This project examines the chronic regulation of mitochondrial enzyme activity in muscle, with a view to understanding the decreased capacity of skeletal muscle to respond to endurance training in old age. Our immediate goal is to identify signals which allow the cell to sense increased energy demand in the form of an increased flux through ATP and respond with a coordinated expression of mitochondrial proteins encoded by the nuclear and mitochondrial genomes. This year we have studied the expression of mitochondrial enzymes in an immortal cell line derived from mouse skeletal muscle (C2 cells) as the cells proliferate in media supplemented with 20% fetal calf serum, and differentiate into myocytes and myotubes in response to serum starvation. Further, we have used uncoupling agents, Ca2+- ionophores and inhibitors of respiratory chain activity to lower cytosol phosphate potential (ATP/ADPxPi) in an attempt to stimulate mitochondrial proliferation. During proliferation, the metabolism of these cells resembles that of tumor cells, with 60% of cell ATP needs met by glycolysis Those mitochondria which are present operate at about 50% of Vmax capacity. Pyruvate dehydrogenase was found to be only 43% in the active, dephospho form (PDHA) - indicative of low energy demands. During differentiation the cells become markedly more oxidative. Mitochondrial enzymes (pyruvate dehydrogenase, citrate synthase, NAD-isocitrate dehydrogenase, s-OH acyl Co dehydrogenase, cytochrome c oxidase) increase 4-fold in activity, and the fraction of ATP flux due to oxidative phosphorylation increases markedly. In contrast, cytosol enzymes (pyruvate kinase, ]-isocitrate dehydrogenase) remain constant in activity. The fraction of pyruvate dehydrogenase presen as PDHA increases to near 100% after 3 days serum starvation, such that maximum flux through this enzyme is increased 10 fold with differentiation. Several pharmacological interventions are being explored as means to replicate these changes in proliferating cells.
