Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle

Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (~20-300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km