Neural regulation of differentiation of rat skeletal muscle cell types.

Three monoclonal antibodies (LM5, F2 and F39) to the fast class of myosin heavy chain (MHC) were used to study the effect of denervation on the differentiation of muscle cell types in some rat skeletal muscles. Antibody LM5 in immunocytochemical investigations did not stain any myotubes during early fetal development but presumptive fast muscle cells started to stain during later fetal development. Unlike antibody LM5, antibodies F2 and F39 stained all myotubes during fetal development. The suppression of fast myosin heavy chains recognised in presumptive slow muscle cells was observed within 1-2 days after birth with antibody F39 but not until 10-14 days after birth with antibody F2. The emergence of subsets of fast muscle fibre types in rat extensor digitorum longus (EDL) and tibialis anteri (TA) detectable by F39 and F2 antibodies was not observed until 2-3 weeks after birth. Denervation of developing muscles led to marked changes in the expression of myosins identified by these antibodies.     

Neural regulation of differentiation of rat skeletal muscle cell types

Summary Three monoclonal antibodies (LM5, F2 and F39) to the fast class of myosin heavy chain (MHC) were used to study the effect of denervation on the differentiation of muscle cell types in some rat skeletal muscles. Antibody LM5 in immunocytochemical investigations did not stain any myotubes during early fetal development but presumptive fast muscle cells started to stain during later fetal development. Unlike antibody LM5, antibodies F2 and F39 stained all myotubes during fetal development. The suppression of fast myosin heavy chains recognised in presumptive slow muscle cells was observed within 1–2 days after birth with antibody F39 but not until 10–14 days after birth with antibody F2. The emergence of subsets of fast muscle fibre types in rat extensor digitorum longus (EDL) and tibialis anteri (TA) detectable by F39 and F2 antibodies was not observed until 2–3 weeks after birth. Denervation of developing muscles led to marked changes in the expression of myosins identified by these antibodies.     

Identification and changes in the pattern of expression of slow-skeletal-muscle-like myosin heavy chains in a developing fast muscle

Immunochemical studies of chicken pectoralis major, a fast muscle, have demonstrated large amounts of myosin heavy chains (MHCs) of the slow-skeletal-muscle type during early stages of embryonic development. A large majority of the myotubes present in early embryonic muscle stained for this class of MHC. As development progressed, its synthesis was suppressed in most of the muscle, except in the deeper presumptive red-strip region. The level of this MHC in the embryonic muscle appeared to be reduced by its suppression in a proportion of the existing cells, by the addition of many presumptive fast cells that never expressed this MHC, and by atrophy or degeneration of a small proportion of the slow MHC-positive cells. Further suppression of this MHC in a proportion of the histochemically typed slow cells present in the red-strip region did not occur until quite late in the post-hatch period.     

Transition of myosin isozymes during development of human masseter muscle. Persistence of developmental isoforms during postnatal stage

Previous results have shown that the adult human masseter muscle contains myosin isoforms that are specific to early stages of development in trunk and limb muscles, i.e. embryonic and fetal (neonatal) myosin heavy chains (MHC) and embryonic myosin light chain (MLC1emb). We wanted to know if this specific pattern is the result of a late maturation or of a distinct evolution during development. We show here that the embryonic and the fetal MHC and the MLC1emb are expressed throughout perinatal and postnatal masseter development. Our results also demonstrate that MLC1emb accumulation increases considerably during the postnatal period. In addition, both the slow MLCs and the slow isoform of tropomyosin are expressed later in the masseter than quadriceps and the fast skeletal muscle isoform MLC3 is not detected during fetal and early postnatal development in the masseter whereas it is expressed throughout fetal development in the quadriceps. Our results thus confirm previous histochemical data and demonstrate that the masseter muscle displays a pattern of myosin and tropomyosin isoform transitions different to that previously described in trunk and limb muscles. This suggests that control of masseter muscle development involves mechanisms distinct from other body muscles, possibly as a result of either its craniofacial innervation or of a possibly different embryonic origin.     

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.     

Myosin heavy chain isoform composition in striated muscle after denervation and self-reinnervation

The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel. After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed. The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.     

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.     

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.     

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.     

Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms

Two types of smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, were recently identified to have different carboxyl termini (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). SM1 and SM2 are considered to be generated from a single gene through alternative RNA splicing. In this study we investigated expression of vascular MHC isoforms during development in rabbits at the mRNA, protein, and histological levels. In adults, all smooth muscle cells reacted with both anti-SM1 and anti-SM2 antibodies on immunofluorescence, suggesting the coexpression of SM1 and SM2 in a single cell. In fetal and perinatal rabbits, however, only anti-SM1 antibody consistently reacted with smooth muscles. Reactivity with anti-SM2 antibody was negative in the fetal and neonatal blood vessels and gradually increased during 30 days after birth. These developmental changes in SM1 and SM2 expression at the histological level coincided with mRNA expression of each MHC isoform as determined by S1 nuclease mapping, indicating that expression of SM1 and SM2 is controlled at the level of RNA splicing. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin from fetal and perinatal aortas revealed the presence of large amount of SM2. Interestingly, fetal SM2 did not cross-react with our anti-SM2 antibody on immunoblotting. We conclude that expression of SM1 and SM2 are differentially regulated during development and that a third type of MHC isoform may exist in embryonic and perinatal vascular smooth muscles.     

Fast skeletal muscle isoforms exhibit the highest incorporation level into myofibrils and stress fibers among members of myosin alkali light chain isoform family

Isoproteins of myosin alkali light chain (LC) were co-expressed in cultured chicken cardiomyocytes and fibroblasts and their incorporation levels into myofibrils and stress fibers were compared among members of the LC isoform family. In order to distinguish each isoform from the other, cDNAs of LC isoforms were tagged with different epitopes. Expressed LCs were detected with antibodies to the tags and their distribution was analyzed by confocal microscopy. In cardiomyocytes, the incorporation level of LC into myofibrils was shown to increase in the order from nonmuscle isoform (LC3nm), to slow skeletal muscle isoform (LC1sa), to slow skeletal/ventricular muscle isoform (LC1sb), and to fast skeletal muscle isoforms (LC1f and LC3f). Thus, the hierarchal order of the LC affinity for the cardiac myosin heavy chain (MHC) is identical to that obtained in the rat (Komiyama et al., 1996. J. Cell Sci., 109: 2089-2099), suggesting that this order may be common for taxonomic animal classes. In fibroblasts, the affinity of LC for the nonmuscle MHC in stress fibers was found to increase in the order from LC3nm, to LC1sb, to LC1sa, and to LC1f and LC3f. This order for the nonmuscle MHC is partly different from that for the cardiac MHC. This indicates that the order of the affinity of LC isoproteins for MHC varies depending on the MHC isoform. Further, for both the cardiac and nonmuscle MHCs, the fast skeletal muscle LCs exhibited the highest affinity. This suggests that the fast skeletal muscle LCs may be evolved isoforms possessing the ability to associate tightly with a variety of MHC isoforms.     

Myosin heavy chain expression during development and following denervation of fast fibers in the red strip of the chicken pectoralis

The myosin heavy chain composition of muscle fibers that comprise the red strip of the pectoralis major was determined at different stages of development and following adult denervation. Using a library of characterized monoclonal antibodies we found that slow fibers of the red strip do not react with antibodies to any of the fast myosin heavy chains of the superficial pectoralis. Immunocytochemical analysis of the fast fibers of the adult red strip revealed that they contain the embryonic fast myosin heavy chain rather than the adult pectoral isoform found throughout the adult white pectoralis. This was confirmed using immunoblot analysis of myosin heavy chain peptide maps. We show that during development of the red strip both neonatal and adult myosin heavy chains appear transiently, but then disappear during maturation. Furthermore, while the fibers of the superficial pectoralis reexpress the neonatal isoform as a result of denervation, the fibers of the red strip reexpress the adult isoform. Our data demonstrate a new developmental program of fast myosin heavy chain expression in the chicken and suggest that the heterogeneity of myosin heavy chain expression in adult fast fibers results from repression of specific isoforms by innervation.     

A myosin isoform repressed in hypertrophied ALD muscle of the chicken reappears during regeneration following cold injury

A library of monoclonal antibodies specific for myosin heavy chain (HC) was used to study myosin expression in regenerating fibers. The response to cold injury of slow skeletal ALD muscle previously induced to eliminate SM1 myosin by weight overload was compared to that of its contralateral control. Native gel electrophoresis combined with immunoblotting demonstrated that slow SM1 myosin HC eliminated from hypertrophic muscle reappeared both at the site of active regeneration and unexpectedly, also distal to the site of injury. The regeneration response of hypertrophied muscles was similar to that of the controls. In addition to SM1 myosin HC, ventricular-like and embryonic/fast isoforms were also expressed in both muscles during the early stages of regeneration and disappeared as the muscle fibers matured. These observations demonstrate that regenerating slow muscle fibers reexpress myosins' characteristic of developing muscle irrespective of the myosin phenotype prior to injury. The reappearance of repressed myosin HC in the hypertrophied ALD muscle is consistent with the presence of newly differentiated myonuclei.     

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.     

The genes and mRNA coding for the heavy chains of chick embryonic skeletal myosin

A size class of polysomes was isolated from chick embryonic leg skeletal muscle which synthesized almost exclusively a polypeptide chain with a molecular weight identical to the myosin heavy chain. The mRNA purified from these polysomes was shown to synthesize the 200,000 dalton polypeptide in the wheat germ cell-free translation system. At least 90% of the polypeptide had properties similar to the myosin heavy chain. Isoelectric focusing indicated that the myosin heavy chain synthesized in vitro contained two chains in equal amounts, as did purified embryonic leg skeletal muscle myosin. The kinetics of hybridization of the complementary DNA with an excess of the myosin heavy chain mRNA (MHC mRNA) indicated the presence of two different mRNA sequences. Reassociation of the cDNA to an excess of the DNA of the genome suggest that there is little, if any, reiteration of the myosin heavy chain genes.     

Developmentally regulated expression of three slow isoforms of myosin heavy chain: diversity among the first fibers to form in avian muscle.

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.     

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.     

Influence of the dwarf mouse mutation on skeletal and cardiac myosin isoforms. Effect of one injection of thyroxine on skeletal and cardiac muscle phenotype

The dwarf mutant is an autosomal recessive mutation of the mouse which causes a defective development of those anterior pituitary cells responsible for the production of thyroid-stimulating hormone, growth hormone, and prolactin. These mice are thus genetically hypothyroid and provide a model system in which one can investigate the influence of thyroid hormone on the transitions of the myosin heavy chain isoforms. We have carried out a qualitative and quantitative investigation of the myosin heavy chain isoforms present at various developmental stages and following one injection of 1 microgram of thyroxine. Myosin heavy chains were identified by nondissociating gel electrophoresis, localized by indirect immunofluorescence, and quantitated by the enzyme-linked immunosorbent assay technique. We find that in skeletal muscle, the appearance of the adult fast myosin heavy chain is severely retarded, that the neonatal myosin heavy chain is never totally eliminated, and that there is an overall increase in the number of fibers containing slow myosin heavy chain. In cardiac tissue the adult phenotype is never attained and beta-cardiac myosin heavy chain remains the predominant isoform. A single injection of 1 microgram of thyroxine was sufficient to cause a slight acceleration in the appearance of the adult fast myosin heavy chain in skeletal muscle, but only after 6-8 days. However, in the cardiac muscle, one injection of thyroxine resulted in a more rapid but transient expression of the alpha-cardiac myosin heavy chain, suggesting that the mechanism of action of thyroid hormone is different in these two tissues.