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 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 {beta}-myosin heavy chain (amino acids 664-915) with alanine-mutated IQ2 domain (r{beta}-MYH(664-915)IQ2(ala4)) 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-1(E56G)) hVLC-1 constructs in neonatal rat cardiomyocytes. Fluorescence lifetime imaging microscopy (FLIM) was applied to map the microenvironment of normal and E56G mutated hVLC-1 in permeabilized muscle fibers. Affinity of M149V, E143K, A57G, and R154H mutated hVLC-1/ r{beta}-MYH(664-915)IQ2(ala4) complexes were significantly lower compared with the normal hVLC-1/ r{beta}-MYH(664-915)IQ2(ala4) complex interaction. In particular the E56G mutation induced an about 30fold lower MYH affinity. Sorting specificity of E56G-mutated hVLC-1 was negligible compared with normal hVLC-1. Fluorescence lifetime of fibers replaced with hVLC-1(E56G) increased significantly compared with hVLC-1 replaced fibers. Conclusion: Disturbed myosin binding of mutated hVLC-1 may provide a pathomechanism for the development of hypertrophic cardiomyopathy