The LRRC8/VRAC anion channel facilitates myogenic differentiation of murine myoblasts by promoting membrane hyperpolarization

Abstract

Skeletal muscle myoblast differentiation involves elaborate signaling networks, including the activity of various ion channels and transporters. Several K⁺ and Ca²⁺ channels have been shown to affect myogenesis, but little is known about roles of Cl^- channels in the associated processes. Here, we report that the leucine-rich repeat containing family 8 (LRRC8)/volume-regulated anion channel (VRAC) promotes mouse myoblast differentiation. All LRRC8 subunits of heteromeric VRAC were expressed during myotube formation of murine C2C12 myoblasts. Pharmacological VRAC inhibitors, siRNA-mediated knockdown of the essential VRAC subunit LRRC8A, or VRAC activity-suppressing overexpression of LRRC8A effectively reduced the expression of the myogenic transcription factor myogenin and suppressed myoblast fusion while not affecting myoblast proliferation. We found that inhibiting VRAC impairs plasma membrane hyperpolarization early during differentiation. At later times (more than 6 h after inducing differentiation), VRAC inhibition no longer suppressed myoblast differentiation, suggesting that VRAC acts upstream of K⁺ channel activation. Consequently, VRAC inhibition prevented the increase of intracellular steady-state Ca²⁺ levels that normally occurs during myogenesis. Our results may explain the mechanism for the thinning of skeletal muscle bundles observed in LRRC8A-deficient mice and highlight the importance of the LRRC8/VRAC anion channel in cell differentiation.

Keywords: C2C12 myoblasts; calcium; cell differentiation; chloride channel; hyperpolarization; membrane potential; myogenesis; skeletal muscle; volume-regulated anion channel (VRAC).

Figure 1.

VRAC inhibitors impair C2C12 myoblast fusion. Cells were stained with an antibody against myosin (red) and DAPI (nuclei, blue) after 4 days in differentiation medium with indicated concentrations of drugs. The fusion index was calculated as the percentage of nuclei in myotubes (with ≥2 nuclei) among all nuclei. Data are presented as mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with the respective controls using one-way ANOVA. Scale bar, 100 μm.

Figure 2.

Expression profile of LRRC8/VRAC subunits during C2C12 cell differentiation. A, Western blot analysis (left) and quantification (right) of LRRC8A, LRRC8B, LRRC8D, and LRRC8E protein levels. B, Western blot analysis of LRRC8C. Arrows in (A) and (B) indicate specific bands as deduced from published knockout controls. C, quantitative PCR analysis of Lrrc8c mRNA, -fold changes relative to day 0. All data are presented as mean ± S.D. from at least three independent experiments. *, p < 0.05 compared with day 0 using one-way ANOVA. DM, differentiation medium.

Figure 3.

LRRC8A knockdown does not affect myoblast proliferation. A, Western blot analysis of LRRC8A expression after siRNA transfection. B, quantitative analysis of C2C12 cell proliferation by measuring dehydrogenase activity at the indicated time points (hours) after siRNA transfection. Cells were plated at the same initial intensities and incubated overnight before transfection. Data are presented as mean ± S.D. from three independent experiments.

Figure 4.

LRRC8A knockdown impairs myoblast differentiation. A, Western blot analysis of LRRC8A, myogenin, and myosin. DM, differentiation medium. B–D, quantification of Western blot analysis. -Fold changes of LRRC8A (B) and myogenin (C) expression are normalized to day 0 with scrambled control. -Fold changes of myosin (D) expression are normalized to day 1 with scrambled control. E, quantitative PCR analysis of myogenin (Myog) on the indicated day of differentiation. -Fold changes of Myog expression are normalized to day 0 with scrambled control. All data are presented as mean ± S.D. from at least three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with the respective controls using one-way ANOVA.

Figure 5.

LRRC8A knockdown impairs myoblast fusion. C2C12 cells were stained with an anti-myosin antibody (red) and DAPI (nuclei, blue) on the indicated day of differentiation. The fusion index was calculated as the percentage of nuclei in myotubes (with ≥2 nuclei) among all nuclei. Data are presented as mean ± S.D. from three independent experiments. *, p < 0.05 and **, p < 0.01 compared with the respective controls using one-way ANOVA. Scale bars, 100 μm.

Figure 6.

Decreased VRAC activity inhibits myoblast differentiation. A, C2C12 cells transfected with plasmid DNA for GFP or LRRC8A-GFP expression (green) were stained with an anti-myosin antibody (red) and DAPI (nuclei, blue) on the indicated day of differentiation. The differentiation index was calculated as the percentage of myosin⁺ cells among GFP⁺ cells. More than 300 GFP⁺ cells were calculated for each group. Scale bar, 100 μm. B, quantitative PCR analysis of myogenin (Myog) after 3 days of cell differentiation in the presence of 20 μm DCPIB or of vehicle (DMSO) alone. -Fold changes of Myog expression are relative to day 0. All data are presented as mean ± S.D. from three independent experiments. *, p < 0.05 compared with the respective controls using a two-tailed unpaired t test.

Figure 7.

VRAC contributes to myoblast hyperpolarization and subsequently increased [Ca²⁺]_i. A, calibration for measuring the resting membrane potential with the fluorescence probe DiBAC₄(3). Left, representative images of C2C12 cells stained with DiBAC₄(3) after differentiation for 6–8 h with or without DCPIB. Different external sodium concentrations and gramicidin were used to set the membrane potential. Fluorescence intensities are depicted in color code, as indicated by the calibration bar. Scale bar, 50 μm. Right, logarithm of the mean fluorescence intensity plotted against the calculated membrane potential (see “Experimental procedures”) and the best linear regression fit, which was used to calculate the membrane potentials in (B). B, average resting membrane potential of C2C12 myoblasts measured after 6–8 h in differentiation medium (blank) supplemented with 20 μm DCPIB, 100 μm NPPB or vehicle (DMSO) only; or DCPIB was added after 6 h. C, resting membrane potential of C2C12 cells measured after 20–24 h of differentiation in the presence or absence of 100 μm CBX by whole-cell patch clamp recording. Symbols represent values of individual cells. D, Fura-2 ratios in C2C12 cells measured on the indicated day of differentiation in the presence or absence of DCPIB. For DCPIB washout, DCPIB-containing medium was replaced by DCPIB-free medium after 24 h. E, differential interference contrast images of C2C12 cells after 3 days in differentiation medium in the presence of DCPIB (for the complete time or only for 24 h in the case of washout) or vehicle (DMSO) only. Scale bar, 200 μm. F, Fura-2 ratios in C2C12 cells expressing RFP or LRRC8A-RFP on the indicated day of differentiation. Numbers in bars indicate the total number of cells for each group. G, C2C12 cells were stained with an anti-myosin antibody (red) and DAPI (nuclei, blue) after 3 days in differentiation medium in the presence of 20 μm DCPIB, vehicle only, or with DCPIB added only 6 h after start of cell differentiation. The differentiation index was calculated as the percentage of nuclei in myosin⁺ cells among all nuclei. Scale bar, 50 μm. All data are presented as mean ± S.D. from three independent experiments. n.s., not significant. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with the respective controls using one-way ANOVA (B and G) or a two-tailed unpaired t test (C, D, and F).