Is Slower Myosin Cross-Bridge Kinetics in Tg-D166V Preparations Due to Decreased Myosin RLC Phosphorylation?

Abstract

In this report we have investigated a particularly malignant phenotype of Familial Hypertrophic Cardiomyopathy (FHC) associated with the 166 Aspartic Acid to Valine (D166V) mutation in the ventricular myosin regulatory light chain (RLC). We show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from transgenic (Tg)-D166V compared to Tg-WT mice. A single molecule approach was taken where the fluorescence anisotropy of rhodamine phalloidin labeled actin protomers was measured in cardiac myofibrils undergoing isometric contraction. Orientation of an actin molecule oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rates of cross-bridge attachment as well as cross-bridge dissociation were significantly decreased in isometrically contracting Tg-D166V myofibrils (binding, 1.4 s−1; detachment, 1.2 s−1) compared to Tg-WT myofibrils (binding, 3 s−1; detachment, 1.3 s−1). The duty ratio of the cross-bridge cycle, equal to the fraction of the total cycle time that cross-bridge remains attached to actin, was 47% inTg-D166V myofibrils and 30% in Tg-WT. Immunoblotting of cardiac myofibrils used for kinetics studies demonstrated a large reduction in RLC phosphorylation in Tg-D166V vs. Tg-WT myofibrils. These data are in accord with our previous findings in skinned and intact papillary muscles showing slower fiber kinetics and prolonged force transients in Tg-D166V fibers compared to Tg-WT preparations (Kerrick et al., FASEB J. 23: 855, 2009). Similarly, the level of RLC phosphorylation in muscle extracts from Tg-D166V ventricles was decreased. Our cellular and single molecule data suggest that a mutation-dependent decrease in RLC phosphorylationcould initiate the slower kinetics of the D166V cross-bridgesand ultimately lead to abnormal cardiac muscle contraction. Supported by NIH-HL071778 (DSC), NIH-AR048622 (JB) and NIH-HL090786 (to JB and DSC).