A Laing distal myopathy-associated proline substitution in the ß-myosin rod perturbs myosin cross-bridging activity.

Proline substitutions within the coiled-coil rod region of the ß-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

Distinct effects of two hearing loss-associated mutations in the sarcomeric myosin MYH7b.

For decades, sarcomeric myosin heavy chain proteins were assumed to be restricted to striated muscle where they function as molecular motors that contract muscle. However, MYH7b, an evolutionarily ancient member of this myosin family, has been detected in mammalian nonmuscle tissues, and mutations in MYH7b are linked to hereditary hearing loss in compound heterozygous patients. These mutations are the first associated with hearing loss rather than a muscle pathology, and because there are no homologous mutations in other myosin isoforms, their functional effects were unknown. We generated recombinant human MYH7b harboring the D515N or R1651Q hearing loss-associated mutation and studied their effects on motor activity and structural and assembly properties, respectively. The D515N mutation had no effect on steady-state actin-activated ATPase rate or load-dependent detachment kinetics but increased actin sliding velocity because of an increased displacement during the myosin working stroke. Furthermore, we found that the D515N mutation caused an increase in the proportion of myosin heads that occupy the disordered-relaxed state, meaning more myosin heads are available to interact with actin. Although we found no impact of the R1651Q mutation on myosin rod secondary structure or solubility, we observed a striking aggregation phenotype when this mutation was introduced into nonmuscle cells. Our results suggest that each mutation independently affects MYH7b function and structure. Together, these results provide the foundation for further study of a role for MYH7b outside the sarcomere.

Expression of Normally Repressed Myosin Heavy Chain 7b in the Mammalian Heart Induces Dilated Cardiomyopathy.

Background In mammals, muscle contraction is controlled by a family of 10 sarcomeric myosin motors. The expression of one of its members, MYH7b, is regulated by alternative splicing, and while the protein is restricted to specialized muscles such as extraocular muscles or muscle spindles, RNA that cannot encode protein is expressed in most skeletal muscles and in the heart. Remarkably, birds and snakes express MYH7b protein in both heart and skeletal muscles. This observation suggests that in the mammalian heart, the motor activity of MYH7b may only be needed during development since its expression is prevented in adult tissue, possibly because it could promote disease by unbalancing myocardial contractility. Methods and Results We have analyzed MYH7b null mice to determine the potential role of MYH7b during cardiac development and also generated transgenic mice with cardiac myocyte expression of MYH7b protein to measure its impact on cardiomyocyte function and contractility. We found that MYH7b null mice are born at expected Mendelian ratios and do not have a baseline cardiac phenotype as adults. In contrast, transgenic cardiac MYH7b protein expression induced early cardiac dilation in males with significantly increased left ventricular mass in both sexes. Cardiac dilation is progressive, leading to early cardiac dysfunction in males, but later dysfunction in females. Conclusions The data presented show that the expression of MYH7b protein in the mammalian heart has been inhibited during the evolution of mammals most likely to prevent the development of a severe cardiomyopathy that is sexually dimorphic.

Two novel MYH7 proline substitutions cause Laing Distal Myopathy-like phenotypes with variable expressivity and neck extensor contracture.

BACKGROUND: Human skeletal muscles express three major myosin heavy chain (MyHC) isoforms: MyHCIIx (MYH1) in fast type 2B muscle fibers, MyHCIIa (MYH2) in fast type 2A fibers and MyHCI/ß-cardiac MyHC (MYH7) in slow type I skeletal fibers and cardiac ventricles. In line with its expression pattern, MYH7 mutations have been reported in association with hypertrophic or dilated cardiomyopathy, skeletal myopathies or a combination of both. We analyzed the clinical and molecular phenotype of two unrelated families of Jewish Moroccan ancestry that presented with apparently autosomal dominant inheritance of progressive Laing-like distal myopathy with non-specific myopathic changes, but uncommon marked contractures and wasting of the neck extensors. METHODS: Clinical phenotyping, whole exome sequencing and restriction analysis, generation of mutants followed by cell culture transfection and imaging. RESULTS: Using whole exome sequencing we identified in both families two novel heterozygous proline substitutions located in exon 31 of MYH7 within its rod domain: c.4309G>C (p.Ala1437Pro) and c.4301G>C (p.Arg1434Pro). Here we show that the phenotype caused by these mutations includes marked cervical muscle contracture, and report that the severity of the phenotype varies significantly, to the extent of non-penetrance in one of the families. Finally, we provide evidence that both proline substitutions impair myosin self-assembly in non-muscle cells transfected with ß-myosin constructs carrying the mutations, but do not prevent incorporation of the mutant molecules into the sarcomere. CONCLUSIONS: This study expands our clinical and molecular knowledge of MYH7 rod mutations causing skeletal myopathies, and underscores the importance of discussing disease penetrance during genetic counseling.

Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly.

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report here that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility. Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. In addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.

Myosin filament assembly requires a cluster of four positive residues located in the rod domain.

Myosin has an intrinsic ability to organize into ordered thick filaments that mediate muscle contraction. Here, we use surface plasmon resonance and light scattering analysis to further characterize the molecular determinants that guide myosin filament assembly. Both assays identify a cluster of lysine and arginine residues as important for myosin polymerization in vitro. Moreover, in cardiomyocytes, replacement of these charged residues by alanine severely affects the incorporation of myosin into the distal ends of the sarcomere. Our findings show that a novel assembly element with a distinct charge profile is present at the C-terminus of sarcomeric myosins.

Effects of pathogenic proline mutations on myosin assembly.

Laing distal myopathy (MPD1) is a genetically dominant myopathy characterized by early and selective weakness of the distal muscles. Mutations in the MYH7 gene encoding for the ß-myosin heavy chain are the underlying genetic cause of MPD1. However, their pathogenic mechanisms are currently unknown. Here, we measure the biological effects of the R1500P and L1706P MPD1 mutations in different cellular systems. We show that, while the two mutations inhibit myosin self-assembly in non-muscle cells, they do not prevent incorporation of the mutant myosin into sarcomeres. Nevertheless, we find that the L1706P mutation affects proper antiparallel myosin association by accumulating in the bare zone of the sarcomere. Furthermore, bimolecular fluorescence complementation assay shows that the α-helix containing the R1500P mutation folds into homodimeric (mutant/mutant) and heterodimeric [mutant/wild type (WT)] myosin molecules that are competent for sarcomere incorporation. Both mutations also form aggregates consisting of cytoplasmic vacuoles surrounding paracrystalline arrays and amorphous rod-like inclusions that sequester WT myosin. Myosin aggregates were also detected in transgenic nematodes expressing the R1500P mutation. By showing that the two MPD1 mutations can have dominant effects on distinct components of the contractile apparatus, our data provide the first insights into the pathogenesis of the disease.

Interplay between exonic splicing enhancers, mRNA processing, and mRNA surveillance in the dystrophic Mdx mouse.

BACKGROUND: Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5' and 3' splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC) often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD), lacks dystrophin by virtue of a premature termination codon (PTC) in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described. METHODOLOGY/PRINCIPAL FINDING: Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE) that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3' splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS), but induces exclusively nonsense-mediated mRNA decay (NMD). Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24. CONCLUSIONS/SIGNIFICANCE: Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon-exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites.