Sudden Cardiac Death (SCD) occurs in more than half a million people every year and arrhythmias, resulting in hemodynamic insufficiency followed by death, account for the majority of SCD cases. Arrhythmias secondary to scar formation are well understood, but mechanisms by which acute ischemia/reperfusion (I/R) injury promotes ventricular tachycardia and fibrillation (VT/VF) are more complex. I/R-related arrhythmias depend on dynamic properties of the tissue, including Ca2+-mediated triggers, functional conduction block, decreased gap junctional conductance, heterogeneous shortening of the action potential (AP) and dispersion of refractoriness. These complex electrophysiological (EP) changes are caused by limitations in ATP supply, changes in reactive oxygen species (ROS), accumulation of detrimental intracellular (Ca2+, Na+, acid) and extracellular (K+, lactate) constituents, all of which can be traced to a common origin, namely impaired mitochondrial function. Our group was the first to recognize the importance of heterogeneous mitochondrial instability, including sustained depolarization or oscillation of the mitochondrial inner membrane potential (??m), across clusters of myocytes or regions within the heart, in setting the stage for VT/VF. However, the mechanisms behind mitochondrial instability during reperfusion are unclear and how they contribute to arrhythmias is not well understood. While inhibition of the mitochondrial permeability transition pore (mPTP) decreases infarct size, cyclosporine A-mediated inhibition of mPTP has little or no effect on arrhythmia incidence after ischemia, suggesting that the early dysfunction and late injury mechanisms may be distinct. In contrast, we found that mitochondrial benzodiazepine receptor (mBzR) ligands are very effective at restoring the action potential and suppressing arrhythmias induced by I/R, in parallel with their ability to prevent or reverse mitochondrial depolarization. Indeed, our exciting preliminary data suggests that mBzR, rather than mPTP, is more important in terms of ??m and electrical stability during the early reperfusion phase. Here, we will use innovative approaches to image the dynamics of ??m, Vm, matrix Ca and ROS during I/R at the cellular and whole 2+ heart scales, combined with powerful genetic models to selectively knockout the key proteins involved in modulating mPTP (cyclophilin D; PPIF), mitochondrial Ca2+ (the mitochondrial Ca2+uniporter; MCU), and the mBzR (translocator protein; TSPO), to define the causal mechanisms underlying mitochondrial instability and arrhythmias on reperfusion. This project will move the field from conclusions based on pharmacological inference to molecular understanding, allowing us to focus our efforts on the correct mitochondrial targets to pursue to prevent I/R-induced arrhythmias with the goal of decreasing the burden of SCD.