Abstract
Rationale: Inhibitors of histone deacetylases (HDACs) reduce pressure-overload–induced left ventricular hypertrophy and dysfunction, but their effects on right ventricular (RV) adaptation to pressure overload are unknown.
Objectives: Determine the effect of the broad-spectrum HDAC inhibitors trichostatin A (TSA) and valproic acid (VPA) on RV function and remodeling after pulmonary artery banding (PAB) in rats.
Methods: Chronic progressive RV pressure-overload was induced in rats by PAB. After establishment of adaptive RV hypertrophy 4 weeks after surgery, rats were treated for 2 weeks with vehicle, TSA, or VPA. RV function and remodeling were determined using echocardiography, invasive hemodynamic measurements, immunohistochemistry, and molecular analyses after 2 weeks of HDAC inhibition. The effects of TSA were determined on the expression of proangiogenic and prohypertrophic genes in human myocardial fibroblasts and microvascular endothelial cells.
Measurements and Main Results: TSA treatment did not prevent the development of RV hypertrophy and was associated with RV dysfunction, capillary rarefaction, fibrosis, and increased rates of myocardial cell death. Similar results were obtained with the structurally unrelated HDAC inhibitor VPA. With TSA treatment, a reduction was found in expression of vascular endothelial growth factor and angiopoietin-1, which proteins are involved in vascular adaptation to pressure-overload. TSA dose-dependently suppressed vascular endothelial growth factor, endothelial nitric oxide synthase, and angiopoietin-1 expression in cultured myocardial endothelial cells, which effects were mimicked by selective gene silencing of several class I and II HDACs.
Conclusions: HDAC inhibition is associated with dysfunction and worsened remodeling of the pressure-overloaded RV. The detrimental effects of HDAC inhibition on the pressure-overloaded RV may come about via antiangiogenic or proapoptotic effects.
Histone deacetylase inhibition has cardioprotective effects in experimental left heart failure, but its effects on right heart adaptation to pressure overload are unknown.
Despite their positive effects in the pressure overloaded left heart, histone deacetylase inhibitors worsen right heart dysfunction and remodeling after pulmonary artery banding in rats, which may be related to suppression of angiogenesis.
Whereas the pathobiology of left-sided heart failure has been systematically explored, very little is known about the cellular and molecular mechanisms that determine the development of right ventricular (RV) failure (10). For example, it is unclear whether a hypertrophic response is of comparable importance in RV and LV adaptation to pressure overload. Under normal conditions, the RV wall thickness is only one fifth of that of the LV; the stress imposed on the RV in patients with pulmonary arterial hypertension (PAH; with doubling or tripling of the afterload) may require for chronic adaptation a considerably larger degree of hypertrophy than the stress imposed by systemic hypertension or, experimentally, after TAC (∼a 50% increase in afterload). Importantly, it was recently shown by Kreymborg and coworkers (11) that the transcriptional control of the pressure-overloaded RV is different from that of the pressure-overloaded LV. At present, there are no known interventions that specifically support the adapting RV in situations of PAH or in pulmonary hypertension related to left-heart disease. In both situations, the development of RV failure is an important determinant of survival (12, 13). The functional, structural, and developmental differences that exist between the RV and LV raise the question whether a treatment that effectively prevents the progression of LV failure is of benefit in RV failure. We have recently shown that adrenergic receptor blockade has cardioprotective effects in the setting of experimental PAH in rats (14). Here we show that altering the RV response to established pressure overload with the broad-spectrum HDAC inhibitors trichostatin A (TSA) and valproic acid (VPA), an intervention that is known to have beneficial effects on the pressure-overloaded LV, results in RV failure in rats subjected to pulmonary arterial banding (PAB). TSA treatment after PAB and established RV hypertrophy did not decrease RV hypertrophy, did not prevent fetal gene reactivation, and caused maladaptive fibrotic RV remodeling. The induction of RV failure by HDAC inhibitors after the compensatory adaptation to pressure overload was associated with decreased expression of the angiogenesis factors vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang-1) in the RV myocardium and likewise in cultured cardiac microvascular endothelial cells. Some of the results of the studies in this manuscript have been previously reported in the form of an abstract (15).
Surgical banding of the pulmonary artery was performed in male Sprague-Dawley rats as described previously (16). Via a left thoracotomy in rats weighing 180–200 g, a silk suture was tied tightly around an 18-gauge needle alongside the pulmonary artery. After subsequent rapid removal of the needle, a fixed constricted opening was created in the lumen equal to the diameter of the needle. Whereas the initial constriction was relatively mild, the combination of a fixed banding around the pulmonary artery and animal growth resulted in a progressive increase in RV systolic pressure and a pressure gradient of about 50 mm Hg after 6 weeks (see online supplement), yet as reported previously by us (16) and others (17, 18) does not result in RV failure. TSA treatment (450 μg/kg intraperitoneally, five times per week) was initiated 4 weeks after surgery, at a time when a robust RV hypertrophy had been established, until the day of tissue harvest. The investigation conforms to 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) and all protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee (protocol AM10157).
Doppler echocardiography was performed using the Vevo770 imaging system (VisualSonics, Toronto, ON, Canada) 6 weeks after PAB and before invasive hemodynamic assessments. Light anesthesia with ketamine and xylazine was used to obtain two-dimensional, M-mode, and Doppler imaging in both long-axis (four-chamber) and short-axis views, using a 30-MHz probe. Measurements were made of the RV inner diameter in diastole (long axis); tricuspid annular plane systolic excursion (long axis); RV free wall thickness in diastole and systole (short axis); and septum thickness in diastole and systole (short axis).
Six weeks after surgical banding of the pulmonary artery and 2 weeks after the first dose of TSA, hemodynamic measurements were made using a 4.5-mm conductance catheter (Millar Instruments, Houston, TX) and the Powerlab data acquisition system (AD Instruments, Colorado Springs, CO). The rats were anesthetized with isoflurane, intubated, and placed in a supine position. After a median sternotomy the RV outflow tract was punctured with a 23-gauge needle and the catheter was introduced antegrade to measure RV and pulmonary artery pressures. In separate groups, using the same anesthesia, intubation, and catheterization techniques, cardiac output was measured by thermodilution. Saline (± 12°C) was injected via the jugular catheter (advanced into the right atrium) and the change in temperature was measured in the aorta using a thermocouple (advanced via the carotid catheter). Data analysis was performed using GraphPad and PVAN software (AD Instruments).
Rat RV, LV, and lung were snap frozen in liquid nitrogen. The FastRNA Pro Green Kit (MPBio, Santa Ana, CA) was used to isolate total RNA from heart tissue. Using the FastPrep 24-instrument (MPBio), 25 mg of tissue was homogenized by Lysing Matrix D (MPBio) in impact-resistant 2-ml tubes. Total RNA released into the proprietary, protective RNApro Solution (MPBio) was extracted with chloroform and precipitated using ethanol. Total RNA (1 μg) was reverse transcribed into complimentary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). First strand cDNA was diluted and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) performed using Power SYBR Green PCR Master Mix (Applied Biosystems) along with murine-specific primers. Cycling parameters were as follows: 95°C, 10 minutes and 45 cycles of 95°C, 15 seconds, 60°C, 1 minute. A dissociation profile was generated after each run to verify specificity of amplification. All PCR assays were performed in triplicate. No template controls and no reverse transcriptase controls were included. Automated gene expression analysis was performed using the Comparative Quantitation module of MxPro QPCR Software (Stratagene, Santa Clara, CA) to compare the levels of a target gene in test samples relative to a sample of reference (calibrator from untreated cells).
Western blots were performed using standard procedures and antibodies commercially available from Santa Cruz Biotechnology (Santa Cruz, CA) (VEGF, #sc-152; collagen I-A1, #sc-25974; Akt, #sc-8312; P-Akt, #sc-7985; Ang1, #sc-6320) and Cell Signaling (Danvers, MA) (cleaved caspase-3, #9661; HIF-1α, #3716).
Masson trichrome stain was used to assess the degree of fibrosis in cardiac sections. Fibrosis was quantified on digitized images, on which blue-stained tissue areas are expressed as percentage of the total surface area. Anti-CD31 and caveolin-1 antibodies were used to stain and quantify the vascular density in the RV in tissue sections with cardiomyocytes that were cut longitudinally. Cell death within the RV myocardium was identified with terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (Tunel stain; DNA fragmentation, Apoptag, Chemicon, CA). The number of Tunel-positive cardiomyocytes was divided by the total number of cardiomyocytes per field at ×400 under a light microscope.
Human cardiac microvascular endothelial cells (HCMVECs) and endothelial growth medium were purchased from Lonza (Walkersville, MD). Human cardiac fibroblasts (HCFs) and fibroblast growth medium were purchased from Cell Applications (San Diego, CA). The cells were cultured in 175-cm2 tissue culture flasks in a cell-culture incubator (37°C, 5% CO2, and 95% air) and used at the sixth passage (HCMVECs) or fourth passage (HCFs) after trypsinization in all the experiments. HCMVECs and HCFs were seeded in six-well culture plate or 10-cm culture dish and cultured until reaching to the confluent. After reaching to the confluent, mediums were changed and various concentrations of TSA (0.01–10 μM) were added to the dishes and cultured for 24 hours. After incubation, the cells were harvested and used for RT-PCR and Western blot analysis. To obtain more specific information regarding the role of individual HDACs in angiogenic gene expression, HCMVECs were transfected with small interfering RNAs (siRNAs) specific for human HDAC1–7 (Invitrogen, Carlsbad, CA) using previously published protocols (19). The efficiency of gene knock-down was verified by qPCR, showing that siRNA transfection resulted in at least 80% reduction HDAC1–7 mRNA expression. The following sequences were used: control siRNA sense, CCUAGAACCUAAGACCCUU, antisense, AAGGGUCUUAGGUUCUAGG; HDAC1 siRNA sense, GCUCCAUCCGUCCAGAUAA, antisense, UUAUCUGGACGGAUGGAGC; HDAC2 siRNA sense, CCAAUG AGUUGCCAUAUAA, antisense, UUAUAUGGCAACUCAUUGG; HDAC3 siRNA sense, CCAAGAGUCUUAAUGCCUU, antisense, AAGGCAUUAAGACUCUUGG; HDAC4 siRNA sense, CCACGAGCACAUCAAGCAA, antisense, UUGCUUGAUGUGCUCGUGG; HDAC5 siRNA sense, GGACUUCUCUGCACAGCAU, antisense, AUGCUGUGCAGAGAAGUCC; HDAC6 siRNA sense, GCAAUGGAAGAAGACCUAA, antisense, UUAGGUCUUCUUCCAUUGC; HDAC7 siRNA sense, GCACCCAGCAAACCUUCUA, antisense, UAGAAGGUUUGCUGGGUGC.
Differences between groups were assessed with analysis of variance (parametric) and Kruskall-Wallis (nonparametric) tests; Bonferroni (parametric) and Dunn (nonparametric) post hoc tests were used to assess for significant differences between pairs of groups. P values less than 0.05 were considered significant. For reasons of clarity, all data are reported as means ± SEM, unless specified otherwise, even if the differences between groups were tested with a nonparametric test that makes no use of means and standard deviations. Six to eight rats were used per group, unless specified otherwise.
Control animals treated with TSA had a normal increase in body weight with aging, but exhibited a mild degree of RV hypertrophy without signs of RV failure (Figures 1 and 2). TSA-treated rats also showed some degree of lung emphysema. TSA treatment of PAB rats was associated with a trend toward lower RV systolic pressures. RV function was maintained after PAB in vehicle-treated rats, as we and others have shown previously (16–18). However, PAB rats treated with TSA showed a decreased cardiac output and overt signs of RV failure on cardiac ultrasound (pericardial fluid, systolic paradox movement of the interventricular septum, and increased RV dilatation) (Figure 2). In contrast to the data obtained with the TAC model, TSA treatment did not decrease the degree of RV hypertrophy (Figure 1) in response to pressure overload. Adaptive myocardial hypertrophy (such as occurs with exercise) is characterized by increased signaling to cardiomyocytes via insulin-like growth factor (IGF)-1, and IGF-1 seems to be at least in part derived from cardiac fibroblasts (20). IGF-1 stimulation of cardiomyocytes is followed by activation (phosphorylation) of Akt, a key progrowth and prosurvival factor in many tissues including the heart (10). In addition to the effects of IGF-1 signaling on Akt, Akt phosphorylation is affected by many other growth factors. Whereas RV IGF-1 mRNA expression was increased after PAB in vehicle-treated rats (without significant changes in the IGF-1 receptor, data not shown), this response was absent in TSA-treated PAB rats (Figure 3). pAkt/Akt ratios were lower in TSA-treated than vehicle-treated PAB rats. The structurally unrelated broad-spectrum HDAC inhibitor VPA had similar but less severe effects on RV function and histology in PAB rats as TSA (see online supplement).
Figure 1. Effects of pulmonary artery banding (PAB) and trichostatin A (TSA) treatment on hemodynamics and hypertrophy. PAB leads to an increase in right ventricular systolic pressure (RVSP) (A), a predictable degree of RV hypertrophy for a given degree of pressure overload (B, C; triangles for vehicle-treated and circles for TSA-treated PAB rats). TSA treatment leads to a small degree of RV hypertrophy in control rats and to disproportional RV hypertrophy (B and C for whole RV weights and E and F for cardiomyocyte cross-sections) in PAB rats. (D) There were no differences between groups in left ventricular (LV) plus septal (S) weights. BW = body weight.
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Figure 2. (A) Cardiac ultrasound shows that pulmonary artery banding (PAB) is associated with an increased right ventricular inner diameter in diastole (RVID) compared with control rats and a maintained geometry of the heart. TSA = trichostatin A. (B) TSA treatment has no effects on RV function of normal rats, but is associated with exaggerated rotation of the heart, RV dilatation, flattening of the interventricular septum (dotted line), and a decreased tricuspid annular plane systolic excursion (TAPSE) in PAB rats. (C) Cardiac output measured by thermodilution was maintained after PAB, but decreased with TSA treatment in PAB animals. Average heart rates in all groups were the same.
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Figure 3. Pulmonary artery banding (PAB) leads to increased gene transcription of insulin-like growth factor (IGF)-1 (A) and activation (phosphorylation) of Akt (B, C; note different order of experimental conditions in Western blot). Trichostatin A (TSA) treatment is associated with increased expression of IGF-1 and Akt phosphorylation in control rats, but paradoxically with repression of IGF-1 and Akt phosphorylation in PAB rats.
[More] [Minimize]Cardiac fibrosis (21, 22) and capillary rarefaction (23) can contribute to the development of LV failure in response to pressure overload. Both histologic features are present in the RV of rats with severe angioproliferative pulmonary hypertension, but not after PAB (16). TSA treatment increased the degree of fibrosis assessed in trichrome-stained RV tissue sections in PAB, but not in control rats (Figures 4A–4E). Similarly, TSA treatment induced significant capillary rarefaction after PAB but did not induce rarefaction in control rats (Figures 4F–4J) (see Figure E2 in the online supplement). Tunel staining showed no or only occasional cell death in the RV of control rats, TSA-treated nonbanded rats, or vehicle-treated PAB rats. In contrast, TSA treatment of PAB rats was associated with increased rates of cell death in the RV (Figures 4K–4O). The increases in fibrosis and rates of cell death in the RVs of TSA-treated PAB rats were paralleled by an increased protein expression of collagen 1A1 and activated caspase 3 (Figure 5). Capillary rarefaction in TSA-treated PAB rats was associated with decreased protein and mRNA expression of VEGF and Ang-1 (Figure 5; no significant changes were observed in VEGF-R1 and VEGF-R2 mRNA expression, data not shown). Importantly, the decreased VEGF gene expression after TSA treatment was associated with increased nuclear HIF-1α expression both in controls and in PAB rats (see online supplement), indicating a transcriptional uncoupling of HIF-1α from the target gene VEGF.
Figure 4. Right ventricular (RV) capillary density (A–D) (capillaries stained with anti–caveolin-1 antibodies) is not affected by either pulmonary artery banding (PAB) or trichostatin A (TSA) alone, but is reduced in TSA-treated PAB rats (quantification in E). Similarly, PAB and TSA alone are not associated with RV fibrosis (F–J; trichrome stain) or myocardial cell death (K–O; Tunel stain, arrows point to apoptotic cells), in contrast to TSA treatment of PAB rats.
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Figure 5. Western blots of right ventricular (RV) whole-cell lysates (A, B; note different order of experimental conditions compared with densitometries) show an increased protein expression of cleaved caspase-3 (densitometry in C) and collagen-I A1 (ColIA1; densitometry in E), and a decreased protein expression of vascular endothelial growth factor (VEGF) (densitometry in D) and angiopoietin (Ang)-1 (densitometry in F) in trichostatin A (TSA)–treated pulmonary artery banding (PAB) rats. Quantitative polymerase chain reaction (qPCR) does not show changes in VEGF gene expression in any of the experimental conditions (G), whereas Ang-1 mRNA expression is reduced by PAB and TSA, alone or in combination (H).
[More] [Minimize]TAC and PAB are both associated with a reactivation of fetal genes, such as β-myosin heavy chain (MHC; at the expense of α-MHC) and atrial natriuretic peptide (ANP) (16). HDAC inhibition has been shown to be associated with an attenuation of fetal gene reactivation in the pressure-overloaded LV (24). Remarkably, TSA did not repress fetal gene reactivation in the pressure-overloaded RV (see online supplement). As expected, TSA treatment increased the ratio of α- to β-MHC in the normal LV (see online supplement) and RV. As reported previously (24), TSA decreased the mRNA expression of ANP in the normal LV. Unexpectedly, TSA increased ANP expression in the normal RV. The increased ANP expression with TSA was associated with an increased expression of the transcription factor Ptix2, which is a major determinant of right–left asymmetry in the heart (25) and one of the controllers of ANP transcription (26).
In cardiomyocytes in vitro, HDAC inhibition suppresses agonist-induced gene expression of β-MHC and ANP (24), and protects hypoxic cells from apoptosis (whereas paradoxically, HDAC inhibition seems to increase apoptotic rates in normoxic cardiomyocytes) (5). Effects of HDAC inhibition on other cell populations in the heart are unknown. Because TSA treatment caused a striking capillary rarefaction in the PAB RV and HDAC inhibitors have well-known antiangiogenic actions (7), we determined the effect of TSA treatment on HMVECs and found a significant reduction in VEGF, endothelial nitric oxide synthase (eNOS), and Ang-1 gene and protein expression (Figure 6). In silencing experiments using siRNAs directed against individual class I and class II HDACs, it seemed that the suppression of angiogenic gene expression by the broad-spectrum HDAC inhibitors TSA and VPA was not the result of the inhibition of any single HDAC (Figure 7). Rather, silencing of several class I and class II HDACs resulted in the suppression of VEGF, eNOS, and Ang-1 gene expression in HMVECs.
Figure 6. Trichostatin A (TSA) dose-dependently represses angiopoietin (Ang)-1 (A), endothelial nitric oxide synthase (eNOS) (B), and vascular endothelial growth factor (VEGF) gene expression (C) in human cardiac microvascular endothelial cells. Accordingly, Ang-1, endothelial nitric oxide synthase (eNOS), and VEGF protein expression in human cardiac microvascular endothelial cells is reduced with TSA treatment (D, Western blot; E, densitometry).
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Figure 7. Transfection of human cardiac microvascular endothelial cells with small interfering RNA against class I (black bars) and II (gray bars) histone deacetylase (HDACs) shows that angiopoietin (Ang)-1 expression is suppressed mainly by HDACs 3 (class I) and 7 (class II; A); that eNOS expression is predominantly suppressed by HDACs 1 (class I) and 4 (class II; B); and that vascular endothelial growth factor (VEGF) expression is suppressed by all class II HDACs (most importantly HDAC6), but none of the class I HDACs (C). Gene silencing of the class I HDACs 2 and 3 activates VEGF and eNOS mRNA expression, respectively. a = P < 0.001; b = P < 0.01.
[More] [Minimize]TSA induced IGF-1 expression in HCFs in a dose-dependent fashion (see online supplement), which deems it unlikely that the reduction in IGF-1 expression in whole RV lysates of banded rats was directly caused by TSA treatment.
HDACs are transcriptional repressors that promote nucleosomal condensation and have recently emerged as important controllers of the LV hypertrophic response and its accompanying fetal gene reactivation (24). When there is no transcription, DNA is wrapped around histone octameres in nucleosomes, which are the basic units of chromatin. The highly compact structure that is formed by interacting nucleosomes limits access of transcriptional enzymes to genomic DNA, thereby repressing gene expression (27). Acetylation of histones by histone acetyltransferases relaxes the nucleosomal structures, thereby facilitating gene expression; HDACs, when activated, have the opposite effect (28). HDAC inhibitors suppress pressure and agonist-dependent cardiac hypertrophy and prevent fetal gene reactivation (2–4, 24), perhaps because class I HDACs (e.g., HDAC2) repress constitutively active inhibitors of hypertrophic pathways, such as GSK-3 (4). However, mice lacking HDAC2 demonstrate hyperplasia and apoptosis of cardiomyocytes and obliteration of the RV cavity (29). Here we provide experimental results that indicate that HDAC inhibitors adversely affect the pressure-overloaded RV. Whereas chronic treatment with TSA did not influence RV systolic pressure, RV weight, or cardiac output in otherwise not stressed control animals, chronic TSA treatment in PAB animals caused a switch from compensated hypertrophy to RV failure. The evidence for TSA-induced RV failure in PAB animals is a decreased cardiac output, increased RV dilatation, and a worsening of myocardial fibrosis and capillary loss. The importance of our findings lies in the categorical difference between the results of HDAC inhibition in the pressure-overloaded LV and RV: TSA treatment in TAC mice reduced LV hypertrophy and improved function (3, 4), whereas TSA treatment in PAB rats did not reduce established RV hypertrophy, reduced RV expression of angiogenic growth factors, and caused the RV to fail. The differential response to pressure overload is perhaps understandable in view of the recent findings by Kreymborg and coworkers (11). Using a microarray approach, these authors showed that the transcriptional adaptation to pressure overload is very different in the RV after pulmonary artery banding than in the LV after aortic constriction.
Whereas TSA reduced hypertrophy, collagen synthesis, fibrosis, and fetal gene reactivation after TAC (2–4, 24), we report opposite effects of TSA treatment in the pressure-overload RV. These findings underscore the fact that with their potential impact on the transcription of a large number of genes in different cell types, broad-spectrum HDAC inhibitors ultimately have effects on organ function and structure that are difficult to predict and that are context-dependent. Remarkably, TSA treatment had different effects on ANP expression in both unstressed cardiac chambers: TSA decreased ANP expression in the control LV (as reported previously [24]), but increased ANP expression in the control RV. This difference was paralleled by a LV–RV differential response in expression of Pitx2, an important controller of the ANP gene and RV morphogenesis (26). This result could indicate intrinsic differences in transcriptional control between both cardiac chambers, both at baseline (i.e., unbanded) and under stress (after PAB). The increase in ANP and IGF-1 transcription that we found at baseline in TSA-treated rats resembled a stress response. It is possible that this was the physiologic result of induction of pulmonary emphysema, but the increase in IGF-1 gene transcription that we found with TSA treatment of cultured cardiac fibroblasts may suggest that TSA can in certain circumstances evoke a transcriptional stress response. It has recently been shown that IGF-1 production by myocardial fibroblasts is essential for LV adaptation to pressure overload (20). The small degree of RV hypertrophy that we found in TSA-treated unbanded rats could be the direct result of IGF-1 up-regulation in cardiac fibroblasts. Absence of this response in the LV could simply be caused by the fact that the context of TSA treatment in the LV is entirely different: normal LV cardiomyocytes are relatively hypertrophic compared with normal RV cardiomyocytes and normal LV cardiomyocytes have a higher ANP transcription than RV cardiomyocytes to begin with. Unfortunately, there is to our knowledge no available method to culture cardiac chamber–specific neonatal cardiomyocytes: such a cell system could explore chamber-specific differences in epigenetic control of gene transcription. Importantly, the degree of emphysema induced by TSA is insufficient to explain the development of RV failure after PAB: we recently showed that PAB rats treated with SU5416 have a comparable degree of pulmonary emphysema, but normal RV function (16).
The antiangiogenic effects of HDAC inhibitors, described previously in cancer cell lines (7), may have been responsible for the extensive capillary rarefaction seen in our experiments. Here we report for the first time that TSA represses gene transcription of eNOS, VEGF, and Ang-1 in HCMVECs. The gene silencing experiments suggested that these effects were the combined result of inhibition of several, predominantly class II, HDACs. HDAC6 seemed to be an important regulator of VEGF expression in HCMVEC endothelial cells. The results from the siRNA experiments underline the fact that the end-result of treatment with broad-spectrum HDAC inhibitors is difficult to predict, which is most likely because of interaction between and partial redundancy of HDACs. In contrast to our findings of extensive RV capillary rarefaction with TSA treatment of PAB rats, there were no effects reported of HDAC inhibition on LV capillary density after TAC (2–4). It is possible that an angiogenic response is much more important for RV adaptation to PAB, than for LV adaptation to TAC. As shown here, PAB is associated with a doubling of RV mass, whereas TAC is usually associated with only a 20–50% increase in LV mass. RV–LV differences in vascular adaptation to pressure overload could be responsible for the differences in the effects of HDAC inhibition after PAB and TAC. Antiangiogenic effects of HDAC inhibition are usually ascribed to a repression of HIF-1α expression (7), which we did not observe in the TSA-treated banded RV. We speculate that the RV dilatation and capillary rarefaction that occurs in the banded RV contributed to the development of myocardial ischemia and hypoxic stabilization of HIF-1α expression. We have reported disconnect of VEGF gene transcription and HIF-1α protein stabilization in the failing RV in experimental pulmonary hypertension (16). HIF-1α expression was increased by TSA in the RV of control rats, which may point to either RV ischemia or hypoxia caused by TSA-related changes of the lung structure. Hypoxia by itself is not sufficient to induce failure of the banded RV (16) and could therefore not account for the detrimental effect of TSA treatment in PAB rats.
An increase in the number of Tunel-positive cells was seen in the failing RV of TSA-treated PAB. HDAC inhibition has been shown to be associated with apoptotic and autophagic cell death of cancer cells (8) and TSA has been shown to induce apoptosis of normoxic (but not ischemic) cardiomyocytes (5). As is true for the effect of TSA on capillary rarefaction, our data do not answer the question whether TSA-related apoptosis was directly responsible for the detrimental effects of HDAC inhibition on the adapting RV. However, data obtained with cultured heart microvascular endothelial cells confirm the antiangiogenic activity of TSA, suggesting that TSA had also exerted an antiangiogenic action in vivo in the pressure-overloaded RV.
Recently, Cho and coworkers (30) reported that VPA prevented the development of RV hypertrophy in young rats where PAB had induced RV failure. The data reported by this group differ in several aspects from the data reported here. Cho and coworkers (30) describe the development of severe RV failure in vehicle-treated PAB rats, as evidenced by an increased inferior vena cava diameter, marked RV hypertrophy (RV/LV+S increasing up to 0.80), a reduced gain in body weight, and the development of RV fibrosis (30). Unfortunately, no hemodynamic data were provided, but it seems that the technique used by this group differed from the more conventional PAB technique, which is known to elicit a compensatory RV hypertrophic response, a limited degree of RV fibrosis, and no evidence of RV failure (16–18). The different findings by Cho and coworkers (30) may have been related to the use of younger rats (3 weeks at the time of surgery), or perhaps to a more severe pulmonary artery constriction at the time of surgery (rendering a model of acute RV pressure overload, not chronic progressive pressure overload as induced by our technique). In addition, Cho and coworkers (30) initiated treatment directly after surgery, whereas we chose to wait until after the generation of adaptive hypertrophy (4 wks after surgery). We believed that this approach would have more resemblance to the clinical situation of TSA treatment of patients with established pulmonary hypertension.
Global HDAC inhibition experiments, such as those based on TSA administration, need to be compared with those of targeted cardiac HDAC gene experiments as performed by Montgomery and coworkers (29). In the latter study it was shown that HDAC1 and HDAC2 redundantly regulate cardiac growth and that deletion of either gene alone was insufficient to reduce LV hypertrophy of the TAC-stressed hearts. The same authors showed in another study that HDAC3 is important for the maintenance of cardiac energy metabolism in mice (31). Because of the technical challenges that are associated with performing PAB in mice and because of the limited possibilities for transgenic manipulations in rats, we were restricted to make use of pharmacologic interventions. We did not initiate TSA treatment immediately after surgery, but rather 4 weeks later. Although this may explain part of the discrepancy between our results and the reported effects of TSA after TAC, we believe that a scenario of late treatment is likely more pertinent to the clinical situation of patients with already established severe PAH.
One important implication of our findings is that concerns are being raised regarding the development of antiangiogenic and proapoptotic drugs for the treatment of severe angioproliferative pulmonary hypertension. Although such treatments may make intuitive sense (32), their consequences for the stressed RV are unpredictable. The second implication is that although HDAC inhibition may seem attractive for the treatment of LV systolic failure, the effects of HDAC inhibition on the RV are again unpredictable. Congestive heart failure almost always involves both ventricles and there is a distinct possibility that what works for the failing LV may not work for the failing RV.
Conception and design: H.J.B., S.M., R.N., and N.F.V. Analysis and interpretation: all authors. Drafting manuscript: H.J.B., R.N., and N.F.V.
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