Human skeletal myopathy myosin mutations disrupt myosin head sequestration

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyze the effects of common MYH7 and MYH2 mutations in the light meromyosin (LMM) region of myosin. Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in silico modeling showed that myosin coiled coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients and fluorescent ATP analog chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with x-ray diffraction measurements to estimate myosin head order, we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofiber mechanics experiments to investigate contractile function showed that myofiber contractility was not affected. These findings indicate that the structural remodeling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.</p

Statistical overview of the exome sequencing results: sporadic case (consanguineous) AHE6.

<p>Statistical overview of the exome sequencing results: sporadic case (consanguineous) AHE6.</p

Statistical overview of the exome sequencing results: sporadic case AHY58.

<p>Statistical overview of the exome sequencing results: sporadic case AHY58.</p

Ultrastructural analysis of muscle biopsies.

<p>Ultrastructural analysis of the biopsy from patient ARX30 revealed myofibrillar disorganization, Z-line streaming of adjacent sarcomeres (arrows), and prominent nemaline rods. The biopsy of patient IM26 showed large disorganized areas around internalized nuclei, the longitudinal muscle section of AKY21 revealed prominent Z-band streaming. Analysis of the biopsy of AHY58 demonstrated marked myofibrillar disorganization, fragmented Z-bands and internalized nuclei. The biopsy of patient AHE6 displayed nuclear centralization, myofibrillar disorganization, fibrosis, lipofuscin granules and myofiber degeneration. R  =  nemaline rods, N  =  nuclei, L  =  lipids, Lp  =  lipofuscin</p

Statistical overview of the exome sequencing results: familial cases (non-consanguineous) AKY21/IM26 and AGT66/AGT67.

<p>Statistical overview of the exome sequencing results: familial cases (non-consanguineous) AKY21/IM26 and AGT66/AGT67.</p

Histological analysis of muscle biopsies from patients ARX30, AKY21, IM26, AHY58, AGT66/AGT67, and AHE6.

<p>The deltoid muscle biopsy of patient ARX30 was performed 2 days after birth and revealed nemaline bodies, fiber size variability and type I fiber predominance. On the deltoid muscle biopsies from AKY21 and IM26 (shortly after birth), nuclear internalization, atrophy, fiber size variability, and areas devoid of oxidative enzyme activity became apparent. Analysis of the deltoid muscle biopsy of patient AHY58 (20 days) demonstrated fiber size variability, atrophy, internal nuclei, and discrete areas of reduced oxidative enzyme activity. Biceps brachii biopsy from AGT66 (8 years) and AGT67 (shortly after birth) revealed nuclear internalization, fiber size variability, multiple minicores devoid of oxidative enzyme activity and type I fiber predominance. Left deltoid muscle biopsy from patient AHE6 (performed at 30 years) revealed internalized nuclei, fiber size variation, radial arrangements of sarcoplasmic strands, necklace fibers, type I fiber predominance, core-like structures, fibrosis and fatty infiltrations.</p

Impact of the<i>NEB/RYR1</i> mutations.

<p>(A) Normal sized cDNA amplicons for exons 44 to 47 from patient ARX30 with the heterozygous c.5574C>G nonsense mutation (exon 45) and from patient ARX33 with the heterozygous c.5783_5784delAT deletion (exon 46). The splice mutation c.8160+1G>A (intron 58, ARX33) resulted in a shorter <i>NEB</i> cDNA amplicon (exons 57–60) compared to the control. The splice mutation c.19101+5G>A (intron 122, ARX30) involved a weak cDNA amplicon (exons 120–124) of normal size and a strong amplicon of smaller size. (B) The <i>NEB</i> c.8160+1G>A splice mutation (ARX33) causes a complete skipping of the in-frame exon 58. The c.5783_5784delAT mutation was not seen in the cDNA, indicating mRNA degradation by nonsense-mediated mRNA decay (NMD) of the allele containing this deletion. The <i>NEB</i> cDNA amplicon of patient ARX30 (exons 122–126) did not contain the in-frame exon 122. The c.5574C>G mutation in exon 46 was not seen by cDNA sequencing, suggesting NMD of the allele harboring this nonsense mutation. (C) Western blot of a deltoid muscle extract revealed a strong reduction of the RYR1 protein level in patient AHE6 compared to a healthy age-matched control. Desmin was used for normalization.</p

Statistical overview of the exome sequencing results: sporadic cases (trio sequencing) ARX 30 and ARX33.

<p>Statistical overview of the exome sequencing results: sporadic cases (trio sequencing) ARX 30 and ARX33.</p

Phenotypic and molecular data.

<p>n.d.  =  not determined.</p><p>Bold  =  new mutations.</p><p>For family 3, the (c.7645-7650dupGCGCTG) change is a potential modifier of the phenotype.</p

Structure and analysis of human obscurin Ig59.

<p><b>A</b>. Cartoon of the Ig59 crystal structure, showing the typical Ig-like fold. <b>B</b>. Comparison between the lowest RMSD Ig59 NMR structure and the X-ray structure. <b>C</b>. CD plot of WT obscurin Ig58-59 (black squares) and p.Arg4444Trp (open circles). <b>D</b>. MD simulated average models of WT (blue) and p.Arg4444Trp (red). Ca position. The side chains for Arg4444/Trp4444 are shown. E. RMSD vs residue number comparison of the mutant model to the wild-type model. The Arg4444Trp site is colored in red.</p