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Mechanisms of Tissue Repair
1University Paris 7; 2Institut National de la Santé et de la Recherche Médicale; 3University Paris 6; 4University Paris 5; and 5University Paris 13, Paris, France
Submitted 6 July 2005 ; accepted in final form 18 January 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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myosin heavy chain; cardiotoxin; denervation; nerve injury; immunohistochemistry
Myosin heavy chain (MyHC) is one of the major components of the contractile apparatus of all striated muscles. It is encoded by a multigene family, the members of which are expressed in a tissue-specific and developmentally regulated manner (3). Several MyHC isoforms have been described in skeletal muscles, including two developmental isoforms (MyHC embryonic and neonatal), one slow isoform (MyHC I), and three fast isoforms (MyHC IIA, IIX/IID, and IIB) (27).
During skeletal muscle regeneration, MyHC expression recapitulates the events observed during development (12). For rats, it has been established that during muscle regeneration, embryonic and neonatal myosin are expressed before the appearance of adult fast myosins (11). In both adult and regenerated rat soleus muscle, nearly all fibers express slow myosin (32). It is well known that for rats, innervation is necessary for the expression of slow myosin, because denervated-regenerating soleus muscles exhibit no slow MyHC and very little IIA MyHC 2 wk after injury (22, 32). During muscle formation, the different MyHC isoforms are expressed in the following order: embryonic, neonatal 
adult fast IIX and IIB IIA and I (20). Recently, the mechanisms by which the motor neuron regulates the expression of these MyHC transitions have been clarified. Calcineurin, a calcium-calmodulin-activated serine-threonine phosphatase, has been shown to mediate the nerve effect on slow-fiber-type gene transcription by dephosphorylating transcription factors, such as nuclear factor of activated T-cells (NFAT) and myocyte enhancer factor 2 (MEF2) (29). Ever since Abbott et al. (1) found that the administration of cyclosporin A (calcineurin inhibitor) immediately after muscle injury inhibits muscle regeneration in rats, it has been well recognized that calcineurin plays a key role during the entire process of rat soleus muscle regeneration. Moreover, when cyclosporin A is administrated three days after muscle injury, it alters the expression of slow MyHC but not muscle growth (30). More recently, Fenyvesi et al. (17) found that calcineurin phosphatase activity (CPA) increases during soleus muscle regeneration to attain the adult level 10 days postinjury, which can explain the reexpression of slow MyHC. They hypothesize further that a high level of calcineurin activity maintains the expression of the slow phenotype. In adult muscles, cyclosporin A induces a decrease in slow MyHC I expression concomitant with an increase of IIA MyHC in the soleus (slow-twitch muscle) but not in the plantaris muscle (fast-twitch muscle) (6). However, in cell culture, calcineurin regulates the preferential activation of the IIA MyHC promoter compared with the IIX and IIB MyHC promoters (4).
All of these studies have been carried out on rats and predominantly on the soleus muscle. Very few studies have been carried out on mice, and Wu et al. (33) propose a schematic model displaying three states (repressing, activating, and permissive) to explain the effects of calcineurin on muscle fiber type. The activating state is a transitory state, during which activated calcineurin induces the translocation of a coactivator of MEF2, resulting in the expression of the slow phenotype. In this study, the authors argue that the activating state is followed by a permissive state, during which CPA is relayed by the presence of transcription factors, which allow the maintenance of a slow phenotype. Maintaining this permissive state requires periodic reinforcement by activated calcineurin. Studies carried out on calcineurin A
and A
gene-targeted mice have shown that the slow phenotype is differentially altered by the loss of function of calcineurin A or A, even if compensation has been observed, as the double mutation calcineurin A/ is lethal (26).
To clarify the discrepancy concerning the respective roles played by CPA and muscle innervation in the reestablishment of the slow muscle phenotype after muscle regeneration in different species, we propose first to determine the chronology of expression of the different MyHC isoforms during mouse muscle regeneration in the presence or absence of innervation and second to examine the correlation between CPA and the expression of adult fast and slow MyHC expression in these muscles.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Myosin heavy-chain gel electrophoresis. The muscles were extracted on ice for 60 min in 4 volumes of extracting buffer (pH 6.5), as previously described (7). After centrifugation, the supernatants were diluted 1:1 (vol/vol) with glycerol and stored at –20°C. MyHC isoforms were separated on 8% polyacrylamide gels, which were made in the Bio-Rad mini-Protean II Dual slab cell system, as described previously (2, 3).
The gels were migrated for 31 h at 72 V (constant voltage) at 4°C. After migration, the gels were silver stained. The positions of the different MyHC bands were confirmed by Western blot analysis using antibodies directed against different MyHC isoforms (3). The gels were scanned using a video acquisition system.
Western-blot analysis. Western blot analysis was carried out using an antislow (I) MyHC antibody (Novocastra, Newcastle, UK). Antibody-reacting bands were visualized after development with peroxidase-labeled horse anti-mouse Ig (Vector Laboratories, Burlingame, CA) and a chemiluminescent detection system (SuperSignal, Pierce Biotechnology, Rockford, IL).
CPA assay. Frozen muscle samples were placed into an ice-cold homogenization buffer (7 vol/wt) containing: 50 mM Tris (pH 8), 1 mM EDTA, 100 µM EGTA, 0.1% beta-mercaptoethanol, 1 mM dithiothreitol, 10 µg/ml leupeptin, 0.2 mM PMSF, and 10 µg/ml soybean trypsin inhibitor. Samples were homogenized using a micro-hand homogenizer and then centrifuged at 12,000 g for 30 min at 0°C. Protein concentration was measured by the method of Bradford using BSA as a standard.
The CPA was measured from the homogenate, according to Dunn et al. (14), with the following modifications. Briefly, CPA was determined photometrically (630 nm) at 30°C after using the RII phosphopeptide substrate (150 µM, Calbiochem, San Diego, CA), which is the most efficient peptide substrate known for calcineurin. Thirty microliters of the muscle homogenate was mixed with 20 µl of reaction solution containing 40 mM Tris (pH 8), 100 mM NaCl, 6 mM MgCl2, 500 µM CaCl2, 500 µM dithiothreitol, 750 nM okadoic acid, 150 µM peptide, and 3 µM calmodulin, and it was incubated for 30 min at 30°C in the presence or absence of 2,500 ng/ml of cyclosporin A. The detection of released phosphate is based on the molybdate:malachite green assay (Promega, Madison, WI). Data are expressed as the number of picomoles of phosphate released per minute per milligram of protein. CPA was assayed in triplicate, and the mean (±SE) values are presented. Assays were performed in conditions that ensure a linear initial rate of reaction.
Immunofluorescence. For immunofluorescence, 5-µm frozen sections were treated with 5% BSA for 20 min and then incubated with the primary antibody for 1 h at room temperature. The primary antibodies, which we used in this study, are as follows: anti-neonatal MyHC (1:50, rabbit polyclonal) (15), anti-slow (I) MyHC (1:10, mouse monoclonal, Novocastra), and anti-IIB MyHC (1:5, mouse monoclonal, BFF3). Binding of primary antibodies was detected by incubating the sections 40 min, with FITC-conjugated anti-mouse IgG (1:40) or anti-rabbit IgG (1:40). Sections were finally washed and mounted in Vectashield medium (Vector Laboratories). Images were made by using a DMRB Leica microscope equipped with epifluorescence optics. Digital images were transferred to a computer equipped with Vision explorer software (Graftek Imaging, Austin, TX).
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RESULTS |
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Adult IIX and IIB MyHC isoforms were expressed in all muscles, including the slow-twitch soleus on day 5 of regeneration. In the soleus muscle, the IIB MyHC progressively decreased until day 28. The presence of IIB MyHC was confirmed by immunohistochemistry, as the presence of this isoform is unusual in the soleus muscle. By immunolabeling, it was demonstrated that the fibers that express IIB MyHC also express neonatal MyHC isoform (data not shown). In the EDL (Fig. 1B), the amount of IIB MyHC isoform increased continuously until by day 10, it was the major myosin isoform. The IIX myosin appeared early (day 5) during regeneration but was accumulated to a lesser extent in the soleus than in the EDL. In the soleus muscle, the IIA MyHC was only weakly detected 5 days postinjury, and it increased, until by day 14, it represented 43.4% of the total MyHC. In the EDL muscle, a very small amount of the IIA MyHC was detected 14 days postinjury, and this amount remained stable.
During regeneration, adult slow (I) MyHC was the predominant isoform to be expressed in the soleus, whereas this isoform was completely absent in the EDL. In the soleus, it was first detected at 10 days of regeneration. The amount of slow (I) MyHC increased until 28 days, after which time, it remained constant. On day 21, the IIA and I MyHC were expressed in equal amounts, and at this stage, the neonatal and the IIB MyHC were only weakly detected. After 28 days of regeneration, the soleus muscle expressed only IIA, IIX, and I MyHC. These results demonstrate that at the end of regeneration, the EDL and soleus muscles had attained a MyHC profile identical to that observed in normal adult mice.
Calcineurin phosphatase activity in the regenerating muscles. To determine whether CPA plays a role in the appearance of the adult muscle phenotype during regeneration, we measured CPA in the regenerating muscles. Experimental points were chosen according to the expression of adult MyHC isoforms. Hence, 10, 12, 16, and 21 days postinjury were chosen for the soleus, whereas 10 and 12 days postinjury were chosen for the EDL.
In the soleus muscle, 10 days postinjury CPA was 211 ± 72 pmol·min–1·mg–1 of protein and increased continuously to 21 days postinjury (745 ± 47 pmol·min–1·mg–1). At 16 and 21 days postinjury, there was a 2.5-fold and 3.5-fold increase in CPA, respectively, compared with 10 days. It should be noted that this value is much higher than that observed in the adult soleus, in which the CPA was 259 ± 66 pmol·min–1·mg–1 of protein (Fig. 1C).
In the EDL muscle, CPA was 319 ± 65 and 107 ± 20 pmol·min–1·mg–1 of protein at 10 and 12 days postinjury, respectively (Fig. 1D).
Muscle regeneration in the absence of innervation. To determine the role of innervation on muscle regeneration in the mouse, we analyzed the MyHC profiles in denervated soleus and EDL muscles. Experiments were carried out by cutting the sciatic nerve high in the thigh before administration of cardiotoxin.
The denervated soleus expressed the developmental embryonic and neonatal, IIX, and IIB MyHC until 10 days postinjury (Fig. 2A). On day 14, embryonic MyHC started to be replaced by the IIA isoform. The neonatal isoform decreased progressively and had disappeared by day 28. At this stage of regeneration, the denervated-regenerated soleus muscle expressed four MyHC isoforms: IIA, IIX, IIB, and I. Between 28 and 49 days postinjury, there was a gradual increase in the accumulation of the I MyHC, after which time its expression became constant (Fig. 2A).
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Calcineurin phosphatase activity in the denervated-regenerating soleus muscles. Our results suggest that CPA is essential for the establishment of a slow MyHC phenotype in the regenerating soleus muscle. To determine the role of calcineurin in the establishment of the slow MyHC in the denervated-regenerating soleus muscle, we measured the activity of this enzyme in the denervated soleus muscle after cardiotoxin administration. Figure 2C shows CPA at 10, 16, 28, and 77 days postinjury in the denervated soleus muscle.
In the denervated-regenerating soleus, we observed a high CPA (841 ± 15 pmol·min–1·mg–1) 10 days postinjury. This activity rapidly decreased and reached 350 ± 40 pmol/min–1·mg–1 of protein 16 days postinjury and, consequently, remained stable in all experimental points (Fig. 2C).
Delayed and continual muscle regeneration in the denervated soleus muscle. Immunohistochemical analyses of denervated soleus muscles were carried out 8 and 77 days postinjury using an antibody directed against the neonatal MyHC (Fig. 2, D–F). Eight days after cardiotoxin treatment, the normal regenerated soleus muscle consisted of centronucleated fibers of a similar diameter, all containing neonatal MyHC (Fig. 2D). By 28 days postinjury, the muscle fibers had increased in diameter, and the neonatal myosin had completely disappeared from soleus muscle (data not shown). In contrast, 8 days after cardiotoxin treatment in the denervated soleus muscle, we still observed inflammatory cells and small newly formed muscle fibers that express neonatal MyHC (Fig. 2E). At 77 days postinjury, we continued to detect small fibers containing neonatal myosin (Fig. 2F). In addition, the muscle fibers expressed four adult MyHC (IIA, IIX, IIB, I) isoforms (Fig. 2, B, G, and H).
Muscle phenotype in denervated-regenerating soleus muscle after cyclosporin A treatment. To determine whether CPA plays a role in the appearance of the slow MyHC in denervated-regenerating mouse soleus muscle, we treated the animals with an inhibitor of calcineurin (cyclosporin A) during the time course of muscle regeneration. Western blot analyses were carried out using an antibody specific for MyHC I (Fig. 3). Our results demonstrate that the profile of MyHC I expression was similar in cyclosporin A-treated denervated-regenerating soleus muscle compared with their noncyclosporin A-treated counterparts (Fig. 3). In contrast, in regenerating soleus muscle, cyclosporin A treatment down-regulated MyHC I expression (Fig. 3).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Calcineurin has been shown to play an important role during muscle regeneration. It is required for muscle differentiation (5, 18, 28) and for the neural control of skeletal muscle maturation and, in particular, the expression of slow MyHC (29, 30). During muscle regeneration, in light of our results, we conclude that in the soleus muscle the appearance of slow (I) MyHC and the disappearance of IIB and neonatal MyHC are concomitant with a transitory increase in CPA. According to Fenyvesi et al. (17), CPA increases during rat soleus muscle regeneration to attain the basal adult level, which can explain the reexpression of slow (I) MyHC. But in our study in contrast to the study by Fenyvesi et al. (17), CPA was lower in the adult soleus muscle compared with regenerating soleus muscle. Two hypotheses can be put forward to explain this discrepancy. 1) Slow myosin is the predominant isoform in the rat soleus, whereas in mice, it is expressed in only 40–50% of the muscle fibers. It cannot be excluded that the high CPA in adult rat soleus is correlated with the predominance of slow MyHC expression, which is not the case in the mouse soleus muscle. 2) In mice, the reexpression of slow (I) MyHC is controlled by a peak of transitory CPA, in contrast to rat soleus muscle. This process was also hypothesized by Wu et al. (33), who described an activating state during which calcineurin is active and induces the slow phenotype followed by a permissive state, which is maintained by periodic reinforcement of transitory active calcineurin. The activating state is due to the presence of MEF2. Our results support this hypothesis, because in the soleus muscle the appearance of slow (I) MyHC is due to an increase of CPA, although the basal activity measured in adult muscle is 3.5 times lower. Furthermore, in contrast to the rat (17), in mice, the increase of calcineurin does not inhibit IIA MyHC expression. It seems that calcineurin triggers activators and/or coactivators that relay this activity via MEF2 and NFAT.
In a previous study, Condon et al. (9) showed that the expression of slow MyHC during primary myogenesis is independent of innervation, whereas the expression of this isoform is nerve dependent during secondary myogenesis. In our study, we have also identified early (until day 28 postinjury) and late events (after day 28 postinjury) during muscle regeneration in the absence of innervation, which are similar to those described during myogenesis. In the early events, fast isoforms were predominantly expressed in both the soleus and EDL muscles. This is in agreement to what has been reported previously (8, 11, 16, 19). However, after 28 days of regeneration, slow (I) MyHC was found to be expressed in both slow- and fast-twitch muscles. In addition, it should be noted that 77 days postinjury, the denervated regenerating EDL muscle was much slower than the normal regenerating EDL muscle, whereas the soleus muscle became faster. At 77 days, four MyHC isoforms were coexpressed in these two skeletal muscles, IIA, IIX, IIB, and I. Similar results have been observed previously in adult denervated muscles in both mouse and rabbit (13, 31). In 1994, d'Albis et al. (13) showed that denervated soleus and gastrocnemius medialis of 8-day-old rabbits expressed slow MyHC. In the mouse, Washabaugh et al. (31) have shown that the denervated soleus expressed slow MyHC 52 days postinjury. Although, to our knowledge, we are the first to show the reexpression of slow MyHC in denervated-regenerating soleus muscles and the expression of slow MyHC in fast EDL muscle of mice.
In the 10-day denervated-regenerating soleus muscle, it should be noted that the CPA is higher than in innervated-regenerating muscle 10 days postinjury. That could be explained by the delay of muscle regeneration and the presence of newly formed myotubes in denervated-regenerating muscles compared with the innervated-regenerating muscles, in which the process of cell fusion is already complete at 10 days postinjury. This is confirmed by the fact that, CPA is required for the fusion of myogenic precursor cells during muscle differentiation (18, 28). In denervated-regenerating soleus muscle, there was no increase of CPA between 10 and 21 days and no expression of MyHC I. Up to 77 days postinjury (the last time point of our study), we measured a slightly higher CPA in denervated-regenerating soleus muscle than in control adult soleus muscle. Furthermore, on day 77 postinjury, we also observed some very small-diameter muscle fibers expressing neonatal MyHC, which could explain the relatively high basal CPA.
Several studies have demonstrated that calcineurin and muscle innervation both play important roles in the reestablishment of the slow-twitch muscle phenotype (29, 30). In the literature, many papers have clearly demonstrated that slow (I) MyHC is expressed during embryonic development in the absence of functional innervation (9, 24). Recently, Oh et al. (25) have also shown that calcineurin is not required for the establishment of a slow-muscle phenotype during embryonic development. However, the authors propose that calcineurin is necessary for the subsequent maintenance of the slow-muscle phenotype. In the present study, we have demonstrated that a similar calcineurin-independent developmental pathway has been used to establish the slow phenotype in long-term denervated-regenerating muscles.
Moreover, our results demonstrate that in long-term denervated-regenerating muscles, the MyHC profile was identical in spite of the functional differences inherent with these muscles. In light of this result, we suggest that in the absence of a functional innervation, satellite cells undergo the same program of maturation after cell differentiation and fusion. In recent experiments performed on the rat, Kalhovde et al. (21) suggested that muscle fibers regenerate from intrinsically different satellite cells in fast- and slow-twitch skeletal muscles. This could appear to be in contrast to our results in the mouse, as in long-term, denervated-regenerating muscles, we were not able to see any difference between the soleus and EDL muscles. It should be noted that in the adult mouse, the soleus contains 60% fast-twitch muscle phenotype in contrast to 40% slow fibers, whereas the rat soleus contains almost exclusively slow-muscle fibers. This could explain the difference in the results obtained in these two studies. In addition, Kalhovde et al. (21) only analyzed muscles up to 28 days postinjury, whereas in our study, the differences only became apparent after 28 days. Therefore, we could hypothesize that in long-term denervated-regenerating muscles, satellite cells are under the control of extrinsic factor(s), which homogenize(s) this cell population.
In conclusion, our results suggest that in innervated muscles, the maintenance of a slow-twitch phenotype could be due to the transitory increases of CPA resulting from the slow motor neuron activity. Therefore, in agreement with the results of Wu et al. (33), CPA would be dependent on an activity/rest cycle. The increase or the decrease in activation of motor neuron firing during repeated exercise or immobilization could alter the peaks of CPA, which would provoke a modification in the muscle phenotype. However, in denervated-regenerating muscles, a slow phenotype becomes established in a calcineurin- and nerve-independent manner. These results show that muscle innervation and calcineurin phosphatase activity are not the only factors controlling slow myosin heavy chain expression in mouse muscles.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* T. Launay and P. Noirez contributed equally to this work. 
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REFERENCES |
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and A gene-targeted mice. Mol Cell Biol 23: 4331–4343, 2003.This article has been cited by other articles:
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  O. Agbulut, A. Vignaud, C. Hourde, E. Mouisel, F. Fougerousse, G. S. Butler-Browne, and A. Ferry Slow myosin heavy chain expression in the absence of muscle activity Am J Physiol Cell Physiol, January 1, 2009; 296(1): C205 - C214. [Abstract] [Full Text] [PDF]
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 A. Szabo, F. Wuytack, and E. Zador The Effect of Passive Movement on Denervated Soleus Highlights a Differential Nerve Control on SERCA and MyHC Isoforms J. Histochem. Cytochem., November 1, 2008; 56(11): 1013 - 1022. [Abstract] [Full Text] [PDF]
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 N. Stupka, J. D. Schertzer, R. Bassel-Duby, E. N. Olson, and G. S. Lynch Calcineurin-A{alpha} activation enhances the structure and function of regenerating muscles after myotoxic injury Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2007; 293(2): R686 - R694. [Abstract] [Full Text] [PDF]
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K. Eizema, D. E. van der Wal, M. M.M. van den Burg, H. W. de Jonge, and M. E. Everts Differential Expression of Calcineurin and SR Ca2+ Handling Proteins in Equine Muscle Fibers During Early Postnatal Growth J. Histochem. Cytochem., March 1, 2007; 55(3): 247 - 254. [Abstract] [Full Text] [PDF]
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 M. I. Lewis Mechanisms underlying myogenesis: complex and likely to become more so! J Appl Physiol, December 1, 2006; 101(6): 1539 - 1540. [Full Text] [PDF]
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H. Scholz Unraveling the basic principles Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2006; 290(6): R1485 - R1487. [Full Text] [PDF]
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