Skeletal muscle fibres in mammalian limb muscles are of four types: slow, 2A, 2X, and 2B, each characterized by a distinct myosin heavy chain (MyHC) isoform. Existing monoclonal antibodies (mabs) against fast MyHCs lack fibre-type specificity across species and could not positively identify 2X fibres. In this work, mabs were raised against each of the fast MyHCs. These mabs were shown to be monospecific by Western blots and immunohistochemistry in the rat. The advantages of using these mabs for identifying the three fast fibre types and hybrid fibres expressing multiple isoforms were illustrated using rat tibialis anterior muscle. Immunohistochemical analyses confirmed the monospecificity of these mabs in the following additional species: mouse, guinea pig, rabbit, cat, and baboon. 2B fibres were absent in limb muscles of the cat and baboon. These mabs constitute a set of powerful tools for studying muscle fibre types and their transformations.

Myosin heavy chain (MyHC) isoforms show a striking diversity of expression patterns during mammalian development. Using a set of monoclonal antibodies that recognize different epitopes on myosin heavy chain isoforms we show that there exist in human and rat skeletal muscle at least three isoforms of slow twitch myosin heavy chain. To facilitate a comparison of our results to others obtained using different antibodies or species, we have identified cDNAs encoding the epitopes recognized by the three slow antibodies. Using these reagents, we show that the onset of expression of three slow MyHC isoforms is temporally distinct during early gestation. This result suggests that a sequence of MyHC transitions plays an important role in determining muscle fiber function at fetal, neonatal, and adult stages.

Myosin heavy chain (MHC) composition of chemically-induced rhabdomyosarcoma (RMS) was analyzed by gel electrophoresis and Western blotting using a panel of monoclonal antimyosin antibodies specific for embryonic-, neonatal-, slow- and adult fast-type MHC isoforms. Myosin extracted from tumours and electrophoresed on 6%-sodium dodecyl sulfate (SDS)glycerol gels was found to migrate as three distinct MHC components. These polypeptides were present in different relative amounts in the five RMS studied. Western blotting experiments revealed that variable proportions of embryonic-, slow- and adult fast-, but not neonatal-type, MHC isoforms are consistently expressed in RMS. Indirect and double immunofluorescence procedures applied to cryosections of tumoral tissue showed that: (a) RMS cells were unreactive with antineonatal-type-MHC antibody, (b) the majority of neoplastic, desmin-positive, cells contained embryonic- as well as adult fast-type MHCs and (c) a minority of cells were labelled by anti-slow MHC antibody. The results of this study indicate that there is no obligatory sequence of MHC isoform expression in the molecular transition (emb----neo----adult) which occurs during rat skeletal myogenesis.

Myosin in adult murine skeletal muscle is composed primarily of three adult fast myosin heavy chain (MyHC) isoforms. These isoforms, MyHC-IIa, -IId, and -IIb, are >93% identical at the amino acid level and are broadly expressed in numerous muscles, and their genes are tightly linked. Mice with a null mutation in the MyHC-IId gene have phenotypes that include growth inhibition, muscle weakness, histological abnormalities, kyphosis (spinal curvature), and aberrant kinetics of muscle contraction and relaxation. Despite the lack of MyHC-IId, IId null mice have normal amounts of myosin in their muscles because of compensation by the MyHC-IIa gene. In each muscle examined from IId null mice, there was an increase in MyHC-IIa- containing fibers. MyHC-IIb content was unaffected in all muscles except the masseter, where its expression was extinguished in the IId null mice. Cross-sectional fiber areas, total muscle cross-sectional area, and total fiber number were affected in ways particular to each muscle. Developmental expression of adult MyHC genes remained unchanged in IId null mice. Despite this universal compensation of MyHC-IIa expression, IId null mice have severe phenotypes. We conclude that despite the similarity in sequence, MyHC-IIa and -IId have unique roles in the development and function of skeletal muscle.

The production of functional myosin heavy chains in many eukaryotic organisms requires the function of proteins containing UCS domains (UNC-45/CRO1/She4), which bind to the myosin head domain and stimulate its folding. UCS proteins are essential for myosin-related functions such as muscle formation, RNA localization and cytokinesis. Here, we show that the Schizosaccharomyces pombe UCS protein Rng3 associates with polysomes, suggesting that UCS proteins might assist myosin folding cotranslationally. To identify Rng3 cotranslational targets systematically, we purified Rng3-associated RNAs and used DNA microarrays to identify the transcripts. Rng3 copurified with only seven transcripts (around 0.1% of S. pombe genes), including all five messenger RNAs encoding myosin heavy chains. These results suggest that every myosin heavy chain in S. pombe is a cotranslational target of Rng3. Furthermore, our data suggest that microarray-based approaches allow the genome-wide identification of cotranslational chaperone targets, and thus pave the way for the dissection of translation-linked chaperone networks.

Exercise training can improve strength and lead to adaptations in the skeletal muscle and nervous systems. Skeletal muscles can develop into two types: fast and slow, depending on the expression pattern of myosin heavy chain (MHC) isoforms. Previous studies reported that exercise altered the distribution of muscle fiber types. It is not currently known what changes in the expression of caveolins and types of muscle fiber occur in response to the intensity of exercise. This study determined the changes in expression of caveolins and MHC type after forced exercise in muscular and non-muscular tissues in rats. A control (Con) group to which forced exercise was not applied and an exercise (Ex) group to which forced exercise was applied. Forced exercise, using a treadmill, was introduced at a speed of 25 m/min for 30 min, 3 times/day (07:00, 15:00, 23:00). Homogenized tissues were applied to extract of total RNA for further gene analysis. The expression of caveolin-3 and MHC2a in the gastrocnemius muscle of female rats significantly increased in the Ex group compared with the Con group (P<0.05). Furthermore, in the gastrocnemius muscle of male rats, the expression of MHC2x was significantly different between the two groups (P<0.05). There was an increased expression in caveolin-3 and a slightly decreased expression in TGFβ-1 in muscular tissues implicating caveolin-3 influences the expression of MHC isoforms and TGFβ-1 expression. Eventually, it implicates that caveolin-3 has positive regulatory function in muscle atrophy induced by neural dysfunction with spinal cord injury or stroke.

In preliminary experiments we noted developmental (i.e., embryonic and neonatal) myosin heavy chains (MHCs) in the diaphragms of patients with severe chronic obstructive pulmonary disease (COPD). We hypothesized that this finding represented new fiber formation secondary to injury associated with the mechanical stress of COPD or previously undescribed MHCs in the human diaphragm. To distinguish between these possibilities, we analyzed diaphragmatic biopsies obtained from 9 patients with severe COPD (forced expiratory volume in 1 s = 21 +/- 2% predicted, residual volume = 283 +/- 22% predicted) and 10 age-matched controls. First, using immunocytochemistry with specific monoclonal antibodies, we noted that control diaphragms had greater proportions of fibers expressing embryonic (50 +/- 2 vs. 28 +/- 3%, P < 0.0001) and neonatal (52 +/- 2 vs. 32 +/- 3%, P < 0.001) MHCs than COPD diaphragms. Second, SDS-PAGE demonstrated that these developmental MHCs represented only a very small fraction of the diaphragmatic MHC content. Third, the RT-PCR demonstrated mRNA coding for embryonic and neonatal MHCs in COPD and control diaphragms. Last, COPD and control diaphragms exhibited normal histology on light microscopy. We conclude that the presence of developmental MHC isoforms does not indicate new fiber formation in diaphragms of patients with severe COPD. Although these results represent the first systematic description of embryonic and neonatal MHCs in normal adult human diaphragms, their function remains to be elucidated.

The purpose of this study was to evaluate the effect of neurotrophin 3 (NT-3) enhanced nerve regeneration on the reinnervation of a target muscle. Muscle fibers can be classified according to their mechanical properties and myosin heavy chain (MHC) isoform composition. MHC1 containing slow-type and MHC2a or 2b fast-type fibers are normally distributed in a mosaic pattern, their phenotype dictated by motor innervation. After denervation, all fibers switch to fast-type MHC2b expression and also undergo atrophy resulting in loss of muscle mass. After regeneration, discrimination between fast and slow fibers returns, but the distribution and fiber size change according to the level of reinnervation. In this study, rat gastrocnemius muscles (ipsilateral and contralateral to the side of nerve injury) were collected up to 8 mo after nerve repair, with or without local delivery of NT-3. The phenotype changes of MHC1, 2a, and 2b were analyzed by immunohistochemistry, and fiber type proportion, diameter, and grouping were assessed by computerized image analysis. At 8 mo, the local delivery of NT-3 resulted in significant improvement in gastrocnemius muscle weight compared with controls (NT-3 group 47%, controls 39% weight of contralateral normal muscle; P < 0.05). NT-3 delivery resulted in a significant increase in the proportion (NT-3 43.3%, controls 35.7%; P < 0.05) and diameter (NT-3 87.8 micron, controls 70.8 micron; P < 0.05) of fast type 2b fibers after reinnervation. This effect was specific to type 2b fibers; no normalization was seen in other fiber types. This study indicates that NT-3-enhanced axonal regeneration has a beneficial effect on the motor target organ. Also, NT-3 may be specifically affecting a subset of motoneurons that determine type 2b muscle fiber phenotype. As NT-3 was topically applied to cut nerves, our data suggest a discriminating effect of the neurotrophin on neuro-muscular interaction. These results would imply that muscle fibers may be differentially responsive to other neurotrophic factors and indicate the potential clinical role of NT-3 in the prevention of muscle atrophy after nerve injury.

Mammalian skeletal muscle is generated by two waves of fiber formation, resulting in primary and secondary fibers. These fibers mature to give rise to several classes of adult muscle fibers with distinct contractile properties. Here we describe fast myosin heavy chain (MyHC) isoforms that are expressed in nascent secondary, but not primary, fibers in the early development of rat and human muscle. These fast MyHCs are distinct from previously described embryonic and neonatal fast MyHCs. To identify these MyHCs, monoclonal antibodies were used whose specificity was determined in western blots of MyHCs on denaturing gels and reactivity with muscle tissue at various stages of development. To facilitate a comparison of our results with those of others obtained using different antibodies or species, we have identified cDNAs that encode the epitopes recognized by our antibodies wherever possible. The results suggest that epitopes characteristic of adult fast MyHCs are expressed very early in muscle fiber development and distinguish newly formed secondary fibers from primary fibers. This marker of secondary fibers, which is detectable at the time of their inception, should prove useful in future studies of the derivation of primary and secondary fibers in mammalian muscle development.

Limb muscles of eutherian (placental) mammals express a slow and three fast isoforms of myosin heavy chain (MyHC), but little is known about marsupial MyHCs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of limb MyHCs from seven marsupial species, spanning two orders, revealed four components, each of which specifically cross-reacted in Western blots with a monoclonal antibody (mAb) against a corresponding eutherian MyHC. For all seven species, the relative mobility of the band identified by each mAb matched that in the rat, suggesting that the four are homologous to eutherian slow, 2B, 2X and 2A MyHCs, respectively, in the order of decreasing mobility. Immunohistochemical analysis of fast marsupial limb muscles identitied four different fiber populations whose relative fiber size spectra (IIA

Aged individuals suffer from multiple dysfunctions during skeletal muscle atrophy. The purpose of this study was to determine differential changes in gene expression in atrophied soleus muscle induced by hindlimb immobilization in young (3-4 months) and old (30-31 months) rats. The hypothesis was that differentially expressed mRNAs with age-atrophy interactions would reveal candidates that induce loss of function responses in aged animals. Each muscle was applied to an independent set of Affymetrix micoarrays, with 385 differentially expressed mRNAs with atrophy and 354 age-atrophy interactions detected by two-factor ANOVA (alpha of 0.05 with a Bonferroni adjustment). Functional trends were observed for 23 and 15 probe sets involved in electron transport and the extracellular matrix, respectively, decreasing more in the young than in the old. Other functional categories with atrophy in both ages included chaperones, glutathione-S-transferases, the tricarboxylic acid cycle, reductions in Z-line-associated proteins and increases in probe sets for protein degradation. Surprisingly, myosin heavy chain IIb and IIx mRNAs were suppressed in the atrophied soleus muscle of old rats as opposed to the large increases in the young animals (16- and 25-fold, respectively, with microarrays, and 61- and 68-fold, respectively, with real-time PCR). No significant changes were observed in myosin heavy chain IIb and IIx mRNA with micoarrays in the atrophied soleus muscles of old rats, but they were found to increase six- and fivefold, respectively, with real-time PCR. Therefore, deficiencies in pre-translational signals that normally upregulate myosin heavy chain IIb and IIx mRNAs during atrophy may exist in the soleus muscle of old animals.

Irisin (a glycosylated protein) is cleaved from fibronectin type III domain-containing protein 5 (FNDC5), which is expressed mainly in animal muscle tissues and has multiple metabolic regulatory activities. However, their roles in controlling myofiber types in skeletal muscle remain unclear.

The myosin II motors are ATP-powered force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta (β-MyHC) is primarily expressed in the ventricular myocardium and in slow-twitch muscle fibers, such as M. soleus. M. soleus-derived myosin II (SolM-II) is often used as an alternative to the ventricular β-cardiac myosin (βM-II); however, the direct assessment of biochemical and mechanical features of the native myosins is limited. By employing optical trapping, we examined the mechanochemical properties of native myosins isolated from the rabbit heart ventricle and soleus muscles at the single-molecule level. We found purified motors from the two tissue sources, despite expressing the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. We demonstrate βM-II was approximately threefold faster in the actin filament-gliding assay than SolM-II. The maximum actomyosin (AM) detachment rate derived in single-molecule assays was also approximately threefold higher in βM-II, while the power stroke size and stiffness of the "AM rigor" crossbridge for both myosins were comparable. Our analysis revealed a higher AM detachment rate for βM-II, corresponding to the enhanced ADP release rates from the crossbridge, likely responsible for the observed differences in the motility driven by these myosins. Finally, we observed a distinct myosin light chain 1 isoform (MLC1sa) that associates with SolM-II, which might contribute to the observed kinetics differences between βM-II and SolM-II. These results have important implications for the choice of tissue sources and justify prerequisites for the correct myosin heavy and light chains to study cardiomyopathies.

Myofibroblasts are unique contractile cells with both muscle and nonmuscle properties. Typically myofibroblasts are identified by the expression of alpha smooth muscle actin (ASMA); however some myofibroblasts also express sarcomeric proteins. In this study, we show that pulmonary myofibroblasts express three of the eight known sarcomeric myosin heavy chains (MyHCs) (IIa, IId, and embryonic) and that skeletal muscle myosin enzymatic activity is required for pulmonary myofibroblast contractility. Furthermore, inhibition of skeletal myosin activity and myofibroblast contraction results in a decrease in both ASMA and skeletal MyHC promoter activity and ASMA protein expression, suggesting a potential coupling of skeletal myosin activity and ASMA expression in myofibroblast differentiation. To understand the molecular mechanisms whereby skeletal muscle genes are regulated in myofibroblasts, we have found that members of the myogenic regulatory factor family of transcription factors and Ca(2+) - regulated pathways are involved in skeletal MyHC promoter activity. Interestingly, the regulation of skeletal myosin expression in myofibroblasts is distinct from that observed in muscle cells and suggests that cell context is important in its control.

As part of a study of cytoskeletal proteins involved in Drosophila embryonic development, we have undertaken the molecular analysis of a 140-kD ATP-sensitive actin-binding protein (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). Analysis of cDNA clones encoding this protein revealed that it represents a new class of unconventional myosin heavy chains. The amino-terminal two thirds of the protein comprises a head domain that is 29-33% identical (60-65% similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chain-binding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain a approximately 100-amino acid region that is predicted to form an alpha-helical coiled-coil. Since the unique gene that encodes this protein maps to the polytene map position 95F, we have named the new gene Drosophila 95F myosin heavy chain (95F MHC). The expression profile of the 95F MHC gene is complex. Examination of multiple cDNAs reveals that transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Developmental Northern and Western blots show that transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of protein expression throughout embryogenesis, but specific tissues appear to contain more protein. We speculate that the 95F MHC protein isoforms are involved in multiple dynamic processes during Drosophila development.

In zebrafish, ventricular myosin heavy chain (vmhc) gene is initially expressed at the anterior lateral mesoderm and thereafter its expression is restricted to the cardiac ventricle. The transcriptional control mechanisms in regulating chamber-specific expression of myosin heavy chains are not well defined. We isolated and analyzed zebrafish vmhc upstream region to examine the spatial and temporal regulation of vmhc using transgenic and transient expression techniques. Promoter deletion analyses defined a basal promoter region sufficient to drive vmhc expression in the ventricle and an upstream fragment necessary for repressing ectopic vmhc expression in the atrium. The transcriptional mechanism that prevents vmhc expression in the atrium is mediated through Nkx2.5 binding elements (NKE). We have further discovered that paired-related homeobox transcriptional factor 2 (Prx2/S8)-like binding elements are required for promoting vmhc expression, and Prrx1b, a Prx-related homeobox protein, participates in the regulation of vmhc expression with other transcriptional factors.

The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying null mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both null strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb null mutants are generally milder than in the MyHC-IId/x null strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for null expression of the two genes. Most striking is that while both null strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb null mice has significantly reduced ability to generate force while IId null mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.

Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.

Using immunofluorescence procedures with specific polyclonal and monoclonal antimyosin antibodies we have found that embryonic and neonatal myosin heavy chains (MHCs), which in rat skeletal muscle disappear during the first weeks after birth, are reexpressed in adult muscle after denervation. Reactivity for embryonic and neonatal MHCs was detected in some fibers as early as 3 days after denervation, became more evident by 7 days, and occurred exclusively in the type 2A fiber population. Paralysis of innervated muscles by tetrodotoxin block of the sciatic nerve also resulted in the reappearance of embryonic and neonatal MHCs in type 2A fibers. Significant variation in the degree of immunoreactivity was observed in different segments of the same muscle fiber, suggesting that coordination of muscle fiber nuclei in the control of myosin heavy chain gene expression is partially lost following denervation.

The alternatively spliced SM1 and SM2 smooth muscle myosin heavy chains differ at their respective carboxyl termini by 43 versus 9 unique amino acids. To determine whether these tailpieces affect filament assembly, SM1 and SM2 myosins, the rod region of these myosin isoforms, and a rod with no tailpiece (tailless), were expressed in Sf 9 cells. Paracrystals formed from SM1 and SM2 rod fragments showed different modes of molecular packing, indicating that the tailpieces can influence filament structure. The SM2 rod was less able to assemble into stable filaments than either SM1 or the tailless rods. Expressed full-length SM1 and SM2 myosins showed solubility differences comparable to the rods, establishing the validity of the latter as a model for filament assembly. Formation of homodimers of SM1 and SM2 rods was favored over the heterodimer in cells coinfected with both viruses, compared with mixtures of the two heavy chains renatured in vitro. These results demonstrate for the first time that the smooth muscle myosin tailpieces differentially affect filament assembly, and suggest that homogeneous thick filaments containing SM1 or SM2 myosin could serve distinct functions within smooth muscle cells.