Mitochondria Isolation and Respiration

Mitochondrial isolation and high resolution respirometry were carried out using modified versions of previously described methods ^(33–35). For mitochondrial isolation, both right and left gastrocnemius were removed, cleaned of extraneous fat and tissue, and placed in mitochondrial isolation buffer (100 mM KCl, 50 mM MOPS, 5 mM MgSO₄, 1 mM EGTA, and 1 mM ATP), on ice. Gastrocnemius muscles were homogenized using a rotor type homogenizer, the homogenate was centrifuged (800xg, 4°C, 10 min), and the supernatant filtered through gauze. The pellet was re-suspended in 5 mL mitochondrial isolation buffer, homogenized using a Potter-Elvehjenm homogenizer (Teflon on glass), and centrifuged (800xg, 4°C, 10 min). The supernatant was filtered, added to the previously filtered supernatant, and centrifuged (12,000xg, 4°C, 10 min). The pellet was re-suspended in 3 mL isolation buffer with glass on glass homogenization, centrifuged (8,000xg, 4°C, 10 min), and the process repeated with 2 mL isolation buffer. 0.2% Bovine Serum Albumin (BSA) was added to 2 mL of the isolation buffer and used to re-suspend the pellet a third time via glass on glass homogenization followed by centrifugation (8,000xg, 4°C, 10 min). Finally, the pellet was re-suspended in 200 µL MiPO₃ buffer (0.5 mM EGTA, 3 mM MgCl2·6H20, 60 mM K-lactobionate, 20 mM Taurine, 10 mM KH2P04, 20 mM HEPES, 110 mM Sucrose, 1g/l BSA, 20 mM Histidine, 20 μM vitamin E succinate, 3 mM glutathione, 1 μM leupeptine, 2 mM glutamate, 2 mM malate, 2 mM Mg-ATP)⁽³³⁾ and allowed to equilibrate on ice for 30 min.

High resolution respirometry was used to measure mitochondrial respiration by the isolated mitochondria using the Orosboros Oxygraph-2k (Orosboros Instruments; Innsbruck, Austria). 30–45 µl of isolated mitochondria were added to the respiration chambers to measure basal respiration. Steady-state oxygen flux was measured by the addition of 2 mM malate and 5 mM glutamate. Oxygen flux through complex I was measured by the titration of 125–375 µM of ADP; through complex I+II by the titration of 1–5 mM succinate, and maximal uncoupled respiration was measured by the addition of 0.125 µM FCCP. Finally, 10 µM cytochrome C was added to assess the quality of mitochondrial isolation. All measurements were normalized to the mitochondrial protein content.