for the induction of general autophagy, but only for the elimination of mitochondria ⁷⁹. Similarly, Bnip3 has been reported to induce mitochondrial autophagy in cardiac myocytes ^{72, 78, 82}. Exactly how Nix and Bnip3 target mitochondria for autophagy is still unclear, but it has been shown that Nix-dependent loss of mitochondrial membrane potential (Δψm) was important in targeting the mitochondria to autophagosomes ⁸¹. In addition, Nix has been proposed to act as an autophagy receptor by interacting directly with the autophagy proteins LC3 and GABARAP ⁸³. Bnip3 has also been reported to interact with LC3 ⁸⁴. However, the importance of Nix and Bnip3 as autophagy receptors is still unclear since mitophagy was restored when Nix^−/− cells were treated with compounds that caused mitochondrial depolarization ⁸¹.

Figure 2.

Induction of mitochondrial autophagy in response to cellular stress. After Drp1-mediated mitochondrial fission, Parkin is recruited to dysfunctional mitochondria where it ubiquitinates proteins. The p62 adaptor protein binds to the ubiquitinated proteins and LC3 on the autophagosomes linking the two organelles. Alternatively, LC3 can directly interact with Bnip3 or Nix on the mitochondria.

The E3 ubiquitin ligase Parkin has also been identified as an important regulator of mitochondrial autophagy in cells. Parkin-mediated clearance of mitochondria plays an important role in cardiac preconditioning ⁸⁵. Narendra et al. discovered that Parkin accumulated on depolarized mitochondria which promoted their removal by autophagy ⁸⁶. Parkin was found to ubiquitinate proteins on dysfunctional mitochondria, which served to recruit HDAC6 and p62/SQSTM1, and promote assembly of the autophagy machinery at the impaired mitochondrion ⁸⁷. Interestingly, p62 was not essential for Parkin-mediated mitophagy as p62 deficiency in cells did not prevent CCCP-mediated mitophagy ⁸⁸. We recently found that Bnip3-mediated mitochondrial autophagy was dependent on Parkin in cardiac myocytes and that Parkin deficient myocytes had reduced induction of autophagy in response to Bnip3 overexpression ⁸⁹. We also found that mitochondria had to undergo Drp1-mediated mitochondrial fission prior to Parkin-translocation and removal by autophagosomes. Also, Ding et al. found that CCCP-induced Parkin translocation was significantly reduced in Nix deficient MEFs ⁹⁰. These studies suggest that Bnip3 and Nix cooperate with the Parkin pathway to clear dysfunctional mitochondria in cells.

Maladaptive role of Autophagy in the Myocardium

Most studies suggest that autophagy is a protective response activated by the cell during stress. However, it is now evident that constitutive and/or excessive autophagy can be detrimental to the heart. Recently, Schips et al. reported that constitutive activation of FoxO3 in mouse hearts led to increased autophagy and atrophy ⁵⁷. These hearts reduced stroke volume and cardiac output, suggesting that constitutive activation of autophagy is harmful for the myocardium. Interestingly, this study discovered that the cardiac atrophy and dysfunction were reversible when FoxO3 expression was turned off ⁵⁷. In addition, studies have reported that excessive autophagy contributes to pathological conditions in response to stress. Upregulation of Beclin1 and activation of autophagy during reperfusion after ischemia have been found to be detrimental to cardiac myocytes ¹³. Interestingly, Beclin1 heterozygous (Beclin1^+/−) mice had reduced levels of autophagy and showed significantly smaller infarcts compared to wild type mice after I/R. This suggests that upregulation of Beclin1 contributes to excessive activation of autophagy which is detrimental to cells. Similarly, it was reported that reduced mTOR activity and enhanced autophagy during reperfusion contributed to I/R injury ¹⁴. Autophagy has also been reported to play a maladaptive role in a mouse model of pressure overload ^{10, 91}. Zhu et al. found that Beclin1 heterozygous mice had decreased induction of autophagy and reduced hypertrophic growth in response to pressure over load, whereas overexpression of Beclin1 led to increased induction of autophagy and enhanced pathological hypertrophy compared to wild type mice ¹⁰. Collectively, these studies suggest that the duration and level of autophagy play an important role in determining whether autophagy will be protective or maladaptive in the heart.

Conclusion

Studies suggest that basal levels of autophagy are important for maintaining cellular homeostasis and for protecting cells against accumulation of toxic protein aggregates or dysfunctional organelles. It is also clear that enhancing autophagy can promote survival in response to stress, such as nutrient deprivation or hypoxia, by recycling macromolecules to maintain energy levels and removing damaged organelles such as mitochondria. In contrast, excessive levels of autophagy contribute to development of pathological conditions, most likely by removal of too many essential organelles and proteins. Manipulation of signaling pathways that regulate autophagy may represent a potential future therapeutic target to treat or prevent development of heart disease. A more thorough understanding of signaling pathways that regulate autophagy will be of great importance for future studies of the heart.