Transitions towards either slow-oxidative or fast-glycolytic phenotype can be induced in the murine WTt myogenic cell line.

Contraction and energy metabolism are functions of skeletal muscles co-regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow-oxidative or fast-glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF-1alpha and PPARdelta. Our data support the idea that HIF-1alpha protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARdelta over-expression appears to enhance the slow-oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC-1alpha, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF-1alpha protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF-1alpha and PPARdelta. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF-1alpha and PPARdelta in the coordinated metabolic and contractile regulations involved in myogenesis.

Immunoneutralization of TGFbeta1 Improves Skeletal Muscle Regeneration: Effects on Myoblast Differentiation and Glycosaminoglycan Content.

When injured by crushing, the repair of the slow-twitch soleus rat muscle, unlike the fast-twitch EDL, is associated with fibrosis. As TGFbeta1, whose activity can be controlled by glycosaminoglycans (GAG), plays a major role in fibrosis, we hypothesized that levels of TGFbeta1 and GAG contents could account for this differential quality of regeneration. Here we show that the regeneration of the soleus was accompanied by elevated and more sustained TGFbeta1 level than in the EDL. Neutralization of TGFbeta1 effects by antibodies to TGFbeta1 or its receptor TGFbeta-R1 improved muscle repair, especially of the soleus muscle, increased in vitro growth of myoblasts, and accelerated their differentiation. These processes were accompanied by alterations of GAG contents. These results indicate that the control of TGFbeta1 activity is important to improve regeneration of injured muscle and accelerate myoblast differentiation, in part through changes in GAG composition of muscle cell environment.

Sustained peripheral arterial insufficiency durably impairs normal and regenerating skeletal muscle function.

Peripheral vascular occlusive diseases are frequently observed in humans, and studies with animal models have been largely used. However the effects of sustained lower limb ischemia on normal and regenerating hindlimb skeletal muscles are not well known in the mouse model. Therefore prolonged unilateral hindlimb ligation was generated by femoral artery ligation. Normal (myotoxic-untreated) and regenerating (myotoxic-reated) ischemic muscles were studied by analyses of the in situ contractile properties and histological parameters. Concerning normal mouse muscles, we found that femoral artery ligation reduced hindlimb perfusion and altered muscle structure and function. Thus 7 days after ligation, maximal tetanic force was reduced by about 70%, (p < 0.05). By 56 days after ligation, muscle weights and cross-section areas of muscle fibers were still reduced (p < 0.05). Concerning myotoxic treated muscles, we report that ligation reduced the recovery of muscle weight and maximal tetanic force and increased fatigue resistance at 56 days (p < 0.05). In conclusion, our results demonstrate that sustained peripheral arterial insufficiency in mice induces long-term as well as acute detrimental effects in both normal and regenerating muscles.

Differential effects of post-natal development, animal strain and long term recovery on the restoration of neuromuscular function after neuromyotoxic injury in rat.

We have analysed the effect of long term recovery, post-natal development and animal strain on the extent of restoration of neuromuscular function after neuromyotoxic injury in the rat (Rattus norvegicus). Muscle isometric contractile properties of soleus muscle in response to nerve stimulation were measured in situ in snake venom injured muscles and compared to contralateral uninjured muscles. We show here that neuromuscular function was not fully recovered until 24 weeks after injury in young adult (2-3 month old) Wistar rats. Moreover, the level of functional recovery 3 weeks after injury induced in juvenile rats (1 month old) was not globally different from that in younger adult, adult (10 month old) and older adult (24 month old) Wistar rats. Furthermore, the level of recovery of some contractile parameters differed between Wistar and Sprague-Dawley strains 3 weeks after injury. In conclusion, a very long time (>12 weeks) is required for full neuromuscular recovery following neuromyotoxic injury of young adult rats. Moreover, neuromuscular recovery during post-natal development is not markedly different from that during adult stage in the Wistar rat strain. Finally, some rat strain differences are observed in the recovery after injury of young adult rats.

Functional, cellular and molecular aspects of skeletal muscle recovery after injury induced by snake venom from Notechis scutatus scutatus.

We have analysed the rate and ultimate extent of muscle functional recovery after snake venom-induced myotoxicity, as well as the relationships between functional, biochemical and structural indices of recovery. We also compared the effects of various injuries leading to muscle necrosis, loss of innervation/vasculature and/or precursors of muscle cells (pmc). We found that several parameters of rat soleus muscle such as maximal isometric force, slow myosin heavy chain, and citrate synthase, were fully and rapidly restored within 6 weeks after treatment with snake Notechis scutatus venom (im, 2 microg/muscle). In contrast, some muscle contractile properties (degree of tetanic fusion, fatigue resistance...) were not fully recovered even by 12 weeks after venom treatment. However, when compared to other injuries, recovery 3 weeks after venom treatment, was better than that observed after severing the terminal nerve and accompanying vessels and after cryodamage known to kill pmc. In conclusion, our studies demonstrate that-contrary to what is commonly believed -- muscle treated by myotoxic agent does not recover rapidly and fully. However, the degree or rate of muscle recovery after snake venom treatment was much better when compared to other types of injury. In addition, histological and biochemical parameters cannot be used as such to easily predict functional recovery following injury.

Effect of anti-inflammatory and antioxidant drugs on the long-term repair of severely injured mouse skeletal muscle.

Non-steroidal anti-inflammatory drugs are frequently prescribed after skeletal muscle injury. It is not known whether this type of medication can interfere with muscle repair, although inflammatory response is thought to play an important role in this process. Tibialis anterior muscles of mice were injured by myotoxic agent (snake venom) or crushed. Then, animals were treated daily for 10-14 days with different types of non-steroidal anti-inflammatory and antioxidant drugs. The long-term repair was studied 10-42 days after injury by analysing the recovery of in situ muscle force production, size of regenerating muscle cells and expression of myosin heavy chain. Our results show that diclofenac, diferuloylmethane (curcumin), dimethylthiourea or pyrrolidine dithiocarbamate treatment did not significantly affect muscle recovery after myotoxic injury (P > 0.05). Similarly, diferuloylmethane, dimethyl sulphoxide or indomethacin administration did not markedly change muscle repair after crush injury. However, we noted that high doses (> 2 mg kg(-1)) of diferuloylmethane or indomethacin increased lethality and reduced muscle repair after crush injury. In conclusion, non-steroidal anti-inflammatory and antioxidant drugs did not exhibit long-term detrimental effects on muscle recovery after injury, except at lethal doses.

Differential changes in protein kinase C associated with regeneration of rat extensor digitorum longus and soleus muscles.

We used a model of crush-induced regeneration in rat in order to characterize biochemically and histologically the implication of protein kinase C (PKC) in muscle repair after damage. In this model, slow soleus and fast extensor digitorum longus (EDL) muscle regeneration proceed differently. PKC activity has been assayed in regenerating muscles and their intact contralateral during the first 14 days following crushing. Degeneration (myolysis) occurring shortly after crush was associated with a marked down-regulation of the enzyme in both wound muscles and notable increase in the corresponding contralateral muscles. Muscle fiber reconstruction in EDL was associated with a rise in PKC activity which peaked at day 7 in regenerating muscle where it was twice higher than in intact muscle. At variance, muscle PKC activity in soleus increased slower than that of EDL and reached later intact level. Western blot analysis and immunohistochemical studies of representative members of the three PKC subfamilies were performed. All the isoform tested were much less expressed in regenerating than in control intact muscles suggesting that the overall PKC activity in regenerating muscles was more activable than in controls. We have shown that PKC isoforms were sequentially expressed during regeneration in both muscle types. PKC theta; being present the earliest, then delta, epsilon and alpha and finally zeta, beta and eta. Some isoforms were differentially expressed according muscle type. PKC delta being more expressed in soleus whereas beta and eta appeared earlier in EDL. Histochemical studies have revealed that the isoforms were differently localized in muscle tissue and that fiber regeneration was associated with PKC alpha translocation from sarcoplasma to sarcolemma. Together these data have shown that multiple PKC isoforms are implicated in the regenerative process acting at different in times and location and suggesting that individual isoform may fulfill distinct functions.

Heparan sulfate mimetics modulate calpain activity during rat Soleus muscle regeneration.

Skeletal muscle regenerates after injury. Tissue remodelling, which takes place during muscle regeneration, is a complex process involving proteolytic enzymes. It is inferred that micro and milli calpains are involved in the protein turnover and structural adaptation associated with muscle myolysis and reconstruction. Using a whole-crush injured skeletal muscle, we previously have shown that in vivo muscle treatment with synthetic heparan sulfate mimetics, called RGTAs (for ReGeneraTing Agents), greatly accelerates and improves muscle regeneration after crushing. This effect was particularly striking in the case of the slow muscle Soleus that otherwise would be atrophied. Therefore, we used this regeneration model to study milli and micro calpain expressions in the regenerating Soleus muscle and to address the question of a possible effect of RGTAs treatment on calpain levels. Micro and milli calpain contents increased by about five times to culminate at days 7 and 14 after crushing respectively, thus during the phases of fibre reconstruction and reinnervation. After 64 days of regeneration, muscles still displayed higher levels of both calpains than an intact uninjured muscle. Milli calpain detected by immunocytochemistry was shown in the cytoplasm whereas micro calpain was in both nuclei and cytoplasm in small myofibres but appeared almost exclusively in nuclei of more mature fibres. Interestingly, the treatment of muscles with RGTA highly reduced the increase of both milli and micro calpain contents in Soleus regenerating muscles. These results suggest that the improvement of muscle regeneration induced by RGTA may be partly mediated by minimising the consequences of calpain activity.

Differential expression of FGF receptors and of myogenic regulatory factors in primary cultures of satellite cells originating from fast (EDL) and slow (Soleus) twitch rat muscles.

In the rat, the fast and slow twitch muscles respectively Extensor digitorum longus (EDL) and Soleus present differential characteristics during regeneration. This suggests that their satellite cells responsible for muscle growth and repair represent distinct cellular populations. We have previously shown that satellite cells dissociated from Soleus and grown in vitro proliferate more readily than those isolated from EDL muscle. Fibroblast growth factors (FGFs) are known as regulators of myoblast proliferation and several studies have revealed a relationship between the response of myoblasts to FGF and the expression of myogenic regulatory factors (MRF) of the MyoD family by myoblasts. Therefore, we presently examined the possibility that the satellite cells isolated from EDL and Soleus muscles differ in the expression of FGF receptors (FGF-R) and of MRF expression. FGF-R1 and -R4 were strongly expressed in proliferating cultures whereas FGF-R2 and R3 were not detected in these cultures. In differentiating cultures, only -R1 was present in EDL satellite cells while FGF-R4 was also still expressed in Soleus cells. Interestingly, the unconventional receptor for FGF called cystein rich FGF receptor (CFR), of yet unknown function, was mainly detected in EDL satellite cell cultures. Soleus and EDL satellite cell cultures also differed in the expression MRFs. These results are consistent with the notion that satellite cells from fast and slow twitch muscles belong to different types of myogenic cells and suggest that satellite cells might play distinct roles in the formation and diversification of fast and slow fibres.

Studies on calpain expression during differentiation of rat satellite cells in primary cultures in the presence of heparin or a mimic compound.

We have synthesized dextran derivatives called RGTAs (for regenerating agents) that were designed to mimic some of the properties of heparin or heparan sulfate to interact with and protect heparin binding growth factors. Some of these growth factors have been described to be involved in myogenesis control. In previous studies, we have shown that muscle regeneration in adults could be greatly enhanced in vivo by treatment with RGTA. Since muscle regeneration occurs through the activation of satellite cells, in the present study we have used primary cultures of rat satellite cells and treated them with the heparan sulfate analogue RGTA or heparin in order to stimulate their growth and differentiation. We also studied the effect of these substances on calpain (calcium-activated neutral proteases) expression in these cultures. Indeed, several reports, principally based on fetal myoblast cultures or myogenic cell lines, have suggested that calpains might be involved in myoblast fusion during myogenic differentiation. We therefore studied the expression of microcalpain (mu-calpain), millicalpain (m-calpain), and calpain 3 in the course of differentiation of these satellite cell cultures in the absence or in the presence of heparin or of a mimic compound (the RGTA RG1282). RGTA and heparin were shown to have a dual effect on satellite cell proliferation and differentiation: RGTA stimulated proliferation with a maximum dose effect at 1 microgam/ml. Heparin used at concentrations similar to those of RGTA was less efficient at stimulating proliferation. Both substances were shown, however, to induce precocious and enhanced differentiation of satellite cells. We showed by quantitative RT-PCR analysis that mu-calpain, m-calpain, and calpain 3 mRNAs were expressed in satellite cell cultures in proliferating myoblasts (day 3) and differentiating cultures (days 7 and 12). The level of mu-calpain mRNA was increased by a factor of 3 during differentiation of satellite cells, whereas the level of m-calpain mRNAs was slightly increased at day 12 only, and calpain 3 mRNA was slightly reduced in these differentiating cultures. Interestingly enough, RGTA and heparin, which both strongly increased differentiation, reduced the expression of the mu- and m-calpains and slightly increased that of calpain 3 in differentiating cultures. These results showed that there was no correlation between the extent of myoblast differentiation and the level of calpain expression in satellite cells grown in primary cultures and underscored the differences between these adult cells and fetal myoblasts.

Dynamic distribution and formation of a para-sarcomeric banding pattern of prosomes during myogenic differentiation of satellite cells in vitro.

Myogenesis proceeds by fusion of proliferating myoblasts into myotubes under the control of various transcription factors. In adult skeletal muscle, myogenic stem cells are represented by the satellite cells which can be cultured and differentiate in vitro. This system was used to investigate the subcellular distribution of a particular type of prosomes at different steps of the myogenic process. Prosomes constitute the MCP core of the 26S proteasomes but were first observed as subcomplexes of the untranslated mRNPs; recently, their RNase activity was discovered. A monoclonal antibody raised against the p27K subunit showed that the p27K subunit-specific prosomes move transiently into the nucleus prior to the onset of myoblast fusion into myotubes; this represents possibly one of the first signs of myoblast switching into the differentiation pathway. Prior to fusion, the prosomes containing the p27K subunit return to the cytoplasm, where they align with the gradually formed lengthwise-running desmin-type intermediate filaments and the microfilaments, co-localizing finally with the actin bundles. The prosomes progressively form discontinuous punctate structures which eventually develop a pseudo-sarcomeric banding pattern. In myotubes just formed in vitro, the formation of this pattern seems to preceed that produced by the muscle-specific sarcomeric (alpha)-actin. Interestingly, this pattern of prosomes of myotubes in terminal in vitro differentiation was very similar to that of prosomes observed in vivo in foetal and adult muscle. These observations are discussed in relation to molecular myogenesis and prosome/proteasome function.

Differential myogenicity of satellite cells isolated from extensor digitorum longus (EDL) and soleus rat muscles revealed in vitro.

Following muscle damage, fast- and slow-contracting fibers regenerate, owing to the activation of their satellite cells. In rats, crush-induced regeneration of extensor digitorum longus (EDL, a fast muscle) and soleus (a slow muscle) present different characteristics, suggesting that intrinsic differences exist among their satellite cells. An in vitro comparative study of the proliferation and differentiation capacities of satellite cells isolated from these muscles is presented there. We observed several differences between soleus and EDL satellite cell cultures plated at high density on gelatin-coated dishes. Soleus satellite cells proliferated more actively and fused into myotubes less efficiently than EDL cells. The rate of muscular creatine kinase enzyme appeared slightly lower in soleus than in EDL cultures at day 11 after plating, when many myotubes were formed, although the levels of muscular creatine kinase mRNA were similar in both cultures. In addition, soleus cultures expressed higher levels of MyoD and myogenin mRNA and of MyoD protein than EDL satellite cell cultures at day 12. A clonal analysis was also carried out on both cell populations in order to determine if distinct lineage features could be detected among satellite cells derived from EDL and soleus muscles. When plated on gelatin at clonal density, cells from both muscles yielded clones within 2 weeks, which stemmed from 3-15 mitotic cycles and were classified into three classes according to their sizes. Myotubes resulting from spontaneous fusion of cells from the progeny of one single cell were seen regardless of the clone size in the standard culture medium we used. The proportion of clones showing myotubes in each class depended on the muscle origin of the cells and was greater in EDL- than in soleus-cell cultures. In addition, soleus cells were shown to improve their differentiation capacity upon changes in the culture condition. Indeed, the proportions of clones showing myotubes, or of cells fusing into myotubes in clones, were increased by treatments with a myotube-conditioned medium, with phorbol ester, and by growth on extra-cellular matrix components (Matrigel). These results, showing differences among satellite cells from fast and slow muscles, might be of importance to muscle repair after trauma and in pathological situations.

Activation of protein kinase C during newt limb regeneration: effect of denervation.

Blastema cell proliferation during newt limb regeneration is a nerve-dependent process. The present study was undertaken to determine whether or not that process is mediated by protein kinase C (PKC) activation during limb regeneration in Pleurodeles walt. Analysis included evaluation of PKC activity and its subcellular localization at various stages of regeneration, both in vivo and in vitro. The data reveal an increase in PKC activity in both the cytosol and particulate fractions of whole blastemas reaching a maximum at the mid-bud stage, which correlates with blastema cell proliferation rate. Denervation significantly reduces blastema cell proliferation and also causes a reduction in membrane-associated PKC activity. The effect of PKC activity appears to be restricted to the blastemal mesenchyme, which exhibits a dramatic reduction in activity 96 h after denervation. In contrast, PKC activity in the epidermal cap did not change. Cultured whole blastemas likewise express a decrease in particulate PKC activity and therefore mimic denervated blastemas in this parameter. Co-culture of blastemas with spinal ganglia partially reduces the decline in PKC activity, and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a direct activator of PKC, also prevents the fall in membrane-bound PKC activity while stimulating blastema cell proliferation, in vitro. These data indicate that blastema cell (mesenchyme) proliferation is related to increased PKC activity and that PKC may therefore be involved in the nerve-dependent signalling pathway regulating the early phase of urodele limb regeneration.

Growth factors in skeletal muscle regeneration.

Adult skeletal muscles are able to regenerate after injury. This process is due to the activation of quiescent muscle precursor cells, also called satellite cells, which proliferate and differentiate to form new myotubes. In this regeneration process, several growth factors which come from the muscle and/or from the motor nerve and inflammatory cells have been shown to play key roles. However, most of our knowledge comes from in vitro studies, where, during myogenesis, proliferation of satellite cells is regulated by FGFs, TGF beta s, PDGF, IGF-I and II, while differentiation appears to be promoted mainly by IGFs. During regeneration in vivo, most of these factors have been shown to operate and interact. Other factors also appear to condition the regeneration process, such as LIF, which acts predominantly as a proliferative factor; and HARP/PTN/HB-GAM and other neurotrophic factors, which may be necessary for the formation of new neuromuscular junctions. TGF beta has a major influence on the reorganisation of the extracellular matrix. This review presents a critical summary of the known effects of growth factors on skeletal muscle regeneration.

The kinase inhibitor iso-H7 stimulates rat satellite cell differentiation through a non-protein kinase C pathway by increasing myogenin expression level.

We analysed the signaling pathways involved in myogenic differentiation of primary cultures of rat satellite cells using substances targeting the protein kinase C (PKC) and the cAMP protein kinase (PKA) pathways. We have previously shown that iso-H7, which putatively inhibits both PKC and PKA, strongly stimulates satellite cell differentiation, as well as the PKA inhibitor HA1004. In the study reported here, the effects of iso-H7 on satellite cell differentiation were compared to those observed in the presence of agents which reduce PKC activity. It was shown that treatments with the highly specific PKC inhibitor GF109203X or with 12-O-tetradecanoylphorbol 13-acetate (TPA) which induced a partial PKC downregulation, did not significantly alter myogenic differentiation. Northern blot analyses showed that iso-H7 activated the expression of myogenin but not that of MyoD mRNA. Concurrently, iso-H7 increased myosin light-chain mRNA expression. In contrast, TPA had no effect on these syntheses. Taken together, these results showed that iso-H7 did not act intracellularly as a PKC inhibitor but rather as a PKA inhibitor as previously suggested. Our results are compatible with the hypothesis that a reduction in PKA activity controls satellite cell myogenesis through an increased myogenin mRNA expression.

Influence of a dextran derivative on myosin heavy chain expression during rat skeletal muscle regeneration.

We recently described dextran derivatives (RGTA) which stimulate tissue repair in several in vivo models. One of them, RGTA11, has been shown to accelerate crush-induced regeneration and reinnervation of rat EDL and Soleus muscles. In this study we wanted to know if RGTA11 alters the pattern of myosin heavy chain expression during regeneration. In both EDL and Soleus muscles, RGTA11, injected at the moment of the crush, was found to accelerate the shift from neonatal to adult myosin heavy chain isoforms within 2 weeks. The proportion of slow fibers increased considerably, especially in the Soleus where RGTA11 induced a precocious and permanent expression of slow myosin isoform, thus confirming that a more efficient innervation had occurred in the presence of RGTA11. These results illustrate the interesting potential pharmacological use of such dextran derivatives in neuromuscular disease.

Thyroid hormone stimulates phosphoglycerate mutase activity and isozyme transition in rat muscle tissues.

Triiodothyronine (T3) increases phosphoglycerate mutase (PGAM) specific activity in rat skeletal and cardiac muscles. This increase is concomitant with an increase in the proportion of phosphoglycerate mutase isozymes which contain type-M subunit. Propylthiouracil (PTU), an anti-hormone, not only decreases phosphoglycerate mutase activity with respect to control rats, but also decreases the total M subunit contents. In liver, which only possesses type-B subunit phosphoglycerate mutase, none of the effects were detected.

Stimulation of rat satellite cell myogenesis by inhibitors of ser/thr protein kinases.

Satellite cells are involved in physiological growth and post-traumatic regeneration of adult skeletal muscle fibres. In this study, it is shown that differentiation of primary cultures of rat satellite cells is increased by inhibitors of ser/thr protein kinases such as iso-H7, which both inhibit cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) activities, and HA1004, a PKA inhibitor. These results, showing a preponderant effect of PKA inhibition on myogenesis in vitro, prompted the effects of iso-H7 on muscular regeneration in vivo to be tested. Preliminary results showed that regeneration of rat muscle EDL was improved by iso-H7 treatment.

Satellite cell myogenesis is highly stimulated by the kinase inhibitor iso-H7: comparison with HA1004 and staurosporine effects.

Differentiation of rat satellite cells, measured by cell fusion into myotubes and isozymic conversion of creatine kinase and phosphoglycerate mutase, was shown to be highly increased in the presence of 1-(5-isoquinolinylsulfonyl)-3-methylpiperazine (iso-H7). This substance inhibited both protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities with similar IC50 between 22 and 34 microM. Iso-H7, as well as the PKA inhibitor HA1004 increased myogenic differentiation without altering the proliferation of satellite cells, whereas the proliferation and the differentiation of these cells were inhibited by the PKC inhibitor staurosporine. Our results suggest a predominant negative control of PKA on satellite cell myogenesis.

Modulation of activities and RNA level of the components of the plasminogen activation system during fusion of human myogenic satellite cells in vitro.

Primary cultures of human myogenic stem cells (satellite cells) mimic myogenic differentiation. During this process, the expression of the components of the plasminogen activation system underwent modulation. Activities and mRNA levels of tissue-type and urokinase-type plasminogen activator were increased in a reproducible pattern during differentiation. A modulation of the mRNA level of PAI-2 was also observed. Human satellite cells expressed a urokinase receptor and also the mRNA level of this component underwent modulation. With the exception of PAI-1 mRNA, the level of all mRNAs increased from Day 4 to Day 8, i.e., just before myoblasts fusion, and then remained high at later stages. The modulation of the plasminogen activating activity indicates that this system is directly involved in the fusion process of myogenic differentiation.