A novel perspective for burn-induced myopathy: Membrane repair defect

Myopathy is a common complication of severe burn patients. One potential cause of this myopathy could be failure of the plasma membrane to undergo repair following injuries generated from toxin or exercise. The aim of this study is to assess systemic effect on muscle membrane repair deficiency in burn injury. Skeletal muscle fibers isolated from burn-injured mice were damaged with a UV laser and dye influx imaged confocally to evaluate membrane repair capacity. Membrane repair failure was also tested in burn-injured mice subjected to myotoxin or treadmill exercise. We further used C2C12 myotubules and animal models to investigate the role of MG53 in development of burn-induced membrane repair defect. We demonstrated that skeletal muscle myofibers in burn-injured mice showed significantly more dye uptake after laser damage than controls, indicating a membrane repair deficiency. Myotoxin or treadmill exercise also resulted in a higher-grade repair defect in burn-injured mice. Furthermore, we observed that burn injury induced a significant decrease in MG53 levels and its dimerization in skeletal muscles. Our findings highlight a new mechanism that implicates membrane repair failure as an underlying cause of burn-induced myopathy. And, the disorders in MG53 expression and MG53 dimerization are involved in this cellular pathology.

membrane permeability, which ultimately result in serious myopathy 15,16 . It has been reported that MG53 is transcriptionally activated by IRS-1/PI3K/Akt signal pathway in skeletal muscle fibers 17 . However, this signal pathway is always inhibited in skeletal muscles for prolonged periods of time after burn injury 18 . Consequently, there is a possibility that skeletal muscle membrane in burn injury would be fragile because of MG53 deficiency.
Here, we hypothesized that sustained musculoskeletal abnormalities that occur in burn patients are caused by on-going membrane injury. As a test of this hypothesis, we extracted skeletal muscles distant from the site of burn injury, and assessed systemic effect on musculoskeletal membrane integrity and musculoskeletal membrane repair deficiency in burn injury using various methodologies. We further used an in vivo and in vitro model of burn injury to investigate the underlying molecular mechanisms associated with defective membrane repair in burn-injured mice.

Results
In vivo analysis of muscle membrane integrity after burn in mice. To determine whether myocytes membrane integrity is affected in distant muscles as a result of burn injury, we monitored gastrocnemius muscle myofibers permeability by exploiting Evans Blue dye (EBD). Mice were intraperitoneally or intramuscularly injected with this dye to identify injured and "leaky" myofibers membrane. Gastrocnemius muscle sections from burn-injured mice displayed significantly more areas of EBD staining when compared with sham-injured mice (Fig. 1a,b). Quantitative analysis of total EBD within extracted gastrocnemius muscle bundles provided direct validation to the above results and displayed a significant increase in dye after burn injury (Table 1). Taken together, these results reveal the presence of musculoskeletal membrane damage in a distant muscle as a result of burn injury. (b) EBD was intraperitoneally injected into control and burn injured (Post Burn Day14) mice. Membraneinjured myofibers in gastrocnemius muscles from sham injury (C) and burn injury (B) in mice are labeled. The number of EBD-positive myofibers counted from these micrographs. *P < 0.05; vs. Control; n = 5 (Scale bar, 500 μm; 200 μm).
Scientific RepoRts | 6:31409 | DOI: 10.1038/srep31409 In vivo/ex-vivo analysis of muscle membrane repair after burn in mice. To test if myocytes membrane injury is exacerbated in burn-injured mice during eccentric contraction, our study measured gastrocnemius muscle myofibers permeability by EBD in mice subjected to treadmill exercise 11,19,20 . Compared with non-exercised controls, sham-injured mice demonstrated no significant changes in EBD staining after downhill running (Fig. 2a,b). In contrast, burn-injured mice showed significantly more EBD staining after downhill running, when compared with non-exercised controls (Fig. 2a,b). These findings were supported by quantitative analysis of total EBD within extracted gastrocnemius muscle bundles (Table 1). We next injected myotoxin from Naja mossambica (CTX) 19 , a toxin that injures myofibers membrane, into the gastrocnemius muscles. At 12 hours post-injection, the serum LDH and CK-MB levels in burn-injured mice significantly increased and provided an effective marker of the disruptions of myocytes membrane (Fig. 2c,d).
To directly evaluate musculoskeletal membrane repair efficiency of burn-injured mice, we subjected flexor digitorum brevis (FDB) myofibers isolated from burn-injured and sham mice to laser-induced injury and monitored membrane permeability via FM1-43 fluorescent dye entry 20 . FM1-43 strongly and selectively stains the plasma membrane of intact myocytes, however, this pattern can be disrupted with laser damage, which causes membrane disruption and diffusion of the dye to the cytosol. We demonstrated that normal myofibers from sham-injury mice could rapidly and effectively reform injured membrane, as they only showed minimal FM1-43 dye influx after ultraviolet laser damage (Fig. 3). In contrast, we observed more FM1-43 fluorescent dye entry in myofibers from burn-injured mice after laser-induced damage (Fig. 3).
Thus, both in vivo and ex-vivo model systems strongly support our hypothesis that muscle membrane repair fails in a distant muscle as a result of burn injury.

Effects of burn injury on MG53 expression and dimerization.
To investigate whether there was a causal relationship between MG53 and burn-induced sarcolemmal membrane damage, we first assessed endogenous MG53 protein levels in gastrocnemius muscle by western blot and immunohistochemistry analysis. Western blot analysis illustrated the statistically significant decline in MG53 expression in gastrocnemius muscle at 10 and 14 d after burn injury (Fig. 4a). Immunohistochemistry analysis validated that the MG53 protein is mainly localized to the myocytes membrane and this staining pattern is significantly decreased after burn injury when compared with sham controls (Fig. 4b). Furthermore, Western blot analysis (in the absence of DTT) showed that burn injury induced a significant decline in MG53 dimer in muscles at 5, 10 and 14 d after burn injury, compared with sham injury control (Fig. 4c).
Protein disulfide isomerase (PDI) inactivation disrupts MG53 dimerization. PDI can catalyze the formation and breakage of disulfide bonds between cysteine residues within many proteins. Lacking functional PDI can increase cellular protein misfolding and thus result in a serious perturbation in dimerization of these proteins 21 . To determine whether the process in MG53 dimerization is affected by PDI, we detected MG53 dimer in C2C12 myotubules after inhibiting PDI activity with 16F16 and thiomuscimol 22 . As illustrated in Fig. 5a,b, inhibition of PDI resulted in a significant decline in MG53 dimer levels in C2C12 myotubules, while showed no significant changes in MG53 protein levels. Furthermore, PDI activity was demonstrated to be reduced in gastrocnemius muscle at 5, 10 and 14 d after burn injury (Fig. 5c), indicating that PDI inactivation could be a possible cause of MG53 dysfunction in burn injury.
Reportedly, NO mediated S-nitrosylation of PDI (SNO-PDI) can inhibit PDI activity and the beneficial effects of PDI 23 . In this study, the NO levels were significantly increased in gastrocnemius muscle after burn injury (Fig. 5d). We further demonstrated that the SNO-PDI levels were significantly increased in gastrocnemius muscle after burn injury (Fig. 5e).
Moreover, it has been established that PDI can be oxidized and lose its activity in presence of endoplasmic reticulum stress (ERS) 24 In this study, we illustrated that CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP), a major ERS marker, was significantly increased in gastrocnemius muscle at 5, 10 and 14 d after burn injury (Fig. 5e). Fig. 6a,b, insulin could upregulate MG53 mRNA and protein levels in C2C12 myotubules when compared with controls, and that these effects could be abolished by inhibition of PI3K via LY294002. These results validate the notion that MG53 expression is regulated by IRS-1/PI3K signal pathway 17 . In this study, burned mice showed that IRS-1 expression in muscles began to decrease at 2 d after burn injury and that this decline remained up to 14 d after the injury, when compared with sham controls (Fig. 7a). The in vitro studies also showed that burn serum induced a significant decline in IRS-1 protein levels in myotubules when compared with the controls (Fig. 7b,c).

Insulin insensitivity induced by Suppressor of cytokine signaling (SOCS). It has been estab-
lished that SOCS-1 and SOCS-3 are transcriptionally regulated by JAK/STAT signal pathway, and have been shown to block IRS-1/PI3K/Akt signal pathway. To investigate the roles of SOCS-1 and SOCS-3 in insulin insensitivity after burn injury, we demonstrated that both SOCS-1 and SOCS-3 were significantly increased in gastrocnemius muscles two weeks after burn injury (Fig. 7a). The in vitro studies also illustrated that burn serum increased the expression of both SOCS-1 and SOCS-3 proteins when compared with control myotubules cultured with sham serum (Fig. 7b,c). Moreover, we demonstrated that the burn serum-induced increase in SOCS-1 and SOCS-3 protein levels both returned to control values by inhibition of STAT with S3I-201, while IRS-1 began to increase significantly (Fig. 7d).

Discussion
Musculoskeletal derangements, a well-known medical complication of burn patients, include muscle atrophy, muscle weakness and bioenergetic disorders, markedly contributing to the incidence of mortality and disability in burn patients 1,25 . This acquired systemic skeletal muscle myopathy, generally speaking, has been viewed as a result of muscles proteolysis causing by systemic inflammatory response syndrome (SIRS) 5 . However, the proteolytic pathways in skeletal muscles display persistent activation after burn injury, even when inflammatory factors in plasma are significantly decreased. Recently, abundant evidence indicates that sarcolemmal membrane injury is a major factor in the development of many myopathies 11,26,27 . However, the roles of sarcolemmal membrane damage in myopathy in burns remain unknown. A striking observation of our study was that burn injury induced a serious failure of plasma membrane repair, resulting in skeletal muscle membrane injury. In this study, myofibers isolated from burn-injured mice displayed a significant increase in EBD staining via morphological and quantitative analyses, indicating that burn injury exacerbated skeletal muscle membrane injury. How could severe burns result in myofiber membranes damage? Membrane repair response is such a crucial process to repair the membrane of the injured myotubes in a timely and accurate manner that membrane damages are transient and rarely cause fatal injuries. In ex-vivo, we found that skeletal muscle myofibers from burn-injured mice displayed a disability to repair membrane disruptions following laser damage. In vivo, we used EBD to show that repair of damages, induced by treadmill exercise, is impaired in burn-injured mice, indicating that the membrane repair defect is not one unique to a UV laser injury, but one that arises under physiological conditions as well. Furthermore, compared with sham controls, the serum LDH and CK-MB levels in burn injury significantly increased after intramuscular injection of CTX, which also provided an effective marker of the skeletal muscle membrane repair defects in burn injury. Therefore, these findings in vivo and ex-vivo models suggest burn injury led to a significant failure of plasma membrane repair in skeletal muscle, which could be a major pathogenesis of burn-induced musculoskeletal membrane injury.
It is convinced that continuous release of intracellular "danger" signals or myokines from myocytes with defective membrane repair induces localized inflammation and exerts autocrine, paracrine and endocrine effects within the muscle itself as well as other organs to aggravate muscular bioenergetic dysfunctions, then resulting in muscle atrophy, muscle weakness, thus leading to myopathy 10,28 . Indeed, multiple forms of muscular diseases are caused by sarcolemmal membrane repair failure, such as DMD and diabetic myopathy. Interestingly, severe burns also induce a significant reduction of muscle mass, capacity, and bioenergetic dysfunctions, and these muscular changes in burn patients are similar to that showed in DMD and diabetic myopathy 11,27 , indicating that How could burn injury result in membrane repair failure? Numerous proteins have been demonstrated to participate in sarcolemmal membrane repair response, one of which is MG53. There are abundant studies showing that MG53 plays a major role in sensing the influx of extracellular oxidants and facilitating "patch vesicles" translocation of membrane repair machinery to initiate this repair response 12,15,16,29,30 . It has been reported that the mice lacking functional MG53 result in the development of muscular atrophy 15,19 . In particular, protein disulfide bond formation mediated by Cys242 is essential for MG53-mediated translocation of "patch vesicles" toward the disruption sites, as expression of cysteine mutation, C242A, results in nearly complete loss of MG53 dimerization and a significant failure of membrane repair 31 . Strikingly, MG53 dimer levels in skeletal muscles began to decrease at 5 d after burn injury, while MG53 expression showed no significant changes in these muscles at this time point. These results indicate that burn injury induces a perturbation in MG53 dimerization, which can be a key molecular mechanism leading to burn-induced musculoskeletal membrane repair failure.
To our knowledge, PDI has been demonstrated to participate in disulfide bonds formation between cysteine residues, and is essential for the dimerization of many proteins 32 . In this study, our in vitro findings showed MG53 dimer levels in myotubules were reduced by inhibition of PDI with 16F16 and thiomuscimol, indicating that PDI was an essential enzyme participating in the formation of disulfide bonds between MG53. Furthermore, we showed that the PDI activity in skeletal muscle was significantly decreased after burn injury. Therefore, these findings together indicate that burn-induced PDI inactivation, can inhibit MG53 dimerization, and finally, result in membrane repair failure. We further demonstrated that the NO levels in skeletal muscle significantly increased, and PDI was mediated by NO to produce a "SNO-PDI". This NO mediated S-nitrosylation of PDI could be a possible cause of PDI inactivation in burn injury 22,23 . In addition, PDI can become oxidized and lose its ability to function as a disulphide bond-rearranging enzyme in the presence of ERS 24 . We demonstrated that CHOP, a major ERS marker, was significantly increased in skeletal muscles after burn injury, indicating that ERS also participated in burn-induced PDI inactivation.
In addition, we showed that the total MG53 content in skeletal muscle was reduced at the beginning of 10 d after burn injury. As a result, MG53-deficiency could be another molecular mechanism causing burn-induced sarcolemmal membrane repair defect 15 . In C2C12 myotubules, MG53 mRNA and protein levels could be elevated by insulin and abolished by PI3K inhibition using LY294002, indicating that burn-induced MG53-deficiency could be caused by insulin insensitivity. Indeed, skeletal muscle insulin insensitivity is a common phenomenon observed for prolonged periods of time after burn injury 18,[33][34][35] . Thus, we believe that this burn-induced chronic insulin insensitivity participates in sarcolemmal membrane repair deficiency via MG53.
It has been established that IRS-1 is a major protein to transport the signal from insulin and its deficiency has been observed to be a common primary cause of persistent insulin insensitivity 18 . In this study, we illustrated that burn injury induced a persistent decline in IRS-1 protein levels in skeletal muscles, which validated the notion that IRS-1 proteins are a main target for the development of chronic insulin insensitivity in burn injury. Meanwhile, we demonstrated that both SOCS-1 and SOCS-3 were significantly increased in skeletal muscles two weeks after burn injury. Further, in our in vitro model system, we observed that burn serum rapidly enhanced SOCS-1 and SOCS-3 expression, and subsequently decreased IRS-1 protein. However, these effects were shown to be abolished by inhibition of STAT with S3I-201. These findings in vivo and in vitro together suggest that a high-grade of inflammation is induced after burn injury and that SOCS-1 and SOCS-3 mediated ubiquitin pathways could be involved in degradation of IRS-1 after burn injury, which finally results in prolonged insulin insensitivity.
The fact that membrane repair failure is a major pathogenetic mechanism of burn-induced myopathy cannot be undermined. In our opinion, promotion of endogenous MG53 and MG53-dimerization is a novel measure to repair sarcolemmal membrane and prevent burn-induced myopathy. In addition, Weisleder and colleagues recently demonstrated that the mice treated with recombinant human MG53 (rhMG53) displayed decreased muscle membrane damage and reduced muscle pathology without toxicity 19 . These results suggest that treatment with recombinant human MG53 could improve muscle membrane damage in burn injuries.
In summary, we provide evidence from in vivo and ex-vivo assays that show that the burn injury induces a significant failure of musculoskeletal membrane repair, which leads to skeletal muscle membrane damage. It is conceivable that this membrane repair failure could negatively affect musculoskeletal functions in burn patients. Furthermore, our results have identified, for the first time that the disorders in MG53 expression and MG53 dimerization are involved in musculoskeletal membrane repair defects as a result of burn injury. However, the pathogenesis of myopathy in burn patients is thought to be complex and multifactorial, and therefore, trials on human subjects are warranted to confirm the findings of our study, and their application to humans.
Animal models. One hundred eight male BALB/C mice (6 weeks old, 25 g to 30 g body weight) were obtained from the Third Military Medical University Laboratory Animal Centre. The mice were raised in individual cages under 22-25 °C and with a 12 h light-dark cycle. The mice were used to the environment with a standard diet for a week prior to the experiment. One hundred eight mice were randomly divided into two groups, control (C) and burn-injury (B) groups. The mice from B group were subjected to 30% total body surface area (TBSA) of full thickness burn as previously described 36 . Briefly, mice were anesthetized with 1% pentobarbital (40 mg/kg of body weight), treated with buprenorphine (1 mg/kg body of weight) for analgesia and their backs were shaven with hair clippers. Mice were placed in a model designed to expose 30% of their dorsa skin to a hot water bath at 90 °C for 10 s. After injury, mice were intraperitoneally injected with lactated ringer's solution (50 ml/kg of body weight). Sham injured mice were anesthetized, shaven, and intraperitoneally injected with lactated ringer's solution. Each treatment group (sham control, 2, 5, 10, and 14 d after burn) employed twelve animals. All animal experimental protocols were approved by the Third Military Medical University Animal Care Committee according to the