Human Molecular Genetics, 2001, Vol. 10, No. 19 2079-2087
© 2001 Oxford University Press
Defective satellite cells in congenital myotonic dystrophy
CNRS UMR 7000, Faculté de Médecine Pitié-Salpêtrière, Université Paris 6, 105 boulevard de l’Hôpital, 75634 Paris Cedex 13, France, 1Département d’Anatomie Pathologique, Hôpital St Vincent de Paul, 74 avenue Denfert-Rochereau, 75014 Paris, France, 2Service d’Oto-Rhino-Laryngologie et Chirurgie de la Face et du Cou, Hôpital Tenon, 4 rue de la Chine, 75020 Paris, France, 3Boston Probes Inc., Bedford, MA, USA and 4INSERM UR383, Hôpital Necker-Enfants Malades, 149–161 rue de Sèvres, 75743 Paris, France
Received April 23, 2001; Revised and Accepted July 12, 2001.
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ABSTRACT |
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In this study we have developed an in vitro cell culture system which displays the majority of the defects previously described for congenital myotonic dystrophy (CDM) muscle in vivo. Human satellite cells were isolated from the quadriceps muscles of three CDM fetuses with different clinical severity. By Southern blot analysis all three cultures were found to have approximately 2300 CTG repeats. This CTG expansion was found to progressively increase in size during the proliferative life span, confirming an instability of this triplet in skeletal muscle cells. The CDM myoblasts and myotubes also showed abnormal retention of mutant RNA in nuclear foci, as well as modifications in their myogenic program. The proliferative capacity of the CDM myoblasts was reduced and a delay in fusion, differentiation and maturation was observed in the CDM cultures compared with unaffected myoblast cultures. The clinical severity and delayed maturation observed in the CDM fetuses were closely reflected by the phenotypic modifications observed in vitro. Since the culture conditions were the same, this suggests that the defects we have described are intrinsic to the program expressed by the myoblasts in the absence of any trophic factors. Altogether, our results demonstrate that satellite cells are defective in CDM and are probably implicated in the delay in maturation and muscle atrophy that has been described previously in CDM fetuses.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Myotonic dystrophy or DM1 is the most common form of inherited neuromuscular disorder in adults with a global incidence of about 1 in 8500 individuals (1). The genetic basis for this autosomal dominant disease is an expanded trinucleotide repeat (CTG)n situated in the 3'-untranslated region (3'-UTR) of the dystrophy myotonic protein kinase (DMPK) gene (2–4). DM1 patients have from 50 to 1000 CTG repeats in the mild and classic forms of the disease and up to several thousand repeats in the severe congenital form. There is usually a good correlation between the size of the expansion, the age of onset and the severity of the disease (5,6). The (CTG)n > 50 expansion is unstable and expands progressively in successive generations of affected families, providing a molecular support for the phenomenon of anticipation which is observed in DM1 (7). Moreover, CTG repeats have been shown to have different sizes in different tissues of the same patient, with one of the largest repeat lengths being present in skeletal muscle (8,9).
The exact physiopathological mechanism leading to DM1 is still not thoroughly understood. The nuclear retention of the mutant DMPK RNA containing large CUG repeats as discrete foci seems to play a key role in the development of the pathology (10–13). This dominant RNA mutation could cause a gain of function at the RNA level possibly by impairing the normal functions of proteins involved in RNA processing (14–16). However, the decreased level of the DMPK protein in DM1 skeletal muscle and myoblasts (17,18), and/or alterations in the expression of the neighbouring genes (DMWD and DMAHP) by the CTG expansions (19–21) could also be involved in the molecular mechanism of DM1.
The classic form of DM1 is characterized by myotonia, progressive muscle weakness and wasting and cataracts as well as many other multisystemic disorders. The congenital form of DM1 (CDM) is more severe and in half of the cases, fatal at birth. CDM is characterized by general hypotonia and respiratory distress at birth (22). If this is overcome, delayed motor development and severe mental retardation are generally observed. Analysis of muscles from CDM patients has shown that the muscle fibres were immature in fetuses and the skeletal muscle maturation was impaired in neonate infants (23,24). In human as in other species, muscle fibres are formed by the fusion of mononucleated myoblasts into multinucleated myotubes. This is a biphasic process with a first generation of myotubes forming in a relative synchronous manner between 8 and 10 weeks of development. A second generation of fibres form progressively and asynchronously around these primary fibres between 11 and 18 weeks of development. By 20 weeks, no new fibres will be formed and the muscle will then undergo maturation (25). Impairment of muscle development in CDM patients was observed in a previous study carried out on muscle from affected fetuses aged between 12 and 40 weeks of pregnancy (J.P.Barbet, D.Furling, O.Agbulut, V.Mouly and G.S.Butler-Browne, manuscript in preparation). In this study we demonstrated that in the early stages, a defect in the proliferation and fusion of myogenic precursor cells occurred in CDM skeletal muscles, resulting in a delayed formation and volumetric growth of the secondary generation myotubes. An abnormal metabolic differentiation and an absence of the secondary generation of slow muscle fibres were also observed. Since muscles from the CDM fetus were frequently atrophic, this suggested that there may be a defect in the property of the satellite cells in these CDM patients. In fact, satellite cells are the only population of myogenic precursor cell which are responsible for the growth of the muscle fibre. Moreover, a decrement in the number of satellite cells has also been described in muscles from CDM fetuses (26).
In the present study, we have developed an in vitro system to analyse the behaviour of CDM satellite cells. We have shown that the proliferative capacity and myogenic program expressed by satellite cells isolated from the skeletal muscles of CDM fetuses were altered in vitro compared with satellite cells isolated from non-affected age matched muscle. Defects implicated in the molecular mechanism of DM1 such as CTG instability and nuclear retention of DMPK RNA containing expanded CUG repeats are already detected in the proliferating CDM satellite cells. Thus the presence of these large CTG repeats altered the various functions of the satellite cells (proliferative capacity, program of differentiation) resulting in modifications in muscle growth and development.
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RESULTS |
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Growth characteristics
The proliferating CDM satellite cells are typically spindle shaped and have a very similar morphology to the proliferating satellite cells isolated from age-matched control biopsies (Table 1). The myogenicity of the muscle cell cultures was calculated from the number of cells expressing desmin, which is only expressed in myogenic cells (27,28). The myogenic purity between CDM and control cultures is slightly different (P < 0.05, t-test) and ranged from 77 to 86% for control cultures and 58 to 69% for the CDM cultures (Table 1). The mean doubling time for each cell strain was calculated at the early stage of the culture and ranged from 37 to 47 h (Table 1) in most of the cultures except for patient 3, in which the doubling time was much longer (84 h). This mean doubling time progressively increased during the time the cells were maintained in culture and finally stopped when the cells reached proliferative senescence.
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Distribution of DMPK mutant RNA
Intracellular localization of DMPK mutant RNA was analysed in differentiated CDM cultures (Fig. 1). Using in situ hybridization, we showed that mutant transcripts were detected as discrete foci in the CDM myotube nuclei (Fig. 1A and B). No cytoplasmic transcripts containing an expanded CUG repeat were detected in the cytoplasm of the CDM myotubes. Moreover, no foci were detected either in the nuclei or in the cytoplasm of control myotubes (Fig. 1C). Nuclear retention of mutant DMPK containing CUG expansion was found in the differentiated muscle cell cultures of all three CDM patients and the foci present in CDM myotube nuclei were brighter and more abundant than those observed in CDM myoblast nuclei (data not shown). Myotubes from patient 1 (Fig. 1A) and from patient 2 (Fig. 1B) showed nuclear foci, but no significant difference in terms of number and size of these foci was found.
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Shortened life span in CDM myoblasts
The life span of the muscle cell cultures was determined by counting the number of times the cells can divide before reaching proliferative arrest, which is defined as the point at which no cell divisions are observed during a period of 3 weeks in growth medium. These results are presented graphically in Figure 2. Unaffected myoblasts isolated from a 29-week-old fetus (control 1) and from a 5-day-old newborn infant (control 2) had replicative life spans of 50 and 60 divisions. In contrast, the proliferative life span of muscle cells isolated from all three CDM patients were shown to be dramatically reduced. Myoblasts isolated from patient 1 had a life span of 31 divisions. For patient 2, muscle cells isolated from the quadriceps had a life span of about 24 divisions. A similar life span was also measured in myoblasts isolated from the masseter muscles of the same patient (data not shown). The muscle cells isolated from patient 3, who had the most severe symptoms, also had the shortest life span, since they could only divide 19 times. Therefore, it is clear that the life span of the CDM cells was always considerably shorter than the life span of control cells isolated at similar stages of development.
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Somatic instability in CDM myoblasts
The instability of the CTG repeats has been studied by southern blot analysis. The CTG expansion length was measured at different time points during the proliferative life span of the CDM cell cultures. Figure 3 illustrates one example of such a DNA analysis. The DNA samples extracted from cultures of patient 1 showed an increase in the number of CTG repeats during the proliferative life span of the cultures. The number of CTG repeats increased by 193 bp over 16 divisions. Similar results were obtained either if DNA samples were digested by AvaI or by BamHI (data not shown). For patient 2, a similar expansion in the number of CTG repeats was measured during the proliferation life span with the number of CTG repeats increasing by 121 bp over 20 divisions (data not shown). Thus, the rate of CTG expansion during the proliferative life span was less in myoblasts isolated from patient 1 than from patient 2, with a mean value of 12 triplets per cell division compared with 6, respectively.
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Defective fusion in CDM myoblasts
The kinetics and quality of the differentiation of CDM muscle cells was compared with unaffected muscle cell cultures. Fusion curves were plotted by determining the fusion index as a function of time in differentiation conditions. The results concerning two of the CDM patients and one of the control cultures are shown in Figure 4. The fusion index of control cultures was
80% after 4 days in differentiation medium. As seen in Figure 4, the kinetics of cell fusion were similar for both CDM and control cultures. Cells began to fuse to form multinucleated myotubes between 2 and 3 days after the cells had been put in differentiation medium. However, the fusion index was much lower in the CDM cultures (30%) than in the controls. In addition, although the proliferating satellite cell cultures of CDM patients and controls had a very similar morphology in growth medium, the myotubes formed by both types of culture showed striking differences. The control satellite cells formed large branched myotubes with as many as 100 nuclei, whereas CDM myotubes were much smaller and thinner and had a relatively small number of nuclei per myotube as shown in Figure 5. Thus, after 6 days in differentiation medium, control cultures showed an average of 15 nuclei per myotube, whereas the CDM myotubes contained an average of 3 to 4 nuclei per myotube (Figure 6).
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Defective maturation in CDM myoblasts
The degree of differentiation and maturation of the CDM myotubes was determined by assaying the expression of four different myosin heavy chain (MHC) isoforms [(two developmental (embryonic and fetal) and two adult (fast and slow)]. After one week of differentiation, all four MyHC isoforms are co-expressed in the large branched myotubes of the control satellite cell cultures from a 29-week-old fetus (Figure 5). This result confirms an earlier study carried out in our laboratory on different clones of human satellite cells (29). The differentiated CDM cultures showed a general decrease in the amount of contractile proteins since it was necessary to reveal the peroxidase activity twice as long to obtain an equivalent staining with the embryonic MyHC. Embryonic MyHC was the predominant species expressed following cell fusion in all three CDM cultures. The fetal isoform was detected at a lower level and not in all CDM myotubes. Expression of the fast MyHC isoforms was greatly decreased in the CDM cells and the slow MHC isoform was almost absent especially in cell cultures from patients 2 and 3. The CDM cells therefore express a very immature phenotype compared with the control cultures.
Phenotypic expression difference in CDM muscle cells with similar CTG expansion
We noted differences between the three CDM cultures, which seemed to be related to the clinical state of each CDM patient since the number of CTG repeats was quasi identical in all cases (2200–2300 CTG). Clinically, patient 1 had only a few features of CDM, i.e. varus feet but only minor anomalies in pulmonary growth. Patient 2 had a more severe condition, with bilateral varus feet, arthrogryposis and muscular hypotrophy. Patient 3 was the most severely affected of the three, with a very low level of fetal activity and arthrogryposis. This fetus was hypotrophic and had major lung hypoplasia. Interestingly this increase in the severity of the disease in these three fetuses was also evident in the phenotype expressed by the cultures.
The life span of cells from patient 3 was the lowest (19 divisions) compared with cells from patient 2 (24 divisions) and patient 1 (31 divisions), suggesting that there is an expressivity effect in vitro in the CDM proliferating satellite cell cultures, very similar to that observed in vivo. Differences were also observed after 1 week of differentiation. As we have stated previously, CDM myotubes are smaller and have fewer nuclei than those from control cultures. As shown in Figure 5, a few myotubes with numerous nuclei may be observed in the differentiated cultures. The proportion of these large myotubes decrease dramatically from patient 1 to patient 3, where only very small myotubes containing 2–5 nuclei were observed. The same phenomenon can be seen for the expression of the fast and slow MHC isoforms. Their expression decreased from patient 1 to patient 3. Altogether, these anomalies support the fact that variability (i.e. different levels of severity) in the CDM phenotype was retained in vitro.
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DISCUSSION |
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In a previous study we have shown that during early stages of development four different classes of myoblasts can be isolated from muscle explants and participate in the formation of human skeletal muscle (30). After 20 weeks of development almost all of the cells isolated from the quadriceps have the same phenotype, which is identical to that expressed by myoblasts isolated from adult muscles (29). Morphologically, differentiated cells from this population form large branched myotubes containing numerous nuclei (up to 100). These myotubes co-express all four MHC isoforms (embryonic, fetal, fast and slow).
In the intact muscle in vivo these cells are quiescent and have been called satellite cells. They are responsible for both the pre- and post-natal growth of the skeletal muscle and play an essential role in the regeneration of skeletal muscle following damage (31,32). Activation of these quiescent satellite cells leads to the generation of myogenic precursor cells, also called myoblasts, which undergo multiple rounds of division prior to fusing with existing or newly formed myofibers (33,34). When myogenic cells were isolated from the skeletal muscle of a 26-week-old fetus and from a 5-day-old newborn infant, the results we obtained corresponded to those described previously for satellite cells isolated from fetal and post-natal muscle (30). In contrast, differentiated CDM myoblast cultures isolated from three affected fetuses (aged 28, 34 and 37 weeks) formed very small myotubes with few nuclei. They expressed mainly embryonic and fetal MHC isoforms but there was little or no expression of the fast and slow MHC isoforms. This phenotype is similar to that expressed by the myoblasts which are predominant in the early limb at between 8 and 10 weeks. A previous report from our laboratory has shown that fusion of myoblasts was not modified until they reach the last few divisions before reaching senescence (35). Since the experiments presented in this report were done when myoblasts were at the middle of their proliferative life span, such an effect can be ruled out. The poor degree of maturation and fusion that was observed in the differentiated CDM cultures could indicate that there was a disturbance in the developmental evolution of the different populations of myoblasts within the CDM skeletal muscle. The muscles of these CDM fetuses still contained early myoblast populations which should have disappeared at this stage of development and been replaced by the later myoblast population, i.e. as found in control muscle after 20 weeks. It is interesting to note that there is an abnormal persistance of mononucleated myoblasts identified by their expression of myoD and myogenin in the skeletal muscles of these fetuses even after 20 weeks, a time at which such cells are no longer observed (J.P.Barbet, D.Furling, O.Agbulut, V.Mouly and G.S.Butler-Browne, manuscript in preparation). In addition there is a delay in the maturation of these muscles which could be correlated with the immaturity observed in vitro. This delay in maturation of the skeletal muscle was characterized by the abnormal presence of myotubes, small fascicles of muscle fibres, thinner myofibres and a delayed differentiation of the second generation slow muscle fibres (24). One cannot exclude the possibility that the perturbations induced by the triplet repeat do not somehow have an influence on the innervation of the developing muscle and consequently on the myoblast populations.
In this study we have demonstrated that there is a reduction in the proliferative capacity of the CDM myoblasts in culture compared with age-matched non-affected myoblasts. This is the first time that a defect in proliferation has been described for CDM myoblasts. It is now well established that human diploid cells such as fibroblasts or myoblasts have limited proliferative life span (36). Satellite cells isolated from a muscle biopsy proliferate rapidly in culture and after a defined number of divisions the growth rate declines and the cells cease to divide when they reached a stage called proliferative senescence (37). The proliferative capacity of myoblasts in culture reflects the capacity of the satellite cells isolated from a muscle biopsy to proliferate. In addition this will reflect their ability to participate in both muscle growth and regeneration. Since CDM myoblasts reached proliferative senescence much earlier than would be expected, it could be suggested that the cells isolated from CDM fetuses have made more divisions in vivo than the age-matched non-affected satellite cells or that unexpected cell death provoked further proliferation of the surviving cells. Although we have not observed increased cell death in the CDM cultures compared with age-matched controls, further investigations must be carried out to explain this reduced proliferative capacity. We would like to propose an alternative mechanism, where large CTG repeats would interfere with mechanisms which regulate the mitotic clock and consequently the life span of the cells. Both hypotheses would result in a decrease in the number of satellite cells that are available to form new muscle fibres or to increase the size of pre-existing muscle fibres. In vivo, a significative number of dividing (KI67 positive) mononucleated muscle cells (MyoD positive) were detected in the CDM muscle biopsies, indicating a dysfunction in the pool of myogenic precursor cells since they have not fused (J.P.Barbet, D.Furling, O.Agbulut, V.Mouly and G.S.Butler-Browne, manuscript in preparation). Fibres were also smaller, which could suggest in fact that there had been less proliferation rather than more. A defect in this pool of muscle cells could also induce a progressive decrease in the number of quiescent satellite cells. Following asymmetric cell division, a percentage of these activated satellites cells will be used to restore the pool of quiescent satellite cells under the basal lamina of the newly formed muscle fibres. It is interesting to note that a decrease in the number of satellite cells has been observed in muscle from CMD patients supporting a defect and/or a premature senescence of this cell population.
Variability in the phenotypic expression in the three different cultures of CDM myoblasts with a similar number of CTG repeats was also demonstrated in this study. Both the proliferative capacity and the myogenic programme were altered to different degrees in the three CDM cultures. This difference in the severity of the modifications observed in the three CDM cultures was also found to be directly correlated with both the morphological and clinical severity of the disease. The most severely affected fetus (patient 3) showed the largest alterations in terms of life span and myogenic differentiation/maturation and the least affected (patient 1) had more minor modifications in vitro. A difference in the severity of the phenotypic expression (expressivity) of this mutation with a similar number of CTG repeats has been previously reported for this disease (38,39). It may, however, have some genetic support since in this study as it was observed in vitro in the absence of epigenetic factors. One of the potential mechanisms involved in this phenotypic variability could be the somatic instability of the mutation. The CTG repeat has been shown to be unstable in somatic cells within an individual and the largest expansion has been observed in skeletal muscle (9,40). Somatic instability could provide an explanation for the variable pleiotropism observed in the DM1 pathology, but the significance of this somatic mosaicism is still unclear. Expansion of the CTG repeat length has been measured in blood cells from DM1 patients (39,41). In this study, we have demonstrated that during continuous serial passaging of the CDM myoblasts, the size of the CTG expansion increased in a linear manner following cell division. The size of the CTG expansion in CDM myogenic precursor cells increased by between 0.25 and 0.5% per division. Thus, mitotic instability of the CTG expansion is present in this pool of muscle cells and could be a mechanism contributing to the defects observed in CDM myoblasts both in vitro and in vivo. Amplification could interfere with the mitotic program that leads to premature senescence of the CDM myoblasts. However, we did not see any correlation between CTG instability and life span shortening or myogenic differentiation/maturation of the different CDM muscle cells in culture. This result suggests that somatic instability could not explain all of the differences in phenotypic expression which were demonstrated in the muscle cell cultures with a comparable number of CTG repeats but isolated from different CDM fetuses. However, the size of the CTG repeat has only been determined from bulk DNA Southern blotting and this technique cannot reveal the differences in the level of instability and range of repeat sizes that have been shown using small pool PCR (42,43). The somatic instability in the CDM muscle cell cultures should be determined more sensitively and quantitatively to confirm our result.
The mutation in myotonic dystrophy corresponds to an expanded CTG repeat situated in the 3'-UTR of the DMPK gene. We have shown in this study that mutations with large CTG expansions in CDM myoblasts considerably modified the in vitro behaviour of these cells. CDM myoblasts isolated from three fetuses all with approximately 2200–2300 repeats showed significative defects in proliferation, fusion, differentiation and maturation compared with age matched non-affected myoblasts. The defects in fusion and differentiation induced by the mutation have also been demonstrated in a muscle cell model overexpressing the CTG expansion and/or the 3' end of the DMPK 3'-UTR (44). The aggregation of mutant DMPK RNA into discrete nuclear foci is at least one of the cellular consequences of the CTG expansion that could lead to disturbances in myogenic differentiation (18). Increasing evidence suggests that the nuclear retention of DMPK RNA containing CUG expansion creates an RNA gain-of-function mutation that disrupt RNA processing in a trans-dominant manner (14,45,46). Discrete foci of mutant DMPK RNA were observed in the nuclei of both the unfused myoblasts as well as the differentiated myoblasts in all three CDM muscle cell cultures. This would suggest that this mechanism is implicated in fusion and differentiation inhibition measured in the CDM cultures. However, we cannot exclude the possibility that other consequences of the CTG expansion such as abnormal expression of SIX5 could also be involved in these alterations.
Altogether, our results indicate that large CTG repeats altered the functions of myogenic precursor cells or myoblasts in CDM. In skeletal muscle development, these cells, after undergoing multiple rounds of division, are responsible for the prenatal growth of the skeletal muscle by fusing with newly formed myofibres. Since both the proliferative capacity and the myogenic differentiation of CDM myoblasts are defective in vitro, this would suggest that anomalies in the pool of activated satellite cells are responsible for the delay in maturation and muscle atrophy that is observed in the DM fetuses. This CDM muscle cell culture model reproduces some of the main features observed in the skeletal muscle of the CDM fetuses and provides a useful model to investigate the molecular mechanisms involved in the physiopathology of CDM.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Materials
Biopsies from quadriceps muscles were obtained during autopsies, in accordance with the French legislation on ethical rules. Three CDM fetuses, one unaffected fetus and one unaffected newborn infant have been included in this study. The three CDM fetuses were affected to different degrees. The 34.5-week-old fetus (patient 1) had mild clinical features, the 28-week-old fetus (patient 2) had more prominent clinical symptoms of CDM and the 37-week-old fetus (patient 3) had a very severe form. The control biopsies showed no sign of neuromuscular disease and were from a 29-week-old fetus (control 1) and a 5-day-old infant (control 2).
Methods
Human cell cultures. Satellite cell populations were isolated from normal and CDM muscle biopsies as described previously (29). Myoblasts were grown in HAM’s F10 medium (Gibco) supplemented with 50 µg/ml gentamycine (Biomedia) and 20% fetal calf serum (Biomedia). All cultures were incubated at 37°C in a humid atmosphere containing 5% CO2. At isolation, all cell populations were considered to be at 1 meaning population doubling (MPD). The number of MPDs carried out at each cell passage was calculated as ln N/ln 2, where N is the number of cells at the time of passage divided by the number of cells initially attached after seeding. For growth curves, cells were seeded at 2500 cells/cm2 in growth medium and cultures were counted in triplicate. The myogenic purity of the population was monitored by immunochemistry using an antibody specific for desmin (D33, DAKO) and was calculated by counting 500 cells for each sample after cell isolation and at different population doublings. For myoblast differentiation, growth medium was removed from sub-confluent cultures and replaced by Dulbecco’s modified Eagle’s medium (Gibco) containing 10 µg/ml insulin and 100 µg/ml transferrin (Sigma). Fusion index was determined by counting the number of nuclei in multinucleated myotubes and it was expressed as a percentage of the total number of nuclei.
Immunocytochemistry. Immunocytochemistry was performed as described previously (29). Briefly, cultures were washed with phosphate-buffered saline (PBS) and fixed in 95% ethanol. Non-specific binding sites were blocked with non-immune serum and then the cultures were incubated independently with the following monoclonal antibodies: embryonic (2B6) (47), fetal, slow and fast (Novocastra) MHCs. Antibodies were used at a dilution of 1/50 for anti-embryonic MHC and 1/10 for anti-fetal, slow and fast MHC. Specific antibody binding was revealed with peroxidase using the avidin-biotin technique (Vectastain kits, Vecta).
In situ hybridization. Muscle cells were cultured on LabTek (NUNC) culture chamber slides. Cells were fixed in 4% paraformaldehyde for 15 min, washed with 70% ethanol and then with PBS. In situ hybridization was performed using a Cyanine-3-labelled peptide nucleic acid (PNA) probe (CAG)5 as described by Taneja (48). Cellular DNA was visualized by inclusion of 4',6-diamino-2-phenylindole in the mounting medium. Immunofluorescence microscopy was performed on a Leica TCS4D confocal microscope (Leica Lazer Tecknik).
Analysis of the CTG repeat length. Genomic DNA was extracted as described previously (37). Ten micrograms of DNA was digested by AvaI or BamHI and subjected to 0.7% agarose gel electrophoresis for 48 h. 
HindIII and 32P-HindIII were used as molecular weight markers. After denaturation (1 N NaOH) and neutralization (1 M Tris, 3 M NaCl pH 8.3), DNA was transferred to a Hybond-N membrane in 6x SSC. Markers migrated on each side of the gel were used to allow a correct alignment of the gel prior to transfer. Hybridization was carried out using a P263-B1.4 32P-labelled probe (49), a 1.4 kb BamHI fragment containing the CTG repeat. The membrane was then washed and analysed by autoradiography. The size of the CTG repeat was measured using NIH Image and the value was determined from the middle of the smear. DNA isolated from a non-affected individual (with no large CTG amplification) was also used as a negative control for CTG amplification hybridization.
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ACKNOWLEDGEMENTS |
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We would like to thank Martin Catala for the many fruitful discussions that we had during the preparation of this manuscript. Financial support has been provided by the Association Française contre les Myopathies (AFM) Association pour la Recherche contre le Cancer (ARC) and the European Community (contract QLK6-1999-02034). L.C. was supported by a grant from the Foundation pour la Recherche Médicale (FRM) and the Assistance Publique-Hôpitaux de Paris. D.F. had a post-doctoral fellowship from the AFM.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +33 1 40 77 98 38; Fax: +33 1 53 60 08 02; Email: butlerb@ext.jussieu.fr The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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REFERENCES |
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