A non-coding GWAS variant impacts anthracycline-induced cardiotoxic phenotypes in human iPSC-derived cardiomyocytes

Figures

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  Fig. 1. rs28714259 risk allele or deletion decreases glucocorticoid receptor (GR)-mediated enhancer activity and disrupts GR binding. a GR:rs28714259 binding efficiency determined by Electrophoretic Mobility Shift Assay (EMSA) in hiPSC-derived cardiomyocyte cells (hiPSC-CMs). Left: Representative gel image. Right: quantification of band intensities. b GR:rs28714259 binding efficiency determined by EMSA in MCF-7 breast cancer cells. Left: Representative gel image. Right: quantification of band intensities. To induce GR binding, cells were treated with 100 nM dexamethasone for 2 h and nuclear extract was incubated with biotin-labeled, G (Major)- or A (Risk)-probes. GR antibody was added to the binding reaction to detect the super shift band. a, b Three independent experiments were performed in hiPSC-CMs or MCF-7 cells for quantification (n = 3). c GR:rs28714259 binding efficiency in hiPSC-CMs of each genotype (intrinsic lines) determined by Chromatin Immunoprecipitation (ChIP) assay. d Schematic of rs28714259 (position 11, yellow highlight) location. GILZ, a known GR-mediated transcriptional enhancer region, was used as positive control. For rabbit IgG control, data from hiPSC-CMs of GG,…

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  Fig. 2. Generation of hiPSC-derived cardiomyocytes (iPSC-CMs) from rs28714259 intrinsically polymorphic or deleted hiPSCs. a Schematic of hiPSC-CMs generation from rs28714259 intrinsically polymorphic or CRISPR-Cas9-mediated deleted hiPSCs. b Schematic of sgRNA design for the deletion of the rs28714259 region in hiPSCs. c Deletion of the variant region generates an amplicon 300 bp shorter than the amplicon from un-edited DNA.+/+, vector transfected clone with intact rs28714259 region. +/−, one clone of heterozygous rs28714259 region deletion. −/−, one clone of homozygous rs28714259 region deletion. At least three independent experiments were repeated with similar results. d Treatment and assay timeline of hiPSC-CMs. Source data for Fig. 2c are provided as a Source Data file.

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  Fig. 3. rs28714259 risk allele or loss of rs28714259 locus decreases dexamethasone -induced protection of viability and contractility after doxorubicin exposure. Cells were pretreated with or without dexamethasone before exposed to doxorubicin. Viability (a), normalized peak height (contractile strength) (b), and normalized beat rate (c) were evaluated in intrinsically polymorphic hiPSC-derived cardiomyocyte cell (hiPSC-CM) lines. Viability (d), normalized peak height (e), and normalized beat rate (f) were also assessed in rs28714259 deleted hiPSC-CM lines. g Representative curves of contraction amplitude in two rs28714259 intrinsically polymorphic hiPSC-CM lines of GG (left) and AA (right) genotypes, respectively. Contractility was analyzed using Cellogy Pulse from recorded movies of beating cardiomyocytes. rs28714259 intrinsically polymorphic lines are labeled as GG, GA, and AA; rs28714259 deletion lines are labeled as +/+, +/−, and −/−. Three lines/genotype for the intrinsically polymorphic hiPSC-CMs (GG/GA/AA) and two lines/genotype for genome-modified hiPSC-CMs [(+/+)/(+/−)/(−/−)] were measured with four technical replicates for each line. Results for each treatment in the intrinsic…

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  Fig. 4. Luciferase activity in hiPSC-derived cardiomyocyte cells (hiPSC-CMs) transfected with empty vector (EV), major allele (G)-, or risk allele (A)-constructs. Schematic of the luciferase construct to test the enhancer activity of rs28714259 locus (Top). Luciferase activity in transfected cells treated with vehicle or dexamethasone (bottom). Three independent experiments were performed with three technical repeats for each construct transfection (n = 9). Data are mean ± sd. Two-tailed unpaired t-tests were used to estimate significance. P-values are indicated in the figure. Source data are provided as a Source Data file.

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  Fig. 5. Transcriptomic changes induced by doxorubicin and dexamethasone/doxorubicin combination treatment and genes/pathways impacted by the rs28714259 risk (A) allele. a, b Volcano plots representation of differential gene expression pattern induced by doxorubicin-only (a) or dexamethasone/doxorubicin combination treatment (b) over vehicle-treated control in nine rs28714259 intrinsically polymorphic hiPSC-derived cardiomyocyte cell (hiPSC-CM) lines. Tests for differential expression were performed with DESeq2 package in R using a negative binomial generalized linear model (two-sided test). The p-values were then corrected for multiple hypothesis testing using the False Discovery rate (FDR) approach of Benjamini–Hochberg. An FDR < 0.05 were considered statistically significant. Significantly differentially expressed genes (FDR < 0.05) of Log2 Fold Change (|log2FC|) > 1 are highlighted in red, while those of |log2FC| < 1 are highlighted in blue. Black and orange highlights represent genes with FDR > 0.05. Top differentially expressed genes are labeled. c Heat map of gene expression patterns in vehicle control, dexamethasone-only, doxorubicin-only, and dexamethasone/doxorubicin combination…

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  Fig. 6. rs28714259 risk (A) allele dysregulates genes following dexamethasone/doxorubicin combination treatment and contributes to an attenuated hypertrophy signaling. Top genes identified by IPA (in red or green blocks with bold black frame) are depicted in the context of canonical pathways. Red: pro-hypertrophic genes AND upregulated under dexamethasone/doxorubicin combination by rs28714259 major (G) allele. Green: anti-hypertrophic genes AND down-regulated under dexamethasone/doxorubicin combination by rs28714259 major (G) allele. Small arrows placed on the right side of identified genes represent the effect of rs28714259 risk (A) allele, which consistently opposes the effect of major (G) allele. Only top genes (P-value <= 2.81 × 10−3) in the cardiac hypertrophy signaling pathway identified by Ingenuity Pathway Analysis are shown for simplicity. A complete list of the 31 genes identified is included in Supplementary Table 6.