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First published online 7 April 2009
doi: 10.1242/jcs.035402

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Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation

¹ Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy
² Department of Physiological Sciences, University of Florence, Florence, Italy
³ Department of Biochemical Sciences, University of Florence, Florence, Italy
⁴ C.S.D.C. Department of Physics, University of Florence, Florence, Italy
⁵ Interuniversity Institute of Miology (IIM), University of Florence, Florence, Italy

View larger version (92K): [in this window] [in a new window]   Fig. 1. Expression and subcellular localization of TRPC1 in C2C12 myoblasts. (A) Transient receptor potential canonical (TRPC) channel mRNA isoforms in C2C12 myoblasts. Lanes show amplified products of RT-PCR reactions. Total RNA (1 µg) obtained from C2C12 myoblasts was retro-transcripted and cDNA amplified as described in Materials and Methods. The PCR products were visualized on an ethidium-bromide-stained agarose gel. Data are representative of three independent experiments. β-actin amplification, used as an internal control, is shown. (B) TRPC1 expression in subcellular fractions of C2C12 myoblasts. Aliquots of proteins (25 µg) from cell lysates (Lys), cytosol (cyt), Triton-soluble (Ms) or Triton-insoluble (Mi) membrane were processed for western blotting analysis. A blot representative of three is shown. (C) Effect of TRPC1 silencing on channel expression. C2C12 myoblasts were transfected with SCR-siRNA (-) and TRPC1-siRNA (+) as described in Materials and Methods. Aliquots of proteins (30 µg) from cell lysate were subjected to western blotting analysis. A blot representative of three independent experiments with similar results is shown. Band intensity is reported as relative percentage with s.e.m. less than 15%. (D) Confocal immunofluorescence analysis of TRPC1 cell localization. C2C12 cells were grown on glass coverslips, fixed and stained with the primary antibody against TRPC1 (green). The cells were counterstained with TRITC-phalloidin (red) to reveal actin filaments. In the inset, a superimposed fluorescence and DIC image shows co-localization of TRPC1 with TRITC-conjugated WGA at the cell surface. Note that the immunostaining is markedly reduced in TRPC1-silenced cells compared with native and SCR-siRNA treated ones. The images are representative of at least three independent experiments with similar results.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Effect of TRPC1 silencing on stretch-activated Ca2+-transients and SAC currents in C2C12 myoblasts. (A) C2C12 myoblasts were pre-loaded with Fluo3-AM and mechanically stretched with the tip of an AFM probe (thick grey line). Fluorescence images were acquired soon after mechanical stimulation at a rate of 1 image/second using a digital camera. The pseudo-colouring represents the global Ca2+ increase as indicated by the colour bar. Note the marked reduction and the absence of the Ca2+ transient in TRPC1 silenced cells. The images are representative of at least five to six independent experiments with similar results (number of stretched cells for each group=5). (B) Two patch pipettes were attached to the cells at (a) resting length and (b) after 20% cell stretching induced by the movement of the upper pipette in the longitudinal direction. (C) Representative total current traces, (a) Im* recorded in bath solution by whole cell path-clamp; (b) leak current, Im,leak, recorded in the presence of GdCl3 added in the bath solution and (c-j) SAC-mediated current; Im, evaluated by detracting Im,leak from Im*; (c-f) SAC-mediated currents (Im) in SCR- and TRPC1-siRNA cells unstimulated and (g-j) S1P-stimulated evaluated before (c,e,g,i) and after (d,f,h,j) the application of the mechanical stretch; (k,l), currents normalized for Cm elicited by ramp voltage pulses from the same cell reported in panels a-j. siTRPC1, TRPC1-siRNA transfected cells; str, stretched cells.

View larger version (65K): [in this window] [in a new window]   Fig. 3. Localization of TRPC1 in lipid microdomains of C2C12 myoblasts: effects of cholesterol depletion. (A) Localization of endogenous TRPC1 in lipid microdomains. Lipid-raft (DRM) and high-density (HDM) fractions were prepared as reported in Materials and Methods. An aliquot of each fraction (20 µl, 1/25 of total volume of 1-11 fractions) was resolved by SDS-PAGE and anti-caveolin 1 (Cav1), anti-TRPC1 and anti-calnexin immunodetected. A blot representative of three independent experiments with similar results is reported. (B) Sphingolipid content measurements. Sucrose density gradient fractions were prepared from [3H]sphingosine-labelled cells as described in Materials and Methods. Total radioactivity of unfractionated [3H]sphingolipids was determined in each fraction and reported as mean ± s.e.m. of a representative experiment performed in duplicate with similar results. (C) Co-immunoprecipitation of TRPC1 and Cav1. Pooled DRM fractions (F3-F4) obtained from C2C12 cell fractionation were subjected to immunoprecipitation with rabbit polyclonal antibodies directed against TRPC1 as described in Materials and Methods and Cav1 immunodetected. A blot, representative of two independent experiments, is reported. (D) Confocal immunofluorescent localization of endogenous TRPC1 in lipid rafts (triple labelling). (a,b) Native C2C12 myoblasts and (d,e) C2C12 cells treated with MβCD for 30 minutes were incubated with Alexa Fluor 488-conjugated CT-B (green) to label lipid rafts, immunostained for TRPC1 (red) and (b,e) counterstained with Alexa Fluor 647-labelled phalloidin to reveal actin filaments. Inset: magnification of outlined area. Yellow spots in panels a and d indicate co-localization of red and green fluorescence signals. (c,f) Scatter plots of the distribution of red and green fluorescence intensity signals. Pixel intensity (PI) for each of the dyes along the lines (AB, CD) in the confocal image g are shown. The degree of co-localization of TRPC1 with CT-B is summarized in the histogram. The images are representative of at least three independent experiments with similar results. (E) Effect of lipid-raft disruption on TRPC1 expression. C2C12 myoblasts were pre-incubated for 30 minutes in media containing vehicle (-) and MβCD (+), collected and processed for lipid-raft (DRM) and high-density membrane fraction (showed fraction 11) purification as described in the Materials and Methods. An equal amount (20 µl) of fractions was evaluated for the presence of TRPC1 and Cav1 by western blotting analysis. A blot representative of three is shown. (F) Effect of MβCD treatment on Gm/Cm. C2C12 cells were incubated for 30 minutes with MβCD in the presence or in the absence of S1P. Transmembrane ion conductance and cell capacitance were analysed by whole cell patch clamp in K+-free bath solution. Data are mean ± s.e.m. of 12-15 different recordings (*P<0.05, **P<0.001).

View larger version (64K): [in this window] [in a new window]   Fig. 4. Effects of stress fibre formation and cytoskeletal integrity on TRPC1-cortactin interaction, channel expression and localization. (A) Co-immunoprecipitation of TRPC1 with cortactin. C2C12 cells were incubated in the presence or absence (-) of 1 µM S1P or of 1 µg/ml DHCB, for 30 minutes and collected in lysis buffer and immunoprecipitation performed as reported in the Materials and Methods and Fig. 3C. A blot representative of three independent experiments of immunoprecipitation and TRPC1 immunoblot is shown (relative percentage is reported as mean ± s.e.m.). (B) Confocal immunofluorescence analysis of TRPC1 lipid microdomain localization (triple labelling). C2C12 myoblasts treated with (a,b) DHCB to inhibit actin polymerization or (d,e) S1P to induce stress fibre formation, were incubated with Alexa Fluor 488-conjugated CT-B (green), processed for TRPC1 immunostaining (red) and (b,e) counterstained with Alexa Fluor 647-labelled phalloidin to reveal actin filaments. Yellow spots in panels a and d indicate co-localization of red and green fluorescence signals. (c,f) Scatter plots indicate the distribution of TRPC1 and lipid-raft fluorescence intensity signals. The images are representative of at least three independent experiments with similar results.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Effect of TRPC1 silencing on skeletal myogenic differentiation of C2C12 myoblasts. (A) Western analysis of TRPC1 silencing on the expression of myogenic markers. Confluent C2C12 myoblasts were transfected with SCR-siRNA (-) or TRPC1-siRNA (+), stimulated with (+) or without (-) 1 µM S1P and differentiation started as described in the Materials and Methods. The content of TRPC1, myogenin and -sarcomeric actin were analysed by western blotting. A blot representative of at least three independent experiments with similar results and the relative percentage is shown (mean ± s.e.m.). (B) Confocal immunofluorescence and phase-contrast analysis of differentiating myoblasts. SCR-siRNA and TRPC1-siRNA cells were cultured on glass coverslips in DM, fixed and stained with the primary antibody against myogenin (green), and counterstained with TRITC-phalloidin to detect actin filaments (red). Parallel experiments were performed to reveal myotube formation by phase contrast. Note that silenced cells reveal reduced nuclear myogenin staining and polyhedral morphology typical of the undifferentiated cells and are unable to form multinucleate myotubes compared to SCR-siRNA cells. The images are representative of at least three separate experiments with similar results.

View larger version (61K): [in this window] [in a new window]   Fig. 6. TRPC1 expression during C2C12 myoblasts differentiation. C2C12 cells were grown until 95% confluence and then cultured for the indicated times in DM. (A) Western blotting analysis of TRPC1 expression. Cell lysates (30 µg) were prepared as described in the Materials and Methods and processed for western blotting analysis. A blot representative of three independent experiments with analogous results is shown. (B) Confocal immunofluorescence analysis showing TRPC1 expression in differentiating myoblasts. Cells at the indicated time were fixed and immunostained for TRPC1 (green). Counterstaining was performed with TRITC-conjugated phalloidin to reveal actin filament organization (red). The images are representative of at least three separate experiments with similar results.

View larger version (61K): [in this window] [in a new window]   Fig. 7. Regulation of TRPC1 expression by known modulators of skeletal myogenesis. (A) Western blotting analysis of TRPC1 expression. C2C12 myoblasts were grown until confluence and cultured in DM in the presence (+) or absence (-) of TGFβ (1 ng/ml). Aliquots of proteins from cell extracts (25 µg) were subjected to western blotting. Proteins were immunodetected using specific anti-TRPC1, myogenin and -sarcomeric actin antibodies. A blot representative of at least three independent experiments with analogous results and the relative percentage (s.e.m. less than 15%) are shown. (B) Confocal immunofluorescence analysis showing the effect of S1P and TGFβ on myogenin expression. Confluent C2C12 cells were cultured in DM in the presence of either 1 µM S1P or TGFβ, and stained with specific antibodies. Representative merged confocal fluorescence and DIC contrast images of C2C12 cells immunostained for myogenin (green) are shown. (C) Confocal immunofluorescence analysis showing the effect of S1P and TGFβ on TRPC1 expression. Cells processed as in B were stained with anti-TRPC1 antibodies (green). Counterstaining was performed with TRITC-phalloidin to detect actin filaments (red). The images are representative of at least three separate experiments with similar results.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Effects of TRPC1-siRNA and 2-APB on Gm/Cm in differentiating unstimulated and S1P-stimulated C2C12 myoblasts. C2C12 differentiating cells were transfected with scrambled-siRNA (SCR) and TRPC1-siRNA (siTRPC1) or treated with 2-APB in the presence or in the absence of S1P and incubated in DM for 24 hours. Significance of differences: *P<0.05, **P<0.01 with respect to relative controls; P<0.01 of TRPC1-siRNA (siTRPC1) with respect to SCR-siRNA (one-way ANOVA). Data are mean ± s.e.m. of 14-18 independent cell recordings.

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