GATA4-targeted compound exhibits cardioprotective actions against doxorubicin-induced toxicity in vitro and in vivo: establishment of a chronic cardiotoxicity model using human iPSC-derived cardiomyocytes

Figures

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  Fig. 1. The effect of long-term doxorubicin (DOX) exposure on the viability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). To study cell viability, the cells were exposed to DOX for 2–21 days after which the MTT assay was performed. a Cell viability after DOX treatment expressed as mean ± SEM (n = 4). b Cell viability after co-exposure to DOX and 3i-1000 expressed as mean + SEM (n = 4). ***P < 0.001 vs. DMSO; **P < 0.01 vs. DMSO; *P < 0.05 vs. DMSO; †††P < 0.001 vs. control; ††P < 0.01 vs. control (randomized block ANOVA followed by Tukey’s HSD)

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  Fig. 2. The effects of doxorubicin and 3i-1000 on DNA content and GATA4 levels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) after long-term exposure. For high-content analysis, the cells were exposed simultaneously to 100 nM doxorubicin and 3i-1000 for 4, 7 or 14 days after which they were fixed and stained. Imaging and analysis was carried out using CellInsight High-Content Screening Platform. a Cell density. b Intensity of DNA staining. c Intensity of GATA4 staining in nucleus. The results are expressed as mean + SEM (n = 3–5). ***P < 0.001 vs. control; **P < 0.01 vs. control (randomized block ANOVA followed by Tukey’s HSD)

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  Fig. 3. The effects of doxorubicin (DOX) and 3i-1000 on expression of pro-B-type natriuretic peptide (proBNP) and caspase activation in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) after long-term exposure. For high-content analysis, the cells were exposed simultaneously to 100 nM DOX and 3i-1000 for 4, 7 or 14 days after which they were fixed and stained. Imaging and analysis was carried out using CellInsight High-Content Screening Platform. a Representative images of proBNP staining after a 4-day exposure. b Proportion of proBNP positive hiPSC-CMs. c Representative images of caspase staining after a 4-day exposure. d Proportion of hiPSC-CMs positive for fluorescent caspase-3/7 activity reporter. Adjustments of individual colour channels to enhance brightness and contrast were made identically to all representative images. The results are expressed as mean + SEM (n = 3–4). ***P < 0.001 vs. control; **P < 0.01 vs. control; *P < 0.05 vs. control (randomized block ANOVA followed by Tukey’s HSD). cTnT cardiac troponin T (colour figure online)

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  Fig. 4. The effect of short-term doxorubicin exposure on the viability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs). To study cell viability, a hiPSC-CMs and b NRVMs were exposed simultaneously to doxorubicin and 3i-1000 for 48 h after which the MTT assay was performed. The results are expressed as mean + SEM (n = 3–4). ***P < 0.001 vs. control; **P < 0.01 vs. control (randomized block ANOVA followed by Tukey’s HSD)

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  Fig. 5. The effects of doxorubicin (DOX) and 3i-1000 on GATA4 levels and caspase activation in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) after short-term exposure. For high-content analysis, the cells were exposed simultaneously to 100 nM DOX and 3i-1000 for 24 h after which they were fixed and stained. Imaging and analysis was carried out using CellInsight High-Content Screening Platform. Representative images of GATA4 staining in a hiPSC-CMs and b primary NRVM cultures. c Average intensity of nuclear GATA4 staining. d Proportion of cardiomyocytes positive for fluorescent caspase-3/7 activity reporter. Adjustments of individual colour channels to enhance brightness and contrast were made identically to all representative images. The results are expressed as mean + SEM (n = 3–4). *P < 0.05 vs. control (randomized block ANOVA followed by Tukey’s HSD) (colour figure online)

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  Fig. 6. The in vivo animal model of doxorubicin (DOX) cardiotoxicity. The rats received saline or DOX 1 mg/kg/day for 10 days and were followed up to 11 weeks. a The experimental design of chronic DOX cardiotoxicity in rats. DOX was administered 1 mg/kg/day i.p. for 10 days. b, c Cardiac function was measured by echocardiography at 2, 7, 9 and 11 weeks. d The survival of the DOX-treated rats decreased quickly after 9 weeks. The results are expressed as mean ± SEM; *P < 0.05 vs. control (Welch’s t test). Number of animals at 2, 7 and 9 week time points: NaCl = 3; DOX = 6, week 11 time point: NaCl = 3; DOX = 3. Panels b–d show data from animals that were not treated with 3i-1000. ECHO echocardiography

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  Fig. 7. The effect of 3i-1000 on doxorubicin (DOX) cardiotoxicity in vivo. The rats received saline or DOX 1 mg/kg/day for 10 days and were then treated with compound 3i-1000 30 mg/kg/day or DMSO vehicle (V) for 2 weeks (weeks 8 and 9). a–d Left ventricular functional and structural changes measured by echocardiography at the end of the experiments. e, f mRNA extracted from left ventricles and measured by RT-PCR. The levels of transcripts were normalised to ribosomal 18S quantified from the same samples. The results are expressed as mean + SEM; *P < 0.05 (independent-samples t test). Number of animals: NaCl + V = 10; DOX + V = 9 (except for e, f DOX + V = 8); DOX + 3i-1000 = 8. LVPW left ventricular posterior wall thickness, LVID left ventricular internal dimension