All-trans retinoic acid (ATRA) regulates key genes in the RARG-TOP2B pathway and reduces anthracycline-induced cardiotoxicity

Abstract

The effectiveness of anthracycline chemotherapeutics (e.g., doxorubicin) is limited by anthracycline-induced cardiotoxicity (ACT). A nonsynonymous variant (S427L) in the retinoic acid receptor-γ (RARG) gene has been associated with ACT. This variant causes reduced RARG activity, which is hypothesized to lead to increased susceptibility to ACT through reduced activation of the retinoic acid pathway. This study explored the effects of activating the retinoic acid pathway using a RAR-agonist, all-trans retinoic acid (ATRA), in human cardiomyocytes and mice treated with doxorubicin. In human cardiomyocytes, ATRA induced the gene expression of RARs (RARG, RARB) and repressed the expression of topoisomerase II enzyme genes (TOP2A, TOP2B), which encode for the molecular targets of anthracyclines and repressed downstream ACT response genes. Importantly, ATRA enhanced cell survival of human cardiomyocytes exposed to doxorubicin. The protective effect of ATRA was also observed in a mouse model (B6C3F1/J) of ACT, in which ATRA treatment improved heart function compared to doxorubicin-only treated mice. Histological analyses of the heart also indicated that ATRA treatment reduced the pathology associated with ACT. These findings provide additional evidence for the retinoic acid pathway's role in ACT and suggest that the RAR activator ATRA can modulate this pathway to reduce ACT.

Fig 1. ATRA protects human cardiomyocytes exposed to doxorubicin.

(a) Dose-response curves from the MTT cell viability assay. The dose-response curves are plotted for control (vehicle-treated) and ATRA (500 nM, 1000 nM) at serial concentrations (0 μM, 1 μM, 3.16 μM, 10 μM, 31.6 μM, and 100 μM) of doxorubicin (N = 12, 4 independent experiments of 3 biological replicates). Data are presented as mean ± standard error of the mean. (b) IC₅₀ values corresponding to each cell-viability curve fit. Data are presented as mean and 95% CI (profile likelihood). Extra-sum-of-squares F test was performed to compare the curve fits (**, and *** denote P = 0.0051 and P < 0.0001, respectively). (c) Relative apoptosis activation measured by caspase 3/7 activity assay. The graph shows apoptosis activation relative to 10 μM doxorubicin vehicle-treated condition. The apoptosis activation was calculated by normalizing to untreated cells in each condition. Relative apoptosis in control (vehicle-treated) and ATRA (500 nM, 1000 nM) at 10 μM doxorubicin treatment is plotted (N = 6 independent biological replicates). Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons correction (*, and ** denote P = 0.0250 and P = 0.0013, respectively).

Fig 2. ATRA regulates RAR genes, TOP2 genes, and downstream ACT response genes in human cardiomyocytes.

(a) Fold-change in gene expression of RARG, RARB, RARA, TOP2A and TOP2B during control (vehicle) or doxorubicin (1 μM) or ATRA (500 nM) treatment. Data are presented as mean ± standard deviation (two independent experiments were performed, the extracted RNA was combined, and gene expression assay was performed as three technical replicates, N = 3). Statistical analysis was performed using two-way ANOVA and Bonferroni multiple comparisons correction (comparisons between the control vs. doxorubicin and control vs. ATRA are shown, *, and **** denote P = 0.012, P < 0.0001, respectively). (b) Fold-change in gene expression of the genes involved in doxorubicin-induced apoptosis (FAS, APAF1, TP53INP1, and BAX) and doxorubicin-induced mitochondrial dysfunction (PPARGC1A, PPARGC1B, and ATP5F1A) during control (vehicle) or doxorubicin (1 μM) or ATRA (500 nM) treatment. Data are presented as mean ± standard deviation (two independent experiments were performed, the extracted RNA was combined, and gene expression assay was performed as three technical replicates, N = 3). Statistical analysis was performed using two-way ANOVA and Bonferroni multiple comparisons correction (comparisons between the control vs. doxorubicin and control vs. ATRA are shown, **, *** and **** denote, P = 0.0059, P < 0.001 and P < 0.0001, respectively).

Fig 3. ATRA regulates Top2a gene expression in heart tissues of doxorubicin-treated mice.

Fold-change in gene expression of Rarg, Rarb, Rara, Top2a, and Top2b in control (saline-treated), doxorubicin-only treated, and ATRA + doxorubicin-treated mice heart tissues. Data are presented as mean ± standard deviation (N = 9, 3 technical replicates of 3 biological replicates). Statistical analysis was performed using two-way ANOVA and Bonferroni multiple comparisons correction (comparisons between the control vs. doxorubicin, control vs. ATRA + doxorubicin and doxorubicin vs. ATRA + doxorubicin are shown, ***, and **** denote P = 0.0009, P < 0.0001, respectively).

Fig 4. ATRA is cardioprotective in doxorubicin-treated mice as demonstrated by echocardiography.

(a) Fractional shortening measurements to evaluate cardiac systolic function in control (saline-treated), doxorubicin-only treated, and ATRA + doxorubicin-treated mice. The graph shows percent fractional shortening, at baseline, after three, six, and eight weeks of weekly doxorubicin/saline treatments (N = 5 mice per group). Data are presented as mean ± standard deviation. Statistical analysis was performed using repeated measures two-way ANOVA with Bonferroni multiple comparisons adjustment (* denotes P = 0.0348). (b) Representative M-Mode echocardiograms in control (saline-treated), doxorubicin-treated, and ATRA + doxorubicin-treated mice after eight weeks of study (cumulative doxorubicin dose of 24 mg/kg in the doxorubicin-treated mice). The y-axis denotes the distance (in mm) from the transducer, and the x-axis denotes time (in ms) [38, 39]. The blue line outlines the left ventricular anterior and posterior walls. The green dotted line represents chamber width during systole, and the red dotted line denotes the chamber width during diastole. (c) Ejection fraction measurements to evaluate cardiac systolic function in control (saline-treated), doxorubicin-only treated, and ATRA + doxorubicin-treated mice. The graph shows percent ejection fraction at baseline, after three, six, and eight weeks of weekly doxorubicin/saline treatments (N = 5 mice per group). Data are presented as mean ± standard deviation. Statistical analysis was performed using repeated measures two-way ANOVA with Bonferroni multiple comparisons adjustment (*, and ** denote P = 0.0193 and P = 0.0020, respectively).

Fig 5. ATRA is cardioprotective in doxorubicin-treated mice as demonstrated by histological analysis.

(a) Representative image of cytoplasmic vacuolization in H&E-stained heart tissue section of doxorubicin-only treated mice. The arrow indicates cytoplasmic vacuolization. A zoomed-in view of the cytoplasmic vacuolization is shown on top. Scale bars, 100 μm. (b) Number of cardiomyocytes with vacuolated cytoplasm in the H&E-stained tissue sections of control (saline-treated), doxorubicin-only-treated, and ATRA + doxorubicin-treated mice (N = 5 mice per group). Data are presented as mean ± standard deviation. Statistical analysis was performed using a one-way ANOVA and Dunnett’s multiple comparisons correction (control vs. doxorubicin: *P = 0.0435, control vs. ATRA + doxorubicin: P = ns). (c) Representative image of TUNEL-stained heart tissue sections of doxorubicin-only treated, and ATRA + doxorubicin-treated mice. The arrow indicates TUNEL-positive cardiomyocyte. Scale bars, 100 μm. (d) TUNEL staining to detect DNA fragmentation during apoptosis. The graph depicts the number of TUNEL-positive cardiomyocytes/mm² in the tissue sections of control (saline-treated), doxorubicin-only treated, and ATRA + doxorubicin-treated mice (N = 5 mice per group). Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA and Bonferroni multiple comparisons adjustment. Statistical comparisons between the saline-treated condition vs. doxorubicin-treated groups are shown above the bar, comparison between doxorubicin-only treated vs. ATRA + doxorubicin is shown with a bracket (*, **, and **** denote P = 0.0376, P = 0.0055 and P < 0.0001, respectively). (e) Representative images of Masson’s trichrome-stained tissue sections from control (saline-treated), doxorubicin-only treated, and ATRA + doxorubicin-treated mice. The arrow indicates focal fibrosis. Scale bars, 200 μm.