223 Potential Association of Burn-Induced NMJ Derrangement and Oxidative Stress-Induced AChR Declustering Signal and the Reversal with AChR alpha7 Agonist

Skeletal muscle wasting and muscle weakness often accompanies critical illnesses including severe burn injury and is associated with a poor prognosis of the patient. Detailed mechanisms remain to be fully clarified. We have previously reported abnormal morphology of neuromuscular junction (NMJ) in the mouse burn model. Burn serum caused inhibition of agrin-induced acetylcholine receptor (AChR)- clustering signal in C2C12 myocytes. What has to be determined has been whether AChR de-clustering signal is involved, and whether it can be prevented. We have previously reported that AChR alpha7 agonist, GTS-21 ameliorates burn-induced muscle wasting, but the precise mechanism was not fully determined. In this study, we tested whether GTS-21 will ameliorate oxidative-stress-induced AChR de-clustering in myocytes. To establish the AChR de-clastering assay, C2C12 cells were cultured into differentiated myocytes. At day 7, AChR clustering was induced by incubating with agrin. To induce AChR de-clustering, agrin was depleted and oxidative stress was induced with hydrogen peroxide (H2O2) in the presence or absence of AChR alpha 7 agonist, GTS-21 (1mM). The chronological changes in the area size of AChR clustering was monitored. Oxidative stress (H2O2) facilitated the AChR de-clustering in the absence of GTS-21 (Fig. a&b, 57% decrease). GTS-21 treatment (Fig. c&d) prevented the de-clustering (p<0.01). It was previously considered that NMJ, once formed, is a solid and stable structure, but according to more recent evidence, AChR clustering is a dynamic process, affected by the balance between the clustering and de-clustering signals. Simple disuse or pharmacological blockade leads to a reversible de-clustering of AChRs at NMJ. In support of our previously report showing that burn injury leads to functional and morphological alterations in NMJ, the current study demonstrated that oxidative stress leads to de-clustering of the AChRs in C2C12 myocytes. GTS-21 ameliorated the de-clustering, suggesting that α7AChR stimulation is effective on mitigating oxidative stress-induced de-clustering of AChRs. The potential efficacy of GTS21 in mitigating oxidative stress-induced AChR de-clustering was documented for the first time, suggesting it can be used for treating burn-induced neuromuscular dysfunction.