Measurement of Mitochondrial Respiratory Capacity in Permeabilized Skeletal Muscle

Mitochondrial oxygen consumption was measured in freshly permeabilized skeletal muscle tissue by high-resolution respirometry (Oxygraph O2k; Oroboros Instruments, Innsbruck, Austria) as previously described [32]. Fiber bundles were separated in ice-cool BIOPS buffer composed of 10 mM Ca-EGTA, 0.1 µM free calcium, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM dithiothreitol, 6.56 mM MgCl₂, 5.77 mM ATP, and 15 mM phosphocreatine (pH 7.1), and then permeabilized with BIOPS containing saponin (5 mg/mL). The tissue (3 mg/mL) was resuspended in MiR05 respiratory liquid composed of 110 mM sucrose, 60 mM K-lactobionate, 0.5 mM EGTA, 3 mM MgCl₂•6H₂O, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, and defatted BSA (1 g/L) at pH 7.1. In the sealed respirometer, different substrates were slowly titrated according to the substrate-uncoupler-inhibitor-titration protocol [32], with 5 mM malate plus 10 mM glutamate as complex I substrates and 5 mM malate plus 0.2 mM octanoylcarnitine as β-oxidation substrates. The above steps were performed in the absence of 10 mM ADP (State 4). State 3 respiration was measured after adding ADP. States 3 and 4 were inhibited by adding 0.5 μM rotenone. The respiration control ratio was calculated by dividing the State 3 respiration rate by State 4 respiration rate. The coupling efficiency was calculated as follows to estimate mitochondrial coupling: j ≈ P = P − L/P, where j ≈ P = FAO/oxidative phosphorylation (Oxphos) coupling efficiency, where P = State 3 respiration rate, and L = State 4 respiration rate. Cytochrome c (10 μM) was added to verify the integrity of the mitochondrial outer membrane. An increase in O₂ consumption of more than 15% following the addition of cytochrome c indicated that the mitochondrial membrane was destroyed [32], and the data on mitochondrial respiratory capacity were omitted from the analysis if it occurred.