Biochemical Phenotypes Associated with the Myosin Essential Light Chain Mutations

Abstract

To investigate the effects of familial hypertrophic cardiomyopathy (FHC) mutations in the ventricular myosin essential light chain (ELC) we have exchanged the human ventricular wild type (WT) and two FHC ELC mutants (A57G, E143K) for the endogenous porcine ELC in isolated and purified pig cardiac myosin.To elucidate the importance of the long N-terminus of cardiac myosin ELC for the actin-myosin interaction, we also exchanged the N-terminal truncation mutant ELC-Δ43 into native porcine myosin. The phenotype associated with the A57G mutation consists of a classic asymmetric hypertrophy with varying pathology and disease progression including sudden cardiac death (SCD) (Lee, et al. (2001) Am Heart J141, 184-9). The E143K mutation is associated with a restrictive cardiomyopathy and SCD phenotype (Olson et al. (2002) Circulation105, 2337-40). We hypothesized that FHC ELC mutants may bind to the myosin heavy chain with a lower affinity than ELC-WT, thus affecting the structural integrity of the thick filaments in muscle. SDS-PAGE demonstrated that indeed the A57G mutation yielded lower percent exchange in cardiac porcine myosin compared to WT. We further hypothesized that as a consequence the interaction of ELC-mutant myosin with actin will be affected. All ELC-mutant myosins were tested for their ability to bind actin using fluorescence spectroscopy and pyrene-labeled F-actin. We observed a significantly decreased binding affinity for both FHC mutants while the binding of the ELC-Δ43 mutant myosin to F-actin was stronger than WT. The latter supports the hypothesis that the N-terminus of ELC acts as a molecular constrain inhibiting the actin-myosin interaction. Lower binding affinity of myosin containing the A57G and E143K mutations might be responsible for the development of the pathologic cardiac phenotype observed in patients carrying these FHC mutations. Supported by NIH HL071778 and NIH HL090786.