Histone deacetylases as therapeutic targets - from cancer to cardiac disease Alon Abend, Izhak Kehat PII: DOI: Reference:

S0163-7258(14)00203-4 doi: 10.1016/j.pharmthera.2014.11.003 JPT 6728

To appear in:

Pharmacology and Therapeutics

Please cite this article as: Abend, A. & Kehat, I., Histone deacetylases as therapeutic targets - from cancer to cardiac disease, Pharmacology and Therapeutics (2014), doi: 10.1016/j.pharmthera.2014.11.003

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ACCEPTED MANUSCRIPT P&T # 22701

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Histone deacetylases as therapeutic targets - from cancer to cardiac disease.

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Alon Abend, Izhak Kehat*

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*The Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, POB 9649, Haifa 31096, Israel.

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Tel: +972-4-8295378

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email: [email protected]

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ACCEPTED MANUSCRIPT Abstract Heart failure is a major public health problem in western society. Recently, agents that inhibit Histone

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Deacetylase (HDAC) enzymes were developed and approved by the FDA as anticancer agents. This

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breakthrough has provided the motivation to develop more potent and more selective HDAC

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inhibitors and to target other pathologic conditions with these drugs. Here we review experimental

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evidence showing that these drugs may be beneficial in preventing cardiac hypertrophy and heart failure. Several lines of evidence show that inhibitors of Class I HDACs can blunt cardiac hypertrophy

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and preserve cardiac function in several small animal models. In contrast, class IIa HDACs appear to be suppressors of hypertrophy, though experimental data with small molecule blockers of this class is

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largely lacking. The effects of HDAC inhibition in cardiac diseases, the cell population in the heart that

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investigation.

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is targeted by HDAC blockers, as well as the relative roles of specific HDACs are still under intense

Key Words: Histone deacetylases, Histone deacetylases inhibitors, Heart failure, Hypertrophy Abbreviations:

DOCA - deoxycorticosterone acetate HAT- histone acetyltransferases HDAC - Histone Deacetylase HDACi - Histone Deacetylase inhibitor MEF2 - myocyte enhancer factor-2

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ACCEPTED MANUSCRIPT MITR - MEF2-interacting transcription repressor

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SAHA - Suberanilohydroxamic acid

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TSA - Trichostatin A

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VPA - valproic acid

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ACCEPTED MANUSCRIPT Table of Contents

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1. Introduction

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2. HDACs structure and classification

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3. HDAC inhibitors

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4. Class I HDACs and hypertrophy

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5. Class II HDACs and hypertrophy 6. HDACs and cardiac fibrosis

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7. HDACs and cardiac ischemia

9. Perspectives

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8. Clinical Experience with HDAC inhibitors in the heart

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ACCEPTED MANUSCRIPT 1. Introduction Heart failure is a major public health problem, and strategies to improve outcomes and

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decrease health care resources use and costs are direly needed. Heart failure is primarily a disease of

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the elderly, and therefore its prevalence increases as the population ages. Despite some

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advancement in diagnosis and treatment, heart failure continues to be associated with a high

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mortality rate and frequent need for hospitalization (Dunlay et al., 2014). For example, each year there are over one million hospitalizations due to heart failure in the United States, with a similar

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number in Western Europe. While these patients may respond to initial therapies, they mostly succumb to the disease and have very high mortality and readmission rates (Sridharan et al., 2013).

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Cardiac hypertrophy occurs when the heart experiences elevated workload, and the heart and

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cardiac cells enlarge resulting in reduced ventricular wall stress. Initially, this pathological

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hypertrophy may involve a compensatory and adaptive phase; however, ultimately the benefit may be lost, resulting in impaired diastolic properties and a decline in ventricular function, often leading to heart failure.

Recently, agents that inhibit histone deacetylases (HDACs) were developed as anticancer agents, and the FDA has approved vorinostat and romidepsin for oncologic indications. This advance has provided the motivation to develop more potent and selective HDAC inhibitors and to target other pathologic conditions with these drugs. Here we will review experimental evidence showing that HDAC inhibitors may be beneficial in preventing cardiac hypertrophy and heart failure.

2. HDACs structure and classification 5

ACCEPTED MANUSCRIPT Histones are the principal proteins of chromatin, and the genomic DNA is looped around them. The core histones are reversibly and dynamically acetylated and deacetylated on multiple

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lysine residues in their amino terminal tails. Broadly, hyperacetylated histones are located in

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transcriptionally active genes and hypoacetylated histones are found in transcriptionally silent

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regions such as heterochromatin; however many patterns of acetylation and histone methylation exist and reflect different states of the chromatin. The level of histone tail acetylation mirrors the

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competing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Eighteen

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distinct human HDACs are grouped into four classes based on their sequence homology to s. cerevisiae HDACs (Figure 1a and b). Class I HDACs (HDAC1, -2, -3, and -8) are homologous to yeast

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RPD3, share a compact structure, and are predominantly nuclear proteins and ubiquitously expressed

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in most tissues and cell lines. Class II HDACs are homologous to yeast HDA1 protein and can be

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subdivided into two subclasses, IIa (HDAC4, -5, -7 and -9 and its splice variant MITR) and IIb (HDAC6 and HDAC10), based on the protein sequence homology and domain organization (Verdin et al., 2003). Class IIa HDACs have one catalytic domain and a long amino-terminal adaptor domain, while class IIb HDACs 6 and 10 contain two catalytic domains. Class III HDACs, known as sirtuins, do not contain zinc and their activity requires nicotinamide adenine dinucleotide (NAD+). Since smallmolecule HDAC inhibitors mostly target the zinc pocket catalytic domain, the sirtuins will not be further discussed here. Class IV HDACs include only HDAC 11, a relatively newly discovered protein, which resembles class I HDACs. Acetylation and deacetylation were characterized for histone tails (Figure 2), where this modification is implicated in processes such as gene expression, DNA replication and DNA repair. Acetylated histones are thought to change chromatin structure and relax it, and to serve as docking 6

ACCEPTED MANUSCRIPT sites for protein complexes that remodel nucleosomes or further modify histones. However, the term “histone acetyltransferases” and “histone deacetylases” for the enzymes that add or remove the

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acetyl group may be misleading, as a screen identified 3600 lysine acetylation sites on 1750

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mammalian proteins that are putatively acetylated and deacetylated by the same group of enzymes

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as histones (Choudhary et al., 2009). The roles of acetylation of non-histone target proteins are only beginning to be understood. Inhibition of HDACs induces global protein acetylation, yet gene

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expression analysis suggests that only 2–10% of genes’ expression is significantly changed, with

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almost similar numbers of genes upregulated and downregulated (Spange et al., 2009). Additionally, analysis of the promoters of the HDAC inhibitor showed that both downregulated and upregulated

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genes promoters displayed increased acetylation, demonstrating that histone acetylation may not be

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an accurate predictor of transcriptional activation (Ellis et al., 2008). These data suggest that the

too narrow.

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notion that HDAC inhibition modulates gene expression mainly by histone acetylation appears to be

3. HDAC inhibitors

A large number of natural and synthetic compounds function as HDAC inhibitors (HDACi). Most of the available HDACi possess a three-part structure consisting of a zinc-binding group that inserts in the active site, a linker and a moiety that interact with residues near the entrance to the active site. HDACi were shown to induce apoptosis, growth arrest, senescence, differentiation, immunogenicity, and to inhibit angiogenesis in different types of cancers (West et al., 2014). The most successful clinical use of HDACi has been with vorinostat and romidepsin in patients suffering 7

ACCEPTED MANUSCRIPT from refractory cutaneous and peripheral T cell lymphoma (Whittaker et al., 2010). HDACi can be divided into four main groups according to their chemistry: hydroxamates, cyclic peptides, aliphatic

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acids, and benzamides. Most of what we know about these molecules originates from cancer biology

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research.

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3.1 Hydroxamates

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Trichostatin A (TSA) - The first natural hydroxamate discovered to inhibit HDACs, was isolated from Streptomyces hygroscopicus as antifungal antibiotics. The (R)-TSA was found to inhibit cell

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proliferation in many cell lines, such as breast and lung cancer cells (Yoshida et al., 1995). Histone

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electrophoresis was instrumental in discovering TSA mechanism of action, showing that TSA

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treatment resulted in histones hyperaceytelation. Pulse-chase experiments revealed that the reason for the hyperacetylation is not augmented acetylation, but rather attenuated deacetylation, demonstrating that TSA was a bona fide HDACi. TSA was originally suggested to be a pan-cellular HDAC inhibitor with activity against HDACs 1-7 and 9 in nanomolar concentrations, and HADC 8 in micromolar concentrations. Despite the high activity of TSA, it was disqualified as a clinical drug due to its many side effects such as non-transformed cells apoptosis and increased DNA damage (Rodriguez-Paredes et al., 2011). Suberanilohydroxamic acid (SAHA) – Known commercially as vorinostat. In October 2006 vorinostat was the first HDAC inhibitor to be approved by the FDA, for the treatment of advanced cutaneous T cell lymphoma (Grozinger et al., 2002). Crystallographic studies revealed that vorinostat binds HDAC catalytic site and blocks its activity. Nanomolar concentrations of vorinostat are sufficient to inhibit 8

ACCEPTED MANUSCRIPT the deacetylation activity of HDACs in the cell and cause accumulation of hyperacetylated histones. The changes in acetyl levels were suggested to inhibit the cell ability to proliferate and avoid

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apoptosis. Suppressing the HDACs deacetylase activity resulted in the accumulation of anti-

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proliferative genes such as p21WAF1, p27 KIP1, DR5 and TNFα on the one hand, and decreased levels

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of pro-growth genes like: CDK2, CDK4, cyclin D1 and cyclin D2. Mechanistically HDAC inhibition also interferes in the cell cycle checkpoint G2 and alters the mitotic spindles in a way that disrupts

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chromosome attachment, causing mitotic accumulation. Vorinostat is a potent anti-cancer agent

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clinically tested in various types of cancers, especially hematological malignancies like lymphoma, myeloma and leukemia, but also in lung carcinoma and other solid tumors. Vorinostat showed

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(Munshi et al., 2006).

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profound but variable degree of inhibition of cell proliferation that appears to affect all cycling cells

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The hydroxamic acids family of HDACi includes many more small molecules that are being evaluated as anti-cancer agents; for example CBHA, LAQ-824, PXD-101, LBH-589, ITF2357, oxamflatin, ABHA, SBHA, Scriptaid, pyroxamide, SK-7041, SK-7068 and tubacin. Most of these molecules have anti-proliferative and pro-apoptotic effects. These different molecules vary in the specific HDACs they inhibit, the type of transformed cell they affect and the clinical concentration in which they are effective (Falkenberg et al., 2014). 3.2 Cyclic peptides Depsipeptide – a natural cyclic peptide product that inhibits HDAC 1 and 2 selectively. When entering the cell, the molecule undergoes glutathione-mediated reduction and becomes active. Once active, it binds to the zinc in the pocket of class I HDACs acting as a zinc chelator and inhibits deacetylation. By 9

ACCEPTED MANUSCRIPT inhibiting class I HDACs, depsipeptide modulates the expression levels of different genes, including cmyc, Hsp90 and p53. The cyclic peptide family includes more HDACi like apicidin, the irreversible

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HDACi - trapoxin A, and CHAP - a hybrid molecule composed of TSA and trapoxin A.

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3.3 Aliphatic acids

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Valporoic acid (VPA, 2-propylpentanoic acid) – a short chain fatty acid that was used for many years

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as an anticonvulsant for the treatment of seizures, epilepsy, and as a mood stabilizer. It was discovered that VPA can inhibit HDACs, especially class I HDACs. VPA inhibits HDAC activity, causing

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accumulation of hyper-acetylated histone tails (H3 and H4 histones) and other protein targets such as

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p53. Like many other HDACi, VPA has anti-proliferative and pro-apoptotic activities that makes it a

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potential anti-cancer agent. Other aliphatic acids that have the ability to inhibit HDAC are butyrate and its derivatives phenyl butyrate and pivanex.

3.4 Benzamides This family includes small molecules that are active mostly against class I HDACs. Like other class I HDACi these molecules were shown to cause apoptosis and block proliferation. The major HDACi of this family are: CI-994 (tacedinaline), MS-275, MGCD0103 and M344.

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3.5 Other HDACi In addition to the 4 families mentioned above there are other small molecules that exhibit

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HDAC inhibition activity. These small molecules cause apoptosis and have anti-proliferative activity,

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and some are being tested as anti-cancer drugs.

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4. Class I HDACs and hypertrophy

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Cardiac hypertrophy occurs when the heart experiences elevated workload; the

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cardiomyocytes adapt by synthesizing new sarcomeric units and enlarge. This cell growth is also

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accompanied by re-expression of fetal genes, alterations in the expression of proteins involved in excitation – contraction coupling, and changes in the energetic and metabolic state of the cardiomyocyte. Collectively, these changes manifest macroscopically as an increase in the heart shape and mass (Kehat et al., 2010). Since the expression of a large number of genes is altered in cardiac hypertrophy, it is not surprising that HDACs were shown to be actively involved in the process. In particular, the roles of class I HDACs in pressure overload- or adrenergic agonist-induced cardiac hypertrophy were studied by several groups. HDAC1 and HDAC2 share 85% amino acid homology (Figure 1b) and are found together in almost all repressive transcriptional complexes (Grozingeret al., 2002). Cardiac specific deletion of either HDAC1 or HDAC2 in knockout mice did not affect the heart baseline structure or function, or the heart response to pressure overload (Montgomery et al., 2007), suggesting that these enzymes may have a redundant function. However,

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ACCEPTED MANUSCRIPT double conditional deletion of both HDAC1 and of HDAC2 by αMHC-Cre resulted in right and left ventricular dilatation and death by two weeks of age. This observation shows that class I HDACs have

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a cardiomyocyte-autonomous role in cardiac structure and functional maintenance. In another

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report, gene-trapped HDAC2 mice were generated and studied (Trivedi et al., 2007). About 50% of

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the mice died at an early post-natal stage, but analysis of the surviving animals showed that they had a blunted hypertrophic response to pressure overload. The difference between these mice and the

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cardiac specific HDAC knockout mice which did not have a cardiac phenotype, may be due to the

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redundant role of HDAC2 in cardiomyocyte versus its non-redundant role in other non-myocytes cardiac cells. Yet, overexpression of HDAC2 specifically in cardiomyocytes (Trivediet al., 2007)

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resulted in cardiac hypertrophy, implying that class I HDACs may also control the hypertrophic

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response in cardiomyocytes. Cardiac-specific deletion of HDAC3 resulted in cardiac hypertrophy and

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fibrosis by 3 months of age and lethality by 4 months (Montgomery et al., 2008). The mice showed upregulation of genes involved in fatty acid uptake and oxidation, suggesting a role for HDAC3 in regulating cardiomyocyte metabolism. In contrast, cardiac transgenic over-expression of HDAC3 resulted in increased muscle mass and wall thickening, but this increase was shown to be due to increased cardiomyocyte hyperplasia without hypertrophy (Trivedi et al., 2008). While these studies clearly show that class I HDACs are actively involved in the heart growth and hypertrophic response, the specific cardiomyocyte autonomous roles versus their roles in non cardiomyocytes cells in the heart are less well defined. The roles of class I HDACs in the heart were also explored using HDACi. Cardiac hypertrophy induced by chronic infusion of isoproterenol was blunted when mice were concomitantly treated by TSA (Kook et al., 2003). Similarly, hypertrophy in rats and mice induced by angiotensin II or aortic 12

ACCEPTED MANUSCRIPT banding was blocked by concomitant administration of TSA or valporoic acid (Kee et al., 2006). The HDAC inhibitor, 3-(4-substituted phenyl)-N-hydroxy-2-propenamide (SK-7041 [SK]), was reported to

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be selective to HDAC1 and HDAC2 (Park et al., 2004). This drug also completely abrogated the

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hypertrophic response to aortic banding (Kooket al., 2003). API-D, a synthetic small-molecule

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derivative of the natural cyclic tetrapeptide apicidin, was reported to be selective to class I HDACs. Administration of this drug decreased myocardial hypertrophy after 1 or 9 weeks of pressure

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overload induced by thoracic aortic constriction in mice (Gallo et al., 2008). Importantly, at 9 weeks

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the mice treated with API-D showed improved cardiac function. Together these studies suggest that the effects of pan-HDAC inhibitors as suppressors of the hypertrophic response mainly resulted from

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inhibition of class I HDACs. In a different study, mice were subjected to aortic banding pressure

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overload for 2 weeks, and then treated with TSA or vehicle for additional 3 weeks (Kooket al., 2003).

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This treatment resulted in partial regression of hypertrophy and prevented further progression, thus showing that HDACi can not only prevent the development of hypertrophy but also reverse it. HDACi also blocked hypertrophy in the more clinically relevant model of post-infarction remodeling. Wistar rats were treated by vehicle or valporoic acid 24 hours after ligation of the left anterior descending artery, and hypertrophy was assessed by measuring the size of enzymatically dissociated cardiomyocyte size from the border zone. Both the cardiomyocyte hypertrophy and collagen formation were significantly reduced by the valporoic acid treatment. Class I HDACs affect the acetylation of large stretches of the genome, and therefore the identification of their targets that control the hypertrophic response is difficult. It is likely that the effect is mediated by altering the expression of many genes, directly or indirectly. Several important HDAC targets were however identified. Class I HDAC inhibition by SK7041 was shown to increase the 13

ACCEPTED MANUSCRIPT acetylation in the promoter region and to up-regulate Krüppel-like factor 4 (KLF4) (Kee et al., 2009). Overexpression of KLF4 blocked cardiac hypertrophy in neonatal cardiomyocytes, suggesting that it

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may be one of the downstream targets of class I HDACs. Brg1 which is the essential ATPase subunit

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of the BAF complex, co-immunoprecipitated with HDAC1, 2, 3 and 9 in the E11.5 mouse heart

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ventricle. Brg1 repressed the α-myosin heavy chain reporter, but failed to do it when cells were treated with TSA , similarly HDACs could not repress the α- myosin heavy chain reporter without

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Brg1 (Hang et al., 2010). Autophagy was also blocked by TSA administration during pressure overload,

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and was suggested as the target of HDACi attenuating the hypertrophic response (Cao et al., 2011). During induction of hypertrophy, myosin isoform acetylation increased progressively, and lysine

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acetylation increased the actin sliding velocity of the myosin. HDAC3 was shown to localize to the A

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band of sarcomeres and was demonstrated to deacetylate myosin heavy chain (Samant et al., 2011).

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These data suggest that HDACs can also control cardiac contractility directly. More recently HDAC inhibitors were shown to stimulate the expression of the phosphatase DUSP5 and block nuclear ERK1/2 localization and activation (Ferguson et al., 2013). In cardiomyocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling was suppressed by DUSP5, but cytosolic ERK1/2 activation was maintained, suggesting that DUSP5 is another HDAC target that controls the hypertrophic response.

5. Class II HDACs and hypertrophy Studies in gene modified animals indicate that class IIa HDACs suppress cardiac hypertrophy. HDAC9 cardiac knockout mice developed spontaneous cardiac hypertrophy by 8 months of age, and 14

ACCEPTED MANUSCRIPT younger mice were sensitized to aortic banding, and displayed a much larger hypertrophic response than wildtype animals (Zhang et al., 2002). HDAC5 knockout mice displayed a similar phenotype to

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HDAC9 knockout mice, with mild spontaneous hypertrophy at 8 months of age, and an exaggerated

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hypertrophic response to thoracic aortic banding (Chang et al., 2004). Interestingly, mice lacking

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either HDAC5 or HDAC9 did not show an exaggerated hypertrophic response to chronic -adrenergic stimulation, suggesting that these HDACs may not block all types of hypertrophic growth (Changet al.,

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2004). However, overexpressing several class IIa HDACs, including HDAC4, 5 and 9 in neonatal rat

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cardiomyocytes, did block adrenergic agonist-dependent cardiac hypertrophy, supporting the antihypertrophic role of class IIa HDACs, but questioning the hypertrophic-pathway specificity of these

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factors.

The function of the class IIa enzymes is not fully understood. Several reports suggest that class

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IIa HDACs functions as transcriptional repressors (McKinsey et al., 2006), although as noted above, many genes are upregulated following HDAC inhibition. Class IIa HDACs can physically associate with transcription factors through their amino terminal domain. HDAC4 was demonstrated to bind the transcription factor Runx2, repressed its activity and regulated chondrocyte hypertrophy and skeletogenesis (Vega et al., 2004). The histone acetyl transferase p300 was shown to mediate Runx2 acetylation, thereby inhibiting the degradation of Runx2 and increasing its transactivation activity (Jeon et al., 2006). Conversely, HDAC4 and HDAC5 were shown to induce deacetylation of Runx2 and to induce degradation of Runx2; however, as these assays were performed using coimmunoprecipitation in cell extract, specific deacetylase activity by these class IIa proteins was not conclusively demonstrated (Jeonet al., 2006). In fact, it was shown that class IIa HDACs are very inefficient enzymes, at least on canonical substrates, and suggested that most reported enzymatic 15

ACCEPTED MANUSCRIPT activity reflects the involvement of endogenous co-purified class I HDACs. To demonstrate this, two structurally dissimilar HDACi, the hydroxamate LAQ824 and the cyclic tetrapeptide apicidin were

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selected as test molecules. Although both LAQ824 and apicidin inhibited purified HDAC4-FLAG

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activity to the same degree, only the LAQ824 derivative significantly and specifically bound HDAC4

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(Lahm et al., 2007). This lack of correlation between binding and inhibition demonstrates that the deacetylase activity of the HDAC4 complex likely originates from other co-purified class I HDACs.

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Moreover, the recombinant HDAC4 catalytic domain was at least 1000 fold less active than class I

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HDAC catalytic domain when tested on acetylated peptides.

Structural analysis of HDAC4 catalytic domain showed a class IIa-specific structural zinc-

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binding domain (Figure 3), and this domain displayed different conformation in the inhibitor-free and

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in the inhibitor-bound forms (Bottomley et al., 2008). It was shown that HDAC4 associates with the N-

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CoR·HDAC3 co-repressor complex via the HDAC’s catalytic zinc-binding domain, which has to be properly folded for the formation of a stable ternary complex. HDAC inhibitor binding may destabilize this domain and disrupt the association of HDAC4 with N-CoR·HDAC3 complex. A more recent analysis showed that at pharmacologically-relevant concentrations class IIa HDACs are not targeted by most HDACi, such as TSA, SAHA and apicidin, (Bradner et al., 2010). Hence, while these agents are usually referred to as ‘non-selective’ or ‘pan-HDAC’, their effects on hypertrophy are likely mediated only by their ability to block other classes of HDACs, most notably class I HDACs. The activity of the transcription factor myocyte enhancer factor-2 (MEF2) is up-regulated in cardiomyocytes in response to phenylephrine (Lu et al., 2000), and cardiac-specific overexpression of constitutively active forms of MEF2A or MEF2C in mice resulted in cardiomyopathy at baseline or

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ACCEPTED MANUSCRIPT predisposed the animals to failure following pressure overload stimulation (Xu et al., 2006). Class IIa HDACs interact with members of the MEF2 transcription factor family through a conserved binding

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domain in the amino terminal tail of class IIa HDACs. The ability of class IIa HDACs such as HDAC4 or 5

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to inhibit MEF2 is blocked by phosphorylation of two conserved serine residues in the amino terminal

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tail of HDACs, which creates docking sites for cytoplasmic proteins such as 14-3-3 (Zhang et al.,

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2001). This cytoplasmic binding, relieves the inhibition from nuclear factors such as MEF2. Angiotensin II, α1-adrenergic agonists and endothelin induce hypertrophy by signals acting

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through a G-protein coupled receptor that couples to the Gq-alpha subunit. These agonists were shown to stimulate protein kinase D1 (PKD1) in neonatal cardiomyocytes cultures (Harrison et al.,

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2006). Ectopic overexpression of constitutively active PKD1 in mouse heart led to dilated

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cardiomyopathy, and PKD1 activation correlated with phosphorylation-dependent nuclear export of

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HDAC5. This mechanism illustratrates how upstream signaling can converge on and regulate class IIa HDACs. Two cysteine residues in HDAC4, Cysteine 667 and Cyteine-669 can be oxidized and form intramolecular disulfide bonds in response to reactive oxygen species. Thioredoxin 1 can reduce these cysteines oxidation, and inhibit the nuclear export of HDAC4, independently of its phosphorylation status, showing that the nucleo-cytoplasmic shuttling of class IIa HDAC4 can also be modulated by their redox modification (Ago et al., 2008). The protein MEF2-interacting transcription repressor (MITR) is encoded by the HDAC9 gene and is homologous to the amino-terminal regions of HDAC4 and -5, but lacks the C-terminal HDAC catalytic domain. Despite lacking the class IIa HDAC catalytic domain, MITR is as potent repressor of MEF2-dependent transcription as the other class IIa HDACs (Zhanget al., 2001). This suggests that

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ACCEPTED MANUSCRIPT the catalytic activity of class IIa HDACs may not be necessary for all of their functions. The ability of MITR to repress MEF2-dependent transcription may be mediated by its ability to directly bind HDAC1

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(Sparrow et al., 1999).

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The Class I HDAC3 was shown to localize to the sarcomere and control contractility (Samantet

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al., 2011). Similarly, Class IIa HDAC4 was shown to be associated with the cardiac sarcomere, and the

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use of HDAC inhibitors or anti-HDAC4 antibody resulted in increased acetylation of sarcomeric proteins, and enhanced the myofilament calcium sensitivity (Gupta et al., 2008). We have shown

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that HDAC4 can also localize to the nuclear pore in cardiomyocytes and modulate the association of selected target genes with nucleoporins through interaction with the nuclear pore protein Nup155

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(Kehat et al., 2011).

In contrast to class IIa HDACs, the class IIb HDAC6 is predominantly localized in the cytoplasm.

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Class IIb HDAC6 contain a full duplication of its deacetylase domain, followed by a specific ubiquitinbinding domain at the C-terminal end (Valenzuela-Fernandez et al., 2008). HDAC6 was shown to interact with misfolded ubiquitinated protein, concentrate toxic protein aggregates and facilitates their aggresome-dependent clearance (Kawaguchi et al., 2003). Further, it was shown that HDAC6 (which is important for angiogenesis) regulates zebrafish vessel formation, and that HDAC6-deficient mice exhibited a reduced formation of perfused vessels (Kaluza et al., 2011). HDAC6 knockout mice displayed cardiac hypertrophy and fibrosis similar to wild type mice in response to angiotensin II infusion. However, following long term infusion or following pressure overload mediated by transverse aortic constriction, the HDAC6 knockout mice displayed improved preservation of cardiac function (Demos-Davies et al., 2014). The treatment with the HDAC6 inhibitor tubastatin A protected

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ACCEPTED MANUSCRIPT the atria of dogs from electric remodeling and cellular dysfunction in atrial tachypacing model (Zhang et al., 2014), suggesting the potential role of this drug in atrial fibrillation.

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Collectively, these data suggest that class II HDACs have a role in cardiac hypertrophy. The

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upstream mechanisms controlling the nucleo-cytoplasmic shuttling of class IIa HDACs in

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cardiomyocytes have been comprehensively explored, although the targets of these enzymes remain

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largely unknown. The apparent lack of significant enzymatic activity in class IIa HDACs is also unexplained. Studies in gene modified animals indicate that specific blockade of this enzyme class

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may be detrimental for the heart. Since specific blockers of class IIa HDACs are just emerging, a pharmacologic proof of this concept is largely lacking. Similar to what has been discussed above

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regarding class I HDACs and their inhibitors, the studies in gene modified animals targeted the

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cardiomyocyte class IIa HDACs, while small molecule inhibitors also affect fibroblasts, endothelial

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cells and other non-myocytes in the heart. 6. HDACs and cardiac fibrosis

Cardiac fibroblasts are the most abundant cells in the mammalian heart, and their main role is secretion and maintenance of the heart extracellular matrix. The accumulation of this matrix can be both adaptive and deleterious, depending on the degree and context. Various pathological conditions trigger the deposition of extracellular matrix in the heart, including myocardial infarction and the development of concentric hypertrophy. In addition, normal aging is also associated with the development of progressive fibrosis (Cieslik et al., 2014). Subcutaneous injections of deoxycorticosterone acetate (DOCA) and salt supplementation in rats results in hypertension, cardiac hypertrophy and interstitial cardiac fibrosis. Administration of the HDACi SAHA to DOCA-salt rats 19

ACCEPTED MANUSCRIPT attenuated the systolic hypertension, the development of left ventricular hypertrophy, the increased inflammatory cell infiltration, and the interstitial collagen deposition (Iyer et al., 2010). Interstitial

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fibrosis was also attenuated by class I HDACi in the aortic banding models, along with the attenuation

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of hypertrophy (Keeet al., 2006). Over-expression of the gene HopX in transgenic mice hearts

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resulted in cardiac hypertrophy, atrial fibrosis and increased susceptibility to develop atrial fibrillation. HDAC inhibition in these mice using TSA reduced the atrial fibrosis, connexin40

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remodeling and atrial arrhythmia vulnerability (Liu et al., 2008).

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HDAC inhibitors may suppress fibrosis in the heart via inhibition of endothelial-tomesenchymal transition, inhibition of fibroblast proliferation, inhibition of fibroblast migration, or

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through the induction of genes that suppress extracellular matrix production from fibroblasts (Figure

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4). However, as cardiac hypertrophy was also blocked in all these models, it is hard to discern

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whether HDAC inhibition primarily affected the cardiomyocytes and suppressed the proinflammatory cues for fibrosis produced by the myocytes, from direct effects on the fibroblasts. Conversely, it is also possible that non-myocytes in the heart are the primary target of HDACi, and that the attenuation in cardiac hypertrophy is secondary to the effect on non-myocytes and the changes in the cardiac milieu. Several lines of evidence however do support direct effects on fibroblasts. Treatment of normal human lung fibroblasts with TSA inhibited TGF-beta1-mediated alpha-smooth muscle actin and alpha1 type I collagen mRNA induction. TSA also blocked the TGFbeta1-driven contractile response (Guo et al., 2009). Similarly, the ability of HDAC inhibitors to block collagen synthesis in isolated cardiac fibroblasts supports a direct effect on fibroblasts (Kong et al., 2006). More recently it was demonstrated that selective inhibition of class I HDACs suppressed angiotensin II mediated cardiac fibrosis by targeting two key effector cell populations, cardiac 20

ACCEPTED MANUSCRIPT fibroblasts and bone marrow-derived fibroblasts (Williams et al., 2014). Here an angiotensin II infusion model that did not trigger significant left ventricular hypertrophy was used, and while pan-

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and class I HDAC-selective inhibitors were effective in blocking cardiac fibrosis, molecules targeting

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blocks cardiac fibroblasts in the G0/G1 phase of the cell cycle.

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class IIa and IIb HDACs were not. Mechanistically it was demonstrated that Class I HDAC inhibition

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7. HDACs and cardiac ischemia

In addition to the effects on cardiac hypertrophy and fibrosis, HDAC inhibition was also shown

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to attenuate ischemic injury in the heart as well as in other tissues. Pre-treatment with TSA resulted

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in improvements in post-ischemic ventricular function, with a reduction in infarct size in both early

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and delayed preconditioning models (Zhao et al., 2007). Mechanistically, the effects of HDAC inhibition by TSA were attenuated in MKK3−/− or Akt-1−/− knockout mice, suggesting that these kinases are downstream targets of HDACs (Zhang et al., 2014). 8. Clinical experience with HDAC inhibitors in the heart So far the clinical experience with HDAC inhibitors in the heart is mostly limited to observations of toxic cardiac side effects of HDACi used in cancer patients. Unfortunately, HDACis such as vorisonstat and romidepsin have been associated with serious cardiac side effects, the most severe of which was QT interval prolongation (Piekarz et al., 2009). The mechanism of QT interval prolongation was explained by aberrant cellular trafficking or function of the ether-ago-go (hERG) potassium channel. Sadly, several cases of sudden cardiac death were attributed to these drugs (Shah

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ACCEPTED MANUSCRIPT et al., 2006). The anti-epileptic drug Sodium valporate has HDAC inhibitory function. The health records of a case control study of patients prescribed this antiepileptic drug suggested that sodium

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valproate exposure was associated with the risk of myocardial infarction (Dregan et al., 2014). The

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raise some concern regarding the cardiac safety of this drug.

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interpretation of this finding is difficult since sodium valproate is not a pure HDAC inhibitor, but does

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9. Perspectives

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Heart failure is one of the largest health challenges we are facing. It is a prevalent disease with very high morbidity and mortality. Despite advances in pharmacologic and non-pharmacologic treatments, new drugs and approaches are direly needed. Class I HDAC inhibitors limit the hypertrophic response, preserve cardiac function and attenuate fibrosis in a variety of small animal models of hypertrophy. However, electrophysiological cardiac toxicities, such as QT prolongation, were also observed in cancer clinical trials. Therefore, despite the promise of these drugs, larger animal studies and the use of more realistic models are still needed before the efficacy of these compounds can be assessed in clinical trials.

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Figure legends

Figure 1: HDAC family enzymes. a. The zinc binding HDACs are grouped into 4 classes based on their

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structure. Class I HDACs (HDAC1, -2, -3, and -8) have a compact structure and are composed mostly of

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the catalytic domain (shown in dark brown). Class II HDACs can be subdivided into 2 subclasses; Class

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IIa HDACs (HDAC4, -5, -7 and -9) have one catalytic domain and a long amino-terminal adaptor and class IIb (HDAC6 and HDAC10), containing two catalytic domains. Class IV HDACs include only HDAC

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11, a relatively newly discovered protein, which resembles class I HDAC's. b. A cladogram of HDACs

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prepared using COBALT (Papadopoulos et al., 2007), showing the relative sequence similarity

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other.

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between the various HDACs. As shown, HDACs 1 and 2 and HDACs 5 and 9 closely resemble each

Figure 2: The balance of histone acetylation and deacetylation. The level of histone tail acetylation which determines the chromatin state is the result of the competing actions of histone acetylase and histone deacetylase enzymes. HDAC inhibitors (HDACi) change this balance by inhibiting HDACs, thereby resulting in increased histone acetylation.

Figure 3: Multiple alignment of the catalytic domain of human HDACs. Multiple alignment of the catalytic domain of human HDACs was performed using T-coffee software (Notredame et al., 2000). This analysis shows a highly conserved domain. The conserved residues involved in the zinc ion 23

ACCEPTED MANUSCRIPT binding are marked with light blue dots. A catalytic tyrosine is conserved in the HDACs except for vertebrate class IIa HDACs where it is replaced by a histidine residue (marked by a black dot). A

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mutation in HDAC4, replacing this histidine back to tyrosine produced an enzyme with a 1,000-fold

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higher catalytic efficiency, suggesting that this residue in class IIa HDACs explain the very low catalytic

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activity.

Figure 4: HDAC inhibitor target cells in the heart. The heart is composed of several cell types and

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HDACi can target all of them. Therefore the net effect of HDAC blockade can stem from the direct

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effect on cardiomyocytes, fibroblasts, endothelial cells and cells in the heart. Since these cells interact

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with each other, effects on one cell type are likely to influence all others as well.

Conflict of Interest Statement: The authors declare that there are no conflicts of interest.

Acknowledgments: This study was supported by the Rappaport Family Institute for Research in the Medical Sciences, The Clinical Research Institute at Rambam, Rambam Medical Center, The MARIE CURIE action Career Integration Grants, The Israel Science Foundation and Israel Ministry of Health.

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