MitoWave: Spatio-temporal analysis of mitochondrial membrane potential fluctuations during ischemia-reperfusion.

Abstract

Mitochondria exhibit unstable inner membrane potentials (ΔΨm) when subjected to stress, such as during Ischemia/Reperfusion (I/R). Understanding the mechanism of ΔΨm instability involves characterizing and quantifying this phenomenon in an unbiased and reproducible manner. Here, we describe a simple analytical workflow called MitoWave that combines wavelet transform methods and image segmentation to unravel dynamic ΔΨm changes in the cardiac mitochondrial network during I/R. In vitro ischemia was effected by placing a glass coverslip on a monolayer of neonatal mouse ventricular myocytes (NMVMs) for 1 hour and removing the coverslip to allowed for reperfusion, revealing complex oscillatory ΔΨm. MitoWave analysis was then used to identify individual mitochondrial clusters within the cells and track their intrinsic oscillation frequencies over the course of reperfusion. Responses segregated into five typical behaviors quantified by MitoWave that were corroborated by visual inspection of the time series. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion showed significant differences between the five different outcomes. Features such as the time-point of ΔΨm depolarization during I/R, area of mitochondrial clusters, and time-resolved frequency components dAuring reperfusion were determined per cell and per mitochondrial cluster. Mitochondria from NMVMs subjected to I/R oscillate in the frequency range of 8.6-45mHz, with a mean of 8.73±4.35mHz. Oscillating clusters had smaller areas ranging from 49.8±1.2 μm2 while non-oscillating clusters had larger areas 66±1.5μm2. A negative correlation between frequency and mitochondrial cluster area was observed. We also observed that late ΔΨm loss during ischemia correlated with early ΔΨm stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a semi-automated method to quantify complex time-resolved mitochondrial behavior in an easy to follow workflow, enabling unbiased, reproducible quantitation of complex non-stationary cellular phenomena.