Abstract 388: High-throughput Single Cell Tracking of Mitochondrial Function in Cardiomyocytes

Abstract

Mitochondria play a significant role in the regulation of multiple functions in the heart, ranging from metabolism to cell death. Most mitochondrial assays require either isolating the organelle, thus disrupting the intracellular signaling or using intact cells with average measurements for the entire population neglecting the ability to distinguish cell heterogeneity. Here we describe a novel method for tracking single cell mitochondrial function in cell populations over time. Adult mouse myocytes were exposed to the mitochondrial uncoupler FCCP and hydrogen peroxide to induce changes in membrane potential and oxidative stress, respectively. TMRM and mitosox fluorescence were used to quantify mitochondrial membrane potential and ROS production, Fluo-4 fluorescence was used to assess intracellular calcium. Tracking of single cells was performed using Matlab and Imaris software. After FCCP exposure TMRM signal intensity decreased before there was a significant change in myocyte length that led to hypercontracture and cell death within 10 minutes. To better understand the dynamics of mitochondrial function and its relationship with cell death a dose response curve was established for hydrogen peroxide at 100, 500 and 1000 μM. The higher doses of hydrogen peroxide induced hypercontracture faster than lower doses. For further studies 100 μM was used to assess how homogenous the response to hydrogen peroxide was in cell populations. We found 3 distinct populations of cells responding at different times, a population of cells hypercontracted between 7-10 min, another between 10-15 min and a third population only after 15 min. Changes in membrane potential, oxidative stress and intracellular calcium were simultaneously assessed for every single cell during these time points. In addition, given the organized structure of the mitochondria in the myocyte we have adapted our technique to track individual mitochondria to study heterogeneous responses at the single mitochondrion level. This method provides a unique tool to simultaneously assess multiple parameters of mitochondrial function in single cells over time. In doing so, we have unmasked a complex heterogeneity of single cell behavior that is lost in methods than only average cell populations.