Membrane Repair Failure Induced By MG53 Dysfunction Plays A Key Role In Postburn Muscle Atrophy

ObjectiveThe aim of this study was to test the hypothesis that burn-induced injury results in skeletal muscle membrane repair defects, and to explore its underlying mechanisms.Methodsa. In vivo, two hundred sixteen BALB/C mice were randomly divided into three groups, namely, normal control(C), burned control(B), and insulin treated(I) groups. The mice in C group were only received anaesthesia and shaved. The mice from other two groups were inflicted with 30% total body surface area of full thickness burn by soaking90 ℃ water for 9 seconds. Burned mice were quickly injected with lactated ringer’s solution via intraperitoneal injection for resuscitation. After burn injury, mice were fed with normal diet and drinking water. The I group was supplemented with 5 IU/kg insulin per day via subcutaneous injections, and the C and B group were supplemented with the same dose of saline via subcutaneous injections. According to different experimental purposes,observation phase include post burn 2 day(PBD2), PBD5, PBD7, PBD10 and PBD14.1.Evans blue dye(EBD) was used to evaluate skeletal muscle membrane integrity.2.Myofibers from mice were injured with a laser and dye uptake imaged confocally to test membrane repair efficiency. Membrane repair defects were also assessed in these mice after downhill running, which induces myocyte plasma membrane disruption injuries in vivo.3.Glycerate-3-phosphate kinase oxidation(GK-GPO) was used to evaluate TG content in skeletal muscle.4.Treadmill was used to evaluate skeletal muscle capacity.5.Total mitsugumin53(MG53) content and oligomerization of MG53 were tested in skeletal musle. Proteins related to MG53 expression and oligomerization, such as AMP activated protein kinase(AMPK-a), p-AMPK-a insulin receptor substance-1(IRS-1),Suppressor of cytokine signaling-1(SOCS-1), SOCS-3, Protein disulfide isomerase(PDI) and CHOP, were also evaluated in skeletal muscle of mice after burn injury.b. In vitro, C2C12 myoblasts were differentiated into myotubes by incubation with DMEM supplemented with 2% horse serum for 48 h.1.Experiment is divided into normal control(Control), insulin treated(INS),insulin+PI3K inhibitors(INS + LY294002) group. Using polymerase chain reaction(PCR)and western blot(WB) method to detect MG53 mRNA and protein expression level.2.Experiment is divided into normal control(Control), AMP activated protein kinase(AMPK) agonist AICAR treated group. Using polymerase chain reaction(PCR) and western blot(WB) method to detect MG53 m RNA and protein expression level.3.Experiment is divided into normal control(Control), burn serum treated(BS), burn serum+STAT inhibitor(BS+S3I-201) group. Using WB and immunofluorescence(IF)method to detect the IRS-1, SOCS-1 and SOCS-3 protein expression.Results1.Compared with sham-injury mice, gastrocnemius muscle from burned mice at 14 d after burn displayed significantly more areas of Evans blue dye staining(P<0.05).Gastrocnemius muscle from mice treated with insulin showed significantly less EBD staining compared with untreated mice, but displayed greater EBD staining than sham injury controls(P<0.05).2.Gastrocnemius muscle from exercised versus non-exercised sham-injured mice showed no obvious changes in EBD staining(P>0.05). However, gastrocnemius muscle from burned mice demonstrated significantly more EBD staining after treadmill exercise when compared with non-exercised controls(P<0.05). We also showed that healthy myofibers from sham-injury mice could effectively reseal sarcolemmal membranes, as they only showed minimal FM1-43 dye entry after laser damage. In contrast, we observed significant FM1-43 fluorescent dye entry in myofibers from burn-injured mice after laser-induced damage(P<0.05). Insulin treatment can decrease the number of eccentric contraction-induced EBD-positive fibers in burned mice. Less entry of FM1-43 fluorescent dye could also be observed after laser-induced damage from insulin treated mice, compared with untreated controls(P<0.05).3.Our research showed that burn injury led to a significant decline in muscle mass at 5,10 and 14 d after burn, increase in TG content in muscle at 10 and 14 d after burn, and decrease in muscle capacity at 2, 5, 10 and 14 d after burn(P<0.05). Insulin treated burnedmice showed a significant decline in TG content and increase in muscle mass and exercise time, compared with untreated mice(P<0.05).4.Immunohistochemistry analysis illustrated that the MG53 protein is mainly localized to the membrane and this staining pattern is significantly decreased on PBD14 compared with sham controls(P<0.05). Western blot analysis highlighted the statistically significant decline in MG53 monomer on PBD14(P<0.05). MG53 polymer showed a significant decrease at 5, 10 and 14 d after burn injury(P<0.05). Insulin treatment could increase MG53 monomer(P<0.05), but did not enhance MG53 multimer at 10 and 14 d after burn when compared with untreated mice(P>0.05).5.Compared with sham controls, PDI and CHOP were significantly increased in gastrocnemius on PBD5, PBD10 and PBD14(P<0.05). Compared with burn controls,insulin have no significant effect on PDI and CHOP(P >0.05).6.Compared with control group, insulin could increase MG53 mRNA and expression in C2C12(P<0.05). Compared with INS group, PI3 K inhibitor could decrease MG53 mRNA and expression(P<0.05). However, AICAR have no effect on MG53 mRNA and expression in C2C12 when compared with control group(P>0.05).7.Compared with sham controls, AMPK-a、p-AMPK-a、IRS-1 protein expression was significantly reduced in gastrocnemius from 2d after burn injury up to 14 d after burn injury(P<0.05). SOCS-1 protein expression was significantly increased in gastrocnemius muscle from 2d after burn injury up to 10 d after burn injury(P<0.05). SOCS-3 protein expression was significantly increased in gastrocnemius muscle at 10 d and 14 d after burn injury(P<0.05).8.Western blot and immunofluorescence microscopy analysis also showed that the burn serum-induced increase in SOCS-1 and SOCS-3 protein levels returned to control values by inhibiting STAT1 and STAT3 with S3I-201, while IRS-1 begun to elevate(P<0.05).Conclusion1.Membrane repair failure could underlie the burn-induced sarcolemmal membrane damage to ultimately result in muscle atrophy.2.MG53 synthesis and oligomerization disorders are involved in postburn sarcolemmal repair failure.3.SOCS-1 and SOCS-3 mediated ubiquitin pathways could be involved in degradation of IRS-1 after burn injury, which finally lead to MG53 deficiency.4.Disorders in PDI caused by endoplasmic reticulum stress(ERS) after burn led to MG53 oligomerization dysfunction.5.Insulin treatment could increase MG53 expression, ameliorate membrane repair,thus alleviate sarcolemmal membrane damage postburn.