Measuring Skeletal Muscle Mitochondrial Function

Oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity was determined by high‐resolution respirometry ex‐vivo in skeletal muscle tissue homogenates. At the time of biopsy, 30–45 mg of muscle tissue was procured and immediately placed into a biopsy preservation solution (BIOPS) for up to 4 h. as previously described (Axelrod et al., 2021 (link)). To accurately calculate the tissue homogenate concentration, a tissue homogenate preparation protocol (Zunica et al., 2021 (link)) was adapted to skeletal muscle tissue as prepared similar to what has been optimized by Ziak et al. (Ziak et al., 2015 (link)). Briefly, tissue was transferred to a mitochondrial respiration medium, (MiR05) blotted on filter paper, and weighed. ~30 mg of tissue was transferred into a chilled glass‐on‐glass dounce homogenizer with 2 mL of MiR05 and homogenized using 8–10 strokes. The homogenate was transferred to a falcon tube and the homogenizer and pestle were washed with additional MiR05 to ensure complete transfer of sample. Non‐homogenized tissue pieces were removed from the homogenate, blotted, and weighed‐ which was subtracted from the initial wet weight to determine the final sample weight. All samples were brought up to a final concentration of 4 mg/mL using additional MiR05 and 2.25 mL were added to the Oxygraph chambers. OXPHOS and ET capacity was determined using the following concentrations of substrates, uncouplers, and inhibitors: malate (2 mM), pyruvate (2.5 mM), ADP (2.5 mM), glutamate (10 mM), succinate (10 mM), tetramethyl‐p‐phenylenediamine (TMPD, 0.5 μM), ascorbate (2 mM), carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP, 0.5 μM increment), rotenone (75 nM), antimycin A (125 nM), and sodium azide (200 mM). Oxygen flux was normalized to wet weight of total homogenized tissue (mg). Cytochrome c (10 μM) was added after the addition of glutamate to confirm mitochondrial outer membrane integrity and to ensure cytochrome c was not limiting for the measurement of each OXPHOS and ET state (Figure S1).