Mitochondrial instability during regional ischemia-reperfusion underlies arrhythmias in monolayers of cardiomyocytes

Regional depolarization of the mitochondrial network can alter cellular electrical excitability and increase the propensity for reentry, in part, through the opening of sarcolemmal K_ATP channels. Mitochondrial inner membrane potential (δΨ_m) instability or oscillation can be induced in myocytes by exposure to reactive oxygen species (ROS), laser excitation, or glutathione depletion, and is thought to be a major factor in arrhythmogenesis during ischemia-reperfusion. Nevertheless, the correlation between δΨ_m recovery kinetics and reperfusion-induced arrhythmias has been difficult to demonstrate experimentally. Here, we investigate the relationship between subcellular changes in δΨ_m, cellular glutathione redox potential, electrical excitability, and wave propagation during coverslip-induced ischemia-reperfusion (IR) in neonatal rat ventricular myocyte (NRVM) monolayers. Ischemia led to decreased action potential amplitude and duration followed by electrical inexcitability after ~15min of ischemia. δΨ_m depolarization occurred in two phases during ischemia: in phase 1 (<30min ischemia), mitochondrial clusters within individual NRVMs depolarized, while phase 2 δΨ_m depolarization (30-60min) was characterized by global functional collapse of the mitochondrial network across the whole ischemic region of the monolayer, typically involving a propagating metabolic wave. Oxidation of the glutathione (GSSG:GSH) redox potential occurred during ischemia, followed by recovery upon reperfusion (i.e., lifting the coverslip). δΨ_m recovered in the mitochondria of individual myocytes quite rapidly upon reperfusion (<5min), but was highly unstable, characterized by subcellular oscillations or flickering of clusters of mitochondria in NRVMs across the reperfused region. Electrical excitability also recovered in a heterogeneous manner, providing an arrhythmogenic substrate which led to formation of sustained reentry. Treatment with 4'-chlorodiazepam, a peripheral benzodiazepine receptor ligand, prevented δΨ_m oscillation, improved GSH recovery rate, and prevented reentry during reperfusion, indicating that stabilization of mitochondrial network dynamics is important for preventing post-ischemic arrhythmias. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".