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AIMS: Following an acute myocardial infarction (MI) the extracellular matrix (ECM) undergoes remodeling in order to prevent dilation of the infarct area and maintain cardiac output. Excessive and prolonged inflammation following an MI exacerbates adverse ventricular remodeling. Macrophages are an integral part of the inflammatory response that contribute to this remodeling. Treatment with histone deacetylase (HDAC) inhibitors preserves LV function and myocardial remodeling in the post-MI heart. This study tested whether inhibition of HDAC activity resulted in preserving post-MI LV function through the regulation of macrophage phenotype and early resolution of inflammation. METHODS AND RESULTS: HDAC inhibition does not affect the recruitment of CD45(+) leukocytes, CD45(+)/CD11b(+) inflammatory monocytes or CD45(+)/CD11b(+)CD86(+) inflammatory macrophages for the first 3 days following infarct. Further, HDAC inhibition does not change the high expression level of the inflammatory cytokines in the first days following MI. However, by day 7, there was a significant reduction in the levels of CD45(+)/Cd11b(+) and CD45(+)/CD11b(+)/CD86(+) cells with HDAC inhibition. Remarkably, HDAC inhibition resulted in the dramatic increase in the recruitment of CD45(+)/CD11b(+)/CD206(+) alternatively activated macrophages as early as 1 Day which remained significantly elevated until 5 days post-MI. qRT-PCR revealed that HDAC inhibitor treatment shifts the cytokine and chemokine environment towards an M2 phenotype with upregulation of M2 markers at 1 and 5 days post-MI. Importantly, HDAC inhibition correlates with significant preservation of both LV ejection fraction and end-diastolic volume and is associated with a significant increase in micro-vessel density in the border zone at 14 days post-MI. CONCLUSION: Inhibition of HDAC activity result in the early recruitment of reparative CD45(+)/CD11b(+)/CD206(+) macrophages in the post-MI heart and correlates with improved ventricular function and remodeling. This work identifies a very promising therapeutic opportunity to manage macrophage phenotype and enhance resolution of inflammation in the post-MI heart.

Background— MicroRNAs (miRNAs) and histone deacetylases (HDACs) serve a significant role in the pathogenesis of a variety of cardiovascular diseases. The transcriptional regulation of miRNAs is poorly understood in cardiac hypertrophy. We investigated whether the expression of miR-133a is epigenetically regulated by class I and IIb HDACs during hypertrophic remodeling. Methods and Results— Transverse aortic constriction (TAC) was performed in CD1 mice to induce pressure overload hypertrophy. Mice were treated with class I and IIb HDAC inhibitor (HDACi) via drinking water for 2 and 4 weeks post TAC. miRNA expression was determined by real-time polymerase chain reaction. Echocardiography was performed at baseline and post TAC end points for structural and functional assessment. Chromatin immunoprecipitation was used to identify HDACs and transcription factors associated with miR-133a promoter. miR-133a expression was downregulated by 0.7- and 0.5-fold at 2 and 4 weeks post TAC, respectively, when compared with vehicle control ( P Conclusions— The results reveal that HDACs play a role in the regulation of pressure overload–induced miR-133a downregulation. This work is the first to provide insight into an epigenetic-miRNA regulatory pathway in pressure overload–induced cardiac fibrosis.

Left ventricular (LV) remodeling, after myocardial infarction (MI), can result in LV dilation and LV pump dysfunction. Post-MI induction of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated as causing deleterious effects on LV and extracellular matrix remodeling in the MI region and within the initially unaffected remote zone. Histone deacetylases (HDACs) are a class of enzymes that affect the transcriptional regulation of genes during pathological conditions. We assessed the efficacy of both class I/IIb- and class I-selective HDAC inhibitors on MMP-2 and MMP-9 abundance and determined if treatment resulted in the attenuation of adverse LV and extracellular matrix remodeling and improved LV pump function post-MI. MI was surgically induced in MMP-9 promoter reporter mice and randomized for treatment with a class I/IIb HDAC inhibitor for 7 days post-MI. After MI, LV dilation, LV pump dysfunction, and activation of the MMP-9 gene promoter were significantly attenuated in mice treated with either the class I/IIb HDAC inhibitor tichostatin A or suberanilohydroxamic acid (voronistat) compared with MI-only mice. Immunohistological staining and zymographic levels of MMP-2 and MMP-9 were reduced with either tichostatin A or suberanilohydroxamic acid treatment. Class I HDAC activity was dramatically increased post-MI. Treatment with the selective class I HDAC inhibitor PD-106 reduced post-MI levels of both MMP-2 and MMP-9 and attenuated LV dilation and LV pump dysfunction post-MI, similar to class I/IIb HDAC inhibition. Taken together, these unique findings demonstrate that selective inhibition of class I HDACs may provide a novel therapeutic means to attenuate adverse LV remodeling post-MI.

The current inability to derive mature cardiomyocytes from human pluripotent stem cells has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of cardiomyocytes derived from human pluripotent stem cells. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments. On the other hand, scaffold-free, 3D cardiac spheroids (i.e., spherical-shaped microtissues) prepared by seeding cardiomyocytes into agarose microwells were shown to improve cardiac functions. However, cardiomyocytes within the spheroids could not assemble in a controlled manner and led to compromised, unsynchronized contractions. Here, we show, for the first time, that incorporation of a trace amount (i.e., ∼0.004% w/v) of electrically conductive silicon nanowires (e-SiNWs) in otherwise scaffold-free cardiac spheroids can form an electrically conductive network, leading to synchronized and significantly enhanced contraction (i.e., >55% increase in average contraction amplitude), resulting in significantly more advanced cellular structural and contractile maturation.

Class IIa histone deacetylases (HDACs) are very important for tissue specific gene regulation in development and pathology. Because class IIa HDAC catalytic activity is low, their exact molecular roles have not been fully elucidated. Studies have suggested that class IIa HDACs may serve as a scaffold to recruit the catalytically active class I HDAC complexes to their substrate. Here we directly address whether the class IIa HDAC, HDAC5 may function as a scaffold to recruit co-repressor complexes to promoters. We examined two well-characterized cardiac promoters, the sodium calcium exchanger (Ncx1) and the brain natriuretic peptide (Bnp) whose hypertrophic upregulation is mediated by both class I and IIa HDACs. Selective inhibition of class IIa HDACs did not prevent adrenergic stimulated Ncx1 upregulation, however HDAC5 knockout prevented pressure overload induced Ncx1 upregulation. Using the HDAC5((-/-)) mouse we show that HDAC5 is required for the interaction of the HDAC1/2/Sin3a co-repressor complexes with the Nkx2.5 and YY1 transcription factors and critical for recruitment of the HDAC1/Sin3a co-repressor complex to either the Ncx1 or Bnp promoter. Our novel findings support a non-canonical role of class IIa HDACs in the scaffolding of transcriptional regulatory complexes, which may be relevant for therapeutic intervention for pathologies.

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a Ca(2+)-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated (n = 6) and untreated (n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [EF decreased from 67 + or - 2% pre-MI to 30 + or - 4% with MI only vs. 41 + or - 2% with MI + calpeptin] and attenuated the increase in EDV [EDV increased from 42 + or - 2 microl pre-MI to 73 + or - 4 microl with MI only vs. 55 + or - 4 microl with MI + calpeptin]. Furthermore, calpeptin treatment resulted in marked reduction in calpain- and caspase-3-associated changes and TUNEL staining. These studies indicate that calpain contributes to MI-induced alterations in myocardial structure and function and that it could be a potential therapeutic target in treating MI patients.

The NCX1 (sodium-calcium exchanger) is up-regulated in human heart failure and in many animal models of heart failure. The potential benefits and risks of therapeutically blocking NCX1 in heart failure and during ischemia-reperfusion are being actively investigated. In this study, we demonstrate that prolonged administration of the NCX1 inhibitor KB-R7943 resulted in the up-regulation of Ncx1 gene expression in both isolated adult cardiomyocytes and intact mouse hearts. Ncx1 up-regulation is mediated by the activation of p38. Importantly, p38 is not activated by KB-R7943 treatment in heart tubes from Ncx1(-/-) mice at 9.5 days postcoitum but is activated in heart tubes from Ncx1(+/+) mice. p38 activation does not appear to be in response to changes in cytosolic calcium concentration, [Ca(2+)](i). Interestingly, chronic KB-R7943 treatment in mice leads to the formation of an NCX1-p38 complex. Our study demonstrates for the first time that the electrogenic sarcolemma membrane cardiac NCX1 can act as a regulator of "activity-dependent signal transduction" leading to changes in gene expression.

It is becoming increasingly evident that histone deacetylases (HDACs) have a prominent role in the alteration of gene expression during the growth remodeling process of cardiac hypertrophy. HDACs are generally viewed as corepressors of gene expression. However, we demonstrate that class I and class II HDACs play an important role in the basal expression and up-regulation of the sodium calcium exchanger (Ncx1) gene in adult cardiomyocytes. Treatment with the HDAC inhibitor trichostatin A (TSA) prevented the pressure-overload-stimulated up-regulation of Ncx1 expression. Overexpression of HDAC5 resulted in the dose-dependent up-regulation of basal and α-adrenergic stimulated Ncx1 expression. We show that Nkx2.5 recruits HDAC5 to the Ncx1 promoter, where HDAC5 complexes with HDAC1. Nkx2.5 also interacts with transcriptional activator p300, which is recruited to the Ncx1 promoter. We demonstrate that when Nkx2.5 is acetylated, it is found associated with HDAC5, whereas deacetylated Nkx2.5 is in complex with p300. Notably, TSA treatment prevents p300 from being recruited to the endogenous Ncx1 promoter, resulting in the repression of Ncx1 expression. We propose a novel model for Ncx1 regulation in which deacetylation of Nkx2.5 is required for the recruitment of p300 and results in up-regulation of exchanger expression.—Chandrasekaran, S., Peterson, R. E., Mani, S. K., Addy, B., Buchholz, A. L., Xu, L., Thiyagarajan, T., Kasiganesan, H., Kern, C. B., Menick, D. R. Histone deacetylases facilitate sodium/calcium exchanger up-regulation in adult cardiomyocytes.

In response to an increased hemodynamic load, such as pressure or volume overload, cardiac hypertrophy ensues as an adaptive mechanism. Although hypertrophy initially maintains ventricular function, a yet undefined derailment in this process eventually leads to compromised function (decompensation) and eventually culminates in congestive heart failure (CHF). Therefore, determining the molecular signatures induced during compensatory growth is important to delineate specific mechanisms responsible for the transition into CHF. Compensatory growth involves multiple processes. At the cardiomyocyte level, one major event is increased protein turnover where enhanced protein synthesis is accompanied by increased removal of deleterious proteins. Many pathways that mediate protein turnover depend on a key molecule, mammalian target of rapamycin (mTOR). In pressure-overloaded myocardium, adrenergic receptors, growth factor receptors, and integrins are known to activate mTOR in a PI3K-dependent and/or independent manner with the involvement of specific PKC isoforms. mTOR, described as a sensor of a cell's nutrition and energy status, is uniquely positioned to activate pathways that regulate translation, cell size, and the ubiquitin-proteasome system (UPS) through rapamycin-sensitive and -insensitive signaling modules. The rapamycin-sensitive complex, known as mTOR complex 1 (mTORC1), consists of mTOR, rapamycin-sensitive adaptor protein of mTOR (Raptor) and mLST8 and promotes protein translation and cell size via molecules such as S6K1. The rapamycin-insensitive complex (mTORC2) consists of mTOR, mLST8, rapamycin-insensitive companion of mTOR (Rictor), mSin1 and Protor. mTORC2 regulates the actin cytoskeleton in addition to activating Akt (Protein kinase B) for the subsequent removal of proapoptotic factors via the UPS for cell survival. In this review, we discuss pathways and key targets of mTOR complexes that mediate growth and survival of hypertrophying cardiomyocytes and the therapeutic potential of mTOR inhibitor, rapamycin.

Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.

Tumor necrosis factor (TNF) family member, the receptor activator of NF-κB ligand (RANKL) is expressed on marrow stromal/osteoblast cells in response to several osteotropic factors and is critical for osteoclast precursor differentiation to form multinucleated osteoclasts, which resorb bone [Lacey et al., 1998; Hsu et al., 1999; Kong et al., 1999]. Enhanced levels of RANKL are associated with pathologic conditions such as Paget's disease of bone [Menaa et al., 2000; Neale et al., 2000] and multiple myeloma [Roux et al., 2002]. Several osteotropic factors such as l,25-(OH)2D3, parathyroid hormone (PTH), interleukin 1β (IL-1β), interleukin-11, and prostaglandin E2 (PGE2) induce osteoclast differentiation through enhanced expression of RANKL in marrow stromal/osteoblast cells [Nakashima et al., 2000; Lee et al., 2002], but the molecular mechanisms which regulate RANKL gene expression are unclear. It has been reported that IL-1β and TNF-α stimulate RANKL expression in human bone marrow stromal cells through activation of p38 MAP kinase pathway [Rossa et al., 2006]. In addition, fibroblast growth factor-2 (FGF-2) has been shown to induce RANKL production through COX-2-mediated prostaglandin synthesis and by suppressing osteoclastogenesis inhibitory factor in mouse osteoblastic cells [Nakagawa et al., 1999]. Similarly, lipopolysaccharide treatment increased the levels of RANKL expression through activation of Toll-like receptors in primary murine osteoblast cells [Kikuchi et al., 2001]. Furthermore, transforming growth factor β (TGF-β) has been shown to increase RANKL expression in activated T-cells by increasing anti-CD3 [Wang et al., 2002]. Recently, it has also been reported that PTH stimulates RANKL expression through cAMP/protein kinase A/CREB cascade [Fu et al., 2006]. We recently demonstrated that heat shock factor-2 (HSF-2) is a downstream target of FGF-2 signaling to induce RANKL expression in bone marrow stromal/preosteoblast cells [Roccisana et al., 2004]. Heat shock proteins (HSP) are molecular chaperones expressed in cells in response to a variety of stimuli such as temperature and stimulation of membrane-bound receptors by hormones/cytokines and other chemical factors. Thus, HSP are an integral part of mammalian development [Christians et al., 2003]. Heat shock transcription factors (HSF), which binds to the heat shock responsive element (HSE), modulate expression of HSP and several other genes including TNF-α family [Mathew et al., 1998; Snoeckx et al., 2001]. Although multiple osteotropic factors including FGF-2 are known to modulate RANKL gene expression in the bone microenvironment, the transcriptional regulatory mechanisms operative in marrow stromal/preosteoblast cells are not well established. DACH1 is a human homolog of the Drosophila dachshund gene, which is a key regulator for organ development and is considered a cell fate determination factor. DACH1 contains a conserved domain (dachshund domain (DS)) in the N-terminal region that is structurally homologous with the Ski and Sno proto-oncogenes and interacts with the nuclear co-repressor NCoR to modulate transcription factor activity. We have previously reported that DACH1 represses TGF-β signaling through binding to Smad4 [Wu et al., 2003]. Two vertebrate homologues, Dach1 and Dach2 are partially functionally redundant, since Dach1−/− mice survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress [Davis et al., 2001]. DACH1 expression is lost in a subset of human breast cancers associated with poor prognosis. Reintroduction of DACH1 inhibits breast tumor cell proliferation and tumor growth through suppression of cyclin D1 [Wu et al., 2006]. DACH1 inhibits TGF-β signaling in ovarian cancer cells [Sunde et al., 2006]. These studies are part of a growing body of evidence that DACH1 may function as a tumor suppressor. Recent evidence indicates that DACH1 is associated with FGF signaling during limb and skeletal development and may regulate cell proliferation or differentiation [Horner et al., 2002]. Dach1 binds with Runx2/Cbfa1 and co-localizes with cyclin-dependent kinase inhibitor p27 (Kip1) and p57 (Kip2) at the growth plate region in chondrocytes [Horner et al., 2002]. In this study, we show that DACH1 negatively regulate RANKL gene expression in human bone marrow-derived stromal/preosteoblast cells and that FGF-2 signaling promotes HSF-2 binding to DACH1 to modulate RANKL gene expression in these cells.

Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival.

Introduction: Alterations in expression and activity of different genes have been implicated in the pathogenesis of heart failure. Our lab has shown that HDAC-repressor complexes play a critical role in the upregulation Sodium Calcium Exchanger ( Ncx1) and HDAC inhibition causes changes that attenuated cardiac remodeling during cardiac hypertrophy and heart failure. Thus, treatment with HDAC inhibitors has been proposed as a potential strategy for treatment of cardiac hypertrophy and heart failure. HDAC inhibitors repress deacetylase activity but we propose that they also affect HDAC confirmation and interaction with other protein factors. We hypothesize that HDAC inhibitors affect the stability of the co-repressor complex with specific transcription factors and that this effect is dependent on the transcription factor. Results: Inhibition of HDACs in adult cardiomyocytes results in the greater stabilization of HDACs with co-repressor molecules that were recruited to the NCX1 promoter through Nkx2.5 transcription factor. HDAC class I specific inhibitor, MS 275 demonstrated stronger association between HDACs and co-repressors while other Class I inhibitors, PD106 and BML 210 failed on showing this phenomenal. The results suggested that class I HDACs inhibitors may affect formations of HDAC-complex via alternated active site interactions other than chelating with zinc binding domain. These results compliment ChIP experiments which also demonstrate the different recruitments of Sin3a at the proximal promoter of NCX1. In vivo analysis on HDAC5 knockout mice reveal that the Sin3a-HDAC1/2 repressor complex is not recruited to the Ncx1 promoter in the absence of HDAC5, indicating not only Class I HDAC but also Class II HDACs play an important role on HDAC-complex formation. Conclusions: This work gives insight into part of the molecular mechanism of how HDAC inhibitors can affect the stability of the HDAC co-repressor complex in cardiac hypertrophy and heart failure. In addition, we demonstrated the Class IIa HDACs are required for the recruitment of the Sin3a/HDAC1/2 co-repressor complex to specific transcription factors on the target promoter.

article i nfo While inhibition of class I/IIb histone deacetylases (HDACs) protects the mammalian heart from ischemia reper- fusion (IR) injury, class selective effects remain unexamined. We hypothesized that selective inhibition of class I HDACs would preserve left ventricular contractile function following IR in isolated hearts. Male Sprague Dawley rats (n = 6 pergroup) wereinjected with vehicle (dimethylsulfoxide, 0.63 mg/kg), the class I/IIb HDAC inhibitor trichostatin A (1 mg/kg), the class I HDAC inhibitor entinostat (MS-275, 10 mg/kg), or the HDAC6 (class IIb) in- hibitor tubastatin A (10 mg/kg). After 24 h, hearts were isolated and perfused in Langendorff mode for 30 min (Sham) or subjected to 30 min global ischemia and 120 min global reperfusion (IR). A saline filled balloon attached to a pressure transducer was placed in the LV to monitor contractile function. After perfusion, LV tissue was collected for measurements of antioxidant protein levels and infarct area. At the conclusion of IR, MS-275 pretreatment was associated with significant preservation of developed pressure, rate of pressure generation, rateofpressurerelaxationandratepressureproduct,ascomparedtovehicletreatedhearts.There wassignificant reduction of infarct area with MS-275 pretreatment. Contractile function was not significantly restored in hearts treated with trichostatinA or tubastatinA. Mitochondrial superoxide dismutase(SOD2) and catalase protein and mRNA in hearts from animals pretreated with MS-275 were increased following IR, as compared to Sham. This was associated with a dramatic enrichment of nuclear FOXO3a transcription factor, which mediates the expres- sion of SOD2 and catalase. Tubastatin A treatment was associated with significantly decreased catalase levels afterIR. Class I HDAC inhibition elicits protection of contractile function following IR, which isassociated with in- creased expression of endogenous antioxidant enzymes. Class I/IIb HDAC inhibition with trichostatin A or selec- tive inhibition of HDAC6 with tubastatin A was not protective. This study highlights the need for the development of new strategies that target specific HDAC isoforms in cardiac ischemia reperfusion.

Background: Following a myocardial infarction (MI) the extracellular matrix (ECM) undergoes massive remodeling to prevent rupture and maintain cardiac output. Large increases in matrix metalloproteinase-9 (MMP-9) are associated with adverse ECM remodeling. We found that treatment with an HDAC inhibitor repressed post-MI upregulation of MMP-9. Significant sources of MMP-9 in the post-MI LV are M1 macrophages. Both phenotypes (M1 and M2) can contribute to MMP expression, but this is dependant on the phase of ECM remodeling. We hypothesize that HDAC inhibition regulates the post-MI expression of MMP-9 by mediating the M1 to M2 macrophage polarization. Methods: CD1 and MMP-9 β-gal reporter mice were induced with MI by LAD ligation then administered HDAC inhibitors: trichostatin A (TSA; class I and IIb), PD106 (class I), or Tubastatin A (HDAC 6) until termination at 5 or 7 days post-MI. Heart function evaluated by echocardiogram and cells or tissue by immunohistochemistry and immunoblotting. Results: The post-MI change in LV end-diastolic volume (49±9%) is significantly lower and ejection fraction (-44±8%) is improved with treatment of TSA vs. control (69±12%; -59±6%) respectively [n=28]. Immunohistochemical analysis revealed that infiltrating macrophages express MMP-9 at 5 and 7 days post-MI. HDAC inhibition decreases this expression and did so without reducing presence of macrophages within infarct. Immunoblotting shows that expression of all class I HDACs are increased following MI; however, in cultured macrophages only HDAC2 and HDAC3 are increased. TSA and PD106, inhibit control levels and lipopolysacharide (LPS) stimulated upregulation of MMP-9 in cultured RAW264.7 and bone marrow derived macrophages. Immunofluorescence revealed that treatment with PD106, Tub A, and TSA leads to M1 to M2 morphology specific polarization and maintenance of anti-inflammatory, M2, phenotype even with LPS stimulation in culture. However, only PD106 and TSA reduced MMP-9 expression in cultured macrophages. Conclusions: Macrophage mediated secretion of MMP-9 contributes to adverse ECM remodeling and loss of LV function post-MI. Class I HDAC inhibition promotes both M2 macrophage polarization and attenuates adverse remodeling by reducing MMP-9 expression.

Background: While dual inhibition of class I/IIb KDACs protects the mammalian heart from ischemia-reperfusion (IR) injury, class-specific effects have not been examined. Hypothesis: We hypothesized that specific inhibition of class I KDACs would preserve left ventricular (LV) function following IR in isolated hearts. Methods: Male Sprague-Dawley rats (n=4 per group) were injected with vehicle, the class I KDAC inhibitor entinostat, the class IIb inhibitor tubastatin A, or the class I/IIb inhibitor trichostatin A (TSA). After 18 h, hearts were isolated and perfused in Langendorff mode. A saline-filled balloon was placed in the LV to monitor contractile function. Following IR (30/120 min), LV free wall tissue was assayed for activation of pro-survival proteins. Group results are reported vs vehicle as mean±SE. Results: There were no differences between groups in LV function before ischemia. At the end of reperfusion, rate pressure product (mm hg/min) was improved with entinostat (27392±3474, pmax and -dP/dt max . Most notably, entinostat alone blunted the rise in end diastolic pressure (mm Hg) during reperfusion (18.6±1.4 vs. 45.3±4.1, p Conclusions: Inhibition of class I KDACs was most effective at preserving contractile function following IR in isolated hearts, notably through prevention of LV contracture during reperfusion. Class I KDAC inhibition may preserve LV function by differentially modulating Src and eNOS activities. These results suggest that the protective effects of KDAC inhibitors in IR can be ascribed to specific inhibition of class I KDACs.

Background: Pressure overload can lead to aberrant gene expression and subsequent adverse hypertrophic changes in cardiac tissue. Histone deacetylases (HDACs) play an important role in alteration of gene expression during cardiac hypertrophy. We observed that class I/IIb HDAC inhibitors, TSA and Vorinostat prevent upregulation of the sodium calcium exchanger gene, Ncx1 , in response to cardiac hypertrophy. Chromatin immunoprecipitation (ChIP) demonstrates the class I HDAC, HDAC1, class IIa HDAC, HDAC5 and co-repressor, Sin3a are recruited to the Ncx1 promoter via Nkx2.5. Class IIa HDACs have very poor catalytic activity and can interact with many specific transcription factors. Thus, we hypothesized , that the class IIa, HDAC5 acts as a scaffold to recruit the class I HDAC/Sin3a complex to the Ncx1 promoter. Methods and Results: To test our hypothesis adult cardiomyocytes were treated with either vehicle (control) or class specific HDAC inhibitors: class I (Entinostat), or class IIa (dPAHA). We found that class I HDAC inhibitor, Entinostat, abrogates adrenergic stimulated upregulation of Ncx1 expression in adult cardiomyocytes to almost control levels (pNcx1 expression (p>0.05 Student’s t; n=4). Also, Entinostat inhibits Ncx1 upregulation in a transaortic constriction (TAC) model of hypertrophy. We assessed the role class IIa HDAC5 in Ncx1 regulation, by subjecting HDAC5-Knockout mice (HDAC5-KO) to TAC. We saw no induction of Ncx1 expression post-TAC (n=5). Furthermore, Co-IP and ChIP analyses show that interactions between the Sin3a/HDAC1 complex and Nkx2.5 are lost in the absence of HDAC5 expression and fail to be recruited to the Ncx1 promoter. To conclude , our data suggests the catalytic activity of HDAC5 is not essential but rather HDAC5 is a scaffold that recruits the class I HDAC1–Sin3a complex to the Ncx1 promoter. Our novel findings provide insight into possible roles of class IIa HDACs in regulation of gene expression and efficacy of HDAC inhibitors to treat cardiac pathologies.

Post myocardial infarction (MI) the extracellular matrix (ECM) undergoes massive remodeling in order to prevent dilation of the infarct area and maintain cardiac output. Integral players in this remodeling are matrix metalloproteinases (MMP). Post-MI macrophages are a significant source of MMP production and have been shown to have temporal expression of the two phenotypes (M1 and M2). Differential expressions of macrophage phenotypes correlate with the different phases of ECM remodeling, ECM composition and MMP expression. Increased expression of MMP-9 is associated with deleterious effects to the ECM and left ventricular (LV) dilation following MI. MMPs major mechanism of expression regulation is transcription. Histone deacetylases (HDAC) are a class of enzymes that can greatly affect transcriptional regulation of genes during pathological conditions. Therefore, we hypothesize that HDAC inhibition controls the post-MI expression of MMP-9 by mediating macrophage polarization to improve LV function. The post-MI change in LV end-diastolic volume is significantly lower with treatment of the class I and IIb HDAC inhibitor trichostatin A (TSA) and ejection fraction is improved in mice. Immunohistochemical analysis and zymography revealed that MMP-9 expression at 7 days post-MI is attributable to infiltrating macrophages. Immunoblotting shows that TSA inhibits control levels and lipopolysacharide (LPS) stimulated upregulation of MMP-9 in cultured macrophages, while the class I specific inhibitor, PD106, only inhibits control. The HDAC 6 inhibitor, Tubastatin A (Tub A), partially inhibits LPS stimulated upregulation of MMP-9, but surprisingly appears to potentiate control levels of MMP-9. Immunofluorescence revealed that direct HDAC inhibition with PD106, Tub A, and TSA leads to M1 to M2 macrophage polarization and maintenance of anti-inflammatory, M2, phenotype even with LPS stimulation in culture. From these data we conclude that macrophage secretion of MMPs is an essential component in the promotion of adverse ECM remodeling post-MI. HDAC inhibition can attenuate this remodeling possibly through macrophage polarization, but HDAC inhibition mediated M2 polarization may not be sufficient to suppress MMP-9 expression.

Background: Histone deacetylases (HDACs) play an important role in the alteration of gene expression during cardiac hypertrophy and failure. Our previous study demonstrated that acetylated Nkx2.5 is associated with the Class I/II HDAC complex, HDAC5/1/2 at the Ncx1 promoter, and deacetylated Nkx2.5 is associated with the transcriptional activator and histone acetylase, p300 in a mutually exclusive manner. Inhibition of HDACs by the Class I/II inhibitor, trichostatin A (TSA) prevents deacetylation of Nkx2.5 and recruitment of p300 to the Ncx1 promoter, thereby repressing its upregulation. Objective: To assess the specific roles of HDAC1, 2 and 5 in the regulation of Ncx1 gene expression and determine the specific Nkx2.5 lysine(s) undergoing acetylation or deacetylation, which mediates Ncx1 upregulation. Results: Treatment of isolated adult cardiomyocytes with the selective class I HDAC inhibitor, BML210, prevented alpha- and beta-adrenergic stimulated upregulation of Ncx1 expression, whereas treatment with the specific class II HDAC inhibitor, dPAHA did not. Interestingly, the HDAC5 knockout prevented NCX1 upregulation after 72 hr trans-aortic constriction (TAC) in mice. In order to determine which Nkx2.5 lysine(s) is deacetylated by HDAC1/2, we performed mass spectrometry analysis. The Nkx2.5 gene contains 15 lysine moieties, and LC-MS/MS analysis demonstrates that Nkx2.5 is acetylated on two conserved lysine residues. Conclusion: Class I HDAC activity is required for Ncx1 expression but not class II HDAC activity (HDAC5). However, the loss of HDAC5 prevents Ncx1 upregulation because it may act as a scaffold to recruit the factors required for Ncx1 promoter activation. These results suggest that HDAC inhibition may represent a novel therapeutic modality for hypertrophy and heart failure.

Protection from coronary heart disease-induced damage of the myocardium during myocardial infarction (MI) injury has been a target of investigation for the development of innovative cardioprotective therapies. Histone deacetylases (HDACs) are a class of enzymes that affect the transcriptional regulation of genes during pathological conditions. We observed that class I/IIb HDAC activity was nearly 5 times greater in the 7-day post-MI LV when compared to the sham ventricles. In vitro inhibition studies indicated that the majority of increased activity was due to class I HDACs. Therefore we hypothesized that suppression of class I HDACs would prevent the pathophysiological changes occurring during MI, thus improving LV pump function in post-MI myocardium. CD-1 mice were administered with the a class I HDAC inhibitor pimelic diphenylamide (PD106) or vehicle immediately after induction of MI and the treatment continued every other day for 7 days post MI. LV end-diastolic volumes, expressed as change from pre-MI values, was significantly lower in the PD106 treated mice compared to vehicle treated mice. Further, the post-MI reduction in LV ejection faction was significantly attenuated in the PD106-treated mice compared to the MI alone group. Similarly, echo cardiac strain analysis showed improved LV strain and better coherence in contractile function among infarct and border zones in PD106 MI group compared to MI only. These unique findings demonstrate that class I HDAC inhibitors may provide a novel therapeutic means to attenuate adverse post-MI LV remodeling.

Cardiovascular diseases are one of the leading causes of morbidity and mortality in the world, underlining the need for innovative therapies and diagnosis. Recent reports have identified microRNAs (miRNAs) as central players in regulating gene expression and showed that several miRNAs are aberrantly expressed in cardiac arrhythmia, hypertrophy, fibrosis, ischemia, vascular atherosclerosis and heart failure. Gene expression is also regulated at the transcriptional level by histone deacetylases (HDACs) under basal and pathological conditions. We previously demonstrated that 1) class I and class II HDACs play an important role in the basal expression and upregulation of the sodium-calcium exchanger (Ncx1) gene in adult cardiomyocytes and pressure-overloaded ventricle and 2) treatment with class I/IIb HDAC inhibitors trichostatin (TSA) or suberoylanilide hydroxamic acid (SAHA) improved ventricular function by suppressing matrix metalloproteinases (MMPs) gene expression in myocardial infarction (MI).Therefore it is possible that protein acetylation regulates the expression of some miRNAs and we hypothesize that HDAC inhibition would attenuate the shift in expression of certain miRNAs that are aberrantly expressed post-MI. In a pilot study, ligation of the left anterior descending (LAD) coronary artery was performed to induce MI with and without SAHA treatment. Because of its misregulation and relevance in cardiac hypertrophy and MI, we examined the expression level of miR-21. qRT-PCR confirmed that miR-21 is increased by 8 fold 7 days post-MI. Interestingly, SAHA treatment significantly attenuated the abnormal expression of miR-21. To our knowledge, it is the first report of the regulation of a miRNA by HDACs in the heart. We anticipate that not only miR-21 but other miRNAs will fall under the same mechanistic control via acetylation. The miR-21 promoter contains binding sites for several transcription factors that get acetylated and we speculate that one or more HDAC mediate the expression of miR-21 by controlling the acetylation state of transcription factors interacting with the miR-21 promoter.

PURPOSE. The pathogenesis of retinal ischemia results from a series of events involving changes in gene expression and inflammatory cytokines. Protein acetylation is an essential mechanism in regulating transcriptional and inflammatory events. The purpose of this study was to investigate the neuroprotective action of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in a retinal ischemic model. METHODS. To investigate whether HDAC inhibition can reduce ischemic injury, rats were treated with TSA (2.5 mg/kg intraperitoneally) twice daily on days 0, 1, 2, and 3. Seven days after ischemic injury, morphometric and electroretinographic (ERG) analyses were used to assess retinal structure and function. Western blot and immunohistochemical analyses were used to evaluate TSA-induced changes in histone-H3 acetylation and MMP secretion. RESULTS. In vehicle-treated animals, ERG a- and b-waves from ischemic eyes were significantly reduced compared with contralateral responses. In addition, histologic examination of these eyes revealed significant degeneration of inner retinal layers. In rats treated with TSA, amplitudes of ERG a- and b-waves from ischemic eyes were significantly increased, and normal inner retina morphology was preserved. Ischemia also increased the levels of retinal TNF-alpha, which was blocked by TSA treatment. In astrocyte cultures, the addition of TNF-alpha (10 ng/mL) stimulated the secretion of MMP-1 and MMP-3, which were blocked by TSA (100 nM). CONCLUSIONS. These studies provide the first evidence that suppressing HDAC activity can protect the retina from ischemic injury. This neuroprotective response is associated with the suppression of retinal TNF-alpha expression and signaling. The use of HDAC inhibitors may provide a novel treatment for ischemic retinal injury.

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation.