Distinct myogenic programs of embryonic and fetal mouse muscle cells: expression of the perinatal myosin heavy chain isoform in vitro.

Early embryonic and late fetal mouse myogenic cells showed distinct patterns of perinatal myosin heavy chain (MHC) isoform expression upon differentiation in vitro. In cultures of somite or limb muscle cells isolated from Day 9 to Day 12 embryos, differentiated cells that expressed perinatal MHC were rare and perinatal MHC was not detectable by immunoblotting. In cultures of limb muscle cells isolated from Day 13 to Day 18 fetuses, in contrast, the perinatal MHC isoform was easily detected and was expressed in a substantial percentage of myocytes and myotubes. Analyses of clonally derived muscle colonies and cytosine arabinoside-treated fetal muscle cell cultures suggested that different fetal muscle cell nuclei initiated perinatal MHC expression at different times. In both embryonic and fetal cell cultures, the embryonic MHC isoform was expressed by all differentiated cells examined. A small number of myotubes in fetal muscle cell cultures showed a mosaic distribution of MHC isoform accumulation in which the perinatal MHC isoform accumulated in a restricted region of the myotube near particular nuclei, whereas the embryonic MHC isoform accumulated throughout the myotube. Thus, the myogenic program of fetal, but not embryonic, mouse myogenic cells includes expression of the perinatal MHC isoform upon differentiation in culture.     

'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.     

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.     

Differences in the Expression and Distribution of Flotillin-2 in Chick,Mice and Human Muscle Cells

Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.     

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.     

Overexpression of nPKC theta is permissive for myogenic differentiation

Although protein kinase C (PKC) has been shown to participate in skeletal myogenic differentiation, the functions of individual isoforms of PKC in myogenesis have not been completely elucidated. These studies focused on the role of nPKC straight theta, an isoform of the PKC family whose expression has been shown to be regulated by commitment to the myogenic lineage, myogenic differentiation and innervation. We used the myogenic cell line C(2)C(12) as a tissue culture model system to explore the role of nPKC straight theta in the formation of multinucleated myotubes. We examined endogenous levels of nPKC straight theta in C(2)C(12) cells and showed that it is expressed at low levels in myoblasts compared to mouse skeletal muscle and that expression is maintained in myotubes. We overexpressed nPKC straight theta in C(2)C(12) myoblasts and examined the ability of overexpressing cells to differentiate into myotubes. Using an nPKC straight theta - green fluorescent protein (GFP) chimera to detect transfected myoblasts, we showed that overexpressed nPKC straight theta-GFP translocates to the plasma membrane in response to phorbol ester treatment of myoblast cultures in situ. nPKC straight theta-GFP was found to be completely extracted into the detergent-soluble fraction of cell lysates and was stably expressed throughout the extent of differentiation into myotubes. No difference was seen in the ability of myoblasts either overexpressing nPKC straight theta - GFP or GFP alone to form myotubes. These studies demonstrate that overexpression of nPKC straight theta does not interfere with fusion of myoblasts into myotubes suggesting that nPKC straight theta activity is not inhibitory for myogenesis. These studies also demonstrate a method for transfecting myoblasts and identifying differentiated cells that overexpress nPKC straight theta-GFP for investigating the function of nPKC straight theta in living myotubes.     

Myogenic potential of human alveolar mucosa derived cells

Difficulties related to the obtainment of stem/progenitor cells from skeletal muscle tissue make the search for new sources of myogenic cells highly relevant. Alveolar mucosa might be considered as a perspective candidate due to availability and high proliferative capacity of its cells. Human alveolar mucosa cells (AMC) were obtained from gingival biopsy samples collected from 10 healthy donors and cultured up to 10 passages. AMC matched the generally accepted multipotent mesenchymal stromal cells criteria and possess population doubling time, caryotype and immunophenotype stability during long-term cultivation. The single myogenic induction of primary cell cultures resulted in differentiation of AMC into multinucleated myotubes. The myogenic differentiation was associated with expression of skeletal muscle markers: skeletal myosin, skeletal actin, myogenin and MyoD1. Efficiency of myogenic differentiation in AMC cultures was similar to that in skeletal muscle cells. Furthermore, some of differentiated myotubes exhibited contractions in vitro. Our data confirms the sufficiently high myogenic potential and proliferative capacity of AMC and their ability to maintain in vitro proliferation-competent myogenic precursor cells regardless of the passage number.     

Static magnetic fields enhance skeletal muscle differentiation in vitro by improving myoblast alignment.

Static magnetic field (SMF) interacts with mammal skeletal muscle; however, SMF effects on skeletal muscle cells are poorly investigated. The myogenic cell line L6, an in vitro model of muscle development, was used to investigate the effect of a 80 +/- mT SMF generated by a custom-made magnet. SMF promoted myogenic cell differentiation and hypertrophy, i.e., increased accumulation of actin and myosin and formation of large multinucleated myotubes. The elevated number of nuclei per myotube was derived from increased cell fusion efficiency, with no changes in cell proliferation upon SMF exposure. No alterations in myogenin expression, a modulator of myogenesis, occurred upon SMF exposure. SMF induced cells to align in parallel bundles, an orientation conserved throughout differentiation. SMF stimulated formation of actin stress-fiber like structures. SMF rescued muscle differentiation in the presence of TNF, a muscle differentiation inhibitor. We believe this is the first report showing that SMF promotes myogenic differentiation and cell alignment, in the absence of any invasive manipulation. SMF-enhanced parallel orientation of myotubes is relevant to tissue engineering of a highly organized tissue such as skeletal muscle. SMF rescue of muscle differentiation in the presence of TNF may have important therapeutic implications.     

Possible application of muscle specific conditional mouse-derived induced pluripotent stem cells for muscle research

The Cre-driver mouse line, which allows for in vivo regulation of target gene(s) in specific cells, is an indispensable tool for recent muscle research. In this study, I aimed to explore new applications of muscle specific Cre-driver mouse line in muscle research. For this purpose, I generated an iPS cells from a myofiber specific conditional mouse with tamoxifen inducible GFP expression, and then I checked whether homologous recombination was induced in the iPS-derived myogenic cells by tamoxifen administration. Fibroblasts were isolated from the tails of Myf6^CE/wt::CAG-EGFP mice, which expressed GFP specifically in Myf6 lineages by tamoxifen injection, and then iPS cells was generated by transfection with a vector based on sendai-virus and containing OSKM genes. Muscle specific conditional mouse-derived iPS cells (mCM-iPSCs) were successfully differentiated to myogenic cells, such as Pax7⁺ muscle progenitors, MyoD⁺ myoblasts, and MHC⁺ myotubes, under myogenic differentiation conditions. Using this model, I examined whether homologous recombination was induced in mCM-iPSC-derived myotubes by 4-hydroxytamoxifen (4OH-TAM) administration. As a result, multinucleated myotubes showed GFP expression, while no GFP signals were detected in both Pax7⁺ muscle progenitor and non-myogenic cells. These results indicated that homologous recombination could be induced in mCM-iPSC–derived myotubes by tamoxifen administration, and that this system operated normally even in reprogrammed cells. Also, I evidenced that GFP reporter was expressed in myoblasts in addition to multinucleated myotubes when tamoxifen-pulse was applied at an early phase of myogenesis. Taken together, Myf6^CE/wt::CAG-EGFP mouse-derived iPS cells reproduced at least in part Myf6 expression during mouse myogenesis. This study demonstrated a novel application of muscle specific conditional mouse in addition to in vivo application, and mCM-iPSCs could also be used in in vitro investigations with muscle specific conditional knock-out mouse.     

IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures

In a previous study investigating the effects of low temperature on skeletal muscle differentiation, we demonstrated that C2C12 mouse myoblasts cultured at 30 °C do not express myogenin, a myogenic regulatory factor (MRF), or fuse into multinucleated myotubes. At this low temperature, the myoblasts continuously express Id3, a negative regulator of MRFs, and do not upregulate muscle-specific microRNAs. In this study, we examined if insulin-like growth factor-I (IGF-I) and a stable form of vitamin C (L-ascorbic acid phosphate) could alleviate the low temperature-induced inhibition of myogenic differentiation in C2C12 cells. Although the addition of either IGF-I or vitamin C alone could promote myogenin expression in C2C12 cells at 30 °C, elongated multinucleated myotubes were not formed unless both IGF-I and vitamin C were continuously administered. In human skeletal muscle cells, low temperature-induced blockage of myogenic differentiation was also ameliorated by exogenous IGF-I and vitamin C. In addition, we demonstrated that satellite cells of IGF-I overexpressing transgenic mice in single-fiber culture expressed myogenin at a higher level than those of wild-type mice at 30 °C. This study suggests that body temperature plays an important role in myogenic differentiation of endotherms, but the sensitivity to low temperature could be buffered by certain factors in vivo, such as IGF-I and vitamin C.     

Insulin receptor substrate protein 53kDa (IRSp53) is a negative regulator of myogenic differentiation

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell-extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes.     

Optimization of bovine satellite cell-derived myotube formation in vitro

Post-natal myogenic satellite cells, isolated from the sternomandibularis muscles of bovine at slaughter were used for primary culture studies. Isolated satellite cells tended to differentiate into multinucleated myotubes more efficiently if initially plated on to a fibronectin substratum. Bovine-derived satellite cells displayed greater fused cell numbers when exposed to Dulbecco's Modified Eagle's Medium (DMEM) supplemented with horse serum than similar supplementation with fetal calf serum (P less than 0.05) or sheep serum (P less than 0.05). In addition, differentiation appeared nearly complete after 4 days exposure to DMEM-1% horse serum as verified by beta-D-arabinofuranosyl-cytosine addition to cultures. Collectively, these data provide the first evidence that satellite cells can be isolated from a bovine skeletal muscle. Furthermore, these data indicate that bovine-derived satellite cells can be induced to undergo substantial morphological differentiation in vitro.     

Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion.

Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient centrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the 200,000-dalton subunit of myosin. When placed in an in vitro protein-synthesizing assay containing [³H]leucine, total polysomes from both mononucleated and multinucleated myogenic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the burst of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with ribosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.     

Creatine kinase, myokinase, and acetylcholinesterase activities in muscle-forming primary cultures of mouse teratocarcinoma cells.

Multipotential mouse teratocarcinoma cells in embryoid bodies were explanted on plastic or collagen substrates. Various modes of cell determination, including myogenesis, occurred. The predominant avenue of differentiation soon became myogenesis: many multinucleated myotubes formed and yielded an extensive network of skeletal muscle fibers. The process does not proceed to normal completion, as the fibers have a paucity of striations and are not contractile. Activities of several enzymes ordinarily associated with muscle differentiation were examined. Acetylcholinesterase activity increases, especially during myotube formation, as in normal myogenesis. However, creatine kinase activity rises during myotube formation and then drops abnormally, and myokinase activity fails to increase appreciably. The fetal isozymic form of creatine kinase is expressed in the cultures, although well differentiated solid tumors taken from mice show attainment of the adult muscle isozyme type if skeletal muscle is demonstrably present. The results are consistent with the interpretation that coordinately regulated changes in gene expressions controlling these functions may be required for later stages of myogenesis.     

Role of muscle fibroblasts in the deposition of type-IV collagen in the basal lamina of myotubes

In cell cultures of quail, chick, or mouse skeletal muscle, both myogenic and fibrogenic cells synthesize and secrete type-IV collagen, a major structural component of the basal lamina. Type-IV collagen, together with laminin, forms characteristic patches and strands on the surface of developing myotubes, marking the onset of basement-membrane formation. The pattern for type-IV collagen and laminin is unique to these proteins and is not paralleled by other matrix proteins, such as fibronectin or type-I or -III collagen. In the present study, we used species-specific antibodies to either mouse or chick type-IV collagen to demonstrate the ability of fibroblast--derived type-IV collagen to incorporate in the basal lamina of myotubes. In combination cultures of embryonic quail skeletal myoblasts and mouse muscle fibroblasts, antibodies specific for mouse type-IV collagen revealed the deposition of type-IV collagen on the surface of quail myotubes in the pattern typical of the beginning of basement-membrane formation. Control cultures consisting of only quail muscle cells containing myoblasts and fibroblasts demonstrated no such reaction with these antibodies. Deposits of mouse type-IV collagen were also observed on the surface of quail myotubes when conditioned medium from mouse muscle fibroblasts was added to quail myoblast cultures. Similarly, in combination cultures of mouse myoblasts and chick muscle fibroblasts, chick type-IV-collagen deposits were identified on the surface of mouse myotubes. These results indicate that type-IV collagen synthesized by muscle fibroblasts may be incorporated into the basal lamina forming on the plasmalemma of myotubes, and may explain ultrastructural studies by Lipton on the contribution of fibroblasts to the formation of basement membranes in skeletal muscle.     

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.     

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.     

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.