Protective effect of Baicalin against doxorubicin-induced cytotoxic and electrophysiological damage in human iPSC-cardiomyocytes

Figures

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  Fig. 1. Cell viability assays in acute and long-term DOX treated hiPSC-CMs. (a) Immunofluorescence staining (left panel) of green Calcein (live) positive hiPSC-CMs treated with DOX 1 µM for 24 h with or without BAI at the indicated doses; quantitative analysis of viability expressed as calcein positive area / total area (right panel). (b) Immunofluorescence staining (left panel) of green Calcein (live) positive hiPSC-CMs treated with DOX 0.3 µM for 7 days, with or without BAI at the indicated doses; quantitative analysis of viability expressed as Calcein positive area / total area (right panel). Data are presented in box plots indicating median (middle line), 25th, 75th percentile (box), and min and max data points (whiskers). n = 12 replicates/group from 3 independent experiments. Statistical analysis was performed using two-way ANOVA and Tukey’s post hoc test: ****p < 0.0001. Phase contrast 60X. Scale bar: 50 μm.

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  Fig. 2. BAI contrasts the acute and long-term DOX-induced apoptosis in hiPSC-CMs in a dose-dependent manner. (a) and (c) Immunofluorescence staining (left panel) and quantitative analysis (right panel) of Tunel-positive cardiomyocytes in acute (a) and long-term (c), DOX and BAI co-treated hiPSC-CMs. (b) and (d) Immunofluorescence staining (left panel) and quantitative analysis (right panel) of cleaved Caspase-3 positive cells in acute (b) and long-term (d) DOX and BAI co-treated hiPSC-CMs. Data are presented in box plots indicating median (middle line), 25th, 75th percentile (box), and min and max data points (whiskers). n = 9 replicates/group from 3 independent experiments. Statistical analysis was performed using two-way ANOVA and Tukey’s post hoc test: **p < 0.01 and ****p < 0.0001. Phase contrast 60X. Scale bar: 50 μm.

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  Fig. 3. BAI contrasts the DOX-induced ROS production in hiPSC-CMs. (a) Representative fluorescent microscopy images (left panel) and quantification (right panel) of CellRox positive hiPSC-CMs treated with DOX 1 µM and BAI at the indicated doses. (b) Representative fluorescent microscopy images (left panel) and quantification (right panel) of CellRox positive hiPSC-CMs treated with long-term DOX 0.3 µM and BAI at the indicated doses. Data are presented in box plots indicating median (middle line), 25th, 75th percentile (box), and min and max data points (whiskers). n = 9 replicates/group from 3 independent experiments conducted in triplicate. Statistical analysis was performed using two-way ANOVA and Tukey’s post hoc test: **p < 0.01 and ****p < 0.0001. Phase contrast 60X. Scale bar: 20 μm.

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  Fig. 4. BAI prevented the long-term DOX-induced sarcomere disorganization in hiPSC-CMs. (a) Immunofluorescence staining of α-actinin (α-act) and c-Tnt protein in DOX 0.3 µM and DOX 0.3 µM plus BAI-treated hiPSC-CMs. (b) Representative fluorescence intensity profile of α-actinin banding showing sarcomere irregularity in cells treated with DOX 0.3 µM. (c) Quantification of sarcomere disarray expressed as % of cells with α-actinin disorganization/total number of cells. The extent of α-actinin disorganization was evaluated in a blinded fashion by two independent observers on at least 50 cells per condition. Data are presented in box plots indicating median (middle line), 25th, 75th percentile (box), and min and max data points (whiskers). n = 12 replicates/group from 3 independent experiments. Statistical analysis was performed using two-way ANOVA and Tukey’s post hoc test: ****p < 0.0001. Phase contrast 100X. Scale bar: 10 μm.

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  Fig. 5. MEA signals recorded in DOX-treated hiPSC-CMs. (a) Representative waveforms recorded at 6 h, 12 h, 18 h, and 24 h from hiPSC-CMs monolayers on MEAs and subjected to DOX 1 µM and BAI at the indicated doses. (b) Representative waveforms recorded at 1d, 3d, 5d and 7d from hiPSC-CMs monolayers on MEAs and subjected to DOX 0,3 µM and DOX 0,3 µM plus BAI at different doses (1 µM, 10 µM, and 25 µM). The line in the waveform identifies the T wave peak, used to evaluate the FPD (the interval from the top of the spike to the peak of the “T-wave-like” repolarization waveform). F= flat T wave; Q= quiescence.

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  Fig. 6. MEA analysis in DOX 0.3 µM-treated hiPSC-CMs. Measurement of FPDc (a), BP (b), Spike Amplitude (c), Conduction Velocity (e), and APD90 (g) across hiPSC-CMs monolayers on MEA after 3d of DOX 0.3 µM and BAI at the indicated doses. (d) Representative heatmaps of field potential (FP) propagation. The color scale indicates the time at which the sodium peak of the FP signal propagates from the start time (0 ms) to the end points (10 ms). (f) Representative spontaneous action potential (AP) traces from the indicated groups acquired after LEAP induction. Data are presented in box plots indicating median (middle line), 25th, 75th percentile (box), and min and max data points (whiskers). For FP analysis n = 20 replicates/group from 5 independent experiments conducted in quadruplicate. For LEAP analysis n = 8 replicates/group from 2 independent experiments conducted in quadruplicate. Statistical analysis was performed using two-way ANOVA and Tukey’s post hoc test; **p < 0.01, ***p < 0.0001, and ****p < 0.0001.