Role Of Cx43 In The Inhibition Of MG53 On Ischemia/Reperfusion Induced Arrhythmias

BackgroundReperfusion arrhythmia(RA) is a common electrophysiological phenomena during ischemia/reperfusion(I/R), it occurred in the coronary artery reperfusion within a short time and it also an important cause of death in patients with reperfusion treatment.The mechanism of RA is complex, including oxygen calcium overload, free radical, reentry generation, gap junction remodeling and free fatty acid increased. They are go hand in hand with cell membrane integrity, ensure the integrity of cell membrane may reduce the incidence of RA.In recent years, Mitsugumin53(MG53) have been found can repair the cell membrane, it’s a muscle specific protein. The repair effect in skeletal muscle cells, myocardial cells and alveolar epithelial cells has been confirmed. The researchers found that MG53 proteins play an important protective role in cardiac I/R, at the same time, ischemic preconditioning(IPC) can promote MG53 expression. Numerous studies report that IPC can reduce the severity and incidence of RA significantly.Gap junction protein Connexin43(Cx43) distributed in the myocardium intercalated disc as phosphorylated form under physiological conditions. The formation of gap junction channel number, size, features and connect shape of myocardial cells is the basis to conduct electrical impulses. During I/R, Cx43 happens dephosphorylation and side to side distribution, lead myocardial cellselectric conduction anisotropic and time inconsistency of depolarization repolarization, they caused arrhythmia. In addition, acidosis and calcium overload caused the Cx43 structure and function change during I/R.Cx43 mainly exist in the cell membrane, while MG53 can repair damage cell membrane. In view of the fact that PKCεsignal pathway can make Cx43 maintains a stable distribution and phosphorylation, while MG53 can activate PKCε signalpathway play a protective role in I/R. These results indicate that PKCε signal pathway may be involved in the process of MG53 inhibit the dephosorylation and redistribution of Cx43.Base on the above results, we hypothesized that during I/R injury: MG53 play a protection role in RA through inhibition of Cx43 redistribution, and enhance the combination ability of Cx43 with PKCε to suppress the Cx43 dephosphorylation.Purpose(1) To make clear that MG53 paly a protect role on myocardial ischemia / reperfusion arrhythmia.(2) Discussion the mechanism on the role of Cx43 in MG53 improve the ischemia / reperfusion arrhythmia.Content(1) Establish SD rat heart Langendorff ischemia/ reperfusion arrhythmia model, observe the effect of MG53 on RA.(2) Establish H9C2 cell hypoxia /reoxygenation model, observe the effect of MG53 on cell viability.(3) Obeseve Cx43 phosphorylation and distribution in RA model when MG53 pretreament.(4) To determine whether MG53 and Cx43 exsit connection,and the influence of the connection as well as MG53 pretreatment.(5) To determine whether PKCε and Cx43 exsit connection,and the influence of the connection as well as MG53 pretreatment.Method(1) Establish SD rat heart Langendorff ischemia/ reperfusion arrhythmia model, observe the occurrence and severity of arrhythmias during reperfusion whenMG53 pretreatmented.(2) Tranfected MG53 plasmid into H9C2 cells, and normal extracted cell protein, Western Blot to observe whether the high expression of MG53 protein. Establish hypoxia / reoxygenation(H/R) model, CCK-8 to observe the protection of MG53.(3) Conventional extract myocardial protein, Western Blot observe the expression of Cx43 and pCx43.(4) Conventional paraffin sections of fixed heart specimen, immunofluoresence observe the distribution change of Cx43.(5) Conventional extract myocardial protein, Co-Immuneprecipitation was used to detect MG53 and Cx43 exist connection, and the influence of the connection as well as MG53 pretreatment.(6) Conventional extract myocardial protein, Co-Immuneprecipitation was used to detect PKCε and Cx43 exist connection, and the influence of the connection as well as MG53 pretreatment.Result(1) Compared with I/R group, MG53 pretreatment group can obviously reduce the reperfusion arrhythmia score, reduce the incidence of VT and VF, shorten the duration of VT and VF.(2) The expression of MG53 protein improved significantly after MG53 plasmid transfection. MG53 can significantly improve the cell vitality during H/R damage.(3) MG53 pretreatment can keep Cx43 protein phosphorylation expression and distributein a stable level, and inhibit its dephosphorylation and redistribute during ischemia / reperfusion injury.(4) MG53 and Cx43 exist connection, after MG53 pretreatment, it can increase the connection during ischemia / reperfusion injury.(5)Cx43 and PKCε exist connection, MG53 pretreatment can enhance its connection during ischemia / reperfusion injury.ConclusionMG53 can improve the incidence of ventricular arrhythmia and the severity during ischemia / reperfusion, Cx43 protein is a critical factor in it. MG53 play a protection role in RA through inhibition of Cx43 redistribution,and enhance the combination ability of Cx43 with PKCε to suppress the Cx43 dephosphorylation.