Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.     

Neural control of the sequence of expression of myosin heavy chain isoforms in foetal mammalian muscles

The expression of myosin isoforms was studied during development of calf muscles in foetal and neonatal rats, using monoclonal antibodies against slow, embryonic and neonatal isoforms of myosin heavy chain (MHC). Primary myotubes had appeared in all prospective rat calf muscles by embryonic day 16 (E16). On both E16 and E17, primary myotubes in all muscles with the exception of soleus stained for slow, embryonic and neonatal MHC isoforms; soleus did not express neonatal MHC. In earlier stages of muscle formation staining for the neonatal isoform was absent or faint. Secondary myotubes were present in all muscles by E18, and these stained for both embryonic and neonatal MHCs, but not slow. In mixed muscles, primary myotubes destined to differentiate into fast muscle fibres began to lose expression of slow MHC, and primary myotubes destined to become slow muscle fibres began to lose expression of neonatal MHC. This pattern was further accentuated by E19, when many primary myotubes stained for only one of these two isoforms. Chronic paralysis or denervation from E15 or earlier did not disrupt the normal sequence of maturation of primary myotubes up until E18, but secondary myotubes did not form. By E19, however, most primary myotubes in aneural or paralyzed tibialis anterior muscles had lost expression of slow MHC and expressed only embryonic and neonatal MHCs. Similar changes occurred in other muscles, except for soleus which never expressed neonatal MHC, as in controls. Paralysis or denervation commencing later than E15 did not have these effects, even though it was initiated well before the period of change in expression of MHC isoforms. In this case, some secondary myotubes appeared in treated muscles. Paralysis initiated on E15, followed by recovery 2 days later so that animals were motile during the period of change in expression of MHC isoforms, was as effective as full paralysis. These experiments define a critical period (E15-17) during which foetuses must be active if slow muscle fibres are to differentiate during E19-20. We suggest that changes in expression of MHC isoforms in primary myotubes depend on different populations of myoblasts fusing with the myotubes, and that the normal sequence of appearance of these myoblasts has a stage-dependent reliance on active innervation of foetal muscles. A critical period of nerve-dependence for these myoblasts occurs several days before their action can be noted.     

Emergence of the mature myosin phenotype in the rat diaphragm muscle

Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2X and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in approximately 27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day greater than or equal to 115, less than 4% of the myofibers expressed MHC2B). Fibers which contained MHC beta/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHC beta/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was greater than or equal to 115 days old.     

The developmental program of fast myosin heavy chain expression in avian skeletal muscles

We have examined the types of fast myosin heavy chains (MHCs) expressed in a number of different developing chicken skeletal muscles by combining peptide mapping and immunoblotting to identify fast MHC-specific peptides among the total mixture of MHC digestion products. Using this technique, we have identified three different fast MHC patterns among the different fast and mixed (i.e., fast and slow) fiber type muscles of the adult. While the different muscles all underwent sequential changes in fast MHC isoform expression during their development, the exact sequence of these changes and the isoform patterns expressed varied from muscle to muscle. During late embryonic or fetal development, all muscles expressed a similar fast MHC pattern (designated here as the fetal pattern) which was replaced shortly after hatching with a different fast MHC pattern (the neonatal pattern). During the transition from the neonatal to the adult state that occurred sometime in the first year after hatching, many of the muscles underwent additional changes in fast MHC isoform expression. In muscles such as the pectoralis major and pectoralis minor, a new fast MHC isoform pattern was seen in the adult so that the developmental program of isoform switching in these muscles involved the sequential appearance of distinct fetal, neonatal, and adult fast MHCs. Other muscles, such as the sartorius and posterior latissimus dorsi, underwent a qualitatively different program of isoform switching and expressed as an adult a fast MHC pattern that was indistinguishable from that expressed during fetal development. Finally, in some muscles, such as the superficial biceps, no change in isoform pattern was detected during the neonatal to adult transition--in these muscles, expression of the neonatal MHC isoform pattern apparently persisted into the adult state. These data indicate that no single scheme or program of fast MHC isoform switching can describe all the developmental changes that occur in fast MHC isoform expression in the chicken and that at least three different programs of isoform switching and expression can be identified.     

Subunit composition of rodent isomyosins and their distribution in hindlimb skeletal muscles

Three adult skeletal muscle sarcomeric myosin heavy chain (MHC) genes have been identified in the rat, suggesting that the expressed native myosin isoforms can be differentiated, in part, on the basis of their MHC composition. This study was undertaken to ascertain whether the five major native isomyosins [3 fast (Fm1, Fm2, Fm3), 1 slow (Sm), and 1 intermediate (Im)], typically expressed in the spectrum of adult rat skeletal muscles comprising the hindlimb, could be further differentiated on the basis of their MHC profiles in addition to their light chain composition. Results show that in muscles comprised exclusively of fast-twitch glycolytic (FG) fibers and consisting of Fm1, Fm2, and Fm3, such as the tensor fasciae latae, only one MHC, designated as fast type IIb, could be resolved. In soleus muscle, comprised of both slow-twitch oxidative and fast-twitch oxidative-glycolytic fibers and expressing Sm and Im, two MHC bands were resolved and designated as slow/cardiac beta-MHC and fast type IIa MHC. In muscles expressing a mixture of all three fiber types and a full complement of isomyosins, as seen in the plantaris, the MHC could be resolved into three bands. Light chain profiles were characterized for each muscle type, as well as for the purified isomyosins. These data suggest that Im (IIa) consists of a mixture of fast and slow light chains, whereas Fm (IIb) and Sm (beta) isoforms consist solely of fast- and slow-type light chains, respectively. Polypeptide mapping of denatured myosin extracted from muscles expressing contrasting isoform phenotypes suggests differences in the MHC primary structure between slow, intermediate, and fast myosin isotypes. These findings demonstrate that 1) Fm, Im, and Sm isoforms are differentiated on the bases of both their heavy and light chain components and 2) each isomyosin is distributed in a characteristic fashion among rat hindlimb skeletal muscles. Furthermore, these data suggest that the ratio of isomyosins in a given muscle or muscle region is of physiological importance to the function of that muscle during muscular activity.     

Temporal and tissue-specific expression of myosin heavy chain isoforms in developing and adult avian muscle

We have raised monoclonal antibodies (Mabs) to myosin heavy chain isoforms (MHCs) that have specific patterns of temporal expression during the development of quail pectoral muscle and that are expressed in very restricted, tissue-specific patterns in adult birds. We find that an early embryonic, a perinatal, and an adult-specific, fast myosin heavy chain are co-expressed at different levels in the pectoral muscle of 8-12 day quail embryos. The early embryonic MHC disappears from the pectoral muscle at approximately 14 days in ovo, whereas the perinatal MHC persists until 26 days post-hatching. The adult-specific MHC accumulates preferentially and eventually completely replaces the other isoforms. These Mabs cross-react with the homologous isoforms of the chick and detect a similar pattern of MHC expression in the pectoral muscle of developing chicks. Although the early embryonic and perinatal MHC isoforms recognized by our Mabs are expressed in the pectoral muscle only during distinct developmental stages, our Mabs also recognize MHC isoforms present in the heart and extraocular muscle of adult quail. Immunofingerprinting using Staphylococcus aureus protease V8 suggests that the early embryonic and perinatal MHC isoforms that we see are strongly homologous with the adult ventricular and extraocular muscle isoforms, respectively. These observations suggest that at least three distinct MHC isoforms, which are normally expressed in adult muscles, are co-expressed during the early development of the pectoral muscle in birds. In this respect, the pattern of expression of the MHCs recognized by our Mabs in developing, fast muscle is very similar to the patterns described for other muscle contractile proteins.     

Expression of type-specific MHC isoforms in rat intrafusal muscle fibers.

Myosin heavy chain (MHC) expression by intrafusal fibers was studied by immunocytochemistry to determine how closely it parallels MHC expression by extrafusal fibers in the soleus and tibialis anterior muscles of the rat. Among the MHC isoforms expressed in extrafusal fibers, only the slow-twitch MHC of Type 1 extrafusal fibers was expressed along much of the fibers. Monoclonal antibodies (MAb) specific for this MHC bound to the entire length of bag2 fibers and the extracapsular region of bag1 fibers. The fast-twitch MHC isoform strongly expressed by bag2 and chain fibers had an epitope not recognized by MAb to the MHC isoforms characteristic of developing muscle fibers or the three subtypes (2A, 2B, 2X) of Type 2 extrafusal fibers. Therefore, intrafusal fibers may express a fast-twitch MHC that is not expressed by extrafusal fibers. Unlike extrafusal fibers, all three intrafusal fiber types bound MAb generated against mammalian heart and chicken limb muscles. The similarity of the fast-twitch MHC of bag2 and chain fibers and the slow-tonic MHC of bag1 and bag2 fibers to the MHC isoforms expressed in avian extrafusal fibers suggests that phylogenetically primitive MHCs might persist in intrafusal fibers. Data are discussed relative to the origin and regional regulation of MHC isoforms in intrafusal and extrafusal fibers of rat hindlimb muscles.     

Distribution of fast myosin heavy chain isoforms in thick filaments of developing chicken pectoral muscle

Colloidal gold-conjugated monoclonal antibodies were prepared to stage-specific fast myosin heavy chain (MHC) isoforms of developing chicken pectoralis major (PM). Native thick filaments from different stages of development were reacted with these antibodies and examined in the electron microscope to determine their myosin isoform composition. Filaments prepared from 12-d embryo, 10-d chick, and 1-yr chicken muscle specifically reacted with the embryonic (EB165), neonatal (2E9), and adult (AB8) antimyosin gold-conjugated monoclonal antibodies, respectively. The myosin isoform composition was more complex in thick filaments from stages of pectoral muscle where more than one isoform was simultaneously expressed. In 19-d embryo muscle where both embryonic and neonatal isoforms were present, three classes of filaments were found. One class of filaments reacted only with the embryonic antibody, a second class reacted only with the neonatal-specific antibody, and a third class of filaments were decorated by both antibodies. Similar results were obtained with filaments prepared from 44-d chicken PM where the neonatal and adult fast MHCs were expressed. These observations demonstrate that two myosin isoforms can exist in an individual thick filament in vivo. Immunoelectron microscopy was also used to determine the specific distribution of different fast MHC isoforms within individual filaments from different stages of development. The anti-embryonic and anti-adult antibodies uniformly decorated both homogeneous and heterogeneous thick filaments. The neonatal specific antibody uniformly decorated homogeneous filaments; however, it preferentially decorated the center of heterogeneous filaments. These observations suggest that neonatal MHC may play a specific role in fibrillogenesis.     

Developmental patterns of expression and coexpression of myosin heavy chains in atria and ventricles of the avian heart

Monoclonal antibodies (mAbs), electrophoresis, immunoblotting, and immunohistochemistry were used to determine the molecular properties of cardiac myosin heavy chain (MHC) isoforms and the regions of the developing chicken heart in which they were expressed. Adult atria expressed three electrophoretically distinct MHCs that reacted specifically with mAbs F18, F59, or S58. During embryonic Days 2-4, when the atrial and ventricular chambers are forming, MHCs that reacted with mAbs F18, F59, or S58 were expressed in both the atria and ventricles. The atria continued to express MHCs that reacted with mAbs F18, F59, or S58 at all stages of development and in the adult. In the ventricles, expression of the MHCs reacting with these mAbs was found to be developmentally regulated. By embryonic Day 16, MHC(s) reacting with mAb F18 had disappeared from the developing ventricles, whereas MHCs reacting with S58 and F59 continued to be expressed throughout the ventricles. As development continued, MHC(s) reacting with S58 in the ventricle became restricted to expression in only the ventricular conducting system. MHC(s) reacting with F59 were expressed in both the ventricular myocytes and the ventricular conducting system throughout development and in the adult. Thus, in contrast to the embryonic chicken heart where at least three MHC isoforms were expressed in both the atria and ventricles, we found in the adult chicken heart that-at a minimum-three MHC isoforms were expressed in the atria, two MHC isoforms were expressed in the ventricular conducting system, and one MHC isoform in the ventricular myocardium. MHC isoform expression in the developing avian heart appears to be more complex than previously recognized.     

Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian embryogenesis

We have utilized a key biochemical determinant of muscle fiber type, myosin isoform expression, to investigate the initial developmental program of future fast and slow skeletal muscle fibers. We examined myosin heavy chain (HC) phenotype from the onset of myogenesis in the limb bud muscle masses of the chick embryo through the differentiation of individual fast and slow muscle masses, as well as in newly formed myotubes generated in adult muscle by weight overload. Myosin HC isoform expression was analyzed by immunofluorescence localization with a battery of anti-myosin antibodies and by electrophoretic separation with SDS-PAGE. Results showed that the initial myosin phenotype in all skeletal muscle cells formed during the embryonic period (until at least 8 days in ovo) consisted of expression of a myosin HC which shares antigenic and electrophoretic migratory properties with ventricular myosin and a distinct myosin HC which shares antigenic and electrophoretic migratory properties with fast skeletal isomyosin. Similar results were observed in newly formed myotubes in adult muscle. Future fast and slow muscle fibers could only be discriminated from each other in developing limb bud muscles by the onset of expression of slow skeletal myosin HC at 6 days in ovo. Slow skeletal myosin HC was expressed only in myotubes which became slow fibers. These findings suggest that the initial commitment of skeletal muscle progenitor cells is to a common skeletal muscle lineage and that commitment to a fiber-specific lineage may not occur until after localization of myogenic cells in appropriate premuscle masses. Thus, the process of localization, or events which occur soon thereafter, may be involved in determining fiber type.     

Skeletal muscle satellite cells appear during late chicken embryogenesis.

The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult 12-16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.     

Immunochemical analysis of protein isoforms in thick myofilaments of regenerating skeletal muscle

The expression of myosin heavy chain (MHC) and C-protein isoforms has been examined immunocytochemically in regenerating skeletal muscles of adult chickens. Two, five, and eight days after focal freeze injury to the anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) muscles, cryostat sections of injured and control tissues were reacted with a series of monoclonal antibodies previously shown to specifically bind MHC or C-protein isoforms in adult or embryonic muscles. We observed that during the course of regeneration in each of these muscles there was a reproducible sequence of antigenic changes consistent with differential isoform expression for these two proteins. These isoform switches appear to be tissue specific; i.e., the isoforms of MHC and C-protein which are expressed during the regeneration of a "slow" muscle (ALD) differ from those which are synthesized in a regenerating "fast" muscle (PLD). Evidence has been obtained for the transient expression of a "fast-type" MHC and C-protein during ALD regeneration. Furthermore, during early stages of PLD regeneration this muscle contains MHCs which antigenically resemble those found in the pectoralis muscle at embryonic and early posthatch stages of development. Both regenerating muscles express an isoform of C-protein which appears immunochemically identical to that normally expressed in embryonic and adult cardiac muscle. These results support the concept that isoform transitions in regenerating skeletal muscles qualitatively resemble those found in developing muscles but differences may exist in temporal and tissue-specific patterns of gene expression.     

Development of chicken intrafusal muscle fibers

The first sign of developing intrafusal fibers in chicken leg muscles appeared on embryonic day (E) 13 when sensory axons contacted undifferentiated myotubes. In sections incubated with monoclonal antibodies against myosin heavy chains (MHC) diverse immunostaining was observed within the developing intrafusal fiber bundle. Large primary intrafusal myotubes immunostained moderately to strongly for embryonic and neonatal MHC, but they were unreactive or reacted only weakly with antibodies against slow MHC. Smaller, secondary intrafusal myotubes reacted only weakly to moderately for embryonic and neonatal MHC, but 1–2 days after their formation they reacted strongly for slow and slow-tonic MHC. In contrast to mammals, slow-tonic MHC was also observed in extrafusal fibers. Intrafusal fibers derived from primary myotubes acquired fast MHC and retained at least a moderate level of embryonic MHC. On the other hand, intrafusal fibers developing from secondary myotubes lost the embryonic and neonatal isoforms prior to hatching and became slow. Based on relative amounts of embryonic, neonatal and slow MHC future fast and slow intrafusal fibers could be first identified at E14. At the polar regions of intrafusal fibers positions of nerve endings and acetylcholinesterase activity were seen to match as early as E16. Approximately equal numbers of slow and fast intrafusal fibers formed prenatally; however, in postnatal muscle spindles fast fibers were usually in the majority, suggesting that some fibers transformed from slow to fast.     

Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms

Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.     

Distribution and properties of myosin isozymes in developing avian and mammalian skeletal muscle fibers

Isozymes of myosin have been localized with respect to individual fibers in differentiating skeletal muscles of the rat and chicken using immunocytochemistry. The myosin light chain pattern has been analyzed in the same muscles by two-dimensional PAGE. In the muscles of both species, the response to antibodies against fast and slow adult myosin is consistent with the speed of contraction of the muscle. During early development, when speed of contraction is slow in future fast and slow muscles, all the fibers react strongly with anti-slow as well as with anti-fast myosin. As adult contractile properties are acquired, the fibers react with antibodies specific for either fast or slow myosin, but few fibers react with both antibodies. The myosin light chain pattern slow shows a change with development: the initial light chains (LC) are principally of the fast type, LC1(f), and LC2(f), independent of whether the embryonic muscle is destined to become a fast or a slow muscle in the adult. The LC3(f), light chain does not appear in significant amounts until after birth, in agreement with earlier reports. The predominance of fast light chains during early stages of development is especially evident in the rat soleus and chicken ALD, both slow muscles, in which LC1(f), is gradually replaced by the slow light chain, LC1(s), as development proceeds. Other features of the light chain pattern include an "embryonic" light chain in fetal and neonatal muscles of the rat, as originally demonstrated by R.G. Whalen, G.S. Butler- Browne, and F. Gros. (1978. J. Mol. Biol. 126:415-431.); and the presence of approximately 10 percent slow light chains in embryonic pectoralis, a fast white muscle in the adult chicken. The response of differentiating muscle fibers to anti-slow myosin antibody cannot, however, be ascribed solely to the presence of slow light chains, since antibody specific for the slow heavy chain continues to react with all the fibers. We conclude that during early development, the myosin consists of a population of molecules in which the heavy chain can be associated with a fast, slow, or embryonic light chain. Biochemical analysis has shown that this embryonic heavy chain (or chains) is distinct from adult fast or slow myosin (R.G. Whalen, K. Schwartz, P. Bouveret, S.M. Sell, and F. Gros. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:5197-5201. J.I. Rushbrook, and A. Stracher. 1979. Proc Natl. Acad. Sci. U.S.A. 76:4331-4334. P.A. Benfield, S. Lowey, and D.D. LeBlanc. 1981. Biophys. J. 33(2, Pt. 2):243a[Abstr.]). Embryonic myosin, therefore, constitutes a unique class of molecules, whose synthesis ceases before the muscle differentiates into an adult pattern of fiber types.     

Nascent muscle fiber appearance in overloaded chicken slow-tonic muscle

The application of a weight overload to the humerus of chickens induces a hypertrophy of anterior latissimus dorsi (ALD) muscle fibers. This growth is accompanied by a rapid and almost complete replacement of one slow-tonic myosin isoform, SM-1, by another slow-tonic isoform, SM-2. In addition, a population of small fibers appears mainly in extrafascicular spaces and, concurrently, three additional myosin bands are detected by gel electrophoresis. Five antibodies against myosin heavy chain (MHC) isoforms were selected as immunocytochemical probes to determine the cellular location and nature of these myosins. The antibodies react with ventricular, fast skeletal muscle and either SM-1 or SM-2, or both the slow-tonic MHCs. The antifast and antiventricular antibodies react with myosin present in the 10-day embryonic ALD muscle but do not react with myosin in posthatch ALD muscle. The small fibers in overloaded muscle contain a myosin isoform characteristically expressed during the embryonic stage of ALD muscle development and therefore are named nascent myofibers. Some of the nascent myofibers do not react with the antibody to both slow-tonic MHCs, indicating the lack of the normal adult slow-tonic myosins which are expressed in 10-day embryos. In order to explore the origin of the nascent fibers, an electron microscopic study was performed. Stereological analysis of the existing fibers shows a stimulation of numbers and sizes of satellite cells. In addition, the volume occupied by nonmuscle and undifferentiated cells increases dramatically. Myotube formation with incipient myofibrils is seen in extrafascicular spaces. These data suggest that new muscle fiber formation accompanies hypertrophy in overloaded chicken ALD muscle and the process may involve satellite cell migration.     

Expression of unique and developmental myosin heavy chain isoforms in adult human digastric muscle.

Digastric muscle (DGM) is a powerful jaw-opening muscle that participates in chewing, swallowing, breathing, and speech. For better understanding of its contractile properties, five pairs of adult human DGMs were obtained from autopsies and processed with immunocytochemistry and/or immunoblotting. Monoclonal antibodies against alpha-cardiac, slow tonic, neonatal, and embryonic myosin heavy chain (MHC) isoforms were employed to determine whether the DGM fibers contain these MHC isoforms, which have previously been demonstrated in restricted specialized craniocervical skeletal muscles but have not been reported in normal adult human trunk and limb muscles. The results showed expression of all these MHC isoforms in adult human DGMs. About half of the fibers reacted positively to the antibody specific for the alpha-cardiac MHC isoform in DGMs, and the number of these fibers decreased with age. Slow tonic MHC isoform containing fibers accounted for 19% of the total fiber population. Both the alpha-cardiac and slow tonic MHC isoforms were found to coexist mainly with the slow twitch MHC isoform in a fiber. A few DGM fibers expressed the embryonic or neonatal MHC isoform. The findings suggest that human DGM fibers may be specialized to facilitate performance of complex motor behaviors in the upper airway and digestive tract.     

Myosin heavy chain composition of single cells from avian slow skeletal muscle is strongly correlated with velocity of shortening during development

We have determined the myosin heavy chain (MHC) composition (using a sensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system) and the maximal velocity of shortening (Vmax) of single cells from neonatal and adult chicken anterior latissimus dorsi (ALD) muscles. In addition, the MHC, myosin light chain, and regulatory protein (i.e., troponin and tropomyosin subunits) compositions of bundles of ALD fibers were determined at late embryonic, neonatal, and adult ages. At young ages, there are two MHCs in ALD muscle, SM1 and SM2, with SM1 decreasing in relative amount with increasing age, as shown previously by others. The mean Vmax of single fibers also decreases from neonatal to adult ages. A strong quantitative correlation is demonstrated between the specific MHC composition and Vmax among individual cells of the ALD muscle at several ages. Since virtually no changes occur in the regulatory protein and myosin light chain compositions of the ALD muscle between late embryonic and adult ages, it appears that the MHC composition of an individual cell in this muscle is the primary determinant of the maximal shortening velocity. These results are the first to illustrate the functional significance of the developmental transition in myosin heavy chain composition of an avian slow skeletal muscle, consistent with our previous findings on mammalian muscle.     

Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression

A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that covered the approximate location of the epitopes. With this method, the epitope recognized by mAb F59 was mapped to amino acids 211-231 of the chicken embryonic fast MHC and the three distinct epitopes recognized by mAbs F18, F30, and F47 were mapped to amino acids approximately 65-92. Each of these epitope sequences is at or near the ATPase active site.     

Adult human upper esophageal sphincter contains specialized muscle fibers expressing unusual myosin heavy chain isoforms.

The functional upper esophageal sphincter (UES) is composed of the cricopharyngeus muscle (CP), the most inferior part of the inferior pharyngeal constrictor (iIPC), and the upper esophagus (UE). This sphincter is collapsed and exhibits sustained muscle activity in the resting state; it only relaxes and opens during swallowing, vomiting, and belching. The tonic contractile properties of the UES suggest that the skeletal muscle fibers in this sphincter differ from those in the limb and trunk muscles. In this study, myosin heavy chain (MHC) composition in the adult human UES muscles obtained from autopsies was investigated using immunocytochemical and immunoblotting techniques. Results showed that the adult human UES muscle fibers expressed unusual MHC isoforms such as slow-tonic (MHC-ton), alpha-cardiac (MHC-alpha), neonatal (MHC-neo), and embryonic (MHC-emb), which coexisted with the major MHCs (i.e., MHCI, IIa, and IIx). MHC-ton and MHC-alpha were coexpressed predominantly with slow-type I MHC isoform, whereas MHC-neo and MHC-emb coexisted mainly with fast-type IIa MHC. A slow inner layer (SIL) and a fast outer layer (FOL) in the iIPC and CP were identified immunocytochemically. MHC-ton- and MHC-alpha-containing fibers were concentrated mainly in the SIL, whereas MHC-neo- and MHC-emb-containing fibers were distributed primarily to the FOL. Identification of the specialized muscle fibers and their distribution patterns in the adult human UES is valuable for a better understanding of the physiological and pathophysiological behaviors of the sphincter.