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Acetylcholine receptors in cultured human muscle cells
Journal oJ the Neurological Sciences, 1980, 47:317-327
317
© Elsevier/North-Holland Biomedical Press
ACETYLCHOLINE RECEPTORS IN CULTURED HUMAN MUSCLE CELLS
GILLIAN I. FRANKLIN*, ROSE YASIN, BEAUMONT P. HUGHES and EDWARD J. THOMPSON
Muscular Dystrophy Research Laboratories, Institute o["Neurology, Queen Square House, Queen Square, London WC1N 3BG (Great Britain) (Received 20 December, 1979) (Accepted I I April, 1980)
SUMMARY
Primary cultures prepared from human muscle biopsies were examined for the presence and distribution of acetylcholine receptors, as measured by the binding of ~2Siodine-labellede-bungarotoxin. The toxin bound to the human muscle cultures with a similar time dependence and specificity as found in muscle cultures from other species. The amount of toxin bound was lower than that obtained for neonatal mouse muscle under similar conditions. The distribution of receptors was similar in cultures derived from the muscles of patients with a variety of neuromuscular disorders. The toxin was located along the myotubes in a fairly even distribution; however, variations in labelling along a single myotube were observed, as well as variations between different myotubes in the same culture. Occasionally, the toxin also bound to other cells, which may have been mononucleated. Cultures prepared from patients with Duchenne muscular dystrophy produced multi-layered cell clusters, instead of the usual monolayer of cells. Within these clusters, only the myotubes bound e-bungarotoxin.
INTRODUCTION
Studies of the initial stages of the development of muscle cells in culture have shown that dividing mononucleated cells lack acetylcholine receptors (AChR) (Patrick et al. 1972; Sytkowski et al. 1973): With increasing differentiation, myoblasts fuse to form multinucleated myotubes which are characterized by a uniform This work was supported by the Muscular Dystrophy Group of Great Britain. * To whom correspondence and reprint requests should be sent.
318 distribution of AChR along their entire surfaces (Vogel et al. 1972; Sytkowski et al. 1973; Prives et al. 1976). This localization resembles that tbund in denervated muscles (Miledi 1960) and embryonic muscle fibres in vivo (Diamond and Miledi 1962). Subsequently, as the myotubes develop further, in culture, the receptor density becomes non-uniform, with randomly-distributed "'hot spots" (Sytkowski et al. 1973; Prives et al. 1976; Jacob and Lentz 1979). The increase in AChR with the onset ofmyoblast fusion is analogous to that described for other muscle specific proteins, e.g. creatine phosphokinase (CPK) (Turner et al. 1976) and therefore both AChR and CPK have been utilized as markers of muscle differentiation (Patrick et al. 1972; Prives et al. 1976; Turner et al. 1976). AChR have also been used as markers for the muscle cell surface membrane (Andrew et al. 1974). Primary cultures initiated from human muscle biopsies develop in a similar manner to those prepared from foetal and neonatal muscles, namely, myoblasts fuse to form myotubes; however, the degree of differentiation obtained varies with individual diseases (Yasin et al. 1977). Primary cultures derived from patients with Duchenne muscular dystrophy (DMD) or Becker dystrophies exhibit an abnormal growth pattern. Instead of growing as a monolayer on the culture dish, some of the cells form multilayered clusters prior to myoblast fusion (Thompson et al. 1977; Yasin et al. 1979). When the myoblasts fuse, the myotubes often emerge from these clusters or span contiguous clusters. The production of cell clusters may be consistent with a defect in the cell surface membrane or in the cytoskeletal structure (Thompson et al. 1977). This investigation was undertaken to ascertain whether human muscle cultures could produce AChR and if the receptor distribution was similar in cultures from patients with D M D as compared with those from patients with various other neuromuscular diseases. The results show that all the muscle cultures produced AChR, as measured by the binding of ~25iodine-labelled ~-bungarotoxin (c~-Bgt), and that these AChR were located mainly in a similar distribution. In the multilayered cell clusters, produced by the D M D cultures, the toxin bound only to the myotubes. MATERIALS AND METHODS Phosphate-buffered saline (PBS) contained 144 mM NaC1, 5.4 mM KCI, 25 mM glucose, 25 mM sucrose and 5 mM sodium phosphate (340 mOsm, pH 7.3 at 37 °C). d-Tubocurarine (15 mg/1.5 ml saline) was purchased from Duncan Flockhardt & Co. Ltd., London. Bovine serum albumin (Sigma Fraction V) was prepared as a 100 (w/v) solution in PBS, hereafter called PBS-A. ~251odine-labelled 7-bungarotoxin (specific activity 300 400 Ci/mmole) was a gift from Dr. P. Darveniza (Institute of Neurology) and was prepared as described elsewhere (Darveniza et al. 1979). The di-iodinated toxin was diluted and stored at - 2 0 ° C , as l0 nM aliquots in PBS-A, for not longer than 80 days. K2 emulsion lbr autoradiography and FF developer contrast 1 were purchased from Ilford Ltd., and Amfix from May & Baker Ltd. Human muscle cultures were dissociated and grown from biopsy specimens
319 as described by Yasin et al. (1977, 1979). Cells were seeded at 6 × 1 0 4 cells per 3.5 cm culture dish. For ~-Bgt binding studies, the culture dishes were washed 3 times with 2 ml of PBS-A and incubated with 1.5 ml of PBS-A for 20 min at 37°C in air. This was removed and replaced with 1.5 ml containing various concentrations of ~-Bgt and further incubated at 37 °C in air. In order to estimate the level of non-specific binding, d-tubocurarine (final concentration, 1 mM in PBS-A), was included in the 20-min pre-incubation and in the incubation mixture with ~-Bgt. After the incubation, unbound 7-Bgt was removed, the cultures washed 3 times with 1.5 ml PBS-A and 3 times with 1.5 ml PBS. The cells were scraped off the surface of the culture dish with a teflon-tape-coated spatula into a small volume of PBS and the amount of toxin bound was measured by counting in a L.K.B. gamma counter. The specific binding was determined from the difference between the toxin bound in the absence of tubocurarine and that bound in its presence. For autoradiography, the cultures were incubated with toxin, in a similar manner to the binding experiments, and then fixed with 2~o (v/v) glutaraldehyde (EM scope, London) in 50 mM cacodylate buffer, pH 7.4 (2 ml per dish) for 1 h at room temperature, dehydrated and dried (Sytkowski et al. 1973). The cultures were coated with K2 emulsion (10 parts K2 : 2 parts H20 : 0.1 part glycerol) and incubated for 3 8 weeks at 4 °C in a light-proof box containing desiccant. The autoradiographs were developed with F F developer and fixed with Amfix and hardener at room temperature. The cultures were photographed under phase contrast and dark field illumination with a Leitz Orthomat microscope. For the CPK assays, cultures were washed 3 times with 1.5 ml PBS and scraped into a small volume of PBS, on ice. The cells were ruptured by sonication with an MSE sonicator for 3 s under nitrogen atmosphere, on ice. This procedure was repeated twice, with two 5-s cooling intervals. CPK activity was measured spectrophotometrically by the method of Rosalki (1967) using a Boehringer UVsystem CPK activated kit (number 124 150). The enzyme activity was expressed as lamole creatine produced/min/culture plate at 30°C. Protein concentrations were determined by the Lowry method (1951) with human serum albumin as standard. RESULTS
Saturation of human muscle A C h R occurred within 20 min (Fig. 1), and remained stable for at least 90 min, under the conditions used. The binding curve was similar to that obtained for neonatal mouse muscle (data not shown) under similar conditions. Between 7 0 ~ and 90~o of the total ~-Bgt binding was inhibited by 1 mM tubocurarine, a known A C h R antagonist. The amount of ~-Bgt bound increased 10-fold with fusion of myoblasts to myotubes (Fig. 2 and Table 1). An autoradiograph of a culture (from a patient with acute polymyositis) corresponding to those used in this binding study is shown in Fig. 3B, after 12 days growth in vitro. A concomitant increase in the CPK levels with myoblast fusion was noted (Table 1), similar to that described for muscle cultures from various sources (Turner et al. 1976; Yasin et al. 1977).
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Fig. 1. Rate o f binding of [12Sl]di_iodo.z~_bungarotoxin to cultures of h u m a n muscle from a patient with minicore myopathy. Culture plates were washed and pre-incubated with PBS-A for 20 min and then incubated at 37°C with 10 nM 2-Bgt in PBS-A. At given time intervals, plates were removed and washed with PBS-A and PBS. Cells were scraped from the dish and the a m o u n t of 2-Bgt binding measured in a ;'-counter. Values for non-specific binding were obtained by preincubation and incubation in the presence of 1 m M d-tubocurarine and were then subtracted from the values for the total binding to yield values of specific toxin binding. Protein concentrations (mg) were determined by the Lowry method.
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Fig. 2. Specific binding of 2-bungarotoxin to h u m a n muscle cultures, from a patient with acute polymyositis, grown in vitro for different periods. The experimental details are given in the text. The arrow denotes the onset of myoblast fusion.
U n d e r saturating conditions the a m o u n t o f :~-Bgt b o u n d specifically was 11.09 +_ 5.93 fmole/culture (for 7 determinations from 5 different biopsies). This was lower than the value obtained for neonatal m o u s e muscle cultures under similar conditions (51.4 + 34.4 fmole/culture) and was near the lower end o f the ranges published for other m a m m a l i a n muscles in culture (Table 2).
TABLE 1 ~ - B U N G A R O T O X I N B I N D I N G A N D C P K LEVELS IN R E P L I C A T E H U M A N M U S C L E CULTURES DERIVED FROM A PATIENT WITH ACUTE POLYMYOSITIS The morphology of this culture on day 12 is shown in Fig. 3A and the corresponding autoradiograph in Fig. 3B. Days in vitro
Specific fmoles [J25I]c~-Bgt bound/culture dish
5 7 8 11 12 14
0.8 0.3 7.4 10.8 10.8
C P K p-moles creatine/min/plate at 30 °C
0.02
0.26
Fig. 3. 2-Bgt binding to a muscle culture from a patient with acute polymyositis after 12 days growth (5 days after the onset of myoblast fusion). A : morphology under phase contrast. B: dark field illumination of silver grains in autoradiograph of the same field of view. The grain density varied along the length of the myotubes, which were of large diameter (50 lam) and branched and had a high level of C P K (specific activity 1.4gmoles/min/mg protein at peak differentiation). Corresponding cultures were utilized for the time-course of receptor appearance shown in Fig. 2. Bar = 50 gin. TABLE 2 A M O U N T S OF :t-Bgt B O U N D T O H U M A N A N D V A R I O U S O T H E R M U S C L E SPECIES IN CULTURE Muscle
Total fmole b o u n d a per mg protein
Specific fmole/cm 2
Reference
Human [ 7] N e o n a t a l m o u s e [19] Embryonic mouse Embryonic chick Embryonic chick G8 mouse line L6 rat line L6 rat line L8 rat line
81.3_+ 51.1 366 +_ 138 -150 - 1000 -20 - 170 52 400 b 14
1.2 5.3 4.6 -10.6 --13.5b (approx.) --
this study this study Christian et al. 1978 Vogel et al. 1972 Prives et al, 1976 Noble et al. 1978 Vogel et al. 1972 Patrick et al. 1972 Vogel et al. 1972
•
a Includes non-specific (curare insensitive) binding. b Specific binding of the closely related Naja naja toxin. N u m b e r of cultures in parentheses.
322 The conditions for a u t o r a d i o g r a p h y were chosen for maximal binding, viz. 1 h at 37°C with 10 n M toxin. Binding was located by silver grains in the emulsion, which were visualized under dark field illumination and phase contrast microscopy was used to view cell morphology. Cultures were examined from 4 patients with D M D , 2 with Becker dystrophy and 12 patients with various other neuromuscular disorders. The a m o u n t s o f ~-Bgt b o u n d to the cultures varied with the individual diseases, and depended on the degree o f differentiation, in a similar fashion to the C P K activity. In all the cultures, the density o f silver grains was greater over the myotubes as c o m p a r e d with the background. The grains were fairly evenly distributed along the myotubes, although patches o f greater intensity sometimes occurred in the more highly differentiated cultures (Fig. 3B). The patches were approximately 20 la m in diameter which lies in the range o f " h o t spots" and A C h R clusters (5--60 lam) described by other workers (Vogel et al. 1972; Prives et al. 1976; Jacob and Lentz 1979). The grain density varied occasionally between different m y o t u b e s in the same culture and more frequently between m y o t u b e s in different cultures. In some cultures, toxin binding was also noted on other cells, which under phase contrast, appeared to be m o n o - or bi-nucleated, although the m o r p h o l o g y was indistinct. Figures 4A and C show a culture, from a patient with peripheral neuropathy, examined shortly after the onset o f myoblast fusion when there were a few thin
Fig. 4. Toxin binding to a human muscle from a patient with peripheral neuropathy grown for 11 days in culture. ,4 and (': two differen! fields of view on the same culture dish, phase contrast. B and D: dark field illuminations after [125I]~-Bgt binding which correspond to the same fields of view on A. C. respectively. Myotubes are labelled with :~-Bgt. Silver grams are also present on other cells, which may be mononucleated and some of these appear to be in the process of fusing. Bar = 50~m.
323 myotubes and many mononucleated cells. Toxin labelling was noted not only on the myotubes but also on other cells, some of which appeared to be aligned before fusion (Figs. 4B and D). The density of silver grains on these cells was comparable to that over the myotubes. Generally, the ~-Bgt binding over the whole plate was low. In a more differentiated culture labelled 5 days after the first myotubes were formed, the toxin binding was greater (Fig. 3A). Here, the myotubes were large, wide and branched with numerous nuclei, some of these having migrated to the sarcolemmal region of the myotubes. The silver grain density varied both along the length of the myotubes (Fig. 3B) and between different myotubes. The corresponding CPK activity was 0.3 lamole/min/plate (specific activity 1.7 ~tmole/min/mg protein, which also indicated a high degree of differentiation (Yasin et al. 1977)). In contrast, the cultures from D M D patients generally exhibited less toxin binding, lower CPK values and poor differentiation. The silver grains were located over the surface of myotubes present within the multi-layered cell clusters or spanning contiguous clusters• The grain density over the majority of the clustered cells was no greater than the background level. Figures 5A and C show such a
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Fig. 5. T o x i n b i n d i n g to a muscle culture from a p a t i e n t with D u c h e n n e m u s c u l a r d y s t r o p h y after 13 days g r o w t h (3 days after the first m y o t u b e s h a d formed). Silver grains are m a i n l y present over the m y o t u b e s in c o n t r a s t to the low level of grains over the m u l t i l a y e r e d cell cluster. T h i s culture e x h i b i t e d very p o o r differentiation, w i t h few m y o t u b e s a n d low levels o f C P K (see text). A a n d C: two different fields o f view on the same culture dish. Phase c o n t r a s t i l l u m i n a t i o n s h o w i n g a m u l t i l a y e r e d cell cluster. B a n d D : c o r r e s p o n d to A , C, respectively. D a r k field i l l u m i n a t i o n . Bar = 50 I,tm, cl = cluster.
324 culture examined 3 days after the commencement of myoblast fusion. This culture was very poorly differentiated and produced a few thin myotubes which were labelled with toxin (Figs. 5B and D). There were fewer silver grains than in the culture illustrated in Fig. 4 and the CPK value was 0.01 gmole/min/plate (specific
O
Fig. 6. Toxin binding to a muscle culture from a patient with D M D after 13 days growth (4 days after the onset of myoblast fusion). :c-Bgt labelling is visible over myotubes present in, emerging from, and spanning contiguous clusters. The clusters are more fully developed than in Fig. 5 but most of the cells do not show greater labelling than the background. A: phase contrast illumination. B: dark field illumination of the same field. Bar = 50 p_m, m t = myotube, cl : cluster.
Fig. 7. Muscle culture from a patient with D M D after 10 days growth (3 days after onset of myoblast fusion). The myotubes are labelled with varying intensity. Some of the nuclei have migrated from the centre of the lateral aspect of the myotubes, indicating a high degree of differentiation. This dystrophic culture did not form multilayered cell clusters. A and C: phase contrast illumination. B and D: dark field illumination. Corresponding to A, C, respectively. Bar = 501am.
325 activity, 0.28 lamole/min/mg protein) which was low in comparison with the values obtained from muscle cultures produced from patients with other diseases (0,53.0 pmoles/min/mg, Yasin et al. 1977). In another DMD muscle culture, labelled 4 days after the onset of myoblast fusion, the clusters had covered a larger surface area of the plate and become denser, although the overall differentiation remained poor. The toxin labelling was located over myotubes in or spanning clusters (Figs. 6A and B). We previously reported (Yasin et al. 1979) that 6 of the 29 DMD cultures prepared in our laboratories, did not exhibit this growth abnormality, although the disorders were diagnosed clinically as Duchenne dystrophies. An example is shown in Figs. 7A and C. The culture was well-differentiated, with large-branched myotubes containing many nuclei, some having migrated away from the centres of the myotubes towards the sarcolemma. The grain density varied along the length of some of the myotubes as well as between different myotubes, (Fig. 7B) in a similar manner to the culture shown in Fig. 3. The CPK value was also higher (0.5 ~tmole/min/plate, 1.8 lamole/min/mg protein) than that usually obtained for clustering DMD cultures (generally, 0.2-0.90 ~tmole/min/mg protein, Thompson et al. 1977). DISCUSSION
:~-Bgt bound to AChR in human muscle cultures with a similar specificity to that described for nicotinic receptors extracted from tissues (Franklin and Potter 1972) and receptors present in cultured muscles (Vogel et al. 1972; Patrick et al. 1977; Christian et al. 1978; Smilowitz and Fischbach 1978). The time course of ~-Bgt binding and the increase in receptor number with increasing differentiation are comparable with those recorded in our laboratories for neonatal mouse muscle cultures and by others for primary chick muscle cultures and for fusing and nonfusing cell lines (Patrick et al. 1972; Sytkowski et al. 1973; Prives et al. 1976; Christian et al. 1978; Smilowitz and Fischbach 1978). The rates of synthesis and degradation of the human muscle AChR were not measured as the amounts of biopsy material were insufficient to carry out such investigations. The level of ~-Bgt binding to cultured human muscle was lower than some of the values reported for other muscle cultures from various sources (Table 2) and was 5-fold lower than the level obtained for neonatal mouse muscle, in our laboratories. The autoradiographs demonstrated that, in the dystrophic cultures, the overall ~-Bgt labelling was low and binding was located on myotubes and occasionally on other cells. The myotubes were present most often within and spanning contiguous cell clusters. The presence of myotubes in these clusters has been confirmed by electron microscopy (Dr. D. Landon, unpublished data). Other cells in the clusters were not labelled with :~-Bgt and although it was difficult to be certain of their morphology, many appeared to be mononucleated. :~-Bgt bound to the myotubes in primary muscle cultures derived from patients with other neuromuscular diseases. The overall amount of labelling was variable and appeared to depend on the degree of differentiation, in parallel with the CPK
326 activity. Occasionally, in highly-differentiated cultures areas o f increased grain intensity were observed, which m a y be the " h o t s p o t s " described by others (Vogel et al. 1972; Prives et al. 1976; J a c o b a n d Lentz 1979). A difference in grain intensity between different m y o t u b e s was also n o t e d in these cultures a n d this c o u l d reflect different degrees o f m y o t u b e d e v e l o p m e n t (Prives et al. 1976). S o m e cells, o t h e r than m y o t u b e s , were labelled with toxin. Similar o b s e r v a t i o n s have been m a d e with m o n o n u c l e a t e d cells, b o t h in p r i m a r y cultures a n d in t r a n s f o r m e d cell lines a n d clones (Sytkowski et al. 1973: Smilowitz a n d F i s c h b a c h 1978; L i n k h a r t a n d H a u s c h k a 1979). In conclusion, m y o t u b e s f o r m e d in muscle cultures p r e p a r e d from biopsies from p a t i e n t s with v a r i o u s n e u r o m u s c u l a r disorders, p r o d u c e d A C h R which b i n d zt-Bgt in a similar d i s t r i b u t i o n to that d e m o n s t r a t e d for c u l t u r e d muscles f r o m o t h e r species. Cultures initiated f r o m D M D muscle exhibit similar toxin labelling a l o n g m y o t u b e s e m e r g i n g from a n d s p a n n i n g the m u l t i l a y e r e d cell clusters, as described previously, but the m a j o r i t y o f the o t h e r m o n o n u c l e a t e d cells in these clusters do not b i n d ~-Bgt. ACKNOWLEDGEMENTS W e w o u l d like to t h a n k Mrs. Elsa Phillips for her careful assistance in p r e p a r i n g the toxin a n d in the p r o t e i n d e t e r m i n a t i o n s a n d Mrs. Gisela Van Beers for her expertise in p r e p a r i n g a n d g r o w i n g the muscle cultures. REFERENCES Andrew, C.G., R. R. Almon and S. H. Appel (1974) Macromolecular characterization of muscle membranes, J. biol. Chem., 249:6163 6165. Christian, C. N., M.P. Daniels, H. Sugiyama, Z. Vogel, L. Jacques and P.G. Nelson (1978) A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells, Proc. Nat. Acad, Sci., U.S.A., 75:4011 4015. Darveniza, P., J.A. Morgan-Hughes, and E.J. Thompson (1979) Interaction of Di-iodinated 1251labelled ~-bungarotoxin and reversible cholinergic ligands with intact synaptic acetylcholine receptors on isolated skeletal muscle fibres from the rat, Biochem. J., 181 : 545 557. Diamond, J. and R. Miledi (1962) A study of foetal and newborn rat muscle fibres, J. Ph)siol. (Lond.), 162:393 408. Fambrough, D. M. and J. E. Rash (1971) Development of acetylcholine sensitivity during myogcnesis, Develop. Biol., 26:55 68. Franklin, G. I. and L.T. Potter (1972) Studies of the binding of ~-bungarotoxin to membrane-bound and detergent-dispersed acetylcholine receptors from Torpedo electric tissue, FEBS Lett., 28:101 106. Hartzell, C. H. and D. M. Fambrough (1973) Acetylcholine receptor production and incorporation into membranes of developing muscle fibres, Develop. Biol., 30:153 165. Jacob, M. and T.L. Lentz (1979) Localization of acetylcholine receptors by means of horseradish peroxidase-~bungarotoxin during formation and development of the neuromuscular junction in the chick embryo, J. Cell Biol., 82:195 211. Linkhart, T.A. and S.D. Hauschka (1979) Clonal analysis of vertebrate myogenesis, Part 6 (Acetylcholinesterase and acetylcholine receptor in myogenic and non-myogenic clones from chick embryo leg cells), Develop. Biol., 69 : 529 548. Lowry, O. H., N.J. Rosebrough, A. L. Far'r and R.J. Randall (1951) Protein measurements with the Folin phenol reagent, J. biol. Chem., 193:265 275.
327 M iledi, R. (1960) The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation, J. Physiol. (Lond.), 151 : 1-23. Noble, M. D., T. H. Brown and J.H. Peacock (1978) Regulation of acetylcholine receptor levels by a cholinergic agonist in mouse muscle cell cultures, Proc. Nat. Acad. Sei,. U.S.A., 75: 3488-3492. Patrick, J., J. McMillan, H. Wolfson and J.C. O'Brien (1977) Acetylcholine receptor metabolism in a non-fusing muscle cell line, J. biol. Chem., 252 : 2143-2153. Patrick, J., S.F. Heinemann, J. Lindstrom, D. Schubert and J.H. Steinback (1972) Appearance of acetylcholine receptors during differentiation of a myogenic cell line, Proc. Nat. Acad. Sci. U.S.A., 10:2762 2766. Prives, J., I. Silman and A. Amsterdam (1976) Appearance and disappearance of acetylcholine receptor during differentiation of chick skeletal muscle in vitro, Cell, 7: 543-550. Rosalki, S. B. (1967) An improved procedure for serum creatine phosphokinase determination, J. Lab. clin. Med., 69:696 705. SmiloWitz, H. and G.D. Fischbach (1978) Acetylcholine receptors on chick mononucleated muscle precursor cells, Develop. Biol., 66: 539-549. Sytkowski, A. J., Z. Vogel and M.W. Nirenberg (1973) Development of acetylcholine receptor clusters on cultured muscle cells, Proc. Nat. Acad. Sci. U.S.A., 70:270 274. Thompson, E.J., R. Yasin, G. Van Beers, K. Nurse and S. Al-Ani (1977) Myogenic defect in human muscular dystrophy, Nature (Lond.), 268 : 241 243. Turner, D. C., R. Gmfir, M. Siegrist, E. Burckhardt and H.M. Eppenberger (1976) Differentiation in cultures derived from embryonic chicken muscle, Develop. Biol., 48 : 258-283. Vogel, Z., A. J. Sytkowski and M. W. Nirenberg (1972) Acetylcholine receptors of muscle grown in vitro, Proe. Nat. Acad. Sci. U.S.A., 69: 3180-3184. Yasin, R., G. Van Beers, K.C.E. Nurse, S. A1-Ani, D.N. Landon and E.J. Thompson (1977) A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells, J. neurol. Sci., 32 : 347 360. Yasin, R., G. Van Beers, P.N. Riddle, D. Brown, G. Widdowson and E.J. Thompson (1979) An abnormality of cell behaviour in human dystrophic muscle cultures - - A time-lapse study, J. Cell Sci., 38:201 210.
317
© Elsevier/North-Holland Biomedical Press
ACETYLCHOLINE RECEPTORS IN CULTURED HUMAN MUSCLE CELLS
GILLIAN I. FRANKLIN*, ROSE YASIN, BEAUMONT P. HUGHES and EDWARD J. THOMPSON
Muscular Dystrophy Research Laboratories, Institute o["Neurology, Queen Square House, Queen Square, London WC1N 3BG (Great Britain) (Received 20 December, 1979) (Accepted I I April, 1980)
SUMMARY
Primary cultures prepared from human muscle biopsies were examined for the presence and distribution of acetylcholine receptors, as measured by the binding of ~2Siodine-labellede-bungarotoxin. The toxin bound to the human muscle cultures with a similar time dependence and specificity as found in muscle cultures from other species. The amount of toxin bound was lower than that obtained for neonatal mouse muscle under similar conditions. The distribution of receptors was similar in cultures derived from the muscles of patients with a variety of neuromuscular disorders. The toxin was located along the myotubes in a fairly even distribution; however, variations in labelling along a single myotube were observed, as well as variations between different myotubes in the same culture. Occasionally, the toxin also bound to other cells, which may have been mononucleated. Cultures prepared from patients with Duchenne muscular dystrophy produced multi-layered cell clusters, instead of the usual monolayer of cells. Within these clusters, only the myotubes bound e-bungarotoxin.
INTRODUCTION
Studies of the initial stages of the development of muscle cells in culture have shown that dividing mononucleated cells lack acetylcholine receptors (AChR) (Patrick et al. 1972; Sytkowski et al. 1973): With increasing differentiation, myoblasts fuse to form multinucleated myotubes which are characterized by a uniform This work was supported by the Muscular Dystrophy Group of Great Britain. * To whom correspondence and reprint requests should be sent.
318 distribution of AChR along their entire surfaces (Vogel et al. 1972; Sytkowski et al. 1973; Prives et al. 1976). This localization resembles that tbund in denervated muscles (Miledi 1960) and embryonic muscle fibres in vivo (Diamond and Miledi 1962). Subsequently, as the myotubes develop further, in culture, the receptor density becomes non-uniform, with randomly-distributed "'hot spots" (Sytkowski et al. 1973; Prives et al. 1976; Jacob and Lentz 1979). The increase in AChR with the onset ofmyoblast fusion is analogous to that described for other muscle specific proteins, e.g. creatine phosphokinase (CPK) (Turner et al. 1976) and therefore both AChR and CPK have been utilized as markers of muscle differentiation (Patrick et al. 1972; Prives et al. 1976; Turner et al. 1976). AChR have also been used as markers for the muscle cell surface membrane (Andrew et al. 1974). Primary cultures initiated from human muscle biopsies develop in a similar manner to those prepared from foetal and neonatal muscles, namely, myoblasts fuse to form myotubes; however, the degree of differentiation obtained varies with individual diseases (Yasin et al. 1977). Primary cultures derived from patients with Duchenne muscular dystrophy (DMD) or Becker dystrophies exhibit an abnormal growth pattern. Instead of growing as a monolayer on the culture dish, some of the cells form multilayered clusters prior to myoblast fusion (Thompson et al. 1977; Yasin et al. 1979). When the myoblasts fuse, the myotubes often emerge from these clusters or span contiguous clusters. The production of cell clusters may be consistent with a defect in the cell surface membrane or in the cytoskeletal structure (Thompson et al. 1977). This investigation was undertaken to ascertain whether human muscle cultures could produce AChR and if the receptor distribution was similar in cultures from patients with D M D as compared with those from patients with various other neuromuscular diseases. The results show that all the muscle cultures produced AChR, as measured by the binding of ~25iodine-labelled ~-bungarotoxin (c~-Bgt), and that these AChR were located mainly in a similar distribution. In the multilayered cell clusters, produced by the D M D cultures, the toxin bound only to the myotubes. MATERIALS AND METHODS Phosphate-buffered saline (PBS) contained 144 mM NaC1, 5.4 mM KCI, 25 mM glucose, 25 mM sucrose and 5 mM sodium phosphate (340 mOsm, pH 7.3 at 37 °C). d-Tubocurarine (15 mg/1.5 ml saline) was purchased from Duncan Flockhardt & Co. Ltd., London. Bovine serum albumin (Sigma Fraction V) was prepared as a 100 (w/v) solution in PBS, hereafter called PBS-A. ~251odine-labelled 7-bungarotoxin (specific activity 300 400 Ci/mmole) was a gift from Dr. P. Darveniza (Institute of Neurology) and was prepared as described elsewhere (Darveniza et al. 1979). The di-iodinated toxin was diluted and stored at - 2 0 ° C , as l0 nM aliquots in PBS-A, for not longer than 80 days. K2 emulsion lbr autoradiography and FF developer contrast 1 were purchased from Ilford Ltd., and Amfix from May & Baker Ltd. Human muscle cultures were dissociated and grown from biopsy specimens
319 as described by Yasin et al. (1977, 1979). Cells were seeded at 6 × 1 0 4 cells per 3.5 cm culture dish. For ~-Bgt binding studies, the culture dishes were washed 3 times with 2 ml of PBS-A and incubated with 1.5 ml of PBS-A for 20 min at 37°C in air. This was removed and replaced with 1.5 ml containing various concentrations of ~-Bgt and further incubated at 37 °C in air. In order to estimate the level of non-specific binding, d-tubocurarine (final concentration, 1 mM in PBS-A), was included in the 20-min pre-incubation and in the incubation mixture with ~-Bgt. After the incubation, unbound 7-Bgt was removed, the cultures washed 3 times with 1.5 ml PBS-A and 3 times with 1.5 ml PBS. The cells were scraped off the surface of the culture dish with a teflon-tape-coated spatula into a small volume of PBS and the amount of toxin bound was measured by counting in a L.K.B. gamma counter. The specific binding was determined from the difference between the toxin bound in the absence of tubocurarine and that bound in its presence. For autoradiography, the cultures were incubated with toxin, in a similar manner to the binding experiments, and then fixed with 2~o (v/v) glutaraldehyde (EM scope, London) in 50 mM cacodylate buffer, pH 7.4 (2 ml per dish) for 1 h at room temperature, dehydrated and dried (Sytkowski et al. 1973). The cultures were coated with K2 emulsion (10 parts K2 : 2 parts H20 : 0.1 part glycerol) and incubated for 3 8 weeks at 4 °C in a light-proof box containing desiccant. The autoradiographs were developed with F F developer and fixed with Amfix and hardener at room temperature. The cultures were photographed under phase contrast and dark field illumination with a Leitz Orthomat microscope. For the CPK assays, cultures were washed 3 times with 1.5 ml PBS and scraped into a small volume of PBS, on ice. The cells were ruptured by sonication with an MSE sonicator for 3 s under nitrogen atmosphere, on ice. This procedure was repeated twice, with two 5-s cooling intervals. CPK activity was measured spectrophotometrically by the method of Rosalki (1967) using a Boehringer UVsystem CPK activated kit (number 124 150). The enzyme activity was expressed as lamole creatine produced/min/culture plate at 30°C. Protein concentrations were determined by the Lowry method (1951) with human serum albumin as standard. RESULTS
Saturation of human muscle A C h R occurred within 20 min (Fig. 1), and remained stable for at least 90 min, under the conditions used. The binding curve was similar to that obtained for neonatal mouse muscle (data not shown) under similar conditions. Between 7 0 ~ and 90~o of the total ~-Bgt binding was inhibited by 1 mM tubocurarine, a known A C h R antagonist. The amount of ~-Bgt bound increased 10-fold with fusion of myoblasts to myotubes (Fig. 2 and Table 1). An autoradiograph of a culture (from a patient with acute polymyositis) corresponding to those used in this binding study is shown in Fig. 3B, after 12 days growth in vitro. A concomitant increase in the CPK levels with myoblast fusion was noted (Table 1), similar to that described for muscle cultures from various sources (Turner et al. 1976; Yasin et al. 1977).
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Fig. 1. Rate o f binding of [12Sl]di_iodo.z~_bungarotoxin to cultures of h u m a n muscle from a patient with minicore myopathy. Culture plates were washed and pre-incubated with PBS-A for 20 min and then incubated at 37°C with 10 nM 2-Bgt in PBS-A. At given time intervals, plates were removed and washed with PBS-A and PBS. Cells were scraped from the dish and the a m o u n t of 2-Bgt binding measured in a ;'-counter. Values for non-specific binding were obtained by preincubation and incubation in the presence of 1 m M d-tubocurarine and were then subtracted from the values for the total binding to yield values of specific toxin binding. Protein concentrations (mg) were determined by the Lowry method.
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Fig. 2. Specific binding of 2-bungarotoxin to h u m a n muscle cultures, from a patient with acute polymyositis, grown in vitro for different periods. The experimental details are given in the text. The arrow denotes the onset of myoblast fusion.
U n d e r saturating conditions the a m o u n t o f :~-Bgt b o u n d specifically was 11.09 +_ 5.93 fmole/culture (for 7 determinations from 5 different biopsies). This was lower than the value obtained for neonatal m o u s e muscle cultures under similar conditions (51.4 + 34.4 fmole/culture) and was near the lower end o f the ranges published for other m a m m a l i a n muscles in culture (Table 2).
TABLE 1 ~ - B U N G A R O T O X I N B I N D I N G A N D C P K LEVELS IN R E P L I C A T E H U M A N M U S C L E CULTURES DERIVED FROM A PATIENT WITH ACUTE POLYMYOSITIS The morphology of this culture on day 12 is shown in Fig. 3A and the corresponding autoradiograph in Fig. 3B. Days in vitro
Specific fmoles [J25I]c~-Bgt bound/culture dish
5 7 8 11 12 14
0.8 0.3 7.4 10.8 10.8
C P K p-moles creatine/min/plate at 30 °C
0.02
0.26
Fig. 3. 2-Bgt binding to a muscle culture from a patient with acute polymyositis after 12 days growth (5 days after the onset of myoblast fusion). A : morphology under phase contrast. B: dark field illumination of silver grains in autoradiograph of the same field of view. The grain density varied along the length of the myotubes, which were of large diameter (50 lam) and branched and had a high level of C P K (specific activity 1.4gmoles/min/mg protein at peak differentiation). Corresponding cultures were utilized for the time-course of receptor appearance shown in Fig. 2. Bar = 50 gin. TABLE 2 A M O U N T S OF :t-Bgt B O U N D T O H U M A N A N D V A R I O U S O T H E R M U S C L E SPECIES IN CULTURE Muscle
Total fmole b o u n d a per mg protein
Specific fmole/cm 2
Reference
Human [ 7] N e o n a t a l m o u s e [19] Embryonic mouse Embryonic chick Embryonic chick G8 mouse line L6 rat line L6 rat line L8 rat line
81.3_+ 51.1 366 +_ 138 -150 - 1000 -20 - 170 52 400 b 14
1.2 5.3 4.6 -10.6 --13.5b (approx.) --
this study this study Christian et al. 1978 Vogel et al. 1972 Prives et al, 1976 Noble et al. 1978 Vogel et al. 1972 Patrick et al. 1972 Vogel et al. 1972
•
a Includes non-specific (curare insensitive) binding. b Specific binding of the closely related Naja naja toxin. N u m b e r of cultures in parentheses.
322 The conditions for a u t o r a d i o g r a p h y were chosen for maximal binding, viz. 1 h at 37°C with 10 n M toxin. Binding was located by silver grains in the emulsion, which were visualized under dark field illumination and phase contrast microscopy was used to view cell morphology. Cultures were examined from 4 patients with D M D , 2 with Becker dystrophy and 12 patients with various other neuromuscular disorders. The a m o u n t s o f ~-Bgt b o u n d to the cultures varied with the individual diseases, and depended on the degree o f differentiation, in a similar fashion to the C P K activity. In all the cultures, the density o f silver grains was greater over the myotubes as c o m p a r e d with the background. The grains were fairly evenly distributed along the myotubes, although patches o f greater intensity sometimes occurred in the more highly differentiated cultures (Fig. 3B). The patches were approximately 20 la m in diameter which lies in the range o f " h o t spots" and A C h R clusters (5--60 lam) described by other workers (Vogel et al. 1972; Prives et al. 1976; Jacob and Lentz 1979). The grain density varied occasionally between different m y o t u b e s in the same culture and more frequently between m y o t u b e s in different cultures. In some cultures, toxin binding was also noted on other cells, which under phase contrast, appeared to be m o n o - or bi-nucleated, although the m o r p h o l o g y was indistinct. Figures 4A and C show a culture, from a patient with peripheral neuropathy, examined shortly after the onset o f myoblast fusion when there were a few thin
Fig. 4. Toxin binding to a human muscle from a patient with peripheral neuropathy grown for 11 days in culture. ,4 and (': two differen! fields of view on the same culture dish, phase contrast. B and D: dark field illuminations after [125I]~-Bgt binding which correspond to the same fields of view on A. C. respectively. Myotubes are labelled with :~-Bgt. Silver grams are also present on other cells, which may be mononucleated and some of these appear to be in the process of fusing. Bar = 50~m.
323 myotubes and many mononucleated cells. Toxin labelling was noted not only on the myotubes but also on other cells, some of which appeared to be aligned before fusion (Figs. 4B and D). The density of silver grains on these cells was comparable to that over the myotubes. Generally, the ~-Bgt binding over the whole plate was low. In a more differentiated culture labelled 5 days after the first myotubes were formed, the toxin binding was greater (Fig. 3A). Here, the myotubes were large, wide and branched with numerous nuclei, some of these having migrated to the sarcolemmal region of the myotubes. The silver grain density varied both along the length of the myotubes (Fig. 3B) and between different myotubes. The corresponding CPK activity was 0.3 lamole/min/plate (specific activity 1.7 ~tmole/min/mg protein, which also indicated a high degree of differentiation (Yasin et al. 1977)). In contrast, the cultures from D M D patients generally exhibited less toxin binding, lower CPK values and poor differentiation. The silver grains were located over the surface of myotubes present within the multi-layered cell clusters or spanning contiguous clusters• The grain density over the majority of the clustered cells was no greater than the background level. Figures 5A and C show such a
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Fig. 5. T o x i n b i n d i n g to a muscle culture from a p a t i e n t with D u c h e n n e m u s c u l a r d y s t r o p h y after 13 days g r o w t h (3 days after the first m y o t u b e s h a d formed). Silver grains are m a i n l y present over the m y o t u b e s in c o n t r a s t to the low level of grains over the m u l t i l a y e r e d cell cluster. T h i s culture e x h i b i t e d very p o o r differentiation, w i t h few m y o t u b e s a n d low levels o f C P K (see text). A a n d C: two different fields o f view on the same culture dish. Phase c o n t r a s t i l l u m i n a t i o n s h o w i n g a m u l t i l a y e r e d cell cluster. B a n d D : c o r r e s p o n d to A , C, respectively. D a r k field i l l u m i n a t i o n . Bar = 50 I,tm, cl = cluster.
324 culture examined 3 days after the commencement of myoblast fusion. This culture was very poorly differentiated and produced a few thin myotubes which were labelled with toxin (Figs. 5B and D). There were fewer silver grains than in the culture illustrated in Fig. 4 and the CPK value was 0.01 gmole/min/plate (specific
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Fig. 6. Toxin binding to a muscle culture from a patient with D M D after 13 days growth (4 days after the onset of myoblast fusion). :c-Bgt labelling is visible over myotubes present in, emerging from, and spanning contiguous clusters. The clusters are more fully developed than in Fig. 5 but most of the cells do not show greater labelling than the background. A: phase contrast illumination. B: dark field illumination of the same field. Bar = 50 p_m, m t = myotube, cl : cluster.
Fig. 7. Muscle culture from a patient with D M D after 10 days growth (3 days after onset of myoblast fusion). The myotubes are labelled with varying intensity. Some of the nuclei have migrated from the centre of the lateral aspect of the myotubes, indicating a high degree of differentiation. This dystrophic culture did not form multilayered cell clusters. A and C: phase contrast illumination. B and D: dark field illumination. Corresponding to A, C, respectively. Bar = 501am.
325 activity, 0.28 lamole/min/mg protein) which was low in comparison with the values obtained from muscle cultures produced from patients with other diseases (0,53.0 pmoles/min/mg, Yasin et al. 1977). In another DMD muscle culture, labelled 4 days after the onset of myoblast fusion, the clusters had covered a larger surface area of the plate and become denser, although the overall differentiation remained poor. The toxin labelling was located over myotubes in or spanning clusters (Figs. 6A and B). We previously reported (Yasin et al. 1979) that 6 of the 29 DMD cultures prepared in our laboratories, did not exhibit this growth abnormality, although the disorders were diagnosed clinically as Duchenne dystrophies. An example is shown in Figs. 7A and C. The culture was well-differentiated, with large-branched myotubes containing many nuclei, some having migrated away from the centres of the myotubes towards the sarcolemma. The grain density varied along the length of some of the myotubes as well as between different myotubes, (Fig. 7B) in a similar manner to the culture shown in Fig. 3. The CPK value was also higher (0.5 ~tmole/min/plate, 1.8 lamole/min/mg protein) than that usually obtained for clustering DMD cultures (generally, 0.2-0.90 ~tmole/min/mg protein, Thompson et al. 1977). DISCUSSION
:~-Bgt bound to AChR in human muscle cultures with a similar specificity to that described for nicotinic receptors extracted from tissues (Franklin and Potter 1972) and receptors present in cultured muscles (Vogel et al. 1972; Patrick et al. 1977; Christian et al. 1978; Smilowitz and Fischbach 1978). The time course of ~-Bgt binding and the increase in receptor number with increasing differentiation are comparable with those recorded in our laboratories for neonatal mouse muscle cultures and by others for primary chick muscle cultures and for fusing and nonfusing cell lines (Patrick et al. 1972; Sytkowski et al. 1973; Prives et al. 1976; Christian et al. 1978; Smilowitz and Fischbach 1978). The rates of synthesis and degradation of the human muscle AChR were not measured as the amounts of biopsy material were insufficient to carry out such investigations. The level of ~-Bgt binding to cultured human muscle was lower than some of the values reported for other muscle cultures from various sources (Table 2) and was 5-fold lower than the level obtained for neonatal mouse muscle, in our laboratories. The autoradiographs demonstrated that, in the dystrophic cultures, the overall ~-Bgt labelling was low and binding was located on myotubes and occasionally on other cells. The myotubes were present most often within and spanning contiguous cell clusters. The presence of myotubes in these clusters has been confirmed by electron microscopy (Dr. D. Landon, unpublished data). Other cells in the clusters were not labelled with :~-Bgt and although it was difficult to be certain of their morphology, many appeared to be mononucleated. :~-Bgt bound to the myotubes in primary muscle cultures derived from patients with other neuromuscular diseases. The overall amount of labelling was variable and appeared to depend on the degree of differentiation, in parallel with the CPK
326 activity. Occasionally, in highly-differentiated cultures areas o f increased grain intensity were observed, which m a y be the " h o t s p o t s " described by others (Vogel et al. 1972; Prives et al. 1976; J a c o b a n d Lentz 1979). A difference in grain intensity between different m y o t u b e s was also n o t e d in these cultures a n d this c o u l d reflect different degrees o f m y o t u b e d e v e l o p m e n t (Prives et al. 1976). S o m e cells, o t h e r than m y o t u b e s , were labelled with toxin. Similar o b s e r v a t i o n s have been m a d e with m o n o n u c l e a t e d cells, b o t h in p r i m a r y cultures a n d in t r a n s f o r m e d cell lines a n d clones (Sytkowski et al. 1973: Smilowitz a n d F i s c h b a c h 1978; L i n k h a r t a n d H a u s c h k a 1979). In conclusion, m y o t u b e s f o r m e d in muscle cultures p r e p a r e d from biopsies from p a t i e n t s with v a r i o u s n e u r o m u s c u l a r disorders, p r o d u c e d A C h R which b i n d zt-Bgt in a similar d i s t r i b u t i o n to that d e m o n s t r a t e d for c u l t u r e d muscles f r o m o t h e r species. Cultures initiated f r o m D M D muscle exhibit similar toxin labelling a l o n g m y o t u b e s e m e r g i n g from a n d s p a n n i n g the m u l t i l a y e r e d cell clusters, as described previously, but the m a j o r i t y o f the o t h e r m o n o n u c l e a t e d cells in these clusters do not b i n d ~-Bgt. ACKNOWLEDGEMENTS W e w o u l d like to t h a n k Mrs. Elsa Phillips for her careful assistance in p r e p a r i n g the toxin a n d in the p r o t e i n d e t e r m i n a t i o n s a n d Mrs. Gisela Van Beers for her expertise in p r e p a r i n g a n d g r o w i n g the muscle cultures. REFERENCES Andrew, C.G., R. R. Almon and S. H. Appel (1974) Macromolecular characterization of muscle membranes, J. biol. Chem., 249:6163 6165. Christian, C. N., M.P. Daniels, H. Sugiyama, Z. Vogel, L. Jacques and P.G. Nelson (1978) A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells, Proc. Nat. Acad, Sci., U.S.A., 75:4011 4015. Darveniza, P., J.A. Morgan-Hughes, and E.J. Thompson (1979) Interaction of Di-iodinated 1251labelled ~-bungarotoxin and reversible cholinergic ligands with intact synaptic acetylcholine receptors on isolated skeletal muscle fibres from the rat, Biochem. J., 181 : 545 557. Diamond, J. and R. Miledi (1962) A study of foetal and newborn rat muscle fibres, J. Ph)siol. (Lond.), 162:393 408. Fambrough, D. M. and J. E. Rash (1971) Development of acetylcholine sensitivity during myogcnesis, Develop. Biol., 26:55 68. Franklin, G. I. and L.T. Potter (1972) Studies of the binding of ~-bungarotoxin to membrane-bound and detergent-dispersed acetylcholine receptors from Torpedo electric tissue, FEBS Lett., 28:101 106. Hartzell, C. H. and D. M. Fambrough (1973) Acetylcholine receptor production and incorporation into membranes of developing muscle fibres, Develop. Biol., 30:153 165. Jacob, M. and T.L. Lentz (1979) Localization of acetylcholine receptors by means of horseradish peroxidase-~bungarotoxin during formation and development of the neuromuscular junction in the chick embryo, J. Cell Biol., 82:195 211. Linkhart, T.A. and S.D. Hauschka (1979) Clonal analysis of vertebrate myogenesis, Part 6 (Acetylcholinesterase and acetylcholine receptor in myogenic and non-myogenic clones from chick embryo leg cells), Develop. Biol., 69 : 529 548. Lowry, O. H., N.J. Rosebrough, A. L. Far'r and R.J. Randall (1951) Protein measurements with the Folin phenol reagent, J. biol. Chem., 193:265 275.
327 M iledi, R. (1960) The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation, J. Physiol. (Lond.), 151 : 1-23. Noble, M. D., T. H. Brown and J.H. Peacock (1978) Regulation of acetylcholine receptor levels by a cholinergic agonist in mouse muscle cell cultures, Proc. Nat. Acad. Sei,. U.S.A., 75: 3488-3492. Patrick, J., J. McMillan, H. Wolfson and J.C. O'Brien (1977) Acetylcholine receptor metabolism in a non-fusing muscle cell line, J. biol. Chem., 252 : 2143-2153. Patrick, J., S.F. Heinemann, J. Lindstrom, D. Schubert and J.H. Steinback (1972) Appearance of acetylcholine receptors during differentiation of a myogenic cell line, Proc. Nat. Acad. Sci. U.S.A., 10:2762 2766. Prives, J., I. Silman and A. Amsterdam (1976) Appearance and disappearance of acetylcholine receptor during differentiation of chick skeletal muscle in vitro, Cell, 7: 543-550. Rosalki, S. B. (1967) An improved procedure for serum creatine phosphokinase determination, J. Lab. clin. Med., 69:696 705. SmiloWitz, H. and G.D. Fischbach (1978) Acetylcholine receptors on chick mononucleated muscle precursor cells, Develop. Biol., 66: 539-549. Sytkowski, A. J., Z. Vogel and M.W. Nirenberg (1973) Development of acetylcholine receptor clusters on cultured muscle cells, Proc. Nat. Acad. Sci. U.S.A., 70:270 274. Thompson, E.J., R. Yasin, G. Van Beers, K. Nurse and S. Al-Ani (1977) Myogenic defect in human muscular dystrophy, Nature (Lond.), 268 : 241 243. Turner, D. C., R. Gmfir, M. Siegrist, E. Burckhardt and H.M. Eppenberger (1976) Differentiation in cultures derived from embryonic chicken muscle, Develop. Biol., 48 : 258-283. Vogel, Z., A. J. Sytkowski and M. W. Nirenberg (1972) Acetylcholine receptors of muscle grown in vitro, Proe. Nat. Acad. Sci. U.S.A., 69: 3180-3184. Yasin, R., G. Van Beers, K.C.E. Nurse, S. A1-Ani, D.N. Landon and E.J. Thompson (1977) A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells, J. neurol. Sci., 32 : 347 360. Yasin, R., G. Van Beers, P.N. Riddle, D. Brown, G. Widdowson and E.J. Thompson (1979) An abnormality of cell behaviour in human dystrophic muscle cultures - - A time-lapse study, J. Cell Sci., 38:201 210.