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

Abstract

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 K m values for ADP of >∼250 and ∼80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the K m 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 <100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ∼1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state. © The Authors Journal compilation © 2011 Biochemical Society.