Fig. 4

Pacing-induced contraction in iPSC-derived myotubes and activation of pSMAD signaling. a xCELLigence® RTCA CardioECR System (Acea Biosciences) was used to pace the cells electrically for 12 h, using 1 Hz, 0.5 ms pulse length, and 2.2 volt intensity. Contraction profiles are shown for each cell line including the negative control (non-excitable cell iPSC). The Y axis represents cell index and the X axis represents time. The contraction profiles indicate that iPSC-derived myotubes can contract after pacing. b Immunofluorescence for pSMAD3 in control iPSC- and DMD iPSC (ex59X)-derived myotubes in control conditions (non-paced) or after 12 h pacing and quantification of pSMAD3 accumulation, expressed as a percentage of pSMAD fluorescence of paced versus non-paced cells. *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student’s t test). c Activation of pro-fibrotic genes (TGFβ1, TGFβ2, IL6, and CTGF) was monitored in control and DMD iPSC-derived myotubes after 12 h pacing (paced), after 12 h release from pacing (paced + release) and compared to control not paced cells (Ctr). The conditions used allow to see a greater and persistent fibrotic response in electrically paced DMD iPSC-derived myotubes as compared to control iPSC-derived myotubes. Data are represented as average ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student’s t test)