Studies have demonstrated at least two major mitophagy pathways: ubiquitin-mediated and receptor-mediated mitophagy (Zimmermann and Reichert, 2017). The ubiquitin-mediated mitophagy pathway is mediated by PINK1/Parkin (Eiyama and Okamoto, 2015; Bingol and Sheng, 2016; Nguyen et al., 2016). PINK1 is a molecular sensor of mitochondrial health that constantly surveys the organelle status. In addition, Parkin is an amplifier of mitophagy. Once mitochondria lose their transmembrane potential, PINK1 accumulates at the OMM of impaired or dysfunctional mitochondria and phosphorylates ubiquitin and Parkin at S65. pS65-Ub (the phosphorylated ubiquitin at S65) binds and activates Parkin by destabilizing Parkin’s autoinhibitory interactions and then recruits Parkin from the cytoplasm to the OMM (Nguyen et al., 2016). As E3 ubiquitin ligase, the activated phosphorylated Parkin ubiquitinates various mitochondrial outer-membrane proteins with less specificity. The elongated PINK1 and Parkin proteins form ubiquitin chains that act as molecular signals to further recruit mitophagy receptors, including optineurin (OPTN), nuclear dot protein 52 (NDP52), Tax1-binding protein 1 (TAX1BP1), neighbor of BRCA1 gene 1 (NBR1), and p62, which link ubiquitin chains with LC3 (Dikic and Elazar, 2018). Thus, ubiquitinated proteins in impaired mitochondria can be recognized by cellular mechanisms and get engulfed by autophagosomes to be transferred to lysosomes for degradation.

The known receptor-mediated mitophagy receptors include BCL2 interacting protein 3 such as NIX, also known as (BNIP3L), BCL2 interacting protein 3 (BNIP3), and FUNDC1 in mammalian systems (Liu et al., 2014; Chen et al., 2016). These receptors are integral proteins of the OMM, possessing LIRs, which are the structural basis for LC3 binding to activate mitophagy (Poole and Macleod, 2021). These receptors can be modified by dephosphorylation or phosphorylation under various stresses to affect their affinity for LC3, effectively regulating mitophagy. For instance, BNIP3L-triggered mitophagy can be reversed by PRKA/PKA (protein kinase, AMP-activated)-induced phosphorylation of BNIP3L at Ser212. Activation of the inhibitory phosphorylation site leads to the translocation of BNIP3L from the mitochondria to the cytosol (da Silva Rosa et al., 2021). Phosphorylation of BNIP3 at Ser17/24 sites or NIX at Ser34/35 sites (Rogov et al., 2017) promotes its binding to LC3 and facilitates subsequent mitophagy (Liu et al., 2014). In the case of FUNDC1, post-transcriptional phosphorylation at Ser17 activates mitophagy, while phosphorylation at Ser13 inhibits the process (Wang et al., 2020a). Thus, mitophagy mediated by these two pathways contributes to the clearance of damaged mitochondria and might be mutually affected.

Proper mitophagy guarantees homeostasis of mitochondria in cells and exerts protective effects on the cardiovascular system, while insufficient or excessive mitophagy may be detrimental. Atherosclerosis, hypertension, ischemia/reperfusion injury, myocardial infarction, cardiac hypertrophy, heart failure, and metabolic cardiomyopathy consistently exhibit mitophagy-involved pathological processes. In vivo experiments have shown that knockout of pivotal mitophagy molecules can affect the phenotype and severity of diseases. For example, deletion of PINK1 leads to more severe cardiac hypertrophy and left ventricle dysfunction in mice than those in wild-type and heterozygous mice (Billia et al., 2011), while Parkin-knockout mice are vulnerable to myocardial infarction induced by ligation of the proximal left anterior descending coronary artery and present a low survival rate (Kubli et al., 2013). Similarly, the mammalian target of rapamycin complex 1 (mTORC1) activation in dietary protein-driven atherosclerotic plaques inhibits mitophagy (its downstream effect) and results in a buildup of dysfunctional mitochondria that contribute to a rise in plaque complexity (Zhang et al., 2020). Likewise, the NIX expression has been found to be decreased in human atherosclerosis. Silencing the NIX expression in murine macrophage cells reduced NIX-mediated mitophagy, enhanced oxidized low-density lipoprotein (ox-LDL)-induced macrophage pyroptosis, and led to formation of unstable plaques (Peng et al., 2020). Therefore, numerous chemicals targeting the modulation of mitophagy may alleviate or exacerbate different cardiovascular dysfunctions (Hsu et al., 2015; Ma et al., 2018; Qiao et al., 2018; Yang et al., 2021).

Cardiomyocytes, cardiac fibroblasts (CFs), endothelial cells (ECs), vascular smooth muscle cells (VMSCs), macrophages, and other cell types need to work in an organized manner to keep the cardiovascular system functioning well and maintain a low disease risk. Improper mitophagy can alter the functions of cells and result in the occurrence and progression of diseases. Inhibited mitophagy aggravates lipid accumulation and leads to heart dysfunction (Tong et al., 2019). A mitophagy imbalance renders cardiomyocytes apoptotic under I/R stress (Li et al., 2019a). PINK1/Parkin-mediated mitophagy is upregulated in endothelial cells under metabolic stress to protect mitochondrial integrity and prevent metabolic stress–induced endothelial injury (Wu et al., 2015). The melatonin-induced suppression of mitophagy protects microvascular endothelial cells against I/R injury (Zhou et al., 2017a). In addition, inhibited mitophagy suppresses activation of cardiac fibroblasts but promotes apoptosis (Gao et al., 2020a), while enhanced mitophagy restrains proliferation and apoptosis of VMSCs (Swiader et al., 2016; Chen et al., 2020). Thus, mitophagy in various cell types contributes to cardiac function.

FUNDC1 is a protein with three α-helix transmembrane domains at the OMM and characteristics similar to those of other mitophagy receptors. Its LIR motif (Y18-E19-V20-L21 at the N-terminal region of FUNDC1 in the cytoplasm) has the classic tetrapeptide W/F/YxxL/I sequence for interaction with LC3, which links it to the ATG5-dependent core autophagic machinery (Liu et al., 2012a; Kuang et al., 2016). Mutants of Y18A, V20A, and L21A or complete deletion of the LIR sequence display a reduced or even abolished affinity of FUNDC1 for LC3 binding that disrupts mitophagy; other FUNDC1 mutations have no effects (Liu et al., 2012a; Kuang et al., 2016). Phosphorylation is the main post-transcriptional modification of FUNDC1 that regulates mitophagy. Three key residues of FUNDC1, Ser13, Ser17, and Tyr18, get phosphorylated and modify the binding affinity of FUNDC1 for LC3 and consequently influence mitophagy (Lv et al., 2017).

Under normal conditions, as depicted in Figure 2, FUNDC1 phosphorylation at Ser13 (Chen et al., 2014; Zhou et al., 2018a) and Tyr-18 (Feng et al., 2013; Zhou et al., 2017b) or dephosphorylation at Ser17 (Wu et al., 2014a) both decrease the affinity of FUNDC1 for LC3 by altering its stereochemical properties. The phosphorylation of FUNDC1 at Ser13 and Tyr18 is mediated by casein kinase 2 (CK2) kinases and Src kinase, respectively (Chen et al., 2014). The affinity of the dephosphorylated FUNDC1 peptide (at Ser17) for LC3 (at Lys49) is ∼3-fold weaker than that of the phosphorylated FUNDC1 peptide (Lv et al., 2017). The direct optic atrophy 1 (OPA1)-FUNDC1 connection and the FUNDC1-calnexin association block other FUNDC1 interactions, and the BCL2L1 (BH3 domain)–PGAM5L (a member of the phosphoglycerate mutase family) complex inhibits FUNDC1 dephosphorylation at Ser13; all these molecular interactions inhibit FUNDC1 activities (Wu et al., 2014b). Additionally, the membrane-associated RING-CH protein 5 (MARCH5), a mitochondrial E3 ligase, mediates FUNDC1 ubiquitylation and degradation by directly interacting with it at lysine 119. Thus, FUNDC1 is inactivated in healthy hearts. Moderately inactivated FUNDC1 may interact with the F-box protein FBXL2 to maintain the mitochondrial integrity or it may form a complex with heat shock protein 70 (HSC70) to promote the mitochondrial translocation of unfolded cytosolic proteins and maintain the cardiac function (Li et al., 2019b; Ren et al., 2020).

Under abnormal conditions like hypoxia, two pathways regulate FUNDC1 to mediate mitophagy: a kinase-mediated pathway and an interactional protein-regulated pathway. Tyr416-phosphorylated Src enhances Tyr18 dephosphorylation of FUNDC1, increasing its binding ability for LC3 and promoting mitophagy (Feng et al., 2013; Zhou et al., 2017b). Similarly, CK2 dissociates from FUNDC1 and allows PGAM5 to dephosphorylate it at Ser13, strengthening its interaction with LC3 (Chen et al., 2014; Zhou et al., 2018a). ULK1 translocates to mitochondria and activates FUNDC1 by phosphorylating its Ser17, and ULK1 deletion inhibits FUNDC1 activation (Wang et al., 2020a). Under stress, phosphorylated FUNDC1 dissociates from OPA1 and interacts instead with dynamin-related protein 1 (DRP1) to enhance mitochondrial fission and mitophagy (Wu et al., 2016b). During mitophagy under hypoxic conditions, the cytosolic loop of FUNDC1 is exposed (due to an attenuated FUNDC1/calnexin association) and interacts with DRP1 (Wu et al., 2016b). Disorders of mitochondrial dynamics, including imbalanced fission and fusion, are prerequisites for mitophagy. Hypoxia induces BCL2L1 degradation, increases PGAM5 dissociation, and then enhances dephosphorylation of FUNDC1 at Ser13 (Ma et al., 2020). Additionally, the decreased endogenous MARCH5 expression significantly inhibits FUNDC1 degradation and promotes mitophagy (Chen et al., 2017). Other regulators of FUNDC1 have been identified: the Nod‐like receptor X1 (NLRX1), in mitochondria, negatively regulates phosphorylated Tyr18 FUNDC1 levels (Li et al., 2021), and the LncRNA MEG3 overexpression induces dephosphorylation of FUNDC1 at Tyr18 by interacting with the 3′UTR of Rac1 to inhibit its expression (Wang et al., 2021a). The transcriptional and post-transcriptional controls of FUNDC1 have also been shown to be important. Under normoxic conditions, the negative regulatory factor microRNA-137 is constitutively expressed; it targets the 3′ UTR of the FUNDC1 mRNA suppressing its translation to attenuate FUNDC1-LC3 associations. Under hypoxic conditions, the microRNA-137 is downregulated. At the transcriptional level, mitophagy is enhanced by the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) protein, which induces the FUNDC1 expression by upregulating the nuclear respiratory factor 1 (NRF1) expression, a factor that binds FUNDC1 at -186/-176 sites (Liu et al., 2021a).

In summary, FUNDC1 is crucial to receptor-mediated mitophagy. Proper mitophagy helps restore cardiac function after hypoxia, I/R, and other stresses. However, a review of FUNDC1-mediated mitophagy in CVDs has not been published.

FUNDC1 has been found enriched at MAMs under stress and to facilitate ER and mitochondrial tethering by interacting with ER proteins such as calnexin and IP3R2 (ER-resided inositol 1,4,5-trisphosphate type 2 receptor) (Wu et al., 2016b; Wu et al., 2017; Wu et al., 2019b). Under hypoxia, FUNDC1 accumulates at the MAM and exhibits a dynamic interaction with the MAM-related protein calnexin (Wu et al., 2016b). In cardiomyocyte-specific FUNDC1-knockout mice, the connection between the ER and mitochondria in cardiomyocytes is disrupted, and the mice present few MAMs and MAM-related proteins (IP3R2 and PACS-2 [phosphofurin acidic cluster sorting protein 2]), a picture consistent with that in the H9C2 cell line (Wu et al., 2017). In a high glucose-induced in vitro model, the FUNDC1 overexpression promoted MAM formation, and FUNDC1 ablation inhibited it (Wu et al., 2019b). Wang et al. found similar phenotypes in FUNDC1-deleted endothelial cells (EC) and EC-specific FUNDC1-knockout mice (Wang et al., 2021b). Based on the evidence, FUNDC1 is a MAM-related protein that participates in the formation and function of MAMs.

During the cardiac cycle, Ca²⁺ is rapidly released to the cytosol from the sarcoplasmic reticulum (SR) and then restored (Gambardella et al., 2018). Appropriate calcium handling is vital for excitation–contraction (EC) coupling of cardiomyocytes, and calcium flux disruption eventually leads to heart dysfunction. Mitochondria can act as Ca²⁺ buffers, and they are also involved in Ca²⁺ reuptake, but Ca²⁺ overload in mitochondria can be harmful and cause heart failure (Brookes et al., 2004; Gambardella et al., 2018). The FUNDC1-mediated MAM is an important structure that regulates intracellular calcium homeostasis. Specific FUNDC1-knockout cardiomyocytes present decreased cytoplasmic and mitochondrial Ca²⁺ and increased ER Ca²⁺, while FUNDC1-overexpressing cardiomyocytes display the opposite effects (which can be abolished by silencing IP3R2) (Wu et al., 2017). Ablation of FUNDC1 decreases mitochondrial Ca²⁺ (via MAMs induced by high glucose) and also inhibits ROS production and cell apoptosis, preventing cardiac dysfunction in vivo (Wu et al., 2019b).

Studies have reported that deletion of FUNDC1 results in elongated mitochondria in cardiomyocytes (Wu et al., 2016b; Chen et al., 2016; Wu et al., 2017). Wu et al. observed both fewer absolute fission events and a decreased ratio of fission to fission and fusion events in specific FUNDC1-knockout cardiomyocytes via time-lapse confocal imaging (Wu et al., 2017). They also found that FUNDC1 loss inhibits the integrity of MAMs, causing increased mitochondrial and intracellular Ca²⁺ concentrations and leading to cardiac dysfunction (Wu et al., 2017). As mentioned, FUNDC1 coordinates mitochondrial dynamics and mitophagy at MAMs by interacting with DRP1 and OPA1. FUNDC1 ablation suppresses the mitochondrial fission 1 protein (Fis1) expression by reducing the binding of the cAMP response element-binding protein (CREB) in the Fis1 promoter and inhibiting mitochondrial fission in cardiomyocytes (Wu et al., 2017). In HeLa cells under hypoxia, FUNDC1 is involved in mitochondrial fission via its Mff interaction (Wu et al., 2016b). In addition, USP19 (an ER-resident deubiquitinase) can bind FUNDC1 and deubiquitinate it at the MAMs leading to DRP1 oligomerization and promotion of mitochondrial division (Chai et al., 2021).