High-resolution respirometry of permeabilized muscle fibers.

Respiratory activity was assessed in soleus fiber bundles using an Oxygraph system (Hansatech Instruments) as described by De Palma et al. (65). Fibers from soleus muscle were prepared in ice-cold BIOPS (50 mM K⁺-MES, 20 mM taurine, 0.5 mM dithiothreitol, 6.56 mM MgCl₂, 5.77 mM ATP, 20 mM imidazole, 10 mM Ca-EGTA buffer, pH 7.1), and then permeabilized in 2 ml of BIOPS solution containing 50 μg/ml saponin by gentle agitation for 30 minutes at 4°C. After rinsing for 10 minutes in ice-cold mitochondrial respiration medium (MiR06; 110 mM sucrose, 60 mM K-lactobionate, 0.5 mM EGTA, 3 mM MgCl₂, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 1 g/l BSA, and 280 U/ml catalase, pH 7.1), the muscle fibers were weighed and added to the Oxygraph respiratory chamber. Respiration was measured in MiR06 at 37°C. With 1 mM ADP, in the presence of 5 mM malate and 5 mM pyruvate, coupled respiration is maximally stimulated. Respiration rates are expressed as pmol O₂/s/mg dried fiber.