Sarcomeric Myosin Heavy Chain Genes Research Articles

Regulation of the Expression of the Myosin Heavy Chain (MYH) Gene myh14 in Zebrafish Development.

The human sarcomeric myosin heavy chain gene MYH14 contains an intronic microRNA, miR-499. Our previous studies demonstrated divergent genomic organization and expression patterns of myh14/miR-499 among teleosts; however, the regulatory mechanism is partly known. In this study, we report the regulation of myh14 expression in zebrafish, Danio rerio. Zebrafish myh14 has three paralogs, myh14-1, myh14-2, and myh14-3. Detailed promoter analysis suggested that a 5710-bp 5'-flanking region of myh14-1 and a 5641-bp region of myh14-3 contain a necessary regulatory region to recapitulate specific expression during embryonic development. The 5'-flanking region of zebrafish myh14-1 and its torafugu ortholog shared two distal and a single proximal conserved region. The two distal conserved regions had no effect on zebrafish myh14-1 expression, in contrast to torafugu expression, suggesting an alternative regulatory mechanism among the myh14 orthologs. Comparison among the 5'-flanking regions of the myh14 paralogs revealed two conserved regions. Deletion of these conserved regions significantly reduced the promoter activity of myh14-3 but had no effect on myh14-1, indicating different cis-regulatory mechanisms of myh14 paralogs. Loss of function of miR-499 resulted in a marked reduction in slow muscle fibers in embryonic development. Our study identified different cis-regulatory mechanisms controlling the expression of myh14/miR-499 and an indispensable role of miR-499 in muscle fiber-type specification in zebrafish.

Evolution of the myosin heavy chain gene MYH14 and its intronic microRNA miR-499: muscle-specific miR-499 expression persists in the absence of the ancestral host gene

BackgroundA novel sarcomeric myosin heavy chain gene, MYH14, was identified following the completion of the human genome project. MYH14 contains an intronic microRNA, miR-499, which is expressed in a slow/cardiac muscle specific manner along with its host gene; it plays a key role in muscle fiber-type specification in mammals. Interestingly, teleost fish genomes contain multiple MYH14 and miR-499 paralogs. However, the evolutionary history of MYH14 and miR-499 has not been studied in detail. In the present study, we identified MYH14/miR-499 loci on various teleost fish genomes and examined their evolutionary history by sequence and expression analyses.ResultsSynteny and phylogenetic analyses depict the evolutionary history of MYH14/miR-499 loci where teleost specific duplication and several subsequent rounds of species-specific gene loss events took place. Interestingly, miR-499 was not located in the MYH14 introns of certain teleost fish. An MYH14 paralog, lacking miR-499, exhibited an accelerated rate of evolution compared with those containing miR-499, suggesting a putative functional relationship between MYH14 and miR-499. In medaka, Oryzias latipes, miR-499 is present where MYH14 is completely absent in the genome. Furthermore, by using in situ hybridization and small RNA sequencing, miR-499 was expressed in the notochord at the medaka embryonic stage and slow/cardiac muscle at the larval and adult stages. Comparing the flanking sequences of MYH14/miR-499 loci between torafugu Takifugu rubripes, zebrafish Danio rerio, and medaka revealed some highly conserved regions, suggesting that cis-regulatory elements have been functionally conserved in medaka miR-499 despite the loss of its host gene.ConclusionsThis study reveals the evolutionary history of the MYH14/miRNA-499 locus in teleost fish, indicating divergent distribution and expression of MYH14 and miR-499 genes in different teleost fish lineages. We also found that medaka miR-499 was even expressed in the absence of its host gene. To our knowledge, this is the first report that shows the conversion of intronic into non-intronic miRNA during the evolution of a teleost fish lineage.

Species-specific expression variation of fish MYH14, an ancient vertebrate myosin heavy chain gene orthologue

MYH14 (MYH7b) is the most recently identified sarcomeric myosin heavy chain gene (MYH) from the human genome. Recent observations have revealed that MYH M5 , a torafugu orthologue of MYH14, is one of the major components of the MYH repertoire expressed in torafugu embryos and adults, suggesting its pivotal role in fish muscle formation. We have examined the expression pattern of MYH14 in zebrafish, a model organism for the study of vertebrate development. In situ hybridization studies revealed that zebrafish MYH14 was expressed in the most myotomal region containing fast muscle fibers in the embryonic stage and in superficial slow muscle fibers in the adult stage. Weak signals of MYH14 transcripts were also detected in intermediate muscle fibers located between superficial slow and inner fast muscle fibers in the adult. Reverse transcription-PCR studies showed that MYH14 was not only expressed in skeletal muscles but also in cardiac muscles. These expression patterns are in a marked contrast to that of torafugu MYH M5 , which is solely expressed in slow and cardiac muscle fibers in both the embryonic and adult stages. Our results imply species-specific functions of MYH14 in fish muscle formation.

A Family of microRNAs Encoded by Myosin Genes Governs Myosin Expression and Muscle Performance

Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.

The sarcomeric myosin heavy chain gene family in the dog: Analysis of isoform diversity and comparison with other mammalian species

Sarcomeric myosin heavy chains (MyHC) are the major contractile proteins of cardiac and skeletal muscles and belong to class II MyHC. In this study the sequences of nine sarcomeric MyHC isoforms were obtained by combining assembled contigs of the dog genome draft available in the NCBI database. With this information available the dog becomes the second species, after human, for which the sequences of all members of the sarcomeric MyHC gene family are identified. The newly determined sequences of canine MyHC isoforms were aligned with their orthologs in mammals, forming a set of 38 isoforms, to search for the molecular features that determine the structural and functional specificity of each type of isoform. In this way the structural motifs that allow identification of each isoform and are likely determinants of functional properties were identified in six specific regions (surface loop 1, loop 2, loop 3, converter, MLC binding region, and S2 proximal segment).

A novel autosomal recessive myopathy with external ophthalmoplegia linked to chromosome 17p13.1-p12

We describe a new autosomal recessive myopathy of early onset and very slow progression distinguished by the prominent external ophthalmoplegia in 16 subjects of eight families from a large and highly inbred Arab community. Characteristic clinical features include mild facial and skeletal muscle weakness and atrophy more pronounced proximally in the upper limbs, facial dysmorphism and scoliosis associated with conjugate, non-restrictive ocular motility impairment greatest in the upgaze and without ptosis or aberrant eye movements. Orbital MRI in the patients demonstrated atrophy with fatty replacement of the oculorotatory muscles. The major pathological alteration on skeletal muscle biopsy was a marked type 1 fibre predominance with core-like formations. A genome wide search for regions of homozygosity in the affected members from two informative families identified linkage with chromosome 17p13.1-p12 markers. Maximum two-point logarithm of odds scores were obtained at loci D17S1803 and AFMA070WD1 (Zmax = 3.74 at = 0). Two independent recombination events at D17S1812 and D17S947 further defined a critical region of 12 cM. Several genes map to this interval, including a cluster of sarcomeric myosin heavy chain genes. One of these genes, MYH2, is involved in inclusion body myopathy 3, but no exonic mutations were found by direct sequencing. The molecular basis for this new myopathy remains to be identified.

Critical regulatory domains in intron 2 of a porcine sarcomeric myosin heavy chain gene.

The porcine sarcomeric fast 2a myosin heavy chain (MyHC) gene was previously found to require a region extending 3' from the transcriptional start site for high levels of expression. Here we established the existence of two novel opposing regulatory domains in intron 2. A positive regulatory element, defined to a 75bp region, resembles a TATA-less intronic promoter, with a consensus transcription initiation element. It can up-regulate its endogenous or a heterologous muscle promoter in a position specific manner, and on its own drive a reporter gene. In tandem with it is a dominant negative regulatory element, localised to a 81bp region, which can down-regulate its native gene and a heterologous muscle promoter. Bandshift and DNase I footprinting assays demonstrated that specific nuclear factors bound to both regulatory elements are distinctly different. Both elements appear to have no counterpart in intron 2 of the porcine fast 2x and 2b MyHC genes. Taken together, we demonstrate for the first time that a 5'-end terminal intron of a sarcomeric MyHC gene contains two critical regulatory domains, which may be involved in the complexity of temporo-spatial expression.

Post-transcriptional regulation of rat alpha cardiac myosin heavy chain gene expression.

The cardiac myosin heavy chain genes, alpha and beta, have been shown to change their patterns of expression rapidly and dramatically in response to a variety of stimuli. A major means of achieving these changes in gene expression is transcriptional control; however, the role of post-transcriptional regulation in cardiac myosin gene expression has not been investigated. We have identified two post-transcriptional events in rat alpha cardiac myosin heavy chain (alpha-MHC) gene expression and investigated their regulatory significance in different developmental and thyroid hormone states. The polyadenylation of alpha-MHC mRNA occurs at three different sites: 12, 18, and 23 bases downstream from a single polyadenylation signal. Hyperthyroid hearts did not demonstrate any change in the proportion of the three alpha-MHC mRNA subspecies. Hypothyroid hearts (which have a decreased amount of total alpha-MHC mRNA) showed a significant increase in the proportion of the longest subspecies and a decrease in the shortest subspecies. The second post-transcriptional event in alpha-MHC gene expression which was demonstrated was the inclusion or exclusion of a codon, CAG, encoding glutamine at position 1931, resulting from alternate splicing of the alpha-MHC transcript. The ratio of CAG+ and CAG- forms of mRNA in the adult euthyroid hearts is 40:60% which was unchanged in hypo- and hyperthyroid states. This is the first example of alternate splicing in a vertebrate sarcomeric myosin heavy chain gene. We conclude that the rat alpha-MHC gene transcript is post-transcriptionally modified.

Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms.

We have investigated the molecular bases of muscle abnormalities in four Drosophila melanogaster heldup mutants. We find that the heldup gene encodes troponin-I, one of the principal regulatory proteins associated with skeletal muscle thin filaments. heldup3, heldup4, and heldup5 mutants, all of which have grossly abnormal flight muscle myofibrils, lack mRNAs encoding one or more troponin-I isoforms. In contrast, heldup2, an especially interesting mutant wherein flight muscles are atrophic, synthesizes the complete mRNA complement. By sequencing mutant troponin-I cDNAs we demonstrate that the molecular basis for muscle degeneration in heldup2 is conversion of an invariant alanine residue to valine. We finally show that degeneration of heldup2 thin filament/Z-disc networks can be prevented by eliminating thick filaments from flight muscles using a null allele of the sarcomeric myosin heavy chain gene. This latter observation suggests that actomyosin interactions exacerbate the structural or functional defect resulting from the troponin-I mutation.

The complete sequence of the human β-myosin heavy chain gene and a comparative analysis of its product

We have isolated and sequenced the gene and the cDNA coding for the human cardiac β-myosin heavy chain (designated MYH7). The gene is 22,883 bp long. The 1935 amino acids of this protein ( M r 223,111) are encoded by 38 exons. The 5′ untranslated region (86 bp) is split by two introns. The 3′ untranslated region is 114 bp long. Three Alu repeats were identified within the gene and a fourth one in the 3′ flanking intergenic region. The molecular organization of this gene reflects the conservative pattern with respect to size, coding ratio, and number or position of introns characteristic of vertebrate sarcomeric myosin heavy chain genes. The protein sequence of the human β-heavy chain was compared with corresponding (homologous) sequences of rabbit, rat, and hamster as well as with the (heterologous) embryonic heavy chain sequences of rat, chicken, and man. The results show that protein subregions responsible for basic functions of myosin heavy chains (nucleotide binding and actin binding) are very similar in homologous and heterologous heavy chains. Regions that differ in their primary sequences in heterologous heavy chains appear to be highly conserved within mammalian β-myosin heavy chains. Constant and variable subregions of heavy chains are discussed in terms of functional significance and evolutionary relatedness.

Disuse and passive stretch cause rapid alterations in expression of developmental and adult contractile protein genes in skeletal muscle

Contractile proteins exist as a number of isoforms that show a developmental and tissue-specific pattern of expression. Using gene-specific cDNA probes, the expression of the sarcomeric myosin heavy chain (MHC) multi-gene family and of cardiac (foetal) alpha-actin was analysed in three different rat hindlimb muscles immobilised for 5 days in either the shortened or lengthened positions. For each of the MHC genes normally expressed in adult muscle (slow, IIA and IIB), the effect of disuse alone (immobilisation in the shortened position) upon expression was markedly different to that of passive stretch (immobilisation in the lengthened position) in each of the three muscles. However, the same adult sarcomeric myosin heavy chain gene can be affected in a different, or even opposite, manner by either disuse or passive stretch depending on the muscle in which it is being expressed. The fast IIB MHC gene, for example, exhibits a rapid induction in the slow postural soleus muscle, in response to disuse but no such induction occurs in the faster plantaris and gastrocnemius muscles. Furthermore, the induction of this gene in the soleus was prevented by passive stretch. The MHC gene, normally only expressed in embryonic skeletal muscle, showed a similar response in all three muscles and was reinduced in adult muscle in response to passive stretch but not by disuse alone. In contrast, the isoform of alpha-actin which is normally only present in significant quantities in embryonic skeletal muscle and which is reduced postnatally, is not reinduced by passive stretch but is reduced still further by immobilisation in the shortened position.

Human nonmuscle myosin heavy chain mRNA: generation of diversity through alternative polyadenylylation.

Myosin is a ubiquitous eukaryotic contractile protein that generates the force responsible for such diverse cellular movements as muscle contraction and cytokinesis. Although there have been numerous studies of sarcomeric myosin heavy chain (MHC) genes, no molecular clones have been reported that encode mammalian nonmuscle MHC. This study presents the molecular genetic characterization of a human nonmuscle MHC that is expressed in fibroblasts, endothelial cells, and macrophages. Human nonmuscle MHC amino acids are weakly homologous (33%) to sarcomeric MHC but are approximately 72% identical to smooth muscle MHC. In contrast to vertebrate sarcomeric MHCs, which generate diversity through the expression of members of a multigene family, an alternative polyadenylylation site is used in the nonmuscle MHC gene to generate multiple transcripts that encode the same protein. We have mapped this gene to chromosome 22. It is thus unlinked to either of the sarcomeric MHC gene clusters on human chromosomes 14 and 17.

The human embryonic myosin heavy chain. Complete primary structure reveals evolutionary relationships with other developmental isoforms.

We have isolated a single 6021-nucleotide cDNA fragment encoding the full length of the myosin heavy chain (MHC) isoform initially expressed in developing human limb muscle. The corresponding transcript is expressed in fetal, but not adult, human muscle, and the corresponding gene maps to human chromosome 17. Comparison of the full length nucleotide sequence with that of the orthologous rat gene transcript reveals 74, 90, and 80% similarities in the 5'-untranslated, coding, and 3'-untranslated regions, respectively. To precisely quantitate the degree of nucleotide sequence divergence between the human embryonic and other developmentally regulated MHC gene transcripts, we utilize the algorithm of Perler et al. (Perler, F., Efstratiadis, A., Lomedico, P., Gilbert, W., Kolodner, R. & Dodgson, J. (1980) Cell 20, 555-566) and make use of the codon-for-codon register attainable in alignments of the MHC rod encoding cDNA fragments. The results allow reconstruction of the order and relative timing of certain gene duplication events involved in the evolution of the multimembered mammalian MHC loci. By this analysis, the principal sarcomeric MHC gene expressed in the 14-day chick embryo is shown to be more distantly related to the mammalian embryonic MHC genes than to those expressed peri- and postnatally. Attention is focused on regional patterns of MHC sequence conservation, ordered with reference to the topology of our phylogenetic tree. We present a composite map depicting the deduced evolutionary age of various primary structural subdomains of the human embryonic MHC.

Ifm(2)2 is a myosin heavy chain allele that disrupts myofibrillar assembly only in the indirect flight muscle of Drosophila melanogaster.

Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an allele of the single-copy sarcomeric myosin heavy chain gene. Flies homozygous for this allele accumulate wild-type levels of mRNA and protein in tubular muscle of adults, but fail to accumulate detectable amounts of myosin heavy chain mRNA or protein in the indirect flight muscle. We propose that the mutation interferes with either transcription of the gene or splicing of the primary transcript in the indirect flight muscle and not in other muscle tissues. Biochemical and electron microscopic analysis of flies homozygous for this mutation has revealed that thick filament assembly is abolished in the indirect flight muscle resulting in the instability of wild-type thick filament proteins. In contrast, thin filament and Z disc assembly are marginally affected. We discuss a working hypothesis for sarcomere assembly and define and experimental approach to test the predictions of this proposed pathway for sarcomere assembly.

Developmental and hormonal regulation of sarcomeric myosin heavy chain gene family.

Sarcomeric myosin heavy chain (MHC), the main component of the sarcomere, contains the ATPase activity that generates the contractile force of cardiac and skeletal muscles. The different MHC isoforms are encoded by a closely related multigene family. Most members (seven) of this gene family have been isolated and characterized in the rat, including the alpha- and beta-cardiac, skeletal embryonic, neonatal, fast IIA, fast IIB, and extraocular specific MHC. The slow type I skeletal MHC is encoded by the same gene that codes for the cardiac beta-MHC. Each MHC gene studied displays a pattern of expression that is tissue and developmental stage specific, both in cardiac and skeletal muscles. Furthermore, more than one MHC gene is expressed in each muscle while each gene is expressed in more than one tissue. The expression of each MHC gene in cardiac and skeletal muscles is modulated by thyroid hormone. Surprisingly, however, the same MHC gene can be regulated by the hormone in a significantly different manner, even in opposite directions, depending on the muscle in which it is expressed. Moreover, the skeletal embryonic and neonatal MHC genes, so far considered specific to these 2 developmental stages, are normally expressed in certain adult muscles and can be reinduced by hypothyroidism in specific muscles. This complex pattern of expression and regulation of the MHC gene family in cardiac and skeletal muscle sheds new light on the mechanisms involved in determining the biochemical basis of the contractile state. It also indicates that the cardiac contractile system needs to be examined in a broader context, including skeletal muscles, in order to understand fully its developmental and physiologic regulation.

Sarcomeric myosin heavy chain gene family: organization and pattern of expression

The sarcomeric myosin heavy chains (MHCs), which exhibit different levels of ATPase activity, are encoded by a closely related multigene family from which seven members have been identified and characterized in the rat. The MHC genes appear to map to a single chromosome, and at least two of them, alpha- and beta-cardiac, are closely linked in the genome. Each of these genes is approximately 25 kilobases long, and their coding sequences are interrupted by 40 introns. Each MHC gene displays a pattern of expression that is tissue-specific and developmentally regulated, with more than one MHC gene expressed in each muscle and developmental stage. Moreover, with the exception of the extra-ocular muscle MHC gene that has a very specific pattern of expression, the other genes are all expressed in more than one tissue. The expression of all MHC genes can be modulated by thyroid hormone. Surprisingly, however, the same myosin heavy chain gene can be regulated by thyroid hormone in highly different modes, even in opposite directions, depending on the tissue in which it is expressed. Furthermore, the skeletal embryonic and neonatal myosin heavy chain genes, so far considered specific to these two developmental stages, can be re-induced by hypothyroidism in specific adult muscles.

Isolation of a non-muscle myosin heavy chain gene from Acanthamoeba.

We have isolated a non-muscle myosin heavy chain gene from Acanthamoeba castellanii using as a heterologous probe a sarcomeric myosin heavy chain gene from Caenorhabditis elegans. The amoeba genomic clone has been tentatively identified as containing a myosin II heavy chain gene based on hybridization to a 5300-nucleotide RNA species, hybrid selection of a mRNA encoding a 185-kDa polypeptide, specific immunoprecipitation of this polypeptide with antiserum to myosin II, and an exact match between the DNA sequence and a carboxyl-terminal myosin II peptide previously sequenced by protein chemical methods (Côté, G.P., Robinson, E.A., Appella, E., and Korn, E. D. (1984) J. Biol. Chem. 259, 12781-12787). We also sequenced a region of the gene whose deduced amino acid sequence shows strong homology with that region of muscle myosins which is thought to be involved in nucleotide binding. These results indicate that the amoeba genomic clone contains at least 90% of the coding information for the 185-kDa heavy chain polypeptide and that the bulk of the gene contains very little intron DNA. Genomic blots of amoeba DNA probed with a portion of this myosin gene indicate the presence of additional highly related sequences within the amoeba genome.

Characterization of diverse forms of myosin heavy chain expressed in adult human skeletal muscle

In an attempt to define myosin heavy chain (MHC) gene organization and expression in adult human skeletal muscle, we have isolated and characterized genomic sequences corresponding to different human sarcomeric MHC genes (1). In this report, we present the complete DNA sequence of two different adult human skeletal muscle MHC cDNA clones, one of which encodes the entire light meromyosin (LMM) segment of MHC and represents the longest described MHC cDNA sequence. Additionally, both clones provide new sequence data from a 228 amino acid segment of the MHC tail for which no protein or DNA sequence has been previously available. One clone encodes a "fast" form of skeletal muscle MHC while the other clone most closely resembles a MHC form described in rat cardiac ventricles. We show that the 3' untranslated region of skeletal MHC cDNAs are homologous from widely separated species as are cardiac MHC cDNAs. However, there is no homology between the 3' untranslated region of cardiac and skeletal muscle MHCs. Isotype-specific preservation of MHC 3' untranslated sequences during evolution suggests a functional role for these regions.

Characterization of cDNA and genomic sequences corresponding to an embryonic myosin heavy chain.

We report here the isolation and characterization of cDNA and genomic sequences corresponding to a rat embryonic myosin heavy chain (MHC) protein. This gene, which is present as a single copy in the rat genome, comprises about 25 kilobase pairs of DNA and contains approximately 80% intronic sequences. The embryonic MHC gene belongs to a highly conserved multigene family, and exhibits a high degree of nucleotide and amino acid sequence conservation with other sarcomeric MHC genes from nematode to man. S1 nuclease mapping experiments using cDNA and genomic probes show that this MHC gene is transiently expressed during skeletal muscle development. Its mRNA is detected in fetal skeletal muscle during early development and persists up to 2 weeks after birth with the overlapping expression of neonatal and adult skeletal MHC mRNAs. However, this MHC is not expressed in the adult skeletal muscle with the exception of extraocular muscle fibers. The transient expression during muscle development of the isoform produced by this gene and its sequential replacement by other MHCs raises interesting questions about the mechanism controlling MHC isozyme transitions and the physiological significance of the individual MHCs in muscle fibers.

Characterization of a human genomic DNA fragment coding for a myosin heavy chain.

A DNA segment from the human genome with information for myosin heavy chain (MHC) was isolated from a human genomic DNA library cloned in lambda Charon 4A phages. The isolation was accomplished by a myosin cDNA probe obtained from rabbit heart muscle mRNA (Sinha et al. 1982). The selected human DNA clone, designated lambda gMHC1, contains a genomic DNA fragment of about 14 kilobase pairs. The transcriptional polarity of this DNA was determined. The 5'-end of the gene is missing from the cloned fragment. This human gene exhibits sequence homology to MHC DNA of rabbit and chicken, but not to an MHC sequence of nematode. The isolated gene fragment is a member of the human MHC multi-gene family, which is presumed to consist of probably more than ten separate sarcomeric MHC genes per haploid genome.