Studies on the control of energy metabolism in mammalian cardiac muscle cells in culture.

Myocardial cells in a monolayer culture are a myogenic model system wihch shows functional differentiation and in which the intracellular metabolism and energy utilization do not differ markedly from those of the fresh tissue. In the myocardial cells, as in numerous other muscle tissues, the concentration of high energy phosphate compounds, primarily phosphorylcreatine, correlates well with the functional integrity of the cells. The decline of adenosine triphosphate (ATP) below a critical level leads to the cessation of rhythmic contractions of the cells in culture, and, conversely, an increased steady state level of ATP correlates with an increased rate of excitability. When creatine, in the concentration range known to be present in the cardiac tissue, is added to the growth medium of the cultured myocardial cells, the intracellular concentration of phosphorylcreatine increases up to 100%. Since the only metabolic path known for phosphorylcreatine synthesis is via creatine phosphokinase, the rate of transphosphorylation and ATP synthesis must have been increased. This stimulation of energy production is due to the regeneration of mitochondrial ADP brought about by the phosphosphorylation of creatine to phosphorylcreatine and possibly, also, to an enhanced rate of glycolysis. No net transfer of approximately P from ATP to creatine was observed under any of the experimental conditions. The high steady state level of phosphorylcreatine was not maintained upon the addition of either oligomycin or 2-deoxyglucose, and the addition of both metabolic inhibitors simultaneously resulted in a depletion of phosphorylcreatine and ATP and in cessation of rhythmic contractions. The excitability was also inhibited upon the addition of 1-fluoro-2,4-dinitrobenzene, a creatine phosphokinase inhibitor, and the accompanying depletion of approximately P was primarily reflected in a decrease of ATP concentration. These findings support the following conclusions: (1) phosphorylcreatine serves as a source of approximately P for the resynthesis of ATP at the site of utilization (i.e., myofibrils, membrane) and thus maintains the optimal energy charge; (2) production site; (3) the regeneration of phosphorylcreatine is coupled to oxidative phosphorylation and possibly to glycolysis. Creatine-phosphorylcreatine system operates as a undirectional shuttle for approximately P and as a control system regulating energy production according to demand. Reports of studies on intact mitochondria and on isoenzymes of creatine phosphokinase give further support to the above conditions.