BET bromodomain inhibition suppresses innate inflammatory ... · these specific transcriptional mechanisms, we found that JQ 1 does not suppress physiological cardiac hypertrophy

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

HEART FA I LURE

1Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA.2Institute for Transformative Molecular Medicine and Department of Medicine, CaseWestern Reserve University School of Medicine and University Hospitals ClevelandMedical Center, Cleveland, OH 44106, USA. 3BioInfo, Plantagenet, Ontario K0B 1L0,Canada. 4Division of Cardiovascular Medicine, Department of Medicine, and De-partment of Molecular Physiology and Biophysics, Vanderbilt University School ofMedicine, Nashville, TN 37232, USA. 5Division of Cardiology, Department of Pedi-atrics, University of California San Francisco School of Medicine, San Francisco, CA94158, USA. 6Department of Medical Oncology, Dana-Farber Cancer Institute andHarvard Medical School, Boston, MA 02215, USA. 7Division of Cardiology, Depart-ment of Medicine, Consortium for Fibrosis Research & Translation, University ofColorado, Anschutz Medical Campus, Denver, CO 80204, USA. 8Division of Cardi-ology, Department of Medicine, and Cardiovascular Research Institute, Universityof California San Francisco School of Medicine, San Francisco, CA 94158, USA.*These authors contributed equally to this work.†Corresponding author. Email: [email protected]

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BET bromodomain inhibition suppresses innateinflammatory and profibrotic transcriptional networksin heart failureQiming Duan,1* Sarah McMahon,1* Priti Anand,1 Hirsh Shah,2 Sean Thomas,1 Hazel T. Salunga,1

Yu Huang,1 Rongli Zhang,2 Aarathi Sahadevan,2 Madeleine E. Lemieux,3 Jonathan D. Brown,4

Deepak Srivastava,1,5 James E. Bradner,6 Timothy A. McKinsey,7 Saptarsi M. Haldar1,8†

Despite current standard of care, the average 5-year mortality after an initial diagnosis of heart failure (HF) isabout 40%, reflecting an urgent need for new therapeutic approaches. Previous studies demonstrated that theepigenetic reader protein bromodomain-containing protein 4 (BRD4), an emerging therapeutic target in cancer,functions as a critical coactivator of pathologic gene transactivation during cardiomyocyte hypertrophy. However,the therapeutic relevance of these findings to human disease remained unknown. We demonstrate that treatmentwith the BET bromodomain inhibitor JQ1 has therapeutic effects during severe, preestablished HF from prolongedpressure overload, aswell as after amassive anteriormyocardial infarction inmice. Furthermore, JQ1potently blocksagonist-induced hypertrophy in human induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs).Integrated transcriptomic analyses across animalmodels andhuman iPSC-CMs reveal that BET inhibition preferentiallyblocks transactivation of a common pathologic gene regulatory program that is robustly enriched for NFkB andTGF-b signaling networks, typified by innate inflammatory and profibrotic myocardial genes. As predicted bythese specific transcriptional mechanisms, we found that JQ1 does not suppress physiological cardiac hypertrophyin a mouse swimming model. These findings establish that pharmacologically targeting innate inflammatoryand profibrotic myocardial signaling networks at the level of chromatin is effective in animal models and humancardiomyocytes, providing the critical rationale for further development of BET inhibitors and other epigenomicmedicines for HF.

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INTRODUCTIONHeart failure (HF) is a leading cause of mortality, hospitalization, andhealth care expenditure in theUnitedStates (1,2). Existingpharmacothera-pies for systolic HF are b-adrenergic receptor and renin-angiotensinaxis antagonists, which dampen the excessive activity of stress neuro-hormones produced in response tomyocardial injury. However, despitethe widespread use of these disease-modifying drugs, the average 5-yearmortality rate after an initial diagnosis of HF is about 40%, which high-lights the urgent need for new therapeutic approaches (1, 2).

In response to persistent hemodynamic and neurohormonal stress,the myocardium undergoes pathological cell state changes character-ized by cardiomyocyte hypertrophy, inflammation, myofibroblast acti-vation, and contractile dysfunction (3–6). Although cardiac remodelingmay provide short-term adaptation in certain settings, sustained orexcessive activation of this process is maladaptive and drives diseaseprogression (3, 4). Studies over the past two decades have established

that inhibition of specific signaling pathways that govern stress-inducedcardiac remodeling has cardioprotective effects even in the face of per-sistent stress (4, 7). Notably, ventricular tissue remodeling is a robustpredictor of HF severity and death in patients, underscoring the de-trimental nature of this process (3, 8–12). Together, these data supportthe contention that targeting the tissue remodeling process itself canbe beneficial without compromising cardiac performance (3, 9, 11–13).

The cell state transitions that underlie pathologic cardiac remodelingare rooted in dynamic changes in gene control (14, 15) and feature ageneral state of transcriptional anabolism (3, 15). In the stressed heart,multiple upstream signaling pathways converge on the transcriptionmachinery, which integrates these signals by transactivating geneprograms that alter cell state (16). Thus, blocking stress-activated signal-ing cascades at the level of chromatin-dependent signaling representsan attractive therapeutic strategy in HF. In response to cardiac stress,a defined set of transcription factors coordinately binds to specificregulatory regions of the genome. These transcription factors recruitlysine acetyltransferases, which hyperacetylate local chromatin andactivate cis-regulatory elements (or enhancers) (16, 17). Subsequenttransactivation of distal target genes occurs via recruitment of epigenetic“reader” proteins (16), such as bromodomain-containing protein 4(BRD4) (14, 18), that bind acetyllysine via bromodomains and coacti-vate transcription by assembling complexes that signal to RNA poly-merase II (Pol II) (19–22). BRD4 belongs to the bromodomain andextraterminal (BET) family of highly conserved acetyllysine recognitionproteins andhas been implicated in themaintenance and progression ofcancer cell state across a broad range of malignancies (18, 23–25). Therecent development of potent, specific, and reversible BET bromodomaininhibitors, such as the first-in-class thienodiazepine small-molecule JQ1(23), has accelerated mechanistic discovery and translation in this field.

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JQ1 binds the bromodomains of BET proteins with exquisite shapecomplementarity and nanomolar affinity, resulting in potent, compe-titive, and transient displacement of BRD4 from acetylated chromatin(23). Studies across a broad range of cell types demonstrate that JQ1preferentially blocks transactivation of specific sets of genes in a dose-dependent and context-specific manner (24). Target gene specificityis achieved, in part, because the BRD4 coactivator protein is asym-metrically enriched at massive cell state–specific enhancers (or super-enhancers) (26, 27). Transcription of super-enhancer–regulatedgenes is disproportionately sensitive to depletion of coactivator pro-teins such as BRD4 (26, 28). Given the tractable therapeutic windowof JQ1 in preclinical studies, there has been intense interest in thedevelopment of BET bromodomain inhibitors as anticancer drugs.These translational efforts have spawned several ongoing early-phasehuman cancer trials using BET bromodomain inhibitors, includingderivatives of JQ1 (29, 30).

Initial studies from our group demonstrated that BRD4 functionsas a critical coactivator of pathologic gene transactivation during car-diomyocyte hypertrophy by a mechanism that involves recruitmentof positive transcription elongation factor b (P-TEFb) activity andpause release of Pol II (31, 32). In cultured neonatal rat ventricularmyocytes (NRVMs), small-interfering RNA (siRNA)–mediated silen-cing of BRD4 or chemical inhibition with JQ1 blocks cardinal featuresof pathologic hypertrophy. In addition, early administration of JQ1 atthe very onset of pressure overload in mice prevented the developmentof cardiac hypertrophy and left ventricular (LV) systolic dysfunction.Although these index studies provided important mechanistic insight,the therapeutic relevance of these early observations remained un-known. In particular, it was not known whether BET bromodomaininhibitors could be used to treat established HF in more clinicallyrelevant experimental settings. Furthermore, it was unknown whetherBETs could be therapeutically targeted in human cardiomyocytes andwhether there were common transcriptional effects of BET bromodo-main inhibitor therapy across pathologic settings and species. Here, weaddress these major knowledge gaps and identify core mechanisms oftherapeutic efficacy that are vital for directing further translation of BETinhibition as a therapeutic strategy for heart disease.

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RESULTSBET bromodomain inhibition is effective in the treatment ofpreestablished HFPatients requiring treatment for HF typically present with preestab-lished disease. Therefore, we first tested whether BET bromodomaininhibition with JQ1 could treat preestablished HF in a mouse modelof LV pressure overload. We subjected adult mice to transverse aorticconstriction (TAC) or sham surgery. On postoperative day 18, a timepoint when robust cardiomegaly and LV dysfunction have alreadydeveloped in this model (31), we randomized mice to receive JQ1[50 mg/kg daily, intraperitoneally (IP)] versus vehicle and continuedout to postoperative week 8 (protocol in Fig. 1A). This dosing schemeof JQ1 has established efficacy and tolerability in previous studies ofcancer and cardiovascular disease in mice and does not cause overttoxicity (23, 24, 31–33). In addition, we have previously demonstratedthat JQ1 administration at these doses does not affect blood pressureor the pressure gradient across the constricted aorta (31). In thistreatment protocol, we found that JQ1 attenuated multiple hallmarkfeatures of HF progression in vivo, including cardiomegaly (Fig. 1B),pulmonary edema (Fig. 1C), LV systolic dysfunction, LV cavity dilation,


LV wall thickening [Fig. 1, D to F, respectively; echocardiographic datafor the final time point (day 53) are shown in fig. S1, A to C], cardio-myocyte hypertrophy (Fig. 1G), and LV fibrosis (Fig. 1H). In addition,JQ1 suppressed the induction of several canonical HF-associated genes(Fig. 1I). Thus, late administration of JQ1 can treat HF even whenstarted at a time when there is robust, preexisting disease.

BET bromodomain inhibition is effective in the treatment ofHF after a large myocardial infarctionAlthough the TACmodel is a widely usedmethod for eliciting hallmarkfeatures of HF pathogenesis, the acute onset of severe hemodynamicoverload in this model does not represent a common clinical scenarioin humans. In contrast, myocardial infarction (MI) is a very commoncause of pathologic cardiac remodeling andHF in humans (1). Further-more, animalmodels of post-MIHF have served as amajor gateway fordrug development, as exemplified by studies of angiotensin-convertingenzyme inhibitors in rodent preclinical studies (13) and subsequenthuman clinical trials (11, 12).

We therefore asked whether JQ1 could be used to treat HF thatdevelops after large, transmural MI in adult mice. We subjectedmiceto permanent surgical ligation of the proximal left anterior descending(LAD) coronary artery to produce a large, dense, anterior wallMI. Asexpected for this anatomically proximal level of LAD occlusion (34),~35% of mice died in the first 5 postoperative days due to LV ruptureor overt HF, confirming the severity of this model in our hands. Onpostoperative day 6, a time point when all infarctions were complete,surviving mice were randomized to receive JQ1 versus vehicle. Weinitially dosed at 25mg/kg daily for 6 days (during the period of earlysurgical recovery) and subsequently ramped up to 50 mg/kg dailyuntil postoperative day 28 (a time point when robust pathologic remo-deling of the remote LVmyocardium is present in thismodel) (protocolin Fig. 2A) (35). Similar to our findings in the pressure overload treat-ment model (Fig. 1), JQ1 attenuated several cardinal features of HF inthe post-MI setting, including cardiomegaly (Fig. 2B), pulmonary con-gestion (Fig. 2C), LV systolic dysfunction (Fig. 2D), LV cavity dilation(Fig. 2E), wall thickening of the remote LV (posterior wall; Fig. 2F),hypertrophy of cardiomyocytes in the remote LV (Fig. 2G), and fibrosisof the remote LV (Fig. 2H). JQ1 also suppressed pathological inductionof canonical HF-associated genes in the remote LV myocardium (Fig.2I). It is possible that therapeutic strategies that attenuate LV remodel-ingmay also prevent normal healing of a transmural infarct, particularlyinmousemodels, which are predisposed to post-MI rupture (34). How-ever, we did not observe any significant excess in mortality or LVrupture between JQ1 and vehicle treatment groups (two mice died/ruptured in each group from n = 12 per group after treatment initiation;c2 = 1), suggesting that this regimen of BET bromodomain inhibitionameliorates HF progression without overt compromise of infarct healing.

BET bromodomain inhibition suppresses transactivation ofshared gene regulatory networks across HF modelsGiven the therapeutic efficacy of JQ1 in two distinct HF models in vivo(Figs. 1 and 2) and our previous observation that BRD4 regulates tran-scriptional pause release in the heart (31), we hypothesized that there wasa common gene-control mechanism mediating these salutary effectsacross disease settings. We performed transcriptomic profiling fromLV tissue using RNA sequencing (RNA-seq) at baseline (sham) and duringdisease (TACandMI) frommice treatedwith vehicle versus JQ1.We firstanalyzed the transcriptomic effect of chronic JQ1 exposure in the baselinestate by comparing the RNA-seq profiles of the sham-vehicle– versus

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Fig. 1. BET bromodomain inhibition treats preestablished HF after prolonged pressure overload. (A) Experimental protocol for preestablished pressure overload modelinduced by TAC inmice. Veh, vehicle. (B) Ratio of heart weight/tibia length (HW/TL) (n = 10) and (C) lung weight/tibia length (LW/TL) (n = 10). (D) LV ejection fraction, (E) LV
diastolic area, and (F) LV wall thickness quantified by echocardiography (n = 10). (IVS + PW)d is the total thickness of the interventricular septum and posterior LVwall at theend-diastole. (G) Representative LV cross sections stained withwheat germ agglutinin (WGA)with quantification ofmean cardiomyocyte (CM) cell area shown below (n = 3 to 4).Scale bars, 20 mm. (H) Representative LV cross sections stained with picrosirius red. Scale bars, 400 mm (top) and 100 mm (bottom). Quantification of fibrotic area below (n =5). (I) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) for indicated genes inmouse heart tissue (n = 5). For (A) to (I), *P < 0.05, **P< 0.01, ***P < 0.001for indicated comparison. Data are shown as means ± SEM.
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Fig. 2. BET bromodomain inhibition ameliorates HF after a massive anterior wall myocardial infarction. (A) Experimental protocol for MI model induced by surgicalligation of the proximal LAD coronary artery. (B and C) Gravimetric measurements on day 28. Ratios of heart weight/tibia length and lung weight/tibia length were measured(n=10). (D) LV ejection fraction, (E) LV diastolic area, and (F) LVposteriorwall thickness quantified by echocardiographyonday 28 [n=10 (MI) and6 (sham)]. PWd is the thickness ofthe posterior LV wall (remote LV) at the end-diastole. The mid-LV interventricular septum was transmurally infarcted in this model and was therefore not relevant to the wallthickness assessment. (G) Representative cross sections from remote LV stained with WGA with quantification of mean cardiomyocyte (CM) cell area shown below (n = 3 to 4).Scale bars, 20 mm. (H) Representative cross sections from remote LV stainedwith picrosirius red. Scale bars, 400 mm(top) and 100 mm(bottom). Quantification of fibrotic area below(n = 5). (I) qRT-PCR for indicated genes in remote LV tissue (n = 5). For (A) to (I), *P < 0.05, **P < 0.01, ***P < 0.001 for indicated comparison. Data are shown as means ± SEM.




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sham-JQ1–treated LV tissue. Differential expression analysis showedthat JQ1 reduced expression of 613 genes and increased expression of125 genes in the left ventricles of sham-treated mice (volcano plot infig. S2A; complete expression data for all detected transcripts and dif-ferential expression calls are provided in table S1).Gene ontology analysisof the JQ1–down-regulated transcripts showed a strong enrichment forinnate immune activation, extracellular matrix production, and celladhesion (fig. S2B). Themuch smaller set of JQ1–up-regulated genesrevealed nomajor enrichment for functional terms, other than three genesthat were cataloged as containing Heat-shock protein 70 binding sites.

Because JQ1 did not alter baseline cardiac structure or function inthe sham group (Figs. 1 and 2 and fig. S1), we next turned our attentionto the global set of transcripts that were differentially expressed duringdisease. Global clustering of transcriptomic profiles by principal com-ponents analysis revealed that the two most significant sources of dif-ferential expression across all samples were the response to stress(such as TAC or MI) and the effect of JQ1 administration (Fig. 3A).When comparing sham versus stress, the dominant transcriptomicfeature was global gene up-regulation (TAC, 725 genes up and 401genes down; MI, 928 genes up and 205 genes down). These globaltranscriptomic profiles are consistent withHF as a state of robust tran-scriptional anabolism (15). For the global set of genes that were differ-entially expressed during stress, hierarchical clustering of samples (Fig.3B; shown above the heat maps) demonstrated that the JQ1-treatedgroup clustered more closely with the sham groups, reflecting broadsuppression of a pathologic state. The dominant effect of JQ1 acrossdisease models was to dampen the transactivation of a broad subsetof stress-inducible genes (Fig. 3B, top third of heat maps). This ob-servation is mechanistically consistent with the function of BRD4 asa key coactivator of enhancer-dependent transcription and effectorof Pol II pause release (26–28, 31). The inhibitory effects of JQ1 werespecific, as exemplified by a subset of stress-inducible genes that wereunaffected by JQ1 (Fig. 3B, middle third of heat maps). Finally, JQ1had no broad effect in rescuing genes that were down-regulated withstress (Fig. 3B, bottom third of heat maps), a finding that is also con-sistent with the primary function of BRD4 as a transcriptional coacti-vator. The volcano plots in Fig. 3C illustrate the potent effects of JQ1in the dampening of stress-mediated gene induction across the TACand MI models (differential expression data from RNA-seq are pro-vided in tables S2 and S3). Together, these data demonstrate that theseHFmodels elicit a state of robust transcriptional anabolism and that thedominant role of JQ1 during HF therapy is to dampen the stress-dependent transactivation of a broad but specific set of genes.

To gain further insight into the therapeutic mechanisms underlyingthe efficacy of JQ1 in HF, we defined a common set of 193 genes whosetransactivation was suppressed by JQ1 in both the TAC andMImodels(see Venn diagram; Fig. 4A) (gene list is provided in table S4). Geneontology analysis of this common set of 193 genes revealed a strongenrichment for specific pathophysiologic processes excessively acti-vated in humanHF (6, 36), including extracellularmatrix deposition,innate immune and inflammatory responses, and cellular growth(Fig. 4B). We next analyzed this gene set using Ingenuity PathwayAnalysis (37, 38) to identify pathologic signaling networks that werespecifically blocked by BET bromodomain inhibitor therapy. Theseunbiased functional analyses revealed striking enrichment for twomyocardial signaling networks that were robustly suppressed by JQ1.First, JQ1 was found to block transactivation of genes that form a mas-sive hub centered on transforming growth factor–b (TGF-b) signaling(P < 1.4 × 10−76; Fig. 4C), a central regulator of pathologic cell state


transformations in the stressed myocardium (39, 40). Genes affectedin this expansive TGF-b network broadly encompassed all major com-partments of the cell. The second observation was that the JQ1 inhibi-tory effect was robustly enriched for genes involved in innate immuneresponses (signified by the “lipopolysaccharide” term at the top level ofthe network), which converged upon NFkB/RelA, AP1, and STAT1 asterminal transcriptional effectors (P < 1 × 10−48; Fig. 4D). The robustenrichment for TGF-b and NFkB targets in this set of JQ1-attenuatedtranscripts was independently corroborated by gene set enrichmentanalysis (GSEA) (fig. S3A). The GSEA demonstrates that genes up-regulated in our TAC and MI models are statistically enriched forTGF-b and NFkB targets, confirming that these signaling pathwaysare activated during disease. Consistent with our Ingenuity PathwayAnalysis, GSEA demonstrated that the set of genes attenuated by JQ1in these HF models was statistically enriched for TGF-b and NFkBtargets. To gain initial insight into myocardial cell types that mightbe contributing to the transcriptional response to JQ1 during HFpathogenesis in vivo, we used fingerprinting analysis (fig. S3B) to com-pare ourRNA-seq profileswith curatedRNA-seq data sets derived frommouse cardiomyocytes (GSE58453 andGSE68509) (41, 42), mouse car-diac fibroblasts (GSE58453) (41), and myeloid cells (GSE57125) (43).These analyses detected a signature for transcriptional activation offibroblasts in TAC/MI when compared to the sham group. There wasa detectable effect of JQ1 in attenuating the transcriptional activationof fibroblast signature. In summary, across in vivo models of HF, BETbromodomain inhibitionwith JQ1 blocks the transactivation of a sharedset ofmyocardial genes that encompass the coordinated output of TGF-bsignaling and proinflammatory transcription factors.

BET bromodomain inhibition does not affect physiologicalcardiac growth during exerciseChronic endurance exercise training leads to a physiological form ofcardiac growth that has been shown to provide protection againstmyocardial injury (44, 45). An important distinguishing feature ofphysiological versus pathological cardiac remodeling is that the formerlacks robust induction of innate inflammatory or profibrotic gene pro-grams (45–47). Because our data in murine models showed that thetranscriptional effects of JQ1 were specifically enriched for the suppres-sion of proinflammatory and profibrotic gene networks (Figs. 3 and 4),we hypothesized that BET bromodomain inhibition with JQ1 might bepermissive for physiological cardiac remodeling during enduranceexercise training. To test this hypothesis, adult mice underwent a well-established, high-intensity endurance swimming protocol inwhichmiceare progressively trained to swim for a total of 3 hours/day for 4 weeks(44). Swimming versus sedentary mice were randomized to receive JQ1(50mg/kg per day, IP) or vehicle injections (protocol in Fig. 5A). In boththe vehicle- and JQ1-treated groups, there were similar increases incardiac mass (Fig. 5B), cardiomyocyte cross-sectional area (Fig. 5C),LV ejection fraction (Fig. 5D), and expression of transcripts inducedwith exercise training in this model (such as Gata4 and Tnni3) (Fig.5E and fig. S4), indicating that JQ1 treatment did not block key featuresof exercise-induced cardiac plasticity. Hence, JQ1 administration toadult mice at the doses used here can treat HF but does not suppressexercise-induced physiological cardiac growth.

BET bromodomain inhibition blocks agonist-inducedhypertrophy in human iPSC-CMsAll previous studies of BET bromodomain inhibition in heart diseasehave been performed in rodent models (31, 32). We therefore asked

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Fig. 3. BETbromodomain inhibition suppresses transactivationofa specific stress-induciblegeneprogramduringHFpathogenesis. (A) Global displayof information contentderived fromprincipal components analysis of RNA-seqdata. Each row in theprincipal components analysis plot is aneigenvector that describes a feature across all samples. Thepurpleto green shading scale reflects the magnitude of the principal components analysis eigenvector for the component of interest. Summary bar plots on the right show that the largestsourceof variance in thedata canbeexplainedby the response to stress (TACandMI)with thenext largest sourceof variation ascribed to JQ1-associated changes in geneexpression. Asmaller source of variation canbe ascribed to differences in gene expression between the TAC andMImodels. (B) Heatmapof genes differentially expressedwith stress in the TAC (left)andMImodel (right). Scale is log2. Statistical criteria fromDESeqanalysiswere≥2-fold changeanda falsediscovery rateof<0.05. The topone-thirdof eachheatmap is the cluster of genesthat are up-regulatedwith stress and dampenedby JQ1. Themiddle one-third of each heatmap is the cluster of genes up-regulatedwith stress and unaffected by JQ1, demonstratingspecificity. The bottom one-third of each heat map shows that genes that are down-regulated with stress are not significantly reversed by JQ1. (C) Volcano plot demonstratingmagnitude and significance of JQ1 on suppressing induction of stress-inducible genes in both the TAC and MI models. Genes up-regulated by TAC (left) or MI (right) versus shamare plotted in red. Plotted in blue is the effect of JQ1 (stress-JQ1 versus stress-vehicle) for each gene shown in red. Representative genes are calledout (black lines) for each volcanoplot.




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whether BET bromodomain inhibition could block pathologic cardio-myocyte hypertrophy in human cells. We tested the effects of JQ1 ina widely used human induced pluripotent stem cell–derived cardio-myocyte (iPSC-CM) culture system that originated from an iPSC


line derived from a healthy human donor [iCell Cardiomyocytes,Cellular Dynamics International Inc. (CDI)] (48). Previous studieshave demonstrated that these cells can mount a hypertrophic responseto 10 nM endothelin-1 (ET-1) (48). We found that JQ1 attenuated

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Category Term % List FDR

GOTERM_CC_FAT GO:0031012~extracellular matrix 18 1.4 × 10–15

GOTERM_BP_FAT GO:0007155~cell adhesion 21 8.1 × 10–6

GOTERM_BP_FAT GO:0006954~inflammatory response 9 0.0014GOTERM_BP_FAT GO:0006952~defense response 16 0.0006

GOTERM_BP_FAT GO:0045087~innate immune response 9 0.003GOTERM_MF_FAT GO:0001558~regulation of cell growth 8 0.01

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Fig. 4. BET bromodomain inhibition suppresses transactivation of shared transcriptional networks across HFmodels. (A) Venn diagram showing significant overlap ofgene sets that are stress-induced and JQ1-suppressed across the TAC and MI models (c2 < 0.001). A common set of 193 genes was identified as being shared targets of BETbromodomain inhibition (gene list is provided in table S4). (B) Gene ontology analysis using DAVID for the Venn overlap genes shown in (A). A false discovery rate (FDR) of<0.05 was considered statistically significant. (C and D) Ingenuity Pathway Analysis. The common gene set was analyzed by Ingenuity Pathway Analysis. These analysesrevealed two dominant signaling networks that were highly enriched in the set of genes suppressed by JQ1. (C) A large TGF-b network (P < 1.0 × 10−76). Each target geneis shown in red, with the darkness of red shading designating the relative degree of inhibition by JQ1 of that particular gene. Although the legend includes a green color scalefor JQ1–up-regulated genes, there are no green-shaded genes on this diagram because all genes were selected a priori to be those that are suppressed by JQ1. TGFB1 as acentral node gene is shown in orange. (D) JQ1 suppressed genes enriched for a network of innate immune signaling nodes with strong convergence on NFkB transcriptionalresponses (P < 1.0 × 10−48). Keys for the Ingenuity Pathway Analysis color shading and line formatting schemes are provided in the figure.

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ET-1–mediated hypertrophic growth in human iPSC-CMs in a dose-dependent manner (Fig. 6, A and B). Consistent with the effects oncellular hypertrophy, qRT-PCR demonstrated that JQ1 suppressedtransactivation of NPPB/BNP (Fig. 6C) and other typical markergenes induced in human iPSC-CMs during ET-1 stimulation (fig. S5A)(48). Quantitative enzyme-linked immunosorbent assay (ELISA) con-firmed that JQ1 attenuated the ET-1–stimulated increase in BNP pro-tein abundance (Fig. 6D).

To gain a broader insight into stress-activated gene expressionprograms affected by JQ1 in human cardiomyocytes, we performedRNA-seq in this experimental system. Similar to the murine RNA-seqprofiles (Fig. 3), a heat map of all ET-1–responsive genes in humaniPSC-CMs illustrates that the dominant effect of JQ1 was to dampeninduction of a subset of ET-1–inducible genes (265 gene subset froma total of 615 ET-1–induced transcripts; Fig. 6E) (differential expressiondata provided as table S5). Functional annotation of this 265 genesubset revealed strong enrichment for genes involved in inflammatoryresponses, cellular growth, and extracellularmatrix production (Fig. 6F),terms that paralleled the cardiac signatures frommurine disease models(Fig. 4). Similar to ourmurine data, Ingenuity PathwayAnalysis revealedthat this set of JQ1-responsive human genes robustly enriched for a


TGF-b signaling hub (P < 1 × 10−25; Fig. 6G) and an innate immunetranscription factor network (signified by the lipopolysaccharide termatthe top level of the network) with a strong convergence on NFkB/RelA(P < 4 × 10−12; Fig. 6H). In addition, the robust enrichment for TGF-band NFkB targets in this set of JQ1-attenuated transcripts in humaniPSC-CM was independently corroborated by GSEA (fig. S5B).Together, these data demonstrate that BET bromodomain inhibitionwith JQ1 blocks agonist-induced hypertrophic responses in humaniPSC-CMs in a cell-autonomous manner, with transcriptional effectsthat parallel our observations in murine models.

DISCUSSIONOurdata provide proof of principle thatBETbromodomain inhibition, anemerging therapeutic strategy in a variety of human cancers (18, 23–25),can treat preestablished HF in vivo. Our observation that nanomolarconcentrations of JQ1 block pathological hypertrophy in human iPSC-CMs suggests that BET inhibitionmay also exert protective effects in thehuman heart. Integrated genome-wide analyses acrossmurinemodelsand human cardiomyocytes reveal that BET inhibition suppresses acommon pathologic transcriptional program that is highly enriched

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Fig. 5. BETbromodomain inhibitiondoesnot suppressphysiologic cardiachypertrophy in response to chronicendurance exercise training. (A) Schematic of swimmingprotocol. (B) Gravimetry; heart weight/body weight ratio on day 35 (n = 7). (C) Representative LV cross sections stained with WGA with quantification of mean cardio-myocyte cell area shownat the right (n=5). Scale bars, 20mm. (D) Echocardiographic LV ejection fractiononday 35 (n=7). (E) qRT-PCR for indicatedgenes inmouse LV tissue (n=5).For (B) to (E), *P < 0.05, **P < 0.01, ***P < 0.001 for indicated comparisons. No statistically significant JQ1 effect was observed. Data are shown as means ± SEM.

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Category Term % List FDR

GOTERM_BP_FAT GO:0030595~leukocyte chemotaxis 6 1.3 × 10–9GOTERM_BP_FAT GO:0006952~defense response 13 0.001

GOTERM_CC_FAT GO:0030198~extracellular matrix organization 5 0.02GOTERM_BP_FAT GO:0001817~reg of cytokine production 7 0.007

GOTERM_BP_FAT GO:0006954~inflammatory response 9 1.1 × 10–5

GOTERM_MF_FAT GO:0008083~growth factor activity 4 4.0 × 10–5

GOTERM_BP_FAT GO:009611~response to wounding 10 8.8 × 10–5

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TGF-β networkInnate immune network

Fig. 6. BET bromodomain inhibition blocks agonist-induced hypertrophy and stress-mediated gene transactivation in human iPSC-CMs. (A) Human iPSC-CMs treated withET-1 (10nM), JQ1, or vehicle for 18hours and stained fora-actinin immunofluorescence for quantificationof cell area (n=12). Bars designatemeans±SEM. (B) Representative imagesofhuman iPSC-CMstained fora-actinin immunofluorescence. ET-1 (10nM)and JQ1 (1000nM); 18hours. Scalebars, 100mm. (C) qRT-PCR forNPPB/BNP in iPSC-CMs (n=12).Dataare shownasmeans ± SEM. (D) ELISA for proBNP protein concentration inmedium of iPSC-CM (n = 11). ET-1 (10 nM) and JQ1 (1000 nM); 18 hours. Bars designatemeans ± SEM. For (A), (C), and(D), ****P<0.0001, ***P<0.0005. (E) Heatmapof genes differentially expressed in iPSC-CMwith ET-1 stimulation. Statistical criteria fromDESeq analysiswere≥2-fold change and a falsediscovery rate of <0.05. The top one-third of each heatmap is the cluster of genes that are up-regulatedwith ET-1 anddampenedby JQ1. Themiddle one-third of each heatmap is thecluster of genesup-regulatedwithET-1andunaffectedby JQ1,demonstrating specificity. Thebottomone-thirdof eachheatmapshows thatgenes that aredown-regulatedwithET-1 arenot significantly reversed by JQ1. Scale is log2. (F) Gene ontology analysis using DAVID for the set of genes that were ET-1–inducible and suppressed by JQ1 (top third of heat map). Afalse discovery rate of <0.05was considered statistically significant. (G andH) Ingenuity Pathway Analysis. The set of ET-1–induced genes that were attenuated by JQ1was analyzed byIngenuity Pathway Analysis. These analyses revealed two dominant signaling networks that were highly enriched in the set of genes suppressed by JQ1 and paralleled the findings inmouseheart tissue fromFig. 4. (G)A largeTGF-b network (P<1.0×10−25). Each targetgene is shown in red,with thedarknessof red shadingdesignating the relativedegreeof inhibitionby JQ1 of that particular gene. Although the legend includes a green color scale for JQ1–up-regulated genes, there are no green-shaded genes on this diagrambecause all geneswereselected apriori tobe those that are suppressedby JQ1. TGFB1 as a central nodegene is shown inorange. (H) JQ1 suppressedgenes enriched for anetwork of innate immune signalingnodes with strong convergence on NFkB transcriptional responses (P < 1.0 × 10−48). Keys for the Ingenuity Pathway Analysis color shading and line formatting schemes are provided.




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for inflammatory and profibrotic signaling effectors in themyocardium.Because inflammation and fibrosis are central to HF pathogenesis,molecular mediators of these processes are intently pursued therapeutictargets (6, 49). Our currentwork demonstrates the efficacy of an epigen-omically based therapeutic approach that targets inflammatory andprofibrotic cell state transitions at the level of chromatin.

Several BET bromodomain inhibitor drugs are currently beingdeveloped in early-phase human cancer trials (29). Our work providesimpetus for next-phase testing of this class of therapeutics in large-animal models of HF, including porcine/ovine MI models. Here, wefocused on the ability of JQ1 to ameliorate severeHF that occurs aftera complete and dense anterior wallMI. Our findings, which highlightthe beneficial effects of JQ1 on the remote/noninfarcted myocardium,are relevant for the treatment of ischemic cardiomyopathy in humans.It will also be interesting to explore whether BET bromodomain in-hibition can protect at-risk myocardium in animal models of ischemia-reperfusion injurywhendelivered at the timeof coronary revascularization,although the mechanisms of acute ischemic myoprotection in thissetting represent vastly different biological processes than the longer-term beneficial effects on the remote LV shown here. One study hasshown that pretreatment with JQ1 in a rat model of permanent coro-nary artery ligation can reduce biomarkers of infarct severity at 24 hours(50), suggesting that BET bromodomain inhibitionmay acutely protectischemicmyocytes during an unrevascularizedMI.However, it remainsunknown whether this strategy provides longer-term benefit or is effec-tive when administered in the perireperfusion phase. In addition tostudying acquired forms of HF, it will also be of interest to study therole of BET proteins in the pathogenesis of cardiomyopathies drivenby genetic mutations (51), some of which feature extensive activationof profibrotic and proinflammatory pathways in the heart. These linesof investigation, guided by the results of ongoing human cancer trials,will provide the critical foundation for developing BET bromodomaininhibition as a potential therapeutic strategy in human heart disease.

Our RNA-seq data demonstrate that BET bromodomain inhibitorssuppress a subset of stress-activatedmyocardial genes. Themechanismsunderlying the selective effects of BET bromodomain inhibition ontarget genes, a phenomenon which has been observed across multiplecell types (24, 26, 33, 52), are just beginning to be understood. Animportant contributor to this selectivity is the observation that potentcoactivators such as BRD4are asymmetrically loaded on a critical subsetof cell state–defining enhancers that have been termed super-enhancers,which often regulate target genes via Pol II pause release (26–28, 33).Super-enhancer–regulated genes are exquisitely sensitive to local deple-tion of coactivator proteins, including BRD4, across a number of celltypes (26–28, 33, 53). Recent in vitro studies using cultured endothelialcells (33) andNRVMs (54) have demonstrated that during pathologicalstress, there is a stimulus-coupled flux of BRD4 frombaseline regulatoryloci to new, stress-activated super-enhancers. In the setting of stress,JQ1 transiently displaces BRD4 from chromatin, prevents the cell’s abil-ity to sustain maximal activity of newly formed super-enhancers, andpreferentially dampens transcription of stress-activated gene programs(33). The precise molecular mechanisms governing locus-specific fluxof BRD4 to these newly formed super-enhancers is not well understoodbut may involve recruitment via protein interactions between BRD4and DNA binding transcription factors (55). Our finding that themyocardial signature of JQ1-suppressed genes is specifically enrichedfor transcription pathways central to inflammatory and fibrotic responsessupports amodel in which specific subsets of DNAbinding transcriptionfactorsmay play a key role in driving genome-wide flux of BRD4during


stress. This feature of BRD4 signalingmay partially explainwhy JQ1 didnot affect exercise-induced cardiac growth, a form of tissue plasticitythat does not typically involve inflammatory or profibrotic transcriptionprograms (45–47).

The preferential inhibitory effect of BET bromodomain inhibitorson stress-dependent enhancer assembly may, in part, contribute totheir therapeutic window (55). The pulsatile exposure to JQ1 used here,which has a relatively short half-life in mice (23), can dampen the as-sembly andnet transcriptional output of stress-activated super-enhancersin the injured myocardium. This reversible, phasic, and dose-titratabledisruption of super-enhancer–dependent transcriptionmay be homeo-statically accommodated in nondiseased tissues in a manner thatallows the adult mammalian organism to guard key aspects of basalorgan physiology. Consistent with this mode of action, JQ1 does notcause overt toxicity in adult mice under the dosing protocols used inthis study (23, 24, 31, 33, 56). Furthermore, JQ1 does not cause majorchanges in cardiac structure or function inmice subject to sham surgery(31). The general tolerability of JQ1 at the doses used here is furthersupported by our observation that mice receiving daily JQ1 injectionsare able to complete a high-intensity, 4-week swimming protocol. How-ever, higher doses of BET inhibitors will bring broader, sustained ex-posure that may produce a wider range of on-target toxicity (57, 58). Amouse model in which total BRD4 protein abundance is ubiquitouslysuppressed by an inducible short hairpin RNA (shRNA) demonstratesthat excessive levels of total BRD4 depletion result in abnormalities inthe intestinal epithelium, bone marrow, and hair follicles—all of whichare reversible after the shRNA expression is turned off (56). Theseobservations indicate that the major toxicity profiles of BET inhibitorsare likely to be on-target, dose-dependent, and reversible (56). Althoughour studies demonstrate efficacy and tolerability of JQ1 in mousemodels, an important hurdle in next-phase preclinical efforts will beto develop compounds and dosing/delivery schemes that have an ap-propriate therapeutic index for large-animal models of HF. In addition,the efficacy of BET inhibition when added on to current standard-of-care HF regimens will need to be established. It will also be of interestto test whether adjunctive BET inhibition can protect at-risk ischemicmyocardium when administered at the time of coronary reperfusion,although the mechanisms of acute myoprotection in such a settingwould likely be different from the chronically beneficial effects onremote myocardium described here. Because several potent BET in-hibitor drugs are progressing in early-phase human cancer trials (29),the collective first-in-human experience with this class of therapeuticswill serve as an important guide for considering cardiac and other non-cancer applications.

An important area of mechanistic inquiry will be to further dissectwhich BET family members (BRD2/3/4) and which cell types (forexample, cardiac myocytes, cardiac fibroblasts) are mediating thetherapeutic effects of small-molecule BET bromodomain inhibitionin vivo. In a previous study, specific siRNA-mediated silencing of BRD4in cultured NRVMs was shown to phenocopy the antihypertrophiceffect of JQ1 (31). BRD4 is also the BET family member that allosteri-cally activates the P-TEFb complex via a C-terminal CDK9-interactingdomain that is not present in BRD2 or BRD3 (19, 20, 59), which isconsistent with our observation that JQ1 suppresses Pol II pause releasein the stressed heart (31). Together, these findings strongly implicatecardiac BRD4 as a major target of JQ1 in this context. With regardto which particular cell types in the myocardium are being affected byBET bromodomain inhibition, our RNA-seq studies reveal that JQ1blocks the induction of a broad program of proinflammatory and

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profibrotic genes. These transcriptomic signatures encompass genesthat are expressed in cardiomyocytes, cardiac fibroblasts, or both. Ourdata in NRVMs (31) and cultured human iPSC-CMs demonstratethat BET inhibition has cell-autonomous effects on inflammatoryand profibrotic gene programs in cardiomyocytes in vitro, suggestingthat the cardiomyocyte is a major cell target of BET inhibitors in vivo.Recent studies also demonstrated an important role for BRD4 in hepaticstellate cells (53), suggesting that JQ1 may also block the pathologicactivation of resident tissue fibroblasts. Our cell type fingerprintinganalysis of mouse left ventricles detected an effect of JQ1 in attenuatingthe transcriptional activation of fibroblasts. Although this observationis consistent with the ability of JQ1 to attenuate profibrotic and innateinflammatory signaling in the heart, it does not exclude importantcontributions from cardiomyocytes, immune cells, and other cells thatpopulate the stressed myocardium in mediating the therapeutic re-sponse to JQ1. More precise annotation of gene- and tissue-specificeffects of BET family members will require the development of condi-tional genetic models for Brd2/3/4, which will be important resourcesfor this field. Such conditional alleles, when coupled with recentlydeveloped Cre drivers (5), will be particularly useful for interrogatingthe role of BRD4 in activated cardiac myofibroblasts, a cell type thatis challenging to manipulate in vivo using viral vectors. It remains im-portant to emphasize that Cre-lox–based conditional gene deletion ispermanent and fundamentally very different from the chemical biologicalapproach leveraged here. Chemical probes like JQ1 transiently and re-versibly disrupt the interaction of BETproteinswith acetylated chroma-tin in a manner that is temporally precise and dose-titratable (23, 55).These features cannot be recapitulated by current genome-targeting orgene-silencing approaches. Therefore, the ability to dissect gene-specificand cell-autonomous responses in a manner that accurately reflects thebiology of these chemical probes would require future development ofboth gene-specific compounds for individual BET family members andmethods for cell type–specific delivery in vivo. The recent developmentof small molecules that promote reversible degradation of BET proteinsmay create future opportunities to potentlymodulate BET function in amore gene- and tissue-restricted manner in vivo, potentially providingan expanded therapeutic window for this pharmacologic strategy (60).Although the delineation of gene- and tissue-specific phenotypes iscertainly an important area of investigation, we postulate that pharma-cologic BET bromodomain inhibitors, like many other drugs, may betherapeutically effective in vivo because they target multiple cell types.

In broader terms, this work highlights that HF, much like cancer,can be considered to be a disease in which there is aberrant transcrip-tional control of cell identity (16). In the case of cancer, progressivemutations in the genome drive transcriptional programs that causeuncontrolled cellular expansion and dissemination (16, 18). In HF,excessive exposure to stress signals converges on the gene regulatoryapparatus in cardiomyocytes and fibroblasts to drive maladaptive cellstate transitions that culminate in progressive organ remodelingand contractile dysfunction. In contrast to several cancer drugs thatcause cardiotoxicity (61), our data indicate that BET bromodomaininhibitors may be a privileged class of anticancer therapeutics thathas cardioprotective properties. The therapeutic efficacy of BET inhibi-tion in both these settings implies that cancer cells and myocardial cellsshare common vulnerabilities at the level of chromatin-dependentsignal transduction from enhancers. This generalized dependence onstimulus-coupled transcription suggests that in addition to BRD4, thecardiac chromatin signaling machinery may contain other druggabletargets that can be exploited for HF therapy.


MATERIALS AND METHODSStudy designThe overall objective of this study was to determine whether BETbromodomain inhibition with the small-molecule JQ1 could treat HFin well-established rodent models (TAC and MI) and attenuate ET-1–induced pathologic remodeling in human iPSC-CMs. RNA-seq studieswere performed to assess transcriptomic effects of JQ1 in both murineand human models in an unbiased, genome-wide manner. For murineHFmodels, mice were randomized to each experimental arm and treat-ment group. Mouse echocardiograms were analyzed by an individualblinded to the experimental group assignment. For each experiment,sample size reflects the number of independent biological replicatesand is provided in the figure legend. The expected effect magnitudeand SD for each experiment were guided by examples from publishedliterature for each model used. Using these parameters, sample sizewas determined by power calculation with a = 0.05 and b = 0.80. Sta-tistical analysis of RNA-seq data was performed in collaboration withM. Lemieux (BioInfo) and the Gladstone Institutes Bioinformatics Core.

Animal modelsAll protocols concerning animal use were approved by the InstitutionalAnimal Care and Use Committees at the University of California SanFrancisco and the Case Western Reserve University and conducted instrict accordance with the National Institutes of Health Guide for theCare and Use of Laboratory Animals (62). Studies were conducted withage-matched male C57Bl/6J mice (The Jackson Laboratory, catalog no.000664). Mice were housed in a temperature- and humidity-controlledpathogen-free facility with 12-hour light/dark cycle and ad libitumaccess to water and standard laboratory rodent chow.

Preparation of JQ1JQ1 was synthesized and purified in the laboratory of J. Bradner (Dana-Farber Cancer Institute), as previously published (23). For in vivoexperiments, a stock solution [50 mg/ml JQ1 in dimethyl sulfoxide(DMSO)] was diluted to a working concentration of 5 mg/ml in anaqueous carrier (10% hydroxypropyl b-cyclodextrin; Sigma C0926)using vigorous vortexing. Mice were injected at a dose of 50 mg/kggiven intraperitoneally once daily. Vehicle control was an equal amountof DMSO dissolved in 10% hydroxypropyl b-cyclodextrin carrier so-lution. All solutions were prepared and administered using steriletechnique. For in vitro experiments, JQ1 was dissolved in DMSOand administered to cells at indicated concentrations using an equalvolume of DMSO as control.

Mouse models of cardiac hypertrophy and HFAll mice were male C57Bl/6J mice aged 10 to 11 weeks from TheJackson Laboratory. Mice were placed on a temperature-controlledsmall-animal surgical table to help maintain body temperature (37°C)during surgery. Mice were anesthetized with ketamine/xylazine, me-chanically ventilated (Harvard Apparatus), and subjected to thora-cotomy. For TAC surgery, the aortic arch was constricted betweenthe left common carotid and the brachiocephalic arteries using a 7-0silk suture and a 27-gauge needle, as previously described (31). Intra-peritoneal injections of JQ1 (50 mg/kg per day) or vehicle wereadministered 18 days after TAC surgery and continued until post-operative day 53 (Fig. 1A). For MI surgery, the proximal LAD waspermanently ligated at the level of a standard anatomic landmark (leftatrial edge) using a 7-0 silk suture, as described previously (63). Oc-clusion of arterial flow was demonstrated by ischemic color changes in

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the anterior wall of the heart. Standard three-lead electrocardiographywas performed to confirm that left coronary artery ligation has resultedin a myocardial injury current (ST-segment elevations). Intraperitonealinjections of JQ1 were started on postoperative day 6 (25 mg/kg perday), increased to 50 mg/kg per day on postoperative day 12, andcontinued until postoperative day 28 (Fig. 2A). For sham surgeries,thoracotomy was performed as above, and the aorta (for TAC) or LAD(for MI) was surgically exposed without any further intervention.

Endurance swimming modelDaily swimming exercise was carried out to induce physiological cardiachypertrophy, as previously described (44) with minor modifications.Swimming exercise started in a ramp-up protocol starting at two ses-sions of 10min, with a 10-min increase each day until two sessions of90minwere reached. Ninetyminutes daily of swimmingwas continuedfor the next 28 days. Injections of JQ1 (50 mg/kg per day, IP) or vehiclestarted on the seventh day of this ramp-up protocol (Fig. 5A). The twoswimming sessions were separated from each other by at least 4 hours.The temperature of the water wasmaintained at 31°C with a heating sys-tem (JBJ True Temp Titanium Heating System, T3-300), and constantflow of water was maintained by a circulation pump (JBJ OceanStream,OS101). Mice in the sedentary group were subjected to the same waterconditions for 5minweekly. Allmicewere closelymonitored during thetraining for signs of struggling or discomfort, and no mice died ofdrowning during the above training protocol.

Transthoracic echocardiographyMice were anesthetized with 1% inhalational isoflurane and imaged usingthe Vevo 770 High Resolution Imaging System (FujiFilm VisualSonicsInc.) and the RMV-707B 30-MHz probe. Measurements were obtainedfrom M-mode sampling and integrated electrocardiogram-gated kilo-hertz visualization (EKV) images taken in the LV short axis at themidpapillary level, as previously described (31, 64). LV areas and ejec-tion fraction were obtained from high-resolution two-dimensionalmeasurements at the end-diastole and end-systole, as previously described(31, 64). For the MI model, measurement of remote LV wall thicknesswas performed on the inferoposterior wall.

Histological analysisShort-axis heart sections from the mid–left ventricle were fixed in 10%buffered formalin and embedded in paraffin. Cardiomyocyte cross-sectional area was determined by staining with rhodamine-conjugatedWGA (Vector Laboratories, RL-1022) and quantified as previouslydescribed (2). Fibrosis was visualized using the picrosirius red stainingkit (Polysciences Inc.). Quantification of fibrotic area was performed aspreviously described (32) with Image-Pro Plus (Media Cybernetics).For the MI model, assessment of remote LV histology was performedon the inferoposterior wall.

Culture, qRT-PCR, and BNP ELISA from human iPSC-CMsiCell human iPSC-CMs (CDI)were thawed andplated on 96-well platescoated with fibronectin (5 mg/ml) (Sigma), as previously described (48).Cells were plated at a density of 4.1 × 104 cells/cm2. After 48 hours in theiCell Cardiomyocyte PlatingMedium (CDI), cells were quiesced by cul-turing in supplementedWilliam’s E (SWE)medium [cocktail B from theHepatocyte Maintenance Supplement Pack (Thermo Fisher Scientific)diluted in William’s E medium at a 1:25 dilution] for 4 days. For subse-quent hypertrophic stimulation, quiesced iPSC-CMswere treatedwith orwithout JQ1 (500 nM) versus DMSO for 3 hours followed by stimulation


with or without ET-1 (10 nM) (Sigma) for 18 hours. RNA was purifiedand reverse-transcribed using the TaqMan Gene Expression Cells-to-CT kit (Life Technologies, AM1278) according to the manufacturer’sinstructions. qRT-PCR for humanNPPB andB2M (normalizer)was per-formedwith theLifeTechnologiesTaqManassays (NPPB,Hs00173590_m1;B2M, Hs000984230_m1). qRT-PCR for other human genes was per-formed using TaqMan chemistry including FastStart Universal ProbeMaster (Roche), labeled probes from theUniversal ProbeLibrary (Roche),and gene-specific oligonucleotide primers (list of qRT-PCR primersand TaqMAN probes are provided in table S6). Relative expression wascalculated using the 2DDCt method with normalization to B2M expres-sion. ELISA for BNP protein was performed on 6 ml of medium fromeach well of a 96-well plate as previously published (48) using thefollowing antibodies: anti-proBNP capture antibody (clone 5B6; Abcamab13111), anti-proBNPHRP conjugate detection antibody (clone 16F3;Abcam ab13124), and purified NT-proBNP (Phoenix Pharmaceuticals,catalog no. 011-42) as a standard.

Cell area measurements for iPSC-CMCells cultured on 96-well plates were fixed in phosphate-bufferedsaline (PBS)with 2%paraformaldehyde for 20min, then permeabilized[PBS–Tween 20 (PBST) + 0.1% Triton X-100] for 20min and blocked(PBST + 5.0% horse serum) for 1 hour at room temperature. Primaryantibody against sarcomeric a-actinin (Sigma A7811) was used at a1:800 dilution in PBST + 5.0% horse serum for 1 hour at room tem-perature. The anti-mouse secondary antibody tagged with Alexa 488was used at a dilution of 1:1,000 for 1 hour at room temperature. Wellswere scanned on a high-content imaging platform (IN Cell Analyzer,GE Healthcare Systems) at ×200 magnification, and quantified as pre-viously described (48).

RNA purification and qRT-PCR from murine samplesA 10- to 20-mg piece of mouse LV tissue was collected and preservedin RNAlater stabilization reagent (Qiagen) followed by mechanicaldisruption/homogenization in PureZOL (Bio-Rad) on a TissueLyserII (Qiagen). For the TAC model, a concentric short-axis slice of theLV was prepared for RNA. For the MI model, a short-axis sector of theremote, noninfarcted left ventricle (infero-posterior wall) was preparedfor RNA. The RNA-containing aqueous phase was extracted withchloroform. RNA was then purified with Aurum purification kit (BioRad,732-6830). qRT-PCRwas performed using TaqMan chemistry includingFastStart Universal Probe Master (Roche), labeled probes from theUniversal Probe Library (Roche), and gene-specific oligonucleotideprimers on aLightCycler 480 system (Roche).A list of qRT-PCRprimersand TaqMAN probes are provided in table S6. Relative expression wascalculated using the 2DDC t method with normalization to constitutivegenes. Mouse data were normalized to Ppib.

Library preparation and next-generation sequencingRNA isolated frommouse tissue andhuman iPSC-CMswere quantifiedusingQubit fluorometric quantitation and assessed for quality usingAgilentBioanalyzer Nano RNA Chip. For human iPSC-CM samples, the 500 nMJQ1 treatment condition was used for RNA-seq. Samples with RNA integ-rity number larger than 8 were considered high-quality and suitable forRNA-seq. Library preparation was performed using the Illumina TruSeqStranded Total RNA kit with Ribo-Zero Gold rRNA depletion, and thelibrary was sequenced on an Illumina HiSeq 2500 at the Genomics CoreFacility of Case Western Reserve University (Cleveland, OH) (paired-end,100 base pairs, >50 M reads per sample; Rapid Run v2 flow cell).

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RNA-seq data analysisAdapters and barcodes were trimmed, and low-quality reads werefiltered using fastq-mcf (https://code.google.com/archive/p/ea-utils/).High-quality sequences were aligned to mm10 (mouse data) and hg38(human data), as appropriate, with TopHat 2.0.13 (65), and assignedto Ensembl genes and counted using “featureCounts” (66), part of theSubread suite (http://subread.sourceforge.net/). (Note: the entry forENSMUSG00000092329/Gm20388 was removed from the murineGencode vM4 annotations we used before counting to avoid the artifac-tual loss of other transcripts, including Acta1, within its ~4-Mb bound-aries.) Raw counts were used as inputs to DESeq (v.1.25.0) (67) fordifferential expression analysis, and Benjamini-Hochberg test wasused to control false discovery of differentially expressed genes (68).Heatmaps were generated using the Bioconductor packageHeatmap(v.1.0.8) using reads per kilobase per million (RPKM) mapped. Valuesshown are log2-transformed and row-normalized. Samples wereclustered using complete linkage and Pearson correlation. Rows wereordered (but not clustered) to highlight patterns between groups. TheVenn diagram shows the overlap of transcript IDs. DAVID (v.6.8;https://david.ncifcrf.gov) (69) was used for functional annotationenrichment analyses with the background universe restricted to genesexpressed in the species and tissue/cell type being interrogated. Log foldchanges were calculated between conditions and imported into theIngenuity Pathway Analysis (37) server (Qiagen) to identify pathwayswith enriched differential expression. GSEA (70) was run on expressiondata preranked by log2 fold change. Curated signatures of TGF-b targets(PLASARI_TGFB1_TARGETS_10HR_UP; Broad Institute C2 dataset) and NFkB targets (NFKB_DIRECT_BU; www.bu.edu/nf-kb/gene-resources/target-genes/) were used in the GSEA. Cell type finger-printing consisted of summing z scores of normalized data sets foreach cell type signature (cardiomyocyte: Nppa, Nppb, Myh7, Fstl1,Actc1, Xirp2, andMybpc2; fibroblast: Postn,Adam12,Wisp2, Col3a1,and Tnc; myeloid: Ly86, Ccr2, Sfpi1/Spi1, Cd80, and Itgam). Thefollowing published RNA-seq data sets were fingerprinted as controls:GSE68509 [n = 3 mouse cardiomyocyte (42)], GSE58453 [n = 3 eachmouse cardiomyocyte and fibroblast (41)], and GSE57125 [n = 2cardiac macrophage precursor cells (called “B-type” cells in this pub-lication) (43)]. All RNA-seq data sets were uploaded to Gene Expres-sion Omnibus (GEO) (accession no. GSE96566).

Statistical analysisData are reported as means ± SEM unless otherwise indicated in thefigure legend. Statistical analyses of murine TAC and MI data wereperformed using analysis of variance (ANOVA) with Tukey’s honestsignificance difference test using GraphPad Prism Plus. Statisticalanalysis of human iPSC-CM area, qRT-PCR, and BNP protein con-centration was performed by ANOVA with Holm-Sidak correctionfor multiple comparisons using GraphPad Prism Plus. For all analy-ses, P < 0.05 was considered significant. The statistical methods usedin the analysis of RNA-seq data are detailed separately above.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/390/eaah5084/DC1Fig. S1. Echocardiographic data from sham- and TAC-treated mice at day 53.Fig. S2. Transcriptomic effects of chronic JQ1 administration in baseline (sham-operated) hearts.Fig. S3. GSEA and fingerprinting analysis of differentially expressed genes in mouse LV.Fig. S4. Additional qRT-PCR of exercise-regulated genes in mouse left ventricle.Fig. S5. qRT-PCR and GSEA in human iPSC-CMs.Table S1. Normalized RNA-seq expression data for sham-vehicle and sham-JQ1 groups.


Table S2. Normalized RNA-seq expression data for sham-vehicle, TAC-vehicle, and TAC-JQ1groups.Table S3. Normalized RNA-seq expression data for sham-vehicle, MI-vehicle, and MI-JQ1groups.Table S4. List of 193 genes in the Venn overlap in Fig. 4A.Table S5. Normalized RNA-seq expression data from human iPSC-CMs for unstimulated, ET-1,and ET-1 + JQ1 groups.Table S6. List of qRT-PCR primers and TaqMan probes.

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Acknowledgments: We thank B. G. Bruneau, C. Y. Lin, and K. S. Pollard for insightfuldiscussions about this work. Funding: This work was supported by NIH grants DK093821 (S.M.H.),HL127240 (S.M.H., J.E.B., and T.A.M.), and HL116848 (T.A.M.). Author contributions: Q.D., S.M.,J.D.B., D.S., J.E.B., T.A.M., and S.M.H. designed the research and analyzed the data. Q.D., S.M., P.A.,H.S., and A.S. performed the research. Q.D., R.Z., Y.H., and H.T.S. performed animal surgeries


and echocardiograms. M.E.L. and S.T. assisted with the bioinformatic analyses. J.E.B. providedthe key reagents. Q.D., S.M., and S.M.H. wrote the manuscript with critical revisions from J.E.B.,J.D.B., D.S., and T.A.M. Competing interests: S.M.H. and D.S. are scientific co-founders andshareholders of Tenaya Therapeutics and serve as consultants for this entity. J.E.B. is now ashareholder and executive of Novartis Pharmaceuticals. S.M.H., J.E.B., and J.D.B. are co-inventorson patent application US20160095867A1 entitled “BET inhibition therapy for heart disease”held jointly by the Dana-Farber Cancer Institute, the Case Western Reserve University, and theBrigham and Women’s Hospital that covers methods for treating cardiac diseases using BETinhibitors. All other authors declare that they have no competing interests. Materials anddata availability: All RNA-seq data sets are available in GEO (accession no. GSE96566).

Submitted 13 July 2016Resubmitted 18 January 2017Accepted 30 March 2017Published 17 May 201710.1126/scitranslmed.aah5084

Citation: Q. Duan, S. McMahon, P. Anand, H. Shah, S. Thomas, H. T. Salunga, Y. Huang,R. Zhang, A. Sahadevan, M. E. Lemieux, J. D. Brown, D. Srivastava, J. E. Bradner,T. A. McKinsey, S. M. Haldar, BET bromodomain inhibition suppresses innate inflammatoryand profibrotic transcriptional networks in heart failure. Sci. Transl. Med. 9, eaah5084 (2017).

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transcriptional networks in heart failureBET bromodomain inhibition suppresses innate inflammatory and profibrotic

McKinsey and Saptarsi M. HaldarAarathi Sahadevan, Madeleine E. Lemieux, Jonathan D. Brown, Deepak Srivastava, James E. Bradner, Timothy A. Qiming Duan, Sarah McMahon, Priti Anand, Hirsh Shah, Sean Thomas, Hazel T. Salunga, Yu Huang, Rongli Zhang,

DOI: 10.1126/scitranslmed.aah5084, eaah5084.9Sci Transl Med

response to exercise, supporting the safety of this approach.severe disease. They also found that JQ1 did not interfere with physiological cardiac hypertrophy that occurs indetrimental effects of this protein and improve cardiac structure and function even in the setting of prolonged and

derived cardiomyocyte models to show that JQ1 can reverse the−mouse and human induced pluripotent stem cell development of heart failure and contributes to cardiomyocyte hypertrophy. The authors used a combination of

protein 4, a member of the BET family of epigenetic regulators and the target of JQ1, plays a role in the originally developed for cancer therapy, may be an effective treatment for heart failure. Bromodomain-containing

determined that a small molecule called JQ1, which was et al.great need for more effective therapeutics. Duan Heart failure remains a common and difficult-to-treat medical condition with a high mortality, and there is a

BETting on a new heart failure treatment

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REFERENCES

http://stm.sciencemag.org/content/9/390/eaah5084#BIBLThis article cites 69 articles, 17 of which you can access for free

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http://stm.sciencemag.org/content/suppl/2017/05/15/9.390.eaah5084.DC1

http://stm.sciencemag.org/content/scitransmed/9/373/eaaf3925.full

http://stm.sciencemag.org/content/scitransmed/8/344/344ra86.full

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http://stm.sciencemag.org/content/scitransmed/9/400/eaal1645.full

http://stm.sciencemag.org/content/scitransmed/10/427/eaao0144.full

http://science.sciencemag.org/content/sci/362/6421/1359.full

http://stm.sciencemag.org/content/scitransmed/11/476/eaat1199.full

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BET bromodomain inhibition suppresses innate inflammatory ... · these specific transcriptional mechanisms, we found that JQ 1 does not suppress physiological cardiac hypertrophy