RARG S427L attenuates the DNA repair response to doxorubicin in induced pluripotent stem cell-derived cardiomyocytes

Figures

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  Figure 1. MD simulation of RARG with ligand (A) Molecular-dynamics-predicted RARG structure with ATRA (purple), highlighting interaction between residue Thr399 (on helix 11) and Ser427 (on F domain) of RARG-WT. Protein structure of RARG-WT is shown in blue and RARG-S427L in yellow. Key components are helix 3 (green), helix 4 (deep blue), helix 10 (light yellow), helix 11 (orange), helix 12 (red), and the variant site 427 (pink). (B) Cα RMSF of F domain (residues 417–454) of RARG-ATRA complex. MD experiments were run as independent triplicates. Analyses are shown as mean ± SEM; n = 3; ∗∗∗∗p < 0.0001; t test.

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  Figure 2. Assessment of retinoic acid signaling in cellular stress response to doxorubicin (A) Flow cytometry for cardiac troponin T (cTnT), indicating the percentage of cardiomyocytes present in different differentiation batches (n = 4). ns, not significant. (B) Gene expression of atrial, ventricular, and pan cardiac markers in two genotypes (n = 8). (C–H) Gene expression of RA receptors RARA, RARB, and RARG measured by qPCR in (C) DIC-case and (F) DIC-control cell lines (n = 3). Luciferase assay results of (D) DIC-case (n = 3) and (G) DIC-control (n = 4) cell lines in response to doxorubicin are shown. Gene expression of RA signaling genes ALDH1A2 and CYP26B1 measured by qPCR in (E) DIC-case and (H) DIC-control cell lines (n = 3). All experiments are biological, independent replicates and are shown as mean ± SEM, ∗p < 0.05; ∗∗p < 0.01; t test.

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  Figure 3. Differentially expressed genes and pathways measured by RNA-seq in doxorubicin-treated iPSC-CMs (A) Summary of enriched pathways and selection process. ChIP, chromatin immunoprecipitation; DOX, doxorubicin. (B) Differentially expressed gene ratio comparing non-treated case-original and case-repaired samples. (C) Summary of up- and down-regulated RARG target genes expression under doxorubicin treatment. (D) Top 10 up and down-regulated pathways that targeted by RARG and responded to doxorubicin. ESR, estrogen receptor; NT, no treatment; PI3K, phosphatidylinositol 3-kinase. (E) Heatmap of RARG-targeted gene expression in DNA-repair-related pathways. (F) Protein association network analysis of 11 highlighted RARG-targeted, DNA-repair-related genes. (G and H) Mitochondrial copy number ratio in iPSC-CM quantified by qPCR after doxorubicin treatment (normalized to DMSO control). All experiments are biological, independent replicates (n = 3-4) and are shown as mean ± SEM, ∗p < 0.05; ns, not significant; t test.

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  Figure 4. Impact of RARG-S427L on doxorubicin-induced DNA damage and repair (A) Comet assay results visualized under fluorescence microscope (scale bars represent 200 μm). (B and C) Relative DNA damage fold change in RARG-WT/WT and RARG-WT/S427L assay (n = 3; 100–150 cells were quantified per sample in each independent experiment). (D–G) Gene expression of DNA repair gene (D and F) RAD9A and (E and G) RARG with 6 and 24 h of recovery after doxorubicin treatment in DIC-case cell lines measured by qPCR (n = 3). (H and I) IC50 and cell viability percentages after 24 h of doxorubicin treatment with no recovery time or with 24 h of recovery (n = 4–8). All experiments are biological, independent replicates and are shown as mean ± SEM; ∗p < 0.05; ∗∗p < 0.01; t test.