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Fetal myosin heavy chains in regenerating muscle.

There are several lines of evidence for the existence of a distinct class of myosins in developing muscle. Using various biochemical and immunological approaches, Whalen et al. recently suggested that two myosin heavy chain isozymes appear sequentially in rat muscle development, preceding the definitive adult myosins. It is unknown whether these myosins are present only in developing fast muscles or whether they also occur in developing slow muscles. Pyrophosphate of gel electrophoresis studies have suggested that fast-twitch and slow-twitch muscles synthesize the same fetal myosin isozymes early in development. Immunocytochemical studies with antibodies directed against adult fast and slow myosins show differences in myosin composition between fetal muscle fibres but interpretation of these findings is complicated by cross-reactions of these antibodies with fetal isomyosins. We have used a more direct immunocytochemical approach to identify the myosin types present in developing muscle fibres. An antibody specific for bovine fetal myosin and cross-reactive with rat fetal myosin has been prepared. We report here that the fetal myosin heavy chains recognized by this antibody show a heterogeneous fibre distribution in fetal and neonatal rat muscle, disappear progressively during postnatal development and are transiently expressed in regenerating muscle.

Animals↗

Connective tissue metabolism in muscular dystrophy. Early amino acid changes in collagen types isolated from the gastrocnemius muscle of developing dystrophic chicken embryos.

The amino acid composition of all collagen types present in the gastrocnemius muscle of dystrophic chick embryos showed an altered profile at both day 14 and day 20 in ovo when compared with the controls. The changes observed at both day 14 and day 20 in ovo suggests that there is a removal of polar side-chains in dystrophic collagen and substitution with non-polar amino acids. The amino acid composition data between day 14 and day 20 indicated: (a) a decrease in hydroxylation (hydroxyproline and hydroxylysine) with a concurrent increase in proline and lysine and a decrease in the levels of arginine; (b) the levels of glycine and alanine did not change with age; and (c) the ratios of glycine to hydroxyproline and proline to hydroxyproline changed significantly in all dystrophic collagen types between day 14 and day 20. Contrast analysis results clearly showed that the changes in amino acid composition observed in each dystrophic type of collagen between day 14 and day 20 were not due to the effect of aging but to some other factor(s). This study provides more evidence that a problem lies in the biosynthesis of collagen present in developing muscles of dystrophic chick embryos, particularly with respect to the transcription or translation of procollagen genes and/or a failure in the processing and differentiation of collagen types.

Amino Acids↗

Expression of fast myosin heavy chain transcripts in developing and dystrophic chicken skeletal muscle.

The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dotblot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation.

Animals↗

Changes in galactosyltransferase activity in chick pectoral muscle during embryonic development.

The two major vertebrate galactosyltransferases have been investigated in developing chick muscle in ovo and in vitro, and in cultured chick fibroblasts. The two enzymes were UDP-galactose-N-acetylglucosamine galactosyltransferase (galactosyltransferase I) and UDP-galactose-N-acetylgalactosamine galactosyltransferase (galactosyltransferase II). Both activities fell during muscle development in ovo. Galactosyltransferase I activity was constant from day 7 to day 16, after which it declined 5-fold, whereas galactosyltransferase II activity fell markedly from day 9 to 13 and 16 to 20, displaying an overall 8-fold decrease. In primary muscle cultures, galactosyltransferase I activity fell slightly during 7 days in culture, whereas galactosyltransferase II increased 2-fold during the same period. No significant change in activity of either galactosyltransferase was observed during intercellular recognition and fusion. Analysis of muscle cultures treated with cytosine arabinoside and of fibroblast cultures revealed that the majority of galactosyltransferase I activity in primary muscle cultures is associated with fibroblasts, whereas the majority of galactosyltransferase II activity is muscle-associated. The addition of 5-bromodeoxyuridine to primary muscle cultures resulted in a 3-fold rise in activities of both transferases.

Animals↗

[Myosin isoforms of skeletal muscles in development].

The latest data are reviewed concerning identification of myosin from the skeletal muscle during embryonic and postnatal development in vertebrates. The data are given on the composition of light subunits and specificity of heavy chains of the early isoforms obtained by electrophoresis, peptide mapping, DNA-RNA hybridization, as well as immunological methods with poly-and monoclonals. The substitution of embryonic heavy chains by neonatal and definitive ones is discussed. The following items are also considered: early isoforms of the fast and slow myosin types and, in particular, endogenous program directing the muscle development and protein synthesis towards the "fast phenotype", which is modulated by neurostimulation and other physiological factors inducing slow myosin type. The enzymatic activity of the early isoforms and its physiological importance in embryogenesis are discussed.

Animals↗

The fine structure of developing locomotor muscles of the pelagic tunicate, cyclosalpa affinis (Thaliacea: Salpidae).

Salps are free-swimming tunicates whose peculiar life history renders them ideal for developmental studies. The solitary salp reproduces asexually by budding a stolon containing the complete developmental sequence of the aggregate generation. The ultrastructure of developing locomotor muscle of the aggregate generation of Cyclosalpa affinis was studied. The early muscle contains essentially non-striated myofibrils. However, in transverse sections, , areas indicating early I-band A-bands can be recognized. As development continues, the number of fibrils increases, the Z-line appear, and the fibrils contain more recognizable striations. The fully developed muscle has the caracteristic structure of striated muscle. Longitudinal sections show sarcomeres with irregular and discontinuous (perforated) Z-lines; H-zones are not apparent. No M-lines are seen. Throughout development, the ratio of thin to thick myofilaments is always 2:1, the ratio found in all vertebrate striated muscle. Other finding in C affinis suggest that: (1) multinucleated muscle cells are formed by the fusion of mononucleated cells, (2) membranes of adjacement mononucleated cells destined to fuse form myelin figures, and (3) these myelin figures become closely associated with mitochondria.

Animals↗

Muscle abnormalities in coeliac disease: studies on gross motor development and muscle fibre composition, size and metabolic substrates.

In 11 children with coeliac disease gross motor development was assessed before and during diet treatment using the gross motor subscale of the Denver developmental screening test. ATP, creatine phosphate (CP), glycogen and lactate concentrations, muscle fibre size and fibre composition were measured in specimens obtained by needle biopsy from the vastus lateralis muscle. Before treatment, gross motor development was delayed. ATP, and to a lesser extent, CP and glycogen concentrations were lowered compared to a control group. After treatment, gross motor development was normal and no differences in ATP, CP or glycogen concentrations were found compared to the control group. Fibre size seemed unaffected by the disease. The percentage of type 1 fibres was significantly lower before treatment, compared to values obtained during treatment and from the control group. Whether these metabolic changes were due to the coeliac disease per se or the inactivity which it causes was not possible to establish. In humans, only altered neurogenic influence on the muscles has been previously shown to give changes in fibre composition.

Adenosine Triphosphate↗

Mechanism for substance P-induced relaxation of precontracted airway smooth muscle during development.

Release of substance P (SP) from sensory nerve endings of the tracheobronchial system modulates airway smooth muscle contraction and may cause relaxation of precontracted airways. We sought to elucidate the effect of postnatal maturation on SP-induced relaxation of precontracted airways and determine the roles of endogenously generated nitric oxide (NO) and prostaglandins (PGs). Cylindrical airway segments were isolated from the midtrachea of rats at four different ages, 1, 2, and 4 wk and 3 mo, and contracted to 50-75% of the maximum response induced by bethanechol. SP was then administered in the absence and presence of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the PG inhibitor indomethacin, or both. Relaxation of airways with SP decreased significantly with advancing postnatal age. SP-induced tracheal relaxation was consistently attenuated by pretreatment with L-NAME, indomethacin, or both. In a different group of animals, L-NAME significantly attenuated the relaxant response of airways to PGE2 exposure, but indomethacin had no significant effect on the relaxant response to exogenous NO. We conclude that SP induces a relaxant effect on precontracted airway smooth muscle, which decreases with advancing age and is mediated via SP-induced release of NO and/or PG.

Aging↗

Muscle acetylcholine receptor biosynthesis. Regulation by transcript availability.

The expression of the muscle nicotinic acetylcholine receptor (AChR) on the cell surface entails a complex biosynthetic pathway, involving the expression and assembly of four subunits. The amount of AChR on the cell surface changes throughout muscle development and upon muscle denervation. We have examined the regulatory role of transcript levels on surface AChR expression by RNA blot analysis. During myogenesis of the fusing mouse muscle cell line C2, which expresses an embryonic type of receptor, changes in the rate of appearance of cell surface AChR have been assayed by 125I-alpha-bungarotoxin binding. The maximal increase in the rate of appearance of cell surface AChR occurs upon cell fusion, closely following the maximal increase in transcript levels for the alpha-, beta-, gamma-, and delta-AChR subunits. AChR alpha-, beta-, gamma-, and delta-subunit gene transcript levels have also been examined in innervated and denervated rat and mouse muscle. Muscle denervation results in an increase of transcripts for all four subunits. However, the amount of beta-subunit transcript in innervated rat skeletal muscle is high relative to the other subunit transcripts, and increases less than the other subunit transcripts upon denervation. Our results indicate that, during myogenesis and upon denervation, the availability of AChR subunit transcripts for translation plays a regulatory role in surface AChR appearance.

Animals↗

Changes in the distribution of slow skeletal myosin heavy chain SM1 in developing avian muscle fibres.

The use of monoclonal antibodies against fast skeletal and slow skeletal myosin heavy chains (MHC) has shown the presence of significant amounts of slow skeletal type MHC in embryonic skeletal muscles of white leghorn chickens. The presence of this slow skeletal myosin heavy chain (SMHC) was not restricted to presumptive slow muscles only, as it was also observed in presumptive fast skeletal muscles. As was the case for embryonic MHC reactive with the antibody against fast skeletal myosin heavy chain (FMHC), the presence of SMHC could be detected at the earliest stages of myogenesis. It appeared to be present in most muscle cells during early embryonic development. The changes in its cellular distribution during subsequent embryonic and post-hatch period indicated its suppression in a certain proportion of the cells in both presumptive fast and slow skeletal muscles. Its time course of suppression, however, was much prolonged, not synchronized, and varied in fast and slow skeletal muscles during both embryonic and post-hatch development.

Animals↗

Ca+2-accumulating components in developing skeletal muscle.

This ultrastructural study on the localization of Ca+2 in developing skeletal muscle indicates that the formation of calcium-accumulating components begins during embryonic development. Both oxalate and pyroantimonate techniques are used to localize Ca+2 in distinct cellular components of chick pectoral and sartorius muscles. Two major sites for Ca+2 accumulation are present in ultrathin sections of embryonic and post-embryonic muscles: the terminal cisternae of the sarcoplasmic reticulum and specific lines in the I-bands. Calcium oxalate-accumulating vesicles are present in the smallest recognizable myotubes at the twelfth day of incubation, but calcium-accumulating components are not seen at myofibrillar I-band sites until the fourteenth to seventeenth days of incubation. The fact that myofibrils first form and later in development accumulate a Ca+2-binding component suggests that this Ca+2-binding component is not necessary for the formation of myofibrils, but is added to myofibrils before hatching to serve a probable regulatory role in contraction.

Age Factors↗

The effects of tenotomy and overload on the postnatal development of muscle fibre histochemistry in the cat triceps surae.

Five to seven day-old kittens were subjected to partial tenotomy of the Achilles (triceps surae) tendon. The effects of tenotomy and overload on the development of muscle fibre histochemistry and fibre sizes were investigated when the cats had reached the adult stage. The examined muscles were the uniform soleus and the mixed medial gastrocnemius. Tenotomized muscles of both types had lower weights than their controls. Tenotomized soleus showed a redistribution of succinic dehydrogenase (SDH) activity, together with signs of muscle fibre death. Tenotomized medial gastrocnemius muscles displayed more severe degenerative signs than the soleus, together with signs of fibre death. The fibre death seemed to affect mainly fibres of type IIb. Moreover, a less distinct differentiation in histochemical staining pattern between muscle fibre types was found in these muscles. Overloaded soleus muscles had greater weights than their controls, while no difference could be shown for the overloaded medial gastrocnemius. The overloaded medial gastrocnemius showed a uniform hypertrophy of all fibre types. Also, overloaded soleus showed a uniform hypertrophy. Both types of muscle showed a normal histology as well as normal staining characteristics (SDH and AcATPase). It is concluded that both soleus and medial gastrocnemius are sensitive to loss of muscle tension during development. The basic features of muscle morphology and histochemistry were normal, though, and it is suggested that other factors account for most of the normally occurring development and differentiation.

Achilles Tendon↗

Early post-hatch fasting induces satellite cell self-renewal.

Early post-hatch satellite cell kinetics are an important aspect of muscle development, and understanding the interplay between fasting and muscle development will lead to improvements in muscle mass following an illness, and optimal meat production. The objective of this experiment was to test the influence of immediate post-hatch fasting on satellite cells in the poult. Male Nicholas poults (Meleagris gallopavo) were placed into two treatments: a fed treatment with immediate access to feed and water upon placement and a fasted treatment without access to feed and water for the first three days post-hatch. 5-bromo-2'-deoxyuridine (BrdU) was injected intra-abdominally in all poults to label mitotically active satellite cells. The pectoralis thoracicus muscle was harvested two hours following the BrdU injection. Immunohistochemistry for BrdU, Pax7, Bcl-2, Pax7 with BrdU, and determining myofiber cross-sectional area along with computer-based image analysis was used to study muscle development. Fed poults had higher body masses throughout the experiment (P< or =0.01), and they had higher pectoralis thoracicus muscle mass (P< or =0.01) at ten days of age than the fasted poults. Fed poults had higher satellite cell mitotic activity at three days and four days of age (P< or =0.01) compared to the fasted poults. However, Pax7 labeling index was higher in the fasted poults (P< or =0.01) at three days, four days, and five days post-hatch than the fed group. Similarly Bcl-2 labeling was higher in the fasted than in the fed group at three days post-hatch. Therefore, fasting depleted proliferating satellite cells indicated by the lower BrdU labeling in the fasted poults compared to the fed poults, and conserved the satellite cell proliferative reserve indicated by the higher level of Pax7 labeling for the fasted poults compared to the fed poults.

Animal Nutritional Physiological Phenomena↗

The developing human muscle: ultrastructural differences between myoblasts and fibroblasts.

Material from foetuses of nine weeks to nine months development was examined by electron microscopy to obtain some information about the various stages in the development of skeletal muscle and structures which are intimately related to it, such as blood vessels, nerves, neuromuscular spindles, fibroblasts and their product, i.e., collagen. Regarding the myoblasts and fibroblasts it was interesting to compare their ultrastructural features since there has been some difficulty in the identification of these two types of mononucleated cells by the authors which deal with light microscopy in the study of developing skeletal muscle. It was possible to note that between nine and eighteen weeks of development the muscle is different from mature skeletal muscle and is composed predominantly of two populations of muscle cells. These are the myoblasts which are mononucleated cells and the myotubes which are multinucleated muscle cells. After eighteen weeks of development there occur only muscle fibres similar to those of adult muscle. Fibroblasts also occur frequently in the developing muscle: they too are mononucleated cells. Fibroblasts and collagen fibrils are seen between the muscle cells and structures related to them as early as nine weeks of development. Bundles of collagen were also seen in the cytoplasm of fibroblasts.

Collagen↗

The importance of competition between motoneurones in developing rat muscle; effects of partial denervation at birth.

1. The number of motor units in developing fourth deep lumbrical muscles was reduced by unilateral partial denervation of the muscle at birth, by cutting the lateral plantar nerve. A minority of the motor axons arrive via the sural nerve, and were thus not cut. Those muscles that contained one motor unit (one-unit muscles) after partial denervation developed in the absence of competition between motoneurones. Muscles with two motor units had little competition. A few four-unit muscles were studied for comparison. 2. Isometric twitch and tetanic tensions of single motor units were recorded in vitro at 60 days of age in response to stimulation of the sural nerve. On average, units in partially denervated muscles generated more tension than normal units. The isometric tension characteristics of the units in the one-unit and two-unit muscles were different from the normal units (e.g. slower contracting and more fatiguable). The units of four-unit muscles had properties similar to those of normal muscles. 3. Fibres of an individual unit were identified by glycogen depletion and S (slow) fibres were identified in cross-section that bound a polyclonal antibody to slow type I myosin. Those fibres that did not bind the antibody were designated F fibres. The units of one-unit muscles had the same total number of fibres and fibre type composition (both S and F fibres in the same unit) as estimated from previous work to exist at birth. The units of two-unit muscles contained the same total number of fibres, but apparently fewer S fibres, though this may have been as a result of incomplete glycogen depletion of some fibres. 4. It is concluded that competition between motoneurone terminals is necessary for the withdrawal of mismatched connections on muscle fibres present at birth; or, alternatively, that such withdrawal cannot take place if it would result in denervation of the muscle fibre.

Animals↗

Myoblast fusion is not a prerequisite for the appearance of calcium current, calcium release, and contraction in rat skeletal muscle cells developing in culture.

During in vitro development of rat skeletal muscle cells, contraction and calcium currents progressively appear after fusion of myoblasts. To investigate whether muscle-specific functions are expressed in the absence of myoblast fusion, rat neonatal muscle cells were cultured in a differentiation medium under conditions that are well known to inhibit fusion: prolonged culture in a low-calcium medium or treatment with cytochalasin B. We have demonstrated that the fusion-arrested cells expressed differentiative properties in L-type calcium current, transient release of calcium ions from internal stores in response to caffeine and depolarizing agents, and contraction elicited by depolarization. Properties and potential-dependence of L-type calcium currents were similar to that in control fused cells, but T-type calcium currents were not observed, while both types coexist in myotubes. Properties of calcium transients and voltage dependence of contraction suggested that the excitation-contraction mechanisms were well established. However, comparing to well-developed myotubes at the same time of culture, the characteristics of calcium transients and contraction of fusion-arrested cells were closer to those of younger myotubes, which can be interpreted in terms of a delay in maturation of excitation-contraction coupling and contractile machinery. All these observations demonstrate that myoblast fusion is not necessary for triggering the establishment of calcium transport and release and contractile functions of rat muscle cells developing in culture. The appearance of muscle-specific functions is consistent with previous results demonstrating that the fusion-arrested cells express muscle-specific proteins and structures.

Acetylcholine↗

Investigating the myopathic effects of 6-mercaptopurine on developing skeletal muscle cells in vitro.

6-Mercaptopurine (6-MP) is an adenine antagonist which has been shown to cause skeletal muscle atrophy in neonatal rats. To investigate the effects of 6-MP on developing muscles, pure populations of myoblasts and myotubes in vitro were treated with 6-MP (5-50 micrograms/ml). The viability and protein content of myotubes, but not myoblasts, was decreased by 6-MP. Microscopic examination of the 6-MP-treated (50 micrograms/ml) myotubes demonstrated that severe degenerative changes had occurred, including extensive necrosis, inhibition of myotube formation, and the appearance of intracellular vacuoles. However, 6-MP treatment decreased the incorporation of radiolabeled leucine and thymidine by myoblasts, but not myotubes. These findings indicate that 6-MP exerted a selective toxicity on myotubes but not myoblasts.

Animals↗

ERK6 is expressed in a developmentally regulated manner in rodent skeletal muscle.

Rat ERK6, also known as SAPK3 and p38gamma, exhibits a distinct pattern of expression during muscle development in vitro and in vivo. Levels of mRNA transcript and protein abundance for ERK6 are increased during the differentiation of 2 rodent myoblast cell lines in culture. This is in contrast to the expression of other MAP kinase family members, namely p42/p44 MAPK and p38 MAPK, whose expression does not change during myogenesis. Similar results are observed in vivo where ERK6 mRNA levels increase with post-natal development in rat hindlimb mixed muscle samples. These results delineate a distinct pattern of ERK6 expression in mature skeletal muscle cells and suggest a specific role for ERK6 in muscle development or muscle function.

Animals↗