Abstract 429: Assessment of Functional Mitochondrial Parameters in Single Cardiomyocytes Reveals Critical Events Not Identified in Cell Populations

Abstract

In cardiomyocytes (CMs), mitochondria play a dual role, maintaining the high energy supply required for rhythmic contraction, but also regulating critical cell death signaling. Impaired mitochondrial function can affect cellular homeostasis and contribute to sub-lethal injury; if mitochondrial impairment is more severe or persistent, pro-death pathways are activated. It is becoming increasingly clear, however, that within a population of cells there is considerable heterogeneity in mitochondrial function between individual CMs; and within a single cell there is also heterogeneity between individual mitochondria/mitochondrial regions. We have developed a high-throughput fluorescence imaging platform to quantitate single CM mitochondrial function in large numbers of cells, yielding information missed using standard assays that evaluate cell populations. When CMs are exposed to H 2 O 2 , they exhibit a dramatic hyperpolarization prior to the loss of mitochondrial membrane potential at the onset of cell contraction and death. There is marked heterogeneity in the timing of this response, with three distinct populations identified using Gaussian mixture models, and the duration of hyperpolarization longer for CMs that hypercontract earlier after H 2 O 2 exposure. This hyperpolarization is accompanied by a simultaneous increase in [Ca 2+ ] i and is preceded by an increase in ROS. Standard methods, which average populations of cells, miss these responses. Blockade of MPT opening with cyclosporine A delays hypercontracture and cell death but does not prevent hyperpolarization. Finally, we have used our platform to track individual mitochondria/mitochondrial regions within a single live CM, identifying mitochondrial heterogeneity within a single cell, which is increased after exposure to H 2 O 2 , isoproterenol or in CMs from mice after ischemia-reperfusion injury. By tracking individual CMs and individual mitochondria within a single CM, we have opened a window to the complex heterogeneities in mitochondrial stress response.