Myotrophic factor(s) in normal and dystrophic chicken skeletal muscle.
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Animals↗
Biomedical subjects
R C Strohman
Publications and source records attributed to R C Strohman.
Ontologic perspective: terminal differentiation and maturation as differently regulated stages in muscle development.
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Cell Differentiation↗
Myosin expression during regeneration and in denervated skeletal muscle.
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Animals↗
Selected muscle and nerve extracts contain an activity which stimulates myoblast proliferation and which is distinct from transferrin.
Extracts from normal chicken anterior latissimus dorsi and dystrophic pectoralis major muscles and from normal chicken sciatic nerves induce a growth stimulation in chicken and rat myogenic cell cultures. Transferrin is only partially responsible for the observed stimulation since the addition of the extracts to transferrin-saturated cultures induces a further growth response and extracts from which transferrin has been removed by immunoabsorption still retain a substantial portion of their stimulation activity. The active fractions of muscle and nerve extracts display heat, acid, and organic solvent inactivation. Gel filtration of ammonium sulfate fractionated activity from the anterior latissimus dorsi muscle suggests the presence of a growth factor in the molecular weight range of 10,000 to 30,000.
Animals↗
Myogenic growth factor present in skeletal muscle is purified by heparin-affinity chromatography.
A myogenic growth factor has been purified from a skeletal muscle, the anterior latissimus dorsi, of adult chickens. In the range of 1-10 ng, this factor stimulates DNA synthesis as well as protein and muscle-specific myosin accumulation in myogenic cell cultures. Purification is achieved through binding of the factor to heparin. The factor is distinct from transferrin and works synergistically with transferrin in stimulating myogenesis in vitro.
Animals↗
Streptomycin retards the phenotypic maturation of chick myogenic cells.
As part of an effort to optimize conditions required for the complete maturation of muscle cells in vitro, we have investigated the effects of the antibiotics penicillin, streptomycin, and Fungizone (amphotericin B) on the development of cultured chick embryo skeletal muscle. It is shown that even low dosages of streptomycin, but not penicillin or Fungizone, retard protein synthesis and accumulation in these cultures. Myosin accumulation was also reduced and the appearance of striations in fused cells was delayed in myotubes formed in medium containing streptomycin. Additional data suggest that this overall retardation of myogenesis is due to the influence of streptomycin on maturing myotubes rather than early proliferation and cell fusion. These results are discussed with regard to recent efforts to promote the full maturation of muscle cells grown in culture.
Amphotericin B↗
There is selective accumulation of a growth factor in chicken skeletal muscle. I. Transferrin accumulation in adult anterior latissimus dorsi.
Chick embryo myoblasts in culture will respond to extracts of adult anterior latissimus dorsi muscle with an increase in cell number and an increase in total protein and in myosin heavy chain in fused myotubes. Extracts of adult pectoralis major and of posterior latissimus muscles are only marginally active. The active adult muscle extracts are fractionated by DEAE-cellulose column chromatography and transferrin is identified as the active component based on the following findings: (1) the active fractions are shown to contain an 80K protein that comigrates with chicken transferrin on SDS-PAGE, (2) the active extract from the anterior latissimus dorsi completely replaced embryo extract in the culture medium and supported normal myogenesis, (3) the active extract requires iron for its ability to support myogenesis, (4) the peptide map of the 80K protein is identical to a peptide map of transferrin. Under conditions where the 80K protein is detected in adult anterior latissimus dorsi muscles it is shown that the protein is nevertheless not synthesized in the muscle. These results support the idea that tissues of selective muscles in the adult chicken accumulate transferrin. An accompanying paper shows that transferrin also accumulates in early developmental stages of fast muscle tissue but that accumulation ceases after hatching in these muscles in normal chickens but not in animals of congenic strains with inherited muscular dystrophy.
Aging↗
There is selective accumulation of a growth factor in chicken skeletal muscle. II. Transferrin accumulation in dystrophic fast muscle.
Transferrin or a transferrin-like protein, with ability to stimulate myogenesis and terminal differentiation in vitro, is found in fast chicken muscle during embryonic development. After hatching, however, transferrin is no longer accumulated or is only weakly accumulated by fast muscles like the pectoralis major and the posterior latissimus dorsi but continues to be accumulated by slow muscles like the anterior latissimus dorsi. In congenic lines of chickens bearing the gene for muscular dystrophy, however, adult fast muscles do not lose the ability to accumulate transferrin. While transferrin is found selectively in adult normal and dystrophic muscle it does not appear to be synthesized by muscle cells. Immunocytochemical localization shows that transferrin is accumulated not so much by muscle fibers as it is by single cells in the muscle interstitial space. The relationship between transferrin presence and growth patterns in adult skeletal muscle is not currently understood but evidence suggests that transferrin stimulation of myogenesis observed in vitro may be mediated in vivo by non-muscle cells dwelling within the muscle interstitial space. These cells may act as transferrin-uptake sources for subsequent satellite cell stimulation.
Animals↗
Denervated skeletal muscle displays discoordinate regulation for the synthesis of several myofibrillar proteins.
Synthesis patterns of myosin heavy- and light-chain isoforms, tropomyosin and troponin, have been studied in chicken fast muscle denervated at both neonatal and adult stages. Denervated neonatal muscle does not synthesize the adult myosin heavy-chain isoform at the time of denervation, but it does synthesize the adult isoform several months after denervation. Thus, innervation does not appear to be necessary for the normal sequential replacement of embryonic and neonatal myosin heavy chain by the adult variant. Nerve is required, however, for the regulation of tropomyosin and troponin expression. Normally the pectoralis major muscle represses synthesis of both beta-tropomyosin and leg-type troponin T during late embryonic development. After denervation, however, the muscle relaxes its ongoing repression of these proteins and significant amounts of both beta-tropomyosin and leg-type troponin T are synthesized by the muscle. Denervation also results in an altered pattern of myosin light-chain synthesis so that the ratio of fast light-chain 3/fast light-chain 1 decreases. Similar results are found in muscle denervated at the adult stage. In denervated muscle, therefore, synthesis of these myofibrillar proteins is not coordinated: ongoing isoform shifts proceed to express an adult pattern of myosin heavy chain while tropomyosin, troponin, and myosin light-chain patterns appear to revert to embryonic configurations.
Aging↗
Human fetal muscle and cultured myotubes derived from it contain a fetal-specific myosin light chain.
Human fetal muscles at ages 110, 125, and 132 days contain a fetal-specific myosin light chain. This light chain is absent in adult human muscle, copurifies with myosin, and is identified as a slow light chain because it reacts with purified antibody to chicken slow muscle light chains and does not react strongly with antibody to fast myosin light chains. This light chain is synthesized in cultures of fetal muscle along with normal myosin light chains. The presence of a fetal light chain in culture provides a marker for studies of human muscle disease in which it is important to know when or if the muscle makes a transition from embryonic or fetal expression to true adult phenotype.
Fetus↗
Regional differences in the expression of myosin light chains and tropomyosin subunits during development of chicken breast muscle.
Types of myosin light chains and tropomyosins present in various regions and at different developmental stages of embryonic and posthatched chicken breast muscle (pectoralis major) have been characterized by two-dimensional gel electrophoresis. In the embryonic muscle all areas appear to accumulate both slow and fast forms of myosin light chains in addition to alpha and beta forms of tropomyosin. During development regional differences in myosin and tropomyosin expression become apparent. Slow myosin subunits become gradually restricted to areas of the anterior region of the muscle and finally become localized to a small red strip found on its anterior deep surface. This red region is characterized by the presence of slow and fast myosin light chains, alpha-fast, alpha-slow, and beta-tropomyosin. In all other areas of the muscle examined only fast myosin light chains, beta-tropomyosin and the alpha-fast form of tropomyosin, are found. In addition, beta-tropomyosin also gradually becomes lost in the posterior regions of the developing breast muscle. In the adult, the red strip area represents less than 1% of the total pectoralis major mass and of the myosin extracted from this area approximately 15% was present as an isozyme that comigrated on nondenaturing gels with myosin from a slow muscle (anterior latissimus dorsi). The red region accumulates therefore fast as well as slow muscle myosin. Thus while the adult chicken pectoralis major is over 99% fast white muscle, the embryonic muscle displays a significant and changing capacity to accumulate both fast and slow muscle peptides.
Animals↗
Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms.
Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.
Animals↗
Inhibition of contraction of cultured muscle fibers results in increased turnover of myofibrillar proteins but not of intermediate-filament proteins.
Muscle fibers are maintained in culture in a fully contractile state and are relaxed by the addition of 10(-7) M tetrodotoxin (TTX). This toxin binds to muscle membrane Na+- channels, abolishes spontaneous contractions and causes failure of the fiber to accumulate myosin heavy chains. These effects are reversible on removal of TTX. Synthesis and accumulation kinetics have been obtained for myofibrillar and for cytoplasmic filament proteins in normal, active muscle and in TTX-relaxed muscle fibers in culture. In relaxed fibers the synthesis of most proteins remained normal or slightly elevated. However, the accumulation of all myofibrillar proteins examined was markedly inhibited in TTX-treated cultures, whereas the accumulation of cytoplasmic filament proteins was normal or slightly elevated. Myofibrillar proteins examined were alpha-actin, troponin-C, myosin fast light chain 1, myosin fast light chain 2, alpha, beta-tropomyosins and the phosphorylated forms of tropomyosin and fast light chain 2. Cytoplasmic filament proteins studied were vimentin, alpha, beta-desmin and beta, alpha-actin. We also examined the synthesis and accumulation of six unidentified muscle-specific proteins and nine unidentified nonmuscle-specific proteins. Most of these proteins showed a normal accumulation pattern in TTX-relaxed fibers. We concluded that muscle fibers made inactive by TTX display an increased instability of all myofibrillar proteins while cytoplasmic filament proteins and cytoplasmic proteins in general are relatively unaffected. We suggest that TTX interferes, in a manner as yet unidentified, with assembly and normal stability of myofibrils. Decreased assembly and/or increased instability of myofibrils would lead to increased rates of myofibrillar protein degradation.
Actins↗
Developmental appearance of myosin heavy and light chain isoforms in vivo and in vitro in chicken skeletal muscle.
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Animals↗
Myosin heavy chains from two different adult fast-twitch muscles have different peptide maps but identical mRNAs.
Myosin heavy chains prepared from the pectoralis major and from the posterior latissimus dorsi of the same adult chicken exhibit different peptide maps when cleaved with Staphylococcus aureus V8 protease. These differences were observed at five different enzyme concentrations and in chickens of various strains. The cleavage pattern of pectoralis major myosin heavy chain from different adult chickens was always identical, as was that of posterior latissimus dorsi myosin heavy chain, demonstrating the reproducibility of the technique. However, when RNAs extracted from the pectoralis major and from the posterior latissimus dorsi were translated in a cell-free reticulocyte lysate, the myosin heavy chain encoded by pectoralis major RNA and the myosin heavy chain encoded by posterior latissimus dorsi RNA exhibited identical peptide maps. These results suggest that the different peptide maps of myosin heavy chains from the pectoralis major and posterior latissimus dorsi may arise from posttranslational modifications.
Animals↗
Increased K+ inhibits spontaneous contractions reduces myosin accumulation in cultured chick myotubes.
Increasing the K+ from 5.4 mM to 12 mM in the culture medium of developing chick myotubes causes an immediate cessation of spontaneous contractions and leads to an inhibition of myosin accumulation. The synthesis of myosin continues at the same rate in 12 mM K+ as in 5.4 mM K+ as measured by [3H]leucine incorporation into myosin corrected for differences in pool specific activity. Total protein synthesis and total protein accumulation are unaffected by growth in 12 mM K+. In addition, growth in 12 mM K+ did not alter the type of myosin heavy-chain isoform expression nor did it alter the pattern of myosin light-chain synthesis. However, the rate of myosin turnover increased threefold in cultures grown in 12 mM K+ compared to cultures grown in 5.4 mM K+, while total protein turnover was only marginally increased. We conclude that suppressed electrical or contractile activity of myotubes leads to an increased rate of myofibrillar protein turnover and that spontaneous mechanical and or electrical activity is required for continued myotube maturation in culture.
Animals↗
The two myosin isoenzymes of chicken anterior latissimus dorsi muscle contain different myosin heavy chains encoded by separate mRNAs.
The two myosin isozymes (SM1 and SM2) of the anterior latissimus dorsi muscle of the chicken change in relative concentration during development. As SM1 decreases from 13 days of embryonic growth through 1 year of adult maturation, SM2 increases. In the adult muscle SM2 accounts for over 95% of the total myosin. The myosin heavy chains of the two isozymes are distinctly different and may be separated from each other by 5% SDS polyacrylamide gel electrophoresis. The faster migrating myosin heavy chain is identified as originating from SM1 and the slower migrating myosin heavy chain from SM2 myosin isozymes. The myosin heavy chains change in relative concentration during development exactly parallel with changes in SM1 and SM2 isozyme levels. Peptide map analysis also reveals that SM1 myosin heavy chains and SM2 myosin heavy chains are distinctly different. When RNA from the ALD muscle is added to reticulocyte lysate protein synthesizing systems the translation products are shown to include both SM1 and SM2 myosin heavy chains. These comigrate exactly on 5% SDS polyacrylamide gels with authentic counterparts from ALD muscle. Finally, when peptide maps of SM1 and SM2 myosin heavy chains synthesized in the reticulocyte lysate are compared they are again found to be distinctly different and each is identical to a peptide map of respective authentic SM1 and SM2 myosin heavy chains. It is concluded that the myosin heavy chains of SM1 and SM2 myosin isozymes of ALD muscle have different primary structures and that they are encoded by two distinctly different mRNAs.
Age Factors↗
Regulation of myosin accumulation by muscle activity in cell culture.
Tetrodotoxin (TTX), at concentrations that do not interfere with normal myogenesis or with myosin synthesis, causes of cultured muscle fibres to accumulate myosin heavy chain peptides. This effect is now shown to be reversible. On removal of TTX, muscle fibres begin to reaccumulate myosin heavy chains and it appears that the myosin heavy chains display a 230% increase in stability when cells are shifted from TTX to a normal medium without TTX. Total protein stability or turnover is not affected by TTX. The ability of TTX to induce failure of accumulation of myosin heavy-chain in cultured muscle fibres does not extend to cultured chick fibroblasts. TTX also does not perturb normal uptake of [3H] leucine during a 1 h pulse and the leucine-specific activity within TTX-treated cells is essentially equivalent to that within normal cells. Finally, limited proteolysis of myosin heavy chain isolated from TTX-treated and normal muscle fibres and display of cleavage products on SDS-polyacrylamide gels does not reveal any significant difference between the two myosins. We conclude that failure of TTX muscle to accumulate myosin heavy chain is not related to impaired synthesis, to changes in myosin heavy-chain primary structure, or to overall changes in muscle fibre proteolytic activity. We speculate that the increase in degradation and resulting failure to accumulate myosin heavy chain in TTX cells is related to an inability of TTX-related muscle fibres to assemble newly synthesized fibrillar proteins into structures such as filaments or fibrils. Failure of assembly would lead to increased exposure to base-line levels of muscle proteolysis and to the observed lack of accumulation of myosin heavy chain.
Animals↗