Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.

We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.     

Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy

The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity-- suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.     

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.     

The methylation state of 2 muscle-specific genes: restriction enzyme analysis did not detect a correlation with expression

To examine the possible role of DNA methylation in the modulation of expression of genes involved in the differentiation of muscle cells, we compared the methylation state of a number of CpG sites in the rat skeletal muscle actin and myosin light chain 2 genes, in muscle and nonmuscle cells, and in proliferating myoblasts and differentiated myotubes of the myogenic cell line L8. No correlation was detected between the state of methylation of these sites and the expression of the two genes. Essentially the same pattern of DNA methylation was observed, in the sites examined, in DNA from muscle, kidney and stomach. In DNA extracted from cultures of proliferating mononucleated myoblasts, as well as from differentiated multinucleated fibers of the myogenic cell line L8, the two genes were more methylated than in other tissues.     

Differentiation-dependent regulation of skeletal myogenesis by neuregulin-1

Neuregulins comprise a group of growth factor proteins that regulate the differentiation of skeletal muscle. Here, we report that neuregulins are regulators of myogenic differentiation and stimulate mitogenesis in L6 skeletal myoblasts. The mitogenic response to neuregulin-1 was differentiation-dependent and observed only in aligned, differentiating cells. Treatment of these cells with neuregulin-1 increased [3H]thymidine incorporation and cell proliferation by 2- to 5-fold, while a minimal increase was seen in proliferating myoblasts. Neuregulin-1 did not induce DNA synthesis in fused, multinucleated myotubes. The increased DNA synthesis correlated with downregulation of myogenin and inhibition of myoblast fusion and myotube formation. These data suggest that neuregulins may regulate skeletal myogenesis in vivo and that this regulation is dependent on the state of differentiation of the myocytes.     

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.     

Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture

Satellite cells represent a cellular source of regeneration in adult skeletal muscle. It remains unclear why a large pool of stem myoblasts in denervated muscle does not compensate for the loss of muscle mass during post-denervation atrophy. In this study, we present evidence that satellite cells in long-term denervated rat muscle are able to activate synthesis of contractile proteins after single fusions in situ. This process of early differentiation leads to formation of abnormally diminutive myotubes. The localization of such dwarf myotubes beneath the intact basal lamina on the surface of differentiated muscle fibers shows that they form by fusion of neighboring satellites or by the progeny of a single satellite cell following one or two mitotic divisions. We demonstrated single fusions of myoblasts using electron microscopy, immunocytochemical labeling and high resolution confocal digital imaging. Sequestration of nascent myotubes by the rapidly forming basal laminae creates a barrier that limits further fusions. The recruitment of satellite cells in the formation of new muscle fibers results in a progressive decrease in their local densities, spatial separation and ultimate exhaustion of the myogenic cell pool. To determine whether the accumulation of aberrant dwarf myotubes is explained by the intrinsic decline of myogenic properties of satellite cells, or depends on their spatial separation and the environment in the tissue, we studied the fusion of myoblasts isolated from normal and denervated muscle in cell culture. The experiments with a culture system demonstrated that the capacity of myoblasts to synthesize contractile proteins without serial fusions depended on cell density and the availability of partners for fusion. Satellite cells isolated from denervated muscle and plated at fusion-permissive densities progressed through the myogenic program and actively formed myotubes, which shows that their myogenic potential is not considerably impaired. The results of this study suggest that under conditions of denervation, progressive spatial separation and confinement of many satellite cells within the endomysial tubes of atrophic muscle fibers and progressive interstitial fibrosis are the important factors that prevent their normal differentiation. Our findings also provide an explanation of why denervated muscle partially and temporarily is able to restore its functional capacity following injury and regeneration: the release of satellite cells from their sublaminal location provides the necessary space for a more active regenerative process.     

Immortalization of murine muscle cells from lysosomal α-glucosidase deficient mice: A new tool to study pathophysiology and assess therapeutic strategies for Pompe disease

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder caused by defects in the acid α-glucosidase (GAA) gene leading to lysosomal glycogen accumulation, mainly in cardiac and muscle tissues. In order to facilitate biological investigation on this disease and to avoid time-consuming direct cell isolation and culture, we have established murine myogenic GSDII cell lines. Lentiviral/retroviral expression of SV40 T antigen, Bmi-1 or cyclin-dependent kinase 4 (CDK4) genes was used to induce the immortalization of primary satellite cells from GSDII mice. The resulting immortalized myoblasts exhibit phenotypic characteristics of their parental cells, including profound GAA deficiency, glycogen accumulation and the ability to fully differentiate into myotubes when placed in proper culture conditions. These cell lines will constitute a powerful tool for both basic and applied studies focused on a better understanding of the pathophysiological mechanisms involved in GSDII and for assessing putative therapeutic strategies.     

Myotube Formation on Micro-patterned Glass: Intracellular Organization and Protein Distribution in C2C12 Skeletal Muscle Cells

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. Studies of myocyte differentiation, cell fusion, and muscle repair are limited by an appropriate in vitro muscle cell culture system. We developed a novel cell culture technique [two-dimensional muscle syncytia (2DMS) technique] that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. This technique is based on UV lithography–produced micro-patterned glass on which conventionally cultured C2C12 myoblasts proliferate, align, and fuse to neatly arranged contractile myotubes in parallel arrays. Combining this technique with fluorescent microscopy, we observed alignment of actin filament bundles and a perinuclear distribution of glucose transporter 4 after myotube formation. Newly formed myotubes contained adjacently located MyoD-positive and MyoD-negative nuclei, suggesting fusion of MyoD-positive and MyoD-negative cells. In comparison, the closely related myogenic factor Myf5 did not exhibit this pattern of distribution. Furthermore, cytoplasmic patches of MyoD colocalized with bundles of filamentous actin near myotube nuclei. At later stages of differentiation, all nuclei in the myotubes were MyoD negative. The 2DMS system is thus a useful tool for studies on muscle alignment, differentiation, fusion, and subcellular protein localization. (J Histochem Cytochem 56:881–892, 2008)     

Activity-dependent gene regulation in conditionally-immortalized muscle precursor cell lines

Skeletal muscle contractile activity has been implicated in many aspects of muscle cell differentiation and maturation. Much of the research in this area has depended upon costly and labor-intensive cultures of isolated primary muscle cells because widely available immortalized muscle cell lines often do not display a high level of either spontaneous or stimulated contractile activity. We sought to develop conditionally-immortalized skeletal muscle cell lines that would provide a source of myofibers that exhibit robust spontaneous contractile activity similar to primary muscle cultures. Using a tetracycline-regulated retroviral vector expressing a temperature-sensitive T-antigen to infect primary myoblasts, we isolated individual clonal muscle precursor cell lines that have characteristics of activated satellite cells during growth and rapidly differentiate into mature myotubes with spontaneous contractile activity after culture in non-transformation-permissive conditions. Comparison of these cell lines (known as rat myoblast-like tetracycline (RMT) cell lines) to primary cell cultures revealed that they share a wide variety of morphological, physiological, and biochemical characteristics. Most importantly, the time-course and extent of activity-dependent gene regulation observed in primary cell culture for all genes tested, including subunits of the nicotinic acetylcholine receptor (nAChR), muscle specific kinase (MuSK), and myogenin, is reproduced in RMT lines. These immortalized cell lines are a useful alternative to primary cultures for studying muscle differentiation and molecular and physiological aspects of electrical activity in muscle fibers.     

Promoting differentiation of cultured myoblasts using biomimetic surfaces that present alpha-laminin-2 peptides

Traditionally, muscle cell lines are cultured on glass coverslips and differentiated to investigate myoblast fusion and differentiation. Efficient differentiation of myoblasts produces a dense network of myotubes with the correct organisation for contraction. Here we have tested the ability of artificially generated, precisely controlled peptide surfaces to enhance the efficiency of myoblast differentiation. We focused on specific short peptides from α-laminin-2 (IKVSV, VQLRNGFPYFSY and GLLFYMARINHA) as well as residues 15–155 from FGF1. We tested if these peptides in isolation, and/or in combination promoted muscle differentiation in culture, by promoting fusion and/or by improving sarcomere organisation. The majority of these peptides promoted fusion and differentiation in two different mouse myogenic cell lines and in primary human myoblasts. The additive effects of all four peptides gave the best results for both mouse cell lines tested, while primary human cell cultures differentiated equally well on most peptide surfaces tested. These data show that a mixture of short biomimetic peptides can reliably promote differentiation in mouse and human myoblasts.     

'Early' mammalian myoblasts are resistant to phorbol ester-induced block of differentiation

Mesenchymal cells were isolated from somites and limbs of mouse embryos at different developmental stages. When grown in tissue culture, some of the cells underwent muscle differentiation as indicated by synthesis of sarcomeric myosin, acetylcholine receptor and, in the case of limb cells, fusion into multinucleated myotubes. When the tumour promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) was added to these cultures, it caused differential effects, depending upon the age of the embryo from which cells were isolated. In cultures of somites or limb bud from embryos up to 12 days post coitum, TPA did not interfere with the appearance of differentiated muscle cells. When TPA was added to cultures from older embryos, it inhibited muscle differentiation with an efficiency which increased with the age of the embryo, reaching about 90% inhibition at 15 days. After this period, a new population of myogenic cells appeared in the limb, which were able to differentiate in the presence of TPA and represented the great majority of myoblasts after day 18 of embryonic development. The simplest interpretation of these data can be based on the existence of three major classes of myogenic cell precursors, which appear sequentially during muscle histogenesis: 'early' myoblasts, which appear resistant to tumour promoters; 'late' myoblasts, whose differentiation is inhibited by tumour promoters and 'satellite' cells which, like early myoblasts, show no sensitivity to TPA.     

Aphidicolin induces myogenic differentiation in the human rhabdomyosarcoma cell line KFR.

The effects of aphidicolin, a specific inhibitor of DNA polymerase α, on cell growth, DNA synthesis and myogenic differentiation in the human alveolar rhabdomyosarcoma cell line KFR were studied. The treatment with aphidicolin at 5 × 10⁻⁶ M concentration, which completely inhibited DNA synthesis and cell growth, induced morphological differentiation of small mononuclear cells to elongated, multinucleated (myotube-like) structures. The morphological differentiation was accompanied by the expression of skeletal muscle myosin; about 30% myosin-positive cells were observed after 14 days of treatment, compared to 2.3% in untreated cultures. The results showed that aphidicolin induces differentiation of human rhabdomyosarcoma cells and that multinucleated myotube-like elements may develop simply by cell fusion without cell division and DNA synthesis.     

Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a "myosheet," was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.     

Calpastatin in rat myoblasts: transient diminution and decreased phosphorylation depend on myogenin-directed myoblast differentiation

The formation of skeletal muscle fibers involves cessation of myoblast division, followed by myoblast differentiation and fusion to multinucleated myofibers. The myogenic regulatory factor myogenin appears at the onset of differentiation; it is required for muscle fiber formation, and cannot be replaced by other factors. The myogenin-dependent pathways and targets are not fully known. Previous studies, indicating an involvement of calpain-calpastatin and caspase in myoblast fusion, were based on the use of various inhibitors. The availability of myogenin deficient cell lines that are incapable of fusion, but regain the ability to differentiate when transfected with myogenin, provide a convenient means to study calpain-calpastatin and caspase in fusing and non-fusing myoblasts without the use of inhibitors. The differentiating wild type myoblasts exhibit decreased calpastatin phosphorylation, transient diminution in calpastatin mRNA, caspase-1 dependent diminution in calpastatin protein, and calpain-promoted proteolysis. In the myogenin-deficient myoblasts, calpastatin phosphorylation is not diminished, caspase-1 is not activated, calpastatin mRNA and protein are not diminished, and protein degradation does not occur. The myogenin-deficient myoblasts transfected with myogenin gene regain the ability to fuse, and exhibit the alterations in calpastatin and proteolysis observed in the wild type cells. Overall, the results demonstrate that the regulation of calpain in these myoblasts is independent of myogenin. In contrast, the regulation of calpastatin depends on myogenin function. The temporary diminution of calpastatin during myogenin-directed differentiation of myoblasts allows calpain activation and calpain-induced protein degradation, required for myoblast differentiation and fusion.     

Partial laminin alpha2 chain restoration in alpha2 chain-deficient dy/dy mouse by primary muscle cell culture transplantation

Laminin-2 is a component of skeletal and cardiac basal lamina expressed in normal mouse and human. Laminin alpha2 chain (LAMA2), however, is absent from muscles of some congenital muscular dystrophy patients and the dystrophia muscularis (dy/dy) mouse model. LAMA2 restoration was investigated following cell transplantation in vivo in dy/dy mouse. Allogeneic primary muscle cell cultures expressing the beta- galactosidase transgene under control of a muscular promoter, or histocompatible primary muscle cell cultures, were transplanted into dy/dy mouse muscles. FK506 immunosuppression was used in noncompatible models. All transplanted animals expressed LAMA2 in these immunologically-controlled models, and the degrees of LAMA2 restoration were shown to depend on the age of the animal at transplantation, on muscle pretreatment, and on duration time after transplantation in some cases. LAMA2 did not always colocalize with new or hybrid muscle fibers formed by the fusion of donor myoblasts. LAMA2 deposition around muscle fibers was often segmental and seemed to radiate from the center to the periphery of the injection site. Allogeneic conditionally immortalized pure myogenic cells expressing the beta-galactosidase transgene were characterized in vitro and in vivo. When injected into FK506- immunosuppressed dy/dy mice, these cells formed new or hybrid muscle fibers but essentially did not express LAMA2 in vivo. These data show that partial LAMA2 restoration is achieved in LAMA2-deficient dy/dy mouse by primary muscle cell culture transplantation. However, not all myoblasts, or myoblasts alone, or the muscle fibers they form are capable of LAMA2 secretion and deposition in vivo.     

Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis

During terminal differentiation of skeletal myoblasts, cells fuse to form postmitotic multinucleated myotubes that cannot reinitiate DNA synthesis. Here we investigated the temporal relationships among these events during in vitro differentiation of C2C12 myoblasts. Cells expressing myogenin, a marker for the entry of myoblasts into the differentiation pathway, were detected first during myogenesis, followed by the appearance of mononucleated cells expressing both myogenin and the cell cycle inhibitor p21. Although expression of both proteins was sustained in mitogen-restimulated myocytes, 5- bromodeoxyuridine incorporation experiments in serum-starved cultures revealed that myogenin-positive cells remained capable of replicating DNA. In contrast, subsequent expression of p21 in differentiating myoblasts correlated with the establishment of the postmitotic state. Later during myogenesis, postmitotic (p21-positive) mononucleated myoblasts activated the expression of the muscle structural protein myosin heavy chain, and then fused to form multinucleated myotubes. Thus, despite the asynchrony in the commitment to differentiation, skeletal myogenesis is a highly ordered process of temporally separable events that begins with myogenin expression, followed by p21 induction and cell cycle arrest, then phenotypic differentiation, and finally, cell fusion.