The Effect of Myosin RLC Phosphorylation in Healthy and Diseased Heart

Abstract

The aim of this study was to understand the mechanism by which the D166V (aspartic acid to valine) mutation in the myosin regulatory light chain (RLC), found to cause hypertrophic cardiomyopathy (HCM) and sudden cardiac death, compromises cardiac contractility and how normal cardiac function could be restored upon phosphorylation of myosin RLC. Our previous studies using papillary muscle fibers from transgenic (Tg) mice expressing the D166V-RLC mutation indicated a diastolic dysfunction caused by a delayed muscle relaxation most likely due to a slower rate of myosin cross-bridge attachment and dissociation. Transmitral Doppler studies confirmed diastolic dysfunction in Tg-D166V mice. Furthermore, invasive hemodynamics examination revealed systolic abnormalities in mutated mice compared with healthy Tg-WT controls. This compromised cardiac function was paralleled by a decrease in RLC phosphorylation in the hearts of Tg-D166V mice compared to Tg-WT. To further pursue this, the effect of myosin light chain kinase (MLCK)-dependent RLC phosphorylation was studied in skinned cardiac muscle preparations from Tg-D166V vs. Tg-WT mice. A phosphorylation-dependent rescue of an increased Ca2+ sensitivity of force was observed in Tg-D166V fibers. Interestingly, upon phosphorylation the maximal force was increased in Tg-WT fibers while it was significantly reduced in Tg-D166V preparations. Myofibrillar ATPase activity was increased in both Tg-WT and Tg-D166V mouse preparations upon MLCK-treatment. Similar results were obtained with recombinant phosphorylation mimics of human cardiac RLC exchanged into porcine cardiac muscle preparations. Beneficial effects of phosphomimetic proteins on the actin-activated myosin ATPase activity and binding affinity of the mutant exchanged myosin to actin were observed. These results suggest that RLC phosphorylation could play an important role in cardiac muscle contraction and may serve as a therapeutic modality in patients with HCM. Supported by American Heart Association-10POST3420009 (PM) and NIH-HL090786 (DSC).