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.

Changes in subcellular localization of fructose 1,6-bisphosphatase during differentiation of isolated muscle satellite cells.

Subcellular localization of FBPase, a regulatory enzyme of glyconeogenesis, was examined inside dividing and differentiating satellite cells from rat muscle. In dividing myoblasts, FBPase was located in cytosol and nuclei. When divisions ceased, FBPase became restricted to the cytosolic compartment and finally was found to associate with the Z-lines, as in adult muscle. Moreover, a 12-fold decrease was observed in the number of FBPase-positive nuclei associated with muscle fibres of adult rat, as compared with young muscle, possibly reflecting the reduction in number of active satellite cells during muscle maturation. The data might suggest that FBPase participates in some nuclear processes during development and regeneration of skeletal muscle.

Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activity.

Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N-acetyl-leu-leu-norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo-1 and Boc-leu-met-MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity.

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.

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.

Differential intracellular distribution and activities of mu- and m-calpains during the differentiation of human myogenic cells in culture.

Calpains are intracellular calcium-dependent cystein proteases active at neutral pH. There have been found in human adult myogenic cells (i.e. satellite cells) 2 forms of calpains requiring either micromolar Ca2+: mu-calpain, or millimolar Ca2+: m-calpain. Calpains could be involved in both intracellular proteolysis and cytoskeleton reorganization required for myogenic cell fusion. We showed significant differences in calpains distribution during differentiation of myogenic cells. Using mono- and polyclonal antibodies against both types of calpains, we localized mu-calpain and m-calpain in cultured human satellite cells. mu-calpain was detected in the nuclei of myoblasts and in the cytoplasm of myotubes. m-calpain was only present in the cytoplasm, and was concentrated near the nuclear envelope. Biochemical assays for calpain activities showed that the amounts of these proteinases were modulated during cell growth and differentiation. m-calpain activity was high at the proliferation phase (day 4 of culture) and reached a maximum with the beginning of fusion (day 8) and decreased slightly when the number of myotubes increased (day 12). This activity profile suggests that m-calpain could play a role in the initiation of fusion of satellite cells. The activity of mu-calpain increased regularly with cell growth, the maximum being reached when the cells differentiate, i.e. when its intracellular localization shifted from the nucleus to the cytoplasm. We conclude that the activity and the intracellular localization of the 2 forms of calpains differ with the state of differentiation of myogenic cells.

Activity of mu- and m-calpain in regenerating fast and slow twitch skeletal muscles.

Calpains--non-lysosomal intracellular calcium-activated neutral proteinases, form a family consisting of several distinct members. Two of the isoenzymes: mu (calpain I) and m (calpain II) responded differently to the injury during complete regeneration of Extensor digitorum longus (EDL) muscle and partial regeneration of Soleus muscle. In the crushed EDL the level of m-calpain on the 3rd and 7th day of regeneration was higher than in non-operated muscles, whereas the activity of this calpain in injured Soleus decreased. The level of mu-calpain in EDL oscillated irregularly during regeneration whereas in Soleus of both injured and contralateral muscles its level rapidly rose. Our results support the hypothesis that m-calpain is involved in the process of fusion of myogenic cells whereas mu-calpain plays a significant but indirect role in muscle regeneration.

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.

Changes in the protein kinase C activity or rat sternomastoid muscle during development and after denervation.

The relationship between the activity of protein kinase C (PKC) and muscle innervation was explored in the rat sternomastoid muscle (SM) from day 18 of gestation (E18) to adult age. Between E18 and birth, PKC activity rose 5-fold, and during the day after birth, diminished to a level characteristic of the mature muscle. The rise chiefly occurred in the neural part of the muscle, in both the membrane and the cytosol fractions. Between E18 and day 5 after birth, the ratios of membrane to cytosol PKC activity rose from 0.5 to 10 and 3 respectively in the neural and aneural parts of the muscle. Denervation of adult SM reduced PKC activity by half in the membrane fraction of the neural part but did not significantly change it in the membrane or cytosol fractions of the aneural parts. These results suggest that innervation plays an important part in determining the level of PKC activity in muscle.

Protein kinase C activity and phorbol ester binding to rat myogenic cells during growth and differentiation.

Phorbol esters have been reported to induce opposite responses in fetal myoblasts and in satellite cells isolated from adult skeletal muscles. We examined the possibility that different levels of protein kinase C (PKC) activity and different phorbol ester binding characteristics account for these responses. For this purpose, the subcellular distributions of PKC were compared in primary cultures of myogenic cells from fetal and adult rat muscles and in the L6 cell line. Cells were used at the proliferative stage or after differentiation into myotubes. Binding of phorbol dibutyrate (PDBu) was assayed. In all three cell types, the levels of PKC specific activity were comparable at the proliferating and the differentiated stages, and partial translocation of PKC activity from the membrane to the cytosolic compartment was observed after differentiation into myotubes. PDBu binding, which had a Kd of 6 to 13 nM in proliferative cells, rose to between 30 and 52 nM in myotubes. Simultaneously, a small increase was observed in the total number of PDBu binding sites. These results suggest that the role of PKC might change with the stage of differentiation. They also imply that the difference described by others between the sensitivity to phorbol esters of fetal myoblasts and satellite cells is not connected with the phorbol ester receptor (i.e., PKC), but might be caused by events subsequent to PKC activation.

Protein kinase C translocation in relation to proliferative state of C3H 10T1/2 cells.

Protein kinase C (PKC) activity of cytosol and membrane fractions of 10T1/2 cells was studied. In cytosol of fast growing cells PKC activity was found in material eluted with 0.150 M NaCl whereas in membrane fractions activity was eluted with 0.065 and 0.150 M NaCl. In the membrane fraction of confluent cells, in contrast to cytosol, very low PKC activity was observed. The translocation pattern of PKC activity eluted with 0.065 M NaCl may be associated with proliferation.

Ultracytochemistry of cyclic 3',5'-nucleotide phosphodiesterase activity in the planarian Dugesia lugubris (O. Schmidt).

The enzyme 3',5'-nucleotide phosphodiesterase was localized in certain tissues of the planarian Dugesia lugubris (O. Schmidt) by means of ultracytochemical methods. This enzyme was found to be active in epithelium, muscles, nerve tissue and in rhabdite-forming cells. The active enzyme was present at the outer or inner side of the membrane, and even in the cytoplasm. Problems of the ultracytochemical localization of PDE are discussed.