Effects of electrical stimulation upon post-hatching development of fibre types in normally innervated fast and slow latissimus dorsii muscles of the chicken

In chicken, the main characteristic properties of muscle fibre types in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii are acquired during post-hatching development. At day 4 it becomes possible to distinguish between alpha' and beta' fibre types in ALD muscle. At the same time, mATPase staining and NADH-TR activity permit recognition of alpha w and alpha R fibres within PLD muscle. During further development, muscle fibre typology progressively changes towards the adult slow and fast type. Chronic stimulation at a slow rhythm (5 Hz) of PLD prevents the change in relative proportions of alpha R and alpha W fibres within the muscle that occurs in normal post-hatching development and increases the number of beta R fibres. Moreover, oxidative activity is increased in all muscle fibre types following stimulation. In ALD muscle, chronic stimulation at a fast rhythm (40 Hz) results in a decrease in oxidative activity and inhibits the differentiation of alpha' and beta' muscle fibre types. This study demonstrates that in young chicken, the pattern of activity influences the differenciation of fibre types in slow and fast muscles.     

Effect of spinal cord stimulation on the metabolism of developing latissimus dorsii muscles in chick embryo

Influence of chronic spinal cord stimulation upon some characteristic enzyme activities of energy metabolism was investigated in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii muscles of the chick embryo. During embryonic life, oxidative metabolism (as evaluated by the activity of malate dehydrogenase (MDH] represents the main energetic pathway in both slow and fast muscles. At the end of embryonic life, an increase in anaerobic (as evaluated by the activity of lactate dehydrogenase (LDH] and creatine phosphokinase (CPK) activities occurs in PLD muscle. Chronic spinal cord stimulation at a low frequency was performed from the 10th day to the 16th day of embryonic development. In ALD, the enzyme activities were unaffected, while in PLD a concomitant decrease in LDH and CPK activities was observed.     

Effects of Electrical Stimulation on Molecular Forms of Butyrylcholinesterase in Denervated Fast and Slow Latissimus Dorsi Muscles of Newly Hatched Chicken

The effects of denervation and direct electrical stimulation upon the activity and the molecular form distribution of butyrylcholinesterase (BuChE) were studied in fast-twitch posterior latissimus dorsi (PLD) and in slow-tonic anterior latissimus dorsi (ALD) muscles of newly hatched chicken. In PLD muscle, denervation performed at day 2 substantially reduced the rate of rapid decrease of BuChE specific activity which takes place during normal development, whereas in the case of ALD muscle little change was observed. Moreover, the asymmetric forms which were dramatically reduced in denervated PLD muscle were virtually absent in denervated ALD muscle at day 14. Denervated PLD and ALD muscles were stimulated from day 4 to day 14 of age. Two patterns of stimulation were applied, either 5-Hz frequency (slow rhythm) or 40-Hz frequency (fast rhythm). Both patterns of stimulation provided the same number of impulses per day (about 61,000). In PLD muscle, electrical stimulation almost totally prevented the postdenervation loss in asymmetric forms and led to a decrease in BuChE specific activity. In ALD muscle, electrical stimulation partially prevented the asymmetric form loss which occurs after denervation. This study emphasizes the role of evoked muscle activity in the regulation of BuChE asymmetric forms in the fast PLD muscle and the differential response of denervated slow and fast muscles to electrical stimulation.     

Developmental changes in myosin isoforms from slow and fast latissimus dorsi muscles in the chicken

In the course of muscle differentiation, changes in fibre-type population and in myosin composition occur. In this work, the expression of native myosin isoforms in developing fast-twitch (posterior latissimus dorsi; PLD) and slow-tonic (anterior latissimus dorsi; ALD) muscles of the chick was examined using electrophoresis under nondissociating conditions. The major isomyosin of 11-day-old embryonic PLD comigrated with the adult fast myosin FM3. Two additional components indistinguishable from adult fast FM2 and FM1 isomyosins appeared successively during the embryonic development. The relative proportion of these latter isoforms increased with age, and the adult pattern was established by the end of the 1st month after hatching. Between day 11 and day 16 of embryonic development, PLD muscle fibres also contained small amounts of slow isomyosins SM1 and SM2. This synthesis of slow isoforms may be related to the presence of slow fibres within the muscle. At all embryonic and posthatch stages, ALD was composed essentially of slow isomyosins that comigrated with the two slow components SM1 and SM2 identified in adult. Several studies have reported that the SM1:SM2 ratio decreases progressively throughout embryonic and posthatching development, SM2 being predominant in the adult. In contrast, we observed a transient increase in SM1:SM2 ratio at the end of embryonic life. This could reflect a transitional neonatal stage in myosin expression. In addition, the presence in trace amounts of fast isomyosins in developing ALD muscle could be related to the presence of a population of fast fibres within this muscle.     

Rat muscle spindle immunocytochemistry revisited.

The expression of myosin heavy chain isoforms in muscle spindle fibres has been the subject of a number of immunocytochemical studies, some of them with discordant results. In order to assess whether these discrepancies are due to differences in the specificity and sensitivity of the antibodies used, we have compared the reactivity of rat muscle spindle fibres to two pairs of antibodies presumed to be directed against slow tonic (ALD 19 and ALD 58) and neonatal (NN5) and neonatal/fast (MF30) myosin heavy chains. Adult, developing and neonatally de-efferented muscle spindles from the rat hind limb muscles were studied in serial cross-sections processed for the peroxidase-antiperoxidase method. Important differences in the staining profiles of intrafusal fibres were noted when ALD 19 and ALD 58 were compared. ALD 19 stained the muscle spindle precursors from the seventeenth day in utero, whereas ALD 58 only did so by the twentieth day of gestation. In adult spindles ALD 19 stained the nuclear bag1 fibres along their entire length, whereas ALD 58 did not stain these fibres towards their ends. ALD 19 stained the nuclear bag2 fibres along the A, B and inner C region, but ALD 58 stained these fibres only in the A and the inner B regions. ALD 19 stained some nuclear chain fibres along a short equatorial segment, whereas ALD 58 did not stain the nuclear chain fibres at all. NN5 stained the nascent nuclear bag1 and chain fibre precursors at earlier stages of development than MF30. Clear differential staining between primary and secondary generation of both extra- and intrafusal myotubes was seen with NN5, whereas MF30 stained all myotubes alike. However, in postnatal spindles, MF30 was a very good negative marker of nuclear bag1 fibres. The staining profile of the adult fibres with NN5 and MF30 was rather similar. The staining pattern of neonatally de-efferented bag fibres obtained with ALD 19 and ALD 58 was practically identical and it differed from that of control spindles, confirming that motor innervation participates in the regulation of the expression of slow tonic MHC along the length of the nuclear bag2 fibres, as we have previously shown with ALD 19. The distinct staining patterns obtained with ALD 19 versus ALD 58 and with NN5 versus MF30 reflect differences in antibody sensitivity and specificity. These differences account, in part, for the discrepancies in the results of previous studies on muscle spindles, published by Kucera and Walro using ALD 58 and MF30, and by us using ALD 19 and NN5.     

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.     

Rat muscle spindle immunocytochemistry revisited

Summary The expression of myosin heavy chain isoforms in muscle spindle fibres has been the subject of a number of immunocytochemical studies, some of them with discordant results. In order to assess whether these discrepancies are due to differences in the specificity and sensitivity of the antibodies used, we have compared the reactivity of rat muscle spindle fibres to two pairs of antibodies presumed to be directed against slow tonic (ALD 19 and ALD 58) and neonatal (NN5) and neonatal/fast (MF30) myosin heavy chains. Adult, developing and neonatally de-efferented muscle spindles from the rat hind limb muscles were studied in serial cross-sections processed for the peroxidase-antiperoxidase method. Important differences in the staining profiles of intrafusal fibres were noted when ALD 19 and ALD 58 were compared. ALD 19 stained the muscle spindle precursors from the seventeenth day in utero, whereas ALD 58 only did so by the twentieth day of gestation. In adult spindles ALD 19 stained the nuclear bag₁ fibres along their entire length, whereas ALD 58 did not stain these fibres towards their ends. ALD 19 stained the nuclear bag₂ fibres along the A, B and inner C region, but ALD 58 stained these fibres only in the A and the inner B regions. ALD 19 stained some nuclear chain fibres along a short equatorial segment, whereas ALD 58 did not stain the nuclear chain fibres at all. NN5 stained the nascent nuclear bag₁ and chain fibre precursors at earlier stages of development than MF30. Clear differential staining between primary and secondary generation of both extra- and intrafusal myotubes was seen with NN5, wheras MF30 stained all myotubes alike. However, in postnatal spindles, MF30 was a very good negative marker of nuclear bag₁ fibres. The staining profile of the adult fibres with NN5 and MF30 was rather similar. The staining pattern of neonatally de-efferented bag fibres obtained with ALD 19 and ALD 58 was practically identical and it differed from that of control spindles, confirming that motor innervation participates in the regulation of the expression of slow tonic MHC along the length of the nuclear bag₂ fibres, as we have previously shown with ALD 19. The distinct staining patterns obtained with ALD 19 versus ALD 58 and with NN5 versus MF30 reflect differences in antibody sensitivity and specificity. These differences account, in part, for the discrepancies in the results of previous studies on muscle spindles, published by Kucera and Walro using ALD 58 and MF30, and by us using ALD 19 and NN5.     

Spinal cord-muscle relations: their role in neuro-muscular development in birds

In the present study we focused our attention on the role of spinal cord-muscle interactions in the development of muscle and spinal cord cells. Four experimental approaches were used: 1) muscle fiber-spinal cord co-culture; 2) chronic spinal cord stimulation in chick embryos; 3) direct electrical stimulation of the denervated chick muscle; 4) skeletal muscle transplantation in close apposition to the spinal cord in chick embryos. The characteristics of mATPase and energetic metabolism enzyme activities and of myosin isoform expression were used as markers for fiber types in two peculiar muscles, the fast-twitch PLD and the slow-tonic ALD. In vitro, in the absence of neurons, myoblasts can express some characteristics of either slow or fast muscle types according to their origin, while in the presence of neurons, muscle fiber differentiation seems to be related to the spontaneous rhythm delivered by the neurons. The in ovo experiments of chronic spinal cord stimulation demonstrate that the differentiation of the fast and slow muscle features appears to be rhythm dependent. In the chick, direct stimulation of denervated muscles shows that the rhythm of the muscle activity is also involved in the control of muscle properties. In chick embryos developing ALD, the changes induced by modifications of muscle tension demonstrate that this factor also influences muscle development. Other experiments show that muscle back-transplantation can alter the early spinal cord development.     

Relationship of primary and secondary myogenesis to fiber type development in embryonic chick muscle.

The formation of fast and slow myotubes was investigated in embryonic chick muscle during primary and secondary myogenesis by immunocytochemistry for myosin heavy chain and Ca2(+)-ATPase. When antibodies to fast or slow isoforms of these two molecules were used to visualize myotubes in the posterior iliotibialis and iliofibularis muscles, one of the isoforms was observed in all primary and secondary myotubes until very late in development. In the case of myosin, the fast antibody stained virtually all myotubes until after stage 40, when fast myosin expression was lost in the slow myotubes of the iliofibularis. In the case of Ca2(+)-ATPase, the slow antibody also stained all myotubes until after stage 40, when staining was lost in secondary myotubes and in the fast primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis. In contrast, the antibodies against slow muscle myosin heavy chain and fast muscle Ca2(+)-ATPase stained mutually exclusive populations of myotubes at all developmental stages investigated. During primary myogenesis, fast Ca2(+)-ATPase staining was restricted to the primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis, whereas slow myosin heavy chain staining was confined to all of the primary myotubes of the slow region of the iliofibularis. During secondary myogenesis, the fast Ca2(+)-ATPase antibody stained nearly all secondary myotubes, while primaries in the slow region of the iliofibularis remained negative. Thus, in the slow region of the iliofibularis muscle, these two antibodies could be used in combination to distinguish primary and secondary myotubes. EM analysis of staining with the fast Ca2(+)-ATPase antibody confirmed that it recognizes only secondary myotubes in this region. This study establishes that antibodies to slow myosin heavy chain and fast Ca2(+)-ATPase are suitable markers for selective labeling of primary and secondary myotubes in the iliofibularis; these markers are used in the following article to describe and quantify the effects that chronic blockade of neuromuscular activity or denervation has on these populations of myotubes.     

Posthatching changes in levels and molecular forms of acetylcholinesterase in slow and fast muscles of the chicken: Effects of denervation and direct electrical stimulation

The evolution of acetylcholinesterase (AChE) activity and AChE molecular form distribution were studied in slow-tonic anterior latissimus dorsi (ALD) and in fast-twitch posterior latissimus dorsi (PLD) muscles of chickens 2-18 days of age. In ALD as well as in PLD muscles, the AChE-specific activity increased transiently from day 2 to day 4; the activity then decreased more rapidly in PLD muscle. During this period asymmetric AChE forms decreased dramatically in ALD muscle and the globular forms increased. In PLD muscle, the most striking change was the decline in A8 form between days 2 and 18 of development. Denervation performed at day 2 delayed the normal decrease in AChE-specific activity in PLD muscle, whereas little change was observed in ALD muscle. Moreover, A forms in these two muscles were virtually absent 8 days after denervation. Direct electrical stimulation depressed the rise in AChE-specific activity in denervated PLD muscle and prevented the loss of the A forms. Furthermore, the different molecular forms varied according to the stimulus pattern. In ALD muscle, electrical stimulation failed to prevent the effect of denervation. This study emphasizes the differential response of denervated slow and fast muscles to electrical stimulation and stresses the importance of the frequency of stimulation in the regulation of AChE molecular forms in PLD muscle during development.     

EFFECTS OF NEUROMUSCULAR BLOCKING AGENTS ON THE DIFFERENTIATION OF NERVE-MUSCLE CONNECTIONS IN SLOW AND FAST CHICK MUSCLES

The effects of neuromuscular blocking drugs on the development of slow and fast muscle fibres and their neuromuscular junctions was studied in chick embryos.
Treatment of embryos with the depolarizing neuromuscular blocking agent suxamethonium affected the development of muscle fibres of the slow anterior latissimus dorsi (ALD) muscle more than that of muscle fibres of the posterior latissimus dorsi (PLD). The differentiation of the presynaptic elements of the neuromuscular junction was delayed and this was particularly obvious in PLD. Normally the number of axon profiles at individual endplates is reduced by 18 days of incubation, but in suxamethonium treated embryos this reduction took place only at 21 days. During earlier stages of development the axon profiles from treated embryos were small with sparse synaptic vesicles. Nevertheless the subsynaptic site of endplates on ALD and PLD muscle fibres became specialized earlier than normal and to a greater extent. Treatment with hemicholinium (HC-3), a drug that reduces the synthesis of acetylcholine (ACh) in nerve terminals affected the development of PLD muscle fibres more than ALD muscle fibres. Although in HC-3 treated embryos nerve-muscle contacts were formed, the axon terminals look immature and remain small even in 18-day old embryos at both ALD and PLD muscle fibres. The reduction of the number of axon profiles normally seen at 18 days failed to take place in treated embryos. At 18 days of incubation many endplates on PLD muscle fibres showed little sign of postsynaptic specilization and resembled endplates usually seen at this stage on ALD muscle fibres.
It is concluded that while neuromuscular activity may be important for the reduction of the number of axon profiles at individual endplates, the specialization of the subsynaptic membrane is brought about by depolarizing effect of ACh.     

Histochemical differentiation of extrafusal muscle fibres of the anterior latissimus dorsi in the chick

Histochemical differentiation of the chick anterior latissimus dorsi (ALD) muscle was studied during embryonic development and after hatching. The two types of adult ALD tonic fibres (alpha' and beta') differentiate from a pool of acid and alkali-stable myofibrillar ATPase fibres. Intermediate stages of the transformation from beta' to alpha' were observed. At all developmental stages studied, a low percentage of formalin-resistant, alkali-stable and acid-labile ATPase fibres were observed. Such fibres have the histochemical properties of the alpha R or fast oxidative-glycolytic fibres and are assumed to be focally innervated.     

Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.     

Formation of primary and secondary myotubes in rat lumbrical muscles

Numbers of myoblasts, primary myotubes and secondary myotubes in developing rat embryo hindlimb IVth lumbrical muscles were counted at daily intervals up until the time of birth, using electron microscopy. Motoneurone death at the spinal cord level supplying the lumbricals was assessed by counting axons in the 4th lumbar ventral root. Death of the motoneurones that supply the intrinsic muscles of the hindfoot was monitored by comparing the timecourse of development of total muscle choline acetyltransferase activity in control embryos with that in embryos where motoneurone death was inhibited by chronic paralysis with TTX, and by counting axons in the mixed nerve trunks at the level of the ankle at daily intervals. Condensations of undifferentiated cells marking the site of formation of the muscle were seen on embryonic day 15 (E15). Primary myotubes began to appear on E16 and reached a stable number (102 +/- 4) by E17. Secondary myotubes first appeared two days later, on E19, and numbered 280 at the time of birth (E22). The adult total of about 1000 muscle fibres, derived from both primary and secondary myotubes, was reached at postnatal day 7 (PN7) so considerable generation of secondary myotubes occurred after birth. There was a linear correlation between the number of undifferentiated mononucleate cells in a muscle and the rate of formation of secondary myotubes. The major period of motoneurone death in lumbar spinal cord was during E16-E17, when axon numbers in the L4 ventral root fell from 12,000 to 4000, but a discontinuity in the curve of muscle ChAT activity versus time indicated that death in the lumbrical motor pool occurred during E17-E19, after all primary myotubes had formed and before generation of secondary myotubes began. We suggest that motoneurone death, by regulating the final size of the motoneurone pool, regulates the ratio of secondary to primary myotube numbers in a muscle.     

The adult fast isozyme of myosin is present in a nerve-muscle tissue culture system

Abstract. Organotypic nerve-muscle cultures were prepared from foetal mouse spinal cord and adult mouse muscle fibres. In this system, the adult fibres degenerate and new myotubes form. The regenerated muscle fibres become innervated, develop cross-striations, and survive for several months. We have investigated the isozymes of myosin present in these muscle fibres using histochemistry and immunocytochemistry with antibodies to rat embryonic, neonatal, and adult fast myosins. We demonstrate that some of the regenerated fibres contain adult fast but not embryonic or neonatal myosin. This is the first demonstration of the production of adult myosin heavy chain in tissue culture. This system therefore offers the possibility for further study of the development of adult myosin isoforms in vitro.     

The adult fast isozyme of myosin is present in a nerve-muscle tissue culture system

Organotypic nerve-muscle cultures were prepared from foetal mouse spinal cord and adult mouse muscle fibres. In this system, the adult fibres degenerate and new myotubes form. The regenerated muscle fibres become innervated, develop cross-striations, and survive for several months. We have investigated the isozymes of myosin present in these muscle fibres using histochemistry and immunocytochemistry with antibodies to rat embryonic, neonatal, and adult fast myosins. We demonstrate that some of the regenerated fibres contain adult fast but not embryonic or neonatal myosin. This is the first demonstration of the production of adult myosin heavy chain in tissue culture. This system therefore offers the possibility for further study of the development of adult myosin isoforms in vitro.     

Effects of electrically induced contractile activity on cultured embryonic chick breast muscle cells

Development of chicken breast muscle is characterized by the sequential appearance of six electrophoretically distinct myosin heavy chain (HC) isoforms. Cultured secondary myotubes, derived from 12-day embryonic chick breast muscle, mainly express the early embryonic HC isoform HCemb/e, normally present in 8-day embryonic breast muscle, and the two fast light chain isoforms LC1f and LC2f. Direct low-frequency (2.5 Hz) stimulation of these myotubes via platinum electrodes leads to a shift in myosin HC expression with increases in the late embryonic HC isoform HCemb/l amounting to 35% of total HC in 19-day-stimulated cultures. Measurements of 35S-methionine incorporation and immunohistochemical analyses demonstrate increases in LC3f. This increase is also seen at the mRNA level. These results indicate that induced contractile activity promotes myotube maturation in vitro. The observation that chronic stimulation enhances the expression of the slow isoform LC2s at the RNA, as well as the protein level, suggests an additional effect consisting of a fast-to-slow change in phenotype expression. In view of the fact that muscle maturation and phenotype expression is under neural control during development in vivo, our results on directly stimulated, aneural myotubes indicate that neurally transmitted contractile activity may be an important factor in modulating phenotype expression of secondary myotubes.     

Differentiation of slow and fast muscles in chickens

1. The development of the characteristic histochemical appearance of the slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) was studied in chickens during embryonic development as well as during regeneration of minced muscle. 2. During embryonic development the activity of the oxidative enzyme succinic dehydrogenase (SDH) is higher in the slow ALD muscle already at 16 days of incubation. At this time the fast PLD has a higher activity of the glycolytic enzyme, phosphorylase. Although the histochemical appearance of the two types of muscle is already different at 16 days, their contractile speeds are still similar. No difference in myosin ATP-ase was found in the two muscles in young embryos but in 20-day old embryos the two muscles became distinctly different when stained for this enzyme. 3. When PLD muscles in hatched chickens redeveloped during regeneration in place of ALD the histochemical characteristics of the regenerated muscle resembled ALD, and when ALD regenerated in place of PLD it resembled PLD. 4. It is concluded that the histochemical characteristics of slow and fast muscles become determined during early development, even before any difference in contractile properties can be detected and that they are determined by the nerve.     

Characterization of a myosin heavy chain in the conductive system of the adult and developing chicken heart

A monoclonal antibody (anterior latissimus dorsi 58 [ALD58]; antimyosin heavy chain, MHC) directed against myosin from slow tonic muscle was found to react specifically with the striated muscle cells of the conductive system in the adult chicken heart. This monoclonal antibody was used to study the expression of myosin in the conductive system of the adult and developing heart. Using immunofluorescence microscopy with ALD58, muscle cells of the conductive system were demonstrated in both the atria and ventricles of the adult heart as previously shown by Sartore et al. (Sartore, S., S. Pierobon-Bormioli, and S. Schiafinno, 1978, Nature (Lond.), 274: 82-83). Radioactive myosin from adult atria and ventricles was precipitated with ALD58 and subjected to limited proteolysis and subsequent peptide mapping. Peptide maps of ALD58 reactive myosin from atria and ventricles were very similar, if not identical, but differed from peptide maps of ordinary atrial and ventricular myosin. The same antibody was used to study cardiac myogenesis in the chick embryo. When ALD58 was reacted with myosin isolated from atria and ventricles at selected stages of development in radioimmunoassays, reactivity was not observed until the last week of embryonic life (greater than 15 d of egg incubation). Thereafter concomitant and progressively increased reactivity was observed in atrial and ventricular preparations. Also, no ALD58 positive cells were observed in immunofluorescence studies of embryonic hearts until 17 d of egg incubation. Primary cell cultures of embryonic hearts also proved to be negative for this antibody. This study demonstrates that an epitope recognized by ALD58 associated with an antimyosin heavy chain of striated muscle cells of the adult heart conductive system is absent or present in only small amounts in the early embryonic heart.     

A critical period for formation of secondary myotubes defined by prenatal undernourishment in rats

Rats fed a restricted diet during gestation and lactation gave birth to pups with about 60% the normal birthweight. Maintaining the undernutrition after birth reduced the rate of growth of the pups so that their body weights were only 40% of control at PN7. Soleus and lumbrical muscles in these animals had reduced numbers of muscle fibres, and quantitative examination of embryonic muscles revealed that this was due solely to a decreased formation of secondary myotubes; the number of primary myotubes remained normal. Undernutrition did not affect the number of motoneurones surviving normal developmental death. Restoration of normal dietary intake on E21, one day before birth, did not correct the deficit in muscle fibre numbers in soleus muscles examined when the animals reached one month of age. Development of the lumbrical muscle lags behind the soleus and unrestricted feeding from E21 onwards allowed a normal number of fibres to develop from this time on, although the initial deficit was never restored. These experiments define a critical period in muscle development during which the potential maximum number of secondary myotubes is determined.