Discovery of Zidovudine as a cardiomyocyte protectant for doxorubicin-induced toxicity through high-throughput phenotypic drug screening

Figures

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  Fig. 1. Discovery of DOX-IC protective drug candidates through hCM-based repurposed drug screening. a, A chemically defined protocol for cardiac differentiation of human embryonic stem cells (hESCs). hCMs, human CMs; CHIR, CHIR99021; EBL, E8 basal lipid. b, Representative immunofluorescence staining of hCMs culture showing high purity (upper) and abundant sarcomere structures (lower). c, Transmission electron microscopic analyses of hCMs. Myofilaments (blue arrows), Z-bands (red arrows), and mitochondria (yellow arrows) are visible. d, Representative traces of spontaneous intracellular calcium transient of hCMs under electrical field stimulation at 1 Hz and 2 Hz, respectively. e and f, The workflow (e) and primary high-throughput drug screening results (f) of DOX-IC protectants. g, List of top ten screened drug hits based on protection levels. h, Chemical structure of the leading hit Zidovudine (ZIDO).

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  Fig. 2. Assessment of in vitro DOX-IC protective effects of ZIDO in hCMs. a and b, Representative (left) and quantitative (right) analysis of cell viability (by Calcein-AM/PI staining (n = 5 experiments; Nine randomly selected images were analyzed for each experiment) and apoptosis by TUNEL assay. (n = 9 experiments; 3 randomly selected images were analyzed for each experiment) in DOX-treated hCMs with or without the co-administration of ZIDO or DRZ at the indicated dose. c–e, Representative traces (c) and averaged parameters (d and e) of intracellular calcium transients recorded from hCMs treated with 0.75 µM DOX alone, or DOX plus 3 µM ZIDO or 10 µM DRZ for 48 h, under 1 Hz or 2 Hz electrical field stimulation. n = 20–36 cells for each group. Data are presented as mean± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant, estimated by one-way ANOVA with Tukey's post hoc test.

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  Fig. 3. Transcriptional changes induced by ZIDO. a, Volcano plot showing the number of differentially expressed genes (p-value <0.05 and fold change (FC) >1.5) in DOX-treated hCMs with or without ZIDO administration. b, Two-way hierarchical clustering of all 508 differentially expressed genes identified in (a). Up- and down-regulated genes are clustered and the representative gene ontology (GO) terms enriched in each cluster are listed along with their p-values and the example genes. c, Gene set enrichment analysis (GSEA) showing terms enriched in up- or down-regulated genes induced by ZIDO. NES, normalized enrichment score. D, Expression levels of the relevant marker genes revealed by RNA-seq.

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  Fig. 4. Assessment of the therapeutic efficacy of ZIDO in a mouse model of DOX-induced chronic heart injury. a, Schematics showing time points when mice received peritoneal injections, subsequently analyzed with echocardiography, and finally sacrificed for heart sample collections. b and c, Representative M-mode echocardiography images (b) and statistical analysis (c) of the ejection fraction and fractional shortening of mouse hearts (n = 7 mice for each group). d, Hematoxylin and eosin (H&E) and Masson staining of heart section samples. e, Statistical analysis of the fibrotic area in Masson-stained heart section samples (n = 5 or 6 mice for each group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant, estimated by one-way ANOVA with Tukey's post hoc test.