Search

The development of small molecule myosin modulators has seen an increased effort in recent years due to their possible use in the treatment of cardiac and skeletal myopathies. Omecamtiv mecarbil (OM) is the first-in-class cardiac myotrope and the first to enter clinical trials. Its selectivity toward slow/beta-cardiac myosin lies at the heart of its function; however, little is known about the underlying reasons for selectivity to this isoform as opposed to other closely related ones such as fast-type skeletal myosins. In this work, we compared the structure and dynamics of the OM binding site in cardiac and in fasttype IIa skeletal myosin to identify possible reasons for OM selectivity. We found that the different shape, size, and composition of the binding pocket in skeletal myosin directly affects the binding mode and related affinity of OM, which is potentially a result of weaker interactions and less optimal molecular recognition. Moreover, we identified a side pocket adjacent to the OM binding site that shows increased accessibility in skeletal myosin compared with the cardiac isoform. These findings could pave the way to the development of skeletal-selective compounds that can target this region of the protein and potentially be used to treat congenital myopathies where muscle weakness is related to myosin loss of function.

Nemaline myopathy is a congenital disorder characterized by muscle weakness. Dominant-negative disease-causing mutations have been identified in various genes that encode sarcomeric thin filament proteins. of these, mutations in skeletal muscle α-actin (ACTA1) account for ∼25% of all reported cases. To investigate the myopathic phenotypes from the level of whole muscle down through the level of single molecules, we generated multiple transgenic Drosophila fly lines expressing three different mutations - V37L, D288G and F352S - in Act88F, the indirect flight muscle (IFM) actin gene. A gradation in flight behavior was observed (V37L>D288G>F352S), which correlated with the observed differences in muscle fiber morphology. While most of these mutations depress muscle function in patients, the F352S lesion elevates myosin cross-bridge strain and steady-state isometric force production and, thereby, apparently increases contractile function. Confocal and electron microscopy revealed breaks in mutant F352S IFM fibers and extensive myofibrillar and myofilamentous disarray. Z-lines showed streaming and formed “zebra bodies”. The microscopic alterations are consistent with excessive acto-myosin interactions, unevenly distributed force, and destructive hypercontraction. Interestingly, in vitro sliding velocities of purified IFM F-actin from wild-type (3.17±0.12 μm/s) vs. F352S mutant heterozygous (2.88±0.09 μm/s, mean±SEM) flies did not significantly differ. Similarly, no differences in maximum calcium-activated velocity (3.88±0.11 vs. 3.77±0.10 μm/s) or in cooperativity (nH = 2.13±0.27 vs. 2.62±0.46) were observed for F-actin in the presence of vertebrate tropomyosin and troponin. However, relative to control, F352S heterozygous filaments showed a significant (p

Hereditary myosinopathies have emerged as a new group of diseases and are characterized by skeletal muscle weakness. These diseases are notably caused by mutations in genes encoding myosin heavy chain (MyHC) isoforms. A new mouse model with a L342Q MyHC IIb mutation has been developed to mimic such myosinopathies. Homozygous mice experience severe skeletal muscle paralysis and, because of ethical reasons, are killed thirteen days after birth. Heterozygous animals have a normal lifespan and no apparent weakness. The underlying mechanisms for such absence of deteriorated phenotype in heterozygous rodents remain unknown. In the current study, we aimed at investigating this particular point by evaluating whether and how the presence of 50% of L342Q MyHC IIb mutants affects the contractile function. We compared isolated membrane-permeabilized muscle fibers from heterozygote (L342Q+/-) and wild-type young adult mice. Results showed, during full activation, at pCa 4.5, an increase in force production in L342Q+/- when compared with wild-type animals (p