Overexpression of interleukin-15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders

Interleukin-15 (IL-15) is a novel anabolic factor for skeletal muscle which inhibits muscle wasting associated with cancer (cachexia) in a rat model. To develop a cell culture system in which the mechanism of the anabolic action of IL-15 on skeletal muscle could be examined, the mouse C2 skeletal myogenic cell line was transduced with a retroviral expression vector for IL-15 and compared to sister cells transduced with a control vector. Overexpression of IL-15 induced fivefold higher levels of sarcomeric myosin heavy chain and alpha-actin accumulation in differentiated myotubes. Secreted factors from IL-15-overexpressing myogenic cells, but not from control cells, induced increased myofibrillar protein accumulation in cocultured control myotubes. IL-15 overexpression induced a hypertrophic myotube morphology similar to that described for cultured myotubes which overexpressed the well-characterized anabolic factor insulin-like growth factor-I (IGF-I). However, in contrast to IGF-I, the hypertrophic action of IL-15 on skeletal myogenic cells did not involve stimulation of skeletal myoblast proliferation or differentiation. IL-15 induced myotube hypertrophy at both low and high IGF-I concentrations. Furthermore, in contrast to IGF-I, which stimulated only protein synthesis under these culture conditions, IL-15 both stimulated protein synthesis and inhibited protein degradation in cultured skeletal myotubes. These findings indicate that IL-15 action on skeletal myogenic cells is distinct from that of IGF-I. Due to the ability of IGF-I to stimulate cell division and its association with several forms of cancer, controversy exists concerning the advisability of treating cachexia or age-associated muscle wasting with IGF-I. Administration of IL-15 or modulation of the IL-15 signaling pathway may represent an alternative strategy for maintaining skeletal muscle mass under these conditions.     

A muscle cell line from dystrophic mice expressing an altered phenotype in vitro

The isolation and characterization of a myogenic cell line from C57BL/6J/dydy mice is described. This line (DyA4) maintains the morphological, biochemical and electrophysiological characteristics of the primary cultured cells, at least for 20 passages. The cells actively divide as long as they are subcultured in media supplemented with horse serum and embryo extract. If the cells are not subcultured for a few days, they fuse into multinucleated contracting myotubes, which readily synthesize specific muscle products such as acetylcholinesterase and acetylcholine receptor. This dystrophic cell line expresses in vitro the same altered phenotype that is characteristic of dystrophic muscle cells in primary cultures, namely reduced acetylcholine sensitivity and reduced acetylcholine receptor expression. Because they can be grown in large amounts, and represent a pure muscle cell population which express an altered phenotype in an in vitro aneural avascular environment, DyA4 cells provide a very useful model system for investigating the pathogenesis of murine muscular dystrophy.     

Cell types required to efficiently innervate human muscle cells in vitro

Previous studies carried out in our laboratory have shown that myofibers formed by fusion of muscle satellite cells from donors with spinal muscular atrophy (SMA) type I or II undergo a characteristic degeneration 1.5-3 weeks after innervation with rat embryonic spinal cord explants. The only cells responsible for degeneration of innervated cocultures are SMA muscle satellite cells. In order to study the kinetics of nerve and muscle cell degeneration in nerve-muscle cocultures implicating SMA muscle cells, we attempted to simplify the nervous component of the coculture and identify the nerve cell types necessary for a successful innervation. We demonstrate here that motoneurons alone were unable to innervate myotubes. However, when three cell types (motoneurons, sensory neurons, and Schwann cells) were added onto a reconstituted muscular component consisting of cloned muscle satellite cells and cloned muscular fibroblasts, myotubes contracted, indicating that functional neuromuscular junctions were formed. We concluded that the three cell types were required for a successful innervation. Moreover, we studied the effects of culture medium conditioned by different combinations of nerve cells on innervation; we observed that physical contacts among sensory neurons, motoneurons, and myotubes are required for a successful innervation; in contrast Schwann cells can be replaced by a Schwann-cell-conditioned medium, indicating that these cells produce a putative soluble "innervation-promoting factor." Obviously such a reconstituted system does not reflect the in vivo situation but it allows the formation of functional motor synapses and could therefore allow us to elucidate neuromuscular disease pathogenesis, especially that of spinal muscular atrophy.     

Differential myogenicity of satellite cells isolated from extensor digitorum longus (EDL) and soleus rat muscles revealed in vitro

Following muscle damage, fast- and slow-contracting fibers regenerate, owing to the activation of their satellite cells. In rats, crush-induced regeneration of extensor digitorum longus (EDL, a fast muscle) and soleus (a slow muscle) present different characteristics, suggesting that intrinsic differences exist among their satellite cells. An in vitro comparative study of the proliferation and differentiation capacities of satellite cells isolated from these muscles is presented there. We observed several differences between soleus and EDL satellite cell cultures plated at high density on gelatin-coated dishes. Soleus satellite cells proliferated more actively and fused into myotubes less efficiently than EDL cells. The rate of muscular creatine kinase enzyme appeared slightly lower in soleus than in EDL cultures at day 11 after plating, when many myotubes were formed, although the levels of muscular creatine kinase mRNA were similar in both cultures. In addition, soleus cultures expressed higher levels of MyoD and myogenin mRNA and of MyoD protein than EDL satellite cell cultures at day 12. A clonal analysis was also carried out on both cell populations in order to determine if distinct lineage features could be detected among satellite cells derived from EDL and soleus muscles. When plated on gelatin at clonal density, cells from both muscles yielded clones within 2 weeks, which stemmed from 3–15 mitotic cycles and were classified into three classes according to their sizes. Myotubes resulting from spontaneous fusion of cells from the progeny of one single cell were seen regardless of the clone size in the standard culture medium we used. The proportion of clones showing myotubes in each class depended on the muscle origin of the cells and was greater in EDL- than in soleus-cell cultures. In addition, soleus cells were shown to improve their differentiation capacity upon changes in the culture condition. Indeed, the proportions of clones showing myotubes, or of cells fusing into myotubes in clones, were increased by treatments with a myotube-conditioned medium, with phorbol ester, and by growth on extra-cellular matrix components (Matrigel). These results, showing differences among satellite cells from fast and slow muscles, might be of importance to muscle repair after trauma and in pathological situations.     

On the non-equivalence of skeletal muscle satellite cells and embryonic myoblasts

Postnatal satellite cells, isolated from normal or previously denervated skeletal muscles of juvenile quails, were tested as to their capacity to participate in embryonic muscle ontogeny. They were grafted into 2-day chick embryo hosts, in place of a piece of brachial somitic mesoderm. Satellite cell implants were prepared from pellets either of freshly isolated cells or of cells precultured in vitro under proliferative conditions. Myogenic capacity of the implanted cells was attested by their ability to fuse into myotubes when cultured under differentiation conditions. In no case did the implanted satellite cells invade the adjacent wing bud or participate in wing muscle morphogenesis. They did not either give rise to myotubes at the site of implantation, nor did they even survive longer than 3 days in the embryonic environment. These negative results indicate that postnatal satellite cells, unlike embryonic myoblasts, are unable to take part in muscle embryogenesis. Although they derive from the same somitic myogenic cell line as the embryonic myoblasts, they therefore represent a differentiated non-totipotent type of myogenic cell.     

Intact satellite cells lead to remarkable protection against Smn gene defect in differentiated skeletal muscle

Deletion of murine Smn exon 7, the most frequent mutation found in spinal muscular atrophy, has been directed to either both satellite cells, the muscle progenitor cells and fused myotubes, or fused myotubes only. When satellite cells were mutated, mutant mice develop severe myopathic process, progressive motor paralysis, and early death at 1 mo of age (severe mutant). Impaired muscle regeneration of severe mutants correlated with defect of myogenic precursor cells both in vitro and in vivo. In contrast, when satellite cells remained intact, mutant mice develop similar myopathic process but exhibit mild phenotype with median survival of 8 mo and motor performance similar to that of controls (mild mutant). High proportion of regenerating myofibers expressing SMN was observed in mild mutants compensating for progressive loss of mature myofibers within the first 6 mo of age. Then, in spite of normal contractile properties of myofibers, mild mutants develop reduction of muscle force and mass. Progressive decline of muscle regeneration process was no more able to counterbalance muscle degeneration leading to dramatic loss of myofibers. These data indicate that intact satellite cells remarkably improve the survival and motor performance of mutant mice suffering from chronic myopathy, and suggest a limited potential of satellite cells to regenerate skeletal muscle.     

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.     

Isolation and characterization of myogenic satellite cells from the muscular dystrophic hamster

Myogenic satellite cells were isolated from control and dystrophic hamster diaphragms to examine cellular mechanisms involved in the physiology of muscular dystrophy. The Bio 14.6 dystrophic hamster, which possesses a defect in the delta-sarcoglycan gene, develops biochemical and physical symptoms of Duchenne-like and limb girdle muscular dystrophies. Because primary cultures of the control and dystrophic satellite cells became extensively contaminated with non-myogenic cells during proliferation, cell clones were developed to provide pure cultures for study. Cell culture conditions were optimized with the use of Ham's F-12K medium containing 10% fetal bovine serum +5% horse serum + 10 ng/mL basic fibroblast growth factor + 50 microg/mL porcine gelatin. Proliferation rates of the two clonal cultures were similar between the two lines. Satellite cell-derived myotubes from both primary cultures and clones differed between control and dystrophic animals. Dystrophic myotubes tended to be long and narrow, while the control-derived myotubes were broader. Measurement of muscle-specific creatine kinase during differentiation revealed that the dystrophic myotubes possessed higher creatine kinase levels than control myotubes (up to 146-fold at 168 h). The results demonstrate that satellite cells can be isolated from the hamster and may provide a useful tool to study muscular dystrophies associated with defects in the sarcoglycan complex and the involvement of sarcoglycans in normal skeletal muscle growth and development.     

Heterogeneity in growth and differentiation characteristics in male and female satellite cells isolated from turkey lines with different growth rates

The effects of growth- and gender-related differences on satellite cell proliferation and differentiation were investigated using satellite cells isolated from the pectoralis major muscle of a turkey line selected for increased 16-week body weight (F-line) and its unselected randombred control (RBC2-line). Proliferation rates within the F- and RBC2-lines did not differ between sexes. The F-line male and female satellite cells when compared to the RBC2-line male and female satellite cells proliferated at a faster rate. Differentiation rates were increased for the F-line male cells compared to both the F-line female and RBC2-line male satellite cells. No difference in differentiation rate was noted within the RBC2-line satellite cells. For satellite cells from females, the RBC2-line differentiated faster than the F-line. Morphological data on myotube length and the number of nuclei per myotube supported the differentiation data in that F-line male satellite cells had the longest myotubes with the most nuclei, there was no significant difference between myotubes within the RBC2-line, and female-derived myotubes from the RBC2-line were longer than those of the F-line by 96 h of fusion. These data are suggestive of both growth- and gender- related differences in satellite cell proliferation and differentiation.     

Emerin expression at the early stages of myogenic differentiation

Emerin is an ubiquitous protein localized at the nuclear membrane of most cell types including muscle cells. The protein is absent in most patients affected by the X-linked form of Emery-Dreifuss muscular dystrophy, a disease characterized by slowly progressive muscle wasting and weakness, early contractures of the elbows, Achilles tendons, and post-cervical muscles, and cardiomyopathy. Besides the nuclear localization, emerin cytoplasmic distribution has been suggested in several cell types. We studied the expression and the subcellular distribution of emerin in mouse cultured C2C12 myoblasts and in primary cultures of human myoblasts induced to differentiate or spontaneously differentiating in the culture medium. In differentiating myoblasts transiently transfected with a cDNA encoding the complete emerin sequence, the protein localized at the nuclear rim of all transfected cells and also in the cytoplasm of some myoblasts and myotubes. Cytoplasmic emerin was also observed in detergent-treated myotubes, as determined by electron microscopy observation. Both immunofluorescence and biochemical analysis showed, that upon differentiation of C2C12 cells, emerin expression was decreased in the resting myoblasts but the protein was highly represented in the developing myotubes at the early stage of cell fusion. Labeling with specific markers of myogenesis such as troponin-T and myogenin permitted the correlation of increased emerin expression with the onset of muscle differentiation. These data suggest a role for emerin during proliferation of activated satellite cells and at the early stages of differentiation.     

Fusion between myoblasts and adult muscle fibers promotes remodeling of fibers into myotubes in vitro

Muscle satellite cells are residual embryonic myoblast precursors responsible for muscle growth and regeneration. In order to examine the role of satellite cells in the initial events of muscle regeneration, we placed individual mature rat muscle fibers in vitro along with their satellite cells. When the satellite cells were allowed to proliferate, they produced populations of myoblasts that fused together to form myotubes on the laminin substrate. These myoblasts and myotubes also fused with the adult fibers. When they did so, the fibers lost their adult morphology, and by 8 days in vitro, essentially all of them were remodeled into structures resembling embryonic myotubes. However, when proliferating satellite cells were eliminated by exposure to cytosine arabinoside (araC), the vast majority of fibers retained their adult shape. Addition of C2C12 cells (a myoblast line derived from adult mouse satellite cells) to araC-treated fiber cultures resulted in their fusion with the rat muscle fibers and restored the ability of the fibers to remodel, whereas addition of either a fibroblast cell line or a transformed, non-fusing variant of C2C12 cells, or addition of conditioned medium from C2C12 cells, failed to do so. These results imply that myoblast fusion is responsible for triggering adult fiber remodeling in vitro.     

Growth factor supplemented matrigel improves ectopic skeletal muscle formation--a cell therapy approach

Following damage to skeletal muscle, satellite cells become activated, migrate towards the injured area, proliferate, and fuse with each other to form myotubes which finally mature into myofibers. We tested a new approach to muscle regeneration by incorporating myoblasts, with or without the exogenous growth factors bFGF or HGF, into three-dimensional gels of reconstituted basement membrane (matrigel). In vitro, bFGF and HGF induced C2C12 myoblast proliferation and migration and were synergistic when used together. In vivo, C2C12 or primary i28 myoblasts were injected subcutaneously together with matrigel and growth factors in the flanks of nude mice. The inclusion of either bFGF or HGF increased the vascularization of the gels. Gels supplemented with bFGF showed myogenesis accompanied by massive mesenchymal cell recruitment and poor organization of the fascicles. Samples containing HGF showed delayed differentiation with respect to controls or bFGF, with increased myoblast proliferation and a significantly higher numbers of cells in myotubes at later time points. HGF samples showed limited mesenchymal cell infiltration and relatively good organization of fascicles. The use of both bFGF and HGF together showed increased numbers of nuclei in myotubes, but with bFGF-mediated fibroblast recruitment dominating. These studies suggest that an appropriate combination of basement membrane components and growth factors could represent a possible approach to enhance survival dispersion, proliferation, and differentiation of myogenic cells during muscle regeneration and/or myoblast transplantation. This model will help develop cell therapy of muscle diseases and open the future to gene therapy approaches.     

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Notch1 enhances B-cell receptor-induced apoptosis in mature activated B cells without affecting cell cycle progression and surface IgM expression

The transmembrane receptor Notch1 plays a crucial role in differentiation and apoptosis of hematopoietic cells. To investigate the influence of Notch1 on apoptosis and cell growth of mature murine B cells, we transduced the murine B-lymphoma line NYC 31.1 with a constitutively active, intracellular form of human Notch1 (Notch1-ICT). NYC cells represent mature activated B cells that can be induced to undergo apoptosis by crosslinking of the B-cell receptor (BCR). In contrast to investigations in immature chicken B-cell lines, transduced Notch1-ICT did not affect cell cycle progression, cell growth or surface IgM levels in NYC cells and resulted only in a slight induction of apoptosis. However, BCR-crosslinking enhanced apoptosis, but did not influence cell cycle progression in Notch1-ICT-positive NYC cells. These data imply a distinct function of Notch1 in mature murine B-cells as compared to immature chicken B cells and provide further evidence for Notch1's involvement in B-cell differentiation and development.     

Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD

Hypoxia alters the biological functions of skeletal muscle cells to proliferate and differentiate into myotubes. However, the cellular responses of myoblasts to hypoxia differ according to the levels of oxygen and the types of cells studied. This study examined the effect of hypoxia (1% oxygen) on bovine satellite cells. Hypoxia significantly increased the proliferation of satellite cells cultured in a growth medium. In addition, the levels of PCNA, cyclin D1, cyclin-dependent kinase-1 (CDK1) and CDK2 expression were increased. Hypoxia facilitated the formation of myotubes as well as the stimulation of MyoD, myogenin, and myosin heavy chain (MHC) expression in differentiating medium (DM) cultures. In particular, satellite cells cultured under hypoxic/DM conditions showed increased p21 expression but not p27. The transfection of satellite cells with antisense MyoD oligonucleotides resulted in a decrease in the MHC, myogenin, MRF4 RNA and protein levels with the concomitant decrease in fused cells to levels similar to those observed under normoxia/DM conditions. This indicates that MyoD up-regulation is closely associated with hypoxia-stimulated myogenic differentiation. In conclusion, hypoxia stimulates the proliferation of satellite cells and promotes their myogenic differentiation with MyoD playing an important role.     

Comparison of ovine and rat muscle-derived satellite cells: Response to insulin

A chemically defined medium has been formulated which supports the growth (proliferation and differentiation) of rat- and ovine-derived myogenic satellite cells in vitro. Utilization of this medium in a direct comparison study in which satellite cells from both species were exposed to insulin resulted in the following observations: (1) insulin promoted the dose-dependent proliferation of primary cultures of ovine-derived but not rat-derived satellite cells and (2) rat-derived satellite cells fused to form multinucleated myotubes when exposed to increasing levels of insulin. Collectively, these observations suggest that the rat satellite cell culture system may not be an appropriate model system for extrapolating in vitro growth data to variables of ruminant skeletal muscle growth.     

Entactin promotes adhesion and long-term maintenance of cultured regenerated skeletal myotubes.

The basal lamina protein, laminin, has been shown to promote migration and proliferation of cultured skeletal myoblasts, resulting in increased myotube formation. However, skeletal myotubes adhere poorly to a laminin substrate, and long-term cultures of skeletal myotubes on laminin have not been achieved. We have found that cultured satellite cells from bupivacaine-damaged rat skeletal muscle actively proliferate and differentiate on a diluted Matrigel substrate composed of laminin, type IV collagen, heparan sulfate proteoglycan, and entactin. Myotubes cultured on diluted Matrigel are contractile and have never been observed to detach from the culture dish; rather, myotubes generally atrophy after 2-3 weeks in culture. Antibodies directed against the various protein components of Matrigel were used to determine the role of each component in enhancing muscle differentiation. Anti-laminin impaired satellite cell adhesion, whereas antibodies against either type IV collagen or heparan sulfate proteoglycan had no effect. Anti-entactin did not inhibit attachment, proliferation, or fusion of cultured satellite cells; however, myotubes exposed to anti-entactin failed to adhere to the culture dish after spontaneous myotube contractions began. We conclude that entactin is responsible for long-term maintenance and maturation of contractile skeletal myotubes on a diluted Matrigel substrate. This is the first study to assign a biological function for entactin in myogenesis.     

Growth and differentiation of permanent and secondary mouse myogenic cell lines on microcarriers

Myogenesis involves the determination of progenitor cells to myoblasts, their fusion to yield multinuclear myotubes, and the maturation of myotubes to muscle fibres. This development is reflected in a time pattern of gene expression, e.g. of genes coding for desmin, the myogenic factors myogenin and myoD, the acetylcholine receptor alpha-subunit and the muscular chloride channel CIC-1. We attempted to improve yields and myogenic differentiation in culture by using three-dimensional microcarrier systems. Out of a variety of carriers tested in stationary cultures, collagen-coated dextran Cytodex3 beads proved optimal for the proliferation and differentiation of the murine myogenic cell line C2C12. With C2C12 myoblasts in stationary and stirred systems (Spinner- and SuperSpinner flasks), surface adherence, differentiation into myotubes and expression of muscle-specific mRNAs on Cytodex3 beads were the same as in conventional cultures. Other carriers tested (DEAE cellulose, glass, plastic, cellulose, polyester) did not support growth and differentiation of C2C12 cells. The secondary mouse myogenic stem cells M12 and M2.7-MDX proliferated and differentiated well in stationary Cytodex3 cultures, but no differentiation occurred in Spinner flasks. As indicated by light and scanning electron microscopy, C2C12 myotubes formed not only on but also in between Cytodex beads. The secondary cell lines may succumb to shear forces under these conditions.