REVIEWS Epigenetic modifications and noncoding RNAs in cardiac hypertrophy and failure Carolina M. Greco and Gianluigi Condorelli Abstract | The regulatory networks governing gene expression in cardiomyocytes are under intense investigation, not least because dysregulation of the gene programme has a fundamental role in the development of a failing myocardium. Epigenetic modifications and functional non-protein-coding RNAs (ncRNAs) are important contributors to this process. The epigenetic modifications that regulate transcription comprise post-translational changes to histones—the proteins around which DNA is wound—as well as modifications to cytosine residues on DNA. The most studied of the histone changes are acetylation and methylation. Histone acetylation is known to be important in cardiac physiology and pathophysiology, but the roles of other histone modifications and of cytosine methylation are only starting to be investigated. Understanding of the role of microRNAs has also seen major advancements, but the function of long ncRNAs is less well defined. Moreover, the connection between ncRNAs and epigenetic modifications is poorly understood in the heart. In this Review, we summarize new insights into how these two layers of gene-expression regulation might be involved in the pathogenesis of cardiac hypertrophy and failure, and how we are only beginning to appreciate the complexity of the interactive network of which they are part. Greco, C. M. & Condorelli, G. Nat. Rev. Cardiol. advance online publication 12 May 2015; doi:10.1038/nrcardio.2015.71

Introduction

Humanitas Clinical and Research Hospital, University of Milan, Via Manzoni 56, Rozzano, 20089 Milan, Italy (C.M.G., G.C.).

Gene expression in postnatal cardiomyocytes—the power units of adult myocardium—was demonstrated 35 years ago to be modifiable on the basis of the specific physiological or pathological state of the organism.1,2 Reprogramming of cardiac gene expression, consisting of upregulation and downregulation of particular sets of genes, is critical in modulating heart function and has a fundamental role in the pathogenesis of cardiac hypertrophy and heart failure.1,2 Mechanisms similar to those that control gene expression in heart development are at the basis of hypertrophy and failure.3 Therefore, the regulatory networks that govern heart development and the adaptation of the adult organ to pathophysiological stimuli are under intense investigation. To date, these studies have highlighted the complex interplay between the various hierarchical levels of gene regulation. Some of the latest advances in the study of geneexpression regulation have been in the fields of epi­ genomic modification and noncoding (nc) RNAs. The former comprises secondary chemical alterations of DNA (methylation of cytosines) and the proteins around which the DNA is wound (post-translational modification of histones),4,5 which induce changes to the state of chromatin. Transcription is modulated by rendering the regulatory elements of genes more or less permissive to the activity of transcription factors (Box 1). Conversely, ncRNAs comprise diverse groups of RNA that do not

Correspondence to: G.C. gianluigi.condorelli@ humanitasresearch.it

Competing interests The authors declare no competing interests.

encode proteins. The established view for many years was that these transcripts did not have a biological function, but they are now recognized to be a fundamental part of gene-expression regulatory mechanisms. In this Review, we provide an overview of epigenetic modifications and ncRNAs, highlighting their essential role in an integrated regulatory network that, when dysregulated, can lead to cardiac hypertrophy and failure.

Epigenetic modifications DNA methylation Methylation of DNA at position 5 of cytosine (5‑mC) is a highly conserved epigenetic mark that signals for transcriptional repression (Figure 1).6 This modification also has important roles in transposon silencing, genomic imprinting, X‑chromosome inactivation, and development.7 In mammals, methylated cytosines are found preferentially on cytosine–(phosphate)–guanine (CpG) dinucleotides, which tend to cluster in regions called CpG islands and on repetitive elements of the genome (such as satellite DNA, centromeric repeats, retrotransposon elements, and ribosomal DNA). In somatic cells, CpG islands are mainly distributed on the 5' end of genes, and the majority (60–80%) of CpG cytosines are methylated.8 Three mammalian DNA methyltransferases (DNMTs) have been identified: DNA (­cytosine‑5)-methyltransferse­ 1 (DNMT1), DNA (cytosine‑5)-methyltransferase 3A (DNMT3A), and DNA (cytosine‑5)-methyltransferase 3B (DNMT3B). DNMT1 is involved in the maintenance of established DNA methylation and binds preferentially to

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REVIEWS Key points ■■ The myocardium adapts to continued stress—either physiological or pathological—by modulating gene expression in its constituent cells ■■ Methylation of cytosine and post-translational chemical alteration of histones —known as epigenetic modification—render regulatory elements of genes more or less permissive to interaction with the transcriptional machinery ■■ These alterations create an epigenetic landscape, or ‘epigenome’, that is probably specific to a particular physiological or pathological state ■■ A plethora of noncoding RNAs, such as microRNAs and long noncoding RNAs, is centrally involved in the regulation of probably all biological processes, including gene expression ■■ Epigenetic modifications and noncoding RNAs form an integrated and highly complex regulatory network to control gene expression; heart failure is associated with disruption of this network ■■ The reversibility of epigenetic modifications and the relative ease with which noncoding RNAs can be manipulated augur well for the development of much‑needed novel pharmaceuticals for treatment of heart failure

Box 1 | Regulatory DNA elements The expression of mammalian protein-coding genes can be regulated by several mechanisms, including transcription, mRNA processing, transport, translation, and stability. Transcription is controlled by RNA polymerase II, which binds to two distinct families of regulatory DNA elements: proximal regulatory elements, otherwise known as promoters; and distal regulatory elements, which include enhancers, insulators, and silencers. The proximal promoter is the region immediately upstream of the transcriptional start site, and usually contains multiple binding sites for activating transcriptional factors. Importantly, about 60% of human genes harbour a cytosine–(phosphate)–guanine (CpG) island,109 a sequence of DNA with a high cytosine/guanine content, and that can be silenced by DNA methylation. Enhancers are distal regions that stimulate the transcription not only of the nearest promoter, but also of distant promoters, even on other chromosomes, by forming DNA ‘loops’.110 Although enhancers can bind to the same transcription factors as promoters, enhancers can be distinguished from promoters by their histone modification profile and trans-acting factors.111,112 These distal regions are particularly enriched in monomethylated lysine 4 on histone H3 (H3K4) and in the transcriptional activator p300. By modulating chromatin structure, epigenetic modifications regulate the accessibility of DNA to promoters and enhancers, and in this way control gene expression. Promoters and enhancers display, and are governed by, specific epigenetic signatures: active promoters are marked by high levels of monoacetylated lysine 9 and lysine 14 (H3K9ac and H3K14ac) and by trimethylated (me3) lysine 4 (H3K4me3); inactive promoters are characterized by methylated DNA, trimethylation of lysine 9 and lysine 27 on histone H3 (H3K9me3 and H3K27me3), and deacetylation of histones.113 By contrast, active enhancers are mainly enriched in H3K27ac, whereas an absence of H3K27ac and a high level of H3K27me3 defines enhancers as being ‘poised’.114

hemi-methylated DNA, allowing symmetrical propagation of DNA methylation during mitosis.7,9 By contrast, DNMT3A and DNMT3B establish de novo DNA methylation patterns,10 generally targeting unmethylated promoters and other epigenetic repressors.7 Moreover, although DNA methylation was historically considered a stable epigenetic mark, the discovery of 5‑hydroxymethylcytosine (5‑hmC; an oxidation product of 5‑mC generated by the ten–eleven translocation family of enzymes) revealed the presence of an active DNA demethylation mechanism in mammalian cells.11 DNA methylation regulates gene expression either by directly blocking transcription-factor binding or by

recruiting methyl-binding proteins, namely methylCpG-binding domain protein (MBD) 1, 2, and 3, and methyl-CpG-binding protein 2 (MECP2), which in turn interact with co-repressor complexes.12 Interestingly, several methyl-binding proteins, including MBD3, MBD4, and MECP2, also bind to hydroxymethylated DNA, suggesting that 5‑hmC is not only an intermediate of DNA demethylation, but also a stable epigenetic mark that can influence gene expression by attracting or precluding the binding of specific proteins.8 To date, knowledge of the function of DNA methy­ lation and the associated enzymes and cofactors in cardiac hypertrophy and failure is limited to the findings of preliminary studies. Immunoprecipitation of methylated DNA from cardiac biopsies, followed by massive DNA sequencing, has indicated that the profile of this epigenetic modification is altered in patients with end-stage cardiomyopathy. 13 DNA methylation differed significantly at the CpGs of promoters and gene bodies in cardiomyopathy compared with healthy hearts; moreover, increased gene expression in failing hearts was associated with promoter demethylation, whereas hypermethylation did not clearly correlate with decreased expression.13 Further analysis revealed that CpG islands associated with double-homeobox protein 4 (DUX4) were heavily methylated in failing hearts, and that hypermethylation of this region was associated with downregulation of the DUX4 transcript.13 Given that DNA methylation has an important role in genome stability by repressing repetitive elements, researchers in another study investigated the effect of differential methylation of repetitive elements in end-stage cardiomyopathy.14 Compared with nonfailing hearts, failing hearts were significantly hypomethylated at satellite regions, and this correlated with a 27‑fold increase of the corresponding transcripts.14 Moreover, a low-resolution DNA methylation microarray analysis of the myocardium of patients with idiopathic dilated cardiomyopathy revealed that altered DNA methylation patterns caused abnormal expression of LY75 (encoding lymphocyte antigen 75) and ADORA2A (encoding adenosine receptor A2a).15 In vivo studies of the zebrafish orthologues of these genes linked them with adaptive and maladaptive heart failure.15 However, cardiomyocytes constitute only 20–30% of the cells of the heart, so these initial studies conducted on human samples are limited because a mixed-cell population was analysed. Investigation of the DNA methylome in specific cardiac cells, such as cardiomyocytes, fibroblasts, endothelial, and immune cells, is needed. A whole-genome bisulfite sequencing study has addressed this problem in an animal model by isolating cardiomyocytes from mouse hearts subjected to pressure overload for 3 weeks.16 Subtle, but significant, differences in the methylation profiles of normal and hypertrophic cells were found, mostly in intergenic regions.16 The investigators also determined that failing cardiomyocytes had a methylation profile that partially resembled that observed in fetal cells.16 The modest magnitude of the differences observed in this study might be explained

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REVIEWS Active enhancer 5hmC H3K27ac H3K4me1 5hmC

5hmC 5hmC

5hmC

5hmC Pol II Core promoter

HMTs

HATs

DNMTs

TETs

HDACs

5mC

5mC

HDMs

5mC

Closed or poised enhancer H3K27me3 H3K4me1

Active chromatin H3K9ac H3K14ac H3K4me3

5mC

5mC

Inactive chromatin H3K27me3 H3K9me3

Figure 1 | DNA and chromatin modifications in gene-expression regulation. Nature Reviews | Cardiology Gene transcription can be regulated by two distinct families of DNA elements: promoters and enhancers. By modulating chromatin structure, epigenetic modifications regulate the accessibility of DNA to these elements, and thereby control gene expression. Chromatin organization is determined by covalent modifications of DNA and the N‑terminal tails of histones. Promoters and enhancers display specific epigenetic signatures governed by DNA and histone modifiers. These chromatin signatures are involved in the control of cardiac genes. Abbreviations: DNMT, DNA methyltransferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; HDM, histone demethylase; HMT, histone methyltransferase; Pol II, RNA polymerase II; TET, ten–eleven translocation; 5hmC, 5‑hydroxymethylcytosine; 5mC, 5‑methylcytosine. Box 2 | Chromatin Chromatin is a macromolecular complex of DNA and histones. The basic unit of chromatin is the nucleosome, an octamer of four core histone proteins (two each of H2A, H2B, H3, and H4) around which 147 base pairs of DNA are wrapped. This allows a fivefold packaging of DNA to be achieved. The nucleosomes are then tightly packed into higher-order structures to condense DNA further. The N‑terminal tails of all eight core histone proteins can undergo posttranslational covalent modifications, among which are acetylation, methylation, phosphorylation, ubiquitination, and poly-ADP-ribosylation.115 Apart from the modification of histones, chromatin structure can also be regulated by DNA methylation and ATP-dependent chromatin remodelling complexes. Transcription, DNA repair, replication, and recombination can all be controlled in this way.116

by the DNA sequencing technology used: although whole-genome bisulfite sequencing allows resolution at the base-pair level, the technique is inefficient at capturing small dynamic changes of 5‑mC.17 In addition, the results do not discriminate between 5‑mC and 5‑hmC, which can lead to overestimation of the 5‑mC level.18 Antibody-based immunoprecipitation approaches might help to elucidate the role of both modifications in failing cardiomyocytes. In summary, these preliminary findings support a role for DNA methylation in dysregulation of gene expression associated with heart failure, but whether this epigenetic mark contributes substantially to pathogenesis remains uncertain, as do the underlying mechanisms of this process.

Histone modifications Acetylation Histone acetylation occurs on lysine residues and promotes relaxation of chromatin structure (Box 2) and, therefore, transcriptional activation. Conversely, histone hypoacetylation leads to chromatin condensation and repression of gene expression. Acetylation is a highly dynamic modification and is regulated by the opposing actions of two families of enzymes: histone acetyltransferases and histone deacetylases (HDACs).19 Histone acetyltransferases use acetyl coenzyme A as a cofactor to catalyse acetylation, and are divided into two major classes. Type A are nuclear and include N‑acetyltransferase 6 (NAT6), the MYST family (named after the founding members MOZ, Ybf2, Sas2, and TIP60), and CBP/p300 proteins. By contrast, type B are predominantly cyto­ plasmic.20 Several reports have indicated the importance of p300 in cardiac development 21,22 and heart failure23–25 through transcription controlled by myocyte-specific enhancer factor (MEF) 2 and transcription factor GATA‑4. Eleven HDAC members have been identified, and are subdivided into four classes (I–IV) according to cellular localization, enzymatic activity, and protein structure. The role of HDACs in cardiac hypertrophy and failure is complex: some HDACs are antihypertrophic whereas others promote hypertrophy (Table 1). For example, loss of function of either HDAC5 or HDAC9 (two class II HDACs) has been associated with increased susceptibility to cardiac hypertrophy and failure, owing to the capacity of these enzymes to bind and silence MEF2C.26,27 HDAC4, another class II HDAC, was found to repress the activities of MEF2 and serum response factor under physiological conditions, but in cardiac hypertrophy became oxidized, causing it to shuttle out of the nucleus and allow derepression of prohyper­ trophy genes.28 Moreover, nuclear export of HDAC4 has been linked to demethylation of H3K9, dissociation of chromobox protein homologue, and activation of the hypertrophy-related gene NPPA, which encodes atrial natriuretic peptide.29 Therefore, cardiac stress induces derepression of class II HDAC target genes by triggering the nuclear export of these enzymes after they are oxidized. In addition, nuclear export can be signalled via phosphorylation of the HDAC. For example, serine/ threonine-protein kinase D1 and calcium/calmodulindependent protein kinase stimulate nuclear export of HDAC5 and HDAC9 by phosphorylating a set of serine residues on their N‑terminal regulatory domain, which can then bind the chaperone 14‑3‑3 protein.30–32 This phosphorylation-dependent nuclear export of HDACs can be blocked by cAMP-dependent protein kinase (protein kinase A; PKA). Indeed, PKA has been reported to induce cleavage of HDAC4, producing an N‑terminal fragment that inhibits the activity of MEF2, but not of serum response factor.33 PKA has also been shown to lead to repression of gene transcription via nuclear accumulation of HDAC5.34 Furthermore, the catalytic activity of the class II HDAC6 has been found to be increased in pathological cardiac hypertrophy,35 and to contribute to cardiac dysfunction in response to angiotensin II,

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REVIEWS Table 1 | Role of chromatin modifiers in cardiac hypertrophy Gene

Animal model

Phenotype

Result

Cardiac hypertrophy and dysfunction

Enhanced acetylation of MEF2 and transcription factor GATA‑423–25

Cardiac hypertrophy

Activation of the Akt/GSK‑3β pathway37

Histone acetyltransferases EP300

Overexpression in the myocardium

Histone deacetylases, class I HDAC2

Cardiac-specific overexpression

Histone deacetylases, class II HDAC4

Cardiac-specific deletion

No described phenotype

Histone deacetylase 4 controls H3K9 demethylation and HP1 dissociation to the NPPA promoter in response to elevated preload29

HDAC5

Histone deacetylase 5 signal-resistant mutant

Sudden death of male mice with loss and morphological changes of cardiac mitochondria

Downregulation of PPARγ coactivator 1α39

HDAC5 and HDAC9

HDAC5 and HDAC9 mutant mice

Enhanced hypertrophy and failure in response to cardiac stress

Histone deacetylase 5/9 bind to MEF2C and inhibit its transcriptional activity26,27

HDAC6

HDAC6 null mice

Maintained cardiac function in response to angiotensin II

Increased myofibril force generation36

Protected from hypertrophic stimuli

Cardiac hypertrophy with abnormal cardiac metabolism owing to excessive activity of PPARα38

Histone deacetylases, class III HDAC3

Cardiac-specific conditional deletion

Histone methyltransferases KDM4A

Overexpression or cardiac-specific deletion

Enhanced or attenuated hypertrophic response

Lysine-specific demethylase 4A activates four and a half LIM domains protein 1 in a myocardin-dependent and serum-response-factor-dependent manner48

PAXIP1

Cardiac-specific conditional deletion

Altered calcium handling and contractility

PAC-interacting protein 1 regulates the expression of KCNIP249

Attenuated cardiac hypertrophy

Transcription activator BRG1 complexes with histone deacetylases and poly [ADP-ribose] polymerases driving the switch from myosin‑6 to myosin‑761

ATP chromatin remodellers SMARCA4 (BRG1)

Cardiac-specific conditional deletion

Abbreviations: MEF, myocyte-specific enhancer factor; PPAR, peroxisome proliferator-activated receptor

probably by regulating sarcomere protein acetylation leading to improved myofibril force generation.36 By contrast, HDAC2 (a class I HDAC) promotes cardiac hypertrophy by repressing the expression of INPP5F, the gene encoding phosphatidylinositide phosphatase SAC2 (also known as inositol polyphosphate‑5-phosphatase F), a negative regulator of the Akt/GSK‑3β pathway.37 HDACs have also been implicated in the regulation of cardiac energy metabolism. Cardiac-specific deletion of HDAC3 caused cardiac hypertrophy associated with excessive activity of the peroxisome proliferator-activated receptor α in the nucleus and increased myocardial lipid accumulation.38 In addition, overexpression of a mutant signal-resistant HDAC5 protein in the heart resulted in sudden death accompanied by downregulation of the mitochondrial regulator peroxisome p­roliferator‑activated­ receptor γ coactivator 1α in male mice.39 Of note, in vivo studies conducted on several models of cardiac hypertrophy and myocardial infarction have identified HDAC inhibition as a possible therapeutic st­rategy.40–42 Indeed, a study in which a derivative of apicidin was identified as a specific class I HDAC inhibitor showed that the drug could decrease myocardial h­ypertrophy (Table 2).43

Methylation Histone methylation occurs on lysine (K) and arginine residues on histones H3 and H4, and can be associated with either activation or repression of transcription, depending on the degree (monomethylation, dimethy­lation, or trimethylation) and location of the modification.44 Methylation at H3K4, H4K36, or H3K79 is associated with transcriptional activation, and methylation at H3K4 and H3K36 is implicated in mRNA el­ongation. Conversely, methylation at H3K9, H3K27, or H3K20 is linked with transcriptional repression.45 As with histone acetylation, histone methylation is a dynamic process regulated by two classes of enzymes: histone methyltransferases and histone demethylases.46,47 Lysine methyltransferases are more s­pecific than the acetyltransferases. Several reports have suggested that histone methylation is involved in cardiac hypertrophy and failure. Lysine-specific demethylase 4A has been shown to promote cardiac hypertrophy under pathological conditions by binding to the promoter of FHL1 and upregulating gene expression in a manner dependent on serum response factor and myocardin. 48 In another study, PAX-interacting protein 1, an important component of the H3K4me complex, was found to be central in

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REVIEWS Table 2 | Therapeutic potential of epigenetic drugs for heart failure Compound

Action

Animal model

Result

Apicidin

Selective class I HDAC inhibitor

TAC

Decreased myocardial hypertrophy43

JQ1

Bet bromodomain inhibitor

TAC

Blunted hypertrophic response and improved cardiac function65,66

SK‑7041

Selective class I HDAC inhibitor

TAC

Decreased myocardial hypertrophy41

Trichostatin A and scriptaid

Pan-HDAC inhibitors

TAC

Blunted hypertrophic response and improved cardiac function40

Trichostatin A or valproic acid

Pan-HDAC inhibitors

Angiotensin II infusion or TAC

Decreased myocardial hypertrophy and improved survival rate41

Vorinostat (SAHA)

Pan-HDAC inhibitor

Ischaemia–reperfusion surgery performed on rabbits

Decreased infarct size and partially restored cardiac function42

Abbreviations: HDAC, histone deacetylase; TAC, transverse aortic constriction.

maintaining the gene-expression profile of fully differentiated cardiomyocytes.49 One of the genes regulated by H3K4me3 is KCNIP2, which encodes a potassium channel that is downregulated in heart failure and involved in arrhythmo­genesis. Hohl and colleagues found that, despite nuclear export of HDAC4, upregulation of the hypertrophy markers atrial and B‑type natriuretic peptide in a mouse model and in patients with heart failure was not caused by altered acetylation of their promoters, but rather by decreased dimethylation and trimethylation of lysine 9 on histone H3.29 H3K79-specific histonelysine N‑methyltranferase has been shown to be critically involved in defining the transcriptional landscape of differentiating cardiomyocytes.50 Of note, expression of this histone methyltransferase was found to be downregulated in patients with idiopathic dilated cardiomyopathy, and its cardiac-specific disruption in mice resulted in dysregulation of dystrophin and the development of a dilated‑cardiomyopathy­-like phenotype.51 Finally, we performed a genome-wide study and defined the profiles of seven histone modifications in cardiomyocytes isolated from mouse heart 1 week after pressure overload. 52 Of the 1,109 genes found to be modulated in hypertrophic cardiomyocytes, 50% were associated with an alteration of at least one histone mark at their promoter region. Changes in histone acetylation and methylation during the hypertrophy response underscored the activation of a specific class of enhancer regulated by MEF2A and MEF2C.52

ATP-dependent chromatin remodelling ATP-dependent chromatin remodelling complexes (ADCRs) use the energy from the hydrolysis of ATP to modulate the distribution of nucleosomes and thereby modify the packaging state of chromatin. These multi­ protein complexes regulate gene expression by controlling the accessibility of transcription factors to regulatory regions of the genome. Based on the identity of the ATPase domain, ADCRs can be divided into four evolutionarily conserved families: switching defective/ sucrose non fermenting (SWI/SNF), imitation switch, chromodomain helicase DNA binding, and inositol requiring 80.53 The SWI/SNF and inositol requiring 80 families of remodellers were purified from Saccharomyces

cerevisiae.54,55 The imitation switch group was identified from Drosophila melanogaster;56 whereas the chromodomain helicase DNA binding group was initially purified from Xenopus laevis.57 Additional subunits establish DNA binding and recognize or modify specific histone modifications. Transcriptional regulation by these remodelling complexes occurs in concert with histone modifiers such as histone acetyltransferases and HDACs.58 The role of ADCRs has been investigated in cardiac development and disease (reviewed previously 59). The most-studied remodellers in the heart are the SWI/SNF complexes. The vertebrate homologue of the yeast SWI/ SNF complex is brahma-associated factor (BAF), which is composed of 12 components, including the ATPase subunit encoded by either SMARCA2 or SMARCA4. These two ATPase subunits are highly homologous, but are involved in specific functions in vivo. The other sub­ units of the BAF complex are cell-specific and, depending on their assembly, can give rise to multiple complexes with distinct functions.60 The transcription activator BRG1 has been shown to be required for cardiac gene expression, and cardiomyocyte proliferation and differentiation.61 BRG1 interacts with HDACs and poly [ADP-ribose] polymerases (PARPs) to repress the adult isoform of myosin heavy chain (myosin‑6) and activate the fetal isoform (myosin‑7) in embryonic heart.61 BRG1 is highly expressed in fetal heart and is inactivated in the adult organ, coinciding with the switch in myosin isoform. Notably, the expression of BRG1 is increased in the hearts of patients with cardiomyopathy; its reactivation by cardiac stress causes the pathological switch from myosin‑6 to myosin‑7 by complexing with its embryonic HDAC and PARP1 partners.61 In another study, the BRG1, BAF180, and BAF60C subunits of the SWI/SNF complex were increased in hyper­t rophic hearts from Dahl salt-sensitive rats. 62 These proteins were enriched at the promoter region of the fetal genes NPPA and NPPB, producing a more accessible chromatin structure and thereby promoting their transcription. Histone modifications are recognized by reader proteins that recruit or stabilize various factors of the nuclear signalling machinery at specific sites.63 A large number of readers matching histone modifications

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REVIEWS specific, gene-expression regulation; moreover, chromatin i­mmunoprecipitation–DNA sequencing showed that BETs coactivated gene transcription by recruiting transcription elongation factor B polypeptide 2 and p­romoting pause release of RNA polymerase II.66

Nucleus

DNA Transcription Drosha Mirtron PremiRNA

Pri-miRNA

mRNA 5'

Debranch

Export

Crop

ORF 3' UTR Dicer

Dice Argonaute

Unwind 3' Ribosome

Cytoplasm

miRISC

5' Target

Translation repression

Figure 2 | miRNA biogenesis and function. Canonical Nature Reviews | Cardiology miRNA genes are transcribed into long pri-miRNAs that can harbour more than one mature miRNA. The endonuclease Drosha cleaves the pri-miRNA into shorter pre-miRNAs that are then transported to the cytoplasm, where the endonuclease Dicer cleaves the loop portion of the premiRNA to form a short duplex. The duplex is unwound and the single-stranded mature miRNA is passed on to argonaute to from a functional miRISC. The miRNA can then direct the miRISC to partially complementary binding sites present on the 3'UTR of targeted mRNAs, which are either transcriptionally silenced or degraded. Parallel to the canonical pathway is that of mirtrons. These are transcribed from pri-miRNA-sized introns within host genes to form looped intermediates that are debranched and refolded into pre-miRNAs, which can enter into the canonical biogenesis pathway. Abbreviations: miRISC, miRNA-mediated silencing complex; miRNA, microRNA; ORF open reading frame; pre-miRNA, precursor microRNA; pri-miRNA, primary microRNA; UTR, untranslated region. Permission obtained from NPG © Latronico, M. V. G. & Condorelli, G. Nat. Rev. Cardiol. 6, 418–429 (2009).

have been discovered.64 The bromodomain and extratermin­al (BET) family of acetyl-lysine readers has a critical role in the pathogenesis of cardiac hypertrophy and failure.65,66 Inhibition of BET with the small molecule JQ1 prevented pathological hypertrophy and improved cardiac dysfunction induced by pressure overload (Table 2). Transcriptomic analyses revealed a broad, but

Noncoding RNAs A large part of the genome is transcribed, but not translated into protein. ‘Canonical’ (protein-coding) genes account for only ~1.5% of the genome, whereas >80% is transcribed into ncRNA. ncRNAs have been divided into two classes based on size: small ncRNA (200 nucleotides and a general lack of protein-coding capacity.67–69 Specific ncRNAs have been linked to the control of many cellular functions, particularly in development and pathological processes.70 Of note, lncRNAs have been found to direct chromatin remodelling complexes to their targets, suggestive of the existence of a complex interacting web of epigenetic modifications and ncRNAs.71 microRNAs miRNAs are ~22‑nucleotide-long oligonucleotides that guide inhibitory protein complexes—called miRNAinduced signalling complexes or miRISCs—to targeted mRNAs (Figure 2). A substantial portion of the proteincoding transcriptome harbours binding sites for one or more miRNAs and, therefore, is sensitive to miRNAmediated post-transcriptional regulation. Consequently, these small RNAs are important regulators of gene expression. The role of miRNAs in cardiovascular physio­logy and disease has been reviewed previously.72–74 We focus on the latest insights into the interplay between miRNAs and epigenetic modification. An important aspect of pathophysiology is the complex, multiple-level network that regulates gene expression. Crosstalk between miRNAs and DNA methylation is a fundamental part of this network: DNA methylation is involved in the regulation of miRNA expression, and particular miRNAs can target DNMTs and affect DNA methylation patterns. A study conducted in 2006 on bladder carcinoma cells treated with a combination of the DNA methylation inhibitor 5‑aza‑2'‑deoxycytidine and the HDAC inhibitor 4‑phenylbutyric acid, demonstrated for the first time that the expression of 17 miRNA genes was regulated by the degree of promoter methylation.75 In the following years, several miRNAs undergoing m­ethylationdependen­t repression in pathological conditions were identified, mainly in tumour cells (reviewed previously76). An alteration in the methylation status of miRNA gene promoters has also been observed in atherosclerotic plaques.77 Howe­­ver, no reports yet exist on this crosstalk in cardiac diseases. Fabbri and colleagues observed that, whereas the expression of miRNA‑29 was reduced in lung cancer, the

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REVIEWS 1 Signaller

2

Decoy

3

Competing endogenous IncRNAs

Transcription factors Transcription factor

LncRNA

cis Chromatin modifiers 4 Guide

microRNA

mRNA

trans

BRVH, FENDRR, MHRT

CHRF

3'UTR

Proteins 5 Scaffold

Figure 3 | Mechanisms of action of lncRNAs. Signal lncRNAs (1)Reviews are transcribed Nature | Cardiology in response to distinct cellular cues and, therefore, interpret cellular context or respond to specific stimuli. Decoy lncRNAs (2) bind and titrate away protein, removing transcription factors and chromatin modifiers from genomic loci. Similarly, competing endogenous lncRNAs (3) compete with mRNAs for microRNAs, regulating the silencing or degradation of the former. Guide lncRNAs (4) act as molecular chaperones for proteins to regulate the expression of genes. Scaffold lncRNAs (5) have multiple domains and can bind distinct proteins to form nucleoprotein complexes with functions such as transcriptional activation or repression. Known cardiac lncRNAs are indicated in boxes. Abbreviations: lncRNA, long noncoding RNA; UTR, untranslated region.

expression of DNMT3A and DNMT3B was increased.78 The enforced expression of miRNA‑29 restored normal DNA methylation patterns and reduced expression of DNMT1, DNMT3A, and DNMT3B.79 Interestingly, miRNA‑29 has been identified as a circulating biomarker in patients with hypertrophic cardiomyopathy.80 In the heart, this miRNA regulates fibrosis, and has been found to be downregulated in myocardial infarction.81 The DNA methylation profile of the diseased heart might, therefore, be influenced by miRNA‑29, through regulation of DNMTs at a post-transcriptional level. DNA methylation is reportedly also regulated by miRNA‑133a: in a model of diabetic cardiomyopathy, this miRNA was found to target all three DNMTs.82 In another study in which miRNA‑133 was identified as a critical determinant of cardiac hypertrophy, NELFA was found to be a specific target of this miRNA.83 Negative elongation factor A is an important regulator of RNA polymerase II pausing and can decrease or increase transcription.84,85 Histone modifications have also been found to interact with miRNAs for transcriptional regulation. For example, the expression of BRG1 was found to be regulated by miRNA‑199a‑5p and miRNA‑199a‑3p in a variety of human cancers.86 In skeletal muscle, miRNA‑1 targets HDAC4 to promote myogenesis.87 In heart, histone-lysine N‑methyltransferase EZH2 and pri-miRNA‑208b were found to interact in the regulation of gene expression during hypertrophy in a mouse model of pressure overload.88 Long noncoding RNAs Unlike miRNAs, lncRNAs constitute a heterogeneous class of transcripts that have been grouped together solely on the basis of their length. Several mechanisms of action have been found, but these cannot be determined

by knowledge of the nucleotide sequence alone. The capacity of lncRNAs to fold into a variety of thermo­ dynamically stable secondary structures—such as double helices, hairpin loops, bulges, and pseudoknots—allows the formation of complex tertiary interactions that enable them to interact with the wide range of macromolecules, including other RNA species, DNA, and p­oteins to ge­nerate ribonucleoprotein particles.42,81 On the basis of their molecular mechanism, lncRNAs can be classified as signallers, protein decoys, guides, or scaffolds (Figure 3).67 In general, lncRNAs are divided into nuclear and cytoplasmic types. The former guide chromatin modifiers to specific genomic loci to prompt a repressive heterochromatin state and, therefore, downregulate transcription.89 Transcriptional activation has also been found to occur, via recruitment of histone-lysine N‑methyltransferase 2A,80 and by lncRNAs acting like enhancers.90 The cytoplasmic lncRNAs repress or stimulate gene expression at the translational level by binding to targeted mRNAs,30,39 or by sequestering miRNAs and preventing them from effecting t­ranslational repression.31 We are just beginning to understand lncRNA function in cardiac development and disease. A breakthrough in the field came with the identification of the lncRNAs BVHT and FENDRR, which are required for cardiomyocyte differentiation.91,92 A deep-sequencing study identified lncRNAs generated from fetal cardiac enhancers during cardiac differentiation and morphogenesis.93 In the diseased heart, 18,480 lncRNAs were detected in the RNA pool from human nonischaemic and ischaemic failing left ventricles; interestingly, the expression signature of lncRNAs, but not of mRNAs or miRNAs, was sufficient to distinguish between the two pathological conditions.94 Another genome-wide sequencing study identified 157 cardiac-specific lncRNAs that were dynamically regulated in the fetal-to-adult transition, suggesting a central role for these lncRNAs in gene expression modulation during heart development.95 The same study, though, found only 17 cardiac-expressed lncRNAs modulated in the adult hypertrophied heart.95 The lncRNA CHRF has been shown to regulate cardiac hypertrophy by acting as an endogenous sponge for miRNA‑489, and downregulating its activity. 96 In a mouse model of myocardial infarction, the expression of specific lncRNAs found to be associated with active enhancers correlated strongly with parameters of cardiac function and dimension.97 LncRNAs have also been shown to modulate the expression of atrial natriuretic peptide (NPPA),98 cardiac muscle troponin I (TNNI3),99 myosin light chain 4 (MYL4),100 and other myosin heavy and light chains.101 LncRNAs can modulate transcription through the control of epigenetic modification. Several lncRNAs associate with chromatin-remodelling complexes to recruit them to specific loci.102 Conversely, the chromatin state can regulate the expression pattern of lncRNAs. Therefore, a complex network links epigenetic m­odifications and lncRNAs.103 An example of lncRNA-mediated epigenetic silencing is the inactivation of the X chromosome by the lncRNA

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REVIEWS XIST. XIST interacts with the polycomb repressive complex (PRC) 2 to promote the formation of a repressive chromatin state across the entire X chromosome.104 Another example is HOTAIR, which represses transcription in trans across the HOXD gene cluster by targeting PRC2 and thereby determines PRC2 occupancy and trimethylation of H3K27 across the locus.105 Importantly, approximately 20% of human lncRNAs expressed in various cell types interact with PRC2, and depletion of several of these lncRNAs has been found to affect gene expression of PRC2-regulated genes.89 A set of lncRNAs has also been implicated in DNA methylation. The lncRNA KCNQ1OT1 regulates the imprinted genes within the KCNQ1 domain by interacting with DNMT1.106 Another report showed that the lncRNA ecCEBPA, which is expressed from the CEBPA gene locus, is critical for the regulation of DNA methylation of the host gene through its association with DNMT1.107 Examples of the interaction between lncRNAs and chromatin remodelling mechanisms in the heart are limited. In differentiating cardiomyocytes, FENDRR was found to regulate several cardiac transcription factors by interacting with PRC2 and trithorax group/mixedlineage leukaemia complexes.92 BVHT has been reported to interact with the SUZ12 subunit of PRC2 and direct cardiovascular cell fate by promoting the activation of core cardiac-specific regulatory networks.91 Investigators in one study identified a cluster of antisense RNAs transcribed from the 5' end of the MYH7 locus: these were termed myosin heavy chain-associated RNA transcripts (Mhrts).108 Interestingly, an anti-MYH7 cardiac-specific 1.

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transcript originating from the 5' end of the gene had been already described in 1992.90 Although the investigators were not able at the time to confer a functional relevance to this finding, they hypothesized that the antisense RNA might have a role in the transcriptional regulation of MYH7. Almost 15 years later, the MYH7 antisense RNA was identified by Chang and colleagues as a Mhrt, a cardioprotective lncRNA that determines the balance of myosin‑6 and myosin‑7 by regulating the BRG1–HDAC–PARP complex.108 Mhrt expression was found to be decreased upon pathological cardiac hypertrophy. Indeed, restoring Mhrt expression in pressureoverloaded mice attenuated hypertrophy and improved cardiac function,108 demonstrating the cardioprotective role of this group of lncRNAs. Mechanistically, the downregulation of Mhrts in hypertrophy is BRG1-dependent; conversely, Mhrts can directly interact with the helicase domain of BRG1 to regulate its occupancy and prevent it recognizing its genomic targets.108

Conclusions

Over the past decade, our understanding of the role of epigenetic modifications and ncRNAs in the regulation of myocardial function in health and disease has improved greatly. The ubiquitous nature of epigenetic modifiers poses a major challenge to the development of cardiac-specific therapeutic applications. Improved understanding of the intimate, tissue-specific epigenetic mechanisms will hopefully lead to the identification of organ-specific targets and, therefore, to new preventive and therapeutic approaches for cardiovascular diseases.

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