Regulation of alpha-smooth muscle actin expression in adult cardiomyocytes through a tyrosine kinase signal transduction pathway.

Adult rat ventricular myocytes assume after 2 weeks in culture a flattened spread morphology and a loss in organized myofibrils. This sequence of phenotypic changes is accompanied by the reexpression of the fetal gene program. Although different signal transduction pathways were recently shown to be involved in cell growth and differentiation, not much is known about tyrosine kinase activation and cardiac myocyte differentiation. We investigated whether the tyrosine kinase signal transduction pathway is involved in the dedifferentiation of adult rat ventricular myocytes in long-term culture using a specific inhibitor of tyrosine phosphorylation, genistein. For this experiment, adult rat ventricular myocytes were cultured as previously described and incubated in culture medium containing different concentrations of genistein (10-250 microM). After 24 hr of incubation and in a concentration-dependent manner genistein prevented cell spreading. However, at high concentration, cells detached from the plates (10% to 100 microM and 95% at 250 microM). The effect of genistein on adult rat ventricular myocyte phenotype in culture was investigated by examining the expression of total actins and alpha-smooth muscle actin and alpha-sarcomeric actin in cells after 6 days of incubation with and without genistein. Myofibrillar proteins were extracted and separated by gel electrophoresis. Expression of alpha-smooth muscle actin and alpha-sarcomeric actin was determined by Western blotting using specific antibodies. While there was an increase in the amount of total actins and no change in the amount of alpha-sarcomeric actin in the cells exposed to genistein, the amount of alpha-smooth muscle actin decreased with increasing concentrations of genistein reaching undetectable levels at 100 microM. These results demonstrate that genistein inhibits cell spreading and the reexpression of alpha-smooth muscle actin in adult rat ventricular myocytes in culture in a dose-dependent manner, therefore, inhibiting the process of dedifferentiation.