Measurement of mitochondrial membrane potential and ROS production

Isolation of single cardiomyocyte from adult mouse heart by Langendorff perfusion was performed as previously described [36]. Briefly, hearts were perfused with collagenase type II (catalog no. {"type":"entrez-nucleotide","attrs":{"text":"LS004177","term_id":"1321650547","term_text":"LS004177"}}LS004177, Worthington) to dissociate individual cardiomyocytes. Isolated cells were then filtered through a 100-μm mesh nylon filter before performing sequential sedimentation to enrich for myocytes, and plated onto glass-covered dishes coated with Laminin. To measure mitochondrial membrane potential of individual cardiomyocytes, cells were incubated with 25 nM Tetramethylrhodamine (TMRM) (catalog no. T668, Thermofisher Scientific) for 60 min at 37 °C, washed, and imaged by confocal microscopy using the light of 543 nm. To determine mitochondrial superoxide production, cells were incubated with 2 μM MitoSOX™ red mitochondrial superoxide indicator (catalog no. {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008, Thermofisher Scientific) for 20 min at 37 °C, washed, and imaged at 514 nm. Data were analyzed using Image J software.