Measurement of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cardiomyocytes

An XF24 extracellular flux analyzer (Agilent Seahorse Bioscience, CA, USA) was used to measure the OCR and ECAR as previously described [9]. Cardiomyocytes were seeded in Seahorse XF24 microplates at a density of 10⁴ cells/well. After treatments, the cells were cultured with XF assay medium in a 37 °C incubator without CO₂ for 1 h before being assayed. Basal respiration was measured before the addition of oligomycin. Proton leak-linked respiration was recorded after oligomycin was added. The increase in the oxygen consumption of the cells after treatment with FCCP indicates the maximal respiratory capacity of these cells. Finally, antimycin A and rotenone were used to evaluate the spare respiratory capacity. Measurement of ECAR was performed in glucose-deprived media with the successive addition of glucose, oligomycin, and 2-deoxyglucose. The OCR and ECAR values were normalized to the protein concentration of the cells and are presented as pmol/min/μg protein and mpH/min/μg protein, respectively.