2005 Volume 51 Issue 1 Pages 67-74
Objective : Gap junctions assembled by connexins mediate cell-cell communication by electrical and chemical coupling. Alterations in electrical uncoupling of the myocardium induce arrhythmogenesis in acute and chronic ischemic heart disease. Recently it has been shown that alterations in the phosphorylation status of the connexins result in a functional alteration of cell-cell communication through the gap junctions. Therefore, we focused on the association between alterations in phosphorylation status of connexin43 (Cx43) and cardiac function using an ex vivo perfusion system of rat heart. Animals : We used adult male Wistar rats weighing 280-320g. All the procedures performed on laboratory animals were approved by the institutional animal care and committee (IACUC) of Juntendo University School of Medicine and all the animal experiments were carried out in compliance with the guidelines for animal experimentation of Juntendo University School of Medicine. Methods : Hearts excised from the rats were transferred to a Langendorff apparatus and perfused via an aortic cannula. After stabilization, the hearts were perfused with hypoxic solution for 0 to 40 min, then reoxygenated for 30 min. During perfusion, we recorded the heart rate, developing pressure of left ventricular and end diastolic pressure. In addition, GOT in the coronary effluent collected at a regular interval was measured. The left ventricle was immediately prepared for immunocytochemistry and immunoblot analysis at the end of the perfusion period. Results : We used three kinds of antibodies (ab) against Cx43 for examining the distribution of Cx43. In control rat heart, all Cx43s were phosphorylated and located at the intercalated disc. There were no signals indicating dephosphorylated Cx43 in the entire section. When the isolated rat hearts were subjected to hypoxia, dephosphorylated Cx43 appeared. The signal for dephosphorylated Cx43 was detected over the entire plasma membranes of myocardial cells. The area of cardiomyocytes that were stained with anti-dephosphorylated Cx43ab increased as the hypoxic perfusion time increased. Immunoblot data also demonstrated that hypoxia induced a time-dependent increase in the amount of dephosphorylated Cx43. We also found that the increase in the area positivefore dephosphorylated Cx43 correlated with the decrease in cardiac function. Conclusions : These data indicate that dephosphorylation and redistribution of Cx43 is an early sign of cardiac injury after hypoxia. The detection of dephosphorylated Cx43 may be useful for examining ischemic heart disease.
