The role of autophagy in cardiovascular pathology

Abstract

Macroautophagy/autophagy is a conserved catabolic recycling pathway in which cytoplasmic components are sequestered, degraded, and recycled to survive various stress conditions. Autophagy dysregulation has been observed and linked with the development and progression of several pathologies, including cardiovascular diseases, the leading cause of death in the developed world. In this review, we aim to provide a broad understanding of the different molecular factors that govern autophagy regulation and how these mechanisms are involved in the development of specific cardiovascular pathologies, including ischemic and reperfusion injury, myocardial infarction, cardiac hypertrophy, cardiac remodelling, and heart failure.

1. Introduction

Autophagy is an evolutionarily conserved catabolic recycling pathway in which different cellular components ranging from protein aggregates to entire organelles are targeted for degradation to promote cell survival under different types of stress.¹ Autophagy induction has been observed under a diverse range of physiological and pathological conditions, including hypoxia,² endoplasmic reticulum (ER) stress,^3,4 oxidative stress and particularly nutrient starvation. All these stimuli are also involved in cardiovascular development, metabolism and disease; thus, autophagy regulation is a relevant subject for cardiovascular pathology.

2. Macroautophagy, microautophagy, and chaperone-mediated autophagy

Autophagy is mainly divided into three main branches, macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA), each of which has been described in detail elsewhere.^5,6 Whereas some studies have shown the importance of microautophagy and CMA in the heart,⁷ the main volume of research points to macroautophagy as the main autophagic branch regulating both the physiological and pathological mechanisms involved with the cardiovascular system. Thus, for the remainder of this review, macroautophagy will be referred to as autophagy. In brief, during macroautophagy, cytoplasmic components targeted for degradation are enclosed by a rapidly expanding cup-shaped membrane called the phagophore. Expansion and maturation of the phagophore lead to the sequestration of the cargo inside double-membrane vesicles (autophagosomes) that ultimately fuse with the vacuole (in yeast and plants) or lysosome (in metazoans). Once fused together, the sequestered cargo is degraded by resident vacuolar/lysosomal hydrolases, generating macromolecules that can be then transported back into the cytosol to be recycled by the cell (Figure 1).¹

Figure 1.

During macroautophagy different cytoplasmic components are enclosed by the expanding phagophore. Maturation of the phagophore results in the sequestration of the cargo inside double-membrane autophagosomes. Fusion between autophagosomes and lysosomes results in the formation of an autolysosome, where the sequestered cargo is degraded by lysosomal hydrolases. Macromolecules obtained from cargo degradation are transported back into the cytosol to be reutilized.

3. Autophagy induction

Autophagy activation is a tightly regulated process that depends on the transcriptional,⁸ post-transcriptional,^8,9 and post-translational⁸ regulation of several autophagy-related (ATG) genes and their corresponding proteins. In nutrient-rich conditions, autophagy is usually kept at low levels; however, during nutrient starvation, autophagy activity is highly upregulated.⁸ Thus, autophagy regulation by nutrient availability depends on several crucial cellular energy sensors such as MTORC1, AKT/PKB, AMPK, and PRKA/PKA.

Activation of the ULK Ser/Thr kinase complex is one of the early events required for autophagy induction. The ULK complex is formed by the catalytic subunits ULK1/2,^10,11 the regulatory scaffold protein ATG13,^12,13 RB1CC1/FIP200,¹⁴ and the stabilizing protein ATG101.¹⁵ As part of the initiation step of the autophagy pathway, the ULK complex phosphorylates downstream ATG proteins leading to their activation and recruitment. Activation of the ULK complex during nutrient starvation conditions leads to ULK1 auto-phosphorylation^13,14 at Thr180¹⁶ and Ser1047¹⁷ which stabilizes its catalytic activity and in turn leads to the phosphorylation of several targets including BECN1 at Ser14¹⁸ and Ser30,¹⁹ ATG14 at Ser29,²⁰ AMBRA1 at Ser465 and Ser635,¹⁹ ATG16L1 at Ser278,²¹ RB1CC1²² at Ser943, Ser986 and Ser1323,¹⁹ ATG13²² at Ser318²³ and Ser389,¹⁹ and ATG101 at Ser11 and Ser203.¹⁹ In nutrient-rich conditions, the ULK complex activity is inhibited by MTORC1-dependent phosphorylation of both ULK1 at Ser757²⁴ and ATG13 at multiple sites.^10,12,22 MTORC1 is a major sensor of the amino acid availability inside the cell, being activated by high amino acid levels through binding to an RRAG GTPase dimer²⁵ and the small GTPase RHEB.²⁶ Thus, MTORC1 is a major autophagy suppressor integrating nutrient and energy signalling into autophagy regulation. Furthermore, MTORC1 signalling has also been implicated in the transcriptional regulation of autophagy. During nutrient-rich conditions, MTORC1 phosphorylates the transcription factor TFEB at Ser211,²⁷ preventing its nuclear translocation and the transcription of several lysosomal²⁸ and ATG genes, including UVRAG, WIPI, MAP1LC3B, SQSTM1, VPS11, VPS18, and ATG9B.²⁹

Closely related to MTORC1 signalling, the AKT/PKB pathway responds to growth factors to inhibit autophagy.³⁰ Activation of INSR (insulin receptor) and IGF1R (insulin like growth factor 1 receptor) triggers the activation of AKT, which in turn phosphorylates the GTPase activating protein TSC2 at Ser393 and Thr1462, preventing RHEB inhibition and leading to autophagy suppression by MTORC1.³¹ Furthermore, AKT directly activates MTORC1 by phosphorylation at Ser2448.³² AKT activation depends on the formation of phosphatidylinositol(3,4,5)trisphosphate (PtdInsP₃) in the plasma membrane; the phosphoinositide phosphatase PTEN is thus able to induce autophagy by dephosphorylating PtdInsP₃ and downregulating AKT signalling.³³ Autophagy is also directly inhibited by AKT, which can directly phosphorylate BECN1 at Ser295 and possibly Ser234, as well as ULK1 at Ser774.^16,34 Similar to MTORC1, AKT can also regulate autophagy at the transcriptional level by phosphorylating the FOXO family of transcription factors, specifically FOXO1 and FOXO3, preventing their translocation to the nucleus and inhibiting the transcription of multiple autophagy genes such as ATG4, ATG12, BECN1, BNIP3, LC3, PIK3C3/VPS34, ULK1, and ULK2.^35–37

Whereas MTORC1 prevents autophagy activation, AMPK has been linked with autophagy induction.²⁴ AMPK is a heterotrimeric Ser/Thr kinase complex that can sense the energy status of the cell by binding AMP and ADP.^38,39 A decrease in the ATP:AMP ratio inside the cell results in more AMP binding to AMPK, which in turn leads to the phosphorylation of PRKAA/AMPK α-subunit at Thr172 by STK11/LBK1, resulting in AMPK activation.^40–42 Once activated, AMPK seeks to restore energy homeostasis inside the cell by upregulating catabolic pathways that can generate ATP and downregulating anabolic processes that consume energy. In this regard, AMPK plays both a direct and indirect role in autophagy regulation. Upon activation, AMPK can directly induce autophagy by phosphorylating ULK1 at Ser555^24,43 and BECN1 at Ser93 and Ser96.⁴⁴ Additionally, AMPK can also indirectly activate autophagy by preventing MTORC1-dependent inhibition of ULK1.^26,45,46 Interestingly, ULK1-dependent phosphorylation of all three subunits of AMPK has been proposed as a negative feedback loop to terminate autophagy induction.^47,48 Recently, a kinase substrate screen discovered that the cyclin-dependent kinase CCNY–CDK16 complex is a novel AMPK phosphorylation target involved in AMPK-mediated autophagy induction.⁴⁹

PRKA/PKA is a cyclic AMP-dependent Ser/Thr protein kinase that phosphorylates LC3, preventing its recruitment to phagophores and inhibiting autophagy.⁵⁰ PRKA/PKA has also been implicated in regulating vascular network formation in endothelial cells by phosphorylating ATG16L1. PRKA/PKA-dependent phosphorylation of ATG16L1 leads to its degradation, reducing autophagy, a mechanism that could be involved in regulating the stabilization of nascent vascular endothelium.⁵¹

Whereas nutrient depletion constitutes the main stimulus for autophagy induction in some organisms, other types of cellular stress are also involved in autophagy regulation. These additional types of stress, relevant components of the autophagy machinery, and mechanism include the following: (i) hypoxia involving HIF1A, BNIP3, and BNIP3L, which can lead to BCL2-BECN1 dissociation;^52,53 MTORC1 inhibition, resulting in BECN1, and ATG14 phosphorylation.⁵⁴ (ii) ER stress can induce autophagy through several mechanisms involving ERN1/IRE1α and MAPK/JNK driving BCL2-BECN1 dissociation;^55–57 EIF2AK3/PERK and EIF2A/eIF2α leading to ATF4-dependent translational activation of ATG genes;^58–64 ATL3, CCPG1, RETREG1, RTN3, SEC62, and TEX264 receptor-dependent reticulophagy.^65,66 (iii) Oxidative stress leading to inactivation of MTORC1 and AKT, along with AMPK-dependent autophagy activation.^67–69

4. Membrane nucleation

Generation and assembly of the phagophore require membrane nucleation by the class III phosphatidylinositol 3-kinase (PtdIns3K). This lipid kinase protein complex catalyzes the formation of phosphatidylinositol-3-phosphate (PtdIns3P), which serves as a recruitment factor for PtdIns3P-binding proteins such as those of the WIPI/WDR45 family.⁷⁰ The PtdIns3K complex is formed by several core proteins, including the catalytic subunit PIK3C3/VPS34, the regulatory subunit PIK3R4/VPS15 and BECN1.⁷¹ Whereas these three proteins constitute the core of the PtdIns3K complex, depending on the other interacting partners, at least three other PtdIns3K subcomplexes involved in autophagy have been described.^72,73 The PtdIns3K complex I is formed by the three core proteins, in addition to the BECN1-binding proteins ATG14 and AMBRA1, and the ATG14-binding protein NRBF2.^74–76 This complex positively regulates autophagy by promoting recruitment of the PtdIns3K complex to the phagophore and inducing the generation of PtsIns3P. In this regard, ATG14 is directly associated with the ability of the PtdIns3K complex I to translocate to the phagophore;⁷⁴ AMBRA1 improves BECN1 and PIK3C3 interaction and catalytic activity;⁷⁶ and NRBF2 modulates PIK3C3 activity by promoting complex I assembly.⁷⁵ The PtdIns3K complex II is formed by the above-mentioned PtdIns3K core proteins in addition to UVRAG.⁷⁷ Although both ATG14 and UVRAG stabilize and directly interact with BECN1, their binding is mutually exclusive, generating two protein complexes with different autophagic functions⁷³ (Figure 2). Furthermore, UVRAG binding partners RUBCN/RUBICON and SH3GLB1/Bif-1 further differentiate the PtdIns3K complex II into two subpopulations with different functions. Whereas the PtdIns3K complex in which UVRAG binds SH3GLB1 stimulates autophagy by promoting the autophagosome maturation step in which autophagosomes and lysosomes fuse,^78,79 UVRAG binding to RUBCN inhibits autophagy.⁷³ UVRAG can be negatively regulated by MTORC1-mediated phosphorylation, which increases UVRAG and RUBCN interaction.⁸⁰

Figure 2.

The proposed structure of the PtdIns3K complex I (top) and PtdIns3K complex II (bottom). Note that not all PtdIns3K complex I and complex II factors, including RUBCN and SH3GLB1, are shown.

5. Autophagosome membrane source

The membrane source from which the phagophore and subsequently the autophagosome are formed have long been a source of debate. Different studies have indicated that the initial phagophore membrane could originate from the ER,⁸¹trans-Golgi,⁸² mitochondria,⁸³ endosomes,⁸⁴ or the plasma membrane.⁸⁵ ATG9A is the only essential transmembrane core autophagy protein, and it plays a key role in phagophore expansion,⁸² in a process regulated by the adaptor complexes AP1, AP2,^86,87 and AP4.⁸⁸ In turn, AP1/2 binding is regulated by the phosphorylation of the ATG9A N terminus by SRC during nutrient-rich conditions, and ULK1 during stress.⁸⁶ Recently, the ATG9A structure has been solved, providing insight into its ability to bend and bind highly curved membranes, consistent with its role as a membrane transporter.⁸⁹ Furthermore, the ATG9A structure also revealed a possible lipid scramblase activity, which could control autophagosome size.⁹⁰ The ATG9A C terminus is responsible for binding ATG2A,⁸⁹ which works as a funnel, tethering the ER membrane to the growing phagophore and mediating lipid transfer from the ER.^91,92

In addition to ATG9A-containing vesicles, the ER-Golgi intermediate compartment (ERGIC) has also been proposed as another possible membrane source for phagophore formation.⁹³ During nutrient-rich conditions coat protein complex II (COPII) vesicles travel from the ER to the Golgi as part of the secretory pathway. However, when nutrients are depleted and the secretory pathway is inhibited, COPII vesicles are repurposed for phagophore formation. During this process, the PtdIns3K complex I is recruited to the ERGIC, leading to the budding of specialized COPII vesicles that serve as a platform for LC3 lipidation, an essential step in phagophore expansion and later autophagosome maturation.^93,94 This process is regulated by ULK1, which during nutrient starvation phosphorylates the COPII essential protein subunit SEC23B, preventing its proteasomal degradation and leading to SEC23B accumulation and the generation of autophagic COPII vesicles from the ERGIC.⁹⁵

Consistent with the idea that the ER is a major membrane source for autophagosome formation, different microscopy and 3D tomography studies have highlighted the connection between ER cisternae and pha-gophore-autophagosome membranes.^96,97 A PtdIns3P-enriched ER-subdomain (the omegasome) is linked to autophagosome biogenesis, providing a dynamic platform for recruiting several ATG proteins.⁹⁸ Omegasome formation is dependent on PtdIns3K complex I ER localization, mediated by ATG14;⁹⁹ subsequent PtdIns3P generation recruits ZFYVE1/DFCP1⁹⁸ and WIPI/WDR45 family proteins.⁷⁰ WIPI2B is required for LC3 lipidation.⁷⁰ This process is regulated by WIPI2 recruitment and direct binding to ATG16L1, which together with ATG12 and ATG5 forms the ATG12–ATG5-ATG16L1 complex, required for efficient LC3 lipidation and phagophore expansion.¹⁰⁰ Other WIPI family members have also been implicated in autophagy regulation. WDR45/WIPI4 forms a complex with ATG2A, allowing the latter to tether PtdIns3P-containing vesicles to non-Ptdns3P-containing membranes.^101,102 Also, the ULK1 complex associates with omegasomes in their early stages in a PtdIns3P-dependent manner.¹⁰³

Multiple studies have indicated that autophagosomes can form at mitochondria-associated ER membranes (MAM), specific sites where the ER and mitochondria are in close proximity to one another.^83,104 During starvation conditions, ATG14 is recruited to the MAM by the ER-resident SNARE protein STX17, which is required for complete autophagosome formation.¹⁰⁴ Furthermore, disrupting ER-mitochondria contacts by depleting the tethering proteins MFN2 or PACS1 impairs ATG14 recruitment and starvation-induced autophagy.^83,104 ATG2A also localizes to the MAM by binding the outer mitochondria membrane protein complex formed by TOMM70–TOMM40, which in turn is required for pha-gophore expansion and autophagic flux.¹⁰⁵ Interestingly, the omegasome marker ZFYVE1 is also enriched in the MAM during starvation conditions, suggesting a unified system in which omegasomes and ER-mitochondria contacts are adjacent to each other.¹⁰⁴

Taken together, an overall model has started to emerge in which the ER and ER-associated membranes serve as the main platform and membrane source for phagophore formation. Membranes from other organelles are very likely to contribute to autophagosome formation by generating membrane contact sites with the ER. Electron microscopy and electron tomography studies have shown phagophores making contact sites with membranes from late endosomes, Golgi, and mitochondria, sometimes even simultaneously.¹⁰⁶ Therefore, it is reasonable to think of the ER as an expanding membranous system across the cell, interconnecting with different organelles that supply the phagophore with their membranes (Figure 3).

Figure 3.

Translocation of the ULK complex to the ER leads to the phosphorylation and activation of the PtdIns3K complex I, which generates PtdIns3P-rich ER subdomains known as omegasomes. ZFYVE1, WDR45/WIPI4, and WIPI2B are recruited to omegasomes by binding PtdIns3P. WIPI4 binds ATG2A, which tethers the ER to the growing phagophore and transports lipids from one side to the other. ATG9A binds ATG2A, and the former moves lipids from one side of the membrane leaflet to the other, expanding the phagophore. WIPI2B recruits the ATG12–ATG5-ATG16L1 complex, inducing the lipidation of LC3 at the phagophore. Different sources provide membranes for phagophore expansion.

6. Expansion of the phagophore membrane

Two interconnected ubiquitin-like conjugation systems are involved in expanding the phagophore membrane, the ATG12–ATG5-ATG16L1 complex and the lipidation of the LC3/GABARAP family (Figure 4). The mechanism involved in the conjugation of LC3/GABARAP to phosphatidylethanolamine (PE), involving the action of the ATG12–ATG5-ATG16L1 complex as an E3 ligase has been described in great detail elsewhere.^107–109 Binding between ATG16L1 and WIPI2 may control the targeting of the ATG12–ATG5-ATG16L1 complex and in turn the site of LC3/GABARAP lipidation.¹⁰⁰ ATG16L1 is also able to interact with the ULK1 complex subunit RB1CC1/FIP200, which plays a role in the recruitment of the ATG12–ATG5-ATG16L1 complex to the phagophore.¹¹⁰ Recently, ATG16L1 has been proposed to bind directly to membranes;¹¹¹ thus, it seems that the localization of ATG12–ATG5-ATG16L1 depends on multiple factors, including protein and lipid interactions.

Figure 4.

The ubiquitin-like proteins ATG12 and the LC3/GABARAP family go through activation by ATG7 and conjugation by ATG10 and ATG3, respectively. Covalent binding between ATG12–ATG5 leads to forming a dimeric ATG12–ATG5-ATG16L1 complex, which is recruited to the phagophore by WIPI2B, promoting the ligation of LC3 to PE. LC3-II/LC3–PE can be deconjugated by ATG4, the same protein involved in LC3 initial processing. GTAMAV, C-terminal amino acid residues; PE, phosphatidylethanolamine.

Beyond their role in the expansion and maturation of autophagosomes, when covalently bound to PE, the LC3/GABARAP family serve as interaction platforms for a diverse range of different proteins, physically linking them to the phagophore membrane. Among these LC3/GABARAP-binding proteins are various autophagy receptors that play an important role in selective autophagy, tethering the cargo targeted for degradation to the growing phagophore. These receptors include SQSTM1/p62, OPTN, TOLLIP, and NBR1, which participate in the autophagic degradation of various types of ubiquitinated cargo; BNIP3L and FUNDC1 which have been linked to mitophagy;¹¹² RETREG1, CCPG1, SEC62, RTN3, and TEX264, which have been connected to reticulophagy;⁶⁵ and CALCOCO2 that has been reported to induce the selective autophagic degradation of intracellular pathogens.¹¹³ Binding between these receptor proteins and LC3/GABARAP is generally mediated by the LC3-interacting region (LIR), a distinct amino acid sequence closely resembling Trp-X-X-Leu/Ile.^109,114 A specific type of LIR termed GABARAP-interacting motif has been described, which resembles the sequence Trp/Phe-Val/Ile-X-V and shows a higher binding affinity for the GABARAP subfamily.¹¹⁵ Besides the autophagy receptor proteins, several other core components of the autophagy machinery such as ULK1, ATG13, RB1CC1,¹¹⁶ BECN1, ATG14,¹¹⁷ ATG4,¹¹⁸ and ATG7¹¹⁹ have been proposed to possess LIR sequences that allow them to interact with LC3/GABARAP and be recruited to the phagophore. Even though most of the research involving LC3/GABARAP and their interacting partners has been focused on the interface between the LIR and its corresponding docking site on the various LC3/GABARAP proteins, a recent publication has reported a novel alternative binding site in this family that binds ubiquitin-interacting motifs, opening the door for the discovery of new LC3/GABARAP-interacting partners and mechanisms of autophagy regulation.¹²⁰

7. Lysosomal fusion and cargo degradation

In the last stages of autophagy, complete autophagosomes must fuse with lysosomes to release their cargo for degradation. This process requires a spatial approach between the two organelles that allow for membrane tethering and fusion to occur. Mature autophagosome trafficking is mediated by microtubules that serve as tracks, allowing the transport of complete autophagosomes to the perinuclear region, where lysosomes are usually located during starvation.^121,122 Once autophagosomes and lysosomes are sufficiently close, tethering and fusion are triggered by multiple factors, including RAB7 and the HOPS complex,^123,124 STX17, SNAP29, VAMP7, and VAMP8.^125–127 Specificity of the lysosome-autophagosome fusion process is provided by several different adaptor proteins; for example, the RAB7 effector protein PLEKHM1 directly interacts with the HOPS complex and LC3/GABARAP through an LIR, providing a bridge between autophagosomes and the different members of the tethering complex.¹²⁸ Once fusion is completed, the inner autophagosomal membrane is degraded inside the newly formed autolysosome, where lipases and acid hydrolases including cathepsins CTSB, CTSD, and CTSL degrade the cargo, generating essential molecules such as amino acids that can be transported back to the cytosol to be recycled by the cell. Recent studies have identified the lysosomal multi-spanning transmembrane protein SLC38A9 as a central amino acid transporter responsible for recycling amino acids from the lysosomal lumen to the cytosol.^129–131 SLC38A9 senses Arg, which plays a modulating role in the lysosomal efflux of Gln and Leu and other essential amino acids to the cytosol.^132,133 SLC38A9-dependent amino acid efflux is sensed by MTORC1,^129–133 which is activated, generating a negative feedback loop that finally inhibits autophagy.

8. Cardiac autophagy

Autophagy has a vital role in the normal and diseased heart.^134–136 Basally, cardiomyocytes display beneficial autophagy to degrade misfolded proteins and damaged organelles.^134,137 Moreover, autophagy is required for normal cardiac development.¹³⁸ Knockdown or knockout of essential autophagy genes, including Atg5, results in defects in cardiac morphogenesis, notably in valve development and chamber septation.¹³⁹ However, this process is altered during metabolic stresses (e.g. diabetes, lipotoxicity), ischemia/reperfusion (I/R) injury, myocardial infarction (MI), cardiac hypertrophy, cardiac remodelling, and heart failure (HF).^134–136 Because our previous review covered cardiovascular autophagy up to 2014,¹³⁴ we focus mainly on the new findings described since 2015. The main discoveries in the cardiovascular area are described in Table 1.

Table 1.

Timeline describing principal findings in cardiovascular autophagy

	Year	Description
Heart	1976	First description of autophagy in cardiomyocytes140
	2000	First description of autophagy in hearts associating accumulation of autophagic vacuoles and cardiomyopathy in LAMP2-deficient mice141
	2001	Autophagic degeneration as a possible mechanism of myocardial cell death in dilated cardiomyopathy142
	2003	Myocytes die by multiple mechanisms in failing human hearts143
	2007	Cardiac autophagy is a maladaptive response to haemodynamic stress
	2007	Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMPK and BECN1 in mediating autophagy144
	2007	The role of autophagy in cardiomyocytes in the basal state and in response to haemodynamic stress145
	2008	Intracellular protein aggregation is a proximal trigger of cardiomyocyte autophagy
	2009	Markers of autophagy are downregulated in failing human heart after mechanical unloading146
	2010	Deacetylation of FOXO by SIRT1 plays an essential role in mediating starvation-induced autophagy in cardiac myocytes147
	2012	Autophagy proteins LC3B, ATG5, and ATG12 participate in quality control after mitochondrial damage and influence lifespan148
	2012	First description of autophagy in cardiac fibroblast assessing the role of ADRB2/beta(2)-adrenergic receptor in the regulation of cardiac fibroblast autophagy and collagen degradation149
	2012	Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure150
	2012	Impaired autophagosome clearance contributes to cardiomyocyte death in ischemia/reperfusion injury151
	2013	Mechanical unloading activates FOXO3 to trigger BNIP3-dependent cardiomyocyte atrophy152
	2013	PINK1-phosphorylated MFN2 is a PRKN receptor for culling damaged mitochondria153
	2013	Autophagy regulates endothelial cell processing, maturation, and secretion of VWF154
	2013	STK4/MST1 inhibits autophagy by promoting the interaction between BECN1 and BCL2155
	2014	Autophagy regulates vascular endothelial cell NOS3 and EDN1 expression induced by laminar shear stress in an ex vivo perfused system156
	2015	PRKN-mediated mitophagy directs perinatal cardiac metabolic maturation in mice157
	2014	PRKN-independent mitophagy requires DNM1L and maintains the integrity of mammalian heart and brain158
	2015	Endogenous DNM1L mediates mitochondrial autophagy and protects the heart against energy stress159
	2015	Interdependence of PRKN-mediated mitophagy and mitochondrial fission in adult mouse hearts160
	2015	SIRT7 contributes to myocardial tissue repair by maintaining the TGFB signaling pathway161
	2016	DNM1L-dependent mitochondrial autophagy plays a protective role against pressure overload-induced mitochondrial dysfunction and heart failure162
	2016	Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling163
	2016	Doxorubicin blocks cardiomyocyte autophagic flux by inhibiting lysosome acidification164
	2016	Cardioprotection and lifespan extension by the natural polyamine spermidine165
	2017	Endothelial-specific deletion of Atg7 attenuates arterial thrombosis in mice166
	2017	Endothelial cell autophagy maintains shear stress-induced nitric oxide generation via glycolysis-dependent purinergic signaling to endothelial NOS3167
	2017	Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow168
	2018	Endothelial autophagic flux hampers atherosclerotic lesion development169
	2018	BECN1-dependent autophagy protects the heart during sepsis170
	2018	TLR4 contributes to a myofibroblast phenotype in cardiac fibroblasts and is associated with autophagy after myocardial infarction in a mouse model171
	2019	Mitophagy is essential for maintaining cardiac function during high fat diet-induced diabetic cardiomyopathy172
	2020	Downregulation of BECN1 promotes direct cardiac reprogramming173
Blood vessels	1997	First description of autophagy in vascular endothelial cells and cardiac endothelial cells obtained from mouse hearts after long-term exposure to non-metabolizable sugars174
	2001	Porphyromonas gingivalis traffics to autophagosomes in human coronary artery endothelial cells175
	2006	First description of autophagy in VSMC by evaluating the effects of IGF1 and TNF in the regulation of autophagy through MAPK/JNK and AKT pathways in human atherosclerotic vascular smooth cells176
	2011	BECN1 deficiency is associated with increased hypoxia-induced angiogenesis177
	2015	Defective autophagy in VSMCs accelerates senescence and promotes neointima formation and atherogenesis178
	2018	Impaired mitochondrial respiration in human carotid plaque atherosclerosis: a potential role for PINK1 in VSMC energetics179
	2019	Altered mitochondrial quality control in ATG7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype180
	2019	VSMC plasticity and autophagy in dissecting aortic aneurysms181
	2020	Defective autophagy in vascular smooth muscle cells increases passive stiffness of the mouse aortic vessel wall182
	2019	FOXO1 inhibits autophagosome-lysosome fusion leading to endothelial autophagic-apoptosis in diabetes183
	2019	Endothelial-specific deficiency of ATG5 attenuates ischemia-related angiogenesis184
	2020	Mitophagy contributes to endothelial adaptation to simulated microgravity185
	2020	Defective autophagy in vascular smooth muscle cells alters vascular reactivity of the mouse femoral artery186
	2020	Endothelial autophagy deficiency induces IL6-dependent endothelial mesenchymal transition and organ fibrosis187
	2020	Laminar flow inhibits the STK3–STK4 (HIPPO)–YAP1 pathway via autophagy and SIRT1-mediated deacetylation against atherosclerosis188
	2020	Ischemia induces autophagy of endothelial cells and stimulates angiogenic effects in a hindlimb ischemia mouse model189

9. Mitochondrial dysfunction and mitophagy

Because of constant beating, the heart is an organ that requires a continuous high supply of ATP. The heart produces and utilizes ∼6 kg ATP per day.¹⁹⁰ Therefore, mitochondrial metabolism and function are essential for cardiac homeostasis.¹⁹¹ Mitophagy, the selective autophagic degradation of mitochondria, is an important mediator of mitochondrial quality control in cardiac myocytes. Removing dysfunctional mitochondria through mitophagy is essential for maintaining cardiomyocyte energetic and metabolic requirements.¹⁹¹ In developing hearts, the MFN2–PINK1–PRKN pathway, which poly-ubiquitinates damaged mitochondria to promote mitophagy, changes the cardiac metabolism by redirecting the mitochondrial substrate preference to fatty acids.¹⁵⁷ Through this mechanism, foetal cardiomyocyte mitochondria are removed by PRKN-mediated mitophagy and replaced by mature adult mitochondria.¹⁵⁷ Cardiac mitophagy is also observed during cardiac ischemia by a complex consisting of ULK1, RAB9, RIPK1, and DNM1L.¹⁹² Downregulation of mitophagy mediates the development of mitochondrial dysfunction and HF.¹⁶² In this model, an isoform shift from PRKAA2/AMPKα2 to PRKAA1/AMPKα1 is observed in mouse hearts and in heart samples from HF patients.¹⁹³ BAG3, a co-chaperone of HSPA/HSP70, is also recruited to depolarized mitochondria, together with PRKN to promote mitophagy,¹⁹⁴ in part by regulating translation of LC3B.¹⁹⁵ Suppression of BAG3 in cardiac myocytes reduces autophagy flux and mitophagy.¹⁹⁴ Conversely, administration in the heart of BAG3 using an adeno-associated virus improves left ventricular ejection fraction (LVEF) after MI.¹⁹⁶ Restoration of PRKAA2 activates the PINK1–PRKN–SQSTM1 pathway that increases cardiac mitophagy associated with the improvement in mitochondrial function, removal of damaged mitochondria, decrease in reactive oxygen species (ROS) production, and cardiomyocyte apoptosis.¹⁹³

Single-nucleotide polymorphisms in the PRKN are linked to blood pressure in Nigerian and Korean families, suggesting an association between mitophagy and hypertension.¹⁹⁷ Moreover, spermidine supplementation in the diet of salt-sensitive rats enhances cardiac mitophagy and delays the development of hypertensive heart disease.¹⁹⁸ Also, in a unilateral renovascular hypertension pig model, valsartan, a well-known antihypertensive drug, reduces left ventricular hypertrophy and increases mitophagy and mitochondrial biogenesis.¹⁹⁸ These data suggest that the increase of mitochondrial turnover decreases hypertensive-dependent cardiac remodelling.

10. Ischemia/reperfusion

Myocardial ischemia is one of the main causes of sudden cardiac death worldwide, and activation of autophagy protects cardiomyocytes from I/R injury.¹³⁴ During ischemia, autophagy is triggered as a pro-survival mechanism in response to nutrient and oxygen deprivation.¹⁹⁹ However, during reperfusion, autophagy has beneficial or detrimental effects depending on the experimental model and whether it involves a BECN1- or an AMPK-MTOR-dependent activation of auto-phagy.^144,199,200 More recent findings indicate that induction of autophagy by simultaneous administration of PT1, a specific activator of AMPK, and 3HOI-BA-01, a potent inhibitor of MTOR, reduces cardiomyocyte death triggered by simulated I/R. In vivo administration of PT1 or 3HOI-BA-01 in a murine I/R injury model stimulates autophagy and reduces infarct size.²⁰¹ Likewise, cardiomyocyte-specific autophagy disruption with a conditional atg7 knockout leads to myofibrillar disarray and severe contractile dysfunction and worsens the I/R injury with cardiac hypertrophy and severe cardiac fibrosis.²⁰² Autosis, a form of cell death due to excessive activation of autophagy, is induced in cardiomyocytes exposed to I/R.²⁰³ This autosis is associated with RUBCN upregulation, autophagic flux attenuation, and autophagosome accumulation.²⁰³ Furthermore, inhibition of excessive I/R-induced autophagy by trimetazidine protects the rat hearts from I/R-induced HF.²⁰⁴

11. Cardiac fibrosis

The differentiation of cardiac fibroblast to myofibroblast is one of the most important features of fibrosis.²⁰⁵ Autophagy is implicated in cardiac fibrosis because autophagy inhibition blocks fibroblast-to-myofibroblast phenoconversion and inhibits myofibroblast cell migration and contractility.²⁰⁶ Moreover, TGFB1 treatment of human atrial fibroblasts induces autophagy and enhances the fibrogenic response, suggesting an association between the myofibroblast phenotype and autophagy.²⁰⁷ In streptozotocin-induced diabetic rats, alterations in autophagy correlate with cardiac fibrosis, indicating a potential synergistic role for autophagy in diabetic cardiac fibrosis.²⁰⁸ Moreover, in vitro and in vivo studies show that inhibition of PARP1 partially decreases autophagy, abrogates cardiac fibrosis, and significantly improves cardiac function post-MI.²⁰⁹ In a mouse model of lipopolysaccharide-induced sepsis, cardiac-specific overexpression of BECN1 promotes autophagy, improves cardiac function, and alleviates inflammation and fibrosis.¹⁷⁰

12. Diabetic cardiomyopathy

Diabetic cardiomyopathy is a cardiovascular disease characterized by morphological, functional, and metabolic changes in the heart produced as a complication of type 2 diabetes mellitus (T2DM).²¹⁰ Excessive autophagy is observed in the right atrial appendages collected from diabetic and non-diabetic patients.²¹¹ Similarly, animal models of T2DM show increased autophagosomes in the heart that is further increased upon chloroquine injection. Importantly, in vitro genetic deletion of Becn1 in adult rat cardiomyocytes exposed to high glucose markedly inhibits autophagy and triggers apoptosis suggesting a pathological role of autophagy in the T2DM heart.²¹¹ Moreover, excessive autophagy in T2DM induces HF aggravation after MI through defective mitophagy, associated with excessive inflammasome activation, secretion of IL18, and cell death.²¹² Prevention of inflammation by NFKB blockade with pyrrolidine dithiocarbamate reduces oxidative stress and improves mitochondrial integrity, thus restoring cardiac function in T2DM.²¹³ In hearts from obese db/db T2DM mice, LC3 lipidation, SQSTM1, and phosphorylated MTOR levels are increased, but CTSD level is decreased, and very few lysosomes are detected, despite the abundance of autophagic vacuoles. In these hearts, AMPK activity and ATP content are decreased. These findings suggest that in this T2DM model, the autophagic flux is blocked at the final step.²¹⁴ In high-fat diet-induced obese mice, a failure to upregulate LC3 lipidation or to clear SQSTM1 in the heart after fasting are observed, although mRNA for Lc3b and Sqstm1 are appropriately upregulated. These data suggest that hearts of diet-induced obese mice also exhibit impaired autophagy.²¹⁵

In hearts from streptozotocin-induced type 1 diabetic mice (T1DM), diastolic dysfunction is observed,^214,216 although autophagic activity is increased, as evidenced by increases in LC3-II and CTSD, and decreased SQSTM1, and by the abundance of autophagic vacuoles and lysosomes.²¹⁴ The increase of blood glucose level in T1DM induces mitochondrial damage by O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins.²¹⁷ Inhibition of autophagic flux by chloroquine decreases autolysosomes, cardiomyocyte apoptosis, and cardiac fibrosis, but increases both Lc3 and Sqstm1 expression.²¹⁸ Surprisingly, mitophagy is inhibited in hearts from T1DM mice.²¹⁹ Moreover, there is substantial attenuation of cardiac damage in mice deficient for BECN1 and ATG16L1 as evidenced by an improvement in cardiac function along with decreased levels of oxidative stress, interstitial fibrosis, and cardiomyocyte apoptosis. In contrast, diabetes-induced cardiac injury is aggravated by BECN1 overexpression.²²⁰ In the same way, administration of 1,25-dihydroxyvitamin-D3 and resveratrol improves diabetic cardiomyopathy by restoring the impaired cardiac autophagy in streptozotocin-induced diabetic rats.^221,222

13. Heart failure

HF is a clinical syndrome due to a structural and/or functional cardiac abnormality, which results in a reduction in cardiac output and/or elevated intracardiac pressure.²²³ HF affects patients ranging from those with normal (≥50%) LVEF, known as HF with preserved EF (HFpEF), to those with reduced (<40%) LVEF, known as HF with reduced EF (HFrEF).²²³ All these patients display signs of HF with evidence of abnormal cardiac structure and function.²²⁴ Epidemiological studies have shown that the incidence of HFrEF and HFpEF are almost equally distributed, but the incidence of HFpEF is increasing with time.²²⁵ The role of autophagy is well described in HFrEF;¹³⁴ however, its role in HFpEF is poorly defined. Recently, a gene expression analysis performed on heart biopsies obtained from HFrEF, HFpEF, and control patients revealed that autophagy genes are strikingly downregulated in HFpEF patients.²²⁶

Initial findings indicated that basal constitutive cardiac-specific autophagy impairment, obtained by a tamoxifen-induced deletion of Atg5, shows disorganized sarcomere structure, mitochondrial misalignment and aggregation in cardiac myocytes, and cardiac hypertrophy, left ventricular dilatation, and contractile dysfunction, indicating the occurrence of HFrEF.¹⁴⁵ HFrEF progression can be prevented by rapamycin.²²⁷ In fact, activation of basal autophagy by rapamycin, improves diastolic function in old mice beginning at 2–4 weeks and progress throughout 10-week treatment. Autophagy was transiently induced during the first week of treatment associated with mitochondrial biogenesis, suggesting the replacement of damaged mitochondria.²²⁸ In a rat model of HFrEF induced by MI, histological and echocardiographic measurements showed that rapamycin treatment improves myocardial function and inhibits cardiac remodelling at 8 weeks post-MI mechanism involving inhibition of the MTORC1 and ER stress pathways.²²⁹ In the same model, the increase of MMP9 (matrix metallopeptidase 9) inhibits cardiomyocyte and cardiac fibroblast autophagy in the peri-infarct site, whereas ablation of MMP9 increases cardiac autophagy.²³⁰ Moreover, in a model of HF induced by pressure overload, nitric oxide and natriuretic peptides induce cardioprotection by activation of autophagy through a PRKG1–TSC2–RHEB-dependent inhibition of MTORC1.²³¹ In patients with dilated cardiomyopathy, autophagic vacuoles in cardiomyocytes are associated with a better HFrEF prognosis, suggesting autophagy could be involved in the prevention of myocardial deterioration.²³² In patients with idiopathic dilated cardiomyopathy, mechanical unloading with a left ventricular assist device decreases markers of autophagy BECN1, ATG12–ATG5, and LC3-II.¹⁴⁶ Because the mechanical unloading decreases the energy demand of the failing heart these results suggest that autophagy is activated as an adaptive mechanism. Moreover, myocardial CTSD, a major lysosomal protease induced by MI, protects against cardiac dysfunction and remodelling, which is in part through promoting cardiac myocyte autophagic flux.²³³

Conversely, autophagy, assessed by anti-LC3-II staining and vacuole formation, is extensively detected in myocardial samples from patients with ischemic cardiomyopathy and idiopathic dilated cardiomyopathy, with the occurrence of necroptosis and oncosis exceeding that of apoptosis.²³⁴ In a mouse model of MI, necroptosis is triggered by autophagy flux dysfunction, and this cell death process contributes to loss of cardiomyocytes, adverse ventricular remodelling, and HFrEF.²³⁵ Moreover, persistent autophagy triggered by upregulation of TLR3 expression and signalling induced by MI, promotes HFrEF and lethality.²³⁶ Likewise, in a model of pressure overload in mice induced by TAC, left ventricular wall thickness is increased at 1 week, associated with a decrease in autophagy.²³⁷ At 9 weeks, this model develops HFrEF characterized by a further increase in left ventricular wall thickness, with the intensification of cardiomyocyte autophagy and apoptosis.²³⁷ In this model, activation of AMPK by injecting AICAR improves cardiac function by attenuating autophagy.²³⁸ These results suggest that in HFrEF, autophagy can be beneficial or detrimental, depending on the context of the disease.

14. Cardiac ageing and regeneration

Cardiac ageing is characterized by fibrosis, hypertrophy, dysfunctional mitochondria, and misfolded protein accumulation, and impaired autophagy.²³⁹ Inhibition of autophagy exacerbates cardiac dysfunction accompanied by the accumulation of dysfunctional organelles and misfolded proteins.²⁴⁰ Reduction of autophagy during ageing correlates with the hypermethylation of Atg5 and Lc3b gene promoter regions in macrophages from aged mice associated with reduced gene expression;²⁴¹ treatment with Trdmt1/Dnmt2 siRNA or a methyltransferase inhibitor restores Atg5 and Lc3 expression.²⁴¹ Interestingly, a homozygous BECN1^F121A knock-in mouse, a mutation that decreases the interaction of BECN1 with the negative regulator BCL2, has increased basal autophagy in several organs, including the heart.²⁴² These mice show an increased life span with decreased cardiac and renal pathological changes and spontaneous tumorigenesis.²⁴²

Conversely, cardiac regeneration is considered a promising tool to treat cardiac diseases. Zebrafish, but not adult mammals, are capable of cardiac tissue regeneration. In an injury model of ventricular apex resection, a tightly regulated autophagic response is observed during the early stages of the regeneration process. Inhibition of autophagy by rapamycin impairs cardiac regeneration.²⁴³ These findings clearly show the double-edged sword effect that autophagy has in cardiac homeostasis.

15. Autophagy in the vascular system

The vascular tissue is mainly constituted by vascular smooth muscle cells (VSMCs) and endothelial cells. VSMCs exhibits several phenotypes in response to endogenous and environmental factors contributing to the genesis and progression of vascular diseases.²⁴⁴ Comprehensive overviews of the role of autophagy in VSMCs were previously and extensively described.^134,135

16. VSMC phenotype

Several studies have shown that autophagy controls VSMC differentiation and dedifferentiation. Dedifferentiated VSMCs are characterized by increased cell migration, proliferation, extracellular matrix protein synthesis, secretion, and decreased contractile protein content.²⁴⁴ This phenotype is responsible for vessel formation and reparation. However, when it is dysregulated, it is responsible for vascular diseases (Figure 5).²⁴⁴ Inhibition of autophagy with 3-methyladenine delays the differentiation of epicardial progenitor cells into coronary VSMCs while its activation with rapamycin induces an early transient differentiation of epicardial progenitor cells and enhanced migration.²⁴⁵ Moreover, GDF11, an inhibitor of cardiac hypertrophy in mice,²⁴⁶ promotes carotid VSMC differentiation and prevents cell dedifferentiation induced by autophagosome accumulation triggered by western diet, lysosome function deficiency, and inflammation.²⁴⁷

Figure 5.

Role of autophagy in the development of vascular diseases using in vivo models and VSMC phenotypic change using in vitro models. AGES, advanced glycation end products; Ang II, angiotensin II; Hhc, hyperhomocysteinemia; PDGF-BB, platelet-derived growth factor-BB; TNF, tumor necrosis factor; VSMC, vascular smooth muscle cell.

Conversely, angiotensin II (Ang II), through an AGTR1–RHOA kinase-dependent signalling pathway, activates autophagy in VSMCs by a mechanism involving the increase of BECN1, PIK3C3, ATG12–ATG5, ATG4, and ATG7 protein levels and BECN1 phosphorylation, suggesting activation of phagophore initiation and expansion.²⁴⁸ Ang II regulates contractile protein content in VSMCs through an autophagy-dependent mechanism.²⁴⁸ Inhibition of autophagy by SEPTIN4,²⁴⁹ and cortistatin, a neuropeptide highly homologous to somatostatin,²⁵⁰ inhibits Ang II-induced VSMC proliferation. Similarly, an AMPK–MTORC1 signalling-dependent induction of autophagy in VSMCs is described for SPARC. SPARC upregulates LC3-II, BECN1, and ATG5, triggering an autophagy-dependent VSMC dedifferentiation.²⁵¹ Regulation of cell migration by autophagy via the degradation of KAT2A is also described for VSMC.²⁵² Therefore, VSMC dedifferentiation processes, i.e. proliferation, migration, contractile, and extracellular matrix protein synthesis, are highly regulated by autophagy.

17. Endothelial function

The endothelium is a critical regulator of vascular health and function. Shear stress generated by the blood flow is one of the most important stimuli that control endothelial function.²⁵³ Steady laminar shear stress (5 or 15 dynes/cm²) induces autophagy in human and rabbit endothelial cells, associated with the induction of endothelial NOS3/eNOS (nitric oxide synthase 3) and inhibition of expression of the vasoconstrictor EDN1/ET-1 (endothelin 1).¹⁵⁶ The mechanism involves the deacetylation of ATG5 and ATG7 in a SIRT1-dependent manner.²⁵⁴ In contrast, oscillatory or low levels of shear stress reduce autophagy, uncouple NOS3, and trigger proatherogenic responses.^168,254,255 From a translational point of view, using primary endothelial cells collected from the radial artery of men, showed that 60 min of rhythmic handgrip exercise activates autophagy, increases NOS3 phosphorylation, and increases nitric oxide and ${\mathrm{O}}_2^{·-}$ generation.²⁵⁶ In diabetic patients, autophagic flux is reduced, which impairs NOS3 activation.¹⁶³ These data suggest that steady laminar shear stress induces autophagy with a beneficial vasodilatory effect, and diabetes disrupts these mechanisms. Moreover, disturbed blood flow promotes atherosclerosis, whereas laminar flow has a protective action by preventing endothelial apoptosis, senescence, and inflammation.¹⁶⁹ The mechanism involves activation of endothelial autophagy that triggers SIRT1 expression that inhibits STK3/MST2–STK4/MST1 (HIPPO)-YAP1 signalling that interrupts atherosclerotic plaque formation.¹⁸⁸

18. Vascular remodelling

Pathological vascular remodelling is characterized by the narrowing of the vessel lumen, mobilization of VSMC to the intima layer, exacerbated extracellular matrix production (fibrosis) and infiltration by immune cells.²⁵⁷ Dedifferentiated VSMCs have been extensively described in the development and progression of atherosclerosis, hypertension, restenosis, and neointimal formation.^258,259 Various rapamycin analogues (rapalogs) have been used in drug-eluting stents for treating arterial restenosis. Two major rapalogs, everolimus, and biolimus strongly inhibit neointimal hyperplasia.²⁶⁰ However, everolimus exerts cytotoxicity by increasing ROS and reducing energy metabolism. In contrast, biolimus preferentially induces autophagy by activating ULK1. The implantation of biolimus-eluting stent reduces endothelial loss and inflammation in porcine coronary arteries.²⁶⁰

VSMCs with specific genetic atg7 deletion show a reduction in serum-induced cell growth and cell proliferation rate and an increase in cell death.²⁶¹ Furthermore, an atg7 conditional knockout mouse crossed with Apoe (apolipoprotein E)-deficient mouse shows a reduction in medial cellularity and an increase in TUNEL-positive cells in the descending aorta. This mouse also shows a reduced survival rate due to aortic rupture associated with increased markers of atherosclerosis.²⁶¹ Using another VSMC-specific atg7 knockout mouse continuously infused with Ang II (1.5 mg/kg/day) also exhibits increased mortality associated with cardiac rupture, MI, end-organ damage, venous distention, pleural effusion, and abdominal aortic dissections.²⁶² Deletion of Atg5 in VSMCs increases cell death susceptibility, enhances ER stress activation, promotes VSMC inflammation, and increases the incidence of fatal rupture of aortic aneurysm (Figure 5).¹⁸¹ TFEB is downregulated in aortic aneurysm lesions, and TFEB deficiency increases VSMC apoptosis and promotes abdominal aortic aneurysm formation in mice.²⁶³ In contrast, TFEB activation has the opposite effect.²⁶³ Additionally, metformin preserves aortic elastin and collagen and reduces aortic cell apoptosis abdominal aortic aneurysms in patients and rat models. The mechanism involves the inactivation of the PI3K–AKT–MTORC1 pathway and the decrease of mRNA and protein levels of LC3B and BECN1.²⁶⁴ Inhibition of vascular inflammation by using BP-1-102, a novel potent STAT3 inhibitor, decreases abdominal aortic aneurysm by preserving autophagy.²⁶⁵ These data suggest that the fine-tuning of autophagy flux is required to maintain aorta wall integrity.

19. Atherosclerosis

VSMC dedifferentiation is a key step in the development of atherosclerotic plaque. Therefore, disruption of autophagy has important consequences on atherosclerosis genesis and progression. Besides macrophages, VSMCs are also an important source of foam cells in atherosclerosis. In a high-fat diet-fed apoe knockout mice model of atherosclerosis, activation of the P2RY12 receptor inhibits autophagy in VSMCs, which decreases cholesterol efflux and promotes VSMC-derived foam cell formation.²⁶⁶

Atherosclerotic plaque instability predisposes to the occurrence of cardiovascular events such as MI and stroke. A mouse model of plaque instability in VSMC-specific atg7 knockout mice crossed with apoe knockout mice, shows that defective autophagy in VSMCs increases plaque instability and the risk of rupture (Figure 5).²⁶⁷ Conversely, enhancement of autophagy in VSMCs or macrophages by using trehalose or by overexpression of TFEB exerts athero-protective effects, reducing plaque formation with a reduction of inflammation.²⁶⁸ Moreover, in the context of proatherogenic stimuli, the deficiency of autophagy induces inflammation in coronary artery VSMCs by promoting NLRP3 inflammasome formation and activation.²⁶⁹ These data suggest that autophagy is also a critical process in maintaining immune homeostasis during atherosclerosis.

20. Pulmonary hypertension

Pulmonary hypertension (PH) is a progressive disease characterized by excessive proliferation of pulmonary arterial vascular smooth muscle cells (PASMC).²⁷⁰ More than 70% of familial PH and 20% of idiopathic PH patients carry heterozygous mutations in BMPR2.²⁷¹ BMPR2 is degraded by autophagy in pulmonary artery endothelial cells and PASMC.²⁷¹ Mutations in BMPR2 are sufficient to trigger an increased autophagic flux.²⁷¹ Similarly, pulmonary microvascular endothelial cells from end-stage idiopathic PH patients present an elevated autophagic flux.²⁷¹ Along these lines, inhibition of autophagy with chloroquine²⁷² or paclitaxel²⁷³ blocks PASMC proliferation (Figure 5).

21. Autophagy as a pharmacological target in cardiovascular diseases and current limitations

Despite several pharmacological agents and regimens that modulate autophagy, very few clinical trials have evaluated their effects in cardiovascular diseases. Of these, only exercise, trehalose, and hydroxychloroquine have been evaluated (Table 2). Another important current limitation in the field is the lack of cell/organ-targeted delivery of exogenous autophagy regulators for cardiac or vascular repair. We are still far from having an effective autophagy therapy. However, insights obtained from such works will provide inspiration for future clinical trials to assess the autophagic response for therapeutic gain. Careful consideration must be kept in mind because of the dual role of autophagy in cytoprotection and cell death.

Table 2.

Clinical trials involving autophagy in the cardiovascular system

Name of the study	Study type	Description	Clinical trial identifier
MUcociliary Clearance IN Stroke (MUCINS)	Observational	Evaluation of autophagy in respiratory tissue (nasal, tracheal, and bronchial)	NCT03884166
Transforming Growth Factor Beta Signalling in the Development of Muscle Weakness in Pulmonary Arterial Hypertension	Observational	Determination of the contribution of atrophy and autophagy to muscle wasting in PAH	NCT01847716
Intermittent Pneumatic Compression With and Without Exercise to Improve Functioning in Peripheral Artery Disease (INTERCEDE)	Interventional	Evaluation among PAD participants whether intermittent pneumatic compression therapy combined with exercise improves autophagy (LC3, LAMP2, PRKN) at 6-month follow-up, compared to exercise alone	NCT03871075
Autophagy and Venous Endothelial Function	Observational	Evaluation of endothelial function in venous samples from patients with venous insufficiency before and after treatment with autophagy enhancer spermidine	NCT04138134
Autophagy Maintains Vascular Function Through a Novel Glycolysis-linked Pathway Regulating eNOS	Interventional	Evaluation of BECN1, ATG3, ATG5, ATG7, LAMP1, LAMP2, and SQSTM1 after rhythmic handgrip exercise or chronic exercise training	NCT04200560
Autophagy, Oxidative Stress, and Hippo Signalling in Human Aortic Aneurysm	Observational	Comparison between the levels of STK3–STK4 (HIPPO) signalling and autophagy markers observed in aortic aneurysms and the levels assessed in the adjacent non-aneurysmatic aortic portions	NCT03211000
Effects of Trehalose and Polyphenols in Vasculopathic Patients	Interventional	Change of autophagy (LC3) in PAD patients after mixed supplementation of trehalose and polyphenols	NCT04061070
Effect of Hydroxychloroquine on Atrial Fibrillation Recurrence	Interventional	Recurrence rate of atrial fibrillation after radiofrequency catheter ablation, anticoagulant therapy, and hydrochloroquine treatment (200 mg, bidpo)	NCT03592823
Potential Impact of Neuroimmune and Autophagic Alterations on the Progression and Severity of Human Atherosclerotic Process	Observational	Characterization of the autophagic process and correlation with neural modulations of the stability/instability plaque	NCT03922698

PAD, peripheral arterial disease; PAH, pulmonary arterial hypertension.

22. Conclusions

As autophagy remains an important factor in maintaining cardiovascular homeostasis, further research is required to pinpoint novel pharmacological targets that will allow us to harness the benefits of controlling autophagy levels to treat different cardiac pathologies.

Funding

This work was supported by grants from the National Institutes of Health (GM131919 to D.J.K.), the Agencia Nacional de Investigacion y Desarrollo (ANID), Chile: Fondo Nacional de Desarrollo Científico y Tecnológico/FONDECYT (1180157 to M.C. and 120049 to S.L.), and Fund for Financing Research Centers in Priority Areas/FONDAP (15130011 to M.C. and S.L.).

Conflict of interest: none declared.