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

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.

Distribution of Connexin43 and Pan-cadherin in Cardiac Myocytes during Intercalated Disc Formation / 대한해부학회지

This experiment was aimed to elucidate about the forming process of intercalated discs. Each ventricular myocardium of Sprague Dawley rats was used at postnatal 3, 7, 14, 21, 35, 84, 120 days and cell lines such as H9c2 cell and C2C12 cell were also cultivated during 1, 3, 5, 7 day. Expressional change of connexin43, a marker of gap junction and pan-cadherin, a protein representing fascia adherence were stained with indirect immuno-fluorescent technique in the cryo-sectioned ventricular tissues and the cells. The cultivated cells were observed under inverted microscope and the myocardial tissues were processed for the recording with transmission electron microscope at each time. Myotubes consisting of multi-nucleated cells similar to skeletal muscle were formed on both of H9c2 cells and C2C12 cells. Connexin43 showed generally punctuate pattern across the cell membrane in the cryo-sectioned specimens at postnatal 3 to 21 days. The reaction at nascent intercalated discs formed linear appearance and thick bands at postnatal 35 and 84 days, respectively, although the protein initially expressed an equal reaction throughout sarcoplasm. On postnatal 120 days, there were dispersed in dots instead of regions of the thick band. Connexin43 in the cytoplasm of H9c2 and C2C12 cells was distributed in punctuate or homogenous reaction, however, the reaction intensity regardless of culture duration was very weak. Pan-cadherin showed mainly stripe-shape in the tissue specimens at postnatal 3 and 7 days. This protein formed more dense stripe-shape at postnatal 14 and 21 days than before. Comparing with connexin43, cadherin was earlier expressed in the sarcoplasm at these time points. A thick band abundantly appeared on the region of nascent intercalated disc at postnatal 35 and 84 days. Comparing with these days, some of the bands changed into mass of dots though the number of band was decreased at postnatal 120 days. Cadherin in H9c2 and C2C12 cells showed stripe- or punctuate- shape, however, the reaction intensity was generally weaker than in the specimens. There were intercalated discs in the ultrastructure of ventricular myocardium from postnatal 3 to 120 days. Gap junction, fascia adherence and desmosome, all constituents of intercalated discs, had their own characteristic structure. In conclusion, these findings may be suggested that the formation of intercalated discs correlate to the distribution pattern of connexin43 and pan-cadherin in the myocardium of Sprague Dawley rat at each postnatal age. Therefore, authors hope that the data would be useful for the explanation of mechanism forming the junctional complexes between grafted cells and normal cardiac myocytes.