Expression Of Fast Myosin Heavy Chain Research Articles

Garcinol Promotes the Formation of Slow-Twitch Muscle Fibers by Inhibiting p300-Dependent Acetylation of PGC-1α.

The conversion of skeletal muscle fiber from fast-twitch to slow-twitch is crucial for sustained contractile and stretchable events, energy homeostasis, and anti-fatigue ability. The purpose of our study was to explore the mechanism and effects of garcinol on the regulation of skeletal muscle fiber type transformation. Forty 21-day-old male C57/BL6J mice (n = 10/diet) were fed a control diet or a control diet plus garcinol at 100 mg/kg (Low Gar), 300 mg/kg (Mid Gar), or 500 mg/kg (High Gar) for 12 weeks. The tibialis anterior (TA) and soleus muscles were collected for protein and immunoprecipitation analyses. Dietary garcinol significantly downregulated (p < 0.05) fast myosin heavy chain (MyHC) expression and upregulated (p < 0.05) slow MyHC expression in the TA and soleus muscles. Garcinol significantly increased (p < 0.05) the activity of peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) and markedly decreased (p < 0.05) the acetylation of PGC-1α. In vitro and in vivo experiments showed that garcinol decreased (p < 0.05) lactate dehydrogenase activity and increased (p < 0.05) the activities of malate dehydrogenase and succinic dehydrogenase. In addition, the results of C2C12 myotubes showed that garcinol treatment increased (p < 0.05) the transformation of glycolytic muscle fiber to oxidative muscle fiber by 45.9%. Garcinol treatment and p300 interference reduced (p < 0.05) the expression of fast MyHC but increased (p < 0.05) the expression of slow MyHC in vitro. Moreover, the acetylation of PGC-1α was significantly decreased (p < 0.05). Garcinol promotes the transformation of skeletal muscle fibers from the fast-glycolytic type to the slow-oxidative type through the p300/PGC-1α signaling pathway in C2C12 myotubes.

Metabolic reprogramming underlies cavefish muscular endurance despite loss of muscle mass and contractility

Physical inactivity is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing an evolutionary perspective into the effect of adaptive physical inactivity on tissue homeostasis. One such example, the Mexican cavefish Astyanax mexicanus, has lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3.7-fold slower than their river-dwelling counterpart. This change in behavior is accompanied by a marked shift in body composition, decreasing total muscle mass and increasing fat mass. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated that this shift is driven by increased expression of pparγ-the master regulator of adipogenesis-with a simultaneous decrease in fast myosin heavy chain expression. Exvivo and invivo analysis confirmed that these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming speed. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omic analysis suggested metabolic reprogramming, specifically phosphorylation of Pgm1-Threonine 19, as a key component enhancing cavefish glycogen metabolism and sustained muscle contraction. Collectively, we reveal broad skeletal muscle changes following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction via enhanced glycogen metabolism.

Ontogeny of myosin isoform expression and prehensile function in the tail of the gray short-tailed opossum (Monodelphis domestica).

Terrestrial opossums use their semiprehensile tail for grasping nesting materials as opposed to arboreal maneuvering. We relate the development of this adaptive behavior with ontogenetic changes in myosin heavy chain (MHC) isoform expression from 21 days to adulthood. Monodelphis domestica is expected to demonstrate a progressive ability to flex the distal tail up to age 7 mo, when it should exhibit routine nest construction. We hypothesize that juvenile stages (3-7 mo) will be characterized by retention of the neonatal isoform (MHC-Neo), along with predominant expression of fast MHC-2X and -2B, which will transition into greater MHC-1β and -2A isoform content as development progresses. This hypothesis was tested using Q-PCR to quantify and compare gene expression of each isoform with its protein content determined by gel electrophoresis and densitometry. These data were correlated with nesting activity in an age-matched sample of each age group studied. Shifts in regulation of MHC gene transcripts matched well with isoform expression. Notably, mRNA for MHC-Neo and -2B decrease, resulting in little-to-no isoform translation after age 7 mo, whereas mRNA for MHC-1β and -2A increase, and this corresponds with subtle increases in content for these isoforms into late adulthood. Despite the tail remaining intrinsically fast-contracting, a critical growth period for isoform transition is observed between 7 and 13 mo, correlating primarily with use of the tail during nesting activities. Functional transitions in MHC isoforms and fiber type properties may be associated with muscle "tuning" repetitive nest remodeling tasks requiring sustained contractions of the caudal flexors.NEW & NOTEWORTHY Little is understood about skeletal muscle development as it pertains to tail prehensility in mammals. This study uses an integrative approach of relating both MHC gene and protein expression with behavioral and morphometric changes to reveal a predominant fast MHC expression with subtle isoform transitions in caudal muscle across ontogeny. The functional shifts observed are most notably correlated with increased tail grasping for nesting activities.

Myostatin facilitates slow and inhibits fast myosin heavy chain expression during myogenic differentiation

Skeletal muscles in the limb and body trunk are composed of heterogeneous myofibers expressing different isoforms of myosin heavy chain (Myh), including type I (slow, Myh7), IIA (intermediate, Myh2), IIX (fast, Myh1), and IIB (very fast, Myh4). While the contraction force and speed of a muscle are known to be determined by the relative abundance of myofibers expressing each Myh isoform, it is unclear how specific combinations of myofiber types are formed and regulated at the cellular and molecular level. We report here that myostatin (Mstn) positively regulates slow but negatively regulates fast Myh isoforms. Mstn was expressed at higher levels in the fast muscle myoblasts and myofibers than in the slow muscle counterparts. Interestingly, Mstn knockout led to a shift of Myh towards faster isoforms, suggesting an inhibitory role of Mstn in fast Myh expression. Consistently, when induced to differentiate, Mstn null myoblasts formed myotubes preferentially expressing fast Myh. Conversely, treatment of myoblasts with a recombinant Mstn protein upregulated Myh7 but downregulated Myh4 gene expression in newly formed myotubes. Importantly, both Mstn antibody and soluble activin type 2B receptor inhibited slow Myh7 and promoted fast Myh4 expression, indicating that myostatin acts through canonical activin receptor to regulate the expression of Myh genes. These results demonstrate a role of myostatin in the specification of myofiber types during myogenic differentiation.

Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, Antechinus flavipes

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC.

De‐phosphorylation of MyoD is linking nerve‐evoked activity to fast myosin heavy chain expression in rodent adult skeletal muscle

Elucidating the molecular pathways linking electrical activity to gene expression is necessary for understanding the effects of exercise on muscle. Fast muscles express higher levels of MyoD and lower levels of myogenin than slow muscles, and we have previously linked myogenin to expression of oxidative enzymes. We here report that in slow muscles, compared with fast, 6 times as much of the MyoD is in an inactive form phosphorylated at T115. In fast muscles, 10 h of slow electrical stimulation had no effect on the total MyoD protein level, but the fraction of phosphorylated MyoD was increased 4-fold. Longer stimulation also decreased the total level of MyoD mRNA and protein, while the level of myogenin protein was increased. Fast patterned stimulation did not have any of these effects. Overexpression of wild type MyoD had variable effects in active slow muscles, but increased expression of fast myosin heavy chain in denervated muscles. In normally active soleus muscles, MyoD mutated at T115 (but not at S200) increased the number of fibres containing fast myosin from 50% to 85% in mice and from 13% to 62% in rats. These data establish de-phosphorylated active MyoD as a link between the pattern of electrical activity and fast fibre type in adult muscles.

TGF-β1 favors the development of fast type identity during soleus muscle regeneration

Transforming growth factor-beta1 (TGF-beta1) is known to be expressed in the environment of developing fast muscle fibres during ontogenesis. In the present study, we have examined effects of administration of either TGF-beta1 or neutralizing TGF-beta1 antibody on the induction of fast type phenotype in regenerating skeletal muscles in rats. Expressions of fast and slow myosin heavy chain (MHC) isoforms were studied using protein electrophoresis, at 3 and 6 weeks after myotoxic treatment. Muscle contractile properties were also measured in situ. The results have shown that a single injection of TGF-beta1 into the regenerating slow soleus muscle increased the expression of fast MHC-2x/d and MHC-2a and decreases that of slow MHC-1 (P<0.05). Moreover, it reduced the degree of tetanic fusion during contraction (P<0.05). Conversely, injection of neutralizing antibody against TGF-beta1 into the regenerating fast EDL muscle increased the expression of MHC-2a and MHC-1 (P<0.05). In conclusion, when the slow muscle was regenerating in the presence of an increased level of TGF-beta1, it induced a shift to a less slow MHC phenotype and contractile characteristics. Conversely, neutralization of TGF-beta1 in the regenerating fast muscle induced a shift to a less fast MHC expression. Together these results suggest that TGF-beta1 influences some aspects of fast muscle-type patterning during skeletal muscle regeneration.

Effect of Endurance Exercise on Myosin Heavy Chain Isoform Expression in Diabetic Rats with Peripheral Neuropathy

This study evaluated the effect of endurance exercise on myosin heavy chain (MHC) isoform expression in soleus muscle of diabetic rats with peripheral neuropathy. Male Sprague Dawley rats were randomly divided into four groups: control sedentary, diabetic sedentary, control exercise, and diabetic exercise. The exercised animals performed treadmill running five times per week. After 12 wks, electrophysiologic testing documented peripheral neuropathy in the diabetic rats. The soleus muscles were then excised and quick-frozen. Cross-sections were immunohistochemically stained for slow, fast, developmental, and neonatal MHCs. Fiber-type composition and fiber cross-sectional areas were then determined. The diabetic groups showed a significantly greater percentage of fast MHC than did the control groups, regardless of exercise status (diabetic sedentary, 22.6%; diabetic exercise, 25.2%; control sedentary, 13.5%; control exercise, 13.1%). The diabetics also showed a significantly lower percentage of slow-only MHC than controls (diabetic sedentary, 77.1%; diabetic exercise, 74.3%; control sedentary, 86.2%; control exercise, 86.1%). No differences in muscle fiber cross-sectional area existed between the groups. The exercised animals showed greater expression of developmental MHC than did the sedentary animals (diabetic sedentary, 1.6%; diabetic exercise, 3.8%; control sedentary, 0.8%; control exercise, 2.0%). The altered slow and fast MHC expression in the diabetic muscle is similar to MHC expression in several other conditions, including decreased neuromuscular activity and denervation. Mechanisms of this MHC expression shift are unknown. Chronic endurance training does not alter adult MHC expression in the diabetic animals. The developmental MHC expression is likely a manifestation of uphill treadmill running due to eccentric contractions in the soleus resulting in myofiber injury and regeneration.

Cardiac-Restricted Ankyrin-Repeated Protein Is Differentially Induced in Duchenne and Congenital Muscular Dystrophy

Cardiac ankyrin-repeated protein (CARP) has been shown to associate with a transcription factor, YB-1, that may activate expression of the ventricular myosin light chain-2 gene during cardiogenesis. CARP is induced in the adult hypertrophic heart subjected to pressure overload, suggesting that CARP may play important functional roles in both embryonic and adult hearts. Although CARP expression was initially believed to be restricted to the heart, we found recently that CARP is induced strongly in human fetal skeletal muscle and in experimentally denervated skeletal muscle, leading us to speculate that CARP may also play important roles in skeletal muscle. In the present study, we found that in rats initially damaged by a single injection of bupivacaine, CARP expression was induced strongly in regenerating muscles with a peak 3 days after the injection, followed by down-regulation to undetectable levels after 28 days. Although CARP was coexpressed with embryonic myosin heavy chain (MHC) in regenerating myofibers, CARP expression persisted even after down-regulation of embryonic MHC expression, whereas it began to decrease before the onset of slow or fast MHC expression, suggesting that CARP is expressed at a specific differentiation stage during muscle regeneration. We analyzed the expression of CARP in muscle biopsy specimens from 14 patients with muscular dystrophy (MD) and detected high expression of CARP in 13 of the 14 cases. CARP-positive myofibers were detected more often in congenital muscular dystrophy (CMD) than in Duchenne muscular dystrophy (DMD). We found that CARP was expressed exclusively, and at a high level, in small regenerating myofibers that express embryonic MHC in DMD, which suggested that CARP could be used as a marker of muscle regeneration in DMD. On the other hand, in CMD, expression of CARP was not limited to regenerating fibers, being detectable in myofibers expressing embryonic MHC and those expressing mature-type MHC. These findings suggest that the differentiation stage of CARP-positive myofibers in DMD and CMD may differ.

Regulation of myosin heavy chain expression during rat skeletal muscle development in vitro.

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and approximately 10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN-nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.

Lack of coordinated changes in metabolic enzymes and myosin heavy chain isoforms in regenerated muscles of trained rats.

We investigated training-induced changes in biochemical properties and myosin heavy chain (MHC) composition of regenerated (cardiotoxin-injected) plantaris muscles (PLA) in rats either maintained sedentary (S, n = 9) or endurance trained on a treadmill over a 8-week period (T, n = 7). Both endurance training and regeneration altered the pattern of fast MHC expression. An analysis of the two-way interaction between training and regeneration showed that the relative content of type IIa MHC was affected (P < 0.05). The 140% increase in type IIa MHC observed in regenerated PLA from T rats compared with nontreated muscle of S rats, exceeded the 102% increase resulting from the combination of regeneration alone (26%) and training alone (61%). A similar interaction between training and regeneration was shown for the percentage of fibres expressing either type IIa or type lIb MHC (P < 0.05). In contrast, a significant increase in the citrate synthase (CS) activity was shown in PLA as a result of endurance training, without specific effect of regeneration. Furthermore, training-induced changes in CK and LDH isoenzyme distribution occurred to a similar extent in regenerated and non-treated PLA muscles, and thus did not follow the changes in MHC isoforms. An increase in the mitochondrial CK isozyme activity (mi-CK) was shown in both non-treated and previously degenerated PLA muscles (123 and 117%, P < 0.01, respectively), without specific effect of regeneration. The ratio of mi-CK to CS activity, an estimate of the mitochondrial specific activity of mi-CK was significantly increased by training (P < 0.02) and decreased by regeneration (P < 0.05). Taken together, these data suggest that while training and regeneration have cumulative effects on the pattern of fast MHC expression, the training-induced changes in the energy metabolism shown in mature non-treated myofibres are similar to those observed in regenerated fibres.

Role of weight-bearing function on expression of myosin isoforms during regeneration of rat soleus muscles

The expression of myosin isoforms was studied in regenerated rat soleus muscle during either normal or altered postural activity. Regeneration was induced following injury by venom from the Notechis scutatus scutatus snake. Immunohistochemical analysis showed that, in regenerating soleus muscle after 3 wk of hindlimb suspension, nearly all fibers reacted positively with the myosin heavy chain (MHC) antibody associated with fast-twitch muscle fibers (fast MHC). When 3 wk of recovery with normal weight-bearing activity followed hindlimb suspension, the regeneration soleus muscle exhibited a nearly homogeneous staining with the MHC antibody associated with the slow-twitch muscle fibers (slow MHC). These findings were in accordance with quantitative analysis of the electrophoretic separation of the native myosin isoforms. Immunohistochemical data showed that removal of weight bearing in the 21-day old regenerated soleus muscles resulted in an increase in fast MHC expression. Together, the results of the present study clearly demonstrate that the postural load is an important component in the induction of slow MHC in regenerating muscle and that the control of the expression of MHC in muscle comprising a homogeneous population of fibers deriving from satellite cells appears more homogeneous and more complete than in a nondegenerated one.

Developmental modulation of myosin expression by thyroid hormone in avian skeletal muscle.

It is well established that a rise in circulating thyroid hormone during the second half of chick embryo development significantly influences muscle weight gain and bone growth. We studied thyroid influence on differentiation in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of embryos rendered hypothyroid by hypophysectomy or administration of an anti-thyroid drug. The expression of native myosins and myosin light chains (MLCs) was studied by electrophoretic analysis, and the myosin heavy chain (MHC) was characterized by immunohistochemistry. The first effects of hypothyroid status were observed at day 21 of embryonic development (stage 46 according to Hamburger and Hamilton). Analysis of myosin isoform expression in PLD muscles of hypothyroid embryos showed persistence of slow migrating native myosins and slow MLCs as well as inhibition of neonatal fast MHC expression, indicating retarded differentiation of this muscle. In ALD muscle, hypothyroidism maintained fast embryonic MHC and induced noticeable amounts of fast MLCs, thus delaying slow muscle differentiation. Our results suggest that thyroid hormones play a role in modulating the appearance of neonatal fast MHC and the disappearance of isomyosins transiently present during embryogenesis. However, T3 supplemental treatment would seem to compensate in part for the effects of hypothyroidism induced by hypophysectomy, suggesting that thyroid hormone might interfere with other factors also accounting for the observed effects.

Fast myosin heavy chain expression during the early and late embryonic stages of chicken skeletal muscle development

The development of embryonic skeletal muscles in the chick can be divided into two periods of fiber specialization—an early one during which the different muscles of the limb are formed and an initial round of fiber specialization occurs and a late or fetal period during which there is extensive growth of this previously established fiber pattern. This latter period of growth is dependent on the establishment and maintenance of functional neuromuscular contacts. As has been described for other developmental stages, we show here that there are different embryonic fast skeletal muscle myosin heavy chain (MHC) isoforms expressed during the different embryonic periods of muscle growth. The identification of these isoforms was based on differences in their reactivity with various fast MHC monoclonal antibodies and on their different peptide banding patterns. The in ovo accumulation of the late embryonic MHC isoform pattern was similar to the time course of the previously described changes in α-actin and troponin T isotype switching during embryogenesis. The appearances of the late embryonic isoforms were blocked by chronic treatment with the neuromuscular blocking agent, d-tubocurarine, and cell cultures of embryonic chicken skeletal muscle which differentiated in the absence of motorneurons expressed little of the late embryonic isoform, indicating that the expression of the late embryonic isoform was dependent on functional nerve-muscle interactions. These different embryonic fast MHC isoforms provide important markers for monitoring the progression of muscle through its embryonic stages and its interaction with motorneurons.

Myosin heavy chain expression in human muscle cocultured with mouse spinal cord

Monoclonal antibodies which specifically recognise the neonatal or adult fast (type 2A and 2B) or slow myosin heavy chain (MHC) were used to investigate the myosin composition of human muscle which had regenerated in culture in the presence or absence of embryonic mouse spinal cord tissue. Adult fast MHC was found in an average of 75% of the cultured fibres, irrespective of the time in culture, the source of the muscle, the presence of neurones or modifications to the growth medium. It was not found alone but was always in association with the neonatal and/or slow myosin isoforms. The expression of fast MHC in human muscle therefore differs from that in mouse muscle in this culture system (Ecob-Prince et al. 1986), but is still strong evidence for the development of at least one aspect of an adult phenotype in culture.

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.

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.

The Determinants of Muscle Fiber Type During Embryonic Development

SYNOPSIS. Most vertebrate skeletal muscles consist of a heterogeneous array of muscle fiber types that are distinguishable, in part, by differences in their contractile protein isoform content. It is often suggested that the information necessary for directing the development of these fiber types is derived from interactions with factors outside the muscle fibers themselves and, in particular, with innervating motoneurons. However, recent data from this and other laboratories indicate that the emergence of fiber specialization within developing muscle is not dependent on innervation at all. These studies recognize two periods of embryonic fiber specialization. The first occurs during early embryonic development as individual muscles are formed from primary generation fibers expressing different myosin isoform types. The formation of these “early” muscle fiber types and their characteristic distributions within and among different muscles are not dependent on interactions with innervating motoneurons. Furthermore, myoblasts isolated from “early” embryonic muscle tissue and cultured in vitro display the same heterogeneity of myosin expression as the primary generation fiber types in ovo , suggesting that the differences in expression among early muscle fiber types are preprogrammed within their myoblasts. The second period occurs “late” in development after the major morphological events of limb formation are complete and the initial pattern of fiber types has been established. It is during this period that massive growth of most muscles occurs which is due, in part, to the formation of a secondary generation of muscle fibers. These secondary generation fibers in ovo and the cultured myotubes derived from “late” embryonic myoblasts exhibit a single myosin phenotype ( e.g. , fast). The transition from “early” to “late” embryonic phases is accompanied by a change in fast myosin heavy chain expression and is blocked by agents that disrupt neuromuscular contacts.

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.