Measurement of activation energy and oxidative phosphorylation onset kinetics in isolated muscle fibers in the absence of cross-bridge cycling

Abstract

This study utilized N-benzyl- p-toluene sulfonamide (BTS), a potent inhibitor of cross-bridge cycling, to measure 1) the relative metabolic costs of cross-bridge cycling and activation energy during contraction, and 2) oxygen uptake kinetics in the presence and absence of myosin ATPase activity, in isolated Xenopus laevis muscle fibers. Isometric tension development and either cytosolic Ca2+concentration ([Ca2+]c) or intracellular Po2(P[Formula: see text]) were measured during contractions at 20°C in control conditions (Con) and after exposure to 12.5 μM BTS. BTS attenuated tension development to 5 ± 0.4% of Con but did not affect either resting or peak [Ca2+]cduring repeated isometric contractions. To determine the relative metabolic cost of cross-bridge cycling, we measured the fall in P[Formula: see text] (ΔP[Formula: see text]; a proxy for V̇o2) during contractions in Con and BTS groups. BTS attenuated ΔP[Formula: see text] by 55 ± 6%, reflecting the relative ATP cost of cross-bridge cycling. Thus, extrapolating ΔP[Formula: see text] to a value that would occur at 0% tension suggests that actomyosin ATP requirement is ∼58% of overall ATP consumption during isometric contractions in mixed fiber types. BTS also slowed the fall in P[Formula: see text] (time to 63% of overall ΔP[Formula: see text]) from 75 ± 9 s (Con) to 101 ± 9 s (BTS) ( P < 0.05), suggesting an important role of the products of ATP hydrolysis in determining the V̇o2onset kinetics. These results demonstrate in isolated skeletal muscle fibers that 1) activation energy accounts for a substantial proportion (∼42%) of total ATP cost during isometric contractions, and 2) despite unchanged [Ca2+]ctransients, a reduced rate of ATP consumption results in slower V̇o2onset kinetics.