The Influence Of CAMP On The Phosphorylation Of Rat Cardiac Connexin 43

Gap junction intercellular communication (GJIC) mediated by connexin43 played an important role in the maintenance of the normal electric activities between cardiac myocytes. Dephosphorylation of connexin43 during cardiac ischemia was closely linked to the occurrence of the malignant arrhythmia complicated in acute myocardial infarction (AMI). It was indicated that the protection effects of myocardial ischemia preconditioning was accompanied by the elevation of the intracellular concentration of cAMP in cardiac myocytes, while the underlying mechanism remained unknown. Besides, there were plenty of evidences that protein kinase A, through which most effect of cAMP was mediated, was implicated in the enhancement of GJIC and the increase of the expression of connexin43 as well as its phosphorylation status. Therefore, it was of great meaning to determine the effect of cAMP on connexin43 phosphorylation status and the underlying molecular mechanism, especially for the further elucidation of the mechanism underlying ischemia arrhythmia complicated in AMI and some possible intervening way.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 cAMP analog 8-Br-cAMP (0molL, 10E-5molL, 10E-4molL, 10E-3molL) were added to the medium of the respective transfected cell types, and cell growth inhibition rates were calculated to determine the effect of 8-Br-cAMP on cell growth. It was observed that the growth of both Wt-Cx43-HeLa cells and S262F-Cx43-HeLa cells were inhibited by 8-Br-cAMP in a dose-dependant manner, while no significant changes were detected in those of S368A -Cx43-HeLa cells. Secondly, with anti specific phosphorylated sites connexin43 antibodies (p-connexin43-Ser368 and p-connexin 43(mSer262-R)) and PKA inhibitor H89, it was observed through immunocellular chemistry that 8-Br-cAMP increased phosphorylation of Ser368 of connexin43 in Wt-Cx43-HeLa cells in a dose-dependant manner but not Ser262. And H89 decreased the phosphorylation of Ser368 of connexin43 by 8-Br-cAMP, suggesting PKA pathway was involved. Therefore, it was concluded that (1) 8-Br-cAMP potentiated the phosphorylation of Cx43 and increased its phosphorylation status. (2) 8-Br-cAMP inhibited the growth of Hela cells which were stably expressing Cx43. (3) the mechanism underlying 8-Br-cAMP potentiated the phosphorylation of Cx43 was mediated by the PKA pathway to increase the phosphorylation of Cx43 on Ser368, and therefore enhancing GJIC.