Normal and high eNOS levels are detrimental in both mild and severe cardiac pressure-overload

Journal of Molecular and Cellular Cardiology 88 (2015) 145–154

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Original article

Normal and high eNOS levels are detrimental in both mild and severe cardiac pressure-overload Elza D. van Deel a, Yanti Octavia a,b, Martine de Boer a, Rio P. Juni b, Dennie Tempel a, Rien van Haperen c, Rini de Crom c, An L. Moens b, Daphne Merkus a, Dirk J. Duncker a,⁎ a b c

Experimental Cardiology, Thorax Center, Cardiovascular Research School COEUR, Erasmus University Medical Center, Rotterdam, The Netherlands Department of Cardiology, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, University of Maastricht, Maastricht, The Netherlands Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands

a r t i c l e

i n f o

Article history: Received 18 July 2015 Received in revised form 28 September 2015 Accepted 1 October 2015 Available online 3 October 2015 Keywords: Cardiac pressure-overload eNOS uncoupling Oxidative stress Cardiac remodeling Cardiac dysfunction

a b s t r a c t Nitric oxide (NO) produced by endothelial NO synthase (eNOS) exerts beneficial effects in a variety of cardiovascular disease states. Studies on the benefit of eNOS activity in pressure-overload cardiac hypertrophy and dysfunction produced by aortic stenosis are equivocal, which may be due to different expression levels of eNOS or different severities of pressure-overload. Consequently, we investigated the effects of eNOS-expression level on cardiac hypertrophy and dysfunction produced by mild or severe pressure-overload. To unravel the impact of eNOS on pressure-overload cardiac dysfunction we subjected eNOS deficient, wildtype and eNOS overexpressing transgenic (eNOS-Tg) mice to 8 weeks of mild or severe transverse aortic constriction (TAC) and studied cardiac geometry and function at the whole organ and tissue level. In both mild and severe TAC, lack of eNOS ameliorated, whereas eNOS overexpression aggravated, TAC-induced cardiac remodeling and dysfunction. Moreover, the detrimental effects of eNOS in severe TAC were associated with aggravation of TAC-induced NOSdependent oxidative stress and by further elevation of eNOS monomer levels, consistent with enhanced eNOS uncoupling. In the presence of TAC, scavenging of reactive oxygen species with N-acetylcysteine reduced eNOS S-glutathionylation, eNOS monomer and NOS-dependent superoxide levels in eNOS-Tg mice to wildtype levels. Accordingly, N-acetylcysteine improved cardiac function in eNOS-Tg but not in wildtype mice with TAC. In conclusion, independent of the severity of TAC, eNOS aggravates cardiac remodeling and dysfunction, which appears due to TAC-induced eNOS uncoupling and superoxide production. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Nitric oxide (NO) exerts a plethora of beneficial effects on the structure and function of the cardiovascular system. For example, NO reduces vascular tone, prevents atherosclerosis, enhances myocyte contractility and mitigates left ventricular (LV) hypertrophy [1,2]. In contrast, reactive oxygen species (ROS), particularly superoxide (O− 2 ), are wellknown contributors to cardiac pathology and neutralize the effects of NO. Consequently, NO and O− 2 are tightly balanced in the cardiovascular system. The importance of this nitroso-redox balance is underlined by studies showing that perturbations in this balance lead to oxidative stress and thereby contribute to diabetes, hypertension, atherosclerosis and the development of heart failure [3–7]. In the heart, NO is principally produced in endothelial cells and cardiomyocytes by endothelial NO synthase (eNOS) [8,9]. Functional ⁎ Corresponding author at: Div. Experimental Cardiology, Dept. Cardiology, Thorax Center, Erasmus University Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail address: [email protected] (D.J. Duncker).

http://dx.doi.org/10.1016/j.yjmcc.2015.10.001 0022-2828/© 2015 Elsevier Ltd. All rights reserved.

eNOS is a dimeric protein composed of 2 eNOS monomers. Pathological conditions, like oxidative stress, induce eNOS uncoupling that is associated with a change from the dimeric to the monomeric form of eNOS. An important inducer of eNOS uncoupling is the shift of glutathione from a reduced (GSH) to an oxidized (GSSG) state which causes Sglutathionylation of redox-sensitive eNOS thiols and concomitant eNOS uncoupling [10]. Uncoupled eNOS no longer produces NO but in− stead forms O− 2 [11]. Although O2 acts as a signaling molecule at low concentrations, excessive production of O− 2 contributes to deterioration of cardiac function and progression toward heart failure [12–14]. This dual role of eNOS as a producer of beneficial NO as well as detrimental O− 2 may explain why the role of eNOS in cardiac disease remains controversial [11]. The beneficial effects of eNOS on cardiac function and structure following myocardial infarction have been well established [15–17]. In contrast, in pressure-overload-induced cardiac pathology, the effects of eNOS remain incompletely understood. For example, in eNOSdeficient (eNOS-Ko) mice, reduced NO bioavailability aggravated LV hypertrophy and dysfunction produced by transverse aortic constriction (TAC) in some studies [18,19], while in another study eNOS-Ko mice

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were protected against TAC-induced cardiac pathology due to the lack of uncoupled eNOS [20]. Although an explanation for these disparate findings is not readily found, it has been suggested that the severity of the pressure-overload [20] and/or the extent of eNOS uncoupling could explain the divergent results between studies, but this has so far not been investigated. A better understanding of the potential benefit or harm of eNOS in pressure-overload derived heart failure is essential for optimizing therapeutic strategies that include the beneficial potential of NO but simultaneously prevent oxidative stress. The aim of the present study was therefore to determine the effect of eNOS expression on pressureoverload induced cardiac pathology. For this purpose we subjected mice with different levels of whole body (general) eNOS expression, i.e. general eNOS-Ko, wildtype (Wt) and general eNOS overexpressing transgenic (eNOS-Tg) mice, to mild TAC (mTAC) and severe TAC (sTAC). Subsequently, we explored the involvement of eNOS uncoupling mediated ROS production in the effects of eNOS in the pressure-overloaded heart.

2. Methods All experiments were performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and with prior approval of the Animal Ethics Committee of the Erasmus University Medical Center Rotterdam (EUR 1614). A total of 282, 18–20 weeks old C57Bl6 mice of either sex entered the study.

2.1. Experimental procedure eNOS-Ko mice in a C57Bl/6 background were originally obtained from Jackson Laboratory (O. Smithies) and bred within our facilities. The eNOS-Tg mice, also in the C57Bl/6 background, were generated as previously described [21]. The transgene was derived from a cosmid and comprises the complete gene including its native promoter, resulting in expression in endothelial cells of tissues examined. Wt littermates were used as controls. In total, 65 eNOS-Ko, 77 Wt and 79 eNOS-Tg mice underwent mTAC (using a 25G needle), severe sTAC (using a 27G needle) or a sham operation as previously described [22]. Additionally, 30 Wt and 31 eNOS-Tg mice underwent sTAC and subsequently received the antioxidant N-acetylcysteine (NAC) (1 mg/ ml in drinking water), to elucidate the effects of eNOS overexpression on ROS production in pressure-overload hypertrophy. Eight weeks after surgery, echocardiographic and hemodynamic measurements were performed under isoflurane anesthesia. All mice were ventilated and anesthetized with 2.5% isoflurane and M-mode LV echocardiography was performed with an Aloka SSD 4000 echo device (Aloka; Tokyo, Japan) using a 12-MHz probe. LV diameters at end diastole (LVEDD) and end systole (LVESD) were measured, and fractional shortening was calculated [22]. Aortic pressure distal to the stenosis was measured through a PE10 catheter in the left carotid artery. A 1.4-Fr microtipped manometer (Millar Instruments; Houston, Texas, USA) was inserted in the right carotid artery to record aortic pressure proximal to the stenosis and subsequently advanced into the LV to measure LV pressure (LVP) and calculate the LVP-derived indexes of LV contractility and relaxation, the maximum rate of rise (LVdP/dtmax) and fall (LVdP/dtmin) of LVP. To minimize the influence of diastolic and systolic aortic pressure, respectively, on the LVP-derived indices of systolic (LVdP/dtmax) and diastolic (LVdP/dtmin) function [23], we also computed the rate of rise of LVP at a pressure of 40 mm Hg (LVdP/dtP40), and the time constant of LV pressure decay (tau) as previously described [22]. At the end of each experiment LV weight and right ventricle (RV) weight, wet and dry lung weights and tibia length (TL) were determined and LV tissue samples were stored for histological and molecular analysis.

2.2. Histomorphometry Paraffin embedded LV tissue was serially sectioned into 4-μm slices. Subsequently, Gomori staining was performed to measure cardiomyocyte cross-sectional area [22]. Capillary density was determined by lectin staining and interstitial fibrosis (collagen content) was measured using picro-sirius red staining [22]. LV sections of 6 mice per group were analyzed with a quantitative image analysis system (Clemex Technologies). 2.3. Gene expression analysis Total mRNA was extracted from 6 frozen LV samples per group using the RNeasy kit (Qiagen, The Netherlands) and quantified using a NanoDrop spectrophotometer (NanoDrop®, Isogen Life Science, The Netherlands). Isolated mRNA was reverse transcribed into cDNA (Iscript, Biorad, The Netherlands) and analyzed by real-time fluorescence assessment of SYBR Green signal in the iCycler iQ Detection system (Bio-Rad, The Netherlands). Primer sets for myocardial hypertrophy marker genes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), α–skeletal actin (α-SKA) and eNOS were employed. mRNA levels were corrected for the housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) and normalized to Wt sham values. 2.4. eNOS protein level and monomer-to-dimer ratio Total eNOS protein levels, phospho-eNOS (p-eNOS) and eNOS monomer and dimer protein expression were determined in 6–8 snap-frozen LV tissue samples per group. For detection of eNOS monomer and dimer fraction, low-temperature SDS-PAGE was performed as previously described [24]. Briefly, gels and buffers were equilibrated at 4 °C before electrophoresis, and the buffer tank was placed in an ice bath during electrophoresis to maintain the low temperature. SDSPAGE for phosphorylated eNOS, total eNOS protein content and housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed at room temperature. Subsequent to SDS-PAGE, the proteins were transferred to nitrocellulose membranes and the blots were probed with primary anti-phospho eNOS (1:1000 Cell Signaling), antieNOS (1:500, Transduction Laboratory) and anti-GAPDH (1:10,000, Imgenex) and secondary rabbit anti-mouse IgG antibody conjugated with HRP (1:1000, Santa Cruz Biotechnology). All blots were analyzed using the Odyssey system (LI-COR). Absolute levels of eNOS monomer and dimer levels were calculated from the monomer/dimer ratio and the total amount of eNOS. For determination of eNOS Sglutathionylation, eNOS was first immunoprecipitated and pulled down with protein G-conjugated (Novex) eNOS antibody (Santa Cruz). Subsequently, standard western blotting was performed using anti-glutathione monoclonal antibody (ViroGen). To confirm that the observed bands indeed detected S-glutathionylation of eNOS, control samples were treated with dithiothreitol (DTT), which removes the glutathionylation modification of eNOS. Rabbit anti-mouse IgG antibody conjugated with HRP (Santa Cruz Biotechnology) was used as isotype control. 2.5. Superoxide levels In order to evaluate cardiac oxidative stress, superoxide generation was measured by lucigenin-enhanced chemiluminescence (5 μmol/L) in 4 homogenized LV samples per group using a single-tube luminometer (Berthold FB12) in which dark-adapted lucigenin was added via an auto-dispenser as previously described [25]. Besides − total O− 2 production, NOS-dependent O2 production was determined by incubated samples from the same homogenized LV stock for 30 min with the NOS inhibitor L-NAME (Sigma-Aldrich) (1 mmol/l). During the entire experiment the temperature was maintained at

E.D. van Deel et al. / Journal of Molecular and Cellular Cardiology 88 (2015) 145–154

37 °C and light emission recorded and expressed as relative light units (RLU) per mg per second. All samples were measured in − duplicate and O− 2 levels were calculated relative to total O2 levels of Wt sham mice. 2.6. Statistical analysis All data were tested using 2-way (eNOS expression level × TAC severity) ANOVA followed by post-hoc testing with the Student–Newman–Keuls test. Multiple comparison linear trend analysis between eNOS-Ko, Wt and eNOS-Tg was performed on the effects of increasing eNOS expression on cardiac geometry and function. A value of P b 0.05 was considered statistically significant (two-tailed). Data are presented as means ± SEM. All groups contained similar numbers of male and female mice. Similar to previous observations [22], we did not observe an influence of sex on the effects of TAC and/or eNOS expression level on the responses of survival and LV hypertrophy or dysfunction. Consequently, data from male and female mice were pooled for final analysis.

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3. Results 3.1. Influence of TAC and eNOS on cardiac pathology In order to assess the effects of eNOS expression level on TACinduced cardiac pathology, survival rate as well as LV dimensions and function were examined 8 weeks after TAC. Lack or overexpression of eNOS did not affect survival in sham-operated animals (Fig. 1A) while mTAC slightly increased mortality in eNOS-Ko (17%) and Wt (19%) mice but not in eNOS-Tg mice (5%). In contrast, sTAC increased mortality in all groups, with mortality being highest in eNOS-Tg (49%) followed by Wt (26%) and eNOS-Ko (15%) mice (Fig. 1A). eNOS expression levels did not affect body weight but tibia length was slightly larger in Wt and eNOS-Tg mice than in age-matched eNOS-Ko sham mice (Table 1). As expected, loss of eNOS resulted in higher LV systolic pressure and mean arterial pressure in sham mice, while eNOS overexpression in eNOS-Tg sham mice resulted in a lower LV systolic pressure and mean arterial pressure compared to Wt littermates (Table 2). In contrast, eNOS expression levels did not affect

Fig. 1. TAC-induced mortality, cardiac remodeling and dysfunction are aggravated with increasing eNOS expression. (A) Kaplan–Meier survival curve of sham, mTAC and sTAC mice showing that sTAC-induced mortality is aggravated in eNOS-Tg mice. Total number of animals entering the study: eNOS-Ko sham (n = 21), Wt sham (n = 22), eNOS-Tg sham (n = 19), eNOSKo mTAC (n = 18), Wt mTAC (n = 17), eNOS-Tg mTAC (n = 19), eNOS-Ko sTAC (n = 26), Wt sTAC (n = 38), eNOS-Tg sTAC (n = 41). (B) Effect of TAC and eNOS on LV mass and geometry, hemodynamic parameters and relative lung fluid weight (lung wet weight minus lung dry weight) in eNOS-Ko (white bars), Wt (gray bars) and eNOS-Tg (black bars) mice. LV, left ventricle; TL, tibia length; LVdP/dtP40, rate of rise of LV pressure at 40 mm Hg; LVdP/dtmin, maximal rate of fall of LV pressure; LVEDD, LV end diastolic diameter. *P b 0.05 vs corresponding sham, §P b 0.05 vs corresponding mTAC, †P b 0.05 vs corresponding Wt; ‡P b 0.05 eNOS-Tg vs corresponding eNOS-Ko. Number of animals is indicated below each bar.

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Table 1 Anatomical data. eNOS-Ko Body weight (g)

Tibia length (cm)

LV weight (mg)

RV weight (mg)

Lung fluid weight (mg)

Sham mTAC sTAC Sham mTAC sTAC Sham mTAC sTAC Sham mTAC sTAC Sham mTAC sTAC

Wt

eNOS-Tg

24.0 ± 0.9 24.2 ± 1.1 23.7 ± 1.0 1.79 ± 0.01† 1.81 ± 0.01 1.79 ± 0.01† 94 ± 4 119 ± 6⁎ 157 ± 7⁎,†,§

26.7 ± 1.2 25.5 ± 1.0 26.7 ± 1.0 1.83 ± 0.01 1.85 ± 0.01 1.87 ± 0.01⁎ 93 ± 3 133 ± 8⁎ 179 ± 7⁎,§

25.3 ± 0.7 25.5 ± 1.1 23.6 ± 0.9§ 1.82 ± 0.01‡ 1.83 ± 0.01 1.82 ± 0.01† 96 ± 3 147 ± 10⁎ 195 ± 8⁎,†,‡,§

22 ± 1 22 ± 1 30 ± 2⁎,§ 103 ± 2 103 ± 3 180 ± 20⁎,§

23 ± 1 23 ± 1 34 ± 2⁎,§ 114 ± 4 135 ± 12 201 ± 22⁎,§

23 ± 1 26 ± 2 39 ± 3⁎,†,‡,§ 126 ± 8 133 ± 10 250 ± 22⁎,†,‡,§

LV, left ventricle; RV, right ventricle. eNOS-Ko sham (n = 20), Wt sham (n = 22), eNOS-Tg sham (n = 18), eNOS-Ko mTAC (n = 15), Wt mTAC (n = 13), eNOS-Tg mTAC (n = 17), eNOS-Ko sTAC (n = 20), Wt sTAC (n = 27), eNOS-Tg sTAC (n = 18). ⁎ P b 0.05 vs corresponding sham. † P b 0.05 vs corresponding Wt. ‡ P b 0.05 eNOS-Tg vs corresponding eNOS-Ko. § P b 0.05 vs corresponding mTAC.

heart rate or LV weight, geometry and function in sham-operated mice (Tables 1 and 2; Fig. 1B). Eight weeks of mTAC and sTAC both produced LV hypertrophy (Fig. 1B). Surprisingly, although arterial blood pressure and LV systolic pressure were markedly reduced by eNOS overexpression (Table 2), LV hypertrophy produced by TAC was most pronounced in eNOS-Tg mice (Fig. 1B). Moreover, mTAC only induced LV dilation in Wt and eNOS-Tg mice but not in eNOS-Ko (Fig. 1B), and whereas mTAC reduced LV systolic (LVdP/dtP40) and diastolic (LVdP/dtmin) function in eNOS-Tg mice, mTAC did not affect LV function in eNOS-Ko and Wt animals. Hence, even though eNOS overexpression resulted in a lower blood pressure, the mTAC-induced pathological changes in LV dimensions and function were most pronounced in eNOS-Tg mice. In contrast to mTAC, sTAC produced LV dysfunction in all mice and resulted in more Table 2 Functional data. eNOS-Ko HR (bpm)

MAPprox (mm Hg)

SPG (mm Hg)

LVSP (mm Hg)

LVdP/dtmax (mm Hg s−1) LVEDP (mm Hg)

Wt

eNOS-Tg

Sham 550 ± 8 558 ± 15 560 ± 18 mTAC 547 ± 10 554 ± 21 539 ± 18 sTAC 541 ± 12 527 ± 10 538 ± 14 Sham 104 ± 3† 87 ± 4 72 ± 4†,‡ mTAC 110 ± 8 99 ± 3⁎ 78 ± 3†,‡ sTAC 94 ± 5 84 ± 4§ 76 ± 3‡ Sham 0±3 0±2 −2 ± 1 mTAC 37 ± 6⁎ 43 ± 4⁎ 37 ± 4⁎ 54 ± 4⁎,§ 49 ± 3⁎,§ sTAC 49 ± 4⁎,† 94 ± 3 83 ± 3†,‡ Sham 116 ± 3† mTAC 140 ± 6⁎ 133 ± 8⁎ 109 ± 4⁎,†,‡ sTAC 131 ± 6⁎ 126 ± 5⁎ 105 ± 5⁎,†,‡ Sham 8650 ± 640 8880 ± 620 9180 ± 760 mTAC 8900 ± 300 8200 ± 510 6870 ± 460⁎,‡ sTAC 7000 ± 470⁎,§ 6490 ± 350⁎,§ 5030 ± 330⁎,†,‡,§ Sham 4.8 ± 0.6 4.4 ± 0.6 3.8 ± 0.6 mTAC 7.0 ± 1.0 7.6 ± 1.6 5.3 ± 0.6 ,§ ,§ ⁎ ⁎ sTAC 14.1 ± 1.5 13.2 ± 1.4 16.9 ± 2.7⁎,§

HR, heart rate; MAPprox, mean arterial pressure proximal to the stenosis; SPG, systolic pressure gradient over the stenosis; LVSP, left ventricular systolic pressure; LVdP/dtmax, maximum rate of rise of LV pressure; LVEDP, LV end diastolic pressure. eNOS-Ko sham (n = 20), Wt sham (n = 22), eNOS-Tg sham (n = 18), eNOS-Ko mTAC (n = 15), Wt mTAC (n = 13), eNOS-Tg mTAC (n = 17), eNOS-Ko sTAC (n = 20), Wt sTAC (n = 19), eNOS-Tg sTAC (n = 14). ⁎ P b 0.05 vs corresponding sham. † P b 0.05 vs corresponding Wt. ‡ P b 0.05 eNOS-Tg vs corresponding eNOS-Ko. § P b 0.05 vs corresponding mTAC.

severe pathological LV remodeling (LV weight and diameter), systolic LV dysfunction (LVdP/dtP40, fractional shortening), diastolic LV dysfunction (LVdP/dtmin, LV end diastolic pressure and tau) and pulmonary congestion (lung fluid weight and RV weight) compared to mTAC (Tables 1 and 2; Fig. 1B). Similar to observations in mTAC, sTACinduced cardiac pathology was more pronounced with increasing eNOS expression levels. This was not simply the result of the detrimental effects of eNOS overexpression compared to Wt, but also of the detrimental effect of normal Wt levels of eNOS expression compared to eNOS-Ko. Consequently, statistical differences were most consistently observed between eNOS-Tg and eNOS-Ko groups (Fig. 1). Furthermore, linear trend analysis of these variables revealed a significant aggravation of TACinduced pathology with increasing eNOS expression in both mTAC and sTAC for most variables (Suppl. Table 1). Taken together these findings suggest that, although eNOS reduces LV systolic pressure and consequently lowers LV afterload at baseline, increased eNOS expression aggravates TAC-induced LV pathology while lack of eNOS protects against LV remodeling and dysfunction following TAC, irrespective of stenosis severity. 3.2. LV histomorphometry and hypertrophic marker genes Since the effects on LV remodeling and dysfunction were most pronounced in sTAC mice, we elected to explore cellular and molecular adaptations to TAC and eNOS expression in sham and sTAC mice only. To determine whether TAC-induced changes in LV morphology and function were accompanied by adaptations at the cellular level, we performed LV histomorphometry. The level of eNOS expression did not affect cardiomyocyte cross-sectional area (CSA), capillary density or interstitial fibrosis in sham-operated animals (Fig. 2A,B). Interestingly, although myocyte hypertrophy in response sTAC was independent of genotype, sTAC only produced capillary rarefaction and interstitial fibrosis in Wt and eNOS-Tg but not in eNOS-Ko mice (Fig. 2A,B), indicating that loss of eNOS expression preserved capillary density and prevented excessive extracellular matrix deposition following sTAC. An additional cellular hallmark of cardiac pathology and hypertrophy is re-expression of fetal genes. To evaluate how eNOS expression levels influence TAC-induced alterations in mRNA levels of fetal genes, we performed qPCR for fetal genes associated with LV hypertrophy, ANP, BNP and α-SKA. eNOS expression levels neither influenced ANP and α-SKA mRNA expression in sham-operated animals nor affected the elevation of ANP and α-SKA mRNA expression following sTAC (Fig. 2C). Similarly, BNP mRNA expression in eNOS-Ko and eNOS-Tg mice was not different from Wt animals but was slightly lower in eNOS-Tg than in eNOS-Ko sham-operated mice. In contrast, sTAC increased BNP expression only in Wt and eNOS-Tg mice but not in eNOS-Ko animals (Fig. 2C), suggesting a beneficial effect of lack of eNOS expression. 3.3. TAC-induced eNOS uncoupling exacerbates oxidative stress in mice overexpressing eNOS To study the impact of eNOS expression on TAC-induced pathology, and assess the role of eNOS uncoupling therein, we not only examined the total amount of eNOS protein expression but additionally measured eNOS monomer and eNOS dimer protein levels. As expected, eNOS protein expression could not be detected in eNOS-Ko mice (Suppl. Fig. 1), while overexpression of eNOS increased eNOS protein levels in both sham and sTAC-operated eNOS-Tg mice approximately 8-fold compared to Wt littermates (Fig. 3A). Interestingly, the increase in eNOS protein expression in eNOS-Tg mice markedly elevated eNOS dimer protein but only slightly increased eNOS monomer levels in shamoperated eNOS-Tg animals (Fig. 3A). Accordingly, the eNOS monomer/ dimer ratio was markedly lower in eNOS-Tg sham mice than in Wt sham littermates (Suppl. Fig. 2), implying that eNOS overexpression promoted eNOS dimerization, likely to maintain a low level of eNOS monomer. In contrast, sTAC induced eNOS uncoupling and increased

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Fig. 2. eNOS aggravates TAC-induced histological and mRNA expression parameters of cardiac pathology. (A) Representative histological images of Gomori, lectin and picro-sirius red staining. (B) TAC-induced capillary rarefaction and cardiac fibrosis are aggravated by eNOS. (C) mRNA expression levels relative to Wt sham samples. CSA, cardiomyocyte cross-sectional area; Cap Dens, capillary density; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; α-SKA, α-skeletal actin. A total of 6 LV samples per group of eNOS-Ko (white bars), Wt (gray bars) and eNOS-Tg (black bars) mice were used. *P b 0.05 vs corresponding sham; †P b 0.05 vs corresponding Wt; ‡P b 0.05 eNOS-Tg vs corresponding eNOS-Ko.

eNOS monomer/dimer ratio ~ 4-fold in both Wt and eNOS-Tg mice (Suppl. Fig. 2). However, since the amount of total eNOS was much higher in mice overexpressing eNOS, sTAC resulted in a more pronounced elevation of the O− 2 producing eNOS monomer in eNOS-Tg mice than in Wt animals (Fig. 3A). Since eNOS uncoupling results in increased O− 2 production, we subsequently assessed to what extent TAC-induced elevation of eNOS monomer protein in eNOS-Tg mice translated into oxidative stress. For this purpose, we measured total and L-NAME inhibitable O− 2 levels in LV tissue samples of Wt and eNOS-Tg animals. In sham-operated mice

total O− 2 levels increased slightly with higher eNOS expression (Fig. 3B). This enhanced O− 2 production with increasing eNOS levels was not affected by inhibition of NOS enzyme activity with L-NAME, suggesting that NOS blockade by L-NAME may have been incomplete, or that O− 2 was produced by a different source than NOS. Strikingly, sTAC did not elevate total O− 2 production in eNOS-Ko mice, whereas sTAC-induced O− 2 production was elevated even more in eNOS-Tg mice than in Wt littermates. Moreover, in eNOS-Tg mice, the elevated O− 2 production following LV pressure-overload was largely L-NAME inhibitable (Fig. 3B), which is in accordance with our observation that

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Fig. 3. TAC-induced elevation of eNOS monomer protein and NOS-dependent O− 2 levels is aggravated by eNOS overexpression. (A) Representative western blots of total as well as eNOS monomer and dimer protein expression. Elevated eNOS expression increases eNOS monomer levels after TAC (n = 6–8 per group). Total eNOS levels are normalized to Wt sham. (B) Elevation of TAC-induced increase in total O− 2 production in eNOS-Tg mouse hearts (normalized to Wt sham) appears largely L-NAME inhibitable (n = 4 per group). RLU, relative light units. *P b 0.05 vs corresponding sham †P b 0.05 vs corresponding Wt (gray bars); ‡P b 0.05 eNOS-Tg (black bars) vs corresponding eNOS-Ko (white bars).

the TAC-induced increase in eNOS monomer levels was most prominent in eNOS-Tg mice. Thus, while eNOS overexpression only marginally affected O− 2 levels in sham-operated animals, sTAC-induced oxidative stress was markedly increased in eNOS-Tg mice. 3.4. ROS scavenging mitigates detrimental effects of eNOS in TAC-induced cardiac pathology To demonstrate that eNOS-mediated oxidative stress was involved in the detrimental effects of eNOS expression in TAC, we investigated the effects of ROS scavenging with NAC. As expected, the ROS scavenger

NAC reduced the TAC-induced increase in total O− 2 production in both Wt and eNOS-Tg mice, but this was most pronounced in eNOS-Tg mice, so that after NAC treatment, TAC-induced O− 2 levels were no longer different between Wt and eNOS-Tg animals (Fig. 4A). This NACinduced reduction in O− 2 was largely inhibitable by L-NAME and more pronounced in eNOS-Tg than in Wt mice (Fig. 4A), suggesting restoration of eNOS coupling. Indeed, NAC reduced eNOS S-glutathionylation (Fig. 4B, D), eNOS monomer levels (Fig. 4C, E) and hence monomer/ dimer ratio (Suppl. Fig. 3) in TAC mice, which was particularly pronounced in eNOS-Tg mice (Fig. 4B, C). Finally, NAC did not affect total eNOS protein expression in Wt or eNOS-Tg mice (Fig. 4F, G), and

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Fig. 4. ROS scavenging through NAC treatment (hatched bars) mitigates detrimental effects of eNOS overexpression in sTAC. (A) Total (normalized to Wt sham), L-NAME inhibitable and LNAME independent O− 2 levels (n = 6 per group). (B) eNOS S-glutathionylation normalized to Wt TAC (n = 4 per group). (C) Amounts of eNOS monomer and dimer protein. (D) Representative blot demonstrating eNOS S-glutathionylation and control samples treated with dithiothreitol (DTT), which removes the glutathionylation modification of eNOS. (E) Representative western blots of eNOS monomer and dimer protein expression. (F) Total eNOS protein expression (eNOS) normalized to Wt sham, and phosphorylated eNOS (peNOS) relative to total eNOS (p-eNOS/eNOS) normalized to Wt TAC; (n = 4 per group). (G) Representative western blots of total eNOS and p-eNOS. RLU, relative light units.†P b 0.05 vs corresponding Wt; #P b 0.05 vs corresponding no treatment.

although NAC reduced p-eNOS following TAC in Wt, it had no effect on p-eNOS levels in eNOS-Tg mice (Fig. 4F, G). ROS scavenging by NAC had no effect on survival or cardiac geometry and function in sham-operated Wt and eNOS-Tg mice (data not shown). However, following NAC treatment, TAC-induced mortality was no longer different between Wt and eNOS-Tg mice (Suppl. Fig. 4). Moreover, in eNOS-Tg mice subjected to sTAC, NAC attenuated LV hypertrophy and dilation, improved systolic function (LVdP/dtP40 and LVdP/dtmax) and blunted pulmonary congestion and secondary RV hypertrophy (Table 3, Fig. 5). Thus, ROS scavenging abolished eNOSrelated aggravated cardiac remodeling and dysfunction so that cardiac function in TAC-operated Wt and eNOS-Tg mice was no longer different from cardiac function of eNOS-Ko mice following TAC (Suppl. Fig. 5).

to a greater extent in eNOS-Tg than in Wt mice. The implications of these findings are discussed below.

Table 3 Anatomical and functional data with and without NAC treatment.

Body weight (g) Tibia length (cm) RV weight (mg) HR (bpm)

4. Discussion The present study demonstrates that eNOS expression levels are directly linked to aggravation of LV hypertrophy and dysfunction in TACinduced pressure-overload. Accordingly, reductions in capillary density, interstitial fibrosis and elevated BNP expression levels following TAC were mitigated in eNOS-Ko mice compared to Wt and eNOS-Tg animals. Importantly, while normal eNOS expression exerted detrimental effects in cardiac pressure-overload, eNOS overexpression significantly further aggravated pressure-overload-induced cardiac pathology. Detrimental effects of eNOS were present in mild as well as severe TAC and thus appeared to be independent of the severity of TAC. Finally, the adverse effects of eNOS in TAC were a likely result of eNOS uncoupling as evidenced by increased eNOS S-glutathionylation and eNOS monomer protein levels that resulted in elevated eNOS-mediated O− 2 production. Accordingly, scavenging of ROS reduced O− 2 superoxide production and eNOS monomer levels and attenuated LV hypertrophy and dysfunction

MAPprox (mm Hg) SPG (mm Hg) LVSP (mm Hg) LVdP/dtmax (mm Hg s−1) LVdP/dtmin (mm Hg s−1) tau (ms)

sTAC sTAC + NAC sTAC sTAC + NAC sTAC sTAC + NAC sTAC sTAC + NAC sTAC sTAC + NAC sTAC sTAC + NAC sTAC sTAC + NAC sTAC sTAC + NAC sTAC sTAC + NAC sTAC sTAC + NAC

Wt

eNOS-Tg

25.6 ± 0.8 23.9 ± 1.0 1.86 ± 0.01 1.81 ± 0.01# 34.7 ± 1.8⁎ 29.5 ± 2.1⁎ 526 ± 9 522 ± 14 83 ± 3 82 ± 4 52 ± 4⁎ 53 ± 5⁎ 123 ± 4⁎ 120 ± 7⁎ 6540 ± 320⁎ 5980 ± 560⁎ −6250 ± 350⁎ −6060 ± 800⁎ 16.5 ± 1.4⁎ 18.9 ± 4.1⁎

23.4 ± 0.7† 23.5 ± 0.6 1.81 ± 0.01 1.80 ± 0.01 36.9 ± 1.9⁎ 28.2 ± 1.7⁎,# 537 ± 12 550 ± 9 73 ± 3† 77 ± 5 46 ± 3⁎ 41 ± 6⁎ 103 ± 4⁎,† 103 ± 4⁎,† 4990 ± 260⁎,† 6150 ± 390⁎,# −4740 ± 280⁎,† −5620 ± 470⁎ 17.2 ± 1.6⁎ 15.3 ± 2.3⁎

RV, right ventricle; HR, heart rate; MAPprox, mean arterial pressure proximal to the stenosis; SPG, systolic pressure gradient over the stenosis; LVSP, LV systolic pressure; LVdP/ dtmax, maximum rate of rise of LV pressure; LVdP/dtmin, maximum rate of fall of LV pressure; tau, time constant of LV pressure decay. Wt sTAC (n = 28), Wt sTAC + NAC (n = 10), eNOS-Tg sTAC (n = 18), eNOS-Tg sTAC + NAC (n = 11). ⁎ P b 0.05 vs corresponding sham. † P b 0.05 vs corresponding Wt. # P b 0.05 vs corresponding sTAC.

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Fig. 5. ROS scavenging with NAC mitigates TAC-induced cardiac remodeling and dysfunction in eNOS-Tg mice. LV, left ventricle; TL, tibia length; LVdP/dtP40, rate of rise of LV pressure at 40 mm Hg; LVEDP, LV end diastolic pressure; LVEDD, LV end diastolic lumen diameter. †P b 0.05 vs corresponding Wt; #P b 0.05 vs corresponding no treatment. Numbers of animals are indicated below the bars.

4.1. Effects of eNOS on pressure-overload LV hypertrophy and dysfunction In agreement with recent data from our laboratory [22], mTAC elevated LV systolic pressure and produced moderate LV hypertrophy and dilation. This was associated with maintained global LV function and minimal effects on fibrosis in Wt mice. Conversely, 8 weeks of sTAC produced robust LV remodeling, and dysfunction with concomitant capillary rarefaction, fibrosis and elevated expression of hypertrophy marker genes. Decompensated LV hypertrophy in sTAC also explains why LV systolic pressure was not further elevated in sTAC compared to mTAC [22]. Consistent with previous observations [15,26], eNOS gene inactivation and overexpression produced hypertension and hypotension, respectively, without influencing basal cardiac function. Moreover, eNOS expression level had no effect on cardiomyocyte cross-sectional area, capillary density and interstitial fibrosis under baseline conditions. Additionally, in accordance with one [27], but not another study [28], loss of eNOS expression did not result in cardiac hypertrophy in shamoperated eNOS-Ko mice. The effects of loss of eNOS expression on the cardiac response to TAC have been previously studied but with equivocal results. One group of investigators found that eNOS deficiency aggravated pressureoverload hypertrophy produced by TAC [18,19], whereas another group observed that loss of eNOS protected the heart against TACinduced pressure-overload [20]. A difference in severity of cardiacoverload was proposed as a possible explanation for this discrepancy [20], but this was not investigated to date. Additionally, differences in genetic background could have influenced the level of eNOS expression in Wt mice. Consequently, in this study we explored the effects of lack as well as overexpression of eNOS in severe and mild pressure-overload through TAC. Interestingly, we found that lack of eNOS attenuated LV dilation, while eNOS overexpression (previously shown to be beneficial in MI [15,16]) aggravated LV dysfunction following mild as well as severe TAC. Our findings additionally demonstrate an inverse relation between eNOS expression level and survival following sTAC and show that eNOS gene inactivation prevented sTAC-induced cardiac fibrosis and capillary rarefaction, as well as elevation in BNP expression. Although mTACinduced mortality was not different between mice with different eNOS expression levels, we found detrimental effects of eNOS on cardiac geometry and function in both mTAC and sTAC. Thus, the described

divergent effects of eNOS on pressure-overload hypertrophy cannot be explained by the severity of pressure-overload. However it should be noted that the same degree of stenosis (i.e. 27 gauge) produced more LV hypertrophy in response to TAC in the present study and in the study of Takimoto et al. [20], in which eNOS deficiency was shown to be protective (LV hypertrophy ~100%), as compared to the studies [18, 19] in which loss of eNOS was reported to be detrimental (LV hypertrophy ~50%). The diverse hypertrophic responses to TAC indicate that perhaps a difference in genetic background influenced the cardiac hypertrophic response but may also have affected TAC-induced eNOS uncoupling and consequently the effects of eNOS on LV dysfunction and remodeling in these studies. The observation that eNOS exerted a detrimental influence in both mild and severe TAC-induced pressure-overload implies that potential differences in severity of cardiac-overload do not explain why eNOS is detrimental in TAC (present study), yet exerts a beneficial influence in cardiac remodeling produced by a myocardial infarction [15,16,29]. In contrast, these findings suggest that the effects of eNOS in cardiac hypertrophy are critically dependent on the type of stimulus for hypertrophy. Likely, the level of eNOS uncoupling is involved in the etiologydependent effect of eNOS but this needs to be further investigated in future studies. Additionally, the divergent effects of eNOS in myocardial infarction and TAC are likely related to the ability to modulate LV afterload. Reduction of systemic vascular resistance by eNOS reduces afterload in myocardial infarction [15,16,29], which facilitates fractional shortening and cardiac output. In contrast, the fixed proximal stenosis in TAC prevents the eNOS-mediated reduction in systemic vascular resistance to influence cardiac afterload, eliminating part of the beneficial effects of eNOS. The nitroso-redox balance appears to be particularly perturbed in pressure-overload produced by aortic stenosis. Consequently in this etiology, uncoupled eNOS and concomitant ROS production severely tilt the balance toward detrimental oxidative stress and nullify the beneficial effects of NO. Under normal physiological conditions, oxidative stress was prevented in eNOS-Tg mice by an increase in eNOS dimerization and concomitant maintenance of a low level of eNOS monomers. In contrast, during pressure-overload, coupling could no longer be maintained and TAC-induced eNOS uncoupling and concomitant disturbance of the nitroso-redox balance resulted in markedly increased ROS production. Consequently, ROS scavenging with NAC had a more

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pronounced effect in eNOS-Tg than in Wt mice and eliminated the negative effects of eNOS on the pressure-overload hypertrophied heart by reducing NOS-mediated oxidative stress. 4.2. Effects of ROS scavenging on pressure-overload-induced LV hypertrophy and dysfunction Excessive ROS generation can contribute to pathological cardiac remodeling [30]. In the normal heart, nicotinamide adenine dinucleotide phosphate oxidase, xanthine oxidase and mitochondrial electron transport, are the main sources of ROS, while detrimental effects of ROS are opposed by antioxidant systems like superoxide dismutases [13,14]. In cardiac pressure-overload, ROS production overwhelms the antioxidant defense system, resulting in oxidative stress [12]. Subsequently, oxidative stress reduces NO bioavailability by directly quenching NO [31] and by promoting uncoupling of eNOS so that eNOS no longer produces NO but instead becomes an important source of O− 2 , thereby instigating a vicious cycle of ROS-induced ROS production. eNOS uncoupling involves both eNOS S-glutathionylation as well as BH4 oxidation and subsequent eNOS monomerization. Additionally, O− 2 production from eNOS following S-glutathionylation also triggers BH4 oxidation and subsequently O− 2 production from the oxygenase domain as well as eNOS monomerization [32]. Because O− 2 production by eNOS following sTAC overruled the beneficial effects of NO production, we subsequently investigated whether prevention of oxidative stress and recoupling of eNOS through ROS scavenging would abolish the observed negative effects of eNOS in sTAC. NAC attenuated eNOS uncoupling and monomerization. Moreover, NAC, as a precursor of de novo GSH synthesis, reduced eNOS Sglutathionylation. Altogether, this resulted in a decrease in L-NAMEinhibitable O− 2 production. However, in accordance with several previous studies [20,24,33], except one [34], we failed to observe significant protection against pressure-overload induced LV remodeling and dysfunction with antioxidant treatment in Wt TAC mice. In contrast to Wt mice, NAC beneficially affected LV geometry and function in eNOS-Tg mice subjected to sTAC, evidenced by a reduction in LV hypertrophy, prevention of LV dilation and improvement of LV function. This was associated with reduced eNOS S-glutathionylation and lower eNOS monomer and O− 2 levels. Nevertheless, ROS scavenging did not unmask a protective effect of eNOS overexpression in eNOS-Tg mice after TAC, as eNOS-Tg mice treated with NAC did not perform better than eNOS-Ko mice. However, since ROS production was not completely abolished by NAC treatment in our mice it seems plausible that this residual O− 2 may have scavenged part of the NO, thereby negating some of its beneficial effects. This would explain why NAC treatment abolished the negative effects of eNOS overexpression but did not unmask beneficial effects of NO. Additionally, counteracting mechanisms in eNOS-Tg mice, such as reduced activity of guanylyl-cyclase [35], may have blunted the beneficial effects of eNOS overexpression in the presence of NAC. It is possible that downstream-factors in the eNOSpathway or indirect stimulation of eNOS may be better therapeutic targets in aortic stenosis-induced LV hypertrophy and dysfunction. Indeed, augmenting the bioavailability of cGMP through PDE5 inhibition [36, 37], or stimulation of eNOS expression through hydrogen sulfide [38], have been shown to protect the heart against pressure-overload induced LV hypertrophy and dysfunction. 4.3. Implications and conclusions Since the detrimental effects of eNOS in TAC appear to be independent of the degree of cardiac overload, beneficial effects of eNOS in other types of cardiac hypertrophy and remodeling cannot simply be explained by differences in severity of hemodynamic overload. Rather, our findings support the concept that the effects of eNOS in cardiac hypertrophy and remodeling are critically dependent on the underlying hypertrophy stimulus, e.g. pressure-overload versus myocardial

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infarction. Clearly, disturbance of the nitroso-redox balance and concomitant eNOS uncoupling and oxidative stress appear to be particularly important in cardiac pressure-overload produced by aortic stenosis. This implies that patients with aortic stenosis could benefit from treatments that target eNOS uncoupling and ameliorate oxidative stress. In conclusion, the present study demonstrates that normal and particularly supranormal eNOS expression levels exert a detrimental influence in TAC-induced LV remodeling and dysfunction, independent of the severity of pressure-overload. This detrimental influence is mediated by eNOS uncoupling through eNOS S-glutathionylation and monomerization, whereas ROS scavenging prevents the eNOS-induced aggravation of LV hypertrophy and dysfunction in TAC. These findings demonstrate that both normal and elevated eNOS expression do not reduce, but rather elevate, pressure-overload induced oxidative stress, and explain why eNOS, which exerts beneficial effects in cardiac remodeling after a myocardial infarction, is detrimental in cardiac hypertrophy and dysfunction resulting from an aortic stenosis. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yjmcc.2015.10.001. Funding This work was supported by grants from the Dutch Heart Foundation [2007B024 to DJD and 2009B023 to AM], and from the Netherlands Organization for Scientific Research (NWO-VIDI 91710383 to AM). Conflict of interest None declared. Acknowledgments The authors gratefully acknowledge Shanti Tel for technical assistance. References [1] U. Forstermann, W.C. Sessa, Nitric oxide synthases: regulation and function, Eur. Heart J. 33 (2012) 829–837. [2] L. Tang, H. Wang, M.T. Ziolo, Targeting NOS as a therapeutic approach for heart failure, Pharmacol. Ther. 142 (2013) 306–315. [3] A.L. Bui, T.B. Horwich, G.C. Fonarow, Epidemiology and risk profile of heart failure, Nat. Rev. 8 (2011) 30–41. [4] J.M. Hare, J.S. Stamler, NO/redox disequilibrium in the failing heart and cardiovascular system, J. Clin. Invest. 115 (2005) 509–517. [5] C. Nediani, L. Raimondi, E. Borchi, E. Cerbai, Nitric oxide/reactive oxygen species generation and nitroso/redox imbalance in heart failure: from molecular mechanisms to therapeutic implications, Antioxid. Redox Signal. 14 (2011) 289–331. [6] W.J. Paulus, C. Tschope, A novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation, J. Am. Coll. Cardiol. 62 (2013) 263–271. [7] Y.J. Taverne, V.J. de Beer, B.A. Hoogteijling, R.P. Juni, A.L. Moens, D.J. Duncker, et al., Nitroso-redox balance in control of coronary vasomotor tone, J. Appl. Physiol. 112 (2012) 1644–1652. [8] P.B. Massion, O. Feron, C. Dessy, J.L. Balligand, Nitric oxide and cardiac function: ten years after, and continuing, Circ. Res. 93 (2003) 388–398. [9] R. Carnicer, M.J. Crabtree, V. Sivakumaran, B. Casadei, D.A. Kass, Nitric oxide synthases in heart failure, Antioxid. Redox Signal. 18 (2013) 1078–1099. [10] J.L. Zweier, C.A. Chen, L.J. Druhan, S-glutathionylation reshapes our understanding of endothelial nitric oxide synthase uncoupling and nitric oxide/reactive oxygen species-mediated signaling, Antioxid. Redox Signal. 14 (2011) 1769–1775. [11] U. Forstermann, T. Munzel, Endothelial nitric oxide synthase in vascular disease: from marvel to menace, Circulation 113 (2006) 1708–1714. [12] A.D. Hafstad, A.A. Nabeebaccus, A.M. Shah, Novel aspects of ROS signalling in heart failure, Basic Res. Cardiol. 108 (2013) 359. [13] H. Tsutsui, S. Kinugawa, S. Matsushima, Oxidative stress and heart failure, Am. J. Physiol. Heart Circ. Physiol. 30 (2011) H2181–H2190. [14] E.D. van Deel, Z. Lu, X. Xu, G. Zhu, X. Hu, T.D. Oury, et al., Extracellular superoxide dismutase protects the heart against oxidative stress and hypertrophy after myocardial infarction, Free Radic. Biol. Med. 44 (2008) 1305–1313. [15] M.C. de Waard, J. van der Velden, N.M. Boontje, D.H. Dekkers, R. van Haperen, D.W. Kuster, et al., Detrimental effect of combined exercise training and eNOS

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