Expression and Functional Characterization of Smyd1a in Myofibril Organization of Skeletal Muscles

Background

Smyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown.

Methodology/Principal Findings

To investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos.

Conclusion/Significance

Collectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.     

Smyd1b_tv1, a key regulator of sarcomere assembly, is localized on the M-line of skeletal muscle fibers

Background

Smyd1b is a member of the Smyd family that plays a key role in sarcomere assembly during myofibrillogenesis. Smyd1b encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2, that are expressed in skeletal and cardiac muscles and play a vital role in myofibrillogenesis in skeletal muscles of zebrafish embryos.

Methodology/Principal Findings

To better understand Smyd1b function in myofibrillogenesis, we analyzed the subcellular localization of Smyd1b_tv1 and Smyd1b_tv2 in transgenic zebrafish expressing a myc-tagged Smyd1b_tv1 or Smyd1b_tv2. The results showed a dynamic change of their subcellular localization during muscle cell differentiation. Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblasts and myotubes at early stage zebrafish embryos. However, in mature myofibers, Smyd1b_tv1, and to a small degree of Smyd1b_tv2, exhibited a sarcomeric localization. Double staining with sarcomeric markers revealed that Smyd1b_tv1was localized on the M-lines. The sarcomeric localization was confirmed in zebrafish embryos expressing the Smyd1b_tv1-GFP or Smyd1b_tv2-GFP fusion proteins. Compared with Smyd1b_tv1, Smyd1b_tv2, however, showed a weak sarcomeric localization. Smyd1b_tv1 differs from Smyd1b_tv2 by a 13 amino acid insertion encoded by exon 5, suggesting that some residues within the 13 aa insertion may be critical for the strong sarcomeric localization of Smyd1b_tv1. Sequence comparison with Smyd1b_tv1 orthologs from other vertebrates revealed several highly conserved residues (Phe223, His224 and Gln226) and two potential phosphorylation sites (Thr221 and Ser225) within the 13 aa insertion. To determine whether these residues are involved in the increased sarcomeric localization of Smyd1b_tv1, we mutated these residues into alanine. Substitution of Phe223 or Ser225 with alanine significantly reduced the sarcomeric localization of Smyd1b_tv1. In contrast, other substitutions had no effect. Moreover, replacing Ser225 with threonine (S225T) retained the strong sarcomeric localization of Smyd1b_tv1.

Conclusion/Significance

Together, these data indicate that Phe223 and Ser225 are required for the M-line localization of Smyd1b_tv1.     

Analysis of skeletal and cardiac muscle from desmin knock-out and normal mice by high resolution separation of myosin heavy-chain isoforms

Summary— In this study, using a modified electrophoretic technique, we have defined in the mouse the myosin heavy-chain composition of both newborn and adult skeletal and cardiac muscles. Using this high resolution technique it was possible to detect modifications in the myosin heavy-chain expression in both cardiac and skeletal muscles of desmin knock-out mice.     

Still Heart Encodes a Structural HMT,SMYD1b,with Chaperone-Like Function during Fast Muscle Sarcomere Assembly

The vertebrate sarcomere is a complex and highly organized contractile structure whose assembly and function requires the coordination of hundreds of proteins. Proteins require proper folding and incorporation into the sarcomere by assembly factors, and they must also be maintained and replaced due to the constant physical stress of muscle contraction. Zebrafish mutants affecting muscle assembly and maintenance have proven to be an ideal tool for identification and analysis of factors necessary for these processes. The still heart mutant was identified due to motility defects and a nonfunctional heart. The cognate gene for the mutant was shown to be smyd1b and the still heart mutation results in an early nonsense codon. SMYD1 mutants show a lack of heart looping and chamber definition due to a lack of expression of heart morphogenesis factors gata4, gata5 and hand2. On a cellular level, fast muscle fibers in homozygous mutants do not form mature sarcomeres due to the lack of fast muscle myosin incorporation by SMYD1b when sarcomeres are first being assembled (19hpf), supporting SMYD1b as an assembly protein during sarcomere formation.     

Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ～20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.     

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

Summary: Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off‐target effects the therapeutic gene should be driven by a tissue‐specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue‐specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle‐specific expression but presents heat‐shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b‐deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle‐specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431–438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Alternative S2 hinge regions of the myosin rod differentially affect muscle function, myofibril dimensions and myosin tail length

Muscle myosin heavy chain (MHC) rod domains intertwine to form alpha-helical coiled-coil dimers; these subsequently multimerize into thick filaments via electrostatic interactions. The subfragment 2/light meromyosin "hinge" region of the MHC rod, located in the C-terminal third of heavy meromyosin, may form a less stable coiled-coil than flanking regions. Partial "melting" of this region has been proposed to result in a helix to random-coil transition. A portion of the Drosophila melanogaster MHC hinge is encoded by mutually exclusive alternative exons 15a and 15b, the use of which correlates with fast (hinge A) or slow (hinge B) muscle physiological properties. To test the functional significance of alternative hinge regions, we constructed transgenic fly lines in which fast muscle isovariant hinge A was switched for slow muscle hinge B in the MHC isoforms of indirect flight and jump muscles. Substitution of the slow muscle hinge B impaired flight ability, increased sarcomere lengths by approximately 13% and resulted in minor disruption to indirect flight muscle sarcomeric structure compared with a transgenic control. With age, residual flight ability decreased rapidly and myofibrils developed peripheral defects. Computational analysis indicates that hinge B has a greater coiled-coil propensity and thus reduced flexibility compared to hinge A. Intriguingly, the MHC rod with hinge B was approximately 5 nm longer than myosin with hinge A, consistent with the more rigid coiled-coil conformation predicted for hinge B. Our study demonstrates that hinge B cannot functionally substitute for hinge A in fast muscle types, likely as a result of differences in the molecular structure of the rod, subtle changes in myofibril structure and decreased ability to maintain sarcomere structure in indirect flight muscle myofibrils. Thus, alternative hinges are important in dictating the distinct functional properties of myosin isoforms and the muscles in which they are expressed.     

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 β-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.     

Partial characterization of the human beta-myosin heavy-chain gene which is expressed in heart and skeletal muscle

A human myosin heavy-chain gene, cloned in gamma Charon 4A phage (and as a clone designated lambda gMHC-1), was shown to code for a cardiac myosin heavy chain of the beta-type. The 5' end of the 14,200-base-pair genomic DNA clone is located in the head region of the myosin chain. The 3' end was shown to extent to the COOH terminus and includes the 3'-nontranslated sequence of the corresponding mRNA. The identification of lambda gMHC-1 as coding for a cardiac beta-myosin heavy chain was achieved by heteroduplex mapping using genomic cardiac myosin heavy-chain DNA of rabbit as a probe and, furthermore, by DNA sequence analysis of three selected subregions of the clones DNA including the 3'-nontranslated sequence. It was demonstrated by the S1 nuclease protection technique that the beta-myosin heavy-chain gene is transcribed in human heart muscle. In addition, we have found by the same technique that it is also expressed in human skeletal muscle.     

Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles

Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.     

Transformation of Drosophila melanogaster with the wild-type myosin heavy-chain gene: rescue of mutant phenotypes and analysis of defects caused by overexpression

We have transformed Drosophila melanogaster with a genomic construct containing the entire wild-type myosin heavy-chain gene, Mhc, together with approximately 9 kb of flanking DNA on each side. Three independent lines stably express myosin heavy-chain protein (MHC) at approximately wild-type levels. The MHC produced is functional since it rescues the mutant phenotypes of a number of different Mhc alleles: the amorphic allele Mhc1, the indirect flight muscle and jump muscle-specific amorphic allele Mhc10, and the hypomorphic allele Mhc2. We show that the Mhc2 mutation is due to the insertion of a transposable element in an intron of Mhc. Since a reduction in MHC in the indirect flight muscles alters the myosin/actin protein ratio and results in myofibrillar defects, we determined the effects of an increase in the effective copy number of Mhc. The presence of four copies of Mhc results in overabundance of the protein and a flightless phenotype. Electron microscopy reveals concomitant defects in the indirect flight muscles, with excess thick filaments at the periphery of the myofibrils. Further increases in copy number are lethal. These results demonstrate the usefulness and potential of the transgenic system to study myosin function in Drosophila. They also show that overexpression of wild-type protein in muscle may disrupt the function of not only the indirect flight but also other muscles of the organism.     

Mutations in Drosophila myosin rod cause defects in myofibril assembly

The roles of myosin during muscle contraction are well studied, but how different domains of this protein are involved in myofibril assembly in vivo is far less understood. The indirect flight muscles (IFMs) of Drosophila melanogaster provide a good model for understanding muscle development and function in vivo. We show that two missense mutations in the rod region of the myosin heavy-chain gene, Mhc, give rise to IFM defects and abnormal myofibrils. These defects likely result from thick filament abnormalities that manifest during early sarcomere development or later by hypercontraction. The thick filament defects are accompanied by marked reduction in accumulation of flightin, a myosin binding protein, and its phosphorylated forms, which are required to stabilise thick filaments. We investigated with purified rod fragments whether the mutations affect the coiled-coil structure, rod aggregate size or rod stability. No significant changes in these parameters were detected, except for rod thermodynamic stability in one mutation. Molecular dynamics simulations suggest that these mutations may produce localised rod instabilities. We conclude that the aberrant myofibrils are a result of thick filament defects, but that these in vivo effects cannot be detected in vitro using the biophysical techniques employed. The in vivo investigation of these mutant phenotypes in IFM development and function provides a useful platform for studying myosin rod and thick filament formation generically, with application to the aetiology of human myosin rod myopathies.     

The evolutionary relationship of avian and mammalian myosin heavy-chain genes

Summary Sequence comparisons of avian and mammalian skeletal and cardiac myosin heavy-chain isoforms are used to examine the evolutionary relationships of sarcomeric myosin multigene families. Mammalian fast-myosin heavy-chain isoforms forms from different species, with comparable developmental expression, are more similar to each other than they are to other fast isoforms within the same genome. In contrast, the developmentally regulated chicken fast isoforms are more similar to each other than they are to myosin heavy-chain isoforms in other species. Extensive regions of nucleotide identity among the chicken fast myosin heavy chains and in the mouse and rat α- and β-cardiac myosin heavy-chain sequences suggest that geneconversion-like mechanisms have played a major role in the concerted evolution of these gene families. We also conclude that the chicken fast myosin heavy-chain multigene family has undergone recent expansion subsequent to the divergence of birds and mammals and that both the developmental regulation and the specialization of myosin isoforms have likely developed independently in birds and mammals.     

粘虫蛾飞行肌超微结构的研究

应用电子显微镜对粘虫雌蛾Mythimna separata(Walker)飞行(背纵)肌的研究结果表明,其肌原纤维由500-700根肌球蛋白丝(粗丝)组成,每根粗丝由6根肌动蛋白丝(细丝)环绕排列成六角形,每根细丝精确地位于两根粗丝间1/2处,从而使粗丝和细丝的比为1：3。肌节较短,长度约2.2-2.6μm。肌原纤维之间充满着线粒体和横管。每个肌节约有线粒体三个,横管二根。线粒体约占肌纤维体积的40%,而横管为7%。每根横管准确地位于肌节的1/4、3/4处,或Z线和中膈的中央,并与肌质网交接形成二位体(dyads)或三位体(triads)。肌质网相当不发达,约占肌纤维体积的2.5%。但其分布很有特色,即除了紧贴于肌原纤维周围的由单层液泡组成的肌质网以外,在中膈处还有一层横穿于肌原纤维的肌质网。和其它同步飞行肌的结构和功能分析比较的结果还表明,粘虫蛾飞行肌具有较善于飞行的结构。     

Expression of MRF4, a myogenic helix-loop-helix protein, produces multiple changes in the myogenic program of BC3H-1 cells.

Expression of MRF4, a myogenic regulatory factor of the basic helix-loop-helix type, produced multiple changes in the myogenic program of the BC3H-1 cell line. BC3H-1 cells that stably expressed exogenous MRF4 were prepared and termed BR cell lines. Upon differentiation, the BR cells were found to have three muscle-specific properties (endogenous MyoD expression, myoblast fusion, and fast myosin light-chain 1 expression) that the parent BC3H-1 cells did not have. Of the four known myogenic regulatory factors (MyoD, myogenin, Myf-5, and MRF4), only MRF4 was capable of activating expression of the endogenous BC3H-1 myoD gene. In addition, the pattern of Myf-5 expression in BR cells was the opposite of that in BC3H-1 cells. Myf-5 expression was low in BR myoblasts and showed a small increase upon myotube formation, whereas Myf-5 expression was high in BC3H-1 myoblasts and decreased upon differentiation. Though the MRF4-transfected BR cells fused to form large myotubes and expressed fast myosin light-chain 1, the pattern of myosin heavy-chain isoform expression was the same in the BR and the nonfusing parent BC3H-1 cells, suggesting that factors in addition to the MyoD family members regulate myosin heavy-chain isoform expression patterns in BC3H-1 cells. In contrast to the changes produced by MRF4 expression, overexpression of Myf-5 did not alter BC3H-1 myogenesis. The results suggest that differential expression of the myogenic regulatory factors of the MyoD family may be one mechanism for generating cells with diverse myogenic phenotypes.     

The length–tension curve in muscle depends on lattice spacing

Classic interpretations of the striated muscle length–tension curve focus on how force varies with overlap of thin (actin) and thick (myosin) filaments. New models of sarcomere geometry and experiments with skinned synchronous insect flight muscle suggest that changes in the radial distance between the actin and myosin filaments, the filament lattice spacing, are responsible for between 20% and 50% of the change in force seen between sarcomere lengths of 1.4 and 3.4 µm. Thus, lattice spacing is a significant force regulator, increasing the slope of muscle''s force–length dependence.