Over 500 disease-causing point mutations have been found in the human b-cardiac myosin heavy chain, many quite recently with modern sequencing techniques. This review shows that clusters of these mutations occur at critical points in the sequence and investigates whether the many studies on these mutants reveal information about the function of this protein.

The myosin 2 family of molecular motors includes isoforms regulated in different ways. Vertebrate smooth-muscle myosin is activated by phosphorylation of the regulatory light chain, whereas scallop striated adductor-muscle myosin is activated by direct calcium binding to its essential light chain. The paired heads of inhibited molecules from myosins regulated by phosphorylation have an asymmetric arrangement with motor-motor interactions. It was unknown whether such interactions were a common motif for inactivation used in other forms of myosin-linked regulation. Using electron microscopy and single-particle image processing, we show that indistinguishable structures are indeed found in myosins and heavy meromyosins isolated from scallop striated adductor muscle and turkey gizzard smooth muscle. The similarities extend beyond the shapes of the heads and interactions between them: In both myosins, the tail folds into three segments, apparently at identical sites; all three segments are in close association outside the head region; and two segments are associated in the same way with one head in the asymmetric arrangement. Thus, these organisms, which have different regulatory mechanisms and diverged from a common ancestor >600 Myr ago, have the same quaternary structure. Conservation across such a large evolutionary distance suggests that this conformation is of fundamental functional importance.electron microscopy ͉ molluscan muscle ͉ regulation ͉ smooth muscle ͉ image processing

The interaction of non-muscle myosins 2A and 2B with actin may drive changes in cell movement, shape and adhesion. To investigate this, we used cultured myoblasts as a model system. These cells characteristically change shape from triangular to bipolar when they form groups of aligned cells. Antisense oligonucleotide knockdown of non-muscle myosin 2A, but not non-muscle myosin 2B, inhibited this shape change, interfered with cell-cell adhesion, had a minor effect on tail retraction and prevented myoblast fusion. By contrast, non-muscle myosin 2B knockdown markedly inhibited tail retraction, increasing cell length by over 200% by 72 hours compared with controls. In addition it interfered with nuclei redistribution in myotubes. Non-muscle myosin 2C is not involved as western analysis showed that it is not expressed in myoblasts, but only in myotubes. To understand why non-muscle myosins 2A and 2B have such different roles, we analysed their distributions by immuno-electron microscopy, and found that non-muscle myosin 2A was more tightly associated with the plasma membrane than non-muscle myosin 2B. This suggests that non-muscle myosin 2A is more important for bipolar shape formation and adhesion owing to its preferential interaction with membrane-associated actin, whereas the role of non-muscle myosin 2B in retraction prevents over-elongation of myoblasts.

Background: It is unclear how mutations in the coiled-coil tail of ␤-cardiac myosin cause heart disease. Results: Effects of disease-causing mutations in the myosin tail were studied in vivo and in vitro. Conclusion: Mutations that reduce helical content in vitro reduce sarcomere incorporation of myosin in vivo. Significance: A change in myosin tail structure can lead to heart disease.

The heart disease familial hypertrophic cardiomyopathy (FHC) a¡ects up to 0.2% of the population and is the largest cause of sudden death in young adults (reviewed in [1]). It is characterised by hypertrophy of cardiac myocytes, disarray of muscle ¢bres, and an increase in connective tissue. It is caused by a mutation in one of a number of cytoskeletal proteins that make up the muscle sarcomere, including myosin, C-protein, troponin-T and I, K-tropomyosin and the ventricular light chains.About 15^30% of families with FHC carry mutations in the gene for L-cardiac myosin heavy chain (L-MHC), for which over 50 di¡erent point mutations are known. In common with other class II conventional myosins, L-cardiac myosin consists of two heavy chains and two pairs of light chains. The Nterminal globular region (catalytic domain) that binds nucleotide and actin is connected to the ¢lament forming tail through a lever arm, consisting of an K-helical region to which the light chains bind. In FHC, mutation hot spots are found in regions of the myosin that bind actin and nucleotide, and in the converter domain, or fulcrum, thought to be important for transmitting conformational changes in the catalytic domain to the lever arm, which rotates during force production.It is not clear whether the L-MHC mutants can directly cause myo¢brillar disarray. Expression of the R403Q mutation, the best studied, causes myo¢brillar disarray in cultured feline cardiomyocytes but not in rat [2]. Mutant mice carrying the equivalent mutation in K-cardiac myosin show the expected FHC phenotype, but do not develop myo¢brillar disarray until about 15 weeks of age (reviewed in [1]). A second FHC mutation, R249Q, assembles normally into muscle sarcomeres in rat cardiomyocytes [2].Given that we know that L-MHC mutants result in myo¢-brillar disarray, it is important to ¢nd out whether any of the known mutations can directly cause disarray as demonstrated for other myosin mutations not associated with FHC [3], by interfering with myo¢brillogenesis in a dominant negative fashion. To address this question, we stably expressed wild type (WT) and three di¡erent FHC mutant myosins that have not been studied in vivo before, in cultured mouse myoblasts (H2k b -tsA58, see [4]), and investigated their e¡ects on myo¢brillogenesis. Two of these were in the region of myosin thought to act as a fulcrum (G741R, D778G) and one was close to the nucleotide binding pocket (N232S). We used myoblasts that did not normally express L-MHC when di¡eren-tiated into myotubes, as determined by Western blotting, immuno£uorescence and enzyme-linked immunosorbent assay using L-MHC speci¢c antibodies [5]. A full length L-MHC cDNA (kind gift of Prof. Vosberg) was used, and expressed using the CMV promoter. We transfected cells by electroporation, and, for WT and each of the mutants, we analysed one out of 24 clones recovered, which had the highest expression levels of L-MHC, in detail. Expression of WT L-MHC using a mouse embryonic MHC promoter, cloned in this laboratory (unpublished res...

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Regulatory myosins are controlled through mechanisms intrinsic to their structures and can alternate between activated and inhibited states. However, the structural difference between these two states is unclear. Scallop (Pecten maximus) striated adductor myosin is activated directly by calcium. It has been proposed that the two heads of scallop myosin are symmetrically arranged and interact through their regulatory light chains [Offer and Knight (1996) J. Mol. Biol. 256, 407-416], the interface being strengthened in the inhibited state. By contrast, vertebrate smooth-muscle myosin is activated by phosphorylation. Its structure in the inhibited state has been determined from two-dimensional crystalline arrays [Wendt, Taylor, Trybus and Taylor (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361-4366] and is asymmetric, requiring no interaction between regulatory light chains. Using site-directed mutagenesis of the scallop regulatory light chain, we have tested the symmetric model for scallop adductor muscle myosin. Specifically, we have made myosin hybrid molecules from scallop (P. maximus) myosin, in which the normal regulatory light chains have been replaced by expressed light chains containing mutations in three residues proposed to participate in the interaction between regulatory light chains. The mutations were R126A (Arg126-->Ala), K130A and E131A; made singly, in pairs or all three together, these mutations were designed to eliminate hydrogen bonding or salt linkages between heads, which are key features of this model. Functional assays to address the competence of these hybrid myosins to bind calcium specifically, to exhibit a calcium-regulated myofibrillar Mg-ATPase and to display calcium-dependent actin sliding were performed. We conclude that the symmetrical model does not describe the inhibited state of scallop regulatory myosin and that an asymmetric structure is a plausible alternative.