MG53 Promotes Membrane Repair of Skeletal Muscle.

MG53 was first reported to be a central component of the plasma membrane repair machinery (Cai et al., 2009a). Since striated muscle cells undergo severe membrane stress in response to muscle contraction, acute muscle membrane repair is particularly important. Deficiency of membrane repair causes muscle cell death, muscular injury, and dystrophy (Bansal et al., 2003; Bansal and Campbell, 2004; Han and Campbell, 2007). Upon muscular membrane rupture, MG53 senses the oxidized milieu from the extracellular space, interacts with phosphatidylserine, and localizes to the site of muscle fiber disruption, where it promotes plasma membrane resealing (Cai et al., 2009a). The MG53-mediated membrane repair process is regulated by multiple factors. Ca²⁺ is required for the fusion of vesicles with the plasma membrane, but not for vesicle trafficking (Cai et al., 2009a). The binding of MG53 to Zn²⁺ is indispensable for the membrane repair machinery (Cai et al., 2015). Mutation at cysteine 242 residue in MG53, which prevents oxidation-mediated oligomerization, impairs its membrane repair ability (Cai et al., 2009a; Hwang et al., 2011). The interaction of MG53 with dysferlin and caveolin-3 (CaV3) is essential for the cell membrane repair (Cai et al., 2009b).

The therapeutic potential of MG53-dependent prevention of skeletal muscle injury has been validated in multiple preclinical animal models. Systemic delivery of an adeno-associated virus overexpressing human MG53 protects skeletal muscle damage in a genetic muscular dystrophy hamster model through enhancement of membrane repair, activation of cell survival kinases, and inhibition of cell apoptosis (He et al., 2012). Furthermore, intravenous injection of MG53 recombinant protein ameliorates skeletal muscle damage in response to multiple insults, including toxic compounds (cardiotoxin VII4), genetic muscle diseases (mdx mice), and ischemia/reperfusion (I/R) and burn injuries (Weisleder et al., 2012; Zhu et al., 2015; Wang et al., 2016).

In contrast, it was reported that recombinant MG53 failed to protect against I/R-induced skeletal muscle injury in rats (Corona et al., 2014). The authors attributed this discrepancy (compared with data from other species) to either the more severe injury in this model, which was beyond therapeutic benefit, or to species differences (Corona et al., 2014). The serum concentrations of MG53 in mouse and human are significantly lower than that in rat, which makes rat muscle less sensitive to the therapeutic effect of recombinant MG53 (Zhu et al., 2015).

Based on the preceding data, although more work is required to fully elucidate its effects and mechanisms, MG53 is a molecular and important target with respect to therapeutic benefit in human skeletal muscle injury. As outlined in the following section, however, in other contexts, MG53 may confer detrimental effects.

MG53 Contributes to Skeletal Muscle Insulin Resistance.

MetS, which is a cluster of disorders including central obesity, dyslipidemia, and hypertension, increases the risk of cardiovascular diseases and type 2 diabetes and has become one of the most serious threats to human health (Grundy, 2006). Insulin resistance has been proven to be central to the mechanism of MetS (Eckel et al., 2005; McMillen and Robinson, 2005). Previous studies on the development of global insulin resistance have focused mainly on liver and adipose tissue; however, the role of skeletal muscle, which accounts for 70%–90% of insulin-stimulated glucose disposal (DeFronzo et al., 1981; Shulman et al., 1990), has been largely neglected.

Our recent studies have demonstrated that skeletal muscle insulin resistance is the initial and central pathologic process during the development of global metabolic disorders. As early as 1 week after high-fat diet (HFD) treatment, skeletal muscle, but not liver and fat, exhibits profound insulin resistance (Song et al., 2013). More importantly, deletion of MG53, which is highly expressed in striated muscle, can prevent systemic insulin resistance and metabolic disorders that are induced by overnutrition, including obesity, hypertension, hyperglycemia, hyperinsulinemia, dyslipidemia, and hepatosteatosis (Song et al., 2013). Furthermore, overexpression of MG53 in striated muscle is sufficient to elicit global insulin resistance and other metabolic disorders (Song et al., 2013), indicating the central role of MG53 in systemic insulin resistance and MetS. The important role of MG53 in skeletal muscle insulin resistance has been independently confirmed by another group (Yi et al., 2013). Also, a recent report has yielded preliminary evidence that MG53 may be involved in the protective effect of exercise against HFD-induced insulin resistance (Qi et al., 2016).

Post-transcriptional downregulation of IR and IRS-1 is a common feature of insulin resistance in both animal models and humans (Olefsky et al., 1976; Goodyear et al., 1995; Kerouz et al., 1997; Song et al., 2013); however, the mechanisms underlying this process remained unclear until recent reports that MG53 acts as an E3 ligase to mediate the degradation of IR and IRS-1 (Song et al., 2013). Ubiquitination is a versatile post-translational modification in eukaryotic cells that controls protein abundance through proteasome-mediated proteolysis (Huibregtse et al., 1995; Scheffner et al., 1995; Hershko and Ciechanover, 1998). Ubiquitin conjugation is catalyzed by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3); E3 ligase is responsible for the recognition of specific substrates. The RING finger domain of MG53 at its N terminal is a characteristic feature of a family of E3 ligases (Joazeiro and Weissman, 2000). Song and colleagues showed that MG53 is both sufficient and necessary for HFD-induced ubiquitination and degradation of IR and IRS-1 in skeletal muscle (Song et al., 2013). The RING domain and the cysteine 14 residue in this domain are indispensable for the E3 ligase activity of MG53 (Fig. 2) (Song et al., 2013). The E2-conjugating enzyme that helps MG53 to degrade IRS-1 was identified as UBE2H by another group, although this study failed to confirm ubiquitination and degradation of IR by MG53 (Yi et al., 2013).

Previous studies have shown that muscle-specific knockout of neither IRS-1 nor insulin receptor substrate-2 in mice causes any obvious dysregulation of glucose homeostasis (Long et al., 2011). Instead, mice with an approximately 50% reduction of both IR and IRS-1, but not either alone, develop profound MetS (Kido et al., 2000). If MG53 selectively downregulates IRS-1, but not IR, in skeletal muscle, it is not expected that transgenic overexpression of MG53 would lead to systemic insulin resistance and metabolic disorders (Song et al., 2013). Instead, the phenotype of MG53 transgenic mice is quite similar to those with a 50% reduction of both IR and IRS-1 (Kido et al., 2000; Song et al., 2013). Therefore, we conclude that MG53 mediates the degradation of both IR and IRS-1, resulting in systemic insulin resistance and subsequently whole-body MetS (Song et al., 2013), similar to the phenotype of the mice with an approximately 50% reduction of both IR and IRS-1.

In summary, MG53 is a novel E3 ligase, highly expressed in skeletal muscle, that targets both IR and IRS-1 for ubiquitin-dependent degradation. Overexpression of MG53 causes systemic insulin resistance and metabolic disorders, constituting a central mechanism responsible for the regulation of insulin sensitivity. Recently, a small molecule that disrupts the interaction between MG53 and IRS-1 and subsequent IRS-1 ubiquitination has been shown to improve insulin sensitivity in cultured myotubes (Lee et al., 2016), highlighting that targeting MG53 E3 ligase activity is a novel strategy to sensitize insulin response and treat MetS.

MG53 Inhibits Myogenesis.

Normal myogenesis is essential for the maintenance of skeletal muscle mass and function. When mature muscle is damaged, myogenesis takes place to repair the tissues through satellite cell activation and differentiation into new fibers (Chargé and Rudnicki, 2004; Rudnicki et al., 2008). Insulin-like growth factor-1 (IGF-1) is a key regulator of the differentiation of satellite cells and of the development, hypertrophy, and regeneration of skeletal muscle (Baker et al., 1993; Powell-Braxton et al., 1993; Glass, 2003). It is noteworthy that MG53 ablation preferentially targets IRS-1 with respect to the role of insulin signaling relative to IGF-1 signaling (Song et al., 2013), although IRS-1 is the shared mediator of both the IR and IGF-1 signaling pathways.

Elevation of MG53 abundance abolishes, whereas decrease of MG53 facilitates, the myogenesis of C2C12 myoblasts through ubiquitination and degradation of IRS-1 and consequent inhibition of IGF-1 signaling (Lee et al., 2010; Yi et al., 2013). Structurally, the middle and C-terminal regions of IRS-1 interact with the coiled-coil domain of MG53 (Lee et al., 2010). MG53-mediated myogenesis is also evidenced by the enlarged cross-sectional area and higher fatigue resistance in the skeletal muscle of mg53^−/− mice compared with wild-type littermates (Yi et al., 2013).

Nevertheless, Cai and coworkers have shown that MG53 deficiency leads to a progressive age-dependent muscular dystrophy in mg53−/− mice (Cai et al., 2009a); however, other studies have failed to detect any defects in the soleus, gastrocnemius, and quadriceps of mg53^−/− mice compared with wt littermates (Song et al., 2013; Yi et al., 2013). In general, the role of MG53 in skeletal muscle cannot be simply defined as “good” or “bad”; rather, it is muscle type- or disease condition-dependent. It merits future investigation to determine whether the lack of MG53-induced myopathy is model- and condition-dependent.

In addition to IR and IRS-1, focal adhesion kinase (FAK) is the third identified substrate of MG53 E3 ligase activity. MG53 causes the ubiquitination and degradation of FAK, subsequently contributing to MG53-mediated inhibition of myogenesis (Nguyen et al., 2014).

In overview, MG53 is a novel regulator of myogenesis, and targeting MG53 is a promising avenue for regenerative medicine in skeletal muscle diseases.

MG53 Induces Skeletal Muscle Stiffness.

Another mechanism underlying MG53-mediated impairment of skeletal muscle function is through its binding with sarcoplasmic reticulum Ca²⁺-ATPase 1a. This binding attenuates sarcoplasmic reticulum Ca²⁺-ATPase 1a activity and subsequently causes muscle stiffness and delayed relaxation (Lee et al., 2012). MG53 has also been found to regulate extracellular Ca²⁺ entry via Orai1 and intracellular Ca²⁺ release through Ry receptor 1 during skeletal muscle contraction (Ahn et al., 2016); however, the role of MG53 in muscle electrophysiology merits further study.

MG53 Role in Cardiac I/R Injury Protection.

The function of MG53 in cardiomyocytes was first reported by Cao and colleagues (2010), who identified MG53 as an important cardioprotective factor. Since adult mammalian cardiomyocytes are terminally differentiated and have a limited ability to regenerate, cardiomyocyte death leads to permanent loss of cardiac functional units and can cause multiple severe cardiac diseases, including myocardial infarction, I/R injury, arrhythmia, cardiac rupture, and heart failure. The most common cause of cardiomyocyte death is blockage of blood supply to the heart (myocardial infarction). Timely restoration of blood flow (i.e., reperfusion) is the best intervention to prevent cardiac cell death and injury induced by myocardial infarction; however, reperfusion causes further damage to the myocardium, termed I/R injury. To date, cardiac I/R injury remains a “neglected therapeutic target,” and only limited effective therapies for myocardial I/R damage are available (Hausenloy and Yellon, 2013).

Multiple lines of evidence have demonstrated that MG53 protects the heart against I/R injury (Wang et al., 2010; Cao et al., 2010; Zhang et al., 2011; Liu et al., 2015b). Cardiac I/R insult decreases myocardial MG53 protein levels (Cao et al., 2010; Zhang et al., 2011). MG53 deficiency exaggerates I/R-induced myocardial damage, whereas elevation of MG53 protects cardiomyocytes from oxidative injury (Cao et al., 2010). Interestingly, S-nitrosylation of MG53 at cysteine 144 abolishes I/R-induced downregulation of MG53 by inhibiting oxidation of cysteine 144 (Kohr et al., 2014). Furthermore, systemic delivery of recombinant MG53 alleviates cardiac I/R injury, suggesting that, in addition to intracellular, extracellular MG53 exerts a cardioprotective effect (Liu et al., 2015b).

MG53 plays an indispensable role in ischemic preconditioning (IPC) and postconditioning protection, both of which are the most powerful intrinsic protective machineries for cardiac I/R injury (Murry et al., 1986; Zhao et al., 2003). A lack of MG53 diminishes the cardioprotection conferred by both IPC and postconditioning (Cao et al., 2010; Zhang et al., 2011). It has recently been shown that MG53 may also participate in remote ischemic preconditioning, which is another form of intrinsic cardiac protection (Ma et al., 2016). MG53 abundance in the heart is also upregulated by anesthetic preconditioning with sevoflurane (Ma et al., 2013), indicating that, in addition to ischemic preconditioning, MG53 may play a role in other forms of cardiac preconditioning. Thus, maintenance of cellular MG53 levels is essential for myocardial integrity.

Several mechanisms have been shown to underlie MG53-elicited cardiac protection. Molecular signaling mediating IPC and PosC protection is classified into two major pathways: reperfusion injury salvage kinase (RISK, composed of PI3K-Akt-GSK3β and ERK1/2 signaling) and survivor activation factor enhancement (SAFE, composed of the activation of tumor necrosis factor-α and the JAK-STAT3 axis) (Crisostomo et al., 2006; Heusch, 2009; Lacerda et al., 2009; Lecour, 2009). MG53 is essential for the activation of RISK pathway, but not the SAFE pathway (Cao et al., 2010; Zhang et al., 2011). MG53 acts as a molecular bridge, binding to the p85 subunit of PI3K with its N terminus and to CaV3 with its C terminus. This dual binding facilitates the formation of the p85-PI3K/MG53/CaV3 survival complex, which is indispensable for activation of the RISK pathway (Cao et al., 2010; Zhang et al., 2011). In this way, in addition to its membrane repair function, MG53 serves as a docking/adapter protein for cell signaling to mediate activation of the cell survival signaling pathway.

Another mechanism of cardioprotection by MG53 involves the facilitation of cardiac plasma membrane repair, which is similar to that seen in skeletal muscle (Wang et al., 2010); however, the mechanisms underlying MG53-mediated membrane resealing in cardiomyocyte are different from those in skeletal muscle. Although MG53 does not bind to cholesterol- binding protein in lipid profiling analysis for skeletal muscle, (Cai et al., 2009a), cholesterol is indispensable for the membrane repair function of MG53 in cardiomyocytes (Wang et al., 2010). It is possible that a cholesterol-dependent secondary membrane structure, rather than the physical interaction of cholesterol with MG53, contributes to the membrane repair process. Additionally, in cardiomyocytes, the redox condition is not required for the translocation of MG53, but it is necessary for the stabilization of the nascent MG53 repair patch (Wang et al., 2010). Since the MG53-mediated membrane repair process and activation of cell survival signaling occur in or adjacent to lipid rafts or caveolae (Cai et al., 2009a; Wang et al., 2010; Cao et al., 2010; Zhang et al., 2011), it is possible that both mechanisms are involved in MG53-mediated cardioprotection.

Upregulation of MG53 Leads to Diabetic Cardiomyopathy.

Similar to the situation in skeletal muscle, MG53 exerts both beneficial and adverse effects in the heart. Diabetic cardiomyopathy is a major complication of diabetes and is the leading cause of morbidity and mortality for diabetic patients (Kannel et al., 1974; Kannel and McGee, 1979a,b,c; Devereux et al., 2000). Recently, it has been demonstrated that MG53 plays a critical role in the development of diabetic cardiomyopathy. In the hearts of multiple animal models with MetS, MG53 protein levels are profoundly upregulated. When specifically overexpressed in the heart, MG53 causes cardiac fibrosis and dysfunction, insulin resistance, depressed glucose uptake, and elevated lipid accumulation, all of which are typical phenotypes of diabetic cardiomyopathy (Liu et al., 2015b). Mechanistically, MG53 not only induces myocardial IR and IRS-1 degradation, insulin resistance, and disorders in glucose metabolism, but it also impaired cardiomyocyte lipid metabolism via transcriptionally upregulated peroxisome proliferation-activated receptor-α (PPAR-α) and its target genes (Liu et al., 2015b). Increased intracellular MG53 levels lead to excessive cardiac lipid accumulation, which contributes to the pathogenesis of diabetic cardiomyopathy (Liu et al., 2015b). Altogether, MG53 is a key regulator of both glucose and lipid metabolism in the heart.

Additionally, in a recent study, MG53 was expressed in cardiac fibroblasts. Inhibition of MG53 has been shown to prevent proliferation and migration of fibroblasts by regulating transforming growth factor-β signaling (Zhao and Lei, 2016). These findings indicate that MG53 may also be involved in cardiac fibrosis and remodeling.