2.15. Measurement of mitochondrial oxygen consumption rate (OCR)

Mitochondria from cardiac tissue were isolated by differential centrifugation as described previously [26]. The OCRs of isolated intact mitochondria or H9c2 cells were measured with a Seahorse XFe24 analyzer (Agilent Technologies, Santa Clara, CA). Freshly isolated cardiac mitochondria or H9c2 cells were transferred to a XFe24 microplate, and the microplate were filled with Mitochondrial Assay Solution (MAS: 220 mM mannitol, 70 mM sucrose, 10 mM MgCl₂, 2 mM HEPES, 1 mM EGTA, 10 mM KH₂PO₄ and BSA 0.02% (w/v) at pH 7.2) to a final amount of 500 μl containing succinate (10 μM; Sigma). The OCR was measured in response to sequential injection of ADP (4 mM; Sigma), oligomycin (2.5 μg/ml; MCE), trifluorocarbonyl cyanide phenylhydrazone (FCCP, 4 μΜ; MCE), and antimycin A (4 μΜ; Sigma), at 37 °C. The respiratory rates are reported as oxygen flux per mass, and all readings are normalized to μg of mitochondrial protein or cell numbers (pmol O₂/min/μg protein or 25000 cells).