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

Abstract

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.

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