BET bromodomain proteins regulate transcriptional reprogramming in genetic dilated cardiomyopathy

Abstract

The bromodomain and extraterminal (BET) family comprises epigenetic reader proteins that are important regulators of inflammatory and hypertrophic gene expression in the heart. We previously identified the activation of proinflammatory gene networks as a key early driver of dilated cardiomyopathy (DCM) in transgenic mice expressing a mutant form of phospholamban (PLNR9C) - a genetic cause of DCM in humans. We hypothesized that BETs coactivate this inflammatory process, representing a critical node in the progression of DCM. To test this hypothesis, we treated PLNR9C or age-matched WT mice longitudinally with the small molecule BET bromodomain inhibitor JQ1 or vehicle. BET inhibition abrogated adverse cardiac remodeling, reduced cardiac fibrosis, and prolonged survival in PLNR9C mice by inhibiting expression of proinflammatory gene networks at all stages of disease. Specifically, JQ1 had profound effects on proinflammatory gene network expression in cardiac fibroblasts, while having little effect on gene expression in cardiomyocytes. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Mechanistically, we demonstrated that BRD4 serves as a direct and essential regulator of NF-κB-mediated proinflammatory gene expression in cardiac fibroblasts. Suppressing proinflammatory gene expression via BET bromodomain inhibition could be a novel therapeutic strategy for chronic DCM in humans.

Keywords: Cardiology; Fibrosis; Heart failure; Inflammation.

Figure 1. Genes suppressed by JQ1 are highly enriched in PLN^R9C hearts.

GSEA for preDCM (A) and DCM (B) PLN^R9C mice against genes that were upregulated in both TAC and MI models and were downregulated by JQ1 treatment in both (15). NES, normalized enrichment score.

Figure 2. BET inhibition delays DCM in PLN^R9C mice.

(A) Experimental protocol. (B) Representative M-mode images of WT and PLN^R9C vehicle- or JQ1-treated mice at 18 weeks of age. Echo, echocardiography. (C–F) Echocardiographic assessment of mice treated with JQ1 or vehicle demonstrates progressive systolic dysfunction and negative LV remodeling in PLN^R9C mice that was significantly blunted by JQ1 (n = 14 PLN^R9C, n = 7 WT mice per group; ANOVA corrected for multiple hypothesis testing). Representative images demonstrating cardiac fibrosis: (G) Masson’s trichrome–stained LV sections from WT and PLN^R9C hearts and (H) false-color images of fibrosis identified by Keyence microscope software (yellow, fibrosis; box, enlarged region). As JQ1 had no effect on fibrosis in WT, a single representative WT image is shown. Scale bar in G: 200 μm. Original magnification in H, ×10.(I) Quantification of scar area demonstrated severe fibrosis in PLN^R9C vehicle-treated hearts at 20-weeks of age that was markedly blunted by JQ1 (n = 3 mice, 36 images from n = 4 levels from apex to base for each mouse). Boxes, IQR; whiskers, 1.5× IQR; black line, median; notches, SD, circles, extreme outlier values).

Figure 3. BET inhibition prolongs survival.

Kaplan-Meier plot of survival in PLN^R9C mice treated with vehicle or JQ1 (n = 16 mice/treatment group, log-rank test).

Figure 4. BETs activate inflammatory gene expression in preDCM hearts.

(A) Experimental protocol (n = 3 mice/treatment group). (B) Heat map of genes significantly differentially expressed in preDCM PLN^R9C vehicle-treated hearts, demonstrating global reduction in gene activation in PLN^R9C JQ1-treated mice. Data plotted are natural log (ln) of fold change values versus those in WT vehicle-treated mice. (C) IPA upstream regulator analysis in preDCM PLN^R9C vehicle-treated compared with WT mice predicted the activation of many inflammatory mediators. (D) IPA upstream regulator analysis demonstrating reduced activity of the majority of proinflammatory mediators in preDCM PLN^R9C JQ1-treated versus PLN^R9C vehicle-treated mice.

Figure 5. Chronic BET inhibition alters inflammatory gene expression in DCM.

(A) Experimental protocol (n = 3 mice/treatment group). Heat maps demonstrating that (B) genes controlling aerobic respiration (tricarboxylic acid cycle and mitochondrial oxidative phosphorylation) were almost uniformly downregulated, while (C) genes controlling cellular glucose utilization were generally upregulated in PLN^R9C hearts. JQ1 had little effect on metabolic gene expression. Data plotted are natural log of fold-change values versus WT vehicle-treated mice. Volcano plots demonstrating the magnitude and significance of JQ1’s alteration of the expression of genes that were (D) upregulated or (E) downregulated in PLN^R9C vehicle-treated hearts. In each plot, blue represents the effect of JQ1 on gene expression for all genes in red. Representative genes (labeled) reveal key processes affected by JQ1, including cardiac stress response signaling (Nppa, Myh6/Myh7), fibrosis and extracellular matrix remodeling (Postn, Col8a1), TGF-β signaling (Tgfb2, Ctgf), cytoskeletal signaling (Mylk4), Wnt signaling (Sfrp1, Dvl2), innate immune activation (Tlr2, Tlr4), and metabolism (Idh2, Atp2a2).

Figure 6. BET inhibition prevents cardiac fibroblast proliferation.

(A) Experimental protocol (n = 4 mice/treatment group). (B) PLN^R9C vehicle-treated mice had 3.7-fold more proliferating non-myocytes than WT mice. This was substantially reduced by JQ1. Boxes show IQR; notches, SD; whiskers, 1.5× IQR; circles, extreme outliers. Student’s 2-tailed t test. (C) Representative confocal images showing staining for EdU, DAPI, WGA, and cardiac troponin I (cTnI), and merged image. Scale bars: 50 μm. (D) Representative images from a multimarker analysis demonstrating costaining of EdU with vimentin (arrows) but not with CD31 or CD45. Scale bars: 50 μm. (E) Quantification of cells staining for both EdU and lineage-specific marker identified the majority of EdU cells as cardiac fibroblasts. Slides were costained with DAPI and WGA (n = 4/group). Boxes show IQR; whiskers, 1.5× IQR; circles, extreme outliers.

Figure 7. BETs primarily affect cardiac non-myocyte gene expression programs.

(A) Experimental protocol (n = 3 mice/treatment group). Non-myocytes: (B) Differentially expressed genes from pooled cardiac non-myocytes (relative to WT vehicle-treated mice) were predominantly downregulated in WT JQ1-treated mice and upregulated in PLN^R9C vehicle-treated mice. Non-myocytes from PLN^R9C mice treated with JQ1 displayed a marked shift in gene expression. (C) Pathways enriched in non-myocytes from PLN^R9C vehicle-treated mice with Z score ±2 were all downregulated or not enriched in mice treated with JQ1. Data represent the Z score versus WT vehicle-treated mice. (D) JQ1 treatment shifted gene expression in these pathways from predominantly up- to predominantly downregulated. Cardiomyocytes: (E) The pattern of differentially expressed genes from pooled cardiomyocytes (relative to WT vehicle-treated mice) was similar in PLN^R9C vehicle-treated and JQ1-treated mice. Further, JQ1 had virtually no effect on gene expression in WT cardiomyocytes. (F) Heat map of genes differentially expressed in PLN^R9C vehicle-treated cardiomyocytes showing little to no change in expression levels with JQ1 treatment. Data plotted are natural log of fold change values versus WT vehicle-treated mice.

Figure 8. NF-κB is activated in a BRD4-dependent manner in PLN^R9C hearts.

(A) Heat map of genes known to be activated by NF-κB demonstrated uniform upregulation in cardiac non-myocytes, with a majority of genes being switched off by JQ1 therapy. Data plotted are natural log of fold change values versus WT vehicle-treated mice. GSEA for genes induced (B) in vehicle-treated or attenuated (C) in PLN^R9C JQ1-treated mice against a curated set of genes known to be directly induced by NF-κB. (D) Western blot showing increased aK310-RELA in PLN^R9C compared with WT hearts irrespective of JQ1 treatment status (representative images of n = 3 experiments). Boxes show IQR and whiskers, 1.5× IQR; individual data points are plotted. Student’s 2-tailed t test. (E) Western blots (IB) from the co-IP of BRD4 with anti-BRD4, anti-RELA, and anti–ak310-RELA demonstrated direct binding of BRD4 and NF-κB in PLN^R9C hearts with DCM. This was completely ablated by treatment with JQ1 (representative images of n = 3 experiments).