Fig. 2

Cav3 mutant myotubes present major defects in membrane tension buffering and integrity. a, b Membrane tension measurement analysis using optical tweezers and nanotube pulling on micropatterned WT, Cav3 P28L, or Cav3 R26Q myotubes. Membrane tethers were pulled in the perpendicular axis of aligned myotubes after micropatterning in resting conditions and 5 min after a 45 mOsm hypo-osmotic shock (a, b, left panels). Membrane tension was analyzed in resting condition (a, right panel) and the difference of membrane tension before and after hypo-osmotic shock was calculated, reflecting the percentage of increase of membrane tension upon mechanical stress (b, right panel). c, e Micropatterned WT, Cav3 P28L, or Cav3 R26Q myotubes (c) or WT ctl (siCtl) and Cav3-depleted (siCav3) myotubes (e) were loaded with calcein-AM (green). The medium was switched with a 30 mOsm medium supplemented with propidium iodide (PI, red). Representative pictures were taken at the indicated times during hypo-osmotic shock. Arrows correspond to myotubes and asterisks correspond to burst myotubes. d, f Quantification of the percentage of burst myotubes (upper panel) and mean time of bursting in minutes (lower panel) in c and e, respectively. a, b Scale bar = 5 µm. c, e Scale bar = 120 µm. Reproducibility of experiments: a Representative pictures and quantifications from 7 independent experiments (WT n = 20 cells, P28L n = 23 cells, and R26Q n = 22 cells). b Representative pictures and quantifications from 7 independent experiments (WT n = 20 cells, P28L n = 27 cells, and R26Q n = 18 cells). c Representative data of 3 independent experiments quantified in d (% burst cells: WT n = 310 cells, P28L n = 299 cells, and R26Q n = 271 cells; mean time of bursting: WT n = 165 cells, P28L n = 233 cells, and R26Q cells n = 240 cells). e Representative data of 3 independent experiments quantified in f (% burst cells: siCtl n = 749 cells and siCav3 n = 569 cells; mean time of bursting: siCtl n = 171 cells and siCav3 n = 506 cells). a, d, f Mean value ± SD. b Mean value ± SEM. a, b Statistical analyses were done using Kruskal–Wallis test. d, f Statistical analysis with two-tailed unpaired t test; *P < 0.05; ***P < 0.001