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First published online 11 November 2008
doi: 10.1242/jcs.037218

Journal of Cell Science 121, 3951-3959 (2008)
Published by The Company of Biologists 2008
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TRPC1 regulates skeletal myoblast migration and differentiation

Magali Louis*, Nadège Zanou*, Monique Van Schoor and Philippe Gailly{ddagger}

Université catholique de Louvain, Institute of Neuroscience, Laboratory of Cell Physiology, 55/40 avenue Hippocrate, 1200 Brussels, Belgium


Figure 1
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  		Fig. 1. Quantification of TRPC channels in control and TRPC1-knockdown myoblasts. (A) Quantification of TRPC channels in C2C12 myoblasts. Each cDNA was amplified in duplicate and Ct values were averaged for each duplicate. The average Ct value for cyclophilin D was subtracted from the average Ct value for the gene of interest, giving a {Delta}Ct. Note that the {Delta}Ct for TRPC1 is at least 7 units below other values, meaning the expression is at least 128 times higher than for any other TRPC. (B) Western blot analysis of TRPC1, MARCKS and β-actin expression in myoblasts transfected with control siRNA (lanes 1 and 3) or with one of two different TRPC1 siRNAs (lanes 2 and 4). TRPC1, MARCKS and β-actin proteins were detected sequentially on the same blot (stripped twice). Blot on left: 12 µg protein/lane; Blot on right: 4 µg/lane. Results are representative of three measurements. (C) Western blot analysis of TRPC1 expression in nontransfected C2C12 cells (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 protein/lane. Left panel, Ponceau Red staining; Right panel, immunodetection with TRPC1 antibody.

 

Figure 2
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  		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 as indicated, inducing a second peak of [Ca2+]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.

 

Figure 3
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  		Fig. 3. Fusion of control and TRPC1-knockdown myoblasts into myotubes. 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 shRNA (lower panels). Left panels, light micrographs; right panels, fluorescence micrographs (nuclei stained with Hoechst 33342). Scale bar: 100 µm. (B) Quantification of fusion process: number of nuclei in myotubes / 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. (C) Comparison (DIC micrographs) of the fusion observed at day 0 (left panels) and day 7 (right panels) in the absence (upper panels) or in the presence (lower panels) of 50 µM ZLL-CHO. Scale bar: 100 µm.

 

Figure 4
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  		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 Bonferroni t-test: *P<0.05 vs control; n=6-15 independent experiments.

 

Figure 5
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  		Fig. 5. Migration of control and TRPC1-knockdown myoblasts. (A) Wound-healing migration assay of C2C12 myoblasts transfected with control shRNA (a) or TRPC1 shRNA (b). Scale bar: 200 µm. (B,C) Number of cells transfected with TRPC1 shRNA (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-test, n=9 independent experiments from three different cultures; in C, one-way ANOVA followed by a Student-Newman-Keuls test, n=6 experiments from three different cultures).

 

Figure 6
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  		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.

 

Figure 7
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  		Fig. 7. Mn2+ influxes in control and in TRPC1-knockdown myoblasts. (A-C) The rate of quenching of Fura-PE3 by Mn2+ was used to estimate the influx of Ca2+ at different stages of differentiation (from day 0 to day 4) in control C2C12 myoblasts (A), in control shRNA (B, black columns) and TRPC1 shRNA-transfected cells (B, grey columns) and in control siRNA (C, black columns) and TRPC1 siRNA-transfected cells (C, grey columns). Statistical analysis: in A, one-way ANOVA, **P<0.01 for D1 vs D0, D2, D3 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 myoblasts at D1 vs controls at D1.

 

Figure 8
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  		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.

 

Figure 9
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  		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 with anti-MARCKS antibody. Cells were observed with a water immersion x63 objective. All four images were taken with the same illumination time (excitation at 488 nm and emission at 515 nm). Note the increased expression of MARCKS and its accumulation in punctuate structures (arrows) both in isolated myoblasts and in the contacts between touching cells.

 

Figure 10
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  		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, +2 µM GsMTx4 toxin or +50 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)

 

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© The Company of Biologists Ltd 2008