Effect Of Diazoxide Preconditioning On The Expression Of Connexin 43 Gene And Its Protein Level In Myocardial Cellular Membrane Of A Myocardial Ischemiareperfusion Model Rat

Objective Myocardial ischemia reperfusion model rats were prepared. The present study was aimed to study the effect of diazoxide preconditioning on the level of myocardial connextin 43 mRNA, phosphorylated protein , nonphosphorylated protein and its intracellual structure, observe the effect of protein kinase C specific inhibitor chelerythrine on the expression of phosphorylated and nonphosphorylated Cx43 protein and its intracellular structure after diazoxide preconditioning thereby to investigate the possible mechanism of diazoxide preconditioning in cardialprotection.Methods A rat models of myocardial ischemia reperfusion injury was established. 82 healthy SD rats were randomly divided into 5 groups: sham operation group(sham group, n=10), ischemia reperfusion injury group(I/R group,n=22), ischemic preconditioning group(IP group, n=22), diazoxide preconditioning group(Dz group, n=22), and diazoxide preconditioning and chelerythrine treatment group(Dz+CH group, n=6). Myocardial ischemia was induced for 30min followed by reperusion for 0min(R 0min), 30min(R30min)or 120min(R30min). Cellular morph was analyzed by light microscope, CK-MB and LDH were assayed by automatic biochemistry analyzer. The infarct area was detected by TTC assay. The level of Cx43 mRNA in myocardium was analyzed by RT-PCR, and the expression of total Cx43 protein and nonphosphorylated Cx43 protein by Western Blot. The intracellular spatial distribution of Cx43 was observed by laser scanning confocal microscopy.Results 1. Dz group and IP group could significantly decrease infarct area and myocardial creatase level as compared to IR group, but with no statistical differences between them. 2. It was observed that in R 0min the levels of Cx43 mRNA in the ischemic area of IR group , IP group and Dz group were significantly lower as compared to sham values, but with a more significant decreasing trend with the reperfusion in IR group(P<0.05) and a increasing trend in IP group and Dz group(P>0.05);There was no significant difference between IP group and Dz group in the Cx43 mRNA level of ischemic area(P>0.05). 3. The expression of TCx43 and PCx43 were significantly decreased in the ischemic area of IP group as compared to the Sham group(P<0.05), but with no significant changes in the IP group and Dz group; Compared with the IR group, TCx43 and PCx43 in the ischemic area of IP group and Dz group were much higher in R 0min and R3 0min (P<0.05). 4. TCx43 fluorescence intensity of celluar membrane in R 0min in IR group was lower and NTCx43 fluorescence intensity higher than that in Sham group, but with no changes in IP group and Dz group. TCx43 fluorescence intensity increased gradually with reperfusion in IR group, IP group and Dz group, and NTCx43 fluorescence intensity decreased. Compared with the IR group, TCx43 fluorescence intensity in R 0min and R3 0min in IP group and Dz group were much higher with a decrease in NTCx43 fluorescence intensity, but there was no difference in R 120min and no difference in TCx43 and NTCx43 fluorescence intensity beween IP group and Dz group . 5. Compared with Dz group , TCx43 protein of ischemic area in R 0min decreased significantly after chelerythrine treatment (P<0.05), but with no significant changes in PTCx43 protein and NTCx43 protein(P>0.05).Conclusion 1. Ischemic preconditioning and diazoxide preconditioning could significantly reduced the ischemia reperfusion injury. 2. IR could decrease Cx43mRNA level of ischemic area and with a descending trend as reperfusion, decrease TCx43 and PTCx43 in R0 but with a increase tendency with reperfusion, also upregulated the NCx43 in R0 but with a descending trend. 3. Ischemic preconditioning and diazoxide preconditioning could increase the Cx43mRNA level and TCx43 and PTCx43 protein content of cellular membrane in ischemic area , decrease NTCx43 protein, but had no effect on that of nonischemic area. 3. Chelerythrine could reverse the increase of TCx43 protein caused by diazoxide, but had no effect on PCx43 and NTCx43 protein. The present study suggest that Cx43 may participate in the cardialprotection of ischemic preconditioning and diazoxide preconditioning, and the mechanism may involve the PKC pathway.