Development of muscle fiber specialization in the rat hindlimb.

The appearance of fast and slow fiber types in the distal hindlimb of the rat was investigated using affinity-purified antibodies specific to adult fast and slow myosins, two-dimensional electrophoresis of myosin light chains, and electron microscope examination of developing muscle cells. As others have noted, muscle histogenesis is not synchronous; rather, a series of muscle fiber generations occurs, each generation forming along the walls of the previous generation. At the onset of myotube formation on the 15th d of gestation, the antimyosin antibodies do not distinguish among fibers. All fibers react strongly with antibody to fast myosin but not with antibody to slow myosin. The initiation of fiber type differentiation can be detected in the 17-d fetus by a gradual increase in the binding of antibody to slow myosin in the primary, but not the secondary, generation myotubes. Moreover, neuromuscular contacts at this crucial time are infrequent, primitive, and restricted predominantly, but not exclusively, to the primary generation cells, the same cells which begin to bind large amounts of antislow myosin at this time. With maturation, the primary generation cells decrease their binding of antifast myosin and become type I fibers. Secondary generation cells are initially all primitive type II fibers. In future fast muscles the secondary generation cells remain type II, while in future slow muscles most of the secondary generation cells eventually change to type I over a prolonged postnatal period. We conclude that the temporal sequence of muscle development is fundamentally important in determining the genetic expression of individual muscle cells.

Slow myosin in developing rat skeletal muscle.

Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC synthesis. Conversely, the content of slow MHC rises in the neonatally denervated EDL. This suggests that as the nerve amplifies fast MHC accumulation in the developing EDL, accumulation of slow MHC is inhibited in an antithetic fashion. Studies with phenylthiouracil-induced hypothyroidism indicate that inhibition of slow MHC accumulation in the EDL and AT is not initially under thyroid regulation. At later stages, the development of thyroid function plays a role in inhibiting slow MHC accumulation in the differentiating EDL and AT.(ABSTRACT TRUNCATED AT 400 WORDS)

Sulfhydryl groups of native myosin and of the myosin heavy chains from Physarum polycephalum compared to vertebrate skeletal, smooth, and non-muscle myosins.

Although a previously reported analysis of Physarum myosin detected no cysteine residues in the molecule, the myosin ATPase activity was inhibited by p-chloromercuribenzoate. We have re-examined this apparently contradictory finding. We found highly purified plasmodial myosin to be very sensitive to N-ethylmaleimide inhibition of the K+, Ca2+ -activated ATPase. An estimate of the number of reactive sulfhydryls of the native myosin using Ellman's reagent showed only 1.5 mol 11 min-reactive sulfhydryl/mol as compared to 4.5 for chicken breast myosin in 5 min. 3H- and 14C-labelled N-ethylmaleimide was used to estimate the total sulfhydryls of the SDS-denatured heavy chains. Plasmodial myosin heavy chains bound 10-13% of the N-ethylmaleimide bound by chicken breast myosin heavy chains. Smooth muscle myosin heavy chains as well as heavy chains of embryonic chicken presumptive myoblasts had 65-70% of the reactive groups of chicken myotube myosin heavy chains. Amino acid analyses of purified Physarum myosin showed that some preparations contained cysteic acid residues even before performic oxidation. After the performic oxidation a mean value of 3 mol cysteic acid per 10(5) g Physarum myosin was found, or less than half that reported for striated muscle myosin. Our results show that in the sulfhydryl-poor plasmodial myosin each heavy chain contains at least two sulfhydryls, and probably more, but that there is variable oxidation of the total sulfhydryls. It has been reported that plasmodial myosin lacks rapidly reacting sulfhydryls groups when tested with an ATP analogue which reacts with light chains of vertebrate muscle myosins. Therefore, the 1-2 sulfhydryls of plasmodial myosin which react rapidly with Ellman's reagent appear to be on the heavy chain. Our results also suggest that during development of myotubes changes occur in the myosin heavy chains.

Thyroidal and neural control of myosin transitions during development of rat fast and slow muscles.

Experiments with developing euthyroid, hypothyroid and hyperthyroid rats show that the transition from neonatal to adult fast myosin is orchestrated by thyroid hormones acting directly upon fast muscle cells. Denervation studies reveal the switch from neonatal to adult fast myosin synthesis is independent of the motoneuron. However the synthesis of slow myosin during development is critically dependent on innervation.

Human embryonic myosin heavy chain cDNA. Interspecies sequence conservation of the myosin rod, chromosomal locus and isoform specific transcription of the gene.

A 3.6 kilobase cDNA clone coding for the human embryonic myosin heavy chain has been isolated and characterized from an expression library prepared from human fetal skeletal muscle. The derived amino acid sequence for the entire rod part of myosin shows 97% sequence homology between human and rat and a striking interspecies sequence conservation among the charged amino acid residues. The single copy gene is localized to human chromosome 17 and its expression in fetal skeletal muscle is developmentally regulated. The sequence information permits the design of isoform-specific probes for studies on the structure of the gene and its role in normal and defective human myogenesis.

The distribution of myosin heavy chain isoforms among rat extraocular muscle fiber types.

PURPOSE: To determine the distribution of myosin heavy chain isoforms in each extraocular muscle (EOM) fiber type. METHODS: Serial sections of adult rat EOMs were stained with isoform-specific monoclonal antibodies against an array of myosin heavy chains. Immunofluorescent antibody staining of whole adult rat EOMs, examined by confocal microscopy, demonstrated the longitudinal variations of isoforms along individual fibers. RESULTS: Each global fiber type reacted predominantly with a single isoform-specific antibody and showed no longitudinal variation. Two major orbital fibers were defined, and both contained multiple myosin heavy chains. Both orbital singly and multiply innervated fibers stained proximal and distal to the neuromuscular junction with antibody to embryonic myosin heavy chain, but this isoform was sharply and completely excluded from the domain of the neuromuscular junction. Orbital singly innervated fibers also contained the EOM-specific isoform at the neuromuscular junction. Orbital multiply innervated fibers did not contain the EOM-specific isoform, but additionally contained a slow isoform along their entire length. CONCLUSIONS: Adult rat EOMs show unique fiber types with arrangements of myosin heavy chain isoforms not seen in other skeletal muscles. Moreover, unique cellular mechanisms must exist to target each isoform to its proper domain along individual orbital fibers.

Isoform-specific cDNAs for human embryonic, neonatal, and slow skeletal myosin heavy chains.

A series of cDNA fragments encoding immunodetectable portions of the human slow/beta, neonatal, and embryonic isoforms of myosin heavy chain (MHC) were isolated from a human fetal muscle cDNA expression library. A 6 kb fragment isolated on a secondary screen represents the first cloned cDNA encoding a full-length vertebrate MHC (the human embryonic isoform). In the 3'-untranslated regions, 70-80% nucleotide sequence homology exists among orthologous human and rat cDNAs, whereas the homology is less than 65% among the paralogous cDNAs. Furthermore, approximately the same level of untranslated sequence conservation is observed at the 5'-terminus of the embryonic transcript. These results suggest that for both the 3'- and the 5'-untranslated domains, the rate of evolutionary sequence divergence is limited by functional constraints.

Effect of hypothyroidism on myosin heavy chain expression in rat pharyngeal dilator muscles.

Although the association between hypothyroidism and obstructive sleep apnea is well established, the effect of thyroid hormone deficiency on contractile proteins in pharyngeal dilator muscles responsible for maintaining upper airway patency is unknown. In the present study, the effects of hypothyroidism on myosin heavy chain (MHC) expression were examined in the sternohyoid, geniohyoid, and genioglossus muscles of adult rats (n = 20). The relative proportions of MHC isoforms present were determined using MHC-specific monoclonal antibodies and oligonucleotide probes. All control muscles showed a paucity of type I MHC fibers, with greater than 90% of fibers containing fast-twitch type II MHCs. In the genioglossus muscle, a population of non-IIa non-IIb fast-twitch type II fibers (putatively identified as type IIx MHC fibers) were detected. Hypothyroidism induced significant changes in MHC expression in all muscles studied. In the sternohyoid, type I fibers increased from 6.2 to 16.9%, whereas type IIa fibers increased from 25.9 to 30.7%. Type I fibers in the geniohyoid increased from 1.2 to 12.8%, whereas type IIa fibers increased from 34.1 to 42.7%. The genioglossus showed the smallest relative increase in type I expression but the greatest induction of type IIa MHC. None of the muscles examined demonstrated reinduction of embryonic or neonatal MHC in response to thyroid hormone deficiency. In summary, hypothyroidism alters the MHC profile of pharyngeal dilators in a muscle-specific manner. These changes may play a role in the pathogenesis of obstructive apnea in hypothyroid patients.

Development of neuromuscular specialization.

This review outlines recent advances in understanding the program of muscle specialization during development and discusses some of the controls over this process. The scheme of fiber heterogeneity is laid down very early in myogenesis and, at least in the chicken, it appears to be intrinsically determined by the limb, independent of neuromuscular contact. In mature muscle, specialized fibers are usually intermingled in a mosaic. This results from the pattern of muscle assembly from primary and secondary generation cells as these distinct generations commonly express different phenotypes. The muscle develops in this way as secondary cells use the walls of primary myotubes as a scaffold to support their differentiation. This process may be regulated by transient expression of N CAMs. Myoblasts left after N CAMs are no longer expressed may become the satellite cells of mature muscle. In the rat, primary cells all initially express slow myosin, whereas most secondary cells are fast in phenotype. Post-partum, the initial plan of fiber specialization is modulated. In developing slow muscles many secondary generation fibers convert from a fast to a slow phenotype, whereas in developing fast muscles, primary slow fibers transform to a fast phenotype. Hormones, particularly thyroid hormone, play a significant role in this modification. The role of the nerve is less well understood.

A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle.

A monoclonal antibody, 2B6, has been prepared against the embryonic myosin heavy chain of rat skeletal muscle. On solid phase radioimmunoassay, 2B6 shows specificity to myosin isozymes known to contain the embryonic myosin heavy chain and on immunoblots of denatured contractile proteins and on competitive radioimmunoassay, it reacts only with the myosin heavy chain of embryonic myosin and not with the myosin heavy chain of neonatal or adult fast and slow myosin isozymes or with other contractile or noncontractile proteins. This specificity is maintained with cat, dog, guinea pig, and human myosins, but not with chicken myosins. 2B6 was used to define which isozymes in the developing animal contained the embryonic myosin heavy chain and to characterize the changes in embryonic myosin heavy chain in fast versus slow muscles during development. Finally, 2B6 was used to demonstrate that thyroid hormone hastens the disappearance of embryonic myosin heavy chain during development, while hypothyroidism retards its decrease. This confirmed our previous conclusion that thyroid hormones orchestrate changes in isozymes during development.

Why are fetal muscles slow?

Differentiating fast and slow mammalian muscles contract slowly at birth and increase their speed during the first few weeks of life. However, only small proportions of slow myosin light chains are found in early developing muscles and the fast type of light chains predominate. In addition, differentiating muscle contains unique, embryonic forms of myosin which may partially determine the early slow responses. The present study suggests additional reasons for these slow twitch times. Most skeletal muscles are initially formed from a small population of primary generation cells which are innervated by pioneering axons early in myogenesis. Subsequently, numerous secondary generation cells develop along the walls of primary myotubes, then separate and become independent units of contraction. Using affinity-purified antibodies to fast and slow myosin, it was found that most primary myotubes react with anti-slow myosin and are destined to become slow, Type I fibres. By contrast, secondary generation cells stain exclusively with anti-fast myosin and develop into Type II, fast fibres. We propose that primary myotubes constitute the fundamental motor units of the developing neuromuscular system and are responsible for early slow movements. Secondary generation cells become organized into large, fast motor units later in development, eclipsing the original slow response.

Hormonal control of myosin heavy chain genes during development of skeletal muscles.

A pattern of myosin heavy chain (MHC) switching is a hallmark of developing muscles. Factors responsible for these changes in gene expression include endogenous signals, motoneurons and hormones, especially thyroid hormones. After perturbing the innervation and/or thyroid hormone levels we have examined the neonatal-IIb MHC transition during rat development. First, denervation does not qualitatively affect the transition at either the transcriptional or translational level. Second, hypothyroidism prevents the appearance of IIb MHC and its mRNA in the innervated limb; in the denervated hypothyroid limb IIb MHC is synthesized at moderately high levels. Third, hyperthyroidism causes a precocious increase in IIb MHC in both innervated and denervated muscles. These results suggest that the transition from neonatal to adult IIb myosin synthesis is endogenously programmed during development, but is closely orchestrated by the changing neuronal and hormonal status of the animal. Thyroid hormone may exert its influence by effects both on the muscle fibre and on the developing motoneuron. In the guinea-pig the temporalis muscle is sexually dimorphic: it contains a fast-red MHC in the female but a fast-white MHC in the male. This dimorphism has been shown to be mediated by testosterone, since the castrated male synthesizes the fast-red MHC while the testosterone-supplemented female contains the fast-white MHC. During development male and female muscles initially synthesize the fast-red isoform. The male switches to the fast-white form at puberty.

Sexual dimorphism in the fibers of a "clasp" muscle of Xenopus laevis.

When antibodies specific to fast-twitch, slow-twitch, and slow-tonic myosins were used to stain the clasp muscle, m. sternoradialis, of Xenopus laevis, three predominant fiber types were identified in both males and females. Three fiber types can also be distinguished by the diameters and conduction velocities of their motor nerves. Significant differences in the numbers of slow-tonic fibers were identified between the genders and between control and castrated male animals. This finding suggests that these slow-tonic fibers, which probably dominate the clasp reflex during mating, may be under hormonal control.

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

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 less than 0.001) in the soleus and by 27% (p less than 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 less than 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 less than 0.0001) and a 50% decrease in the number of slow fibres (p less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Testosterone-induced changes in contractile protein isoforms in the sexually dimorphic temporalis muscle of the guinea pig.

The guinea pig temporalis muscle is sexually dimorphic, classified histochemically as a fast-red muscle in the female, but as a fast-white muscle in the adult male. Since this sexual difference in metabolic properties is related to plasma testosterone levels, we asked if testosterone also affected the contractile protein isoforms. In the newborn guinea pig, both male and female temporalis muscles contained a fast-red isoform of the myosin heavy chain and approximately equal amounts of alpha- and beta-tropomyosins. At puberty, the male began to replace the fast-red isoform with a fast-white isoform of the heavy chain and by 120 days the muscle contained predominantly the fast-white myosin heavy chain. This transition in myosins in the male was accompanied by a shift to greater than 90% alpha-tropomyosin. No changes in myosins or tropomyosins were observed in the female. The changes in the male could be reversed by castration and could be mimicked in the female by the injection of testosterone. Although these same myosins and tropomyosins could be detected in other fast-twitch muscles, postpartum transitions in contractile protein isoforms in those muscles were testosterone-insensitive, and no sexual dimorphism of these proteins was seen in other muscles.

Myosin types during the development of embryonic chicken fast and slow muscles.

We have studied the myosin types present in developing fast and slow muscles of the chicken embryo. Myosin light chains were characterized by their mobility on sodium dodecyl sulfate/polyacrylamide gels; myosin heavy chains were identified by their reaction with antibodies specific for adult fast or adult slow myosin heavy chains. During development, the pectoralis muscle, a fast muscle in the adult, contains heavy chains and two of the three light chains characteristic of adult fast muscle myosin. However, the anterior latissimus dorsi muscle, a slow muscle in the adult, also contains fast myosin light and heavy chains during early development. Only after the time of innervation does this muscle begin synthesizing predominantly the slow myosin heavy and light chains. We hypothesize that the synthesis of fast myosin in both early fast and slow muscles is the result of the endogenous program for muscle development; initiation of the synthesis of slow myosin, however, is dependent upon exogenous factors.

Myosin transitions in chronic stimulation do not involve embryonic isozymes.

Chronic low-frequency stimulation of a fast skeletal muscle effects a transition of myosin isozymes from those characteristic of a fast muscle to those characteristic of a slow muscle. This transformation involves changes in both myosin heavy and light chains. During development, muscles usually change directly from embryonic to neonatal to fast or from embryonic to neonatal to slow isozymes. We have questioned whether chronic stimulation of the adult fast muscle results in a direct fast-to-slow isozyme shift or whether transformation requires reexpression of the developmental isozymes prior to the synthesis of adult slow isozymes. We have examined these alternatives in the chronically stimulated dog diaphragm using adenosine triphosphatase (ATPase) histochemistry, pyrophosphate gel electrophoresis of native isozymes, peptide mapping of myosin heavy chains, immunoblotting with an antibody specific to embryonic myosin heavy chain, and solid-phase radioimmunoassay. We have demonstrated that a transition from adult fast to adult slow isozymes during chronic stimulation does not involve a recapitulation of embryonic isozymes.

Histochemical and fatigue characteristics of conditioned canine latissimus dorsi muscle.

To induce fatigue resistance in the latissimus dorsi muscle of the dog in preparation for possible myocardial assistance, eight adult male beagles underwent unilateral electrical stimulation of the thoracodorsal nerve at a frequency of 2 Hz (120 stimuli/min) and 10 Hz (600 stimuli/min) for a 6-week period. The conditioned muscles were compared with their unconditioned contralateral controls by fiber typing, pyrophosphate gel electrophoresis, isometric characteristics, and fatigue rates. At the end of the period of stimulation, the conditioned muscles had a greater percentage of slow-twitch, fatigue-resistant fibers on acid and alkaline stains (100 +/- 0.7% and 83 +/- 15.3%), respectively, than did their contralateral controls (45 +/- 7.6% and 43 +/- 7.0). Pyrophosphate gel electrophoresis revealed an increase in the slow myosin and a decrease in the fast myosin content in the conditioned muscles; the stimulated muscles also demonstrated a slower contraction time (87 +/- 20 msec vs. 57 +/- 17.9 msec), a lower initial tension (4.4 +/- 1.45 kg vs. 7.2 +/- 2.11 kg), and a slower fatigue rate during a 30-minute fatigue test than did their contralateral controls. The muscles stimulated at 2 Hz had fatigue rates similar to those stimulated at 10 Hz, but generally had less diminution in muscle fiber diameters and less interfiber connective tissue. Thus, it is possible to make canine latissimus dorsi muscles more fatigue resistant, and, theoretically, more capable of myocardial assistance by electrical stimulation of the thoracodorsal nerve at a frequency as low as 2 Hz--the natural canine heart rate.

Up-regulation of type XIX collagen in rhabdomyosarcoma cells accompanies myogenic differentiation.

Rhabdomyosarcomas are known to recapitulate some of the early events in skeletal muscle embryogenesis, and cultures derived from these tumors have been extensively used to elucidate processes associated with the differentiation of primitive mesenchymal cells. These neoplasms have also provided important systems for studying different collagen types. This aspect is particularly relevant to type XIX collagen, which was originally identified from rhabdomyosarcoma cDNA clones. Although this collagen has been localized in vivo to basement membrane zones in a wide variety of tissues, including skeletal muscle, the tumor cells appear to be a unique source of its expression in vitro. We have found that one particular cell line-derived from a peritesticular embryonal rhabdomyosarcoma-produced relatively large amounts of type XIX collagen, especially in those rare instances in which these cells appear to spontaneously differentiate. To characterize this phenomenon, tumor cells were grown under conditions known to induce differentiation in normal myoblast cultures. In response to this treatment, the typical tumor cell morphology consistently and reproducibly switched from polygonal to round/spindle-shaped with the subsequent appearance of some structures resembling myotubes. Concurrently, the cultures commenced a dramatic up-regulation of type XIX collagen and skeletal muscle myosin heavy chain and alpha-actinin in a time-dependent fashion, whereas protein and mRNA levels of other matrix proteins were either decreased or unchanged. Moreover, immunocytochemical analysis revealed that only a subpopulation of the cells was responsible for the increased synthesis of type XIX collagen, alpha-actinin, and myosin, and that the same cells which stained positive for the collagen also stained positive for the muscle proteins. Taken together, the results suggested that type XIX collagen may be involved in the initial stages of skeletal muscle cell differentiation.