Actin isoform synthesis by cultured cardiac myocytes: effects of actinomycin D and mithramycin

Cultured neonatal rat myocardial cells (CMC) were incubated with the Actinomycin D (ACT) and mithramycin (MIT) in concentrations of 1 x 10-8M to 1 x 10-5M in medium containing 35μCi of 35S-methionine to determine incorporation into myocardial contractile proteins. After 24h, cells were harvested in buffer with Triton X-100, homogenized and subjected to centrifugation. Protein content of the centrifuged extracts was determined and equivalent amounts of protein were applied to 8-15% gradient sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE). Similar aliquots were subjected to isoelectric focusing and to gel electrophoresis in the second dimension. Electrophoretic gels were autoradiographed and were quantified densitometrically. A concentration-dependent effect of ACT and MIT on protein and on actin synthesis was found in cultured myocytes with decreased intensity of the radiolabeled actin band in CMC exposed to 1 x 10-8M to 1 x 10-5M ACT of 1 x 10-7M to 1 x 10-5M MIT. Autoradiography of gels showed focusing of actin isoforms but decreased intensity of iso-actin spots in extracts from CMC exposed to 1 x 10-7M ACT. ACT and MIT decreased CMC protein and synthesis by 20% at 1 x 10-8M respectively. The effect of MIT and ACT on CMC actin synthesis and synthesis of other proteins in heart cells is not as specific as the effect of anthracycline antineoplastics on cardiac myocyte proteins and actin