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

Abstract

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 (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 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 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 mCa²⁺ import during I/R, nor does it affect ΔΨ_m recovery during reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCE) suppressed the mCa²⁺ 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 mCa²⁺. Interestingly, inhibition of mitochondrial electron transport or supplementation with antioxidants stabilized I/R-induced ΔΨ_m 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 ΔΨ_m instability during reperfusion injury.

Keywords: image processing; ischemia; mitochondrial membrane potential; oscillation; oxidative phosphorylation; reperfusion; time-series analysis; wavelet.