Systems proteomics of cardiac chromatin identifies nucleolin as a regulator of growth and cellular plasticity in cardiomyocytes

Myocyte hypertrophy antecedent to heart failure involves changes in global gene expression, although the preceding mechanisms to coordinate DNA accessibility on a genomic scale are unknown. Chromatin-associated proteins alter chromatin structure by changing their association with DNA, thereby altering the gene expression profile. Little is known about the global changes in chromatin subproteomes that accompany heart failure, and the mechanisms by which these proteins alter chromatin structure. The present study tests the fundamental hypothesis that cardiac growth and plasticity in the setting of disease recapitulates conserved developmental chromatin remodeling events. We used quantitative proteomics to identify chromatin-associated proteins extracted via detergent and to quantify changes in their abundance during disease. Our study identified 321 proteins in this subproteome, demonstrating it to have modest conservation (37%) with that revealed using strong acid. Of these proteins, 176 exhibited altered expression during cardiac hypertrophy and failure; we conducted extensive functional characterization of one of these proteins, Nucleolin. Morpholino-based knockdown of nucleolin nearly abolished protein expression but surprisingly had little impact on gross morphological development. However, hearts of fish lacking Nucleolin displayed severe developmental impairment, abnormal chamber patterning and functional deficits, ostensibly due to defects in cardiac looping and myocyte differentiation. The mechanisms underlying these defects involve perturbed bone morphogenetic protein 4 expression, decreased rRNA transcription, and a shift to more heterochromatic chromatin. This study reports the quantitative analysis of a new chromatin subproteome in the normal and diseased mouse heart. Validation studies in the complementary model system of zebrafish examine the role of Nucleolin to orchestrate genomic reprogramming events shared between development and disease.

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Abstract 137: Ctcf is Essential for Transcriptome Regulation in the Healthy Heart

Circulation Research ◽

10.1161/res.119.suppl_1.137 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Manuel Rosa-Garrido ◽

Todd Kimball ◽

Douglas J Chapski ◽

Tsai-Ting Shih ◽

Elizabeth Soehalim ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Severe Heart Failure ◽

Surgical Techniques ◽

Ctcf Binding ◽

Global Changes ◽

Mouse Heart ◽

Precise Control ◽

Transcriptome Regulation ◽

Heart Size

Heart failure is a syndrome resulting from complex genetic predisposition and multiple environmental factors. It is well established that global changes in gene expression accompany the transition through hypertrophy and on to overt failure producing deterioration of function at the organ level. Unknown, however, are the global changes in chromatin structure that allows for pathological gene expression. The epigenetic factor CTCF is a ubiquitous protein thought to be involved in maintenance of chromatin microenvironments. We found that hypertrophic stimulation down-regulates CTCF in the heart, and CTCF expression is significantly correlated with heart size in disease across genetic backgrounds. To test this relationship further, we have generated an inducible cardiac-specific CTCF knockout animal. These animals quickly develop severe heart failure characterized by a dilated left ventricle, decreased ejection fraction, and reactivation of the fetal gene program. Our analysis of published CTCF ChIP-seq data performed in mouse heart has also shown CTCF binding peaks flanking the fetal genes. Furthermore, we have found that CTCF abundance at these sites changes after hypertrophic agonist isoproterenol treatment, suggesting that CTCF protects from the development of cardiac disease by regulating gene expression. We also used RNA-seq to compare the transcriptome of adult myocytes isolated from control, CTCF KO mice, and mice that underwent transverse aortic constriction (TAC) to induce heart failure. These analyses showed that CTCF depletion remodels gene expression in a manner that mimics aspects of the pathological transcriptome in heart failure. Our data demonstrate that precise control of CTCF levels is required for normal transcriptome regulation in the adult heart and that alterations in CTCF levels (either via genetic, pharmacologic or surgical techniques) lead to cardiac pathology.

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Abstract 1: Nucleolar Proteins Controlling Cardiac Phenotype in Rat Myocytes and Zebrafish Revealed by Chromatin Proteomics

Circulation Research ◽

10.1161/res.111.suppl_1.a1 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Emma Monte ◽

Kevin Mouillesseaux ◽

Haodong Chen ◽

Shuxun Ren ◽

Yibin Wang ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Cardiac Hypertrophy ◽

Ribosome Biogenesis ◽

Cardiac Cells ◽

Global Changes ◽

Cellular Plasticity ◽

Rdna Transcription ◽

Altered Expression ◽

Cardiac Phenotype

Cardiac hypertrophy is a common precursor to heart failure, during which cardiomyocytes grow to compensate for an increased workload. Hypertrophic cardiomyocytes undergo significant changes in cellular plasticity by adopting the expression profile and some phenotypic aspects of more primitive (embryonic or fetal) cardiac cells. These global changes in gene expression, conserved between humans and animal models of heart failure, must be preceded by structural alterations to the chromatin. Specifically, chromatin regions must be architecturally modified to allow or deny access for transcriptional machinery. However, the proteins responsible for remodeling chromatin to accomplish these gene expression changes during cardiac hypertrophy and failure are largely unknown. We used a proteomics approach to identify proteins bound to cardiac chromatin and to quantify changes in their abundance during disease. Quantitative mass spectrometry and bioinformatics revealed that 366 of the chromatin-bound proteins detected in this study displayed altered expression in a mouse model of pressure overload cardiac hypertrophy and failure. This included the chromatin remodeling protein Nucleolin (Ncl), which exhibited increased association with chromatin in the hypertrophic heart. To examine its role in regulating cardiac morphology and function we performed morpholino based knockdown of Ncl in zebrafish embryos. Ncl knockdown promoted the expression of bmp4 (a fetal marker), inhibited normal cardiomyocyte differentiation and resulted in abnormal heart chamber formation and looping. Hearts in surviving fish exhibited functional deficits as measured by fluorescence imaging and line-scanning analysis. To investigate the actions of Ncl in the mammalian cardiomyocyte, knockdown was carried out in isolated rat ventricular myocytes using siRNA. Loss of Ncl induced heterochromatin formation (increased Histone H3 K9-trimethylation), suppressed rDNA transcription (52% decrease in pre-rRNA via qPCR) and promoted fetal gene expression (65% increase in ANF; 41% increase in β-MHC transcripts). Overall, this study identifies Ncl as a regulator of chromatin structure, cell growth via ribosome biogenesis and cellular plasticity in the cardiomyocyte.

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Abstract 835: Downregulation Of Cardiomyocyte-enriched Micrornas Contributes To Altered Gene Expression In Heart Failure

Circulation ◽

10.1161/circ.116.suppl_16.ii_162-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Sadakatsu Ikeda ◽

Sek Won Kong ◽

Jun Lu ◽

Egbert Bisping ◽

Natalya Bodyak ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Calcium Signaling ◽

Target Genes ◽

Heart Function ◽

Altered Expression ◽

Regulate Gene Expression ◽

False Discovery ◽

Altered Gene ◽

Calmodulin Signaling

Background: MicroRNAs (miRNAs) are a novel class of non-coding RNAs that regulate gene expression posttransciptionally. Altered miRNA expression has been implicated in diverse human diseases such as cancer. Accumulating evidence suggests the importance of miRNAs in the heart. However, the contribution of miRNAs to heart disease remains incompletely understood. Methods and Results: We measured the expression of 261 miRNAs in heart failure resulting from transgenic overexpression of calcineurin. 59 miRNAs were confidently detected in the heart, and 11 miRNAs belonging to 6 families (miR-1, -15, -30, -133, -195, -208) were downregulated compared to non-transgenic control (Welch’s t-test nominal p<0.05, false discovery rate <0.001). The results were validated by qRTPCR. There was no upregulated miRNA. Four of these miRNAs (miR-1, -30, -133, -208) were enriched in a purified cardiomyocyte preparation, compared to non-myocytes. Downregulation of these four miRNAs was reproduced in purified failing versus non-failing cardiomyocytes. This excluded artifactual downregulation from reduced myocyte fraction in failing hearts. The remaining two miRNAs (miR-15, and -195) were exclusively expressed in non-cardiomyocytes and did not changed in failing cardiomyocytes. Next, we used Affymetrix expression profiling to show that the predicted targets of these downregulated miRNAs were disproportionately upregulated compared to the entire transcriptome (Fisher’s exact p < 0.001). This suggests an association between downregulation of these miRNAs and upregulation of predicted target genes in heart failure. One particularly intriguing target of the predominant cardiac microRNA miR-1 is calmodulin, a key regulator of calcium signaling. We showed that calmodulin and downstream calmodulin signaling to NFAT is regulated by miR-1 in cultured cardiomyocytes. Conclusion: Our results indicate that altered expression of cardiomyocyte-enriched miRNAs may contribute to abnormal gene expression in heart failure. The regulation of calmodulin and calcium signaling by miR-1 suggests a mechanism by which miR-1 may regulate heart function.

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Altered expression of a limited number of genes contributes to cardiac decompensation during chronic ventricular tachypacing in dogs

Physiological Genomics ◽

10.1152/physiolgenomics.00159.2006 ◽

2007 ◽

Vol 29 (1) ◽

pp. 76-83 ◽

Cited By ~ 23

Author(s):

Caroline Ojaimi ◽

Khaled Qanud ◽

Thomas H. Hintze ◽

Fabio A. Recchia

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Decompensated Heart Failure ◽

Left Ventricular ◽

Fold Change ◽

Differentially Expressed ◽

Altered Expression ◽

Cardiac Decompensation ◽

Number Of Genes ◽

End Stage

Our aim was to determine the changes in the gene expression profile occurring during the transition from compensated dysfunction (CD) to decompensated heart failure (HF) in pacing-induced dilated cardiomyopathy. Twelve chronically instrumented dogs underwent left ventricular pacing at 210 beats/min for 3 wk and at 240 beats thereafter, and four normal dogs were used as control. The transition from CD to HF occurred between the 3rd and 4th wk of pacing, with end-stage HF at 28 ± 1 days. RNA was extracted from left ventricular tissue at control and 3 and 4 wk of pacing ( n = 4) and tested with the Affymetrix Canine Array. We found 509 genes differentially expressed in CD vs. control ( P ≤ 0.05, fold change ≥±2), with 362 increasing and 147 decreasing; 526 genes were differentially expressed in HF vs. control ( P ≤ 0.05; fold change ≥±2), with 439 increasing and 87 decreasing. To better understand the transition, we compared gene alterations at 3 vs. 4 wk pacing and found that only 30 genes differed ( P ≤ 0.05; fold change of ±2). We conclude that a number of processes including normalization of gene regulation during decompensation, appearance of new upregulated genes and maintenance of gene expression all contribute to the transition to overt heart failure with an unexpectedly small number of genes differentially regulated.

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AGO1 in association with NEAT1 lncRNA contributes to nuclear and 3D chromatin architecture in human cells

10.1101/525527 ◽

2019 ◽

Cited By ~ 3

Author(s):

Muhammad Shuaib ◽

Krishna Mohan Parsi ◽

Hideya Kawaji ◽

Manjula Thimma ◽

Sabir Abdu Adroub ◽

...

Keyword(s):

Gene Expression ◽

Genome Organization ◽

Global Gene Expression ◽

Human Cells ◽

Nuclear Bodies ◽

Genome Architecture ◽

Global Changes ◽

Active Chromatin ◽

Altered Expression ◽

3D Genome

AbstractAside from their roles in the cytoplasm, RNA-interference components have been reported to localize also in the nucleus of human cells. In particular, AGO1 associates with active chromatin and appears to influence global gene expression. However, the mechanistic aspects remain elusive. Here, we identify AGO1 as a paraspeckle component that in combination with the NEAT1 lncRNA maintains 3D genome architecture. We demonstrate that AGO1 interacts with NEAT1 lncRNA and its depletion affects NEAT1 expression and the formation of paraspeckles. By Hi-C analysis in AGO1 knockdown cells, we observed global changes in chromatin organization, including TADs configuration, and A/B compartment mixing. Consistently, distinct groups of genes located within the differential interacting loci showed altered expression upon AGO1 depletion. NEAT1 knockout cells displayed similar changes in TADs and higher-order A/B compartmentalization. We propose that AGO1 in association with NEAT1 lncRNA can act as a scaffold that bridges chromatin and nuclear bodies to regulate genome organization and gene expression in human cells.

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Distinct gene expression profiles in adult mouse heart following targeted MAP kinase activation

Physiological Genomics ◽

10.1152/physiolgenomics.00224.2005 ◽

2006 ◽

Vol 25 (1) ◽

pp. 50-59 ◽

Cited By ~ 31

Author(s):

Scherise Mitchell ◽

Asuka Ota ◽

William Foster ◽

Bin Zhang ◽

Zixing Fang ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Map Kinase ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Mouse Heart ◽

Matrix Remodeling ◽

Long Term Effects ◽

Kinase Activation

Three major MAP kinase signaling cascades, ERK, p38, and JNK, play significant roles in the development of cardiac hypertrophy and heart failure in response to external stress and neural/hormonal stimuli. To study the specific function of each MAP kinase branch in adult heart, we have generated three transgenic mouse models with cardiac-specific and temporally regulated expression of activated mutants of Ras, MAP kinase kinase (MKK)3, and MKK7, which are selective upstream activators for ERK, p38, and JNK, respectively. Gene expression profiles in transgenic adult hearts were determined using cDNA microarrays at both early (4–7 days) and late (2–4 wk) time points following transgene induction. From this study, we revealed common changes in gene expression among the three models, particularly involving extracellular matrix remodeling. However, distinct expression patterns characteristic for each pathway were also identified in cell signaling, growth, and physiology. In addition, genes with dynamic expression differences between early vs. late stages illustrated primary vs. secondary changes on MAP kinase activation in adult hearts. These results provide an overview to both short-term and long-term effects of MAP kinase activation in heart and support some common as well as unique roles for each MAP kinase cascade in the development of heart failure.

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Abstract 355: Systems Epigenomics of Chromatin Structure in the Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a355 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Sarah Franklin ◽

Haodong Chen ◽

Elaheh Karbassi ◽

Emma Monte ◽

Thomas M Vondriska

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Chromatin Structure ◽

Genomic Structure ◽

Structural Proteins ◽

Basic Question ◽

Cell Type ◽

Chromatin Structural ◽

Genomic Scale ◽

Cell Type Specific

Except during metaphase, endogenous chromatin structure is unknown. DNA - invariant between cells - and the cell type-specific modifiers of the genome establish chromatin structural features, both local (e.g. at the scale of individual nucleosomes) and global (e.g. chromosomal territories). A fundamental question is how these cell type-specific modifiers, including DNA modification, non-coding RNAs, and proteins, establish the chromatin environment conducive to gene expression for the correct cell type: in cardiac muscle, how is the genome structurally poised to confer cardiac (and not, say, renal) transcriptomes and proteomes, and what physical reprogramming events occur during disease? To address these questions, we are conducting a systems analysis of the epigenetic features of the healthy and diseased heart. In adult mice, we have used quantitative mass spectrometry to dissect distinct fractions of the nucleus and reveal the itineraries of chromatin structural proteins, enzymatic nucleosome remodelers, histone molecules and histone post-translational modifications. These studies have revealed rules for global reprogramming of gene expression, which involve altered abundance of non-histone chromatin structural proteins, a shift from hetero- towards euchromatic post-translational marks and a decreased linker to core histone ratio in heart failure. Furthermore, interrogation of genome-wide binding patterns of known cardiac transcription factors within the genes that encode proteins operative in cardiac genomic structure reveals hierarchical relationships between these protein, transcript and gene networks. Complementary investigations in isolated myocytes are characterizing the global rearrangement of chromatin following hypertrophic agonist treatment using conventional and super-resolution microscopy to directly visualize the chromatin backbone. Lastly, a combination of multiple genomic scale sequencing studies have revealed regions under control of specific chromatin structural proteins. Together, these studies aim to address the basic question of how global chromatin structure is maintained in cardiac myocytes and how diseases like heart failure result from deranged chromatin structure on a genomic scale.

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Cardiac gene expression profile in rats with terminal heart failure and cachexia

Physiological Genomics ◽

10.1152/physiolgenomics.00165.2004 ◽

2005 ◽

Vol 20 (3) ◽

pp. 256-267 ◽

Cited By ~ 38

Author(s):

Maren Wellner ◽

Ralf Dechend ◽

Joon-Keun Park ◽

Erdenechimeg Shagdarsuren ◽

Nidal Al-Saadi ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Cardiac Hypertrophy ◽

Expression Profile ◽

Left Ventricular ◽

Altered Expression ◽

Cardiac Gene Expression ◽

Terminal Heart Failure ◽

Cardiac Gene ◽

Blood Pressures

About one-half of double transgenic rats (dTGR) overexpressing the human renin and angiotensinogen genes die by age 7 wk of terminal heart failure (THF); the other (preterminal) one-half develop cardiac damage but survive. Our study’s aim was to elucidate cardiac gene expression differences in dTGR-THF compared with dTGR showing compensated cardiac hypertrophy but not yet THF. dTGR treated with losartan (LOS) and nontransgenic rats (SD) served as controls. THF-dTGR body weight was significantly lower than for all other groups. At death, THF-dTGR had blood pressures of 228 ± 7 mmHg (cardiac hypertrophy index 6.2 ± 0.1 mg/g). Tissue Doppler showed reduced peak early (Ea) to late (Aa) diastolic expansion in THF-dTGR, indicating diastolic function. Preterminal dTGR had blood pressures of 197 ± 5 mmHg (cardiac hypertrophy index 5.1 ± 0.1 mg/g); Ea < Aa compared with LOS-dTGR (141 ± 6 mmHg; 3.7±0.1 mg/g; Ea > Aa) and SD (112 ± 4 mmHg; 3.6 ± 0.1 mg/g; Ea > Aa). Left ventricular RNA was isolated for the Affymetrix system and TaqMan RT-PCR. THF-dTGR and dTGR showed upregulation of hypertrophy markers and α/β-myosin heavy chain switch to the fetal isoform. THF-dTGR (vs. dTGR) showed upregulation of 239 and downregulation of 150 genes. Various genes of mitochodrial respiratory chain and lipid catabolism were reduced. In addition, genes encoding transcription factors (CEBP-β, c-fos, Fra-1), coagulation, remodeling/repair components (HSP70, HSP27, heme oxygenase), immune system (complement components, IL-6), and metabolic pathway were differentially expressed. In contrast, LOS-dTGR and SD had similar expression profiles. These data demonstrate that THF-dTGR show an altered expression profile compared with preterminal dTGR.

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Abstract 240: Differential Impact of RBFox Family Proteins in Adult Murine Heart

Circulation Research ◽

10.1161/res.119.suppl_1.240 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Chen Gao ◽

Shuxun Ren ◽

Yibin Wang

Keyword(s):

Heart Failure ◽

Alternative Splicing ◽

Rna Splicing ◽

Adult Mouse ◽

Regulatory Elements ◽

Global Changes ◽

Mouse Heart ◽

Cardiac Physiology ◽

Differential Impact ◽

Adult Heart

Background: The complexity of cardiac transcriptome and proteome is significantly contributed by alternative splicing of mRNA. Alternative splicing is regulated by the cis-regulatory elements located in pre-mRNA together with the trans-activating factors guiding the assembling and function of the spliceosome. In our earlier study, we have observed global changes of alternative splicing events during pressure-overload induced heart failure, and identified RBFox1 as a key regulator for cardiac RNA splicing regulation during postnatal development and pathological remodeling. Both RBFox1 and RBFox2 are highly enriched in cardiomyocytes, and their expression are both significantly repressed in response to pathological stress. Loss-of-function studies for RBFox1 and RBFox2 are achieved using cardiac specific but constitutively active Cre. Therefore, the isoform specific contribution of RBFox1 vs. RBFox2 in maintaining cardiac physiology and homeostasis in adult heart is unknown. Methods and Results: We generated mouse models of cardiac specific and inducible knockout of RBFox1 and RBFox2 individually in adult hearts by breeding the individual floxed alleles with the αMHC-Mer-Cre-Mer mice. At baseline, inactivating RBFox1 in adult heart caused a slight but significant decrease of cardiac function without activating hypertrophy gene expression. However, following myocardial infarction, the RBFox1 deficient hearts showed enhanced global fibrosis in non-infarcted areas comparing to the control animals. In contrast, inactivating RBFox2 in adult mouse heart caused overt heart failure associated with chamber dilation without external stress as early as 2 weeks post tamoxifen administration. Conclusion: We have identified differential impact of RBFox1 and RBFox2 deficiency in adult mouse heart. Our in vivo study illustrate the functional importance of the RBFox family RNA splicing regulators in normal physiology of adult heart, and support the pathogenic contribution of loss of RBFox expression to heart failure. Further analysis focusing on the underlying molecular mechanisms for their differential impact would yield new insights on transcriptome regulation and complexity in cardiac physiology and diseases.

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Abstract 471: Paradigm Shift to Epigenetic Memory for the Pathological Understanding of Chronic Heart Failure

Circulation ◽

10.1161/circ.118.suppl_18.s_304-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Yoshihiro Asano ◽

Seiji Takashima ◽

Yulin Liao ◽

Masafumi Kitakaze

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Transcription Factors ◽

Chronic Heart Failure ◽

Chromatin Structure ◽

Epigenetic Modification ◽

Heart Function ◽

Epigenetic Mechanism ◽

Epigenetic Markers

Background- Epigenetic modification becomes a popular regulatory mechanism of gene expression in development or tumor progression. However its role in cardiovascular diseases has not been elucidated. Since reactivation of cardiac fetal gene expression in chronic heart failure (CHF) is more remarkable than that expected by the increased transcriptional factors, the concept of epigenetic modification may be required for the explanation of such a phenomenon. We tested this idea. Methods- To test the epigenetic potency in cardiomyocytes, we focused on the expression of both BNP and ANP genes. As epigenetic markers, nucleic chromatin structure by electron microscopy and the entire-regional histone modifications were evaluated using our newly developed in vivo chromatin immunoprecipitation (ChIP) assay. Results- In the developmental stage of hearts, we observed a parallel movement of fetal gene expression and epigenetic markers. On the other hand, in the short time stimuli of GPCR agonists in murine cardiomyocytes, fetal gene expression was controlled only by the amount of transcription factors, Gata4 and Nkx2.5, but epigenetic maker was not altered. In contrast, in in vivo murine model of CHF both Gata4 and Nkx2.5 depressed despite elevated expression of both BNP and ANP. Nucleic chromatin structure in failing cardiomyocytes was changed to less condensed forms and increased accumulation of both histone H3K9-acetylation and H3K4-trimethylation was observed across these gene loci. Also in human failing heart, similar changes of nucleic chromatin structure were observed as that in murine CHF model and interestingly these changes was reversed when the heart function was recovered along with proper treatment. Conclusions- We for the first time clarified that both BNP and ANP reactivation mechanisms in chronic heart failure link to the alteration of histone modification by the in vivo ChIP method. Different from acute stimuli, this change was coincided with nucleic chromatin structure and was explicable of paradoxical depression of transcription factors, suggesting epigenetic potency. Epigenetic mechanism may play a pivotal role in the nuclear memory-mediating failing myocardium, raising the hope for a novel pathological understanding for human CHF.

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