Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in culture

Differentiat ion

Differentiation (1987) 36: 194-198

0 Springer-Verlag 1987

Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in culture M. Immacolata Senni Francesco Castrignano 2 , Giancarlo Poiana 2 , Giulio Cossu ’*, Gianfranco Scarsella 2 , and Stefan0 Biagioni Istituto di Istologia ed Embriologia Generale, Universiti di Roma “La Sapienza”,Via A. Scarpa 14, 00161 Rome, Italy Dipartimento di Biologia Cellulare e dello Sviluppo, Universiti di Roma “La Sapienza”, Piazza A. Moro 5, 00185 Rome, Italy Abstract. The pattern of acetylcholinesterase (AChE) molecular forms, obtained by sucrose gradient sedimentation, was studied at different in vitro developmental stages of myogenic cells isolated from adult mouse skeletal muscle. Only the globular forms were present in rapidly dividing satellite cells during the first days in culture. After myotube formation, a pattern similar to that described in mammalian fast-twitch skeletal muscle was observed. This pattern did not change during the following period in culture (up to 1 month) nor could it be modified by co-culturing with spinal cord motoneurons or by addition of brain-derived extracts. The internal-external localization of AChE molecular forms has been determined by the use of echothiophate iodide, a membrane-impermeant irreversible inhibitor of AChE. Echothiophate-treated cultures showed about 40% of both asymmetric and globular forms localized on the sarcolemma, with their active sites oriented outward. Analysis of culture medium from untreated cultures revealed the presence of both asymmetric and globular forms. When the same analysis was repeated on cultures of myoblasts derived from 16-day-old mouse embryos, the pattern of AChE forms was different. The myotubes derived from these cells exhibit a very small proportion of asymmetric form, which was not released into the medium. This pattern was not further modified during the following days of culture, nor by co-cultures with spinal cord motoneurons or by incubations with brain-derived extracts. Thus, the myotubes derived from myoblasts express in culture a clear phenotypic difference when compared to the corresponding myotubes from satellite cells, supporting the view that these two myogenic cells are endowed with different developmental programs.

Introduction

Satellite cells are a class of quiescent mononucleated elements located between the sarcolemma and the basal lamina of the muscle fiber in normal adult skeletal muscle [18]. Upon any injury to the tissue, they regain proliferative activity and then differentiate into new muscle fibers which replace the damaged ones [3]. Despite much work on satellite cells, the mechanisms regulating their quiescence during adult life and their activa-

* To whom offprint requests should be sent

tion following muscle injury are still obscure [recent review, 51. Similarly unknown are both the developmental origin of satellite cells and the differentiative program with which they are endowed (beside the obvious fact that they form myotubes in culture). In other words we do not know whether they are part of a common population of myoblasts or rather represent a unique myogenic cell, characterized by its own specific developmental program. Recently, however, evidence has been accumulated showing the existence of phenotypic differences among myogenic cells, which allow us to divide them into three major classes: (1) early myoblasts, characterized by need for conditioned medium for differentiation at clonal density [32] and by differential expression of slow and fast myosins [19-211; (2) late myoblasts, which differentiate at clonal density independently from the medium [32], synthesize only fast myosin [21, 221 and are sensitive to phorbol esterinduced block of differentiation 141; (3) satellite cells, which differ from late myoblasts in that they are resistant to phorbol esters [6, 101 and express acetylcholine receptors during their mitotic phase 181. In this context, it is important to analyze other specific muscle gene products expressed by satellite cells under a controlled environment, and to compare these products with those expressed by other myogenic cells, such as early or late myoblasts, under the same environment. One of the most extensively studied markers of muscle cell differentiation is represented by the molecular forms of AChE [l]. This enzyme undergoes a peculiar process of synthesis and assembly which eventually leads to the formation of increasingly complex molecular forms. These are distinguished by their sedimentation coefficient on sucrose gradient analysis, as well as by their solubility properties [17]. In cultured muscle cells, AChE is present as membranebound, low salt-soluble and asymmetric forms [16, 261. Although Vallette et al. [30] have demonstrated that in chick myoblasts the expression of A12 asymmetric AChE form is, at least partially, affected by the composition of culture medium, the synthesis of this form seems to be specifically controlled by the establishment of precise interactions between nerve and muscle. Detectable amounts of asymmetric forms, in fact, can only be observed in myotubes derived from embryonic muscle already innervated in situ, but can be induced in myotubes from earlier myoblasts by co-cultures with spinal motoneurons [16]. Inasmuch as satellite cells are isolated from an adult, innervated and contracting muscle tissue, we investigated the patterns of AChE molecular forms expressed by these cells in culture.

195

Here we report the activity and the characterization of the molecular forms of AChE of satellite cells at different stages of their in vitro differentiation, as well as a comparison of these forms with those expressed by late embryonic myoblasts under the same experimental environment.

Table 1. Acetylcholinesterase activity of myogenic cells in culture Cell type

2 DIV

7 DIV

14DIV

9DIV BE

Satellite cells

0.82 +0.13 (4) 0.34 0.02 (4)

16.80 k1.81 (6) 2.03 10.19 (4)

40.76 - 2.93 (4) 5.32 +0.12 (4)

31.77 *9.15 (3) 8.26

Methods Myoblasts

Reagents

Acetylthiocholine iodide, 5,5-dithiobis-(2-nitrobenzoic) acid (DTNB), ethopropazine hydrochloride, pepstatin, leupeptin, collagen, collagenase, hyaluronidase, Triton X-100 and trypsin were obtained from Sigma, St. Louis, Mo.; beef liver catalase and E. coli P-galactosidase from BoehringerMannheim, FRG; Dulbecco's minimum essential medium (DMEM), horse serum and penicillin-streptomycin from GIBCO, Paisley, Scotland ; sucrose for density gradient ultracentrifugation from Merck, Darmstadt, FRG ; Sephadex G-25 from Pharmacia, Upsala, Sweden. Echothiophate (Echo) was kindly provided by Ayerst Laboratories Incorporated, New York, NY. Cell cultures

Myoblasts and satellite cells were cultured as described before [6]. Briefly, myoblasts were isolated from the limbs of 16 days postcoitum mouse embryos and, after removal of the skin, digested with 0.05% trypsin in phosphate buffered saline (PBS) for 15 min at 37" C with occasional shaking. To isolate satellite cells, the hind limbs were dissected from adult mice (1-2 months). In this case a predigestion with 0.1 % collagenase (w/v) and 0.1 % hyaluronidase and three sequential digestions with trypsin were required. After the proteolytic digestions, the tissues were fragmented by repeated pipetting, the debris was removed by filtration through a sterile nylon gauze, and the cells were collected by centrifugation. All cultures were grown in DMEM supplemented with 10% horse serum and 3% chick embryo extract. Spinal cords from 15 days postcoitum mouse embryos were dissected, the cells were isolated by digestion with 0.1% collagenase (w/v) in PBS for 15 min, and plated onto myotube cultures. After 2 days in the medium described above, many cells showed long neurite-like extensions which made contacts with myotubes and with each other. Immunostaining with an antibody against neurofilaments revealed that about 30% of the plated cells were neurons [31]. Brain extract, prepared from 18 days postcoitum mouse embryos, was added to the medium for 2 days before harvesting, as described before [7]. The AChE released into the medium was studied by incubating cells for 24 h in DMEM. The medium was then collected, centrifuged, dialyzed extensively against the buffer used for enzyme extraction and then against solid Sephadex G-25 in order to concentrate enzyme activity. Echo M) was added to the cells for 15 rnin at 4" C in PBS. Enzyme extraction

Cultures were rinsed with PBS, scraped from the dishes with a rubber policeman and collected in a conical glass homogenizer. For AChE extraction cells were homogenized

+

+

(1)

+

AChE activity is expressed in mU(mean SEM)/90 mm dish. At 2 days in vitro (DIV) most of the cells are still mitotic and undifferentiated; under the culture conditions used, fusion starts after 4 days in culture and is practically completed within the next 3 days. Numbers in parentheses indicate the number of experiments performed. Brain extract (BE) was added at 7 DIV

with 20 strokes in a conical glass-glass homogenizer, in 0.01 M phosphate buffer pH 7.0, 1 M NaCl, 1% Triton X-3 00,lO m M EDTA, 40 pg/ml leupeptin, 20 pg/ml pepstatin (complete buffer), and then centrifuged at 27000 g for 30 min at 4" C; the supernatant was used for the enzyme assay. Sequential extractions were performed as described before [24]. Enzyme activity

The AChE activity was assayed by Ellman's method [I21 in 0.1 Mphosphate buffer pH 8.0 with 1 m M acetylthiocholine iodide as substrate. All determinations of true AChE activity were performed in the presence of 1.4 x 10- A4 ethopropazine. Enzyme activity is expressed in milliunits, 1 mU corresponding to 1 n M substrate hydrolyzed per min at pH 8.0 and 30" C. Sucrose gradient sedimentation

Samples of 0.2 ml containing 8-10 mU true AChE activity were loaded onto 5%-20% (w/v) linear sucrose gradients. Bacterial P-galactosidase (16s) and beef liver catalase (11.3s) were added as markers. Centrifugation was at 38000rpm for 18 h and 30min in an SW41 Ti rotor at 4" C in a Beckman L5-50B ultracentrifuge. Results

The levels of total AChE activity at different stages of differentiation in cultures of satellite cells and of myoblasts from 16 days postcoitum embryos are shown in Table 1. In cultures of satellite cells, total AChE activity increased about 20-fold between proliferating cells (2 days in vitro) and 7-day-old myotubes and twice between 7-dayold and 14-day-old myotubes. At longer times of culture, the level of AChE activity remained constant up to the 30th day. Addition of brain extract to 7-day-old myotubes resulted in doubling of the activity within 48 h of treatment (31.77f9.15 mU in treated vs 14.75f2.13 mU in control cultures). The AChE activity of myoblasts in culture showed a pattern similar to that observed in satellite cell culture, with a steep increase (although somewhat lower) between proliferating cells and myotubes and a further doubling of activity during the week after fusion. Here too, the addition of

196

brain extract caused a faster increase of activity within 48 h of treatment. It should be noted that, at any time in culture examined, the AChE activity was considerably (from 3 to 8 times) lower in cultures of myoblasts than in cultures of satellite cells. It is difficult to estimate quantitatively the amount of AChE expressed by these two cell types, because cultures are contaminated by different proportions of nonmuscle cells and therefore expressing the data per protein would not be informative. When the total level of AChE was expressed per number of nuclei present in myotubes (per microscopic field), the magnitude of the difference in activity between myotubes from satellite cells and myotubes from myoblasts was considerably reduced. However, the activity of myotubes derived from satellite cells was still about 50% higher than that of myotubes derived from myoblasts. When differential extractions of AChE were performed, the activity of low salt-soluble, membrane-bound and high salt-soluble forms were 15%, 40% and 45% of the total AChE activity respectively for both myoblast-derived and satellite cell-derived myotubes (data not shown). The sedimentation profiles of AChE molecular forms extracted from 7-day-old myotubes derived from satellite cells and myoblasts are shown in Fig. 1 . In both cases AChE activity was resolved into three major peaks with sedimentation coefficients of 16S, 10.5s and 4 s probably representing the A12, G4 and G I molecular forms. Comparison of the two profiles showed that the 16s peak, which accounts for about 25% of the AChE activity in myotubes from satellite cells (Fig. 1 A), represents only 4%-5% of activity in myotubes from embryonic muscles (Fig. 1B). On the other hand, the peak corresponding to the G4 molecular form appeared considerably larger in myoblast-derived myotubes. Reported in Fig. 1 is one of four independent experiments. While there was some variability in total activity, the relative proportion of the different peaks did not vary among experiments (less than 10% of the average value). The pattern of molecular forms did not change during the following period in culture (up to 1 month) and was not affected by co-culturing with spinal cord motoneurons or supplementing the culture medium with brain extract (data not shown). The AChE activity released into the culture medium (during a 24-h period) by 7-day-old myotubes derived from satellite cells was found to be 7 mU/dish. Comparing the AChE content of the myotubes with the activity released into the medium, we calculated that the cells release about 2% of their total activity per hour. The AChE activity released by myoblast-derived myotubes under the same culture conditions was about 5 times higher (IIYo of total activity). Analysis of the released AChE on sucrose gradients revealed the presence of two major peaks of activity sedimenting at 4 s and 10.5s for both satellite cell-derived and myoblast-derived myotubes (Fig. I C , D). A minor peak of activity, which exhibited a sedimentation coefficient of 16S, was clearly detectable in the case of myotubes from satellite cells (Fig. 1C) but not in the case of myoblastderived myotubes (Fig. 1 D). In order to inhibit the extracellular enzyme, we treated 7-day-old myotubes with echo, which is a membrane-impermeant irreversible inhibitor of AChE. After this treatment the cellular activity was 9.6 5 1.7 mU/dish (mean fSEM of four observations) revealing that about 60% of the enzyme is located intracellularly.

p

z

c

Q

z

c

Fraction Number Fig. 1. A, B. Typical sucrose gradient profiles of AChE molecular forms extracted from satellite cell-derived (A) and myoblast-derived (B) myotubes on day 7 of culture. C, D Molecular form profiles of AChE released into the medium (during a 24-h period), after 7 days of culture, by satellite cell-derived and myoblast-derived myotubes respectively. The arrows indicate the position of sedimentation markers : Z, j?-galactosidase (16s); C, catalase

(11.3s)

I

z

c

10 20 30 40 Fraction Number

!

Fig. 2. Typical sucrose gradient profiles of AChE molecular forms extracted from satellite cell-derived myotubes. Solid lines, control; broken lines, myotubes treated with echothiophate lo-' M for 15 min at 4" C in PBS. The arrows indicate the position of sedimentation markers: 2, 8-galactosidase (16s);C, catalase (11.3s)

The comparison of AChE molecular forms extracted from control and Echo-treated myotubes from satellite cells showed that about 40% of both asymmetric and globular forms was localized on the sarcolemma, with their active side oriented outward (Fig. 2). Similar results were obtained with myotubes derived from myoblasts (data not shown).

3 97

Discussion The data reported in this paper show the existence of a phenotypic difference between the myotubes derived from satellite cells and those derived from embryonic myoblasts. The former synthesize, express on their surface and secrete considerable amounts of the asymmetric, collagen-tailed molecular form of AChE; the latter express only a very small proportion of this form. The difference, though quantitative in nature, clearly reflects a difference intrinsic to the genetic program of these two cell types, since it cannot be modified by time in culture, nerve cells or nerve-derived extracts, or by hormones such as thyroxine (unpublished results). In rat embryos, Koenig and Vigny [16] reported that myoblasts cultured from 14-day-old embryos (likely “ early ” myoblasts, according to current terminology [ 191) give rise to myotubes which do not synthesize any asymmetric form, unless co-cultured with spinal cord cells. Conversely, myoblasts cultured from an already innervated limb of 16-day-old embryos (likely “late” myoblasts) give rise to myotubes which do synthesize detectable amounts of asymmetric AChE in the absence of nerve cells. In both cases the activity of the asymmetric form represented only a minute proportion of total activity in comparison with globular forms. In a later paper, the same authors reported that contractile activity was necessary for the expression of the asymmetric form in rat cultured myotubes. In this case the activity of this form was much higher than that reported previously, reaching a proportion comparable with the other forms. It is worth noting that in this case, cells were cultured from 18-day-old rat embryos, a developmental stage when basal lamina had formed [ l l ] and satellite cells have already appeared in mice [6], humans [9] and rats (Cossu et al., unpublished). Thus the cell population used in this study contained a substantial proportion of satellite cells. In fact, Silberstein and co-workers [13, 291 using the C2 muscle cell line derived from satellite cells [33] reported a high proportion of asymmetric AChE, thus showing that this phenotypic trait was faithfully preserved in established cell lines after a high number of divisions. On the other hand, studies on chick myoblasts showed either no synthesis of asymmetric AChE [27] or synthesis of trace amounts of this form [14,25,28], probably depending on the composition of the culture medium, as suggested by Vallette et al. [30]. Our data are in good agreement with previous observations, and, together with these, allow us to conclude that the three major classes of myogenic cells so far described differ in their phenotypic expression of the synaptic form of AChE: “early” myoblasts do not synthesize it, unless induced by motoneurons; ‘‘late’’ myoblasts synthesize small amounts (which cannot be further stimulated by motoneurons); and satellite cells synthesize a large proportion of asymmetric AChE in a nerve-independent manner. As to the physiological significance of this form in satellite cell-derived myotubes, the most obvious consideration which may relate to these observations is that satellite cells are located in a special environment, between the basal lamina and the sarcolemma, where they are exposed to low but continuous release of acetylcholine from the synaptic region. In this context, it is interesting to note that the density of satellite cells is higher around the synapse [I51 and that undifferentiated satellite cells, freshly isolated from

skeletal muscle, already express functional nicotinic receptor channels [8]. Thus it appears that some strict, as yet unexplained relationship must exist between satellite cells (and their progeny) and the synaptic apparatus of skeletal muscle. For example, denervation, which does not initially alter muscle membranes, leads to satellite cell activation ~31. It is difficult, however, to envision direct experiments to solve these problems, since in situ synapses are not easily accessible to experimental manipulation, whereas studies in culture begin with the destruction of the complex apparatus to be analyzed. Hopefully, newly developed methods for muscle fiber isolation [2], coupled with in vitro innervation in an opportune extracellular matrix environment, might help to shed light on this aspect of satellite cell physiology. Acknowledgements. We thank Prof. M. Molinaro and Prof. G. Toschi for helpful discussion. This research has been supported by Minister0 P.I. grants (Progetti Nazionali and Progetti di Ateneo, Universita “La Sapienza”, Rome) and by a C.N.R. grant (86.00421.04).

References 1. Barnard EA, Barnard PJ, Jarvis J , Jedrzejc7yk J, Lai J, Pizxey JA, Randall WR, Silman I (1984) Multiple molecular forms of acetylcholinesterase and their relationship to muscle function. In: Brzin M, Barnard EA, Sket D (eds) Cholinesterdses, fundamental and applied aspects. De Gruyter, Berlin, pp 437496 2. Bischoff R (1986) Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115:129-139 3. Campion D R (1984) The muscle satellite cell: a review. Int Rev Cytol 87:225-231 4. Cohen R, Pacifici M, Rubinstein N, Biehl J, Holtzer H (1977) Effect of a tumor promoter on myogenesis. Nature 266:538-539 5. Cossu G, Molinaro M (1987) Cell heterogeneity in the myogenic lineage. Curr Top Dev Biol (in press) 6. Cossu G, Molinaro M, Pacifici M (1983) Differential response of satellite cells and embryonic myoblasts to a tumor promoter. Dev Biol98 : 520-524 7. Cossu G, Eusebi F, Senni MI, M o h d r o M (1985) Increased endocytosis of acetylcholine receptor in dystrophic mouse myotubes in vitro. Dev Biol 110:362-368 8. Cossu G, Cicinelli P, Fieri C, Coletta M, Molinaro M (1985) Emergence of TPA-resistant satellite cells during histogenesis of human limbs. Exp Cell Res 160:403-411 9. Cossu G, Senni MI, Eusebi F, Giacomoni D, Molinaro M (1986) Effect of phorbol esters and liposome-delivered phospholipids on the differentiation program of normal and dystrophic satellite cells. Dev Biol 118 : 182-189 10. Cossu G, Eusebi F, Grassi F, Wanke E (1987) Acetylcholine receptor channels are present in undifferentiated satellite cells but not in embryonic myoblasts in culture. Dev Biol 123:43-50 11. Dennis MJ, Ziskind-Conhaim L, Harris AJ (1981) Development of neuromuscular junction in rat embryos. Dev Biol 81 :266279 12. Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7: 88-95 13. Inestrosa NC, Miller JB, Silberstein L, Ziskind-Conhaim L, Hall ZW (1983) Developmental regulation of 16s acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line. Exp Cell Res 147: 393-405 14. Kato AC, Vrachliotis A, Fulpius B, Dunant Y (1980) Molecular forms of acetylcholinesterase in chick muscle and ciliary

198 ganglion: embryonic tissues and cultured cells. Develop Biol 76:222-228 15. Kelly AM (1978) Perisynaptic satellite cells in the developing and mature rat soleus muscle. Anat Rec 190:891-904 16. Koenig J, Vigny M (1978) Neural induction of 16s acetylcholinesterase in muscle cell cultures. Nature 271 :75-77 17. Massoulit: J, Bon S (1982) The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. Ann Rev Neurosci 5: 57-106 18. Mauro A (1961) Satellite cells of skeletal muscle fibres. J Biophys Biochem Cytol9: 493495 19. Miller JB, Stockdale FE (1986) Developmental origin of skeletal muscle fibers : clonal analysis of myogenic cell lineages based on fast and slow myosin heavy chain expression. Proc Natl Acad Sci USA 83 :386c3864 20. Miller JB, Stockdale FE (1986) Developmental regulation of multiple myogenic cell lineages of the avian embryo. J Cell Biol 103:2197-2208 21. Miller JB, Crow MT, Stockdale FE (1985) Slow and fast myosin heavy chain contcnt defines three types of myotubes in early muscle cell cultures. J Cell Biol 101 :1643-1650 22. Mouly V, Toutant M, Fiszman MY (1987) Chick and quail limb bud myoblasts, isolated at different time during muscle development, express stage specific phenotypes when differentiated in culture. Cell Differ 20: 17-25 23. Ontell M (1973) Muscle satellite cells: a validated technique for light microscopic identification and a quantitative study of changes in the population following denervation. Anat Rec 178:211-226 24. Poiana G, Scarsella G , Biagioni S, Senni MI, Cossu G (1985) Membrane acetylcholinesterase in murine muscular dystrophy in vivo and in cultured myotubes. Int J Dev Neurosci 3: 331-340 25. Popiela H, Beach RL, Festoff BW (1983) Appearance of acetyl-

,

cholinesterase molecular forms in not innervated cultured primary chick muscle cells. Cell Mol Neurobiol 3 :263-277 26. Rieger F, Koenig J, Vigny M (1980) Spontaneous contractile activity and the presence of 16s form of acetylcholinesterase in rat muscle cells in culture: reversible suppressive action of tetrodotoxin. Dev Biol 76: 358-365 27. Rotundo RL, Fambrough DM (1979) Molecular forms of chicken embryo acetylcholinesterase in vitro and in vivo. J Biol Chem 254:47904799 28. Rubin LL, Schuetze SM, Weill CL, Fischbach G D (1980) Regulation of acetylcholinesterase appearance at neuromuscular junctions in vitro. Nature 283 :264267 29. Silberstein L, Inestrosa NC, Hall ZW (1982) Aneural muscle cell cultures make synaptic basal lamina components. Nature 295~143-145 30. Vallette FM, Vigny M, Massoulit. J (1986) Muscular differentiation of chicken myotubes in a simple defined synthetic culture medium and in serum supplemented media: expression of the molecular forms of acetylcholinesterase. Neurochem Int 8: 121-133 31. Vivarelli E, Cossu G (1986) Neural control of early myogenic differentiation in cultures of mouse somites. Dev Biol 117 :319-325 32. White NK, Bonner PH, Nelson DR, Hauschka SD (1975) Clonal analysis of vertebrate myogenesis. IV. Medium-dependent classification of colony-forming cells. Dev Biol 44: 346361 33. Yaffe D, Saxel 0 (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725-727

Received August 1987 / Accepted in revised form October 21,1987