REGULATED EXPRESSION OF A CONTRACTILE PROTEIN GENE CORRELATES WITH RECOVERY OF CONTRACTILE FUNCTION AFTER REVERSIBLE METABOLIC INHIBITION IN CULTURED MYOCYTES

Little is known of the relation between recovery of contraction and th e regulation of contractile protein gene expression in ventricular myo cytes after severe ATP depletion, We have examined alterations in acti vation of an MLC-2 luciferase fusion gene in cultured neonatal rat ven tricular myocytes produced by exposure to 2 mM Na CN and 20 mM 2-deoxy glucose, and after recovery in serum or serum free medium, The effects of metabolic inhibition followed by recovery on expression on an RSV- luciferase activity were also investigated, Myocytes were co-transfect ed with a CMV beta-galactosidase fusion gene, and luciferase activitie s were normalized relative to beta-galactosidase activity to control f or transfection efficiency. Two hours of metabolic inhibition produced significant cell injury, as documented by disorganization of myofilam ents, and reduction in luciferase and beta-galactosidase activity with in transfected cells. Cells allowed to recover for 48 h in serum free hormone supplemented medium showed a further decline in corrected luci ferase activity, consistent with a marked reduction in MLC-2 gene tran scription. Cells recovered from severe metabolic inhibition in serum f ree medium also showed failure to redevelop contractile activity, and failure of redevelopment of organized myofibrils. In contrast, myocyte s exposed to serum during the 48 h recovery period had a marked increa se in luciferase activity, resumed contractile activity and re-establi shed organized myofilaments. There were no significant differences bet ween RSV luciferase activities in cells recovered in serum versus seru m free media. In ventricular myocytes in which contraction was inhibit ed by exposure to 10 mu M verapamil, MLC-2 luciferase activity decline d by 87%, However, even when contractile activity was inhibited by exp osure to verapamil during recovery from metabolic inhibition, exposure to serum containing medium caused a significantly greater increase in MLC-2 luciferase activity than did serum free medium, Thus, the effec ts of serum on MLC-2 gene expression were not solely due to an effect of serum on recovery of contractile activity. Verapamil had no consist ent effect on expression of RSV luciferase, These results suggest that expression of the MLC-2 gene is markedly reduced following recovery f rom severe metabolic inhibition, an effect largely due to cessation of myocyte contractile activity. Resupply of growth factors present in f etal calf serum reactivate expression of this gene, and this is associ ated with resumption of contractile activity and redevelopment of orga nized myofibrils. These results suggest that reactivation of contracti le protein gene expression during recovery from metabolic inhibition m ay be beneficial in allowing cells to recover from this insult.