Impact of Familial Hypertrophic Cardiomyopathy-Linked Mutations in the N-Terminus of the RLC on β-Myosin Cross-Bridge Mechanics

Abstract

Familial Hypertrophic Cardiomyopathy (FHC) is associated with mutations in sarcomeric proteins of which myosin regulatory light chain (RLC) is included. Here we studied the impact of the N-terminal FHC mutations on the molecular mechanism of β-Myosin heavy chain cross-bridge mechanics using the in vitro motility assay. To generate mutant β-Myosin, native pig RLC was depleted from porcine cardiac myosin heavy chain and reconstituted with mutant (A13T, F18L & E22K) or wild-type human RLC. We characterized the mutant myosin force and motion generation capability in the presence of a frictional load and thin filament regulatory proteins. All three mutants exhibited reductions in maximal filament velocity when tested with both calcium-regulated (40.5% ± 1.07, 28.9% ± 1.11, & 24.5% ± 1.14 A13T, F18L & E22K resp.) and unregulated filament (34.4% ± 0.8, 19.1% ± 0.9 & 5.6% ± 0.9 resp.) compared to wild-type. Furthermore, all three mutants displayed reductions in calcium sensitivity (pCa 50) (∼5.13%, ∼6.52% & ∼4.50% resp.) as well as cooperative activation (∼22%, ∼42% & ∼34% resp.) of regulated velocity. These results suggest that the known FHC mutants on the N-terminus of the RLC affect formation of strong binding cross-bridges thereby reducing maximal force production. This work is supported by grants to the following people HL077280 (JM) HL071778 (D S-C).