Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes

Abstract

Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response.

Figure 1. Characterization of hPSC-derived cardiomyocytes.

(a,b) Relative gene expression of pluripotency (a) and cardiac (b) markers. The mean Ct values of duplicate measurements were normalized against the values for β actin for the same sample. After normalization, the means of three independent experiments were plotted, data represented are the mean ± SD. (c) Immunostaining of hPSC-derived CMs with cTnT and SAA antibodies followed by counterstaining with DAPI. (d) Flow cytometry analysis of cTnT and MLC2a expression. cTnT = cardiac Troponin T; MYH7 = myosin heavy chain beta; SAA = sarcomeric actinin alpha; MLC2a = myosin light chain 2a. *p < 0.05; ****p < 0.0001. Scale bar: 10 μm.

Figure 2. Characterization of doxorubicin-induced cell death in hPSC-derived cardiomyocytes.

(a) Cell viability of hPSC-derived cardiomyocytes was assessed by measurement of ATP levels using CellTiter-Glo^® after 24 h of doxorubicin treatment. (b,c) hPSC-derived cardiomyocytes were treated with 3 μM of doxorubicin for 16 h and stained with Annexin V (b) or Propidium Iodide (c). Cells were counterstained with DAPI and analyzed by confocal microscopy. The total numbers of Annexin V and PI-positive cells as a percentage of control are shown in the bar graphs representing the means of three independent experiments ± SD. **p < 0.01; ***p < 0.001. Scale bar: 10 μm.

Figure 3. Doxorubicin triggers mitochondrial perturbations and reactive oxygen species (ROS) production in hPSC-derived cardiomyocytes.

(a) Cells were incubated with doxorubicin or antimycin A (positive control) for 1 h and intra-mitochondrial O₂⁻ production was detected using the fluorescent dye MitoSOX^TM Red. The total numbers of MitoSOX^TM Red-positive cells as a percentage of control is shown in the bar graph. (b) After 4 h of doxorubicin treatment, cells were washed and incubated with H2DCFHDA for intracellular H₂O₂ detection by confocal microscopy. The total numbers of H2DCFHDA-positive cells as a percentage of control is shown in the bar graph. (c) Doxorubicin and FCCP (positive control) treated cells were incubated with the potential-sensitive probe JC-1 and ΔΨm was analyzed in a plate reader with excitation set at 535 nm and emission at 590 nm. Graphs represent the means of three independent experiments ± SD. AA = antimycin A; FCCP = carbonyl cyanide p-trifluoromethoxyphenylhydrazone. ***p < 0.001; ****p < 0.0001. Scale bar: 30 μm.

Figure 4. Doxorubicin induces intracellular Ca^2 +increase and double-strand DNA breaks (DSBs).

(a) hPSC-derived cardiomyocytes were incubated with 5 μM of doxorubicin for 16 h and intracellular Ca²⁺ was measured using the cell-permeant fluorescent indicator Fluo-4, AM. The total numbers of Fluo-4, AM-positive cells as a percentage of control is shown in the bar graph. (b) Representative images of DSBs in hPSC-derived cardiomyocytes. γ-H2AX staining is shown in red, and DAPI nuclear staining in blue. γ-H2AX fluorescence intensity as a percentage of control is shown in the bar graph. ****p < 0.0001. Scale bar: 30 μm.

Figure 5. Doxorubicin alters the electrophysiological properties of hPSC-derived cardiomyocytes.

(a–c) iCell^® cardiomyocytes were treated with doxorubicin in the indicated doses, and assessed by multi-electrode array technology immediately after treatment, and 2 h and 20 h after treatment. Doxorubicin treatment resulted in dose-dependent effects on beat period (a), spike amplitude (b), and FPDc (c). Graphs represent the means of three biological replicates ± SEM. (d) Activity maps displaying the beat rate at baseline and 20 h post-DOX treatment. (e) Activity map showing the spike amplitude at baseline and 20 h post-DOX treatment. (f) Beat waveform trace overlay at baseline and after 2 h of 1 μM DOX treatment. FPDc = corrected field potential duration. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Figure 6. Whole genome transcript profiling identifies doxorubicin-dependent changes in gene expression in human cardiomyocytes.

(a) Mean-average plots displaying the number of differentially expressed genes in hPSC-derived cardiomyocytes at indicated concentrations vs. control (analysis in edgeR). (b) Focused global test analysis of ROS, DNA damage and mitochondrial pathways summarizing the pathways significance in the groups 1 μM vs. 0 μM and 2.5 μM vs. 0 μM. (c) Spatially ordered clustering of gene expression profiles across increasing doxorubicin concentrations as determined by self-organizing maps (SOM). The clusters highlighted in red and blue were selected for pathway over-representation analysis via DAVID. (d,e) Comparative analysis of the number of significantly up- (d) and down (e) -regulated KEGG pathways (FDR < 0.05) determined either by PreRanked GSEA (1 μM drug vs. control and 2.5 μM drug vs. control) or via DAVID (dose-dependent changes, SOM cluster 6 for up-regulated genes and clusters 3, 4 and 9 for down-regulated genes). Number of pathways up- and down-regulated by doxorubicin were determined by PreRankedGSEA analysis.

Figure 7. Disruption of TOP2B in hPSC-derived cardiomyocytes decreases sensitivity to doxorubicin-induced cell death.

(a) Schematic of the TOP2B gene showing location of single guide RNAs (sgRNAs) designed to recognize the 3′ region of exon 3. Exonic sequence is shown in uppercase and intronic sequence in lowercase. (b) Sequences of two clones transfected with CRISPR-Cas9 targeting TOP2B gene showing either wild type sequence (WT), or bi-allelic deletions resulting in gene knock-out (KO). (c,d) Flow cytometry analysis of cTnT expression in wild type (c) and KO (d) clones. (e) Relative gene expression levels of TOP2B mRNA in the genome edited-cardiomyocytes. Data represent the mean Ct values of duplicate measurements normalized against the values obtained for β actin for the same sample. (f) After cardiomyocyte differentiation, cell viability of TOP2B edited-clones treated with doxorubicin for 24 hours was measured using CellTiter-Glo^® luminescent assay. (g) Wild type and knock out TOP2B cardiomyocytes were treated with doxorubicin and stained with γ-H2AX to identify double strand DNA breaks. (h) Quantification of data in panel (g). Graphs represent the means of three independent experiments ± SD **p < 0.01 Scale bar: 10 μ m.