Mutations of ventricular essential myosin light chain disturb myosin binding and sarcomeric sorting

Aims We tested the hypothesis that mutations in the human ventricular essential myosin light chain (hVLC-1) that are associated with hypertrophic cardiomyopathy (HCM) affect protein structure, binding to the IQ1 motif of cardiac myosin heavy chain (MYH) and sarcomeric sorting in neonatal cardiomyocytes. Methods and results We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties and protein-protein interactions of a recombinant head-rod fragment of rat cardiac β-MYH (amino acids 664-915) with alanine-mutated IQ2 domain (rβ-MYH664-915IQ2ala4) and normal or five mutated (M149V, E143K, A57G, E56G, R154H) hVLC-1 forms. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced normal and E56G-mutated (hVLC-1E56G) hVLC-1 constructs in neonatal rat cardiomyocytes. Fluorescence lifetime imaging microscopy was applied to map the microenvironment of normal and E56G-mutated hVLC-1 in permeabilized muscle fibres. Affinity of M149V, E143K, A57G, and R154H mutated hVLC-1/rβ-MYH664-915IQ2ala4 complexes was significantly lower compared with the normal hVLC-1/rβ-MYH664-915IQ2ala4 complex interaction. In particular, the E56G mutation induced an ∼30-fold lower MYH affinity. Sorting specificity of E56G-mutated hVLC-1 was negligible compared with normal hVLC-1. Fluorescence lifetime of fibres replaced with hVLC-1E56G increased significantly compared with hVLC-1-replaced fibres. Conclusion Disturbed myosin binding of mutated hVLC-1 may provide a pathomechanism for the development of HCM.