Mitochondrial membrane potential instability on reperfusion after ischemia does not depend on mitochondrial Ca2+uptake

Physiologic Ca2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload but may also contribute to cell death during ischemia/reperfusion (I/R) injury. The MCU has been identified as the primary mode of Ca2+ import into mitochondria. Several groups have tested the hypothesis that Ca2+ import via MCU is detrimental during I/R injury using genetically-engineered mouse models, yet the results from these studies are inconclusive. Furthermore, mitochondria exhibit unstable or oscillatory membrane potentials (AWm) when subjected to stress, such as during I/R, but it is unclear if the primary trigger is an excess influx of mitochondrial Ca2+(mCa2+), reactive oxygen species (ROS) accumulation, or other factors. Here, we critically examine whether MCU-mediated mitochondrial Ca2+ uptake during I/R is involved in AWm instability, or sustained mitochondrial depolarization, during reperfusion by acutely knocking out MCU in neonatal mouse ventricular myocyte (NMVM) monolayers subjected to simulated I/R. Unexpectedly, we find that MCU knockout does not significantly alter mCa2+ import during I/R, nor does it affect AWm recovery during reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCE) suppressed the mCa2+ increase during Ischemia but did not affect AWm recovery or the frequency of AWm oscillations during reperfusion, indicating that mitochondrial AWm instability on reperfusion is not triggered by mCa2+. Interestingly, inhibition of mitochondrial electron transport or supplementation with antioxidants stabilized I/R-induced AWm oscillations. The findings are consistent with mCa2+ overload being mediated by reverse-mode mNCE activity and supporting ROS-induced ROS release as the primary trigger of AWm instability during reperfusion injury.