Human cardiac myosin heavy chain genes and their linkage in the genome.

Human myosin heavy chains are encoded by a multigene family consisting of at least 10 members. A gene-specific oligonucleotide has been used to isolate the human beta myosin heavy chain gene from a group of twelve nonoverlapping genomic clones. We have shown that this gene (which is expressed in both cardiac and skeletal muscle) is located 3.6kb upstream of the alpha cardiac myosin gene. We find that DNA sequences located upstream of rat and human alpha cardiac myosin heavy chain genes are very homologous over a 300bp region. Analogous regions of two other myosin genes expressed in different muscles (cardiac and skeletal) show no such homology to each other. While a human skeletal muscle myosin heavy chain gene cluster is located on chromosome 17, we show that the beta and alpha human cardiac myosin heavy chain genes are located on chromosome 14.     

Concerted evolution of mammalian cardiac myosin heavy chain genes

We have recently determined the complete nucleotide sequences of the cardiac - and -myosin heavy chain (MyHC) genes from both human and Syrian hamster. These genomic sequence data were used to study the molecular evolution of the cardiac MyHC genes.Between the - and -MyHC genes, multiple gene conversion events were detected by (1) maximum parsimony tree analyses, (2) synonymous substitution analyses, and (3) detection of pairwise identity of intron sequences. Approximately half of the 40 cardiac MyHC exons have undergone concerted evolution through the process of gene conversion with the other half undergoing divergent evolution. Gene conversion occurred more often in exons encoding the a-helical myosin rod domain than in the globular head domain, and an apparent directional bias was also observed, with transfer of genetic material occurring more often from to .     

Regulation of myosin heavy chain expression during rat skeletal muscle development in vitro

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and approximately 10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN-nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.     

Regulation of human cardiac myosin heavy chain genes: the effect of catecholamine.

The 5'-flanking regions of the alpha- and beta-cardiac myosin heavy chain (MyHC) genes were excised from the cosmid human genomic clones using Hind III and Xbal for the alpha-MyHC gene, and the Hind III and Hind III sites for the beta-MyHC gene. These fragments were linked to chloramphenicol acetyl transferase (CAT) vector to generate a chimeric fusion gene. These fusion genes were subsequently transfected to neonatal rat cardiac cultured cells to analyze the CAT activity. The alpha-MyHC gene is preferentially expressed as compared to the beta-MyHC. In the presence of norepinephrine (NE) the beta-MyHC gene is remarkably induced (within 24 hours following the addition of norepinephrine to the cardiocyte culture). However, the alpha-MyHC is also induced. Specific alpha andrenergic antagonists such as terazosin (Tz) partially suppressed both the alpha- and beta-MyHC genes as revealed by the CAT activity. These findings suggest that catecholamine does activate the human cardiac MyHC genes but does not differentiate the specific expression of either the alpha- or beta-MyHC genes.     

TRs have common and isoform-specific functions in regulation of the cardiac myosin heavy chain genes.

TRalpha1 and TRbeta mediate the regulatory effects of T3 and have profound effects on the cardiovascular system. We have analyzed the expression of the cardiac myosin heavy chain (MyHC) genes alpha and beta in mouse strains deficient for one or several TR genes to identify specific regulatory functions of TRalpha1 and TRbeta. The results show that TRalpha1 deficiency, which slows the heart rate, causes chronic overexpression of MyHCbeta. However, MyHCbeta was still suppressible by T3 in both TRalpha1- and TRbeta-deficient mice, indicating that either receptor can mediate repression of MyHCbeta. T3-dependent induction of the positively regulated MyHCalpha gene was similar in both TRalpha1- and TRbeta-deficient mice. The data identify a specific role for TRalpha1 in the negative regulation of MyHCbeta, whereas TRalpha1 and TRbeta appear interchangeable for hormone-dependent induction of MyHCalpha. This suggests that TR isoforms exhibit distinct specificities in the genes that they regulate within a given tissue type. Thus, dysregulation of MyHCbeta is likely to contribute to the critical role of TRalpha1 in cardiac function.     

Fast myosin heavy chain expression during the early and late embryonic stages of chicken skeletal muscle development

The development of embryonic skeletal muscles in the chick can be divided into two periods of fiber specialization--an early one during which the different muscles of the limb are formed and an initial round of fiber specialization occurs and a late or fetal period during which there is extensive growth of this previously established fiber pattern. This latter period of growth is dependent on the establishment and maintenance of functional neuromuscular contacts. As has been described for other developmental stages, we show here that there are different embryonic fast skeletal muscle myosin heavy chain (MHC) isoforms expressed during the different embryonic periods of muscle growth. The identification of these isoforms was based on differences in their reactivity with various fast MHC monoclonal antibodies and on their different peptide banding patterns. The in ovo accumulation of the late embryonic MHC isoform pattern was similar to the time course of the previously described changes in alpha-actin and troponin T isotype switching during embryogenesis. The appearances of the late embryonic isoforms were blocked by chronic treatment with the neuromuscular blocking agent, d-tubocurarine, and cell cultures of embryonic chicken skeletal muscle which differentiated in the absence of motorneurons expressed little of the late embryonic isoform, indicating that the expression of the late embryonic isoform was dependent on functional nerve-muscle interactions. These different embryonic fast MHC isoforms provide important markers for monitoring the progression of muscle through its embryonic stages and its interaction with motorneurons.     

Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development

Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or intrinsic "clock." Taken together, these results suggest that, unlike embryonic and neonatal MyHCs, the expression of slow MyHC in vivo at different developmental stages during gestation is not the result of commitment to a distinct myoblast lineage, but is largely determined by the environment.     

Characterization of sarcomeric myosin heavy chain genes

Myosin heavy chain is encoded by a large multigene family. Using pMHC-25, a recombinant cDNA clone isolated from the rat myogenic cell line L6E9, four members of this family in the rat have been isolated and shown to be tissue-specific and developmentally regulated. The coding regions of these genes share regions of homology interspaced with regions of non-homology. Detailed analysis of one embryonic and one adult myosin heavy chain gene shows that the coding sequences are interrupted by numerous intervening sequences whose number, size, and distribution do not appear to be conserved in the same organism or between species.     

Dictyostelium myosin heavy chain kinase A regulates myosin localization during growth and development

Phosphorylation of the Dictyostelium myosin II heavy chain (MHC) has a key role in regulating myosin localization in vivo and drives filament disassembly in vitro. Previous molecular analysis of the Dictyostelium myosin II heavy chain kinase (MHCK A) gene has demonstrated that the catalytic domain of this enzyme is extremely novel, showing no significant similarity to the known classes of protein kinases (Futey, L. M., Q. G. Medley, G. P. Cote, and T. T. Egelhoff. 1995. J. Biol. Chem. 270:523-529). To address the physiological roles of this enzyme, we have analyzed the cellular consequences of MHCK A gene disruption (mhck A- cells) and MHCK A overexpression (MHCK A++ cells). The mhck A- cells are viable and competent for tested myosin-based contractile events, but display partial defects in myosin localization. Both growth phase and developed mhck A- cells show substantially reduced MHC kinase activity in crude lysates, as well as significant overassembly of myosin into the Triton-resistant cytoskeletal fractions. MHCK A++ cells display elevated levels of MHC kinase activity in crude extracts, and show reduced assembly of myosin into Triton-resistant cytoskeletal fractions. MHCK A++ cells show reduced growth rates in suspension, becoming large and multinucleated, and arrest at the mound stage during development. These results demonstrate that MHCK A functions in vivo as a protein kinase with physiological roles in regulating myosin II localization and assembly in Dictyostelium cells during both growth and developmental stages.     

Distinct myogenic programs of embryonic and fetal mouse muscle cells: expression of the perinatal myosin heavy chain isoform in vitro.

Early embryonic and late fetal mouse myogenic cells showed distinct patterns of perinatal myosin heavy chain (MHC) isoform expression upon differentiation in vitro. In cultures of somite or limb muscle cells isolated from Day 9 to Day 12 embryos, differentiated cells that expressed perinatal MHC were rare and perinatal MHC was not detectable by immunoblotting. In cultures of limb muscle cells isolated from Day 13 to Day 18 fetuses, in contrast, the perinatal MHC isoform was easily detected and was expressed in a substantial percentage of myocytes and myotubes. Analyses of clonally derived muscle colonies and cytosine arabinoside-treated fetal muscle cell cultures suggested that different fetal muscle cell nuclei initiated perinatal MHC expression at different times. In both embryonic and fetal cell cultures, the embryonic MHC isoform was expressed by all differentiated cells examined. A small number of myotubes in fetal muscle cell cultures showed a mosaic distribution of MHC isoform accumulation in which the perinatal MHC isoform accumulated in a restricted region of the myotube near particular nuclei, whereas the embryonic MHC isoform accumulated throughout the myotube. Thus, the myogenic program of fetal, but not embryonic, mouse myogenic cells includes expression of the perinatal MHC isoform upon differentiation in culture.