Myotubes myoblast differentiation

Cell proliferation and differentiation Electric conductivity was introduced by electrospinning carbon nanotubes with polyurethane. Myotube formation and myoblast differentiation were significantly increased with electrical stimulation apphed on the scaffolds. Sirivisoot et al. Composition... 

The added attraction of the transition from myoblast to myotube is the synchrony with which differentiation occurs in vitro. Myogen-esis will occur in primary cultures of skeletal muscle (e.g. 6.12) but can also be induced in diploid myoblast lines (Richter and Yaffe, 1970) which has allowed the selection of mutants (Chapter 13) that exhibit drug resistance or temperature-sensitive differentiation (Loomis et al., 1973 Somers et al., 1975). Holtzer et al. (1975) and Fiszman and Fuchs (1975) have developed a myoblast line transformed with a temperature-sensitive virus. At the permissive temper-... 

GH also has actions on the differentiation of muscle. It stimulates the conversion of myoblasts to myotubes in vitro, with associated changes in the levels of creatine phosphokinase [99]. It is also possible that GH has direct effects on the differentiation of cartilage, acting synergistically with somatomedins to promote cartilage growth [72],... 

Bechet DM, Ferrara MJ, Mordier SB, Roux MP, Deval CD, Obled A. Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation, characterization of a cDNA encoding bovine cathepsin B. J Biol Chem. 1991 266 14104-14112. 

Prenylation also influences the balance between myocyte viability and apoptosis. Statin-induced apoptosis has been demonstrated in vitro, using myotubes [84], myoblasts [85], and differentiated primary human skeletal muscle cells [86], This effect can be reproduced by geranyl-geranyl-transferase inhibitors, and rescued by replacement of mevalonic acid [84], Compelling evidence suggests that statins cause apoptosis in skeletal muscle by disrupting the prenylation of small G proteins like Rho [85],... 

Most cell lines have lost some or many of the functions characteristic of the differentiated cells from which they were derived. Such relatively undifferentiated cells are poor models for investigating the normal functions of specific cell types. Better in this regard are several more-differentiated cell lines that exhibit many properties of normal nontransformed cells. These lines Include the liver tumor (hepatoma) HepG2 line, which synthesizes most of the serum proteins made by normal liver cells (hepatocytes). Another example consists of cells from a certain cultured fibroblast line, which under certain experimental conditions behave as muscle precursor cells, or myoblasts. These cells can be Induced to fuse to form myotubes, which resemble differentiated multlnucleated muscle cells and synthesize many of the specialized proteins associated with contraction. The results of studies with this cell line have provided valuable information about the differentiation of muscle (Chapter 22). Finally, as discussed previously, the MDCK cell line retains many properties of highly differentiated epithelial cells and forms well-defined epithelial sheets in culture (see Figure 6-6). 

Figure 8.22 Fluorescence images of differentiated primary myotubes, aligned along medium density fibers of poly(OTE] (A and B] or poly(HET) (C]. A and B are the same image without (A] and with [B] fibers shown. Myotuhes differentiated on the gold mylar substrate (D] or on low densities of poly(OTE] (E] or poly(HET) [F] fibers were randomly oriented and branched. Myotube nuclei were stained with DAPI (blue] whereas the myoblast protein desmin was immunostained with an anti-Desmin primary antibody and Alexa 546-ta ed (red] secondary antibody. Scale bars represent 100 micrometer. Reprinted with permission from Ref. 246, Copyright 2013, American Chemical Society.

First, we sought to develop an effective assay that could enable rapid measurement of statin-induced toxicity and its suppression by small molecules [10]. Our previous work to generate a mitochondrial compendium in muscle cells provided us the preliminary data needed for this assay. We seeded C2C12 myoblasts in optical 384-well plates, and when the cells reached confluence, induced differentiation to myotubes by incubating with 2% horse serum for 4-6 days. Differentiation was confirmed visually by observing cell fusion and syncytia formation characteristic of myotubes. We then treated differentiated myotubes for 48 h with 10 pM simvastatin, and measured cellular ATP levels with a commercially available kit (CellTiter-Glo, Promega). As expected, we observed a twofold reduction in ATP levels compared to dimethyl sulfoxide (DMSO) controls. 

There seems to be simultaneous synthesis of embryonic and adult subunits in early stages of myotube differentiation. This is not unexpected since complete hybrid sets of both isoenzymes apparently formed by subunit association are found during normal in situ development (Eppen-berger et al., 1964 Lebherz, 1972a). Addition of actinomycin D at various intervals prior to fusion or shortly after fusion shows, on the one hand, that the required RNA is present in the cell at least 6 hours before fusion, and, on the other hand, that in fused cells the CPK increase proceeds after actinomycin D addition for about 12 more hours (Shainberg et al., 1971). This constitutes evidence that the cell is able to continue protein synthesis and thus indicates that stable mRNA must be present (Fig. 6). Transcription of at least part of the information required for subsequent differentiation must have already occurred when myoblasts entered the phase of fusion. 

Myogenesis in vivo and in vitro involves conversion of cycling and undifferentiated myoblasts to terminally differentiated and noncycling myotubes [1,2]. The sequence of events associated with differentiation of muscle cells in culture has been well defined and involves myoblast proliferation to confluency, cessation of DNA synthesis with irreversible withdrawal of myoblasts from the cell cycle, plasma membrane fusion and formation of multinucleated myotubes, and induction of muscle specific proteins and isoenzymes (e.g., myosin, a-actin, a-tropomysin, creatine phosphokinase, etc.) at the time of onset of fusion [3-5]. 

The isolation of established lines of myoblasts which retain the abUity to fuse and form myotubes has provided a very useful system for studying muscle differentiation [14]. These cells display the same characteristic morphological and biochemical changes as freshly explanted muscle cells in culture and offer the advantage that pure populations of myoblasts and myotubes can be obtained. 

The formation of skeletal muscle tissue in vivo involves the determination of precursor cells to the myoblastic pathway, fusion of myoblasts into multinucleated myotubes, and maturation into muscle fibers. Tourovskaia and colleagues [10] have developed a microfluidic cell culture system for myogenesis studies that provides control over the external environment to fuUy recapitulate myogenic differentiation in vivo and allows the manipulation of subcellular domains within mature myotubes for simulation of postsynaptic differentiation and further modula-... 

It is well known that proliferation or differentiation of nerve or muscle tissue can be enhanced by electrical stimulation, which is in part attributed to the propagation of action potentials upon stimulation. To exploit these physiological events, composites of biodegradable polymers with electroconduc-tive materials have been investigated for effective transmission of electrical signals to the cells cultured on them. PLCL and polyaniline, one of the widely used electrically conductive materials, were mixed to fabricate the nanofiber meshes, illustrating that myotube formation was accelerated when the myoblasts were cultured on polyaniline-containing nanofibers compared to that of PLCL-only nanofibers, even without electrical stimulation (Jun et ah, 2009). Another electroconductive polymer, polypyrrole, was also used as a composite with other synthetic polymers. For example, polypyrrole was incorporated as particles into polylactide scaffolds, and the fibroblasts cultured on them with various intensities of DC current showed controlled proliferation in a current-dependent manner (Shi et al., 2004). 

With respect to muscle differentiation in the limb, mononucleated myoblasts are also initially separated from each other by a hyaluronan-rich matrix in vivo and express pericellular matrices in culture [49] (Figure 2E). However during the processes of condensation and fusion to give myotubes these coats are lost [49] (Figure 2F), thus allowing the interactions required for these cells to differentiate. 

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