Research Article 3951 TRPC1 regulates skeletal myoblast migration and differentiation

Magali Louis*, Nadège Zanou*, Monique Van Schoor and Philippe Gailly‡ Université catholique de Louvain, Institute of Neuroscience, Laboratory of Cell Physiology, 55/40 avenue Hippocrate, 1200 Brussels, Belgium *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 2 September 2008 Journal of Cell Science 121, 3951-3959 Published by The Company of Biologists 2008 doi:10.1242/jcs.037218

Summary Myoblast migration is a key step in myogenesis and myristoylated alanine-rich C-kinase substrate (MARCKS), an regeneration. It allows myoblast alignment and their fusion into actin-binding protein and substrate of calpain. Their fusion into myotubes. The process has been shown to involve m-calpain or myotubes was significantly slowed down as a result of the μ-calpain, two Ca2+-dependent cysteine proteases. Here we reduced speed of cell migration. Accordingly, migration of measure calpain activity in cultured cells and show a peak of control myoblasts was inhibited by 2-5 μM GsMTx4 toxin, an activity at the beginning of the differentiation process. We also inhibitor of TRP channels or by 50 μM Z-Leu-Leu, an inhibitor observed a concomitant and transient increase of the influx of of calpain. By contrast, stimulation of control myoblasts with Ca2+ and expression of TRPC1 protein. Calpains are specifically IGF-1 increased the basal influx of Ca2+, activated calpain and activated by a store-operated entry of Ca2+ in adult skeletal accelerated migration. These effects were not observed in muscle fibres. We therefore repressed the expression of TRPC1 TRPC1-knockdown cells. We therefore suggest that entry of in myoblasts and studied the effects on Ca2+ fluxes and on Ca2+ through TRPC1 channels induces a transient activation differentiation. TRPC1-depleted myoblasts presented a largely of calpain and subsequent proteolysis of MARCKS, which reduced store-operated entry of Ca2+ and a significantly allows in turn, myoblast migration and fusion. diminished transient influx of Ca2+ at the beginning of differentiation. The concomitant peak of calpain activity was Key words: Myoblast, Calcium, Calpain, Differentiation, Migration, abolished. TRPC1-knockdown myoblasts also accumulated TRPC1, MARCKS

Introduction skeletal muscles (Boittin et al., 2006; Ducret et al., 2006; During myogenesis and muscle regeneration, myogenic stem cells Vandebrouck et al., 2002) and in myotubes (Gutierrez-Martin et al.,

Journal of Cell Science (called satellite cells) proliferate as myoblasts. These myoblasts 2005; Vandebrouck et al., 2006). The identification of the membrane then withdraw from the cell cycle and begin to express muscle- channels involved in this store-dependent Ca2+ entry has been specific genes. They migrate and align with each other, and finally largely investigated in non-excitable cells (Gailly, 1998; Gailly and they fuse either with other myoblasts to form multinucleated Colson-Van Schoor, 2001). The best candidate molecules seem to myotubes, or with pre-existing muscle fibres. The fusion process belong to the superfamily of transient receptor potential (TRP) is known to be Ca2+ dependent. Indeed, formation of myotubes channels (for reviews, see Owsianik et al., 2006; Venkatachalam requires the presence of extracellular Ca2+ (Schmid et al., 1984), and Montell, 2007). These TRP channels present a similarity of and is preceded by an increase of cytosolic concentration of Ca2+ structure (six transmembrane domains) and some sequence 2+ 2+ ([Ca ]i) (Przybylski et al., 1994) because of a net influx of Ca homology. In mammals, the TRP superfamily contains six into the cell (David et al., 1981). The Ca2+ influx occurs through subfamilies. Four of them share substantial sequence identity in the T-type Ca2+ channels (Bijlenga et al., 2000) and most probably transmembrane domains: classical (TRPC), vanilloid (TRPV), through other not clearly identified Ca2+ channels, possibly melastatin (TRPM) and ANKTM1 (TRPA). The remaining two including store-operated and stretch-activated channels subgroups, muclopins (TRPML) and polycystins (TRPP) are only (Arnaudeau et al., 2006). distantly related to the other subfamilies. During the initial phase of differentiation, Ca2+ activates several So far, the TRP channels identified as possibly involved in store- myogenic transcription factors such as myogenin and MEF2 (Black operated influxes of Ca2+ belong to the TRPC and TRPV and Olson, 1998; Molkentin and Olson, 1996), triggering the subfamilies. In particular, studies reporting that the endogenous expression of muscle-specific genes. Calpain is another possible TRPC1 channel constitutes a component of store-operated channels Ca2+-sensitive target. Indeed, calpains are Ca2+-sensitive are consistent (for reviews, see Ambudkar, 2007; Beech et al., 2003; intracellular proteases implicated in spreading and locomotion of Rychkov and Barritt, 2007). TRPC1 might be associated in a ternary adherent cells (Glading et al., 2002; Huttenlocher et al., 1997; complex with another channel called Orai1 (Feske et al., 2006; Mazeres et al., 2006), and it has been reported that calpain inhibition Soboloff et al., 2006) and with STIM1, an intrareticular Ca2+ sensor, completely prevents myoblast differentiation into myotubes (Dedieu to contribute to store-operated channels (Ambudkar et al., 2007; 2+ et al., 2004). Sensitivity of calpains to [Ca ]i is quite low (μM to Hewavitharana et al., 2007; Liao et al., 2008; Worley et al., 2007; mM range). However, we previously showed that a store-operated Yuan et al., 2007). TRPC1 has also been reported to be entry of Ca2+ was sufficient, in adult skeletal muscle fibres, to mechanosensitive (Maroto et al., 2005), although this is still a matter activate calpains. Store-dependent entry of Ca2+ is present in adult of controversy (Dietrich et al., 2007; Gottlieb et al., 2008). 3952 Journal of Cell Science 121 (23)

In this paper, we studied the involvement of TRPC1 in myoblasts migration and differentiation. We first show that endogenous TRPC1 is indeed a necessary component for store-operated entry of Ca2+ in myoblasts. Second, we show that myoblast differentiation requires a transient increase of expression of TRPC1 channels with a consecutive transient increase of Ca2+ influx in the cytosol, resulting in activation of calpains. Calpain activation in turn allows myoblast migration and fusion into myotubes.

Results TRPC1 repression reduces store-operated entry of Ca2+ C2C12 myoblasts express several isoforms of the classical subfamily of TRP channels. Among these, TRPC1 is by far the most abundantly expressed (its mRNA content being at least 100 times higher than for any other isoform) (Fig. 1A). Several investigators have reported that TRPC1 is responsible for store-operated entry of Ca2+. Accordingly, we previously showed that in adult skeletal muscle fibres, there was a Ca2+ leak channel that could be activated by store depletion or by membrane stretching (hypo-osmotic shock); this channel is abnormally open in dystrophin-deficient fibres and seems to be constituted of TRPC1 and/or TRPC4 isoforms (Vandebrouck et al., 2002). We therefore repressed the expression of TRPC1 in C2C12 myoblasts by using two different siRNAs (see Materials and Methods). By quantitative RT-PCR, we could measure a repression of 72±2% (n=5, both siRNAs were similarly effective). Accordingly, western blot analysis showed an important decrease of the expression of TRPC1 protein (Fig. 1B); pooling the results for both TRPC1 siRNAs, we measured a repression of 70.5±11%, n=6. For long-term experiments (such as differentiation for 7 days, see below), we used a shRNA plasmid strategy (bicistronic vector coding for a silencing hairpin RNA and for EGFP) to select the cells stably expressing the shRNA (selection by resistance to antibiotics and by EGFP fluorescence). Myoblasts Fig. 1. Quantification of TRPC channels in control and TRPC1-knockdown stably transfected with TRPC1 shRNA showed a significant myoblasts. (A) Quantification of TRPC channels in C2C12 myoblasts. Each cDNA was amplified in duplicate and Ct values were averaged for each

Journal of Cell Science decrease of TRPC1 expression in comparison with cells transfected duplicate. The average Ct value for cyclophilin D was subtracted from the with control shRNA (Fig. 1C). These transfected myoblasts were average Ct value for the gene of interest, giving a ΔCt. Note that the ΔCt for then studied functionally. As expected, we observed that TRPC1 TRPC1 is at least 7 units below other values, meaning the expression is at least repression significantly reduced the store-operated entry of Ca2+. 128 times higher than for any other TRPC. (B) Western blot analysis of β Indeed, in the absence of external Ca2+, treatment of the cells with TRPC1, MARCKS and -actin expression in myoblasts transfected with μ 2+ control siRNA (lanes 1 and 3) or with one of two different TRPC1 siRNAs 1 M thapsigargin, a SERCA inhibitor, induced a [Ca ]i transient (lanes 2 and 4). TRPC1, MARCKS and β-actin proteins were detected (as a result of the release of the stores) that was similar in myoblasts sequentially on the same blot (stripped twice). Blot on left: 12 μg protein/lane; transfected with control shRNA and TRPC1 shRNA. However, re- Blot on right: 4 μg/lane. Results are representative of three measurements. addition of external Ca2+ triggered a much weaker store-depletion- (C) Western blot analysis of TRPC1 expression in nontransfected C2C12 cells induced entry of Ca2+ in TRPC1 shRNA transfected cells than in (lane 1) and in C2C12 cells stably expressing TRPC1 shRNA (lane 2) or control shRNA (lane 3), after 7 days of differentiation. Loading: 10 μg the control (Fig. 2). Similar results were obtained in TRPC1 siRNA protein/lane. Left panel, Ponceau Red staining; Right panel, immunodetection knockdown myoblasts and in myoblasts treated with GsMTx4 toxin with TRPC1 antibody. (Fig. 2), an inhibitor of mechanosensitive and store-operated channels, and in particular, an inhibitor of TRPC1 (Bowman et al., 2007). tip on a 600-μm-wide area and myoblast migration into this acellular area was quantified and expressed as the percentage of TRPC1 repression reduces myoblast migration and the number of cells having migrated in control conditions. We first differentiation studied the effect of GsMTx4 toxin. We observed that treatment of Interestingly, we observed that TRPC1 shRNA silenced myoblasts C2C12 myoblasts with GsMTx4 dose-dependently reduced cell fused much more slowly into myotubes than the controls (Fig. 3). migration (Fig. 4). We then confirmed the involvement of TRPC1 Indeed, after 7 days of differentiation, we observed that the by showing that repression of TRC1 expression by two different proportion of cellular nuclei in myotubes (being defined as having siRNAs or by a shRNA significantly reduced the speed of cell at least three nuclei) was three times lower in TRPC1-knockdown migration (Fig. 5). cells than in controls (Fig. 3B). This effect seemed to be due to the Leloup and colleagues have recently demonstrated the absence of pre-fusion alignment of myoblasts. Therefore, we used involvement of calpains in the migration of myogenic cell lines a wound-healing assay to investigate the role of TRPC1 in cell (Leloup et al., 2006). We confirmed here that inhibition of calpains migration (Fig. 4). Cells in culture were scraped off with a pipette with 50 μM Z-Leu-Leu-CHO dramatically inhibited migration (Fig. TRPC1 channels control myoblast migration 3953

Fig. 2. Store-operated entry of Ca2+ in control and TRPC1-knockdown myoblasts. Influence of GsMTx4. (A) Store-operated entry of Ca2+ was triggered by depletion of the stores with 1 μM thapsigargin (TG) in the absence of external Ca2+ (0.1 mM EGTA). 1.8 mM Ca2+ was then re-added to the extracellular medium 2+ as indicated, inducing a second peak of [Ca ]i. Comparison of the response in C2C12 myoblasts (1 day post differentiation) transfected with control shRNA or TRPC1 shRNA (efficiency of transfection checked by EGFP fluorescence emission). Traces are representative of up to 11 experiments. (B-D) Results of similar experiments obtained in myoblasts treated with TRPC1 shRNA or TRPC1 siRNA and myoblasts treated with 5 μM GsMTx4. SOCE, store-operated Ca2+ entry. Results are mean ± s.e.m. Student’s t-test: *P<0.05 vs control; n=6-12.

4). In addition, we also observed an important Z-Leu-Leu-CHO- in myoblasts incubated for 10 minutes in the presence of 5 μM mediated deceleration of differentiation (fusion into myotubes) (Fig. GsMTx4 toxin (reduction by a factor of 1.6 in the presence 3C). compared with in the absence of GsMTx4, n=7; P<0.001). In C2C12

Journal of Cell Science myoblasts stably transfected with control plasmid shRNA or Myoblast differentiation is accompanied by a transient increase transiently transfected with control siRNA, a similar peak of Ca2+ of calpain activity and a concomitant peak of Ca2+ influx entry was observed, emphasizing the absence of toxicity of 2+ The possible involvement of calpain in the migration and fusion transfections. However, this peak of [Ca ]i was abolished in processes prompted us to measure its activity after inducing myoblasts transfected with TRPC1 shRNA and TRPC1 siRNA, differentiation by replacing the proliferation medium (10% fetal calf confirming the involvement of TRPC1 in this differentiation- serum) with a differentiation medium (adult horse serum). In order induced transient increase of Ca2+ influx (Fig. 7B,C). We therefore to measure calpain activity in situ, we used a fluorescent technique investigated the possibility that the transient increase of Ca2+ influx based on the detection of the cleavage of Boc-Leu-Met-CMAC observed at day 1 of differentiation might be explained by the peptide, a membrane-permeant fluorogenic substrate of calpain (see presence of a larger number of TRPC1 channels. TRPC1 expression Materials and Methods). We observed that the activity of calpain was quantified by real-time RT-PCR. We observed that the increased from day 0 to day 1 and then return to its basal value expression of TRPC1 increased by 55±12% at day 1 of (Fig. 6). Interestingly, this peak of activity was not observed in differentiation in comparison with day 0 (n=5, P<0.01) and then myoblasts transfected with TRPC1 shRNA. Therefore, we returned to a basal value at day 4 to day 6. Western blot analysis investigated whether this transient increase of calpain activity was supported this finding (Fig. 8). Incidentally, we also observed a large the result of activation by a transient increase of Ca2+ entry through increase of expression of TRPC3 during differentiation (data not TRPC1 channels. We evaluated this influx of Ca2+ by measuring shown), confirming previous observations (Lee et al., 2006). the passive influx of Mn2+ ions used as surrogates for Ca2+ ions. However, this occurred after 3 to 4 days of differentiation, after the This technique is based on the fact that Mn2+ entering the cells initial peak of Ca2+ influx in the cell, after the transient activation through Ca2+ channels is not sequestered by SERCA pumps and of calpains and after their effects on migration. quenches Fura-PE3 fluorescence; the rate of quenching therefore directly reflects the rate of Ca2+ influx (Merritt et al., 1989). Using TRPC1-knockdown myoblasts accumulate MARCKS this Mn2+ quenching technique, we observed that indeed, control Myristoylated alanine-rich C kinase substrate (MARCKS) is an C2C12 cells presented a differentiation-induced transient increase actin-binding protein that is reported to be involved in myoblast of Ca2+ influx that was concomitant with the peak of calpain activity fusion through its regulation by calpain proteolytic cleavage (significant increase from day 0 to day 1, then return to basal values) (Dulong et al., 2004). It accumulates in cells showing a reduced (Fig. 7A). This peak of Ca2+ entry at day 1 was significantly reduced calpain activity, such as in calpastatin-overexpressing cells (Dedieu 3954 Journal of Cell Science 121 (23) Journal of Cell Science

Fig. 4. Involvement of Ca2+ channels and calpain in the migration of myoblasts. (A) Wound-healing migration assay of C2C12 myoblasts. Some cells were scraped off with a pipette tip to obtain a 600-μm-wide acellular area (visualized in DIC microscopy, panel a). 15 hours later, cells that had migrated into the acellular area were stained and counted. Cells were migrated in control conditions (b), or in the presence of 2 μM GsMTx4 toxin (c) or 50 μM Z-Leu- Leu-CHO (ZLL) (d). Scale bar: 250 μm. (B) Results expressed as a percentage of cells migrating in control conditions. Two-way ANOVA followed by a = Fig. 3. Fusion of control and TRPC1-knockdown myoblasts into myotubes. Bonferroni t-test: *P<0.05 vs control; n 6-15 independent experiments. C2C12 myoblasts were submitted to a differentiation medium (fetal serum replaced by adult serum in the culture) and were followed during 7 days in culture (D0 to D7). (A) Comparison of the fusion observed at day 7 in C2C12 myoblasts transfected with control shRNA (upper panels) or with TRPC1 myoblasts transfected with siRNA-TRPC1 compared with shRNA (lower panels). Left panels, light micrographs; right panels, myoblasts treated with control siRNA (n=6, Mann-Whitney Rank fluorescence micrographs (nuclei stained with Hoechst 33342). Scale bar: Sum Test P<0.01). Fig. 9 shows MARCKS localization in myoblasts μ 100 m. (B) Quantification of fusion process: number of nuclei in myotubes / treated with control siRNA and TRPC1 siRNA (1 day of total number of nuclei (where a myotube is defined as having at least three nuclei). Results submitted to Student’s t-test: ***P<0.001 vs control, n=6. differentiation). MARCKS presented a diffuse cytosolic pattern in (C) Comparison (DIC micrographs) of the fusion observed at day 0 (left control myoblasts. Interestingly, in TRPC1-knockdown myoblasts, panels) and day 7 (right panels) in the absence (upper panels) or in the MARCKS expression increased and appeared both as punctate presence (lower panels) of 50 μM ZLL-CHO. Scale bar: 100 μm. structures and diffusely in the cytosol.

IGF-1 increases the influx of Ca2+ and the activity of calpain et al., 2004). We therefore compared the amount of MARCKS in and accelerates migration control and TRPC1-knockdown myoblasts. Using western blot Stimulation of myoblasts with growth factors such as IGF-1 is analysis (Fig. 1B), we detected a large band around 80 kDa and known to accelerate their migration. The effect requires calpain showed that the expression was increased by a factor of 2.25 in activity because it is inhibited by 80 μM calpeptin, a calpain inhibitor TRPC1 channels control myoblast migration 3955

(Leloup et al., 2006). Here, we first confirmed that stimulation of C2C12 myoblasts with 5 nM IGF-1 for 12 hours increased by about twice their migration capacity. As expected, this effect was inhibited by the presence of 50 μM Z-Leu-Leu-CHO (Fig. 10A). Interestingly, the effect of IGF-1 on myoblast migration was also totally abolished by a low dose (2 μM) of GsMTx4. We therefore investigated whether IGF-1 stimulation influenced transmembrane Ca2+ influxes and calpain activity. Stimulation of control myoblasts maintained in proliferation medium with 5 nM IGF-1 for 10 minutes significantly increased the rate of Mn2+ quenching of Fura- PE3 from 0.18±0.01%, n=18 to 0.453±0.030%, n=7 (P<0.001). When stimulated for longer periods of time (1-4 hours), the effect was less pronounced but the influx of Mn2+ nevertheless remained significantly higher than in basal conditions (Fig. 10B). A similar effect was observed in myoblasts transfected with control siRNA or shRNA, but the effect was completely lost in cells treated with TRPC1 siRNA and shRNA (not shown). In parallel, stimulation of C2C12 cells with 5 nM IGF-1 significantly increased calpain activity (Fig. 10C). A trend was observed within 10 minutes, but the effect was significant only after 4 hours. In myoblasts transfected with TRPC1 siRNA, the effect of IGF-1 on calpain activity was completely abolished (not shown), again suggesting a causal relation between the influx of Ca2+ through TRPC1 channel and the activation of calpain. Fig. 5. Migration of control and TRPC1-knockdown myoblasts. (A) Wound-healing Discussion migration assay of C2C12 myoblasts transfected with control shRNA (a) or TRPC1 Calpains are members of a large family of Ca2+-dependent shRNA (b). Scale bar: 200 μm. (B,C) Number of cells transfected with TRPC1 shRNA cysteine proteases. Both μ-calpain and m-calpain, which (B) and TRPC1 siRNA (C) that had migrated into the acellular area compared with control siRNA and shRNA transfected cells. *** P<0.001 vs control (in B, Student’s t- are activated by micromolar or millimolar ranges of test, n=9 independent experiments from three different cultures; in C, one-way ANOVA 2+ [Ca ]I, respectively, have been implicated in cell followed by a Student-Newman-Keuls test, n=6 experiments from three different

Journal of Cell Science spreading by modifying adhesion sites, and in promoting cultures ). locomotion of adherent cells by facilitating rear-end detachment (Glading et al., 2002; Huttenlocher et al., 1997; Mazeres et al., 2006). In particular, calpains are implicated found accumulation of MARCKS only in cells showing a reduced in growth-factor-mediated migration of myoblasts (Leloup et al., calpain activity, suggesting the involvement of this actin-binding 2006). Migration is inhibited by pharmacological inhibitors of protein in cell migration (Dedieu et al., 2003; Dedieu et al., 2004). calpains, such as calpeptin, leupeptin or calpain inhibitor II, and Dulong and colleagues demonstrated the direct cleavage of migration speed is reduced in cells overexpressing calpastatin, an MARCKS by calpain and its involvement in myoblast migration endogenous inhibitor of calpains (Dedieu et al., 2004). When (Dulong et al., 2004). Calpain-dependent MARCKS proteolysis was myoblast differentiation is promoted by removing fetal serum from also shown to activate its actin binding activity and therefore its the culture medium, the expression of m-calpain increases; ability to modulate actin dynamics and cell migration (Tapp et al., conversely, the expression of μ-calpain decreases (Leloup et al., 2005). This proteolysis seems to be PKCα dependent (Goudenege 2006). The resulting intracellular calpain activity is unpredictable et al., 2005). In the present study, we show that MARCKS because it depends not only on the expression of the proteins but accumulates in TRPC1-knockdown myoblasts. In these cells, also on the presence of intracellular regulators (calpastatin, Ca2+). MARCKS partially appears as spotted structures. We propose that In the present study, we measured the activity of calpain in situ and accumulation of MARCKS could stabilize actin structures, possibly show that differentiation of myoblasts is accompanied by a transient focal adhesion plaques, and that the entry of Ca2+ through TRPC1 increase of calpain activity. This activity peaks after 1 day of (see below) and the subsequent activation of calpain would induce differentiation and then returns to basal values. As expected, MARCKS proteolysis and allow migration. stimulation with IGF-1, which increases migration speed, also If the substrate targets of calpain are identified, a question increases calpain activity, whereas Z-Leu-Leu-CHO, which inhibits remains concerning the mechanisms involved in their activation calpain activity, reduces migration accordingly. at the beginning of differentiation. The most important regulator Calpain exerts its action on cell motility by cleaving different of calpain is Ca2+. The m-calpain isoform requires millimolar levels substrates of the adhesion complex (e.g. desmin, vinculin, talin, focal of [Ca2+] for activation, a concentration unattainable inside cells adhesion kinase) (Glading et al., 2002). However, comparing the under physiological conditions. The μ-calpain isoform is more proteolytic pattern of these proteins in normal myoblasts and in sensitive to Ca2+ but nevertheless requires a half-activating [Ca2+] myoblasts overexpressing calpastatin, Dedieu and collaborators as high as 34 μM (Kapprell and Goll, 1989), which is also largely 3956 Journal of Cell Science 121 (23)

Fig. 6. Calpain activity during differentiation of control and TRPC1- knockdown myoblasts. Calpain activity was measured as the rate of fluorescence increase of cells incubated with 10 μM Boc-Leu-Met-CMAC, a permeant and nonfluorescent substrate that, upon cleavage by calpain, becomes fluorescent. Calpain activity was measured at different stages of differentiation (day 0 to day 4) and all measurements were expressed relative to the calpain activity measured in control myoblasts at day 0 of differentiation, during the same session of measurements (n=10 or more controls per session). Two-way ANOVA followed by a Student-Newmann- Keuls test: **P<0.01 for controls at D1 vs control at D0, D2, D3 and D4; §P<0.05 for TRPC1-knockdown at D1 vs control cells at D1; n=12-48 measurements from four different cultures.

2+ above the [Ca ]i occurring within cells in physiological conditions. However, conditions that lower the Ca2+ requirement have been identified: binding of calpain to membrane phosphatidylinositol Fig. 7. Mn2+ influxes in control and in TRPC1-knockdown myoblasts. (Arthur and Crawford, 1996; Melloni et al., 1996) and autolysis (A-C) The rate of quenching of Fura-PE3 by Mn2+ was used to estimate the 2+ that occurs in activated calpain (Baki et al., 1996; Suzuki and influx of Ca at different stages of differentiation (from day 0 to day 4) in control C2C12 myoblasts (A), in control shRNA (B, black columns) and Sorimachi, 1998). In addition, we recently reported that calpain TRPC1 shRNA-transfected cells (B, grey columns) and in control siRNA was partially autolyzed and specifically activated by a store (C, black columns) and TRPC1 siRNA-transfected cells (C, grey columns). 2+

Journal of Cell Science operated and/or mechanosensitive entry of Ca in adult skeletal Statistical analysis: in A, one-way ANOVA, **P<0.01 for D1 vs D0, D2, D3 muscle fibres (Gailly et al., 2007). Previously, we had suggested and D4; in B,C, two-way ANOVA followed by Student-Newmann-Keuls tests, *P<0.05 D1 control vs control at D0 and D2; §P<0.05 TRPC1-knockdown that the channels responsible for store-dependent and myoblasts at D1 vs controls at D1. mechanosensitive entry of Ca2+ in adult skeletal muscle might belong to the TRPC family (TRPC1 and/or TRPC4) (Ducret et al., 2+ 2006; Vandebrouck et al., 2002). We also showed that these subsarcolemmal) increase of [Ca ]i as recently suggested (Gailly channels are involved in the deregulation of intracellular Ca2+ et al., 2007). homeostasis observed in dystrophin-deficient fibres (Gailly, 2002; Finally, we showed that normal differentiation of myoblasts is Gailly et al., 1993a; Gailly et al., 1993b). Here, we therefore accompanied by a transient overexpression of TRPC1 and by repressed the expression of TRPC1 in C2C12 myoblasts. As concomitant TRPC1-dependent transient increases of Ca2+ influxes expected, we observed an important decrease of store-operated and of calpain activity. We therefore suggest that the following entry of Ca2+. Interestingly, these TRPC1-knockdown myoblasts sequence of events takes place during myoblasts differentiation: (1) migrated more slowly and therefore presented a retarded fusion transient increase of the expression of TRPC1 channels; (2) influx process into myotubes. of Ca2+; (3) activation of calpains; (4) cleavage of MARCKS (and By contrast, stimulation of control cells with IGF-1, which is possibly other substrates); (5) migration of myoblasts; (6) alignment; known to increase store-operated influx of Ca2+ (Ju et al., 2003), and (7) fusion into myotubes. increased the basal influx of Ca2+, activated calpain activity and Ca2+ influx has long been known to be important for triggering accelerated migration. These effects were not observed in TRPC1- myoblast differentiation or fusion. This influx of Ca2+ is activated knockdown cells, confirming the involvement of TRPC1 in this by a hyperpolarization of the plasma membrane induced by a process. The rapidity of the effect of IGF-1 on Ca2+ influx suggests progressive expression of the Kir2.1 K+ channel (Bernheim and a translocation of TRPC1 protein from a pre-existing intracellular Bader, 2002; Lory et al., 2006; Park et al., 2002). The pool to the plasma membrane, as shown for other TRP channels hyperpolarization induces a very small but permanent current of (Iwata et al., 2003; Rolland et al., 2006). Alternatively, IGF-1 Ca2+ through T-type Ca2+ channels in human myoblasts (Bijlenga stimulation might activate or induce translocation of K+ channels, et al., 2000). However, it was recently shown that T-type Ca2+ leading to a rapid hyperpolarization. The time lag between the IGF- currents are not detectable in C2C12 cells and are detected only 1-induced influx of Ca2+ and the activation of calpain might indicate subsequent to differentiation process in mouse primary satellite cells the necessity of a prolonged and localized (probably (Bidaud et al., 2006). RT-PCR confirms that T-type Ca2+ channels TRPC1 channels control myoblast migration 3957

Fig. 8. Time course of TRPC1 expression during myoblast differentiation. Western blot analysis of TRPC1 showing the increase of expression of TRPC1 protein at day 1. Loading: 10 μg protein/lane. Upper panel, immunodetection with TRPC1 antibody; lower panel, Ponceau Red staining.

Fig. 10. Effect of IGF-1 on myoblast migration, Mn2+ entry and calpain activity of control C2C12 myoblasts. (A) Wound-healing migration assay of C2C12 myoblasts in control conditions or in the presence of 5 nM IGF-1, ϩ2 μM GsMTx4 toxin or ϩ50 μM Z-Leu-Leu-CHO. (B) Rate of quenching of Fura-PE3 by Mn2+ (used to estimate the influx of Ca2+) in control conditions or after stimulation for 10 minutes, 1 hour or 4 hours with 5 nM IGF-1. Statistical analysis: one-way ANOVA followed by Student-Newmann-Keuls test, ***P<0.001 and ** P<0.01 compared with control conditions (n=5-14). (C) Calpain activity measured in control conditions or after stimulation for 10 minutes, 1 hour or 4 hours with 5 nM IGF-1. Statistical analysis: one way ANOVA followed by Student-Newmann-Keuls test, * P<0.05 vs control (n=6- 15)

Journal of Cell Science Fig. 9. Immunolocalisation of MARCKS in myoblasts. (a-d) siRNA control (a,b) and TRPC1 siRNA (c,d) treated myoblasts were kept for 48 hours in culture, submitted to differentiation medium for 1 day and immunostained Materials and Methods with anti-MARCKS antibody. Cells were observed with a water immersion Cell culture ϫ63 objective. All four images were taken with the same illumination time C2C12 mouse skeletal myoblasts were obtained from American Type Culture (excitation at 488 nm and emission at 515 nm). Note the increased expression Collection and grown in DMEM supplemented with 10% fetal bovine serum and 1% of MARCKS and its accumulation in punctuate structures (arrows) both in non essential amino-acids, and maintained at 37°C in a humidified atmosphere of isolated myoblasts and in the contacts between touching cells. 5% CO2. To induce differentiation, myoblasts were grown to about 50% confluency; the growth medium was then replaced by DMEM supplemented with 1% horse serum 2+ (differentiation medium). For [Ca ]i measurements, cells were cultured on glass coverslips. are not expressed in these cells; they are therefore not involved in the early stages of myogenic differentiation (Bidaud et al., 2006). Transfection and mRNA quantification Other channels might be involved, such as nicotinic acetylcholine For transient transfections, two different TRPC1 siRNAs were used: (1) 5Ј- GCUCAGCUUUGUUAUGAAU-3Ј and (2) 5Ј-GAUGGAUGUCGCACCUGUUA- receptors (Constantin et al., 1996; Krause et al., 1995), store- 3Ј. A final concentration of 100 nM of each siRNA and their controls (sequence with operated channels (Arnaudeau et al., 2006) or even stretch-activated no homology with any known eukaryotic gene; Eurogentec, Seraing, Belgium) were channels (Park et al., 2002). Here, we show the involvement of transfected into C2C12 myoblasts using Lipofectamine 2000 reagent according to the manufacturer’s instructions (Invitrogen). For stable transfections, we used a TRPC1 protein, a component of the store-operated channel, in the Ј 2+ bicistronic shRNA-TRPC1 plasmid encoding a short hairpin silencing sequence (5 - influx of Ca linked to the initial phase of differentiation of C2C12 GATGGATGTCGCACCTGTTA-3Ј) (psiRNA-zsk-qz-mTRPC1, Invitrogen) with cells. Interestingly, the group of Bernheim recently demonstrated same protocol of transfection at 4 μg plasmid per 3.5 cm2 well. As a control, we that the hyperpolarization-induced Ca2+ signal was linked to used a bicistronic shRNA plasmid (5Ј-GACTTACGCTGAGTACTTCA-3Ј) encoding a luciferase-silencing sequence and a GFP (psiRNA-Luc-GL3, Invitrogen). The cells myoblast differentiation through a calcineurin pathway. We showed 2+ were selected for a few days using 300 μM zeocin. For calpain and [Ca ]i previously that the expression of TRPC1 was under control of the measurements, GFP-related fluorescence emission was used to select transfected cells. calcineurin-NFAT pathway (Pigozzi et al., 2006). It is therefore For mRNA quantification, C2C12 cells were extracted with a Ribopure kit (Ambion) and reversed-transcribed using SuperScript II RNase H (Invitrogen). Gene-specific possible that the transient expression of TRPC1 channels is triggered PCR primers were designed using Primer3 (http://biotools.umassmed.edu/bioapps/ 2+ 2+ by hyperpolarization-induced entry of Ca through other Ca primer3_www.cgi). To avoid amplification of genomic DNA, primers were chosen channels and that this process would amplify Ca2+ entry, allowing in different exons: TRPC1 (NM_011643) F, 5Ј- CAGCCTCAGACATTCCAGGT-3Ј Ј Ј Ј the necessary threshold for target activation to be reached (myogenin and R, 5 -CTCCACAAGGCTGAGTTCCT-3 ; TRPC2 (NM_011644) 5 - ACTTCCTGGACGTGGTCATC-3Ј and R, 5Ј-CTGAGCATGCTGGTGACAGT-3Ј; and MEF2 expression, calpain activation). TRPC3 (NM_019510) 5Ј-TGGATTGCACCTTGTAGCAG-3 Ј and R, 5Ј-ACGTG- 3958 Journal of Cell Science 121 (23)

AACTGGGTGGTCTTC-3Ј; TRPC4 (NM_016984) 5Ј-AAGCCAAGTGGAGAGA - use. Pellets were extracted, sonicated once for 2 seconds and incubated for 30 minutes AGCA-3Ј and R, 5Ј-ATCCGAGCTGGAGACACACT-3 Ј; TRPC5 (NM_009428) 5Ј- at 0°C in 100-200 μl lysis buffer pH 7.2 containing 158 mM NaCl, 1 mM Tris, 1 GCTGAAGGTGGCA ATCAAAT-3Ј and R, 5Ј-AAGGTTGCTTCTGGGTGAGA-3Ј; mM EGTA, 1 mM PMSF, 0.1% SDS, 1% deoxycholate, 1% NP40 and a protease TRPC6 (NM_013838) 5Ј-CCTCCCTAATGAAACCAGCA-3Ј and R, 5Ј-GATTG - inhibitor cocktail for mammalian cell and tissue extracts (Sigma P8340). CCAGCATTCCAAAGT-3Ј; TRPC7 (NM_012035) 5Ј-CTGTGAAAACGACCG - Protein concentration was determined by the BCA protein assay kit (Pierce) on 10 GAAAT-3Ј and R, 5Ј-CCCTTCATTGCTTCATCGTT-3Ј; Cyclophilin D (BC019778) μl sample in a total reaction volume of 50 μl and concentration was measured on 2 5Ј-CGTCCAGA TGAGGAGTCGGA-3Ј and R, 5Ј-TAAGCATGATCGGGAGGGTT- μl with a nanodrop spectrophotometer. Samples were heated for 5 minutes at 95°C 3Ј. All primers were purchased from Eurogentec. The cyclophilin D housekeeping in Laemmli sample buffer containing SDS and β-mercaptoethanol. Equal protein gene and the genes of interest were amplified in parallel. Real-time RT-PCR was quantities (5-20 μg) were separated on 7.5% SDS-polyacrylamide gels and transferred performed using 5 μl cDNA, 12.5 μl SybrGreen Mix (Bio-Rad) and 300 nM of each on nitrocellulose membranes for 7 minutes at 20 V with the dry iBlotting system primer in a total reaction volume of 25 μl. The reaction was initiated at 95°C for 3 (Invitrogen). After blocking with 5% non-fat milk, blots were incubated with anti- minutes, and followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing TRPC1 (Alomone labs; dilution 1:200) for at least 48 hours at 4°C, anti- at 60°C for 1 minute and extension at 72°C for 10 seconds. Data were recorded on MARCKS(N19) (Santa Cruz-6454; 1:200) or anti β-actin antibodies (Sigma). After a MyiQ Real-Time PCR Detection System (BioRad) and cycle threshold (Ct) values incubation with the secondary antibody coupled to peroxidase (Dako), peroxidase for each reaction were determined using analytical software from the same was detected with ECL+ (Amersham) on ECL hyperfilm. manufacturer. TRPC1 and MARCKS expression was quantified by densitometry. Three batches Each cDNA was amplified in duplicate and Ct values were averaged for each of polyclonal anti-TRPC1 antibodies from Alomone were tested. Only the batch duplicate. The average Ct value for cyclophilin D was subtracted from the average number AN-06 allowed the detection of a single band (between 100 kDa and 130 Ct value for the gene of interest. This ΔCt value obtained in myoblasts silenced with kDa), which disappeared in TRPC1 shRNA-treated cells (Fig. 1). Moreover, TRPC1 TRPC1 siRNA or shRNA, or at different stages of differentiation, was then subtracted protein seemed very sensitive to contaminant proteases. Indeed, in the absence of from the ΔCt value obtained in control conditions (siRNA or shRNA treated cells, protease inhibitors, two degradation products were regularly observed (around 65 or at day 0 for the time course) giving a ΔΔCt value. As amplification efficiencies kDa and 50 kDa). Actin staining was used as a control of gel loading (Fig. 1). In of the genes of interest and cyclophilin D were comparable, the amount of mRNA, case of differentiation, where the expression of α-actin and β-actin is upregulated ΔΔ normalized to cyclophilin, was given by the relationship 2– Ct. and downregulated, respectively, Ponceau Red staining was used to check gel loading and transfer. 2+ [Ca ]i measurements Myoblasts were loaded with 1 μM Fura-PE3/AM for 60 minutes at room temperature. Immunohistochemistry Fura-2-loaded cells were alternatively excited at 340 nm and 380 nm and fluorescence siRNA-transfected cells were cultured on glass coverslips. Cells were fixed with 4% emission was monitored at 510 nm using a Deltascan spectrofluorimeter (Photon paraformaldehyde (w/v) in phosphate-buffered saline, permeabilized with Triton X- Technology International, dichroic mirror at 400 nm) coupled to an inverted 100 (0.5% w/v in phosphate-buffered saline) and stained with MARCKS antibody microscope (Nikon Diaphot, oil-immersion objective ϫ40 NA 1.3). Fluorescence (goat polyclonal sc-6454, Santa Cruz Biotechnology). Primary antibody was revealed intensity was recorded over the entire surface of single cells. In cells expressing EGFP, with donkey anti goat IgG-FITC. Images were obtained using a ϫ63 objective (water signal from EGFP contributed less than 3% of the signal from Fura-PE3 at resting immersion) on a Zeiss S100 inverted microscope equipped with an Axiocam HR 2+ calcium concentration; no correction was made for this small signal. [Ca ]i was camera. calculated from the ratio of the fluorescence intensities excited at the two wavelengths, using an intracellular calibration procedure performed after cell permeabilization with Solutions and drugs 5 μM ionomycin (calibration performed in another series of cells, because no The Krebs solution contained 135 mM NaCl, 5.9 mM KCl, 1.8 mM CaCl2, 1.2 mM significant interference could be detected between Fura-2 and EGFP fluorescence) 2+ MgCl2, 11.6 mM HEPES and 10 mM glucose (pH 7.3). In Ca free solution, CaCl2 (De Backer et al., 2002; Gailly et al., 1993a). was omitted and 0.1 mM EGTA was added. Thapsigargin and ionomycin were 2+ purchased from Sigma. Fura-PE3/AM was obtained from Calbiochem (Darmstadt, Mn quenching measurements Germany) DMEM, serum and streptomycin-penicillin solutions were purchased from 2+ 2+ Passive Ca influx was estimated by measuring the passive influx of Mn ions used Invitrogen. The GsMTx-4 toxin, isolated from Grammostola spatulata spider 2+ 2+ as a replacement for Ca ions. Influx of Mn into fibres loaded with Fura-PE3, (Suchyna et al., 2000), was obtained from PeptaNova (Sandhausen, Germany), Boc- quenches the fluorescence of the dye and the quenching rate reflects the influx rate Leu-Met-CMAC from Molecular Probes; Z-Leu-Leu-CHO from Bio Mol Labs and

Journal of Cell Science 2+ (Gailly et al., 1996; Merritt et al., 1989). To avoid any interference of Ca , Fura- SKF-96365 from Alexis Corporation (Lausen, Switzerland). PE3 fluorescence was excited at 360 nm where the dye is insensitive to changes of 2+ 2+ μ [Ca ] (isosbestic point) and the intensity obtained before Mn perfusion (500 M) Statistical analysis was set to 100%. Data are presented as mean ± s.e.m. ANOVA and Student’s t-tests were used to determine statistical significance. In situ measurements of calpain activity The activities of m-calpain and μ-calpain were measured in situ as described (Gailly et al., 2007). Briefly, myoblasts were incubated in a Krebs medium containing 10 We thank J. Lebacq, N. Tajeddine and N. Morel for helpful μM Boc-Leu-Met-CMAC (7-amino-4-chloromethylcoumarin-t-butoxycarbonyl-L- discussions. This work was supported by the Association française leucyl-L-methionine amide), a permeant and nonfluorescent synthetic peptide that contre les myopathies (AFM), the Association belge contre les maladies becomes impermeant and fluorescent upon cleavage of the Boc-Leu-Met moiety by neuro-musculaires (ABMM), and by a grant ARC 05/10-328 from the calpain. The rate at which fluorescence increases therefore reflects the intracellular General Direction of Scientific Research of the French Community of accumulation rate of the cleavage product resulting from the enzymatic activity (excitation and emission wavelengths: 380 and 480 nm, respectively). Fluorescence Belgium. was detected with a photon counter and restricted to an adjustable rectangular aperture of the size of a single cell. The settings for fluorescence recordings were maintained References rigorously constant for all measurements. To avoid photobleaching, the cells were Ambudkar, I. S. (2007). TRPC1: a core component of store-operated calcium channels. illuminated for only 6 seconds every minute for a period of 10-20 minutes. The Biochem. Soc. Trans. 35, 96-100. quasilinear increase of the signal made the calculation of the slope straightforward. Ambudkar, I. S., Ong, H. L., Liu, X., Bandyopadhyay, B. and Cheng, K. T. (2007). These fluorescence measurements were not ratiometric. Therefore, all measurements TRPC1: the link between functionally distinct store-operated calcium channels. Cell were expressed relative to the calpain activity measured in control myoblasts at day Calcium 42, 213-223. 0 of differentiation, during the same session of measurements (n=10 or more controls Arnaudeau, S., Holzer, N., Konig, S., Bader, C. R. and Bernheim, L. (2006). Calcium per session). sources used by post-natal human myoblasts during initial differentiation. J. Cell Physiol. 208, 435-445. In vitro wound-healing assay Arthur, J. S. and Crawford, C. (1996). Investigation of the interaction of m-calpain with After incubation for 24 hours in a culture medium containing 0.1% fetal bovine serum, phospholipids: calpain-phospholipid interactions. Biochim. Biophys. Acta 1293, 201-206. Baki, A., Tompa, P., Alexa, A., Molnar, O. and Friedrich, P. (1996). Autolysis parallels some cells were scraped off with a pipette tip to obtain a 600-μm-wide acellular area activation of mu-calpain. Biochem. J. 318, 897-901. (controlled under DIC microscopy) (Fig. 4A). The medium was then replaced and, Beech, D. J., Xu, S. Z., McHugh, D. and Flemming, R. (2003). TRPC1 store-operated after 15 hours, the cells were stained with Hansen’s hemalun for 10 minutes and cells cationic channel subunit. Cell Calcium 33, 433-440. that migrated into the acellular area were counted. Bernheim, L. and Bader, C. R. (2002). Human myoblast differentiation: Ca2+ channels are activated by K+ channels. News Physiol. Sci. 17, 22-26. Western blot analysis Bidaud, I., Monteil, A., Nargeot, J. and Lory, P. (2006). Properties and role of voltage- Cells of six-well tissue culture dishes were rinsed twice with PBS, scraped off in dependent calcium channels during mouse skeletal muscle differentiation. J. Muscle Res. PBS and centrifuged for 5 minutes at 5000 g. Dry pellets were kept at –80°C until Cell. Motil. 27, 75-81. TRPC1 channels control myoblast migration 3959

Bijlenga, P., Liu, J. H., Espinos, E., Haenggeli, C. A., Fischer-Lougheed, J., Bader, C. Kapprell, H. P. and Goll, D. E. (1989). Effect of Ca2+ on binding of the calpains to calpastatin. R. and Bernheim, L. (2000). T-type alpha 1H Ca2+ channels are involved in Ca2+ signaling J. Biol. Chem. 264, 17888-17896. during terminal differentiation (fusion) of human myoblasts. Proc. Natl. Acad. Sci. USA Krause, R. M., Hamann, M., Bader, C. R., Liu, J. H., Baroffio, A. and Bernheim, L. 97, 7627-7632. (1995). Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured Black, B. L. and Olson, E. N. (1998). Transcriptional control of muscle development by human myoblasts. J. Physiol. 489, 779-790. myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell Dev. Biol. 14, 167-196. Lee, E. H., Cherednichenko, G., Pessah, I. N. and Allen, P. D. (2006). Functional coupling Boittin, F. X., Petermann, O., Hirn, C., Mittaud, P., Dorchies, O. M., Roulet, E. and between TRPC3 and RyR1 regulates the expressions of key triadic proteins. J. Biol. Chem. 2+ 2+ Ruegg, U. T. (2006). Ca -independent phospholipase A2 enhances store-operated Ca 281, 10042-10048. entry in dystrophic skeletal muscle fibers. J. Cell Sci. 119, 3733-3742. Leloup, L., Mazeres, G., Daury, L., Cottin, P. and Brustis, J. J. (2006). Involvement Bowman, C. L., Gottlieb, P. A., Suchyna, T. M., Murphy, Y. K. and Sachs, F. (2007). of calpains in growth factor-mediated migration. Int. J. Biochem. Cell Biol. 38, 2049- Mechanosensitive ion channels and the peptide inhibitor GsMTx-4: history, properties, 2063. mechanisms and pharmacology. Toxicon 49, 249-270. Liao, Y., Erxleben, C., Abramowitz, J., Flockerzi, V., Zhu, M. X., Armstrong, D. L. and Constantin, B., Cognard, C. and Raymond, G. (1996). Myoblast fusion requires cytosolic Birnbaumer, L. (2008). Functional interactions among Orai1, TRPCs, and STIM1 suggest calcium elevation but not activation of voltage-dependent calcium channels. Cell Calcium a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels. Proc. Natl. 19, 365-374. Acad. Sci. USA 105, 2895-2900. David, J. D., See, W. M. and Higginbotham, C. A. (1981). Fusion of chick embryo skeletal Lory, P., Bidaud, I. and Chemin, J. (2006). T-type calcium channels in differentiation and myoblasts: role of calcium influx preceding membrane union. Dev. Biol. 82, 297-307. proliferation. Cell Calcium 40, 135-146. De Backer, F., Vandebrouck, C., Gailly, P. and Gillis, J. M. (2002). Long-term study of Maroto, R., Raso, A., Wood, T. G., Kurosky, A., Martinac, B. and Hamill, O. P. (2005). Ca2+ homeostasis and of survival in collagenase-isolated muscle fibres from normal and TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat. Cell Biol. 7, mdx mice. J. Physiol. 542, 855-865. 179-185. Dedieu, S., Mazeres, G., Poussard, S., Brustis, J. J. and Cottin, P. (2003). Myoblast Mazeres, G., Leloup, L., Daury, L., Cottin, P. and Brustis, J. J. (2006). Myoblast attachment migration is prevented by a calpain-dependent accumulation of MARCKS. Biol. Cell 95, and spreading are regulated by different patterns by ubiquitous calpains. Cell Motil. 615-623. Cytoskeleton 63, 193-207. Dedieu, S., Poussard, S., Mazeres, G., Grise, F., Dargelos, E., Cottin, P. and Brustis, J. Melloni, E., Michetti, M., Salamino, F., Minafra, R. and Pontremoli, S. (1996). Modulation J. (2004). Myoblast migration is regulated by calpain through its involvement in cell of the calpain autoproteolysis by calpastatin and phospholipids. Biochem. Biophys. Res. attachment and cytoskeletal organization. Exp. Cell Res. 292, 187-200. Commun. 229, 193-197. Dietrich, A., Kalwa, H., Storch, U., Mederos, Y. S. M., Salanova, B., Pinkenburg, O., Merritt, J. E., Jacob, R. and Hallam, T. J. (1989). Use of manganese to discriminate between Dubrovska, G., Essin, K., Gollasch, M., Birnbaumer, L. et al. (2007). Pressure-induced calcium influx and mobilization from internal stores in stimulated human neutrophils. J. and store-operated cation influx in vascular smooth muscle cells is independent of TRPC1. Biol. Chem. 264, 1522-1527. Pflugers Arch. 455, 465-477. Molkentin, J. D. and Olson, E. N. (1996). Combinatorial control of muscle development Ducret, T., Vandebrouck, C., Cao, M. L., Lebacq, J. and Gailly, P. (2006). Functional by basic helix-loop-helix and MADS-box transcription factors. Proc. Natl. Acad. Sci. USA role of store-operated and stretch-activated channels in murine adult skeletal muscle fibres. 93, 9366-9373. J. Physiol. 575, 913-924. Owsianik, G., D’Hoedt, D., Voets, T. and Nilius, B. (2006). Structure-function relationship Dulong, S., Goudenege, S., Vuillier-Devillers, K., Manenti, S., Poussard, S. and Cottin, of the TRP channel superfamily. Rev. Physiol. Biochem. Pharmacol. 156, 61-90. P. (2004). Myristoylated alanine-rich C kinase substrate (MARCKS) is involved in myoblast Park, J. Y., Lee, D., Maeng, J. U., Koh, D. S. and Kim, K. (2002). Hyperpolarization, but fusion through its regulation by protein kinase Calpha and calpain proteolytic cleavage. not depolarization, increases intracellular Ca2+ level in cultured chick myoblasts. Biochem. Biochem. J. 382, 1015-1023. Biophys. Res. Commun. 290, 1176-1182. Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S. H., Tanasa, B., Hogan, P. Pigozzi, D., Ducret, T., Tajeddine, N., Gala, J. L., Tombal, B. and Gailly, P. (2006). Calcium G., Lewis, R. S., Daly, M. and Rao, A. (2006). A mutation in Orai1 causes immune store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP deficiency by abrogating CRAC channel function. Nature 441, 179-185. prostate cancer cell line. Cell Calcium 39, 401-415. Gailly, P. (1998). Ca2+ entry in CHO cells, after Ca2+ stores depletion, is mediated by Przybylski, R. J., Szigeti, V., Davidheiser, S. and Kirby, A. C. (1994). Calcium regulation arachidonic acid. Cell Calcium 24, 293-304. of skeletal myogenesis. II. Extracellular and cell surface effects. Cell Calcium 15, 132- Gailly, P. (2002). New aspects of calcium signaling in skeletal muscle cells: implications in 142. Duchenne muscular dystrophy. Biochim. Biophys. Acta 1600, 38-44. Rolland, J. F., De Luca, A., Burdi, R., Andreetta, F., Confalonieri, P. and Conte Camerino, Gailly, P. and Colson-Van Schoor, M. (2001). Involvement of trp-2 protein in store-operated D. (2006). Overactivity of exercise-sensitive cation channels and their impaired modulation influx of calcium in fibroblasts. Cell Calcium 30, 157-165. by IGF-1 in mdx native muscle fibers: beneficial effect of pentoxifylline. Neurobiol. Dis. Gailly, P., Boland, B., Himpens, B., Casteels, R. and Gillis, J. M. (1993a). Critical evaluation 24, 466-474. 2+

Journal of Cell Science of cytosolic calcium determination in resting muscle fibres from normal and dystrophic Rychkov, G. and Barritt, G. J. (2007). TRPC1 Ca -permeable channels in animal cells. (mdx) mice. Cell Calcium 14, 473-483. Handb. Exp. Pharmacol. 23-52. Gailly, P., Hermans, E., Octave, J. N. and Gillis, J. M. (1993b). Specific increase of genetic Schmid, A., Renaud, J. F., Fosset, M., Meaux, J. P. and Lazdunski, M. (1984). The expression of parvalbumin in fast skeletal muscles of mdx mice. FEBS Lett. 326, 272- nitrendipine-sensitive Ca2+ channel in chick muscle cells and its appearance during 274. myogenesis in vitro and in vivo. J. Biol. Chem. 259, 11366-11372. 2+ 2+ Gailly, P., Hermans, E. and Gillis, J. M. (1996). Role of [Ca ]i in “Ca stores depletion- Soboloff, J., Spassova, M. A., Dziadek, M. A. and Gill, D. L. (2006). Calcium signals Ca2+ entry coupling’ in fibroblasts expressing the rat neurotensin receptor. J. Physiol. 491, mediated by STIM and Orai proteins-a new paradigm in inter-organelle communication. 635-646. Biochim. Biophys. Acta 1763, 1161-1168. Gailly, P., De Backer, F., Van Schoor, M. and Gillis, J. M. (2007). In situ measurements Suchyna, T. M., Johnson, J. H., Hamer, K., Leykam, J. F., Gage, D. A., Clemo, H. F., of calpain activity in isolated muscle fibres from normal and dystrophin-lacking mdx mice. Baumgarten, C. M. and Sachs, F. (2000). Identification of a peptide toxin from J. Physiol. 582, 1261-1275. Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels. Glading, A., Lauffenburger, D. A. and Wells, A. (2002). Cutting to the chase: calpain J. Gen. Physiol. 115, 583-598. proteases in cell motility. Trends Cell Biol. 12, 46-54. Suzuki, K. and Sorimachi, H. (1998). A novel aspect of calpain activation. FEBS Lett. 433, Gottlieb, P., Folgering, J., Maroto, R., Raso, A., Wood, T. G., Kurosky, A., Bowman, 1-4. C., Bichet, D., Patel, A., Sachs, F. et al. (2008). Revisiting TRPC1 and TRPC6 Tapp, H., Al-Naggar, I. M., Yarmola, E. G., Harrison, A., Shaw, G., Edison, A. S. and mechanosensitivity. Pflugers Arch. 455, 1097-1103. Bubb, M. R. (2005). MARCKS is a natively unfolded protein with an inaccessible actin- Goudenege, S., Poussard, S., Dulong, S. and Cottin, P. (2005). Biologically active milli- binding site: evidence for long-range intramolecular interactions. J. Biol. Chem. 280, 9946- calpain associated with caveolae is involved in a spatially compartmentalised signalling 9956. involving protein kinase C alpha and myristoylated alanine-rich C-kinase substrate Vandebrouck, A., Ducret, T., Basset, O., Sebille, S., Raymond, G., Ruegg, U., Gailly, P., (MARCKS). Int. J. Biochem. Cell Biol. 37, 1900-1910. Cognard, C. and Constantin, B. (2006). Regulation of store-operated calcium entries Gutierrez-Martin, Y., Martin-Romero, F. J. and Henao, F. (2005). Store-operated calcium and mitochondrial uptake by minidystrophin expression in cultured myotubes. FASEB J. entry in differentiated C2C12 skeletal muscle cells. Biochim. Biophys. Acta 1711, 33-40. 20, 136-138. Hewavitharana, T., Deng, X., Soboloff, J. and Gill, D. L. (2007). Role of STIM and Orai Vandebrouck, C., Martin, D., Colson-Van Schoor, M., Debaix, H. and Gailly, P. (2002). proteins in the store-operated calcium signaling pathway. Cell Calcium 42, 173-182. Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse Huttenlocher, A., Palecek, S. P., Lu, Q., Zhang, W., Mellgren, R. L., Lauffenburger, D. skeletal muscle fibers. J. Cell Biol. 158, 1089-1096. A., Ginsberg, M. H. and Horwitz, A. F. (1997). Regulation of cell migration by the Venkatachalam, K. and Montell, C. (2007). TRP channels. Annu. Rev. Biochem. 76, 387- calcium-dependent protease calpain. J. Biol. Chem. 272, 32719-32722. 417. Iwata, Y., Katanosaka, Y., Arai, Y., Komamura, K., Miyatake, K. and Shigekawa, M. Worley, P. F., Zeng, W., Huang, G. N., Yuan, J. P., Kim, J. Y., Lee, M. G. and Muallem, (2003). A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth S. (2007). TRPC channels as STIM1-regulated store-operated channels. Cell Calcium 42, factor-regulated channel. J. Cell Biol. 161, 957-967. 205-211. Ju, Y. K., Wu, M. J., Chaulet, H., Marciniec, T., Graham, R. M. and Allen, D. G. (2003). Yuan, J. P., Zeng, W., Huang, G. N., Worley, P. F. and Muallem, S. (2007). STIM1 IGF-1 enhances a store-operated Ca2+ channel in skeletal muscle myoblasts: involvement heteromultimerizes TRPC channels to determine their function as store-operated of a CD20-like protein. J. Cell Physiol. 197, 53-60. channels. Nat. Cell Biol. 9, 636-645.