Mitochondrial Respiration Analysis Protocol

Muscle samples collected into ice-cold BIOPS and stored on ice or at 4 °C were analyzed for mitochondrial oxidative phosphorylation (P) and electron transfer system capacities (E) using high-resolution respirometry within 24 h of collection. Immediately prior to analysis, samples were saponin permeabilized as described previously [5 (link)]. Permeabilized fibers were then rinsed in mitochondrial respiration solution (MiR05; 110 mM sucrose, 60 mM potassium lactobionate, 0.5 mM EGTA, 3 mM MgCl₂∙6H₂O, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 1 g/L BSA, pH 7.1) for 10 min at 4 °C. Approximately 1.5–2.5 mg (wet weight) of rinsed fibers were then immediately added to each chamber of an Oroboros Oxygraph-2k (O2k; Oroboros, Innsbruck, Austria) containing MiR06 (MiR05 + 280 U/mL catalase) and 20 mM creatine. Chambers were maintained at 37 °C and in hyperoxic conditions (200 to 650 µM O₂) through the addition of 200 mM H₂O₂. The previously described [13 (link)] substrate uncoupler inhibitor titration protocol for this study was as follows: (1) complex I substrates, pyruvate (5 mM), and malate (1 mM), to determine mitochondrial proton leak (LEAK); (2) adenosine diphosphate (ADP; 2.5 mM), to quantify complex I-supported P (P_CI); (3) glutamate (10 mM), an additional complex I substrate (P_CIG); (4) cytochrome c (cyt c, 10 µM), to measure integrity of the outer mitochondrial membrane; (5) the complex II substrate, succinate (10 mM), to measure maximal P (P_CI+II); (6) uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 0.5 µM steps), to attain maximal noncoupled E (E_CI+II); (7) a complex I inhibitor, rotenone (0.5 µM), to measure complex II-supported E (E_CII); and (8) a complex III inhibitor, antimycin A (2.5 µM), to quantify non-mitochondrial residual O₂ consumption. All data were normalized to residual O₂ consumption. Respiration data are presented either relative to tissue weight (integrative), CS activity (mitochondrial volume density; intrinsic), or as a ratio of contribution to maximal electron transfer capacity (flux control ratio, FCR).