Expression of the Na(+)/Ca(2+) exchanger in skeletal muscle.

The expression of the Na(+)/Ca(2+) exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na(+)/Ca(2+) exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.

Staging of the commitment of murine cardiac cell progenitors.

The staging of murine cardiomyocyte specification and determination was investigated in cultures of tissue explants from pre- and postgastrulated embryos and after transplantation of cardiac or cardiogenic tissues from mouse embryos into 2-day-old chick embryos in different locations. The development of transplanted and cultured cells in cardiomyocytes was evaluated by testing the expression of several cardiac transcription factor genes (Nkx 2.5, eHAND, dHAND, GATA-4), alpha-cardiac actin mRNA, and beta-myosin heavy chain protein. In vitro analyses showed that cells with the potential to form cardiac muscle were present prior to gastrulation in 6.5-day postconception (dpc) epiblasts, as indicated by the expression of Nkx 2.5, eHAND, dHAND, and GATA-4 cardiac transcription factors; desmin transgene; alpha-cardiac actin; and beta-myosin heavy chain. Conversely, epiblasts transplanted into the chicken somitic environment did not exhibit full cardiogenic cell differentiation. It was determined that chick host axial structures did not influence cardiogenesis in transplants. Mesoderm from late streak explants was capable of differentiating into the cardiac phenotype in the avian heterotopic environment, indicating that the specification of cardiac precursors (under way by 6.5 dpc) became irreversible at around the late streak stage in mouse embryo. Although in vitro analyses showed that interaction with endoderm is not required for the specification of murine cardiac cells, the presence of endoderm in explant cultures between mid- and late streak stages stimulated emerging mesodermal cells to adopt a myocardial pathway, whereas ectoderm had no influence on cardiomyogenesis.

Macrophages enhance muscle satellite cell proliferation and delay their differentiation.

This study investigated the effect of macrophages on in vitro satellite cell myogenesis in the turkey and mouse. Macrophages are considered to act as scavengers of tissue debris during the muscle degeneration-regeneration process. The number of dividing cells and of myoblasts expressing the myogenic regulatory factor MyoD indicated that macrophages enhanced satellite cell proliferation in both species. This was confirmed by observations with cultures treated for bromodeoxyuridine (BrdU) incorporation. In mouse and turkey macrophage-satellite cell cocultures, the number of differentiated myoblasts, the frequency of myogenin-positive cells, and the expression of developmental myosin isoforms were reduced as compared with control cultures, indicating that macrophages delayed satellite cell differentiation. The possibility that macrophages facilitate muscle fiber reconstitution by enhancing satellite cell proliferation should be taken into consideration in designing future strategies of satellite cell transplantation as a treatment for muscular dystrophies.

Comparative analysis of satellite cell properties in heavy- and lightweight strains of turkey.

The growth of muscle during postnatal development results partly from the proliferation of satellite cells and their fusion with muscle fibres. We analysed the properties of satellite cells in a heavyweight (HW) turkey strain characterized by high body weight and a fast growth rate, and in a lightweight farm strain (LW) characterized by low body weight and a slow growth rate. Satellite cell activation was then examined in stretched-overloaded anterior latissimus dorsi (ALD) muscle by weighting one wing in young turkeys from both strains. As early as day 1 of stretching for HW and day 2 for LW, small embryonic-like fibres expressing ventricular cardiac myosin heavy chain (MHC) isoform were observed. Following four days of stretching, the number of nascent fibres had increased in both strains but was significantly greater in HW than LW ALD muscle. The proliferation and differentiation capacities of satellite cells from HW and LW strains were investigated in culture. As judged by in vitro measurements of 3H-thymidine incorporation and DNA content, satellite cells of HW turkey exhibited a greater proliferative capability than those of LW turkey. No differences in the temporal appearance of muscle markers (desmin, MHC isoforms) were noted in vitro between the two strains. These data confirm our in vivo observations indicating that selection based on growth rate does not modify muscle fibre maturation. Our in vivo and in vitro observations suggest that variations in the postnatal muscle growth pattern between HW and LW strains may be related to a difference in the capacity of their satellite cells to proliferate.

Innervation regulates myosin heavy chain isoform expression in developing skeletal muscle fibers.

The influence of innervation on primary and secondary myogenesis and its relation to fiber type diversity were investigated in two specific wing muscles of quail embryo, the posterior (PLD) and anterior latissimus dorsi (ALD). In the adult, these muscles are composed almost exclusively of pure populations of fast and slow fibers, respectively. When slow ALD and fast PLD muscles developed in ovo in an aneurogenic environment induced after neural tube ablation, the cardiac ventricular myosin heavy chain (MHC) isoform was not expressed. The adult slow MHC isoform, SM2, appeared by embryonic day 7 (ED 7) in normal innervated slow ALD but was not expressed in denervated muscle. Analysis of in vitro differentiation of myoblasts from fast PLD and slow ALD muscles isolated from ED 7 control and neuralectomized quail embryos showed no fundamental differences in the pattern of MHC isoform expression. Newly differentiated fibers accumulated cardiac ventricular, embryonic fast, slow SM1 and SM3 MHC isoforms. Nevertheless, the expression of slow SM2 isoform in myotubes formed from slow ALD myoblasts only occurred when myoblasts were cultured in the presence of embryonic spinal cord. Our studies demonstrate that the neural tube influences primary as well as secondary myotube differentiation in avian forelimb and facilitates the expression of different MHC, particularly slow SM2 MHC gene expression in slow myoblasts.

Selection for rapid growth increases the number and the size of muscle fibres without changing their typing in chickens.

Quantitative (muscle fibre number and cross-sectional areas) and qualitative (myosin isoforms and metabolic enzyme activities) characteristics of two muscles, M. pectoralis major and M. anterior latissimus dorsi, were compared among male chickens of two lines during growth from hatching to adulthood. The lines were derived from a divergent selection based on growth rate. The two muscles were chosen on the basis of their histochemical profile. Pectoralis major muscle contains only fast contracting muscle fibres whereas anterior latissimus dorsi muscle is almost entirely made up with slow contracting fibres. At both ages, the two lines showed similar fibre type distributions. At hatching, fibre cross-sectional areas were equivalent in the two lines, but after the first week, animals from the fast growing line exhibited wider fibre areas, whatever the muscle, than animals from the slow growing line. The total number of fibres in a muscle was found greater in the fast growing line, irrespective of whether it was exactly determined (anterior latissimus dorsi muscle, + 20%) or only estimated (pectoralis major muscle). This number remains constant in the two lines throughout the growth. Myosin isoform profiles and metabolic enzyme activities were similar in the two lines, at both ages, and were in good agreement with the histochemical muscle fibre profiles.

Neural tube can induce fast myosin heavy chain isoform expression during embryonic development.

We investigated the role of the neural tube in muscle cell differentiation in developing somitic myotome of chick embryo, particularly through fast myosin heavy chain (MHC) isoform expression. An embryonic fast MHC labeled with EB165 mAb was expressed in somitic cells from stage 15 of Hamburger and Hamilton (H.H.) (24 somites). Moreover, a distinct early embryonic fast MHC was expressed only from stage 15 of H.H. to stage 36 (E10). Like neonatal MHC, this isoform was labeled with 2E9 mAb but differed in its immunopeptide mapping. Expression of EB165-labeled embryonic fast MHC occurred in somitic myotomes deprived of neural tube influence by in ovo ablation as well as in somite explants cultured alone in vitro. Conversely, ablation of the neural tube prevented somitic expression of MHC labeled with 2E9 mAb. The neural tube induced in vitro expression of this MHC in explants of somites which failed to express it when cultured alone. These results indicate that signals emanating from the neural tube are required for the expression of early embryonic fast MHC isoform in developing somitic myotome.

Comparison of postnatal development of anterior latissimus dorsi (ALD) muscle in heavy- and light-weight strains of turkey (Meleagris gallopavo).

ALD muscle development was studied from day 2 to week 15 in males of two turkey strains. At 15 weeks, the heavy-weight (HW) strain weighted 2.2 times as much as the light-weight strain (LW). Morphometric and immunocytochemical analysis showed the presence of small fibers in HW ALD muscle which simultaneously accumulated ventricular and embryonic fast myosin heavy chain isoforms. The appearance of these nascent myofibers suggests that hyperplasia contributes to the growth of HW ALD muscle.

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.

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.

Role of nerve and tension in maturation of posthatching slow-tonic muscle in chicken.

The role of motor innervation and muscle tension in the posthatching maturation of the slow-tonic anterior latissimus dorsi (ALD) muscle of the chicken has been investigated. Modification of the muscle tension was obtained either by maintaining ALD in a shortened state or by stretching, after or without denervation. In denervated as well as in innervated ALD, shortening resulted in atrophy and inhibition of developmental change in muscle fiber population. In contrast, stretch causes hypertrophy, transformation of all 3B fibers, increase in SM2 isomyosin expression, and decrease in Ca2+-activated myosin ATPase in innervated or denervated ALD. On the other hand oxidative activity in ALD fibers was strikingly reduced after denervation even in presence of stretch-induced hypertrophy. This study suggests that a passive stretch can be involved in some, but not all, changes in ALD characteristics occurring after denervation and may be also involved in normal posthatching development of the slow-tonic muscle. Possible clinical implications of these results in relation to treatments for preventing muscle atrophy resulting from immobilization or disuse are suggested.

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.

[Innervation, activity rhythm and accumulation of myosin light chains in a fast muscle of chickens]. / Innervation, rythme d'activité et accumulation des chaînes légères de myosine d'un muscle rapide de poulet.

The fast posterior latissimus dorsi muscle (PLD) of the chick ceases to accumulate slow myosin light chains (MLC) during neonatal development. On day 18 of embryonic life slow MLC represented only 2% of total MLC, and LC3F was first detected. In chick embryo, spinal cord stimulation at a slow rhythm modifies PLD differentiation toward the slow type: LC3F did not accumulate and slow MLC increased. In contrast, stimulation at a fast rhythm accelerated LC3F accumulation. PLD denervation on day 2 after hatching inhibited the synthesis of LC3F. Direct stimulation at a fast rhythm led to post-hatching development into normal fast type while a slow rhythm influenced the development of denervated PLD towards the slow type. In innervated PLD, the effect of stimulation at a slow rhythm was less important than in denervated PLD. These results suggest that the rhythm of the neural and/or contractile activity plays an important role in the MLC expression during embryonic and post-natal development of the chicken fast muscle.

Effects of low and high frequency patterns of stimulation on contractile properties, enzyme activities and myosin light chain accumulation in slow and fast denervated muscles of the chicken.

The effects of denervation and direct stimulation in fast and slow latissimus dorsii muscles were investigated in chicken. In slow ALD muscle, denervation resulted in an incompleteness of the relaxation, a decrease in MDH and CPK activities and an increase in fast myosin light chains (MLC) accumulation. Direct stimulation at either fast or slow rhythm prevented the effects of denervation on relaxation and CPK activity but was ineffective on MDH activity and fast MLC accumulation. Moreover, direct stimulation of denervated ALD caused rhythm-dependent change in tetanic contraction. In fast PLD muscle, the main changes in muscle properties following denervation were a slowing down of the time course of the twitch and an incompleteness of the relaxation, a decrease in LDH and CPK activities and in LC3F accumulation. Stimulation at a high frequency partly prevented the effects of denervation and resulted in a large accumulation of LC3F, while a low frequency stimulation did not restore the twitch time to peak, increased MDH activity and induced synthesis of slow MLC. This study emphasizes the role of muscle activity and its pattern in some properties of slow and fast chicken muscles following denervation.

Influence of spinal cord stimulation upon myosin light chain and tropomyosin subunit expression in a fast muscle (posterior latissimus dorsi) of the chick embryo.

Latissimus dorsi muscles of the chick consist of a slow (ALD) and a fast (PLD) muscle. The influence of chronic spinal cord stimulation in the chick embryo upon the expression of myosin light chains and tropomyosin subunits was investigated. Early in development the two muscles exhibited the same ratio of alpha- and beta-tropomyosin subunits. Later, in the slow muscle the ratio beta:alpha decreased and in chicken the amounts of the two components were about the same. In the fast muscle, the alpha-subunit increased and reached 66% in young chicken. In the fast muscle, the alpha-subunit increased and reached 66% in young chicken. In the In the early stages of embryonic development, both muscles accumulated slow and fast light chains. However, in ALD the amount of slow light chains was greater than that of fast light chains and the reverse was observed in PLD muscle. Later during development, the slow components decreased in PLD while the fast components increased; the reverse was observed in ALD muscle. The fast myosin LC3f has been detected in 18-day-old embryonic PLD. Chronic spinal cord stimulation at a low rhythm was performed from day 10 of embryonic development to day 15 or 16. In both muscles from spinal cord-stimulated embryos, the beta-tropomyosin subunit was lower than in control embryos. In ALD, the pattern of light chains was unaffected by chronic stimulation while in PLD muscle the slow and fast components were modified. In particular the ratio LCs:LCf was increased in spinal cord-stimulated embryos with regard to controls.

Influence of neurons on the occurrence of fibre types and myosin light chains in cultured presumptive slow and fast myoblasts.

Myoblasts from 9-day-old quail embryo slow anterior latissimus dorsi (ALD) and fast posterior and latissimus dorsi (PLD) muscles were co-cultured with neurons. The presence of neurons allowed ALD-derived muscle fibres to express characteristic features of a slow muscle (occurrence of alpha' and of beta' fibres and predominance of slow myosin light chains). On the contrary, PLD-derived fibres did not differentiate into normal fast fibres (occurrence of alpha'-like fibres and absence of LC3f). These results are compared with the differentiation of ALD and PLD myoblasts in aneural condition. It is suggested that neurons can modify some phenotypic expression of presumptive slow or fast myoblasts.

Developmental change in choline acetyltransferase activity in nerve endings of latissimus dorsii muscles in the chick embryo: influence of chronic spinal cord stimulation.

Choline acetyltransferase (CAT) activity of chick latissimus dorsii muscles was studied during embryonic development and at post-hatching states. CAT activity was always higher in anterior (ALD) than in posterior (PLD) muscles. At embryonic stages, chronic spinal cord stimulation at a low rhythm did not modify CAT activity in ALD nerve endings but caused a transient increase in PLD terminals. This increase in CAT activity seems to be related to an acceleration of neuronal maturation rather than to the occurrence of the multiple innervation that results from the central stimulation.

Influence of chronic spinal cord stimulation upon differentiation of beta muscle fibers in a fast muscle (posterior latissimus dorsi) of the chick embryo.

The aim of this work was to study the influence upon differentiation of muscle fiber types of the multiple innervation induced in a fast muscle by chronic spinal cord stimulation. In previous work, we showed that low-frequency stimulation applied to the spinal cord of the chick embryo caused a distributed innervation of muscle fibers in the posterior latissimus dorsi (PLD). In normal development, some beta fibers differentiate within this muscle, the maximal number being attained by 14 to 15 days of embryonic development. Later, the numbers of beta fibers decreased with age. In spinal cord-stimulated embryos the beta muscle fibers within the PLD were stabilized and did not disappear. After the cessation of spinal cord stimulation, the number of beta fibers within the PLD muscle did not decrease. There are possible explanations of the influence of chronic spinal cord stimulation and muscular activity upon formation and persistence of beta fibers within a fast muscle.