Kazunobu Ban

On the mechanism of the failure of mitochondrial function in isolated guinea-pig myocytes subjected to a Ca2+ overload

OBJECTIVE The influence of agents that inhibit the movement of Ca2+ across the mitochondrial membrane or Ca2+ dependent changes to this membrane upon the response of isolated ventricular myocytes to a Ca2+ overload has been investigated. METHODS The changes of intracellular Ca2+ and Mg2+ ([Ca2+]i, [Mg2+]i) (as reflected by cellular ATP), mitochondrial membrane potential (psi m) and NADH was measured upon the response of isolated ventricular myocytes to a Ca2+ overload. RESULTS A slow depolarization of psi m during Ca2+ depletion and its prompt recovery on Ca2+ repletion were unaffected by ruthenium red, clonazepam, CGP-37157 which is a high potent inhibitor of the mitochondrial Na+/Ca2+ antiport or cyclosporin A but a large delayed sustained depolarization was inhibited. The slow small fall in [Mg2+]i on Ca2+ depletion and a rapid recovery on Ca2+ repletion were unaffected by ruthenium red, clonazepam, CGP-37157 or cyclosporin A. A delayed sustained larger rise in [Mg2+]i was inhibited. The marked sustained fall in NADH autofluorescence that occurs on Ca2+ overload was attenuated and transient in the presence of ruthenium red, CGP-37157 and cyclosporin A. CONCLUSION These results are consistent with an increase in Ca2+ cycling across the mitochondrial membrane provoked by the combined Na+ and Ca2+ overload of cardiac myocytes, causing a depolarization sufficient to uncouple respiration and lead to the depletion of cellular ATP.

Identification of Free Radical Species in the Myocardium

With the increasing interest in the role of oxygen-derived free radical generation in various diseases, several attempts to directly evaluate free radicals in tissue have been performed using EPR.1–8 However, none of the previous studies has fully determined the species of the radicals and none has deliberated on the possible existence of artifactual radicals which are likely to be generated during sample handling. The purposes of this study were 1) to establish an artifact-free sampling method for the quantification of freeze-trapped free radicals in myocardium by EPR, 2) to identify artifact radicals and the radicals native to the myocardium and 3) to demonstrate the relationship of these radicals to reperfusion injury.