Mitochondrial membrane potential instability on reperfusion after ischemia does not depend on mitochondrial Ca uptake.

Physiologic Ca 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 Ca import into mitochondria. Several groups have tested the hypothesis that Ca 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 (ΔΨ) when subjected to stress, such as during I/R, but it is unclear if the primary trigger is an excess influx of mitochondrial Ca (mCa), reactive oxygen species (ROS) accumulation, or other factors. Here, we critically examine whether MCU-mediated mitochondrial Ca uptake during I/R is involved in ΔΨ 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 mCa import during I/R, nor does it affect ΔΨ recovery during reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCE) suppressed the mCa increase during Ischemia but did not affect ΔΨ recovery or the frequency of ΔΨ oscillations during reperfusion, indicating that mitochondrial ΔΨ instability on reperfusion is not triggered by mCa. Interestingly, inhibition of mitochondrial electron transport or supplementation with antioxidants stabilized I/R-induced ΔΨ oscillations. The findings are consistent with mCa overload being mediated by reverse-mode mNCE activity and supporting ROS-induced ROS release as the primary trigger of ΔΨ instability during reperfusion injury.