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. 2020 Jun 19;8:535. doi: 10.3389/fbioe.2020.00535

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Copyright © 2020 Jelinkova, Vilotic, Pribyl, Aimond, Salykin, Acimovic, Pesl, Caluori, Klimovic, Urban, Dobrovolna, Soska, Skladal, Lacampagne, Dvorak, Meli and Rotrekl.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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DMD-CCs show remodeled β-adrenergic response and increased death. The contraction force and beat rate were measured using an AFM-based method and tested for β adrenergic response with isoproterenol (ISO, activator) and metoprolol (METO, inhibitor) (A). WT-CMs show increase in contraction force (n = ISO/METO: n = 14/13 for WT hESC, n = 6/7 for WT-hiPSC-EBs, n = 6/10 for DMD02 EBs, n = 5/6 for DMD03 EBs, n = 17/17 for cDMD-EBs, exact value for each is represented by • in each graph) and (B) beat rate (n = ISO/METO: n = 6/11 for WT hESC, n = 14/8 for WT-hiPSC-EBs, n = 7/11 for DMD02 EBs, n = 5/6 for DMD03 EBs, n = 15/15 for cDMD-EBs, exact value for each is represented by • in each graph) after ISO treatment and decrease in both parameters under METO treatment. The DMD-hiPSC-EBs remained unresponsive to any of these stimulations. cDMD derived EBs show similar response in contraction force to its isogenic WT control. The red line represents the ratio of 1 for better orientation in change. Significance calculated by Wilcoxon test (*p < 0.05, **p < 0.01, ***p < 0.01, ^#p < 0.1) is visualized as asterisks. (C–E) Western blot analysis of β receptors shows higher expression of β1 in DMD-hiPSC derived EBs compared to WT-EBs; cDMD line shows similar expression to its isogenic WT control (calculates as β1 signal/actinin signal). β2 receptor showed increased levels in DMD-hiPSC-EBs. Human heart (HH) sample was used as positive control, human foreskin fibroblasts (HFF) as CM negative sample. Lamin B was used as loading control. Exact value for each is represented by • in each graph. Statistical difference was calculated by Mann-Whitney test for both β1 and β2 receptor expression. (F) CM death in the EBs was evaluated using free troponin level analysis in the medium. All DMD-EBs show significantly increased free cTnT levels compared to WT-EBs. Statistical difference was calculated using two-way ANOVA with uncorrected Fisher's post hoc test. ****p < 0.0001 according to cell line factor (WT vs. DMD) and ++++p < 0.0001 according to day of differentiation factor.