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. 2005 Jul 21;568(Pt 1):171–180. doi: 10.1113/jphysiol.2005.091439

Autocrine activation of nicotinic acetylcholine receptors contributes to Ca2+ spikes in mouse myotubes during myogenesis

Elena Bandi 1, Annalisa Bernareggi 1, Micaela Grandolfo 2, Chiara Mozzetta 3, Gabriella Augusti-Tocco 3, Fabio Ruzzier 1, Paola Lorenzon 1
PMCID: PMC1474771  PMID: 16037088

Abstract

It is widely accepted that nicotinic acetylcholine receptor (nAChR) channel activity controls myoblast fusion into myotubes during myogenesis. In this study we explored the possible role of nAChR channels after cell fusion in a murine cell model. Using videoimaging techniques we showed that embryonic muscle nAChR channel openings contribute to the spontaneous transients of intracellular concentration of Ca2+ ([Ca2+]i) and to twitches characteristic of developing myotubes before innervation. Moreover, we observed a choline acetyltransferase immunoreactivity in the myotubes and we detected an acetylcholine-like compound in the extracellular solution. Therefore, we suggest that the autocrine activation of nAChR channels gives rise to [Ca2+]i spikes and contractions. Spontaneous openings of the nAChR channels may be an alternative, although less efficient, mechanism. We report also that blocking the nAChRs causes a significant reduction in cell survival, detectable as a decreased number of myotubes in culture. This led us to hypothesize a possible functional role for the autocrine activation of the nAChRs. By triggering mechanical activity, such activation could represent a strategy to ensure the trophism of myotubes in the absence of nerves.

It is widely accepted that nicotinic acetylcholine receptor (nAChR) channels play an important role during myogenesis. In the first phase of the myogenic process, it has been demonstrated in vitro that acetylcholine (ACh) exogenously applied, and cholinergic agonists in general, promote the fusion of myoblasts into myotubes via the activation of the nAChR channels (Entwistle et al. 1988a, b; Krause et al. 1995). More recent experiments have suggested a similar role for the ACh synthesized and secreted by the motor nerve in vivo (Misgeld et al. 2002). In particular, skeletal muscle tissue of mutant mice lacking the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) revealed an incomplete development, at least in part due to an impaired cell fusion. Interestingly, during skeletal muscle development, muscle cells by themselves could represent a source of cholinergic agonists. The content and release of ACh in skeletal muscle fibres was first suggested many years ago (Miledi et al. 1977, 1982). More recently, it has been reported that ACh-like compounds (ACh-lc; Entwistle et al. 1988b; Hamann et al. 1995) are synthesized and released by cultured myoblasts differentiating in vitro. Therefore, it has been proposed that fusion of muscle cells could be controlled by an autocrine activation of nAChRs induced by endogenous cholinergic agonists (Entwistle et al. 1988a, b; Krause et al. 1995).

After cell fusion, the expression of the embryonic nAChR channels is enhanced (reviewed by Hall & Sanes, 1993) and myotubes retain the capability to produce an ACh-lc (Hamann et al. 1995) suggesting that nAChR channels could be involved even in the later phases of myogenesis. As, in myotubes, the embryonic nAChR are evenly distributed on the membrane, it has been proposed that such a diffuse expression of the receptors might make the muscle cells particularly receptive to innervation and improve the likelihood of synaptic formation (Nakajima et al. 1987; Brehm & Henderson, 1988; see also Lin et al. 2001; Yang et al. 2001; Misgeld et al. 2002). Furthermore, it has been demonstrated that openings of the embryonic nAChR channels occur as spontaneous events in myotubes (Jackson, 1984, 1986; Franco-Obregón & Lansman, 1995). Therefore, after cell fusion, the nAChRs could be important not only for their localization at the cell membrane level but also for their own activity. Accordingly, the persistent release of an endogenous agonist in myotubes could be an additional strategy to sustain the activation of nAChR channels.

Interestingly, developing murine primary myotubes have been shown to contract in culture, independently from nerve activity, as a consequence of spontaneous variations of the intracellular concentration of Ca2+ ([Ca2+]i; Grouselle et al. 1991; Cognard et al. 1993; Lorenzon et al. 2002a). In the light of this evidence, it may be speculated that, in skeletal muscles before innervation, the spontaneous openings of the nAChR channels and/or their autocrine activation via endogenous ACh-lc could mimic the presence of the motor nerve and promote muscle contractions. The aim of the present work was to test such hypothesis. For that purpose, we used mouse primary myotubes developing in culture. In these cells we have already described the presence of embryonic nAChR channels and the existence of ‘spontaneous’ contractile activity triggered by membrane depolarizations and variations of the [Ca2+]i (Lorenzon et al. 2002a). The presence of an endogenous ACh-lc in the extracellular fluid, and the effects that changes in the content of ACh-lc have on the [Ca2+]i transients, led us to conclude that an autocrine activation of nAChR channels is the most probable mechanism responsible for the spontaneous contractions of developing myotubes. However, we cannot exclude that, in the absence of the endogenous agonist, even truly spontaneous openings of the nAChR channels could play a similar role but with lower efficiency. Finally, we suggest that such an intrinsic activation of nAChR channels might contribute to the survival of developing myotubes, sustaining the mechanical activity in the absence of the nerve. A preliminary report of some of these results has been presented in abstract form (Lorenzon et al. 2002b).

Methods

Cell culture

Cell cultures were established from mouse satellite cells (Irintchev et al. 1997). Briefly, myoblasts were derived from the hind-leg muscles of a 7-day-old male Balb/c mouse killed by cervical dislocation as approved by local Animal Care Committee and in agreement with the European legislation. Muscular tissue was minced and then dissociated in a solution containing collagenase and trypsin. Expansion and enrichment in myogenic cells (desmin positive) was achieved by replating and culturing in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal calf serum. Myogenic cells, termed i28, could be maintained as exponentially growing myoblasts in a medium consisting of HAM'S F-10 containing 20% fetal calf serum, l-glutamine (2 mm), penicillin (100 units ml−1) and streptomycin (100 μg ml−1). Cell differentiation and myoblast fusion were obtained switching to DMEM supplemented with 2% horse serum and l-glutamine, penicillin and streptomycin as above (differentiation medium). Cells were maintained at 37°C in a humidified atmosphere, 95% air and 5% CO2. A complete medium change was carried out routinely every 2 days of culture. Experiments were performed on myotubes maintained in culture up to day 7 of differentiation, after which time the cells started to peel off because of their contractions. When the cell differentiation was performed in the presence of toxins (α-bungarotoxin and tetrodotoxin, see text for further details), the medium supplemented with drugs was renewed every day.

Fusion index, mean number of myotubes and mean number of contracting myotubes

The efficiency of cell fusion was assessed by determining the fusion index; which was established by dividing the number of nuclei in the myotubes (i.e. cells having more than two nuclei) by the total number of nuclei observed in 50 fields randomly chosen. The mean number of myotubes was calculated by counting the number of multinucleated cells per optical field. One microscope field (×40 objective) contained between 60 and 120 nuclei (30–60 cells). The number of nuclei in the observed cells was evaluated using fluorescent staining with 4,6-diamino-2-phenylindole (DAPI). Briefly, cells were fixed in freshly prepared 3.7% paraformaldehyde in phosphate-buffer saline (PBS) for 15 min at room temperature (20°C) and permeabilized by a 5 min incubation in 100% methanol at room temperature. Staining was then carried out by incubating the cells for 10 min at room temperature in PBS containing 10 μm DAPI. Cells were then rinsed in PBS and the fluorescence images were compared to the corresponding bright-field images.

The mean number of contracting myotubes was evaluated in bright-field as the number of myotubes exhibiting twitches divided by the number of cells per optical field (×40 objective). In particular, 50 optical fields were randomly chosen and each was observed for 2 min.

Videoimaging

Videoimaging experiments were carried out on cells plated on glass coverslips coated with matrigel (kindly given to us by Drs A. Albini and D. Noonan, CBA, Genova, Italy). Fura-2 pentacetoxymethylester (fura-2 AM) was used as Ca2+ indicator. The cells were loaded by incubating (30 min, 37°C) in a physiological external saline solution (NES, in mm: 2.8 KCl, 140 NaCl, 2 CaCl2, 10 Hepes, 2 MgCl2, 10 glucose, pH 7.3) supplemented with 10 mg ml−1 bovine serum albumin and 5 μm fura-2 AM. After loading, the cells were rinsed and maintained in NES for an additional 15 min at 37°C to allow de-esterification of the dye. The digital fluorescence-imaging microscopy system was built around an inverted microscope (Zeiss Axiovert 135, Oberkochen, Germany) equipped with an intensified CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). Loaded cells were alternatively excited at 340 and 380 nm using a modified dual wavelength microfluorimeter (Jasco CAM-230, Tokyo, Japan). Fluorescence images were collected with the CCD camera and the analog output was digitized and integrated in real time using an image processor. Image acquisition was performed at 4 frames/s. Calculations of ratio and the temporal plots of the fluorescence signal were calculated off line (Grynkiewicz et al. 1985). The temporal plots, i.e. the variations in the mean value of fluorescence intensity, were calculated from ratio images in areas of interest. A temperature-controlled microincubator chamber (Medical System Corporation, Greenvale, NY, USA) maintained the temperature at 37°C during the videoimaging experiments. Drugs were gently applied to the bathing solution by loading appropriate volumes (×100) of concentrated solution into a 2-ml syringe connected to the microincubator chamber via a small tube.

Immunocytochemistry

Cells were fixed in freshly prepared 4% paraformaldehyde in PBS for 15 min at room temperature, rinsed three times in PBS and stored at 4°C until ready to use. Cultures were incubated in 5% normal rabbit serum, 3% bovine serum albumin, 0.1% Triton X-100 in PBS (blocking solution), for 30 min at room temperature, and then incubated with goat polyclonal antibodies against choline acetyl transferase (ChAT; Chemicon Temecula, CA, USA), diluted 1: 50 in blocking solution, for 16 h, at 4°C in a humid chamber. Subsequently, the cultures were washed four times in PBS, and incubated in blocking solution containing a 1: 100 dilution of horseradish peroxidase conjugated secondary antibodies for 2 h at room temperature. Cultures were then washed three times in PBS and incubated in developing solution consisting of 0.4 mg ml−1 diaminobenzidine, 2 mg ml−1d-glucose (Merk, Whitehouse Station, NJ, USA) and 0.02 mg ml−1 glucose oxidase in 100 mm Tris-HCl pH 7.5. The reaction was stopped by washing with water. Cultures were then dehydrated in graded ethanol and mounted with Eukitt (Bio-Optica, Milan, Italy).

Chemiluminescence assay

The ACh content was detected using the chemiluminescence method described by Israel & Lesbats (1981). The experiments were carried out on 10 different cell cultures. The assay was performed after 7 days of differentiation when the fusion index was 60–80% and the number of cells per 35 mm Petri dish was 1 200 000 ± 58 000 (n = 44). For determination of ACh content, the culture medium was carefully removed and cells were washed with NES in order to remove any choline present in the medium. Then, 850 μl fresh NES was added to each 35 mm dish and incubated for 15 min at 37°C after which the fluid was collected and stored at −20°C until the assay. After adjusting the pH to 8.6, by adding NaOH, a 750 μl aliquot from each sample was added to the chemiluminescent reaction mixture containing 10 μl luminol (1 mm stock solution), 5 μl horseradish peroxidase (2 mg ml−1 stock solution) and 5 μl acetylcholinesterase (AChE; 1000 IU ml−1 stock solution purified on a Sephadex G-50 column). Chemiluminescence was recorded using a Lumat LB9507 (EG & G Berthold, Bad Wilbad, Germany); when the light emission reached a stable baseline, 50 μl choline oxidase (50 IU ml−1 stock solution) were added. The ACh in the samples was calculated from each recording by comparison with a standard ACh solution of known concentration. The ACh assay was shown to be linear over a concentration range of 0.1–150 pmol.

Electrophysiological recordings

Patch clamp recordings were performed, at room temperature, in the cell-attached configuration. For recording, the dish was placed on the stage of an inverted microscope (Zeiss IM 35, Oberkochen, Germany). Cells were bathed in the NES. Electrodes (5–8 MΩ tip resistance) were prepared from borosilicate glass (Harvard Apparatus, Holliston, MA, USA). According to the experimental requirements, the electrodes were filled with NES alone, or NES supplemented with ACh (100 nm) or α-BuTX (2–5 μm). Patch clamp recordings were performed using the Axopatch 200 amplifier (Axon Instruments, Union City, CA, USA). Signals were filtered at 5 kHz (3-pole internal Bessel filter), digitized at 20 kHz using the DigiData 1200 interface and stored on hard disk. Acquisition and analysis were performed using the pCLAMP6 software (Axon Instruments). Transitions were detected by a threshold-crossing algorithm with thresholds for open and closed states set at ∼ 50% of the mean channel current level. Amplitude histograms were fitted with Gaussian curves using the least-squares method. Channel conductance was estimated from the slope of the regression line obtained by plotting the current amplitude against the pipette potentials at +40, +60 and +80 mV. Dwell time histograms were constructed for > 450 events and fitted with the least-squares method. Kinetic analysis was performed at +60 mV pipette potential. The single channel open probability (Popen) was calculated using the formula:

graphic file with name tjp0568-0171-m1.jpg

where to is the total open time, ti is the time interval over which Popen is measured and N is the number of events detected in the same interval. The frequency of channel openings was estimated dividing the number of events detected by the time interval of recording (events s−1). The graphical representation was performed using the pCLAMP6 and Prism 3.0 (GraphPad Software, San Diego, CA, USA) software.

Chemicals

Dulbecco's modified Eagle's medium, horse serum, antibiotics and l-glutamine were purchased from ICN Biomedicals (Costa Mesa, CA, USA); fetal calf serum from PAA Laboratories (Linz, Austria); tetrodotoxin from Calbiochem Novabiochem (La Jolla, CA, USA); and all other chemicals were from Sigma (St Louis, MO, USA).

Statistics

Results obtained were expressed as means ±s.e.m. Student's unpaired t test was used to examine statistical significance. Values were considered significantly different when P < 0.05.

Results

Experiments were carried out on i28 myotubes at 7 days of differentiation (if not otherwise stated), at which time we observed the highest fusion index (60–80%; n = 50 fields) and the highest percentage of contracting cells (28.32 ± 2.44%; n = 50 fields; see also Lorenzon et al. 2002a). In addition, at this differentiation time, the spontaneous contracting activity could last > 90 min making possible the evaluation of the effects of pharmacological agents.

Muscle nAChR channel activity is involved in the [Ca2+]i spiking activity accompanying spontaneous contractions

In the skeletal muscle cell, a twitch requires membrane depolarization and an increase of the [Ca2+]i. We have previously shown that the spontaneous twitches, exhibited by i28 cells differentiated in vitro, are the result of [Ca2+]i spikes triggered by spontaneous membrane depolarizations via the activation of the excitation–contraction coupling mechanism (Lorenzon et al. 2002a). In our experimental conditions, the event frequency varied from 0.05 to 0.45 Hz depending on the cell, resulting in a mean event frequency of 0.16 ± 0.02 Hz (n = 35).

A series of experiments was carried out in order to explore whether nAChR channel activity could be involved in the generation of spontaneous twitches. In view of the fact that contraction always reflects [Ca2+]i variations, the possible role of AChRs was studied evaluating the effects of specific antagonists on the spontaneous [Ca2+]i spikes accompanying the contractions.

We first investigated the effect of the specific nAChR blocker α-bungarotoxin (α-BuTX). The addition of 5 μmα-BuTX stopped the spontaneous [Ca2+]i spikes in ∼ 70% of myotubes (19 cells out of 28; Fig. 1A). Then, as it has been demonstrated that the α7 neuronal isoform of the nAChR channels is also expressed during skeletal myogenesis (Fischer et al. 1999), we studied the effect of the α7 nAChR blocker methyllycaconitine (MLA; Alkondon et al. 1994; López et al. 1998). After treating myotubes with 100 nm MLA (n = 9), no significant change in the pattern of [Ca2+]i spikes was observed (Fig. 1B), excluding any role for the α7 nAChR isoform in the generation of spontaneous twitches.

Figure 1. Effects of antagonists of nAChRs on the spontaneous [Ca2+]i spiking activity.

Figure 1

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Representative traces showing the effects of α-BuTX (A) and MLA (B) on the [Ca2+]i spikes exhibited by developing mouse myotubes during myogenesis in vitro. In A and B, each trace interruption represents an interval of 15 min. Results are from two different cells.

Presence of ChAT and endogenous ACh-lc

The synthesis of endogenous cholinergic agonists has been documented in chick (Entwistle et al. 1988b) and in human developing muscle cells (Hamann et al. 1995). In order to asses whether an endogenous ACh-lc was also synthesized in the murine cell model under our experimental conditions, we tested for the expression of the ACh synthesizing enzyme, ChAT, in the myotubes and looked for the presence of ACh-lc in the extracellular medium.

The expression of ChAT was assessed by immunocytochemistry and the staining revealed the presence of the enzyme in all the myotubes examined, as well as in the few myoblasts still present in cultures (10 fields, 347 cells; Fig. 2). The specificity of the staining for ChAT was checked by control experiments, carried out in the absence of ChAT antibody, in which only a slight background immunolabelling was observed (data not shown).

Figure 2. ChAT immunocytochemistry.

Figure 2

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Immunocytochemical staining with goat polyclonal primary antibodies against ChAT and horseradish peroxidase conjugated secondary antibodies (see Methods). Note the cytoplasmic labelling in all the myotubes as well as in myoblasts (arrows). Phase-contrast micrograph. The scale bar represents 50 μm.

To determine the amount of ACh-lc released, the cells were incubated for 15 min at 37°C in the physiological external saline solution, the supernatant was then removed and added to the chemiluminescent reaction solution (for more details see Methods). Using this method we detected the presence of molecules hydrolysed to choline by AChE, suggesting the release of the endogenous agonist also in our mouse cell model. The content of the endogenous agonist was estimated to be 124.60 ± 27.11 pmol per well (n = 10), corresponding to a concentration of 165.82 ± 35.97 nm. Taking into account the fact that AChE can hydrolyse compounds similar to ACh, we consider the term ACh-lc more appropriate than ACh in referring to the endogenous agonist (see also Hamann et al. 1995). Accordingly, we refer to the endogenous cholinergic compound as ACh-lc throughout the text.

Endogenous ACh-lc content modulates the [Ca2+]i spiking activity

In view of the presence of an ACh-lc in our cell cultures, it was of interest to see if the endogenous ACh-lc could influence [Ca2+]i spiking activity. We took advantage of the fact that AChE is expressed by mouse myotubes differentiated and cultured in vitro particularly when, as in our experimental conditions, myotubes are electrically active (Powell et al. 1986; Senni et al. 1987). Firstly, we examined the effect of a decrease in the ACh-lc content on the [Ca2+]i spiking activity by adding purified AChE to the extracellular solution, and in other experiments, we investigated the result of increasing the ACh-lc content by inhibiting the hydrolitic action of AChE using edrophonium. The addition of exogenous AChE (200 IU) caused, after 2–4 min, a block of the [Ca2+]i spiking activity in ∼ 45% of the cells (14 cells out of 33; Fig. 3A). Furthermore, the AChE inhibitor edrophonium (500 nm) increased the frequency of the [Ca2+]i spiking activity by 282.61 ± 59.34% in 75% of the cells (6 out of 8 cells; Fig. 3B). Altogether, these results provide further evidence for a correlation between increased extracellular ACh-lc and the occurrence of [Ca2+]i spikes.

Figure 3. Changes in the extracellular concentration of the ACh-lc affect spontaneous [Ca2+]i spiking activity.

Figure 3

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AChE blocked spiking activity (A), whereas edrophonium increased its frequency (B). Trace C is representative of the cell population in which [Ca2+]i spiking activity was not affected by AChE but it was blocked by subsequent addition of α-BuTX. In A and C each trace interruption represents an interval of 15 min. Results are from three different cells.

It is worth noting that in 20% of the cells unaltered by incubation with exogenous AChE (4 out of 19), the [Ca2+]i spikes were stopped by a further addition of 5 μmα-BuTX (Fig. 3C). This confirmed the involvement of nAChR channels in the spiking activity even in some of the cells apparently ‘resistant’ to the decrease in the endogenous ACh-lc content.

Embryonic muscle nAChR channel activity is detectable in the absence of exogenously applied ACh

Interestingly, patch clamp recordings performed in the cell-attached configuration detected single channel openings in myotubes even when the patch pipettes were filled with the physiological saline solution alone. In particular, after 7 days of culture in differentiation medium, patch clamp recordings revealed the presence of such openings in 31% of mouse myotubes (n = 32 recordings). In these conditions, the single channel open probability was 0.02 ± 0.01, corresponding to a frequency of channel openings of ∼ 2 events s−1 (n = 7; Fig. 4). In accordance with previous reports (Jackson, 1984, 1986; Franco-Obregón & Lansman, 1995), the biophysical and pharmacological properties of such openings partially resembled those of the embryonic muscle nAChR channels recorded in the presence of ACh in the pipette. Indeed, the single channel openings were characterized by a conductance of 27.79 ± 2.62 pS, and the open-time histograms were best fitted with two exponentials: brief and long time constants were of 0.21 ± 0.02 ms (72.2% of all the events) and 1.78 ± 0.24 ms, respectively (n = 8; data not shown). When recordings were performed in the presence of 2–5 μmα-BuTX in the pipette, such channel openings were invariably abolished in ∼ 20 min (n = 4; Fig. 4), suggesting that the activity recorded in the absence of ACh can be ascribed to openings of nAChR channels. In addition, as the single channel open probability did not change during the recording, it is unlikely that such openings were induced by the release of the endogenous ACh-lc. In fact, in this case, the single channel open probability should increase because of the continuous secretion of the ACh-lc from the membrane patch inside the pipette, leading to a progressive accumulation of the ACh-lc. From this evidence, we concluded that the single channel events observed in the absence of exogenous applied ACh were probably due to the spontaneous opening of the nAChR channels.

Figure 4. Openings of the embryonic nAChR channels occur spontaneously in developing mouse myotubes.

Figure 4

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In A, examples of single-channel activity recorded when the patch pipettes were filled with physiological external saline solution alone (No ACh) and with physiological external saline solution containing α-BuTX (No ACh +α-BuTX 5 μm) or acetylcholine (ACh 100 nm). The recordings were from the same cell with a holding potential of +60 mV. The histogram in B shows the proportions of cells in which nAChR channel openings were detected in the three different experimental conditions. The corresponding single channel open probabilities are shown in C. *P < 0.05.

When control experiments were performed with patch pipettes filled with 100 nm ACh (see also Lorenzon et al. 2002a), the single channel open probability increased to 0.13 ± 0.03 (∼ 7 events s−1, n = 7; Fig. 4). Moreover, the I–V relationship gave a similar slope conductance of 27.52 ± 2.35 pS. However, although the brief time constant was similar, being 0.19 ± 0.02 ms (33% of all the events), the long time constant was significantly longer being 12.24 ± 1.42 ms (n = 7; data not shown).

It should be noted that the recordings were acquired at 20 kHz, and channel kinetics are expressed as open time constants instead of mean open time. This approach allows an accurate analysis of the biophysical properties of the spontaneous openings. However, it makes a quantitative comparison of our data with those already published (e.g. Jackson, 1986; Franco-Obregón & Lansman, 1995) more difficult. Therefore, we also evaluated the mean open time values in the different experimental conditions. In the absence of exogenous applied ACh the mean open time value was 0.78 ± 0.15 (n = 5), whereas it increased to 7.55 ± 1.07 (n = 8) when 100 nm ACh was present in the pipette. In our cell model, as previously reported, nAChR openings tended to be less frequent and briefer in the physiological saline than in the presence of the exogenous ACh.

Blocking the nAChR activity decreases the number of myotubes in culture

Exogenously applied cholinergic agonists have been shown to promote the fusion of myoblasts into myotubes by activation of the nAChRs (Entwistle et al. 1988a, b; Krause et al. 1995). Therefore, experiments were aimed at investigating the role of nAChR channel activity on the number of myotubes formed; cells were differentiated in the presence of the nAChR blocker α-BuTX and the numbers of myotubes were evaluated after 2, 4 and 7 days of differentiation, and compared to controls. The toxin was applied at 5 μm, i.e the concentration found to block both the nAChR openings and the [Ca2+]i spiking activity (see above).

In control cultures, the mean number of myotubes per field was 2.20 ± 1.17 (n = 60 fields) on day 2 and significantly increased to 4.47 ± 0.17 (n = 48) on day 4 and remained at that level up to day 7 of differentiation when it was 4.70 ± 0.35 (n = 15). This shows that, in our culture conditions cell fusion occurred by day 4 (Fig. 5).

Figure 5. Blocking the nAChR activity decreases the number of myotubes in culture.

Figure 5

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Myoblasts were differentiated in control conditions (grey bars) and in the presence of α-BuTX (black bars). The mean number of myotubes per field was evaluated after 2, 4 and 7 days of differentiation. In cultures treated with α-BuTX the number of myotubes observed after 7 days of differentiation was significantly lower than in control conditions. The effect of α-BuTX was mimicked by 7-days treatment with tetrodotoxin (TTX; white bars). *P < 0.05.

In cultures chronically treated with 5 μmα-BuTX, on days 2 and 4, the mean number of myotubes per field were similar to those in controls, being 2.52 ± 0.35 (n = 18) and 4.23 ± 0.32 (n = 14). However, after 7 days of treatment with α-BuTX, the number of myotubes per field was reduced to 3.14 ± 0.33 (n = 14; Fig. 5). Interestingly, this effect of α-BuTX was mimicked by 7 days culturing in a medium supplemented with 1 μm tetrodotoxin (TTX), which abolishes spikes and twitches. In this case the number of formed myotubes per field was 3.30 ± 0.27 (n = 30; Fig. 5). As the number of myoblasts in culture was similar to the control at any time of differentiation (data not shown), a nonspecific effect of the two toxins was excluded.

Discussion

So far, nAChRs have been demonstrated to play essentially two important roles in the development of skeletal muscle fibres and their innervation. Chronologically, the first one consists of favouring the fusion of myoblasts into myotubes (Entwistle et al. 1988a, b; Krause et al. 1995). The second one manifests during the process of synaptogenesis, where nAChRs have been proposed to mark membrane sites for nerve-muscle contacts (Nakajima et al. 1987; Brehm & Henderson, 1988; see also Lin et al. 2001; Yang et al. 2001; Misgeld et al. 2002).

The main finding of the present study is the characterization of a possible new role for muscle nAChRs during the myogenesis of mouse skeletal muscle cells; and demonstrates that activation of the embryonic muscle nAChRs contributes significantly to the spontaneous contractile activity exhibited by developing myotubes in vitro. The main evidence supporting the involvement of cholinergic channels in the spontaneous contraction derives from the effects of well known cholinergic antagonists on the [Ca2+]i spiking activity sustaining the twitches. Briefly, the inhibitory effect of the blocker α-BuTX on the [Ca2+]i spikes make evident the contribution of the nAChR channel activity. Furthermore, the lack of effect of the α7 receptor blocker MLA on the [Ca2+]i spikes excludes a role for the α7 neuronal isoform expressed during myogenesis (Fischer et al. 1999), and confirms the exclusive role of the muscle type nAChRs. The effect of α-BuTX was observed in the majority of myotubes (∼ 70%), but not in all of them. This evidence suggests that in some cells the [Ca2+]i spiking may be caused by another oscillatory mechanism, such as an interplay between different voltage-dependent channels (Sciancalepore et al. 2005). It has been already demonstrated that different oscillatory mechanisms could be related to different phases of myogenesis (Lorenzon et al. 1997). Thus, one possible explanation for the less than total effect of cholinergic antagonists could be the heterogeneity of myotube population in terms of functional differentiation.

The mechanism by which the nAChRs may be activated, in the absence of a nerve, was also investigated. Immunocytochemical and chemiluminescence experiments detected ChAT activity in the developing myotubes as well as the presence of an ACh-lc in the conditioned medium. This shows that the myotubes themselves synthesize and release an endogenous cholinergic agonist, in a concentration similar to that estimated in human myotube cultures (Hamann et al. 1995). This points to an autocrine activation of the nAChRs in the mouse cell model, similar to that of chick (Entwistle et al. 1988a, b) and human cells (Hamann et al. 1995; Krause et al. 1995). The notion of an autocrine mechanism in mouse myotubes is greatly strengthened by the effects of AChE and edrophonium on [Ca2+]i spikes and twitches. Thus, the inhibitory effect of additional AChE and the enhancing effect of edrophonium strongly suggest that the endogenous agonist controls the frequency of [Ca2+]i transients and twitches. Altogether, these results provide new insights on a possible physiological role for the autocrine activation of the nAChRs. It must be pointed out that, in accordance with the hypothesis of the key role of autocrine activation of the nAChR on the [Ca2+]i transients and contractions, the addition of α-BuTX and AChE to contracting cells should block [Ca2+]i spikes and twitches with the same efficiency. At the concentrations we used, both toxin and enzyme should block the autocrine activation of the channel: α-BuTX (5 μm) by a direct action on the channel molecule, and AChE (200 IU) by the chemical inactivation of the endogenous cholinergic agonist (estimated to be in the nanomolar range). In fact, we observed a discrepancy in preventing [Ca2+]i spikes: the toxin blocked the [Ca2+]i spiking by ∼ 70%, whereas the enzyme blocked it by only 45%. The patch clamp experiments may provide an explanation for that. Single channel recordings confirm the presence of spontaneous openings of the embryonic nAChR channel, as previously described (Jackson, 1984, 1986; Franco-Obregón & Lansman, 1995). Consequently, when the amount of ACh-lc is reduced in the extracellular fluid through the addition of AChE, the spontaneous nAChR channel openings themselves might trigger [Ca2+]i spikes and contraction. Accordingly, we observed that in ∼ 20% of developing myoblasts ‘resistant’ to the administration of exogenous AChE, the [Ca2+]i spiking and twitches did stop after subsequent addition of α-BuTX.

It has been suggested that nAChRs can promote myoblast fusion into myotubes. However, in mammals, this was found only in developing muscle cells, and under appropriate conditions, i.e. when the concentration of the cholinergic agonist in the medium was artificially increased by blocking the cholinesterase (Hamann et al. 1995), or when the AChRs channels were activated by the addition of cholinergic agonists (Krause et al. 1995; see also Constantin et al. 1996). In this study, we investigated whether activation of the nAChRs has a functional role in the absence of any external action on ACh-lc. To do this, we evaluated the number of forming myotubes while cells were differentiated in the presence of 5 μmα-BuTX, i.e. when any possible intrinsic activation of the nAChRs had been removed (spontaneous openings included). The results revealed that α-BuTX treatment did not affect the number of myotubes up to day 4 of differentiation, when cell fusion occurred. Nevertheless, a 30% decrease in the number of myotubes was present on day 7 of differentiation. Considering that the effect of α-BuTX was reproduced by TTX, which in these cells prevented the membrane depolarization preceding each [Ca2+]i spike and twitch (Lorenzon et al. 2002a), it may be speculated that the autocrine activation of nAChRs causes depolarization and turns on the excitation–contraction coupling mechanism, before the arrival of the nerve. This can be a strategy of the skeletal muscle cell developing in vitro to guarantee its own cell trophism by mechanical activity (Miledi & Slater, 1970) in order to survive until innervation.

Whether a similar mechanism is needed during myogenesis in vivo is a matter of discussion. During embryogenesis, most of the myotubes form as soon as motor axons enter the developing muscle and, within a few hours, synaptic transmission is physiologically detectable in vivo (reviewed by Hall & Sanes, 1993). Therefore, in vivo the activity-dependent trophism could be ensured by the nerve from the beginning of the myotube life. In light of our results, it would be interesting to see if autocrine activation of nAChRs contributes to the fibrillatory activity (i.e. trains of spontaneous action potentials resulting in visible contractions) of denervated mammalian muscle (Tower, 1939); whose origin is still not well understood (Belmar & Eyzaguirre, 1966; Purves & Sakmann, 1974; Smith & Thesleff, 1976; Dryden et al. 1983).

Acknowledgments

The authors are particularly grateful to Professor Anton Wernig (University of Bonn, Germany) for introducing us to the study of i28 cells and for providing them. They wish to thank Professor Tomoyuki Takahashi for the helpful comments. This work was supported by grants from Fondazione Benefica Kathleen Foreman-Casali of Trieste to P.L., from the University of Trieste, MIUR-Italy (PRIN and FIRB projects) and Regione F.-V.G. to F.R.

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