Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases

JPT-06664; No of Pages 13 Pharmacology & Therapeutics xxx (2014) xxx–xxx

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Associate editor: P. Molenaar

Posttranslational modifications of histone deacetylases: Implications for cardiovascular 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

a r t i c l e

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Keywords: Histone deacetylases Cardiovascular diseases Posttranslational modifications Histone deacetylase inhibitors Therapeutics

a b s t r a c t Posttranslational modification (PTM) is a term that implies dynamic modification of proteins after their translation. 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 posttranslational modifiers with diverse roles in human pathophysiology, including cardiovascular diseases. In experimental models, HDAC inhibitors are beneficial in supraventricular arrhythmia, myocardial infarction, cardiac remodeling, hypertension, and fibrosis. In addition, HDACs are closely related to other vascular diseases such as neointima formation, 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 factors to their target binding elements in a phosphorylation-dependent manner. Class I HDACs, by contrast, have much greater enzymatic activity than the class II HDACs and target various non-histone proteins as well as the histone-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 the HDACs themselves as well as HDAC-mediated PTM of their targets should be considered for future potential therapeutic targets. In this review, we discuss 1) the roles of each HDAC in specific cardiovascular diseases and 2) the PTM of HDACs, 3) and the implications of such modifications for cardiovascular diseases. © 2014 Elsevier Inc. All rights reserved.

Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Histone deacetylases in cardiovascular disease . . . . . . . . . . . . . . . 3. Posttranslational modifications in cardiovascular diseases . . . . . . . . . . 4. Possible limitations of histone deacetylase modifiers in therapeutic application 5. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: CaMK, Ca2+/calmodulin-dependent protein kinase; CK2, Casein kinase 2; COPD, Chronic obstructive pulmonary disease; CTCL, Cutaneous T cell lymphoma; CVD, Cardiovascular diseases; Cys, Cysteine; Hif-1, Hypoxia-inducible factor-1; HAT, Histone acetyltransferase; HDAC, Histone deacetylase; HDACi, Histone deacetylase inhibitors; I/R, Ischemia–reperfusion; KLF, Krüppel-like factor; LDLR, Low-density lipoprotein receptor; Lys, Lysine; MEF, Myocyte enhancer factor; MI, Myocardial infarction; MITR, MEF-2 interacting transcription repressor; NAD, Nicotinamide adenine dinucleotide; PKA, Protein kinase A; PKC, Protein kinase C; PKD, Protein kinase D; PTM, Posttranslational modification; SAHA, Suberoylanilide hydroxamic acid; SCFA, Short chain fatty acid; Ser, Serine; siRNA, Small interfering RNA; SUMO, Small ubiquitin-like modifier; TSA, Trichostatin A; VEGF, Vascular endothelial growth factor; VPA, Valproic acid. ⁎ Corresponding author at: Department of Pharmacology and Medical Research Center for Gene Regulation, Chonnam National University Medical School, 5 Hak-dong, Dong-ku, Gwangju, 501-746, Republic of Korea. Tel.: +82 62 220 4243; fax: +82 62 232 6974. E-mail address: [email protected] (H. Kook).

http://dx.doi.org/10.1016/j.pharmthera.2014.02.012 0163-7258/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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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 leading cause of death throughout the world (Hunter & Reddy, 2013). Although great efforts have been made by physicians, researchers, and even primary care practitioners to reduce the mortality due to CVD, the disease is still a major cause of death in developed countries. In large part because of unbalanced, high-fat diets, the median age of patients with CVD has been decreasing (McGill et al., 2008). “Cardiovascular disease” is a term indicating medical problems in the heart, the blood vessels, or both. Sometimes CVD indicates “heart disease” in a limited sense; usually, however, vascular problems in the brain or kidney or other peripheral arterial disease is also included. The most common CVDs are hypertension and atherosclerosis (Ross, 1999). Even in healthy individuals, aging followed by morphological and physiological changes affects cardiovascular structures and function, which subsequently leads to a high risk of CVD. Therefore, risk-factor-reducing efforts such as consuming a balanced healthy diet, getting adequate amounts of exercise, increasing lean body mass, and stopping smoking are strongly recommended to reduce the development of CVD (McGill et al., 2008). Besides a preventive approach, therapeutic interventions to halt disease progress or to recover a healthy state are necessary as well, and such interventions require a fundamental understanding of disease development and progress. Considering that most CVD-related pathologic events are caused by malfunction of normal proteins, PTMs that might result in those abnormal behaviors should be extensively studied. Thus, understanding the PTMs associated with CVD may offer opportunities for the development of ideal therapeutics with maximal efficacy and minimal unwanted effects. 1.2. Posttranslational modifications Proteins are not stable but are dynamically modified by other proteins, such as kinases, acetyltransferases, methyltransferases, ubiquitinylases, and carboxylases. These changes are finely balanced by opposing enzymes such as phosphatases, deacetylases, demethylases, deubiquitinylases, and decarboxylases. These dynamic changes are called PTMs and are closely linked to diverse cellular functions and human diseases. For example, when ligands occupy their binding sites in receptors, receptor tyrosine kinases phosphorylate target molecules and an extracellular signal is delivered to cytoplasmic or nuclear targets (Hubbard & Till, 2000). Phosphorylation is an essential modification in the regulation of enzyme activation (Stambolic & Woodgett, 1994; Dimmeler et al., 1999), DNA-binding capacity (Beg et al., 1993), formation of complexes (Maudsley et al., 2000), and cell cycle regulation (Serrano et al., 1993). Besides phosphorylation, ubiquitination is an important modification that has been intensively investigated recently. Ubiquitin is a small protein with a molecular mass of just 8.5 kDa. It has 7 lysine residues in its structure. Ubiquitination indicates the covalent binding of ubiquitin to a substrate. This process generally involves binding of glycine 76 at the C-terminus of ubiquitin to a lysine of the substrate. Polyubiquitination refers to additional ligation of ubiquitin to another ubiquitin that has already been conjugated with a protein, which implies that ubiquitin works as a substrate for further ubiquitination. Two lysines of ubiquitin are involved in polyubiquitination: Lys-48 and Lys-63. Lys48-linked polyubiquitination is associated with protein degradation and recycling by proteolysis (Glickman & Ciechanover, 2002), whereas Lys63-linked polyubiquitination is atypical and is involved in other processes such 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. Although monoubiquitination is sometimes regarded as a beginning step of polyubiquitination, most monoubiquitination solely affects cellular

events such as endocytosis, trafficking, and signal transduction such as phosphorylation (Miranda & Sorkin, 2007). Small ubiquitin-like modifier (SUMO) proteins are analogous to ubiquitin, and the characteristics of SUMO modification, which is termed sumoylation, resemble those of monoubiquitination (Melchior, 2000). Likewise, protein acetylation, an alternate well-known PTM, has a unique biological function. Perhaps one of the best documented targets of acetylation involves histone H3 and H4 proteins; acetylation of the histone tail is closely associated with transcriptional activation. The positive charge of the histone core is neutralized by adding an acetyl moiety, which thereby loosens the tight interaction between the negative charge of the phosphate group in DNA and the histone tail (de Ruijter et al., 2003). The nucleosome is then opened to the transcriptional machinery, which initiates gene expression. Non-histone proteins are also susceptible to acetylation, which affects enzyme activity (Santos-Rosa et al., 2003), protein–protein interaction (Levy et al., 2004), DNA recruitment (Gu & Roeder, 1997), and transcriptional activity (Evans et al., 2007). It is noteworthy that each PTM can affect other modifications; indeed, we can easily find multiple modifications at different residues in a single molecule or even at a single residue, which seems like “competition” between the various modifications. For example, acetylation, methylation, and ubiquitination commonly occur at a lysine residue, and these modifications sometimes regulate the target protein function in a competitive manner. Histone H3 Lys-9 is a target site for both acetylation and methylation, and trimethylated Lys-9 is found in constitutively repressed genes (Barski et al., 2007). In contrast, acetylation on this residue activates gene expression (Koch et al., 2007). Following the removal of the methyl group by specific demethylases, histone acetyltransferase (HAT) enzyme acetylates H3 Lys-9. By contrast, after the acetyl moiety is removed by histone deacetylase (HDAC), the remaining unmodified lysine residue is subject to mono-, di-, and tri-methyl modification (Guillemette et al., 2011). Indeed, growing evidence suggests that HDAC works in conjunction with histone methyltransferase (Wysocka et al., 2003). Similarly, acetylation increases protein stability by competition with polyubiquitination (Li et al., 2002). Furthermore, PTM-associated PTMs such as acetylation-dependent phosphorylation (Park et al., 2003), phosphorylation-dependent acetylation (Corre et al., 2009), or phosphorylation-dependent ubiquitination (Koepp et al., 2001; Lin et al., 2002) are also reported. Protein acetylation is finely regulated by two different groups of enzymes: HATs and HDACs. At least 18 different HDACs in mammals have been 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 are class II HDACs; the sirtuin family members, Sirt1, Sirt2, Sirt3, Sirt4, Sirt5, Sirt6, and Sirt7, are class III HDACs; and HDAC11 is the only class IV HDAC. Class I, II, and IV HDACs contain and require zinc ion for their enzyme activity (Minucci & Pelicci, 2006); however, class III HDACs are NAD+-dependent (Blander & Guarente, 2004). Like the HATs, 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 the only substrate and these non-histone targets have more diverse biological functions than transcriptional regulation (Choudhary et al., 2009). In the present review, we discuss the roles and PTMs of the class I and class II HDACs and their mechanisms of regulation in association with CVD. 2. Histone deacetylases in cardiovascular disease 2.1. Histone deacetylases The criterion applied to divide the class I and class II HDACs is based on the homology of each HDAC to yeast HDACs (Blander & Guarente, 2004). Class II HDACs consist of 1) a large N terminus regulatory region,

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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2) an HDAC domain, and 3) a short tail in the C terminus. Class II HDACs form a huge complex by interaction with distinct corepressors or epigenetic regulators and thereby suppress the gene expression of downstream targets (McKinsey et al., 2000a). The class I HDACs, however, do not have a “large N terminus regulatory region” like the class II HDACs; they have only the HDAC domain and a short regulatory site in the C terminus (Taunton et al., 1996). Indeed, class I HDACs look like a truncated form of class II HDACs and the molecular weight of class II HDACs is more than double that of the class I HDACs. Class II HDACs are divided into two subgroups, IIa (4, 5, 7, and 9) and IIb (6 and 10), by structure homology. Unlike the IIa HDACs, which share common molecular structures, IIb HDACs are somewhat atypical. Two HDAC domains exist in a single HDAC6 molecule (Verdel et al., 2000), whereas an 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 intrinsic deacetylase activity. By in vitro analysis, Fischle et al. (2002) and Jones et al. (2008) reported that the deacetylase activity of class II HDACs is an artifact that results from co-purification of class I HDACs. Indeed, class II HDAC-mediated transcriptional repression is thought to be mediated by class I HDACs, which are recruited as a repressor complex in vivo. Nebbioso et al. (2009) however, reported that class IIa HDACs do have intrinsic activity in vivo. Indeed, we observed the small but significant magnitude of intrinsic activity of HDAC5 under the condition of treatment with class I HDAC-selective inhibitors. We also observed the absence of deacetylase activity of HDAC5 by substitution of histidine 833 with alanine, which is known to disrupt the pocket structure (Eom, unpublished observation). It is noteworthy that some chemical inhibitors 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 is expected to elucidate whether the intrinsic activity of class II HDACs is required for the maintenance of homeostasis or pathophysiologic

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processes. The molecular structures of the HDACs, the known PTM sites, 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 their unique properties to induce growth arrest, differentiation, and apoptosis (Marks et al., 2001). Class III HDACs require NAD+ for intrinsic activity, whereas class I, II, and IV HDACs are zinc-dependent. Thus, most class I and II HDACi are targeted to zinc ion. These zinc-targeted HDACi are mainly divided into four classes according to their structure homology: hydroxamic acid, cyclic peptides, short chain fatty acids (SCFAs), and benzamides. Hydroxamic acid binds to zinc ion in the catalytic site of HDACs and thereby inactivates enzymes (Villar-Garea & Esteller, 2004). For this reason, hydroxamic acid derivatives are nonselective inhibitors because both class I and class II HDACs are zinc-dependent. The first example of this type of inhibitor, which was approved for the treatment of human cancer by the U.S. Food and Drug Administration in 2006, is suberoylanilide hydroxamic acid (SAHA). Other well-known hydroxamic acid-derived HDAC inhibitors are trichostatin A (TSA), scriptaid, panobinostat, and givinostat. Among these inhibitors, TSA and scriptaid have been widely studied. Cyclic peptides are another class of zinc-ion-dependent HDAC inhibitors (Furumai et al., 2002). Interestingly, however, their various chemical structures enable the inhibitors to have relative selectivity on specific HDACs. For example, apicidin and romidepsin preferentially inhibit class I HDACs. Romidepsin was approved by the U.S. Food and Drug Administration in 2009 for the treatment of cutaneous T cell lymphoma (CTCL). SCFAs are relatively small HDACi with a simple structure. Although SCFAs are less potent, they can easily cross the blood–brain-barrier owing to their small molecular weight (Xu et al., 2007). For example,

Fig. 1. Molecular structure of histone deacetylases and information for inhibitors. Color bars indicate: red, phosphorylation; blue, acetylation; yellow, sumoylation; gray, di-sulfide bond; white, S-nitrosylation. Abbreviations: a, acetylation; di-S, disulfide bond; HDAC, histone deacetylase; s, sumoylation; S-NO, S-nitrosylation; p, phosphorylation; ZnF, zinc-finger domain

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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Table 1 Posttranslational 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, K441 p: S421, S423 s: K444, K476 p: S394, S407, S411, S422, S424 s: K462 S-NO: C262, C274 p: S424 p: S39 p: S210, S246, S265, S298, S302, S350, S467, S632 s: K559 di-S: C667, C669 p: S259, S279, T292, S498, S611, S661, S755, S1108 di-S: C696, C698 p: S155, S181, S358, S486 p: S220, S239, S253, S451, S554 p: S22, S458, Y570 ND ND

HDAC2

IIa

HDAC3 HDAC8 HDAC4

HDAC5

IIb IV

HDAC7 HDAC9 HDAC6 HDAC10 HDAC11

valproic acid (VPA) has been widely used for a long time to control various types of epilepsy. Thus, SCFAs are emerging HDACi for the treatment of neurologic disorders, such as Parkinson's disease, Huntington's disease, and Alzheimer's disease (Harrison & Dexter, 2013). Unlike other HDACi, benzamides are considered to be specific inhibitors with long half-lives (Glozak et al., 2005). MS-275 is currently in phase II trials for Hodgkin lymphoma, lung cancer, and breast cancer. 2.3. Vascular diseases 2.3.1. Angiogenesis The anti-neoplasmic potential of HDACi is well established. For example, SAHA was approved by the U.S. Food and Drug Administration in 2006 for the advantageous modulation of disease progression of CTCL. Besides the induction of apoptosis of tumor cells, these inhibitors also block angiogenesis; SAHA and TSA prevent sprouting of capillaries from rat aorta (Deroanne et al., 2002). HDACi block angiogenesis by activating anti-mitotic proteins like p53 and von Hippel–Lindau and by suppressing angiogenic factors such as vascular endothelial growth factors (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 tumor angiogenesis as well as the growth of the cancer itself. Considering that multiple steps are involved in tumor growth and metastasis and that diverse HDAC subtypes might be involved in the different steps, the use of specific HDACi may make it possible to alter specific pathways in the development of cancer. Even in a single phenomenon such as angiogenesis, different HDAC subtypes may result in diverse physiological events. For example, contradictory results have been reported for the roles of individual HDACs in the regulation of angiogenesis. HDAC4 was reported to negatively regulate angiogenesis by reducing VEGF expression (Sun et al., 2009), whereas another group reported that HDAC4 induces angiogenesis through an increase in stability of Hif-1α (Geng et al., 2011). Phosphorylation-dependent nuclear export of class II HDACs such as HDAC5 (Urbich et al., 2009) and HDAC7 (Mottet et al., 2007; Margariti et al., 2010) and thereby de-repression of target genes is crucial for the 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 induces cell migration by the deacetylation of cytoskeletal proteins (Kaluza et al., 2011; Li et al., 2011). Recently, it was reported that HDAC9 induces angiogenesis by repressing the miR-17-92 cluster at

the transcriptional level (Kaluza et al., 2013). Thus, even though HDACs may have diverse physiologic actions, it is widely accepted that class II HDACs are pro-angiogenic. In contrast with these observations of HDACi as anti-angiogenic mediators, studies showing a positive correlation have also been reported. Long-term treatment of VPA in a rodent model of cerebral infarction enhances neovascularization, reduces infarction size, and promotes functional recovery (Wang et al., 2012). As a mechanism, up-regulation of VEGF and matrix metalloproteinase-2/9 is suggested for the protective effect of VPA in ischemic injury. For these contradictory reports, the duration of HDAC inhibition should be considered. Many reports show that long-term administration of HDACi induces neovascularization (Zhang et al., 2012a,b). In contrast, however, HDAC inhibition blocks angiogenesis especially in the acute phase (Granger et al., 2008). In summary, HDACi inhibit angiogenesis as well as induce cytotoxicity in tumor cells. Considering that HDACi may provoke angiogenesis in certain circumstances, such as long-term treatment, more supportive data on normal versus cancer vessels or short- versus long-term effects are required. The contradictory effect of HDAC inhibition in angiogenesis is discussed again below in the Myocardial infarction (MI) section. 2.3.2. Atherosclerosis Atherosclerosis is a progressive disease that is characterized by chronic inflammation, accumulation of lipids, generation of a fibrous cap, proliferation of smooth muscle cells, calcification in vascular smooth muscle layers, and resultant loss of elasticity of arteries. As a result of the growth of atheroma, the lumen of the artery is gradually narrowed, 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 overlay the fibrous cap of the atheroma (Ross, 1999). The fibrous cap is vulnerable to rupture (Finn et al., 2010), which further blocks blood flow. When blood flow in the coronary artery is blocked, the myocardium suffers from the lack of oxygen and nutrients. If blood flow is not reinitiated at an appropriate time point, the myocardium rapidly dies. Thus, atherosclerosis in a coronary artery is the most common independent risk factor of MI (Van de Werf et al., 2008). HDACs are closely associated with multiple steps in the development of atherosclerosis. As also shown in a previous report from our group (Kee et al., 2011), global HDAC inhibition by use of scriptaid or TSA successfully prevents neointima formation after injury (Okamoto et al., 2006; Findeisen et al., 2011). When primary cultured vascular smooth muscle cells or injured carotid arteries are exposed to TSA, p21WAF1/Cip1, a potent negative regulator of the cell cycle, is significantly increased (Okamoto et al., 2006). A decrease in the transcriptional activity of KLF4 is involved in the HDACi-induced upregulation of p21WAF1/Cip1 and thereby repression of atherosclerosis (Yoshida et al., 2008; Kee et al., 2011). However, alteration of activity of p53, a well-known upstream regulator of p21WAF1/Cip1, is not observed. In contrast, however, several reports have elucidated the proatherogenic effects of TSA (Choi et al., 2005; Song et al., 2010); TSA potentiates vascular smooth muscle cell proliferation both by inhibition of thioredoxin 1 and by activation of Akt signaling (Song et al., 2010). In Ldlr null mice that lack the LDL receptor and thereby develop hypercholesterolemia (Ishibashi et al., 1993), TSA exacerbates the disease through upregulation of scavenger receptor A in macrophages, an increase in macrophage infiltration, and synergistic accumulation of oxidized LDL in atheroma (Choi et al., 2005). Although a few reports showed pro-atherogenic effects of HDACi, at least for vascular smooth muscle cell proliferation (one of the most important components in atherogenesis), HDACi seem to have a beneficial effect to prevent atherosclerosis. It should also be noted that characteristics of the individual HDACi used might result in these contradictory results at least in part. Nonspecific HDACi, such as TSA and SAHA, differently regulate proinflammatory gene expression in bone marrow macrophages; they

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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down-regulate the expression of Ccl-7, Edn-1, and Il-12p40, but have no effect on the induction of Cox-2 and Pai-1 (Halili et al., 2010). Interestingly, TSA shows a biphasic response: anti-inflammatory action at low concentrations and pro-inflammatory activity at high concentrations (Halili et al., 2010). Inhibition of class I HDACs by their specific inhibitor MS-275 potentiates inflammatory gene expression (Halili et al., 2010), which suggests that thrombosis, the most common and serious side effect of SAHA (Duvic & Vu, 2007), is caused by preferential inhibition of class I HDACs. Targeted studies against a specific subtype of HDACs would be necessary to unveil the secret of the discrepancy of the effect of TSA on atherosclerosis. Recent advances in atherosclerosis research on the specific roles of individual HDACs should be also noted. For example, restoration of HDAC2 in the development of atherosclerosis seems to be noteworthy as a target to modulate disease prognosis. HDAC2 significantly reduces both the stability and the activity of class II transactivators in macrophages and vascular smooth muscle cells (Kong et al., 2009). HDAC3 is essential for survival of endothelial cells in shear-stress conditions, and knock-down of HDAC3 aggravates neointima formation (Zampetaki et al., 2010). Thus, these beneficial effects of HDAC2 and HDAC3 are consistent with the observations that selective class I HDACi MS-275 aggravates the inflammatory response. The HDAC4/MEF2 complex suppresses c-jun expression in a PKA-dependent manner in the nonstimulated condition (Gordon et al., 2009). Shear stress induces cytoplasmic shuttling of phosphor-HDAC5, which results in the increases in both KLF4 and eNOS levels (Wang et al., 2010). HDAC7 interacts with CtBP1 to prevent toll-like receptor-driven inflammatory events (Shakespear et al., 2013). Taken together, these data suggest that either restoration of class I HDACs or nuclear enrichment of class IIa HDACs could be a therapeutic target for atherosclerosis via modulation of the inflammatory response. It should be noted that regardless of the general beneficial effects of HDACi in the cardiovascular diseases, at least in some conditions, HDAC inhibition may cause detrimental results. For example, HDAC inhibition seems to cause exaggeration of vascular calcification. Inorganicphosphate-induced calcification of human arterial smooth muscle cells is significantly increased by simultaneous treatment with TSA, a classes I and II inhibitor. TSA-mediated hypercalcification induces both expression and activity of alkaline phosphatase, and down-regulation of alkaline phosphatase blunts the hyper-calcification phenotype (Azechi et al., 2013). Indeed, according to our preliminary studies, one or two class I HDACs seem to be involved in the development of vascular calcification. Loss of activities of class I HDACs may relay phosphate-induced vascular calcification (Kwon, unpublished observation). Considering that many cellular and noncellular components are involved in the atherogenic process, investigations of the functional roles of each HDAC or HDACi in each component in the development of atherogenesis should be carried out for further delineations of the mechanisms. 2.4. Cardiac diseases In the following sections, we review the functional roles of HDACs in cardiac diseases such as arrhythmia, MI, cardiac hypertrophy, hypertension, and cardiac fibrosis. 2.4.1. Arrhythmia Only a few studies have focused on the relevance of HDACs in the conduction system. Consistent with previous reports that genetic disruption of HopX causes shortening of the PR interval, whereas overexpression of HopX induces its prolongation (Ismat et al., 2005), Liu et al. also reported atrium-origin arrhythmia in the α-myosin heavy chain (encoding myosin heavy chain 6) promoter-driven HopX transgenic mice (Liu et al., 2008). TSA successfully reverses the conduction abnormality by suppression of connexin 40. It is not clear, however, whether the anti-arrhythmic effect of TSA in their report was mediated by direct regulation of the expression of connexin 40 or whether it was a

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secondary effect after attenuation of pathologic defects such as atrial fibrosis. Considering that HopX directly associates with HDAC2 (Kook et al., 2003), this beneficial effect of TSA might be caused by inhibition of HopX-associated HDAC2. More direct relation of HDACs to cardiac arrhythmia has been delineated; gene ontology tests obtained from cardiac-specific deletion of both cardiac HDAC1 and HDAC2 reveal a dramatic increase in calcium channel subunits (Montgomery et al., 2007). Our group also performed a DNA microarray after over-expression of HDAC2 in the heart and found that several channels for ion current were dysregulated: increase in Scn2a1 (sodium) and Cacnb2 (calcium) and decrease in Scn3b (sodium) and Kcne1 (potassium). Recent studies on hereditary familial arrhythmia reveal that loss of function of Scn3b is associated with Burgada syndrome and dysfunction of Kcne1 was also responsible for Long QT syndrome (Monteforte et al., 2012), which suggests that alteration of HDAC2 function may contribute to these types of cardiac arrhythmias. Other evidence shows that HDAC3 also plays functional roles in the conduction system. Prx1 recruits HDAC3 and this Prx1/ HDAC3-complex negatively regulates NKX2.5, which ensures the maintenance of both anatomical structures and integrity of the conduction system environment (Risebro et al., 2012). Taken together, these data suggest that class I HDACs may play an important role in ion handing and in maintaining the integrity of conduction systems for synchronized contraction of the myocardium either by regulation of transcription of the target channel protein or by direct association with other proteins.

2.4.2. Myocardial infarction Oxygen and nutrients are supplied to the ventricular myocytes by the coronary arteries. In the pathologic condition, the coronary artery is often occluded by various pathologic conditions, such as growth of atheroma in the coronary artery, rupture of vulnerable plaque, thrombi from proximal lesions, emboli secondary to atrial fibrillation, or vegetation after endocarditis. Even if blood flow is resumed a relatively short time after ischemia, the myocytes are vulnerable to damage by the alternate insults of reperfusion. This type of reversible injury is called ischemia–reperfusion (I/R) injury. HDACi have emerged as novel agents for minimizing I/R injury, for restoring cardiac function, and for reducing infarct size (Lee et al., 2007; Zhao et al., 2007; Granger et al., 2008). According to an ex vivo study in mouse heart using the Langendorff perfusion system, TSA preserves cardiac performance. Intraperitoneal injection of TSA 24 h before I/R injury reduces infarct size and ameliorates contractile function (Zhao et al., 2007). Possible mechanisms are that HDACi block ischemiainduced gene regulation, including Hif-1α and VEGF, and thereby stabilize vascular permeability (Granger et al., 2008). HDAC4 may induce an increase in vascular permeability and subsequent I/R injury. Besides, HDACi retain down-regulation of PGC-1α, a master regulator of fatty acid oxidation and mitochondrial biogenesis, in I/R injury (Ramjiawan et al., 2013). HDAC5/MEF2 could serve as a negative regulator of PGC1α expression (Czubryt et al., 2003). Thus, the main beneficial effects of HDACi in I/R injury seem to be caused by suppression of incomplete vascularization 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 significantly reduced by nonselective HDACi such as VPA, tributyrin, or TSA (Lee et al., 2007; Granger et al., 2008; Zhang et al., 2012a). In contrast, however, it has also been reported that administration of TSA for 8 weeks significantly ameliorates cardiac function and prevents cardiac remodeling, which is mediated by recruitment of c-kit-positive stem cells and thereby increases both in neovascularization and in new myocytes (Zhang et al., 2012a). The obvious inconsistencies regarding the effects of HDACi on angiogenesis remain to be clarified by use of different concentrations and various time windows as well as specific HDACi. Despite some contrasting reports about HDACi in MI, it is more commonly accepted that HDACi are beneficial for conserving cardiac function in MI.

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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2.4.3. Cardiac hypertrophy Cardiac hypertrophy is a form of remodeling and is an adaptive response to the request for high workload from peripheral tissue or from intrinsic underlying disease conditions such as valvular dysfunction, hypertension, and MI (Frey & Olson, 2003). Although the initial process might be physiologic, sustained stimuli as the result of uncontrolled underlying disease lead to pathologic changes (Hill & Olson, 2008). Pathologic hypertrophy is characterized by diastolic dysfunction and massive interstitial fibrosis, whereas physiologic hypertrophy shows relatively normal cardiac performance and the absence of fibrosis (Bernardo et al., 2010). Pathologically hypertrophied heart enters a maladaptive phase and hemodynamic function abruptly falls off, which is termed heart failure. Heart failure is still the most common cause of death worldwide. The roles of the HDACs in pressure overload- or adrenergic agonistinduced cardiac hypertrophy are being extensively investigated by numerous 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 development of cardiac hypertrophy, their roles are quite opposite. Global deletion of HDAC2 allows resistance to exogenous stresses (Trivedi et al., 2007). Transgenic overexpression of HDAC2 provokes cardiac hypertrophy, whereas HDAC1 and HDAC3, other class I HDACs, have no effect on heart size (Trivedi et al., 2007; Eom et al., 2011). Despite the role of HDAC2 in cardiac hypertrophy, the expression level of HDAC2 is not changed (Kee et al., 2008; Eom et al., 2011). Instead, the intrinsic enzyme activity of HDAC2 is significantly increased in response to hypertrophic stress, which has been shown both in an in vivo mouse model (angiotensin II, isoproterenol, or transverse aortic constriction) and through in vitro experiments with primary cultured rat ventricular cardiomyocytes (angiotensin II, endothelin-1, phenylephrine, or fetal bovine serum) (Kee et al., 2008; Eom et al., 2011). However, the enzyme activity of other class I HDACs, such as HDAC1, HDAC3, or HDAC8, is not altered (Kee et al., 2008). HDAC2 is activated by phosphorylation of the Ser-394 residue, which is mediated by casein kinase 2α1 (CK2α1) (Eom et al., 2011). The phosphorylation is then protected from phosphatase by a phosphor-S394-specific physical interaction with HSP70 (Kee et al., 2008; unpublished data). Then, the activated HDAC2 represses the fetal gene program by inhibition of anti-hypertrophic mediators, such as KLF4 (Kee & Kook, 2009) and inositol polyphosphate-5phosphatase F (Trivedi et al., 2007; Zhu et al., 2009) (Fig. 2). Very recently we demonstrated that acetylation of HDAC2 K75 is an alternate PTM to regulate its activity (Eom et al., 2014). As for class I HDACs other than HDAC2, although no clear evidence regarding cardiac hypertrophy has been reported, HDAC3 seems to be associated with cardiomyocyte proliferation in the perinatal period (Trivedi et al., 2008). By contrast, the functional roles of class IIa HDACs such as HDAC5 and HDAC9 are opposite to those of HDAC2. Targeted deletion of HDAC9 (Zhang et al., 2002) or HDAC5 (Chang et al., 2004) results in an exaggeration of hypertrophic phenotypes when the knockout mice are crossed with the calcineurin-transgenic mouse, which suggests that class IIa HDACs are negative regulators of cardiac hypertrophy. In normal conditions, class IIa HDACs inhibit the transcriptional activity of MEF2 by interfering with the binding of MEF2 to its motif. After phosphorylation of HDAC5 and HDAC9 by protein kinases such as PKC, PKD, or CaMK, a molecule named 14-3-3 recognizes the phosphorylated class IIa HDACs and causes shuttling of the HDACs from the nucleus to the cytoplasm. Because MEF2 binds to DNA and activates transcription of the fetal gene program, the redistribution of class IIa HDACs from the nucleus to the cytosol causes de-repression of those genes, resulting in cardiac 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 may question 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 of pan-HDACi such as TSA (Kee et al., 2006; Kong et al., 2006), VPA (Kee et al., 2006; Cho et al., 2010), and scriptaid (Kong et al., 2006). Strikingly, even selective class I HDACi, such as SK7041 (Kee et al., 2006), apicidin (Gallo et al., 2008), and MS-275 (Cavasin et al., 2012), prevent cardiac hypertrophy. Considering that those class I HDAC-selective inhibitors can attenuate cardiac hypertrophy similar to the pan-HDACi, the anti-hypertrophic effect of the pan-HDACi is likely to be mediated by inhibition of class I HDACs. It is noteworthy that pre-established hypertrophy can also be prevented by HDACi (Kee et al., 2006). Thus, on the basis of these observations, we speculate that the enzyme activity of class I HDACs is critical in the development of cardiac hypertrophy. On the other hand, class IIa HDACs show anti-hypertrophic action in an enzyme-activity-independent manner. This is supported by other reports regarding the function of MEF-2 interacting transcription repressor (MITR) in cardiac physiology. MITR is an endogenous splicing form of HDAC9 that lacks an HDAC domain and has only an extended Nterminal regulatory region (Sparrow et al., 1999). Even though MITR cannot function as an “enzyme,” MITR shows anti-hypertrophic effects similar to those of full-length HDAC9 (Zhang et al., 2002). Thus, class IIa HDACs are believed to work as simple transcriptional modulators rather than as histone deacetylase enzymes, at least in association with the development of cardiac hypertrophy. One report argued that HDACi might aggravate right ventricular dysfunction (Bogaard et al., 2011); however, our previous result, which focused on right ventricular hypertrophy (Cho et al., 2010), tightened the general concept about HDACi in cardiac hypertrophy. HDACi are thus an effective treatment modality for restoration of cardiac function in ventricular remodeling. 2.4.4. Miscellaneous Compared 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 hypertension and the benefit of treatment with VPA for blood pressure control (Lee et al., 2013). Those authors showed shown that mineralocorticoid receptor induces blood volume retention through increases in expression of its target genes, such as ATP1a1, GILZ, and SGK-1. Mineralocorticoid receptor undergoes deacetylation by HDAC3, which is its transcriptionally active state. VPA significantly reduces the transcriptional activity of the receptor through acetylation, which thereby regulates blood pressure within the normal range. Usui et al. (2012) also reported that HDAC4 is a responsible factor for the development of hypertension through vascular inflammation. HDAC4 relays the inflammatory response via upregulation of VCAM-1, and TSA treatment completely blocks the increase in blood pressure. Taken together, these data suggest HDAC as an alternate therapeutic target for regulation of hypertension. Cardiac fibrosis is also dramatically suppressed by HDACi as demonstrated by many groups including ours (Kee et al., 2006; Kong et al., 2006; Eom et al., 2011). More direct evidence supports the concept that HDAC induces trans-differentiation of fibroblasts to myofibroblasts in either cardiac or noncardiac fibrosis (Niki et al., 1999; Guo et al., 2009). The HDAC2 protein amount is significantly increased during scar formation (Fitzgerald O'Connor et al., 2012) and it is considered to be responsible for renal fibrosis (Noh et al., 2009; Marumo et al., 2010). In addition, Sarrazy et al. (2011) reported the functional importance of myofibroblasts in pathological scar formation such as a keloid. These studies suggest that HDACs do play important roles in scar formation. Therefore, HDAC modification with HDACi would be a promising target for suppressing extensive fibrosis, because conventional therapeutic 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 rejuvenation therapy in patients with MI (Strauer et al., 2002). Delivery of pluripotent stem cells, however, may result in uncontrolled proliferation. Moreover,

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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Quiescent state Cytoplasm

CK2α1 Regulation of Class IIa HDACs

Regulation of Class I HDACs

Nucleus

HAT Class IIa HDAC

KLF4

PP? MEF2 Myocardin

HDAC2

CAMTA NKX2.5

SRF

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

Pro-hypertrophic gene

Hypertrophic stresses

Cytoplasm

Inpp5f

Activated state Class IIa HDAC

Class II HDAC

Regulation of Class IIa HDACs

Regulation of Class I HDACs

Nucleus

PP? CK2α1 Myocardin SRF

CAMTA MEF2

HAT

HDAC2

KLF4 Inpp5f

Ac

NKX2.5

Pro-hypertrophic gene

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

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 suppressed 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 is arrested by the inhibitory activity of KLF4 and Inpp5f (left). When hypertrophic stresses insult the myocardium, several posttranslational modifications of proteins take places. Class II HDACs are shuttled out in a phosphorylation-dependent manner. HDAC2 is recognized by an unidentified HAT and thereby acetylated. This acetylation induces phosphorylation of HDAC2. At the same time, inducible heat shock protein, HSP70, dramatically increases in the nucleus. CK2α1 is phosphorylated by hypertrophic stimuli and is activated. Activated CK2α1 undergoes nuclear redistribution and phosphorylates HDAC2 at Ser-394, which induces hyper-phosphorylation of HDAC2. HSP70 preferentially binds to hyper-phosphorylatedHDAC2. Functionally activated HDAC2 inhibits anti-hypertrophic mediators and the transcription factors that were arrested by class IIa HDACs are re-activated. Both of these transcriptional regulations synergistically induce reactivation of the fetal gene program (right). Black stars indicate phosphorylation. Abbreviations: Ac, acetylation. CAMTA, calmodulin binding transcription activators. CK2α1, casein kinase 2 α1. HAT, histone acetyltransferase. HDAC, histone deacetylase. HSP70, heat shock protein 70. Inpp5f, inositol polyphosphate-5phosphatase 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 induce 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 enhances the differentiation of embryonic stem cells into cardiomyocytes. Acetylation of GATA4 is increased by treatment with TSA, which thereby

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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induces cardiac-specific genes such as NKX2.5, ANF, and cardiac MHC (Kawamura et al., 2005). Considering that HDAC2 deacetylases acetylGATA4 (Trivedi et al., 2010), targeted inhibition of HDAC2 may accelerate differentiation of pluripotent stem cells into the cardiac lineage. As described briefly in the Myocardial infarction section, TSA also enhances regeneration of cardiomyocytes by recruitment of c-kit-positive stem cells in vivo. Cardiac rejuvenation studies by use of various HDACi will be necessary to test this hypothesis. Macrophage differentiation and polarization play a pivotal role in the wound-healing process. Polarized macrophages are roughly classified into two groups according to their predominant expressing pattern: M1, the killer phenotype, and M2, the repair phenotype (Martinez et al., 2008). M2-polarization is deeply involved in chronic inflammation and consequent immune-tolerance. Mullican et al. (2011) reported HDAC3 as a regulator of the balance between M1 and M2 macrophages. M2 polarization, which is driven by IL-4 exposure, is potentiated in the HDAC3-deleted macrophage. Bacterial infection-mediated inflammation in lung parenchyma is significantly reduced in HDAC3-deletion mice, which seems to be the M2 polarization effect. HDACi are thus a potential modality for control of macrophage polarization and thereby the wound-healing process in specific tissues. 3. Posttranslational modifications in cardiovascular diseases 3.1. Non-histone targets of histone acetyltransferase and histone deacetylase in association with cardiovascular disease Adult heart is a typical organ whose cell cycle is arrested. Proliferation ability, however, is transiently observed, although rapidly abolished in the perinatal period. Very interestingly, rebirth of the fetal gene programs that are arrested in adult heart has drawn much interest in regard to adult CVD. This process is closely related to development during the embryonic period. Thus, the role of acetyl-protein and its modifiers in fetal development may have applications in adult cardiac pathophysiology. GATA4, a member of the GATA family, is a cardiac-specific transcription factor. GATA4 is intimately associated with both cardiac development and adult cardiac disease. It is a crucial factor in the formation of the 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 diverse stress. Acetyl-GATA4 acquires DNA-binding affinity and transcription activity (Takaya et al., 2008). In addition, acetyl-GATA4 is subjected to deacetylation by HDAC2, which is controlled by HopX (Trivedi et al., 2010). Thus, cardiac homeostasis is regulated by dynamic modulation of GATA4 acetylation. We suggested KLF4 as a negative regulator of cardiac hypertrophy (Kee & Kook, 2009) or vascular smooth muscle cell proliferation (Kee et al., 2011). KLF4 is also regulated by PTMs such as acetylation. For example, P300/CBP-mediated acetylation of KLF4 induces binding to DNA and transactivates expression of its target genes, such p21Cip1/WAF1 (Evans et al., 2007). Other groups have suggested that HDAC2 physically interacts with KLF4 and regulates transcriptional activity by deacetylation (Meng et al., 2009). Taken together, these findings suggest that the anti-hypertrophic or anti-proliferative effect of KLF4 might be executed in an acetylation-dependent manner. p53, the so-called guardian of the genome, has been highlighted in cancer pathophysiology because of its tumor suppressor function. The target genes of activated p53 are mainly associated with DNA repair, cell cycle arrest, and apoptosis. p21Cip1/WAF1, a powerful cell cycle arrestor, is also regulated by p53. Rebirth of the arrested fetal gene program might be the result of escape from p53; therefore, what we have learned about the PTM of p53 in cancer biology might be adopted in the cardiac field. Sano et al. (2007) suggested the role of p53 in pressureoverloaded mouse hearts. Cardiac angiogenesis is well maintained in the physiologic phase; however, sustained overload induces failure of neovascularization. Uncoupling between cardiac burden and

vasculature plays a major role in the transition to heart failure. According to numerous studies in cancer biology, acetylation of p53 induces transcriptional activation and thereby apoptosis (Gu & Roeder, 1997). The deacetylation process is mediated by both class I and class III HDACs. Thus, the functional role of p53 should be investigated in the development of CVD with respect to its PTM and modifiers of PTM such as HDACs. In addition to acetylation dynamics in non-histone targets of activated HATs and HDACs, the HATs and HDACs themselves are actively modified by other epigenetic molecules and those activities are dynamically regulated. 3.2. Regulation mechanism of class IIa histone deacetylases The mechanism of regulation of class IIa HDACs has been well established by many different groups (McKinsey et al., 2000a,b; Zhang et 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 HDACs in response to exogenous hypertrophic signals. Because two serines that are susceptible to phosphorylation flank the nuclear localization signal, phosphorylation-specific binding of 14-3-3 molecules to class IIa HDACs causes masking of that signal. As a result, the class IIa HDACs are exported from the nucleus by the nuclear export signal that resides in their molecular structures. This results in de-repression of MEF2, which then leads to reactivation of fetal gene reprogramming and ensuing hypertrophic growth. In addition to phosphorylation, intramolecular disulfide bond formation also regulates subcellular localization. Reactive oxygen species induce oxidation of cysteine residues in HDAC5 (Haworth et al., 2012) (Cys-696 and Cys-698) or in HDAC4 (Ago et al., 2008) (Cys-667 and Cys-669), which actively promotes cytoplasmic shuttling, in a phosphorylation-independent manner (for more detailed review, please see Bush & McKinsey, 2009 or Haberland et al., 2009). In contrast, phosphorylation by different kinases results in quite the opposite outcome (Ha et al., 2010; Chang et al., 2013). PKA stimulated by either forskolin (Ha et al., 2010) or isoproterenol (Chang et al., 2013) can also phosphorylate HDAC5 at Ser-279. This PKA-dependent phosphorylation of HDAC5 causes nuclear relocalization. Sumoylation is also a notable regulation mechanism of class IIa HDACs. HDAC4 and MITR are substrates for SUMO-modifiers (Kirsh et al., 2002). For example, RanBP2-mediated sumoylation of the HDAC4 at Lys-599 induces nuclear accumulation against CaMK-driven export stimuli, thereby suppressing MEF2 expression (Kirsh et al., 2002). Thus, sumoylation of class IIa HDACs looks like a triggering modification for transcriptional repression. In summary, the subcellular localization and thereby transcriptional activity of class II HDACs are finely regulated by diverse PTMs, which suggests that specific enzymes modifying class II HDACs are promising targets for drug discovery. 3.3. Regulation mechanism of class I histone deacetylases HDAC1, 2, and 8 are found mainly in the nucleus. In contrast, HDAC3 is sometimes detected in the cytoplasm. As enzymes, the class I HDACs are definitely involved in the deacetylation of histone and non-histone proteins. Therefore, PTMs are focused on either promoter accumulation or alteration of enzyme activity. Although class I HDACs have only a short regulatory domain compared with class IIa HDACs, class I HDACs themselves 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 molecules. To have enzymatic activity, HDAC1/2 should be phosphorylated in vivo even in the absence of any phosphorylation stresses; we observed that purified HDAC2 obtained from a bacterial overexpression system does not have intrinsic activity, whereas it gains intrinsic activity when it is basally phosphorylated by the addition of cell lysates (Kee et al., 2008). Basal phosphorylation of HDAC1 at Ser-421 and Ser-423

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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was reported in Jurket cells by Pflum et al. (2001). Likewise, in HeLa cells, it has been known that Ser-422 and Ser-424 are responsible for the basal phosphorylation of HDAC2 (Tsai & Seto, 2002). Thus, basal phosphorylation is believed to be a prerequisite for intrinsic activity or for the assembly of repressor complex such as NuRD, Sin3A, or CoREST (Yang & Seto, 2008). Interestingly, further phosphorylation on other serine residues by specific kinases upon certain stimuli is associated with disease prognosis (Walters et al., 2009; Eom et al., 2011). We have demonstrated the role of HDAC2 and its PTM in the development 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-specific transgenic mouse system that expressing the atypical homeodomain protein HopX induces cardiac hypertrophy, which is dependent on whether HDAC2 is recruited. Furthermore HopX-driven cardiac hypertrophy is completely blocked by HDAC inhibition (Kook et al., 2003). As described above, we and others have also demonstrated that either nonspecific HDAC inhibition or selective class I HDACi successfully block cardiac hypertrophy. Thus, HDAC2, a class I HDAC, specifically functions like a cornerstone in the development of cardiac hypertrophy. In addition to the role of HDAC2 in CVD, we have also delineated the PTM of HDAC2 in hypertrophy (Eom et al., 2011). First, we demonstrated the role of CK2α1 and its target residue (Eom et al., 2011). Second, we reported the role of molecular chaperone heat shock protein 70 as a regulator of HDAC2 activity (Kee et al., 2008). Recently, we found evidence of another PTM, HDAC2 acetylation, and the role of phopho-HDAC2specific binding of HSP70 in association with cardiac hypertrophy and heart failure. HDAC2 acetylation and phosphor-specific binding to HSP70 result in the activation of HDAC2 (Eom, unpublished observation). As in cancer cells as described above, in the heart, HDAC2 is basally phosphorylated at Ser-422 and Ser-424 and is mainly located in the nucleus in the quiescent state (Eom et al., 2011). When the myocardium is stimulated by various hypertrophic stresses, a diverse signal cascade, including a phosphorylation stream, is activated. For example, activation of PKD and CaMK (Vega et al., 2004; Bossuyt et al., 2008), phosphorylation of cytoplasm-localized CK2α1 (Eom et al., 2011), and an increase in inducible HSP70 (Kee et al., 2008) take place. PKD and CaMK induce redistribution of class IIa HDACs as discussed previously. We first observed that diverse hypertrophic stresses induce phosphorylation of HDAC2 Ser-394 and Ser-411. Interestingly, activation of HDAC2 is induced by phosphorylation of HDAC2 Ser-394, Ser422, and Ser-424, but not by that of HDAC2 Ser-411. It is noteworthy that HDAC2 Ser-422 and Ser-424 are phosphorylated in nonstimulated states to maintain the basal activity but that they are not hypertrophy-responsive. These findings suggest that only Ser394 is “enzyme-activity-associated” as well as “hypertrophy-responsive.” We also found that phosphorylation-dependent activation and thereby shuttling of CK2α1 into the nucleus are responsible for the HDAC2 Ser-394 phosphorylation. Phosphorylation of HDAC2 at Ser394 is inhibited by both siRNA against CK2α1 and CK2 blockers. This CK2α1-mediated phosphorylation of HDAC2 increases the enzyme activity (Eom et al., 2011). Furthermore, a molecular chaperone HSP70 preferentially binds to Ser-394-phosphorylated HDAC2 and further potentiates its activity by protecting HDAC2 (Kee et al., 2008). In addition to phosphorylation, we recently identified that acetylation of HDAC2 takes place in response to hypertrophic stresses and that this acetylation is also required for the HDAC2 activation (Eom, unpublished observation). This observation raises the further question of interrelationship between phosphorylation and acetylation in the regulation of the enzymatic activity of HDAC2 in response to hypertrophic stresses, which remains to be clarified. Moreover, it should be clarified which HDAC and HAT is responsible for the regulation of acetylation of HDAC2. Perhaps in the future, studies will more clearly identify the role of acetylation as an independent regulation mechanism of HDACs, such as protein stability, subcellular localization, or enzymatic activity.

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3.4. Ideas for future studies on posttranslational modification of histone deacetylases in cardiovascular diseases HDAC1 undergoes SUMO modification in the C-terminus, which regulates transcription-repression ability (David et al., 2002; Kirsh et al., 2002). It is not clear, however, whether sumoylation directly affects the intrinsic activity of HDAC1 or regulates the formation of a corepressor complex. HDAC2 Lys-462 is also known to be subject to sumoylation and SUMO-HDAC2 is catalytically active (Brandl et al., 2012). As shown in the case of p53, Lys-320 in p53 is a target of SUMO-HDAC2, and deacetylation of p53 fails to control apoptosis of DNA-damaged cells (Brandl et al., 2012). These findings on the sumoylation of HDAC2 suggest de-sumoylation as a novel target for cell cycle arrest and thereby cancer treatment. S-Nitrosylation is an alternate important modification of HDAC2. SNitrosylation of HDAC2 was first reported in neuronal development. Nott et al. (2008) found that S-nitrosylation occurs in Cys-262 and Cys-274, which regulates chromatin recruitment rather than intrinsic activity. Colussi et al. (2008) also reported the therapeutic potential of HDAC2 S-nitrosylation in a Duchenne muscular dystrophy model. In that study, the intrinsic activity of HDAC2 was dramatically suppressed by S-nitrosylation and endothelial nitric oxide synthase was sufficient to produce nitric oxide to repress HDAC2. In addition, Malhotra et al. (2011) clearly demonstrated HDAC2 S-nitrosylation and its functional relevance to the failure in patients with chronic obstructive pulmonary disease (COPD) to control underlying chronic inflammation despite treatment with glucocorticoid. S-Nitrosylation directly suppresses the activity of HDAC2, whereas de-nitrosylation restores it. In summary, Snitrosylation seems to be an independent regulatory mechanism of the intrinsic activity of HDAC2 to rearrange the transcriptional profiles of HDAC2 target genes. The functional role and biological significance of the S-nitrosylation of HDAC2 in CVD remain to be elucidated. 4. Possible limitations of histone deacetylase modifiers in therapeutic application Early tissue distribution studies that used serial analysis of gene expression (SAGE) profiles suggested that class IIa HDACs are expressed in limited organs such as the muscles, brain, or bone, whereas class I HDACs exist ubiquitously. Indeed, class I HDACs are expressed in most cells. Thus, one may question the specificity and adverse effects of HDACi when they are used for therapeutics. According to HDAC2 whole-body deletion study, however, it should be noted that the expression profile of HDAC2 is not even throughout the body, at least during developmental stages (Trivedi et al., 2007) or in diseased states. In addition, considering that class I HDACs bind to tissue-specific transcription factors, the combination of components of the repression complex will vary in each tissue, which will determine the specific effects 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) through changing of binding partner in certain diseases. It is noteworthy that tissue distribution or expression of PTM modifiers varies depending on the cellular type, organ, or even diseases conditions. Most importantly, regulations of HDAC function are also finely accomplished in a combinatorial manner by diverse types of PTMs. Thus, simultaneous modification of PTM of class I HDAC as well as regulation of HDAC activity would provide better tissue-selectivity for the therapeutics targeting class I HDACs. 5. Conclusions and future perspectives In this review, we summarize 1) the role of HDACs in CVD, 2) the PTMs of HDACs in cardiovascular diseases, and 3) the implications of the PTM of the HDACs. According to HDACi studies, HDAC inhibition has a beneficial outcome in supraventricular tachyarrhythmia, MI,

Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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cardiac remodeling including eccentric hypertrophy, hypertension, cardiac fibrosis, and muscular dystrophy. However, it may worsen neointimal proliferation, atherosclerosis, vascular calcifications, and COPD. Actually, thrombus formation is reported as the most common and serious side effect of SAHA in clinical use for anti-cancer therapy. Various evidence including ours suggest that phosphorylation of serine residues of HDACs is a common mechanism of regulation of subcellular localization or enzyme activity, which is an emerging target for drug discovery. It is unavoidable that drugs that can control the enzyme activity of modifiers of PTM may also have numerous unwanted adverse effects. Therefore, it is necessary to develop an ideal inhibitor that blocks a single subtype or a specific agent for targeting an upstream modifier that regulates each subtype of HDAC as a substrate. For example, we suggest the therapeutic potential of CK2 inhibitors to modulate cardiac remodeling. CK2 blockers are equivalently effective as HDACi in the development of cardiac hypertrophy (Eom et al., 2011). In addition to HDACi, HDAC activators should also be developed. Glucocorticoid is a widely used regimen to control chronic inflammation, but some COPD patients show a tolerance to glucocorticoid (Sundar et al., 2013). By restoration of the activity of HDAC2 through de-S-nitrosylation, resistance against steroid is dramatically improved (Malhotra et al., 2011). According to our preliminary results, HDAC2 itself is regulated by acetylation dynamics (Eom, unpublished observation), which suggests the possibility of crosstalk between HDAC classes in addition to crosstalk between PTMs. Yet, it remains unclear whether interaction between classes would be an alternate mechanism for the regulation of HDAC activity in vivo. It would be noteworthy and interesting to understand the discrepancies of the effect of pan-HDACi in a single disease. The intrinsic activity of class IIa HDACs is quite low, and class IIa HDACs recruit class I HDACs for actual regulation of gene expression. Thus, studies to delineate the mechanism by which the class IIa HDACs recruit class I HDACs would be required. To date, several HDACi are in clinical trials, especially for cancer treatment, 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 and VPA for solid tumor are in phase III trials. MGCD0103, PCI-24781, MS275, SB939, 4SC-201, and ITF2357 are in phase II trials. Besides cancer treatment, a Danish group has studied HDACi for HIV/AIDS treatment. More precise information on HDACi and their patent status is reviewed elsewhere (Thaler, 2012). HDACi are also beneficial for controlling several CVDs. Thus, as a novel modality for treatment, HDACi study aimed at CVD is mandatory for patients in conventional therapy-resistant state. A novel regimen that modulates HDAC or an HDAC-modifying molecule 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 by a National Research Foundation of Korea grant funded by the Korean government (MEST, #2012-0005602), by the National Research Foundation of Korea grant (MRC, 2011–0030132) funded by the Korea government (MSIP), and by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A121561). References Ago, T., Liu, T., Zhai, P., Chen, W., Li, H., Molkentin, J.D., et al. (2008). A redoxdependent pathway for regulating class II HDACs and cardiac hypertrophy. Cell 133, 978–993.

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Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012

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Please cite this article as: Eom, G.H., & Kook, H., Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.012