Myoblast Motility Research Articles

The extracellular matrix differentially directs myoblast motility and differentiation in distinct forms of muscular dystrophy

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated “on-slide” decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.

Assessment of laser-synthesized Si nanoparticle effects on myoblast motility, proliferation and differentiation: towards potential tissue engineering applications.

Due to their biocompatibility and biodegradability and their unique structural and physicochemical properties, laser-synthesized silicon nanoparticles (Si-NPs) are one of the nanomaterials which have been most studied as potential theragnostic tools for non-invasive therapeutic modalities. However, their ability to modulate cell behavior and to promote proliferation and differentiation is still very little investigated or unknown. In this work, ultrapure ligand free Si-NPs of 50 ± 11.5 nm were prepared by femtosecond (fs) laser ablation in liquid. After showing the ability of Si-NPs to be internalized by murine C2C12 myoblasts, the cytotoxicity of the Si-NPs on these cells was evaluated at concentrations ranging from 14 to 224 μg mL-1. Based on these findings, three concentrations of 14, 28 and 56 μg mL-1 were thus considered to study the effect on myoblast differentiation, proliferation and motility at the molecular and phenotypical levels. It was demonstrated that up to 28 μg mL-1, the Si-NPs are able to promote the proliferation of myoblasts and their subsequent differentiation. Scratch tests were also performed revealing the positive Si-NP effect on cellular motility at 14 and 28 μg mL-1. Finally, gene expression analysis confirmed the ability of Si-NPs to promote proliferation, differentiation and motility of myoblasts even at very low concentration. This work opens up novel exciting prospects for Si-NPs made by the laser process as innovative tools for skeletal muscle tissue engineering in view of developing novel therapeutic protocols for regenerative medicine.

Role of H2 Calponin in Myoblast Differentiation, Fusion and Myogenesis

Actin cytoskeleton plays a pivotal role in myoblast motility, adhesion, fusion and differentiation into myotubes. However, how the actin cytoskeleton is regulated during myogenesis is largely unknown. H2-calponin is a regulatory protein that stabilizes the actin cytoskeleton and regulates cell motility and adhesion. In the present study, we investigated the role of h2 calponin in the regulation of myoblast migration, differentiation and fusion. The results showed that h2-calponin is expressed at significant levels in undifferentiated mouse primary satellite cells and C2C12 myoblasts. In migrating satellite cells and C2C12 myoblasts, h2-calponin co-localizes with actin stress fibers at both the leading edge and the tail regions. In newly fused myotubes derived from primary satellite cells, h2-calponin is mainly present at the ends of the myotubes. The level of h2-calponin significantly decreases during satellite cell and myoblast differentiation and fusion, and becomes undetectable in mature myocytes. Primary satellite cells isolated from h2-calponin gene knockout mice had significantly decreased cell spreading area indicating reduced substrate adhesion and increased rate of cell proliferation as compared with wild type controls. Biochemical and histological studies revealed that skeletal muscles of h2-calponin knockout mice retain normal muscle weight and myofilament protein compositions. Myotubes derived from h2-calponin-null satellite cells showed apparently normal sarcomere striations and expression of myofilament proteins. Therefore, deletion of h2-calponin does not abolish the maximum capacity of myoblast function and myogenesis. However, in vitro differentiation studies revealed that h2-calponin-hull myoblasts had higher efficiency of fusion, as indicated by increased fusion index, than wild type controls. The data suggest that the function of h2-calponin provides a novel mechanism in regulating actin cytoskeleton activity during myoblast differentiation, fusion and myogenesis.

C-MET Regulates Myoblast Motility and Myocyte Fusion during Adult Skeletal Muscle Regeneration

Adult muscle stem cells, satellite cells (SCs), endow skeletal muscle with tremendous regenerative capacity. Upon injury, SCs activate, proliferate, and migrate as myoblasts to the injury site where they become myocytes that fuse to form new muscle. How migration is regulated, though, remains largely unknown. Additionally, how migration and fusion, which both require dynamic rearrangement of the cytoskeleton, might be related is not well understood. c-MET, a receptor tyrosine kinase, is required for myogenic precursor cell migration into the limb for muscle development during embryogenesis. Using a genetic system to eliminate c-MET function specifically in adult mouse SCs, we found that c-MET was required for muscle regeneration in response to acute muscle injury. c-MET mutant myoblasts were defective in lamellipodia formation, had shorter ranges of migration, and migrated slower compared to control myoblasts. Surprisingly, c-MET was also required for efficient myocyte fusion, implicating c-MET in dual functions of regulating myoblast migration and myocyte fusion.

In vitro myoblast motility models: investigating migration dynamics for the study of skeletal muscle repair

Skeletal muscle repair requires the migration of myoblasts (activated satellite cells) both to the injury site and then within the wound to facilitate cellular alignment in preparation for differentiation, fusion and eventual healing. Along this journey, the cells encounter a range of soluble and extracellular matrix factors which regulate their movement and ultimately determine how successful the repair process will be. Sub-optimal migration can lead to a number of scenarios, including reduced myoblast numbers entering the wound, poor alignment and insufficient differentiation to correctly repair the damage. It is therefore critical that all aspects of myoblast migration are understood, particularly in response to the changing growth and matrix factor profile prevalent following skeletal muscle injury. Since 1962, when Boyden first introduced his chemotactic chamber, numerous in vitro migration assays have been developed to mimic the wound more closely. These have increased in complexity to account for the complex micro-environment found in vivo during muscle repair and include a range of modified cell exclusion, chemotactic and three-dimensional assays. This review describes and discusses these advances and highlights the importance they have in expanding our understanding of myoblast migration dynamics.

ELF-MF transiently increases skeletal myoblast migration: Possible role of calpain system

Purpose: Cell migration is crucial for myogenesis since it is required for the alignment and fusion of myoblast. Ca2+ signals are involved in regulating myoblast migration and an extremely low frequency (ELF) magnetic field (MF) increases intracellular calcium levels in C2C12 myoblast. This study was aimed at investigating whether ELF-MF could affect myoblast migration. As calpains contribute to the regulation of myoblast motility, the effect of ELF-MF on μ- and m-calpain was also investigated.Materials and methods: The effect of ELF-MF (1 mT; 50 Hz) on C2C12 cell motility was observed by wound-healing assay. Protein expression of calpains, calpastatin, myristoylated alanine-rich C-kinase substrate (MARCKS) and vinculin were examined by Western blot analysis. Casein zymography and immunofluorescence analysis were carried out to evaluate, respectively, activity levels of calpains and intracellular distribution of calpains, calpastatin and actin.Results: Exposure to ELF-MF resulted in a transient but significant increase of myoblast migration. This stimulatory effect was associated with a marked increase of μ- and m-calpain activity followed by the concomitant variation in their subcellular localization. No significant changes in intracellular distribution and protein levels of calpastatin were detected. Finally, a significant decrease of MARCKS expression and modifications of actin dynamics were reported.Conclusions: This study clearly outlines an involvement of calpains in ELF-MF-mediated myoblast migration.

Defining Akt actions in muscle differentiation

Muscle development in childhood and muscle regeneration in adults are highly regulated processes that are necessary for reaching and maintaining optimal muscle mass and strength throughout life. Muscle repair after injury relies on stem cells, termed satellite cells, whose activity is controlled by complex signals mediated by cell-cell contact, by growth factors, and by hormones, which interact with genetic programs controlled by myogenic transcription factors. Insulin-like growth factors (IGFs) play key roles in muscle development and help coordinate muscle repair after injury, primarily by stimulating the phosphatidylinositol 3-kinase-Akt signaling pathway, and both in vitro and in vivo studies have shown that Akt kinase activity is critical for optimal muscle growth and regeneration. Here we find that of the two Akts expressed in muscle, Akt1 is essential for initiation of differentiation in culture and is required for normal myoblast motility, while Akt2 is dispensable. Although Akt2 deficiency did lead to diminished myotube maturation, as assessed by a decline in myofiber area and in fusion index, either Akt1 or Akt2 could restore these processes toward normal. Thus levels of Akt expression rather than distinct actions of individual Akt species are critical for normal myofiber development during the later stages of muscle differentiation.

The actin regulator N-WASp is required for muscle-cell fusion in mice

A fundamental aspect of skeletal myogenesis involves extensive rounds of cell fusion, in which individual myoblasts are incorporated into growing muscle fibers. Here we demonstrate that N-WASp, a ubiquitous nucleation-promoting factor of branched microfilament arrays, is an essential contributor to skeletal muscle-cell fusion in developing mouse embryos. Analysis both in vivo and in primary satellite-cell cultures, shows that disruption of N-WASp function does not interfere with the program of skeletal myogenic differentiation, and does not affect myoblast motility, morphogenesis and attachment capacity. N-WASp-deficient myoblasts, however, fail to fuse. Furthermore, our analysis suggests that myoblast fusion requires N-WASp activity in both partners of a fusing myoblast pair. These findings reveal a specific role for N-WASp during mammalian myogenesis. WASp-family elements appear therefore to act as universal mediators of the myogenic cell-cell fusion mechanism underlying formation of functional muscle fibers, in both vertebrate and invertebrate species.

Examination of FGFRL1 as a candidate gene for diaphragmatic defects at chromosome 4p16.3 shows that Fgfrl1 null mice have reduced expression of Tpm3, sarcomere genes and Lrtm1 in the diaphragm

Fgfrl1 (also known as Fgfr5; OMIM 605830) homozygous null mice have thin, amuscular diaphragms and die at birth because of diaphragm hypoplasia. FGFRL1 is located at 4p16.3, and this chromosome region can be deleted in patients with congenital diaphragmatic hernia (CDH). We examined FGFRL1 as a candidate gene for the diaphragmatic defects associated with 4p16.3 deletions and re-sequenced this gene in 54 patients with CDH. We confirmed six known coding single nucleotide polymorphisms (SNPs): c.209G > A (p.Pro20Pro), c.977G > A (p.Pro276Pro), c.1040T > C (p.Asp297Asp), c.1234C > A (p.Pro362Gln), c.1420G > T (p.Arg424Leu), and c.1540C > T (p.Pro464Leu), but we did not identify any gene mutations. We genotyped additional CDH patients for four of these six SNPs, including the three non-synonymous SNPs, to make a total of 200 chromosomes, and found that the allele frequency for the four SNPs, did not differ significantly between patients and normal controls (p > or = 0.05). We then used Affymetrix Genechip Mouse Gene 1.0 ST arrays and found eight genes with significantly reduced expression levels in the diaphragms of Fgfrl1 homozygous null mice when compared with wildtype mice-Tpm3, Fgfrl1 (p = 0.004), Myl2, Lrtm1, Myh4, Myl3, Myh7 and Hephl1. Lrtm1 is closely related to Slit3, a protein associated with herniation of the central tendon of the diaphragm in mice. The Slit proteins are known to regulate axon branching and cell migration, and inhibition of Slit3 reduces cell motility and decreases the expression of Rac and Cdc42, two genes that are essential for myoblast fusion. Further studies to determine if Lrtm1 has a similar function to Slit3 and if reduced Fgfrl1 expression can cause diaphragm hypoplasia through a mechanism involving decreased myoblast motility and/or myoblast fusion, seem indicated.

Regulation of Myoblast Motility and Fusion by the CXCR4-associated Sialomucin, CD164

Myoblast fusion is fundamental to the development and regeneration of skeletal muscle. To fuse, myoblasts undergo cell-cell recognition and adhesion and merger of membranes between apposing cells. Cell migration must occur in advance of these events to bring myoblasts into proximity, but the factors that regulate myoblast motility are not fully understood. CD164 is a cell surface sialomucin that is targeted to endosomes and lysosomes via its intracellular region. In hematopoietic progenitor cells, CD164 forms complexes with the motility-stimulating chemokine receptor, CXCR4, in response to the CXCR4 ligand, CXCL12/SDF-1 (Forde, S., Tye, B. J., Newey, S. E., Roubelakis, M., Smythe, J., McGuckin, C. P., Pettengell, R., and Watt, S. M. (2007) Blood 109, 1825-1833). We have previously shown that CD164 stimulates myotube formation in vitro. We report here that CD164 is associated with CXCR4 in C2C12 myoblasts. Cells in which CD164 levels are increased or decreased via overexpression or RNA interference-mediated knockdown, respectively, show enhanced or reduced myotube formation and cell migration, the latter both basally and in response to CXCL12/SDF-1. Furthermore, expression of CD164 cytoplasmic tail mutants that alter the endosome/lysosome targeting sequence and, consequently, the subcellular localization in myoblasts, reveals a similar correlation between cell motility and myotube formation. Finally, Cd164 mRNA is expressed in the dorsal somite (the early myogenic compartment of the mouse embryo) and in premuscle masses. Taken together, these results suggest that CD164 is a regulator of myoblast motility and that this property contributes to its ability to promote myoblast fusion into myotubes.

Mannose receptor regulation of macrophage cell migration

The migration of macrophages through peripheral tissues is an essential step in the host response to infection, inflammation, and ischemia as well as in tumor progression and tissue repair. The mannose receptor (MR; CD206, previously known as the macrophage MR) is a 175-kDa type I transmembrane glycoprotein and is a member of a family of four recycling endocytic receptors, which share a common extracellular domain structure but distinct ligand-binding properties and cell type expression patterns. MR has been shown to bind and internalize carbohydrate and collagen ligands and more recently, to have a role in myoblast motility and muscle growth. Given that the related Endo180 (CD280) receptor has also been shown to have a promigratory role, we hypothesized that MR may be involved in regulating macrophage migration and/or chemotaxis. Contrary to expectation, bone marrow-derived macrophages (BMM) from MR-deficient mice showed an increase in random cell migration and no impairment in chemotactic response to a gradient of CSF-1. To investigate whether the related promigratory Endo180 receptor might compensate for lack of MR, mice with homozygous deletions in MR and Endo180 were generated. These animals showed no obvious phenotypic abnormality, and their BMM, like those from MR-deficient mice, retained an enhanced migratory behavior. As MR is down-regulated during macrophage activation, these findings have implications for the regulation of macrophage migration during different stages of pathogenesis.

Mannose receptor regulates myoblast motility and muscle growth

Myoblast fusion is critical for the formation, growth, and maintenance of skeletal muscle. The initial formation of nascent myotubes requires myoblast–myoblast fusion, but further growth involves myoblast–myotube fusion. We demonstrate that the mannose receptor (MR), a type I transmembrane protein, is required for myoblast–myotube fusion. Mannose receptor (MR)–null myotubes were small in size and contained a decreased myonuclear number both in vitro and in vivo. We hypothesized that this defect may arise from a possible role of MR in cell migration. Time-lapse microscopy revealed that MR-null myoblasts migrated with decreased velocity during myotube growth and were unable to migrate in a directed manner up a chemoattractant gradient. Furthermore, collagen uptake was impaired in MR-null myoblasts, suggesting a role in extracellular matrix remodeling during cell motility. These data identify a novel function for MR during skeletal muscle growth and suggest that myoblast motility may be a key aspect of regulating myotube growth.

Involvement of Ras and Ral in Chemotactic Migration of Skeletal Myoblasts

In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca(2+)-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors.

Hepatocyte growth factor stimulates chemotactic response in mouse embryonic limb myogenic cells in vitro.

In this study we investigate the influence of Hepatocyte Growth Factor (HGF) on the motility of embryonic forelimb myoblasts. Using Blindwell chemotactic chambers, it was found that HGF at concentrations of 1-50 ng/ml dramatically enhanced the ability of myogenic cells to migrate. This stimulatory effect was elicited in a dose-dependent fashion and the effect was reversed with the addition of HGF neutralizing antibodies. A checkerboard analysis was performed and it revealed that HGF's effect on limb myoblast motility was through both chemokinesis and chemotaxis. HGF was also examined for its ability to stimulate myogenic cell proliferation, using MF20 antibody as the myogenic marker. At all concentrations tested, HGF did not stimulate an overall increase in the numbers of MF20-positive myoblasts in culture. To examine the chemokinetic effect of HGF on cell migration in the limb, cells were isolated from the proximal regions of the limb (areas rich in myogenic cells), exposed to HGF, labeled with DiI and transplanted into 11.5 day mouse forelimbs. After 36 h of culture, it was found that DiI-labeled limb cells, pretreated with HGF, migrated significantly further in the limb than labeled cells that have not been exposed to HGF. The chemotactic effect of HGF was also investigated by implanting beads loaded with and without HGF into the 11.5 day limb. Proximal to the beads, DiI-labeled limb cells were also transplanted. It was found that HGF was able to chemotactically attract and direct the migration of DiI-labeled limb cells. Immunohistological staining was performed with HGF antibodies to determine the distribution of HGF in the 11.5 day mouse forelimb. It was found that HGF was strongly expressed by the apical ectodermal ridge (AER), the ectoderm and the mesenchyme directly beneath the AER. Positive staining was also obtained for the myogenic regions. However, the pattern was heterogeneous--punctuated with myogenic cells expressing and not expressing HGF.