Cardiac Mitochondrial Function Assessment

Heart mitochondrial function was assessed as previously described [49 (link)]. Briefly, cardiac samples were minced in isolation buffer (300 mM sucrose, 10 mM HEPES, 2 mM EGTA, pH 7.2, 4 ℃ ), containing type I protease (bovine pancreas; Sigma, P4630), centrifuged at 500× g to pellet cell debris, followed by centrifugation of supernatant at 9000× g, resulting in a mitochondrial-enriched pellet, which was resuspended in a minimal volume of isolation buffer. Mitochondrial O₂ consumption and H₂O₂ release were measured in 0.125 mg/mL of isolated mitochondria in experimental buffer (125 mM sucrose, 65 mM KCl, 10 mM HEPES, 2 mM inorganic phosphate, 2 mM MgCl₂, 100 μM EGTA, 0.01% BSA, pH 7.2), containing succinate 2 mM (Sigma, S3674), malate 2 mM (Sigma, M1000) and glutamate 2 mM (Sigma, G1251) substrates, with continuous stirring at 37 °C. ADP 1 mM (Amresco, 0160) was added to induce state 3 respiratory rate. Mitochondrial O₂ consumption was monitored using a computer-interfaced Clark-type electrode (OROBOROS Oxygraph−2k). Mitochondrial H₂O₂ release was measured using Amplex Red 25 μM (Molecular Probes A12222) horseradish peroxidase 0.5 U/mL (Sigma P8125) system, and detected using a fluorescence spectrophotometer (ʎex = 563/ʎem = 587 nm) (F-2500 Hitachi—Hitachi).