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Skeletal muscle demonstrates a high degree of regenerative capacity repeating the embryonic myogenic program under strict control. Rhabdomyosarcoma is the most common sarcoma in childhood and is characterized by impaired muscle differentiation. In this study, we observed that silencing the expression of syndecan-4, the ubiquitously expressed transmembrane heparan sulfate proteoglycan, significantly enhanced myoblast differentiation, and fusion. During muscle differentiation, the gradually decreasing expression of syndecan-4 allows the activation of Rac1, thereby mediating myoblast fusion. Single-molecule localized superresolution direct stochastic optical reconstruction microscopy (dSTORM) imaging revealed nanoscale changes in actin cytoskeletal architecture, and atomic force microscopy showed reduced elasticity of syndecan-4-knockdown cells during fusion. Syndecan-4 copy-number amplification was observed in 28% of human fusion-negative rhabdomyosarcoma tumors and was accompanied by increased syndecan-4 expression based on RNA sequencing data. Our study suggests that syndecan-4 can serve as a tumor driver gene in promoting rabdomyosarcoma tumor development. Our results contribute to the understanding of the role of syndecan-4 in skeletal muscle development, regeneration, and tumorigenesis. [ABSTRACT FROM AUTHOR]

Inherited fatty acid oxidation diseases in their mild forms often present as metabolic myopathies. Carnitine Palmitoyl Transferase 2 (CPT2) deficiency, one such prototypical disorder is associated with compromised myotube differentiation. Here, we show that CPT2-deficient myotubes exhibit defects in focal adhesions and redox balance, exemplified by increased SOD2 expression. We document unprecedented alterations in the cellular prion protein PrPC, which directly arise from the failure in CPT2 enzymatic activity. We also demonstrate that the loss of PrPC function in normal myotubes recapitulates the defects in focal adhesion, redox balance and differentiation hallmarks monitored in CPT2-deficient cells. These results are further corroborated by studies performed in muscles from Prnp−/− mice. Altogether, our results unveil a molecular scenario, whereby PrPC dysfunction governed by faulty CPT2 activity may drive aberrant focal adhesion turnover and hinder proper myotube differentiation. Our study adds a novel facet to the involvement of PrPC in diverse physiopathological situations. [ABSTRACT FROM AUTHOR]

The myostatin (MSTN) gene is of interest in the livestock industry because mutations in this gene are closely related to growth performance and muscle differentiation. Thus, in this study, we established MSTN knockout (KO) quail myoblasts (QM7) and investigated the regulatory pathway of the myogenic differentiation process. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate MSTN KO QM7 cells and subsequently isolated a single cell-derived MSTN KO QM7 subline with 10- and 16-nucleotide deletions that induced translational frameshift mutations. The differentiation capacity and proliferation rate of MSTN KO QM7 cells were enhanced. We conducted next-generation-sequencing (NGS) analysis to compare the global gene expression profiles of wild-type (WT) QM7 and MSTN KO QM7 cells. Intriguingly, NGS expression profiles showed different expression patterns of p21 and p53 in MSTN KO QM7 cells. Moreover, we identified downregulated expression patterns of leukemia inhibitory factor and DNA Damage Inducible Transcript 4, which are genes in the p53 signaling pathway. Using quantitative RT-PCR (qRT-PCR) analysis and western blotting, we concluded that p53-related genes promote the cell cycle by upregulating p21 and enhancing muscle differentiation in MSTN KO QM7 cells. These results could be applied to improve economic traits in commercial poultry by regulating MSTN-related networks. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study is to investigate Ranolazine action on skeletal muscle differentiation and mitochondrial oxidative phenomena. Ranolazine, an antianginal drug, which acts blocking the late INaL current, was shown to lower hemoglobin A1c in patients with diabetes. In the present study, we hypothesized an action of Ranolazine on skeletal muscle cells regeneration and oxidative process, leading to a reduction of insulin resistance. Methods: 10 μM Ranolazine was added to C2C12 murine myoblastic cells during proliferation, differentiation and newly formed myotubes. Results: Ranolazine promoted the development of a specific myogenic phenotype: increasing the expression of myogenic regulator factors and inhibiting cell cycle progression factor (p21). Ranolazine stimulated calcium signaling (calmodulin-dependent kinases) and reduced reactive oxygen species levels. Furthermore, Ranolazine maintained mitochondrial homeostasis. During the differentiation phase, Ranolazine promoted myotubes formation. Ranolazine did not modify kinases involved in skeletal muscle differentiation and glucose uptake (extracellular signal-regulated kinases 1/2 and AKT pathways), but activated calcium signaling pathways. During proliferation, Ranolazine did not modify the number of mitochondria while decreasing osteopontin protein levels. Lastly, neo-formed myotubes treated with Ranolazine showed typical hypertrophic phenotype. Conclusion: In conclusion, our results indicate that Ranolazine stimulates myogenesis and reduces a pro-oxidant inflammation/oxidative condition, activating a calcium signaling pathway. These newly described mechanisms may partially explain the glucose lowering effect of the drug. [ABSTRACT FROM AUTHOR]

Nuclear lamins form nuclear lamina located under the inner nuclear membrane. It was believed that the nuclear lamina plays predominantly a structural role. Recently, its involvement in regulatory processes have been described, e.g., chromatin organization and gene transcription. It is known that mutations in the LMNA gene lead to development of laminopathies, primarily affecting tissues of mesenchymal origin. Today, the mechanisms of the lamina regulation of cell differentiation are largely unknown. In the present work, we studied the effect of LMNA gene mutations on the process of muscle differentiation of primary satellite cells and in С2С12 cell line. The genome of satellite and С2С12 cells was modified by cell transduction via lentiviral constructs encoding LMNA G232E associated with the development of Emery-Dreyfus muscular dystrophy and LMNA R571S associated with the development of dilated cardiomyopathy. Cell morphology was assessed with immunofluorescence, and expression of myogenic genes was analyzed by qPCR. We showed that the analyzed mutations reduced the cell ability to differentiate (to fuse and to form myotubes). We proposed that these mutations enhanced expression of early and reduced expression of late markers of myogenesis. Thus, mutations in nuclear lamins can modify the process of muscle differentiation. [ABSTRACT FROM AUTHOR]

Cyclin D3 is important for muscle development and regeneration, and is involved in post-mitotic arrest of muscle cells. Cyclin D3 also has cell-cycle-independent functions such as regulation of specific genes in other tissues. Ectopic expression of cyclin D3 in myoblasts, where it is normally undetectable, promotes muscle gene expression and faster differentiation kinetics upon serum depletion. In the present study, we investigated the mechanistic role of cyclin D3 in muscle gene regulation. We initially showed by mutational analysis that a stable and functional cyclin D3 was required for promoting muscle differentiation. Using chromatin immunoprecipitation assays, we demonstrated that expression of cyclin D3 in undifferentiated myoblasts altered histone epigenetic marks at promoters of muscle-specific genes like MyoD, Pax7, myogenin and muscle creatine kinase but not non-muscle genes. Cyclin D3 expression also reduced the mRNA levels of certain epigenetic modifier genes. Our data suggest that epigenetic modulation of muscle-specific genes in cyclin-D3-expressing myoblasts may be responsible for faster differentiation kinetics upon serum depletion. Our results have implications for a regulatory role for cyclin D3 in muscle-specific gene activation. [ABSTRACT FROM AUTHOR]

Myostatin, a member of the transforming growth factor-β superfamily, is a potent negative regulator of skeletal muscle growth and is conserved in many species, from rodents to humans. Myostatin inactivation can induce skeletal muscle hypertrophy, while its overexpression or systemic administration causes muscle atrophy. As it represents a potential target for stimulating muscle growth and/or preventing muscle wasting, myostatin regulation and functions in the control of muscle mass have been extensively studied. A wealth of data strongly suggests that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression. Moreover, myostatin plays a central role in integrating/mediating anabolic and catabolic responses. Myostatin negatively regulates the activity of the Akt pathway, which promotes protein synthesis, and increases the activity of the ubiquitin-proteasome system to induce atrophy. Several new studies have brought new information on how myostatin may affect both ribosomal biogenesis and translation efficiency of specific mRNA subclasses. In addition, although myostatin has been identified as a modulator of the major catabolic pathways, including the ubiquitin-proteasome and the autophagy-lysosome systems, the underlying mechanisms are only partially understood. The goal of this review is to highlight outstanding questions about myostatin-mediated regulation of the anabolic and catabolic signaling pathways in skeletal muscle. Particular emphasis has been placed on (1) the cross-regulation between myostatin, the growth-promoting pathways and the proteolytic systems; (2) how myostatin inhibition leads to muscle hypertrophy; and (3) the regulation of translation by myostatin. [ABSTRACT FROM AUTHOR]

Three cases of primary lipomatous tumors of the anterior mediastinum with prominent muscle differentiation are presented. The patients were two women and one man between the age of 52 and 68 years. All patients presented with progressive shortness of breath. Radiographically, all patients demonstrated anterior mediastinal tumors which were surgically resected. In two cases, the gross findings were those of circumscribed tumors, while one lesion was described as an ill-defined and unencapsulated neoplasm. Histologically, two cases corresponded to a well-differentiated liposarcoma, while the third was a thymolipoma. Extensive areas of smooth muscle were identified in one of the liposarcomas, while the other contained areas of mature skeletal muscle. The thymolipoma displayed a prominent myoid component. Immunohistochemical studies for muscle markers including smooth muscle actin, desmin, and myoglobin showed positive staining in the corresponding components. Follow-up information showed that one patient with liposarcoma died 60 months after initial diagnosis, while the other two patients remain alive and well 16 and 36 months after diagnosis, respectively. The current cases highlight the spectrum of muscle differentiation that can be seen in adipose tumors of the anterior mediastinum and also emphasize the difficulty that such a diagnosis can pose when confronted with limited mediastinoscopic biopsies. [ABSTRACT FROM AUTHOR]

The aim of this review article is to evaluate the current knowledge on associations between muscle formation and regeneration and components of the nuclear lamina. Lamins and their partners have become particularly intriguing objects of scientific interest since it has been observed that mutations in genes coding for these proteins lead to a wide range of diseases called laminopathies. For over the last 10 years, various laboratories worldwide have tried to explain the pathogenesis of these rare disorders. Analyses of the distinct aspects of laminopathies resulted in formulation of different hypotheses regarding the mechanisms of the development of these diseases. In the light of recent discoveries, A-type lamins-the main building blocks of the nuclear lamina-together with other key elements, such as emerin, LAP2α and nesprins, seem to be of great importance in the modulation of various signaling pathways responsible for cellular differentiation and proliferation. [ABSTRACT FROM AUTHOR]

Terminal sialic acid residues are found in abundance in glycan chains of glycoproteins and glycolipids on the surface of all live cells forming an outer layer of the cell originally known as glycocalyx. Their presence affects the molecular properties and structure of glycoconjugates, modifying their function and interactions with other molecules. Consequently, the sialylation state of glycoproteins and glycolipids has been recognized as a critical factor modulating molecular recognitions inside the cell, between the cells, between the cells and the extracellular matrix, and between the cells and certain exogenous pathogens. Sialyltransferases that attach sialic acid residues to the glycan chains in the process of their initial synthesis were thought to be mainly responsible for the creation and maintenance of a temporal and spatial diversity of sialylated moieties. However, the growing evidence also suggests that in mammalian cells, at least equally important roles belong to sialidases/neuraminidases, which are located on the cell surface and in intracellular compartments, and may either initiate the catabolism of sialoglycoconjugates or just cleave their sialic acid residues, and thereby contribute to temporal changes in their structure and functions. The current review summarizes emerging data demonstrating that neuraminidase 1 (NEU1), well known for its lysosomal catabolic function, can be also targeted to the cell surface and assume the previously unrecognized role as a structural and functional modulator of cellular receptors. [ABSTRACT FROM AUTHOR]

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms. [ABSTRACT FROM AUTHOR]

Mitochondrial reactive oxygen species (mROS) have been considered detrimental to cells. However, their physiological roles as signaling mediators have not been thoroughly explored. Here, we investigated whether mROS generated from mitochondrial electron transport chain (mETC) complex I stimulated muscle differentiation. Our results showed that the quantity of mROS was increased and that manganese superoxide dismutase (MnSOD) was induced via NF-κB activation during muscle differentiation. Mitochondria-targeted antioxidants (MitoQ and MitoTEMPOL) and mitochondria-targeted catalase decreased mROS quantity and suppressed muscle differentiation without affecting the amount of ATP. Mitochondrial alterations, including the induction of mitochondrial transcription factor A and an increase in the number and size of mitochondria, and functional activations were observed during muscle differentiation. In particular, increased expression levels of mETC complex I subunits and a higher activity of complex I than other complexes were observed. Rotenone, an inhibitor of mETC complex I, decreased the mitochondrial NADH/NAD+ ratio and mROS levels during muscle differentiation. The inhibition of complex I using small interfering RNAs and rotenone reduced mROS levels, suppressed muscle differentiation, and depleted ATP levels with a concomitant increase in glycolysis. From these results, we conclude that complex I-derived O2·−, produced through reverse electron transport due to enhanced metabolism and a high activity of complex I, was dismutated into H2O2 by MnSOD induced via NF-κB activation and that the dismutated mH2O2 stimulated muscle differentiation as a signaling messenger. [ABSTRACT FROM AUTHOR]

Localization of acetylcholine receptors (AChRs) to the postsynaptic region of muscle is mediated in part by transcriptional mechanisms. An important way of regulating transcription is through targeting histone modifications on chromatin to distinct gene loci. Using chromatin immunoprecipitation, we examined the developmental regulation of certain histone modifications at the AChR epsilon subunit locus, including methylations at lysine residues K4 and K27 and acetylations at K9 and K14. We modeled various stages of muscle development in cell culture, including pre-determined cells, committed but undifferentiated myoblasts, and differentiated myotubes, and modeled synaptic myotube nuclei by stimulating myotubes with neuregulin (NRG) 1. We found that a pattern of histone modifications associated with transcriptional activation is targeted to the AChR epsilon subunit locus in myotubes prior to stimulation with NRG1 and does not change upon addition of NRG1. Instead, we found that during muscle cell determination and differentiation, specific histone modifications are targeted to the AChR epsilon subunit locus. Within the gene, at K4, dimethylation is induced during muscle cell determination, while trimethylation is induced during differentiation. At K27, loss of trimethylation and appearance of monomethylation occurs during determination and differentiation. In addition, in a region upstream of the gene, K4 di- and trimethylation, and K9/14 acetylation are induced in a distinct developmental pattern, which may reflect a functional regulatory element. These results suggest synaptic signaling does not directly target histone modifications but rather the histone modification pattern necessary for transcriptional activation is previously established in a series of steps during muscle development. [ABSTRACT FROM AUTHOR]

Oncogenic H-Ras G12V and its variants have been shown to inhibit muscle differentiation. However, the role of proto-oncogenic Ras (c-Ras) in muscle differentiation remains unclear. The active GTP-bound form of Ras has been known to associate with diverse effectors including Raf, phosphatidylinositol 3-kinase (PI3K), Ral-GDS, and other molecules to transmit downstream signals. We hypothesize that c-Ras may stimulate muscle differentiation by selectively activating PI3K, an important mediator for muscle differentiation. In our experiments, inhibition of c-Ras by farnesyltransferase inhibitors and a dominant negative form of H-Ras (Ras S17N) suppressed muscle differentiation. Consistently, individual knockdown of H-Ras, K-Ras, and N-Ras by siRNAs all blocked muscle differentiation. Interestingly, we found that c-Ras preferentially interacts with PI3K rather than its major binding partner c-Raf, during myogenic differentiation, with total c-Ras activity remaining unchanged. PI3K and its downstream myogenic pathway, the Nox2/NF-κB/inducible nitric oxide synthase (iNOS) pathway, were found to be suppressed by inhibition of c-Ras activity during differentiation. Furthermore, expression of a constitutively active form of PI3K completely rescued the differentiation block and reactivated the Nox2/NF-κB/iNOS pathway in c-Ras-inhibited cells. On the basis of our results, we conclude that contrary to oncogenic Ras, proto-oncogenic H-Ras, K-Ras, and N-Ras are directly involved in the promotion of muscle differentiation via PI3K and its downstream signaling pathways. In addition, PI3K pathway activation is associated with a concurrent suppression of the otherwise predominantly activated Raf/Mek/Erk pathway. [ABSTRACT FROM AUTHOR]

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines—such as putrescine, spermidine and spermine—are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion—by means of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase—abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells. [ABSTRACT FROM AUTHOR]

This work describes the step-by-step development of a novel, serum-free, in vitro cell culture system resulting in the formation of robust, contracting, multinucleate myotubes from dissociated skeletal muscle cells obtained from the hind limbs of fetal rats. This defined system consisted of a serum-free medium formulation developed by the systematic addition of different growth factors as well as a nonbiological cell growth promoting substrate, N-1[3-(trimethoxysilyl) propyl] diethylenetriamine. Each growth factor in the medium was experimentally evaluated for its effect on myotube formation. The resulting myotubes were evaluated immunocytochemically using embryonic skeletal muscle, specifically the myosin heavy chain antibody. Based upon this analysis, we propose a new skeletal muscle differentiation protocol that reflects the roles of the various growth factors which promote robust myotube formation. Further observation noted that the proposed skeletal muscle differentiation technique also supported muscle–nerve coculture. Immunocytochemical evidence of nerve–muscle coculture has also been documented. Applications for this novel culture system include biocompatibility and skeletal muscle differentiation studies, understanding myopathies, neuromuscular disorders, and skeletal muscle tissue engineering. [ABSTRACT FROM AUTHOR]

Ubiquitin-mediated protein degradation is the main mechanism for controlled proteolysis, which is crucial for muscle development and maintenance. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 gene (ASB2) encodes the specificity subunit of an E3 ubiquitin ligase complex involved in differentiation of hematopoietic cells. Here, we provide the first evidence that a novel ASB2 isoform, ASB2β, is important for muscle differentiation. ASB2β is expressed in muscle cells during embryogenesis and in adult tissues. ASB2β is part of an active E3 ubiquitin ligase complex and targets the actin-binding protein filamin B (FLNb) for proteasomal degradation. Thus, ASB2β regulates FLNb functions by controlling its degradation. Knockdown of endogenous ASB2β by shRNAs during induced differentiation of C2C12 cells delayed FLNb degradation as well as myoblast fusion and expression of muscle contractile proteins. Finally, knockdown of FLNb in ASB2β knockdown cells restores myogenic differentiation. Altogether, our results suggest that ASB2β is involved in muscle differentiation through the targeting of FLNb to destruction by the proteasome.Cell Death and Differentiation (2009) 16, 921–932; doi:10.1038/cdd.2009.27; published online 20 March 2009 [ABSTRACT FROM AUTHOR]

The E2A proteins are basic helix–loop–helix transcription factors that regulate proliferation and differentiation in many cell types. In muscle cells, the E2A proteins form heterodimers with muscle regulatory factors such as MyoD, which then bind to DNA and regulate the transcription of target genes essential for muscle differentiation. We now demonstrate that E2A proteins are primarily localized in the nucleus in both C2C12 myoblasts and myotubes, and are degraded by the ubiquitin proteasome system evidenced by stabilization following treatment with the proteasome inhibitor, MG132. During the differentiation from myoblast to myotube, the cellular abundance of E2A proteins is relatively unaltered, despite significant changes (each ∼5-fold) in the relative rates of protein synthesis and protein degradation via the ubiquitin-proteasome system. The rate of ubiquitin-proteasome-mediated E2A protein degradation depends on the myogenic differentiation state (t½∼2 h in proliferating myoblasts versus t½>10 h in differentiated myotubes), and is also associated with cell cycle in non-muscle cells. Our findings reveal an important role for both translational and post-translational regulatory mechanisms in mediating the complex program of muscle differentiation determined by the E2A proteins.Oncogene (2007) 26, 441–448. doi:10.1038/sj.onc.1209793; published online 7 August 2006 [ABSTRACT FROM AUTHOR]

Lamins are the major structural proteins of the nucleus in an animal cell. In addition to being essential for nuclear integrity and assembly, lamins are involved in the organization of nuclear processes such as DNA replication, transcription and repair. Mutations in the human lamin A gene lead to highly debilitating genetic disorders that primarily affect muscle, adipose, bone or neuronal tissues and also cause premature ageing syndromes. Mutant lamins alter nuclear integrity and hinder signalling pathways involved in muscle differentiation and adipocyte differentiation, suggesting tissue-specific roles for lamins. Furthermore, cells expressing mutant lamins are impaired in their response to DNA damaging agents. Recent reports indicate that certain lamin mutations act in a dominant negative manner to cause nuclear defects and cellular toxicity, and suggest a possible role for aberrant lamins in normal ageing processes. [ABSTRACT FROM AUTHOR]

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease characterized by a typical regional distribution, featuring composed patterns of clinically affected and unaffected muscles. No treatment is available for this condition, in which the pathophysiological mechanism is still unknown. Autologous transfer of myoblasts from unaffected to affected territories could be considered as a potential strategy to delay or stop muscle degeneration. To evaluate the feasibility of this concept, we explored and compared the growth and differentiation characteristics of myoblasts prepared from phenotypically unaffected muscles of five FSHD patients and 10 control donors. According to a clinically approved procedure, 109 cells of a high degree of purity were obtained within 16–23 days. More than 80% of these cells were myoblasts, as demonstrated by labeling of the muscle markers CD56 and desmin. FSHD myoblasts presented a doubling time equivalent to that of control cells; they kept high proliferation ability and did not show early telomere shortening. In vitro, these cells were able to differentiate and to express muscle-specific antigens. In vivo, they participated to muscle structures when injected into immunodeficient mice. These data suggest that myoblasts expanded from unaffected FSHD muscles may be suitable tools in view of autologous cell transplantation clinical trials.Gene Therapy (2005) 12, 1651–1662. doi:10.1038/sj.gt.3302565; published online 16 June 2005 [ABSTRACT FROM AUTHOR]

We describe a new approach for studying insulin-induced exocytosis in individual, well-differentiated, innervated human muscle fibres. We used an in vitro system in which motor axons extending from embryonic rat spinal cord explants functionally innervate co-cultured human muscle fibres. Under such conditions, the human muscle fibres reach a high degree of differentiation that is never observed in non-innervated, cultured human muscle fibres. To monitor insulin-induced membrane dynamics, we used confocal microscopy to measure the fluorescence intensity changes of the styryl dye FM1-43, a marker for membrane area. The fluorescence intensity increased after insulin stimulation. This increase, as well as the intensity of staining for the glucose transporter 4 (GLUT4), was significantly higher in the innervated and contracting fibres than in myoblasts and myotubes. This shows that in vitro innervation of human muscle cells not only enhances the differentiation stage but also improves the insulin response. Our approach allows continuous monitoring and quantitative assessment of insulin-induced increase in cumulative exocytosis in individual human muscle fibres at a differentiation level practically corresponding to that of adult muscle. It is therefore a suitable system for studying various parameters affecting the mechanisms underlying insulin-induced GLUT4 translocation in human skeletal muscle. [ABSTRACT FROM AUTHOR]

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-ß-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric a-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and ß-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation. [ABSTRACT FROM AUTHOR]

Under normal conditions in situ, muscle fibers and motoneurons, the main partners of motor units, are strongly dependent on each other. This interdependence hinders ex vivo studies of neuromuscular disorders where nervous or muscular components are considered separately. To allow in vitro access to complex nerve–muscle relationships, we developed a novel nerve-muscle co-culture system where mouse muscle innervation is assured by rat spinal cord explants. The degree of muscular maturation during co-culture was evaluated using the distribution of nicotinic acetylcholine receptors (AChRs) and their electrophysiological characteristics before and after innervation. In myotubes from non-innervated cultures, AChRs were diffusely distributed over the entire myotube surface. Their single-channel conductance (33.5±0.6 pS) and mean open time (8.1±0.7 ms) are characteristic of AChRs described in embryonic or denervated skeletal muscles. In innervated muscle fibers from co-cultures, AChRs appear as discrete aggregates and co-localize with synaptotagmin. In addition to the embryonic type currents, in innervated fibers AChR currents having high conductance (53.3±5.9 pS) and short mean open time (2.6±0.1 ms), characteristic of AChRs at mature neuromuscular junctions, were observed. Our data support the use of this new nerve–muscle co-culture system as a reliable model for the study of murine muscular differentiation and function. [ABSTRACT FROM AUTHOR]

Differentiation of skeletal muscle cells involves two distinct events: exit from the cell cycle and expression of musclespecific contractile genes and formation of multinucleated myocytes. Although many studies have shown that growth factors regulate the initial step of differentiation, little is known about regulation of fusion. BC3H1 cells are a skeletal muscle cell line characterized by a nonfusing phenotype and an ability to dedifferentiate. When subjected to serum or growth factors, differentiated BC3H1 cells lose muscle-specific gene expression and re-enter the cell cycle. In this study, we describe a spontaneously fusing clone of BC3H1 cells. We demonstrate that this fusion capability is not due to altered muscle regulatory factor or adhesion molecule expression. Furthermore, we show that fusion inhibits dedifferentiation. Multinucleated BC3H1 cells do not lose myosin expression, nor do they re-enter the cell cycle. Fused BC3H1 cells react to serum stimulation with a hypertrophic response. Our results suggest that the state of differentiation, mono- or multinucleated, is essential to how myocytes react to growth stimulation and may provide a mechanism for how differentiation, fusion, and hypertrophy are regulated in vivo. [ABSTRACT FROM AUTHOR]

A mechanistic definition of the dystrophic process is proposed, and the effects of growth factors vs. down-regulation of growth are critically analyzed. A conceptual scheme is presented to illustrate the steps leading to pathology, and various compensatory systems which ameliorate the pathology are examined, particularly in regards to the mdx mouse which is resistant to the deficiency of dystrophin, the main protein product of the Duchenne and Becker muscular dystrophy (DMD/BMD) gene. These compensatory systems are analyzed in terms of the differential resistance of fiber types to pathogenesis. The generation of a stable population of maturationally arrested centronucleated fibers which express the mature adult myosin isoforms is proposed to be the main strategy of mdx muscle to minimize apoptosis. Physiological properties of these fibers, such as utrophin expression, and high mitochondrial and endoplasmic reticulum content, together with probable increased glycerophosphorylcholine concentrations and facile access to the vascular system, are hypothesized to be instrumental in their resistance to pathogenesis. It is proposed that the major element that determines the susceptibility of most human muscles to the dystrophic process is their inability to arrest the maturation of regenerated fibers at the centronucleated stage with a concomitant expression of the adult myosins. [ABSTRACT FROM AUTHOR]

Molecular components of basal lamina, such as laminin, stimulate the differentiation of skeletal muscle cells in culture, while interstitial matrix components such as fibronectin are inhibitory. However, the role of extracellular matrix (ECM) molecules in muscle cell differentiation in the embryo is less well understood. As a first step toward understanding the role of the ECM in embryonic myogenesis, the localization of basal lamina molecules in the mouse limb bud before and during muscle cell differentiation was determined by immunofluorescence. Laminin, collagen type IV and nidogen (entactin) were concentrated in myogenic regions of the limb bud both before and during differentiation of skeletal muscle cells. Punctate immunofluorescence for basal lamina molecules was concentrated in dorsal and ventral premuscle and muscle masses, when compared with other regions of limb mesenchyme. In contrast, immunofluorescence for fibronectin, an interstitial extracellular matrix molecule, was decreased in premuscle and muscle masses. These results suggest that basal lamina components play an important stimulatory role in early stages of skeletal muscle differentiation in the developing mouse limb bud. [ABSTRACT FROM AUTHOR]

Cell lines corresponding to various cell lineages of the mouse embryo have been isolated from murine teratocarcinomas. Embryonal carcinoma cell lines are developmentally equivalent to the embryonic ectoderm or inner cell mass. Most of these cell lines have a modal chromosome number equal or close to 40, the normal mouse complement. However, cell lines corresponding to more advanced cell lineages (e. g., endoderm) are tetraploid or hypotetraploid and display multiple chromosomal rearrangements. This paper describes the isolation of a near-diploid differentiated cell line (LT-D) from an LT teratocarcinoma. The modal chromosome number of LT-D is 40, and this number is stable during at least 12 mo of continuous culture. LT-D cells are morphologically distinct from embryonal carcinoma cells and no longer express the SSEA-1 cell surface antigen or high alkaline phosphatase activity characteristic of embryonal carcinoma cells. LT-D cells can be induced to fuse into structures resembling myotubes. The formation of these structures is accompanied by expression of the muscle-specific isozyme of creatine phosphokinase and desmin, a muscle-specific component of intermediate filaments. Lastly, LT-D cells do not form tumors in syngenetic mice. [ABSTRACT FROM AUTHOR]

The development and differentiation of the avian myotome was studied after removal of the neural tube, including neural crest, and after replacement of dorsal half-somites by ventral half-somites. Results show that in the absence of neural tissue myoblast differentiation within the somites does not take place. Ventral halfsomites are able to undergo muscle differentiation if they were grafted in place of dorsal half somites. It is suggested that local signals must be responsible for the dorsalisation of the newly formed somite including myoblast differentiation. Neural crest cells are discussed as possible sources of these signals. [ABSTRACT FROM AUTHOR]

Muscle spindles form de novo in reinnervated muscles of neonatal rats treated with nerve growth factor. Whether the spindles can also form in muscle reinnervated only by afferents was investigated by removing the lumbosacral segment of the spinal cord immediately after crushing the nerve to the medial gastrocnemius muscle at birth, and administering nerve growth factor for 10 days afterwards. As predicted, the medial gastrocnemius muscles were reinnervated by afferents, but not efferents. No motor endplates were visible on any muscle fibers, and extrafusal fibers were atrophied. The reinnervated muscles contained spindle-like encapsulations of one to four fibers at 5, 7, 9 and 30 days after the nerve crush. The number of spindles as well as encapsulated fibers exceeded that of normal medial gastrocnemius muscles. The encapsulated fibers resembled typical intrafusal fibers. They had normal sensory-muscle contacts, but no motor endings. The fibers displayed equatorial clusters of myonuclei and expressed the spindle-specific slow-tonic myosin heavy chain isoform at postnatal day 30. Thus, efferents are not essential for the formation and differentiation of muscle spindles in reinnervated muscles of neonatal rats. [ABSTRACT FROM AUTHOR]

Mice embryos from day 9 to 15 p.c. have been studied by Nile blue sulfate staining revealed segmental necroses in the back of mice embryos centrally between the intersegmental arteries. Necroses appeared in the cranial myotomes and extended to the caudal ones during the days 9 to 13 p.c. By light microscopy the necroses extended in the centre of the myotome to the ventromedial margin. Electron microscopy revealed in this area myoblast-like cells rich in filaments, arranged in parallel without striation. A part of these myoblasts were electron-opaque and the nucleus was no longer evident. Forming vortices of filaments, these cells on the cranial and caudal end of the myotomes ceased fusion with the more centrally situated myoblasts. Vortices of filaments were also found in phagolysosomes of macrophages on day 12 p.c. On the same day of gestation we found an unsegmented myoblastema with irregular long myoblasts and striated polynuclear muscle fibres, arranged parallel to the neural tube. Between and parallel to the muscle fibres elongated individual cells could be seen, growing necrotic. Their nuclei were electron-dense with the chromatin clumping cap-like on the inner nuclear membrane. Fragments of myofibrils, corresponding approximately to sarcomeres, were dispersed irregularly as filament logs in the cytoplasm. Logs of filaments were bent at an obtuse angle, partly showing remnants of Z line material. In this stage of necrosis cells were phagocytized by macrophages. After disintegration of the segmental arrangement in scleroblastemata and myoblastemata a segmental rearrangement was not achieved by segmental shifting or by necrotic liquefaction zones. Whether the described cell necroses are due to differentiation, or even to phylogenetic causes is discussed. [ABSTRACT FROM AUTHOR]

The intracellular distributions of major muscle proteins, myosin, actin, tropomyosin, α-actinin, and desmin, in smooth muscle cells of chicken gizzard at various stages of embryogenesis were investigated by immunofluorescence-labeling of enzyme-dispersed cells cultured up to three hours. These muscle proteins, except some part of myosin, were organized into fibrous structures as soon as synthesis and accumulation of proteins started. As for myosin, a considerable amount of it was dispersed in soluble cytoplasm as well. On the other hand, Ca-dependent contractility was detected with detergent-extracted myoblasts and glycerinated tissue from embryos older than 7 days. Although the nascent myofibrils bear a resemblance to 'stress fibers,' the former could be distinguished from the latter by their high stability in dispersed, spherical cells. The above findings, therefore, show that the synthesis of contractile proteins is followed by immediate assembly of them into functional myofibrils without undergoing any intermediate structure. Based on these findings, the mechanism of myofibril formation in developing smooth muscle cells is discussed. [ABSTRACT FROM AUTHOR]

The myotome of early chick embryos was investigated histochemically by means of the acetylcholinesterase (AChE) reaction. Light-microscopically, at the cervical level, the myotome was first recognized and AChE activity demonstrated at stage 13 (2 day-old embryo). Subsequently, the myotome elongated ventro-laterally along the inner surface of the dermomyotome and reached the ventro-lateral end of the dermomyotome at stage 17 to 18 (3 day-old embryo). AChE activity in the myotome showed subsequent increase in intensity during the course of development. The myotome consisted mainly of AChE-positive cells displaying enzymatic activity along the nuclear membrane and within the cytoplasm. In contrast, almost all cells of the dermomyotome and the interstitial cells were AChE-negative. Electron-microscopically, the myotome cells of the 2 day-old embryo and the cells in the dorso-medial portion of the myotome of the 3 day-old embryo were morphologically undifferentiated; AChE activity was detected in the nuclear envelope and in single short profiles of the endoplasmic reticulum (ER). On the other hand, in the 3 day-old embryo the cells in the ventro-lateral portion of the myotome showed AChE activity in the nuclear envelope, numerous profiles of the ER and some Golgi complexes. These AChE-positive cells were regarded as developing myogenic cells based on their morphological characteristics. The present findings indicate (i) that the appearance of AChE activity in the cytoplasm is the first sign of the differentiation of myogenic cells, and (ii) that in these myogenic cells the increase in AChE activity is based on the development of the ER. [ABSTRACT FROM AUTHOR]

Methyl-bupivacaine is a local anaesthetic with a selective myotoxic action. A single subcutaneous injection of the drug into the hind leg of adult rats produces a uniform, complete and irreversible destruction of superficial layers of fibres in the underlying extensor digitorum longus muscle. The degeneration of muscle fibres is followed by phagocytosis and a rapid and complete regeneration. The first stage in the regeneration process is the appearance of presumptive myoblasts within the original basement membrane of the sarcolemmal tube. On the second day after injury aggregates of myoblasts are present and fusion is observed between the cells. The myotubes thus formed increase in size by fusing with additional myoblasts. Myotubes are also observed to fuse with one another. On the fifth day after injury the regeneration process has proceeded to the stage of early muscle fibres with fully differentiated myofibrils with typical sarcomere structures. By ten days only mature muscle fibres of about normal size are present and regeneration appears complete. In previously denervated and methyl-bupivacaine treated muscles the stages of regeneration are similar to those observed in innervated muscles, the only apparent difference being a slowing of cell differentiation and incomplete maturation. An electrophysiological study shows that the motor nerve at the third day after injury forms synaptic contacts with regenerating muscle cells. At that stage of myogenesis the myotubes are highly sensitive to applied acetylcholine. 1 (1-n-butyl-DL-piperidine-2-carboxylic acid-2,6-dimethyl-anilide-hydrochloride); Marcaine®, manufactured by AB Bofors, Nobel-Pharma, Mölndal, Sweden. [ABSTRACT FROM AUTHOR]

The uncommon case is described of a girl severely affected with Duchenne muscular dystrophy. Cytogenetic analysis revealed no numerical or structural abnormalities of the X-chromosome in any of the cells examined (leucocytes and myoblasts). No abnormality in morphology, growth pattern or differentiation was observed in the dystrophic muscle cultures as compared with control cultures. [ABSTRACT FROM AUTHOR]