Bioenergetic Assessment of Cardiomyocytes

We used XF Cell Mito Stress Test and XF Glycolytic Rate Assay kit to measure the oxygen consumption rate (OCR) for the mitochondrial respiratory activity and proton efflux rate (PER) for the glycolytic levels in the cardiomyocytes, by using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent, CA). Cells (45,000) were plated into an Xfe96 cell culture microplate (Agilent) containing RPMI/B27 supplemented with 10% FBS and 10 μM ROCK inhibitor. After 48 h to allow recovery, we conducted the metabolic profiling using the XFe96 Seahorse analyzer with two kits according to the manufacture’s manual. Briefly, 1 day prior to the experiment, the Xfe96 sensor cartridges were hydrated in XF calibrator solution and incubated overnight at 37°C in a non-CO₂ incubator. 1 hour prior to the experiment, the cells were incubated at 37°C (non-CO₂) in 200 μl of Seahorse assay medium, containing XF base medium supplemented 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose (pH 7.4). OCR was measured with sequential injections of 2 μM oligomycin, 2 μM FCCP and each 0.5 μM of rotenone/antimycin A. PER was measured with sequential injections of 0.5 μM of rotenone/antimycin A and 50 mM of 2-deoxy-D-glucose (2-DG). Data were normalized by fluorescence of cell viability using PrestoBlue reagent (Thermo Fisher).