Expression of extraocular myosin heavy chain in rabbit laryngeal muscle

Summary The intrinsic laryngeal muscles of mammals are functionally heterogeneous, some of these muscles (e.g. the thyroarytenoid) contract extremely rapidly, like extraocular muscle, whilst others (e.g. the cricothyroid) contract as fast as limb fast muscle. The extraordinarily rapid contraction speed of extraocular muscles is associated with a fast myosin not found in limb muscles. In this work we explored the possibility that the thyroarytenoid muscle may also express this extraocular-specific fast myosin by raising a monoclonal antibody (mab 4A6) against its heavy chain. Electrophoretic separation of native isomyosins revealed that both the extraocular and the thyroarytenoid have two similar bands migrating ahead of bands found in limb fast or cricothyroid myosins. These two bands bound mab 4A6. The thyroarytenoid muscle can be divided into two divisions, a vocalis division which is important in phonation and an external division which functions in closing the glottis. Fibres in the vocalis are heterogeneous, some stain with mab 4A6, whilst others stain with mabs against limb myosin heavy chains. Fibres in the external division stain almost homogeneous with mab 4A6. The immunohistochemical staining pattern in the cricothyroid muscle resembled that of fast limb muscle: no fibres stained with mab 4A6. Thus, the high speed of contraction of the thyroarytenoid is associated with the same myosin heavy chain found in extraocular muscles, this characteristic is presumably an evolutionary adaptation for rapid closure of the glottis to enhance airway defense mechanisms.     

Expression of a fast myosin heavy chain mRNA in individual rabbit skeletal muscle fibers with intermediate oxidative capacity.

In situ hybridization (ISH) of myosin heavy chain (MHC) mRNA, immunofluorescent detection of MHC protein, and oxidative enzyme histochemistry were performed on the same fibers in serially sectioned rabbit skeletal muscle. By combining these three techniques quantitatively, on a fiber-by-fiber basis, fibers that expressed mRNA complementary to a fast MHC cDNA pMHC24-79 of unknown subtype (Maeda et al., 1987) were classified into fiber types with respect to slow myosin expression and oxidative capacity. As expected, slow fibers had low hybridization to pMHC24-79. Fast fibers were divided into three subtypes. mRNA from the low oxidative fibers (fast-glycolytic, IIB) did not hybridize with pMHC24-79. Fast fibers whose mRNA hybridized best to pMHC24-79 were mainly in the intermediate range of oxidative capacity (probably IIX). The fast fibers with the highest oxidative capacity had low hybridization to this MHC mRNA (probably IIA). Thus, pMHC24-79 was identified as a clone of a fast isomyosin, tentatively designated as the fast IIX with intermediate oxidative capacity. The expression of more than a single species of fast and slow isomyosin mRNAs in classically defined fiber type was considered in interpreting these results.     

Distinct myosin heavy chain isoform transitions in developing slow and fast cat hindlimb muscles

The expression of myosin heavy chain (MHC) isoforms leading to adult fiber phenotypes in the tibialis anterior (TA) and soleus muscles of the cat were investigated from embryonic day 35 to 1 year after birth. Electrophoresis and immunoblotting of myofibrils demonstrated the expression of 5 different MHC isoforms, i.e. I, IIa, IIx, embryonic, and neonatal, during development. Based on electrophoresis, the adult-like MHC composition of the soleus and TA were not observed until postnatal day 40 (P40) and 120 (P120), respectively. In contrast, immunohistochemical analyses revealed that the adult-like fiber phenotype composition was attained much later (P120) in the soleus. The existence of multiple MHC isoforms in individual fibers suggested that transitions occurred until P120 in both muscles. Adult type I fibers were first observed at P1. Adult IIA fibers were first observed at P30 in the TA and P40 in the soleus. IIX fibers were not identified until P40 in the TA. The transition to the predominantly slow phenotype of the soleus involved a gradual loss of embryonic and fast isoforms accompanied by an accumulation of slow MHC. In contrast, the expression of slow and fast MHC in the fast TA muscle was relatively unchanged throughout development. These results show that the establishment of a given MHC-based fiber phenotype varies significantly between slow and fast muscles in the kitten.     

Expression of desmin and smooth muscle myosin heavy chain in dermatofibromas

BACKGROUND: The histopathologic features of dermatofibroma vary remarkably, and this diversity may occasionally cause problems in differentiating between benign and malignant mesenchymal lesions, including smooth muscle neoplasms. Immunohistochemical stains are sometimes necessary to clarify the histogenesis of a lesion. OBJECTIVE: To evaluate dermatofibromas for expression of desmin and smooth muscle myosin heavy chain (SM-MHC) antigens, which are commonly used as evidence of smooth muscle differentiation. METHODS: We studied 100 consecutive cases of dermatofibroma using hematoxylin-eosin-stained sections and immunoperoxidase staining with antibodies against desmin, SM-MHC, and smooth muscle actin. RESULTS: We found focal positivity for desmin in 9 cases, and in 2 of these cases, at least 10% of lesional cells showed strong expression. We found focal staining for SM-MHC in 10 cases, and in 2 of these cases, at least 10% of the lesional cells were positive. Regions positive for desmin and/or SM-MHC did not show definite histologic features of myogenous differentiation on hematoxylin-eosin-stained sections. All dermatofibromas expressing desmin and SM-MHC were also strongly positive for smooth muscle actin. CONCLUSIONS: About 10% of dermatofibromas show focal expression of desmin and SM-MHC, and this expression may be present in up to 10% to 15% of lesional cells. Thus, in dermal spindle cell lesions, focal expression of these muscle antigens, like that of smooth muscle actin, is not diagnostic of a smooth muscle tumor.     

Expression of slow and fast myosin heavy chains in overloaded muscles of the developing rat

Summary The present study examines the developmental accumulation of slow myosin heavy chain in the extensor digitorum longus, soleus and plantaris muscles of rats after early post-natal imposition of mechanical overload by removal of synergistic muscles. The proportions of slow and fast myosin heavy chain were measured in each muscle by ELISA. Fibres expressing slow myosin were examined immunocytochemically using a monoclonal antibody specific for slow MHC. Between 30 and 60 days of age, MHC increases by 15% (p<0.001) in the soleus and by 27% (p<0.001) in the plantaris of normally developing, unoperated animals. The effect of overload on the soleus and plantaris is to accelerate the rate of increase in slow MHC accumulation so that levels are respectively 16 and 39% higher than controls by 30 days of age (p<0.001). By 60 days, the control soleus and plantaris attain levels of slow MHC roughly equivalent to their overloaded counterparts. In overloaded plantaris, the increase in levels of slow myosin does not occur at the expense of fast myosin expression. In the EDL there is a normal developmentally regulated decrease in slow MHC accumulation, reflected by a 40% decrease in levels of slow MHC (p<0.0001) and a 50% decrease in the number of slow fibres (p<0.001), between 30 days and 20 weeks of age. This elimination of slow myosin accumulation in the EDL is unimpeded by chronic overload. Thus, muscles react to mechanical overload in a tissue specific manner. The pattern of response is conservative and potentiates normal, long term maturational shifts in myosin heavy chain expression characteristic of each muscle.     

Evidence for distinct fast and slow myogenic cell lineages in human foetal skeletal muscle

To analyse the myogenic cell lineages in human foetal skeletal muscle, muscle cell cultures were prepared from different foetal stages of development. The in vitro muscle cell phenotype was defined by staining the myotubes with antibodies to fast and slow skeletal muscle type myosin heavy chains using immunoperoxidase or double immunofluorescence procedures. The antibodies to fast skeletal muscle myosin heavy chains stained nearly all myotubes dark in cell cultures prepared from quadriceps muscles at 10–18 weeks of gestation. The antibodies to slow skeletal muscle myosin heavy chains, in contrast, stained only 10–40% of the myotubes very dark. The remaining myotubes were further subdivided into two populations, one of which was unstained while the other stained with variable intensity for slow myosin heavy chain. The slow myosin heavy chain staining was not influenced by the nature of the substratum used to culture these cells, although the growth of muscle cell cultures was greatly improved on matrigel-coated dishes. The presence of both slow and fast myosin heavy chains was detected even when myotubes were grown on uncoated petri dishes. The myotube diversity was further investigated by analysing the clonal populations of human foetal skeletal muscle cells in vitro. When cultured at clonal densities, two types of myogenic clones were identified by their differential staining with antibodies to slow myosin heavy chain. As was the case with the high density muscle cell cultures, virtually all myotubes in both groups of clones stained with antibodies to fast myosin heavy chains. Antibodies to slow myosin heavy chains stained nearly all myotubes dark in one group of myogenic clones, but only a subset of the myotubes stained dark for slow myosin heavy chain in the second group of clones. The proportion of slow myosin heavy chain positive myotubes in this group varied in different clones. The myogenic diversity was thus apparent in both high density and clonal human muscle cell cultures, and myogenic cells retained their ability to modify their muscle cell phenotype. This revised version was published online in September 2006 with corrections to the Cover Date.     

Heterogeneous postnatal transitions in myosin heavy chain isoforms within the rabbit temporalis muscle

Postnatal changes in the fiber type composition and fiber cross-sectional area were investigated in the superficial (TEM1) and deep (TEM23) temporalis of male rabbits. It was hypothesized that, due to the transition from suckling to chewing during early postnatal development, the proportion of fast fiber types would decrease, while the proportion of fibers positive for myosin heavy chain (MyHC) cardiac alpha would increase, and that, due to the influence of testosterone during late postnatal development, the proportion of these alpha fibers would decrease again. Classification of the fibers types was performed by immunohistochemistry according to their MyHC content. The proportion of alpha fiber types significantly increased in both muscle portions from 2% and 8% for TEM1 and TEM23 at week 1 to 29% and 54% at week 8, respectively,. While in TEM1 the proportion of this fiber type did not change thereafter, it decreased again to 27% in TEM23 at week 20. The change for the fast fiber types was opposite to that of the alpha fiber types. Significantly more MyHC IIX fibers were found in TEM1 than in TEM23 in adult rabbits. In the first 8 weeks, the cross-sectional areas of all fibers increased. After this period, only MyHC cardiac alpha + I fibers continued to increase significantly. It was concluded that there are developmental differences in the myosin heavy chain transitions of the two portions of the temporalis muscle.     

Postnatal transitions in myosin heavy chain isoforms of the rabbit superficial masseter and digastric muscle

We investigated the early (< 8 weeks) and late (> 8 weeks) postnatal development of the fibre type composition and fibre cross-sectional area in the superficial masseter and digastric muscle of male rabbits. It was hypothesized, first, that due to the transition between suckling and chewing, during early postnatal development the increase in the proportion of slow fibre types and in fibre cross-sectional areas would be larger in the masseter than in the digastric; and second, that due to the supposed influence of testosterone during late postnatal development, the proportion of slow fibre types in both muscles would decrease. Fibre types were classified by immunostaining according to their myosin heavy chain (MyHC) content. The proportion of slow fibre types significantly increased in the masseter, from 7% at week 1 to 47% at week 8, and then decreased to 21% at week 20, while in the digastric it increased from 5% in week 1 to 19% at week 8 and remained the same thereafter. The changes in the proportion of fast fibre types were the opposite. The remarkable increase and decrease in the proportion of slow fibre types in the masseter was attributed predominantly to MyHC-cardiac alpha fibres. During early development, the cross-sectional area of all fibres in both muscles increased. However, only the fast fibre types in the masseter continued to grow further after week 8. Before weaning, the fast fibre types in the digastric were larger than those in the masseter, but after week 8, they became larger in the masseter than in the digastric. In adult animals, masseter and digastric had the same percentage of fast fibre types, but these fibres were almost twice as large in masseter as in digastric.     

Differential modification of myosin heavy chain expression by tenotomy in regenerating fast and slow muscles of the rat

We have examined the effect of tenotomy on the expression of myosin heavy chains (MyHC) in regenerating fast and slow skeletal muscles. Degeneration/regeneration of the left soleus and plantaris of Wistar male rats was induced by an injection into the muscle belly of a myotoxin (snake venom: Notechis scutatus scutatus). MyHC isoform content of regenerating plantaris and soleus muscles were studied 21 days after muscle injury using an electrophoretic technique. Tenotomy of the regenerating plantaris (mechanical underload) did not alter its MyHC expression (P > 0.05). In contrast, tenotomy of the regenerating soleus increased its relative levels of MyHC-2b (P < 0.05) and MyHC-2x/d (P < 0.01), and decreased its relative level of MyHC-1 (P < 0.01). Tenotomy of the synergistic gastrocnemius (overload) tended to decrease the relative level of MyHC-2b in regenerating plantaris (P < 0.07). The effect of tenotomy of the synergistic gastronecmius on the regenerating soleus was different: a decrease in the relative levels of MyHC-1 (P < 0.05) and an increase in the relative level of MyHC-neonatal (P < 0.01). In conclusion, and in contrast to a regenerating slow muscle, a change of mechanical loading by tenotomy did not seem to markedly alter the expression of mature MyHC phenotype in a fast regenerating muscle.     

Evidence for differential post-translational modifications of slow myosin heavy chain during murine skeletal muscle development

The contractile properties of muscle fibres are, in part, determined by the myosin heavy chain (MyHC) isoforms they express. Using monoclonal antibodies, we show that at least three forms of slow twitch MyHC accumulate sequentially during mouse fetal development and that slow MyHC maturation in slow fibres occurs before expression of the adult fast MyHCs in fast fibres. Expression of deletion derivatives of -cardiac MyHC cDNA shows that the slow MyHC epitopes that are detected in adult but not in young animals are located near the N-terminus. The same N-terminal region of various fast MyHC molecules contains a conserved epitope that can, on occasions, be observed when slow MyHC cDNA is expressed in non-muscle cells. The results raise the possibility that the N-terminal epitopes result from post-translational modification of the MyHC and that a sequence of slow and fast MyHC isoform post-translational modifications plays a significant role during development of murine muscle fibres.     

Protein kinase C activity regulates slow myosin heavy chain 2 gene expression in slow lineage skeletal muscle fibers.

Expression of the slow myosin heavy chain (MyHC) 2 gene defines slow versus fast avian skeletal muscle fiber types. Fetal, or secondary, skeletal muscle fibers express slow MyHC isoform genes in developmentally regulated patterns within the embryo, and this patterning is at least partly dependent on innervation in vivo. We have previously shown that slow MyHC 2 gene expression in vitro is regulated by a combination of innervation and cell lineage. This pattern of gene expression was indistinguishable from the pattern observed in vivo in that it was restricted to innervated muscle fibers of slow muscle origin. We show here that slow MyHC 2 gene expression in the slow muscle fiber lineage is regulated by protein kinase C (PKC) activity. Inhibition of PKC activity induced slow MyHC 2 gene expression, and the capacity to express the slow MyHC 2 gene was restricted to muscle fibers of slow muscle (medial adductor) origin. Fast muscle fibers derived from the pectoralis major did not express significant levels of slow MyHC 2 with or without inhibitors of PKC activity. This differential expression pattern coincided with different inherent PKC activities in fast versus slow muscle fiber types. Furthermore, over-expression of an unregulated PKCalpha mutant suppressed slow MyHC 2 gene expression in muscle fibers of the slow lineage. Lastly, denervation of skeletal muscles caused an increase in PKC activity, particularly in the slow medial adductor muscle. This increase in PKC activity was associated with lack of slow MyHC 2 gene expression in vivo. These results provide a mechanistic link between innervation, an intracellular signaling pathway mediated by PKC, and expression of a muscle fiber type-specific contractile protein gene. Dev Dyn 1999;216:177-189.     

Identification of alpha-cardiac myosin heavy chain mRNA and protein in extraocular muscle of the adult rabbit

Summary Extraocular muscles contain both fast-twitch and multiply-innervated, tonic-contracting fibres. In rat, these fibres collectively express numerous myosin heavy chain isoforms including fast-type embryonic and neonatal, adult slow twitch type I and fast twitch type II, and a fast isoform unique to extraocular muscle. Immunocytochemical and Western blotting results are presented which suggest that, in rabbit, an additional species, the -cardiac myosin heavy chain, is present. The immunoreactive species is found in all rabbit extraocular muscles and in the rotatory extraocular muscles is expressed in almost all fibres which do not contain a fast myosin heavy chain. Positive identification of this isoform as the -cardiac myosin heavy chain was obtained by sequencing a cloned PCR product derived from extraocular muscle mRNA unique to the 3-end of rabbit -cardiac myosin heavy chain mRNA. This is the first unequivocal demonstration of -cardiac myosin heavy chain expression in extraocular muscle.     

Changes in the distribution of slow skeletal myosin heavy chain SM1 in developing avian muscle fibres

Summary The use of monoclonal antibodies against fast skeletal and slow skeletal myosin heavy chains (MHC) has shown the presence of significant amounts of slow skeletal type MHC in embryonic skeletal muscles of white leghorn chickens. The presence of this slow skeletal myosin heavy chain (SMHC) was not restricted to presumptive slow muscles only, as it was also observed in presumptive fast skeletal muscles. As was the case for embryonic MHC reactive with the antibody against fast skeletal myosin heavy chain (FMHC), the presence of SMHC could be detected at the earliest stages of myogenesis. It appeared to be present in most muscle cells during early embryonic development. The changes in its cellular distribution during subsequent embryonic and post-hatch period indicated its suppression in a certain proportion of the cells in both presumptive fast and slow skeletal muscles. Its time course of suppression, however, was much prolonged, not synchronized, and varied in fast and slow skeletal muscles during both embryonic and post-hatch development.     

Stretch activation and myosin heavy chain isoforms of rat,rabbit and human skeletal muscle fibres

The underlying mechanism of stretch-induced delayed force increase (stretch activation) of activated muscles is unknown. To assess the molecular correlate of this phenomenon, we measured stretch activation of single, Ca2+-activated skinned muscle fibres from rat, rabbit and the human and analysed their myosin heavy chain complement by SDS gradient gel electrophoresis. Stretch activation kinetics was found to be closely correlated with the myosin heavy chain isoform complement (I, IIa, IId/x and IIb). In hybrid fibres containing two myosin heavy chain isoforms (especially IId and IIb), the kinetics of stretch activation depended on the percentage distribution of the two isoforms. Muscle fibres of the same type but originating from different mammalian species exhibited similar kinetics of stretch activation. Considering the differing unloaded shortening velocities of these fibres, the time-limiting factors for stretch activation and unloaded shortening velocity appear not to be the same. The stretch activation kinetics of the fibre types IIB, IID and IIA more likely seemed to follow a Normal Gaussian distribution than that of type I fibres. Several type I fibres had extraordinarily slow kinetics. This observation corroborates biochemical data indicating the possible existence of more than one slow myosin heavy chain isoform     

Maximum shortening velocity and coexistence of myosin heavy chain isoforms in single skinned fast fibres of rat skeletal muscle

Summary Myosin heavy chain composition of a large number (288) of single fibres from slow (soleus), and fast (superficial part of tibialis anterior, and plantaris) muscles of adult (3–5-month-old) Wistar rats was determined. A combination of SDS-PAGE and monoclonal antibodies against myosin heavy chains allowed to identify four myosin heavy chain isoforms (1, 2A, 2X, and 2B) and to detect myosin heavy chain coexistence. Four groups of fibres containing only one myosin heavy chain (1 myosin heavy chain, 2A myosin heavy chain, 2X myosin heavy chain, and 2B myosin heavy chain), and five groups containing more than one myosin heavy chain 1 and 2A myosin heavy chains, 2A and 2X myosin heavy chains, 2X and minor amounts of 2B (2X-2B fibres), 2B and minor amounts of 2X (2B-2X fibres), and 2A, 2X, and 2B myosin heavy chain were identified and their relative percentages were assessed. Coexistence of fast myosin heavy chain isoforms was found to be very frequent (50% of the fibres in plantaris, and 30% in tibialis anterior), whereas coexistence of slow and fast (2A) myosin heavy chain was very rare. Maximum shortening velocity (V₀) was determined using the slack-test procedure in a subset of 109 fast fibres from the above population. The values of V₀ formed a continuum extending from 2A to 2X to 2X-2B to 2B-2X to 2B fibres. 2A fibres had the lowest value of V₀ and 2B fibres the highest. Only the differences between 2A and 2B and 2A and 2B-2X fibres were statistically significant. Importantly, the variability of V₀ in fibres containing only one myosin heavy chain and in fibres containing a variable proportion of two myosin heavy chain isoforms was similar and, in some case (e.g. 2B fibres), such to encompass the whole range of variation of fast fibres shortening velocities. The results of this study demonstrate that the large variability in maximum shortening velocity of fast fibres is not due to myosin heavy chain coexistence, and therefore suggest that it cannot be explained on the basis of myosin heavy chain composition.     

Myosin heavy chain expression in zebrafish and slow muscle composition.

In the zebrafish embryo, two distinct classes of muscle fibers have been described in the forming myotome that arise from topographically separable precursor populations. Based entirely on cross-reactivity with antibodies raised against mammalian and chick myosin heavy chain isoforms slow twitch muscle has been shown to arise exclusively from "adaxial" myoblasts, which migrate from their origin flanking the notochord to form a single layer of subcutaneous differentiated muscle cells. The remainder of the myotome differentiates behind this migration as muscle fibers recognized by anti-fast myosin heavy chain (MyHC) antibodies. To identify unambiguous molecular markers of cell fate in the myotome, we have characterized genes encoding zebrafish fast and slow MyHC. Using phylogenetic and expression analysis, we demonstrate that these genes are definitive molecular markers of slow and fast twitch fates. We also demonstrate that zebrafish embryonic slow twitch muscle co-expresses both slow and fast twitch MyHC isoforms, a property that they share with primary fibers of the amniote myotome.