Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts.

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.     

Chemokine-like receptor 1 regulates skeletal muscle cell myogenesis

The chemokine-like receptor-1 (CMKLR1) is a G protein-coupled receptor that is activated by chemerin, a secreted plasma leukocyte attractant and adipokine. Previous studies identified that CMKLR1 is expressed in skeletal muscle in a stage-specific fashion during embryogenesis and in adult mice; however, its function in skeletal muscle remains unclear. Based on the established function of CMKLR1 in cell migration and differentiation, we investigated the hypothesis that CMKLR1 regulates the differentiation of myoblasts into myotubes. In C(2)C(12) mouse myoblasts, CMKLR1 expression increased threefold with differentiation into multinucleated myotubes. Decreasing CMKLR1 expression by adenoviral-delivered small-hairpin RNA (shRNA) impaired the differentiation of C(2)C(12) myoblasts into mature myotubes and reduced the mRNA expression of myogenic regulatory factors myogenin and MyoD while increasing Myf5 and Mrf4. At embryonic day 12.5 (E12.5), CMKLR1 knockout (CMKLR1(-/-)) mice appeared developmentally delayed and displayed significantly lower wet weights and a considerably diminished myotomal component of somites as revealed by immunolocalization of myosin heavy chain protein compared with wild-type (CMKLR1(+/+)) mouse embryos. These changes were associated with increased Myf5 and decreased MyoD protein expression in the somites of E12.5 CMKLR1(-/-) mouse embryos. Adult male CMKLR1(-/-) mice had significantly reduced bone-free lean mass and weighed less than the CMKLR1(+/+) mice. We conclude that CMKLR1 is essential for myogenic differentiation of C(2)C(12) cells in vitro, and the CMKLR1 null mice have a subtle skeletal muscle deficit beginning from embryonic life that persists during postnatal life.     

Protein synthesis and secretion in a myogenic cell line

Myogenesis in a clonal myoblast cell line is accompanied by an increase in the specific activities of creatine phosphokinase and myokinase and in the rates of synthesis and accumulation of myosin heavy chain. Exponentially dividing myoblasts synthesize myosin heavy chain at a rate of about 1% of their rate of total protein synthesis; this rate increases 7-fold during the differentiation process. Both myoblasts and myotubes secrete a minimum of 12-soluble proteins. Although there is a quantitative change in the rates of appearance of five of these proteins during myogenesis, no qualitative changes in the profile of the secreted proteins are detected. Three of the secreted proteins share several properties of soluble collagen molecules. Basal laminae and polymerized collagen fibrils are associated with myotubes, but not with exponentially dividing myoblasts.     

Activation of myosin synthesis in fusing and mononucleated myoblasts

The synthesis of the heavy chain subunit of myosin has been studied in breast muscle myoblasts from embryos of the Japanese quail, Coturnix coturnix japonica, during differentiation of these cells in culture. Specifically, these experiments were done to examine the roles of myoblast fusion and the regulation of myoblast cell division in the control of myosin heavy chain synthesis.The rates of myosin heavy chain synthesis have been quantitated in cultures of fusing myoblasts by measurement of the incorporation of radioactive leucine and valine precursors into myosin heavy chain, and simultaneous determination of the intracellular specific activities of these radioactive amino acids. These measurements demonstrate that, prior to fusion, dividing myoblasts synthesize little, if any, myosin heavy chain, but that during the period of myoblast fusion, myosin heavy chain synthesis becomes activated at least 50 to 100-fold. Myosin heavy chain synthesis was also measured in mononucleated myoblasts inhibited from fusing by the presence of EGTA in the culture medium. These experiments demonstrate that myosin synthesis can be activated in mononucleated myoblasts to reach rates similar to those attained in fused myoblasts. This activation occurs under conditions in which EGTA-inhibited myoblasts were induced to withdraw from the cell division cycle by reducing the concentrations of the serum and embryo extract components of the culture medium or by prior “conditioning” of standard growth medium.These experiments, therefore, establish that the activation of myosin synthesis in breast muscle myoblasts does not require fusion, but indicate that activation is co-ordinated with the withdrawal of myoblasts from the cell division cycle.     

Opposing Effects of Activin A and Follistatin on Developing Skeletal Muscle Cells

Activin and the activin-binding protein follistatin modulate a variety of biological processes and are abundant at sites of muscle development. Activin and follistatin were expressed in developing chick pectoral musclein vivoand in primary cell culture. Addition of recombinant activin inhibited muscle development in a dose-dependent manner as measured by the number of nuclei in myosin heavy chain positive cells and creatine phosphokinase activity. Conversely, follistatin potentiated muscle development. The effects of activin were found to be distinct from those of the related protein transforming growth factor (TGF) β1. Muscle development was repressed by activin at all time points investigated and did not recover with the removal of activin following a limited exposure. In contrast, while myogenic differentiation in TGFβ1 was initially repressed, muscle marker expression recovered to control levels—even in the continued presence of TGFβ1. Fibroblast growth factor (FGF) had little effect on inhibiton of muscle development caused by activin A. However, inhibition of development produced by TGFβ increased with increasing concentrations of FGF. Finally, early expression of myoD and myf5 mRNA by muscle cultures in the presence of activin and follistatin was analyzed. Activin-treated cultures expressed reduced myoD and myf5 levels at 1.5 days after plating. Myf5 levels in follistatin-treated cultures were elevated, but, surprisingly, these cultures showed a reduction in myoD levels. These data suggest that endogenously expressed activin and follistatin are important modulators of muscle development.     

Regulation of proliferation and differentiation of myoblasts derived from adult mouse skeletal muscle by specific isoforms of PDGF

The expression of receptors and the mitogenic response to PDGF by C2 myoblasts, derived from adult mouse skeletal muscle, was investigated. Employing 125I-PDGF binding assays, we showed that the cells exhibit high level binding of PDGF-BB (approximately 165 x 10(3) molecules/cell at saturation) and much lower binding of the PDGF-AA and PDGF-AB (6-12 x 10(3) molecules/cell at saturation). This indicates that the C2 myoblasts express high levels of PDGF receptor beta-subunits and low levels of alpha-subunits. PDGF-BB enhances the proliferation of C2 cells maintained in 2% FCS by about fivefold. PDGF-AB had a moderate effect on cell proliferation (less than twofold) and PDGF-AA had no effect. Inverse effects of PDGF isoforms on the frequency of differentiated myoblasts were observed; the frequency of myosin-positive cells was reduced in the presence of PDGF-BB while PDGF-AA and PDGF-AB had no effect. PDGF may thus act to increase the number of myoblasts that participate in muscle regeneration following muscle trauma by stimulating the proliferation and by inhibiting the differentiation of myogenic cells.     

In vitro myogenesis : Expression of muscle specific function in the absence of cell fusion

Correlation of cell fusion with other muscle specific functions observed in vivo and in vitro has suggested the possibility that the fusion process itself might be involved in the mechanism of controlled expression of differentiated function. In order to test this possibility in primary cultures of fetal calf myoblasts, we have established conditions of Ca²⁺ depletion which block cell fusion. Under these conditions, three separate markers of muscle specific differentiation were measured: creatine phosphokinase, a soluble enzyme; myosin heavy chain, a component of the contractile apparatus; and acetylcholine receptor, an integral membrane protein. The data indicate that the expression of the three functions studied is independent of the fusion process, and therefore that cell fusion plays no major role in the switch on of differentiated gene expression.     

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.     

The CLIC5 (chloride intracellular channel 5) involved in C2C12 myoblasts proliferation and differentiation

CLIC5 (chloride intracellular channel 5) is a CLIC (chloride intracellular channel) with various functions. Its high expression in skeletal muscle and association with actin‐based cytoskeleton suggests that it may play an important role in muscle tissue. This study was conducted to examine whether CLIC5 regulates the proliferation and differentiation of C2C12 myoblasts into myotubes. Differentiation of C2C12 myoblasts induced by switching to a differentiation culture medium was accompanied by a significant increase of CLIC5 protein expression level. Constitutive overexpression of CLIC5 was associated with reduced cell proliferation and more cells from G2/M phase into G0/G1 phase, followed by increased number and size of myotubes and up‐regulation of muscle‐specific proteins of myosin heavy chain, myogenin and desmin. These results demonstrate that CLIC5 is involved in C2C12 proliferation and myogenic differentiation in vitro.     

Inhibition of miR-214 expression represses proliferation and differentiation of C2C12 myoblasts

MicroRNAs (miRNAs) are small non-coding RNAs that participate in diverse biological processes including skeletal muscle development. MiR-214 is an miRNA that is differentially expressed in porcine embryonic muscle and adult skeletal muscle, suggesting that miR-214 may be related to embryonic myogenesis. In this study, the myoblast cell line C2C12 was used for functional analysis of miR-214 in vitro. The results showed that miR-214 was expressed both in myoblasts and in myotubes and was upregulated during differentiation. After treatment with an miR-214 inhibitor and culturing in differentiation medium, myoblast differentiation was repressed, as indicated by the significant downregulation of expression of the myogenic markers myogenin and myosin heavy chain (MyHC). Interestingly, myoblast proliferation was also repressed when cells were transfected with an miR-214 inhibitor and cultured in growth medium by real-time proliferation assay and cell cycle analysis. Our results showed that miR-214 regulates both proliferation and differentiation of myoblasts depending on the conditions.     

1α,25-dihydroxyvitamin D₃ enhances fast-myosin heavy chain expression in differentiated C2C12 myoblasts

We investigated the effect of VD3 (1α,25-dihydroxyvitamin D3) on the proliferating, differentiating and differentiated phases of C2C12 myoblasts, a mouse skeletal muscle cell line. VD3 treatment in 10% FBS (fetal bovine serum) inhibited the proliferation and viability of the cells in a dose-dependent manner. It also dose-dependently increased the percentage of cells in the G0/G1 phase as shown by flow cytometry. In the differentiating phase, VD3 treatment inhibited the formation of myotubes and the expression of total myosin heavy chain at both the mRNA and protein levels. In the differentiated phase, treatment had no significant effect on the amount of total myosin heavy chain, as Western blot analysis with MF20 antibody [DSHB (Developmental Studies Hybridoma Bank)] showed. However, significantly greater expression of fast myosin heavy chain in 1 nM VD3 was found by Western blot analysis with MY-32 (Sigma). Thus VD3 inhibited the proliferation of myoblasts during proliferating and differentiating phases, whereas it increased the expression of the fast myosin heavy chain isoform in the differentiated phase. The data indicate that an adequate concentration of VD3 might have an anabolic effect on differentiated skeletal muscle.     

Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.