Interrelationships between ATP levels, enzyme leakage, gluconate uptake, trypan blue exclusion and length/width ratio of isolated rat cardiac myocytes subjected to anoxia and reoxygenation

Abstract

Rat cardiac myocytes were isolated by perfusion of the heart with collagenase and subsequently incubated in the absence or presence of oxygen. As a result of anoxia, there was a gradual increase in plasma membrane permeability, as indicated by a decrease in trypan blue exclusion, leakage of cytosolic lactate dehydrogenase, and intracellular accumulation of the isotope [ 99Tc m]gluconate. These changes in plasma membrane permeability were preceded by a marked decrease in cellular ATP levels and an increased proportion of contracted myocytes. After an initial anaerobic incubation for 10 min, there was complete replenishment of ATP followed by signs of recovery from anoxic cell injury upon reoxygenation. Cell viability could also be followed on the basis of changes in the length/width ratio of myocytes upon anoxia and reoxygenation. The ability of myocytes to resynthesize ATP and to recover from anoxic injury upon reoxygenation decreased in proportion to the length of the initial anaerobiosis during the first 25 min and disappeared completely after 30 min of anoxia. The present model system is a convenient tool for evaluation of interrelationships between energy metabolism, plasma membrane integrity, and the morphology of myocytes subjected to anoxia and reoxygenation.