Effects of Myosin Light Chain Phosphorylation on Length-Dependent Myosin Kinetics in Rat Cardiac Ventricles

Abstract

Myosin's capacity to generate force is Ca2+-regulated by thin-filament proteins and sarcomere length, which dictates the number of potential actin-myosin cross-bridge interactions throughout a heartbeat. However, the ventricular form of myosin regulatory light chain-2 (RLC) binds to the neck of myosin and is thought to modulate contraction via its phosphorylation state. Previous studies have shown that RLC phosphorylation can vary throughout the left ventricle wall, prompting the hypothesis that regional effects of RLC phosphorylation could alter myosin behavior throughout the heart. Thus, we performed biochemical analyses of flash-frozen myocardial samples to assess the relative percentage of RLC phosphorylation in the left and right ventricles. We found that RLC phosphorylation varied across the left ventricle, being significantly greater near the outside vs. the inside of the left ventricle free wall, and average RLC phosphorylation was greater in the right vs. left ventricle free wall. To assess the functional consequences of RLC phosphorylation on Ca2+-regulated contractility as sarcomere length varied, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure myosin cross-bridge kinetics as RLC phosphorylation varied with myosin light chain kinase incubation. Increases in RLC phosphorylation and sarcomere length both led to increased Ca2+-sensitivity of the force-pCa relationship, but RLC phosphorylation influenced this relationship more greatly at 2.2 vs. 1.9 µm sarcomere length. We found that RLC-phosphorylation slowed myosin rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 µm sarcomere length, whereas RLC-phosphorylation only slowed myosin MgATP binding rate (∼55%) at 2.2 µm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles.