Mitochondrial membrane potential instability on reperfusion after ischemia does not depend on mitochondrial Ca(2+) uptake

Physiologic Ca(2+) 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(2+) import into mitochondria. Several groups have tested the hypothesis that Ca(2+) 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(m)) when subjected to stress, such as during I/R, but it is unclear if the primary trigger is an excess influx of mitochondrial Ca(2+) (mCa(2+)), reactive oxygen species (ROS) accumulation, or other factors. Here, we critically examine whether MCU-mediated mitochondrial Ca(2+) uptake during I/R is involved in ΔΨ(m) 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(2+) import during I/R, nor does it affect ΔΨ(m) recovery during reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCE) suppressed the mCa(2+) increase during Ischemia but did not affect ΔΨ(m) recovery or the frequency of ΔΨ(m) oscillations during reperfusion, indicating that mitochondrial ΔΨ(m) instability on reperfusion is not triggered by mCa(2+). Interestingly, inhibition of mitochondrial electron transport or supplementation with antioxidants stabilized I/R-induced ΔΨ(m) oscillations. The findings are consistent with mCa(2+) overload being mediated by reverse-mode mNCE activity and supporting ROS-induced ROS release as the primary trigger of ΔΨ(m) instability during reperfusion injury.