Growth factors and TPA stimulate DNA synthesis and alter the morphology of cultured terminally differentiated adult rat cardiac muscle cells

Abstract

Previous studies have established that the terminally differentiated ventricular cardiac muscle cell of the adult rat reinitiates semiconservative DNA replication when grown in culture (W. C. Claycomb and H. D. Bradshaw, Jr., 1983, Dev. Biol. 90, 331-337). Work reported here shows that several growth factors and chemicals will stimulate this DNA synthetic activity in a concentration-dependent manner. Autoradiographic experiments establish that this stimulated DNA synthesis is due to cells not previously synthesizing DNA being induced to enter the S phase of the cell cycle. By far the greatest stimulation (250%) is observed with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Fifty ng/ml is the optimal concentration, and the maximal effect is observed 5 days after adding TPA. TPA also substantially increases the protein content of the cultured myocytes. Diacylyglcerols (DAG) induce these same changes, indicating that the effect of TPA is mediated by protein kinase C. The morphology of the cultured cardiac muscle cells is profoundly altered by TPA and DAG. TPA- and DAG-treated myocytes spread more thinly on the surface of the culture flask, acquire multiple nuclei, and undergo nucleolar fragmentation. The myofibrillar ultrastructure of the treated cells becomes almost totally disorganized, and intermediate filaments and rough endoplasmic reticulum accumulate in the cytoplasm. These TPA results suggest a possible relationship between the degree of ultrastructural differentiation of the ventricular cardiac muscle cell and DNA synthetic activity. This easily altered cellular plasticity should be very useful for studies of the regulation of cardiac muscle cell proliferation and cell differentiation.