Figure 4.

Characterization of the cardiomyopathic phenotype in vitro of DMD hiPSC-CMs, showing premature cell death, depolarized mitochondria, and increased intracellular ROS levels, which were counteracted by NAC, ataluren (PTC124), and idebenone

(A–C) Flow cytometric quantification at day 15 of cardiac differentiation showing the percentage of cell death of signal-regulatory protein alpha (SIRPA)-positive hiPSC-CMs (A), the percentage of CMs with high intracellular ROS levels (B), and the MFI of intracellular ROS in CMs (C) in conditions with (NAC, PTC124, and idebenone) or without (untreated) treatments. Data are representative of four independent experiments (n = 4), and values are reported as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

(D) Immunostaining of the fluorescent voltage-sensitive dye JC-1 was used to determine ΔΨ_m and mitochondrial health in 15-day-old differentiated hiPSC-CMs. Untreated DMD hiPSC-CMs were characterized by mitochondrial depolarization, as indicated by the decrease in mitochondrial aggregates (JC-1 red, top panels) and the increase in mitochondrial monomers (JC-1 green, middle panels) with respect to treated DMD hiPSC-CMs and controls. Corresponding histograms (bottom panels) showed the JC-1 fluorescence intensity ratios (aggregates/monomers). Scale bar: 5 μm.

(E) Representative flow cytometric analyses at day 15 of differentiation for JC-1 aggregates (phycoerythrin [PE]) and JC-1 monomers (fluorescein isothiocyanate [FITC]) in DMD hiPSC-CMs upon treatment. Data are representative of four independent experiments (n = 4).

(F) Flow cytometric analyses at day 15 of differentiation showing the mitochondrial superoxide production (MitoSOX; PE) in depolarized DMD mitochondria compared with in DMD isogenic and healthy controls. Data are representative of four independent experiments (n = 4). Flow cytometry data are reported as mean ± SEM.

See also Figures S3A–S3E and S4A–S4H.