Chimeras vs X inactivation mosaics: significance of differences in pigment distribution

Cell cultures of cardiac, pectoral, and thigh muscle of chick embryos synthesized myoglobin, as measured by incorporation of radioactive lysine detected by radioimmunoprecipitation. Liver and skin cultures, although active in protein synthesis, failed to demonstrate myoglobin synthesis. Puromycin inhibited myoglobin synthesis by the cell cultures. The electrophoretic characteristics of the myoglobin antigen synthesized by thigh and pectoral muscle were identical. Myglobin synthesizing progenitor cells attached to plastic dishes in 1 hr, but not completely in 0.5 hr. Cells, unattached at 0.5 hr, were enriched in myoglobin synthesizing cells. Incorporation of lysine-U-¹⁴C into myoglobin was maximal in confluent cultures and its increase paralleled the increase of cell fusion in the cultures. The ability of pectoral, white muscle to synthesize myoglobin in a manner equivalent to that of cardiac tissue was unexpected because of its failure to synthesize myoglobin in vivo and may indicate that factors in the whole organism may regulate the expression of this muscle cell's capabilities.     

Effect of serum from chickens treated with clenbuterol on myosin accumulation, β-adrenergic receptor population, and cyclic AMP synthesis in embryonic chicken skeletal muscle cell cultures

Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 microM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The beta-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25-30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 microM isoproterenol, limited increases of 12-20% in cAMP concentration above the basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 microM isoproterenol, cAMP concentration was stimulated 5- to 9-fold above the basal levels. Thus, not only did cells grown in horse serum have a higher betaAR population, but also each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration was tested. No correlation was apparent between basal cAMP concentration and MHC content.     

Metabolic differentiation of red and white skeletal muscle in vivo and in monolayer culture

Cytochrome oxidase, succinate oxidase and lactate dehydrogenase were compared in: (a) leg and breast muscle from 11-19-day-old chick embryos; and (b) 2, 6, 10 and 14-day-old primary cell cultures established from myoblasts of embryonic leg and breast muscle. Cytochrome oxidase, succinate oxidase and lactate dehydrogenase activities were higher (48.8, 65.4, 277.6%, respectively) in leg muscle after 19 days in ovo. Cytochrome and succinate oxidase activities were higher (111.3, 48.1%, respectively) in leg muscle cell cultures after 14 days in vitro. The data represent evidence for intrinsic developmental patterns for certain enzymes.     

Glutamine metabolism in uricotelic species: variation in skeletal muscle glutamine synthetase, glutaminase, glutamine levels and rates of protein synthesis

High intracellular glutamine levels have been implicated in promoting net protein synthesis and accretion in mammalian skeletal muscle. Little is known regarding glutamine metabolism in uricotelic species but chicken breast muscle exhibits high rates of protein accretion and would be predicted to maintain high glutamine levels. However, chicken breast muscle expresses high glutaminase activity and here we report that chicken breast muscle also expresses low glutamine synthetase activity (0.07±0.01 U/g) when compared to leg muscle (0.50±0.04 U/g). Free glutamine levels were 1.38±0.09 and 9.69±0.12 nmol/mg wet weight in breast and leg muscles of fed chickens, respectively. Glutamine levels were also lower in dove breast muscle (4.82±0.35 nmol/mg wet weight) when compared to leg muscle (16.2±1.0 nmol/mg wet weight) and much lower (1.80±0.46 nmol/mg wet weight) in lizard leg muscle. In fed chickens, rates of fractional protein synthesis were higher in leg than in breast muscle, and starvation (48 h) resulted in a decrease in both glutamine content and rate of protein synthesis in leg muscle. Thus, although tissue-specific glutamine metabolism in uricotelic species differs markedly from that in ureotelic animals, differences in rates of skeletal muscle protein synthesis are associated with corresponding differences in intramuscular glutamine content.     

Myoglobin expression in L6 muscle cells. Role of differentiation and heme

Analysis of myoglobin levels in L6 cells (derived from rat skeletal muscle) by radioimmunoassay shows that myoglobin is not synthesized until after the cells differentiate to form multinucleated myotubes. Thereafter, myoglobin accumulates in a linear fashion for up to 20 days, the longest time for which the cultures may be reliably maintained. Treatment of cultures with hemin increased myoglobin levels in a dose-dependent manner resulting in a 70% increase in myoglobin with 20 microM hemin. Succinyl acetone, a heme synthesis inhibitor, reduced myoglobin levels by 40% while simultaneous treatment with hemin restored myoglobin levels to control values. Treatment of cultures with a variety of Fe(III) chelates known to enhance both iron accumulation and ferritin synthesis in L6 cells had no effect on myoglobin levels. delta-Aminolevulinic acid also had no effect on myoglobin levels. None of the treatments had any effect on either the total soluble protein or DNA content of the cultures, and, therefore, the observed effects appear to be specific for myoglobin. These results suggest that myoglobin is expressed as a function of differentiation and that intracellular heme exerts a regulatory effect on myoglobin levels.     

Coordinate regulation of contractile protein synthesis during myoblast differentiation

The synthesis of contractile proteins has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Myoblast differentiation was synchronized by transferring secondary cultures of rapidly dividing myoblasts into medium lacking cell division-promoting factors. Cultures at various stages of differentiation were then pulse-labeled with 35S-methionine, and cell extracts were resolved by electrophoresis on two-dimensional gels. Incorporation into specific proteins was quantitated by autoradiography and fluorography using a scanning densitometer. Contractile proteins synthesized by muscle cultures were identified by their co-electrophoresis on two-dimensional gels with contracile proteins purified from quail breast muscle. Our results show that the synthesis of myosin heavy chain, two myosin light chains, two subunits of troponin and two subunits of tropomyosin is first detected at the time of myoblast fusion and then rapidly increase at least 500 fold to maximum rates which remain constant in muscle fibers. Both the kinetics of activation and the molar rates of synthesis of these contractile proteins are virtually identical. Muscle-specific actin (alpha) synthesis also increases at the time of myoblast fusion, but this actin (alpha) is synthesized at 3 times the rate of other contractile proteins. The synthesis of 30 other muscle cell proteins was quantitated, and most of these are shown to follow different patterns of regulation. From these results, we conclude that the contractile proteins are regulated coordinately during myoblast differentiation.     

Biosynthesis of tropomyosin and troponin during development of the chicken skeletal muscle

The level of functional mRNA coding for myofibrillar proteins was studied during development of the chicken skeletal muscle. RNA isolated from the developing chicken muscle directed protein synthesis in a wheat germ cell-free system. By means of polyacrylamide gel electrophoresis and immunological analysis, tropomyosin subunits and troponin components were identified among the cell-free translation products. The mRNA activities for alpha- and beta-subunit of tropomyosin were prominent in the embryonic breast muscle as well as in the embryonic leg muscle. At the early post-embryonic stage, the mRNA activity for beta-subunit disappeared from the breast muscle, while those for alpha- and beta-subunit were detectable in the leg muscle. Troponin-C and troponin-I synthesized in vitro in response to the muscle RNA formed a binary complex in the presence of calcium ion. Despite the observed difference in molecular weight between troponin-Ts in the breast and leg muscle, RNA preparations from the two muscles encoded identical troponin-Ts whose molecular weights were indistinguishable from that of troponin-T present in the breast muscle of adult chicken. It is suggested from these results that the biosynthesis of tropomyosin is regulated at the pre-translational level during the development of the chicken skeletal muscle, whereas post-translational (or co-translational) events may produce the tissue-specific form of troponin-T.     

Metabolism of myosin heavy chain in steady-state chick skeletal muscle cultures.

Synthesis, accumulation and breakdown of the 200000-mol.wt. heavy subunit of myosin were analysed over an 11 day period in muscle cell cultures isolated from the leg muscle of 12-day chick embryos. Muscle cells accumulated myosin heavy chain rapidly from days 2 to 5 and maintained a maximum, constant myosin-heavy-chain concentration between days 7 and 11. Myosin-heavy-chain content and breakdown rate were compared in steady-state muscle cultures grown either in the presence of an optimum batch of horse serum (control) or in the presence of horse serum that had been pre-selected for its ability to inhibit several-fold the rate of synthesis of myosin heavy chain (inhibitory). The quantity of myosin heavy chain in the inhibited cultures was decreased in direct proportion to the decrease in the rate of synthesis of myosin heavy chain; however, the half-lives of myosin heavy chain (control, 17.7h; inhibitory, 17.0h) were virtually identical. In contrast, the absolute rate of breakdown of myosin heavy chain, expressed as molecules/min per nucleus, was approx. 5-fold lower in the inhibited cultures (4.3 X 10(3) molecules/min per nucleus) than in the control cultures (21.7 X 10(3) molecules/min per nucleus). Thus, inhibition of myosin-heavy-chain synthesis in this case was accompanied by diminished myosin-heavy-chain concentration and absolute breakdown rate at the altered steady state, but relative myosin-heavy-chain breakdown rates were unchanged.     

Growth and Sporulation of Clostridium welchii in Breast and Leg Muscle of Poultry

Growth of a heat resistant, food poisoning strain of Clostridium welchii was followed in raw, minced breast and leg muscle of the chicken. Within the range 22–50° growth was slightly more rapid in the leg (pH 6·5–6·7) than in the breast (pH 5·6–5·7) and was fastest in leg muscle at 50°. No growth occurred at 15 or 52°.
In a comparison between chicken and turkey, inoculated breast and leg muscle were cooked for 1 h at 85° and held at 37°. Multiplication of surviving organisms was initiated much more rapidly in chicken than in turkey meat, though the growth rates were comparable in each case.
Sporulation of several strains of CI. welchii , including other heat resistant, food poisoning types, was generally 10–100 times greater in leg than in breast muscle of the chicken. Differences in sporulation could be attributed both to differences in pH and type of meat.     

Leucine pools in normal and dystrophic chicken skeletal muscle cells in culture

The specific radioactivity of [3H]Leu in the extracellular, intracellular, and Leu-tRNA pools of normal (white leghorn) and dystrophic (line 307) embryonic chick breast muscle cultures was analyzed as a function of equilibration time and extracellular Leu concentration (0.05-5 mM). The primary results were the following 1) [3H]Leu equilibrated to a constant specific radioactivity in the intracellular and Leu-tRNA pools within 2 min after addition to both normal and dystrophic cultures. 2) After equilibration, the extracellular [3H] Leu specific radioactivity in dystrophic cell culture medium was lower than that of medium exposed to normal cells (especially at low Leu concentrations), probably because of increased release of unlabeled Leu from the dystrophic cells as a result of faster protein breakdown. Accordingly, the specific radioactivities in the intracellular and the Leu-tRNA pools were also lower in dystrophic cells. 3) At 5 mM extracellular Leu, the specific radioactivity in the Leu-tRNA pool was approximately 40% lower than the specific radioactivity in the intracellular pool in both normal and dystrophic cells. Thus, high concentrations of extracellular Leu cannot be used to "flood out" reutilization of unlabeled Leu (released by protein degradation) during protein synthesis. 4) At 5.0 mM extracellular Leu, the specific radioactivity of [3H]Leu in the intracellular pool was comparable to that in the extracellular pool in normal and dystrophic cells; however, the specific radioactivity of Leu-tRNA (i.e. the immediate precursor to protein synthesis) was only 55-65% of the extracellular specific radioactivity in normal and dystrophic cells. In conclusion, reutilization of Leu from protein degradation is higher in dystrophic muscle cell cultures than in normal muscle cell cultures, and accurate rates of protein synthesis in cell cultures can only be obtained if specific radioactivity of amino acid in tRNA is measured.     

Molecular forms of pigeon skeletal muscle AMP deaminase

Phosphocellulose chromatography of pigeon leg muscle extract revealed the existence of two well-separated forms of AMP deaminase. This was in contrast to the pigeon breast muscle extract, which yielded only one form. The two leg muscle enzyme isoforms manifested similar kinetic and regulatory properties. They were activated by very low concentration of potassium ions and demonstrated similar patterns of pH and effector dependence. At pH 6.5, as well as at other pH values tested. ADP and ATP slightly stimulated, whereas GTP and orthophosphate inhibited the two molecular forms of pigeons leg muscle enzyme. Surprisingly, the molecular form of AMP deaminase present in pigeon breast muscle was inhibited by ATP at all pH values tested. The kinetic and regulatory properties of the three molecular forms of pigeon skeletal muscle AMP deaminase examined do not resemble those which have been described for pigeon heart muscle enzyme.     

Inhibition of muscle cell development in culture by cells from spinal cord due to production of low molecular weight factor.

Relatively small numbers of cells cultured from chick embryo spinal cord had the property of inhibiting muscle cell growth and differentiation, as measured by protein synthesis, myoglobin synthesis, and myotube formation, when they had been in culture 4 days before the addition of dispersed muscles cells. Inhibition of pectoral white muscle and thigh red muscle development in culture was similar. Inhibition of this sort was not brought about by similar cocultivation with cells from liver, gizzard, intestine, lung, or skin, although skin cultures were slightly inhibitory. Simultaneous cocultivation of muscle and cord cells failed to result in inhibition of myogenesis. The inhibitory property was present in the medium, and inhibition was reduced by removal of conditioned medium and replenishment with fresh medium before introduction of myoblasts. Medium obtained from other tissues, similarly cultured, did not possess inhibitory properties. The inhibitiory properties of “cord-conditioned” medium were related to a factor or factors able to be concentrated by lyophilization and of relatively low molecular weight, as measured by membrane ultrafiltration and gel filtration chromatography. The nature of the cell type in spinal cord, e.g., neuronal glial, responsible for the production of this factor is not known.     

Cell density and proliferation modulate collagen synthesis and procollagen mRNA levels in arterial smooth muscle cells.

Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.     

Role of myoglobin in the oxygen supply to red skeletal muscle.

The contribution of nyoglobin to the oxygen uptake of red skeletal muscle was estimated from the difference in oxygen uptake with and without functional myoglobin. The oxygen uptake of bundles (25 mm long, 0.5 mm mean diameter) of muscle fibers teased from pigeon breast muscle was measured in families of steady states of oxygen pressure from 0 to 250 mm Hg. The oxygen-binding function of myoglobin, in situ in muscle fiber bundles, was abolished by treatment with nitrite of hydroxylamine, which convert oxymyoglobin in situ to high spin ferric myoglobin, or with phenylhydrazine, which converts oxymyoglobin to denatured products, or with 2-hydroxyethylhydrazine, which appears to remove myoglobin from the muslce. The oxygen uptake was again measured. At higher oxygen pressure, where oxygen availability does not limit the respiration of the fiber bundles, oxygen uptake is not affected by any of the four reagents, which is evidence that mitochondrial oxygen uptake is not impaired. At lower oxygen pressure, where oxygen uptake is one-half maximal, the steady state oxygen consumption is roughly halved by abolishing functional myoglobin. Under the steady state conditions studied, the storage function of myoglobin, being static, vanishes and the transport function stands revealed. We conclude from these experiments that myoglobin may transport a significant fraction of the oxygen consumed by muscle mitochondria.     

Embryonic synthesis of myoglobin in vivo estimated by radioimmunoassay

Chick embryos in ovo incorporated radioactivity from lysine-U-¹⁴C into myoglobin, as measured by an immunoprecipitation technique. The most consistent results were obtained by injection of the precursor into the yolk sac fluid.Incorporation, or apparent myoblobin synthesis, occurred in cardiac and skeletal muscle but not in liver, although incorporation of amino acid into total soluble proteins was equivalent in all tissues studied. Synthesis was highest in cardiac muscle and appeared there first in younger embryos. Myoglobin synthesis was detectable in the heart of embryos as early as 6 days of age and rose with age thereafter. Myoglobin synthesis appeared later and at lower levels in skeletal muscle.In vitro at neutral pH, tissue extracts of liver and muscle possessed only slight properties of myoglobin degradation.Using nonradioactive precipitin techniques, sensitive to 5–10 μg/ml, myoglobin was detected in embryonic heart muscle by week 2 of life and rose in content thereafter. Two of 8 embryos had trace amounts in thigh muscle near the time of hatching, and no embryos possessed measurable amounts of myoglobin in liver tissue or in pectoral skeletal muscle. Adult birds possessed equivalent amounts of myoglobin in heart and thigh muscle while pectoral muscle and liver tissue had no detectable myoglobin content.     

The expression of multiple forms of troponin T in chicken-fast-skeletal muscle may result from differential splicing of a single gene

Troponin T isolated from chicken fast skeletal muscle has been shown to be present in three different molecular forms, one in breast and two in leg muscle. The three forms differ in both size and charge. Troponin T from breast muscle has a molecular mass of 33.5 kDa and a pI of about 7. Of the two leg muscle forms the larger has a molecular mass of 30.5 kDa and a pI of about 8.5 and the smaller a molecular mass of 29.8 kDa and a pI of about 10. Considerably more heterogeneity has been found in the leg than in the breast muscle proteins although this is not reflected in their N-terminal sequences. The reason for this is not clear. Troponin T from breast or leg muscle can be phosphorylated with troponin T kinase at the single serine residue at the N-terminus. No difference in the rate or extent of phosphorylation could be found between proteins from breast or leg muscle. The three proteins have been shown to differ only in the amino acid sequence of their N-terminal tryptic peptides. These peptides are of different length, that from breast troponin T being 58 residues and those from leg troponin T being 36 and 42 residues, these differences account for the difference in molecular mass of the parent proteins. Despite this difference the sequence of the first 12 and last 14 residues is identical in all three N-terminal peptides. The remainder of the sequence of the smallest peptide is also repeated in the other two but they each contain an extra piece of unique sequence. On the basis of these sequences it is proposed that chicken troponin T is coded for by a single gene containing, at the 5' end, a number of small exons and that three different mRNA molecules may be produced by alternative pathways of RNA splicing. The possible significance of these N-terminal sequence variations is discussed.     

Ultrastructure and mechanical activity expressed by striated muscle in culture

Newly devised assay procedures for quantitating the mechanical capabilities of striated muscle fibers grown in cell culture have permitted the correlation of cytological features with the ability to respond mechanically to electrical and chemical stimuli during development. By developmental timing and by physiological characteristics, three distinct mechanical activities can be distinguished: : TWITCH, contracture and wave propagation (escalation). Parallel electron microscopy studies suggest that contracture and escalation require significantly greater internal membrane development than twitch. The assay procedures have revealed that fibers developed in culture from genetically dystrophic chick muscle cells display a heightened electrical threshold for a twich response, but are otherwise similar to normal fibers. Cultured chick fibers, whether of leg or breast origin, exhibit similar ultrastructural and mechanical properties; yet these are different from those of in vivo adult muscle and may represent the avian striated muscle archetype expressed in the absence of innervation. Primary or cell line cultures of rat muscle produce far fewer mechanically active fibers than do avian cell cultures. The influence of culture conditions and cell source, whether avian or mammalian, on the extent of differentiation expressed in culture is so great that our understanding of studies on cultured muscle fibers would benefit from some characterization of both morphological and contractile properties of the fibers being used.