Selective Estrogen Receptor Degraders Induce Bradycardia by Modulating Nuclear Estrogen Signaling

Figures

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  Figure 1. Giredestrant and Camizestrant Induce Bradycardia in Zebrafish Embryos (A and B) Chemical structures of giredestrant (GDC-9545, or GDC) and camizestrant (AZD-9833, or AZD). (C). Schematic overview of zebrafish chemical treatments and heart rate assessment. (D) Brightfield images of wild-type (WT) zebrafish embryos at 72 hours post-treatment (hpt) with DMSO, giredestrant at a dose of 33 μmol/L, or camizestrant at a dose of 100 μmol/L. (E to G) Heart rate measurements of zebrafish embryos treated with the highest nontoxic doses of giredestrant (33 μmol/L), camizestrant (100 μmol/L), fulvestrant (100 μmol/L), or amcenestrant (SAR439859) (3.3 μmol/L) at 24 hpt (E), 48 hpt (F), and 72 hpt (G). Heart rates were normalized to control embryos from the same experimental batch. Sample size: n = 12 embryos per condition, with a minimum of 3 independent replicates. Statistical significance was determined using ordinary 1-way analysis of variance followed by Tukey's post hoc test or the Kruskal-Wallis test followed by Dunn’s multiple comparison test for data that did not meet normality criteria. Data are expressed as mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. ns = not significant.

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  Figure 2. Giredestrant Does Not Alter Thyroid Signaling or Expression of Cardiac Conduction Genes (A) Schematic illustration of the timeline of zebrafish chemical treatment and subsequent enzyme linked immunosorbent assay. (B) Quantification of tri-iodothyronine (T3) from giredestrant-, camizestrant-, fulvestrant- and G1-treated embryos by enzyme linked immunosorbent assay. (C to I) RNA expression levels of hcn4l (C), hcn4 (D), tbx3 (E), shox2 (F), isl1 (G), ryr2b (H), and cacna1g (I) in giredestrant-treated 4 dpf embryos as measured by RT-qPCR. Statistical significance was determined using an unpaired Student's t-test. Data are expressed as mean ± SEM. Abbreviations as in Figure 1.

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  Figure 3. Estrogen Receptor Agonists Alleviate Giredestrant-Induced Bradycardia (A to C) Normalized heart rates of zebrafish embryos treated with giredestrant, estrogen (E2) at a dose of 1 or 10 μmol/L, or giredestrant in combination with E2. (D to F) Normalized heart rates of embryos treated with giredestrant, diethylstilbestrol (DES) at a dose of 3.3 or 10 nmol/L, or giredestrant in combination with DES. (G to I) Normalized heart rates of embryos treated with giredestrant, G1 at a dose of 300 nmol/L or 1 μmol/L, or giredestrant in combination with G1. (J to L) Normalized heart rates of embryos treated with giredestrant, 4-nonylphenol (4NP) 33 or 100 nmol/L, or giredestrant in combination with 4NP. Statistical significance was determined using ordinary 1-way analysis of variance followed by Tukey's post hoc test or the Kruskal-Wallis test followed by Dunn’s multiple comparison test for data that did not meet normality criteria. Data are expressed as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figure 1.

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  Figure 4. Giredestrant-Induced Bradycardia Is Independent of GPER (A) Schematic illustrating the timeline for zebrafish chemical treatments, protein isolation for Western blot analysis, and heart rate assessment. (B) Western blot analysis showing phospho-ERK1/2 (top) relative to total ERK1/2 (bottom) in 50 hpf WT embryos treated with giredestrant, camizestrant, fulvestrant, G1, and the ERK inhibitor mirdametinib for 4 hours. (C) Absolute heart rate of WT embryos treated with giredestrant, camizestrant, fulvestrant, G1, and the ERK inhibitor mirdametinib at 24 hpt. D) Schematic illustration of the timeline for zebrafish chemical treatments in gper−/− mutant embryos. (E and F) Absolute heart rate of gper−/− mutant embryos treated with giredestrant, camizestrant, fulvestrant, and G1 at 24 and 48 hpt, respectively. Statistical significance was determined using ordinary 1-way analysis of variance followed by Tukey's post hoc test or the Kruskal-Wallis test followed by Dunn’s multiple comparison test for data that did not meet normality criteria. Data are expressed as mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Abbreviations as in Figure 1.

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  Figure 5. Giredestrant-Induced Bradycardia Requires Nuclear Estrogen Receptors (A) Schematic representation of the timeline of zebrafish chemical treatment and heart rate assessment. (B) Absolute heart rate of WT and Esr1 mutant embryos treated with giredestrant (GDC-9545, or GDC). (C) Absolute heart rate of WT and Esr2a mutant embryos treated with giredestrant. Statistical significance was determined using ordinary 1-way analysis of variance followed by Tukey's post hoc test or the Kruskal-Wallis test followed by Dunn’s multiple comparison test for data that did not meet normality criteria. Data are expressed as mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Abbreviations as in Figure 1.