The Effect of Myosin Regulatory Light Chain Phosphorylation on N47K Mutant Myosin Mechanics

Abstract

Familial Hypertrophic Cardiomyopathy (FHC) is characterized by left ventricular hypertrophy that can often be preceded by diastolic dysfunction. The clinical presentation of the disease varies from asymptomatic to progressive heart failure to sudden cardiac death. FHC is caused by mutations in genes that encode for all major sarcomeric proteins. There are 12 known FHC-linked mutations in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. Two FHC mutations, N47K and R58Q, located in the RLC Ca2+-Mg2+ site have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg et al., PNAS add details 2010). In contrast, phosphorylation of the RLC can impart stiffness to the myosin lever arm. We hypothesized that phosphorylation of the N47K-RLC may mitigate distinct mutation-induced structural and functional abnormalities. To generate mutant β-myosin, native pig RLC was depleted from porcine cardiac myosin heavy chain and reconstituted with mutant N47K or wild-type human RLC. In the work presented here, in vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the N47K-RLC mutant phenotype in the presence of an α-actinin frictional load. Consistent with previous findings, myosin bearing the N47K mutation reduced actin sliding velocity compared to WT when incubated with α-actinin, resulting in a 25% reduction in force production. Phosphorylation of N47K mutant myosin increased sliding velocity and restored force production to WT values. These results point to RLC phosphorylation as a potential target to ameliorate the FHC RLC phenotype at the molecular level. Supported by AHA- 12PRE11910009 (AK), 10POST3420009 (PM), NIH- HL071778 & HL108343 (DSC) and HL077280 (JM).