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SummaryTuning of genome structure and function is accomplished by chromatin binding proteins, which determine the transcriptome and phenotype of the cell. We sought to investigate how communication between extracellular stress and chromatin structure may regulate cellular mechanical behaviors. We demonstrate that the linker histone H1.0, which compacts nucleosomes into higher order chromatin fibers, controls genome organization and cellular stress response. Histone H1.0 has privileged expression in fibroblasts across tissue types in mice and humans, and modulation of its expression is necessary and sufficient to mount a myofibroblast phenotype in these cells. Depletion of histone H1.0 prevents transforming growth factor beta (TGF-β)-induced fibroblast contraction, proliferation and migration in a histone H1 isoform-specific manner via inhibition of a transcriptome comprised of extracellular matrix, cytoskeletal and contractile genes. Histone H1.0 is associated with local regulation of gene expression via mechanisms involving chromatin fiber compaction and reprogramming of histone acetylation, rendering the cell stiffer in response to cytokine stimulation. Knockdown of histone H1.0 prevented locus-specific histone H3 lysine 27 acetylation by TGF-βand decreased levels of both HDAC1 and the chromatin reader BRD4, thereby preventing transcription of a fibrotic gene program. Transient depletion of histone H1.0in vivodecompacts chromatin and prevents fibrosis in cardiac muscle, thereby linking chromatin structure with fibroblast phenotype in response to extracellular stress. Our work identifies an unexpected role of linker histones to orchestrate cellular mechanical behaviors, directly coupling cellular force generation, nuclear organization and gene transcription.Graphical Abstract

Background: During neonatal cardiac development, myocytes integrate cues such as changes in blood pressure and circulating hormones to execute a temporal genetic response that drives physiological cardiac hypertrophy. Chromatin remodeling precedes the adult heart’s response to growth stimuli, yet the temporal nature of neonatal chromatin remodeling is unclear. In other tissues, genes regulated by the master circadian rhythm factor BMAL1 display circadian oscillations in histone acetylation and nucleosome and RNAP II occupancy. We hypothesized that BMAL1 regulates temporal chromatin remodeling and expression of genes critical for neonatal cardiac hypertrophy. Methods: BMAL1 chromatin immunoprecipitation sequencing (ChIP-Seq) and assay for transposase-accessible chromatin and sequencing (ATAC-Seq) data from murine hearts were analyzed to identify genes at which BMAL1 may drive chromatin remodeling. To interrogate myocyte time-of-day dependent response to growth stimuli, neonatal rat ventricular myocytes (NRVM) were subjected to serum shock synchronization to drive oscillatory BMAL1 expression, followed by treatment with the growth stimulus phenylephrine (PE) at the peak and trough of BMAL1 expression. BMAL1 was knocked down via siRNA and hypertrophy was assessed by measurement of cell size and of RT-PCR of fetal genes (Nppa and Nppb). Results: BMAL1 ChIP-Seq data revealed myocyte-specific BMAL1 localization to regions exhibiting an open chromatin signature based on ATAC-seq, including the pro-hypertrophic gene salt-inducible kinase 1 ( Sik1 ). Serum shock induced oscillations of BMAL1 and histone H3.3: administration of PE at the peak of BMAL1 expression induced a 30% increase in cell size and 2-fold increase in fetal gene expression. PE given at the trough of BMAL1 expression affected neither cell size nor fetal genes, demonstrating a critical role for the circadian clock in myocyte growth. BMAL1 knockdown: decreased expression of its known target and partner clock gene, Per2 , as well as expression of Sik1, Nppa , and Nppb ; decreased myocyte size by 30%; and increased expression of histone H3.3. Conclusions: These data indicate that BMAL1 temporally regulates myocyte growth by modulating histone stoichiometry and expression of Sik1 .

Background: Heart Failure (HF) is driven by the interactions of multiple genetic, epigenetic, and environmental factors both within and between the cell types of the heart. Our lab uses the Hybrid Mouse Diversity Panel (HMDP), a large cohort of inbred mouse strains, to perform systems genetics analyses of isoproterenol-induced cardiac hypertrophy and failure by leveraging the benefits of a curated model organism population to identify key drivers of phenotype. We are now expanding this study into the DNA methylome, which has been convincingly linked to cardiac-associated phenotypic changes in prior studies. Methods and Results: Using Reduced Representational Bisulfite Sequencing, we profiled left ventricular tissue samples from 88 HMDP strains subjected to isoproterenol challenge (30 mg/kg/day for 21 days) and matched control animals. We identified nearly 170,000 CpGs whose methylation status varies across the HMDP and 179 significant associations (FDR of 5%) between CpG methylation and phenotypic variation using the epigenome-wide association study algorithm MACAU, including 37 associations linking CpG methylation in unchallenged hearts to 19 post-challenge phenotypes. To identify high-confidence candidate genes, we combined our loci with data from the Wellcome Trust Mouse Genomes Resource, transcriptomic and metabolomic data from the HMDP, Hi-C data, and cell-type-specific gene expression and methylation data from healthy and failing mouse hearts. We are systematically querying the resulting 78 candidates using in silico and in vitro approaches. These genes include Mospd3 , which is associated with right ventricular hypertrophy and whose knockdown in vitro results in reduced cardiomyocyte hypertrophy and changes to hypertrophy-related gene expression. Also observed were Atp9a , whose expression levels are associated with significant global DNA methylation changes between control and ISO-treated animals and whose knockdown likewise causes a reduction in observed cellular hypertrophy, and Mdga1 , whose promoter methylation status is linked to changes in 5% of expressed genes of the heart during heart failure. Further analysis and in vivo study of these loci will further our understanding of the role of DNA methylation in heart failure.

BackgroundAtrial fibrillation (AF) is the most common sustained cardiac arrhythmia and post-operative atrial fibrillation (POAF) is a major healthcare burden, contributing to an increased risk of stroke, kidney failure, heart attack and death. Genetic studies have identified associations with AF, but no molecular diagnostic exists to predict POAF based on pre-operative measurements. Such a tool would be of great value for perioperative planning to improve patient care and reduce healthcare costs. In this pilot study of epigenetic precision medicine in the perioperative period, we carried out bisulfite sequencing to measure DNA methylation status in blood collected from patients prior to cardiac surgery to identify biosignatures of POAF.MethodsWe enrolled 221 patients undergoing cardiac surgery in this prospective observational study. DNA methylation measurements were obtained from blood samples drawn from awake patients prior to surgery. After controlling for clinical and methylation covariates, we analyzed DNA methylation loci in the discovery cohort of 110 patients for association with POAF. We also constructed predictive models for POAF using clinical and DNA methylation data. We subsequently performed targeted analyses of a separate cohort of 101 cardiac surgical patients to measure the methylation status solely of significant methylation loci in the discovery cohort.ResultsA total of 47 patients in the discovery cohort (42.7%) and 43 patients in the validation cohort (42.6%) developed POAF. We identified 12 CpGs that were statistically significant in the discovery cohort after correcting for multiple hypothesis testing. Of these sites, 6 were amenable to targeted bisulfite sequencing and chr16:24640902 was statistically significant in the validation cohort. In addition, the methylation POAF prediction model had an AUC of 0.79 in the validation cohort.ConclusionsWe have identified DNA methylation biomarkers that can predict future occurrence of POAF associated with cardiac surgery. This research demonstrates the use of precision medicine to develop models combining epigenomic and clinical data to predict disease.

Modulating the number of muscle stems cells, called satellite cells, during early postnatal development produces long-term effects on muscle growth. We tested the hypothesis that high expression levels of the anti-aging protein Klotho in early postnatal myogenesis increase satellite cell numbers by influencing the epigenetic regulation of genes that regulate myogenesis. Our findings show that elevated klotho expression caused a transient increase in satellite cell numbers and slowed muscle fiber growth, followed by a period of accelerated muscle growth that leads to larger fibers. Klotho also transcriptionally downregulated the H3K27 demethylase Jmjd3, leading to increased H3K27 methylation and decreased expression of genes in the canonical Wnt pathway, which was associated with a delay in muscle differentiation. In addition, Klotho stimulation and Jmjd3 downregulation produced similar but not additive reductions in the expression of Wnt4, Wnt9a, and Wnt10a in myogenic cells, indicating that inhibition occurred through a common pathway. Together, our results identify a novel pathway through which Klotho influences myogenesis by reducing the expression of Jmjd3, leading to reductions in the expression of Wnt genes and inhibition of canonical Wnt signaling.

The response of an organ to stimuli emerges from the actions of individual cells. Recent cardiac single-cell RNA-sequencing studies of development, injury, and reprogramming have uncovered heterogeneous populations even among previously well-defined cell types, raising questions about what level of experimental resolution corresponds to disease-relevant, tissue-level phenotypes. In this review, we explore the biological meaning behind this cellular heterogeneity by undertaking an exhaustive analysis of single-cell transcriptomics in the heart (including a comprehensive, annotated compendium of studies published to date) and evaluating new models for the cardiac function that have emerged from these studies (including discussion and schematics that depict new hypotheses in the field). We evaluate the evidence to support the biological actions of newly identified cell populations and debate questions related to the role of cell-to-cell variability in development and disease. Finally, we present emerging epigenomic approaches that, when combined with single-cell RNA-sequencing, can resolve basic mechanisms of gene regulation and variability in cell phenotype.

Post-transcriptional regulation plays a key role in transcriptome reprogramming during cardiac pathogenesis. In previous studies, we have identified that cardiac enriched RNA-binding protein, RBFox1 plays key role in cardiac hypertrophy through mRNA alternative splicing regulation in nuclei. However, RBFox1 gene also generates a cytosolic isoform (RBFox1c), suggesting additional functions of post-transcriptional regulation in heart. In adult heart, RBFox1c mRNA constituted ~ 40% of total RBFox1 level but was significantly repressed in pressure-overloaded failing mouse heart. Using CRISPR-Cas9 technology, we have established an isoform specific RBFox1c-cKO mouse. At baseline inactivation of RBFox1c led to decreased cardiac function along with induction of cardiac fibrosis. RBFox1c-cKO mice also showed macrophages infiltration into myocardium post 7days MI. In contrast, restoration of RBFox1c expression in adult intact hearts significantly reduced cardiac fibrosis post stress. RNA-seq analyses in RBFox1c expressing cardiomyocytes showed that RBFox1c specifically suppressed the expression of pro-inflammatory genes. Secondly, CLIP-Seq analysis and targeted RNA-IP showed that RBFox1c could directly interact with inflammatory pathway mRNAs. These results suggested the inflammatory mRNAs are direct downstream targets regulated by RBFox1c. Using both in vitro cultured cardiomyocytes and intact mouse hearts, we demonstrated that expression of RBFox1c reduces pro-inflammatory mRNA expression at baseline and upon hypertrophy stimulation. Lastly, we characterized the interactome of RBFox1c through proteomic analysis and found RBFox1c specifically interacted with a component of the RNA NMD machinery-Upf1. RBFox1c interaction with Upf1 in cardiomyocytes was diminished upon hypertrophic stress. Furthermore, by inactivation of Upf1 via siRNA, we demonstrated that RBFox1c mediated repression of proinflammatory genes was Upf1 dependent.RBFox1 regulates cardiac transcriptome reprogramming in two post-transcriptional processes via distinct isoforms. While the RBFox1n regulates RNA splicing, the RBFox1c functions through targeted mRNA repression of proinflammatory genes by recruitment of Upf1 mediated RNA degradation.

Background: Heart failure (HF) is a highly heterogeneous disorder characterized by the interactions of multiple genetic and environmental factors as well as the interaction of different cell types in the heart. Although reductionistic approaches have successfully identified many genes involved in HF, heritability studies suggest that many genes have resisted discovery through these approaches. By utilizing cell-type-specific gene expression paired with transcriptomic data from a large cohort of mice, we sought to identify important drivers of HF using a systems genetics approach. Methods and Results: Mice from 93 unique inbred lines of the Hybrid Mouse Diversity Panel were given 30 ug/g/day of isoproterenol for three weeks via osmotic minipump to induce heart failure. Transcriptomes were generated from these mice and the weighted Maximal Information Component Analysis (wMICA) algorithm was applied to generate transcriptomic gene networks. Cardiomyocytes and Fibroblasts were isolated from both control and isoproterenol-treated adult C57BL/6J hearts using a Langendorff apparatus (n=3 per sex/treatment) and transcriptomes were generated. Significantly differentially expressed genes were identified using DESEQ2 and used to query the wMICA-derived network, identifying the gene Adamts2 as a potential regulator of cardiac hypertrophy. Follow-up in vitro and in vivo work has demonstrated that Adamts2 knockdown significantly blunts the hypertrophic effect of isoproterenol on cardiomyocytes while simultaneously reducing fibroblast proliferation and increasing apoptosis as measured by TUNEL staining. Careful examination of the gene network reveals evidence of paracrine signaling between cardiomyocytes and fibroblasts and suggests a key trans-cell-type role of Adamts2 in the regulation of HF after catecholamine stimulation. Conclusion: Co-expression network algorithms combined with cell-type-specific transcriptomics identified Adamts2 as a driver of HF. Adamts2 plays an important role via paracrine signaling in the proliferative response of fibroblasts and the hypertrophic response of cardiomyocytes to catecholamines. Further mechanistic analysis of Adamts2 will further reveal its role in the progression of heart failure.

Heart failure with preserved ejection fraction (HFpEF) exhibits a sex bias, being more common in women than men, and we hypothesize that mitochondrial sex differences might underlie this bias. As part of genetic studies of heart failure in mice, we observe that heart mitochondrial DNA levels and function tend to be reduced in females as compared to males. We also observe that expression of genes encoding mitochondrial proteins are higher in males than females in human cohorts. We test our hypothesis in a panel of genetically diverse inbred strains of mice, termed the Hybrid Mouse Diversity Panel (HMDP). Indeed, we find that mitochondrial gene expression is highly correlated with diastolic function, a key trait in HFpEF. Consistent with this, studies of a “two-hit” mouse model of HFpEF confirm that mitochondrial function differs between sexes and is strongly associated with a number of HFpEF traits. By integrating data from human heart failure and the mouse HMDP cohort, we identify the mitochondrial gene Acsl6 as a genetic determinant of diastolic function. We validate its role in HFpEF using adenoviral over-expression in the heart. We conclude that sex differences in mitochondrial function underlie, in part, the sex bias in diastolic function.

The temporal nature of chromatin structural changes underpinning pathologic transcription are poorly understood. We measured chromatin accessibility and DNA methylation to study the contribution of chromatin remodeling at different stages of cardiac hypertrophy and failure. ATAC-seq and reduced representation bisulfite sequencing were performed in cardiac myocytes after transverse aortic constriction (TAC) or depletion of the chromatin structural protein CTCF. Early compensation to pressure overload showed changes in chromatin accessibility and DNA methylation preferentially localized to intergenic and intronic regions. Most methylation and accessibility changes observed in enhancers and promoters at the late phase (3 weeks after TAC) were established at an earlier time point (3 days after TAC), before heart failure manifests. Enhancers were paired with genes based on chromatin conformation capture data: while enhancer accessibility generally correlated with changes in gene expression, this feature, nor DNA methylation, was alone sufficient to predict transcription of all enhancer interacting genes. Enrichment of transcription factors and active histone marks at these regions suggests that enhancer activity coordinates with other epigenetic factors to determine gene transcription. In support of this hypothesis, ChIP-qPCR demonstrated increased enhancer and promoter occupancy of GATA4 and NKX2.5 at Itga9 and Nppa, respectively, concomitant with increased transcription of these genes in the diseased heart. Lastly, we demonstrate that accessibility and DNA methylation are imperfect predictors of chromatin structure at the scale of A/B compartmentalization—rather, accessibility, DNA methylation, transcription factors and other histone marks work within these domains to determine gene expression. These studies establish that chromatin reorganization during early compensation after pathologic stimuli is maintained into the later decompensatory phases of heart failure. The findings reveal the rules for how local chromatin features govern gene expression in the context of global genomic structure and identify chromatin remodeling events for therapeutic targeting in disease.

The study of epigenomics has advanced in recent years to span the regulation of a single genetic locus to the structure and orientation of entire chromosomes within the nucleus. In this review, we focus on the challenges and opportunities of clinical epigenomics in cardiovascular disease. As an integrator of genetic and environmental inputs, and because of advances in measurement techniques that are highly reproducible and provide sequence information, the epigenome is a rich source of potential biosignatures of cardiovascular health and disease. Most of the studies to date have focused on the latter, and herein we discuss observations on epigenomic changes in human cardiovascular disease, examining the role of protein modifiers of chromatin, noncoding RNAs and DNA modification. We provide an overview of cardiovascular epigenomics, discussing the challenges of data sovereignty, data analysis, doctor-patient ethics and innovations necessary to implement precision health.

PURPOSE OF REVIEW: Technical advances have facilitated high-throughput measurements of the genome in the context of cardiovascular biology. These techniques bring a deluge of gargantuan datasets, which in turn present two fundamentally new opportunities for innovation—data processing and knowledge integration—toward the goal of meaningful basic and translational discoveries. RECENT FINDINGS: Big data, integrative analyses, and machine learning have brought cardiac investigations to the cutting edge of chromatin biology, not only to reveal basic principles of gene regulation in the heart, but also to aid in the design of targeted epigenetic therapies. SUMMARY: Cardiac studies using big data are only beginning to integrate the millions of recorded data points and the tools of machine learning are aiding this process. Future experimental design should take into consideration insights from existing genomic datasets, thereby focusing on heretofore unexplored epigenomic contributions to disease pathology.

Heart failure can be induced or ameliorated in animal models by regulation of chromatin modifying enzymes, yet the chromatin level actions of these enzymes during pathogenesis is unknown. Because many histone modifiers and transcription factors regulate gene expression, we sought to directly measure chromatin accessibility through an unbiased method (ATAC-seq) that reports the status of a given locus at any time—the sum total of all epigenetic modifiers—in a mouse model of pressure overload hypertrophy. Early compensation of pressure overload at 3 days was associated with widespread changes in chromatin accessibility and DNA methylation, primarily in noncoding regions. The majority of changes that persisted to the decompensated phase (3weeks) were already established at the earlier time point, revealing a temporal nature of epigenomic compensation to pathologic stimuli. A cardiac-specific CTCF depletion model was used to examine basal cardiac chromatin function and revealed that disruption of this structure by loss of CTCF causes widespread changes in accessibility and methylation distinct from those in pressure overload. Less than half of the gene expression changes occurring at either time point after pressure overload were explained by DNA methylation alone and accessibility was likewise an imperfect predictor of transcription. Distal enhancers were paired with genes based on chromatin structural data and the regulatory actions of these elements examined in the context of DNA methylation and accessibility: enhancer actions require specific combinations of transcription factors and histone modifications at different stages of disease and to execute aspecific transcriptional event (methylation or accessibility alone was insufficient to predict the behavior). For example, the subset of differentially accessible enhancers in both 3 weeks TACand CTCF depletion significantly overlaps with cardiac transcription factors Gata4 (p=4.13x10 -6 ),Nkx2-5 (p=2.49x10 -5 ) and P300 (p=8.38x10 -7 ). In summary, these studies characterize the logic employed at coding, regulatory, and noncoding regions to regulate chromatin accessibility and transcription, providing a resource of epigenomic data at distinct temporal stages of heart failure.

Background & Aims A holistic insight on the relationship between obesity and metabolic dysfunction-associated fatty liver disease is an unmet clinical need. Omics investigations can be used to investigate the multifaceted role of altered mitochondrial pathways to promote nonalcoholic steatohepatitis, a major risk factor for liver disease-associated death. There are no specific treatments but remission via surgery might offer an opportunity to examine the signaling processes that govern the complex spectrum of chronic liver diseases observed in extreme obesity. We aim to assess the emerging relationship between metabolism, methylation and liver disease. Methods We tailed the flow of information, before and after steatohepatitis remission, from biochemical, histological, and multi-omics analyses in liver biopsies from patients with extreme obesity and successful bariatric surgery. Functional studies were performed in HepG2 cells and primary hepatocytes. Results The reversal of hepatic mitochondrial dysfunction and the control of oxidative stress and inflammatory responses revealed the regulatory role of mitogen-activated protein kinases. The reversible metabolic rearrangements leading to steatohepatitis increased the glutaminolysis-induced production of α-ketoglutarate and the hyperactivation of mammalian target of rapamycin complex 1. These changes were crucial for the adenosine monophosphate-activated protein kinase/mammalian target of rapamycin-driven pathways that modulated hepatocyte survival by coordinating apoptosis and autophagy. The signaling activity of α-ketoglutarate and the associated metabolites also affected methylation-related epigenomic remodeling enzymes. Integrative analysis of hepatic transcriptome signatures and differentially methylated genomic regions distinguished patients with and without steatohepatitis. Conclusion We provide evidence supporting the multifaceted potential of the increased glutaminolysis-induced α-ketoglutarate production and the mammalian target of rapamycin complex 1 dysregulation as a conceivable source of the inefficient adaptive responses leading to steatohepatitis. Lay summary Steatohepatitis is a frequent and threatening complication of extreme obesity without specific treatment. Omics technologies can be used to identify therapeutic targets. We highlight increased glutaminolysis-induced α-ketoglutarate production as a potential source of signals promoting and exacerbating steatohepatitis.

Unraveling the principles of cardiac chromatin organization for next generation therapies in heart disease

Supplemental Digital Content is available in the text., Background: Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. Methods: To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload–induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Results: Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. Conclusions: These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy.

Eukaryotes must balance the metabolic and cell death actions of mitochondria via control of gene expression and cell fate by chromatin, thereby functionally binding the metabolome and epigenome. This interaction has far-reaching implications for chronic diseases in humans, the most common of which are those of the cardiovascular system. The most devastating consequence of cardiovascular disease, heart failure, is not a single disease, diagnosis, or endpoint. Human and animal studies have revealed that, regardless of etiology and symptoms, heart failure is universally associated with abnormal metabolism and gene expression – to frame this as cause or consequence, however, may be to wrongfoot the question. This essay aims to challenge current thinking on metabolic–epigenetic crosstalk in heart failure, presenting hypotheses for how chronic diseases arise, take hold, and persist. We unpack assumptions about the order of operations for gene expression and metabolism, exploring recent findings in noncardiac systems that link metabolic intermediates directly to chromatin remodeling. Lastly, we discuss potential mechanisms by which chromatin may serve as a substrate for metabolic memory, and how changes in cellular transcriptomes (and hence in cellular behavior) in response to stress correspond to global changes in chromatin accessibility and structure.

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. As a potential epigenetic biomarker, DNA methylation differs between healthy and diseased hearts. However, its association according to disease severity has not yet been studied. Here, we sought to investigate how DNA methylation (DNAm) differs across HF disease severity, and study the use of a DNMT methyltransferase inhibitor RG108 on DNAm and its effect on heart function. A fixed dose of Isoprenaline (ISO), or saline control (SAL), administered to a hybrid mouse diversity panel (HMDP) consisting of 85 classical and recombinant inbred mouse strains produced a range of cardiac hypertrophy and/or LV dilatation. Left ventricles were harvested and subjected to genome-wide cardiac DNAm profiling by Reduced Representation Bisulfite Sequencing (RRBS). Unsupervised clustering of the top 1% most variable CpG methylation segregated strains to their genetic origin. Disregarding strain-specific methylation differences, differential methylation between ISO and SAL unexpectedly categorised mice into mild and severe disease responders. In the severe-responder strain BTBRT, the pharmacological DNMT methyltransferase inhibitor, RG108, rescued disease from ISO-response, with accompanying evidence of gene expression recovery. This work establishes the range of cardiac differential DNAm correlating according to disease severity. It displays the involvement of DNA methylation-dependent gene expression changes that turns out to be unique, despite different mouse strain backgrounds. This gives further proof of principle that cardiac DNAm changes represent novel biomarkers for disease stratification and consequent targeted therapy.

Heart failure is associated with hypertrophying of cardiomyocytes and changes in transcriptional activity. Studies from rapidly dividing cells in culture have suggested that transcription may be compartmentalized into factories within the nucleus, but this phenomenon has not been tested in vivo and the role of nuclear architecture in cardiac gene regulation is unknown. While alterations to transcription have been linked to disease, little is known about the regulation of the spatial organization of transcription and its properties in the pathological setting. In the present study, we investigate the structural features of endogenous transcription factories in the heart and determine the principles connecting chromatin structure to transcriptional regulation in vivo. Super-resolution imaging of endogenous RNA polymerase II clusters in neonatal and adult cardiomyocytes revealed distinct properties of transcription factories in response to pathological stress: neonatal nuclei demonstrated changes in number of clusters, with parallel increases in nuclear area, while the adult nuclei underwent changes in size and intensity of RNA polymerase II foci. Fluorescence in situ hybridization-based labeling of genes revealed locus-specific relationships between expression change and anatomical localization-with respect to nuclear periphery and heterochromatin regions, both sites associated with gene silencing-in the nuclei of cardiomyocytes in hearts (but not liver hepatocytes) of mice subjected to pathologic stimuli that induce heart failure. These findings demonstrate a role for chromatin organization and rearrangement of nuclear architecture for cell type-specific transcription in vivo during disease. RNA polymerase II ChIP and chromatin conformation capture studies in the same model system demonstrate formation and reorganization of distinct nuclear compartments regulating gene expression. These findings reveal locus-specific compartmentalization of stress-activated, housekeeping and silenced genes in the anatomical context of the endogenous nucleus, revealing basic principles of global chromatin structure and nuclear architecture in the regulation of gene expression in healthy and diseased conditions.

The past two decades have witnessed a rapid evolution in our ability to measure RNA and protein from biological systems. As a result, new principles have arisen regarding how information is processed in cells, how decisions are made, and the role of networks in biology. This essay examines this technological evolution, reviewing (and critiquing) the conceptual framework that has emerged to explain how RNA and protein networks control cellular function. We identify how future investigations into transcriptomes, proteomes, and other cellular networks will enable development of more robust, quantitative models of cellular behavior whilst also providing new avenues to use knowledge of biological networks to improve human health. © 2016 American Physiological Society. Compr Physiol 6:1851-1872, 2016.

Transcriptome remodeling in heart disease occurs through the coordinated actions of transcription factors, histone modifications, and other chromatin features at pathology-associated genes. The extent to which genome-wide chromatin reorganization also contributes to the resultant changes in gene expression remains unknown. We examined the roles of two chromatin structural proteins, Ctcf (CCCTC-binding factor) and Hmgb2 (high mobility group protein B2), in regulating pathologic transcription and chromatin remodeling. Our data demonstrate a reciprocal relationship between Hmgb2 and Ctcf in controlling aspects of chromatin structure and gene expression. Both proteins regulate each others' expression as well as transcription in cardiac myocytes; however, only Hmgb2 does so in a manner that involves global reprogramming of chromatin accessibility. We demonstrate that the actions of Hmgb2 on local chromatin accessibility are conserved across genomic loci, whereas the effects on transcription are loci-dependent and emerge in concert with histone modification and other chromatin features. Finally, although both proteins share gene targets, Hmgb2 and Ctcf, neither binds these genes simultaneously nor do they physically colocalize in myocyte nuclei. Our study uncovers a previously unknown relationship between these two ubiquitous chromatin proteins and provides a mechanistic explanation for how Hmgb2 regulates gene expression and cellular phenotype. Furthermore, we provide direct evidence for structural remodeling of chromatin on a genome-wide scale in the setting of cardiac disease.

Article, see p 1528 Heart failure is a growing public health concern, affecting 20% of individuals at some point in their lifetime and contributing to 11% of deaths, with an incidence that is expected to rise by 25% over the next 15 years.1 As a condition that can develop asymptomatically for years, after which the efficacies of most interventions are modest and palliative, heart failure is a prime target for the development of novel biomarkers that could be used in a clinical setting for stratification or as prognostic markers detectable before clinical presentation (the ultimate prize). Are the tools of high-throughput biology, so-called ’omics investigations, up to the task? Discovery technologies and their analytic platforms, especially for genomics, epigenomics, and transcriptomics, are comprehensive and, if not fully mature, rapidly approaching that point, whereas other ’omics techniques such as proteomics, metabolomics, and lipidomics are increasingly quantitative, reproducible, and amenable to application outside specialized laboratories. These approaches have been widely applied to lower organisms and, at an increasing rate, in the clinical setting. Get on with it then, one might argue, and use these methods to improve human cardiovascular health on a population scale. If only it were so easy. Genetic variability is a massive confounder in the study of common disease in large human populations. Many of the most successful and largest cohort studies are characterized by modest ethnic and genetic diversity. Syndromes such as heart failure are polygenic, and genome-wide association studies have identified only a few causal variants, each of which explains only a small fraction of the genetic basis of the syndrome. Add to this the contribution of environmental factors, and it is easy to see why current …

Loss of the chromatin structural protein CTCF induces heart failure. Chromatin capture experiments (Hi-C), which detect genomic interactions, revealed global remodeling of chromatin structure after cardiac-specific depletion of CTCF or pressure overload (TAC). In the present study, we investigated epigenomic features (histone marks and DNA methylation) contributing to distinct behaviors of chromatin microenvironments. CTCF-KO or TAC induced gene expression changes mediated by a shift in compartmentalization between active and inactive chromatin (~4% of the genome). At the transcriptome level, genes significantly dysregulated in disease were involved in cardiac function ( e.g. , Mybpc3, Lmna , Hand2 ), chromatin structure ( e.g. , Hmgb2 , Rad21 ), and histone modification ( e.g. , Hdac4 , Mapk14, Foxo1 ). Our Hi-C data allowed us to correlate changes in gene expression with localization within the nucleus, showing efficient segregation of differentially expressed genes into specific neighborhoods with similar expression behavior. Analyses of histone marks in these neighborhoods reveal enrichment of activating and inhibiting marks, H3K9ac and H3K9me3, respectively, in the specialized chromatin environments associated with gene expression or silencing. DNA methylation studies showed that transcriptionally active neighborhoods tended to have unmethylated CpGs, while repressive environments were methylated. Our Hi-C data also showed how active environments positively influence the expression of the genes that physically interact with them. For example, 351 unique genes with interacting partner genes had increased H3K9ac after TAC, and their 3D interacting partners included 200 upregulated genes and 104 downregulated genes, demonstrating similar transcriptional behavior in areas with similar epigenomic features and anatomical distribution in the nucleus. Heart failure involves conserved structural reprogramming of these chromatin microenvironments.

Supplemental Digital Content is available in the text., If unifying principles could be revealed for how the same genome encodes different eukaryotic cells and for how genetic variability and environmental input are integrated to impact cardiovascular health, grand challenges in basic cell biology and translational medicine may succumb to experimental dissection. A rich body of work in model systems has implicated chromatin-modifying enzymes, DNA methylation, noncoding RNAs, and other transcriptome-shaping factors in adult health and in the development, progression, and mitigation of cardiovascular disease. Meanwhile, deployment of epigenomic tools, powered by next-generation sequencing technologies in cardiovascular models and human populations, has enabled description of epigenomic landscapes underpinning cellular function in the cardiovascular system. This essay aims to unpack the conceptual framework in which epigenomes are studied and to stimulate discussion on how principles of chromatin function may inform investigations of cardiovascular disease and the development of new therapies.

Epigenetic processes are known to have powerful roles in organ development across biology. It has recently been found that some of the chromatin modulatory machinery essential for proper development plays a previously unappreciated role in the pathogenesis of cardiac disease in adults. Investigations using genetic and pharmacologic gain- and loss-of-function approaches have interrogated the function of distinct epigenetic regulators, while the increased deployment of the suite of next-generation sequencing technologies have fundamentally altered our understanding of the genomic targets of these chromatin modifiers. Here, we review recent developments in basic and translational research that have provided tantalizing clues that may be used to unlock the therapeutic potential of the epigenome in heart failure. Additionally, we provide a hypothesis to explain how signal-induced crosstalk between histone tail modifications and long non-coding RNAs triggers chromatin architectural remodeling and culminates in cardiac hypertrophy and fibrosis.

Astrocytes are ubiquitous in the brain and are widely held to be largely identical. However, this view has not been fully tested, and the possibility that astrocytes are neural circuit specialized remains largely unexplored. Here, we used multiple integrated approaches, including RNA sequencing (RNA-seq), mass spectrometry, electrophysiology, immunohistochemistry, serial block-face-scanning electron microscopy, morphological reconstructions, pharmacogenetics, and diffusible dye, calcium, and glutamate imaging, to directly compare adult striatal and hippocampal astrocytes under identical conditions. We found significant differences in electrophysiological properties, Ca2+ signaling, morphology, and astrocyte-synapse proximity between striatal and hippocampal astrocytes. Unbiased evaluation of actively translated RNA and proteomic data confirmed significant astrocyte diversity between hippocampal and striatal circuits. We thus report core astrocyte properties, reveal evidence for specialized astrocytes within neural circuits, and provide new, integrated database resources and approaches to explore astrocyte diversity and function throughout the adult brain. VIDEO ABSTRACT.

The devastating impact of congenital heart defects has made mechanisms of vertebrate heart and vascular development an active area of study. Because myocyte death is a common feature of acquired cardiovascular diseases and the adult heart does not regenerate, the need exists to understand whether features of the developing heart and vasculature—which are more plastic—can be exploited therapeutically in the disease setting. We know that a core network of transcription factors governs commitment to the cardiovascular lineage, and recent studies using genetic loss-of-function approaches and unbiased genomic studies have revealed the role for various chromatin modulatory events. We reason that chromatin structure itself is a causal feature that influences transcriptome complexity along a developmental continuum, and the purpose of this article is to highlight the areas in which ‘omics technologies have the potential to reveal new principles of phenotypic plasticity in development. We review the major mechanisms of chromatin structural regulation, highlighting what is known about their actions to control cardiovascular differentiation. We discuss emergent mechanisms of regulation that have been identified on the basis of genomic and proteomic studies of cardiac nuclei and identify current challenges to an integrated understanding of chromatin structure and cardiovascular phenotype.

Transcriptome and proteome dynamics in higher eukaryotes are precisely controlled in a cell type-specific manner. Epigenetic regulation of development in mammals is not a new field; however, there have been several advances in the last decade that have profoundly altered our understanding of the mechanisms of epigenetic regulation and the extent to which genome function is differentially modified in the adult during disease. As the leading cause of death in the developed world, cardiovascular disease has been an area where novel findings about transcriptome regulation in lower organisms are rapidly investigated for their relevance to human health. Furthermore, the cardiovascular field has itself been an area of innovation and discovery, with new principles of biological networks arising that have fundamental, as well as translational importance (Barabasi et al. 2010; Chang & Bruneau, 2012; Weiss et al. 2012). To discuss the state-of-the-art in this area, The Physiological Society, at its meeting ‘Physiology 2014’, hosted a symposium entitled ‘Epigenetic regulation of cardiovascular development and disease’ on 1 July 2014 in the Queen Elizabeth II Conference Centre, overlooking Westminster Abbey, in London.

It has been appreciated for some time that cardiovascular disease involves large-scale transcriptional changes in various cell types. What has become increasingly clear only in the past few years, however, is the role of chromatin remodeling in cardiovascular phenotypes in normal physiology, as well as in development and disease. This review summarizes the state of the chromatin field in terms of distinct mechanisms to regulate chromatin structure in vivo, identifying when these modes of regulation have been demonstrated in cardiovascular tissues. We describe areas in which a better understanding of chromatin structure is leading to new insights into the fundamental biology of cardiovascular disease.

For precision medicine to become a reality, we propose 3 changes. First, healthcare deliverables must be prioritized, enabling translation of knowledge to the clinic. Second, physicians and patients must be convinced to participate, requiring additional infrastructure in health systems. Third, discovery science must evolve to shift the preclinical landscape for innovation. We propose a change in the fundamental relationship between basic and clinical science: rather than 2 distinct entities between which concepts must be translated, we envision a natural hybrid of these approaches, wherein discovery science and clinical trials coincide in the same health systems and patient populations. Even the generally progressive physician can be unsure and perhaps even skeptical about how precision medicine will fit into daily practice. In our view, precision medicine is personalized clinical care informed (primarily) by measurements of the genome, epigenome, proteome, transcriptome, metabolome, and microbiome. Precision medicine should result in a more healthy life with less disease burden, and it should be delivered on an individualized basis. Many therapies fail when they are tested in large animal models1,2 or when transitioning from phase I to phase II clinical trials,3,4 leading to a decrease in the number of new drugs making it to market.5 Despite improvements based on guideline adherence, cardiovascular disease remains the leading killer and a tremendous financial burden on the nation. Implementation of precision medicine will require collaboration across the spectrum of research and healthcare delivery, including funding agencies, insurers, academic medical centers, private hospitals and consumer ‘omics providers, and the potentially underappreciated relationship between patient and physician. Where to begin? How to convince patients and families to enroll? How to persuade clinicians to participate? How to add value to the health system? Who should pay? In answering these questions, we considered a new model …

The heart’s response to stress entails precise gene expression changes to affect the metabolic and structural features of the cardiomyocyte. The changes in gene expression are mediated by structural alterations in the packaging of the genome. However, the manner in which the three-dimensional architecture of the genome is established is unknown. In non-cardiac cells, genes that are actively transcribed are thought to reside in transcriptionally permissive compartments called transcription factories. The structural principles for achieving cardiac-specific transcription are not understood. We sought to understand the functional nature of cardiac transcription factories: whether they are stable structures (to which genes move in and out of) or are transiently formed around genes in response to cardiac stimuli. Using 5-fluorouridine incorporation into nascent RNA, we quantified changes in RNA polymerase II-mediated transcription in cardiomyocytes upon hypertrophic stress. Furthermore, we characterized the spatial distribution of transcription factories, marked by RNA polymerase II, from adult mice subjected to pressure overload. Using super-resolution microscopy, our analyses revealed reorganization of RNA polymerase II, evidenced by a significant increase in the distance between clusters (130nm in sham to 132.5nm in failing hearts, p=0.02) and a 38% increase in cluster intensity in failing hearts. To understand regulation of cardiac gene expression, we used DNA fluorescence in situ hybridization to map the nuclear position of the gene for SERCA2a (atp2a2), which is down regulated in disease. In failing hearts, we measured increased association of atp2a2 with the nuclear envelope (0/159 loci in sham to 11/278 loci in failure) and increased colocalization with heterochromatin (53/160 loci in sham versus 139/290 loci in failure), providing a structural mechanism for the decrease in SERCA2a expression. In contrast, atp2a2 positioning in the liver remained unaffected, with the majority of loci colocalizing with heterochromatin. These findings show that RNA polymerase II is redistributed to affect transcriptional programming and characterize for the first time the structural rearrangements in chromatin that underpin cardiac pathology.

Post-operative atrial fibrillation (AF) is the most common complication for cardiac surgery, with an incidence of 27-40% and is associated with increased 30-day mortality. Several models exist to predict post-op AF using clinical data, however none have been widely implemented, and all have room for improvement (average area under ROC curve is ~0.70). We tested whether DNA methylation, assayed from pre-operative blood draws, could add predictive value. We enrolled a cohort of 55 adult patients undergoing cardiopulmonary bypass, with a 36% incidence of post-op AF. Reduced representative bisulfite sequencing provided single-base resolution for an average of 3 million CpGs with 10x sequencing coverage per sample. We first tested whether these CpGs were correlated with AF. Of the 809,569 CpGs that passed thresholds in all samples, 16,774 were significantly correlated with AF (corrected p-value

Expression of a cohort of disease-associated genes, some of which are active in fetal myocardium, is considered a hallmark of transcriptional change in cardiac hypertrophy models. How this transcriptome remodeling is affected by the common genetic variation present in populations is unknown. We examined the role of genetics, as well as contributions of chromatin proteins, to regulate cardiac gene expression and heart failure susceptibility. We examined gene expression in 84 genetically distinct inbred strains of control and isoproterenol-treated mice, which exhibited varying degrees of disease. Unexpectedly, fetal gene expression was not correlated with hypertrophic phenotypes. Unbiased modeling identified 74 predictors of heart mass after isoproterenol-induced stress, but these predictors did not enrich for any cardiac pathways. However, expanded analysis of fetal genes and chromatin remodelers as groups correlated significantly with individual systemic phenotypes. Yet, cardiac transcription factors and genes shown by gain-/loss-of-function studies to contribute to hypertrophic signaling did not correlate with cardiac mass or function in disease. Because the relationship between gene expression and phenotype was strain specific, we examined genetic contribution to expression. Strikingly, strains with similar transcriptomes in the basal heart did not cluster together in the isoproterenol state, providing comprehensive evidence that there are different genetic contributors to physiological and pathological gene expression. Furthermore, the divergence in transcriptome similarity versus genetic similarity between strains is organ specific and genome-wide, suggesting chromatin is a critical buffer between genetics and gene expression.