Multinuclear Myotubes Research Articles

VAMP2 is expressed in myogenic cells during rat development

Vesicle-associated membrane protein 2 (VAMP2) is a member of the SNARE family of proteins that regulate the intracellular vesicle fusion process. This study investigated the developmental expression of VAMP2 in the rat embryo. In the trunk, VAMP2 was primarily found in the heart on embryonic day (E) 10. On E12.5, VAMP2 expression was found in nerve fibers, somites, and heart. In somites, epithelial cells in the dorsomedial lip, and elongated myoblasts in myotome were positive for VAMP2. On E16.5, VAMP2 was expressed in the heart, nerve fibers, and skeletal muscles. In skeletal muscles, multinuclear myotubes were positive for VAMP2. In the head, where muscles are derived both from somitic and non-somitic origin, VAMP2 was found in myotubes of the extrinsic ocular muscles and masseter muscle on E16.5. These findings suggest the involvement of VAMP2 in the development of skeletal muscles of somitic and non-somitic origins.

M-cadherin and β-catenin participate in differentiation of rat satellite cells

Cadherins belong to a large family of membrane glycoprotein adhesion receptors that mediate homophilic, calcium-dependent cell adhesion. During myogenesis, cadherins are involved in initial cell-to-cell recognition; and it has also been suggested that they play a role in the initiation of myoblast fusion into multinuclear myotubes. One of the members of the cadherin family, M-cadherin, has been detected during embryogenesis in myogenic cells of somitic origin and in adult muscles. We investigated the distribution and function of M-cadherin and β-catenin during differentiation of myoblasts in primary cultures of rat satellite cells. We found that M-cadherin was accumulated at the areas of contact between fusing myoblasts and that it colocalized with β-catenin. Moreover, β-catenin colocalized with actin in pre-fusing myoblasts. We show that myoblast differentiation is accompanied by an increase in the amounts of M-cadherin and β-catenin both at the mRNA and the protein level. Flow cytometry analysis showed that M-cadherin expression was highest in fusing myoblasts. In addition, an antibody specific for the extracellular domain of M-cadherin inhibited the fusion of cultured myoblasts. These data suggest that regulation of the M-cadherin level plays an important role in the differentiation of satellite cells and in myoblast fusion in primary cultures.

Reduced expression of nicotinic AChRs in myotubes from spinal muscular atrophy I patients

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degeneration of motoneurons and skeletal muscle atrophy. In its most severe form, it leads to death before the age of 2 years. While primary degeneration of motor neurons is well established in this disease, and this results in neurogenic atrophy of skeletal muscle, we have previously reported evidence for a primary muscle defect. In this study, we used primary cultures of embryonic human skeletal muscle cells from patients with SMA and from controls to examine the effects of muscle fiber differentiation in the absence of a nerve component. Cultured SMA skeletal muscle cells are unable to fuse correctly to form multinuclear myotubes, the precursors of the myofibers. We also show that agrin-induced aggregates of nicotinic acetylcholine receptors, one of the earliest steps of neuromuscular junction formation, cannot be visualized by confocal microscopy on cells from SMA patients. In binding experiments, we demonstrate that this lack of clustering is due to defective expression of the nicotinic acetylcholine receptors in the myotubes of SMA patients whereas the affinity of α-bungarotoxin for its receptor remains unchanged regardless of muscle cell type (SMA or control). These observations suggest that muscle cells from SMA patients have intrinsic abnormalities that may affect proper formation of the neuromuscular junction.

Selective cell dissemination into the heart by retrograde intracoronary infusion in the rat.

Cell transplantation through the intracoronary route has theoretical advantages over direct intramuscular injection in global cell dissemination into the myocardium with less injury. The authors have established a novel experimental model of retrograde intracoronary infusion in the rat by which the feasibility of this infusion for cell transplantation and the dynamics of myoblasts grafted by this route were investigated. After retrograde intracoronary implantation of beta-galactosidase-expressing myoblasts, myoblasts were observed to disseminate to all layers of only the left ventricular free wall, with little myocardial damage, and to differentiate into multinuclear myotubes by day 28. Beta-galactosidase enzyme activity estimated that 31.4% of initially grafted myoblasts existed within the heart at 10 min after implantation. The number slightly increased by day 3, increased to 117.3% at day 7, and reached a plateau of 132.1% at day 14. These data suggest the utility of retrograde intracoronary infusion for selective cell dissemination into the myocardium.

Smad proteins function as co-modulators for MEF2 transcriptional regulatory proteins.

An emerging theme in transforming growth factor-ss (TGF-ss) signalling is the association of the Smad proteins with diverse groups of transcriptional regulatory proteins. Several Smad cofactors have been identified to date but the diversity of TGF-ss effects on gene transcription suggests that interactions with other co-regulators must occur. In these studies we addressed the possible interaction of Smad proteins with the myocyte enhancer-binding factor 2 (MEF2) transcriptional regulators. Our studies indicate that Smad2 and 4 (Smad2/4) complexes cooperate with MEF2 regulatory proteins in a GAL4-based one-hybrid reporter gene assay. We have also observed in vivo interactions between Smad2 and MEF2A using co-immunoprecipitation assays. This interaction is confirmed by glutathione S:-transferase pull-down analysis. Immunofluorescence studies in C2C12 myotubes show that Smad2 and MEF2A co-localise in the nucleus of multinuclear myotubes during differentiation. Interestingly, phospho-acceptor site mutations of MEF2 that render it unresponsive to p38 MAP kinase signalling abrogate the cooperativity with the Smads suggesting that p38 MAP Kinase-catalysed phosphorylation of MEF2 is a prerequisite for the Smad-MEF2 interaction. Thus, the association between Smad2 and MEF2A may subserve a physical link between TGF-ss signalling and a diverse array of genes controlled by the MEF2 cis element.

Identification of skeletal muscle satellite cells by transfecting EGFP driven by skeletal alpha-actin promoter.

In isolating skeletal muscle satellite cells, sometimes a problem is encountered in removing contaminating nonmyogenic cells. In the present study, we constructed a novel vector, pSKA-EGFP, which achieves the expression of enhanced green fluorescent protein (EGFP) exclusively in myogenic cells under the control of skeletal alpha-actin promoter when transfected to primary cultured cells from skeletal muscle. Cells from rat skeletal muscle positive for EGFP after transfecting with pSKA-EGFP were all positive for desmin and none of the nonmyogenic cells expressed EGFP, indicating that the expression of EGFP is specific to myogenic cells. Among the cells positive for EGFP were proliferating cells, presumably satellite cells. In addition, EGFP positive cells derived from horse skeletal muscle after transfecting pSKA-EGFP in vitro formed multinuclear myotubes, indicating that myogenic expression of EGFP driven by skeletal alpha-actin was achieved also in the equine cells. These results indicated that pSKA-EGFP vector will be useful in identifying and following up the satellite cells in real time, and also permit us to isolate satellite cells in combination with fluorescence-activated cell sorting (FACS).

Expression of the E6 and E7 Genes of Human Papillomavirus (HPV16) Extends the Life Span of Human Myoblasts

Primary human myoblasts (satellite cells), like other human cells, have a limited life spanin vitro.Here we show that expression of the E6E7 early region from human papillomavirus type 16 can greatly extend the life span of both fetal and satellite cell-derived myoblasts and release them from dependence on the growth factors normally necessary for their proliferation. Expression of either the E6 or the E7 gene alone was not sufficient to confer this phenotype, although expression of E7 did delay cellular senescence. The steady-state level of E6E7 transcripts in clonal cultures correlated with proliferative capacity and inversely with the capacity to differentiate into multinuclear myotubes. The expression of E7 alone markedly inhibited cell fusion in both adult and fetal cultures. These effects on myoblast differentiation could be related in part to the level of retinoblastoma protein (pRb), the major cellular target of E7. Terminal differentiation of skeletal myoblasts is associated with permanent withdrawal from the cell cycle; however, continued expression of E6E7 in differentiated myotubes permits reentry of myotube nuclei into S phase in response to growth factor stimulation. These results support a key role for pRb in the acquisition and maintenance of the differentiated state in human skeletal muscle and, in cooperation with p53, in the control of proliferative capacity and response to external growth factors.

Myoblast Fusion Promotes the Appearance of Active Protease Nexin I on Human Muscle Cell Surfaces

Protease nexin I (PNI) is a 43- to 50-kDa glycoprotein capable of inhibiting a number of serine proteases and belongs to the serpin superfamily. PNI is identical to glia-derived nexin, a neurite outgrowth promoter by virtue of its thrombin-inhibiting activity. Of particular relevance to neuromuscular biology and pathology, PNI was the first serpin shown to be highly localized to the neuromuscular junction and it maps to precisely the same locus as autosomal recessive amyotrophic lateral sclerosis (ALSJ) at chromosome 2q33–35. In the present report, we now show that in cultures of human skeletal muscle, PNI protein is expressed only after myoblast fusion into multinuclear myotubes and is localized in patches on their surfaces. We performed complex formation experiments with labeled thrombin, another target protease for PNI, with intact human muscle cells in culture. We detected specific SDS-stable PNI/thrombin complexes in myotube extracts only, indicating that active PNI was bound to their surfaces. We studied the gene expression of PNI mRNA using a 300-bp cDNA synthesized from the published sequence of human PNI. Confirming the protein data, upregulation of PNI appears in myotubes using Northern blot analysis. The current results reinforce the hypothesis that the regulation of the balance of serine proteases and serpins, such as PNI, is involved in muscle differentiation. They also prompt us to explore PNI abnormalities in several neuromuscular diseases, including ALSJ.

Transient expression of the intermediate filament nestin during skeletal muscle development

It has previously been established that skeletal muscle development is accompanied by changes in the composition of intermediate filaments: vimentin is expressed predominantly in myoblasts and desmin in adult myotubes. We show that the intermediate filament transitions during muscle development are more complex, and involve a transient expression of the recently discovered intermediate filament nestin. Nestin RNA is expressed predominantly early, in a biphasic pattern, and is markedly downregulated in adult rat muscle, whereas desmin RNA becomes more abundant throughout development. Nestin protein was found up to the postnatal myotube stage, where it colocalized with desmin in Z bands. The intracellular distribution of nestin, vimentin and desmin was analysed in the human myogenic cell line G6 before and after in vitro differentiation. Despite its more distant evolutionary and structural relationship to the other two intermediate filaments, nestin formed a cytoplasmic filamentous network indistinguishable from that of desmin and vimentin, both in undifferentiated myoblasts and after differentiation to multinuclear myotubes. In conclusion, our data suggest that nestin is an integrated component of the dynamic intermediate filament network during muscle development and that nestin copolymerizes with desmin and vimentin at stages of coexpression.

Preparation of Poly(2-hydroxyethyl methacrylate)-Collagen Composites

Linear and three-dimensional polymer composites were prepared on the basis of poly(2-hydroxyethyl methacrylate)--pHEMA--and collagen. Their biological properties were tested by in vitro as well as in vivo methods. The composite material (unlike pure pHEMA) supported myoblast adhesion as well as their fusion into multinuclear myotubes in vitro. The three-dimensional polymer composites stimulated a new bone formation after their intraosseal implantation in dogs and pigs. The biomaterial itself was degraded in the host organism, in contrast to stable pHEMA.

Na,K-ATPase expression in C2C12 cells during myogenesis: Minimal contribution of α2 Isoform to Na,K transport

Cells of the murine skeletal muscle line, C2C12, undergo differentiation from mononuclear myoblasts to multinuclear myotubes that express a number of proteins associated with striated muscle. We examined the relationship between the abundance of the mRNAs encoding the fast-twitch Ca-ATPase and the alpha isoforms of Na,K-ATPase and the subsequent expression of their respective polypeptides. Both the mRNA and protein levels of the alpha 1 isoform remained constant throughout differentiation. In contrast, the content of mRNAs encoding the alpha 2 isoform and fast-twitch Ca-ATPase increased coordinately with the abundance of their corresponding polypeptides during myotube development. Despite the dramatic increase in alpha 2 expression, estimates of in vitro Na,K-ATPase activity and assessments of in vivo transport activity suggest that alpha 2 contributes little to ionic homeostasis in C2C12 myotubes.

The formation of somites and early myotomal myogenesis in Xenopus laevis, Bombina variegata and Pelobates fuscus*

ABSTRACT Myogenesis in Xenopus laevis and in Bombina variegata is similar despite differences in the structure of the nonsegmented mesoderm and in the formation of the myotomes. In X. laevis the nonsegmented mesoderm consists of two cell layers with the premyocoel between them. During somitogenesis the premyoblasts rotate covering subsequently the whole myotome length. In B. variegata the premyocoel is absent. The myotomal cells change their shape and elongate, attaining ultimately the whole myotome length. The morphologically mature mononuclear muscle cells in both species result from myogenesis beginning in similarly arranged myoblasts. The multinuclear myotubes arise in the swimming tadpole (stage 45). The structure of the nonsegmented mesoderm and of the newly formed myotomes in Pelobates fuscus is similar to that of B. variegata, while the process of myogenesis is different. It begins in the multinuclear myotubes. The stage of morphologically mature mononuclear muscle cells was not observed in the light microscope. The results suggest that myotomal myogenesis is related neither to any particular type of nonsegmented mesoderm structure nor to any specific mode of myotome formation.