Myotube orientation using strong static magnetic fields

In this experiment, we evaluated the effects of strong static magnetic fields (SMF) on the orientation of myotubes formed from a mouse-derived myoblast cell line, C2C12. Myogenic differentiation of C2C12 cells was conducted under exposure to SMF at a magnetic flux density of 0-10 T and a magnetic gradient of 0-41.7 T/m. Exposure to SMF at 10 T led to significant formation of oriented myotubes. Under the high magnetic field gradient and a high value of the product of the magnetic flux density and magnetic field gradient, myotube orientation increased as the myogenic differentiation period increased. At the 3 T exposure position, where there was a moderate magnetic flux density and moderate magnetic field gradient, myotube orientation was not observed. We demonstrated that SMF induced the formation of oriented myotubes depending on the magnetic flux density, and that a high magnetic field gradient and a high value of the product of the magnetic flux density and magnetic field gradient induced the formation of oriented myotubes 6 days after myogenic differentiation. We did not detect any effect of the static magnetic fields on myogenic differentiation or cell number. To the best of our knowledge, this is the first report to demonstrate that myotubes orient to each other under a SMF without affecting the cell number and myogenic differentiation.     

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)     

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.     

Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle

In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle-specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal alpha isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle gamma actin to stain cultured myotubes and adult skeletal myofibrils by double-indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double-stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin-containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.     

A subpopulation of murine bone marrow cells fully differentiates along the myogenic pathway and participates in muscle repair in the mdx dystrophic mouse

Bone marrow (BM) transplantation in mice suggests the existence of pluripotent cells able to differentiate into skeletal muscle tissue, although sustained myofiber reconstitution has not yet been achieved. We investigated the myogenic potential of mouse BM cells and evaluated whether a BM fraction enriched for cells expressing skeletal muscle markers would ameliorate muscle repair, when compared to whole BM, into the dystrophic mdx mouse. We demonstrate that cells expressing striated-muscle-specific proteins are already present in the BM independently from experimentally forced myogenic conversion. We observed the presence of both markers of early myogenic program such as Pax3, Myf5, MyoD, desmin, and late myogenesis such as myosin heavy chain and alpha-sarcomeric actin. These myogenic cells are more represented in the early nonadherent BM fraction, which generates clones able to fully differentiate into myotubes. Transplantation in mdx mice by intravenous injection of whole BM and a tenfold BM myogenic enriched fraction resulted in BM reconstitution and limited dystrophin restoration. Taken together, these data show that a fraction of BM cells have a definite potential for differentiation along the skeletal muscle pathway and can be recruited by muscle repair mechanisms. They also indicate that factors limiting the degree of muscle recruitment and the host stem cell competition should be assessed in order to evaluate the usefulness of BM-derived myogenic cells into the context of cell-mediated gene therapy of inherited muscle diseases.     

Myogenic differentiation of mesenchymal stem cells co‐cultured with primary myoblasts

TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co‐cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)‐transduced rat MSC co‐cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative PCR). MSC—myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and α‐sarcomeric actin, and MEF2 expression was up‐regulated upon co‐cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co‐cultures. In summary, we have determined optimal co‐culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell‐based therapies.     

Transient production of alpha-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation

alpha-smooth muscle actin (SMA) is typically not present in post-embryonic skeletal muscle myoblasts or skeletal muscle fibers. However, both primary myoblasts isolated from neonatal mouse muscle tissue, and C2C12, an established myoblast cell line, produced SMA in culture within hours of exposure to differentiation medium. The SMA appeared during the cells' initial elongation, persisted through differentiation and fusion into myotubes, remained abundant in early myotubes, and was occasionally observed in a striated pattern. SMA continued to be present during the initial appearance of sarcomeric actin, but disappeared shortly thereafter leaving only sarcomeric actin in contractile myotubes derived from primary myoblasts. Within one day after implantation of primary myoblasts into mouse skeletal muscle, SMA was observed in the myoblasts; but by 9 days post-implantation, no SMA was detectable in myoblasts or muscle fibers. Thus, both neonatal primary myoblasts and an established myoblast cell line appear to similarly reprise an embryonic developmental program during differentiation in culture as well as differentiation within adult mouse muscles.     

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

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

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.     

Switching of actin isoforms in skeletal muscle differentiation using mouse ES cells

Among six actin isoforms, α-skeletal and α-cardiac actins have similar amino acid components and are highly conserved. Although skeletal muscles essentially express α-skeletal actins in the adult tissue, α-cardiac isoform actin is prominent in the embryonic muscle tissue. Switching of actin isoforms from α-cardiac to α-skeletal actin occurs during skeletal muscle differentiation. The cardiac type α-actin is expressed in the regeneration and patho-physiological states of the skeletal muscles as well. In the present study, we demonstrate the morphological switching of α-type actin isoforms from α-cardiac to α-skeletal actin in vitro using mouse ES cells for the first time. Immunofluorescent double staining with two specific antibodies revealed that α-cardiac actin appeared first in myoblasts. After cell fusion to form myotubes, the cardiac type actin decreased and α-skeletal actin conversely increased. Finally, the α-skeletal isoform remained as a main actin component in the fully mature skeletal muscle fibers. The exchange of isoforms is not directly linked to the sarcomere formation. As a result, ES cells provide a useful in vitro system for exploring skeletal muscle differentiation.     

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.     

Transforming Growth Factor-��-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

Satellite cells/myoblasts account for the majority of muscle regenerative potential in response to injury and muscular adaptation to exercise. Although the ability to influence this process would provide valuable benefits for treating a variety of patients suffering from muscle loss, the regulatory mechanisms of myogenesis are not completely understood. We have tested the hypothesis that transforming growth factor-β-activated kinase 1 (TAK1) is an important regulator of skeletal muscle formation. TAK1 is expressed in proliferating C2C12 myoblasts, and its levels are reduced upon differentiation of myoblasts into myotubes. In vivo, TAK1 is predominantly expressed in developing skeletal muscle of young mice. However, the expression of TAK1 was significantly up-regulated in regenerating skeletal muscle of adult mice. Overexpression of a dominant negative mutant of TAK1 or knockdown of TAK1 inhibited the proliferation and differentiation of C2C12 myoblasts. TAK1 was required for the expression of myogenic regulatory factors in differentiating myoblasts. Genetic ablation of TAK1 also inhibited the MyoD-driven transformation of mouse embryonic fibroblasts into myotubes. Inhibition of TAK1 suppressed the differentiation-associated activation of p38 mitogen-activated protein kinase (MAPK) and Akt kinase. Overexpression of a constitutively active mutant of MAPK kinase 6 (MKK6, an upstream activator of p38 MAPK) but not constitutive active Akt restored the myogenic differentiation in TAK1-deficient mouse embryonic fibroblasts. Insulin growth factor 1-induced myogenic differentiation was also found to involve TAK1. Collectively, our results suggest that TAK1 is an important upstream regulator of skeletal muscle cell differentiation.     

Coexpression of α-sarcomeric actin, α-smooth muscle actin and desmin during myogenesis in rat and mouse embryos I. Skeletal muscle

Expression of vimentin, desmin, alpha-sarcomeric and alpha-smooth muscle actins in embryonic tissues of rat and mice was examined using an immunohistochemical approach. The results showed a similarity in the expression of desmin and alpha-actin isoforms (alpha-sr and alpha-sm) in skeletal muscle cells during murine feto-embryonic development. In the two species, coexpression of alpha-sr and alpha-sm actins has been observed in cardiomyoblasts, myotomal myoblasts and myotubes. The intensity of alpha-sm actin expression decreased during the terminal steps of myogenesis and disappeared completely in mature cardiomyocytes and myofibres. Desmin was expressed in all prefusion myoblasts (type 1 and 2 myoblasts), myotubes, and in myofibres. The appearance of desmin in myoblasts of somites preceded by a few hours the expression of the alpha-actins (alpha-sr and alpha-sm). Our study on vimentin expression, limited to rat embryos, revealed that somite premyoblasts expressed only vimentin, type 1 myoblasts expressed vimentin and desmin, and type 2 myoblasts (rhabdomyoblasts) expressed desmin and alpha-actins (alpha-sr and alpha-sm). Our study demonstrates the resemblance between feto-embryonic myogenesis and myogenic neoplastic differentiation: desmin appears before the alpha-actins in embryonic myoblasts, and can be considered as a marker of an initial step in myogenic differentiation. alpha-sm actin, considered as a striated muscle cell feto-embryonic actin, is expressed transiently in skeletal myoblasts and cardiomyoblasts during development and reappears during neoplastic transformation of skeletal muscle.     

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.     

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.