ROLE OF MITOCHONDRIAL CALCIUM UNIPORTER IN MITOCHONDRIAL MEMBRANE POTENTIAL INSTABILITY IN ISCHEMIA-REPERFUSION INJURY

Mitochondria exhibit non-stationary unstable membrane potential (ΔΨm) when subjected to stress, such as during Ischemia/Reperfusion (I/R). Understanding mitochondrial instability in Ischemia Reperfusion injury is key to determining efficacy of interventions. Excess influx of mitochondrial Ca2+ and reactive oxygen species (ROS) accumulation are thought to be primary triggers of ΔΨm instability, but the underlying molecular mechanisms are still unclear. The goal of this thesis is to understand the contributions of mCa2+ and ROS in triggering ΔΨm instability. For this purpose, it was important to first define and characterize oscillatory patterns of non-stationary mitochondrial ΔΨm instability. A data analysis tool was developed based on wavelet transform functions to automate analysis of time-series data from microscopy images to detect ΔΨm changes in an unbiased and reproducible manner. It is an ImageJ-MATLAB-based workflow called ‘MitoWave’ to unravel dynamic mitochondrial ΔΨm changes that occur during ischemia and reperfusion. Features such as, time-points of ΔΨm depolarization during I/R, area of mitochondrial clusters and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster with this tool. We then used this tool to understand the role of Ca2+ and ROS in triggering ΔΨm instability. Physiologic Ca2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload but is thought to contribute to cell death during I/R injury. We genetically knocked out the MCU to examine whether MCU-mediated mCa2+ uptake is required to trigger ΔΨm loss or oscillation during reperfusion in neonatal mouse ventricular myocyte (NMVM) monolayers. Our findings demonstrate that MCU knockout does not significantly alter mCa2+ import during I/R, nor does it affect ΔΨm recovery during Reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchange (mNCE) with CGP-37157 suppressed mCa2+ increase during Ischemia but did not affect ΔΨm recovery during reperfusion or the frequency of ∆Ψm oscillations, confirming that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa2+. Interestingly, inhibition of mitochondrial electron transport and supplementation with antioxidants stabilized ΔΨm oscillations. The findings are consistent with mCa2+ overload being mediated by reverse-mode mNCE activity and support ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury