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Cationic channels in normal and dystrophic human myotubes
Neuromuscular Disorders 11 (2001) 72±79
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Cationic channels in normal and dystrophic human myotubes C. Vandebrouck a,*, G. Duport b, C. Cognard a, G. Raymond a a
Laboratoire de Biomembranes et Signalisation Cellulaire, UMR CNRS/Universite de Poitiers 6558, 40 Avenue du Recteur Pineau, F-86022 Poitiers Cedex, France b Service de Chirurgie GeÂneÂrale, Centre Hospitalier Universitaire, Poitiers, France Received 16 October 1999; received in revised form 22 March 2000; accepted 3 May 2000
Abstract Human skeletal muscle cells obtained from normal and Duchenne muscular dystrophy patients were cocultured with explants of rat dorsal root ganglions. Single-channel recordings were performed with the cell-attached con®guration of the patch-clamp technique and negative pressure was applied via the patch-pipette in order to mechanically stimulate the membrane patch. Inward elementary current activity was recorded under control or negative pressure conditions. Its occurrence and mean open probability were higher in Duchenne muscular dystrophy. Amplitude histograms reveal that these channels have a small unitary conductance of around 10 pS in 110 mM Ca 21 and could be inhibited in a dose-dependent manner by gadolinium. Results show that the membrane stress favoured calcium permeation through these channels. Taken together these data provide arguments for the involvement of such channels in calcium overload previously observed in cocultured dystrophic human (Duchenne muscular dystrophy) muscle cells. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Duchenne muscular dystrophy; Human muscle; Calcium; Cationic channels; Mechanosensitive-channels
1. Introduction Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease affecting 1/3300 male births. This pathology is due to a defect in the p21 band of the X-chromosome [1] affecting dystrophin, a 427 kDa protein located at the cytoplasmic face of the sarcolemma [2±4]. In normal skeletal muscle cells, dystrophin is associated with a complex of proteins and glycoproteins, called dystrophinassociated-proteins (DAP), at its carboxy-terminal domain, and with the F-actin ®lament at its amino-terminal domain [5±8]. It is now clear that an alteration of dystrophin expression leads to a reduction in the expression of DAPs, causing disruption of the link between the cytoskeleton and the extracellular matrix [9±11]. Consequently, dystrophin could play a role in membrane stabilization and protection from mechanical damage during contractile activity of muscle [12], in addition to a regulatory role on other membrane proteins such as channels and in particular mechanosensitive channels [13±16]. Another aspect of the disease is an increase of the intracellular free calcium concentration in muscle of DMD patients reported by Bodensteiner and Engel [17] and * Corresponding author. Tel.: 133-5-49-45-35-27: fax: 133-5-49-45-4014. E-mail address: [email protected] (C. Vandebrouck).
Bertorini et al. [18]. This abnormal calcium homeostasis could be responsible for the activation of proteases, with subsequent proteolysis [19] and ultimately cell necrosis. Previous studies had shown that the elevated level of resting calcium could also be observed in human DMD cells developing in vitro, but only when the muscle cells were cocultured with nervous tissue and had reached a contracting state [20±23]. Taken together, these data and other reports progressively led to the `mechanical' hypothesis in which mechanical activity is required to trigger the appearance of the pathological phenotype. In this way, the alteration of calcium homeostasis in DMD cells could result, among numerous hypotheses, from an excessive entry of calcium via nonspeci®c channels or mechanosensitive cationic channels. Such ionic channels have been shown to be present in the sarcolemma of skeletal muscle cells [13±15,24,25]. This idea was reinforced by studies of Menke et al. [26,27] and by Imbert et al. [28] who used primary cultured cells to demonstrate that mechanical stress applied to the sarcolemma produced an increase in the resting calcium level that develops more frequently and more rapidly in human cocultured DMD cells than in control myotubes. The present study was designed for looking for evidence of the presence of cationic channels in cocultured control and DMD myotubes using the single-channel cell-attached
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C. Vandebrouck et al. / Neuromuscular Disorders 11 (2001) 72±79
con®guration of the patch-clamp technique. This con®guration was chosen instead of excised patches in order to lessen the disruption of cytoskeletal elements in the patched membrane since they may have functional links to channels. Moreover, this con®guration allowed to directly apply mechanical stimuli onto the membrane through negative pressure inside the patch pipette. The activity of these calcium permeant cationic channels was recorded in normal and dystrophin-de®cient cells with or without membrane mechanical stress. The data analysis suggests the elevated level of cytosolic calcium of DMD myotubes could be related to increased calcium entry through these channels displaying mechanosensitive properties. 2. Methods 2.1. Primary culture and coculture of human skeletal muscle cells Muscle cultures were initiated from satellite cells obtained by trypsinization of human biopsies (for details see [21,23,29]) obtained during orthopaedic surgery of patients without muscular disease (n 15, 16±51 years old) and of DMD patients (n 13, 4±15 years old) in accordance with French government regulations. When myoblasts reached the alignment stage following 8±15 days of proliferation, the growth medium was exchanged for a medium containing F-14 medium (Gibco BRL, Life Technologies, Cergy Pontoise, France) supplemented with 10% foetal bovine serum (Sigma Chemical, St. Louis, MO) and 10 mg/ml insulin (Sigma) to favour fusion of myoblasts into myotubes. The medium was renewed twice weekly. Following fusion, transverse slices of 13-day-old rat embryo spinal cord with dorsal root ganglia were added (four or ®ve per Petri dish) to the monolayer culture of muscle soon after fusion [23,30,31]. Cocultures were then maintained for 3 weeks in the same medium which was renewed twice weekly. All culture media contained penicillin G (10 units/ml, Sigma) and streptomycin (50 mg/ml, Sigma). 2.2. Electrophysiology Single-channel activity was recorded from patches of the surface membrane of normal and DMD cells using the cellattached mode of the patch-clamp technique [32]. Patchpipettes were pulled in two steps from glass haematocrit tubes (soft glass; Assistent, Bardram, Denmark) coated with Sylgard (Dow Corning, Wiesbaden, Germany). Pipettes (2±4 MV) were used to record membrane currents with a RK400 patch-clamp ampli®er (Biologic, Claix, France) at constant holding potentials. Data were ®ltered at 1 kHz and stored on a digital tape recorder (DTR 1204, Biologic). All experiments were performed at room temperature (228C). The application of membrane stress is realized after the formation of a giga-seal. A negative pres-
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sure, (i.e. suction) of 210 to 240 mmHg was applied inside the patch-pipette, and monitored by a mercury manometer. 2.3. Solutions The compositions of solutions used in these experiments are summarized in Table 1. The depolarising external solution (bath solution) contained 155 mM potassium aspartate and was designed to achieve a resting membrane potential close to 0 mV. MgCl2 (5 mM) was used in order to maintain a physiological level of divalent cations on the external membrane surface. Pipettes were ®lled with a 110 mM CaCl2 solution (pipette solution) containing DIDS to block possible chloride conductances. Electrode-®lling solutions for recording single-channel activity in the presence of monovalent cations contained a 155 mM concentration of the corresponding chloride salt in place of 110 mM CaCl2. The pH of all solutions was adjusted to 7.4 by addition of TEAOH, and the osmolarity adjusted to 300 mOsM with glucose and mannitol. 2.4. Data analysis Current records stored on DTR were replayed and digitized through an A/D converter (Digidata 1200, Axon Instruments, Foster City, CA) with appropriate records saved onto the hard disc of a PC using Biopatch software version 3.30 (Biologic, Claix, France). Current records were analyzed after ®ltering by means of the built-in analogue ®ve-pole Bessel ®lter of the patch-ampli®er. For kinetic analysis, current records were digitized at 5 kHz. Amplitude distributions were ®tted by single Gaussian curves. Statistical data analysis was performed using GraphPad Prism version.2.00 (GraphPad Software, San Diego, CA, www.graphpad.com). Unless stated, statistical signi®cance between the different mean values was determined using the unpaired Student's t-test. Table 1 Composition of bath and pipette solutions used in cell-attached patch-clamp con®guration a Bath solution mM
150 KOH 150 Aspartic acid 5 MgCl2 10 HEPES
Pipette solutions mM 110 Ca
X b solution
110 CaCl2 10 HEPES 0.01 DIDS
155 XCl 10 HEPES 0.01 DIDS
a The osmolarity was 300 mOsm/l in all cases and was adjusted with glucose and mannitol and the pH was adjusted with TEAOH. DIDS: 4.4 0 -Diisothyocyanate stilbene-2.2 0 disulfonic acid, HEPES: hydroxyl ethyl piperazine ethane sulfonic acid, TEAOH: tetraethylammonium hydroxide. b with X Na or K or Cs.
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Fig. 1. Single-channel current recordings from cell-attached patches of human cocultured normal (top record) and DMD (bottom) myotubes in presence or absence of mechanical stress. One min recordings were made at a constant holding potential of 260 mV. Current records were ®ltered at 1 kHz and sampled at 5 kHz. c represents the single-channel closed state.
3. Results 3.1. Channel activity and characteristics Recordings were made from cell-attached patches on normal and DMD cocultured human myotubes at a constant holding membrane potential. Current records shown in Fig. 1 illustrate the single-channel activities observed at 260 mV as inward current de¯ections on the two cell types. Under control conditions (110 mM Ca 21 solution; left panel), a spontaneous ion channel activity was recorded both in dystrophic and control cells. When a negative pressure of 210 mmHg (mechanical stress; right panel) was applied, both types of cells displayed larger inward de¯ections. Regardless of the characteristics, channel activity was more frequently observed in DMD cells than in normal cells (Fig. 2). In the absence of stress, only 50% of patch recordings of normal cells displayed an activity as compared to 73% in DMD cells (signi®cantly different, P 0:013 chisquare test). Under conditions of stress, 60% of patches from DMD cells displayed channel activity during application of suction vs. 50% in normal myotubes. Therefore, irrespective of the experimental conditions for inducing current activity, the fraction of patches displaying channel activity was signi®cantly greater in DMD cells. Nevertheless, in DMD cells the fraction of patches displaying channel activity was slightly reduced under mechanical stress (10 mmHg). It can be hypothesized that stress conditions could, in addition to enhancing open probabilities (see below), reduce the activity of stretch-inactivated channels demonstrated to be present in muscle (see [14]). The amplitude distribution histograms (Fig. 3A) reveal that current activity displayed a multi-level behaviour.
This probably corresponds to multiple open-state levels of a channel or to the activity of several similar channels since the different amplitudes were n-fold the unitary one (see Fig. 3A:b). As can be seen in Fig. 3B the unitary conductance was slightly larger in normal cells (around 11 pS) than in DMD cells (around 10 pS). With or without stress, channels displayed a reversal potential of around 0 mV for normal as well as for DMD cells. In order to quantify these multi-level openings, areas under the ®tted gaussian curves were computed for each activity level for normal and DMD cells (Fig. 4). Clearly, mechanical stimulation favoured the appearance of higher levels of openings. The relative selectivity for divalent over monovalent cations was computed from reversal potential measurements using the Goldman±Hodgkin±Katz equation modi®ed for
Fig. 2. Occurrence of channel activity in normal and DMD myotubes in presence or absence of mechanical stress. The number inside each bar corresponds to the membrane patches in which channel activity was present, (i.e. an inward current de¯ection greater than 0.4 pA and longer than 10 ms) divided by the total number of succeeding patches tested. *Difference considered as signi®cant at P , 0:05.
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Cs 1); the order of selectivity was Cs 1 K 1 Na 1 ( < 1) . Ca 21(0.65) and Cs 1(1.20) . Na 1(1.10) . K 1(1) . Ca 21(0.60) for spontaneous channel activity under control conditions for normal as well as DMD myotubes. Under mechanical stress the sequences were Na 1 K 1 Cs 1 ( < 1) . Ca 21(0.79) and Na 1 K 1 Cs 1 ( < 1) . Ca 21(0.88) for normal or DMD myotubes. Thus under mechanical stimulation, the relative permeability for calcium appeared to be only slightly increased in dystrophic cells (not signi®cantly different at P , 0:05). 3.2. Open probability
Fig. 3. Analysis of unitary current conductances on normal and DMD myotubes. (A) Example of a 400 ms unitary current recording in a DMD cell exposed to mechanical stress (a); amplitude histogram corresponding to the whole 1 min record at 260 mV (b). Numbers above each peak correspond to the peak amplitude currents obtained from a multi-Gaussian curve ®tted to the data (black trace). (B) Current-voltage relationships obtained from recordings of channel activity with 110 mM CaCl2 in the pipette without stress and in the presence of mechanical stress. Mean values of single-channel current amplitude were determinated by ®tting the amplitude histograms with Gaussian curves obtained for normal and DMD cells (mean ^ SEM). For the determination of the single-channel conductance g and the reversal potential Erev, the data, in a voltage range between 280 and 220 mV, were approximated by a straight line.
divalent cations [33]. With 110 mM Ca 21 medium in the electrode pipette and K 1 as predominant intracellular cation (around 155 mM; [34]), the PCa 21/PK 1 ratios for normal and DMD myotubes were 0.65 ^ 0.03 (n 16) and 0.60 ^ 0.05 (n 14), respectively, in the absence of stress. Under negative pressure (210 mmHg), these values were 0.79 ^ 0.06 (n 12) and 0.88 ^ 0.07 (n 13), respectively, and significantly different. The ion selectivity of the channels was investigated for a variety of cations (K 1, Na 1, Ca 21 and
Fig. 5 shows the mean open probability (®rst level) at 260 mV for each type of cell in the presence or absence of stress. The open probability appeared to be always significantly higher in DMD cells than in normal cells whatever were the conditions (Fig. 5A). Without stress the open probability were 0.61 ^ 0.04 and 0.75 ^ 0.03 in normal and DMD cells, respectively, (P 0:043). Under mechanical stress the corresponding mean values were 0.60 ^ 0.04 and 0.74 ^ 0.03 (P 0:042). As shown in Fig. 5B, this increase was observed at all membrane potentials tested but with a low voltage-dependence. The probability increased with negative transmembrane potential. The mean slopes for normal and DMD myotubes were 20.40 ^ 0.01 and 20.50 ^ 0.06 mV 21 under control conditions and 20.40 ^ 0.06 and 20.50 ^ 0.09 mV 21 under mechanical stress, respectively. These values were not signi®cantly different (P 0:135 and P 0:137, respectively). The single-channel open probability slightly increased with suction in both types of myotubes (Fig. 5C) indicating that channels exhibited moderate mechanosensitivity. Differences in slopes of the mean open probability/suction amplitude relationship for the two types of cells were not signi®cantly different (0.17 ^ 0.02 mmHg 21 for normal cells (n 14) and 0.14 ^ 0.05 mmHg 21 for DMD cells (n 16)). 3.3. Inhibition by inorganic cations As the lanthanide cation gadolinium (Gd 31) is known to block mechanosensitive channels [35], we tested its effect on unitary currents (Fig. 6A). The concentration of Gd 31 producing half-inhibition (IC50) of single-channel unitary current amplitude was 8.8 ^ 0.5 mM for normal (n 17) and 12.4 ^ 0.6 mM for DMD (n 15) cells (P , 0:0001). Under suction applied via the pipette, the IC50 was 7.1 ^ 0.4 mM for normal (n 16) and 11.1 ^ 0.2 mM for DMD (n 15) cells (P , 0:0001) (Fig. 6B). Thus, it appears that the channel activity can be blocked by gadolinium with a slightly smaller sensitivity in DMD cells than in control cells. In addition, it can be noted that gadolinium ions blocking effect on current amplitude was accompanied by an obvious reduction of the channel open-probability (Fig. 6A).
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Fig. 4. Multi-level opening behaviour of cationic channels on normal and DMD cells. Examples of amplitude histograms for each experimental condition and for the two types of cells ®tted by multi-peak Gaussian curves. The area under the derived single gaussian curves was centred at mean amplitude values (n 10) obtained at 260 mV for each type of cells in presence or absence of mechanical stress (vertical dashed lines) and was calculated for each level (number indicated below each dashed line) of detected activity. The mean values (n 10) of these areas (in event £ pA) are indicated for each level at the top of the dashed lines.
4. Discussion The main ®nding of this study is that normal and DMD skeletal human muscle myotubes possess a distinct class of cation channels permeable to calcium. These channels can be detected by recordings from cell-attached patches either as spontaneous activity or by applying suction via the electrode. The corresponding ion channel activity appears to be higher in dystrophic cells than in control cells, under control condition and mechanical stimulation. This conclusion was supported by the following data. (1) The occurrence of active patches was higher in DMD cells regardless of the conditions. (2) The multi-level openings (number of levels and number of events for each level) were favoured in DMD cells and under mechanical stimulation. (3) The calcium ion permeability relative to potassium was increased in mechanically stimulated DMD cells. (4) The opening probability (®rst level) increased in DMD cells with negative membrane patch potentials and slightly with mechanical stimulation. The existence of similar types of channels has been shown on chick and mouse muscle cells in primary culture at different stages of myogenesis as well as on cell lines [13± 16,36]. The human channels studied here share some properties with these channels such as a small conductance
(around 10 pS) and a permeability to calcium ions in addition to monovalent cations. Nevertheless, some important properties distinguish the human channels from those of other preparations. These include a moderate mechanosensitivity compared to stretch-activated channels observed on mdx myotubes, a Ca 21/K 1 permeability ratio lower than the one calculated on mdx cells, an open probability changing with the membrane potential in the reverse direction (increasing with more negative membrane potentials) and a weak dependence on negative pressure inside the pipette as compared to ion channels in mdx myotubes [13±15]. Taken together, these data suggest that the type of these cationic channels is different from those already described or, at least, that the type of interaction with the membrane lipid bilayer or the cytoskeleton elements gives them an anomalous mechanosensitivity in the human skeletal cells. The question raised by the data is how the presence of these channels in human DMD cells could be related to the pathological phenotype. In particular, the elevated channel activity in these cells could be related to the calcium overload observed in adult DMD muscle [18] and in primary coculture [23]. From this point of view, one of the properties of these channels is particularly relevant. That is, calcium ions can permeate through these channels not only under mechanical stress but also at rest. In previous reports,
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Fig. 5. Open probability on normal and DMD cells. (A) n Indicates the number of patches for each type of cell in each condition. The inset shows how Po was calculated, with T representing the recording time and tn the successive open-state durations. *Indicates that the difference between DMD and corresponding normal cells is signi®cant (Student's t-test P , 0:05). HP 260 mV. (B) Voltage-dependence of open probability. The straight line corresponds to stress conditions and the dashed line to resting conditions (n 12±15). (C) Effects of the level of negative pressure (suction) applied to the patch electrode on channel open probability in normal and DMD cells (n 10). HP 260 mV.
various authors have suggested such an explanation for the increased intracellular calcium level of dystrophin de®cient muscle cells. Mechanosensitive channels in mdx myotubes could permit an in¯ux of calcium suf®cient to elevate intracellular calcium to `pathological levels' [15] as a greater proportion of stretch-inactivated channels is seen in mdx muscle than in normal muscle [14]. Fong et al. [24] reported an increased activity of calcium leak channels in myotubes
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of a clonal Duchenne human cell line and in mdx mouse cells. These channels have a low conductance similar to that described here for DMD and normal cocultured human myotubes. In cultured chick heart, Sigurdson et al. [37] have shown that mechanosensitive channels under physiological condition pass up to 20 fA of calcium current which could increase the intracellular free calcium concentration to a maximum concentration of 12.5 mM. This is more than suf®cient to trigger calcium-induced calcium release [35]. Similarly the channels studied here could permit the passage of enough calcium at rest and under the membrane stress caused by a contraction to contribute to an elevation of intracellular calcium concentration. Nevertheless, the technical con®guration which allowed to directly apply mechanical stimulation to the membrane patch precluded to measure the quantity of calcium ions entering the whole cell and contributing to the calcium elevation in physiological conditions. Imbert et al. [28] and Leijendekker et al. [38] had previously shown that another type of mechanical stimulation (hypoosmotic shock) was able to induce intracellular calcium elevation. It must be noted that in spite of the ability to directly stimulate the membrane patch, a technical limitation was brought by the patch-clamp technique because of formation of the giga-seal which could per se (with the resulting classical ohm-shape of the patched membrane) mechanically induce channel activity. The giga-seal conditions did therefore not represent a true zero mechanical stimulation level. Furthermore the mechanosensitivity could be reduced or only detected for high stimulations. Nevertheless these data ®t well with the hypothesis that an absence of dystrophin could modify channel conformation and/or facilitate the activation of channels which, in turn, could increase calcium ion in¯ux. Since the studied channels carrying this current are activated in the potential range at which contraction develops, the contraction of human DMD myotubes could induce or boost cationic channel activity and permit more calcium to enter those cells. The use of gadolinium, a non-speci®c inhibitor of these channels [35,39], indicated that normal cells are slightly more sensitive to this inhibitor than DMD cells. Gadolinium has been shown to interact strongly with lipid bilayers and is capable of changing the physical environment of membrane channel proteins [40]. Since DMD myotubes have an unstable environment due to the lack of dystrophin, the application of gadolinium to the membrane could produce unexpected effects that could explain the difference of gadolinium-sensitivity observed between the two types of cells studied here. In conclusion, normal and DMD human cocultured myotubes exhibit cationic channels that spontaneously activate at physiological resting transmembrane potentials in the absence of stress. This greater activity in DMD cells is probably related to the lack of membrane stability in dystrophin-free cells. The second observation is that these cells possess cationic channels which can be opened by negative
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Fig. 6. Effect of Gd 31 on normal and DMD myotube single-channel activity with or without mechanical stress at 260 mV. (A) Examples of spontaneous unitary current traces recorded in DMD cells in the presence of increasing gadolinium concentrations in the pipette medium. (B) Inhibition percentage of the single-channel unitary current amplitude against the gadolinium concentration in the absence and in the presence of mechanical stress.
pressure applied through the pipette. Mechanical stimulation could be responsible for signi®cant calcium entry and we postulate that the high level of cytosolic calcium concentrations observed in DMD cells could be related, at least partly, to an elevated in¯ux of calcium through these channels. These results provide a strong case in relation to the important role of dystrophin and the consequences of its absence in DMD myotubes.
Acknowledgements This work was supported by grants from the CNRS (UMR 6558), the University of Poitiers, and the Association FrancËaise contre les Myopathies. We thank FrancËoise Mazin for expert technical assistance.
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Cationic channels in normal and dystrophic human myotubes C. Vandebrouck a,*, G. Duport b, C. Cognard a, G. Raymond a a
Laboratoire de Biomembranes et Signalisation Cellulaire, UMR CNRS/Universite de Poitiers 6558, 40 Avenue du Recteur Pineau, F-86022 Poitiers Cedex, France b Service de Chirurgie GeÂneÂrale, Centre Hospitalier Universitaire, Poitiers, France Received 16 October 1999; received in revised form 22 March 2000; accepted 3 May 2000
Abstract Human skeletal muscle cells obtained from normal and Duchenne muscular dystrophy patients were cocultured with explants of rat dorsal root ganglions. Single-channel recordings were performed with the cell-attached con®guration of the patch-clamp technique and negative pressure was applied via the patch-pipette in order to mechanically stimulate the membrane patch. Inward elementary current activity was recorded under control or negative pressure conditions. Its occurrence and mean open probability were higher in Duchenne muscular dystrophy. Amplitude histograms reveal that these channels have a small unitary conductance of around 10 pS in 110 mM Ca 21 and could be inhibited in a dose-dependent manner by gadolinium. Results show that the membrane stress favoured calcium permeation through these channels. Taken together these data provide arguments for the involvement of such channels in calcium overload previously observed in cocultured dystrophic human (Duchenne muscular dystrophy) muscle cells. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Duchenne muscular dystrophy; Human muscle; Calcium; Cationic channels; Mechanosensitive-channels
1. Introduction Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease affecting 1/3300 male births. This pathology is due to a defect in the p21 band of the X-chromosome [1] affecting dystrophin, a 427 kDa protein located at the cytoplasmic face of the sarcolemma [2±4]. In normal skeletal muscle cells, dystrophin is associated with a complex of proteins and glycoproteins, called dystrophinassociated-proteins (DAP), at its carboxy-terminal domain, and with the F-actin ®lament at its amino-terminal domain [5±8]. It is now clear that an alteration of dystrophin expression leads to a reduction in the expression of DAPs, causing disruption of the link between the cytoskeleton and the extracellular matrix [9±11]. Consequently, dystrophin could play a role in membrane stabilization and protection from mechanical damage during contractile activity of muscle [12], in addition to a regulatory role on other membrane proteins such as channels and in particular mechanosensitive channels [13±16]. Another aspect of the disease is an increase of the intracellular free calcium concentration in muscle of DMD patients reported by Bodensteiner and Engel [17] and * Corresponding author. Tel.: 133-5-49-45-35-27: fax: 133-5-49-45-4014. E-mail address: [email protected] (C. Vandebrouck).
Bertorini et al. [18]. This abnormal calcium homeostasis could be responsible for the activation of proteases, with subsequent proteolysis [19] and ultimately cell necrosis. Previous studies had shown that the elevated level of resting calcium could also be observed in human DMD cells developing in vitro, but only when the muscle cells were cocultured with nervous tissue and had reached a contracting state [20±23]. Taken together, these data and other reports progressively led to the `mechanical' hypothesis in which mechanical activity is required to trigger the appearance of the pathological phenotype. In this way, the alteration of calcium homeostasis in DMD cells could result, among numerous hypotheses, from an excessive entry of calcium via nonspeci®c channels or mechanosensitive cationic channels. Such ionic channels have been shown to be present in the sarcolemma of skeletal muscle cells [13±15,24,25]. This idea was reinforced by studies of Menke et al. [26,27] and by Imbert et al. [28] who used primary cultured cells to demonstrate that mechanical stress applied to the sarcolemma produced an increase in the resting calcium level that develops more frequently and more rapidly in human cocultured DMD cells than in control myotubes. The present study was designed for looking for evidence of the presence of cationic channels in cocultured control and DMD myotubes using the single-channel cell-attached
0960-8966/01/$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S09 60-8966(00)0015 3-X
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con®guration of the patch-clamp technique. This con®guration was chosen instead of excised patches in order to lessen the disruption of cytoskeletal elements in the patched membrane since they may have functional links to channels. Moreover, this con®guration allowed to directly apply mechanical stimuli onto the membrane through negative pressure inside the patch pipette. The activity of these calcium permeant cationic channels was recorded in normal and dystrophin-de®cient cells with or without membrane mechanical stress. The data analysis suggests the elevated level of cytosolic calcium of DMD myotubes could be related to increased calcium entry through these channels displaying mechanosensitive properties. 2. Methods 2.1. Primary culture and coculture of human skeletal muscle cells Muscle cultures were initiated from satellite cells obtained by trypsinization of human biopsies (for details see [21,23,29]) obtained during orthopaedic surgery of patients without muscular disease (n 15, 16±51 years old) and of DMD patients (n 13, 4±15 years old) in accordance with French government regulations. When myoblasts reached the alignment stage following 8±15 days of proliferation, the growth medium was exchanged for a medium containing F-14 medium (Gibco BRL, Life Technologies, Cergy Pontoise, France) supplemented with 10% foetal bovine serum (Sigma Chemical, St. Louis, MO) and 10 mg/ml insulin (Sigma) to favour fusion of myoblasts into myotubes. The medium was renewed twice weekly. Following fusion, transverse slices of 13-day-old rat embryo spinal cord with dorsal root ganglia were added (four or ®ve per Petri dish) to the monolayer culture of muscle soon after fusion [23,30,31]. Cocultures were then maintained for 3 weeks in the same medium which was renewed twice weekly. All culture media contained penicillin G (10 units/ml, Sigma) and streptomycin (50 mg/ml, Sigma). 2.2. Electrophysiology Single-channel activity was recorded from patches of the surface membrane of normal and DMD cells using the cellattached mode of the patch-clamp technique [32]. Patchpipettes were pulled in two steps from glass haematocrit tubes (soft glass; Assistent, Bardram, Denmark) coated with Sylgard (Dow Corning, Wiesbaden, Germany). Pipettes (2±4 MV) were used to record membrane currents with a RK400 patch-clamp ampli®er (Biologic, Claix, France) at constant holding potentials. Data were ®ltered at 1 kHz and stored on a digital tape recorder (DTR 1204, Biologic). All experiments were performed at room temperature (228C). The application of membrane stress is realized after the formation of a giga-seal. A negative pres-
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sure, (i.e. suction) of 210 to 240 mmHg was applied inside the patch-pipette, and monitored by a mercury manometer. 2.3. Solutions The compositions of solutions used in these experiments are summarized in Table 1. The depolarising external solution (bath solution) contained 155 mM potassium aspartate and was designed to achieve a resting membrane potential close to 0 mV. MgCl2 (5 mM) was used in order to maintain a physiological level of divalent cations on the external membrane surface. Pipettes were ®lled with a 110 mM CaCl2 solution (pipette solution) containing DIDS to block possible chloride conductances. Electrode-®lling solutions for recording single-channel activity in the presence of monovalent cations contained a 155 mM concentration of the corresponding chloride salt in place of 110 mM CaCl2. The pH of all solutions was adjusted to 7.4 by addition of TEAOH, and the osmolarity adjusted to 300 mOsM with glucose and mannitol. 2.4. Data analysis Current records stored on DTR were replayed and digitized through an A/D converter (Digidata 1200, Axon Instruments, Foster City, CA) with appropriate records saved onto the hard disc of a PC using Biopatch software version 3.30 (Biologic, Claix, France). Current records were analyzed after ®ltering by means of the built-in analogue ®ve-pole Bessel ®lter of the patch-ampli®er. For kinetic analysis, current records were digitized at 5 kHz. Amplitude distributions were ®tted by single Gaussian curves. Statistical data analysis was performed using GraphPad Prism version.2.00 (GraphPad Software, San Diego, CA, www.graphpad.com). Unless stated, statistical signi®cance between the different mean values was determined using the unpaired Student's t-test. Table 1 Composition of bath and pipette solutions used in cell-attached patch-clamp con®guration a Bath solution mM
150 KOH 150 Aspartic acid 5 MgCl2 10 HEPES
Pipette solutions mM 110 Ca
X b solution
110 CaCl2 10 HEPES 0.01 DIDS
155 XCl 10 HEPES 0.01 DIDS
a The osmolarity was 300 mOsm/l in all cases and was adjusted with glucose and mannitol and the pH was adjusted with TEAOH. DIDS: 4.4 0 -Diisothyocyanate stilbene-2.2 0 disulfonic acid, HEPES: hydroxyl ethyl piperazine ethane sulfonic acid, TEAOH: tetraethylammonium hydroxide. b with X Na or K or Cs.
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Fig. 1. Single-channel current recordings from cell-attached patches of human cocultured normal (top record) and DMD (bottom) myotubes in presence or absence of mechanical stress. One min recordings were made at a constant holding potential of 260 mV. Current records were ®ltered at 1 kHz and sampled at 5 kHz. c represents the single-channel closed state.
3. Results 3.1. Channel activity and characteristics Recordings were made from cell-attached patches on normal and DMD cocultured human myotubes at a constant holding membrane potential. Current records shown in Fig. 1 illustrate the single-channel activities observed at 260 mV as inward current de¯ections on the two cell types. Under control conditions (110 mM Ca 21 solution; left panel), a spontaneous ion channel activity was recorded both in dystrophic and control cells. When a negative pressure of 210 mmHg (mechanical stress; right panel) was applied, both types of cells displayed larger inward de¯ections. Regardless of the characteristics, channel activity was more frequently observed in DMD cells than in normal cells (Fig. 2). In the absence of stress, only 50% of patch recordings of normal cells displayed an activity as compared to 73% in DMD cells (signi®cantly different, P 0:013 chisquare test). Under conditions of stress, 60% of patches from DMD cells displayed channel activity during application of suction vs. 50% in normal myotubes. Therefore, irrespective of the experimental conditions for inducing current activity, the fraction of patches displaying channel activity was signi®cantly greater in DMD cells. Nevertheless, in DMD cells the fraction of patches displaying channel activity was slightly reduced under mechanical stress (10 mmHg). It can be hypothesized that stress conditions could, in addition to enhancing open probabilities (see below), reduce the activity of stretch-inactivated channels demonstrated to be present in muscle (see [14]). The amplitude distribution histograms (Fig. 3A) reveal that current activity displayed a multi-level behaviour.
This probably corresponds to multiple open-state levels of a channel or to the activity of several similar channels since the different amplitudes were n-fold the unitary one (see Fig. 3A:b). As can be seen in Fig. 3B the unitary conductance was slightly larger in normal cells (around 11 pS) than in DMD cells (around 10 pS). With or without stress, channels displayed a reversal potential of around 0 mV for normal as well as for DMD cells. In order to quantify these multi-level openings, areas under the ®tted gaussian curves were computed for each activity level for normal and DMD cells (Fig. 4). Clearly, mechanical stimulation favoured the appearance of higher levels of openings. The relative selectivity for divalent over monovalent cations was computed from reversal potential measurements using the Goldman±Hodgkin±Katz equation modi®ed for
Fig. 2. Occurrence of channel activity in normal and DMD myotubes in presence or absence of mechanical stress. The number inside each bar corresponds to the membrane patches in which channel activity was present, (i.e. an inward current de¯ection greater than 0.4 pA and longer than 10 ms) divided by the total number of succeeding patches tested. *Difference considered as signi®cant at P , 0:05.
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Cs 1); the order of selectivity was Cs 1 K 1 Na 1 ( < 1) . Ca 21(0.65) and Cs 1(1.20) . Na 1(1.10) . K 1(1) . Ca 21(0.60) for spontaneous channel activity under control conditions for normal as well as DMD myotubes. Under mechanical stress the sequences were Na 1 K 1 Cs 1 ( < 1) . Ca 21(0.79) and Na 1 K 1 Cs 1 ( < 1) . Ca 21(0.88) for normal or DMD myotubes. Thus under mechanical stimulation, the relative permeability for calcium appeared to be only slightly increased in dystrophic cells (not signi®cantly different at P , 0:05). 3.2. Open probability
Fig. 3. Analysis of unitary current conductances on normal and DMD myotubes. (A) Example of a 400 ms unitary current recording in a DMD cell exposed to mechanical stress (a); amplitude histogram corresponding to the whole 1 min record at 260 mV (b). Numbers above each peak correspond to the peak amplitude currents obtained from a multi-Gaussian curve ®tted to the data (black trace). (B) Current-voltage relationships obtained from recordings of channel activity with 110 mM CaCl2 in the pipette without stress and in the presence of mechanical stress. Mean values of single-channel current amplitude were determinated by ®tting the amplitude histograms with Gaussian curves obtained for normal and DMD cells (mean ^ SEM). For the determination of the single-channel conductance g and the reversal potential Erev, the data, in a voltage range between 280 and 220 mV, were approximated by a straight line.
divalent cations [33]. With 110 mM Ca 21 medium in the electrode pipette and K 1 as predominant intracellular cation (around 155 mM; [34]), the PCa 21/PK 1 ratios for normal and DMD myotubes were 0.65 ^ 0.03 (n 16) and 0.60 ^ 0.05 (n 14), respectively, in the absence of stress. Under negative pressure (210 mmHg), these values were 0.79 ^ 0.06 (n 12) and 0.88 ^ 0.07 (n 13), respectively, and significantly different. The ion selectivity of the channels was investigated for a variety of cations (K 1, Na 1, Ca 21 and
Fig. 5 shows the mean open probability (®rst level) at 260 mV for each type of cell in the presence or absence of stress. The open probability appeared to be always significantly higher in DMD cells than in normal cells whatever were the conditions (Fig. 5A). Without stress the open probability were 0.61 ^ 0.04 and 0.75 ^ 0.03 in normal and DMD cells, respectively, (P 0:043). Under mechanical stress the corresponding mean values were 0.60 ^ 0.04 and 0.74 ^ 0.03 (P 0:042). As shown in Fig. 5B, this increase was observed at all membrane potentials tested but with a low voltage-dependence. The probability increased with negative transmembrane potential. The mean slopes for normal and DMD myotubes were 20.40 ^ 0.01 and 20.50 ^ 0.06 mV 21 under control conditions and 20.40 ^ 0.06 and 20.50 ^ 0.09 mV 21 under mechanical stress, respectively. These values were not signi®cantly different (P 0:135 and P 0:137, respectively). The single-channel open probability slightly increased with suction in both types of myotubes (Fig. 5C) indicating that channels exhibited moderate mechanosensitivity. Differences in slopes of the mean open probability/suction amplitude relationship for the two types of cells were not signi®cantly different (0.17 ^ 0.02 mmHg 21 for normal cells (n 14) and 0.14 ^ 0.05 mmHg 21 for DMD cells (n 16)). 3.3. Inhibition by inorganic cations As the lanthanide cation gadolinium (Gd 31) is known to block mechanosensitive channels [35], we tested its effect on unitary currents (Fig. 6A). The concentration of Gd 31 producing half-inhibition (IC50) of single-channel unitary current amplitude was 8.8 ^ 0.5 mM for normal (n 17) and 12.4 ^ 0.6 mM for DMD (n 15) cells (P , 0:0001). Under suction applied via the pipette, the IC50 was 7.1 ^ 0.4 mM for normal (n 16) and 11.1 ^ 0.2 mM for DMD (n 15) cells (P , 0:0001) (Fig. 6B). Thus, it appears that the channel activity can be blocked by gadolinium with a slightly smaller sensitivity in DMD cells than in control cells. In addition, it can be noted that gadolinium ions blocking effect on current amplitude was accompanied by an obvious reduction of the channel open-probability (Fig. 6A).
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Fig. 4. Multi-level opening behaviour of cationic channels on normal and DMD cells. Examples of amplitude histograms for each experimental condition and for the two types of cells ®tted by multi-peak Gaussian curves. The area under the derived single gaussian curves was centred at mean amplitude values (n 10) obtained at 260 mV for each type of cells in presence or absence of mechanical stress (vertical dashed lines) and was calculated for each level (number indicated below each dashed line) of detected activity. The mean values (n 10) of these areas (in event £ pA) are indicated for each level at the top of the dashed lines.
4. Discussion The main ®nding of this study is that normal and DMD skeletal human muscle myotubes possess a distinct class of cation channels permeable to calcium. These channels can be detected by recordings from cell-attached patches either as spontaneous activity or by applying suction via the electrode. The corresponding ion channel activity appears to be higher in dystrophic cells than in control cells, under control condition and mechanical stimulation. This conclusion was supported by the following data. (1) The occurrence of active patches was higher in DMD cells regardless of the conditions. (2) The multi-level openings (number of levels and number of events for each level) were favoured in DMD cells and under mechanical stimulation. (3) The calcium ion permeability relative to potassium was increased in mechanically stimulated DMD cells. (4) The opening probability (®rst level) increased in DMD cells with negative membrane patch potentials and slightly with mechanical stimulation. The existence of similar types of channels has been shown on chick and mouse muscle cells in primary culture at different stages of myogenesis as well as on cell lines [13± 16,36]. The human channels studied here share some properties with these channels such as a small conductance
(around 10 pS) and a permeability to calcium ions in addition to monovalent cations. Nevertheless, some important properties distinguish the human channels from those of other preparations. These include a moderate mechanosensitivity compared to stretch-activated channels observed on mdx myotubes, a Ca 21/K 1 permeability ratio lower than the one calculated on mdx cells, an open probability changing with the membrane potential in the reverse direction (increasing with more negative membrane potentials) and a weak dependence on negative pressure inside the pipette as compared to ion channels in mdx myotubes [13±15]. Taken together, these data suggest that the type of these cationic channels is different from those already described or, at least, that the type of interaction with the membrane lipid bilayer or the cytoskeleton elements gives them an anomalous mechanosensitivity in the human skeletal cells. The question raised by the data is how the presence of these channels in human DMD cells could be related to the pathological phenotype. In particular, the elevated channel activity in these cells could be related to the calcium overload observed in adult DMD muscle [18] and in primary coculture [23]. From this point of view, one of the properties of these channels is particularly relevant. That is, calcium ions can permeate through these channels not only under mechanical stress but also at rest. In previous reports,
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Fig. 5. Open probability on normal and DMD cells. (A) n Indicates the number of patches for each type of cell in each condition. The inset shows how Po was calculated, with T representing the recording time and tn the successive open-state durations. *Indicates that the difference between DMD and corresponding normal cells is signi®cant (Student's t-test P , 0:05). HP 260 mV. (B) Voltage-dependence of open probability. The straight line corresponds to stress conditions and the dashed line to resting conditions (n 12±15). (C) Effects of the level of negative pressure (suction) applied to the patch electrode on channel open probability in normal and DMD cells (n 10). HP 260 mV.
various authors have suggested such an explanation for the increased intracellular calcium level of dystrophin de®cient muscle cells. Mechanosensitive channels in mdx myotubes could permit an in¯ux of calcium suf®cient to elevate intracellular calcium to `pathological levels' [15] as a greater proportion of stretch-inactivated channels is seen in mdx muscle than in normal muscle [14]. Fong et al. [24] reported an increased activity of calcium leak channels in myotubes
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of a clonal Duchenne human cell line and in mdx mouse cells. These channels have a low conductance similar to that described here for DMD and normal cocultured human myotubes. In cultured chick heart, Sigurdson et al. [37] have shown that mechanosensitive channels under physiological condition pass up to 20 fA of calcium current which could increase the intracellular free calcium concentration to a maximum concentration of 12.5 mM. This is more than suf®cient to trigger calcium-induced calcium release [35]. Similarly the channels studied here could permit the passage of enough calcium at rest and under the membrane stress caused by a contraction to contribute to an elevation of intracellular calcium concentration. Nevertheless, the technical con®guration which allowed to directly apply mechanical stimulation to the membrane patch precluded to measure the quantity of calcium ions entering the whole cell and contributing to the calcium elevation in physiological conditions. Imbert et al. [28] and Leijendekker et al. [38] had previously shown that another type of mechanical stimulation (hypoosmotic shock) was able to induce intracellular calcium elevation. It must be noted that in spite of the ability to directly stimulate the membrane patch, a technical limitation was brought by the patch-clamp technique because of formation of the giga-seal which could per se (with the resulting classical ohm-shape of the patched membrane) mechanically induce channel activity. The giga-seal conditions did therefore not represent a true zero mechanical stimulation level. Furthermore the mechanosensitivity could be reduced or only detected for high stimulations. Nevertheless these data ®t well with the hypothesis that an absence of dystrophin could modify channel conformation and/or facilitate the activation of channels which, in turn, could increase calcium ion in¯ux. Since the studied channels carrying this current are activated in the potential range at which contraction develops, the contraction of human DMD myotubes could induce or boost cationic channel activity and permit more calcium to enter those cells. The use of gadolinium, a non-speci®c inhibitor of these channels [35,39], indicated that normal cells are slightly more sensitive to this inhibitor than DMD cells. Gadolinium has been shown to interact strongly with lipid bilayers and is capable of changing the physical environment of membrane channel proteins [40]. Since DMD myotubes have an unstable environment due to the lack of dystrophin, the application of gadolinium to the membrane could produce unexpected effects that could explain the difference of gadolinium-sensitivity observed between the two types of cells studied here. In conclusion, normal and DMD human cocultured myotubes exhibit cationic channels that spontaneously activate at physiological resting transmembrane potentials in the absence of stress. This greater activity in DMD cells is probably related to the lack of membrane stability in dystrophin-free cells. The second observation is that these cells possess cationic channels which can be opened by negative
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Fig. 6. Effect of Gd 31 on normal and DMD myotube single-channel activity with or without mechanical stress at 260 mV. (A) Examples of spontaneous unitary current traces recorded in DMD cells in the presence of increasing gadolinium concentrations in the pipette medium. (B) Inhibition percentage of the single-channel unitary current amplitude against the gadolinium concentration in the absence and in the presence of mechanical stress.
pressure applied through the pipette. Mechanical stimulation could be responsible for signi®cant calcium entry and we postulate that the high level of cytosolic calcium concentrations observed in DMD cells could be related, at least partly, to an elevated in¯ux of calcium through these channels. These results provide a strong case in relation to the important role of dystrophin and the consequences of its absence in DMD myotubes.
Acknowledgements This work was supported by grants from the CNRS (UMR 6558), the University of Poitiers, and the Association FrancËaise contre les Myopathies. We thank FrancËoise Mazin for expert technical assistance.
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