HDAC class I inhibitor, Mocetinostat, reverses cardiac fibrosis in heart failure and diminishes CD90+ cardiac myofibroblast activation
© Nural-Guvener et al.; licensee BioMed Central Ltd. 2014
Received: 22 January 2014
Accepted: 2 June 2014
Published: 2 July 2014
Interstitial fibrosis and fibrotic scar formation contribute to cardiac remodeling and loss of cardiac function in myocardial infarction (MI) and heart failure. Recent studies showed that histone deacetylase (HDAC) inhibitors retard fibrosis formation in acute MI settings. However, it is unknown whether HDAC inhibition can reverse cardiac fibrosis in ischemic heart failure. In addition, specific HDAC isoforms involved in cardiac fibrosis and myofibroblast activation are not well defined. Thus, the purpose of this study is to determine the effects of selective class I HDAC inhibition on cardiac fibroblasts activation and cardiac fibrosis in a congestive heart failure (CHF) model secondary to MI.
MI was created by left anterior descending (LAD) coronary artery occlusion. Class I HDACs were selectively inhibited via Mocetinostat in CD90+ fibroblasts isolated from atrial and ventricular heart tissue in vitro. In vivo, Class I HDACs were inhibited in 3 weeks post MI rats by injecting Mocetinostat for the duration of 3 weeks. Cardiac function and heart tissue were analyzed at 6 weeks post MI.
In sham hearts, HDAC1 and HDAC2 displayed differential expression patterns where HDAC1 mainly expressed in cardiac fibroblast and HDAC2 in cardiomyocytes. On the other hand, we showed that HDAC1 and 2 were upregulated in CHF hearts, and were found to co-localize with CD90+ cardiac fibroblasts. In vivo treatment of CHF animals with Mocetinostat improved left ventricle end diastolic pressure and dp/dt max and decreased the total collagen amount. In vitro treatment of CD90+ cells with Mocetinostat reversed myofibroblast phenotype as indicated by a decrease in α-Smooth muscle actin (α-SMA), Collagen III, and Matrix metalloproteinase-2 (MMP2). Furthermore, Mocetinostat increased E-cadherin, induced β-catenin localization to the membrane, and reduced Akt/GSK3β signaling in atrial cardiac fibroblasts. In addition, Mocetinostat treatment of atrial CD90+ cells upregulated cleaved-Caspase3 and activated the p53/p21 axis.
Taken together, our results demonstrate upregulation of HDAC1 and 2 in CHF. In addition, HDAC inhibition reverses interstitial fibrosis in CHF. Possible anti-fibrotic actions of HDAC inhibition include reversal of myofibroblast activation and induction of cell cycle arrest/apoptosis.
KeywordsCongestive heart failure Myocardial Infarction Myofibroblast Mocetinostat Fibrosis HDAC Rat
Interstitial fibrosis and fibrotic scar formation contribute to cardiac remodeling and loss of cardiac function in myocardial infarction (MI) and heart failure. In response to myocardial injury, the number of fibroblasts increased by replication of resident cardiac fibroblasts, recruitment of bone marrow cells and transformation of endothelial/epicardial cells to fibroblasts. Cardiac fibroblasts are activated leading to myofibroblasts, which are contractile, invasive, and high producers of extracellular matrix (ECM) proteins [1–3]. While myofibroblasts serve an important role in wound healing and scar formation to repair areas of cardiomyocyte loss, further deposition of interstitial ECM causes myocardial stiffness and loss of ventricular function. Therefore, elucidating the mechanism(s) that regulates cardiac fibroblasts may provide a therapeutic strategy to reduce the amount of fibrosis in heart failure.
Epigenetic alterations, such as histone modifications, have been shown to be involved in tissue fibrosis in multiple organs including kidney, lung, and heart . Histone acetylation (by histone acetyl-transferases) relaxes normally tight chromatin super-coiling, enhancing accessibility of transcriptional regulatory proteins to promoter regions. On the other hand, histone deacetylases remove acetyl groups from lysine residues of histones and other proteins, which remodel chromatin, resulting in inhibition of gene expression. To date, 18 mammalian HDACs were identified and categorized into four classes. Class I HDACs (HDAC1, 2, 3, and 8) are widely expressed and have pro-hypertrophic function in heart disease. Among class I HDACs, HDAC1 and HDAC2 have been shown to play redundant roles in cardiac growth and function [4, 5]. Several studies had reported that HDAC1 and 2 have pro-fibrotic roles in renal injury disease models [6, 7]. Recently, an idiopathic pulmonary fibrosis study linked HDACs to myofibroblast differentiation and extracellular matrix deposition . Moreover, small molecule HDAC inhibitors, especially Class I and II inhibitors, were shown to effectively retard myocardial remodeling, decrease interstitial fibrosis, and improve cardiac function in pathological heart conditions [9–13]. In most of these studies, HDAC inhibitors were applied in acute MI settings rather than after development of interstitial fibrosis in CHF. Thus, here we aim to investigate whether HDAC inhibition could be effective in reversing fibrosis in chronic conditions like ischemic heart failure. Here we explore the anti-fibrotic mechanisms of Mocetinostat, a highly specific HDAC1, 2, and 3 inhibitor, in cardiac fibroblasts.
In the present study, we demonstrated that HDAC 1/2 were upregulated in CHF infarcted areas and this upregulation progressed to non-infarcted myocardium including the left atrium when CHF advanced to a de-compensated stage. Using a small molecule HDAC inhibitor, Mocetinostat, we showed that inhibition of class I HDACs reversed interstitial collagen deposition and improved heart function in CHF. Moreover, Mocetinostat promoted reversal of myofibroblasts activation in vitro. In parallel, Mocetinostat treatment activated a p53/p21 axis and Caspase-3. Thus, the present study suggests that, in advanced heart failure settings, class I HDAC inhibition can reverse fibrosis. Anti-fibrotic activity of Mocetinostat includes reversal of myofibroblast phenotype and regulation of cell proliferation/apoptosis in cardiac fibroblasts.
HDAC1 and 2 levels are elevated in CHF
First, we investigated the time course of HDAC 1 and 2 protein levels using western blot analysis at three time points following MI: at 3 days (Acute MI-AMI), 3 weeks (3w CHF) where the scar was formed and animals progressed into heart failure, and 6 weeks (6w CHF) where animals were in de-compensated heart failure  that is in junction with development of prominent fibrosis in the ventricles and atria. In AMI, both HDAC1 and HDAC2 showed a trend of increase in the infarcted myocardium (Figure 1A). At 3 weeks and 6 weeks after MI, HDAC1 and HDAC2 levels were upregulated in the infarcted LV (scar) (Figure 1B and C).
In the non-infarcted myocardium, there was a trend of increase in HDAC1/2 levels in the LV of 3 w CHF (Figure 2A). The increase in HDAC1 and 2 levels in non-infarcted LV was significant for 6w CHF (Figure 2A). Moreover, only HDAC1 was upregulated in the right ventricle (RV) at 6w CHF (Figure 2B) whereas there were no changes in HDAC2 level at 3w CHF or 6w CHF (Figure 2B). HDAC1 levels were also unaltered in the RV at 3w CHF (Figure 2B).
HDAC1 and 2 are co-localized with cardiac fibroblast in CHF
HDAC inhibition reversed myofibroblast activation in vitro
Activation of fibroblasts to myofibroblasts is a hallmark of cardiac fibrosis . The upregulation of HDAC1 and HDAC2 primarily in cardiac fibroblasts in CHF suggests a role for HDAC1 and 2 in this process. Thus, we investigated whether HDAC inhibition reduces activation of myofibroblasts in vitro. We isolated CD90+/ cKit- cardiac fibroblasts from ventricles and atria. CD90 is a surface protein expressed in cardiac fibroblasts in vivo and in vitro regardless of their activation to myofibroblasts . Flow cytometry analysis confirmed that 91% of cultured CD90+ cells co-expressed α-SMA suggesting their activation to myofibroblasts in culture. In addition, they expressed myofibroblast markers; smooth muscle embryonic myosin (SMemb) and fibronectin-EIIIA variant (Additional file 2) confirming their myofibroblast phenotype. Therefore, we concluded that cultured CD90+ cells represent a suitable model to study effects of HDAC inhibition on myofibroblasts in vitro.
To investigate effects of class I HDAC inhibition on cardiac fibroblasts, we treated atrial and ventricular CD90+ cells with Mocetinostat, a benzamide class I HDAC inhibitor (HDAC1 (IC50 = 0.15 μM), HDAC2 (IC50 = 0.29 μM), and HDAC3 (IC50 = 1.66 μM) for 7 days in culture. In atrial CD90+ cells, Mocetinostat treatment resulted in downregulation of α-SMA and upregulation of E-cadherin gene expression. Gene expressions of Collagen III and MMP2, which are markers for fibrosis, were also downregulated with Mocetinostat treatment (Figure 7A).
Similar to atrial CD90+ cells, Mocetinostat treatment of ventricular CD90+ cells resulted in downregulation of α-SMA, MMP2, and Collagen III gene expressions. In contrast to atrial CD90+ cells, level of E-cadherin experssion did not change with Mocetinostat treatment (Figure 7B).
HDAC inhibition induced E-cadherin/β-catenin expression in atrial fibroblasts
HDAC inhibition downregulates Akt signaling
To elucidate cell signaling components associated with HDAC inhibition in cardiac fibroblasts, we measured levels of GSK3β and Akt proteins and their phosphorylation status in atrial fibroblasts in vitro. Akt is an important regulator of mesenchymal cell differentiation into smooth muscle cells  and in addition have key roles in promotion of cell proliferation, survival, and motility [19, 20]. On the other hand, GSK3β is a regulator of β-catenin, where GSK3β (in unphosphorylated state) results in cytoplasmic β-catenin stabilization allowing its nuclear translocation .
Mocetinostat treatment inactivated Akt as indicated by downregulation of p-Akt levels (Figure 8D). The level of phosphorylated GSK3β (p-GSK3β) was unchanged with Mocetinostat treatment (Figure 8D). In contrast, the total level of GSK3β was reduced. Overall, the ratio of p-GSK3β to GSK3β was elevated with Mocetinostat treatment indicating less activation of GSK3β. Thus, reduction of GSK3 β levels could be implicated in upregulation of β-catenin levels with HDAC inhibition. Thus, effects of HDAC inhibition on cardiac fibroblast could be via de-activation of Akt/GSK3 β signaling.
Mocetinostat treatment elevates levels of p21/p53 and cleaved-caspase-3 in cardiac fibroblasts
Mocetinostat treatment improved cardiac function and reduced fibrosis in CHF in vivo
Mocetinostat improved left ventricular function in CHF
Heart rate (bpm)
Maximum pressure (mmHg)
End-diastolic pressure (mmHg)
Ejection fraction (%)
Cardiac output (μL/min)
LV dP/dt max (mmHg/s)
CHF + MOCE
243.7 ± 21.1
110.5 ± 12.1
9.4 ± 3.4a
42.1 ± 14.3
23,234 ± 12,996
5,573 ± 689a
235.8 ± 16.1
102.7 ± 12.3b
28.7 ± 6.2b
33.6 ± 8.1b
19,001 ± 6,082b
3,921 ± 816b
261.1 ± 39.4
122.5 ± 13.8
7.2 ± 2.3
63.0 ± 10.2
40,162 ± 16,010
6,928 ± 1,090
In addition, we examined whether Mocetinostat treatment promotes apoptosis in CHF myocardium using CardioTACS in situ apoptosis detection kit. We did not observe increased apoptosis in scar and LV of Mocetinostat-treated CHF hearts compared to untreated CHF group (Additional file 3).
In this study, we demonstrated class I HDAC isoforms HDAC1 and HDAC2 are upregulated in cardiac fibroblasts as the animals progressed through heart failure. Interestingly, inhibition of Class I HDACs with Mocetinostat reversed the cardiac fibrosis in CHF animals suggesting an association between cardiac fibrosis and HDAC1/2 upregulation. Investigation of anti-fibrotic effects of HDAC inhibition on cardiac fibroblasts suggested several mechanisms including reversal of myofibroblast phenotype to fibroblast and induction of cell cycle arrest/apoptosis.
Analysis of HDAC1 and HDAC2 expression patterns in sham hearts showed that HDAC1 is mainly expressed by fibroblasts/interstitial cells while HDAC2 is more prominent in cardiomyocytes with low levels of expression in fibroblasts/interstitial cells (Figure 3). In addition, in CHF, both HDAC1 and HDAC2 showed strong staining in fibroblasts while HDAC2 maintained its expression in cardiomyocytes. Although our conclusions were based on confocal microscopy analysis, we cannot exclude the possibility of low level of expression of HDAC1 in cardiomyocytes. Altogether, these data suggest that in addition to their roles in cardiomyocyte regulation and hypertrophy , HDAC1 and 2 are associated with regulation of cardiac fibroblast biology in heart.
We showed that class I HDAC inhibitor, Mocetinostat improved cardiac function in parameters of ventricular contractility and reduced total collagen amount in heart failure animals. Recent studies showed that HDAC inhibition retards ventricular and atrial fibrosis formation when applied immediately after injury [23–27]. Moreover, in most of these studies pan-HDAC inhibitors such as TSA (Class I and Class II HDAC inhibitor) have been administrated for systematic HDAC inhibition. Our results highlight two important findings. First, we showed that selective inhibition of class I HDACs alone is effective in reducing fibrosis in CHF. Second, we started the treatments at 3w after MI, where animals already developed cardiac fibrosis. Thus, HDAC inhibition could effectively reverse interstitial fibrosis in heart failure and improve cardiac function.
Through HDAC inhibition with Mocetinostat in vitro, we observed a reduction in α-SMA, a myofibroblast marker. We showed that Mocetinostat treatment downregulated collagen III in CD90+ cells, which is consistent with previous work where TSA blocked TGFβ-induced collagen synthesis in rat cardiac fibroblasts . Thus, our results suggest that the anti-fibrotic effect of Class I HDAC inhibition involves a reduction in myofibroblast activation and downregulation of one the most abundant ECM proteins, Collagen-III .
As existing fibroblasts differentiate into myofibroblasts, epithelial, endothelial, and smooth muscle cells can also contribute to fibrosis via EMT, EndoMT, and mechanical tension. Recent study showed that HDAC inhibition blocked TGFβ-induced EMT via upregulation of E-cadherin and downregulation of α-SMA and ECM proteins in renal injury . Consistently, we showed that Mocetinostat application induced E-cadherin expression and surface translocation of β-catenin associated with morphological transformation of atrial CD90+ cells toward epithelial-like phenotype. The decrease in cell density in Mocetinostat-treated samples could induce changes in morphology of the cells as well. However, E-cadherin upregulation in these cells suggests that changes cell morphology does not solely depend on cell density. In addition, we demonstrated that in CD90+ fibroblasts, Mocetinostat reduced phosphorylation of Akt, an important EMT/EndoMT player [29–33]. Thus, another possible anti-fibrotic effect of Class I HDAC inhibition could be reversal of EMT-induced fibrosis in cardiac fibroblasts.
Other effects of HDAC inhibitors are their ability to induce cell cycle arrest and apoptosis . Here, we showed a reduction in cell number, elevation of p21and p53 gene expression, and upregulation of Cleaved Caspase-3 protein with Mocetinostat treatment. p21 is a cyclin-dependent kinase inhibitor, which is involved in cell growth arrest , while p53 induces apoptosis. Our data are supported by the recent studies where HDAC inhibition reduced cardiac fibroblast proliferation in isoproterenol induced heart failure  and induced cell cycle arrest in cardiac fibroblast in model of angiotensin-II mediated fibrosis . On the other hand, we did not observe an increase in overall apoptosis in CHF myocardium with Mocetinostat treatment suggesting that systemic Mocetinostat administration does not induce excessive cell death in myocardium. However, rates of apoptosis in different cells types needs to be examined to determine whether Mocetinostat induces apoptosis selectively in certain cell types, that is, fibroblasts in vivo. Altogether, these data suggest that regulation of fibroblast proliferation and apoptosis could be another possible mechanism of HDAC inhibition in control of fibrosis.
One of the limitations in our study is that we isolated cardiac fibroblast from atrial explants and ventricular tissue based on CD90 (Thy1) surface marker. Even though the majority of cardiac fibroblasts express CD90, this marker is not fibroblast-exclusive. Thus, other cells types such as blood originated cells could be included as well. However, a fibroblast specific marker representing majority of the fibroblasts in the heart is not currently available. In addition, CD90+ cells do not represent entire fibroblast population in heart; this marker is rather expressed on a subset of fibroblasts. Therefore, we used another marker, Vimentin (also not fibroblast specific), to confirm expression of HDAC1 and HDAC2 in cardiac fibroblasts. Thus, expression of HDAC1/2 in both CD90 and Vimentin + cells suggests that cardiac fibroblasts express HDAC1/2 in CHF hearts.
Our results show that cardiac fibroblasts activated HDAC1 and 2 in infarcted/non-infarcted myocardium and atrium in CHF. In addition, inhibition of Class I HDACs in cardiac fibroblasts reduces myofibroblasts activation. This knowledge is important to further understand more fibroblast-specific biology HDAC inhibition and develop specific therapeutic agents to reduce fibrosis.
This study was performed in an accredited facility by the American Association for Accreditation of Laboratory Animal Care and was approved by the Institutional Animal Care and Use Committee at Banner Sun Health Research Institute. Animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).
Myocardial infarction and treatments
MI was created by ligation of the left coronary artery as previously performed by our laboratory . In brief, rats were anesthetized using 1 mL/kg of an MI cocktail composed of ketamine (50 mg/mL), xylazine (15 mg/mL), acepromazine (2 mg/mL), and atropine (1 mg/mL). Animals were intubated and ventilated using a small animal ventilator (Harvard Apparatus). A left thoracotomy was performed via the third intercostal rib, and the left coronary artery was ligated. In sham operated animals, the chest was closed without ligation of the artery. The rats were sacrificed at 3 days (Acute MI-AMI), 3 weeks, or 6 weeks (congestive heart failure (CHF)) post surgery. At 3 weeks or 6 weeks after successful infarction, rats exhibited CHF indicated by elevation of left ventricular end-diastolic pressure (LVEDP), LV remodeling, and fluid accumulation in the chest [36, 37]. In addition, prominent fibrosis in the ventricles and left atrium was evident at 6 weeks post surgery. Closed-chest in-vivo cardiac function was measured using a Millar pressure conductance catheter system (Millar instruments, Houston, TX, USA) as previously described . A transverse cut was made to postmortem hearts to assess scar size and LV remodeling. Only animals with visible scar, LV remodeling, and LVEDP greater than 20 mmHg were included in the 3-week or 6-week CHF group.
Three weeks post surgery, animals in the CHF group were further divided into two groups; the first group received 10 mg/kg Mocetinostat dissolved in 0.1 N HCl PBS solution daily for the duration of 3 weeks (n = 8). The second group received only vehicle for the same duration (n = 6). In addition, animals that underwent sham surgery were injected with vehicle only and served as control group (n = 5).
Cell isolation and culture
Atrial explant outgrowth was generated as previously described [38, 39]. Briefly, tissue was cut into 1 to 2 mm3 pieces and digested with 0.2% trypsin (Life Technologies, Carlsbad, CA, USA) and 0.1% collagenase IV (Life Technologies) for a total of 10 min. The remaining tissue fragments were cultured as ‘explants’ in explants medium (CEM), which was composed of IMDM supplemented with 10% fetal bovine serum (FBS, Lonza), 100 U/mL penicillin G, 100 μg/mL streptomycin, and 2 mmol/L L-glutamine (Sigma-Aldrich). After 21 days in culture, cells were collected by trypsinization. CD90+ cells were separated from the cell outgrowths using magnetic beads (MACS, Miltenyi Biotec) according to manufacturer protocol and analyzed by flow cytometry for purity assessment. CD90 cells from ventricles were isolated by enzymatic digestion with a Dispase II (2.4 mg/mL, Roche)/Collagenase II (0.05 mg/mL, Gibco) mix in PBS. Ventricles were cut into small pieces and digested for 10 min at 37°C with agitation. The supernatant spun at 1,200 g for 7 min to collect dissociated cells. The last two steps were repeated five times and cells were pooled. Dissociated cells were plated and media was changed after 2 h to discard non-attached cells and debris. Then, attached cells were trypsinized and collected for CD90 isolation as described above.
Isolated atrial or ventricular CD90+ cells were seeded on six well plates at a density of 0.2 × 106 cells/well in CEM. Cells were treated with 1 μM and 2 μM of Mocetinostat (SelleckChem). Control cells were treated with 0.005% DMSO.
Heart tissue was embedded in tissue freezing media (Triangle Biomedical Science) snap-frozen in liquid nitrogen and sectioned in the coronal plane using Leica CM1900 cryostat (Leica Microsystems, Bannockburn, IL, USA). Coronal and axial tissue sections (5 to 7 μm thickness) were mounted on positively charged glass slides and fixed/permeabilized in a 1:1 mixture of acetone and ethanol. Sections were blocked with 3% BSA in PBS and incubated with primary antibodies against α-MHC (Abcam), HDAC1, HDAC2, α-SMA (Abcam), CD90 (BD), and Vimentin (Abcam). Specific staining was visualized using corresponding secondary antibodies conjugated with Alexa 488 or 568 (Molecular Probes). Nuclei were stained with DAPI, 4′ 6-diamidino-2-phenylindole (Life Technologies).
Cells were fixed/permeabilized in a 1:1 mixture of acetone and ethanol. Cells were blocked with 3% BSA in PBS and stained with primary antibodies. Corresponding secondary antibodies were conjugated with Alexa-488 or Alexa 568 (Molecular Probes). Nuclei were stained with DAPI, 4′ 6-diamidino-2-phenylindole (Life Technologies).
Fluorescent images were captured using Leica TCS SPE confocal system configured with Leica DM 2500 microscope. Excitation maximums of 488 nm, 532 nm, and 405 nm, were used for image acquisition. Images were processed using LAS AF software (Leica Microsystems).
Scar size assessment and collagen assay
Heart tissue was processed as described above and sections of 7 or 20 μm were mounted on positively charged glass slides. Routine staining was performed with the Hematoxylin and Eosin kit (H&E, Sigma) according to manufacturer instructions. For scar size assessment, sections were stained with Masson’s Trichrome kit (Sigma-Aldrich) according to the manufacturer’s protocol. Transmitted light images of heart sections were processed using DP2-BSW software (Olympus Corp). Scar percentage was calculated as a ratio of collagen enriched scar area (blue staining) to the whole left ventricle area (red staining).
Total collagen amount was measured in 20 μm cross-sections using Sirus Red Fast Green Collagen staining kit (Chondrex) according to manufacturer instructions. Absorbance of collagen (540 nm) and non-collagenous protein (605 nm) was assessed using BioTek Synergy HT Microplate Reader. Collagen percentage was calculated as a ratio of OD540collagen to OD605non-collagenous protein.
RNA isolation and quantitative real-time RT-PCR
Total RNA was extracted from CD90+ cells using PureLink™ RNA Mini Kit (Life Technologies) according to the manufacturer’s protocol. RNA was then quantified with the Quanti-iT™ RiboGreen® RNA Assay Kit, and assessed using BioTek Synergy HT Microplate Reader (excitation/emission 480 nm/520 nm). Total RNA (200 ng) was reverse transcribed with QuantiTect Reverse Transcription kit (Qiagen). Real-time RT-PCR was conducted using the Rower SYBR Green Master Mix (Applied Biosystems) on a StepOnePlus Real-time PCR System (Applied Biosystems). Specific primers were synthesized by Life Technologies (sequences are available upon request). CYP A was used as a reference gene. Data analysis was performed on StepOne software version 2.1 (Applied Biosystems) using the comparative Ct (ΔΔCt) quantitation method.
Scar and LV tissue were dissected from AMI, CHF, and sham hearts, respectively. Dissected tissues and isolated cells were homogenized in lysis buffer (50 mM Tris- HCl pH7.5, 150 mM NaCl, 0.5% NP-40 (Sigma), 0.5% Triton-X (Sigma), 1 mM EDTA (Sigma), and complete mini protease inhibitor (Roche). Protein concentrations were determined by BCA assay (Thermo Scientific). Typically, 40 μg of protein was loaded on 4% to 12% Tris-Bis gels (Life Technologies), separated in MOPS running buffer, and transferred to a PVDF membrane (Millipore). After blocking with 5% non-fat dry milk in 1× TBS, membranes were probed with HDAC1, HDAC2 (Abcam, 1:1,000), α-SMA (Sigma, 1:5,000), E-cadherin, GSK3β (Santa Cruz, 1:500), p-GSK3β, Cleaved Caspase 3, p-Akt, Akt (Cell Signaling, 1:1,000), β-Catenin active (Millipore,1:1,000), and β-Actin (Sigma, 1:50,000) in TBS with 5% milk overnight at 4°C. Following three washes in TBS, membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz, 1:50,000) for 1 h at room temperature. An ECL (Millipore) system was used for detection of the bands and exposed to X-ray film (Thermo Scientific) in a dark room. Densitometry analysis was performed with Alpha Ease FC software.
All datasets are represented as mean ± S.E. Significance (P <0.05) was determined using Student’s t-test. Statistical analysis was conducted using Sigma Stat 3.5 software.
Congestive heart failure
Epithelial/endothelial mesenchymal transition
α-smooth muscle actin
Myosin heavy chain.
We thank Lorraine Feehery for her contribution in animal surgeries and cardiac function analysis and proofreading the manuscript. The study was supported by the NIH RO1 AG027263 grant and Sun Health Foundation.
- Porter KE, Turner NA: Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol Ther. 2009, 132: 255-278.View ArticleGoogle Scholar
- Squires CE, Escobar GP, Payne JF, Leonardi RA, Goshorn DK, Sheats NJ, Mains IM, Mingoia JT, Flack EC, Lindsey ML: Altered fibroblast function following myocardial infarction. J Mol Cell Cardiol. 2005, 39: 699-707.View ArticlePubMedGoogle Scholar
- van den Borne SW, Diez J, Blankesteijn WM, Verjans J, Hofstra L, Narula J: Myocardial remodeling after infarction: the role of myofibroblasts. Nat Rev Cardiol. 2010, 7: 30-37.View ArticlePubMedGoogle Scholar
- Xie M, Hill JA: HDAC-dependent ventricular remodeling. Trends Cardiovasc Med. 2013, 23: 229-235.PubMed CentralView ArticlePubMedGoogle Scholar
- Montgomery RL, Davis CA, Potthoff MJ, Haberland M, Fielitz J, Qi X, Hill JA, Richardson JA, Olson EN: Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis, growth, and contractility. Genes Dev. 2007, 21: 1790-1802.PubMed CentralView ArticlePubMedGoogle Scholar
- Marumo T, Hishikawa K, Yoshikawa M, Hirahashi J, Kawachi S, Fujita T: Histone deacetylase modulates the proinflammatory and -fibrotic changes in tubulointerstitial injury. Am J Physiol Renal Physiol. 2010, 298: F133-F141.View ArticlePubMedGoogle Scholar
- Noh H, Oh EY, Seo JY, Yu MR, Kim YO, Ha H, Lee HB: Histone deacetylase-2 is a key regulator of diabetes- and transforming growth factor-beta1-induced renal injury. Am J Physiol Renal Physiol. 2009, 297: F729-F739.View ArticlePubMedGoogle Scholar
- Guo W, Shan B, Klingsberg RC, Qin X, Lasky JA: Abrogation of TGF-beta1-induced fibroblast-myofibroblast differentiation by histone deacetylase inhibition. Am J Physiol Lung Cell Mol Physiol. 2009, 297: L864-L870.PubMed CentralView ArticlePubMedGoogle Scholar
- Kee HJ, Sohn IS, Nam KI, Park JE, Qian YR, Yin Z, Ahn Y, Jeong MH, Bang YJ, Kim N, Kim JK, Kim KK, Epstein JA, Kook H: Inhibition of histone deacetylation blocks cardiac hypertrophy induced by angiotensin II infusion and aortic banding. Circulation. 2006, 113: 51-59.View ArticlePubMedGoogle Scholar
- Kook H, Lepore JJ, Gitler AD, Lu MM, Wing-Man Yung W, Mackay J, Zhou R, Ferrari V, Gruber P, Epstein JA: Cardiac hypertrophy and histone deacetylase-dependent transcriptional repression mediated by the atypical homeodomain protein Hop. J Clin Invest. 2003, 112: 863-871.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee TM, Lin MS, Chang NC: Inhibition of histone deacetylase on ventricular remodeling in infarcted rats. Am J Physiol Heart Circ Physiol. 2007, 293: H968-H977.View ArticlePubMedGoogle Scholar
- Zhang L, Chen B, Zhao Y, Dubielecka PM, Wei L, Qin GJ, Chin YE, Wang Y, Zhao TC: Inhibition of histone deacetylase-induced myocardial repair is mediated by c-kit in infarcted hearts. J Biol Chem. 2012, 287: 39338-39348.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang L, Qin X, Zhao Y, Fast L, Zhuang S, Liu P, Cheng G, Zhao TC: Inhibition of histone deacetylases preserves myocardial performance and prevents cardiac remodeling through stimulation of endogenous angiomyogenesis. J Pharmacol Exp Ther. 2012, 341: 285-293.PubMed CentralView ArticlePubMedGoogle Scholar
- Francis J, Weiss RM, Wei SG, Johnson AK, Felder RB: Progression of heart failure after myocardial infarction in the rat. Am J Physiol Regul Integr Comp Physiol. 2001, 281: R1734-R1745.PubMedGoogle Scholar
- Baudino TA, Carver W, Giles W, Borg TK: Cardiac fibroblasts: friend or foe?. Am J Physiol Heart Circ Physiol. 2006, 291: H1015-H1026.View ArticlePubMedGoogle Scholar
- Hudon-David F, Bouzeghrane F, Couture P, Thibault G: Thy-1 expression by cardiac fibroblasts: lack of association with myofibroblast contractile markers. J Mol Cell Cardiol. 2007, 42: 991-1000.View ArticlePubMedGoogle Scholar
- Orsulic S, Huber O, Aberle H, Arnold S, Kemler R: E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. J Cell Sci. 1999, 112: 1237-1245.PubMedGoogle Scholar
- Lien SC, Usami S, Chien S, Chiu JJ: Phosphatidylinositol 3-kinase/Akt pathway is involved in transforming growth factor-beta1-induced phenotypic modulation of 10T1/2 cells to smooth muscle cells. Cell Signal. 2006, 18: 1270-1278.View ArticlePubMedGoogle Scholar
- Willems L, Tamburini J, Chapuis N, Lacombe C, Mayeux P, Bouscary D: PI3K and mTOR signaling pathways in cancer: new data on targeted therapies. Curr Oncol Rep. 2012, 14: 129-138.View ArticlePubMedGoogle Scholar
- Kim EK, Choi EJ: Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta. 2010, 1802: 396-405.View ArticlePubMedGoogle Scholar
- Gosens R, Meurs H, Schmidt M: The GSK-3/beta-catenin-signalling axis in smooth muscle and its relationship with remodelling. Naunyn Schmiedebergs Arch Pharmacol. 2008, 378: 185-191.PubMed CentralView ArticlePubMedGoogle Scholar
- Hewitson R, Dargan J, Collis D, Green A, Moorjani N, Ohri S, Townsend PA: Heart failure: the pivotal role of histone deacetylases. Int J Biochem Cell Biol. 2013, 45: 448-453.View ArticlePubMedGoogle Scholar
- Iyer A, Fenning A, Lim J, Le GT, Reid RC, Halili MA, Fairlie DP, Brown L: Antifibrotic activity of an inhibitor of histone deacetylases in DOCA-salt hypertensive rats. Br J Pharmacol. 2010, 159: 1408-1417.PubMed CentralView ArticlePubMedGoogle Scholar
- Kao YH, Liou JP, Chung CC, Lien GS, Kuo CC, Chen SA, Chen YJ: Histone deacetylase inhibition improved cardiac functions with direct antifibrotic activity in heart failure. Int J Cardiol. 2013, 168: 4178-4183.View ArticlePubMedGoogle Scholar
- Kong Y, Tannous P, Lu G, Berenji K, Rothermel BA, Olson EN, Hill JA: Suppression of class I and II histone deacetylases blunts pressure-overload cardiac hypertrophy. Circulation. 2006, 113: 2579-2588.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu F, Levin MD, Petrenko NB, Lu MM, Wang T, Yuan LJ, Stout AL, Epstein JA, Patel VV: Histone-deacetylase inhibition reverses atrial arrhythmia inducibility and fibrosis in cardiac hypertrophy independent of angiotensin. J Mol Cell Cardiol. 2008, 45: 715-723.PubMed CentralView ArticlePubMedGoogle Scholar
- Williams SM, Golden-Mason L, Ferguson BS, Douglas KB, Cavasin MA, Demos-Davies K, Yeager ME, Stenmark KR, McKinsey TA: Class I HDACs regulate angiotensin II-dependent cardiac fibrosis via fibroblasts and circulating fibrocytes. J Mol Cell Cardiol. 2013, 67: 112-125.PubMed CentralView ArticlePubMedGoogle Scholar
- Weber KT: Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol. 1989, 13: 1637-1652.View ArticlePubMedGoogle Scholar
- Julien S, Puig I, Caretti E, Bonaventure J, Nelles L, van Roy F, Dargemont C, de Herreros AG, Bellacosa A, Larue L: Activation of NF-kappaB by Akt upregulates Snail expression and induces epithelium mesenchyme transition. Oncogene. 2007, 26: 7445-7456.View ArticlePubMedGoogle Scholar
- Kattla JJ, Carew RM, Heljic M, Godson C, Brazil DP: Protein kinase B/Akt activity is involved in renal TGF-beta1-driven epithelial-mesenchymal transition in vitro and in vivo. Am J Physiol Renal Physiol. 2008, 295: F215-F225.PubMed CentralView ArticlePubMedGoogle Scholar
- Kumarswamy R, Volkmann I, Jazbutyte V, Dangwal S, Park DH, Thum T: Transforming growth factor-beta-induced endothelial-to-mesenchymal transition is partly mediated by microRNA-21. Arterioscler Thromb Vasc Biol. 2012, 32: 361-369.View ArticlePubMedGoogle Scholar
- Meadows KN, Iyer S, Stevens MV, Wang D, Shechter S, Perruzzi C, Camenisch TD, Benjamin LE: Akt promotes endocardial-mesenchyme transition. J Angiogenes Res. 2009, 1: 2.PubMed CentralView ArticlePubMedGoogle Scholar
- Widyantoro B, Emoto N, Nakayama K, Anggrahini DW, Adiarto S, Iwasa N, Yagi K, Miyagawa K, Rikitake Y, Suzuki T, Kisanuki YY, Yanagisawa M, Hirata K: Endothelial cell-derived endothelin-1 promotes cardiac fibrosis in diabetic hearts through stimulation of endothelial-to-mesenchymal transition. Circulation. 2010, 121: 2407-2418.View ArticlePubMedGoogle Scholar
- Condorelli F, Gnemmi I, Vallario A, Genazzani AA, Canonico PL: Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells. Br J Pharmacol. 2008, 153: 657-668.PubMed CentralView ArticlePubMedGoogle Scholar
- Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D: p21 is a universal inhibitor of cyclin kinases. Nature. 1993, 366: 701-704.View ArticlePubMedGoogle Scholar
- Gaballa MA, Raya TE, Goldman S: Large artery remodeling after myocardial infarction. Am J Physiol. 1995, 268: H2092-H2103.PubMedGoogle Scholar
- Gaballa MA, Goldman S: Gene transfer of endothelial nitric oxide isoform decreases rat hindlimb vascular resistance in vivo. Hum Gene Ther. 2000, 11: 1637-1646.View ArticlePubMedGoogle Scholar
- Zakharova L, Mastroeni D, Mutlu N, Molina M, Goldman S, Diethrich E, Gaballa MA: Transplantation of cardiac progenitor cell sheet onto infarcted heart promotes cardiogenesis and improves function. Cardiovasc Res. 2010, 87: 40-49.PubMed CentralView ArticlePubMedGoogle Scholar
- Messina E, De Angelis L, Frati G, Morrone S, Chimenti S, Fiordaliso F, Salio M, Battaglia M, Latronico MV, Coletta M, Vivarelli E, Frati L, Cossu G, Giacomello A: Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res. 2004, 95: 911-921.View ArticlePubMedGoogle Scholar
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