Phospholipase D1 facilitates second-phase myoblast fusion and skeletal muscle regeneration

Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes.     

Versican Processing by a Disintegrin-like and Metalloproteinase Domain with Thrombospondin-1 Repeats Proteinases-5 and -15 Facilitates Myoblast Fusion

Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.     

A 5-Bromodeoxyuridine-sensitive Interval during Drosophila Myogenesis

Drosophila myogenesis was monitored in vitro and the cells were treated with 5-bromodeoxyuridine (BrdU) or with thymidine at certain intervals. Muscle cells were scored for survival, contractility, and the uptake of thymidine and BrdU. Results indicated that the final S period for myoblasts takes place in vitro between 1.3 and 3.3 h following the initiation of gastrulation in the donor embryos. Treatment with 10^-4 M BrdU during this interval inhibited myogenesis, but later treatment did not. Thymidine reversed or prevented the BrdU effect if given before the final myoblast division, but not if given afterwards. All results support the hypothesis that BrdU inhibits Drosophila myogenesis through its incorporation into DNA.     

Differentiation in cultures derived from embryonic chicken muscle: II. Phosphorylase histochemistry and fluorescent antibody staining for creatine kinase and aldolase

The presence or absence of five proteins (glycogen phosphorylase, aldolase A, aldolase C, creatine kinase M, creatine kinase B) in the various classes of cells found in primary cultures derived from embryonic chick breast muscle was investigated using cytological staining methods. Histochemical staining for phosphorylase and indirect fluorescent antibody staining for aldolase A and C as well as for creatine kinases M and B showed the following: All five proteins were found in the many myotubes present in standard medium cultures and in the very few myotubes found in cultures containing 5-bromodeoxyuridine (10^?5M). The elongated bipolar cells prevented from fusing in medium containing EGTA also contain all five proteins. The flattened myogenic cells that predominate in the 5-bromodeoxyuridine-treated cultures contain no phosphorylase or creatine kinase M, though many of them contain creatine kinase B and aldolases A and C. These results are interpreted as indicating that: (1) phosphorylase and creatine kinase M, but not aldolase A, are suitable all-or-none markers for terminal muscle differentiation; (2) the small amounts of creatine kinase M detected in electrophoreses of 5-bromodeoxyruridine-treated cultures can be accounted for by the few myotubes present and are not due to “protodifferentiation” of large numbers of cells; (3) proteins typical of differentiated muscle are produced only in cells that have passed through the last step in myogenesis that is susceptible to 5-bromodeoxyuridine inhibition, and (4) if fusion is blocked by reducing the concentration of calcium ions, accumulation of characteristic muscle proteins can continue in those cells that have initiated terminal differentiation.     

The role of myonuclei in muscle regeneration: An in vitro study

It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell division to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or “freed” as a result of the trauma, play any role in muscle regeneration remains unresolved. In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub-cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H³-thymidine and their ultimate fate, either as “free” myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub-cultured. In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re-enter the mitotic cycle.     

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti-genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.     

M-cadherin activates Rac1 GTPase through the Rho-GEF trio during myoblast fusion

Cadherins are transmembrane glycoproteins that mediate Ca²⁺-dependent homophilic cell–cell adhesion and play crucial role during skeletal myogenesis. M-cadherin is required for myoblast fusion into myotubes, but its mechanisms of action remain unknown. The goal of this study was to cast some light on the nature of the M-cadherin–mediated signals involved in myoblast fusion into myotubes. We found that the Rac1 GTPase activity is increased at the time of myoblast fusion and it is required for this process. Moreover, we showed that M-cadherin–dependent adhesion activates Rac1 and demonstrated the formation of a multiproteic complex containing M-cadherin, the Rho-GEF Trio, and Rac1 at the onset of myoblast fusion. Interestingly, Trio knockdown efficiently blocked both the increase in Rac1-GTP levels, observed after M-cadherin–dependent contact formation, and myoblast fusion. We conclude that M-cadherin–dependent adhesion can activate Rac1 via the Rho-GEF Trio at the time of myoblast fusion.     

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.     

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.     

Differentiation-dependent regulation of skeletal myogenesis by neuregulin-1

Neuregulins comprise a group of growth factor proteins that regulate the differentiation of skeletal muscle. Here, we report that neuregulins are regulators of myogenic differentiation and stimulate mitogenesis in L6 skeletal myoblasts. The mitogenic response to neuregulin-1 was differentiation-dependent and observed only in aligned, differentiating cells. Treatment of these cells with neuregulin-1 increased [3H]thymidine incorporation and cell proliferation by 2- to 5-fold, while a minimal increase was seen in proliferating myoblasts. Neuregulin-1 did not induce DNA synthesis in fused, multinucleated myotubes. The increased DNA synthesis correlated with downregulation of myogenin and inhibition of myoblast fusion and myotube formation. These data suggest that neuregulins may regulate skeletal myogenesis in vivo and that this regulation is dependent on the state of differentiation of the myocytes.     

Expression of a developmentally regulated antigen on the surface of skeletal and cardiac muscle cells

H36 is a species-specific, cell-surface antigen on differentiating newborn rat skeletal myoblasts and myogenic lines. This membrane antigen has been defined by a monoclonal antibody raised by the fusion of SP 2/0-Ag14 myeloma cells with spleen cells from mice immunized with myotubes derived from the myogenic E63 line. H36 antigen, isolated by immunoaffinity chromatography, is comprised of two polypeptides with apparent molecular weights of 98,000 and 117,000. Fluorescence photometry and radioimmunoassays have been used to follow quantitative and topographic changes in the H36 determinant during myogenesis. H36 is present at a basal level on replicating myoblasts; it increases on prefusion myoblasts and persists on myotubes. At or near the time of prefusion, it becomes concentrated between adjacent aligned myoblasts and localized on membrane "blebs". H36 is present on both skeletal and cardiac cells but absent from a variety of cells that include fibroblasts, neuronal cells, and smooth muscle. There are approximately 4 x 10(5) determinants per myoblast, and the Ka of the antibody is 3.8 x 10(8) liters/mol. The distributions of H36 on the top and attached surfaces of myoblasts and myotubes are distinct, which suggests localized specialization of these surfaces. H36 is an integral membrane component and upon cross-linking, it associates with the detergent-insoluble cytoskeletal framework. Inhibition of myogenesis by 5-bromodeoxyuridine or by calcium deprivation prevents the developmentally associated changes in the expression of H36. H36 is also absent or markedly reduced on the fu- and Ama102 developmentally defective mutant myoblast lines. We conclude that H36 is a muscle-specific, developmentally regulated cell-surface antigen that may have a role in myoblast differentiation and that can be used to determine the embryonic lineages of skeletal and cardiac muscle.     

MITOSIS AND THE PROCESSES OF DIFFERENTIATION OF MYOGENIC CELLS IN VITRO

The relation between the mitotic cycle and myoblast fusion has been studied in chick skeletal muscle in vitro. The duration of the cell cycle phases was the same in both early and late cultures. By tracing a cohort of pulse-labeled cells, it was found that myoblast fusion does not occur in S, G₂, or M. Cell surface alterations required for fusion are dependent upon the position of the cell in the division cycle. In early cultures, fusion takes place only after a minimum delay of 5 hr from the time the cell has entered G₁. The mitosis preceding fusion may condition the cell for the abrupt shift in synthetic activity that occurs in the subsequent G₁. In older cultures fusion of labeled cells is diminished. Two factors account for the cessation of fusion in older cultures. First, the number of myogenic stem cells declines, but these cells do not disappear as the cultures mature. Their persistence was demonstrated by labeling dividing mononucleated cells in older cultures and challenging them with nascent myotubes. Some of these labeled cells were incorporated into the forming myotubes. Second, a block to fusion develops during myotube maturation. Well developed myotubes challenged with labeled competent myogenic cells failed to incorporate the labeled nuclei.     

Dual Roles of Palladin Protein in In Vitro Myogenesis: Inhibition of Early Induction but Promotion of Myotube Maturation

Palladin is a microfilament-associated phosphoprotein whose function in skeletal muscle has rarely been studied. Therefore, we investigate whether myogenesis is influenced by the depletion of palladin expression known to interfere with the actin cytoskeleton dynamic required for skeletal muscle differentiation. The inhibition of palladin in C2C12 myoblasts leads to precocious myogenic differentiation with a concomitant reduction in cell apoptosis. This premature myogenesis is caused, in part, by an accelerated induction of p21, myogenin, and myosin heavy chain, suggesting that palladin acts as a negative regulator in early differentiation phases. Paradoxically, palladin-knockdown myoblasts are unable to differentiate terminally, despite their ability to perform some initial steps of differentiation. Cells with attenuated palladin expression form thinner myotubes with fewer myonuclei compared to those of the control. It is noteworthy that a negative regulator of myogenesis, myostatin, is activated in palladin-deficient myotubes, suggesting the palladin-mediated impairment of late-stage myogenesis. Additionally, overexpression of 140-kDa palladin inhibits myoblast differentiation while 200-kDa and 90-kDa palladin-overexpressed cells display an enhanced differentiation rate. Together, our data suggest that palladin might have both positive and negative roles in maintaining the proper skeletal myogenic differentiation in vitro.     

Muscle differentiation: how two cells become one

A key feature of myogenesis is the fusion of myoblasts to form multinucleate myotubes. Recent work in Drosophila has uncovered a collection of genes that operate at different stages of this process. Some interactions between them have been described that begin to define links from outside the cell via the plasma membrane to the cytoskeleton. Future studies will establish the extent to which the molecular mechanisms of myoblast fusion are conserved between Drosophila and other animals, as found in other aspects of myogenesis.