Targeting histone acetylation in pulmonary hypertension and right ventricular hypertrophy

Epigenetic mechanisms, including DNA methylation and histone post‐translational modifications (PTMs), have been known to regulate chromatin structure and lineage‐specific gene expression during cardiovascular development and disease. However, alterations in the landscape of histone PTMs and their contribution to the pathogenesis of incurable cardiovascular diseases such as pulmonary hypertension (PH) and associated right heart failure (RHF) remain largely unexplored. This review focusses on the studies in PH and RHF that investigated the gene families that write (histone acetyltransferases), read (bromodomain‐containing proteins) or erase (histone deacetylases [HDACs] and sirtuins [SIRT]) acetyl moieties from the ε‐amino group of lysine residues of histones and non‐histone proteins. Analysis of cells and tissues isolated from the in vivo preclinical models of PH and human pulmonary arterial hypertension not only confirmed significant alterations in the expression levels of multiple HDACs, SIRT1, SIRT3 and BRD4 proteins but also demonstrated their strong association to proliferative, inflammatory and fibrotic phenotypes linked to the pathological vascular remodelling process. Due to the reversible nature of post‐translational protein acetylation, the therapeutic efficacy of numerous small‐molecule inhibitors (vorinostat, valproic acid, sodium butyrate, mocetinostat, entinostat, tubastatin A, apabetalone, JQ1 and resveratrol) have been evaluated in different preclinical models of cardiovascular disease, which revealed the promising therapeutic benefits of targeting histone acetylation pathways in the attenuation of cardiac hypertrophy, fibrosis, left heart dysfunction, PH and RHF. This review also emphasizes the need for deeper molecular insights into the contribution of epigenetic changes to PH pathogenesis and therapeutic evaluation of isoform‐specific modulation in ex vivo and in vivo models of PH and RHF.


Funding information
Pulmonary arterial hypertension (PAH) is a rapidly progressive vascular disease with multifactorial aetiology mediated by the interplay of a susceptible genetic background, epigenetic changes, and injurious events . Despite our increased understanding of the pathomechanisms of PAH and the recent therapeutic advances, PAH remains an incurable disease. Notably, preliminary findings support the hypothesis that there is a significant contribution of epigenetic mechanisms to PAH.

| EPIGENETIC MECHANISMS
An epigenetic trait is a stably inherited phenotype resulting from changes in a chromosome without alterations in the DNA sequence (Berger, Kouzarides, Shiekhattar, & Shilatifard, 2009) (Bonisch & Hake, 2012). Regulation of the compaction or relaxation of chromatin at specific genes can lead to their repression or activation, respectively. Histone PTMs such as lysine acetylation, lysine or arginine methylation, serine/threonine phosphorylation and lysine ubiquitination on their N-terminal tails have been shown to modulate the chromatin structure by changing protein-DNA or protein-protein interactions (Lalonde, Cheng, & Cote, 2014).

| HISTONE ACETYLATION
Histone lysine acetylation plays a fundamental role in the epigenetic regulation of gene expression. Acetylated histones tend to be less compact and more accessible to RNA polymerase and the transcriptional machinery, thereby enabling transcription of nearby genes (Cohen, Poreba, Kamieniarz, & Schneider, 2011). Acetylated histones also serve as binding sites for bromodomain-containing proteins (BRDPs) to recruit transcriptional machinery and other chromatinmodifying elements (Bannister & Kouzarides, 2011). Conversely, histone deacetylation causes transcriptional repression via chromatin compaction. Acetylation of non-histone targets can lead to changes in enzyme activity and protein-protein interactions (Kim & Workman, 2010).

| REGULATORS OF HISTONE ACETYLATION
Histone PTMs can be deposited on or removed from chromatin by different enzyme families. These "writers" and "erasers" of histone acetylation include lysine acetyltransferases (HATs) and deacetylases (HDACs) gene families (Lalonde et al., 2014). The balance between the actions of these enzyme families serves as a critical regulatory mechanism for gene expression and governs numerous developmental processes and disease states (Lalonde et al., 2014). Notably, the recruitment of proteins to macromolecular complexes by acetylated lysine residues is mediated by BRDPs, which are the principal readers of ε-N-acetyl-lysine (Kac; Muller, Filippakopoulos, & Knapp, 2011). All three enzyme families are an integral component of gene regulatory complexes, which are well known to regulate transcription through modulation of chromatin modification states (Lalonde et al., 2014). Of the three enzyme families regulating histone acetylation, the HDAC family has been the most studied in the context of pulmonary hypertension (PH) and right ventricular hypertrophy (RVH; Figure 1, Table 1).

| Family and molecular functions
HDACs are a large family of enzymes that contain a highly conserved deacetylase domain and are responsible for the removal of acetyl groups and maintenance of the equilibrium of lysine acetylation in histones (Peserico & Simone, 2011). So far, 18 human HDACs have been identified and grouped into four classes according to functional and phylogenetic criteria. They are class I HDACs (HDAC1, 2, 3, and 8), class IIa HDACs (HDAC4, 5, 7 and 9), class IIb HDACs (HDAC6 and 10) and class IV HDAC (HDAC11) which are Zn 2+ -dependent, while the class III HDACs, also called sirtuins (SIRT1, 2, 3, 4, 5, 6, and 7), are NAD + -dependent enzymes (Haberland, Montgomery, & Olson, 2009). HDAC enzymes differ in structure, enzymatic function, subcellular localization and expression patterns. In addition to the nuclear roles of HDACs, HDAC isoforms also exhibit essential cytoplasmic functions by controlling the acetylation status and activity of numerous cytoplasmic proteins, including transcription factors (Glozak & Seto, 2007).

| Class I histone deacetylases: Expression and preclinical studies
Although in vivo studies on the pulmonary vascular cell-specific roles of class I HDAC isoforms are lacking, the contribution of aberrant HDAC activity to the pathogenesis of PH and RVH is mainly based on the promising therapeutic benefits observed upon the application of small-molecule HDAC inhibitors in different animal models of PH (Chelladurai, Seeger, & Pullamsetti, 2016). Table 1 summarizes all research findings that reported the aberrant expression and activity of HDACs and the therapeutic effects of non-selective HDAC inhibitors targeting multiple HDAC isoforms (pan-HDAC). In PH, Li et al. (2011)) first reported the association between the persistent proinflammatory phenotype exhibited by activated pulmonary adventitial fibroblasts isolated from a bovine model of hypoxia-induced PH and the abnormal activity of class I HDACs. Elevated class I HDAC F I G U R E 1 Regulatory mechanisms associated with post-translational protein acetylation in the pathogenesis of PH. PH is a complex disease with multifactorial aetiology. Besides genetic predisposition, vascular injury caused by hypertensive stimuli such as shear stress, autoimmunity, hypoxia, infection, drugs and toxins may disrupt the cellular homeostasis. The persistence of injurious events alters the existing epigenetic state of the healthy pulmonary vascular cells and re-establishes an aberrant epigenetic signature that favours the acquisition of altered cellular phenotypes that aggravate the vascular remodelling process. Vascular cells isolated from PAH pulmonary vasculature exhibit stable proproliferative, pro-migratory, anti-apoptotic, pro-inflammatory and pro-fibrotic vascular cell phenotypes, which correlate with the observed changes in the transcriptional levels of genes associated with respective phenotypes. Histone acetylation plays a fundamental role in the epigenetic regulation of gene expression. Besides the recent focus on the BRD4 that recognizes and binds acetylated histones, the most investigated epigenetic regulators in the context of PH were the enzyme isoforms from HDAC and SIRT families. Mechanistically, aberrant expression of HDACs or HATs in vascular cells may cause an imbalance in acetylome (hypoacetylation or hyperacetylation) of histone tails and cellular proteins that consequently modulate DNA accessibility and transcriptional state (activation or repression) of critical PAH-associated genes that aggravate the vascular remodelling process in PAH. Ac, acetylation; BET, bromodomain and extra-terminal domain family; BRD, bromodomain; HATs, histone acetyltransferases; HDACs, histone deacetylases; POL2, RNA polymerase II; SIRT, sirtuins catalytic activity also correlated with an increased abundance of HDAC1, HDAC2 and HDAC3 protein levels in PH fibroblasts (Li et al., 2011). Further, six HDACs (1, 2, 3, 4, 5, and 7) were screened in human idiopathic PAH (IPAH) lung homogenates and elevated expression of HDAC1 and HDAC5 was reported (Zhao et al., 2012b).
A large number of preclinical studies using different HDAC inhibitors have shown their promising antitumour responses in vivo, of which few of them have been approved by U.S. FDA for the treatment of human lymphoma (Segre & Chiocca, 2011;Suraweera, O'Byrne, & Richard, 2018). These compounds are generally well tolerated, with the most notable adverse effects being thrombocytopenia, nausea and fatigue (Tan, Cang, Ma, Petrillo, & Liu, 2010). In addition to their anticancer actions, several studies have reported beneficial effects of HDAC inhibitors in preclinical models of left ventricular (LV) dysfunction by attenuating pathological cardiac hypertrophy, inflammation, fibrosis and restenosis thereby improving ventricular function (Cavasin, Stenmark, & McKinsey, 2015).
Based on these promising findings, the therapeutic potential of commercially available broad-spectrum HDAC inhibitors, valproic acid

| Class II and IV histone deacetylases: Expression and preclinical studies
The class IIa HDACs (subtypes 4, 5, 7, and 9) are characterized by tissue-specific expression and stimulus-dependent nucleo-cytoplasmic shuttling (Witt, Deubzer, Milde, & Oehme, 2009 Recently, ) reported up-regulation of HDAC6 protein levels in lungs, distal PAs and isolated PA smooth muscle cells (PASMCs), PA endothelial cells (PAECs) from PAH patients as well as in the RV of rats exposed to SU5416-hypoxia and MCT. Exogenous expression of HDAC10 in cervical cancer cells significantly inhibited cell motility and invasiveness in vitro and metastasis in vivo, by transcriptional repression of MMP2 and MMP9 genes (Song, Zhu, Wu, & Kang, 2013). HDAC11 expression is enriched in the brain, heart, muscle, kidney and testis (Gao, Cueto, Asselbergs, & Atadja, 2002), but little is known about its function.
Specifically, MEF2 is a transcription factor family that plays a prominent role in cardiovascular development and differentiation and is constitutively associated with class IIa HDACs, which maintain MEF2 in a transcriptionally inactive state (Desjardins & Naya, 2016).
In the context of PH, ) demonstrated that impaired MEF2 transcriptional activity found in PAH PAECs was mediated by excess nuclear accumulation of HDAC4 and HDAC5. Selective, pharmacological inhibition of class IIa HDACs using MC1568 restored MEF2 activity in IPAH PAECs, as demonstrated by increased expression of its transcriptional targets, decreased cell migration and proliferation, and rescue of experimental PH models (MCT and SU5416 + hypoxia). Importantly, class IIa HDAC inhibition did not promote RV fibrosis or coronary artery endothelial cell apoptosis (Kim, Hwangbo, et al., 2015). However, a recent study (Lemon et al., 2015) reported that the commercially available compound MC1568 (used in the above study by Kim, Hwangbo, et al.) failed to inhibit class IIa HDAC catalytic activity in vitro. Expression patterns of class II HDAC isoforms were proposed to be tissue specific, and therefore, thorough in vivo evaluation of their gene-specific functional roles in development and disease of the cardiovascular system is warranted.  . TSA treatment in the setting of PAB was also associated with exaggerated RV fibrosis, increased numbers of apoptotic cells in the RV, and induced capillary rarefaction in the RV, but not in control rats

| Expression and preclinical studies
The most extensively studied member among the sirtuin family is nic- deacetylase, whose activity is regulated in a context-dependent manner by cellular stress, redox state, transcription factors, and PTMs (Yu & Auwerx, 2010). Although neither the expression levels or activity status of SIRT1 nor the lysine acetylation state in human PAH setting have been explored in detail, a few studies have reported a potential protective role for SIRT1 in rodent models of PH and RVH ( Figure 2, Table 2).
A recent study demonstrated that inactivation of Sirt1 aggravates chronic hypoxia-induced PA muscularization and cardiac remodelling in vivo (Zurlo et al., 2018). Sirt1 global knockout mice displayed a more intense vascular remodelling and RVH upon exposure to chronic hypoxia. Aggravated vascular remodelling exhibited by Sirt1 ablation was associated with a significant increase in the percentage of fully muscular pulmonary arteries and α-smooth muscle actin expression, paralleled by a decreased percentage of non-muscular arteries. However, at the molecular level, the authors observed no changes in both mRNA and protein levels of SIRT1 in human PAH PASMCs compared to control PASMCs (Zurlo et al., 2018). Even though the catalytic activity of SIRT1 was not quantified in PAH or hypoxia, there is an imbalance in the acetylation/deacetylation state of the non-histone targets of SIRT1 in PH, wherein the acetylated forms of PGC-1α, histone H1, and FOXO1 were observed in PAH PASMCs. Pharmacological activation of SIRT1 using Stac-3 reduced not only PDGF-induced proliferation in PASMCs from PAH patients but also reduced mitochondrial fragmentation and increased mitochondrial biogenesis (Zurlo et al., 2018). Multiple observations confirm the proposition that SIRT1 is a negative regulator of PASMC proliferation and promotes mitochondrial biogenesis, and SIRT1 inactivation may be strongly associated with the pathogenesis of PH. Although the study used global Sirt1 knockout mice, investigating vascular cell-specific roles of Sirt1 may yield more in-depth insights into the complex regulatory mechanisms associated with Sirt1 activity regulation.
Along the same lines, short-term calorie restriction (CR) ameliorated MCT-induced mean pulmonary arterial pressure and reduced vascular remodelling and RVH that was accompanied by a significant increase in Sirt1 expression (Ding et al., 2015). Remarkably, Sirt1 overexpression in the rat model of MCT-induced PAH and hypoxia-induced PH is sufficient to mimic the reduction in mean pulmonary arterial pressure (mPAP) that was achieved merely with short-term CR (Ding et al., 2015). These studies demonstrated the At the cellular level, Sirt3-deficient PASMCs exhibited suppressed mitochondrial oxidative phosphorylation and had increased levels of mitochondrial protein lysine acetylation compared to wild type . Furthermore, the authors confirmed that suppression of SIRT3 activity in IPAH can be either due to the down-regulation of the expressed protein or due to the presence of a loss-of-function single nucleotide polymorphism in the SIRT3 gene (rs11246020) within the conserved catalytic deacetylase domain or due to the occurrence of both in a PAH patient . These findings support the metabolic basis of PAH and confirm the central role of the SIRT family of deacetylases both during the physiological maintenance of metabolic and mitochondrial homeostasis and during the pathogenesis of PH, thereby rapidly emerging as potential therapeutic targets.
On the contrary to cardioprotective role of Sirt3 described by Paulin et al., observed in the transgenic mice from 129/Sv strain, Waypa et al. (2013)) revealed that their Sirt3-deficient mice from C57/BL6 background did not exhibit any differences from their wildtype littermates in PA wall remodelling, and RVH, when exposed to chronic hypoxia. The disparity in the outcome between the two studies carried out by Waypa et al. and Paulin et al. led to the supposition that Sirt3 responses may be cell type specific or restricted to specific genetic backgrounds, which has to be taken into account and correlated with the status of expression pattern in cells and tissues from human PH setting, in all the studies to be carried out in future.

| Expression and preclinical studies
Dysregulated HAT activity has been largely linked to cancer formation and progression (Avvakumov & Cote, 2007). Although in-depth studies dissecting the contribution of HAT isoforms in vascular remodelling associated with PH are lacking, few studies provide insights into the role of HATs in development and disease in cardiovascular development and disease setting. The Ep300/Crebbp family has mainly been studied and found to play critical roles in the physiological and pathological growth of cardiac myocytes. With regard to heart development, Ep300 knockout mice displayed defects like pericardial effusion, weaker heart contractions, reduced trabeculation, proliferation and reduced expression of cardiac muscle structural proteins such as β-myosin heavy chain and α-actinin (Yao et al., 1998). A knock-in approach was further used to demonstrate the significance of the HAT domain of Ep300 in heart development and coronary vascularization (Shikama et al., 2003). Mice overexpressing Ep300 in the heart exhibit increased mortality and marked eccentric dilatation and systolic dysfunction of the LV. Cardiomyocyte-specific overexpression further established the crucial role of Ep300 in cardiac hypertrophy and heart failure in vivo (Miyamoto et al., 2006;Yanazume et al., 2003). In a murine model of myocardial infarction, cardiac overexpression of Ep300 promoted LV remodelling after myocardial infarction in adult mice in vivo (Miyamoto et al., 2006). Acetylation mediated by Ep300 increased DNA-binding activity of hypertrophyresponsive transcription factor Gata4, which preceded the development of LV dilatation and dysfunction (Yanazume et al., 2003).
With regard to the therapeutic evaluation, only limited numbers of HAT inhibitors have been investigated in cardiovascular diseases.
Although an increased level of Ep300 is documented in multiple fibrotic tissues (Rai et al., 2017), only a few studies have evaluated pharmacological modulation of Ep300 in fibrosis-associated organ dysfunction (Ghosh, 2014;Rai et al., 2017). For instance, the acetyltransferase activity of Ep300 was shown to enhance Smaddependent Tgf-β stimulation of collagen gene expression in fibroblasts (Ghosh, Yuan, Mori, & Varga, 2000). In a murine model of hypertensive cardio-renal fibrosis, EP300 inhibitor (L002) suppresses profibrotic processes and associated cell proliferation, migration, myofibroblast differentiation and collagen synthesis. Systemic administration of L002 in angiotensin II infused mice reduced angiotensin II-induced perivascular and interstitial collagen deposition and hypertension-associated pathological hypertrophy, cardiac fibrosis and renal fibrosis. However, these anti-hypertrophic and anti-fibrotic benefits of L002 were independent of vasodilatory effects (Rai et al., 2017). Some natural products such as anacardic acid, garcinol and curcumin have been reported as potent EP300 and PCAF inhibitors, and γ-butyrolactone (MB-3) and a series of isothiazolones have been revealed as inhibitors of both EP300 and PCAF HAT activities (Mai et al., 2006). Considering the extent of biological roles of HAT enzymes in cardiovascular system development and disease, both mechanistic and pharmacological studies targeting the acetyltransferases in the context of PH and RVH have to be evaluated in detail.

| Family and molecular functions
The bromodomain (BRD) is a conserved structural module composed of several α-helices connected by two loops forming a hydrophobic cavity that selectively recognizes and binds acetyl-lysine residues present on histone and non-histone proteins (Filippakopoulos et al., 2012;Fujisawa & Filippakopoulos, 2017). Given that they act in concert with proteins responsible for writing (HATs) and erasing (HDACs) histone acetylation marks, BRDPs mainly serve as epigenetic telomere elongation (Wang et al., 2017) as well as the DNA damage response by stimulating expression of DNA repair factors (Li et al., 2018;Zhang et al., 2018). Taken together, these experimental findings illustrate the multifaceted roles of BRDPs and their importance in regulating major biological processes.

| Expression and preclinical studies
Altered expression levels of BRDPs have been linked to various disease states (Sanchez, Meslamani, & Zhou, 2014). Among them, a particular attention has been paid to BET proteins, especially BRD4, essential for embryonic viability (Houzelstein et al., 2002) and found to be overexpressed in many types of cancers (Dawson et al., 2011;Delmore et al., 2011;Liao et al., 2016;Segura et al., 2013;Zhang et al., 2016). Small-molecule inhibitors have been developed, causing displacement of BET proteins from chromatin by competing with the acetyl-binding pockets present in the BRDs (Filippakopoulos et al., 2010;Nicodeme et al., 2010). Pharmacological inhibition of BETs repeatedly reduced cancer cell growth and tumour formation in multiple preclinical cancer models (Dawson et al., 2011;Delmore et al., 2011;Segura et al., 2013). Considering that PAH may be viewed as a chronic inflammatory, proliferative disease with a cancer-like nature Pullamsetti, Savai, Seeger, & Goncharova, 2017), the possible implication of BET proteins, especially BRD4, in the process of pulmonary vascular remodelling was recently explored ( Figure 2, Table 1).
Increased expression of BRD4 was found in RV, lungs, dissected PAs and isolated PASMCs from PAH patients compared to controls (Meloche et al., 2015). The authors demonstrated that reduced miR-204 expression accounts, at least in part, for the increased level of  (Liu, Zhang, Joo, & Sun, 2017). Additionally, BRD4 has emerged as a critical regulatory factor of inflammatory and immune responses. Specifically, BRD4 interacts with acetylated RelA (a subunit of nuclear factor κ light chain enhancer of activated B cells, NF-κB) and enhances the transcriptional activity of the latter (Huang, Yang, Zhou, Ozato, & Chen, 2009;Nicodeme et al., 2010). Chromatin immunoprecipitation analyses showed that BRD2 and BRD4 physically associate with the promoters of the pro-inflammatory cytokines genes IL-6, TNFα and MCP-1 increasing their expression in activated macrophages (Belkina, Nikolajczyk, & Denis, 2013). Similarly, JQ1 was reported to inhibit serum-stimulated proliferation and migration of human pulmonary microvascular ECs as well as expression of proinflammatory cytokines (IL-6 and IL-8) by preventing the recruitment of RelA to these gene promoters (Mumby et al., 2017). Based on these data, the potential benefit of intratracheal nebulization of JQ1 and siBRD4 was explored in the SU5416/hypoxia rat model with established PAH. Both approaches significantly reduced pulmonary vascular remodelling and improved pulmonary haemodynamic parameters and cardiac function (Meloche et al., 2015), underscoring the pivotal role of BET proteins, particularly BRD4, in the acquired abnormal phenotype of PA cells. In support of this, the pan-BET inhibitor I-BET151 also reduced RV hypertrophy and PH in rats exposed to chronic hypoxia combined with pulmonary inflammation (Chabert et al., 2018).
In a translational perspective, a preclinical multicentre study was recently conducted to evaluate the therapeutic potential of the clini-  preclinical studies (Provencher et al., 2018) and the development of predictive biomarkers to identify poor-and good-responder patients, a deeper mechanistic understanding of the altered epigenetic landscape is warranted to reduce the gap between preclinical animal studies and clinical trials.
Lessons gleaned from cancer biology can be harnessed for optimizing epigenetic therapy in PAH. It is important to note that the pathogenesis of PAH and RVH involves the complex interaction between resident vascular cells (e.g, PAECs, PASMCs, and PAAFs), infiltrating immune cell types, vasoconstrictive, pro-proliferative, proinflammatory and pro-fibrotic mediators. Therefore, treatment with a single epigenetic drug may not completely reverse the complex disease process of PAH but may require a combination of drugs to promote reverse remodelling of severe human PAH. For instance, it has been demonstrated that BET inhibitors synergize with HDAC or PARP inhibitors to combat the growth of cancer cells (Karakashev et al., 2017;Zhao, Okhovat, Hong, Kim, & Wood, 2019). Given that PARP1 inhibitor shows beneficial effects in different animal models mimicking PAH (Meloche et al., 2014), it can be envisaged that this drug combination maximizes the desired intracellular effects. Besides, small chimeric molecules have been recently designed to target BET proteins for degradation providing an alternative approach to inhibit BET functions (Choi et al., 2019). This strategy allows us to circumvent a possible compensatory accumulation of BET protein in response to their inhibition and to abolish the kinase-and HAT-mediated functions of BRD4 (potentially involved in PAH development and progression) in addition to the interaction between the BRD and the acetyl group.
Another line of active research involves strategies to improve drug delivery for specifically targeting diseased cells while minimizing the potential unwanted toxicity within healthy tissues. Notably, the majority of HDAC inhibitors currently evaluated in PAH nonselectively modulate the activities of HDAC isoforms and also exert off-target effects, which may limit the clinical application. Moreover, the mixed results of HDAC inhibition on RV function highlight the need for identification of specific HDAC isoforms dysregulated in human PAH. The expression pattern of individual enzyme isoforms in different tissues and vascular cells of the cardiopulmonary system in rodent and human PAH should be profiled in order to circumvent the harmful side effects of broad-spectrum epigenetic modulation.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMA-COLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/2020 Alexander, Keely, et al., 2019).