Hyperglycemia Induce Phosphorylation Of Cardiac Connexin 43 And Its Influence On GJIC

Gap junctions are collections of plasma membrane channels that enable adjacent cells to exchange cytoplasmic components directly without transit through the extracellular space. These channels allow passage of small molecules (generally less than 1000 Da) such as ions, individual amino acids or short peptides,second messengers (e.g. Ca2+, IP3, cAMP) and other metabolites.Gap junction intercellular communication (GJIC) mediated by connexin43 played an important role in the maintenance of the normal electric activities between cardiac myocytes. The increase in gap junctional communication correlated with the phosphorylation of Cx43. Gap junctional communication was also dependent upon extracellular calcium,entracellular PH value, phosphorylation status of Cx43. Recent work has also illustrated that hyperglycemia can induce phosphorylation of cardiac connexin 43 and change gap junction intercellular communication, but little is known about the mechanism and phosphorylation site of Cx43 hy hyperglycemia. Other investigators previously demonstrated that hyperglycemia induces a persistent actvation of protein kinase C(PKC) in vascular tissues and cells in culture,and actvation of protein kinase C(PKC) lead to serines phosphorylation of connexin 43.So we suggest that hyperglycemia may induces alteration of gap junction intercellular communication throught serines phosphorylation of connexin 43.In the present research, wild-type connexin43-pcDNA3 (Wt-Cx43-pcDNA3) and site-directed mutant connexin43-pcDNA3 (S262F-Cx43-pcDNA3 , S368A-Cx43-pcDNA3) plasmids were constructed and then transfected into HeLa cells respectively, which undergo further selection with G418 to purify those transfected cell types (Wt-Cx43-HeLa cell, S262F-Cx43-HeLa cell, S368A -Cx43-HeLa cell) respectively. Further experiments were carried out in two parts with these cell types. Firstly, different concentrations of glucose (5mmolL, 25mmolL) were added to the medium of the respective transfected cell types, and phosphorylation status and phosphorylation site were detected to determine the effect of hyperglycemia on Cx43. Secondly,Dye-coupling(evaluated by scrape-loading) was used to detect the gap junction permeability of the respective transfected cell types to evaluate the effect of hyperglycemia on gap junction permeability . Firstly,it was observed that hyperglycemia can induce phosphorylation of cardiac connexin 43, and hyperglycemia can lead to S368 phosphorylation of Cx43 in those of Wt-Cx43-HeLa cells [with anti specific phosphorylated sites connexin43 antibodies (p-connexin43-Ser368)], while no S262 phosphorylation(in those of S262F-Cx43-Hela cells) or S368 phosphorylation of Cx43 (in those of Wt-Cx43-Hela cells or S368A -Cx43-HeLa cells) was detected. Secondly, Dye-coupling(evaluated by scrape-loading) indicated that hyperglycemia decreased gap junction permeability in Wt-Cx43-Hela cells(compared to S368A-Cx43-Hela cells and Wt-Cx43-Hela cells cultured with normal glucose ).Therefore, it was concluded that (1) Hyperglycemia can induce phosphorylation of connexin 43, (2) hyperglycemia can lead to S368 phosphorylation of Cx43. (3)Hyperglycemia decreased gap junction permeability in Wt-Cx43-Hela cells .