Role of the Mitochondrial Protein Import Machinery and Protein Processing in Heart Disease

Mitochondria are essential organelles for cellular energy production, metabolic homeostasis, calcium homeostasis, cell proliferation, and apoptosis. About 99% of mammalian mitochondrial proteins are encoded by the nuclear genome, synthesized as precursors in the cytosol, and imported into mitochondria by mitochondrial protein import machinery. Mitochondrial protein import systems function not only as independent units for protein translocation, but also are deeply integrated into a functional network of mitochondrial bioenergetics, protein quality control, mitochondrial dynamics and morphology, and interaction with other organelles. Mitochondrial protein import deficiency is linked to various diseases, including cardiovascular disease. In this review, we describe an emerging class of protein or genetic variations of components of the mitochondrial import machinery involved in heart disease. The major protein import pathways, including the presequence pathway (TIM23 pathway), the carrier pathway (TIM22 pathway), and the mitochondrial intermembrane space import and assembly machinery, related translocases, proteinases, and chaperones, are discussed here. This review highlights the importance of mitochondrial import machinery in heart disease, which deserves considerable attention, and further studies are urgently needed. Ultimately, this knowledge may be critical for the development of therapeutic strategies in heart disease.


INTRODUCTION
Mitochondria are vital for energy production in eukaryotic cells, generating cellular ATP through oxidative phosphorylation (OXPHOS) (1). Importantly, mitochondria are also crucial for numerous metabolic pathways, maintenance of calcium homeostasis, and regulation of cell proliferation and apoptosis (2). However, only 13 proteins involved in OXPHOS are encoded by the mitochondrial genome in mammals. About 99%-more than 1,500-mammalian mitochondrial proteins are encoded by the nuclear genome, synthesized as precursors in the cytosol, and need to be imported into mitochondria by mitochondrial protein import machinery (3). To date, six translocases of the mitochondrial protein import machinery have been discovered. The TOM complex serves as the entry gate for most precursors at the outer membrane (OM); the TIM22 and TIM23 complexes at the inner membrane (IM) are responsible for the insertion of carrier precursors into the IM and the translocation of presequence-carrying precursors into the mitochondrial matrix or IM individually; the mitochondrial intermembrane space (IMS) import and assembly machinery (MIA) complex mediates the import of cysteine-rich proteins to the IMS; the SAM and MIM complexes are responsible for insertion of β-barrel proteins and α-helical proteins, respectively, into the OM (Figure 1 and Table 1) (3). The dynamic interaction and cooperation of these mitochondrial protein import pathways enable cells to respond to environmental stress and energy demands rapidly and with plasticity. Further, mitochondrial protein import pathways function not only as independent units for protein translocation, but also are deeply integrated into a functional network of mitochondrial bioenergetics, protein quality control, mitochondrial dynamics and morphology, and interaction with other organelles (4). Mitochondrial protein import deficiency is FIGURE 1 | Overview of the major mitochondrial protein import pathways in yeast. First, the presequence pathway transports presequence-carrying cleavable preproteins to the mitochondrial matrix (1) or IM (2, 3) (in green). Presequence-carrying precursors to the mitochondrial matrix (1) are typically recognized by TOM at the OM, passage the IM through TIM23, and are driven into the matrix assisted by PAM. ψ across the IM is essential for the entry of presequences into the matrix. The presequences are cleaved by MPP, and additional proteolytic processing occurs by intermediate cleaving peptidases. Presequence-carrying precursors that integrate into IM follow two distinct routes. IM proteins are either directly laterally released from the TIM23 complex (2) or transported into the matrix first followed by further insertion into the IM with the help of Oxa1 (3). Oxa1 also is responsible for insertion of IM proteins synthesized on mitochondrial ribosomes. Second, in the carrier pathway for the import of precursor proteins without a cleavable presequence, yet with internal targeting signals into the IM (in yellow), carrier precursors are accompanied by cytosolic chaperones, delivered to the Tom70 receptor of the TOM complex, bound by small TIM chaperones in the IMS, and eventually integrated into the IM by the TIM22 complex in a ψ-dependent manner. Third, in the MIA pathway for cysteine-rich proteins to the IMS (in blue), the precursors are kept in a reduced state in the cytosol, imported by the TOM complex, oxidized by the MIA machinery, and stay in an oxidized state in the IMS. Fourth, in the SAM complex for β-barrel proteins to the OM (in red), the precursors of β-barrel proteins are imported by the TOM complex at the OM, bound to small TIM chaperones in the IMS, and inserted into the OM by the SAM complex. Fifth, some proteins with α-helical transmembrane segments are inserted into the OM by the MIM complex (in purple). Typically, the Tom40 channel is not involved in this route, but Tom70 is indispensable. In addition, ERMES is a complex that connects ER and mitochondrial OM, facilitating the dynamic substrate exchange between ER and mitochondrion. Mdm10 is a protein with dual localization in SAM and ERMES. OM, outer membrane; IM, inner membrane; IMS, intermembrane space; TOM, the translocase of the outer membrane; TIM23, the inner membrane translocase TIM23; PAM, presequence translocase-associated motor; ψ, membrane potential; MPP, mitochondrial processing peptidase; Oxa1, oxidase assembly protein 1; TIM22, the carrier translocase of the inner membrane TIM22; MIA, mitochondrial intermembrane space import and assembly machinery; SAM, sorting and assembly machinery; MIM, mitochondrial import complex; ERMES, endoplasmic reticulum (ER)-mitochondria encounter structure. linked to various diseases, including neuropathies, myopathies, neurodegenerative diseases, cancer, and cardiovascular disorders (5,6). The heart is a high-energy-requiring organ that depends heavily on mitochondrial activity and the efficient import of mitochondrial proteins. However, in heart disease, the roles of the mitochondrial protein import machinery have not been well-studied. Here, we summarize the current knowledge on mitochondrial protein import in heart disease for the first time. This review highlights the importance of the mitochondrial import machinery in heart disease, which deserves considerable attention, and further studies are urgently needed. Ultimately, this knowledge may be critical for the development of therapeutic strategies in heart disease. The presequence pathway is the best characterized pathway, responsible for the import of ∼60% of all mitochondrial proteins (7). The precursor proteins in this pathway carry a cleavable N-terminal presequence that functions as a targeting signal (7)(8)(9). This unique feature of the pathway distinguishes it from all the others, where the precursor proteins do not have cleavable presequences, but possess different kinds of internal targeting signals. Presequence-carrying precursors to the mitochondrial matrix are typically recognized by the translocase of the outer membrane (TOM) (9)(10)(11), passaged through the IM by the translocase of the inner membrane (TIM23) (12)(13)(14)(15)(16), and driven into the matrix assisted by the presequence translocaseassociated motor (PAM) (Figure 1) (17)(18)(19)(20)(21)(22)(23)(24). The membrane potential ( ψ) across the IM is essential for the activation of the TIM23 channel and the translocation of presequences into the matrix (25-28). The presequences are cleaved by the mitochondrial processing peptidase (MPP) (3, 29-31), and additional proteolytic processing occurs by intermediate cleaving peptidases (7,(32)(33)(34). Presequence-carrying precursors that integrate into the IM follow two distinct routes (Figure 1). IM proteins are either directly released laterally from the TIM23 complex (35-37) or transported first into the matrix, followed by further insertion into the IM with the help of the oxidase assembly protein 1 (Oxa1) insertase (38-41). Oxa1 also is responsible for insertion of IM proteins synthesized on mitochondrial ribosomes (42). The presequences are located at the N-termini of preproteins and typically consist of ∼15-50 amino acids. An essential characteristic of mitochondrial presequences is the formation of an amphipathic α-helix that is specifically recognized by mitochondrial import receptors and other mitochondrial import components during preprotein translocation by TOM, TIM23, and PAM (3).
The TOM complex is the main gate for precursors entering mitochondria. The TOM complex is composed of Tom20, Tom22, and Tom70 as receptors, β-barrel protein Tom40 forming the channel, and three small associated proteins, Tom5, Tom6, and Tom7 (11,(43)(44)(45). Tom20 and Tom22 function cooperatively as the receptors for presequence-carrying precursors (9,10,46). Tom70 mainly functions as the receptor for preproteins with internal targeting sequences, such as carrier precursors (47-52). Presequence-carrying precursors cross the Tom40 channel as linear polypeptide chains (44, 45, 53-57), and interact with the tail of the Tom22 receptor in the IMS (57). Tom22 has presequence binding sites on both the cytoplasmic and IMS sides of the OM. The exact roles of the three small subunits, Tom5, Tom6, and Tom7, have not been well-clarified. It has been proposed that they are not essential for TOM functions but are involved in the assembly and stability of the TOM complex (58-61).
The TIM23 complex translocates cleavable preproteins into mitochondrial matrix or IM. Tim50, Tim23, Tim17, and Tim21 compose the main elements of the TIM23 complex (12,13,16,(62)(63)(64)(65). Tim50 functions as a presequence receptor that binds preproteins emerging in the IMS (63) and a channel blocker that closes out the TIM23 channel in the absence of preproteins (25, [66][67][68][69]. Tim23 and associated partner Tim17 form the channel (16,25,28,64,70,71). Tim21, Tim50, and Tim23 expose domains to the IMS that transiently connect the TOM and TIM23 complexes to facilitate the preprotein transfer (16,67,(72)(73)(74). Additionally, Tim21 also physically links the TIM23 complex to the respiratory chain III-IV supercomplex [bc1 complex and cytochrome c oxidase (COX)] (65,75,76). Tim21 thus plays a dual role in TOM-TIM23 transfer and the recruitment of respiratory chain complexes. A small membrane protein, Mgr2, functions as a lateral gatekeeper for preproteins that are sorted into the IM (35, 76). The ψ across the IM is crucial for translocation of the presequences through the Tim23 channel, which is negative at the matrix side and positive at the IMS side of the IM, whereas presequences are mostly positively charged. Two roles have been assigned to ψ: activation of TIM23 channel (25) and an electrophoretic effect that drives the import of presequences (77,78).
PAM. The ψ is a prerequisite for translocation of the presequence across the TIM23 channel. Nevertheless, it is not sufficient to import the entire protein into the matrix. PAM is necessary for the translocation of matrix proteins. The core of PAM is formed by the molecular chaperone mitochondrial 70 kDa heat shock protein (mtHsp70) (17,18) and its cochaperones (Tim44, Mge1, Pam16, Pam17, and Pam18) (16,21,79,80). mtHsp70 binds the unfolded polypeptide chain and drives its translocation into the matrix in an ATP-dependent manner (17,18). The peripheral membrane protein Tim44 is a docking site for mtHsp70 at the TIM23 complex (21). Mge1 (also known as mitochondrial GrpE) stimulates the release of ADP from mtHsp70 (81). Pam16, Pam17, and Pam18 are three membrane-associated co-chaperones. Pam18 (also termed Tim14) is a J-type co-chaperone that stimulates the ATPase activity of mtHsp70 (82,83). The J-related Pam16 (Tim16) forms a complex with Pam18 and functions as a negative regulator (82)(83)(84)(85)(86). Pam17 mediates the organization of the TIM23-PAM interaction (79,87).
OXA translocase is vital for exporting proteins from the mitochondrial matrix into the IM. OXA has three different roles. (1) Proteins encoded by the mitochondrial genome are exported into the IM by Oxa1 (42, [95][96][97]. (2) Some presequence-carrying proteins imported into the matrix via the TOM-TIM23 machinery are exported into the IM via Oxa1. This import-export pathway is termed conservative sorting of nuclear-encoded IM proteins (38-41, 98-100). (3) Oxa1 is also vital for the assembly of the carrier translocase TIM22 (38, 101).

Carrier Pathway Into the IM
The carrier pathway is the second mitochondrial protein import pathway to be discovered, and is responsible for importing precursor proteins without a cleavable presequence, yet with different kinds of internal targeting signals (3,(102)(103)(104). The carrier precursors are accompanied by cytosolic chaperones, such as the HSP70 and HSP90 classes in the cytosol, directly delivered to the Tom70 receptor of the TOM complex (47, 105,106), and then bound by small TIM chaperones in the IMS (107)(108)(109)(110) and eventually integrated into the IM by the carrier translocase of the IM (TIM22) complex in a ψ-dependent manner (Figure 1) (109,(111)(112)(113)(114)(115)(116).
Chaperone-guided transport of carrier precursors (including chaperones in the cytosol and IMS). The carrier import pathway uses the same mitochondrial entry gate as the presequence pathway, the TOM complex. However, the mechanisms of translocation differ significantly. The involvement of cytosolic (47, 105, 106) and mitochondrial IMS chaperones, which is crucial to prevent aggregation of the hydrophobic carrier precursors in the aqueous environment, is the main feature distinguishing this from the presequence pathway. Chaperones of the Hsp70 and Hsp90 classes directly participate in delivering the precursors to Tom70 (47, 105,106). The receptor Tom70 possesses two distinct binding sites, one for the precursor and another for a chaperone (47, 49, 117), ATP is needed to release the precursor proteins from the cytosolic chaperones (47, 48). Upon binding to Tom70, the carrier precursors are transferred to the central receptor Tom22, followed by insertion into the Tom40 channel in a loop conformation (118, 119), and transferred to small TIM chaperones in the IMS (107)(108)(109)(110). These small TIM heterohexameric chaperone complexes, like the Tim9-Tim10 complex (120,121) and the homologous Tim8-Tim13 complex (122), bind to the precursor proteins and transfer them through the aqueous IMS to IM.
Insertion of carrier precursors into the IM. The TIM22 complex consists of the receptor-like protein Tim54, the channelforming protein Tim22, the Tim9-Tim10-Tim12 chaperone complex, and the Tim18-Sdh3 module. The majority of Tim54 domain is exposed to the IMS and probably functions as the binding site for the Tim9-Tim10-Tim12 complex (123,124). The Tim9-Tim10-Tim12 complex is a modified form of the IMS chaperone, docking onto the TIM22 complex (123,125). Carrier precursors are inserted into the Tim22 channel in a ψdependent manner (115). The Tim18-Sdh3 module is involved in the assembly of the TIM22 complex (126,127). The carrier precursors are first bound to the Tim9-Tim10-Tim12 chaperone complex on the surface of the translocase. Upon activation of the Tim22 channel ( ψ-dependent), the precursors are inserted into the translocase, probably in a loop structure. Finally, the proteins are laterally released into the lipid phase of the IM.

MIA Complex
Many IMS proteins contain internal targeting signals and characteristic cysteine motifs. In the cytosol, the precursors are kept in a reduced and unfolded state (128). Upon import by the TOM complex (60), they are oxidized by the MIA machinery, and stay in the IMS in an oxidized state (Figure 1). The MIA system consists of two main components: the oxidoreductase Mia40 and the sulfhydryl oxidase Erv1 (129)(130)(131)(132)(133).
Mia40 serves as a receptor and protein disulfide carrier. Most IMS proteins are synthesized without cleavable presequences but contain cysteine motifs. Unlike presequencecarrying precursors and carrier precursors, none of the Tom receptors is necessary for the import of MIA substrates (60, 61). Instead, upon passage through the Tom40 channel (60), Mia40 functions as a receptor on the IMS side of the Tom40 channel (133)(134)(135)(136)(137)(138). It recognizes an internal signal of the precursor proteins, typically consisting of a hydrophobic element flanked by a cysteine residue (133,139,140). Mia40 binds to precursors via hydrophobic interaction and catalyzes the formation of disulfide bonds in imported proteins (133,138). The disulfide bonds facilitate the conformational stabilization and assembly of many IMS proteins.
Erv1 cooperates with Mia40 in a disulfide relay. Mia40 does not form disulfide bonds de novo. Disulfide bonds are generated by Erv1 and transferred to Mia40 by the formation of transient intermolecular disulfide bonds (141). Mia40 then transfers the disulfides onto the imported protein. Upon transfer of disulfide bonds to proteins, cysteines of Mia40 become reduced and are re-oxidized by Erv1. Electrons originating from the oxidation of imported proteins flow in the opposite direction. They flow from Mia40 to Erv1 and then to O 2 or cytochrome c of the respiratory chain (141)(142)(143)(144). In addition to most IMS proteins, some IM and matrix proteins are also MIA-system-dependent (28, 71,113,114,145).

Sorting Pathways of Mitochondrial OM Proteins
All the mitochondrial OM proteins are imported from the cytosol. The membrane contains two different types of integral protein: β-barrel proteins, which are integrated into the membrane by multiple transmembrane β-strands, and α-helical proteins, which are membrane-anchored by one or more αhelical transmembrane segments.
Sorting and Assembly Machinery for β-Barrel Proteins. The precursors of β-barrel proteins initially pass through the TOM complex at the OM (146), then bind to small TIM chaperones in the IMS (110), like the carrier precursors, to avoid aggregation (Figure 1). Subsequent insertion of the β-barrel proteins into the OM is performed by the SAM complex, which consists of a membrane-integrated protein, Sam50 (Tob55), and two peripheral membrane proteins exposed to the cytosol, Sam35 and Sam37 (147)(148)(149). Folding of the β-barrel proteins occurs at Sam50-Sam35, followed by lateral release into the lipid phase of the OM (148,150,151). Sam37 directly interacts with Tom22, coupling the TOM and SAM complexes into a transient supercomplex that promotes the efficient transfer of precursor proteins (54, 152).
OM Insertion of α-Helical Proteins. The main α-helical proteins are classified as signal-anchored proteins (containing an α-helical transmembrane segment at the N-terminus), tailanchored proteins (containing an α-helical transmembrane segment at the C-terminus), and polytopic (multi-spanning) OM proteins. α-helical OM proteins are imported via distinct routes that do not involve the Tom40 channel, in contrast to most mitochondrial proteins. The insertion of some signalanchored and polytopic OM proteins is performed by the MIM complex (153)(154)(155)(156), which consists of multiple copies of Mim1 and one copy of Mim2, both of which are small single-spanning OM proteins (Figure 1) (157,158). Tom70 is required for the insertion of some polytopic proteins into MIM (155,156). In the case of tail-anchored proteins and some multi-spanning proteins, import is aided by the lipid composition of the membrane, and no proteinaceous machinery has been identified (159)(160)(161)(162). However, the exact mechanism for sorting and insertion of αhelical outer membrane proteins is only partially understood, and further studies are urgently needed.

INTEGRATION OF MITOCHONDRIAL PROTEIN IMPORT INTO FUNCTIONAL NETWORKS
Mitochondrial protein import pathways not only function as independent units for protein translocation, but also are deeply integrated into a functional network of mitochondrial bioenergetics, protein quality control, mitochondrial dynamics and morphology, and interaction with other organelles.
The protein import activity serves as a sensor for the fitness and quality of mitochondria. The protein import activity is determined by the energetic state ( ψ, ATP levels) and protein homeostasis of mitochondria. Both the translocation of precursor proteins through the TIM23 complex and the TIM22 complex require the ψ (25, 77,78,113,115,116). The ATP-dependent chaperones play essential roles in delivering carrier precursors to Tom70 receptor (47, 48), driving presequence precursor translocation to the matrix (17,18) and folding in the matrix (92). Impairment of respiratory chain activity, reduction of ATP levels, and accumulation of misfolded proteins or reactive oxygen species (ROS) in the matrix (163) will directly affect the importdriving activity of the translocases. The protein import activity of mitochondria is thus a sensitive indicator of their energetic state and fitness.
Mitochondrial protein import machinery and respiratory chain assembly. Both the insertion of mitochondrial-encoded proteins from the matrix into the IM and the import of nuclear-encoded precursors from the cytosol into mitochondria rely heavily on the mitochondrial protein import machinery. Increased mitochondrial ROS levels generated by the respiratory chain contribute to decreased mitochondrial translation efficiency (164). In addition, the TIM23 complex forms supercomplexes with respiratory complexes III and IV as well as with the ADP/ATP carrier. These interactions of the TIM23 complex facilitate protein import under energy-limiting conditions (65,75,165) and can also promote the assembly of respiratory complexes (166)(167)(168). The respiratory chain complexes also function as assembly platforms for some PAM subunits (75).
Mitochondrial protein import machinery associated with protein quality control, specifically in the following aspects: (1) Mitochondrial unfolded protein response (UPR mt ): the stressactivated transcription factor ATFS-1 contains a mitochondrial presequence and a nuclear localization signal. In healthy mitochondria, ATFS-1 is imported into the mitochondrial matrix and degraded by the LON AAA+ protease. When mitochondrial import is mildly impaired, ATFS-1 is translocated into the nucleus, where it functions as a transcription factor and induces expression of mitochondrial chaperones, proteases, and other elements to promote recovery of impaired mitochondria (169)(170)(171). (2) Unfolded protein response activated by mistargeted mitochondrial proteins (UPR [am]): upon mild damage to mitochondrial protein import, some mitochondrial precursors fail to enter mitochondria and accumulate in the cytosol, thus triggering mitochondrial Precursor Over-accumulation Stress (mPOS). This is followed by a stress response termed UPR [am] that reduces cytosolic protein synthesis and increases proteasome activity to clear the mistargeted proteins from the cytosol (172,173). (3) PTEN-induced kinase 1 (PINK1)/Parkin-induced mitophagy: in healthy conditions, PINK1 is imported into the IM by the presequence pathway and processed by MPP and the presenilin-associated rhomboid-like protease PARL, following the retrotranslocation into the cytosol and degradation by the proteasome (174,175). Upon severe damage to mitochondrial protein import, PINK1 remains at the OM bound to the TOM complex, where it phosphorylates ubiquitin and the E3 ubiquitin ligase Parkin, triggering the removal of damaged mitochondria by mitophagy (176,177). (4) Mitochondria as guardian in the cytosol (MAGIC): in certain conditions, some aggregationprone or misfolded cytosolic proteins may be imported into mitochondria for further degradation. This process is termed MAGIC, suggesting a crucial role of mitochondria in cytosolic proteostasis (178). (5) Proteolysis of mitochondrial proteins: upon removal of the presequence by MPP, destabilizing Nterminal amino acid residues of the imported proteins can be further removed by the intermediate cleaving peptidase Icp55 (which removes a single amino acid) or the mitochondrial intermediate peptidase Oct1 (which removes an octapeptide) (7,34). The matrix AAA+ proteases, CLPXP and LON, degrade misfolded proteins and prevent protein aggregation in the matrix (179)(180)(181)(182). The IM contains two AAA+ proteases: the i-AAA protease removes misfolded proteins from the IM, IMS, and OM (183)(184)(185), whereas the m-AAA protease degrades proteins from the matrix and IM (186,187). Thus, the process of mitochondrial protein import is connected to protein turnover and quality control.
Mitochondrial protein import machinery connected to mitochondrial membrane architecture and dynamics. The mitochondrial contact site and cristae organizing system (MICOS) is a large protein complex enriched at crista junctions of the IM (188)(189)(190). It is crucial for the maintenance of inner membrane cristae organization and is embedded into an interactional network with protein translocases, including TOM, SAM, and MIA. Thus, it provides a dynamic link between protein import, mitochondrial membrane dynamics, and membrane contact sites (136,188,189,191).
The inner-membrane fusion protein optic atrophy (OPA1) is an example of how protein import and processing are connected to mitochondrial membrane dynamics. OPA1 is first processed by matrix MPP, generating a long isoform, and further processed by different IM proteases, AAA+ protease, or OMA1 in mammals and Pcp1 in yeast, yielding a short isoform (192)(193)(194)(195). The balance between long OPA1 and the short isoform is essential for membrane fusion and fission, which is modulated by stress and mitochondrial energetic state. Thus, the processing of imported mitochondrial protein is linked to mitochondrial fragmentation, mitophagy, or even cell death.
Endoplasmic reticulum-mitochondria encounter structure (ERMES). ERMES is a multi-subunit protein complex that connects the endoplasmic reticulum and mitochondrial OM, mainly formed by the MDM complex (196,197). Other molecules, such as voltage-dependent anion-selective channel (VDAC) (198), TOM70 (199,200), and inositol trisphosphate (inositol 1,4,5-trisphosphate) receptors (194) also play crucial roles in forming ER-mitochondria contact sites. The outer membrane protein Mdm10 is a subunit of both SAM and MDM complexes (196). The shuttling of Mdm10 between SAM and MDM is regulated by the small protein Tom7 (58,196,197,201). Therefore, TOM, SAM, and ERMES are linked as a functional network, involved in the maintenance of mitochondrial morphology and the transport of lipids and calcium (202)(203)(204)(205).

MITOCHONDRIAL PROTEIN IMPORT MACHINERY AND HEART DISEASE
Mitochondrial dynamics have become a key topic in the field of heart disease. However, only limited studies investigated the involvement of mitochondrial protein import machinery in these diseases (Summarized in Table 2). Moreover, most of these studies were related to the presequence pathway, which undertakes the import of ∼60% of all mitochondrial proteins.

TOM Complex in Heart Disease
Tom20 is an essential receptor subunit of the TOM complex that recognizes mitochondrial precursor proteins with cleavable N-terminal presequences. Tom20 expression was reduced by ischemic insults (206,207), and showed a cardioprotective role against ischemia/reperfusion (I/R) injury through enhancing the mitochondrial import of PKCepsilon (PKCε) in an HSP90-dependent manner (208). PKCε, a member of the serine/threonine kinase family, has been demonstrated to play a protective role against cardiac I/R injury (208,216,293). Additionally, calcium homeostasis, which is closely related to Lon seemed beneficial to cardiomyocytes upon high-fat diet and hypertrophic stresses but harmful to cardiomyocytes upon hypoxia insults; In aged hearts, Lon expression increased, but proteolytic efficiency declined; In cardiac-specific frataxin-deletion mice, Lon expression and activity were increased in the heart. Cardiac-specific ablation of YME1L in mice led to DCM and heart failure; YME1L was also crucial for the progression of experimental autoimmune myocarditis to DCM and the therapeutic efficacy of mesenchymal stem cells in myocardial infarction. (195,(277)(278)(279) (Continued) Frontiers in Cardiovascular Medicine | www.frontiersin.org cardiac health, is influenced by Tom20. For example, Wattamon recently reported that the protective role of fibroblast growth factor 2 (FGF-2) against calcium overload was partially mediated by mitochondrial connexin 43 (Cx43) (introduced below), probably in a Tom20-dependent manner (209). Cx43 was imported into mitochondria via a Tom20-dependent pathway (257). In another study, Tom20 was reported to be responsible for the direct insertion of VDAC, a protein crucial in regulating cardiac calcium homeostasis (210) through mitochondrial permeability transition pore (mPTP) (294) and mitochondriaassociated endoplasmic reticulum membranes (MAMs), into the OM of heart mitochondria in rats (295). Thus, Tom20 could be a crucial adaptor of cardiac calcium homeostasis in a Cx43-or VDAC-associated manner. Tom22 serves as the central receptor for both presequence precursors and carrier precursors. Furthermore, it is also the key factor linking the TOM and SAM complex to a supercomplex by interaction with Sam37, which promotes the efficient transfer of β-barrel precursors. Tom22 was recently identified as a potential receptor for cardiac mitochondrial large conductance voltage and Ca 2+ -dependent K [+] (mitoBK Ca ) channels, facilitating the import of mitoBK Ca via the presequence pathway (211). Tom22 deficiency might induce cardiomyocyte dysfunction by interfering with cardiac mitochondrial Ca 2+ import (211). Additionally, Tom22 has been reported as a potential regulator of heart function through assisting the synthesis of cardiac aldosterone in mitochondria (212). Finally, in chronically hypoxic rat hearts, the level of Tom22 mRNA was increased in cardiac ventricles (213), suggesting a potential role of Tom22 in ischemic heart disease.
Small Tom Proteins (Tom5, Tom6, and Tom7). An allele of TOMM5 (the gene encoding Tom5) intronic variant (rs57578064) was correlated with a significant increase in lipoprotein-associated phospholipase A2 activity, which is associated with increased risk of cardiovascular events (239). Tom7 deficit in endothelial cells particularly damaged formation of the cerebrovascular network, but not cardiac vasculature, in zebrafish and mice (296).

TIM23 Complex in Heart Disease
Tim23, the channel-forming protein of the TIM23 complex, responsible for the translocation of presequence precursors into mitochondrial matrix or IM, was reduced by hypoxia/reoxygenation (H/R) or I/R (207,242,243). Restoring expression of Tim23 by various treatments seemed protective (207,242,243) against H/R or I/R injuries. However, a controversial study from Bian showed that the protective role of zinc against I/R injury was mediated by enhanced mitophagy, accompanied by downregulation of Tom20 and Tim23 expression (244). In addition, another study reported the association of decreased Tim23 expression in patients with DCM (238).
Tim50 is the receptor of the TIM23 complex that recognizes presequence carrying proteins. Guo et al. demonstrated that the loss of Tim50 during early zebrafish embryonic development caused neurodegeneration, cardiovascular defects (dysmorphic heart, reduced heartbeat, and decreased circulating blood), and impaired motility. These pathological changes might result from increased cell death, which was mediated by mitochondrial membrane permeabilization and acceleration of cytochrome c release (240). Tang et al. further identified that Tim50 was downregulated in both human DCM heart and transverse aortic constriction (TAC)-induced murine hypertrophic heart (241). Meanwhile, global Tim50 knockout mice showed more severe cardiac hypertrophy than wild-type mice, which was alleviated by cardiac-specific overexpression of Tim50 via reducing ROS accumulation and ASK1 activity (241).
The Presequence-Pathway-Associated Chaperones in Heart Disease mtHSP70 (also known as GRP75/ mortalin/ PBP74) is an essential ATP-dependent chaperone of the PAM complex. It drives the translocation of preproteins into the matrix in the membrane-bound motor form and exhibits typical chaperone activity to prevent protein misfolding and aggregation in the soluble form. In vitro studies, mtHSP70 was identified as a cardioprotective chaperone against H/R-induced oxidative stress (245,246), potentially via increased import of nuclear genome-encoded antioxidant defense proteins, such as DJ-1 (246). Expression of mtHSP70 was significantly decreased in the interfibrillar mitochondria (IFM) of type 1 diabetes mellitus (T1DM) (247) and the subsarcolemmal mitochondria (SSM) of type 2 diabetes mellitus (T2DM) (248). Cardiacspecific mtHSP70 overexpression restored cardiac function and nuclear-encoded mitochondrial protein import, contributing to a beneficial impact on proteome signature and enhanced mitochondrial function during T2DM (248). Further, mtHSP70 expression was increased in myocardial samples from patients with chronic atrial fibrillation, which suggested an adaptive heat shock response to restore cellular homeostasis (249).
HSP60 and HSP10 are mitochondrial matrix chaperones, playing pivotal roles in implementing protein folding and preventing protein aggregation. Cardiac-specific HSP60 deficiency in mice led to DCM, heart failure, and lethality. Interestingly, the import of preproteins into mitochondria was unaffected by HSP60 deficiency. However, the imported proteins processed by HSP60 underwent further degradation, suggesting lower stability of those proteins (263). HSP10 showed a similar cardioprotective role in I/R-induced myocyte death (264). Both the beneficial roles of HSP60 and HSP10 in cardiomyocytes were related to the preserved function of Complex I and Complex II. Clinical evidence found upregulated expression of both mitochondrial HSP60 and HSP10 in myocardial samples from patients with chronic atrial fibrillation (265). Agsteribbe et al. reported a single case of a girl who had facial dysmorphic features and breathing difficulties upon birth and died at 2 days of age of heart failure (266). The post-mortem examination revealed that the amount of mitochondrial HSP60 was only about 1/5 of the normal level (266).
Tim14 (encoded by DNAJC19), human homolog to yeast Pam18/Tim14, is a mitochondrial IM co-chaperone of the TIM23 complex. Mutations in DNAJC19 were related to DCM and cerebellar ataxia (DCMA) syndrome, a novel autosomal recessive syndrome characterized by early-onset DCM with conduction defects, non-progressive cerebellar ataxia, testicular dysgenesis, growth failure, mild developmental delay, and 3methylglutaconic aciduria, with or without sensorineural hearing loss and basal ganglia lesions (250)(251)(252)(253)(254)(255). The pathogenic mechanism of DCMA was associated with protein import inefficiency and cardiolipin remodeling. Experimental evidence suggested DNAJC19 forms a complex with prohibitins (PHBs). Furthermore, the loss of PHB/DNAJC19 complexes affected cardiolipin acylation and led to the accumulation of cardiolipin species with altered acyl chains (297).
MAGMAS (mitochondria-associated granulocyte macrophage colony stimulating factor signaling molecule), also termed PAM16/Tim16, forms a stable subcomplex with J-protein Pam18 or DnaJC19. It tethers to the TIM23 complex in yeast and humans (298). Cybel et al. reported that two patients from a family with MAGMAS mutation died at 2 years of age of heart failure (256).

The Presequence-Pathway-Associated Proteinases and Peptidases in Heart Disease
Mitochondrial Lon Protease is crucial for the clearance of oxidized or misfolded proteins in the matrix. It played a beneficial role in improving cardiac metabolic flexibility via degradation of pyruvate dehydrogenase kinase 4 in mice fed a high-fat diet (269). Moreover, it was also verified to enhance cardiac function via improving mitochondrial respiration capacity in pressure overload-induced heart failure in mice (270). However, it seemed harmful to cardiomyocytes upon hypoxia insults, which was associated with enhanced ROS production (271,272) and accelerated degradation of phosphorylated complex IV subunits (273). In murine heart, mitochondrial Lon protease levels rose with age, but proteolytic efficiency and adaptation to stress were compromised in older animals (274,275). Mitochondrial Lon protease was also found to be involved in Friedreich's ataxia (FRDA), a rare hereditary neurodegenerative disease characterized by progressive ataxia and cardiomyopathy due to mitochondrial frataxin defect. In cardiac-specific frataxindeletion mice, a progressive increase in mitochondrial Lon and ClpP protease expression and activity were found in the heart, accompanied by the loss of mitochondrial Fe-S proteins (276).
YME1L. As we mentioned above, the balance between long OPA1 (L-OPA1) and short OPA1 (S-OPA1), which is crucial for mitochondrial fusion and fission, is modulated by two mitochondrial proteases, OMA1 and the AAA+ protease YME1L. Cardiac-specific ablation of YME1L in mice led to DCM and heart failure via activated OMA1 and accelerated OPA1 proteolysis, which triggered mitochondrial fragmentation and altered cardiac metabolism (195). Moreover, cardiac function and mitochondrial morphology were rescued by Oma1 deletion by preventing OPA1 cleavage (195). The regulation of YME1L in mitochondrial fusion via OPA1 proteolysis was further verified in experimental autoimmune myocarditis animals (277) and YME1L-overexpressing or deficit cells (278). Furthermore, it was related to the therapeutic efficacy of mesenchymal stem cells for myocardial infarction (279).
MPPα. MPP is a dimeric protease in the matrix that removes N-terminal presequences and consists of MPPα and MPPβ. Mugdha reported that a patient with mutations in the PMPCA gene, which encodes MPPα, had multisystem impairments, including developmental delay, severe hypotonia, ataxia, lactic acidemia, and severe hypertrophic left ventricular cardiomyopathy and died at 14 months from respiratory failure (267). This phenotype may be related to reduced MPPα levels and impaired processing of frataxin and other mitochondrial proteins. Downregulation of MPPα was found linked to the protective role of GSK inhibitor SB216763 in I/R injury (258).
MIP/Oct1. Upon removal of the presequence by MPP, some mitochondrial precursor proteins undergo secondary processing carried out by the mitochondrial intermediate peptidase MIP/Oct1 or intermediate cleaving peptidase Icp55/XPNPEP3 to remove destabilizing N-terminal amino acid residues of the imported proteins. Mutations in the MIPEP gene, which encodes MIP, causes COXPD31/Eldomery-Sutton syndrome with developmental delay, cardiomyopathy, left ventricular noncompaction, hypotonia, and infantile death (268).
CLPP (mitochondrial ATP-dependent Clp proteolytic subunit), a mitochondrial matrix proteinase, has a central role in protein homeostasis. Loss of CLPP in the heart was found to alleviate mitochondrial cardiomyopathy induced by DARS2 deficiency, potentially mediated by increased de novo synthesis of individual OXPHOS subunits, without affecting the mammalian UPR [mt] (280).

Carrier Pathway Involved in Heart Disease
Tom70 mainly serves as the receptor for hydrophobic precursors without a cleavable presequence, such as carrier precursors. Tom70 protein was downregulated in hypertrophic heart of animals and humans, which was associated with increased oxidative stress. Furthermore, upregulation of Tom70 provided cardiomyocytes with full resistance to diverse pro-hypertrophic insults (218). Tom70 expression was also reduced by I/R insult in cardiomyocytes (219)(220)(221)(222). Supplementation of Tom70 significantly attenuated I/R injury by promoting translocation of PKCε (216,217) [to increase the expression of KATP channel pore-forming subunit Kir6.2 (223), augment mitochondrial respiratory capacity, and modulate cardiac glucose metabolism (224)], MICU1 (to reduce mitochondrial Ca 2+ overload), (222) and PINK1 (associated with mitophagy) (221, 225) into mitochondria. Increased expression of PGC-1α/Tom70 was also involved in melatonin-induced cardiac protection against post-myocardial infarction, which was associated with inhibited mitochondrial impairment and reduced ROS generation (219,220). In the hearts of diabetic db/db mice, Tom70 expression was suppressed. Reconstitution of Tom70 protected against diabetic cardiomyopathy through its antioxidant and antiapoptotic properties (226). Moreover, in aging hearts of diabetic db/db mice, only the expression of mitochondrial membrane proteins like Tom70 and VDAC, but not respiratory enzymes, could be increased by short-term exercise (227). Further, phosphoproteome mapping in a rat model of heart failure revealed phosphorylation of several import machinery proteins (Tom70, HSP90, and Tim8a), suggesting that the modification of mitochondrial protein import was involved in heart failure (228).
AGK (acylglycerol kinase). AGK is a mitochondrial lipid kinase that was recently identified as a subunit of the TIM22 complex. It plays an indispensable role in the import and assembly of mitochondrial carrier proteins in the IM (299). It has been shown that loss-of-function mutations in the AGK gene cause Sengers syndrome (281)(282)(283)(284)(285)(286), an autosomal recessive mitochondrial disorder characterized by hypertrophic cardiomyopathy, congenital cataracts, skeletal myopathy, exercise intolerance, and lactic acidosis. Loss of AGK led to destabilized TIM22 complex; defects in the biogenesis of carrier substrates (such as adenine nucleotide translocator); lower complex I, III, and IV activities; perturbed tricarboxylic acid (TCA) cycle; and higher citrate synthase activity (300).
Tim8a (Tim8a/DDP1 and Tim8b/DDP2 are human homologs of yeast Tim8) is a small TIM chaperone in the IMS. Tim8a expression in ischemic rat heart was downregulated by treatment with GSK inhibitor SB216763, which showed a protective effect against I/R stress (258), suggesting a potential role of Tim8a in ischemic heart disease.

MIA Machinery Related to Heart Disease
FAD-linked sulfhydryl oxidase ALR is the human homolog of yeast Erv1. The interaction between Mia40 and Erv1/ALR facilitates the import of the small Tim proteins and cysteinerich proteins. Inhibition of ALR activity by MitoBloCK-6 or a translation initiation codon (ATG) morpholino targeted to ALR in zebrafish embryos led to retarded cardiac development and impaired cardiac function (287).
Apoptosis-Inducing Factor (AIF) was initially characterized as a pro-apoptotic factor. It translocates from the mitochondrial IMS to the nucleus in the presence of apoptotic insults. It is also critical for the mitochondrial import and maturation of CHCHD4 (in human)/Mia40 (in yeast) (301)(302)(303). Mutations of the AIF-encoding gene AIFM1 led to early prenatal ventriculomegaly (288) and childhood cardiomyopathy (289), accompanied by respiratory chain complex I and IV deficiency. Global loss of AIF in mice during embryogenesis resulted in embryonic growth retardation and death during mid-gestation. Muscle-specific loss of AIF in mice led to severe DCM and skeletal muscle atrophy, associated with a significant defect in respiratory chain complex I activity (290). The Harlequin (Hq) mice, a genetic model with an 80% reduction in mitochondrial AIF, displayed more severe ischemic damage than wild-type hearts after acute I/R injury (291,292).
Other Molecules Involved in Heart Disease by Impacting Mitochondrial Protein Import Efficiency or Altering Mitochondrial Protein Translocation Cardiolipin (CL) is a unique phospholipid that is localized and synthesized in mitochondrial IM. CL plays a central role in many biological processes, such as mitochondrial biogenesis, protein import, morphology and mitophagy, oxidative phosphorylation, and apoptosis (304)(305)(306). Defective remodeling of CL due to genetic mutations of TAZ-1 causes Barth syndrome, a rare, X-linked recessive, infantile-onset debilitating disorder characterized by early-onset cardiomyopathy, skeletal myopathy, growth delay, and neutropenia (304)(305)(306)(307)(308)(309). The molecular mechanisms were partially mediated by impaired mitochondrial import machinery. CL was verified to play a central role in the structural integrity and functions of mitochondrial translocases, such as TOM Complex (44, 310), TIM22 Complex, and TIM23 Complex (26, 311,312).
Connexin 43 (Cx43) is the predominant protein forming gap junctions and non-junctional hemichannels in ventricular cardiomyocytes and is also localized at the IM of cardiomyocyte mitochondria (313)(314)(315). The translocation of Cx43 to the IM was TOM-HSP90-dependent and was enhanced by ischemic preconditioning (IP). The beneficial role of mitochondrial Cx43 in I/R stress was associated with its regulation of mitochondrial potassium influx and ROS production (257,(313)(314)(315). The cardioprotection of IP was abolished by genetic ablation of Cx43, blockade of mitochondrial Cx43 import, or age-related loss of mitochondrial Cx43 (257,(316)(317)(318).
PINK1 is imported into the mitochondrial matrix in healthy conditions, followed by retrotranslocation into cytosol and degradation by the proteasome. Perturbation of this process causes mitophagy, which plays a vital role in the quality control of mitochondria in heart disease. Given that it has been well-studied and summarized in many other reviews (319, 320), we do not discuss it here in detail.
NDUFB10, an accessory subunit of complex I, is a substrate of the MIA machinery (CHCHD4/ Mia40) for oxidationdependent protein import into the mitochondrial IMS. Mutation of cysteine 107 of NDUFB10 impaired its mitochondrial import via CHCHD4 and resulted in complex I assembly defect, led to fatal infantile lactic acidosis and cardiomyopathy in a single-case report of an infant (321).
In addition, some other proteins also showed a protective effect against I/R or H/R stress through enhancing their translocation from the cytosol to the mitochondria; these include α-crystallin B (cryAB, the major small heat shock protein in cardiomyocytes) via VDAC-Tom20 (322) and DJ-1 via mtHSP70 (246,(323)(324)(325).

Other Conditions That Affect Mitochondrial Protein Import Efficiency in Heart Disease
According to their subcellular spatial arrangement in cardiomyocytes, mitochondria are classified into three groups: subsarcolemmal mitochondria (SSM) existing below the cell membrane, interfibrillar mitochondria (IFM) residing in rows between the myofibrils, and perinuclear mitochondria located at the nuclear poles. Mitochondrial subpopulations vary in structure and function and appear to be influenced disparately in different cardiac pathologies, including I/R, heart failure, aging, exercise, and diabetes mellitus. According to recent studies, the mitochondrial import machinery in IFM of T1DM hearts (247,326) and SSM of T2DM hearts (248,327) were more susceptible to inefficiency. Further, many studies reported the downregulation of mitochondrial importmachinery components in heart disease, such as heart failure, DCM, ischemic cardiomyopathy, diabetic cardiomyopathy, etc. Supplements of corresponding components could partially recover cardiac function.
However, some studies pointed out an enhancement of mitochondrial protein import in aging animals. Craig et al. found mitochondria from senescent animals exhibited a higher import rate of precursors into the matrix than mitochondria from young animals (328). Later studies showed that, although expression of some key import machinery components was upregulated in the aging heart, import efficiency was compromised (238). The mechanism and significance need to be determined in future studies.
In addition, the import rate of matrix-localized proteins was found to be increased in heart of hyperthyroid animals or by T3 treatment (329)(330)(331)(332), which was partially mediated by elevated levels of the OM receptor Tom20 and mtHSP70. Meanwhile, the proteolysis of matrix proteins was unaffected.

CONCLUSION
Mitochondrial import machinery pathways are involved in various heart diseases, including heart failure, DCM, hypertrophic cardiomyopathy, ischemic cardiomyopathy, and diabetic cardiomyopathy. Mutants of genes encoding components of the mitochondrial import machinery in humans or genetic deficiency of those genes in animals usually cause severe mitochondrial cardiomyopathy and are lethal, highlighting the critical role of mitochondrial import machinery in heart disease. However, compared with neurodegenerative diseases, in which the functions of mitochondrial import machinery are relatively well-studied and established, research in heart disease is still fairly limited. Although many studies detected some components of mitochondrial import machinery, most studies simply regarded those components as indicators of mitochondrial content to evaluate mitochondrial biogenesis or mitophagy. The function of mitochondrial import machinery was highly neglected. Actually, even from the perspective of mitochondrial biogenesis or mitophagy, under different stimuli, import machinery components are able to adapt to diverse cellular functions, which are not always proportional to mitochondrial quantity.
Furthermore, our current understanding of mitochondrial import machinery in heart disease is still widely based on experimental evidence from yeast. Nevertheless, recent research in higher eukaryotes has identified more complex and diverse functions in some conserved components of mitochondrial import machinery. Furthermore, with the development of high-throughput sequencing in genomics, transcriptomics, proteomics, and metabolomics, more and more novel import machinery components have been revealed in mammals. The roles of mitochondrial import machinery in heart disease deserve considerable attention, and future studies are urgently needed.

AUTHOR CONTRIBUTIONS
FZ devised the original idea of the work, searched the literature, and wrote the manuscript. M-HZ edited the manuscript. All authors made significant contributions to this work and approved it for publication.

FUNDING
M-HZ's lab was supported by funding from the following agencies: National Heart, Lung, and Blood Institute (HL079584, HL080499, HL089920, HL110488, HL128014, HL132500, HL137371, and HL142287) and National Cancer Institute (CA213022). M-HZ is the Eminent Scholar in Molecular and Translational Medicine of the Georgia Research Alliance.