The Effects And Mechanisms Of Hypoglycemia On Cardiomyocytes Connexin43 Expression

Objective: To investigate the effects of hypoglycemia on ventricular arrhythmia and cardiac connexin43 expression in type 1 diabetic rat,and the effects of different glucose concentrations on cardiac connexin43 expression in H9c2 cells.Methods: The successful models were subsequently randomized to: 1)Diabetes mellitus(DM group,n=12);2)Diabetes mellitus hypoglycemia(DMH group,n=12).Another 12 rats served as sham controls(Con group,n=12).Hypoglycemia was induced by intraperitoneally administering short-acting insulin.Animal with glucose level ? 3.0mmol/l were considered hypoglycemia.2 hours after hypoglycemia,electrophysiology study was performed for rats to assess the incidence of inducible ventricular arrhythmias(VAs)and ventricular fibrillation threshold(VFT).The H9c2 cells were divided into high glucose group(HG group,25.0mmol/l),low glucose group(LG group,5.5mmol/l)and low glucose plus chloroquine(Chq group,5.5mmol/l),no glucose group(NG group,0mmol/l)and no glucose plus U0126(U0126 group,0mmol/l)randomly.The expression and distribution of Cx43 was assessed by cytofluorometry.Hematoxylin and eosin(H&E)staining was used to demonstrate myocardial morphology.The level of total Cx43 and phosphorylated Cx43 of myocardium were evaluated by immunohistochemistry.Western blot analysis was performed to assess related protein expression.Ultrastructural alteration in myocyte and autophagic vacuole were assessed by transmission electron microscopy.Then the cells were incubation for 2 h and assessed proliferation ability and cytotoxicity by MTT assay and LDH.Results: Compared with Con group,the DM group was significantly prolonged QTc interval,the inducible rate of ventricular arrhythmias was increased,VFT was significantly decreased.Western blot showed that the total Connexin43,phosphorylation of Cx43,p62 and LC3 protein expression was significantly increased in DM group compared with Con group.Compared with DM group,the QTc interval prolongation more obvious in DMH group,the inducibility of ventricular arrhythmias was significantly higher,while VFT value was further decreased.Hypoglycemia exacerbated the cardiac ultrastructural changes observed in diabetic rats,as indicated by the complete lack of structural organization of myofilaments and mitochondria vacuolization and the discontinuous intercalated discs,the levels of T-Cx43,P-Cx43 and p62 expression were all significantly decreased,while no significant changes was observed in LC3 protein in DMH group.Under low glucose conditions,the expression of Cx43 and p-Cx43 were significantly decreased in LG group than HG group.The LC3-II/LC3-I ratio was increased,while the amount of p62 was decreased in LG group.Compared with LG group,administration chloroquine could effectively inhibited low glucose-induced Cx43 downregulation,together with increased the LC3-II/LC3-I ratio and p62 protein.Under no glucose conditions,the expression of Cx43 was significantly increased in NG group than HG group,together with increased p-ERK protein.The proliferation ability was decreased,while LDH leakage was increased.The distribution of Cx43 was increased in cell surface.Compared with NG group,administration U0126 could effectively inhibited no glucose-induced Cx43 upregulation,together with decreased p-ERK protein and Cx43 distribution.No significant difference about proliferation ability and LDH leakage between NG group and U0126 group.Conclusion: Hypoglycemia increased the inducibility of VAs and decreased VFT in diabetic rats,which may be related with down-regulation of cardiac Cx43 and damage cardiac ultrastructure.The reduced Cx43 may be associated with increased the activity of autophagy.There exists a discrepancy regarding Cx43 expression under different glucose concentrations,as well the regulation mechanisms.