Journal of the Neurological Sciences, 1979, 41 : 115-122
© Elsevier/North-Holland Biomedical Press
115
M A T U R A T I O N OF H U M A N S K E L E T A L M U S C L E FIBRES I N E X P L A N T TISSUE C U L T U R E
A. L. HARVEY, J. G. ROBERTSON and J. A. WITKOWSKI Department of Physiology and Pharmacology (ALH, JGR), University of Strathclyde, Glasgow, G1 1XW and Jerry Lewis Muscle Research Centre (JAW), Department of Paediatrics, Hammersmith Hospital, London, W12 OHS (Great Britain)
(Received 9 October, 1978) (Accepted 19 October, 1978)
SUMMARY H u m a n foetal skeletal muscle was grown in explant cultures and the development of myotubes was monitored morphologically and by the development of membrane potential and acetylcholine sensitivity. Migration of uninucleate cells from the explants occurred in the first day of culture and formation of multinucleated myotubes took place between 7 to 10 days. Early myotubes were variable in appearance, being either flat and nonrefractile or narrow and cylindrical. Some cross-striated cells were observed. Resting membrane potentials were around - - 2 5 mV and there was great variation in myotube sensitivity to acetylcholine. After about 6 weeks in culture most myotubes were of the refractile type. Many had hypolemmal nuclei and were cross-striated; some contracted spontaneously. All myotubes had developed high sensitivity to acetylcholine although there had been no increase in resting membrane potential.
INTRODUCTION Use of tissue culture techniques would facilitate the study of human skeletal muscle if mature muscle fibres could develop in culture conditions. Earlier reports have concentrated on morphological or biochemical studies (for review see Witkowski 1977). Although it has been claimed that mature human muscle cells develop in culture This study was supported by grants from the Muscular Dystrophy Association Inc. (to ALH) and the Muscular Dystrophy Group of Great Britain (to JAW). The authors thank the Medical Research Council for a training award to JGR and the Smith Kline and French Foundation for an equipment grant (to ALH).
116 (Askanas and Engel 1976: Yasin et al. 1977), these reports have been based on cell morphology and the presence of muscle-specific enzymes e.g. creatine phosphokinase. It is clearly important to determine if cells that appear mature by these criteria have developed electrophysiological properties. Recent reports (Bevan et al. 1977, 1978) have demonstrated that human muscle cells in culture respond to the physiological neurotransmitter acetylcholine, but these authors made no attempt to relate their results with the structural development of the cells. We have prepared cultures from explants of human foetal skeletal muscle and have correlated morphological development of myotubes with functional maturation as revealed by resting membrane potential and sensitivity to acetylcholine. Our results show that fibres appeared morphologically well differentiated before the appearance of high acetylcholine sensitivity and that despite the subsequent development of high acetylcholine sensitivity, the fibres remained relatively immature since their resting membrane potentials did not reach normal levels (about - - 3 0 mV compared to about - - 8 0 mV). METHODS Tissue culture H u m a n foetal skeletal muscle was obtained from the M R C Tissue Bank at the Royal Marsden Hospital. Samples were stored overnight at 14 °C in complete growth medium; storage at 14 °C has no effect on cell viability (Witkowski et al. 1976). Details of muscle samples are presented in Table 1. Growth medium was Medium 199 (with Hanks' salt solution), with 10 ~ foetal calf serum, penicillin (50 IU/ml), streptomycin (50/~g/ml) and 15 m M HEPES. After myotube formation the cultures were maintained in medium containing 2.5 ~o foetal calf serum. All cultures were explant cultures prepared in gelatin-coated (Hauschka 1972) TABLE 1 RESTING MEMBRANE POTENTIALS AND ACETYLCHOLINE SENSITIVITY OF HUMAN MUSCLE CULTURES AT DIFFERENT AGES AND FROM DIFFERENT SOURCES Preparation
Days in culture
No. of cells Mean RMP ~ sensitive Mean maximum tested (-mV) cells response (depolarization as ~ of RMP)
17.5-week foetus (R.6) ll.5-week foetus (R.5) 12-week foetus (R.3)
13 18 24
15 18 17
20.7 i 2.3 28.4 -4- 2.5 28.3 ± 2.8
86 56 76
26 J_ 5.3 38 :t: 8.4 35 :t: 5.5
12-week foetus (R.3)
31
13
21.9 ± 2.8
61
29:5 7.7
21-week foetus (R.4)
36
23
18.t ± 1.7
52
30 ~ 5.7
12-week foetus (R.3) ll.5-week foetus (R.5)
47 77
12 17
27.0 :L 2.6 29.2 ~ 2.6
100 100
89 & 3.1 56 ~ 4.9
117 35-mm petri dishes. Muscle samples were cut into 1-mm a pieces and up to 16 explants were arranged in a grid-pattern in the dish and covered with a 22 mm x 22 mm glass coverslip. 1.5 ml of medium were added to each dish and medium was changed once weekly. With cultures that were sent to Glasgow by post, coverslips were removed from each petri dish, the dishes filled with medium and the lids sealed with sterile silicone grease. The cells did not appear to be adversely affected by their treatment, and cultures were incubated at 37 °C for at least 2 hr before electrophysiological studies began.
Electrophysiology Conventional electrophysiological techniques were used to measure muscle cell resting membrane potentials and responses to iontophoretic application of acetylcholine. Microelectrodes for iontophoresis were filled with 0.5 M acetylcholine chloride and connected to a stimulator through one channel of a WP1 750 electrometer. The resistance of the acetylcholine electrode was 100-200 megohm and care was taken to select electrodes that did not leak spontaneously. Cultures were maintained at 37 °C and p H 7.4 during experiments. RESULTS
Cell growth Migration of uninucleate cells from explants was observed after 24 hr culture (Fig. 1A) and the first multinucleate myotubes were seen after 7-10 days in vitro. Fusion of myoblasts was partially synchronised and took place in a 48-hr period. The first myotubes formed were broad and non-refractile with clearly visible nuclei (Fig. 1B) but these subsequently gave rise to long narrow refractile myotubes (Fig. 1C). When medium containing only 2.5 70 foetal calf serum was used, division of uninucleate cells was inhibited so that the myotubes were not overgrown but appeared to lie on a layer of uninucleate cells. After 3-4 weeks in culture, both types of myotube were present, together with a small number showing a mixed morphology, i.e. at either end of the cell there was a fiat portion containing nuclei while the central region was narrow and refractile (Fig. 1D). In 4-6 week cultures many of the myotubes were cross-striated (Fig. 1E) and some cells contracted spontaneously. The organization of myofibrils into cross-striations appeared to be greater in refractile myotubes than in flat myotubes, although cells of both forms were seen to contract. Cultures were maintained for up to 11 weeks by which time the morphology of the myotubes had changed from the straight, refractile form to a more flattened form (Fig. 1F). The myotubes were no longer straight but were thrown into waves. Contracting myotubes were also seen in these "old" cultures.
Membrane potential and acetylcholine sensitivity Uninucleated cells with the bipolar appearance of myoblasts had low resting
118
Fig. 1. Phase contrast micrographs of cultures of human foetal muscle. Scale bar represents 50 fro1. A: uninucleate cells; myoblasts cannot be distinguished from fibroblasts with any certainty. (7 days in vitro). B: early stages of myotube formation with broad flat myotubes (MT) and centrally placed nuclei (arrows). C: a number of refractile myotubes with cross-striations and hypolemmal nuclei. These myotubes were typical of the muscle cells found in cultures older than 6 weeks in vitro. D: myotube showing intermediate morphology with flattened area (a) and refractile region (b). (18 days in vitro). E: portion of a refractile myotube with cross-striations and hypolemmal nuclei. This cell was about 2.3 mm long and was contracting spontaneously (18 days in vitro). F: a flattened, non-refractile myotube in an I 1-week culture. Hypolemmal nuclei were still present (N).
119 0mV
-23mV
~
0.5 2
,
8
32
,
~
'
64-34mV
o'.s
;
;2nC ACh
10 mV l 2s
Fig. 2. Responses of two myotubes to iontophoretic application of acetylcholine. Increasing doses of acetylcholine (expressed as nanocoulombs, nC, charge passed) produced increasing depolarizations. Note that both cells depolarized close to 0 mV although the resting membrane potentials differed by 11 mV. m e m b r a n e potentials (--8.0 4- 0.6 mV; mean i S E M of 26 cells in 2 cultures) and did not respond to iontophoretic application of acetylcholine. In the 4-6 week old cultures in which the two morphological types o f myotubes were found, there was no consistent difference in m e m b r a n e potentials between either group o f cells: the average for the flat myotubes was - - 2 4 . 7 4- 2.6 mV (n = 27) and for the rounded m y o t u b e s the mean was - - 2 1 . 0 4- 1.8 mV (n = 30). The sensitivity to acetylcholine was tested in b o t h types o f cells and although the difference was n o t absolute, the retractile myotubes exhibited greater responsiveness than the fiat cells. In 3 cultures 10 out of 23 fiat cells tested (43 ~ ) responded to acetylcholine whereas 21 out o f 29 rounded cells (72 ~ ) responded. The cells which were sensitive responded to increasing doses o f acetylcholine with increasing depolarizations (Fig. 2) although there was a wide variation in the level of sensitivity between individual responsive cells (Fig. 3). The variation in response was not correlated with the resting m e m b r a n e potential as fibres with potentials as low as - - 2 2 mV could show a 22 mV depolarization but some fibres with higher po-
100 o.
so -
~
60 -
u 40 g 2O '=
~
0
I
0'5
I
1.0
I
10 Acetylcholine
f
lOOnC
Fig. 3. Responses to acetylcholine of myotubes in a 24-day culture of muscle explanted from a 12-week human foetus. Responses of the cell with the greatest sensitivity (V), and the cell with the lowest detectable sensitivity (A) and the mean level of sensitivity (A) are shown. Standard errors of the means are shown unless smaller than the symbol (n = 13). Acetylcholine dose is expressed as nanocoulombs (nC) of charge passed.
120
F
-: '°1•~
t
0 I._
Fig. 4. Responses to acetylcholine of myotubes in explant cultures from a 12-week human foetus at 24 days (~), 31 days (V) and 47 days (0) in culture. Standard errors bars are shown unless smaller than the symbols (n = 8-13). tentials, in the range o f - - 4 0 to - - 5 0 mV, were insensitive or depolarized by only one or two mV. The distribution of receptors was apparently uniform along the length of the cell as the variation in sensitivity to acetylcholine was usually less than a factor of two. The greatest variation in maximum response between 3 points on a single fibre was in a cell with resting potential o f - - 3 0 mV where the maximum depolarizations obtained were 8, 15 and 18 mV. Insensitive cells were apparently insensitive over their entire surface as applications of transmitter were generally tested at 4 or 5 different sites along the length of each fibre. In the older cultures (8-10 weeks in vitro) all myotubes responded when challenged with acetylcholine and the variation between fibres was less than in younger cultures (Table 1). The overall sensitivity was much higher than in younger cultures, the maximum response being about 90 ~o depolarization (Fig. 4). Despite this increased responsivenes to acetylcholine, the mean resting membrane potential was not greater than that of younger cultures (Table 1). DISCUSSION Studies of human foetal muscle cells in culture have been made using both dissociated (Hauschka 1974; Emery and McGregor 1977; Bevan et al. 1977, 1978) and explant (Bevan et al. 1977, 1978) cultures. Cultures prepared by dissociation of human foetal muscle (Hauschka 1974) or by trypsinisation of the outgrowths from explants (Emery and McGregor 1977) contained myotubes that were not reported as being cross-striated or spontaneously contracting. Bevan et al. (1977, 1978) did not describe their explant cultures in detail but commented that myotubes formed after about 3 weeks in vitro, considerably later than in our cultures (7-10 days). Myotube formation took place over about a 48-hr period, and continued fusion of myoblasts to form new myotubes was not observed, the number of myotubes remaining fairly constant. Hauschka (1974) has reported that variation in the time of myotube formation also occurs in clones of dissociated human foetal muscle.
121 The early formation of myotubes in our cultures was followed by a period of further development in which the myotubes became narrow and phase-refractile, developed cross-striations and some spontaneously contracted. This degree of differentiation resembles that found in cultures of embryonic animal muscle but appears to be more advanced than that previously described for human foetal muscle cells in culture. No previous study has reported the occurrence of spontaneously contracting human foetal myotubes. After another 3 or 4 weeks growth in culture all fibres were highly sensitive to acetylcholine, indicating a continued synthesis of receptor molecules. The apparent lag in the appearance of receptors is in marked contrast to the behaviour of chick, rat and mouse skeletal muscle in culture. Here a rapid increase in receptor numbers is coincident with fusion and early myotube formation (Fambrough and Rash 1971; Powell and Fambrough 1973; Prives et al. 1976). Although it has been reported that receptor n umbers on cultured chick myotubes decline if the cells are contracting (Cohen and Fischbach 1973; Prives et al. 1976), it is unlikely that such a mechanism would be significant in the human cultures where the proportion of spontaneously contracting fibres is much smaller. Despite the additional development implied by the appearance of high acetylcholine sensitivity, some further maturation would appear to be required: the average resting membrane potential in the most mature cultures of about - - 3 0 mV is still much lower than values of around - - 6 0 to - - 8 0 mV reported for adult human muscle in vitro or in vivo (McComas and Mrozek 1969; Norris 1976); and for chick embryo muscle in culture (Kano et al. 1972; Harvey and Dryden 1977; Harvey et al. 1978). However, a recent study on h u m a n foetal muscle in culture has reported membrane potential values o f - - 5 0 mV (Bevan et al. 1977). Culture conditions used by Bevan et al. (1977) were almost identical with those in our cultures and we are unable to account for the difference in resting membrane potential. The lack of correlation between the apparent morphological maturity and appearance of developed membrane characteristics suggests that one should be cautious of using any single parameter such as morphology or presence of muscle specific enzymes as an indication of muscle development in culture.
REFERENCES Askanas, V. and W. K. Engel (1976) Diseased human muscle in tissue culture. A new approach to the pathogenesis of human neuromuscular disorders. In: L. P. Rowland (Ed.), Pathogenesis of Human Muscular Dystrophies, Excerpta Medica, Amsterdam, pp. 856-871. Bevan, S., R. W. Kullberg and S. F. Heinemann (1977) Human myasthenic sera reduce acetylcholine sensitivity of human cells in tissue culture, Nature (Lond.), 267: 263-265. Bevan, S., R. W. Kullberg and J. Rice (1978) Acetylcholine-induced conductance fluctuations in cultured human myotubes, Nature (Lond.), 273: 469-471. Cohen, S. A. and G. D. Fischbach (1973) Regulation of muscle acetylcholine sensitivity by muscle activity in cell culture, Science, 181 : 76-78. Emery, A. E. H. and L. McGregor (1977) The foetus in Duchenne muscular dystrophy - - Muscle growth in tissue culture, Clin. Genet., 12: 183-187. Fambrough, D. and J. E. Rash (1971) Development of acetylcholine sensitivity during myogenesis, Develop. BioL, 26: 55-68.
t22 Harvey, A. L. and W. F. Dryden (1977) Electrophysiological and pharmacological properties of skeletal muscle in tissue culture, J. Pharm. Sci., 66:913-922. Harvey, A. L., T. Barkas, R. Harrison and G. G. Lunt (1978) Inhibition of receptor function in cultured chick myotubes by antiserum to purified acetylcholine receptor (from Torpedo marmorata) and myasthenic sera. In: G. G. Lunt and R. M. Marchbanks (Eds.), The Biochemistry of Myasthenia Gravis and Muscular Dystrophy, Academic Press, London, pp. 167-175. Hauschka, S. D. (1972) Cultivation of muscle tissue. In: G. H. Rothblat and V. J. Cristafalo (Eds.), Growth, Nutrition and Metabolism of Cells, Vol. 1, Academic Press, New York, pp. 67-130. Hauschka, S. D. (1974) Clonal analysis of vertebrate myogenesis, Part 2 (Environmental influences upon human muscle differentiation), Develop. Biol., 37: 329-344. Kano, M., Y. Shimada and K. lshikawa (1972) Electrogenesis of embryonic chick skeletal muscle cells differentiated in vitro, J. cell. Physiol., 79: 363-366. McComas, A. J. and K. Mrozek (1969) An in vivo microelectrode study of muscle fibres in normal subjects and in patients with myotonia. In : N. Canal, G. Scarlato and J. N. Walton (Eds.), Muscle Diseases, Excerpta Medica, Amsterdam, pp. 151-158. Norris, F. H. (1976) Micropipette recording from human striated muscle, J. NeuroL, 213: 1-15. Powell, J. E. and D. M. Fambrough (1973) Electrical properties of normal and dysgenic mouse skeletal muscle in culture, J. cell. PhysioL, 82: 21-38. Prives, J., 1. Silman and A. Amsterdam (1976) Appearance and disappearance of acetylcholine receptor during differentiation of chick skeletal muscle in vitro, Cell, 7: 543-550. Witkowski, J. A. (1978) Diseased muscle cells in culture, Biol. Rev., 52: 431-476. Witkowski, J. A., M. Durbridge and V. Dubowitz (1976) Growth of human muscle in tissue c u l t u r e An improved technique, In vitro, 12: 98-106. 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.
© Elsevier/North-Holland Biomedical Press
115
M A T U R A T I O N OF H U M A N S K E L E T A L M U S C L E FIBRES I N E X P L A N T TISSUE C U L T U R E
A. L. HARVEY, J. G. ROBERTSON and J. A. WITKOWSKI Department of Physiology and Pharmacology (ALH, JGR), University of Strathclyde, Glasgow, G1 1XW and Jerry Lewis Muscle Research Centre (JAW), Department of Paediatrics, Hammersmith Hospital, London, W12 OHS (Great Britain)
(Received 9 October, 1978) (Accepted 19 October, 1978)
SUMMARY H u m a n foetal skeletal muscle was grown in explant cultures and the development of myotubes was monitored morphologically and by the development of membrane potential and acetylcholine sensitivity. Migration of uninucleate cells from the explants occurred in the first day of culture and formation of multinucleated myotubes took place between 7 to 10 days. Early myotubes were variable in appearance, being either flat and nonrefractile or narrow and cylindrical. Some cross-striated cells were observed. Resting membrane potentials were around - - 2 5 mV and there was great variation in myotube sensitivity to acetylcholine. After about 6 weeks in culture most myotubes were of the refractile type. Many had hypolemmal nuclei and were cross-striated; some contracted spontaneously. All myotubes had developed high sensitivity to acetylcholine although there had been no increase in resting membrane potential.
INTRODUCTION Use of tissue culture techniques would facilitate the study of human skeletal muscle if mature muscle fibres could develop in culture conditions. Earlier reports have concentrated on morphological or biochemical studies (for review see Witkowski 1977). Although it has been claimed that mature human muscle cells develop in culture This study was supported by grants from the Muscular Dystrophy Association Inc. (to ALH) and the Muscular Dystrophy Group of Great Britain (to JAW). The authors thank the Medical Research Council for a training award to JGR and the Smith Kline and French Foundation for an equipment grant (to ALH).
116 (Askanas and Engel 1976: Yasin et al. 1977), these reports have been based on cell morphology and the presence of muscle-specific enzymes e.g. creatine phosphokinase. It is clearly important to determine if cells that appear mature by these criteria have developed electrophysiological properties. Recent reports (Bevan et al. 1977, 1978) have demonstrated that human muscle cells in culture respond to the physiological neurotransmitter acetylcholine, but these authors made no attempt to relate their results with the structural development of the cells. We have prepared cultures from explants of human foetal skeletal muscle and have correlated morphological development of myotubes with functional maturation as revealed by resting membrane potential and sensitivity to acetylcholine. Our results show that fibres appeared morphologically well differentiated before the appearance of high acetylcholine sensitivity and that despite the subsequent development of high acetylcholine sensitivity, the fibres remained relatively immature since their resting membrane potentials did not reach normal levels (about - - 3 0 mV compared to about - - 8 0 mV). METHODS Tissue culture H u m a n foetal skeletal muscle was obtained from the M R C Tissue Bank at the Royal Marsden Hospital. Samples were stored overnight at 14 °C in complete growth medium; storage at 14 °C has no effect on cell viability (Witkowski et al. 1976). Details of muscle samples are presented in Table 1. Growth medium was Medium 199 (with Hanks' salt solution), with 10 ~ foetal calf serum, penicillin (50 IU/ml), streptomycin (50/~g/ml) and 15 m M HEPES. After myotube formation the cultures were maintained in medium containing 2.5 ~o foetal calf serum. All cultures were explant cultures prepared in gelatin-coated (Hauschka 1972) TABLE 1 RESTING MEMBRANE POTENTIALS AND ACETYLCHOLINE SENSITIVITY OF HUMAN MUSCLE CULTURES AT DIFFERENT AGES AND FROM DIFFERENT SOURCES Preparation
Days in culture
No. of cells Mean RMP ~ sensitive Mean maximum tested (-mV) cells response (depolarization as ~ of RMP)
17.5-week foetus (R.6) ll.5-week foetus (R.5) 12-week foetus (R.3)
13 18 24
15 18 17
20.7 i 2.3 28.4 -4- 2.5 28.3 ± 2.8
86 56 76
26 J_ 5.3 38 :t: 8.4 35 :t: 5.5
12-week foetus (R.3)
31
13
21.9 ± 2.8
61
29:5 7.7
21-week foetus (R.4)
36
23
18.t ± 1.7
52
30 ~ 5.7
12-week foetus (R.3) ll.5-week foetus (R.5)
47 77
12 17
27.0 :L 2.6 29.2 ~ 2.6
100 100
89 & 3.1 56 ~ 4.9
117 35-mm petri dishes. Muscle samples were cut into 1-mm a pieces and up to 16 explants were arranged in a grid-pattern in the dish and covered with a 22 mm x 22 mm glass coverslip. 1.5 ml of medium were added to each dish and medium was changed once weekly. With cultures that were sent to Glasgow by post, coverslips were removed from each petri dish, the dishes filled with medium and the lids sealed with sterile silicone grease. The cells did not appear to be adversely affected by their treatment, and cultures were incubated at 37 °C for at least 2 hr before electrophysiological studies began.
Electrophysiology Conventional electrophysiological techniques were used to measure muscle cell resting membrane potentials and responses to iontophoretic application of acetylcholine. Microelectrodes for iontophoresis were filled with 0.5 M acetylcholine chloride and connected to a stimulator through one channel of a WP1 750 electrometer. The resistance of the acetylcholine electrode was 100-200 megohm and care was taken to select electrodes that did not leak spontaneously. Cultures were maintained at 37 °C and p H 7.4 during experiments. RESULTS
Cell growth Migration of uninucleate cells from explants was observed after 24 hr culture (Fig. 1A) and the first multinucleate myotubes were seen after 7-10 days in vitro. Fusion of myoblasts was partially synchronised and took place in a 48-hr period. The first myotubes formed were broad and non-refractile with clearly visible nuclei (Fig. 1B) but these subsequently gave rise to long narrow refractile myotubes (Fig. 1C). When medium containing only 2.5 70 foetal calf serum was used, division of uninucleate cells was inhibited so that the myotubes were not overgrown but appeared to lie on a layer of uninucleate cells. After 3-4 weeks in culture, both types of myotube were present, together with a small number showing a mixed morphology, i.e. at either end of the cell there was a fiat portion containing nuclei while the central region was narrow and refractile (Fig. 1D). In 4-6 week cultures many of the myotubes were cross-striated (Fig. 1E) and some cells contracted spontaneously. The organization of myofibrils into cross-striations appeared to be greater in refractile myotubes than in flat myotubes, although cells of both forms were seen to contract. Cultures were maintained for up to 11 weeks by which time the morphology of the myotubes had changed from the straight, refractile form to a more flattened form (Fig. 1F). The myotubes were no longer straight but were thrown into waves. Contracting myotubes were also seen in these "old" cultures.
Membrane potential and acetylcholine sensitivity Uninucleated cells with the bipolar appearance of myoblasts had low resting
118
Fig. 1. Phase contrast micrographs of cultures of human foetal muscle. Scale bar represents 50 fro1. A: uninucleate cells; myoblasts cannot be distinguished from fibroblasts with any certainty. (7 days in vitro). B: early stages of myotube formation with broad flat myotubes (MT) and centrally placed nuclei (arrows). C: a number of refractile myotubes with cross-striations and hypolemmal nuclei. These myotubes were typical of the muscle cells found in cultures older than 6 weeks in vitro. D: myotube showing intermediate morphology with flattened area (a) and refractile region (b). (18 days in vitro). E: portion of a refractile myotube with cross-striations and hypolemmal nuclei. This cell was about 2.3 mm long and was contracting spontaneously (18 days in vitro). F: a flattened, non-refractile myotube in an I 1-week culture. Hypolemmal nuclei were still present (N).
119 0mV
-23mV
~
0.5 2
,
8
32
,
~
'
64-34mV
o'.s
;
;2nC ACh
10 mV l 2s
Fig. 2. Responses of two myotubes to iontophoretic application of acetylcholine. Increasing doses of acetylcholine (expressed as nanocoulombs, nC, charge passed) produced increasing depolarizations. Note that both cells depolarized close to 0 mV although the resting membrane potentials differed by 11 mV. m e m b r a n e potentials (--8.0 4- 0.6 mV; mean i S E M of 26 cells in 2 cultures) and did not respond to iontophoretic application of acetylcholine. In the 4-6 week old cultures in which the two morphological types o f myotubes were found, there was no consistent difference in m e m b r a n e potentials between either group o f cells: the average for the flat myotubes was - - 2 4 . 7 4- 2.6 mV (n = 27) and for the rounded m y o t u b e s the mean was - - 2 1 . 0 4- 1.8 mV (n = 30). The sensitivity to acetylcholine was tested in b o t h types o f cells and although the difference was n o t absolute, the retractile myotubes exhibited greater responsiveness than the fiat cells. In 3 cultures 10 out of 23 fiat cells tested (43 ~ ) responded to acetylcholine whereas 21 out o f 29 rounded cells (72 ~ ) responded. The cells which were sensitive responded to increasing doses o f acetylcholine with increasing depolarizations (Fig. 2) although there was a wide variation in the level of sensitivity between individual responsive cells (Fig. 3). The variation in response was not correlated with the resting m e m b r a n e potential as fibres with potentials as low as - - 2 2 mV could show a 22 mV depolarization but some fibres with higher po-
100 o.
so -
~
60 -
u 40 g 2O '=
~
0
I
0'5
I
1.0
I
10 Acetylcholine
f
lOOnC
Fig. 3. Responses to acetylcholine of myotubes in a 24-day culture of muscle explanted from a 12-week human foetus. Responses of the cell with the greatest sensitivity (V), and the cell with the lowest detectable sensitivity (A) and the mean level of sensitivity (A) are shown. Standard errors of the means are shown unless smaller than the symbol (n = 13). Acetylcholine dose is expressed as nanocoulombs (nC) of charge passed.
120
F
-: '°1•~
t
0 I._
Fig. 4. Responses to acetylcholine of myotubes in explant cultures from a 12-week human foetus at 24 days (~), 31 days (V) and 47 days (0) in culture. Standard errors bars are shown unless smaller than the symbols (n = 8-13). tentials, in the range o f - - 4 0 to - - 5 0 mV, were insensitive or depolarized by only one or two mV. The distribution of receptors was apparently uniform along the length of the cell as the variation in sensitivity to acetylcholine was usually less than a factor of two. The greatest variation in maximum response between 3 points on a single fibre was in a cell with resting potential o f - - 3 0 mV where the maximum depolarizations obtained were 8, 15 and 18 mV. Insensitive cells were apparently insensitive over their entire surface as applications of transmitter were generally tested at 4 or 5 different sites along the length of each fibre. In the older cultures (8-10 weeks in vitro) all myotubes responded when challenged with acetylcholine and the variation between fibres was less than in younger cultures (Table 1). The overall sensitivity was much higher than in younger cultures, the maximum response being about 90 ~o depolarization (Fig. 4). Despite this increased responsivenes to acetylcholine, the mean resting membrane potential was not greater than that of younger cultures (Table 1). DISCUSSION Studies of human foetal muscle cells in culture have been made using both dissociated (Hauschka 1974; Emery and McGregor 1977; Bevan et al. 1977, 1978) and explant (Bevan et al. 1977, 1978) cultures. Cultures prepared by dissociation of human foetal muscle (Hauschka 1974) or by trypsinisation of the outgrowths from explants (Emery and McGregor 1977) contained myotubes that were not reported as being cross-striated or spontaneously contracting. Bevan et al. (1977, 1978) did not describe their explant cultures in detail but commented that myotubes formed after about 3 weeks in vitro, considerably later than in our cultures (7-10 days). Myotube formation took place over about a 48-hr period, and continued fusion of myoblasts to form new myotubes was not observed, the number of myotubes remaining fairly constant. Hauschka (1974) has reported that variation in the time of myotube formation also occurs in clones of dissociated human foetal muscle.
121 The early formation of myotubes in our cultures was followed by a period of further development in which the myotubes became narrow and phase-refractile, developed cross-striations and some spontaneously contracted. This degree of differentiation resembles that found in cultures of embryonic animal muscle but appears to be more advanced than that previously described for human foetal muscle cells in culture. No previous study has reported the occurrence of spontaneously contracting human foetal myotubes. After another 3 or 4 weeks growth in culture all fibres were highly sensitive to acetylcholine, indicating a continued synthesis of receptor molecules. The apparent lag in the appearance of receptors is in marked contrast to the behaviour of chick, rat and mouse skeletal muscle in culture. Here a rapid increase in receptor numbers is coincident with fusion and early myotube formation (Fambrough and Rash 1971; Powell and Fambrough 1973; Prives et al. 1976). Although it has been reported that receptor n umbers on cultured chick myotubes decline if the cells are contracting (Cohen and Fischbach 1973; Prives et al. 1976), it is unlikely that such a mechanism would be significant in the human cultures where the proportion of spontaneously contracting fibres is much smaller. Despite the additional development implied by the appearance of high acetylcholine sensitivity, some further maturation would appear to be required: the average resting membrane potential in the most mature cultures of about - - 3 0 mV is still much lower than values of around - - 6 0 to - - 8 0 mV reported for adult human muscle in vitro or in vivo (McComas and Mrozek 1969; Norris 1976); and for chick embryo muscle in culture (Kano et al. 1972; Harvey and Dryden 1977; Harvey et al. 1978). However, a recent study on h u m a n foetal muscle in culture has reported membrane potential values o f - - 5 0 mV (Bevan et al. 1977). Culture conditions used by Bevan et al. (1977) were almost identical with those in our cultures and we are unable to account for the difference in resting membrane potential. The lack of correlation between the apparent morphological maturity and appearance of developed membrane characteristics suggests that one should be cautious of using any single parameter such as morphology or presence of muscle specific enzymes as an indication of muscle development in culture.
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