Phosphorylation of Myosin Increases the ATPase Activity of Relaxed Skeletal Muscle Fibers

Abstract

Myosin in skeletal muscle is phosphorylated by a calcium-calmodulin dependent kinase during periods of sustained activity. Phosphorylation of myosin is known to destabilize the array of myosin heads bound to the core of the thick filament in relaxed muscle, known as the interacting heads motif. Myosin heads in this array are associated with a state with a highly inhibited ATPase activity, known as the Super Relaxed State (SRX). To determine whether phosphorylation of myosin increases the ATPase activity, we measured the ATPase activity of relaxed, skinned skeletal fibers in the presence and absence of myosin phosphorylation. Fibers were phosphorylated, >50%, by including phosphatase inhibitors in the glycerinating solution. Control fibers had an ATPase activity of 0.040 ± 0.004 s−1. Phosphorylated fibers had an ATPase activity of 0.060 ± 0.002 s−1, a 50 % increase. We have recently found a small molecule, piperine that destabilizes the SRX, increasing relaxed fiber ATPase activity. In the presence of 100μM piperine the control fibers had an ATPase activity of 0.064 ± 0.004 s−1, the phosphorylated fibers had an ATPase activity of 0.057 ± 0.001 s−1. Thus both phosphorylation and piperine increase the ATPase activity of resting fibers by destabilizing a fraction of myosin heads in the SRX, 30-50%, but their effects are not additive, suggesting that they are each affecting the same population of myosin heads. One candidate for this population may be the “free” head of the interacting heads motif, which is known to be less stable than the other head. As myosin remains in a phosphorylated state for several minutes following a period of activity, the effect described above will contribute to the energetic cost of physical activity, particularly during light activity.