Search

Skeletal muscle regeneration is a complex process, involving the proliferation, migration, and differentiation of myoblasts. Recent studies suggest that some natural flavanones stimulate myogenesis. However, the effect of plant estrogen, silibinin, on the regulation of myoblast behaviors is unclarified. In this study, we investigated the effects of silibinin on immortalized murine myoblast C2C12 in the aspects of proliferation, migration, differentiation along with underlying mechanisms. The results show that silibinin at concentrations below 50 μM enhanced the migration and differentiation of C2C12 cells, but had no effect on cell proliferation. Silibinin significantly promoted the production of ROS, which appeared to play important roles in the migration and differentiation of the myoblasts. Interestingly, among ROS, the superoxide anion and hydroxyl radical were associated with the migration, whereas hydrogen peroxide contributed to the myogenic differentiation. We used ER agonist and antagonist to explore whether estrogen receptors (ERs), which are affected by silibinin treatment in the silibinin-enhanced C2C12 migration and differentiation. Migration was independent of ERs, whereas the differentiation was associated with decreased ERα activity. In summary, silibinin treatment increases ROS levels, leading to the promotion of migration and myogenic differentiation. Negative regulation ERα of differentiation but not of migration may suggest that ERα represses hydrogen peroxide generation. The effect of silibinin on myoblast migration and differentiation suggests that silibinin may have therapeutic benefits for muscle regeneration.

Skeletal muscle regeneration implies the coordination of myogenesis with the recruitment of myeloid cells and extracellular matrix (ECM) remodelling. Currently, there are no specific biomarkers to diagnose the severity and prognosis of muscle lesions. In order to investigate the gene expression profile of extracellular matrix and adhesion molecules, as premises of homo‐ or heterocellular cooperation and milestones for skeletal muscle regeneration, we performed a gene expression analysis for genes involved in cellular cooperation, migration and ECM remodelling in a mouse model of acute crush injury. The results obtained at two early time‐points post‐injury were compared to a GSE5413 data set from two other trauma models. Third day post‐injury, when inflammatory cells invaded, genes associated with cell‐matrix interactions and migration were up‐regulated. After day 5, as myoblast migration and differentiation started, genes for basement membrane constituents were found down‐regulated, whereas genes for ECM molecules, macrophage, myoblast adhesion, and migration receptors were up‐regulated. However, the profile and the induction time varied according to the experimental model, with only few genes being constantly up‐regulated. Gene up‐regulation was higher, delayed and more diverse following more severe trauma. Moreover, one of the most up‐regulated genes was periostin, suggestive for severe muscle damage and unfavourable architecture restoration.

The migration and invasion of myoblast cells derive from the occipital somites is critical for tongue morphogenesis. However, the molecular mechanisms underlying myoblast migration and invasion remain elusive. In this study, we report that transcription factor Isl1 is required for tongue development. Loss of Isl1 in the distal mandibular epithelium results in aglossia due to the defect of myoblast cells invasion. In the absence of Isl1 expression, myoblast cells in the first branchial arch fail to migrate into the presumptive tongue primordium. Down-regulation of Shh was detected in the distal mandibular epithelium after Isl1 deletion. As a downstream target gene of Shh, Wnt5a attracts the migration of mesenchymal cells expressing chemokine ligand 12 (CXCL12). CXCL12+ mesenchymal cells in the Isl1 mutant embryo fail to migrate to the distal region but accumulate in a relatively small proximal domain of the first branchial arch. CXCL12 serves as a bidirectional cue for myoblasts that express its receptor CXCR4, attracting CXCR4+ myoblasts invasion at low concentration but repelling at high concentration. The defect in the distal migration of CXCL12+ mesenchymal cells leads to a high local concentration of CXCL12, thereby preventing the invasion of CXCR4+ myoblasts. In addition, transgenic activation of Ihh alleviates the defect in tongue development and rescues the invasion of myoblasts, corroborating the functional involvement of Hedgehog signaling in tongue development. In summary, this study provides the first line of genetic evidence for the Isl1/Shh/Wnt5a cascade to regulate myoblasts invasion by tuning the CXCL12/CXCR4 axis.

Muscle is one of the important economic traits in poultry production, and its production depends on the increased number of muscle fibers during the embryonic stage. Chicken GHR gene can transcribe in double directions, possessing not only GHR-S but also GHR-AS. The 2 kinds of transcripts are partially complementation in sequences and interact with each other. Until now, the roles and mechanisms of GHR-AS in myoblast differentiation was still unknown. In this study, we not only analyzed the GHR-AS expression patterns in myoblast differentiation phase but also clarified that GHR-AS promoted myoblast differentiation via GH-GHR-IGF1 signal pathway. Quantitative PCR analysis indicated that GHR-AS was increased during myoblast differentiation. Sub-cellular localization showed that GHR-AS and GHR-S were expressed at a higher level in the nucleus than that in the cytoplasm. The expression of MyoD and MyHC and the myoblast differentiation significantly increased after GHR-AS overexpression, while the distance between wounds decreased, suggesting that GHR-AS repressed myoblast migration and promoted differentiation. Additionally, the expression of GHR-AS, IGF1 and MyHC increased after GH protein treated, and the myoblast differentiation also increased. In conclusion, GHR-AS promoted myoblast differentiation by enhancing fusion and inhibiting migration possibly via GH-GHR-IGF1 signal pathway.

The Qianhua Mutton Merino (QHMM) is a new variety of sheep (Ovis aries) with improved meat performance compared with the traditional Small Tail Han (STH) sheep variety. We recently reported the transcriptome profiling of longissimus muscle tissues between QHMM and STH sheep. In the present study, we aimed to evaluate key micro (mi)RNA-mRNA networks associated with sheep muscle growth and development. We used miRNA sequencing to obtain longissimus muscle miRNA profiles from QHMM and STH sheep. We identified a total of 153 known sheep miRNAs, of which 4 were differentially expressed (DE) between the 2 sheep varieties. We combined these results with mRNA library data to build an miRNA-mRNA network, including 26 target genes of the 4 DE miRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that 26 target genes were significantly enriched in 86 biological processes, including muscle organogenesis, myoblast migration, cell proliferation, and adipose tissue development, and in 9 metabolic pathways, including carbohydrate, nucleotide, and amino acid metabolic pathways. oar-miR-655-3p and its target gene ACSM3 and oar-miR-381-5p and its target gene ABAT were selected for subsequent analysis based on GO and KEGG analyses. The binding sites of oar-miR-655-3p with ACSM3 and oar-miR-381-5p with ABAT were validated by a dual-luciferase reporter gene detection system. This represents the first integrative analysis of miRNA-mRNA networks in QHMM and STH muscles and suggests that DE miRNAs, especially oar-miR-655-3p and oar-miR-381-5p, play crucial roles in muscle growth and development.

Methylmercury (MeHg) is a ubiquitous environmental contaminant and developmental toxicant known to cause a variety of persistent motor and cognitive deficits. While previous research has focused predominantly on neurotoxic MeHg effects, emerging evidence points to a myotoxic role whereby MeHg induces defects in muscle development and maintenance. A genome wide association study for developmental sensitivity to MeHg in Drosophila has revealed several conserved muscle morphogenesis candidate genes that function in an array of processes from myoblast migration and fusion to myotendinous junction (MTJ) formation and myofibrillogenesis. Here, we investigated candidates for a role in mediating MeHg disruption of muscle development by evaluating morphological and functional phenotypes of the indirect flight muscles (IFMs) in pupal and adult flies following 0, 5, 10, and 15 μM MeHg exposure via feeding at the larval stage. Developmental MeHg exposure induced a dose-dependent increase in muscle detachments (myospheres) within dorsal bundles of the IFMs, which paralleled reductions eclosion and adult flight behaviors. These effects were selectively phenocopied by altered expression of kon-tiki (kon), a chondroitin sulfate proteoglycan 4/NG2 homologue and a central component of MTJ formation. MeHg elevated kon transcript expression at a crucial window of IFM development and transgene overexpression of kon could also phenocopy myosphere phenotypes and eclosion and flight deficits. Finally, the myosphere phenotype resulting from 10 μM MeHg was partially rescued in a background of reduced kon expression using a targeted RNAi approach. Our findings implicate a component of the MTJ as a MeHg toxicity target which broaden the understanding of how motor deficits can emerge from early life MeHg exposure.

Mounting evidence suggests that site-appropriate loading of implanted extracellular matrix (ECM) bioscaffolds and the surrounding microenvironment is an important tissue remodeling determinant, although the role at the cellular level in ECM-mediated skeletal muscle remodeling remains unknown. This study evaluates crosstalk between progenitor cells and macrophages during mechanical loading in ECM-mediated skeletal muscle repair. Myoblasts were exposed to solubilized ECM bioscaffolds and were mechanically loaded at 10% strain, 1 Hz for 5 h. Conditioned media was collected and applied to bone marrow-derived macrophages followed by immunolabeling for proinflammatory M1-like markers and proremodeling M2-like markers. Macrophages were subjected to the same loading protocol and their secreted products were collected for myoblast migration, proliferation, and differentiation analysis. A mouse hind limb unloading volumetric muscle loss model was used to evaluate the effect of loading upon the skeletal muscle microenvironment after ECM implantation. Animals were sacrificed at 14 or 180 days. Isometric torque production was tested and tissue sections were immunolabeled for macrophage phenotype and muscle fiber content. Results show that loading augments the ability of myoblasts to promote an M2-like macrophage phenotype following exposure to ECM bioscaffolds. Mechanically loaded macrophages promote myoblast chemotaxis and differentiation. Lack of weight bearing impaired muscle remodeling as indicated by Masson's Trichrome stain. Isometric torque was significantly increased following ECM implantation when compared to controls, a response not present in the hind limb-unloaded group. This work provides an important mechanistic insight of the effects of rehabilitation upon ECM-mediated remodeling and could have broader implications in clinical practice, advocating multidisciplinary approaches to regenerative medicine, emphasizing rehabilitation.

Platelet-derived growth factors (PDGFs) regulate embryonic development, tissue regeneration, and wound healing through their binding to PDGF receptors, PDGFRα and PDGFRβ. However, the role of PDGF signaling in regulating muscle development and regeneration remains elusive, and the cellular and molecular responses of myogenic cells are understudied. Here, we explore the PDGF-PDGFR gene expression changes and their involvement in skeletal muscle myogenesis and myogenic fate. By surveying bulk RNA sequencing and single-cell profiling data of skeletal muscle stem cells, we show that myogenic progenitors and muscle stem cells differentially express PDGF ligands and PDGF receptors during myogenesis. Quiescent adult muscle stem cells and myoblasts preferentially express PDGFRβ over PDGFRα. Remarkably, cell culture- and injury-induced muscle stem cell activation altered PDGF family gene expression. In myoblasts, PDGF-AB and PDGF-BB treatments activate two pro-chemotactic and pro-mitogenic downstream transducers, RAS-ERK1/2 and PI3K-AKT. PDGFRs inhibitor AG1296 inhibited ERK1/2 and AKT activation, myoblast migration, proliferation, and cell cycle progression induced by PDGF-AB and PDGF-BB. We also found that AG1296 causes myoblast G0/G1 cell cycle arrest. Remarkably, PDGF-AA did not promote a noticeable ERK1/2 or AKT activation, myoblast migration, or expansion. Also, myogenic differentiation reduced the expression of both PDGFRα and PDGFRβ, whereas forced PDGFRα expression impaired myogenesis. Thus, our data highlight PDGF signaling pathway to stimulate satellite cell proliferation aiming to enhance skeletal muscle regeneration and provide a deeper understanding of the role of PDGF signaling in non-fibroblastic cells.

The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud.

Powered flight was fundamental to the establishment and radiation of birds. However, flight has been lost multiple times throughout avian evolution. Convergent losses of flight within the ratites (flightless paleognaths, including the emu and ostrich) often coincide with reduced wings. Although there is a wealth of anatomical knowledge for several ratites, the genetic mechanisms causing these changes remain debated. Here, we use a multidisciplinary approach employing embryological, genetic, and genomic techniques to interrogate the mechanisms underlying forelimb heterochrony in emu embryos. We show that the initiation of limb formation, an epithelial to mesenchymal transition (EMT) in the lateral plate mesoderm (LPM) and myoblast migration into the LPM, occur at equivalent stages in the emu and chick. However, the emu forelimb fails to subsequently proliferate. The unique emu forelimb expression of Nkx2.5, previously associated with diminished wing development, initiates after this stage (concomitant with myoblast migration into the LPM) and is therefore unlikely to cause this developmental delay. In contrast, RNA sequencing of limb tissue reveals significantly lower Fgf10 expression in the emu forelimb. Artificially increasing Fgf10 expression in the emu LPM induces ectodermal Fgf8 expression and a limb bud. Analyzing open chromatin reveals differentially active regulatory elements near Fgf10 and Sall-1 in the emu wing, and the Sall-1 enhancer activity is dependent on a likely Fgf-mediated Ets transcription factor-binding site. Taken together, our results suggest that regulatory changes result in lower expression of Fgf10 and a concomitant failure to express genes required for limb proliferation in the early emu wing bud.

The proper function of the skeletal muscle is essential for the survival of most animals. Thus, efficient and rapid repair of muscular damage following injury is crucial. In recent years, satellite cells have emerged as key players of muscle repair, capable of undergoing extensive proliferation after injury, fusing into myotubes and restoring muscle function. Furthermore, it has been shown that Ca(2+)/calmodulin-dependent generation of nitric oxide (NO) is an important regulator of muscle repair. Here, we demonstrate the functional expression of transient receptor potential, subfamily A1 (TRPA1) channel in human primary myoblasts. Stimulation of these cells with well-known TRPA1 ligands led to robust intracellular Ca(2+) rises which could be inhibited by specific TRPA1 antagonists. Moreover, we show that TRPA1 activation enhances important aspects of skeletal muscle repair such as cell migration and myoblast fusion in vitro. Interestingly, TRPA1 levels and inducible Ca(2+) transients decline with ongoing myoblast differentiation. We suggest that TRPA1 might serve as a physiological mediator for inflammatory signals and appears to have a functional role in promoting myoblast migration, fusion, and potentially also in activating satellite cells in humans.

Myoblast transplantation (MT) is a method to introduce healthy genes into abnormal skeletal muscle. It has been considered as a therapeutic modality in the last few decades for diseases such as Duchenne Muscular Dystrophy (DMD). However, challenges including cell death and poor graft engraftment have limited its application. The current experiment utilizes MMP1 gene transfer to improve the efficacy of myoblast transplantation into the diseased dystrophic skeletal muscle of mdx mice. Our results indicated that MMP1 expression can promote myogenic differentiation and fusion capacities, increase migration of MMP1 expressing myoblasts in vitro, as well as improve engraftment of dystrophin positive myofibers in vivo. Taken together, our observation suggests that the addition of MMP1 can overcome limitations in MT and improve its clinical efficacy.

Myoblast migration is essential for muscle development and repair; however, the factors that contribute to the polarity of migrating myoblasts are relatively unknown. We find that randomly migrating C2C12 myoblasts orient their centrosomes in the direction of migration. Using wounded monolayers, we further show that centrosome orientation is stimulated by the serum factor lysophosphatidic acid (LPA) and involves the rearward movement of the nucleus while the centrosome is maintained at the cell centroid. The rate of nuclear movement correlated with that of actin retrograde flow and both cytochalasin D and blebbistatin prevented nuclear movement and centrosome orientation. Actin-dependent rearward nuclear movement in fibroblasts is mediated by assembly of nuclear membrane nesprin-2G and SUN2 LINC complexes into transmembrane actin-associated nuclear (TAN) lines anchored by A-type lamins and emerin. In C2C12 myoblasts, depletion of nesprin-2G, SUN2 or lamin A/C prevented nuclear movement and endogenous nesprin-2G and a chimeric GFP-mini-nesprin-2G formed TAN lines during nuclear movement. Depleting nesprin-2G strongly interfered with directed cell migration and reduced the efficiency of myoblast fusion into multinucleated myotubes. Our results show that nuclear movement contributes to centrosome orientation and polarity for efficient migration and fusion of myoblasts. Given that mutations in the genes encoding A-type lamins, nesprin-2 and SUN2 cause Emery-Dreifuss muscular dystrophy and related myopathies, our results have implications for understanding the mechanism of disease pathogenesis.

Skeletal muscle injury elicits the activation of satellite cells and their migration to the wound area for subsequent terminal differentiation and tissue integration. However, interstitial fibroblasts recruited to the site of injury promote deposition of fibrotic tissue, which hampers myoblast-mediated muscle regeneration. Currently, analysis of myoblast migration in vitro can be accomplished using chemotactic, cell-exclusion, or wound healing assays. Yet, to investigate cell motility following skeletal muscle damage more accurately, migration assays need to better simulate the repair process. Here we present a protocol for the simultaneous isolation of myoblasts and fibroblasts from the same muscle tissue, ensuring the consistent generation of enriched, purified, and matched cell populations at a low passage number. We then describe a wound assay that uses a novel approach to the co-culture of myoblasts and fibroblasts to mimic the injured environment more closely than other established methods. Using this assay, we demonstrate that fibroblasts are able to increase myoblast migration significantly, validating our new in vitro method. As the observed effect on migration is most likely mediated by secreted factors, our assay could easily be extended to include antibody-based protein analysis of secreted factors in animal or human systems.

Exposing myoblasts to basic fibroblast growth factor (bFGF), which is released after muscle injury, results in receptor phosphorylation, faster migration, and increased proliferation. These effects occur on time scales that extend across three orders of magnitude (10(0)-10(3) minutes). Finite element modeling of Transwell assays, which are traditionally used to assess chemotaxis, revealed that the bFGF gradient formed across the membrane pore is short-lived and diminishes 45% within the first minute. Thus, to evaluate bFGF-induced migration over 10(2) minutes, we employed a microfluidic assay capable of producing a stable, linear concentration gradient to perform single-cell analyses of chemokinesis and chemotaxis. We hypothesized that the composition of the underlying extracellular matrix (ECM) may affect the behavioral response of myoblasts to soluble bFGF, as previous work with other cell types has suggested crosstalk between integrin and fibroblast growth factor (FGF) receptors. Consistent with this notion, we found that bFGF significantly reduced the doubling time of myoblasts cultured on laminin but not fibronectin or collagen. Laminin also promoted significantly faster migration speeds (13.4 μm h(-1)) than either fibronectin (10.6 μm h(-1)) or collagen (7.6 μm h(-1)) without bFGF stimulation. Chemokinesis driven by bFGF further increased migration speed in a strictly additive manner, resulting in an average increase of 2.3 μm h(-1) across all ECMs tested. We observed relatively mild chemoattraction (∼67% of myoblast population) in response to bFGF gradients of 3.2 ng mL(-1) mm(-1) regardless of ECM identity. Thus, while ECM-bFGF crosstalk did impact chemoproliferation, it did not have a significant effect on chemokinesis or chemotaxis. These data suggest that the main physiological effect of bFGF on myoblast migration is chemokinesis and that changes in the surrounding ECM, resulting from aging and/or disease may impact muscle regeneration by altering myoblast migration and proliferation.

The communication between nonmyogenic cells, such as macrophages and fibroblasts, and myoblasts is crucial for successful skeletal muscle repair. In vitro co-culture methods can be used to increase our understanding of these cellular interactions; however, current protocols are restricted to two, often physically separate, cell populations. Here, we demonstrate a novel, inexpensive in vitro triple co-culture method that facilitates the co-culture of at least three cell populations with some degree of cell–cell contact. Using this method, we determined the effect of macrophages and fibroblasts on myoblast proliferation and migration. A significant increase in myoblast proliferation and migration was observed following co-culture with either macrophages or fibroblasts. However, triple co-culture of macrophages, fibroblasts, and myoblasts revealed that the presence of macrophages prevented fibroblasts from maintaining this positive effect on myoblast migration. Macrophages, on the other hand, continued to promote myoblast proliferation whether in the presence of fibroblasts or not. Our triple co-culture system highlights the significance of multicellular communication in regulating myoblast proliferation and migration and emphasizes the importance of more complex co-culture systems when investigating myogenesis in vitro.

Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed a heretofore unexplored role of resident myogenic stem cell (satellite cell)-derived myoblasts as a key presenter of a secreted neural chemorepellent semaphorin 3A (Sema3A); hepatocyte growth factor (HGF) triggered its expression exclusively at the early-differentiation phase. In order to verify this concept, the present study was designed to clarify a paracrine source of HGF release. In vitro experiments demonstrated that activated anti-inflammatory macrophages (CD206-positive M2) produce HGF and thereby promote myoblast chemoattraction and Sema3A expression. Media from pro-inflammatory macrophage cultures (M1) did not show any significant effect. M2 also enhanced the expression of myoblast-differentiation markers in culture, and infiltrated predominantly at the early-differentiation phase (3-5 days post-injury); M2 were confirmed to produce HGF as monitored by in vivo/ex vivo immunocytochemistry of CD11b/CD206/HGF-positive cells and by HGF in situ hybridization of cardiotoxin- or crush-injured tibialis anterior muscle, respectively. These studies advance our understanding of the stage-specific activation of Sema3A expression signaling. Findings, therefore, encourage the idea that M2 contribute to spatiotemporal up-regulation of extracellular Sema3A concentrations by producing HGF that, in turn, stimulates a burst of Sema3A secretion by myoblasts that are recruited to site of injury. This model may ensure a coordinated delay in re-attachment of motoneuron terminals onto damaged fibers early in muscle regeneration, and thus synchronize the recovery of muscle-fiber integrity and the early resolution of inflammation after injury.

Satellite cell migration is critical for skeletal muscle growth and regeneration. Controlled cell migration is dependent on the formation of mature focal adhesions between the cell and the underlying extracellular matrix (ECM). These cell-ECM interactions trigger the activation of signalling events such as the Rho/ROCK pathway. We have previously identified a specific role for ROCK-2 during myoblast migration. In this study we report that ROCK inhibition with Y-27632 increases C2C12 myoblast velocity, but at the expense of directional migration. In response to Y-27632 an increased number of smaller focal adhesions were distributed across adhesion sites that in turn were clearly larger than sites in untreated cells, suggesting a reduction in focal adhesion maturation. We also confirm ROCK-2 localisation to the focal adhesion sites in migrating myoblasts and demonstrate a change in the distribution of these ROCK-2 containing adhesions in response to Y-27632. Taken together, our observations provide further proof that ROCK-2 regulates directional myoblast migration through focal adhesion formation and maturation.

In vitro systems comprised of wells interconnected by microchannels have emerged as a platform for the study of cell migration or multicellular models. In the present study, we systematically evaluated the effect of microchannel width on spontaneous myoblast migration across these microchannels&mdash, from the proximal to the distal chamber. Myoblast migration was examined in microfluidic devices with varying microchannel widths of 1.5&ndash, 20 &micro, m, and in chips with uniform microchannel widths over time spans that are relevant for myoblast-to-myofiber differentiation in vitro. We found that the likelihood of spontaneous myoblast migration was microchannel width dependent and that a width of 3 &micro, m was necessary to limit spontaneous migration below 5% of cells in the seeded well after 48 h. These results inform the future design of Polydimethylsiloxane (PDMS) microchannel-based co-culture platforms as well as future in vitro studies of myoblast migration.

Efficient skeletal muscle repair and regeneration require coordinated remodeling of the extracellular matrix (ECM). Previous reports have indicated that matrix metalloproteinases (MMPs) play the pivotal role in ECM remodeling during muscle regeneration. The goal of the current study was to determine if the interstitial collagenase MMP-13 was involved in the muscle repair process. Using intramuscular cardiotoxin injections to induce acute muscle injury, we found that MMP-13 expression and activity transiently increased during the regeneration process. In addition, in muscles from mdx mice, which exhibit chronic injury, MMP-13 expression and protein levels were elevated. In differentiating C2C12 cells, a murine myoblast cell line, Mmp13 expression was most pronounced after myoblast fusion and during myotube formation. Using pharmacological inhibition of MMP-13 to test whether MMP-13 activity is necessary for the proliferation, differentiation, migration, and fusion of C2C12 cells, we found a dramatic blockade of myoblast migration, as well as a delay in differentiation. In contrast, C2C12 cells with stable overexpression of MMP-13 showed enhanced migration, without affecting myoblast maturation. Taken together, these results support a primary role for MMP-13 in myoblast migration that leads to secondary effects on differentiation.

The transient receptor potential canonical (TRPC) family consists of seven isoforms that have been proposed as molecular components essential for nonselective calcium (Ca2+) entry. TRPC1 proteins are expressed in the sarcolemma of skeletal muscles, and TRPC1 is important and necessary for stretch-activated and store-operated channels for Ca2+ entry. Studies have established the essential role of TRPC1 in maintaining Ca2+ homeostasis, regulating myoblast migration and differentiation, regenerating muscle, and contributing to the pathogenesis of muscular dystrophy. This chapter summarizes the evidence for the regulation of TRPC1 to fulfill specific physiological functions in skeletal muscles.

Made available in DSpace on 2018-11-29T03:50:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-11-21 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3'-untranslated region (3' UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80-90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3' UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3' UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells. Sao Paulo State Univ, Inst Biosci, Dept Morphol, Sao Paulo, Brazil Univ Sao Paulo, Inst Biomed Sci, Dept Cell & Dev Biol, Sao Paulo, Brazil Sao Paulo State Univ, Inst Biosci, Dept Morphol, Sao Paulo, Brazil FAPESP: 2012/13961-6 FAPESP: 2014/13783-6

Muscular injuries that destroy the basal lamina result in poor functional recovery of skeletal muscle. This is due, in part, to the deposition of structural fibrotic proteins such as fibronectin and collagen by fibroblasts and other cells. Transforming growth factor-β (TGF-β) promotes fibrosis, whereas the proteoglycan decorin is known to act as an anti-fibrotic agent, in part via the binding and neutralization of TGF-β. We have previously established that decorin can alter the migratory response of skeletal muscle myoblasts to the extracellular matrix (ECM) factor collagen, but not fibronectin. We have also shown that TGF-β reduces myoblast migration. In the current study we demonstrate that decorin can dramatically alter the inhibitory role of TGF-β on human myoblast migration and go on to shown that the extracellular matrix can significantly modify this effect. Decorin and TGF-β2 in combination were observed to significantly increase the rate of human myoblast migration, despite the inhibitory effect of TGF-β2 on its own. Furthermore, in the presence of fibronectin, TGF-β2 and decorin no longer acted synergistically to promote migration; while in the presence of collagen I, TGF-β2 failed to inhibit migration. These studies show, for the first time, that decorin can alter the bioactivity of TGF-β2 on human myoblast migration and emphasize the crucial regulatory role of the extracellular matrix in determining the response of skeletal muscle myoblasts to migratory cues.

During vertebrate somitogenesis, retinoic acid is known to establish the position of the determination wavefront, controlling where new somites are permitted to form along the anteroposterior body axis. Less is understood about how RAR regulates somite patterning, rostral-caudal boundary setting, specialization of myotome subdivisions, or the specific RAR subtype that is required for somite patterning. Characterizing the function of RARβ has been challenging due to the absence of embryonic phenotypes in murine loss-of-function studies. Using the Xenopus system, we show that RARβ2 plays a specific role in somite number and size, restriction of the presomitic mesoderm anterior border, somite chevron morphology and hypaxial myoblast migration. Rarβ2 is the RAR subtype whose expression is most up-regulated in response to ligand and its localization in the trunk somites positions it at the right time and place to respond to embryonic retinoid levels during somitogenesis. RARβ2 positively regulates Tbx3 a marker of hypaxial muscle, and negatively regulates Tbx6 via Ripply2 to restrict the anterior boundaries of the presomitic mesoderm and caudal progenitor pool. These results demonstrate for the first time an early and essential role for RARβ2 in vertebrate somitogenesis.

Aligned poly( l -lactide-r-glycolide) (PLGA) nanofibrous meshes were prepared by electrospinning technique. The surface patterns of the meshes were observed by SEM and the diameter distribution of the fibers was tested. C2C12 myoblasts were cultured on the electrospun fibers. The cell proliferations and migration of the patterns of aligned and random-oriented fibrous PLGA meshes were investigated. The results showed that the two surface patterns were favorable for cell adhesion and proliferation. The aligned fibers could guide the cells to migrate the fibrous direction. The aligned surface pattern was more efficient to accelerate the excretion of extracellular matrix. It provided a promising pattern technique to control the migration of cells.

Syntrophins are adaptor proteins that link intracellular signaling molecules to the dystrophin based scaffold. In this study, we investigated the function of syntrophins in cell migration, one of the early steps in myogenic differentiation and in regeneration of adult muscle. Hepatocyte growth factor (HGF) stimulates migration and lamellipodia formation in cultured C2 myoblasts. In the migrating cells, syntrophins concentrated in the rear-lateral region of the cell, opposite of the lamellipodia, instead of being diffusely present throughout the cytoplasm of non-migrating cells. When the expression of α-syntrophin, the major syntrophin isoform of skeletal muscle, was reduced by transfection with the α-syntrophin-specific siRNA, HGF stimulation of lamellipodia formation was prevented. Likewise, migration of myoblasts from α-syntrophin knockout mice could not be stimulated by HGF. However, HGF-induced migration was restored in myoblasts isolated from a transgenic mouse expressing α-syntrophin only in muscle cells. Treatment of C2 myoblasts with inhibitors of PI3-kinase not only reduced the rate of cell migration, but also impaired the accumulation of syntrophins in the rear-lateral region of the migrating cells. Phosphorylation of Akt was reduced in the α-syntrophin siRNA-treated C2 cells. These results suggest that α-syntrophin is required for HGF-induced migration of myoblasts and for proper PI3-kinase/Akt signaling.

The BTB-Kelch protein Krp1 is highly and specifically expressed in skeletal muscle, where it is proposed to have a role in myofibril formation. We observed significant upregulation of Krp1 in C2 cells early in myoblast differentiation, well before myofibrillogenesis. Krp1 has a role in cytoskeletal organization and cell motility; since myoblast migration and elongation/alignment are important events in early myogenesis, we hypothesized that Krp1 is involved with earlier regulation of differentiation. Krp1 protein levels were detectable by 24 h after induction of differentiation in C2 cells and were significantly upregulated by 48 h, i.e., following the onset myogenin expression and preceding myosin heavy chain (MHC) upregulation. Upregulation of Krp1 required a myogenic stimulus as signaling derived from increased myoblast cell density was insufficient to activate Krp1 expression. Examination of putative Krp1 proximal promoter regions revealed consensus E box elements associated with myogenic basic helix-loop-helix binding. The activity of a luciferase promoter-reporter construct encompassing this 2,000-bp region increased in differentiating C2 myoblasts and in C2 cells transfected with myogenin and/or MyoD. Knockdown of Krp1 via short hairpin RNA resulted in increased C2 cell number and proliferation rate as assessed by bromodeoxyuridine incorporation, whereas overexpression of Krp1-myc had the opposite effect; apoptosis was unchanged. No effects of changed Krp1 protein levels on cell migration were observed, either by scratch wound assay or live cell imaging. Paradoxically, both knockdown and overexpression of Krp1 inhibited myoblast differentiation assessed by expression of myogenin, MEF2C, MHC, and cell fusion.

Extracellular matrix factors, specifically fibronectin and collagen I, are essential for structural support during muscle regeneration. Decorin has been identified as an anti-fibrotic agent with binding sites located on both fibronectin and collagen I. Upon injury, activated myoblasts are required to migrate through the extracellular matrix factors deposited by the myofibroblasts to facilitate skeletal muscle regeneration. In this study we looked at the effects decorin on fibronectin- and collagen I-stimulated myoblast migration. Dose response studies demonstrated 10 μg/ml, 5 μg/ml and 25 μg/ml as the optimal stimulatory concentrations of decorin (1.2 fold increase), fibronectin (3.5 fold increase) and collagen I (2.4 fold increase), when compared with control respectively. A synergistic effect was identified when decorin and collagen I were added in combination; this effect was not evident when decorin was added with fibronectin. The effects of these factors on the ROCK signalling pathway were also analyzed. ROCK-2 was identified as the key Rho-activated kinase isoform involved in migration, due to its higher expression levels and localisation to focal points within migrating C2C12 myoblasts. Decorin and collagen I in combination stimulated an increase in the number of ROCK-2 localized focal points when compared with control, decorin and collagen I added separately. Fibronectin did not show any increase in ROCK-2 focal points when compared with control. These results show for the first time that decorin can modify collagen I-stimulated, but not fibronectin-stimulated myoblast migration in vitro. Furthermore, the synergistic, rather than additive, effect observed suggests a direct modification of collagen I signalling by decorin mediated, at least in part, by ROCK-2 rather than ROCK-1.

The coordinated and dynamic regulation of adhesions is required for cell migration. We demonstrated previously that limited proteolysis of talin1 by the calcium-dependent protease calpain 2 plays a critical role in adhesion disassembly in fibroblasts (Franco, S. J., Rodgers, M. A., Perrin, B. J., Han, J., Bennin, D. A., Critchley, D. R., and Huttenlocher, A. (2004) Nat. Cell Biol. 6, 977-983). However, little is known about the contribution of other calpain substrates to the regulation of adhesion dynamics. We now provide evidence that calpain 2-mediated proteolysis of focal adhesion kinase (FAK) regulates adhesion dynamics in motile cells. We mapped the preferred calpain cleavage site between the two C-terminal proline-rich regions after Ser-745, resulting in a C-terminal fragment similar in size to the FAK-related non-kinase (FRNK). We generated mutant FAK with a point mutation (V744G) that renders FAK resistant to calpain proteolysis but retains other biochemical properties of FAK. Using time-lapse microscopy, we show that the dynamics of green fluorescent protein-talin1 are impaired in FAK-deficient cells. Expression of wild-type but not calpain-resistant FAK rescues talin dynamics in FAK-deficient cells. Taken together, our findings suggest a novel role for calpain proteolysis of FAK in regulating adhesion dynamics in motile cells.

The dynamic behaviors of human skeletal muscle myoblasts were investigated in the culture on a laminin-coated surface in the presence of 100 ng/ml epidermal growth factor (EGF) in medium. The coexistence of laminin and EGF caused the enhancement of myoblast migration, giving an average migration rate of 62.0 μm/h, which was 2.7 times that on a plain surface. This encouraged migration could be a driving force to separate the dividing cells from each other, accompanied by shortened disjunction time of daughter cells to complete cytokinesis. In addition, the synergic effect of laminin and EGF led to the promotion of myoblast growth with keeping a relatively high fraction of proliferative cells during the culture for 150 h, which is considered to arise from the reduced frequency of cell–cell contacts during cytokinesis and thereby suppressing the process towards myotube formation after cell division.

OBJECTIVES To investigate the optimal microenvironment for efficient myoblast transplantation in vivo. MATERIALS AND METHODS The effects of co-culture with growth factors, including basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor-I) and platelet-derived growth factor (PDGF), on in vitro growth, migration and proteolytic activity of mouse skeletal myoblasts were investigated. Myoblasts were co-injected with growth factors into the subcutis and bladder wall of nude mice, and its impact on the growth patterns of myoblasts in vivo assessed. RESULTS There was dose-dependent stimulation of in vitro myoblast growth after treatment with each of the four growth factors, but bFGF induced the most marked increase in the growth of myoblasts. Treatment of myoblasts with all types of growth factors also resulted in a dose-dependent increase in the in vitro migration of myoblasts, and PDGF had the most prominent effect on myoblast migration. Increased secretion of matrix metalloproteinase-9 (MMP-9) in myoblasts induced by growth factors was proportional to their increased migration capacity, which was partly inhibited by SB-3CT, an inhibitor of MMP-9. The in vivo growth of myoblasts was significantly enhanced by co-injection with all types of growth factor into both the subcutis and bladder wall, but this effect was most marked 1 and 2 weeks after co-injection with bFGF and PDGF, respectively. Furthermore, there was synergistic in vivo growth of myoblasts by co-injection of both bFGF and PDGF compared with that achieved with either agent alone. CONCLUSIONS These findings suggest that modulation of the microenvironment using growth factors, particularly bFGF and PDGF, could provide the optimum condition for effective myoblast transplantation in vivo.

The Drosophila embryonic salivary gland is an epithelial organ formed by the coordinated invagination and migration of primordial cells. To identify genes that regulate gland migration we performed a deficiency screen of the third chromosome. Here, we report on the analysis of the beta 2 tubulin isoform (β2t) that maps at 85D15. We show that, in β2t mutant embryos, salivary glands did not complete their posterior migration and that migration of fusion competent myoblasts and longitudinal visceral muscle founder cells between the gland and circular visceral mesoderm was delayed. We also demonstrate that gland migration defects correlate with reduced βPS and αPS2 integrin expression in the surrounding mesoderm and that β2t genetically interacts with genes encoding integrin αPS1 and αPS2 subunits. Our studies reveal for the first time that β2t is expressed in embryogenesis and that β2t plays an important role in salivary gland and myoblast migration, possibly through proper regulation of integrin adhesion proteins. Developmental Dynamics 238:853–863, 2009. © 2009 Wiley-Liss, Inc.

Myoblast migration is a key step in myogenesis and regeneration. It allows myoblast alignment and their fusion into myotubes. The process has been shown to involve m-calpain or mu-calpain, two Ca(2+)-dependent cysteine proteases. Here we measure calpain activity in cultured cells and show a peak of activity at the beginning of the differentiation process. We also observed a concomitant and transient increase of the influx of Ca(2+) and expression of TRPC1 protein. Calpains are specifically activated by a store-operated entry of Ca(2+) in adult skeletal muscle fibres. We therefore repressed the expression of TRPC1 in myoblasts and studied the effects on Ca(2+) fluxes and on differentiation. TRPC1-depleted myoblasts presented a largely reduced store-operated entry of Ca(2+) and a significantly diminished transient influx of Ca(2+) at the beginning of differentiation. The concomitant peak of calpain activity was abolished. TRPC1-knockdown myoblasts also accumulated myristoylated alanine-rich C-kinase substrate (MARCKS), an actin-binding protein and substrate of calpain. Their fusion into myotubes was significantly slowed down as a result of the reduced speed of cell migration. Accordingly, migration of control myoblasts was inhibited by 2-5 microM GsMTx4 toxin, an inhibitor of TRP channels or by 50 microM Z-Leu-Leu, an inhibitor of calpain. By contrast, stimulation of control myoblasts with IGF-1 increased the basal influx of Ca(2+), activated calpain and accelerated migration. These effects were not observed in TRPC1-knockdown cells. We therefore suggest that entry of Ca(2+) through TRPC1 channels induces a transient activation of calpain and subsequent proteolysis of MARCKS, which allows in turn, myoblast migration and fusion.

Many pre-mRNAs are alternatively spliced in a tissue-specific manner in multicellular organisms. The Fox-1 family of RNA-binding proteins regulate alternative splicing by either activating or repressing exon inclusion through specific binding to UGCAUG stretches. However, the precise cellular contexts that determine the action of the Fox-1 family in vivo remain to be elucidated. We have recently demonstrated that ASD-1 and FOX-1, members of the Fox-1 family in Caenorhabditis elegans, regulate tissue-specific alternative splicing of the fibroblast growth factor receptor gene, egl-15, which eventually determines the ligand specificity of the receptor in vivo. Here we report that another RNA-binding protein, SUP-12, coregulates the egl-15 alternative splicing. By screening for mutants defective in the muscle-specific expression of our alternative splicing reporter, we identified the muscle-specific RNA-binding protein SUP-12. We identified juxtaposed conserved stretches as the cis elements responsible for the regulation. The Fox-1 family and the SUP-12 proteins form a stable complex with egl-15 RNA, depending on the cis elements. Furthermore, the asd-1; sup-12 double mutant is defective in sex myoblast migration, phenocopying the isoform-specific egl-15(5A) mutant. These results establish an in vivo model that coordination of the two families of RNA-binding proteins regulates tissue-specific alternative splicing of a specific target gene.

Duchenne muscular dystrophy (l is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of l The expression of uPA is induced in mdx cl strophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular [unction. Bone marrow (BM) transplantation experiments ravealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy. ispartof: Journal of Cell Biology vol:178 issue:6 pages:1039-1051 ispartof: location:United States status: published

The first wave of myoblasts which constitutes the post-mitotic myotome stems from the medial epithelial somite. Whereas medial pioneers extend throughout the entire mediolateral myotome at cervical and limb levels, at flank regions they are complemented laterally by a population of early myoblasts emerging from the lateral epithelial somite. These myoblasts delaminate underneath the nascent dermomyotome and become post-mitotic. They are Myf5-positive but express MyoD and desmin only a day later while differentiating into fibers. Overexpression of Noggin in the lateral somite triggers their premature differentiation suggesting that lateral plate-BMP4 maintains them in an undifferentiated state. Moreover, directly accelerating their differentiation by MyoD overexpression prior to arrival of medial fibers, generates a severely mispatterned lateral myotome. This is in contrast to medial pioneers that have the capacity for self-organization. Furthermore, inhibiting differentiation of medial pioneers with dominant-negative MyoD also disrupts lateral myoblast patterning and differentiation. Thus, we propose that medial pioneers are needed for proper morphogenesis of the lateral population which is kept as undifferentiated mesenchyme by BMP4 until their arrival. In addition, medial pioneers also organize dermomyotome lip-derived fibers suggesting that they have a general role in patterning myotome development.

Skeletal muscle regeneration depends on myoblast migration, differentiation and myofiber formation. Isoforms of the nuclear factor of activated T cells (NFAT) family of transcription factors display nonredundant roles in skeletal muscle. NFAT5, a new isoform of NFAT, displays many differences from NFATc1-c4. Here, we examine the role of NFAT5 in myogenesis. NFAT5+/- mice displayed a defect in muscle regeneration with fewer myofibers formed at early times after injury. NFAT5 has a muscle-intrinsic function because inhibition of NFAT5 transcriptional activity caused both a migratory and differentiation defect in cultured myoblasts. We identified Cyr61 as a target of NFAT5 signaling in skeletal muscle cells. Addition of Cyr61 to cells expressing inhibitory forms of NFAT5 rescued the migratory phenotype. These results demonstrate a role for NFAT5 in skeletal muscle cell migration and differentiation. Furthermore, as cell-cell interactions are crucial for myoblast differentiation, these data suggest that myoblast migration and differentiation are coupled and that NFAT5 is a key regulator.

International audience; Previous research in our laboratory has already shown the importance of the role played by ubiquitous calpains during myoblast migration. The aim of this study was to investigate calpain expression during myoblast migration and, to enhance this phenomenon via calpain stimulation. Ubiquitous calpains are members of a large family of calcium-dependent cysteine proteases. They play an important role in numerous biological and pathological phenomena, such as signal transduction, apoptosis, cell-cycle regulation, cell spreading, adhesion, invasion, myogenesis, and motility. Myoblast migration is a crucial step in myogenesis, as it is necessary for myoblast alignment and fusion to form myotubes. This study started by examining changes in calpain expression during migration, then investigated the possibility of activating myoblast migration via the stimulation of calpain expression and/or activity. The migration rate of myoblasts overexpressing mu- or milli-calpain was quantified. The results showed that calpain overexpression dramatically inhibited myoblast migration. Growth-factor treatments were then used to enhance myoblast migration. The results showed that treatment with IGF-1, TGF-beta1, or insulin induced a major increase in migration and caused a significant increase in m-calpain expression and activity. The increase in migration was totally inhibited by adding calpeptin, a calpain-specific inhibitor. These findings suggest that milli-calpain is involved in growth factor-mediated migration.

CD44 is a transmembrane protein that plays a role in cell–cell interactions and motility in a number of cell types. Cell–cell interactions are critical for myoblast differentiation and fusion but whether CD44 regulates myogenesis is unknown. Here, we show that CD44 plays a functional role in early myogenesis. Analyses of myofiber cross-sectional area, after local injury in mouse tibialis anterior (TA) muscles, revealed that growth was transiently delayed in the absence of CD44. A muscle-intrinsic role for CD44 is suggested as primary myoblasts from CD44−/− mice displayed attenuated differentiation and subsequent myotube formation at early times in a differentiation-inducing in vitro environment. Chemotaxis of CD44−/− myoblasts toward hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) was totally abrogated, although expression of their respective receptors did not appear to differ from wild-type. Furthermore, motility of CD44−/− myoblasts was decreased at early stages of differentiation as determined by time-lapse microscopy. Wild-type myoblasts contained two subpopulations of slow- and fast-migrating cells, whereas CD44−/− myoblasts were composed predominantly of the slower migrating subpopulation. Taken together, these data suggest that myoblast migration and differentiation are closely linked and CD44 is a key regulator. J. Cell. Physiol. 209: 314–321, 2006. © 2006 Wiley-Liss, Inc.

Cell migration is a fundamental cellular function particularly during skeletal muscle development. Ubiquitous calpains are well known to play a pivotal role during muscle differentiation, especially at the onset of fusion. In this study, the possible positive regulation of myoblast migration by calpains, a crucial step required to align myoblasts to permit them to fuse, was investigated. Inhibition of calpain activity by different pharmacological inhibitors argues for the involvement of these proteinases during the migration of myoblasts. Moreover, a clonal cell line that fourfold overexpresses calpastatin, the endogenous inhibitor of calpains, and that exhibits deficient calpain activities was obtained. The results showed that the migratory capacity of C2C12 and fusion into multinucleated myotubes were completely prevented in these clonal cells. Calpastatin-overexpressing myoblasts unable to migrate were characterized by rounded morphology, the loss of membrane extensions, the disorganization of stress fibers and exhibited a major defect in new adhesion formation. Surprisingly, the proteolytic patterns of desmin, talin, vinculin, focal adhesion kinase (FAK) and ezrin, radixin, moesin (ERM) proteins are the same in calpastatin-overexpressing myoblasts as compared to control cells. However, an important accumulation of myristoylated alanine-rich C kinase substrate (MARCKS) was observed in cells showing a reduced calpain activity, suggesting that the proteolysis of this actin-binding protein is calpain-dependent and could be involved in both myoblast adhesion and migration.

Vascular endothelial growth factor (VEGF) expression is enhanced in ischemic skeletal muscle and is thought to play a key role in the angiogenic response to ischemia. However, it is still unknown whether, in addition to new blood vessel growth, VEGF modulates skeletal muscle cell function. In the present study immunohistochemical analysis showed that, in normoperfused mouse hindlimb, VEGF and its receptors Flk-1 and Flt-1 were expressed mostly in quiescent satellite cells. Unilateral hindlimb ischemia was induced by left femoral artery ligation. At day 3 and day 7 after the induction of ischemia, Flk-1 and Flt-1 were expressed in regenerating muscle fibers and VEGF expression by these fibers was markedly enhanced. Additional in vitro experiments showed that in growing medium both cultured satellite cells and myoblast cell line C2C12 expressed VEGF and its receptors. Under these conditions, Flk-1 receptor exhibited constitutive tyrosine phosphorylation that was increased by VEGF treatment. During myogenic differentiation Flk-1 and Flt-1 were down-regulated. In a modified Boyden Chamber assay, VEGF enhanced C2C12 myoblasts migration approximately fivefold. Moreover, VEGF administration to differentiating C2C12 myoblasts prevented apoptosis, while inhibition of VEGF signaling either with selective VEGF receptor inhibitors (SU1498 and CB676475) or a neutralizing Flk-1 antibody, enhanced cell death approximately 3.5-fold. Finally, adenovirus-mediated VEGF(165) gene transfer inhibited ischemia-induced apoptosis in skeletal muscle. These results support a role for VEGF in myoblast migration and survival, and suggest a novel autocrine role of VEGF in skeletal muscle repair during ischemia.

During limb skeletal muscle formation, committed muscle cells proliferate and differentiate in the presence of extracellular signals that stimulate or repress each process. Proteoglycans are extracellular matrix organizers and modulators of growth factor activities, regulating muscle differentiation in vitro. Previously, we characterized proteoglycan expression during early limb muscle formation and showed a spatiotemporal relation between the onset of myogenesis and the expression of decorin, an important muscle extracellular matrix component and potent regulator of TGF-β activity. To evaluate decorin’s role during in vivo differentiation in committed muscle cells, we grafted wild type and decorin-null myoblasts onto chick limb buds. The absence of decorin enhanced the migration and distribution of myoblasts in the limb, correlating with the inhibition of skeletal muscle differentiation. Both phenotypes were reverted by de novo decorin expression. In vitro, we determined that both decorin core protein and its glycosaminoglycan chain were required to reverse the migration phenotype. Results presented here suggest that the enhanced migration observed in decorin-null myoblasts may not be dependent on chemotactic growth factor signaling nor the differentiation status of the cells. Decorin may be involved in the establishment and/or coordination of a critical myoblast density, through inhibition of migration, that permits normal muscle differentiation during embryonic myogenesis.

Skeletal muscle regeneration is a complex process in which many agents are involved. When skeletal muscle suffers an injury, quiescent resident myoblasts called satellite cells are activated to proliferate, migrate, and finally differentiate. This whole process occurs in the presence of growth factors, the extracellular matrix (ECM), and infiltrating macrophages. We have shown previously that different proteoglycans, either present at the plasma membrane or the ECM, are involved in the differentiation process by regulating growth factor activity. In this article, we evaluated the role of glycosaminoglycans (GAGs) in myoblast proliferation and migration, using C2C12, a satellite cell-derived cell line. A synergic stimulatory effect on myoblast proliferation was observed with hepatocyte growth factor (HGF) and fibroblast growth factor type 2 (FGF-2), which was dependent on cell sulfation. The GAG dermatan sulfate (DS) enhanced HGF/FGF-2-dependent proliferation at 1–10 ng/ml. However, decorin, a proteoglycan containing DS, was unable to reproduce this enhanced proliferative effect. On the other hand, HGF strongly increased myoblast migration. The HGF-dependent migratory process required the presence of sulfated proteoglycans/GAGs present on the myoblast surface, as inhibition of both cell sulfation, and heparitinase (Hase) and chondroitinase ABC (Chabc) treatment of myoblasts, resulted in a very strong inhibition of cell migration. Among the GAGs analyzed, DS most increased HGF-dependent myoblast migration. Taken together, these findings showed that DS is an enhancer of growth factor-dependent proliferation and migration, two critical processes involved in skeletal muscle formation. J. Cell. Physiol. 198: 169–178, 2004© 2003 Wiley-Liss, Inc.

Migration of myoblasts is an important component of the reparative response to muscle injury, and furthermore may be a key determinant of the success of myoblast transplantation for the treatment of genetic muscle diseases. The present study examined the hypothesis that K+ channels modulate myoblast migration. The migration of cultured L-6 myoblasts was assessed in vitro on confluent cultures with the razor wound method, in the absence and presence of the following agents: 3,4-diaminopyridine and tetraethylammonium (which block several types of K+ channels), apamin and charybdotoxin (which block Ca++-activated K+ channels), glibenclamide (which blocks ATP-sensitive K+ channels), and α-, β-, γ-, and δ-dendrotoxin (which block voltage-gated K+ channels). Migration was assessed with respect to number of migrated cells, average distance migrated, and total distance migrated. Overall, myoblast migration was stimulated in response to low concentrations of tetraethylammonium, apamin, glibenclamide, and α-, β- and δ-dendrotoxin. With these agents, the number of migrated cells increased by 28–47%, the average distance migrated increased by 22–35%, and the total distance migrated increased by 60–85%. Conversely, migration was inhibited by high concentrations of 3,4-diaminopyridine, tetraethylammonium, and all dendrotoxins. These data indicate that in L-6 myoblasts migration is regulated by K+ channels, and that several types of K+ channels appear to participate in cell migration.

Myoblast migration is essential for muscle development, regeneration, and repair. Various assays are available for the study of migration; however, many are not suitable for the analysis of myoblast migration during skeletal muscle wound repair. Furthermore, versatility (e.g., to investigate the effect of the extracellular matrix on cell movement) is limited within these methods. Here we report the optimization of the scratch assay specifically for two-dimensional myoblast migration and discuss the advantages of this assay in comparison with other commercially available methods.

Background Via the hepatocyte growth factor receptor (Met), hepatocyte growth factor (HGF) exerts key roles involving skeletal muscle development and regeneration. Heparan sulfate proteoglycans (HSPGs) are critical modulators of HGF activity, but the role of specific HSPGs in HGF regulation is poorly understood. Glypican-1 is the only HSPG expressed in myoblasts that localize in lipid raft membrane domains, controlling cell responses to extracellular stimuli. We determined if glypican-1 in these domains is necessary to stabilize the HGF-Met signaling complex and myoblast response to HGF. Methods C2C12 myoblasts and a derived clone (C6) with low glypican-1 expression were used as an experimental model. The activation of Met, ERK1/2 and AKT in response to HGF was evaluated. The distribution of Met and its activated form in lipid raft domains, as well as its dependence on glypican-1, were characterized by sucrose density gradient fractionation in both cell types. Rescue experiments reexpressing glypican-1 or a chimeric glypican-1 fused to the transmembrane and cytoplasmic domains of mouse syndecan-1 or myoblast pretreatment with MβCD were conducted. In vitro and in vivo myoblast migration assays in response to HGF were also performed. Results Glypican-1 localization in membrane raft domains was required for a maximum cell response to HGF. It stabilized Met and HGF in lipid raft domains, forming a signaling complex where the active phospho-Met receptor was concentrated. Glypican-1 also stabilized CD44 in a HGF-dependent manner. In addition, glypican-1 was required for in vitro and in vivo HGF-dependent myoblast migration. Conclusions Glypican-1 is a regulator of HGF-dependent signaling via Met in lipid raft domains.

Recent reports revealed that myogenic progenitors, derived from either bone marrow or muscle can migrate into muscle tissue and participate in myofiber regeneration, when injected in the peripheral circulation. This observation might open a new strategy for the treatment of muscular dystrophies. The signals involved in myoblast recruitment from circulation are at present poorly understood. To investigate myoblast migration we used a transwell assay in which murine myoblasts and myogenic cell lines were seeded on microporous membrane covered by an endothelial monolayer and chemotactic factors were added in the lower chamber. We demonstrated that myoblasts are able to cross the endothelium and that this process can be modulated. In particular among tested factors, we observed a gradient of chemotactic activity as follows: HGF >> RANTES > PDGF-A > PDGF-B > FGF >> TNF-alpha > IFN-gamma > EGF. Endothelial and myoblast expression of Pax3 (a transcription factor expressed by embryonic migrating myogenic cells) and cytokine transcripts (TNF-alpha, IFN-gamma) was also monitored either at the basal level and after transmigration. We observed increased Pax3 expression after interaction of C2C12 myoblasts with endothelial cells. We consider that any new report elucidating the molecular signals involved in myoblast migration may be useful toward the development of systemic cellular-mediated gene therapy of muscle diseases.

Donor myoblast migration is a major limiting factor in the success of myoblast transfer therapy, a potential treatment for Duchenne muscular dystrophy. A possible strategy to promote the migration of donor myoblasts into host muscle is to enhance their proliferation and delay their fusion, two properties that are major characteristics of myoblasts in regenerating skeletal muscle in MyoD null (2/2) mice. Here we investigate whether the migration of MyoD (2/2) donor myoblasts into host muscle is enhanced in vivo. Sliced muscle grafts from male MyoD (2/2) or normal control (Balb/c) mice were transplanted into the muscles of female normal (Balb/c) host mice. Muscles were sampled at 1, 3, and 12 weeks after grafting, and the fate of male donor myoblasts within female host muscles determined by in situ hybridization with the mouse Ychromosome-specific Y-1 probe. MyoD (2/2) donor myoblasts migrated into host muscle continuously over 1, 3, and 12 weeks after grafting, in contrast with Balb/c donor myoblasts, whose overall numbers and migratory distances did not increase significantly after 1 week. These results strongly support a role for elevated donor myoblast proliferation and/or their delayed fusion in enhancing migration into host muscle in vivo, and endorse the use of either genetically engineered donor myoblasts, or the administration of exogenous myoblast mitogens to improve donor myoblast migration in myoblast transfer therapy. © 2001 Academic Press