Oxidative stress in the mitochondrial matrix underlies ischemia/reperfusion-induced mitochondrial instability

Ischemia and reperfusion affect multiple elements of cardiomyocyte electrophysiology, especially within the mitochondria. We previously showed that in cardiac monolayers, upon reperfusion after coverslip-induced ischemia, mitochondrial inner membrane potential (Delta Psi) unstably oscillates between polarized and depolarized states, and Delta Psi instability corresponds with arrhythmias. Here, through confocal microscopy of compartment-specific molecular probes, we investigate the mechanisms underlying the postischemic Delta Psi oscillations, focusing on the role of Ca2+ and oxidative stress. During reperfusion, transient Delta Psi depolarizations occurred concurrently with periods of increased mitochondrial oxidative stress (5.07 +/- 1.71 oscillations/15 min, N = 100). Supplementing the antioxidant system with GSH monoethyl ester suppressed.. oscillations (1.84 +/- 1.07 oscillations/15 min, N = 119, t test p = 0.027) with 37% of mitochondrial clusters showing no Delta Psi oscillations (versus 4% in control, odds ratio = 14.08, Fisher's exact test p < 0.001). We found that limiting the production of reactive oxygen species using cyanide inhibited postischemic Delta Psi oscillations (N = 15, t test p < 10-5). Furthermore, Delta Psi oscillations were not associated with any discernable pattern in cell-wide oxidative stress or with the changes in cytosolic or mitochondrial Ca2+. Sustained Delta Psi depolarization followed cytosolic and mitochondrial Ca2+ increase and was associated with increased cell-wide oxidative stress. Collectively, these findings suggest that transient bouts of increased mitochondrial oxidative stress underlie postischemic Delta Psi oscillations, regardless of Ca2+ dynamics.