Fast Myosin Heavy Chain Research Articles

Muscle after spinal cord injury

The morphological and contractile changes of muscles below the level of the lesion after spinal cord injury (SCI) are dramatic. In humans with SCI, a fiber-type transformation away from type I begins 4-7 months post-SCI and reaches a new steady state with predominantly fast glycolytic IIX fibers years after the injury. There is a progressive drop in the proportion of slow myosin heavy chain (MHC) isoform fibers and a rise in the proportion of fibers that coexpress both the fast and slow MHC isoforms. The oxidative enzymatic activity starts to decline after the first few months post-SCI. Muscles from individuals with chronic SCI show less resistance to fatigue, and the speed-related contractile properties change, becoming faster. These findings are also present in animals. Future studies should longitudinally examine changes in muscles from early SCI until steady state is reached in order to determine optimal training protocols for maintaining skeletal muscle after paralysis.

NFAT isoforms control activity-dependent muscle fiber type specification

The intracellular signals that convert fast and slow motor neuron activity into muscle fiber type specific transcriptional programs have only been partially defined. The calcium/calmodulin-dependent phosphatase calcineurin (Cn) has been shown to mediate the transcriptional effects of motor neuron activity, but precisely how 4 distinct muscle fiber types are composed and maintained in response to activity is largely unknown. Here, we show that 4 nuclear factor of activated T cell (NFAT) family members act coordinately downstream of Cn in the specification of muscle fiber types. We analyzed the role of NFAT family members in vivo by transient transfection in skeletal muscle using a loss-of-function approach by RNAi. Our results show that, depending on the applied activity pattern, different combinations of NFAT family members translocate to the nucleus contributing to the transcription of fiber type specific genes. We provide evidence that the transcription of slow and fast myosin heavy chain (MyHC) genes uses different combinations of NFAT family members, ranging from MyHC-slow, which uses all 4 NFAT isoforms, to MyHC-2B, which only uses NFATc4. Our data contribute to the elucidation of the mechanisms whereby activity can modulate the phenotype and performance of skeletal muscle.

The effects of repeated allergen challenge on airway smooth muscle structural and molecular remodeling in a rat model of allergic asthma

The effects of remodeling of airway smooth muscle (SM) by hyperplasia on airway SM contractility in vivo are poorly explored. The aim of this study was to investigate the relationship between allergen-induced airway SM hyperplasia and its contractile phenotype. Brown Norway rats were sensitized with ovalbumin (OVA) or saline on day 0 and then either OVA-challenged once on day 14 and killed 24 h later or OVA-challenged 3 times (on days 14, 19, and 24) and killed 2 or 7 days later. Changes in SM mass, expression of total myosin, SM myosin heavy chain fast isoform (SM-B) and myosin light chain kinase (MLCK), tracheal contractions ex vivo, and airway responsiveness to methacholine (MCh) in vivo were assessed. One day after a single OVA challenge, the number of SM cells positive for PCNA was greater than for control animals, whereas the SM mass, contractile phenotype, and tracheal contractility were unchanged. Two days after three challenges, SM mass and PCNA immunoreactive cells were increased (3- and 10-fold, respectively; P < 0.05), but airway responsiveness to MCh was unaffected. Lower expression in total myosin, SM-B, and MLCK was observed at the mRNA level (P < 0.05), and total myosin and MLCK expression were lower at the protein level (P < 0.05) after normalization for SM mass. Normalized tracheal SM force generation was also significantly lower 2 days after repeated challenges (P < 0.05). Seven days after repeated challenges, features of remodeling were restored toward control levels. Allergen-induced hyperplasia of SM cells was associated with a loss of contractile phenotype, which was offset by the increase in mass.

Rapid muscle atrophy response to unloading: pretranslational processes involving MHC and actin

Skeletal muscles, especially weight-bearing muscles, are very sensitive to changes in loading state. The aim of this paper was to characterize the dynamic changes in the unloaded soleus muscle in vivo following a short bout of hindlimb suspension (HS), testing the hypothesis that transcriptional events respond early to the atrophic stimulus. In fact, we observed that after only 1 day of HS, primary transcript levels of skeletal alpha-actin and type I myosin heavy chain (MHC) genes were significantly reduced by more than 50% compared with ground control levels. The degree of the decline for the mRNA expression of actin and type I MHC lagged behind that of the pre-mRNA levels after 1 day of HS, but by 2 and 7 days of HS, large decreases were observed. Although the faster MHC isoforms, IIx and IIb, began to be expressed in soleus after 1 day of HS, a relatively significant shift in mRNA expression from the slow MHC isoform type I toward these fast MHC isoforms did not emerge until 7 days of HS. One day of HS was sufficient to show significant decreases in mRNA levels of putative signaling factors serum response factor (SRF), suppressor of cytokine signaling-3 (SOCS3), and striated muscle activator of Rho signaling (STARS), although transcription factors yin-yang-1 (YY1) and transcriptional enhancing factor-1 (TEF-1) were not significantly affected by HS. The protein levels of actin and type I MHC were significantly decreased after 2 days of HS, and SRF protein was significantly decreased after 7 days HS. Our results show that after only 1 day of unloading, pre-mRNA and mRNA expression of muscle proteins and muscle-specific signaling factors are significantly reduced, suggesting that the downregulation of the synthesis side of the protein balance equation that occurs in atrophying muscle is initiated rapidly.

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

The age-dependent decline in skeletal muscle mass and function is believed to be due to a multi-factorial pathology and represents a major factor that blocks healthy aging by increasing physical disability, frailty and loss of independence in the elderly. This study has focused on the comparative proteomic analysis of contractile elements and revealed that the most striking age-related changes seem to occur in the protein family representing myosin light chains (MLCs). Comparative screening of total muscle extracts suggests a fast-to-slow transition in the aged MLC population. The mass spectrometric analysis of the myofibril-enriched fraction identified the MLC2 isoform of the slow-type MLC as the contractile protein with the most drastically changed expression during aging. Immunoblotting confirmed an increased abundance of slow MLC2, concomitant with a switch in fast versus slow myosin heavy chains. Staining of two-dimensional gels of crude extracts with the phospho-specific fluorescent dye ProQ-Diamond identified the increased MLC2 spot as a muscle protein with a drastically enhanced phosphorylation level in aged fibres. Comparative immunofluorescence microscopy, using antibodies to fast and slow myosin isoforms, confirmed a fast-to-slow transformation process during muscle aging. Interestingly, the dramatic increase in slow MLC2 expression was restricted to individual senescent fibres. These findings agree with the idea that aged skeletal muscles undergo a shift to more aerobic-oxidative metabolism in a slower-twitching fibre population and suggest the slow MLC2 isoform as a potential biomarker for fibre type shifting in sarcopenia of old age.

Muscular dystrophy begins early in embryonic development deriving from stem cell loss and disrupted skeletal muscle formation

Examination of embryonic myogenesis of two distinct, but functionally related, skeletal muscle dystrophy mutants (mdx and cav-3(-/-)) establishes for the first time that key elements of the pathology of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy type 1C (LGMD-1c) originate in the disruption of the embryonic cardiac and skeletal muscle patterning processes. Disruption of myogenesis occurs earlier in mdx mutants, which lack a functional form of dystrophin, than in cav-3(-/-) mutants, which lack the Cav3 gene that encodes the protein caveolin-3; this finding is consistent with the milder phenotype of LGMD-1c, a condition caused by mutations in Cav3, and the earlier [embryonic day (E)9.5] expression of dystrophin. Myogenesis is severely disrupted in mdx embryos, which display developmental delays; myotube morphology and displacement defects; and aberrant stem cell behaviour. In addition, the caveolin-3 protein is elevated in mdx embryos. Both cav-3(-/-) and mdx mutants (from E15.5 and E11.5, respectively) exhibit hyperproliferation and apoptosis of Myf5-positive embryonic myoblasts; attrition of Pax7-positive myoblasts in situ; and depletion of total Pax7 protein in late gestation. Furthermore, both cav-3(-/-) and mdx mutants have cardiac defects. In cav-3(-/-) mutants, there is a more restricted phenotype comprising hypaxial muscle defects, an excess of malformed hypertrophic myotubes, a twofold increase in myonuclei, and reduced fast myosin heavy chain (FMyHC) content. Several mdx mutant embryo pathologies, including myotube hypotrophy, reduced myotube numbers and increased FMyHC, have reciprocity with cav-3(-/-) mutants. In double mutant (mdxcav-3(+/-)) embryos that are deficient in dystrophin (mdx) and heterozygous for caveolin-3 (cav-3(+/-)), whereby caveolin-3 is reduced to 50% of wild-type (WT) levels, these phenotypes are severely exacerbated: intercostal muscle fibre density is reduced by 71%, and Pax7-positive cells are depleted entirely from the lower limbs and severely attenuated elsewhere; these data suggest a compensatory rather than a contributory role for the elevated caveolin-3 levels that are found in mdx embryos. These data establish a key role for dystrophin in early muscle formation and demonstrate that caveolin-3 and dystrophin are essential for correct fibre-type specification and emergent stem cell function. These data plug a significant gap in the natural history of muscular dystrophy and will be invaluable in establishing an earlier diagnosis for DMD/LGMD and in designing earlier treatment protocols, leading to better clinical outcome for these patients.

Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading

Recent advances in chromatin biology have enhanced our understanding of gene regulation. It is now widely appreciated that gene regulation is dependent upon post-translational modifications to the histones which package genes in the nucleus of cells. Active genes are known to be associated with acetylation of histones (H3ac) and trimethylation of lysine 4 in histone H3 (H3K4me3). Using chromatin immunoprecipitation (ChIP), we examined histone modifications at the myosin heavy chain (MHC) genes expressed in fast vs. slow fiber-type skeletal muscle, and in a model of muscle unloading, which results in a shift to fast MHC gene expression in slow muscles. Both H3ac and H3K4me3 varied directly with the transcriptional activity of the MHC genes in fast fiber-type plantaris and slow fiber-type soleus. During MHC transitions with muscle unloading, histone H3 at the type I MHC becomes de-acetylated in correspondence with down-regulation of that gene, while upregulation of the fast type IIx and IIb MHCs occurs in conjunction with enhanced H3ac in those MHCs. Enrichment of H3K4me3 is also increased at the type IIx and IIb MHCs when these genes are induced with muscle unloading. Downregulation of IIa MHC, however, was not associated with corresponding loss of H3ac or H3K4me3. These observations demonstrate the feasibility of using the ChIP assay to understand the native chromatin environment in adult skeletal muscle, and also suggest that the transcriptional state of types I, IIx and IIb MHC genes are sensitive to histone modifications both in different muscle fiber-types and in response to altered loading states.

Differential Epigenetic Modifications of Histones at the Myosin Heavy Chain Genes in Fast and Slow Skeletal Muscle Fibers and in Response to Muscle Unloading

In order to delineate the relationship between epigenetic modifications and myosin heavy chain (MHC) gene regulation, we examined histone modifications of the four MHC genes (I, IIa, IIx, IIb) in fast vs. slow fiber‐type rat skeletal muscle, and in a model of muscle unloading, which causes a coordinated shift from slow to fast MHC gene expression in the soleus (sol). We used chromatin immunoprecipitation (ChIP) to examine acetylation of histone H3 (AcH3) and histone H3 lysine 4 trimethylation (H3K4me3). We report that both AcH3 and H3K4me3 correlate with the transcriptional activity, assessed via pre‐ and mRNA, of each of the MHC genes in both fast plantaris and slow sol. Likewise, during dynamic MHC remodeling of sol with unloading, the slow (I, IIa) to fast (IIx, IIb) transition in mRNA parallels alterations in histone modifications (p&lt;0.05). Thus, I and IIa MHC genes become de‐acetylated when their transcription is down‐regulated, while induction of the fast IIx and IIb MHCs occurs in conjunction with enhanced acetylation at those genes. H3K4me3 is similarly altered. These observations demonstrate the feasibility of using the ChIP assay to understand the native chromatin environment in adult skeletal muscle, and also suggest that the transcriptional state of MHC genes are sensitive to histone modifications both in different muscle fiber‐types and in response to altered loading states. Supported by NIH AR30346‐25

Improved myogenic differentiation and myoblast fusion under physiological amino acid concentrations

Myoblast cell lines have been applied to model myogenesis in vitro. Muscle growth can be modulated by altering insulin, IGF‐I, as well as amino acids (AA) levels. However, little attention has been paid to the fact that most culture media contain supra‐physiological AA levels. This raises the question whether such conditions are suitable for AA related research questions and whether the data are relevant to in vivo experiments. The aim of this study was to determine if myogenesis of C2C12 myoblasts differed in the presence of physiological AA levels compared to supra‐physiological levels. Myoblasts were grown in DMEM with low glucose and 10% FBS. DMEM was diluted with HBSS supplemented with NaHCO3 to yield culture media of similar composition containing 5‐100% of AA levels in unmodified DMEM. Physiological levels of AA were reached at ~15% of unmodified medium. Differentiation was induced by culturing the cells in DMEM with 1% FBS and decreasing levels of AA. Myogenesis was assessed 3 and 5 days after differentiation initiation. Myoblast fusion, creatine kinase activity and myosin heavy chain‐fast were increased in cells cultured in DMEM containing physiological levels of AA compared to unmodified medium. These findings show that supra‐physiological AA levels result in sub‐optimal differentiation, emphasizing the use of physiological AA levels in muscle cell culture for more meaningful in vivo extrapolation of data.

ALT-C, a disintegrin-like Cys-rich protein from Bothrops alternatus, increases skeletal myoblast viability

ALT-C, an ECD motif (glutamic acid, cysteine, aspartic acid) disintegrin from Bothrops alternatus snake venom, induces α2β1 integrin-mediated signaling and neutrophil chemotaxis. In vitro, in human umbilical vein endothelial cells (HUVEC), ALT-C induces cell proliferation, thus showing an interesting potential for tissue regeneration studies. This work aimed to evaluate the influence of ALT-C in myoblast viability and differentiation. Myoblasts were obtained from hind limb muscles of 3 to 4-day old Wistar rats. The cells were incubated with ALT-C at different concentrations and incubation periods were followed by total RNA isolation. cDNA synthesis and real time polymerase chain reaction (PCR) were performed with primers of myoD as well as of both (slow and fast) myosin heavy chain isoforms (MHC). ECD-disintegrin increased myoblast viability in a dose-dependent way, mostly with 50 to 100 nM concentrations, and such effect was more prevalent after 48 hours. No changes in gene expression of both MHC isoforms were observed in ALT-C-treated cells. MyoD expression was not detected, which suggests that myoblasts were in mature stages. Protease activity and cytokine array tested in a medium of 50 nM ALT-C-treated cells after 48 hours were not different from controls. In conclusion, it was shown that myoblats are sensitive to ALT-C indicating an integrin-mediated intracellular signaling that increases cell viability.

Myonuclear domain size and myosin isoform expression in muscle fibres from mammals representing a 100 000‐fold difference in body size

This comparative study of myonuclear domain (MND) size in mammalian species representing a 100,000-fold difference in body mass, ranging from 25 g to 2500 kg, was undertaken to improve our understanding of myonuclear organization in skeletal muscle fibres. Myonuclear domain size was calculated from three-dimensional reconstructions in a total of 235 single muscle fibre segments at a fixed sarcomere length. Irrespective of species, the largest MND size was observed in muscle fibres expressing fast myosin heavy chain (MyHC) isoforms, but in the two smallest mammalian species studied (mouse and rat), MND size was not larger in the fast-twitch fibres expressing the IIA MyHC isofom than in the slow-twitch type I fibres. In the larger mammals, the type I fibres always had the smallest average MND size, but contrary to mouse and rat muscles, type IIA fibres had lower mitochondrial enzyme activities than type I fibres. Myonuclear domain size was highly dependent on body mass in the two muscle fibre types expressed in all species, i.e. types I and IIA. Myonuclear domain size increased in muscle fibres expressing both the beta/slow (type I; r = 0.84, P < 0.001) and the fast IIA MyHC isoform (r = 0.90; P < 0.001). Thus, MND size scales with body size and is highly dependent on muscle fibre type, independent of species. However, myosin isoform expression is not the sole protein determining MND size, and other protein systems, such as mitochondrial proteins, may be equally or more important determinants of MND size.

Skeletal muscle changes after hemiparetic stroke and potential beneficial effects of exercise intervention strategies.

Stroke is the leading cause of disability in the United States. New evidence reveals significant structural and metabolic changes in skeletal muscle after stroke. Muscle alterations include gross atrophy and shift to fast myosin heavy chain in the hemiparetic (contralateral) leg muscle; both are related to gait deficit severity. The underlying molecular mechanisms of this atrophy and muscle phenotype shift are not known. Inflammatory markers are also present in contralateral leg muscle after stroke. Individuals with stroke have a high prevalence of insulin resistance and diabetes. Skeletal muscle is a major site for insulin-glucose metabolism. Increasing evidence suggests that inflammatory pathway activation and oxidative injury could lead to wasting, altered function, and impaired insulin action in skeletal muscle. The health benefits of exercise in disabled populations have now been recognized. Aerobic exercise improves fitness, strength, and ambulatory performance in subjects with chronic stroke. Therapeutic exercise may modify or reverse skeletal muscle abnormalities.

Nerve‐dependent changes in skeletal muscle myosin heavy chain after experimental denervation and cross‐reinnervation and in a demyelinating mouse model of Charcot–Marie–Tooth disease type 1A

Innervation regulates the contractile properties of vertebrate muscle fibers, in part through the effect of electrical activity on expression of distinct myosins. Herein we analyze the role of innervation in regulating the accumulation of the general, maturational, and adult forms of rodent slow myosin heavy chain (MyHC) that are defined by the presence of distinct antigenic epitopes. Denervation increases the number of fibers that express general slow MyHC, but it decreases the adult slow MyHC epitope. Cross-reinnervation of slow muscle by a fast nerve leads to an increase in the number of fibers that express fast MyHC. In both cases, there is an increase in the number of fibers that express slow and fast IIA MyHCs, but without the adult slow MyHC epitope. The data suggest that innervation is required for maturation and maintenance of diversity of both slow and fast fibers. The sequence of slow MyHC epitope transitions is a useful biomarker, and it may play a significant role during nerve-dependent changes in muscle fiber function. We applied this detailed muscle analysis to a transgenic mouse model of human motor and sensory neuropathy IA, also known as Charcot-Marie-Tooth disease type 1A (CMT1A), in which electrical conduction in some motor nerves is poor due to demyelination. The mice display atrophy of some muscle fibers and changes in slow and fast MyHC epitope expression, suggestive of a progressive increase in innervation of muscle fibers by fast motor neurons, even at early stages. The potential role of these early changes in disease pathogenesis is assessed.

Myosin, Transgelin, and Myosin Light Chain Kinase

Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma.

Fiber Type Composition of Cadaveric Human Rotator Cuff Muscles

Descriptive cadaveric laboratory study. To identify the fiber type composition of the rotator cuff and teres major muscles in human subjects. The rotator cuff is commonly injured in athletics and is a major focus of sports medicine. Although the anatomy and architecture of each muscle have been described in great detail, these muscles have never been fiber typed using immunohistochemistry or gel electrophoresis. Fiber typing is important in modeling function, exercise training, and rehabilitation. We harvested tissue samples for all 4 rotator cuff muscles, as well as the teres major muscle from cadavers. Tissues were frozen in liquid nitrogen and sectioned. Cryosections were labeled with commercially available antibodies against fast and slow isoforms of myosin heavy chain (MHC). We also harvested fresh (unembalmed) tissue from deceased subjects and labeled tissue sections with antibodies against fast or slow MHC and wheat germ agglutinin. Gel electrophoresis followed by silver staining was also used to identify and quantify MHC isoforms in fresh tissue samples. All of the muscles were of mixed fiber type composition. As a whole, 44% of rotator cuff fibers labeled positively for slow MHC, with slow MHC content of 54% in supraspinatus, 41% in infraspinatus, 49% in teres minor, 38% in subscapularis, and 40% in teres major. Mixed MHC isoform distribution was confirmed by SDS-PAGE, which also indicated that the IIa and IIx isoforms were roughly equally present across the muscles. Human rotator cuff muscles, at least in older subjects, have a mixed fiber type. Because we only examined older subjects, we must limit our interpretation to this population.

Myostatin preferentially down-regulates the expression of fast 2x myosin heavy chain in cattle

Myostatin is involved in an inhibitor of muscular growth and differentiation. Myoblasts derived from double-muscled Japanese shorthorn cattle (DM myoblasts) with absence of functional myostatin had higher abilities to proliferate and differentiate than myoblasts derived from normal-muscled cattle (NM myoblasts). In DM myoblasts, mRNA expressions of fetal myosin heavy chain (MyHC) in growth medium and of fast 2a and 2x MyHC in fusion medium were significantly greater than that in NM myoblasts. No significant difference existed in expressions of embryonic and slow MyHC mRNA between DM and NM myoblasts. The expression of MyoD mRNA was suppressed in myoblasts by administration of myostatin. Two cloned DM myoblast strains (DMc) were established. Addition of myostatin for DMc resulted in less myotube formation and suppression of mRNA expression of fast 2x MyHC. These findings suggest that the endogenous myostatin preferentially down-regulates the expression of the fast 2x MyHC and participates in differentiation of myofiber types during early bovine myogenesis.

Modulation of skeletal muscle fiber type by mitogen‐activated protein kinase signaling

Skeletal muscle is composed of diverse fiber types, yet the underlying molecular mechanisms responsible for this diversification remain unclear. Herein, we report that the extracellular signal-regulated kinase (ERK) 1/2 pathway, but not p38 or c-Jun NH(2)-terminal kinase (JNK), is preferentially activated in fast-twitch muscles. Pharmacological blocking of ERK1/2 pathway increased slow-twitch fiber type-specific reporter activity and repressed those associated with the fast-twitch fiber phenotype in vitro. Overexpression of a constitutively active ERK2 had an opposite effect. Inhibition of ERK signaling in cultured myotubes increased slow-twitch fiber-specific protein accumulation while repressing those characteristic of fast-twitch fibers. Overexpression of MAP kinase phosphatase-1 (MKP1) in mouse and rat muscle fibers containing almost exclusively type IIb or IIx fast myosin heavy chain (MyHC) isoforms induced de novo synthesis of the slower, more oxidative type IIa and I MyHCs in a time-dependent manner. Conversion to the slower phenotype was confirmed by up-regulation of slow reporter gene activity and down-regulation of fast reporter activities in response to forced MKP1 expression in vivo. In addition, activation of ERK2 signaling induced up-regulation of fast-twitch fiber program in soleus. These data suggest that the MAPK signaling, most likely the ERK1/2 pathway, is necessary to preserve the fast-twitch fiber phenotype with a concomitant repression of slow-twitch fiber program.

Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMDJ) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle

BackgroundSkeletal muscles are composed of heterogeneous collections of muscle fiber types, the arrangement of which contributes to a variety of functional capabilities in many muscle types. Furthermore, skeletal muscles can adapt individual myofibers under various circumstances, such as disease and exercise, by changing fiber types. This study was performed to examine the influence of dystrophin deficiency on fiber type composition of skeletal muscles in canine X-linked muscular dystrophy in Japan (CXMDJ), a large animal model for Duchenne muscular dystrophy.MethodsWe used tibialis cranialis (TC) muscles and diaphragms of normal dogs and those with CXMDJ at various ages from 1 month to 3 years old. For classification of fiber types, muscle sections were immunostained with antibodies against fast, slow, or developmental myosin heavy chain (MHC), and the number and size of these fibers were analyzed. In addition, MHC isoforms were detected by gel electrophoresis.ResultsIn comparison with TC muscles of CXMDJ, the number of fibers expressing slow MHC increased markedly and the number of fibers expressing fast MHC decreased with growth in the affected diaphragm. In populations of muscle fibers expressing fast and/or slow MHC(s) but not developmental MHC of CXMDJ muscles, slow MHC fibers were predominant in number and showed selective enlargement. Especially, in CXMDJ diaphragms, the proportions of slow MHC fibers were significantly larger in populations of myofibers with non-expression of developmental MHC. Analyses of MHC isoforms also indicated a marked increase of type I and decrease of type IIA isoforms in the affected diaphragm at ages over 6 months. In addition, expression of developmental (embryonic and/or neonatal) MHC decreased in the CXMDJ diaphragm in adults, in contrast to continuous high-level expression in affected TC muscle.ConclusionThe CXMDJ diaphragm showed marked changes in fiber type composition unlike TC muscles, suggesting that the affected diaphragm may be effectively adapted toward dystrophic stress by switching to predominantly slow fibers. Furthermore, the MHC expression profile in the CXMDJ diaphragm was markedly different from that in mdx mice, indicating that the dystrophic dog is a more appropriate model than a murine one, to investigate the mechanisms of respiratory failure in DMD.

Myostatin preferentially down-regulates the expression of fast 2x myosin heavy chain in cattle

Myostatin is involved in an inhibitor of muscular growth and differentiation. Myoblasts derived from double-muscled Japanese shorthorn cattle (DM myoblasts) with absence of functional myostatin had higher abilities to proliferate and differentiate than myoblasts derived from normal-muscled cattle (NM myoblasts). In DM myoblasts, mRNA expressions of fetal myosin heavy chain (MyHC) in growth medium and of fast 2a and 2x MyHC in fusion medium were significantly greater than that in NM myoblasts. No significant difference existed in expressions of embryonic and slow MyHC mRNA between DM and NM myoblasts. The expression of MyoD mRNA was suppressed in myoblasts by administration of myostatin. Two cloned DM myoblast strains (DMc) were established. Addition of myostatin for DMc resulted in less myotube formation and suppression of mRNA expression of fast 2x MyHC. These findings suggest that the endogenous myostatin preferentially down-regulates the expression of the fast 2x MyHC and participates in differentiation of myofiber types during early bovine myogenesis.

Molecular Mechanics of Smooth Muscle Contractile Proteins in Airway Hyperresponsiveness and Asthma

Airway hyperresponsiveness (AH) is a hallmark of asthma. The dynamics of the airway smooth muscle (SM) contraction, rather than its force-generating capacity, have been postulated to be key features of AH. Two mechanisms were proposed whereby an increased velocity of shortening (Vmax) of the airway SM leads to excessive bronchoconstriction. The first mechanism involves a greater Vmax during the initial portion of contraction, whereas the second mechanism implicates a greater Vmax after muscle stretches, such as after each tidal breath. This review focuses on the components of the contractile apparatus that have so far been reported to enhance the mechanics of the myosin molecular motor, thus leading to a greater Vmax. A greater activation of myosin, via increased phosphorylation of its regulatory light chain (LC20) by myosin light chain kinase, correlates with an increased Vmax in models of AH and in human asthmatic bronchial SM cells. However, poor correlations between these two parameters have also been reported in other models. A greater expression of the fast SM myosin heavy chain isoform [(+)insert or SM-B] also correlates with the greater Vmax measured in models of AH and in human asthmatic bronchial SM cells. However, the (+)insert isoform can only explain a twofold increase in Vmax, as extrapolated from its velocity of actin filament propulsion in the in vitro motility assay. Further considerations are given to the combination of these two factors with other components of the contractile machinery, thereby leading to the enhancement of airway SM function.