SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Role of Ca2+ and Ca2+-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca2+ transport into myoblasts. We have also demonstrated that a Ca2+-activated protease, CAF (mM), appears to relocate in response to the Ca2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

Quantal Division and a Postmitotic State in Myoblast Differentiation

The reversible arrest of myoblast differentiation by ethidium bromide (EB) has been used to examine the nature of the transition from the proliferative state to terminal differentiation resulting in fusion into muscle fibers. If EB is introduced at the time that myoblasts are shifted to medium that induces fusion, all apparent cytodifferentiation is suspended. When such EB arrested myoblasts are released from EB inhibition they fuse without reentering the cell cycle. If EB arrested myoblasts are released into proliferation promoting medium rather than medium that induces fusion they neither fuse nor proliferate. In this case they remain quiescent in the proliferating medium for an extended period, however, if these myoblasts are subsequently shifted to medium that induces fusion, they fuse without reentering the cell cycle. Apparently the myoblasts have become postmitotic and competent to fuse into muscle fibers during their initial exposure to fusion inducing medium, even though cytodifferentiation has been blocked. Exposure of these postmitotic fusion competent myoblasts to proliferation promoting medium does not stimulate them to reenter the cell cycle but does prevent fusion into muscle fibers. These results are most consistent with a quantal division model of myoblast differentiation rather than a gradual transition from the proliferative state to a state in which fusion occurs.     

Quantal division and a postmitotic state in myoblast differentiation.

The reversible arrest of myoblast differentiation by ethidium bromide (EB) has been used to examine the nature of the transition from the proliferative state to terminal differentiation resulting in fusion into muscle fibers. If EB is introduced at the time that myoblasts are shifted to medium that induces fusion, all apparent cytodifferentiation is suspended. When such EB arrested myoblasts are released from EB inhibition they fuse without reentering the cell cycle. If EB arrested myoblasts are released into proliferation promoting medium rather than medium that induces fusion they neither fuse nor proliferate. In this case they remain quiescent in the proliferating medium for an extended period, however, if these myoblasts are subsequently shifted to medium that induces fusion, they fuse without reentering the cell cycle. Apparently the myoblasts have become postmitotic and competent to fuse into muscle fibers during their initial exposure to fusion inducing medium, even though cytodifferentiation has been blocked. Exposure of these postmitotic fusion competent myoblasts to proliferation promoting medium does not stimulate them to reenter the cell cycle but does prevent fusion into muscle fibers. These results are most consistent with a quantal division model of myoblast differentiation rather than a gradual transition from the proliferative state to a state in which fusion occurs.     

A cell surface phosphoprotein of 48 kDa specific for myoblast fusion

The data we present here permit us to affirm that a 48 kDa phosphoprotein is the target of extracellular Ca2+ during fusion. It is detected only in fusion-competent L6 myoblasts and not in the fusion-defective spontaneous stable variants we isolated. The phosphorylation of this protein species can be totally inhibited by culturing myoblasts in a medium containing low Ca2+ concentrations (0.250 mM). However, under such conditions myoblasts do not fuse, but withdraw from the cell cycle and accumulate the muscle isoform of creatine kinase (M-CK). The results we have obtained support the following conclusions: (1) in fusion-competent cells, overall Ca2+-dependent phosphorylation of cell surface proteins appears to be necessary, but is not sufficient by itself for myoblast fusion; (2) the phosphorylation of a 48 kDa protein species is required for cell fusion; and (3) the phosphorylation of this 48 kDa protein is independent of other main events of cellular differentiation.     

Influence of external diffusible factors on myogenesis of the cells of the line L6

The influence of external diffusible factors on the terminal differentiation of cells of the myogenic line L₆ has been studied. The cultures were fed either with medium which had been depleted of mitogenic factors by previous incubation in the presence of myogenic cells, or with standard medium to which proteins secreted by myoblasts had been added. We present evidence that the length of the proliferative phase of the cultures is largely dependent upon environmental cues. However, by inhibiting DNA replication by a variety of means during this phase, we show that in order to differentiate, DNA synthesis is needed for myogenic cells of this line.Once the myoblasts have initiated their last presumptive round of DNA synthesis, they cannot be induced to undergo further DNA replication by environmental factors. Cloning experiments showed that, at this time, the cells lose their proliferative capacity. Our data strongly suggest that, at this stage, cells of line L₆ become irreversibly committed for differentiation. The fusion rate of the committed myoblasts could be significantly increased by proteins secreted by proliferating myogenic cells, but not by those secreted by myotubes.     

Specific features of satellite cells and myoblasts at different stages of rat postnatal development

A cell culture consisting mainly of satellite cells and mononuclear myoblasts was derived from femoral muscles of infant (aged 3–7 days) and adult rats. Satellite cells identified by expression of the specific marker Pax7 accounted for approximately 80% of the isolated cell fraction. Mononuclear myoblasts represented by proliferating and postmitotic cell pools were identified immunocytochemically by the expression of markers Ki67 and desmin. Differentiation of satellite cells and myoblasts in the culture depended on the concentration of Ca²⁺ in the culture medium (F12 with different Ca²⁺ concentrations or DMEM). Differentiation of myogenic cells manifested in myoblasts fusion, formation of myotubes, and expression of myosin in myofibrils was observed only in the medium with a high Ca²⁺ concentration (2mM). Satellite cells and myoblasts from the muscles of newborn and adult rats did not differ noticeably in their capacity for differentiation.     

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.     

Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression

Specific DNA fragments complementary to the 3' untranslated regions of the beta-, alpha-cardiac, and alpha-skeletal actin mRNAs were used as in situ hybridization probes to examine differential expression and distribution of these mRNAs in primary myogenic cultures. We demonstrated that prefusion bipolar-shaped cells derived from day 3 dissociated embryonic somites were equivalent to myoblasts derived from embryonic day 11-12 pectoral tissue with respect to the expression of the alpha-cardiac actin gene. Fibroblasts present in primary muscle cultures were not labeled by the alpha-cardiac actin gene probe. Since virtually all of the bipolar cells express alpha-cardiac actin mRNA before fusion, we suggest that the bipolar phenotype may distinguish a committed myogenic cell type. In contrast, alpha-skeletal actin mRNA accumulates only in multinucleated myotubes and appears to be regulated independently from the alpha-cardiac actin gene. Accumulation of alpha- skeletal but not alpha-cardiac actin mRNA can be blocked by growth in Ca2+-deficient medium which arrests myoblast fusion. Thus, the sequential appearance of alpha-cardiac and then alpha-skeletal actin mRNA may result from factors that arise during terminal differentiation. Finally, the beta-actin mRNA was located in both fibroblasts and myoblasts but diminished in content during myoblast fusion and was absent from differentiated myotubes. It appears that in primary myogenic cultures, an asynchronous stage-dependent induction of two different alpha-striated actin mRNA species occurs concomitant with the deinduction of the nonmuscle beta-actin gene.     

An alternate protocol for establishment of primary caprine fetal myoblast cell culture: an in vitro model for muscle growth study

Cultured myoblasts have been used extensively as an in vitro model in understanding the underlying mechanisms of myogenesis. Various protocols for establishing a pure myoblast culture have been reported which involve the use of special procedures like flow cytometry and density gradient centrifugation. In goat, only a few protocols for establishing a myogenic cell culture are available and these protocols use adult muscle tissues which often does not yield sufficient numbers of precursor cells with adequate proliferative capacity. Considering the disadvantages of adult myoblasts, we are proposing an alternate protocol using caprine fetus which does not require any special procedures. In the present study, more than 90–95% fetal-derived cell populations had the typical spindle to polyhedral shape of myoblast cell and stained positive for desmin, hence confirming their myogenic origin. These cells attained the maximum confluency as early as 3–4 d against 3 wk by adult myoblasts indicating a better growth potential. Further, quantitative real-time PCR analysis revealed a higher expression (p?<?0.01) of myogenic regulatory factors (i.e., myogenic determination factor 1, myogenic factor 5, and myogenin) and myostatin (MSTN) in the fetal as compared to the adult myoblasts. Consequently, higher proliferation and differentiation ability along with higher abundance of myogenic markers and MSTN make the fetal myoblasts a better in vitro model.     

Cell cycle withdrawal without concomitant differentiation: analysis of a G1-specific temperature-sensitive murine myoblast cell line

Skeletal muscle differentiation is accompanied by the withdrawal of the proliferating myoblasts from the cell cycle in the G1 phase. We showed earlier that the length of G1 and the timing of the differentiative transition could be controlled in large part by the composition of the culture medium. In this study we have asked whether a G1 arrest imposed independently of the culture medium is sufficient to elicit the differentiative response. To examine this possibility we have characterized a new G1-specific ts murine myoblast line. This line, ts-36, was identified as a G1-specific mutant on the basis of four criteria: prolonged viability at the nonpermissive temperature (npt), the kinetics of cell cycle withdrawal and reentry in temperature shift experiments, the ability of the cells to differentiate at the npt in low-growth medium, and, finally, the observation that, by the criterion of flow microfluorometry, the mutant cells block at the G1 landmark in the cell cycle. A ts-imposed G1 arrest of up to 96-hr duration is by itself insufficient to activate the differentiative program in ts-36 cells cultured in complete growth medium. The differentiated phenotype is expressed, however, in temperature-arrested cells cultured either in low-growth (conditioned) medium or in a medium from which mitogens have been removed by ultrafiltration. Differentiation can be reversed by refeeding with complete growth medium. The effects of growth medium can be mimicked by FGF to the extent of inhibiting activation of the differentiative program in temperature-arrested ts-36 cells and in eliciting downregulation of muscle-specific contractile protein synthesis. Extrapolating from these observations suggests that growth factors may have more than one role in myogenesis in vitro. They not only stimulate proliferation, but also inhibit differentiation in the absence of proliferation. Examining the kinetics of withdrawal from the cell cycle indicates that ts-36, cultured in conditioned medium blocks at the npt restriction point rather than the conditioned medium block. Our results suggest that two conditions must be met to trigger myogenic differentiation in vitro. Withdrawal from the cell cycle in G1 alone is not sufficient. Reduction of the mitogen level in the medium below a threshold level is an obligate condition for phenotypic expression.     

Skeletal myogenesis : Control of proliferation in a normal cell lineage

The fates of 10-day chick embryo myogenic cell populations cultured in conditions that maximize or suppress proliferation or fusion were examined. The repression of the cell cycle, prevalence of fusible cells, or expression of myofibril formation were monitored by autoradiography, counts of myotube nuclei, staining with fluorescein-labelled antibody against skeletal myosin heavy chain, or electron microscopy. Using a calcium chelator (EGTA) to block cell fusion did not prevent the accumulation of myoblasts blocked in G1 of the cell cycle that initiated skeletal myosin synthesis and myofibril formation. Trypsinization, dilution, and daily feeding with fresh medium and serum did not reverse this cell cycle block. Using cytochalasin B (CB) as an alternative fusion block confirmed these results. Using fluorodeoxyuridine (FUdR) to prevent the cycling of myogenic cells that normally would have multiplied in vitro did not prompt these cells to fuse. Muscle-conditioned medium could not prompt a switch in the commitment of replicating cells when in G1 to terminal differentiation. The indications are that myogenic cell populations contain definite mixtures of precursor phenotypes. The terminal phenotype is initiated coordinately with a relatively stable cue for the repression of the cell cycle. Differentiation-specified growth control is discussed within the context of a set of growth control mechanisms known to operate in culture.     

DNA polymerase activity in muscle cultures

Nuclei within myotubes do not synthesize DNA for replication. Accordingly, cultures of myotubes display low levels of DNA polymerase activity. The coincidental decline in DNA polymerase activity and increased formation of multinucleated myotubes during culture does not prove that the loss of capacity to synthesize DNA is a consequence of fusion. Tne experiments described demonstrate that myogenic cells prevented from fusing have low levels of DNA polymerase activity. This is consistent with the notion that, in myogenic cultures, there is a population of mononucleated cells, the myoblasts, which have withdrawn from the mitotic cycle before fusion.     

INHIBITION OF CELLULAR DIFFERENTIATION BY PHOSPHOLIPASE C : I. Effects of the Enzyme on Myogenesis and Chondrogenesis In Vitro

In cell culture, a partially purified commercial preparation of phospholipase C (PLC) from Clostridium welchii inhibited fusion of myoblasts at concentrations of 12–50 µg per ml. At lower concentrations, PLC-treated cultures were indistinguishable from controls, and at concentrations above 100 µg per ml, PLC-treated cells detached from their substrates. The effect was reversible and fusion resumed approximately one cell cycle time after removal of the enzyme. Neither the percent of cells in the mitotic cycle nor the duration of the different phases of the cycle were altered by PLC at concentrations which inhibited fusion. Cell motility was not reduced by the enzyme. Unfused, PLC-treated myoblasts were virtually indistinguishable in ultrastructure from untreated cells just before fusion. In the presence of PLC, mononucleated myogenic cells did not synthesize thick (150 Å) filaments. Treatment of culture medium with insolubilized commercial PLC did not abolish the capacity of the medium to support myogenesis. Chondrocytes treated with PLC divided repeatedly but failed to synthesize metachromatic matrix and failed to incorporate labeled sulfate into chondroitin sulfate. PLC was further purified by chromatography on Sephadex G-100. The resulting preparation was free of detectable protease, yielded one band on SDS-acrylamide gel electrophoresis, and displayed all of the biological activities of the less pure material.     

In vitro characterization of proliferation and differentiation of trout satellite cells

Fish satellite cells have been extracted from various species, but the myogenic characteristics of these cells in culture remain largely unknown. We show here that 60%-70% of the adherent cells are myogenic based on their immunoreactivity for the myogenic regulatory factor MyoD. In DMEM containing 10% fetal calf serum (FCS), trout myoblasts display rapid expression of myogenin (18% of myogenin-positive cells at day 2) combined with rapid fusion into myotubes (50% of myogenin-positive nuclei and 30% nuclei in myosin heavy chain [MyHC]-positive cells at day 7). These kinetics of differentiation are reminiscent of the behavior of fetal myoblasts in mammals. However, not all the myogenic cells differentiate; this subpopulation of cells might correspond to the previously named “reserve” cells. More than 90% of the BrdU-positive cells are also positive for MyoD, indicating that myogenic cells proliferate in vitro. By contrast, less than 1% of myogenin-positive cells are positive for BrdU suggesting that myogenin expression occurs only in post-mitotic cells. In order to maximize either the proliferation or the differentiation of cells, we have defined new culture conditions based on the use of a proliferation medium (F10+10%FCS) and a differentiation medium (DMEM+2%FCS). Three days after switching the medium, the differentiation index (% MyHC-positive nuclei) is 40-fold higher than that in proliferation medium, whereas the proliferation index (% BrdU-positive nuclei) is three-fold lower. Stimulation of cell proliferation by insulin-like growth factor 1 (IGF1), IGF2, and FGF2 is greater in F10 medium. The characterization of these extracted muscle cells thus validates the use of this in vitro system of myogenesis in further studies of the myogenic activity of growth factors in trout.     

Role of Ca and Ca-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L₆. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca²⁺ transport into myoblasts. We have also demonstrated that a Ca²⁺-activated protease, CAP (mM), appears to relocate in response to the Ca²⁺ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca²⁺ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

Conversion of dermal fibroblasts to a myogenic lineage is induced by a soluble factor derived from myoblasts

The limb and axial skeletal muscles of mammals originate from somitic dermomyotome, which during early development separates to form two discrete structures, the dermatome and the myotome. The latter cell mass gives rise to the muscle-forming lineage while cells of the dermatome will form the skin dermal fibroblast population of the dorsal regions of the body. It has been generally accepted for some time that myotome-derived myoblasts were the sole source of muscle fibre nuclei, but evidence has recently been presented from several laboratories that fibroblasts can fuse with myoblasts to contribute active nuclei to the resulting myotubes. We report here an investigation into the myogenic capacity of fibroblasts. Confluent monocultures of mouse dermal fibroblasts, muscle fibroblasts, and C2C12 myoblasts each retain their individual phenotype when maintained for periods up to 7 days in culture. We also grew isolated colonies of fibroblasts and myoblasts in an arrangement which allowed free exchange of tissue culture medium between the 2 cell types. We found evidence of the conversion of dermal fibroblasts to a myogenic lineage as measured by the appearance of MyoD-positive cells expressing the muscle-specific intermediate filament desmin. In addition, dermal fibroblast cultures contained multinucleate syncytia positive for MyoD and containing sarcomeric myosin heavy chain. In contrast, muscle-derived fibroblasts showed no evidence of myogenic conversion when maintained in identical culture conditions. We prepared conditioned medium from confluent cultures of C2C12 myoblasts and added this material to confluent monocultures of either dermal or muscle fibroblasts. While muscle fibroblasts showed no phenotypic alterations, cultures of dermal fibroblasts responded to myoblast conditioned medium by converting to a myogenic lineage as judged by expression of MyoD and desmin. We conclude that a proportion of dermal fibroblasts retain a myogenic capacity into stages well beyond their early association with myoblasts in the dermomyotome. © 1996 Wiley-Liss, Inc.