Muscle-specific Protein Synthesis Research Articles

Effects of Creatine in Skeletal Muscle Cells and in Myoblasts Differentiating Under Normal or Oxidatively Stressing Conditions.

Creatine (Cr) - along with the Cr kinase (CK) system - plays a fundamental role in muscle biochemistry and physiology not limited to its ergogenic role. Indeed, Cr has been shown to exert pleiotropic effects, which promote protein accretion, muscle-specific protein synthesis, growth in cultured myogenic cells and favour the myogenic process either in normal or stressing conditions. This review focuses on the effects of Cr supplementation on cellular and mitochondrial biochemistry and function in the course of skeletal muscle differentiation, either in normal or oxidatively stressing conditions, and on the ensuing nutraceutical/therapeutic perspectives.

Actions of Short-Term Fasting on Human Skeletal Muscle Myogenic and Atrogenic Gene Expression

Background: Skeletal muscle mass is governed by multiple IGF-1-sensitive positive regulators of muscle-specific protein synthesis (myogenic regulatory factors which includes myoD, myogenin and Myf5) and negative regulators, including the atrogenic proteins myostatin, atrogin-1 and muscle ring finger 1 (MuRF-1). The coordinated control of these myogenic and atrogenic factors in human skeletal muscle following short-term fasting is currently unknown. Method: Healthy adults (n = 6, age 27.6 years) undertook a 40-hour fast. Skeletal muscle biopsy (vastus lateralis) and venous blood samples were taken 3, 15 and 40 h into the fast after an initial standard high-carbohydrate meal. Gene expression of the myogenic regulator factors (myoD, myogenin and Myf5) and the atrogenic factors (myostatin, atrogin-1 and MuRF-1) were determined by real-time PCR analysis. Plasma myostatin and IGF-1 were determined by ELISA. Results: There were no significant alterations in either the positive or negative regulators of muscle mass at either 15 or 40 h, when compared to gene expression measured 3 h after a meal. Similarly, plasma myostatin and IGF-1 were also unaltered at these times. Conclusions: Unlike previous observations in catabolic and cachexic diseased states, short-term fasting (40 h) fails to elicit marked alteration of the genes regulating both muscle-specific protein synthesis or atrophy. Greater periods of fasting may be required to initiate coordinated inhibition of myogenic and atrogenic gene expression.

Muscle Hypertrophy Models: Applications for Research on Aging

Muscle hypertrophy is an adaptive response to overload that requires increasing gene transcription and synthesis of muscle-specific proteins resulting in increased protein accumulation. Progressive resistance training (P(RT)) is thought to be among the best means for achieving hypertrophy in humans. However, hypertrophy and functional adaptations to P(RT) in the muscles of humans are often difficult to evaluate because adaptations can take weeks, months, or even years before they become evident, and there is a large variability in response to P(RT) among humans. In contrast, various animal models have been developed which quickly result in extensive muscle hypertrophy. Several such models allow precise control of the loading parameters and records of muscle activation and performance throughout overload. Scientists using animal models of muscle hypertrophy should be familiar with the advantages and disadvantages of each and thereby choose the model that best addresses their research question. The purposes of this paper are to review animal models currently being used in basic research laboratories, discuss the hypertrophic and functional outcomes as well as applications of these models to aging, and highlight a few mechanisms involved in regulating hypertrophy as a result of applying these animal models to questions in research on aging.

Purothionin from wheat endosperm reversibly blocks myogenic differentiation of chick embryonic muscle cells in culture

Purothionin from wheat endosperm is a cysteine-rich, basic polypeptide of about 5000 Da, which modifies membrane permeability of cultured mammalian cells. This peptide was found to block fusion of chick embryonic muscle cells in culture but allows proliferation and alignment. A purothionin concentration of 6 μg/ml (1.2 μ M) was necessary for the complete prevention of myotube formation. Under similar conditions, incorporation of [ 35S]methionine occurred normally but the synthesis of muscle-specific proteins including creatine kinase and acetylcholine receptor was strongly inhibited. In addition, purothionin blocked the uptake of 86Rb +, immediately after its addition to the cultured myoblasts. No such effects were found with the purothionin chemically modified with acetic or succinic anhydride. Thus, the basic residues in purothionin appear to be associated with the inhibition of myogenic differentiation. These results suggest that purothionin exerts its regulatory effect on the transition from proliferative to differentiative myoblasts by interfering with membrane permeability or intercellular contact and recognition, which are necessary for the initiation of muscle differentiation.

Control of differentiation in BC3H1 muscle cells.

BC3H1 is a cell line that undergoes a musclelike pattern of differentiation under the appropriate conditions. We have examined the control of the synthesis of proteins characteristic of differentiated muscle in these cells as a function of their position in the cell cycle. We define two positions in the cell cycle where BC3H1 cells can remain stably quiescent. G1d is a restriction point early in the G1 portion of the cell cycle that permits the synthesis of muscle-specific proteins and is probably identical to G0. The second restriction point, G1q, occurs approximately 4 hr later in the G1 portion of the cell cycle and does not permit the synthesis of muscle-specific proteins. Movement of the cells from G1d to G1q occurs when fibroblast growth factor is added to the cells and is reversed when this growth factor is removed. Repression of the synthesis of muscle-specific proteins occurs when fibroblast growth factor is added to cells in G1d. In the case of the muscle form of creatine phosphokinase (M-CPK), the decline in the rate synthesis of this protein is a consequence of a decreased level of its mRNA. By contrast, the repression of alpha-actin synthesis, a protein synthesized only in differentiated cells, appears to be controlled at the translational level. The effect of fibroblast growth factor and other mitogens in these cells require activation of tyrosine kinase(s), but the intracellular targets of these kinases are not known. Studies by others suggest that activation of the ras oncogene can mimic the action of mitogenic polypeptides on these and other muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta.

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with transforming growth factor-beta (TGF-beta). Muscle-specific protein synthesis and satellite cell fusion were used as indicators of muscle differentiation; a dose-dependent inhibition of differentiation was observed in response to TGF-beta. In addition, TGF-beta depressed cell proliferation in a dose-dependent manner. Half-maximal inhibition of differentiation was seen with a TGF-beta concentration of approximately 0.1 ng/ml. Although proliferation was not inhibited, it was depressed and half-maximal suppression of proliferation occurred in response to 0.1-0.5 ng TGF-beta/ml. Neonatal rat myoblasts were also subjected to TGF-beta treatment, and similar results were observed. Neonatal cells, however, were more sensitive to TGF-beta than satellite cells, as indicated by the reduced concentrations of TGF-beta required to inhibit differentiation and reduce the rate of proliferation. Under identical culture conditions proliferation of muscle-derived fibroblasts were also depressed. The differentiation inhibiting effect of TGF-beta on satellite cells was reversible. It has been suggested that TGF-beta could be an important regulator of tissue repair, and its in vitro effects on satellite cells suggest a possible role in regulation of muscle regeneration.

Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis

Primary isolates of chick leg muscle myoblasts cultured on hyaluronic acid substrates have been examined by transmission electron microscopy for evidence of myoblast fusion and subsequent differentiation. Even though these cells form close contacts, no evidence of multinucleated myotubes is found in these cultures. Two-dimensional SDS-polyacrylamide gel electrophoresis shows that the muscle macromolecular biosynthetic program is not initiated in these hyaluronic acid fusion-blocked cells. Further, these fusion-blocked myoblasts continue replicating while cultured on hyaluronic acid surfaces. The inhibition of both fusion and the myogenic expressional program is reversed by replating these myoblasts onto a denatured collagen (gelatin) substrate; both the synthesis of muscle-specific proteins and the formation of multinucleated myotubes are observed when these subcultured cells are introduced onto gelatin substrates. These observations indicate that the hyaluronic acid inhibition of fusion is not permanent and is manifested in a way different from other fusion blockers in that hyaluronic acid inhibits both fusion and the myogenic expressional program.

Transcriptional-translational regulation of muscle-specific protein synthesis and its relationship to chondrogenic stimuli.

Demineralized bone (bone matrix) has a well-characterized ability to evoke the re-differentiation of cells derived from skeletal muscle into chondrocytes. Recent investigations in this laboratory have shown that muscle-specific (alpha) actin synthesis continues throughout redifferentiation. Conversely, expression of the cartilage phenotype is associated with repression of muscle-specific enzyme synthesis. The present experiments were undertaken to determine the mode of genomic regulation responsible for control of these muscle-specific syntheses. As part of these experiments, we investigated the ability of embryonic and adult RNA to direct translation in vitro. The results indicate that unfractionated (total) RNA is capable of directing the efficient synthesis of actin, but not myosin heavy or light chains. Decreased abundance of polyadenylated mRNA cannot account for lack of myosin synthesis. Polyadenylated mRNA, however, directed synthesis of actin and myosin with an efficiency greater than that of total RNA. This data suggested that embryonic total RNA was subject to translational control. Dot blot hybridization against cDNA probes for alpha-actin, myosin heavy chain, and fast light chains demonstrated that myogenic cells were subject to a pattern of mixed transcriptional and translational control. It is hypothesized that full expression of the muscle phenotype involves sequential release of transcriptional, and subsequently, the translational controls. We have also observed that cultures of skeletal muscle on bone matrix contain mRNA for muscle-specific proteins, even through the period normally characterized by chondrogenesis. In the absence of concurrent enzyme protein synthesis, it appears that one action of bone matrix is to continue genomic controls that in the source skeletal muscle maintain the genome in an embryonic (translationally repressed) state.

Manipulation of medium conditions and differentiation in the rat myogenic cell line L6

Myoblasts of the L6 rat cell line were grown in Ham's F12 nutrient medium containing 10% fetal calf serum (F12 + FCS). Although the cells were confluent by 6 days in culture, fusion was not observed even if cultures were maintained for 10–14 days. At least 80% of the cells in such confluent unfused cultures were in the G 1 phase of the cell cycle and less than 5% of the cells in confluent cultures synthesized DNA during a 4-day period. The synthesis of muscle-specific proteins (α-actin, β-tropomyosin, and myosin light chains LC1 emb and LC2 F) was negligible when compared to fused cultures of L6 cells grown for a similar time in Dulbecco's medium with 10% FCS (DME + FCS). When the unfused cultures were shifted from F12 + FCS to DME + FCS, DNA synthesis could be demonstrated in more than 95% of the cells and fusion occurred, indicating that neither proliferative nor myogenic capacity had been irreversibly lost. Raising the levels of calcium, varying the serum concentration from 0 to 20%, or the addition of medium components (present in DME but reduced or absent in F12) all failed to induce fusion in the L6 cells grown in F12. However, L6 cells will fuse in mixtures of F12 + FCS and DME + FCS. Fusion will also occur if L6 cells are grown at clonal density in F12 + FCS supplemented with calcium. While it has not been possible to determine why F12 + FCS is nonpermissive for L6 cells in confluent mass cultures, the results demonstrate that prolonged residence in the G 1 phase of the cell cycle is not a sufficient condition for L6 myoblast differentiation to occur.

Evidence for transcriptional regulation of the myosin heavy chain gene during myogenesis.

One of the changes accompanying skeletal muscle cell (myoblast) fusion is a dramatic increase in synthesis of muscle specific proteins, one of which is myosin. The underlying mechanism for this burst in synthesis is not yet understood but may occur by two mechanisms: (a) gradual storage of mRNA and translational control as found by others or (b) gene activation and rapid synthesis of mRNA for immediate translation. In this paper we show that the myosin gene changes its organization such that postfusion skeletal muscle cells show an increased susceptibility to DNase I, a recognized probe for gene activation. We also show that this change accompanies an increase in rate of transcription and an increased cell content of myosin heavy chain mRNA. This work shows that transcriptional control is an important mechanism during muscle cell development in addition to the translational control shown by other workers.

Multiple controls for the synthesis of muscle-specific proteins in BC3H1 cells.

The regulation of the synthesis of muscle-specific proteins has been examined in BC3H1 cells, a smooth muscle-like cell line isolated by Schubert et al. (J. Cell Biol., 1974, 61: 398-413.). The synthesis of both creatine kinase and the acetylcholine receptor appear to be under dual control, a positive control due to cell-cell contact which increases the rate of synthesis of this protein, and a negative signal, elicited by serum components, that decreases the rate of synthesis of these proteins. Induction of muscle-specific proteins in BC3H1 cells is a reversible process and can be arrested after partial induction has taken place by the addition of serum or high-molecular-weight protein fraction from serum to these cells. The high-molecular-weight protein fraction from serum is not by itself mitogenic for Bc3H1 cells and cannot be replaced by a variety of known hormones (mitogenic factors).

A reexamination of the effects of creatine on muscle protein synthesis in tissue culture.

Experiments designed to test the hypothesis that intracellular creatine level regulates the synthesis of muscle specific proteins have failed to demonstrate any creatine regulatory effect. Manipulation of the extracellular creatine in culture medium over a 5,700-fold range (1.3-7.4 mM) was successful in altering intracellular total creatine by only a factor of 20 (1.4-42 mg creatine/mg protein), an indication that muscle cells are able to regulate intracellular creatine levels over a wide range of external creatine concentrations. Alterations of cell creatine had no effect on either total protein synthesis or synthesis of myosin heavy chain. Methods were perfected to measure total creatine, and incorporation of [3H]leucine into total protein and purified myosin heavy chain from the same culture dish to avoid the possibility of variation between dishes. The creatine analog 1-carboxymethyl-2-iminohexahydropyrimidine (CMIP) previously reported to stimulate myosin synthesis in culture was found to depress creatine accumulation by cells and depressed total protein synthesis and synthesis of myosin heavy chain. This inhibitory action of CMIP is consistent with the reported competitive inhibition of creatine kinase and presumed interference with energy metabolism.

Manipulation of myogenesis in vitro: Reversible inhibition by DMSO

A system has been developed for the detailed analysis of the transition from proliferative myoblast to differentiated muscle cell. Dimethylsulfoxide (DMSO) prevents the terminal differentiation of L8 myoblasts in vitro, and its effect is reversible. DMSO (2%) inhibits the fusion of myoblasts to form multinucleate myotubes, the normal increases in activity of creatine phosphokinase (CPK) and acetylcholinesterase, and the synthesis of α-actin and acetylcholine receptor protein. Upon removal of DMSO from the medium, a lag precedes the onset of differentiation. The potential to inhibit muscle differentiation reversibly is not specific to DMSO, but is shared by a number of compounds, including dimethylformamide, hexamethylbisacetamide and butyric acid, all potent inducers of gene expression in Friend erythroleukemia cells. L8 cells routinely cease DNA synthesis and initiate fusion and muscle protein synthesis once they are confluent. In the presence of DMSO, however, nearly all cells continue DNA synthesis, even several days after reaching confluence. Protein synthetic patterns of DMSO-inhibited cells are almost indistinguishable from those of untreated myoblasts and distinct from differentiated myotubes. It appears that cells exposed to DMSO are locked indefinitely in a proliferative myoblast stage of development and are unable to enter the G 0 phase of the cell cycle necessary for initiation of differentiation. DMSO coordinately inhibits all the differentiative parameters measured. In contrast, cytochalasin B uncouples normally linked differentiative events so that fusion is inhibited while muscle-specific protein synthesis proceeds. DMSO has similar effects on both cytochalasin B-treated and fusing control cultures, suggesting that its primary effect is exerted not at the level of fusion but earlier in the differentiative timetable. Once fusion and the synthesis of muscle-specific proteins are well under way, the addition of DMSO is ineffective and differentiation continues in its presence. The potential to manipulate muscle gene expression in vitro makes this system particularly useful for the detailed analysis of the processes involved in the transition to the differentiated state and for determining the linkage of developmental events.

Calcium and the control of muscle-specific creatine kinase accumulation during skeletal muscle differentiation in vitro

A polyacrylamide gel separation method for creatine kinase (CPK) isoenzymes is described, and its use to determine muscle-specific CPK (M-CPK) levels in skeletal muscle cultures is illustrated. In cultures in which cell fusion has been prevented by very low Ca 2+ concentrations, the increases in M-CPK after 96 hr are similar to those in control cultures. Slightly higher concentrations of Ca 2+, however, inhibit both cell fusion and M-CPK accumulation. As the calcium concentration is gradually increased further, cell fusion is permitted, followed, at even higher Ca 2+ levels, by M-CPK accumulation. These effects can be obtained both by adding EGTA to the culture medium and by using Ca 2+-free culture medium and varying the Ca 2+ concentration directly. The latter method has the advantage that deleterious effects of EGTA on cell attachment and cell numbers do not occur, even at the lowest Ca 2+ concentrations. By revealing dramatic effects on CPK levels of small changes in external Ca 2+ concentrations, these observations may resolve conflicting data in the literature on the question of whether cell fusion is a prerequisite for muscle-specific protein synthesis. Possible mechanisms for the two effects of Ca 2+ on CPK specific activity (permissive at very low, but inhibitory at intermediate, concentrations) are considered, including membrane mediation, mediation by changes in ionized cytoplasmic Ca 2+ levels, and possible involvement of cyclic nucleotides.

Changes in the mRNA population of chick myoblasts during myogenesis in vitro

We have analyzed the sequence complexity, frequency distribution and coding capacity of the mRNA populations of primary chick embryo muscle cultures at different stages of myogenesis. Prefusion cultures, fused myofibrillar cultures and cultures blocked for both fusion and myogenesis all contain about 17,000 different mRNA sequences, arranged in three or four abundance classes. The myofibril (96 hr) cultures, however, contain about 2500 sequences in higher concentration and six sequences in exceptionally high concentration, each present in about 15,000 copies per nucleus. These sequences are shown to be 10 times less common in premyogenic (26 hr) cultures and 40 times less common in cultures that have been blocked by BUdR against both fusion and myogenesis. The concentration of these sequences in cultures developing toward myofibril formation correlates well with the capacity of the mRNA to stimulate the cell-free synthesis of muscle-specific proteins. A more direct approach to the identity of the abundant class of myofibril mRNA indicates that it contains the templates for the synthesis of seven polypeptides that are synthesized in particularly large amounts in myogenic cultures, including myosin, actin and tropomyosin. Between 20 and 30% of the abundant mRNA is transcribed from moderately repetitive DNA sequences. The remainder of the abundant, and all of the less-abundant, mRNA is transcribed from single-copy DNA.

Apparent dominance of serine auxotrophy and the absence of expression of muscle-specific proteins in rat myoblast � mouse L-cell hybrids

Hybrids obtained from crosses of rat myoblasts and mouse L-cells are unable to differentiate into myotubes. The synthesis of muscle-specific proteins, creatine phosphokinase (CPK), myosin, and adenyl cyclase is also suppressed in the hybrids. Adenyl cyclase produced in the hybrids is not activated by isoproterenol. The glycine auxotrophy of myoblasts is phenotypically expressed as a dominant trait in hybrids. Genotypically, however, all the enzymes of the phosphorylated pathway of serine biosynthesis and serine hydroxymethyltransferase, the enzyme inter converting serine and glycine, are present in normal concentrations in the hybrids, just as they are in the mouse L-cells. Unlike either of the parental lines, the hybrid cells are unable to grow on serine-supplemented medium, but do so when glycine is also added. Glycine by itself supports growth of the hybrids in the absence of added serine. All the curious effects of glycine/serine on growth of hybrids are rationalized and accounted for by the hypothesis that in these cells the enzyme serine hydroxy-methyltransferase, for unknown reasons, does not function in vivo.

Do increases in enzyme activities during muscle differentiation reflect expression of new genes?

TRANSITIONS from embryonic isoenzymic forms to mature adult forms are known to occur during in vitro myogenesis for creatine phosphokinase1 (CPK) and other muscle enzymes2. More recently, similar transitions have been shown to occur for myosin3,4, and actin5,6. Both CPK activity7,8 and amino acid incorporation into myosin9–11 increase during the early stages of differentiation in vitro when cells fuse to form multinucleate myotubes, and it is tempting to assume that these increases require the production of adult form mRNA after activation of a “battery” of genes for myogenesis2. Yaffe and Dym11 have proposed a model of myogenesis in which mRNAs for characteristic muscle proteins are synthesised before cell fusion and stored in a “masked” form until activated by the fusion process, thus giving rise to simultaneous increases in muscle-specific protein synthesis and cell fusion. We describe here experiments which show large increases in CPK activity at the time of early cell fusion which cannot result from activation of “masked” muscle-specific mRNAs and which do not require adult form mRNAs at all. Using a new technique which allows measurement of low levels of CPK isoenzymes, we have shown that the early increase in CPK activity during the first 2 d of culture is of the embryonic, or brain, form, and only later does the adult muscle form make a major contribution to the total increase in enzyme activity.