Mitochondrial quality surveillance: mitophagy in cardiovascular health and disease.

Selective autophagy of mitochondria, known as mitophagy, is a major quality control pathway in the heart that is involved in removing unwanted or dysfunctional mitochondria from the cell. Baseline mitophagy is critical for maintaining fitness of the mitochondrial network by continuous turnover of aged and less-functional mitochondria. Mitophagy is also critical in adapting to stress associated with mitochondrial damage or dysfunction. The removal of damaged mitochondria prevents reactive oxygen species-mediated damage to proteins and DNA and suppresses activation of inflammation and cell death. Impairments in mitophagy are associated with the pathogenesis of many diseases, including cancers, inflammatory diseases, neurodegeneration, and cardiovascular disease. Mitophagy is a highly regulated and complex process that requires the coordination of labeling dysfunctional mitochondria for degradation while simultaneously promoting de novo autophagosome biogenesis adjacent to the cargo. In this review, we provide an update on our current understanding of these steps in mitophagy induction and discuss the physiological and pathophysiological consequences of altered mitophagy in the heart.

INTRODUCTION

Mitochondria are essential for many metabolic processes, including oxidative phosphorylation, fatty acid oxidation, and calcium homeostasis (1). Mitochondria are also key participants in other processes, including apoptosis, necrosis, and initiation of inflammation (1). They are a major source of reactive oxygen species (ROS), a byproduct of oxidative phosphorylation (2). Although ROS are not inherently detrimental, excessive ROS production can cause oxidative damage to mitochondrial lipids, proteins, and DNA, as well as cytosolic macromolecules and other organelles (2, 3). Excessive ROS can also lead to opening of the mitochondrial permeability transition pore, an initiating step of outer membrane rupture and necrotic cell death (4). In addition, release of mitochondrial DNA (mtDNA) in response to stress or injury can activate proinflammatory responses that can persist as chronic inflammation and subsequently increase susceptibility to disease (5, 6).

Maintaining a healthy functional mitochondrial network is critical for cellular homeostasis, especially in terminally differentiated cells such as neurons and cardiac myocytes that are enriched in mitochondria. Mitochondrial dysfunction is associated with both neurodegenerative and cardiovascular diseases (4). Several mitochondrial quality control mechanisms are in place to facilitate repair or degradation of damaged mitochondria to ensure that they do not pose a hazard to the rest of the network. Mitochondrial chaperones ensure proper folding of proteins, whereas resident proteases are responsible for protein degradation (7). When these mechanisms are overwhelmed, the cell can activate a mitochondrial unfolded protein response that initiates transcription of mitochondrial chaperones and proteases. If the local repair mechanism is unable to restore mitochondrial protein homeostasis, then the cell activates autophagy as a mechanism to remove the damaged organelle from the cellular environment.

In general, autophagy is a catabolic degradation pathway through which cargos such as damaged organelles and protein aggregates are broken down and recycled. In this process, the cargo is engulfed by a double-membraned vesicle called the autophagosome and degraded upon fusion with the lysosome (Fig. 1). Mitochondrial autophagy (mitophagy) is a selective form of autophagy and the main pathway responsible for degrading mitochondria in cells (8). Mitophagy is essential in ensuring mitochondrial quality and quantity at baseline and in response to stress, and impairments in mitophagy are linked to aging as well as many diseases (9). Mitophagy involves both labeling of the mitochondrion for degradation and de novo biogenesis of the autophagosome membrane near the cargo. Although decreased mitophagy will lead to accumulation of harmful mitochondria that can activate cell death, excessive degradation of mitochondria can lead to energy deficiency and death. Cardiac myocytes are terminally differentiated cells that rely on mitochondria to sustain contraction. They are therefore highly enriched in mitochondria and rely on mitophagy for both baseline turnover and adaptation to stress. Thus, the pathways that regulate mitophagy are tightly controlled to safeguard against unnecessary removal of this key organelle. In this review, we discuss the current understanding of the proteins and pathways involved in regulating mitophagy. We also discuss the importance of functional mitophagy in cardiovascular physiology and its role in suppressing disease development and aging.

Figure 1.

Overview of mitophagy. When damaged mitochondria are labeled for degradation, de novo autophagosome formation is initiated at the mitochondrion. The phagophore, a precursor of the autophagosome, forms and elongates around its cargo. Once fully enclosed around the mitochondrion, the mature autophagosome fuses with the lysosome and the contents are degraded. (Created with Biorender.com)

MECHANISMS OF MITOPHAGY

PINK1/Parkin-Mediated Mitophagy

The regulation of mitophagy by PINK1 and Parkin is one of the most well-studied pathways in the field. There is a strong interest in these proteins because loss-of-function mutations in the genes encoding PINK1 or Parkin (PARK2) are associated with development of juvenile recessive Parkinson’s disease (PD) (10, 11). Activation of PINK1/Parkin-mediated mitophagy is linked to a loss of mitochondrial membrane potential, a common consequence of stress and damage. In the absence of mitochondrial damage, the serine/threonine kinase PINK1 is partially imported into mitochondria, where it is cleaved by mitochondrial proteases and then released into cytosol for proteasomal degradation (12, 13) (Fig. 2). When mitochondria become depolarized, import of PINK1 is abrogated and it accumulates on the outer mitochondrial membrane (14, 15). PINK1 then phosphorylates ubiquitin, which leads to recruitment and activation of the E3 ubiquitin ligase Parkin (16, 17) (Fig. 2). Parkin is also phosphorylated by PINK1 on Ser65 in its ubiquitin-like domain (8, 18, 19). Parkin then proceeds to ubiquitinate different proteins on the outer mitochondrial membrane, which functions as a degradation signal for the autophagic machinery. The polyubiquitin chains are recognized by different autophagy adaptor proteins that are recruited to mitochondria by PINK1 (20). These adaptors tether mitochondria to autophagosomes via motifs that bind to ubiquitin on mitochondria and to microtubule-associated protein 1A/1B-light chain 3 (LC3) on the autophagosome membranes (21).

Figure 2.

Mechanisms of mitophagy. A: under normal conditions, PINK1 is imported into mitochondria where it is cleaved by proteases. Upon loss of mitochondrial membrane potential, PINK1 is stabilized on the outer mitochondrial membrane due to inhibition of import. Activated PINK1 phosphorylates ubiquitin molecules and the E3 ubiquitin ligase Parkin. Once activated, Parkin ubiquitinates specific substrates in the outer mitochondrial membrane. Autophagy adaptor proteins subsequently bind to the poly-ubiquitin chains and to LC3 on the growing phagophore adjacent to mitochondria. B: mitophagy receptors BNIP3, NIX, FUNDC1, BCL2L13, and FKBP8 are anchored in the outer mitochondrial membrane via their transmembrane domains and contain an LC3-interacting region (LIR), a motif that binds LC3. Upon induction of mitophagy, these proteins directly bind to LC3 in an ubiquitin-independent manner to link the mitochondrion to the autophagosome membrane. Cardiolipin, a lipid that relocalizes to the outer mitochondrial membrane during stress, can also function as a mitophagy receptor by interacting with LC3. PHB2 is a mitophagy receptor that localizes to the inner mitochondrial membrane and mainly functions in the event of mitochondrial rupture. LC3, 1A/1B-light chain 3; PHB2, prohibitin 2. (Created with Biorender.com)

Because aberrant mitophagy can have severe consequences for the cell, there are several mechanisms in place to fine-tune mitophagy and prevent excessive removal of mitochondria. Deubiquitinating enzymes (DUBs) at the mitochondria are responsible for counteracting Parkin-mediated ubiquitination and diminishing mitophagic activity. Usp15 and Usp30 have been identified as DUBs that specifically antagonize mitophagy by opposing Parkin-mediated ubiquitination of proteins in the outer mitochondrial membrane (22, 23). Parkin activation is also associated with auto-ubiquitination (24, 25). Niu and colleagues (26) recently identified Usp33 as a DUB that specifically targets Parkin to antagonize its activity. Mitophagy is also limited by the mitochondrial ubiquitin ligase (MITOL; also known as MARCH5). Specifically, MITOL mediates ubiquitination of Parkin, which promotes its proteasomal degradation (27). Thus, limiting protein ubiquitination and Parkin levels are key steps in suppressing mitophagy. Regulating mitophagy at the level of Parkin is clearly important, as overexpression of Parkin in cells can lead to degradation of the majority of the mitochondrial population upon activation of mitophagy (8, 28). This also suggests that Parkin might represent a rate-limiting step in this pathway. PINK1 can also limit the level of mitophagy by activating a noncanonical function of the mitochondrial Tu translocation elongation factor (TUFm) (29). Lin and colleagues discovered that PINK1 phosphorylates TUFm at Ser222, restricting its localization to the cytosol where it then blocks the formation of the Atg5-12 lipid conjugation complex and subsequent elongation of the autophagosome membrane.

Mitophagy Receptors

Mitophagy receptors are anchored in the outer mitochondrial membrane and facilitate mitophagy by directly interacting with LC3 on the autophagosome membrane via an LC3-interacting region (LIR) motif (Fig. 2) (21). Thus, unlike PINK1/Parkin-mediated mitophagy, these receptors facilitate removal of mitochondria in a ubiquitin-independent manner. Many proteins, including Nix, Bnip3, Fundc1, BCL2L13, FKBP8, and PHB2, and even lipids (cardiolipin) can function as mitophagy receptors depending on the context (30–38). For instance, Nix is critical for programmed mitophagy and plays an important role in eliminating mitochondria during maturation of erythroid cells (39, 40), whereas Bnip3 and Fundc1 induce mitophagy in response to oxygen deprivation and ischemia/reperfusion injury (36, 37, 41). Although some of these receptors are activated by similar conditions, it is currently unclear why such redundancies are in place. Prohibitin 2 (PHB2) is the only mitophagy receptor that is localized in the inner membrane and is responsible for removal of mitochondria after outer membrane rupture (33). PHB2 can also promote PINK1/Parkin-mediated mitophagy by suppressing proteolytic cleavage of PINK1 by mitochondrial protease PARL, which leads to increased recruitment of Parkin to mitochondria (42). Many of these mitophagy receptors have alternative functions in other pathways, which has made it challenging to dissect how they regulate mitophagy in cells. For instance, Bnip3 and Nix were initially identified as proapoptotic BH3-only proteins (43–45), whereas PHB2 is a mitochondrial scaffolding protein that is important for mitochondrial stabilization and function (46). Altering expression of mitophagy receptors that are critical effectors of other mitochondrial signaling pathways could result in off-target effects that are not a direct result of these proteins’ role in mitophagy.

AMPK-Ulk1 Axis in Mitophagy

A key step in mitophagy is signal emission from damaged mitochondria to the autophagic machinery to initiate autophagosome formation. One well-studied signaling pathway that is activated by defective mitochondria involves the cellular energy sensor AMP-activated protein kinase (AMPK) (47). Damage to mitochondria often leads to decreased oxidative phosphorylation and diminished ATP generation. The resulting increase in cellular AMP and ADP levels leads to activation of AMPK and phosphorylation of its downstream targets function to shift metabolism toward decreased anabolism and increased catabolism (47). Several studies have also linked AMPK activation to mitophagy (48–51). This function was initially reported by Egan et al. (48), who discovered that AMPK-deficient cells have increased numbers of mitochondria due to impaired mitophagy. Subsequent studies have identified that AMPK can selectively regulate the induction of mitophagy through at least three different mechanisms, including 1) activation of the serine/threonine kinase unc-51-like kinase 1 (Ulk1) (48, 49), 2) interaction with the Atg16L complex (50), and 3) induction of mitochondrial fission factor (Mff)-mediated mitochondrial fission (51).

Ulk1

The primary pathway through which AMPK regulates mitophagy appears to be via Ulk1. This specific function of Ulk1 in mitophagy was discovered when Kundu and colleagues (52) noted that Ulk1 deficiency is associated with abrogation of mitochondrial clearance in red blood cells during maturation. Additional studies using tissue-specific Ulk1-deficient mice have confirmed that Ulk1 is required for functional mitophagy (48, 53, 54). Mechanistically, AMPK initiates mitophagy by phosphorylating Ulk1 on Ser555 (48), which leads to translocation of Ulk1 to damaged mitochondria (55). Ulk1 then recruits the Beclin1-Vps34-Vps15 complex, a downstream effector involved in nucleation of the phagophore membrane, ensuring that autophagosome formation is initiated at the cargo for more efficient removal of mitochondria (56). Although Ulk1 is activated by AMPK during nutrient starvation (48), activation of mitophagy is not strictly dependent on AMPK. A recent study reported that while Parkin-mediated mitophagy of depolarized mitochondria is impaired in Ulk1/2-deficient cells, this process remains intact in AMPK α 1/2 double knockout cells (57).

Moreover, autophagy adaptor proteins facilitate autophagy by linking ubiquitinated cargo to LC3 at the growing autophagosome membrane. Recently, these proteins have also been linked to the recruitment and retention of Ulk1 at the mitochondria during mitophagy (57). To date, at least five adaptor proteins have been reported to facilitate mitophagy by tethering mitochondria and the autophagosome membrane together, and HeLa cells lacking all five adaptor proteins exhibit impairments in mitophagy (20). The authors also discovered that these cells have a defect in Ulk1 recruitment to damaged mitochondria, suggesting the adaptor proteins also play a role in recruiting Ulk1 to mitochondria during mitophagy. In a subsequent study, this group found that the presence of the adaptor protein NDP52 on mitochondria is sufficient to recruit and activate Ulk1 independently of AMPK (57).

In addition to initiating autophagosome formation at the mitochondria, Ulk1 also phosphorylates the mitophagy receptor FUNDC1 (FUN14 domain-containing protein 1), facilitating the interaction between FUNDC1 and LC3 on the autophagosome (58). More recently, Hung et al. (59) reported that Ulk1 is also involved in promoting translocation of Parkin to mitochondria, where phosphorylation of Parkin at Ser108 by Ulk1 in the cytosol represents one of the initial events in mitophagy induction. Inhibition of Ulk1 or mutation of this phosphorylation site leads to delayed activation of Parkin and defects in mitophagy. These findings suggest that Ulk1 functions as a central regulator of several distinct steps of mitophagy: 1) initiating autophagosome formation by activating the Beclin1-Vps34-Vps15 complex, 2) enhancing the tethering between mitochondria and the autophagosome membrane via FUNDC1, and 3) promoting Parkin translocation to mitochondria.

AMPK and Atg16

The second mechanism by which AMPK can regulate the induction of mitophagy is via Atg16 (Atg16L1 in mammals), a core autophagy protein that forms a complex with Atg12 and Atg5 during autophagosome biogenesis (60, 61). Atg12-Atg5 exhibits the E3-like ligase activity responsible for conjugating phosphatidylethanolamine (PE) to LC3. The main function of Atg16 is to bring the conjugation complex to the phagophore (62, 63), and Atg16-deficiency leads to abrogation of LC3 lipidation due to a lack of Atg12-Atg5 complex at the phagophore (64). When AMPK associates with damaged mitochondria during mitophagy, it also recruits the Atg16-Atg5-Atg12 complex to the site of phagophore formation to initiate mitophagy (50). The same study reported that treating cells with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) leads to increased association between AMPK and the Atg16-Atg12-Atg5 complex at depolarized mitochondria. Interestingly, this study also discovered that AMPK is responsible for retaining the conjugation complex at the mitochondria. Overall, the recruitment and retention of the Atg16-Atg12-Atg5 complex at the mitochondria by AMPK ensure the localized formation of autophagosome near the cargo.

AMPK and Mff-Induced Mitochondrial Fission

Finally, AMPK can facilitate mitophagy by promoting mitochondrial fission via phosphorylation of Mff (51, 65, 66). Mitochondria can undergo symmetric or asymmetric division depending on the physiological context. Symmetric division of mitochondria occurs during cell division to facilitate mitochondrial biogenesis, whereas asymmetric division is associated with clearance of damaged mitochondria through mitophagy (67, 68). Thus, once separated from the healthy mitochondrial network, these dysfunctional mitochondrial fragments can be degraded via mitophagy (67, 69). AMPK-mediated phosphorylation of Mff leads to recruitment of the large GTPase Drp1 from the cytosol to the outer mitochondrial membrane. Upon recruitment, Drp1 promotes the constriction and subsequent fission of mitochondria (51). Abrogation of Mff phosphorylation leads to inhibition of both mitochondrial fission and mitophagy, confirming the importance of the AMPK-Mff/Drp1 pathway for functional mitophagy (51).

Although the mechanisms regulating asymmetric and symmetric division of mitochondria are mostly unclear, a recent study reported that mitochondria have distinct fission signatures that dictate their fate (68). This study discovered that division at the periphery of the mitochondria (asymmetrical fission) generates fragments that are destined for mitophagy, whereas division at the midzone (symmetrical fission) is associated with mitochondrial biogenesis. Although both peripheral and midzone divisions require Drp1, the authors found that Mff is primarily associated with midzone division while Fis1 is enriched in peripheral sites (68). Thus, the findings in this study contradict the report that the AMPK-Mff axis is involved in regulating mitophagy. Moreover, Mff-deficient mice develop lethal heart failure within 13 wk of birth, and these hearts have reduced mitochondrial respiration along with decreased mitochondrial content and enhanced mitophagy (70). Clearly, the exact functions of Mff in regulating mitochondrial fission and mitophagy need to be investigated further to determine if these contrasting reports are due to differences in experimental systems. Although these studies indicate that Mff-mediated fission might not be required for mitophagy, it is well accepted that functional mitochondrial fission is important for mitochondrial and cardiac health (71–73).

Mitochondria-Associated Membranes and Mitophagy

Autophagosome formation requires a source of lipids, and various cellular compartments have been reported to supply lipids to the growing autophagosome. It has been reported that the lipids can come from the plasma membrane, Golgi apparatus, or nuclear membrane, but the endoplasmic reticulum (ER) seems to be the major contributor of membrane lipids to the autophagosome (74). The phagophore originates from a subcompartment of the ER called the omegasome that is enriched in phosphatidylinositol 3-phosphate (PI3P) (75). In addition, the ER forms contact sites with mitochondria called mitochondria-associated ER membranes (MAMs), and these MAMs have been shown to be instrumental in autophagosome formation (76). MAMs mark the site of phagophore nucleation, which is where the autophagic machinery assemble to initiate formation and expansion of the autophagosome (Fig. 3) (76). The Atg2-Atg18 complex accumulates at MAMs and is responsible for tethering the phagophore membrane to the ER to facilitate membrane elongation (77). Atg2 is also crucial for autophagosome growth by promoting lipid transfer from donor membranes to the growing phagophore (78–80). Abrogating Atg2 recruitment to MAMs leads to impaired phagophore expansion and autophagic flux, which suggests that Atg2 may source lipids from the ER and mitochondria for autophagosome biogenesis at these contact sites (81). In mitophagy, the localized formation of the autophagosome at MAMs on dysfunctional mitochondria ensures their efficient removal. However, the mechanism through which autophagic machinery is selectively recruited to MAMs on damaged mitochondria is unclear and needs to be investigated further.

Figure 3.

Mitophagy initiation at MAMs. MAMs are contact sites between the ER and mitochondria that are maintained by a variety of tethering proteins. When mitophagy is initiated, the initiation machinery, which includes the Beclin1-Vps34-Vps15 complex and Atg9-containing vesicles, translocates to MAMs to promote autophagosome biogenesis. The Beclin1-Vps34-Vps15 complex generates PI3P, which promotes the recruitment of downstream autophagy regulators. Atg9-positive vesicles also localize to MAMs, where they serve as platforms for autophagosome nucleation and subsequent membrane elongation. ER, endoplasmic reticulum; MAMs, mitochondria-associated ER membranes; PI3P, phosphatidylinositol 3-phosphate. (Created with Biorender.com)

Moreover, fission of mitochondria has been reported to occur at MAMs (82). The fission protein Drp1 localizes to MAMs to induce mitochondrial fission, which allows for more efficient degradation of these fragments (82, 83). Interestingly, the mitophagy receptor FUNDC1 also localizes to MAMs (84), where it may regulate fission (85, 86). Wu et al. (86) reported that FUNDC1 accumulates at MAMs and facilitates fission by directly interacting with DRP1 during hypoxia in HeLa cells. Additional key proteins involved in mitophagy have also been shown to localize to MAMs. For instance, both Beclin1 and PINK1 relocalize to MAMs during mitophagy to facilitate enhancement of ER-mitochondria contacts and initiation of autophagy (87). Parkin is also recruited to the MAMs by PINK1 upon induction of mitophagy and may play a role in regulating MAM formation and stability during mitophagy (87, 88). Specifically, Parkin enhances the ER-mitochondria connection by ubiquitinating the mitochondria-ER tether protein Mfn2 (89). Overall, these findings demonstrate the important role of MAMs as sites of both autophagosome formation and mitochondrial fission for more efficient removal of dysfunctional mitochondria.

Beclin1 and Mitophagy

Beclin1 is the mammalian ortholog of Atg6 in yeast and a positive regulator of autophagy (87, 90). Early studies identified Beclin1 as a Bcl-2-interacting protein and tumor suppressor in cancer cells (90, 91). Subsequent studies revealed that Beclin1 is a scaffold protein that interacts with the lipid kinase Vps34 and its regulatory subunit Vps15 to form the Class III phosphatidylinositol 3-kinase (PI3K) complex. This core complex binds to additional proteins to activate its proautophagic functions, including initiation of autophagosome formation and autophagosome maturation. Binding of Atg14L to the Beclin1-Vps34-Vps15 complex facilitates proper localization of the complex to the site of autophagosome biogenesis and local generation of PI3P (56, 75, 76, 92). This pool of PI3P promotes the recruitment of downstream autophagic regulators containing motifs that directly bind to PI3P (56, 75, 92). The Beclin1-Vps34-Vps15 complex also functions at later stages of autophagy through binding with UV radiation resistance-associated gene protein (UVRAG) to facilitate autophagosome maturation (93).

Studies have also reported that Beclin1 plays a selective role in mitophagy. For instance, Beclin1 interacts with Parkin and facilitates its translocation to mitochondria upon induction of mitophagy (94, 95). However, a different study found that although silencing of Becn1 leads to reduced mitophagy, it does not alter Parkin recruitment to mitochondria (87). Beclin1 also interacts with PINK1, and this interaction is essential for both MAM and autophagosome formation during general autophagy and mitophagy (87, 96). The importance of Beclin1 in MAM formation or stability in the heart has also been confirmed in vivo, where Becn1 mice exhibit decreased MAM levels and Becn1 transgenic mice exhibit enhanced MAM formation in hearts (97). Altogether, these studies implicate Beclin1 in autophagosome formation at MAMs during mitophagy.

Despite the findings implicating Beclin1 in mitophagy, it is important to note that other groups have reported that loss of Beclin1 does not significantly impair mitochondrial clearance or LC3 lipidation during mitophagy (98, 99). These findings suggest the existence of Beclin1-independent mechanisms of autophagy. However, considering the importance of mitophagy for cellular survival, it is not surprising that redundancies in these pathways would exist.

Atg9-Positive Lipid Vesicles in Mitophagy

Atg9 is the only membrane-spanning core autophagy protein and is embedded in single-membrane vesicles 30–60 nm in diameter derived from the Golgi apparatus (100). The Atg9-positive vesicles cycle between the trans-Golgi network, endosomal compartments, and plasma membrane under basal conditions (101–103). Upon induction of autophagy, Atg9-positive vesicles redistribute to sites of autophagosome formation (104). Interestingly, these vesicles associate only transiently with the growing autophagosome and are not integrated into the autophagosome membrane (104). Although it was initially reported that Atg9-positive vesicles are responsible for promoting autophagosome membrane expansion by delivering lipids, it is now clear that these small vesicles are not able to transport enough lipids to form mature autophagosomes. Instead, the ER is likely the main source of lipids for the autophagosome. It has been reported that the Atg9-positive vesicles can supply proteins that are involved in autophagosome biogenesis. For instance, these vesicles contain proteins involved in lipid synthesis, such as acetyl-coA synthetases Faa1 and Faa4 (105), and have been shown to deliver phosphatidylinositol 4 kinases PI4KIIIβ and PI4KIIα to the forming autophagosome (106). More recently, a study demonstrated that Atg9-positive vesicles serve as platforms for the recruitment and assembly of downstream autophagic machinery to initiate autophagosome formation and elongation (105). Atg9 can also serve as an acceptor of Atg2-mediated lipid transfer from ER to the phagophore (105). Indeed, Tang et al. (81) found that interactions between Atg9a and MAM-localized Atg2 are required for proper phagophore expansion.

For proper maturation of the autophagosome, there is also a need for lipid scramblase activity to facilitate the transfer of lipids from the cytoplasmic leaflet of the autophagosome membrane to its luminal leaflet (78). Recent structural studies have discovered that ATG9A exhibits lipid scramblase activity (107, 108). Proteomic analysis also found that flippases Drs2 and Neo1 are associated with Atg9-positive vesicles isolated from yeast (105). Thus, it is possible that these flippases are transferred to the growing phagophore. Because the interaction between Atg9-positive vesicles and the growing autophagosome is transient (104), this redundancy could ensure that there is machinery in place to continue performing the functions of Atg9 at the autophagosomes once the vesicles are recycled elsewhere.

Specific roles for Atg9a in Parkin-mediated mitophagy have also been identified. First, Atg9a-positive vesicles have been observed to localize to depolarized mitochondria in a Ulk1-independent manner (109). Atg9a deficiency also suppresses autophagosome formation during mitophagy, which subsequently leads to accumulation of depolarized mitochondria (109). The fact that autophagosome formation is not completely abrogated suggests that the paralog Atg9b can potentially compensate for the loss of Atg9a in mammalian cells. Atg9a and Atg9b are often expressed in the same cells (110), but whether they have distinct functions in regulating autophagy and mitophagy are unknown. A recent study also found that the autophagy adaptor optineurin (OPTN) forms a complex with ATG9A and LC3 at ubiquitinated mitochondria during mitophagy. Disrupting this complex results in loss of Parkin-mediated mitophagy (111). Thus, it is likely that the OPTN-ATG9A complex serves as a platform for the recruitment of downstream autophagic machinery to damaged mitochondria for localized autophagosome formation.

MITOCHONDRIAL QUALITY CONTROL IN THE HEART

The heart is enriched in mitochondria due to its high energy demand, and mitophagy plays an important function in maintaining cardiac homeostasis. Cardiac myocytes rely on mitophagy to sustain the quality and quantity of mitochondria at baseline. They also need mitophagy to adapt to stressors that are associated with mitochondrial damage to suppress activation of inflammation and protect against cell death. Studies in mice have demonstrated the impact of defective mitophagy on baseline cardiac homeostasis. For example, both Parkin-deficient and Bnip3/Nix double knockout mice accumulate dysfunctional mitochondria in the heart at an accelerated rate with aging (112, 113). Similarly, PINK1-deficient mice display abnormal mitochondrial function and develop cardiac hypertrophy and contractile dysfunction at an early age (114). PINK1 protein levels are also reduced in human heart failure (114), but whether this is part of the underlying cause or a consequence of heart failure in humans is still unknown. In contrast, overexpression of Parkin in the heart ameliorates the decline in mitochondrial and cardiac function observed in aging mouse hearts (115, 116).

Decreases in autophagy and mitophagy levels in the myocardium are observed with aging (116, 117), and interventions that enhance these processes are associated with improved cardiac function and extended longevity (115, 116, 118, 119). For instance, the compromised myocardial function and mitochondrial morphology observed in aged (>24 mo old) mice is preventable in mice with transgenic overexpression of Parkin (116). Although this study found that Parkin overexpression had no effect on cardiac function in young mice (116), another study reported increased cardiac fibrosis with aging in transgenic mice with cardiac-specific overexpression of Parkin (120). It is likely that differences in the level of Parkin overexpression are responsible for the different findings in these two studies. Although Parkin is likely a good therapeutic target in the heart, its levels must be carefully regulated to prevent any detrimental outcomes and effects.

Interestingly, mitochondrial activity and turnover in tissues closely follow circadian rhythms, and disruptions to the circadian clock lead to altered mitochondrial morphology and impaired function (121–123). Fis1, Pink1, and Bnip3, key regulators of mitochondrial dynamics and mitophagy, are transcriptional targets of regulators of the circadian clock (124). Therefore, it is not surprising that disruptions to these cycles lead to accumulation of enlarged and dysfunctional mitochondria. Rabinovich-Nikitin et al. (125) recently identified a link between transcription factor circadian locomotor output cycles kaput (CLOCK) activity and susceptibility to cardiac injury. They demonstrated that loss of CLOCK activity in myocytes leads to decreased expression of various autophagy and mitophagy regulators, including Atg7, Rab7a, Tfeb, and Sqstm1. The authors also found that disruption of CLOCK is associated with impaired activation of mitophagy and increased susceptibility to cardiac stress (125). Because it is well established that circadian disruption is associated with increased risk of developing heart disease and increased damage after cardiac injury in humans, it is possible that dysregulation of mitophagy is a major underlying factor.

Moreover, there is strong evidence that mitophagy is important for adapting to various cardiac stressors. For instance, Parkin-mediated mitophagy protects against myocardial infarction (126) and hemodynamic overload (127). Mitochondrial dysfunction is also often observed in diabetic cardiomyopathy, but how mitophagy is altered and whether this contributes to or is a consequence of the pathology are still unclear. Depending on the model being studied, autophagy and mitophagy have been reported to be either downregulated (128–130) or upregulated (131). However, a more recent study performed by Junichi Sadoshima’s group using new tools and mouse models demonstrated that a high-fat diet (HFD) in mice is associated with activation of autophagy and mitophagy in the heart. They also reported that Parkin-deficient mice with impaired mitophagy are more susceptible to HFD-induced cardiac impairment and exhibit increased mitochondrial dysfunction and lipid accumulation. In contrast, activating mitophagy in wild-type mice by administering the Tat-Beclin1 peptide (TB1), a potent autophagy activator, provided protection against diabetic cardiomyopathy induced by HFD (132). Overall, the findings in this study suggest that mitophagy is activated as an adaptive and cardioprotective response to high fat intake.

Myocardial damage induced by inflammation is a key feature of various cardiovascular diseases, and mitophagy has been reported to play an important role in suppressing inflammation. Excessive generation of mitochondrial ROS and release of mtDNA can stimulate the assembly and activation of the NLRP3 inflammasome (5, 133). Thus, mitophagy plays a critical role in suppressing inflammation by eliminating ROS-producing and leaky mitochondria. The importance of mitophagy in suppressing inflammation in the heart was demonstrated by Sun and colleagues (134), who reported that cardiac-specific overexpression of Beclin1 protects mice against LPS exposure by reducing inflammation and fibrosis. Specifically, Beclin1 overexpression or activation with the TB1 peptide leads to enhanced activation of PINK1/Parkin-mediated mitophagy and reduced release of mitochondrial danger-associated molecular patterns (mtDAMPs). Similarly, another group discovered that activation of FUNDC1-mediated mitophagy protects the heart against LPS-induced sepsis by preserving mitochondrial function and attenuating inflammation (6). Thus, both PINK1/Parkin- and mitophagy receptor-mediated mitophagy can suppress inflammation in the heart during LPS challenge, suggesting that there is redundancy in these pathways’ function. Finally, Kawasaki disease (KD) is associated with inflammation in blood vessels and is the most common cause of acquired heart disease among children (135). Studies in both mouse models and human patients indicate that activation of the NLRP-IL-1β pathway is the main factor underlying the pathology of KD (136). A recent study reported that autophagy and mitophagy are both impaired in a mouse model of KD vasculitis and that activating autophagy and mitophagy attenuates NLRP3 activation and cardiovascular inflammation (137). This study also found that Parkin-deficient mice are more susceptible to KD development and exhibit increased heart inflammation relative to wild-type controls. Overall, these studies clearly demonstrate the importance of mitophagy in suppressing NLRP3 activation and inflammation.

MITOCHONDRIAL QUALITY CONTROL IN OTHER DISEASES

Neurodegeneration

Accumulation of dysfunctional mitochondria is a hallmark of many neurodegenerative disorders, and studies have demonstrated that this is, at least in part, due to decreased mitophagy (138). Loss-of-function mutations in PINK1 and PARK2, two key mitophagy regulators, lead to development of juvenile recessive PD (10, 11). Many neurodegenerative diseases are also age-dependent, and mitochondrial quality control mechanisms, including mitophagy, are known to be impaired with aging, leaving cells with diminished ability to efficiently remove dysfunctional mitochondria (9, 110). For example, Alzheimer’s disease (AD), which is typically characterized by the presence of amyloid-β plaques and hyperphosphorylation of tau protein, has also been associated with accumulation of dysfunctional mitochondria and impaired mitophagy (138). A recent study reported that restoration of mitophagy reduces the size of amyloid-β plaques, inhibits tau hyperphosphorylation, and improves cognition in Caenorhabditis elegans and in mouse models of AD (139). The pathophysiology of many neurodegenerative diseases is poorly understood, which has made the development of treatments extremely challenging (138). However, therapeutic interventions that restore or enhance mitochondrial quality control mechanisms represent a promising path forward in the search for treatments.

Cancer

Mitochondria play an important role in the commitment of cancer cells to either proliferation or apoptosis. During the earlier stages of tumorigenesis, accumulation of dysfunctional mitochondria due to impaired mitophagy can lead to a metabolic switch to glycolysis, hence promoting tumor growth and survival (140, 141). In more established tumors, mitophagy is vital for survival, potentially by preventing activation of apoptosis in the hypoxic conditions of the tumor microenvironment (142). Loss of function or downregulation of Parkin has been identified as a contributor to tumorigenesis in several different cancers (143). The tumor-suppressing functions of Parkin include its positive regulation of mitophagy and its suppression of metabolic reprogramming, which shifts energy production toward glycolysis (144). Bnip3-mediated mitophagy is also responsible for suppressing tumor progression by preventing accumulation of dysfunctional mitochondria and metabolic reprogramming (140). However, the roles that many other core mitophagy effector proteins play in cancer are still unclear and seem to be either stage- or cell type-dependent. Downregulation of many mitophagy regulators has been observed in different cancers, but they have also been identified as biomarkers of poor prognosis because of their tumor-protective functions (145). Clearly, the role of mitophagy in cancer is highly complex, and further studies are required to understand the importance of mitophagy during different stages and types of cancer.

COVID-19 Infection

Although mitochondria are directly involved in cellular responses to infection by stimulating inflammation and cytokine production, many pathogens can directly target mitochondria to evade host immunity and use them as a base for expansion (146). Interestingly, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA contains mitochondrial localization sequences, and computational modeling predicts that the virus is likely trafficked to the mitochondria (147). Other coronaviruses have been shown to interfere with mitochondrial function, “hijacking” the mitochondria to optimize conditions for replication and suppress viral immunity (148, 149). For example, SARS-CoV-1, the virus responsible for the 2002 SARS outbreak, encodes a protein that promotes proteasomal degradation of Drp1 (150), thereby inhibiting mitochondrial fission and potentially limiting mitophagy (68). Analysis of the SARS-CoV-2-host interactome also revealed that proteins encoded by SARS-CoV-2 can interact with host mitochondrial proteins, suggesting that SARS-CoV-2 may use a similar strategy (151). In addition, there are predictions that SARS-CoV-2 may indirectly impair mitochondrial function through its binding to the angiotensin-converting enzyme carboxypeptidase 2 (ACE2) receptor, a regulator of calcium signaling, mitochondrial respiration, and ATP production (148). Because aging is associated with accumulation of dysfunctional mitochondria and inefficient mitophagy (9), studies on mitochondria and SARS-CoV-2 might provide insights into why aged individuals are more susceptible to COVID-19 infection and at increased risk for hospitalization and death. Also, if the virus is indeed hijacking mitochondria for proliferation, enhancing mitophagy might then represent a therapeutic intervention to limit the infection.

CONCLUSIONS

It is clear that functional mitophagy in cardiac myocytes is necessary to preserve a healthy mitochondrial network. There is overwhelming evidence in the literature that functional and properly regulated mitophagy is essential for cellular homeostasis and survival and that impaired mitophagy contributes to both aging and disease development, raising the exciting possibility that this pathway represents a desirable future therapeutic target. However, much still needs to be learned before proteins in this pathway can be safely manipulated pharmacologically. For instance, it is unknown how this process can be safely targeted without causing excessive mitochondrial clearance or undesirable off-target effects due to the involvement of many key mitophagy proteins in other critical cellular processes. In the heart, most efforts to date have focused on investigating autophagy and mitophagy in cardiac myocytes, but there are still very few studies on mitophagy in the other cell populations in the heart, such as fibroblasts, smooth muscle cells, and endothelial cells. Therapies aimed at targeting mitophagy will also affect this process in these cells. Therefore, it will be important to determine how targeting mitophagy will impact their function.

GRANTS

Å. B. Gustafsson is supported by NIH Grants R01HL138560, R01HL132300, R01HL155281, and HL157265. R. Y. Diao is supported by the University of California, San Diego Graduate Training Program in Cellular and Molecular Pharmacology Grant T32GM007752.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.Y.D. prepared figures; R.Y.D. and A.B.G. drafted manuscript; R.Y.D. and A.B.G. edited and revised manuscript; R.Y.D. and A.B.G. approved final version of manuscript.