Mitochondrial autophagy: molecular mechanisms and implications for cardiovascular disease

Abstract

Mitochondria are highly dynamic organelles that participate in ATP generation and involve calcium homeostasis, oxidative stress response, and apoptosis. Dysfunctional or damaged mitochondria could cause serious consequences even lead to cell death. Therefore, maintaining the homeostasis of mitochondria is critical for cellular functions. Mitophagy is a process of selectively degrading damaged mitochondria under mitochondrial toxicity conditions, which plays an essential role in mitochondrial quality control. The abnormal mitophagy that aggravates mitochondrial dysfunction is closely related to the pathogenesis of many diseases. As the myocardium is a highly oxidative metabolic tissue, mitochondria play a central role in maintaining optimal performance of the heart. Dysfunctional mitochondria accumulation is involved in the pathophysiology of cardiovascular diseases, such as myocardial infarction, cardiomyopathy and heart failure. This review discusses the most recent progress on mitophagy and its role in cardiovascular disease.

Full text

REVIEW ARTICLE OPEN
Mitochondrial autophagy: molecular mechanisms and
implications for cardiovascular disease
Anqi Li
1
, Meng Gao
1
, Bilin Liu
1
, Yuan Qin
2
, Lei chen
1
, Hanyu Liu
1
, Huayan Wu
1
and Guohua Gong
1
✉
© The Author(s) 2022
Mitochondria are highly dynamic organelles that participate in ATP generation and involve calcium homeostasis, oxidative stress
response, and apoptosis. Dysfunctional or damaged mitochondria could cause serious consequences even lead to cell death.
Therefore, maintaining the homeostasis of mitochondria is critical for cellular functions. Mitophagy is a process of selectively
degrading damaged mitochondria under mitochondrial toxicity conditions, which plays an essential role in mitochondrial quality
control. The abnormal mitophagy that aggravates mitochondrial dysfunction is closely related to the pathogenesis of many
diseases. As the myocardium is a highly oxidative metabolic tissue, mitochondria play a central role in maintaining optimal
performance of the heart. Dysfunctional mitochondria accumulation is involved in the pathophysiology of cardiovascular diseases,
such as myocardial infarction, cardiomyopathy and heart failure. This review discusses the most recent progress on mitophagy and
its role in cardiovascular disease.
Cell Death and Disease (2022) 13:444 ; https://doi.org/10.1038/s41419-022-04906-6
FACTS
Several distinguished molecular pathways mediate mitophagy.
Mitophagy selectively degrades damaged mitochondria is
essential for mitochondrial homeostasis.
The mitochondrial fragment is generally a prerequisite for
mitophagy.
Mitophagy involves various cardiovascular diseases, including
atherosclerosis, ischemia-reperfusion injury, cardiomyopathy,
hypertrophy, and heart failure.
OPEN QUESTIONS
What is the relationship between mitophagy and cell autophagy?
How to control the optimal regulation of mitophagy?
How to restore the homeostasis of dysfunctional mitochondria?
INTRODUCTION
Cellular homeostasis is a prerequisite for the optimal functional
performance of cells [1]. Autophagy, as a conventional regulation
mechanism of the homeostasis of eukaryotic cells, is highly
conserved throughout the evolutionary process. It is responsible
for removing cellular components such as accumulated proteins
and damaged organelles in cells to maintain intracellular home-
ostasis [1,2]. As a degradation pathway, autophagy balances the
biosynthesis and catabolism of macromolecules to protect
organisms against diverse pathologies, including cancer, aging,
neurodegeneration, and heart disease [1,3–5].
Mitochondria, as the most important energetic cellular orga-
nelles, play a pivotal role in intracellular homeostasis [6].
Maintenance of mitochondrial function and integrity is crucial
for normal cell physiology [7]. As an energy-rich compound,
adenosine triphosphate (ATP) is mainly produced by mitochon-
dria. In addition, mitochondria are also involved in modulating
second messenger levels, such as calcium ions (Ca
2+
), cAMP and
reactive oxygen species (ROS) [8]. It has been proved that
dysfunctional mitochondria not only in response to decrease
ATP production but also increase oxidative stress [9,10]. Dysfunc-
tional mitochondria can also perturb calcium homeostasis due to
the closed contact between mitochondria and the endoplasmic
reticulum. The Ca
2+
signal converted from physiological into a
pathological is concerned as a pathological marker [11]. Thus,
mitochondrial quality needs to be well controlled in cells. Nuclear
and mitochondrial DNA jointly regulate mitochondria, which
makes them sensitive to environmental stimulation. The different
contexts of mutations in mtDNA or nuclear DNA will result in
different clinical phenotypes of disease [12]. Damaged mitochon-
dria need to be selectively removed through mitochondrial
autophagy (mitophagy), the process of which is controlled by
nuclear coding proteins [13].
Mitophagy and mitochondrial biogenesis are two opposite
processes in determining the number of mitochondria, both of
which also are key regulators of mitochondrial quality and steady-
state mitochondrial turnover [14]. Mitophagy as targeting degrade
severely damaged mitochondria plays a more important role. It
has been known that reactive oxygen species (ROS) are generated
from the electron transport chain, which challenges mitochondrial
structure and function [8]. If damaged mitochondria are not
eliminated in time, they would contaminate the healthy mito-
chondria through reactive oxygen species (ROS)-induced ROS
Received: 20 January 2022 Revised: 27 April 2022 Accepted: 3 May 2022
1
Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
2
Department of Pharmacy,
Shanghai East Hospital, Tongji University, Shanghai 200120, China. ✉email: guohgong@tongji.edu.cn
Edited by Professor Paolo Pinton
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release (RIRR) [15]. RRIR is a vicious downward spiral, amplifying
the ROS signal, thus eventually causing irreversible damage to
cells. Excessive ROS impacts cellular proliferation and triggers the
peroxidation of lipids, impairment of DNA and apoptosis [8].
Mitophagy, as a selective autophagy pathway to mediate the
clearance of damaged or senescent mitochondria, is critical for
maintaining cellular function [16]. Abnormal mitophagy is closely
related to various diseases, particularly in organisms with high
energy demand heart, including heart failure, hypertrophy and
ischemia-reperfusion [17].
In this review, we focus on the current understanding of
mitophagy machinery and how mitophagy is regulated in
cardiovascular disease.
Mitophagy
Mitochondria, as the powerhouse, play a pivotal role in cells.
Damaged mitochondria need to be precisely removed timely to
keep proper mitochondrial functions. Selectively removal or
degradation of damaged mitochondria by autophagy are termed
mitophagy [17,18]. As a double-membrane organelle, Mitochon-
dria are mainly responsible for intracellular aerobic respiration and
producing energy in oxidative phosphorylation. The normal
oxidative phosphorylated process will generate adenosine tripho-
sphate (ATP) and reactive oxygen species (ROS) [19]. Not only do
mitochondria play a vital role in maintaining cell homeostasis and
regulating cell proliferation, but they are also involved in many cell
activities, such as calcium signal transduction [20], metabolic
synthesis, programmed death [21], and tumorigenesis [22,23].
As a defense mechanism, mitophagy can selectively remove
damaged and dysfunctional mitochondria in cells to maintain
the quality of mitochondria, thereby keeping mitochondrial
physiological functions. In a word, mitophagy is indispensable for
cells to clear abnormal mitochondria in response to stress.
Mitochondrial damage is usually associated with programmed
cell death, inflammation and aging. Increased or accumulated
damaged mitochondria will aggravate the occurrence and the
pathogenesis of many diseases [9]. Under normal physiological
conditions, the basal level of mitophagy in cells can make
dysfunctional mitochondria timely recognized and removed,
thereby providing sufficient raw materials for fresh mitochondria
and ensuring the energy supply of cells to preserve cellular
homeostasis. In turn, insufficient mitophagy will lead to the
accumulation of dysfunctional mitochondria. Consequently,
intracellular ATP level decreases, and ROS level will elevate.
Excess mitochondrial ROS reacts with proteins, lipids, and nucleic
acids, causing oxidative damage and apoptosis. Impaired
mitophagy can lead to various diseases, including cancer,
neurodegenerative diseases, and cardiovascular diseases.
Increasing mitophagy in response to different stress conditions
can preserve mitochondrial quality by maintaining cellular ATP
level, reducing the oxidative damage caused by ROS, and
selectively removing the damaged mitochondria in the cell.
Healthy mitochondrial quality control is crucial for the metabo-
lism of cells.
In recent studies, it has been demonstrated that accelerating
cellular metabolism can prevent the development of many
metabolic diseases. However, too much water drowned the miller.
Excessive clearance of mitochondria leads to loss of mitochondria
and increases oxidative species such as ROS, which is deleterious
in terms of normal cellular requirements. Therefore, precise and
proper regulation of mitophagy is helpful for cells to keep
homeostasis.
Pathways of mitophagy
Mitophagy can be divided into ubiquitin-mediated mitophagy and
receptor-mediated mitophagy. The ubiquitin-mediated mitophagy
includes PINK/Parkin, and other ubiquitin-mediated pathway. The
receptor-mediated mitophagy pathways include BNIP3 (Bcl-2 and
adenovirus E1B19 kDa-interacting protein 3) mediated mitophagy,
FUNDC1-mediated mitophagy, and lipid-mediated mitophagy.
PINK1/Parkin-mediated mitophagy
In 1998, PRKN was discovered as the causative gene of autosomal
recessive juvenile parkinsonism (AR-JP) [24]. Parkin protein is
composed of 465 amino acids, including ubiquitin-like domain
(UBL), repressor element of Parkin (REP), four zinc-coordinating
RING-like domains: RING0, RING1 (H302 to R305 motif), in-between
RING (IBR), and RING2 [25] (Fig. 1A). As an E3 ubiquitin ligase,
Parkin mainly consists of three families: homologous to the
C-terminus of E6-AP (HECT ligase) can react with ubiquitin to form
thioester-linked intermediates. Really Interesting New Gene (RING)
ligase can transfer ubiquitin from the E2 ubiquitin-conjugating
enzyme onto the substrate by binding E2 enzyme. RING-Between-
RING (RBR) ligase is referred to as a RING-HECT hybrid because it
combines the characteristics of RING-type ligases and HECT-type
ligases. Parkin is a member of RBR E3 ubiquitin ligases that can
modulate ubiquitination of various proteins in cytosolic and outer
mitochondrial membrane. Parkin is closely related to mitochon-
drial morphology, mitochondrial dynamics, mitochondrial quality
control, autophagy and other physiological activities in cells.
Under normal conditions, Parkin can maintain its structural
stability and inhibit its ubiquitin ligase activity due to its closed
conformation. In “closed”Parkin, C431 site, the catalytic center of
RING2 region is blocked, and the E2 ubiquitin-conjugating enzyme
binding site in the RING1 region is also blocked; both of which can
inhibit ubiquitin–thioester formation. In the PINK1/Parkin mito-
chondrial autophagy pathway, Parkin acts as a downstream
protein of PINK1 [25,26].
In 2004, PINK1 is also reported as another pathogenic gene of
Parkinson’s disease. Similar to PRKN, PINK1 is inherited in an
autosomal recessive manner [27]. PINK1 is a serine/threonine-
protein kinase composed of 581 amino acids. It is mainly involved
in oxidative stress in cells, releasing transmitters, autophagy,
apoptosis and other physiological activities. As a mitochondrial
kinase, PINK1 has been discovered as the primary detector of
mitochondrial damage. The N-terminal domain of PINK1 is
regarded as a mitochondrial target signal (MTS), which is followed
by a hydrophobic transmembrane domain (TM) as a terminal
transfer signal of the mitochondrial inner membrane. Resides 156
to 509 constitutes a serine/threonine kinase domain, and to the
end followed by C-terminal domain that serves as a retention
signal or others for the outer mitochondrial membrane (OMM).
(Fig. 1B)
Physiological PINK1 protein is continuously imported into
healthy mitochondria through MTS and the mitochondrial
membrane potential (ΔΨm) [28,29]. Subsequently, the
N-terminal MTS of PINK1 was cleaved by matrix processing
peptidase (MPP) including MPPαand MPPβwhile MTS reaching
matrix. Hydrophobic transmembrane helix within TM was cleaved
by (PINK1/PGAM5-associated rhomboid-like protease) PARL, the
catalytic ability of which is indispensable for Parkin recruitment in
mitophagy [30]. After this series processing, a 64 kDa full-length
forms of PINK became 52 kDa shorter forms, which are released
into the cytoplasm. The cytoplasmic PINK1 is rapidly degraded by
the Ubiquitin-Proteasome system (UPS) via the N end–rule
pathway. In damaged depolarized mitochondrial, PINK1 cannot
be transported to IMM to be cleaved, causing the accumulation of
PINK1 on OMM to form dimers. PINK1 dimers are activated by
phosphorylation at S228 and S402 [31], which is required for
recruiting Parkin to OMM to initiate mitophagy. Ser65-
phosphorylated ubiquitin (pUb) on PINK1 interacts with RING0,
RING1, and IBR regions of Parkin, which contributes to straighten-
ing a helix in the RING1 domain. Such conformational changes in
“opened”Parkin cause the UBL domain to be released from its
core structure (Fig. 1A, B) [32,33]. Subsequently, PINK1
phosphorylates Parkin on its UBL domain at S65, which promotes
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the interaction between UBL and RING0 to release the catalytic
RING2 domain that is mutually inhibited with RING0, thereby
further activating E3 ubiquitin ligase activity of Parkin [34,35]. The
subsequent Parkin activation and ubiquitination proteins of OMM
would provide extra substrates for PINK1, recruits more Parkin.
Such a positive cycle amplifies mitophagy signals [36] (Fig. 1C, D).
Mitochondrial fusion protein 2 (Mfn2) is localized on OMM and
phosphorylated at S442 and T111 by PINK1. The phosphorylated
Mfn2 act as a Parkin ubiquitination substrate to prevent impaired
mitochondria fusing with the healthy organelle [37]. Increasing
evidence implicates that the fusion machinery components on
damaged mitochondria were degraded by UPS. Parkin will
furtherly utilizes UPS to promote the division of damaged
mitochondria. The Miro GTPases also are substrates of Parkin,
and the damaged mitochondria were segregated from the
mitochondrial network by Miro to form an insulating membrane,
which is necessary for subsequent mitophagy.
The formation of isolation membrane by Lc3 largely depend on
two kinds of ubiquitin-like reaction. The carboxyl-terminal region
of pro-Lc3 form is cleaved by cysteine protease Atg4 to form Lc3-I
by exposing its glycine residues. The Lc3-II formation is dependent
on the binding PE to Lc3- I by Atg7 (an E1-like enzyme) and Atg3
(a specific E2-like ligase). The second ubiquitin-like reaction is
driven by Atg7 and Atg10 (an E2-like enzyme) to combine Atg12
with Atg5 to form Atg12~5 conjugates, which subsequently bind
to Atg16 dimer to form Atg12~5/16 complex. Finally, the
Atg12~5/16 complex promotes Atg8 bind with (PE) on the
expanding autophagosomal membrane via its E3-like activity. It
is thought that the isolation membranes recognize the damaged
mitochondria via the LIR motif (Lc3-interacting region), which will
wrap the mitochondria to form autophagosomes [38,39].
The process of linking isolation membrane and damaged
mitochondria is mediated by autophagic adaptor proteins,
including sequestosome1 (P62 /SQSTM1), a neighbor of Brca1
gene (NBR1), Nuclear dot protein 52 kDa (NDP52), Optineurin
(Optn), Tax1-binding protein 1(TAX1BP1) [40]. OMM proteins
degradation is induced by K63-linked ubiquitin that recruits
mitochondrial adaptor proteins and interacts with Lc3 anchored to
the autophagosome membrane. It has been claimed that p62
preferentially locates between adjacent mitochondria and pro-
motes the aggregation of damaged mitochondria through
polymerization [41,42]. But other scientists argued that OPTN
and NDP52 are the main adaptors in PINK1/ Parkin-mediated
mitophagy, rather than P62 and NBR1 [5,43,44]. NDP52 and OPTN
are mainly located in the OMM of damaged mitochondria. TBK1 is
activated and anchored to p62 via phosphorylation at S172 by
OPTN. TBK1 phosphorylates P62 at S403 and OPTN at S177 to
promote their binding with UB chains or Lc3. OPTN and NDP52
then initiate the autophagy process by recruiting autophagy-
related unc-51-like autophagy-activating kinase1 (ULK1), Double
FYVE-containing protein 1(DFCP1) and WD repeat domain
phosphoinositide-interacting protein 1(wipi-1). AMBRA1 as a
Parkin interactor can localize to OMM to enhance mitochondrial
clearance via binding to Lc3 through its LIR motif [45]. In addition,
AMBRA1 also function despite of Parkin and p62 [46]. TBC1D15/17
(TBC1 domain family member 15/17) also assists in elongating the
phagophore membrane, both of which can interact with OMM-
anchored mitochondrial fission protein 1 (Fis1) [47–49].
Finally, the damaged or senescent mitochondria are enclosed
into autophagosomes and then delivered to lysosomes for
degradation via fusing with lysosomes to form autolysosomes.
Mitophagy is a highly conserved process, which also requires
Fig. 1 PINK1/Parkin-mediated mitophagy. A Under normal conditions, the conformation of Parkin is closed, which contains an N-terminal
ubiquitin-like (UBL) domain, repressor element of Parkin (REP), four zinc-coordinating RING-like domains: RING0, RING1 (H302 to R305 motif),
in-between RING (IBR), and RING2. Once Parkin is activated, its conformation becomes open and the UBL domain is released. BDomain
features of PINK1: PINK1 possesses a Ser/Thr kinase domain (Kinase) close to C-terminal, followed by an α-helical transmembrane ( TM)
segment, and N-terminal mitochondrial targeting sequence (MTS). CPINK1 is continuously imported into healthy mitochondria then
degraded. DPositive cycles for PINK1 and Parkin Recruitment on damaged mitochondria.
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traffic GTPase Rab7 to regulate lysosomal transport [47,50]. PINK1
and Parkin are degraded after the lysosome-dependent degrada-
tion process (Fig. 2A).
Other ubiquitin-mediated mitophagy
Mitochondrial ubiquitination plays a central role in mitophagy.
Apart from Parkin, other E3 ubiquitin ligases are also involved in
the clearance of damaged mitochondria. Mitochondrial ubiquitin
ligase activator of mitochondrial E3 ubiquitin-protein ligase 1
(MUL1), an E3 ubiquitin ligase, contains two TM domains anchored
to OMM and contains a RING finger (RNF) domain facing the
cytoplasm [51]. It also has multiple common mitochondrial
substrates with Parkin, including Drp1, Mfn1, and Mfn2. Studies
on Drosophila and mice have shown that MUL1 is parallel to
PINK1/ Parkin-mediated mitophagy and can compensate for the
deletion of PINK1/ Parkin to rescue its phenotype [51]. Meanwhile,
studies in HeLa cells have suggested that MUL1 can directly
interact with GABARAP protein via the LIR-like motifs in the RING
finger domain [52].
MUL1 not only plays a ubiquitination role as E3 ubiquitin ligase
but also directly participates in mitophagy as a mitochondrial
receptor. ULK1 is a newly discovered substrate of MUL1. The
MUL1-regulated mitophagy process is independent of Parkin and
FUNDC1, but needs ATG5 and ULK1 [53]. There is growing
evidence revealed that mitophagy can still proceed without Parkin
[40]. It is widely believed that the main regulators of mitophagy
are tumor suppressors. Still, the presence of RBR E3 ubiquitin-
protein ligase 1 (ARIH1) challenges this view because it has been
shown to protect cancer cells from chemotherapy-induced death
[54]. ARIH1 is mainly expressed in pluripotent stem cells and
various cancer cells, especially lung cancer cells [55]. It belongs to
the RBR E3 ubiquitin ligase, which shows many similar structures
and substrates with Parkin. Both Parkin and ARIH1 act with the
ubiquitin-conjugating enzyme UBCH7 (UBE2L3), but they lack
intrinsic E3-independent reactivity with lysine. Parkin substrates
including Mfn2, NPD52 or OPTN are not required in ARIH1-
mediated mitophagy, suggesting that ARIH1 can target a group of
mitochondrial proteins different from Parkin substrates to induce
mitophagy (Fig. 2B). Mitochondrial-derived vesicle (MDV) is
discrete vesicles from OMM or IMM, which are targeted to
peroxisomes, lysosomes, endosomes, and phagosomes. MDV buds
off from mitochondria and incorporates specific mitochondrial
cargo especially oxidized cargo to the late endosome. So far, MDV
has only been found in some cell types, such as hepatic cells and
cardiac cells. An increased number of MDVs can be found in H9C2
cardiac myoblasts in response to stress [56]. MDVs in the healthy
heart and acute stress heart can trigger mitophagy. MDVs are
involved in the clearance of mildly damaged mitochondria, and its
Fig. 2 Pathways of mitophagy. A Parkin-dependent mitophagy: While mitochondria became damaged (pink), PINK1 is accumulated on the
OMM, promoting Parkin recruitment to ubiquitinate several outer membrane components. Phosphorylated Poly-Ub chains on mitochondrial
proteins modify other proteins and serve as an ‘eat me’signal for the autophagic machinery. TBK1 interacts with Ub chains by
phosphorylating OPTN, which promotes mitochondrial clearance. The PINK1-Parkin pathway modulates mitochondrial dynamics and inhibits
damaged mitochondria entering the mitochondrial network by targeting Mfn and Miro for proteasomal degradation. BOther pathways of
Parkin-independent mitophagy: (1) Bnip3, Bnip3L/Nix, FUNDC1 and other receptor-mediated mitophagy; (2) Lipid-mediated mitophagy; (3) E3
ubiquitin ligases or ubiquitin-mediated mitophagy; (4) Mitophagy and mitochondrial dynamics. Mitophagy receptors such as BNIP3 and
FUNDC1 promote the fission of damaged organelles through the disassembly and release of OPA1 and the recruitment of DRP1.
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formation depends on PINK1 and Parkin but is independent of
Drp1 [57]. Whether MDVs are present in every cell type remains to
be explored.
BNIP3 and BNIP3L/Nix-mediated mitophagy
Bcl-2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and
BNIP3-like (BNIP3L, also called Nix) are homologous members of
Bcl-2 family proteins [58]. They are constitutively expressed on the
MOM and are known as an apoptotic protein at first. Both play a
role as mitophagy receptors to regulate PRKN/PARK2-independent
mitophagy. BNIP3L has two LIR domains, a minimal essential
region (MER), a TM domain, a BCL2 homolog 3 domain (BH3)
inducing apoptosis. The C-terminal LIR domain was considered as
an essential component to initiate mitophagy and interact with
Lc3 due to phosphorylation. Recent studies in HeLa cells
demonstrate that BNIP3L/Nix can increase its interaction with
Lc3/GABARAP by phosphorylation at S34 and 35 adjacent to the
LIR motif [59]. In particular, phosphorylation of the LIR domain can
promote mitophagy and deletion of LIR has a reverse result.
However, some scientists argued that is MER domain rather than
LIR domain closed to the C-terminus is essential for BNIP3L
-mediated mitophagy [60]. The deletion of the MER domain in
BNIP3L failed to induce mitophagy. TM domain facilitates BNIP3L
OMM location and promotes BNIP3L homodimerization through
phosphorylation, which is essential for mitophagic activity [13].
Currently, the understanding of BNIP3L regulated mitophagy
remains limited. BNIP3L helps BECN release from the Bcl-BECN
complex, which promotes autophagosome formation. Besides,
BNIP3 and BNIP3L /Nix can bind to the Rheb proteins to inhibit
mTOR activation through their N-terminal and enhance mito-
phagy. BNIP3L relies on LIR to directly interact with the Atg8
family, such as Lc3, after phosphorylation and ubiquitination
under hypoxia (Fig. 2B) [61]. When BNIP3L is accumulated to a
certain level on OMM, it will lead to mitochondrial membrane
potential loss and thus initiate mitophagy [62,63], but this causal
relationship has been questioned. Some scientists argued that
different from Parkin, the translocation of BNIP3 is independent of
membrane potential loss. It has been found that the membrane
potential still decreased when programmed mitophagy occurred
in cardiac Progenitor cells (CPS) lacking BNIP3 [64]. Other scientists
demonstrated that hypoxia does not seem to be the only
condition that triggers BNIP3L-mediated mitophagy. In hypoxic
colon carcinoma, hypoxia-induced mitophagy is independent of
BNIP3L but activated by AMPK [65]. The relationship between
hypoxia and BNIP3-mediated mitophagy needs further research.
The mechanism of BNIP3L-mediated mitophagy is much con-
troversial and needs to be explored in more detail for well
understood. What’s more, specific diseases caused by BNIP3L
mutations have also been rarely reported.
FUNDC1-mediated mitophagy
FUNDC1 (FUN14 domain-containing protein 1) is another widely
studied mitophagy receptor localized to OMM, which was first
proposed in 2012 by Chen’slab[66]. It contains three transmem-
brane domains consisting of three conserved α-helical stretches, a
LIR motif closed to N-terminus in the mitochondrial cytoplasmic
face and a C-terminus in the intermembrane space. Similar to
BNIP3 and BNIP3L/Nix, FUNDC1 also interacts with Lc3-II through
phosphorylation and dephosphorylation of LIR. FUNDC1 is
dependent on hypoxia-induced dephosphorylation to promote
mitophagy. In contrast, other proteins containing LIR motifs need
phosphorylation to increase Lc3 binding affinity. What’more,
FUNDC1 also can interact with other Lc3 paralogues such as
GABARAP, but the binding affinity between them is not as high as
with Lc3-II. Under normal conditions, LIR activity of FUNDC1 is
inhibited due to phosphorylation at tyrosine 18 (Tyr18) and Ser13
by Src kinase and Casein kinase 2 (CK2 kinase), respectively [66,67].
Phosphoglycerate mutase family Member 5 (PGAM5) can
dephosphorylate FUNDC1 at Ser13 under hypoxia or mitochondrial
uncoupling. It has been suggested that phosphorylation of Tyr18
played a key role and phosphorylation of Ser13 plays an auxiliary
role in FUNDC1-mediated mitophagy [68]. Phosphorylation of
Tyr18 significantly reduced the binding affinity of LIR to Lc3, but
phosphorylation of Ser13 had only a slight effect. UNC-51, like
autophagy-activating kinase 1(ULK1), can also phosphorylate
FUNDC1 at Ser17 on the LIR motif in HeLa cells and promote the
interaction between FUNDC1 and Lc3 [69]. Intriguingly, Ser17
phosphorylation has a stimulative effect compared with Ser13 and
Tyr18 phosphorylation on mitophagy in response to hypoxia or
FCCP treatment. Unlike Parkin, ULK1 migrates to damaged
mitochondria in response to hypoxia. The substrates of ULK1 and
subcellular location remain unknown and need more research.
On the other hand, the E3 ubiquitin-ligase membrane-associated
RING finger protein 5 (MARCH5) has been found as a supervisor to
avoid excessive and inappropriate clearance of mitochondria by
FUNDC1 degradation, which is a new substrate for MARCH5 [70].
Similar to Parkin, both of them were able to ubiquitinate OMM
proteins. Nevertheless, the purpose of Parkin is recruit p62 and the
isolation membrane, while MARCH5 specifically targets FUNDC1 for
degradation to fine-tune mitophagy. It has been reported that
MARCH5 ubiquitinated Lys119 of FUNDC1 for proteasome
degradation, which was independent of FUNDC1 phosphorylation.
In addition, MARCH5 knockdown significantly inhibited FUNDC1
degradation and enhanced mitophagy signals [71]. It has also been
suggested that FUNDC1 regulates mitochondrial fusion and fission
by interacting with optic atrophy protein 1(OPA1) and Drp1, which
is a critical quality control event upstream of mitophagy (Fig. 2B)
[72]. Other mitochondrial fusion-related protein such as Mfn1,
Mfn2, and fission-related protein Fis1, MiD49 can also be
ubiquitylated by MARCH5 [73]. These results imply that the role
of E3 ubiquitin ligase is also varied in mitophagy.
Lipid-mediated mitophagy
Some OMM-localized lipids, such as ceramide and cardiolipin,
induce mitophagy via their LIR motif. Ceramide is a bioactive
sphingolipid synthesized by ceramide synthase (CerS), including
six isoforms (CerS1- CerS 6). The production of ceramides in the
hepatic mitochondria relies on mitochondrial thioesterase and
neutral ceramidase (NCDase) [74]. Under ER stress, ceramides are
translocated from ER to OMM in preparation for mitophagy [75].
When they accumulate in cells to a certain extent, accompanied
by accumulation of Beclin-1 and inhibition of Akt phosphorylation,
cell death will happen. Ceramides located on the OMM interact
with the ceramide-binding domain of Lc3-II to selectively remove
damaged mitochondria. It has been reported that Lc3-II lipidation
is necessary for ceramide binding during the mitophagy process
[76]. C18-ceramides are preferentially synthesized from 18 carbon
(C18) fatty acids catalyzed by CerS1. Overexpression of CerS1 or
adding exogenous C18-ceramide can enhance ceramide-
mediated mitophagy depending on Drp1 [77]. Drp1 determines
OMM localization of ceramide to target autophagolysosomes.
Drp1 knockdown disrupts ceramide localization in OMM and
autophagolysosomes recruitment in tumor cells [78]. Mitochon-
drial fission is essential for ceramide-induced mitophagy due to
Drp1 being the crucial regulator of mitochondrial fission.
Moreover, ceramides, along with activated BAX, form a
ceramide channel in the phospholipid bilayer to release cyto-
chrome c to activate apoptosis. Cardiolipin, as a negatively
charged phospholipid similar to ceramide, is synthesized by
cardiolipin synthase (CRD1). It is synthesized by mitochondria and
makes up about 25 mol% of mitochondrial membrane lipids,
which is also important in mitochondrial cristae formation and
other mitochondrial functions and mitochondrial fusion [79]. The
distribution of cardiolipin is highly asymmetric between IMM and
OMM in healthy mitochondria, most of which about 96.5 mol% are
confined to the IMM [80]. The cardiolipin binding site in Lc3 is
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believed to be at the N-terminal α-helices through computational
modeling. Under normal conditions, cardiolipin locates at IMM
and interacts with OPA1 to promote IMM protein fusion and
regulate mitochondrial networks. Cardiac phospholipid cardiolipin
is transferred to OMM via phospholipid scramblase-3 (PLS3) to
bind to Lc3-II under stress (Fig. 2B) [81].
PLS3 is a newly recognized protein is responsible for
phospholipid translocation between bilayer structures. Overex-
pressing PLS3 in HEK293 cells results in cardiolipin accumulation
in OMM and enhanced intracellular ATP and mitochondrial
respiration [81]. Nucleoside diphosphate kinase D (NDPK-D) is a
hexameric intermembrane space protein that also assists
cardiolipin-translocating. Knockdown endogenous NDPK-D
reduced cardiolipin on OMM inducef by CCCP in Hela cells (Fig.
2B) [80].
Mitophagy and mitochondrial dynamics
Mitochondria are highly active organelles undergoing continuous
movement, fission, and fusion activity and response to environ-
mental stimuli. Before being selectively eliminated by mitopahgy,
a damaged mitochondrial will be asymmetrically divided into a
healthy daughter and an impaired organelle via fission proteins.
The mitochondrial fission proteins are constitutive of Drp1, FIS1,
MFF proteins. The impaired organelle is degraded via mitophagy.
The healthy daughter can fuse with other mitochondria to enable
content mixing and maintain mitochondrial fusion proteins’
genetic integrity. Severely damaged mitochondria will be replaced
by fresh mitochondria generated by mitochondrial biogenesis.
Mitochondrial fusion consists of the outer membrane fusion and
the inner membrane fusion induced by mitofusins (Mfn1, Mfn2)
and OPA1. Such a dynamic process is termed mitochondrial
dynamics (Fig. 3).
Mitochondrial dynamics and mitophagy are two key processes
to maintain mitochondrial homeostasis and normal physiological
function. Ample evidence proved that inhibiting mitochondrial
fission or enhancing mitochondrial fusion will weaken mitophagy
[82,83].
Many studies have shown that mitochondrial fission is a
prerequisite for mitophagy [82,84,85]. Downregulation of Drp1
via transfection of Ad-shDrp1 can lead to a significant decrease in
Lc3 protein in cardiomyocytes [85,86]. Lc3 protein is a direct
indicator of evaluating the degree of autophagy. Hence,
insufficient autophagy due to downregulation of Lc3 leads to
the accumulation of dysfunctional mitochondria in cardiomyo-
cytes. BNIP3 is another mitophagy-related protein that induces
autophagy. Overexpression of BNIP3 leads to an increase in Drp1-
mediated mitochondrial division [84]. FUNDC1 can regulate
mitochondrial fission by interacting with OPA1. Overexpression
of FUNDC1 will promote mitochondrial fission and mitophagy,
while knocking down FUNDC1 will induce mitochondrial fusion
[72]. Mitochondrial fission and fusion counterbalance with each
other under normal mitochondrial dynamic network. Some other
studies have demonstrated that regulating mitochondrial fusion
will have a similar effect on mitophagy like mitochondrial fission.
For example, the reduction in fusion caused by OPA1 knockout
also helps the elimination of dysfunctional mitochondria [87,88].
However, conditional knockout of Mfn1/2 in mouse cardiomyo-
cytes lead to an inversed result where dysfunctional mitochondria
increased and cardiac hypertrophy happened due to impaired
mitochondrial autophagy [89–91]. Thus, mitochondrial dynamics is
an important part of mitophagy. However, mitochondrial
dynamics also can regulate mitophagy.
Mitophagy and mitochondrial biogenesis
Mitophagy and mitochondrial biogenesis are two interrelated and
interactive processes, which have exactly the reverse effect in
regulate mitochondrial number and content. Excessive mitophagy
or elevated mitochondrial biogenesis will break up the delicate
balance of mitochondrial homeostasis, inducing (mitophagic) cell
death or necrosis eventually [92].
AMPK and CaMK are two compounds which can regulate
mitophagy and directly activate PGC-1αto improve mitochondrial
biogenesis. In addition, PGC-1αcan also be stimulated increased
NAD+induced by AMPK. The effects of these proteins on PGC-1α
are post-translational modifications. It has been reported that NAD
+can activate SIRT1 to deacetylates PGC-1αin response to the
need of mitochondrial metabolism. As a bridge between
mitophagy and mitochondrial biogenesis, AMPK also phosphor-
ylates ULK1/2 to initiate the formation of autophagic vacuole.
CaMK enzymes are also involved in mitophagy by stimulating
AMPK when Ca2+increased. Overexpression SIRT1 can stimulate
the formation of autophagosomes [93]. Lysosomes act as final
Fig. 3 Consequences of mitochondrial dynamics. Damaged or senescent mitochondria divide into one healthy organelle that fuses with
other healthy mitochondria to regenerate the mitochondrial pool and one severely depolarized organelle that is timely eliminated by
autophagosomal engulfment. Thereby new mitochondria are generated by biogenesis to replace degraded mitochondria parts. The balance
offset between mitochondrial fusion and fission leads to either hyperelongated or hyper fragmented mitochondria.
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“incinerators stoker”to degrade mitochondria during mitochon-
drial clearance. It has been proved that PGC1a directly interacted
with TFEB to enhance autophagy-lysosome pathway [94]. More-
over, there is a positive cycle between TFEB and PGC-1α
interaction via CLEAR (coordinated lysosomal expression and
regulation) motif to keep mitochondrial number and content in
stable [94,95]. CLEAR motif exists in mitochondrial genes of
human or mouse, it also exists in other genes involved in
autophagy [96]. It has been demonstrated that TFEB was a
therapeutic target in kidney, muscle, heart diseases by regulating
mitochondrial biogenesis via PGC-1α[97]. Moreover, PGC-1α
activator (ZLN005) alleviated kidney injury by accelerating
mitochondrial clearance in cisplatin-induced AKI mice, where the
co-localization between LC3 and damaged mitochondria was
increased [98,99]. PGC-1αalso show a protective effect against
myocardial ischaemia-reperfusion injury, heart failure, but the
drug for cardiac PGC-1αagonist is still immature [100,101]. It has
been reported that mice lacking cardiac PGC-1αwere more easily
developed heart failure than mice lacking systemic PGC-1α, but
the reasons for this are unclear [100]. A breakthrough may come if
mitophagy is taken into account.
The major mitophagic component-Parkin can also activate PGC-
1αby inhibit Parkin-interacting substrate (PARIS), which is tended
to bind to PGC-1αpromotor to repress its expression in brain.
Whether the system of Parkin-PARIS-regulated PGC-1αexists in
heart need to be furtherly investigated. It is worth considering and
studying whether PARIS is involved in the inconsistent levels of
autophagy in different stage of ischemia reperfusion. Both mTOR
and sirt1 are like balance weights on a scale, able to modify the
regulators of mitophagy and mitochondrial biogenesis at either
end of the scale in their own way. Proteins related with mitophagy
(Atg families including LC3) or mitochondrial biogenesis (PGC-1α)
can be deacetylated to induce mitophagy or mitochondrial
biogenesis. But not all proteins interact with PGC-1αin a positive
feedback loop as TFEB does. It’s well known that inhibiting mTOR
will activate autophagy or mitophagy, but it has been reported
that the action of mTOR will improve mitochondrial biogenesis
accompanied with increased lysosomal biogenesis with repressed
autophagy [102]. That means the regulation of mitophagy and
mitochondrial biogenesis by mTOR occurs antagonistically.
MITOPHAGY AND CARDIOVASCULAR DISEASE
Atherosclerosis
Atherosclerosis (AS) is, a chronic inflammatory disease of the large
arteries initiated by lipid entry. In industrial countries, athero-
sclerosis is a primary disease among causes of heart disease and
stroke. Endothelial adhesion molecules are highly expressed in
arterial endothelial cells promoting differentiation of monocytes
into macrophages, which subsequently turn into foam cells with
lipid accumulation [103]. The pathogenesis of atherosclerosis is
characterized by the accumulation of these macrophages, lipids,
cholesterol, the migration and proliferation of vascular smooth
muscle cells (VSMCs) [104]. Autophagy or mitophagy, as a cleaner,
plays an indispensable role in removing this “accumulated waste”
mentioned above. The role of autophagy or mitophagy does not
have a single effect on these cells involved in atherosclerosis.
Hypoactive autophagy will contribute to increased plaque
formation in VSMCs, and increased ROS levels from damaged
mitochondria in all cell types, on the contrary, hyperactive
autophagy will generate autophagy-induced cytotoxicity in
macrophage and VSMCs [105]. Atherosclerosis begins in the fetal
aorta and slowly spreads to the coronary arteries and cerebral
arteries in turn. During this complex process, plaque is formed
[106].
Plaque is also termed fatty deposit, and its formation is related
to matrix metabolism, calcification, and inflammation. Many
scientists argue that plaques build up when the artery’s intima
becomes damaged. Accumulated plaques narrow the arterial
lumen and reduce blood flow, which leads to the occurrence and
development of atherosclerosis [107]. Plaque rupture is the
highest fatal cause in atherosclerosis, which is related with
autophagy and mitophagy [108]. There were many articles
indicated that it was insufficient autophagy and mitophagy that
triggered inflammasome activation and eventually lead to the
accumulation of plaque [109,110]. Enhancing autophagy may
help to delay cell death or ensure that the dysfunctional cells are
efficiently cleared. Tacrolimus, as a mTOR inhibitor, helped to
macrophage content in plaques, which was related with the
unstable plaque [111]. Moreover, melatonin also shows a positive
function in stabilizing atherosclerotic plaques by activating SIRT3/
FOXO3a/Parkin-dependent mitophagy pathway and reducing
inflammation [112]. Yu et al. put forward that autophagy and
mitophagy was upregulated, on the contrary, mitochondrial
respiration and mtDNA was reduced in human atherosclerotic
plaques compared with normal arteries, which indicating impaired
mitochondrial turnover [113]. In addition, Swaminathan et al.
suggested that the autophagy marked by Lc3 was profoundly
decreased in carotid plaques of symptomatic patient compared
with asymptomatic patients [114]. The stabilization of plaque was
weakened by impaired mitophagy. Their findings are not contra-
dictory because of their different study background. The impaired
mitochondrial renewal and turnover caused by imbalance
between mitophagy and mitochondrial biogenesis will contribute
to the ROS production.
ROS and inflammation play a vital role in the development of
atherosclerosis. There is evidence that overproduction of ROS can
damage mitochondrial DNA and lipid, which directly contributes
to atherosclerosis and increase inflammation [115]. Excessive ROS
also leads to endothelial dysfunction, with proliferation and
apoptosis of VSMCs and macrophages, leading to atherosclerotic
progression and possible plaque rupture. Worsely, excessive ROS
will in turn damage other normal mitochondria, thus a negative
cycle is created called ROS-induced ROS release (RIRR). Mitochon-
drial DNA damage with resultant mitochondrial dysfunction has
been associated with the degree of atherosclerosis in early human
atherosclerotic specimens and apoE
−/−
mice with reduced LDL
absorption from blood due to lacking apolipoprotein E [116].
Manganese superoxide dismutase (Mn-SOD) deficiency can
accelerate mtDNA damage and enhance atherosclerosis pheno-
type in apoE
−/−
mice [117]. Melatonin was found as a potential
therapeutic drug to reduce inflammation and ROS by inhibiting
JNK/Mff signaling and sustained mitochondrial homeostasis,
thereby protecting endothelial cells against ox-LDL-induced
damage [118].
Mitophagy and mitochondrial dynamics are cornerstones of
mitochondrial quality control by removing damaged mitochondria
to ensure normal mitochondrial function and cell homeostasis,
which is disturbed in the pathogenesis of vascular diseases. The
increased mitochondrial fragmentation and FIS1 expression have
been found in venous endothelial cells of patients with type 2
diabetes [119]. What’s more, the expressions of FIS1 and Drp1 are
also increased in human aortic endothelial cells cultured in high
glucose medium. Fragmented mitochondria resulted from a 50%
decrease of Mfn2 protein, also existed in synthetic VSMCs induced
by platelet-derived growth factor-BB (PDGF) [120,121]. Mitochon-
drial division inhibitor 1 (mdivi-1) is considered as an effective
drug to protect against synthetic VSMCs by inhibiting mitochon-
dria fission and attenuating cell proliferation [122]. On the other
hand, Melatonin regulates mitophagy. It attenuates leukocyte
hormone-1 β(IL-1β) secretion through SIRT3/FOXO3a/ Parkin-
dependent signaling pathways to decrease the inflammatory
factors secretion, preventing atherosclerotic plaques rupture [112].
Still, the protective effects were partially abolished by the
autophagy inhibitor-3-MA. Atg7 deletion in VSMCs or mouse
models has accelerated atherosclerosis development due to
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dysfunctional autophagy [123]. Defective autophagy in athero-
sclerosis is mainly due to dysfunctional lysosomes. Activating
autophagy by elevating the autophagosomal marker Lc3-II can
relieve asymptomatic patients with carotid plaques. Although
enhancing autophagy and mitophagy show a protective effect on
cardiac function, everolimus (mTOR inhibitor) is currently
approved to reduce graft vasculopathy in heart transplant
patients, not in atherosclerotic patients [124]. Interestingly, it has
been reported that sirolimus and everolimus-eluting coronary
artery stents show a protection in inhibition VSMCs proliferation
[125]. We hope more analogs of rapamycin will be found to treat
against atherosclerosis effectively and prevent coronary acute
syndrome.
Myocardial ischemia-reperfusion injury and myocardial
infarction
Ischemic heart disease (IHD) is a major cause of death and
disability worldwide, with a clinical manifestation of myocardial
infarction and ischemic cardiomyopathy. During myocardial
infarction and I/R, temporary or permanent occlusion of coronary
arteries can cause multi-component consequences including
insufficient oxygen and nutrient supply, intracellular acidification,
mitochondrial Ca
2+
overload, abnormal metabolism, the mito-
chondrial permeability transition pore complex (mPTP) opening.
The mentioned above further resulted in mitochondrial dysfunc-
tion and increased ROS, ultimately leading to myocardial cell
death and myocardial injury. Most of this myocardial damage is
irreversible and it is difficult to achieve complete or partial
recovery.
Mitochondrial function plays a decisive role in the sensitivity
and recovery of I/R and MI injury. Excessive mitochondrial fission
caused by I/R injury induces extrinsic apoptotic cell death; this
process may contribute to the pathogenesis of postischemic cell
death [126]. Inhibition of excessive mitochondrial fission is
considered to be potential cardioprotection of I/R injury. For
example, Drp1K38A shows a protective mitochondrial uncoupling
effect against I/R in Drp1K38A-treated cardiomyocytes [127].
Myocardial infarct size was also significantly decreased in
Drp1K38A-treated rats compared with controls. Mdivi-1 was
considered as a beneficial drug for I/R injury by reducing infarct
size, increasing the proportion of elongated interfibrillar mito-
chondria and delaying mPTP opening in the ischemic adult
murine heart [128]. Another mitochondrial inhibitor, P110, has
been shown to reduce myocardial infarction size during reperfu-
sion and protect against poor left ventricular remodeling after
myocardial infarction in adult rats [129]. Inhibiting Fis1-mediated
fission in cardiomyocytes by miR-484 has also shown a reduced
infarct size in a mouse myocardial infarction model [130].
Autophagy plays a pivotal role in “housekeeping”for disposal of
damaged or obsolete organelles such as mitochondria (mito-
phagy) and it is markedly upregulated during I/R. It should be
pointed out that regulating autophagy may play a different role in
different stages of I/R. For example, it has been suggested that the
autophagy pathway may be protective by maintaining basal
metabolic needs when the heart experiences an energy loss, such
as ischemia, which is in contrast to reperfusion [131,132]. In the
ischemic heart, AMPK was activated to induce cardiomyocyte
autophagy to remove malfunctioning mitochondria that other-
wise lead to oxidative stress (Fig. 4). Both Park2
−/−
mice and
Pgam5
−/−
mice show mitophagy inhibition after MI injury, which
leads to increased heart infarction area, aggravated heart injury,
and reduced cellular survival rate [2]. Unlike PINK1
−/−
mice with
increasingly susceptible to I/R, overexpression of PINK1 in cardiac
cells protects against death by delaying the initiation of mPTP
opening [4]. However, just one-sided seems to be the case since
inhibition of autophagy actually alleviates heart injury in other
situations. Inhibition of Beclin1 has been shown a protective effect
for cardiomyocytes by preventing cell death in vivo [133]. What’s
more, Becn1
+/−
mice are more resistant to heart injury during
reperfusion than WT mice. Similarly, Bnip3
−/−
mice also show
reduced myocardial injury and maintenance of cardiac function
during ischemia/reperfusion [134]. Studies on Ulk1/Rab9/Rip1/
Drp1 mitophagy mediated pathway have also shown a beneficial
role in protecting the heart against ischemia by maintaining
healthy mitochondria [135]. Autophagy was also induced by
repetitive myocardial ischemia in chronically instrumented pigs,
and cardiac function can also be restored after the coronary
arteries return to normal flow, suggesting that autophagy may be
vital for the survival of hibernating myocardia [136,137].
It has been reported that the calcified aortic valve stenosis
(CAVS) contributed to the myocardial infarction [138]. The trend of
autophagy and mitophagy in CAVS was reverse in the articles of
Deng et al. and Carracedo et al., which is because of their different
compared group to the CAVS [139,140]. Compared with the
normal aortic valve referred by Deng et al., the autophagy level in
CAVS was reduced, which was increased while compared with
aortic regurgitation referred by Carracedo et al. The different result
between them may indicate that the severity of the disease may
have a different effect on autophagy. Marciano et al. confirmed
this suppose in 2021. They argued that calcification aroused
Fig. 4 I/R induces autophagic degradation of mitochondria in cardiac. White arrow: Swollen mitochondria induced by the mPTP opening of
the depolarized mitochondria that triggered by reperfusion after glucose deprivation. Red arrow: Depolarized mitochondria have been
eliminated by mitophagy.
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autophagic cell death, which was proved by the inconsistency
between the decreased ATP production caused by impaired
mitochondrial turnover and the enhancement of autophagy and
mitophagy [141]. This result was also supported by Somers et al. in
2006, who revealed that it was autophagy in calcific aortic valve
stenosis not apoptosis lead to cell death proved by immunohis-
tochemical data [142]. Mitochondrial biogenesis also influences
mitochondrial renewal and turnover. PGC-1αas the mitochondrial
biogenesis regulator also has been proved to be downregulated in
the failing heart [143].
Cardiomyopathy
Cardiomyopathy (CM), is a group of myocardial diseases
characterized by abnormal structure and function of the
myocardium. Cardiovascular complications are the major risk
factor of morbidity and mortality in T2DM. The main factors
promoting the development of CM include hyperglycemia, insulin
resistance of the whole body and heart, and the increase of free
fatty acid (FFA) level, myocardial inflammation, oxidative stress,
myocardial remodeling and fibrosis. In clinical common cardio-
myopathy, autophagy and mitophagy have been frequently
reported in the following kinds of diseases: dilated cardiomyo-
pathy (DCM); hypertrophic cardiomyopathy (HCM), desmin-related
cardiomyopathy (DRM) and restrictive cardiomyopathy (RCM).
More and more evidence indicated that the alteration of
autophagy and mitophagy is closely related to cardiomyopathy
progression.
Beclin1 and Atg5 are two autophagy-related (Atg) proteins,
however they show a different result between Becn1
+/−
mice with
alleviative myocardial remodeling and Atg5
+/−
mice with
aggravating myocardial hypertrophy in pathological myocardial
remodeling [144,145]. It’s known that Atg5 and Beclin1 both are
related with autophagosome formation, but Beclin1 can induce
autophagy independent of Atg5 indicating a complicated function
in cardiomyopathy. It has been reported Beclin knockdown
stimulated Rab-9 as a alternative autophagy and mitophagy with
increased BNIP3, but Parkin was downregulated [146]. What’more,
mitochondrial biogenesis was also rescued by Beclin knockdown
to relieve cardiomyopathy induced by high-fat diet. These results
suggested that Beclin1 knockout did not inhibit autophagy and
mitophagy, but instead activated other autophagy and BNIP3-
mediated mitophagy, thus protecting heart function via enhance
mitochondrial turnover. But the role of autophagy is not always an
angel. It has been reported that decreased autophagy is an
adaptive and protective result to protect heart function in type1
diabetes, while overexpressed Beclin1 contributed to aggravated
cardiac injury [147]. Both genes are upstream of autophagy, Beclin
and ATG5 knockout both reduced autophagy, but why were the
results different? Unfortunately, the reason has not been found,
and more research need to further explore. The pathobiology of
DCM also exhibited partially heritability and reduced autophagy
level. There were about 25% titin mutations and 6% Lamin A/C
(LMNA) mutations in DCM [148]. Whether it was the increased
formation of autophagosome or decreased (mitophagy) clearance
contributed to Lc3 II increase has not been elaborated. The
inhibition of cardiac autophagy or mitophagy in T1DM and T2DM
may also lead to diabetic cardiomyopathy. Studies have shown
that PINK1 and Parkin protein levels were significantly lower in
T1DM hearts than in healthy hearts, consistent with reduced
cardiac mitophagy [149].
Transferrin receptor (Tfrc) is responsible for iron uptake, which is
important for cardiac function. Cardiac iron deficit by Tfrc deletion
leads to severely ineffective mitophagy and develop lethal CM,
indicating that mitophagy may be an effective therapeutic target
for Tfrc-dependent CM [150]. Mitochondrial DNA (mtDNA) is
generally degraded by DNase II in the mitophagy-lysosome
system. Incomplete mtDNA degradation can stimulate cardiac
inflammation, leading to heart failure. It has been proved that
DNase II deficiency can lead to severe myocarditis and DCM, and
result in premature death under pressure overload conditions
[151]. Lc3 overexpression and enhancing Parkin-mediated mito-
phagy by inhibiting MST1 phosphorylation can protect against CM
[152].
The autophagy level labeled by Lc3 significantly increased in
HCM induced by Mybpc3 mutations and diabetic CM (type 2
diabetes). Rapamycin treated in Mybpc3 mice increased autop-
hagy flux and reduced hypertrophy. It’s not just genetic mutations
that can lead to cardiac hypertrophy, nurture or postnatal
conditions are also included. It has been reported that the
expression of PINK1 and Parkin was reduced in TAC-induced HCM,
which indicating a downregulation of mitophagy. Moreover, it has
been proved that loss of PINK1 lead to cardiac hypertrophy and
mitochondrial dysfunction in mice at 2 months of age [153]. Xiong
et al. demonstrated that accelerated PINK1-mediated mitophagy
played a protective role in angiotensin-induced cardiac hyper-
trophy [154].
Tacrolimus treatment was also beneficial for attenuating
myocardium damage and mitochondria function by preserving
mitochondrial transmembrane potential [155]. However, Noda
et al. has reported that tacrolimus triggered HCM in a patient with
dermatomyositis [156]. The different result of tacrolimus treatment
may be associated with inflammatory reaction and need to be
further explored. Knocking out Parkin aggravated cardiac lipo-
toxicity and mitochondrial dysfunction under high-fat diet, which
was reversed by Tat-Beclin1-activated mitophagy [157]. In
addition, ALCAT1 show an abnormal increase in hypertrophic
cardiomyopathy. Inhibiting ALCAT1 expression reduced ventricu-
lar fibrosis and mitigated HCM by increasing mitophagy level
labeled with upregulation of PINK1, Lc3, and downregulation of
p62 [158]. In conclusion, enhanced autophagy and mitophagy has
shown a protective effect in cardiac hypertrophy, whether due to
genetic mutations or other causes.
DRM is a kind of genetic cardiomyopathy caused by desmin- or
αB-crystallin- mutations [159]. It has been reported that there was
a significant increase of the desmin and insoluble CryAB levels in
hearts of patients, which was also found in the end-stage ischemic
HF [160]. In the early days, it was thought that the aberrant protein
aggregation in DRM would inhibit autophagy, but in 2019, Pan
et al. demonstrated that the level autophagy varies with TFEB
during disease progression [161]. As the symptoms of the cardiac
phenotypes become more apparent, the regulation of autophagy
declined more and more, which changed from positive to
negative when impaired TFEB signaling occurred as an inflection
point. Knocking out Beclin1 further increasing interstitial fibrosis
and accumulation of polyubiquitinated proteins in CryAB
R120G
mice [162]. In contrast, accelerated autophagy via overexpressing
Atg7, treating with SAHA, or overexpressing UBC9 has enhanced
elimination of aggregated proteins in heart, and restored heart
functions in CryAB
R120G
mice [163–165]. Although mitochondria
function was abnormal or the content of mitochondria is low in
DRM, unfortunately, there are few studies on mitophagy in DRM
[159]. Similar with DRM, dysfunctional autophagy and mitophagy
has been associated with cardiomyopathy in the patient with a
BAG3-Pro209Leu mutation and mice with abnormal BAG3
[166,167].
Myocardial hypertrophy and heart failure
Myocardial hypertrophy is a typical early adaptive response of the
heart to increased pressure and mechanical stress. The direct
cause of myocardial hypertrophy is the enlargement of cardio-
myocyte size. Not all such increases in the cardiomyocyte area are
harmful. Compensatory hypertrophy is a beneficial case in
response to stress. The increased cardiomyocyte sarcomeres and
cardiac mass were adaptive to normalize ventricular wall stress.
However, chronic pressure will lead to cardiomyocyte cell death
and irreversible damage to the heart and finally develop into
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Heart Failure (HF). The incidence of HF is related to age and it is as
high as 20–30% in people aged 70–80 [168]. With age, the injured
mitochondria cannot be cleared in time, caused by decreased
autophagy and mitophagy in the heart, which leads to excessive
ROS production and oxidative damage of various mitochondrial
proteins.
Increased autophagy and mitophagy in multiple HF models was
considered a protective response in cardiomyocytes [3].
Mitochondria-derived ROS activated AMPK, and then autophagy
was activated. In addition, the cAMP/PKA and MAPK/ERK1/
2 signaling pathways can also activate autophagy. Increased
proteostasis caused by enhanced autophagy can alleviate cardiac
hypertrophy and delay HF progression. Increased mitophagy is
adaptive to the nutrient and energy requirements to pressure
overload. Cytosolic p53 impairs mitophagy by binding with Parkin
to disturb subsequent clearance of damaged mitochondria and
further facilitates cardiac dysfunction and HF [169]. Mfn2
deficiency impeded Parkin-mediated mitophagy and contractility,
which finally lead to myocardial hypertrophy and HF [170]. At the
end-stage human HF, the expression level of PINK1 protein
decreased significantly, indicating weakened mitophagy [171].
However, some scientists hold the opposite view, they argue that
impaired mitophagy will contribute to negative pathological
remodeling of myocardium. The activation of mitophagy leads
to the transformation of the heart from adaptive compensatory
hypertrophy to myocardial fibrosis, then develops into HF. There
were only mild systolic dysfunction and no significant change in
heart size of Becn1
+/−
mice and cardiac-specific BECLIN 1
knockout mice. However, cardiac-specific overexpression of
Beclin-1 resulted in converse effects [144]. Upregulated mitophagy
under pressure overload aggravates myocardial hypertrophy and
systolic dysfunction. In addition, cardiac-specific NIX knockout
reduces myocardial fibrosis and apoptosis, and systolic dysfunc-
tion after TAC [172].
Over the years, many studies have suggested a link between
heart failure and cancer sharing similar causes of disease, such as
smoking and unhealthy diets. Researchers have shown that
cardiac excreted factors such as serpinA3 and A1 during heart
failure caused intestinal precancerous polyp growth [173]. More-
over, doxorubicin as an effective anti-cancer drug, has significant
side effects in leading to cardiomyopathy and heart failure.
Abdullah et al. reported that the cardiotoxicity of doxorubicin is
mainly due to its inhibition of autophagolysosome degradation,
accompanied by the backlog of damaged mitochondria and Lc3
[174]. This backlog of garbage, if not removed in time, can
accelerate ROS generation and decrease ATP production by
OXPHOS. A patient with dilated cardiomyopathy supported this
conclusion, who show an excessive autophagy but abnormal
lysosome function due to LAMP-2 lack [175].
Mitophagy is an essential factor for maintaining mitochondrial
homeostasis. Insufficient mitochondrial clearance by mitophagy
deficiency results in the accumulation of myocardial ROS, which
triggers apoptosis. Lavandero et al. reported that the level of
autophagy and Lc3 expression was decreased in patients with
postoperative atrial fibrillation. However, excessive mitophagy
leads to a decline in mitochondrial number and ATP production,
leading to insufficient contraction of cardiomyocytes and HF
deterioration. Although autophagy and mitophagy in HF are still
controversial, they will be the new focus of HF therapy.
“mPTP and mitophagy”in cardiac diseases
Long-lasting mPTP opening can cause mitochondrial dysfunction
and cell death, which was closely related to the occurrence and
development of various cardiac diseases. Understanding the link
between mPTP and mitophagy in cardiac diseases may help to a
better understanding of the mechanisms of cardiac disease. mPTP
consist of a group of proteins that is responsible for transporting
substances between the mitochondrial matrix and cytoplasmic.
Currently recognized proteins include cyclophilin-D (CyP-D)
located in matrix, voltage-dependent anion-selective channel 1
(VDAC1) located at OMM, the adenine nucleotide translocase
(ANT) and the phosphate carrier (PiC) both located in inner
membrane.
There are many similarities between mPTP opening and
mitophagy occurrence, for example, the decrease of mitochon-
drial membrane potential, calcium overload, the change of ROS
production. These similarities mean that there may be a certain
connection between them. Excessive calcium can impair mito-
chondrial function and increase ROS, eventually triggering
mitochondrial scavenging system. More seriously, calcium over-
load in clinic is often associated with arrhythmias and
sudden death.
It has been demonstrated that a small portion of mPTP opening
can initiate mitophagy to repair mitochondrial homeostasis via
cleaning abnormal mitochondria, while excessive mPTP opening
will induce mitophagy [176]. Both autophagy and mitophagy were
increased in mouse hearts after myocardial infarction, while there
was also accompanied by mPTP opening [177,178]. It is not
simple cause-and-effect relationship between mPTP opening and
mitophagy, and the change between them do not always
coincide. It has been shown that CyP-D knockout mice had a
smaller myocardial infarct size, better preserved LV systolic
function, and less mortality in post-myocardial infarction (MI)
heart failure [179]. Moreover, other inhibitors of CyP-D also show a
protective effect in post-MI heart failure. In addition, it has been
proved that enhancing mitophagy provided a stronger protection
against cardiac injury after acute MI [177]. In conclusion, either
enhanced mitophagy or repressed mPTP opening can alleviate
cardiac injury. Cyclosporine A, as a mPTPs inhibitor by binding
with CyP-D, can also inhibit parkin recruitment when mitochon-
drial damaged [180]. The relationship between mPTP and
mitophagy in MI injury needs to be further explored.
As the only ADP/ATP translocase in mitochondria, ANT also
participate in mitophagy, which is independent of its role in ATP
production and nucleotide exchange [181]. It has been demon-
strated that ANT is required in pink/parkin-mediated mitophagy
via stabilizing pink located in mitochondria [181]. Mice lacking
ANT show an accumulation of abnormal mitochondria induced by
blunted mitophagy indicating that ANT was important in keeping
mitochondrial quality [181]. In addition, mice lacking ANT more
easily developed concentric hypertrophy with dilation and had a
higher oxidative phosphorylation level [182]. Clinically, patients
with ANT1 deficiency often show cardiomyopathy, mitochondrial
myopathy, lactic academia and other clinical cases [183]. Over-
expressing myocardial ANT1 improved myocardial function and
protected against hypertension-induced cardiac pathology with
improved mitochondrial structure and function [184].
Repressing PiC expression protect against cardiac ischemic-
reperfusion injury. The effect of PIC on autophagy is rarely
reported [185]. Both ANT and PiC are important in ATP generation,
which strongly supported cardiac work. It has been found that
decreased ATP production and abnormal energetic metabolism in
the failing heart [186].
It’s well-known that Vdac1 interacts with pro-apoptotic Bcl-2
members or anti-apoptotic Bcl-2 members, the former process is
involved with the cytochrome C release, while the later inhibits
VDAC1 oligomerization. Marked overexpression of VDAC1 has
been found in post-myocardial infarction patients, as well as in
patients with chronic ventricular dilatation\dysfunction, which
means an important role of vdac1 [187]. The overexpressed
VDAC1 will result in an excessive mPTP opening and the release of
proapoptotic proteins including cytochrome C. It has been
revealed that the loss of cytochrome C via vdac1 channel in
mitochondria impaired and diminished mitophagy level [188]. The
relationship between vdac1 and parkin-mediated mitophagy is
still controversial. Some scientist thought that vdac1 was
A. Li et al.
10
Cell Death and Disease (2022) 13:444

irrelevant to pink-parkin-mediated mitophagy. Others demon-
strated that vdac1 is the critical substrate of parkin. According to
the description by Jongkyeong Chung, there are two different
ubiquitinated structures of VDAC1: VDAC1 monoubiquitination
and VDAC1 polyubiquitination [189]. The former monoubiquitina-
tion inhibits apoptosis, the later polyubiquitination is crucial in
parkin-mediated mitophagy. the absence of polyubiquitination
impaired mitophagy and hindered parkin translocation to
mitochondria. Parkin regulates mitophagy and apoptosis through
different modifications of VDAC1. The mitophagy level is different
in different stages of ischemia-reperfusion, but the mechanism is
unknown, exploring vdac1 mono- or ploy-ubiquitination may help
to better understand its role in mitophagy.
Conclusion and perspectives
As essential organelles for eukaryotic cells, Mitochondria are
involved in multiple cellular functions, including ATP production,
free radical production, calcium homeostasis, and cell apoptosis.
Thus, mitochondrial quality needs to be well maintained for the
optimal performance of mitochondria. Mitophagy is an essential
mitochondrial quality control mechanism that eliminates
damaged mitochondria, coordinating with mitochondrial biogen-
esis to control mitochondrial homeostasis. Abnormal mitophagy is
related to many cardiovascular diseases, including atherosclerosis,
ischemia-reperfusion injury, cardiomyopathy, hypertrophy, and
heart failure. Modulating mitophagy can be a target for relieving
pathologies of illness. Mitochondria occupy ~40% of the volume
of adult cardiomyocytes that could not proliferate. Each cardio-
myocyte is valuable for the heart. Overregulation of mitophagy
would cause cell death. How to control the optimal regulation of
mitophagy? Maintaining the time and level of regulated
mitophagy is possible using compounds.
Furthermore, mitophagy regulation can not completely restore
mitochondrial homeostasis, a prerequisite for cellular functions.
How to restore the homeostasis of dysfunctional mitochondria?
The coordination between mitochondrial biogenesis and mito-
phagy has been destroyed under pathological conditions. The
turnover of mitochondria could not efficiently work. Simulta-
neously regulating mitophagy and mitochondrial biogenesis may
be necessary to restore mitochondrial homeostasis via promoting
mitochondrial turnover.
DATA AVAILABILITY
The data that supports the findings of this study is available from the corresponding
author upon reasonable request.
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AUTHOR CONTRIBUTIONS
AL and GG wrote the manuscript. MG, BL, YQ, LC, HL, HW and helped in manuscript
writing and editing. AL and GG prepared the images. All authors contributed to the
article and approved the submitted version.
A. Li et al.
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FUNDING
This work was partially supported by the National Key Research and Development
Program of China (2018YFA0107102, 2017YFA0105601) and the National Natural
Science Foundation of China (81970333, 31901044), the Program for Professor of
Special Appointment at Shanghai Institutions of Higher Learning (Grant No.
GZ2020008) and the Fundamental Research Funds for the Central Universities.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41419-022-04906-6.
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