posttranslational modifications of histone deacetylases: implications for cardiovascular diseases

Pharmacology & Therapeutics xxx (2014) xxx–xxx

JPT-06664; No of Pages 13

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Pharmacology & Therapeutics

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Associate editor: P. Molenaar

Posttranslational modifications of histone deacetylases: Implications forcardiovascular diseases

Gwang Hyeon Eom, Hyun Kook ⁎Department of Pharmacology and Medical Research Center for Gene Regulation, Chonnam National University Medical School, Gwangju 501-746, Republic of Korea

Abbreviations: CaMK, Ca2+/calmodulin-dependent pCardiovascular diseases; Cys, Cysteine; Hif-1, Hypoxia-iIschemia–reperfusion; KLF, Krüppel-like factor; LDLR, Lowtranscription repressor; NAD, Nicotinamide adenine dinSuberoylanilide hydroxamic acid; SCFA, Short chain fattythelial growth factor; VPA, Valproic acid.⁎ Corresponding author at: Department of Pharmacol

Gwangju, 501-746, Republic of Korea. Tel.: +82 62 220 4E-mail address: [email protected] (H. Kook).

http://dx.doi.org/10.1016/j.pharmthera.2014.02.0120163-7258/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Eom, G.H., & Kook,Pharmacology & Therapeutics (2014), http://

a b s t r a c t
a r t i c l e i n f o
Keywords:
Histone deacetylasesCardiovascular diseasesPosttranslational modificationsHistone deacetylase inhibitorsTherapeutics
Posttranslational modification (PTM) is a term that implies dynamic modification of proteins after their transla-tion. PTM is involved not only in homeostasis but also in pathologic conditions related to diverse diseases.Histone deacetylases (HDACs), which are known as transcriptional regulators, are one example of posttransla-tionalmodifierswith diverse roles in human pathophysiology, including cardiovascular diseases. In experimentalmodels, HDAC inhibitors are beneficial in supraventricular arrhythmia, myocardial infarction, cardiac remodel-ing, hypertension, andfibrosis. In addition, HDACs are closely related to other vascular diseases such as neointimaformation, atherosclerosis, and vascular calcification. Currently, HDACs are classified into four different classes.The class IIa HDACs work as transcriptional regulators mainly by direct association with other transcription fac-tors to their target binding elements in a phosphorylation-dependent manner. Class I HDACs, by contrast, havemuch greater enzymatic activity than the class II HDACs and target various non-histone proteins as well as thehistone-core complex. Class I HDACs undergo PTMs such as phosphorylation, sumoylation, and S-nitrosylation.Considering the growing evidence for the role of HDACs in cardiovascular diseases, the PTMs of theHDACs them-selves as well as HDAC-mediated PTM of their targets should be considered for future potential therapeutictargets. In this review, we discuss 1) the roles of each HDAC in specific cardiovascular diseases and 2) the PTMof HDACs, 3) and the implications of such modifications for cardiovascular diseases.

© 2014 Elsevier Inc. All rights reserved.

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Histone deacetylases in cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Posttranslational modifications in cardiovascular diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. Possible limitations of histone deacetylase modifiers in therapeutic application . . . . . . . . . . . . . . . . . 05. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
rotein kinase; CK2, Casein kinase 2; COPD, Chronic obstructive pulmonary disease; CTCL, Cutaneous T cell lymphoma; CVD,nducible factor-1; HAT, Histone acetyltransferase; HDAC, Histone deacetylase; HDACi, Histone deacetylase inhibitors; I/R,-density lipoprotein receptor; Lys, Lysine; MEF, Myocyte enhancer factor; MI, Myocardial infarction; MITR, MEF-2 interactingucleotide; PKA, Protein kinase A; PKC, Protein kinase C; PKD, Protein kinase D; PTM, Posttranslational modification; SAHA,acid; Ser, Serine; siRNA, Small interfering RNA; SUMO, Small ubiquitin-like modifier; TSA, Trichostatin A; VEGF, Vascular endo-

ogy and Medical Research Center for Gene Regulation, Chonnam National University Medical School, 5 Hak-dong, Dong-ku,243; fax: +82 62 232 6974.

H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases,dx.doi.org/10.1016/j.pharmthera.2014.02.012

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2 G.H. Eom, H. Kook / Pharmacology & Therapeutics xxx (2014) xxx–xxx

1. Introduction

1.1. Cardiovascular diseases

Since the 1970s, cardiovascular disease (CVD) has been a leadingcause of death throughout the world (Hunter & Reddy, 2013). Althoughgreat efforts have been made by physicians, researchers, and even pri-mary care practitioners to reduce the mortality due to CVD, the diseaseis still a major cause of death in developed countries. In large part be-cause of unbalanced, high-fat diets, the median age of patients withCVD has been decreasing (McGill et al., 2008). “Cardiovascular disease”is a term indicating medical problems in the heart, the blood vessels, orboth. Sometimes CVD indicates “heart disease” in a limited sense;usually, however, vascular problems in the brain or kidney or other pe-ripheral arterial disease is also included. The most common CVDs arehypertension and atherosclerosis (Ross, 1999). Even in healthy individ-uals, aging followed bymorphological and physiological changes affectscardiovascular structures and function, which subsequently leads toa high risk of CVD. Therefore, risk-factor-reducing efforts such asconsuming a balanced healthy diet, getting adequate amounts of exer-cise, increasing lean body mass, and stopping smoking are strongly rec-ommended to reduce the development of CVD (McGill et al., 2008).Besides a preventive approach, therapeutic interventions to halt diseaseprogress or to recover a healthy state are necessary as well, and such in-terventions require a fundamental understanding of disease develop-ment and progress. Considering that most CVD-related pathologicevents are caused by malfunction of normal proteins, PTMs that mightresult in those abnormal behaviors should be extensively studied.Thus, understanding the PTMs associated with CVD may offer opportu-nities for the development of ideal therapeutics with maximal efficacyand minimal unwanted effects.

1.2. Posttranslational modifications

Proteins are not stable but are dynamically modified by otherproteins, such as kinases, acetyltransferases, methyltransferases,ubiquitinylases, and carboxylases. These changes are finely balanced byopposing enzymes such as phosphatases, deacetylases, demethylases,deubiquitinylases, and decarboxylases. These dynamic changes arecalled PTMs and are closely linked to diverse cellular functions andhuman diseases. For example, when ligands occupy their binding sitesin receptors, receptor tyrosine kinases phosphorylate target moleculesand an extracellular signal is delivered to cytoplasmic or nuclear targets(Hubbard & Till, 2000). Phosphorylation is an essential modificationin the regulation of enzyme activation (Stambolic & Woodgett, 1994;Dimmeler et al., 1999), DNA-binding capacity (Beg et al., 1993), forma-tion of complexes (Maudsley et al., 2000), and cell cycle regulation(Serrano et al., 1993).

Besides phosphorylation, ubiquitination is an important modifica-tion that has been intensively investigated recently. Ubiquitin is asmall protein with a molecular mass of just 8.5 kDa. It has 7 lysine resi-dues in its structure. Ubiquitination indicates the covalent binding ofubiquitin to a substrate. This process generally involves binding of gly-cine 76 at the C-terminus of ubiquitin to a lysine of the substrate.Polyubiquitination refers to additional ligation of ubiquitin to anotherubiquitin that has already been conjugated with a protein, which impliesthat ubiquitinworks as a substrate for further ubiquitination. Two lysinesof ubiquitin are involved in polyubiquitination: Lys-48 and Lys-63. Lys-48-linked polyubiquitination is associated with protein degradation andrecycling by proteolysis (Glickman & Ciechanover, 2002), whereas Lys-63-linkedpolyubiquitination is atypical and is involved in other processessuch as inflammation, DNA repair, and endocytic trafficking (Miranda &Sorkin, 2007). In contrast to polyubiquitination, monoubiquitination,which is also frequently observed, has quite different biological functions.Althoughmonoubiquitination is sometimes regarded as a beginning stepof polyubiquitination, most monoubiquitination solely affects cellular

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modificaPharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmth

events such as endocytosis, trafficking, and signal transduction such asphosphorylation (Miranda & Sorkin, 2007). Small ubiquitin-likemodifi-er (SUMO) proteins are analogous to ubiquitin, and the characteristicsof SUMO modification, which is termed sumoylation, resemble thoseof monoubiquitination (Melchior, 2000).

Likewise, protein acetylation, an alternate well-known PTM, has aunique biological function. Perhaps one of the best documented targetsof acetylation involves histone H3 and H4 proteins; acetylation of thehistone tail is closely associatedwith transcriptional activation. The pos-itive charge of the histone core is neutralized by adding an acetyl moie-ty, which thereby loosens the tight interaction between the negativecharge of the phosphate group in DNA and the histone tail (de Ruijteret al., 2003). The nucleosome is then opened to the transcriptional ma-chinery, which initiates gene expression. Non-histone proteins are alsosusceptible to acetylation, which affects enzyme activity (Santos-Rosaet al., 2003), protein–protein interaction (Levy et al., 2004), DNA re-cruitment (Gu & Roeder, 1997), and transcriptional activity (Evanset al., 2007).

It is noteworthy that each PTM can affect other modifications; in-deed, we can easily find multiple modifications at different residues ina single molecule or even at a single residue, which seems like “compe-tition” between the various modifications. For example, acetylation,methylation, and ubiquitination commonly occur at a lysine residue,and these modifications sometimes regulate the target protein functionin a competitive manner. Histone H3 Lys-9 is a target site for both acet-ylation and methylation, and trimethylated Lys-9 is found in constitu-tively repressed genes (Barski et al., 2007). In contrast, acetylation onthis residue activates gene expression (Koch et al., 2007). Followingthe removal of the methyl group by specific demethylases, histone ace-tyltransferase (HAT) enzyme acetylates H3 Lys-9. By contrast, after theacetyl moiety is removed by histone deacetylase (HDAC), the remainingunmodified lysine residue is subject to mono-, di-, and tri-methyl mod-ification (Guillemette et al., 2011). Indeed, growing evidence suggeststhat HDAC works in conjunction with histone methyltransferase(Wysocka et al., 2003). Similarly, acetylation increases protein stabilityby competition with polyubiquitination (Li et al., 2002). Furthermore,PTM-associated PTMs such as acetylation-dependent phosphorylation(Park et al., 2003), phosphorylation-dependent acetylation (Correet al., 2009), or phosphorylation-dependent ubiquitination (Koeppet al., 2001; Lin et al., 2002) are also reported.

Protein acetylation is finely regulated by two different groups of en-zymes: HATs and HDACs. At least 18 different HDACs in mammals havebeen discovered, which are categorized into four classes. HDAC1, 2, 3,and 8 are members of the class I HDACs; HDAC4, 5, 6, 7, 9, and 10 areclass II HDACs; the sirtuin family members, Sirt1, Sirt2, Sirt3, Sirt4,Sirt5, Sirt6, and Sirt7, are class III HDACs; and HDAC11 is the only classIV HDAC. Class I, II, and IV HDACs contain and require zinc ion fortheir enzyme activity (Minucci & Pelicci, 2006); however, class IIIHDACs are NAD+-dependent (Blander & Guarente, 2004). Like theHATs, HDACs also have non-histone substrates (Chen et al., 2002;Hubbert et al., 2002; Ito et al., 2002; Watamoto et al., 2003; Ito et al.,2006). Thus, it has been suggested that lysine deacetylase, or KDAC,would be more appropriate nomenclature because histone is not theonly substrate and these non-histone targets have more diverse biolog-ical functions than transcriptional regulation (Choudhary et al., 2009).In the present review, we discuss the roles and PTMs of the class I andclass II HDACs and their mechanisms of regulation in association withCVD.

2. Histone deacetylases in cardiovascular disease

2.1. Histone deacetylases

The criterion applied to divide the class I and class II HDACs is basedon the homology of each HDAC to yeast HDACs (Blander & Guarente,2004). Class II HDACs consist of 1) a large N terminus regulatory region,

tions of histone deacetylases: Implications for cardiovascular diseases,era.2014.02.012


3G.H. Eom, H. Kook / Pharmacology & Therapeutics xxx (2014) xxx–xxx

2) an HDAC domain, and 3) a short tail in the C terminus. Class II HDACsform a huge complex by interactionwith distinct corepressors or epige-netic regulators and thereby suppress the gene expression of down-stream targets (McKinsey et al., 2000a). The class I HDACs, however,do not have a “large N terminus regulatory region” like the class IIHDACs; they have only the HDAC domain and a short regulatory sitein the C terminus (Taunton et al., 1996). Indeed, class I HDACs looklike a truncated form of class II HDACs and the molecular weight ofclass II HDACs is more than double that of the class I HDACs. Class IIHDACs are divided into two subgroups, IIa (4, 5, 7, and 9) and IIb (6and 10), by structure homology. Unlike the IIaHDACs,which share com-monmolecular structures, IIb HDACs are somewhat atypical. TwoHDACdomains exist in a single HDAC6molecule (Verdel et al., 2000),whereasan eccentric leucine-rich region is located in HDAC10 (Fischer et al.,2002; Kao et al., 2002).

It is still controversial whether the class II HDACs have intrinsicdeacetylase activity. By in vitro analysis, Fischle et al. (2002) and Joneset al. (2008) reported that the deacetylase activity of class II HDACs isan artifact that results from co-purification of class I HDACs. Indeed,class II HDAC-mediated transcriptional repression is thought to be me-diated by class I HDACs, which are recruited as a repressor complexin vivo. Nebbioso et al. (2009) however, reported that class IIa HDACsdo have intrinsic activity in vivo. Indeed, we observed the small but sig-nificantmagnitude of intrinsic activity of HDAC5 under the condition oftreatment with class I HDAC-selective inhibitors. We also observed theabsence of deacetylase activity of HDAC5 by substitution of histidine833 with alanine, which is known to disrupt the pocket structure(Eom, unpublished observation). It is noteworthy that some chemicalinhibitors with high class selectivity, such as class I-selective inhibitors,class IIa HDACi, and class IIb HDACi, have been recently developed(Jones et al., 2006; Kim et al., 2006). Use of these selective inhibitors isexpected to elucidate whether the intrinsic activity of class II HDACs isrequired for the maintenance of homeostasis or pathophysiologic

Fig. 1.Molecular structure of histone deacetylases and information for inhibitors. Color bars indwhite, S-nitrosylation. Abbreviations: a, acetylation; di-S, disulfide bond; HDAC, histone deacet


processes. The molecular structures of the HDACs, the known PTMsites, and inhibitor information are summarized in Fig. 1 and Table 1.

2.2. Structures and classification of histone deacetylase inhibitors

HDACi have been highlighted as anti-cancer drugs because of theirunique properties to induce growth arrest, differentiation, and apopto-sis (Marks et al., 2001). Class III HDACs require NAD+ for intrinsic activ-ity, whereas class I, II, and IV HDACs are zinc-dependent. Thus, mostclass I and II HDACi are targeted to zinc ion. These zinc-targetedHDACi are mainly divided into four classes according to their structurehomology: hydroxamic acid, cyclic peptides, short chain fatty acids(SCFAs), and benzamides.

Hydroxamic acid binds to zinc ion in the catalytic site of HDACsand thereby inactivates enzymes (Villar-Garea & Esteller, 2004).For this reason, hydroxamic acid derivatives are nonselective inhib-itors because both class I and class II HDACs are zinc-dependent. Thefirst example of this type of inhibitor, whichwas approved for the treat-ment of human cancer by the U.S. Food and Drug Administration in2006, is suberoylanilide hydroxamic acid (SAHA). Other well-knownhydroxamic acid-derived HDAC inhibitors are trichostatin A (TSA),scriptaid, panobinostat, and givinostat. Among these inhibitors, TSAand scriptaid have been widely studied.

Cyclic peptides are another class of zinc-ion-dependent HDAC inhib-itors (Furumai et al., 2002). Interestingly, however, their various chem-ical structures enable the inhibitors to have relative selectivity onspecific HDACs. For example, apicidin and romidepsin preferentially in-hibit class I HDACs. Romidepsinwas approved by theU.S. Food andDrugAdministration in 2009 for the treatment of cutaneous T cell lymphoma(CTCL).

SCFAs are relatively small HDACi with a simple structure. AlthoughSCFAs are less potent, they can easily cross the blood–brain-barrierowing to their small molecular weight (Xu et al., 2007). For example,

icate: red, phosphorylation; blue, acetylation; yellow, sumoylation; gray, di-sulfide bond;ylase; s, sumoylation; S-NO, S-nitrosylation; p, phosphorylation; ZnF, zinc-finger domain



Table 1Posttranslational modification sites of histone deacetylases. Abbreviations: a, acetylation.C, cysteine. di-S, disulfide bond. HDAC, histone deacetylase. K, lysine. ND, not determined.s, sumoylation. S, serine. S-NO, S-nitrosylation. p, phosphorylation. T, threonine. Y,tyrosine.

Class Subtype PTM sites

I HDAC1 a: K218, K220, K432, K438, K439, K441p: S421, S423s: K444, K476

HDAC2 p: S394, S407, S411, S422, S424s: K462S-NO: C262, C274

HDAC3 p: S424HDAC8 p: S39

IIa HDAC4 p: S210, S246, S265, S298, S302, S350, S467, S632s: K559di-S: C667, C669

HDAC5 p: S259, S279, T292, S498, S611, S661, S755, S1108di-S: C696, C698

HDAC7 p: S155, S181, S358, S486HDAC9 p: S220, S239, S253, S451, S554

IIb HDAC6 p: S22, S458, Y570HDAC10 ND

IV HDAC11 ND


valproic acid (VPA) has been widely used for a long time to control var-ious types of epilepsy. Thus, SCFAs are emerging HDACi for the treat-ment of neurologic disorders, such as Parkinson's disease, Huntington'sdisease, and Alzheimer's disease (Harrison & Dexter, 2013).

Unlike other HDACi, benzamides are considered to be specific inhib-itors with long half-lives (Glozak et al., 2005). MS-275 is currently inphase II trials for Hodgkin lymphoma, lung cancer, and breast cancer.

2.3. Vascular diseases

2.3.1. AngiogenesisThe anti-neoplasmic potential of HDACi is well established. For ex-

ample, SAHA was approved by the U.S. Food and Drug Administrationin 2006 for the advantageous modulation of disease progression ofCTCL. Besides the induction of apoptosis of tumor cells, these inhibitorsalso block angiogenesis; SAHA and TSA prevent sprouting of capillariesfrom rat aorta (Deroanne et al., 2002). HDACi block angiogenesis by ac-tivating anti-mitotic proteins like p53 and von Hippel–Lindau and bysuppressing angiogenic factors such as vascular endothelial growth fac-tors (VEGFs), hypoxia-inducible factor-1α (Hif-1α) (Kim et al., 2001),and endothelial nitric oxide synthase (Rossig et al., 2002; Fish et al.,2005). These reports support that HDACs are closely related to tumorangiogenesis as well as the growth of the cancer itself. Consideringthat multiple steps are involved in tumor growth and metastasis andthat diverse HDAC subtypes might be involved in the different steps,the use of specificHDACimaymake it possible to alter specific pathwaysin the development of cancer.

Even in a single phenomenon such as angiogenesis, different HDACsubtypes may result in diverse physiological events. For example, con-tradictory results have been reported for the roles of individual HDACsin the regulation of angiogenesis. HDAC4 was reported to negativelyregulate angiogenesis by reducing VEGF expression (Sun et al., 2009),whereas another group reported that HDAC4 induces angiogenesisthrough an increase in stability of Hif-1α (Geng et al., 2011).Phosphorylation-dependent nuclear export of class II HDACs such asHDAC5 (Urbich et al., 2009) and HDAC7 (Mottet et al., 2007; Margaritiet al., 2010) and thereby de-repression of target genes is crucial forthe expression of VEGF or matrix metalloproteinase-10 (Ha et al.,2008a), which results in an increase in angiogenesis (Martin et al.,2008). HDAC6 can be classified as a pro-angiogenic factor because it in-duces cell migration by the deacetylation of cytoskeletal proteins(Kaluza et al., 2011; Li et al., 2011). Recently, it was reported thatHDAC9 induces angiogenesis by repressing the miR-17-92 cluster at


the transcriptional level (Kaluza et al., 2013). Thus, even thoughHDACs may have diverse physiologic actions, it is widely accepted thatclass II HDACs are pro-angiogenic.

In contrast with these observations of HDACi as anti-angiogenic me-diators, studies showing a positive correlation have also been reported.Long-term treatment of VPA in a rodentmodel of cerebral infarction en-hances neovascularization, reduces infarction size, and promotes func-tional recovery (Wang et al., 2012). As a mechanism, up-regulation ofVEGF and matrix metalloproteinase-2/9 is suggested for the protectiveeffect of VPA in ischemic injury. For these contradictory reports, the du-ration ofHDAC inhibition should be considered.Many reports show thatlong-term administration of HDACi induces neovascularization (Zhanget al., 2012a,b). In contrast, however, HDAC inhibition blocks angiogen-esis especially in the acute phase (Granger et al., 2008). In summary,HDACi inhibit angiogenesis as well as induce cytotoxicity in tumorcells. Considering that HDACi may provoke angiogenesis in certain cir-cumstances, such as long-term treatment,more supportive data on nor-mal versus cancer vessels or short- versus long-term effects arerequired. The contradictory effect of HDAC inhibition in angiogenesisis discussed again below in the Myocardial infarction (MI) section.

2.3.2. AtherosclerosisAtherosclerosis is a progressive disease that is characterized by

chronic inflammation, accumulation of lipids, generation of a fibrouscap, proliferation of smooth muscle cells, calcification in vascularsmoothmuscle layers, and resultant loss of elasticity of arteries. As a re-sult of the growth of atheroma, the lumen of the artery is graduallynarrowed, which changes the local environment of hemodynamics;for example, laminar flow turns into turbulence (Fox & Hugh, 1966).Platelets broken down by turbulence make thrombi, which overlaythe fibrous cap of the atheroma (Ross, 1999). The fibrous cap is vulner-able to rupture (Finn et al., 2010), which further blocks blood flow.When bloodflow in the coronary artery is blocked, themyocardium suf-fers from the lack of oxygen and nutrients. If bloodflow is not reinitiatedat an appropriate time point, the myocardium rapidly dies. Thus, ath-erosclerosis in a coronary artery is the most common independent riskfactor of MI (Van de Werf et al., 2008).

HDACs are closely associated with multiple steps in the develop-ment of atherosclerosis. As also shown in a previous report from ourgroup (Kee et al., 2011), global HDAC inhibition by use of scriptaid orTSA successfully prevents neointima formation after injury (Okamotoet al., 2006; Findeisen et al., 2011). When primary cultured vascularsmooth muscle cells or injured carotid arteries are exposed to TSA,p21WAF1/Cip1, a potent negative regulator of the cell cycle, is significantlyincreased (Okamoto et al., 2006). A decrease in the transcriptional activityof KLF4 is involved in the HDACi-induced upregulation of p21WAF1/Cip1

and thereby repression of atherosclerosis (Yoshida et al., 2008; Keeet al., 2011). However, alteration of activity of p53, a well-known up-stream regulator of p21WAF1/Cip1, is not observed.

In contrast, however, several reports have elucidated the pro-atherogenic effects of TSA (Choi et al., 2005; Song et al., 2010); TSA po-tentiates vascular smooth muscle cell proliferation both by inhibition ofthioredoxin 1 and by activation of Akt signaling (Song et al., 2010). InLdlr null mice that lack the LDL receptor and thereby develop hypercho-lesterolemia (Ishibashi et al., 1993), TSA exacerbates the diseasethrough upregulation of scavenger receptor A in macrophages, anincrease in macrophage infiltration, and synergistic accumulation ofoxidized LDL in atheroma (Choi et al., 2005). Although a few reportsshowed pro-atherogenic effects of HDACi, at least for vascular smoothmuscle cell proliferation (one of the most important components inatherogenesis), HDACi seem to have a beneficial effect to preventatherosclerosis.

It should also be noted that characteristics of the individual HDACiused might result in these contradictory results at least in part. Non-specific HDACi, such as TSA and SAHA, differently regulate pro-inflammatory gene expression in bone marrow macrophages; they




down-regulate the expression of Ccl-7, Edn-1, and Il-12p40, but have noeffect on the induction of Cox-2 and Pai-1 (Halili et al., 2010). Interest-ingly, TSA shows a biphasic response: anti-inflammatory action at lowconcentrations and pro-inflammatory activity at high concentrations(Halili et al., 2010). Inhibition of class I HDACs by their specific inhibitorMS-275 potentiates inflammatory gene expression (Halili et al., 2010),which suggests that thrombosis, the most common and serious side ef-fect of SAHA (Duvic & Vu, 2007), is caused by preferential inhibition ofclass I HDACs. Targeted studies against a specific subtype of HDACswould be necessary to unveil the secret of the discrepancy of the effectof TSA on atherosclerosis.

Recent advances in atherosclerosis research on the specific roles ofindividual HDACs should be also noted. For example, restoration ofHDAC2 in the development of atherosclerosis seems to be noteworthyas a target to modulate disease prognosis. HDAC2 significantly reducesboth the stability and the activity of class II transactivators in macro-phages and vascular smooth muscle cells (Kong et al., 2009). HDAC3 isessential for survival of endothelial cells in shear-stress conditions, andknock-down of HDAC3 aggravates neointima formation (Zampetakiet al., 2010). Thus, these beneficial effects of HDAC2 and HDAC3 areconsistent with the observations that selective class I HDACi MS-275aggravates the inflammatory response. The HDAC4/MEF2 complexsuppresses c-jun expression in a PKA-dependent manner in the non-stimulated condition (Gordon et al., 2009). Shear stress induces cyto-plasmic shuttling of phosphor-HDAC5, which results in the increasesin both KLF4 and eNOS levels (Wang et al., 2010). HDAC7 interactswith CtBP1 to prevent toll-like receptor-driven inflammatory events(Shakespear et al., 2013). Taken together, these data suggest that eitherrestoration of class I HDACs or nuclear enrichment of class IIa HDACscould be a therapeutic target for atherosclerosis via modulation of theinflammatory response.

It should be noted that regardless of the general beneficial effects ofHDACi in the cardiovascular diseases, at least in some conditions, HDACinhibition may cause detrimental results. For example, HDAC inhibitionseems to cause exaggeration of vascular calcification. Inorganic-phosphate-induced calcification of human arterial smooth muscle cellsis significantly increased by simultaneous treatment with TSA, a classesI and II inhibitor. TSA-mediated hypercalcification induces both expres-sion and activity of alkaline phosphatase, and down-regulation of alka-line phosphatase blunts the hyper-calcification phenotype (Azechiet al., 2013). Indeed, according to our preliminary studies, one or twoclass I HDACs seem to be involved in the development of vascular calci-fication. Loss of activities of class I HDACsmay relay phosphate-inducedvascular calcification (Kwon, unpublished observation). Consideringthat many cellular and noncellular components are involved in the ath-erogenic process, investigations of the functional roles of each HDAC orHDACi in each component in the development of atherogenesis shouldbe carried out for further delineations of the mechanisms.

2.4. Cardiac diseases

In the following sections, we review the functional roles of HDACs incardiac diseases such as arrhythmia,MI, cardiac hypertrophy, hyperten-sion, and cardiac fibrosis.

2.4.1. ArrhythmiaOnly a few studies have focused on the relevance of HDACs in the

conduction system. Consistent with previous reports that genetic dis-ruption of HopX causes shortening of the PR interval, whereas overex-pression of HopX induces its prolongation (Ismat et al., 2005), Liuet al. also reported atrium-origin arrhythmia in the α-myosin heavychain (encoding myosin heavy chain 6) promoter-driven HopX trans-genic mice (Liu et al., 2008). TSA successfully reverses the conductionabnormality by suppression of connexin 40. It is not clear, however,whether the anti-arrhythmic effect of TSA in their report was mediatedbydirect regulation of the expression of connexin 40 orwhether itwas a


secondary effect after attenuation of pathologic defects such as atrial fi-brosis. Considering that HopX directly associates with HDAC2 (Kooket al., 2003), this beneficial effect of TSA might be caused by inhibitionof HopX-associated HDAC2.

More direct relation of HDACs to cardiac arrhythmia has been delin-eated; gene ontology tests obtained from cardiac-specific deletion ofboth cardiac HDAC1 and HDAC2 reveal a dramatic increase in calciumchannel subunits (Montgomery et al., 2007). Our group also performeda DNA microarray after over-expression of HDAC2 in the heart andfound that several channels for ion current were dysregulated: increasein Scn2a1 (sodium) and Cacnb2 (calcium) and decrease in Scn3b (sodi-um) and Kcne1 (potassium). Recent studies on hereditary familial ar-rhythmia reveal that loss of function of Scn3b is associated withBurgada syndrome and dysfunction of Kcne1 was also responsible forLong QT syndrome (Monteforte et al., 2012), which suggests that alter-ation of HDAC2 function may contribute to these types of cardiac ar-rhythmias. Other evidence shows that HDAC3 also plays functionalroles in the conduction system. Prx1 recruits HDAC3 and this Prx1/HDAC3-complex negatively regulates NKX2.5, which ensures themain-tenance of both anatomical structures and integrity of the conductionsystem environment (Risebro et al., 2012). Taken together, these datasuggest that class I HDACs may play an important role in ion handingand inmaintaining the integrity of conduction systems for synchronizedcontraction of the myocardium either by regulation of transcription ofthe target channel protein or by direct association with other proteins.

2.4.2. Myocardial infarctionOxygen and nutrients are supplied to the ventricular myocytes by

the coronary arteries. In the pathologic condition, the coronary arteryis often occluded by various pathologic conditions, such as growth ofatheroma in the coronary artery, rupture of vulnerable plaque, thrombifrom proximal lesions, emboli secondary to atrial fibrillation, or vegeta-tion after endocarditis. Even if blood flow is resumed a relatively shorttime after ischemia, themyocytes are vulnerable to damage by the alter-nate insults of reperfusion. This type of reversible injury is called ische-mia–reperfusion (I/R) injury.

HDACi have emerged as novel agents for minimizing I/R injury, forrestoring cardiac function, and for reducing infarct size (Lee et al.,2007; Zhao et al., 2007; Granger et al., 2008). According to an ex vivostudy in mouse heart using the Langendorff perfusion system, TSA pre-serves cardiac performance. Intraperitoneal injection of TSA 24 h beforeI/R injury reduces infarct size and ameliorates contractile function (Zhaoet al., 2007). Possible mechanisms are that HDACi block ischemia-induced gene regulation, including Hif-1α and VEGF, and thereby stabi-lize vascular permeability (Granger et al., 2008). HDAC4may induce anincrease in vascular permeability and subsequent I/R injury. Besides,HDACi retain down-regulation of PGC-1α, a master regulator of fattyacid oxidation and mitochondrial biogenesis, in I/R injury (Ramjiawanet al., 2013). HDAC5/MEF2 could serve as a negative regulator of PGC-1α expression (Czubryt et al., 2003). Thus, the main beneficial effectsof HDACi in I/R injury seem to be caused by suppression of incompletevascularization or by preservation of energy metabolism.

HDAC inhibition is also effective in the prevention of permanent MI.Scar size by ligation of the left anterior descending artery is significantlyreduced by nonselective HDACi such as VPA, tributyrin, or TSA (Leeet al., 2007; Granger et al., 2008; Zhang et al., 2012a). In contrast, how-ever, it has also been reported that administration of TSA for 8 weekssignificantly ameliorates cardiac function and prevents cardiac remod-eling, which is mediated by recruitment of c-kit-positive stem cellsand thereby increases both in neovascularization and in new myocytes(Zhang et al., 2012a). The obvious inconsistencies regarding the effectsof HDACi on angiogenesis remain to be clarified by use of different con-centrations and various timewindows aswell as specific HDACi. Despitesome contrasting reports about HDACi in MI, it is more commonly ac-cepted that HDACi are beneficial for conserving cardiac function in MI.




2.4.3. Cardiac hypertrophyCardiac hypertrophy is a form of remodeling and is an adaptive re-

sponse to the request for high workload from peripheral tissue orfrom intrinsic underlying disease conditions such as valvular dysfunc-tion, hypertension, and MI (Frey & Olson, 2003). Although the initialprocess might be physiologic, sustained stimuli as the result of uncon-trolled underlying disease lead to pathologic changes (Hill & Olson,2008). Pathologic hypertrophy is characterized by diastolic dysfunctionand massive interstitial fibrosis, whereas physiologic hypertrophyshows relatively normal cardiac performance and the absence offibrosis(Bernardo et al., 2010). Pathologically hypertrophied heart enters amal-adaptive phase and hemodynamic function abruptly falls off, which istermed heart failure. Heart failure is still the most common cause ofdeath worldwide.

The roles of the HDACs in pressure overload- or adrenergic agonist-induced cardiac hypertrophy are being extensively investigated by nu-merous research groups including ours (Zhang et al., 2002; Kook et al.,2003; Chang et al., 2004; Vega et al., 2004; Kee et al., 2006; Kong et al.,2006; Montgomery et al., 2007; Trivedi et al., 2007; Gallo et al., 2008;Kee et al., 2008; Kee & Kook, 2009; Cho et al., 2010; Eom et al., 2011).Although both class I and class IIa HDACs are involved in the develop-ment of cardiac hypertrophy, their roles are quite opposite. Global dele-tion of HDAC2 allows resistance to exogenous stresses (Trivedi et al.,2007). Transgenic overexpression of HDAC2 provokes cardiac hypertro-phy, whereas HDAC1 and HDAC3, other class I HDACs, have no effect onheart size (Trivedi et al., 2007; Eom et al., 2011). Despite the role ofHDAC2 in cardiac hypertrophy, the expression level of HDAC2 is notchanged (Kee et al., 2008; Eom et al., 2011). Instead, the intrinsic en-zyme activity of HDAC2 is significantly increased in response to hyper-trophic stress, which has been shown both in an in vivo mouse model(angiotensin II, isoproterenol, or transverse aortic constriction) andthrough in vitro experiments with primary cultured rat ventricularcardiomyocytes (angiotensin II, endothelin-1, phenylephrine, or fetalbovine serum) (Kee et al., 2008; Eomet al., 2011). However, the enzymeactivity of other class I HDACs, such as HDAC1, HDAC3, or HDAC8, is notaltered (Kee et al., 2008). HDAC2 is activated by phosphorylation of theSer-394 residue,which ismediated by casein kinase 2α1 (CK2α1) (Eomet al., 2011). The phosphorylation is then protected from phosphataseby a phosphor-S394-specific physical interaction with HSP70 (Keeet al., 2008; unpublished data). Then, the activated HDAC2 repressesthe fetal gene program by inhibition of anti-hypertrophic mediators,such as KLF4 (Kee & Kook, 2009) and inositol polyphosphate-5-phosphatase F (Trivedi et al., 2007; Zhu et al., 2009) (Fig. 2). Very re-cently we demonstrated that acetylation of HDAC2 K75 is an alternatePTM to regulate its activity (Eom et al., 2014). As for class I HDACsother than HDAC2, although no clear evidence regarding cardiac hyper-trophy has been reported, HDAC3 seems to be associated with cardio-myocyte proliferation in the perinatal period (Trivedi et al., 2008).

By contrast, the functional roles of class IIa HDACs such as HDAC5and HDAC9 are opposite to those of HDAC2. Targeted deletion ofHDAC9 (Zhang et al., 2002) or HDAC5 (Chang et al., 2004) results inan exaggeration of hypertrophic phenotypes when the knockout miceare crossed with the calcineurin-transgenic mouse, which suggeststhat class IIa HDACs are negative regulators of cardiac hypertrophy. Innormal conditions, class IIa HDACs inhibit the transcriptional activityofMEF2 by interferingwith the binding ofMEF2 to itsmotif. After phos-phorylation of HDAC5 and HDAC9 by protein kinases such as PKC, PKD,or CaMK, amolecule named 14-3-3 recognizes the phosphorylated classIIa HDACs and causes shuttling of theHDACs from the nucleus to the cy-toplasm. Because MEF2 binds to DNA and activates transcription of thefetal gene program, the redistribution of class IIa HDACs from the nucle-us to the cytosol causes de-repression of those genes, resulting in cardi-ac hypertrophy (McKinsey et al., 2000b; Chang et al., 2004; Vega et al.,2004) (briefly summarized in Fig. 2).

Because those two classes of HDACs have opposite actions, one mayquestion the overall effects of nonspecific HDACi in the development of


cardiac hypertrophy. The answer is that HDACi have “anti-hypertrophic”action. We (Kee et al., 2006) and other research groups (Kong et al.,2006; Gallo et al., 2008) have shown the anti-hypertrophic effect ofpan-HDACi such as TSA (Kee et al., 2006; Kong et al., 2006), VPA (Keeet al., 2006; Cho et al., 2010), and scriptaid (Kong et al., 2006). Striking-ly, even selective class I HDACi, such as SK7041 (Kee et al., 2006),apicidin (Gallo et al., 2008), and MS-275 (Cavasin et al., 2012), preventcardiac hypertrophy. Considering that those class I HDAC-selective in-hibitors can attenuate cardiac hypertrophy similar to the pan-HDACi,the anti-hypertrophic effect of the pan-HDACi is likely to be mediatedby inhibition of class I HDACs. It is noteworthy that pre-established hy-pertrophy can also be prevented by HDACi (Kee et al., 2006). Thus, onthe basis of these observations, we speculate that the enzyme activityof class I HDACs is critical in the development of cardiac hypertrophy.

On the other hand, class IIa HDACs show anti-hypertrophic action inan enzyme-activity-independentmanner. This is supported by other re-ports regarding the function of MEF-2 interacting transcription repres-sor (MITR) in cardiac physiology. MITR is an endogenous splicing formof HDAC9 that lacks an HDAC domain and has only an extended N-terminal regulatory region (Sparrow et al., 1999). Even though MITRcannot function as an “enzyme,” MITR shows anti-hypertrophic effectssimilar to those of full-length HDAC9 (Zhang et al., 2002). Thus, classIIa HDACs are believed to work as simple transcriptional modulatorsrather than as histone deacetylase enzymes, at least in associationwith the development of cardiac hypertrophy. One report argued thatHDACi might aggravate right ventricular dysfunction (Bogaard et al.,2011); however, our previous result, which focused on right ventricularhypertrophy (Cho et al., 2010), tightened the general concept aboutHDACi in cardiac hypertrophy. HDACi are thus an effective treatmentmodality for restoration of cardiac function in ventricular remodeling.

2.4.4. MiscellaneousCompared with effects on cardiac hypertrophy or MI, the effects of

HDACi on hypertension are not well described. A few elegant reports,however, clearly demonstrated the functional role of HDAC3 in hyperten-sion and the benefit of treatment with VPA for blood pressure control(Lee et al., 2013). Those authors showed shown that mineralocorticoidreceptor induces blood volume retention through increases in expressionof its target genes, such as ATP1a1, GILZ, and SGK-1.Mineralocorticoid re-ceptor undergoes deacetylation by HDAC3, which is its transcriptionallyactive state. VPA significantly reduces the transcriptional activity of thereceptor through acetylation, which thereby regulates blood pressurewithin the normal range. Usui et al. (2012) also reported that HDAC4 isa responsible factor for the development of hypertension through vascu-lar inflammation. HDAC4 relays the inflammatory response via up-regulation of VCAM-1, and TSA treatment completely blocks the increasein blood pressure. Taken together, these data suggest HDAC as an alter-nate therapeutic target for regulation of hypertension.

Cardiac fibrosis is also dramatically suppressed by HDACi as demon-strated by many groups including ours (Kee et al., 2006; Kong et al.,2006; Eom et al., 2011). More direct evidence supports the conceptthat HDAC induces trans-differentiation of fibroblasts to myofibroblastsin either cardiac or noncardiac fibrosis (Niki et al., 1999; Guo et al.,2009). The HDAC2 protein amount is significantly increased duringscar formation (Fitzgerald O'Connor et al., 2012) and it is consideredto be responsible for renal fibrosis (Noh et al., 2009; Marumo et al.,2010). In addition, Sarrazy et al. (2011) reported the functional impor-tance of myofibroblasts in pathological scar formation such as a keloid.These studies suggest that HDACs do play important roles in scar forma-tion. Therefore, HDAC modification with HDACi would be a promisingtarget for suppressing extensive fibrosis, because conventional thera-peutic strategies to date are not as effective at controlling tissue fibrosis,keloid, or hypertrophic scars.

Stem cell transplantation is regarded as a new era of rejuvenationtherapy in patientswithMI (Strauer et al., 2002). Delivery of pluripotentstem cells, however, may result in uncontrolled proliferation.Moreover,



Quiescent state

Class IIaHDAC

HDAC2

HAT

Inpp5f

KLF4

Cytoplasm

Nucleus

Pro-hypertrophic gene

Class II HDAC

MEF2

SRF NKX2.5

CAMTAMyocardin

Anti-hypertrophic gene (e.g. Inpp5f, KLF4, and etc)

Regulation of Class IIa HDACs Regulation of Class I HDACs

Anti-hypertrophic gene (e.g. Inpp5f, KLF4, and etc)

HDAC2 Inpp5f

KLF4

Cytoplasm

Nucleus

Pro-hypertrophic gene

MEF2SRF NKX2.5

CAMTAMyocardin

CK2α1

CK2α1

Class II HDAC

HAT

Hypertrophic stresses

Ac

Class IIaHDAC

Activated state

Regulation of Class IIa HDACs Regulation of Class I HDACs

PP?

PP?

Fig. 2.Mechanism of regulation of HDACs in the development of cardiac hypertrophy. In the absence of hypertrophic stresses, HDAC2 and prohypertrophic transcription factors are sup-pressed by class II HDAC. The suppressed HDAC2 fails to repress the expression of the anti-hypertrophic regulators such as KLF4 and Inpp5f, and consequently the fetal gene program isarrested by the inhibitory activity of KLF4 and Inpp5f (left). When hypertrophic stresses insult the myocardium, several posttranslational modifications of proteins take places. Class IIHDACs are shuttled out in a phosphorylation-dependent manner. HDAC2 is recognized by an unidentified HAT and thereby acetylated. This acetylation induces phosphorylation ofHDAC2. At the same time, inducible heat shock protein, HSP70, dramatically increases in the nucleus. CK2α1 is phosphorylated by hypertrophic stimuli and is activated. ActivatedCK2α1 undergoes nuclear redistribution and phosphorylates HDAC2 at Ser-394, which induces hyper-phosphorylation of HDAC2. HSP70 preferentially binds to hyper-phosphorylated-HDAC2. Functionally activated HDAC2 inhibits anti-hypertrophic mediators and the transcription factors that were arrested by class IIa HDACs are re-activated. Both of these transcrip-tional regulations synergistically induce reactivation of the fetal gene program (right). Black stars indicate phosphorylation. Abbreviations: Ac, acetylation. CAMTA, calmodulin bindingtranscription activators. CK2α1, casein kinase 2 α1. HAT, histone acetyltransferase. HDAC, histone deacetylase. HSP70, heat shock protein 70. Inpp5f, inositol polyphosphate-5-phosphatase F. KLF4, Krüppel-like factors 4. MEF2, myocyte enhancing factor 2. NKX2.5, NK2 homeobox 5. PP, protein phosphatase. SRF, serum response factor.


Murry et al. (2004) reported that hematopoietic stem cells cannot in-duce trans-differentiation into cardiac myocytes in the infarct zone.Thus, utilizing cardiac stem cells that are obtained in vitro is noteworthy


for future trials and to prove therapeutic potential. TSA dramatically en-hances the differentiation of embryonic stem cells into cardiomyocytes.Acetylation of GATA4 is increased by treatment with TSA, which thereby




induces cardiac-specific genes such as NKX2.5, ANF, and cardiac MHC(Kawamura et al., 2005). Considering that HDAC2 deacetylases acetyl-GATA4 (Trivedi et al., 2010), targeted inhibition of HDAC2may acceler-ate differentiation of pluripotent stem cells into the cardiac lineage. Asdescribedbriefly in theMyocardial infarction section, TSA also enhancesregeneration of cardiomyocytes by recruitment of c-kit-positive stemcells in vivo. Cardiac rejuvenation studies by use of various HDACi willbe necessary to test this hypothesis.

Macrophage differentiation and polarization play a pivotal role inthe wound-healing process. Polarized macrophages are roughly classi-fied into two groups according to their predominant expressingpattern:M1, the killer phenotype, andM2, the repair phenotype (Martinez et al.,2008). M2-polarization is deeply involved in chronic inflammation andconsequent immune-tolerance. Mullican et al. (2011) reported HDAC3as a regulator of the balance between M1 and M2 macrophages. M2polarization, which is driven by IL-4 exposure, is potentiated in theHDAC3-deleted macrophage. Bacterial infection-mediated inflamma-tion in lung parenchyma is significantly reduced in HDAC3-deletionmice,which seems to be theM2polarization effect. HDACi are thus a po-tential modality for control of macrophage polarization and thereby thewound-healing process in specific tissues.

3. Posttranslational modifications in cardiovascular diseases

3.1. Non-histone targets of histone acetyltransferase andhistone deacetylase in association with cardiovascular disease

Adult heart is a typical organwhose cell cycle is arrested. Proliferationability, however, is transiently observed, although rapidly abolished inthe perinatal period. Very interestingly, rebirth of the fetal gene programsthat are arrested in adult heart has drawnmuch interest in regard to adultCVD. This process is closely related to development during the embryonicperiod. Thus, the role of acetyl-protein and its modifiers in fetal develop-ment may have applications in adult cardiac pathophysiology.

GATA4, a member of the GATA family, is a cardiac-specific transcrip-tion factor. GATA4 is intimately associated with both cardiac develop-ment and adult cardiac disease. It is a crucial factor in the formation ofthe proepicardium and subsequent cardiogenesis (Watt et al., 2004)and in the development of cardiac hypertrophy (Molkentin et al.,1998). GATA4 undergoes acetylation by p300 in response to diversestress. Acetyl-GATA4 acquires DNA-binding affinity and transcriptionactivity (Takaya et al., 2008). In addition, acetyl-GATA4 is subjected todeacetylation by HDAC2, which is controlled by HopX (Trivedi et al.,2010). Thus, cardiac homeostasis is regulated by dynamic modulationof GATA4 acetylation.

We suggested KLF4 as a negative regulator of cardiac hypertrophy(Kee & Kook, 2009) or vascular smooth muscle cell proliferation (Keeet al., 2011). KLF4 is also regulated by PTMs such as acetylation. For ex-ample, P300/CBP-mediated acetylation of KLF4 induces binding to DNAand transactivates expression of its target genes, such p21Cip1/WAF1

(Evans et al., 2007). Other groups have suggested that HDAC2 phys-ically interacts with KLF4 and regulates transcriptional activity bydeacetylation (Meng et al., 2009). Taken together, these findingssuggest that the anti-hypertrophic or anti-proliferative effect ofKLF4 might be executed in an acetylation-dependent manner.

p53, the so-called guardian of the genome, has been highlighted incancer pathophysiology because of its tumor suppressor function. Thetarget genes of activated p53 are mainly associated with DNA repair,cell cycle arrest, and apoptosis. p21Cip1/WAF1, a powerful cell cycle arres-tor, is also regulated by p53. Rebirth of the arrested fetal gene programmight be the result of escape fromp53; therefore, whatwe have learnedabout the PTM of p53 in cancer biology might be adopted in the car-diac field. Sano et al. (2007) suggested the role of p53 in pressure-overloaded mouse hearts. Cardiac angiogenesis is well maintainedin the physiologic phase; however, sustained overload induces fail-ure of neovascularization. Uncoupling between cardiac burden and


vasculature plays a major role in the transition to heart failure. Ac-cording to numerous studies in cancer biology, acetylation of p53 in-duces transcriptional activation and thereby apoptosis (Gu & Roeder,1997). The deacetylation process is mediated by both class I and classIII HDACs. Thus, the functional role of p53 should be investigated inthe development of CVD with respect to its PTM and modifiers of PTMsuch as HDACs. In addition to acetylation dynamics in non-histone tar-gets of activated HATs and HDACs, the HATs and HDACs themselves areactively modified by other epigenetic molecules and those activities aredynamically regulated.

3.2. Regulation mechanism of class IIa histone deacetylases

The mechanism of regulation of class IIa HDACs has been wellestablished by many different groups (McKinsey et al., 2000a,b; Zhanget al., 2002; Chang et al., 2004; Vega et al., 2004; Ha et al., 2008b).Activated CaMKII (Bossuyt et al., 2008)/CaMKIV (Sucharov et al.,2006) or PKC/PKD (Vega et al., 2004) phosphorylates class IIa HDACsin response to exogenous hypertrophic signals. Because two serinesthat are susceptible to phosphorylation flank the nuclear localizationsignal, phosphorylation-specific binding of 14-3-3 molecules to classIIa HDACs causes masking of that signal. As a result, the class IIaHDACs are exported from the nucleus by the nuclear export signalthat resides in their molecular structures. This results in de-repressionof MEF2, which then leads to reactivation of fetal gene reprogrammingand ensuinghypertrophic growth. In addition to phosphorylation, intra-molecular disulfide bond formation also regulates subcellular localiza-tion. Reactive oxygen species induce oxidation of cysteine residues inHDAC5 (Haworth et al., 2012) (Cys-696 and Cys-698) or in HDAC4(Ago et al., 2008) (Cys-667 and Cys-669),which actively promotes cyto-plasmic shuttling, in a phosphorylation-independentmanner (for moredetailed review, please see Bush & McKinsey, 2009 or Haberland et al.,2009).

In contrast, phosphorylation by different kinases results in quite theopposite outcome (Ha et al., 2010; Chang et al., 2013). PKA stimulatedby either forskolin (Ha et al., 2010) or isoproterenol (Chang et al.,2013) can also phosphorylate HDAC5 at Ser-279. This PKA-dependentphosphorylation of HDAC5 causes nuclear relocalization. Sumoylationis also a notable regulation mechanism of class IIa HDACs. HDAC4 andMITR are substrates for SUMO-modifiers (Kirsh et al., 2002). For exam-ple, RanBP2-mediated sumoylation of the HDAC4 at Lys-599 inducesnuclear accumulation against CaMK-driven export stimuli, thereby sup-pressingMEF2 expression (Kirsh et al., 2002). Thus, sumoylation of classIIa HDACs looks like a triggeringmodification for transcriptional repres-sion. In summary, the subcellular localization and thereby transcription-al activity of class II HDACs are finely regulated by diverse PTMs, whichsuggests that specific enzymes modifying class II HDACs are promisingtargets for drug discovery.

3.3. Regulation mechanism of class I histone deacetylases

HDAC1, 2, and 8 are foundmainly in the nucleus. In contrast, HDAC3is sometimes detected in the cytoplasm. As enzymes, the class I HDACsare definitely involved in the deacetylation of histone and non-histoneproteins. Therefore, PTMs are focused on either promoter accumulationor alteration of enzyme activity. Although class I HDACs have only ashort regulatory domain compared with class IIa HDACs, class I HDACsthemselves are also actively modified. Among the class I HDACs,HDAC1 and HDAC2 share an extremely well-conserved architecture.Therefore, PTMs in HDAC1 or HDAC2 are also mediated by similar mol-ecules. To have enzymatic activity, HDAC1/2 should be phosphorylatedin vivo even in the absence of any phosphorylation stresses; we ob-served that purified HDAC2 obtained from a bacterial overexpressionsystemdoes not have intrinsic activity,whereas it gains intrinsic activitywhen it is basally phosphorylated by the addition of cell lysates (Keeet al., 2008). Basal phosphorylation of HDAC1 at Ser-421 and Ser-423




was reported in Jurket cells by Pflum et al. (2001). Likewise, in HeLacells, it has been known that Ser-422 and Ser-424 are responsible forthe basal phosphorylation of HDAC2 (Tsai & Seto, 2002). Thus, basalphosphorylation is believed to be a prerequisite for intrinsic activity orfor the assembly of repressor complex such as NuRD, Sin3A, or CoREST(Yang & Seto, 2008). Interestingly, further phosphorylation on otherserine residues by specific kinases upon certain stimuli is associatedwith disease prognosis (Walters et al., 2009; Eom et al., 2011).

We have demonstrated the role of HDAC2 and its PTM in the devel-opment of cardiac hypertrophy (Kook et al., 2003; Kee et al., 2006, 2008;Eom et al., 2011). Kook et al. (2003) reported by use of a heart-specifictransgenic mouse system that expressing the atypical homeodomainprotein HopX induces cardiac hypertrophy, which is dependent onwhether HDAC2 is recruited. Furthermore HopX-driven cardiac hyper-trophy is completely blocked by HDAC inhibition (Kook et al., 2003).As described above, we and others have also demonstrated thateither nonspecific HDAC inhibition or selective class I HDACi success-fully block cardiac hypertrophy. Thus, HDAC2, a class I HDAC, specif-ically functions like a cornerstone in the development of cardiachypertrophy.

In addition to the role of HDAC2 in CVD, we have also delineated thePTM of HDAC2 in hypertrophy (Eom et al., 2011). First, we demonstratedthe role of CK2α1 and its target residue (Eom et al., 2011). Second, we re-ported the role ofmolecular chaperone heat shock protein 70 as a regula-tor of HDAC2 activity (Kee et al., 2008). Recently, we found evidence ofanother PTM, HDAC2 acetylation, and the role of phopho-HDAC2-specific binding of HSP70 in association with cardiac hypertrophy andheart failure. HDAC2 acetylation and phosphor-specific binding toHSP70 result in the activation of HDAC2 (Eom, unpublished observation).

As in cancer cells as described above, in the heart, HDAC2 is basal-ly phosphorylated at Ser-422 and Ser-424 and is mainly located inthe nucleus in the quiescent state (Eom et al., 2011). When the myo-cardium is stimulated by various hypertrophic stresses, a diverse sig-nal cascade, including a phosphorylation stream, is activated. Forexample, activation of PKD and CaMK (Vega et al., 2004; Bossuytet al., 2008), phosphorylation of cytoplasm-localized CK2α1 (Eomet al., 2011), and an increase in inducible HSP70 (Kee et al., 2008)take place. PKD and CaMK induce redistribution of class IIa HDACsas discussed previously.

We first observed that diverse hypertrophic stresses inducephosphorylation of HDAC2 Ser-394 and Ser-411. Interestingly, activa-tion of HDAC2 is induced by phosphorylation of HDAC2 Ser-394, Ser-422, and Ser-424, but not by that of HDAC2 Ser-411. It is noteworthythat HDAC2 Ser-422 and Ser-424 are phosphorylated in non-stimulated states to maintain the basal activity but that they arenot hypertrophy-responsive. These findings suggest that only Ser-394 is “enzyme-activity-associated” as well as “hypertrophy-respon-sive.”We also found that phosphorylation-dependent activation andthereby shuttling of CK2α1 into the nucleus are responsible for theHDAC2 Ser-394 phosphorylation. Phosphorylation of HDAC2 at Ser-394 is inhibited by both siRNA against CK2α1 and CK2 blockers. ThisCK2α1-mediated phosphorylation of HDAC2 increases the enzyme ac-tivity (Eom et al., 2011). Furthermore, a molecular chaperone HSP70preferentially binds to Ser-394-phosphorylated HDAC2 and further po-tentiates its activity by protecting HDAC2 (Kee et al., 2008). In additionto phosphorylation, we recently identified that acetylation of HDAC2takes place in response to hypertrophic stresses and that this acetyla-tion is also required for the HDAC2 activation (Eom, unpublishedobservation). This observation raises the further question of inter-relationship between phosphorylation and acetylation in the regulationof the enzymatic activity of HDAC2 in response to hypertrophic stresses,which remains to be clarified. Moreover, it should be clarified whichHDAC and HAT is responsible for the regulation of acetylation ofHDAC2. Perhaps in the future, studies will more clearly identify therole of acetylation as an independent regulation mechanism of HDACs,such as protein stability, subcellular localization, or enzymatic activity.


3.4. Ideas for future studies on posttranslationalmodification of histone deacetylases in cardiovascular diseases

HDAC1undergoes SUMOmodification in the C-terminus,which reg-ulates transcription-repression ability (David et al., 2002; Kirsh et al.,2002). It is not clear, however, whether sumoylation directly affectsthe intrinsic activity of HDAC1 or regulates the formation of a co-repressor complex. HDAC2 Lys-462 is also known to be subject tosumoylation and SUMO-HDAC2 is catalytically active (Brandl et al.,2012). As shown in the case of p53, Lys-320 in p53 is a target ofSUMO-HDAC2, and deacetylation of p53 fails to control apoptosis ofDNA-damaged cells (Brandl et al., 2012). These findings on thesumoylation of HDAC2 suggest de-sumoylation as a novel target forcell cycle arrest and thereby cancer treatment.

S-Nitrosylation is an alternate important modification of HDAC2. S-Nitrosylation of HDAC2 was first reported in neuronal development.Nott et al. (2008) found that S-nitrosylation occurs in Cys-262 andCys-274, which regulates chromatin recruitment rather than intrinsicactivity. Colussi et al. (2008) also reported the therapeutic potential ofHDAC2 S-nitrosylation in a Duchenne muscular dystrophy model. Inthat study, the intrinsic activity of HDAC2 was dramatically suppressedby S-nitrosylation and endothelial nitric oxide synthasewas sufficient toproduce nitric oxide to repress HDAC2. In addition, Malhotra et al.(2011) clearly demonstrated HDAC2 S-nitrosylation and its functionalrelevance to the failure in patients with chronic obstructive pulmonarydisease (COPD) to control underlying chronic inflammation despitetreatment with glucocorticoid. S-Nitrosylation directly suppresses theactivity of HDAC2, whereas de-nitrosylation restores it. In summary, S-nitrosylation seems to be an independent regulatory mechanism ofthe intrinsic activity of HDAC2 to rearrange the transcriptional profilesof HDAC2 target genes. The functional role and biological significanceof the S-nitrosylation of HDAC2 in CVD remain to be elucidated.

4. Possible limitations of histonedeacetylase modifiers in therapeutic application

Early tissue distribution studies that used serial analysis of gene ex-pression (SAGE) profiles suggested that class IIa HDACs are expressed inlimited organs such as the muscles, brain, or bone, whereas class IHDACs exist ubiquitously. Indeed, class I HDACs are expressed in mostcells. Thus, one may question the specificity and adverse effects ofHDACi when they are used for therapeutics. According to HDAC2whole-body deletion study, however, it should be noted that the ex-pression profile of HDAC2 is not even throughout the body, at least dur-ing developmental stages (Trivedi et al., 2007) or in diseased states. Inaddition, considering that class I HDACs bind to tissue-specific tran-scription factors, the combination of components of the repressioncomplex will vary in each tissue, which will determine the specific ef-fects of the class I HDAC in a tissue-dependent manner. For example,specific subtypes of class I HDACs are uniquely modified and regulated(Trivedi et al., 2007; Walters et al., 2009; Eom et al., 2011) throughchanging of binding partner in certain diseases. It is noteworthy that tis-sue distribution or expression of PTMmodifiers varies depending on thecellular type, organ, or even diseases conditions. Most importantly, reg-ulations of HDAC function are also finely accomplished in a combinato-rial manner by diverse types of PTMs. Thus, simultaneous modificationof PTM of class I HDAC as well as regulation of HDAC activity wouldprovide better tissue-selectivity for the therapeutics targeting class IHDACs.

5. Conclusions and future perspectives

In this review, we summarize 1) the role of HDACs in CVD, 2) thePTMs of HDACs in cardiovascular diseases, and 3) the implications ofthe PTM of the HDACs. According to HDACi studies, HDAC inhibitionhas a beneficial outcome in supraventricular tachyarrhythmia, MI,




cardiac remodeling including eccentric hypertrophy, hypertension, car-diac fibrosis, andmuscular dystrophy. However, it may worsen neointi-mal proliferation, atherosclerosis, vascular calcifications, and COPD.Actually, thrombus formation is reported as themost common and seri-ous side effect of SAHA in clinical use for anti-cancer therapy. Variousevidence including ours suggest that phosphorylation of serine residuesof HDACs is a common mechanism of regulation of subcellular localiza-tion or enzyme activity, which is an emerging target for drug discovery.

It is unavoidable that drugs that can control the enzyme activity ofmodifiers of PTM may also have numerous unwanted adverse effects.Therefore, it is necessary to develop an ideal inhibitor that blocks a sin-gle subtype or a specific agent for targeting an upstream modifier thatregulates each subtype of HDAC as a substrate. For example, we suggestthe therapeutic potential of CK2 inhibitors to modulate cardiac remod-eling. CK2 blockers are equivalently effective as HDACi in the develop-ment of cardiac hypertrophy (Eom et al., 2011). In addition to HDACi,HDAC activators should also be developed. Glucocorticoid is a widelyused regimen to control chronic inflammation, but some COPD patientsshow a tolerance to glucocorticoid (Sundar et al., 2013). By restorationof the activity of HDAC2 through de-S-nitrosylation, resistance againststeroid is dramatically improved (Malhotra et al., 2011).

According to our preliminary results, HDAC2 itself is regulated byacetylation dynamics (Eom, unpublished observation), which suggeststhe possibility of crosstalk between HDAC classes in addition tocrosstalk between PTMs. Yet, it remains unclearwhether interaction be-tween classes would be an alternate mechanism for the regulation ofHDAC activity in vivo. It would be noteworthy and interesting to under-stand the discrepancies of the effect of pan-HDACi in a single disease.The intrinsic activity of class IIa HDACs is quite low, and class IIaHDACs recruit class I HDACs for actual regulation of gene expression.Thus, studies to delineate the mechanism by which the class IIaHDACs recruit class I HDACs would be required.

To date, several HDACi are in clinical trials, especially for cancertreatment, even though the exact anti-neoplasmic effect is not clear.Two drugs, Vorinostat and Romidepsin, are now approved by the U.S.Food and Drug Administration for CTCL. Panobinostat for CTCL andVPA for solid tumor are in phase III trials. MGCD0103, PCI-24781, MS-275, SB939, 4SC-201, and ITF2357 are in phase II trials. Besides cancertreatment, a Danish group has studied HDACi for HIV/AIDS treatment.More precise information on HDACi and their patent status is reviewedelsewhere (Thaler, 2012). HDACi are also beneficial for controlling sev-eral CVDs. Thus, as a novel modality for treatment, HDACi study aimedat CVD is mandatory for patients in conventional therapy-resistantstate. A novel regimen that modulates HDAC or an HDAC-modifyingmolecule would be a promising target.

Conflict of interest statement

The authors declare that there are no conflicts of interests.

Acknowledgments

The authors are grateful for critical comments by Dr. Jonathan A.Epstein of the University of Pennsylvania. This study was supported bya National Research Foundation of Korea grant funded by the Koreangovernment (MEST, #2012-0005602), by the National Research Foun-dation of Korea grant (MRC, 2011–0030132) funded by the Korea gov-ernment (MSIP), and by a grant of the Korean Health Technology R&DProject, Ministry of Health & Welfare, Republic of Korea (A121561).

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posttranslational modifications of histone deacetylases: implications for cardiovascular diseases

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