Variation in RARG increases susceptibility to doxorubicin-induced cardiotoxicity in patient specific induced pluripotent stem cell-derived cardiomyocytes

Figure 1. Generation of patient specific induced pluripotent stem cell-derived cardiomyocytes. (a) Representative photomicrographs of peripheral blood mononuclear cells and induced pluripotent stem cell (iPSC) colonies. (b) Confocal microscopy images showing presence of Tra1–60, NANOG and DAPI, scale bar = 50μm. (c) Cardiac Troponin T and Myosin heavy chain 7 (cTnT, MYH7) mRNA expression in iPSC-derived cardiomyocytes (CM), n = 10. Data are shown as mean ± s.e.m. (d) Confocal microscopy images showing the presence of cTnT and Sarcomeric α-actinin (SAA), scale bar = 20μm. (e) Flow cytometry analysis of cTnT expression in iPSC-derived cardiomyocytes.

Figure 2. Modelling sensitivity to doxorubicin in patient specific induced pluripotent stem cell-derived cardiomyocytes from cases and controls. (a) Cell viability and (b) IC50 of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from cases and controls using Cell Titer-Glo after 24 h of doxorubicin treatment. Graphs show pooled data from n = 4 cases and n = 3 controls, each patient cell line was assessed three independent times, in technical triplicates. (c,d) Representative optical mapping experiments showing the action potential of one case (c) and one control (d) cell line in response to increasing doses of doxorubicin, upon 20 mins of doxorubicin incubation. (e) Change of action potential duration at 50% of repolarization 1 μΜ and 10 μΜ doxorubicin n = 3 cases, n = 3 controls, relative to 0μΜ doxorubicin. Data points represent independent, biological replicates of pooled data of cases and controls measured in multiple differentiation batches of one cell line per patient. Data are shown as mean ± s.e.m. *p < 0.05, **p < 0.01, ****p < 0.0001, t-test.

Figure 3. Genome editing experimental approach and comparison of edited and unedited isogenic cell lines. (a) Experimental approach of CRISPR/Cas9 experiments, correction of RARG-S427L/WT in a case (DIC-003) patient cell line and insertion of RARG-S427L in a control (DIC-023) patient cell line. Images have been acquired from Servier Medical Art by Servier licensed under a Creative Commons Attribution 3.0 Unported License. (b) Schematic overview of RARG locus and CRISPR/Cas9 genome editing approach. (c) Sanger sequencing results showing correction of RARG-S427L/WT to WT/WT (DIC-003). (d) Sanger sequencing results showing introduction of RARG-S427L/WT to a control WT/WT patient cell line (DIC-023). Flow cytometry results of cTnT expression in (e) RARG-WT/WT and in (f) its isogenic genome edited S427L/WT cell line. (g) RT-qPCR results of RARG expression, in the heterozygous case (DIC-003) and the isogenic corrected WT/WT cell line (h) RT-qPCR results of RARG expression in the homozygous control (DIC-023) cell line (WT/WT) and the isogenic edited S427L/WT cell line. Data are shown as mean ± s.e.m., n = 3, representing biological, independent replicates *p < 0.05, t-test.

Figure 4. Genetic correction of RARG-S427L in iPSC-CMs decreases sensitivity to doxorubicin-induced cardiotoxicity. (a) Cell viability curve and (b) IC50 of RARG-S427L/WT and its corrected isogenic control (WT/WT) assessed using Cell Titer-Glo after 24 h of doxorubicin incubation (n = 3). (c) Representative images showing staining for γH2A.X. Scale bar = 20 μm. (d) Relative to vehicle control fluorescent intensity of γH2A.X staining (n = 3). (e) Representative images showing reactive oxygen species (ROS) production, scale bar = 50 μm. (f) Relative to vehicle control fluorescent intensity of ROS production (n = 3). All experiments represent biological, independent replicates, each experiment was conducted in triplicates and are shown as mean ± s.e.m., *p < 0.05, ****p < 0.0001, t-test.

Figure 5. Introduction of RARG-S427L in iPSC-CMs increases sensitivity to doxorubicin-induced cardiotoxicity. (a) Cell viability curve and (b) IC50 of RARG-WT/WT and its isogenic cell line with genome edited S427L/WT pair (n = 5). (c) Representative images showing staining for γH2A.x. Scale bar = 20μm. (d) Relative to vehicle control fluorescent intensity of γH2A.X (n = 5). (e) Representative images showing ROS production. Scale bar = 50 μm. (f) Relative to vehicle control ROS production (n = 4). All experiments represent biological, independent replicates, each experiment was conducted in triplicates and are shown as mean ± s.e.m., *p < 0.05, ****p < 0.0001, t-test.

Figure 6. Effect of doxorubicin treatment on TOP2B and RARG in RARG-S427L/WT and corrected RARG-WT/WT. RT-qPCR gene expression of (a) TOP2B and (b) RARG relative to vehicle control in RARG-S427L/WT, (c) TOP2B and (d) RARG expression relative to vehicle control in corrected RARG-WT/WT. Data are biological, independent replicates and are shown as mean ± s.e.m., n = 3, *p < 0.05, **p < 0.01, n.s. = not significant, t-test.

Figure 7. Disruption of RARG protects from doxorubicin induced cardiotoxicity. (a) Schematic overview of RARG locus and CRISPR/Cas9 knock out in ESC-CMs. (b) RARG-KO Sanger sequencing results in ESCs, showing a 35 base pair (bp) deletion in one allele and a 4 bp insertion in the other allele. (c) Cell viability curve and (d) IC50 of RARG-WT/WT and its isogenic cell line with disruption of RARG gene in ESC-CMs (n = 6). (e) Schematic overview of RARG locus and CRISPR/Cas9 knock out in iPSC-CMs from a control patient. (f) RARG-KO Sanger sequencing results in iPSCs, showing a 476 bp deletion. (g) Cell viability curve and (h) IC50 of RARG-WT/WT cell line and KO in iPSC-CMs (n = 4). Data are biological, independent replicates shown as mean ± s.e.m, *p < 0.05, ****p < 0.0001, t-test.