Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Glucocorticoid Inhibition of Estrogen Regulation of the Serotonin Receptor 2B in Cardiomyocytes Exacerbates Cell Death in Hypoxia/Reoxygenation Injury

doi: 10.1161/JAHA.120.015868

Figure Lengend Snippet: HL‐1 cells were treated with vehicle (Veh), 10 nmol/L estradiol (estrogen [E2]), 100 nmol/L dexamethasone (Dex), or 100 nmol/L dexamethasone+10 nmol/L estrogen (Dex+E2). mRNA was isolated and analyzed using a whole mouse genome 4×44 multiplex format oligo array (Agilent) for gene expression. A , Heat maps were generated with the online software Heatmapper ( http://www2.heatmapper.ca ) to visualize the differences in the gene expression of cells treated with each hormone and their combination. Blue indicates downregulation, and red indicates upregulation. B , The estrogen‐regulated, dexamethasone‐regulated, and estrogen+dexamethasone–regulated genes within each group were sorted by Venn diagrams that were generated using the Ingenuity Pathway Analysis software. Each hormone treatment regulated a similar number of genes; however, each treatment led to the regulation of a significant number of unique genes, and dexamethasone+estrogen significantly altered the transcriptional response to estrogen and dexamethasone alone. The hormone combination treatment inhibited the effects of dexamethasone in the regulation of 438 genes and of estrogen in the regulation of 257 genes (circled in red). In addition, dexamethasone+estrogen had unique effects on the expression of 360 genes. C , Results from microarray analysis of each hormone treatment were loaded into Ingenuity Pathway Analysis software. Estrogen treatment significantly regulated an important number of genes associated with serotonin receptor (5‐HTR) signaling (ranked top 1). Dexamethasone treatment led to the regulation of genes involved in different cellular and molecular biological pathways compared with estrogen. Dexamethasone+estrogen cotreatment alter the pathways that are regulated by a single estrogen treatment in cardiomyocytes. D , Estrogen altered the expression of 4 subtypes of 5‐HTRs in HL‐1 cardiomyocytes: 5‐HTR2B (5‐HTR2 subtype B), 5‐HTR2C (5‐HTR2 subtype C), and 5‐HTR5 (5‐HTR2 subtype A) and 5‐HTR6 . The figure displays the effects of each hormone treatment on the expression of each gene. Estrogen also affects the expression of ADCY1 and ADCY4. Red indicates upregulation, and green indicates downregulation. ADCY1 indicates adenylate cyclase 1; ADCY4, adenylate cyclase 4; cAMP, cyclic adenosine monophosphate; EGF, endothelial growth factor; FGF, fibroblast growth factor; Th1, T helper 1; and Th2, T helper 2.

Article Snippet: Dexamethasone (1,4‐pregnadien‐9α‐fluoro‐16α‐methyl‐11β, 17, 21‐triol‐3, 20‐dione; ≥98% by thin‐layer chromatography) and estrogen (17‐β‐estradiol; ≥98% thin‐layer chromatography) were purchased from Steraloids (Newport, RI).

Techniques: Isolation, Multiplex Assay, Gene Expression, Generated, Software, Expressing, Microarray

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Glucocorticoid Inhibition of Estrogen Regulation of the Serotonin Receptor 2B in Cardiomyocytes Exacerbates Cell Death in Hypoxia/Reoxygenation Injury

doi: 10.1161/JAHA.120.015868

Figure Lengend Snippet: A , HL‐1 cells were treated with vehicle (Veh; gray line), 100 nmol/L dexamethasone (Dex; blue), estradiol (estrogen [E2]; red), and dexamethasone+estrogen (Dex+E2; pink) for the indicated times (0–24 hours), and 5‐HTR2B levels were analyzed by quantitative real‐time polymerase chain reaction and Western blotting. Estrogen steadily increased the mRNA expression of 5‐HTR2B in the time course experiment from 3 to 12 hours. Dexamethasone led to 5‐HTR2B increased expression at the 3‐hour time point. Dexamethasone+estrogen prevented estrogen‐induced upregulation of 5‐HTR2B expression at all time points. No significant changes in protein levels were observed at baseline (0 hours) or 6 hours following hormone treatment. The 5‐HTR2B protein levels were significantly increased in response to estrogen at 24 hours. Cotreatment of HL‐1 cells with dexamethasone+estrogen abolished estrogen effects on 5‐HTR2B at the protein level. Levels of 5‐HTR2B were normalized to the loading control GAPDH. B , HL‐1 cells were pretreated for 3 hours with vehicle (control) or 10 μg/mL of cycloheximide. Then cells were exposed to 100 nmol/L dexamethasone, 10 nmol/L estrogen, or dexamethasone+estrogen for 6 hours. Cycloheximide treatment did not have significant effects on 5‐HTR2B regulation by estrogen, suggesting that 5‐HTR2B is a direct target of estrogen. C , HL‐1 cells were transfected with NTC siRNA or glucocorticoid receptor (GR) siRNA (‐GR). At 48 hours after transfection, cells were treated with vehicle, 100 nmol/L dexamethasone, 10 nmol/L estrogen, or dexamethasone+estrogen for 3 hours. Downregulation of GR levels significantly blocked dexamethasone effects on 5‐HTR2B mRNA levels, showing that GR is required for dexamethasone effects. D , HL‐1 cells were also transfected with ERα (estrogen receptor α) siRNA (‐ERα), ERβ (‐ERβ) siRNA, and GPR30 (G protein‐coupled estrogen receptor 1) siRNA (‐GPR30) as described previously. Knocking down ERα abolished estrogen regulation of 5‐HTR2B , demonstrating that 5‐HTR2B regulation by estrogen is dependent on ERα expression. E , HL‐1 cells were treated with the following 3 estrogen receptor antagonists: ICI (182,780; fulvestrant; ERα antagonist), PHTPP (4‐[2‐Phenyl‐5,7‐bis(trifluoromethyl) pyrazolo[1,5‐a]pyrimidin‐3‐yl]phenol; ERβ antagonist), and G‐15 ([3aS,4R,9bR]‐4‐[6‐bromo‐1,3‐benzodioxol‐5‐yl]‐3a,4,5,9b‐tetrahydro‐3H‐cyclopenta[c]quinoline; G‐protein‐coupled estrogen receptor [GPER/GPR30] antagonist). After 1 hour of antagonist treatment, cells were subjected to hormone treatments. ICI significantly inhibited the estrogen upregulation of 5‐HTR2B . For the cycloheximide, siRNA, and antagonists experiments, 5‐HTR2B levels were measured by quantitative real‐time polymerase chain reaction. mRNA was normalized to cyclophillin B for all mRNA experiments. We used the same doses of hormones for all treatments. The figure includes representative Western blots confirming the changes in 5‐HTR2B protein levels and the knockdown of each individual receptor. Ordinary 1‐way ANOVA with Dunnett multiple comparisons analysis was used to evaluate differences among group treatments unless otherwise specified. Data represent the mean±SE (n=3–5 independent samples per group). * P <0.05; ** P <0.01; *** P <0.001, ns=not significant.

Article Snippet: Dexamethasone (1,4‐pregnadien‐9α‐fluoro‐16α‐methyl‐11β, 17, 21‐triol‐3, 20‐dione; ≥98% by thin‐layer chromatography) and estrogen (17‐β‐estradiol; ≥98% thin‐layer chromatography) were purchased from Steraloids (Newport, RI).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control, Transfection, Knockdown

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Glucocorticoid Inhibition of Estrogen Regulation of the Serotonin Receptor 2B in Cardiomyocytes Exacerbates Cell Death in Hypoxia/Reoxygenation Injury

doi: 10.1161/JAHA.120.015868

Figure Lengend Snippet: A , Schematic representation of the mouse 5‐HTR2B gene. Sequences of the consensus EREs and GREs identified within the predicted promoter, targeting areas of the designed primers, and the gene sequence of 5‐HTR2B . The table displaying the sequences of the primers and probes used for the assay. B , HL‐1 cells were treated with vehicle (Veh; black bars), 100 nmol/L dexamethasone (Dex; gray bars), 10 nmol/L estrogen (E2; dark gray bars), or dexamethasone+estrogen (Dex+E2; light gray bars) for 2 hours, and chromatin immunoprecipitation (ChIP) assays were performed with equivalent amounts of rabbit immunoglobulin G or rabbit anti‐GR or ERα antibody. Coimmunoprecipitated DNA was analyzed by real‐time polymerase chain reaction using primers and probes to the identified EREs and GREs located in the 5‐HTR2B promoter and gene sequence. Sheared DNA fragment sizes were between 200 and 500 bp (left panel). Results are plotted as a function of input DNA. Percentage input was calculated using the following equations: adjusted input 100%=(Ct input−6.64) and 100*2 (Adjusted input−Ct (input)) . Ordinary 1‐way ANOVA with Dunnett multiple comparisons analysis was used to evaluate differences among group treatments unless otherwise specified. Data represent mean±SE for 4 independent experiments per treatment group. * P <0.05 and *** P <0.001 vs vehicle in each group.

Article Snippet: Dexamethasone (1,4‐pregnadien‐9α‐fluoro‐16α‐methyl‐11β, 17, 21‐triol‐3, 20‐dione; ≥98% by thin‐layer chromatography) and estrogen (17‐β‐estradiol; ≥98% thin‐layer chromatography) were purchased from Steraloids (Newport, RI).

Techniques: Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Glucocorticoid Inhibition of Estrogen Regulation of the Serotonin Receptor 2B in Cardiomyocytes Exacerbates Cell Death in Hypoxia/Reoxygenation Injury

doi: 10.1161/JAHA.120.015868

Figure Lengend Snippet: Gonadectomized and adrenalectomized C57BL/6 male and female mice were injected with vehicle (Veh), 1 mg/kg of dexamethasone (Dex), or 10 μg/kg estradiol (estrogen [E2]). RNA and protein were isolated at 6 hours after the treatment. mRNA and representative Western blots of 5‐HTR2B in each treatment group for male ( A ) and female ( B ) hearts. mRNA was normalized to cyclophillin B, and protein was normalized to GAPDH. Ordinary 1‐way ANOVA with Dunnett multiple comparisons analysis was used to evaluate differences among group treatments unless otherwise specified. Data represent the mean±SE (n=3–5 mice per group). * P <0.05; ns=not significant.

Article Snippet: Dexamethasone (1,4‐pregnadien‐9α‐fluoro‐16α‐methyl‐11β, 17, 21‐triol‐3, 20‐dione; ≥98% by thin‐layer chromatography) and estrogen (17‐β‐estradiol; ≥98% thin‐layer chromatography) were purchased from Steraloids (Newport, RI).

Techniques: Injection, Isolation, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Glucocorticoid Inhibition of Estrogen Regulation of the Serotonin Receptor 2B in Cardiomyocytes Exacerbates Cell Death in Hypoxia/Reoxygenation Injury

doi: 10.1161/JAHA.120.015868

Figure Lengend Snippet: HL‐1 cells were pretreated for 6 hours with vehicle (Veh), 100 nmol/L dexamethasone (Dex), 10 nmol/L estradiol (estrogen [E2]), or dexamethasone+estrogen (Dex+E2). Following these treatments, the cells were subjected to 17 hours of hypoxia followed by 1 hour of reoxygenation in the presence and absence of hormones. A , Cell viability was then analyzed by flow cytometry using propidium iodide staining. The plot represents the percentage of dead cells in each treatment group. B , Apoptotic cardiomyocytes stained with TUNEL (terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling; red) and 4´,6‐diamidino‐2‐phenylindole (blue nuclear staining). The percentage of TUNEL‐positive cells was calculated by quantifying the TUNEL‐positive cells in ImageJ using the TUNEL Cell Counter method as described in the Methods section. C , mRNA levels of 5‐HTR2B (5‐HTR2 subtype B) and SLC25A4 (adenine nucleotide translocase 1) in each treatment group after hypoxia/reoxygenation. mRNA was normalized to cyclophillin B. Ordinary 1‐way ANOVA with Dunnett multiple comparisons analysis was used to evaluate differences among group treatments unless otherwise specified. Data are represented as the mean±SE from 3 independent experiments. * P <0.05; ** P <0.01; ns=not significant vs vehicle in each group.

Article Snippet: Dexamethasone (1,4‐pregnadien‐9α‐fluoro‐16α‐methyl‐11β, 17, 21‐triol‐3, 20‐dione; ≥98% by thin‐layer chromatography) and estrogen (17‐β‐estradiol; ≥98% thin‐layer chromatography) were purchased from Steraloids (Newport, RI).

Techniques: Flow Cytometry, Staining, TUNEL Assay, End Labeling

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Glucocorticoid Inhibition of Estrogen Regulation of the Serotonin Receptor 2B in Cardiomyocytes Exacerbates Cell Death in Hypoxia/Reoxygenation Injury

doi: 10.1161/JAHA.120.015868

Figure Lengend Snippet: 5‐HTR2B siRNA was used to knockdown 5‐HTR2B expression in HL‐1 cardiomyocytes. HL‐1 cells were transfected with nontarget (NTC) siRNA or 5‐HTR2B siRNA. At 48 hours after transfection, cells were treated with vehicle, 100 nmol/L dexamethasone (DEX), 10 nmol/L estrogen (E2), or dexamethasone+estrogen (D+E2) for 6 hours and then subjected to hypoxia/reoxygenation. A , 5‐HTR2B levels were knocked down to 50% at the mRNA and protein levels. Live and dead cells were quantified using the LIVE/DEAD Cell viability assay as described in the Methods section. Estrogen failed to protect HL‐1 cells from dead in response hypoxia/reoxygenation in 5‐HTR2B knockdown cells. B , 5‐HTR2B and SLC25A4 mRNA levels in NTC and 5‐HTR2B siRNA cells. mRNA was normalized to cyclophillin B. Ordinary 1‐way ANOVA with Dunnett multiple comparisons analysis was used to evaluate differences among group treatments unless otherwise specified. Data are represented as the mean±SE from 3 independent experiments. * P <0.05; ** P <0.01; *** P <0.001; ns=not significant vs vehicle in each group.

Article Snippet: Dexamethasone (1,4‐pregnadien‐9α‐fluoro‐16α‐methyl‐11β, 17, 21‐triol‐3, 20‐dione; ≥98% by thin‐layer chromatography) and estrogen (17‐β‐estradiol; ≥98% thin‐layer chromatography) were purchased from Steraloids (Newport, RI).

Techniques: Knockdown, Expressing, Transfection, Viability Assay