Change in the Beating Rate and Intercellular Adhesion Molecule of Cardiomyocytes in Culture

Hong, Jong Wook; Park, Jeong Hyun; Kim, Dae Kwang; Song, In Hwan; Sung, Eon Gi; Lee, Yungchang; Kim, Joo Young

Abstract

This experiment was designed for the elucidation of relationships between the cell adhesion molecules and synchronous beating rates of cardiomyocytes isolated from ventricles of 3-day-old rats. These cells were grown on the culture vessel coated or non-coated with cardiogel for 1, 3, 5, and 7 days. The synchronous beating rate and morphologic changes of cells were investigated under the inverted microscope. Those changes of cardiomyocytes were observed by transmission elcectron microscopy (TEM). Connexin43, pan-cadherin, and alpha-sarcomeric actin were stained by indirect immunofluorescent (IF) technique. Western blotting were used for identifying unique bands of connexin43 and pan-cadherin in the regions of specific size. The synchronous beating numbers of cardiomyocytes in each group on the dish coated or non-coated with cardiogel were significantly different for 3, 5, and 7 days in culture. The maximum values of synchronized beating in the cells appeared on the day 5. The beating numbers of cells grown on coated dish comparing with non-coated dish were significantly increased on the day 5 and day 7. The proliferation of cardiomyocytes increased markedly in the cardiogel-coated dish on the day 5, while the number of fibroblasts in non-coated dish were obviously increased on the day 7. In indirect IF studies, a normal redistribution of connexin43, pan-cadherin, and alpha-sarcomeric actin of the cells was expressed in day 3 throughout day 5. The reaction intensity of those proteins were increased or decreased in the proportion to the beating numbers. The lack of reaction was appeared in the fibroblasts consisting of monolayer on the day 7. Unique bands of connexin43 and pan-cadherin having a specific size were marked on Western Blotting. The structures such as gap junction, fascia adherence and desmosome compatible with intercellular adhesion in cardiac muscle were abundant on day 3 to 7. In conclusion, the amount or reaction intensity of connexin43 and pan-cadherin in cardiomyocytes were stronger simultaneously with the increase of synchronous beating numbers, particularly for 3 and 5 days. The maximum beating rates of cardiomyocytes reached on the day 5, while the maximum beating rates of them were markedly decreased owing to the proliferation of fibroblasts on day 7. TEM findings consisting of intercalated discs might be explained regardless of coating with cardiogel on why the close relationships between the beating rates and the connexin43 and pan-cadherin of those cells in culture exist.