Regulatory Light Chain Phosphorylation Mimic S15D Causes Partial Rescue of Isometric Force Production in FHC Causing Mutation D166V

Abstract

Familial hypertrophic cardiomyopathy (FHC) is characterized by a pathological thickening of the muscle surrounding the heart and is the leading cause for sudden cardiac death in young people. There are numerous mutations in sarcomeric proteins that have been implicated in causing FHC. Myosin, the molecular motor that powers cardiac muscle contraction, consists of a globular domain and an elongated α-helical neck region, which is thought to undergo large conformational changes during muscle contraction. The myosin regulatory light chain (RLC) functions to support the neck region, therefore it is not surprising that several single amino acid substitutions in the RLC have been implicated in FHC.Here we studied D166V, an RLC point mutation that is associated with increased left ventricular wall thickness, abnormal electrocardiogram, and decreased isometric force and ATPase in skinned fibers from transgenic mouse hearts. We have expressed porcine cardiac β myosin (the same isoform found in humans) and replaced the endogenous RLC with a human RLC. The exchanged RLC contained either wild type (WT), D166V, S15A, or a double mutant D166V/S15A, or D166V/S15D. The S15D mutation served as a phosphorylation mimic. We performed frictional loading assays using a modified in vitro motility assay and determined the average force produced by a bed of monomeric myosin. While maximal unloaded velocity was unchanged for all of the mutants studied, the force of D166V and D166V/S15A was significantly reduced compared to WT, with an additive decrease in the double mutant. D166V/S15D resulted in a significant increase in force compared to D166V/S15A and was restored to near WT levels. These results suggest that D166V causes a reduction in myosin isometric force production and phosphorylation may act to recover it.