Glutathione oxidation as a trigger of mitochondrial depolarization and oscillation in intact hearts.

Abstract Depolarization of the mitochondrial inner membrane potential (ΔΨ m ) associated with oxidative stress is thought to be a critical factor in cardiac dysfunction and cell injury following ischemia–reperfusion or exposure to cardiotoxic agents. In isolated cardiomyocytes, mitochondrially-generated reactive oxygen species (ROS) can readily trigger cell-wide collapse or oscillations of ΔΨ m but it is not known whether these phenomena scale to the level of the whole heart. Here we utilize two-photon laser scanning fluorescence microscopy to track ΔΨ m , ROS, and reduced glutathione (GSH) levels in intact perfused guinea-pig hearts subjected to simulated ischemia reperfusion or GSH depletion with the thiol oxidizing agent diamide. Exposure to oxidative stress by either method provoked heterogeneous ΔΨ m depolarization and occasional oscillation in clusters of myocytes in the epicardium in association with increased mitochondrial ROS production. Furthermore, the whole-heart oxidative stress dramatically increased the sensitivity of seemingly quiescent cells to ΔΨ m depolarization induced by a localized laser flash. These effects were directly correlated with depletion of the intracellular GSH pool. Unexpectedly, hearts perfused with nominally Ca 2+ -free solution or those switched from 0.5 mM Ca 2+ to nominally Ca 2+ -free solution also displayed heterogeneous ΔΨ m depolarization and oscillation, in parallel with net oxidation of the GSH pool. The findings demonstrate that metabolic heterogeneity initiated by mitochondrial ROS-induced ROS release is present in the intact heart, and that the redox state of the glutathione pool is a key determinant of loss of ΔΨ m .