Surface Of Myoblasts Research Articles

Pannexin 1 Regulates Skeletal Muscle Regeneration by Promoting Bleb-Based Myoblast Migration and Fusion Through a Novel Lipid Based Signaling Mechanism.

Adult skeletal muscle has robust regenerative capabilities due to the presence of a resident stem cell population called satellite cells. Muscle injury leads to these normally quiescent cells becoming molecularly and metabolically activated and embarking on a program of proliferation, migration, differentiation, and fusion culminating in the repair of damaged tissue. These processes are highly coordinated by paracrine signaling events that drive cytoskeletal rearrangement and cell-cell communication. Pannexins are a family of transmembrane channel proteins that mediate paracrine signaling by ATP release. It is known that Pannexin1 (Panx1) is expressed in skeletal muscle, however, the role of Panx1 during skeletal muscle development and regeneration remains poorly understood. Here we show that Panx1 is expressed on the surface of myoblasts and its expression is rapidly increased upon induction of differentiation and that Panx1–/– mice exhibit impaired muscle regeneration after injury. Panx1–/– myoblasts activate the myogenic differentiation program normally, but display marked deficits in migration and fusion. Mechanistically, we show that Panx1 activates P2 class purinergic receptors, which in turn mediate a lipid signaling cascade in myoblasts. This signaling induces bleb-driven amoeboid movement that in turn supports myoblast migration and fusion. Finally, we show that Panx1 is involved in the regulation of cell-matrix interaction through the induction of ADAMTS (Disintegrin-like and Metalloprotease domain with Thrombospondin-type 5) proteins that help remodel the extracellular matrix. These studies reveal a novel role for lipid-based signaling pathways activated by Panx1 in the coordination of myoblast activities essential for skeletal muscle regeneration.

COMP-Angiopoietin-1 accelerates muscle regeneration through N-cadherin activation

Angiopoietin-1 modulates vascular stability via Tie2 on endothelial cells. In our previous study, we also showed it acts as an inhibitor of cardiomyocyte death. However, it remains poorly understood how Ang1 regulates myogenesis during muscle regeneration. Here we found that COMP-Ang1 (cAng1) enhances muscle regeneration through N-cadherin activation. Muscle fiber regeneration after limb muscle damage by ischemic injury was enhanced with cAng1 treatment. Mechanistically cAng1 directly bound to N-cadherin on the myoblast surface in a Ca2+ dependent manner. The interaction enhanced N-cadherin activation via N-cadherin/p120-catenin complex formation, which in turn activated p38MAPK (but not AKT or ERK) and myogenin expression (but not myoD) as well as increasing myogenin+ cells in/ex vivo. After transplantation of GFP-expressing myoblasts (GFP-MB), we showed an increased generation of GFP+ myotubes with adenovirus cAng1 (Adv-cAng1) injection. Adv-cAng1, however, could not stimulate myotube formation in N-cadherin-depleted GFP-MB. Taken together, this study uncovers the mechanism of how cAng1 promotes myoblast differentiation and muscle regeneration through the N-cadherin/p120-catenin/p38MAPK/myogenin axis.

Productive infection of human skeletal muscle cells by pandemic and seasonal influenza A(H1N1) viruses.

Besides the classical respiratory and systemic symptoms, unusual complications of influenza A infection in humans involve the skeletal muscles. Numerous cases of acute myopathy and/or rhabdomyolysis have been reported, particularly following the outbreak of pandemic influenza A(H1N1) in 2009. The pathogenesis of these influenza-associated myopathies (IAM) remains unkown, although the direct infection of muscle cells is suspected. Here, we studied the susceptibility of cultured human primary muscle cells to a 2009 pandemic and a 2008 seasonal influenza A(H1N1) isolate. Using cells from different donors, we found that differentiated muscle cells (i. e. myotubes) were highly susceptible to infection by both influenza A(H1N1) isolates, whereas undifferentiated cells (i. e. myoblasts) were partially resistant. The receptors for influenza viruses, α2-6 and α2-3 linked sialic acids, were detected on the surface of myotubes and myoblasts. Time line of viral nucleoprotein (NP) expression and nuclear export showed that the first steps of the viral replication cycle could take place in muscle cells. Infected myotubes and myoblasts exhibited budding virions and nuclear inclusions as observed by transmission electron microscopy and correlative light and electron microscopy. Myotubes, but not myoblasts, yielded infectious virus progeny that could further infect naive muscle cells after proteolytic treatment. Infection led to a cytopathic effect with the lysis of muscle cells, as characterized by the release of lactate dehydrogenase. The secretion of proinflammatory cytokines by muscle cells was not affected following infection. Our results are compatible with the hypothesis of a direct muscle infection causing rhabdomyolysis in IAM patients.

Methylglyoxal Impairs GLUT4 Trafficking and Leads to Increased Glucose Uptake in L6 Myoblasts

Methylglyoxal (MG) is a highly reactive dicarbonyl compound derived mainly from glucose degradation pathways, but also from protein and fatty acid metabolism. MG modifies structure and function of different biomolecules and thus plays an important role in the pathogenesis of diabetic complications. Hyperglycemia-associated accumulation of MG might be associated with generation of oxidative stress and subsequently insulin resistance. Therefore, the effects of MG on insulin signaling and on translocation of glucose transporter 4 (GLUT4) were investigated in the rat skeletal muscle cell line L6-GLUT4myc stably expressing myc-tagged GLUT4. Twenty four-hour MG treatment resulted in elevated GLUT4 presentation on the surface of L6 myoblasts and in an increased uptake of glucose even without insulin stimulation. Exogenously added MG neither effected IRS-1 expression nor IRS-1 phosphorylation. A decreased expression of Akt1 but not Akt2 and concomitantly increased apoptosis were detected following MG treatment. To exclude that oxidative stress caused by MG treatment leads to increased GLUT4 translocation, effects of pretreatment with 2 antioxidants were investigated. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. In contrast, tiron, a well-known antioxidant that does not exert MG-scavenger function, had no impact on MG-induced GLUT4 translocation supporting the hypothesis of a direct effect of MG on GLUT4 trafficking. In conclusion, prolonged treatment with MG augments GLUT4 level on the surface of L6 myoblasts, at least in part through a higher translocation of GLUT4 from the intracellular compartment as well as a reduction of GLUT4 internalization, resulting in increased glucose uptake.

Myomaker is a membrane activator of myoblast fusion and muscle formation

SummaryFusion of myoblasts is essential for the formation of multi-nucleated muscle fibers. However, the identity of myogenic proteins that directly govern this fusion process has remained elusive. Here, we discovered a muscle-specific membrane protein, named Myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is down-regulated thereafter. Over-expression of Myomaker in myoblasts dramatically enhances fusion and genetic disruption of Myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibers. Remarkably, forced expression of Myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacologic perturbation of the actin cytoskeleton abolishes the activity of Myomaker, consistent with prior studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein both necessary and sufficient for mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation.

Impact of GLO1 Knock Down on GLUT4 Trafficking and Glucose Uptake in L6 Myoblasts

Methylglyoxal (MG), a highly reactive α-dicarbonyl metabolite of glucose degradation pathways, protein and fatty acid metabolism, plays an important role in the pathogenesis of diabetic complications. Hyperglycemia triggers enhanced production of MG and increased generation of advanced glycation endproducts (AGEs). In non-enzymatic reactions, MG reacts with arginine residues of proteins to form the AGEs argpyrimidine and hydroimidazolone. Glyoxalase 1 (GLO1), in combination with glyoxalase 2 and the co-factor glutathione constitute the glyoxalase system, which is responsible for the detoxification of MG. A GLO1 specific knock down results in accumulation of MG in targeted cells. The aim of this study was to investigate the effect of intracellularly accumulated MG on insulin signaling and on the translocation of the glucose transporter 4 (GLUT4). Therefore, L6 cells stably expressing a myc-tagged GLUT4 were examined. For the intracellular accumulation of MG, GLO1, the first enzyme of the glyoxalase pathway, was down regulated by siRNA knock down and cells were cultivated under hyperglycemic conditions (25 mM glucose) for 48 h. Here we show that GLO1 knock down augmented GLUT4 level on the cell surface of L6 myoblasts at least in part through reduction of GLUT4 internalization, resulting in increased glucose uptake. However, intracellular accumulation of MG had no effect on GLUT4 concentration or modification. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. Tiron, which is also a well-known antioxidant, had no impact on MG-induced GLUT4 translocation.

Shuttling glucose across brain microvessels, with a little help from GLUT1 and AMP kinase. Focus on “AMP kinase regulation of sugar transport in brain capillary endothelial cells during acute metabolic stress”

The Blood-Brain Barrier: a Gatekeeper of Brain “Individuality” The blood-brain barrier (BBB) maintains the individuality of brain fluid, while allowing it to selectively import nutrients and process toxic products. Its major constituent is a single layer of continuous, nonfenestrated endothelium lining the microvessels, whose unique features account in large part for the integrity of the barrier. The endothelial cell monolayer is the major determinant, but other cells in the central nervous system (e.g., pericytes and astrocytes) contribute to the integrity of the BBB. The interendothelial tight junctions of the BBB are one of its signature elements that greatly restrict paracellular permeability to solutes, fluid, and transmigrating cells. This is markedly distinct from the leakier, discontinuous, and fenestrated endothelium of liver microvessels (sinusoids). For example, the tight junctions of the cerebral endothelium (pial vessels) confer an electrical resistance (1,300 ·cm 2 ) far higher than other endothelia (6). Therefore, communication between blood and the brain interstitial fluid requires individually tailored molecular mechanisms. Transcytosis is the movement of molecules through a cellular barrier. Whereas proteins can traverse through vesicular carrier processes that could carry solutes through fluid phase endocytosis, the brain microvasculature is uniquely poor in plasmalemmal and cytoplasmic caveolae (1). Instead, bidirectional passage of small molecules across the BBB is stringently regulated by numerous transporters and receptors. These typically work in tandem across the luminal and abluminal membranes of the endothelial cell.

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell–cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.

Fusogenic Activity of Annexins A1 and A5

Annexins are a family of proteins that bind to anionic phospholipids in a calcium-dependent manner and have been implicated in a number of cell biological processes including intracellular fusion. Recently we found that extracellular annexins (A1 and A5) play important roles in myoblast fusion. In this study we explored fusogenic activity of these proteins in the absence of other proteins and found both A1 and A5 to aggregate phophatidylserine-containing liposomes and to induce lipid mixing between these liposomes. Annexins A1 and A5 drive fusion beyond early hemifusion stage, as evidenced by lipid mixing between the inner leaflets of liposomal membranes. The annexin-mediated vesicle aggregation and lipid mixing depended on the presence of calcium, anionic lipids and were inhibited by specific antibodies and, in the case of annexin A1, by a synthetic peptide derived from its N-terminal domain. Annexins A1 and A5 applied together were more effective in inducing fusion than either of the proteins on its own. This synergistic activity can be important in fusion between myoblasts since both of these proteins are present at myoblast surface under fusion conditions. Our results support the hypothesis that annexins directly mediate myoblast fusion by mechanisms similar to those discussed for viral and intracellular protein fusogens rather than regulate cell fusion via other proteins.

Glycogenome expression dynamics during mouse C2C12 myoblast differentiation suggests a sequential reorganization of membrane glycoconjugates

BackgroundSeveral global transcriptomic and proteomic approaches have been applied in order to obtain new molecular insights on skeletal myogenesis, but none has generated any specific data on glycogenome expression, and thus on the role of glycan structures in this process, despite the involvement of glycoconjugates in various biological events including differentiation and development. In the present study, a quantitative real-time RT-PCR technology was used to profile the dynamic expression of 375 glycogenes during the differentiation of C2C12 myoblasts into myotubes.ResultsOf the 276 genes expressed, 95 exhibited altered mRNA expression when C2C12 cells differentiated and 37 displayed more than 4-fold up- or down-regulations. Principal Component Analysis and Hierarchical Component Analysis of the expression dynamics identified three groups of coordinately and sequentially regulated genes. The first group included 12 down-regulated genes, the second group four genes with an expression peak at 24 h of differentiation, and the last 21 up-regulated genes. These genes mainly encode cell adhesion molecules and key enzymes involved in the biosynthesis of glycosaminoglycans and glycolipids (neolactoseries, lactoseries and ganglioseries), providing a clearer indication of how the plasma membrane and extracellular matrix may be modified prior to cell fusion. In particular, an increase in the quantity of ganglioside GM3 at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation.ConclusionFor the first time, these results provide a broad description of the expression dynamics of glycogenes during C2C12 differentiation. Among the 37 highly deregulated glycogenes, 29 had never been associated with myogenesis. Their biological functions suggest new roles for glycans in skeletal myogenesis.

Genetic Complementation of Human Muscle Cells via Directed Stem Cell Fusion

Duchenne muscular dystrophy (DMD) is caused by mutations in the X chromosome-linked DMD gene, which encodes the sarcolemma-stabilizing protein-dystrophin. Initial attempts at DMD therapy deployed muscle progenitor cells from healthy donors. The utilization of these cells is, however, hampered by their immunogenicity, while those from DMD patients are scarce and display limited ex vivo replication. Nonmuscle cells with myogenic capacity may offer valuable alternatives especially if, to allow autologous transplantation, they are amenable to genetic intervention. As a paradigm for therapeutic gene transfer by heterotypic cell fusion we are investigating whether human mesenchymal stem cells (hMSCs) can serve as donors of recombinant DMD genes for recipient human muscle cells. Here, we show that forced MyoD expression in hMSCs greatly increases their tendency to participate in human myotube formation turning them into improved DNA delivery vehicles. Efficient loading of hMSCs with recombinant DMD was achieved through a new tropism-modified high-capacity adenoviral (hcAd) vector directing striated muscle-specific synthesis of full-length dystrophin. This study introduces the principle of genetic complementation of gene-defective cells via directed cell fusion and provides an initial framework to test whether transient MyoD synthesis in autologous, gene-corrected hMSCs increases their potential for treating DMD and, possibly, other muscular dystrophies.

Nogo-A, -B, and -C Are Found on the Cell Surface and Interact Together in Many Different Cell Types

Nogo-A, -B, and -C are generated from the Nogo/RTN-4 gene and share a highly conserved C-terminal domain. They lack an N-terminal signal sequence and are predominantly localized to the endoplasmic reticulum (ER). We found the N terminus of endogenous Nogo-A exposed on the surface of fibroblasts, DRG neurons, and myoblasts. Surface-expressed Nogo-A was also present on presynaptic terminals of the neuromuscular junction and on DRG neurons in vivo. Surface biotinylations confirmed the presence of all Nogo isoforms on the surface. To search for proteins that interact with Nogo-A and suggest a function for the large intracellular pool of Nogo-A, immunoprecipitations were performed. Surprisingly, the most predominant proteins that interact with Nogo-A are Nogo-B and Nogo-C as seen with radiolabeled lysates and as confirmed by Western blotting in multiple cell lines. Nogo-A, -B, and -C share a 180-amino acid C-terminal domain with two highly conserved hydrophobic stretches that could form a channel or transporter in the ER and/or on the cell surface.

Alpha-enolase plasminogen receptor in myogenesis

Plasmin is a potent extracellular protease specialized in the degradation of fibrin (fibrinolysis). Active plasmin is generated by proteolytic activation of the zymogen plasminogen (Plg) by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Alpha-enolase, although traditionally considered a glycolytic enzyme, constitutes a receptor for plasminogen on several cell types, serving to localize and promote plasminogen activation pericellularly. Localization of plasmin activity on the cell surface plays a critical role in fibrinolysis and in physiopathological processes involving extracellular matrix remodelling. Previous studies have unambiguously demonstrated that uPA-dependent plasmin generation is necessary for myogenesis in vitro and for muscle regeneration in vivo. However, the implication of alpha-enolase plasminogen receptor in myogenesis had never been investigated. This review focuses on the recently reported expression and function of alpha-enolase plasminogen receptor during myogenesis. Skeletal myoblasts express alpha-enolase plasminogen receptor, being its expression greatly induced during the differentiation process in vitro. MAb 11G1, a monoclonal antibody against anti-alpha-enolase plasminogen receptor, that inhibits plasmin generation, was able to fully abrogate myoblast fusion and differentiation. Moreover, both plasmin activity and alpha-enolase plasminogen receptor expression were significantly augmented in injury-induced regenerating muscle of wild type mice and in the dystrophic muscle of mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Altogether, these results indicate that the plasminogen activation (PA) system is an important component of skeletal myogenesis in vitro and in vivo. In particular, the expression of alpha-enolase plasminogen receptor may serve to concentrate and enhance plasmin generation on the cell surface of migratory myoblasts contributing to efficient muscle repair.

β1 Integrins Regulate Myoblast Fusion and Sarcomere Assembly

The mechanisms that regulate the formation of multinucleated muscle fibers from mononucleated myoblasts are not well understood. We show here that extracellular matrix (ECM) receptors of the β1 integrin family regulate myoblast fusion. β1-deficient myoblasts adhere to each other, but plasma membrane breakdown is defective. The integrin-associated tetraspanin CD9 that regulates cell fusion is no longer expressed at the cell surface of β1-deficient myoblasts, suggesting that β1 integrins regulate the formation of a protein complex important for fusion. Subsequent to fusion, β1 integrins are required for the assembly of sarcomeres. Other ECM receptors such as the dystrophin glycoprotein complex are still expressed but cannot compensate for the loss of β1 integrins, providing evidence that different ECM receptors have nonredundant functions in skeletal muscle fibers.

Interaction of skeletal muscle cells with collagen type IV is mediated by perlecan associated with the cell surface.

We have previously shown that the expression of perlecan, a heparan sulfate proteoglycan localized on the myoblast surface, is down-regulated during terminal differentiation of skeletal muscle myoblasts (Larraín et al. [1997] Exp. Cell Res. 234:405-412). In this study, we have evaluated the biochemical characteristics of perlecan, its association with the myoblast surface, and its involvement in C(2)C(12) myoblast adhesion to different substrates. Perlecan associated with myoblasts was solubilized by Triton X-100, whereas heparin, high salt, and RGD peptides were unable to solubilize perlecan. Pre-incubation of myoblasts with [(35)S]-Na(2)SO(4), followed by solubilization with Triton X-100 and immunoprecipitation with antibodies against murine perlecan, demonstrated that this proteoglycan present on the cell surface has a heterogeneous size profile with a K(av) value of 0.45, determined by Sepharose CL-4B chromatography. Myoblasts were found to adhere with decreasing affinities to collagen type IV, type I, laminin, fibronectin, perlecan, and matrigel. We found that cell adhesion to collagen type IV was inhibited by blocking this substrate with exogenous perlecan prior to cell plating, whereas no effect was observed for laminin. Furthermore, adhesion of myoblasts to collagen type IV was inhibited by the perlecan core protein obtained by treatment of perlecan with heparitinase, as well as by pre-incubation of the cells with antibodies against murine perlecan. These data support the idea that skeletal muscle cells interact with collagen type IV through the perlecan core protein present on the surface of undifferentiated myoblasts.

Antisense Inhibition of Syndecan-3 Expression during Skeletal Muscle Differentiation Accelerates Myogenesis through a Basic Fibroblast Growth Factor-dependent Mechanism

Syndecan-3 is a member of a family of transmembrane proteoglycans that posses highly homologous cytoplasmic and transmembrane domains and function as extracellular matrix receptors and low-affinity receptors for signaling molecules such as basic fibroblasts growth factor (FGF-2). Syndecan-3 is transiently expressed in developing limb bud and absent in adult skeletal muscle. In this study we investigated the expression of syndecan-3 and its role on FGF-2-dependent inhibition of myogenesis. Syndecan-3 expression was down-regulated during skeletal muscle differentiation of C(2)C(12) myoblasts, as determined by Northern blot analyses and immunoprecipitation. To probe the function of syndecan-3 during myogenesis, C(2)C(12) myoblasts were stably transfected with a plasmid coding for antisense syndecan-3 mRNA. The resulting inhibition of syndecan-3 expression caused accelerated skeletal muscle differentiation, as determined by expression of creatine kinase and myosin and myoblast fusion. Expression of a master transcription factor for muscle differentiation, myogenin, was also accelerated in antisense syndecan-3-transfected myoblasts compared with control transfected and wild type cells. Reduced expression of syndecan-3 resulted in a 13-fold decrease in sensitivity to FGF-2-dependent inhibition of myogenin expression. Addition of heparin partially reversed this effect. These results demonstrate that syndecan-3 expression is down-regulated during differentiation and the level of expression of membrane-bound heparan sulfate on myoblast surface is critical for fine modulation of responsiveness to FGF-2. These findings strongly suggest a role for syndecan-3 in regulation of skeletal muscle terminal differentiation.

The alpha7beta1 integrin in muscle development and disease.

The alpha7beta1 integrin is a laminin receptor on the surface of skeletal myoblasts and myofibers. Alternative forms of both the alpha7 and beta1 chains are expressed in a developmentally regulated fashion during myogenesis. These different alpha7beta1 isoforms localize at specific sites on myofibers and appear to have distinct functions in skeletal muscle. These functions include the migration and proliferation of developing myoblasts, the formation and integrity of neuromuscular and myotendinous junctions, and the "gluing" together of muscle fibers that is essential to the generation of contractile force. The alpha7beta1 integrin appears to be both directly and indirectly causally related to several muscle diseases. Enhanced expression of alpha7beta1-mediated linkage of the extracellular matrix is seen in Duchenne muscular dystrophy and may compensate for the absence of the dystrophin-mediated linkage. Downregulation of expression of the integrin may contribute to the development of pathology in congenital laminin deficiencies. Mutations in the alpha7 integrin gene underlie additional congenital muscle diseases. The functional roles of this integrin in the formation and stability of the neuromuscular and myotendinous junctions and its localization between fibers suggest that altered expression or function of this integrin may have widespread involvement in other myopathies. The localization of the alpha7 gene at human chromosome 12q13 is a useful clue for focusing such studies.

Expression of Perlecan, a Proteoglycan That Binds Myogenic Inhibitory Basic Fibroblast Growth Factor, Is Down Regulated during Skeletal Muscle Differentiation

Heparan sulfate proteoglycans (HSPG) have been shown to be involved in the activation of tyrosine kinase receptors by basic fibroblasts growth factor (bFGF), a strong inhibitor of skeletal muscle differentiation. Skeletal muscle fibers contact extracellular matrix (ECM) that surrounds individual fibers (endomysium) and bundles of several fibers (perimysium). Perlecan is a HSPG present in the majority of basement membranes. In this study we evaluated the expression and localization of perlecan during differentiation of C2C12skeletal muscle cells. C2C12myoblasts incubated with [35S]Na2SO4synthesize a HSPG that can be specifically immunoprecipitated with antibodies against murine perlecan. The immunoprecipitated HSPG eluted from a Sepharose CL-4B with a Kav of 0.44. Analysis of the core protein of the HSPG immunoprecipitated from [35S]methionine-labeled C2C12after treatment with heparitinase revealed two polypeptides of 170 and over 300 kDa. The amount of polypeptides immunoprecipitated decreased with muscle differentiation. Immunocytolocalization studies indicate that perlecan is localized on the myoblast surface and by immunogold staining we have demonstrated that it is associated with patches of incipient extracellular matrix. The expression of perlecan mRNA decreased substantially during skeletal muscle differentiation, in contrast to the increase in transcripts for specific skeletal muscle proteins such as myogenin and creatine kinase. By immunofluorescence microscopy almost no perlecan staining associated with the surface of myotubes was observed. All these results suggests that perlecan, a HSPG that binds myogenic inhibitory bFGF, normally associated with basement membranes in adult tissues is present on the surface of myoblasts and its expression is down regulated during skeletal muscle differentiation.

Serine proteinase inhibitors in human skeletal muscle: expression of beta-amyloid protein precursor and alpha 1-antichymotrypsin in vivo and during myogenesis in vitro.

The balance of serine proteases and inhibitors in nerve and muscle is altered during programmed- and injury-induced remodeling. A serpin, alpha 1-antichymotrypsin (alpha 1-ACT), and Kunitz-inhibitor containing forms of the beta-amyloid precursor protein (beta APP) may be important components of this balance. In the present study, we analyzed their expression in primary cultures of human myogenic (satellite) cells that mimic myogenic differentiation using Western blotting and immunocytochemistry. In vitro results were compared to in vivo results from normal adult human skeletal muscle biopsies. Using an anti-alpha 1-ACT polyclonal antibody, we detected a 62 kDa immunoreactive band both in cultured human myogenic cells (mononucleated myoblasts as well as multi-nucleated myotubes) and in extracts of human muscle biopsies. With a polyclonal anti-beta APP antibody we found two bands (105 and 120 kDa) in myoblasts and myotubes in culture. However, the same antibody recognized only a single band at 92 kDa in biopsies. By immunocytochemistry, both alpha 1-ACT and beta APP were indistinctly present on localized to the surface of myoblasts in culture. In contrast, these inhibitors were dense on myotube surfaces, where they often formed distinct aggregates and frequently co-localized. In permeabilized muscle cells, alpha 1-ACT and beta APP appeared to be localized to the perikarya of both myoblasts and myotubes. Confirming previous results, both alpha 1-ACT and beta APP were present at the neuromuscular junction in human muscle sections. These developmental changes found during in vitro myogenesis for alpha 1-ACT and beta APP, both serine protease inhibitors, reinforce the hypothesis that regulation of the serine proteases and serine protease inhibitors plays an important role in neuromuscular differentiation.

Temporal and spatial appearance of α‐dystroglycan in differentiated mouse myoblasts in culture

The dystrophin-glycoprotein complex plays an important role in muscle function. One of the components of the complex, a 156-kDa cell surface glycoprotein (alpha-dystroglycan) binds to laminin, thereby connecting the basal lamina and muscle cells. We have examined the progressive appearance of alpha-dystroglycan and laminin in muscle cells that differentiate in culture. We find that nondifferentiated cultures of C2C12 myoblasts express low amounts of dystroglycan mRNA and, in contrast, this gene is prominently expressed in differentiated myotubes. Immunofluorescence analysis with a monoclonal antibody against alpha-dystroglycan shows its progressive appearance during myoblast differentiation into myotubes. Immunostaining with a monoclonal antibody against laminin shows that it is not present on the surface of undifferentiated myoblasts. Subsequently, laminin becomes apparent on the surface of differentiated myotubes where it codistributes with immunostained alpha-dystroglycan. Immunoblotting of isolated surface membrane proteins of differentiated myotubes with antibodies against alpha-dystroglycan identifies a broad band of about 140-160 kDa, resembling alpha-dystroglycan from rabbit muscle. The composite results indicate that alpha-dystroglycan and laminin appear and become co-distributed on the surface of cultured C2C12 during the progression of differentiation.