Novel model of distal myopathy caused by the myosin rod mutation R1500P disrupts acto-myosin binding

IntroductionMore than 400 mutations in β-myosin, a slow myosin motor, can cause both cardiac and skeletal myopathy in humans. A small subset of these mutations, mostly located in the myosin rod, leads to a progressive skeletal muscle disease known as Laing distal myopathy (MPD1). While this disease has previously been studied using a variety of systems, it has never been studied in the mammalian muscle environment. Here, we describe a mouse model for the MPD1-causing mutation R1500P to elucidate disease pathogenesis and to act as a future platform for testing therapeutic interventions.MethodsBecause mice have very few slow skeletal muscles compared to humans, we generated mice expressing the β-myosin R1500P mutation or WT β-myosin in fast skeletal muscle fibers and determined the structural and functional consequences of the R1500P mutation.ResultsThe mutant R1500P myosin affects both muscle histological structure and function and the mice exhibit a number of the histological hallmarks that are often identified in patients with MPD1. Furthermore, R1500P mice show decreased muscle strength and endurance, as well as ultrastructural abnormalities in the SR & t-tubules. Somewhat surprisingly because of its location in the rod, the R1500P mutation weakens acto-myosin binding by affecting cross-bridge detachment rate.ConclusionsWhile each group of MPD1-causing mutations most likely operates through distinct mechanisms, our model provides new insight into how a mutation in the rod domain impacts muscle structure and function and leads to disease.

THERMAL STABILITY OF SYNTHETIC PEPTIDES MIMICKING THE SEQUENCE OF THE REGION CONTAINING THE SKIP RESIDUES IN SQUID MYOSIN ROD

Myosin is the major protein in skeletal muscles including those of fish and shellfish. The characteristics of this protein are closely related to the biological function and the quality and physical properties of muscle<br />food. In the myosin rod (the coiled-coil region of myosin), several amino acid residues, known as skip residues, seem to destabilize the ordered structure (heptad repeat). These residues might be responsible for reducing thermal stability. Attempts were thus made to examine the role of these residues in the rod of squid myosin, based on the thermodynamic properties of synthetic peptides which have been designed to mimic the partial sequence of myosin heavy chain from the squid Todarodes pacificus mantle muscle. Five peptides, namely, with the sequence of Trp1343 -Ala1372 having the skip residue Glu1357 at the center (Peptide WT), without the skip residue (Peptide Δ), with the replacements of the skip residue (Glu) by Ile, Gln and Pro (Peptides E/I, E/Q, and E/P, respectively) to modify the helix forming propensity, were synthesized. The results obtained showed that the stability of the peptides as measured by circular dichroism spectrometry was in the order of Peptide Δ &gt; Peptide WT &gt; Peptide E/Q &gt; Peptide E/P &gt; Peptide E/I. It is suggested that the presence of the skip residues dexterously tunes the stability or flexibility of the coiled-coil structure, thus possibly regulating thick filament formation and further gel formation ability of myosin.

Enucleation of human erythroblasts involves non-muscle myosin IIB

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34+ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.