Mitochondrial Membrane Potential Oscillations Persist during Reperfusion After Ischemia in MCU Knockout Cardiomyocytes

Abstract

Ca2+ entry via Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload under physiological conditions but is thought to contribute to cell death during ischemia-reperfusion (I/R) injury. Previous work has shown that mitochondrial membrane potential (ΔΨm) instability contributes to early-reperfusion arrhythmias and contractile dysfunction; however, the role of mitochondrial Ca2+ uptake in triggering ΔΨm oscillation versus sustained permeability transition pore (PTP) opening is unclear. We hypothesized that MCU-mediated Ca2+ uptake is required to trigger irreversible ΔΨm loss mediated by PTP but is not required for ΔΨm oscillation during early reperfusion, involving reactive oxygen species (ROS)-induced ROS release (RIRR). A spinning-disk confocal microscope was used to image ΔΨm(with TMRM) in monolayers of neonatal mouse ventricular myocytes (NMVM) during I/R (1hr/1hr). Acute MCU knockout (KO) was achieved by transducing floxed MCU NMVMs with an adenovirus expressing CRE-recombinase. ΔΨm response to I/R was analyzed at cellular level by segmentation analysis (ImageJ) and a new wavelet transform method was developed to characterize the dominant frequency of ΔΨm oscillation (Matlab). We found that surprisingly, the rate of ΔΨm loss during ischemia was greater in MCU-KO compared to control, yet the recovery of ΔΨm was more sustained, lacking a late decline after 30 min reperfusion. ΔΨm oscillations during reperfusion persisted in MCU-KO myocytes and wavelet analysis revealed a dominant frequency ranging from 0.022Hz-0.0088Hz in both WT and KO. These results suggest that MCU-mediated Ca2+ uptake is not required for RIRR induced ΔΨm oscillation but is involved in late reperfusion-induced sustained ΔΨm collapse; and that MCU-mediated Ca2+ uptake during reperfusion contributes to PTP-mediated ΔΨm collapse during reperfusion. However, ΔΨm oscillations during early reperfusion are independent of MCU-mediated Ca2+ entry, and likely to underlie mechanical and electrophysiological dysfunction observed during this period.