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Up-regulation of p27kip1 contributes to Nrf2-mediated protection against angiotensin II-induced cardiac hypertrophy

Jinqing Li, Cheng Zhang, Yifan Xing, Joseph S. Janicki, Masayuki Yamamoto, Xing Li Wang, Dong-Qi Tang, Taixing Cui
DOI: http://dx.doi.org/10.1093/cvr/cvr010 315-324 First published online: 18 January 2011

Abstract

Aims Nuclear factor erythroid-2-related factor 2 (Nrf2) appears to be a negative regulator of maladaptive cardiac remodelling and dysfunction; however, a potential of the Nrf2-mediated cardiac protection in diverse pathological settings remains to be determined. This study was aimed to explore the role of Nrf2 in angiotensin II (Ang II)-induced cardiac hypertrophy.

Methods and results Littermate wild-type (WT) and Nrf2 knockout (Nrf2−/−) mice were administered Ang II via osmotic mini-pumps for 2 weeks to induce cardiac hypertrophy. Elevation of blood pressure by the continuous Ang II infusion was comparable between WT and Nrf2−/− mice. Relative to WT mice, however, Nrf2−/− mice exhibited exaggerated myocardial oxidative stress with an impaired induction of a group of antioxidant genes and increased cardiac hypertrophy in response to the sustained Ang II stimulation. In cultured cardiomyocytes, adenoviral overexpression of Nrf2 shRNA enhanced Ang II-induced reactive oxygen species (ROS) production and protein synthesis, whereas adenoviral overexpression of Nrf2 exerted opposite effects. Moreover, Nrf2 deficiency exacerbated Ang II-induced down-regulation of p27kip1 expression in the heart via a mechanism of post-transcriptional regulation. In contrast, adenoviral overexpression of Nrf2 increased p27kip1 protein but not mRNA expression and reversed Ang II-induced down-regulation of p27kip1 protein expression in cultured cardiomyocytes by suppressing ROS formation. Finally, the enhancement of Ang II-induced hypertrophic growth due to the Nrf2 deficiency was negated by overexpressing p27kip1 in cultured cardiomyocytes.

Conclusion The Nrf2-p27kip1 pathway serves as a novel negative feedback mechanism in Ang II-induced pathogenesis of cardiac hypertrophy, independent of changes in blood pressure.

  • Nrf2
  • p27kip1
  • Oxidative stress
  • Angiotensin II
  • Cardiac hypertrophy

1. Introduction

Angiotensin II (Ang II), a key effector of the renin–angiotensin system (RAS), plays a critical role in the regulation of vascular tone, blood pressure, and blood volume homeostasis.1,2 There are at least two G-protein-coupled receptors with seven transmembrane domains to mediate the actions of Ang II, designated as Ang II type 1 (AT1R) and type 2 receptors. Most of the known effects of Ang II are mediated by the AT1 receptor. However, sustained activation of the Ang II-AT1R axis contributes to cardiac remodelling, hypertrophy, and dysfunction.3,4 Accordingly, the inhibition of Ang II production by angiotensin-converting enzyme (ACE) inhibitors or the blockade of Ang II-mediated actions by AT1R blockers (ARBs) has been demonstrated to be beneficial in patients with heart failure or other forms of cardiovascular disease. Unfortunately, the blockade of RAS by ACE inhibitors and ARBs leads to Ang II escape and AT1 receptor up-regulation. Moreover, the utilization of ACE inhibitors and ARBs frequently causes adverse skin reactions, including angiooedema, pruritus, bullous eruptions, urticaria, rashes, photosensitivity, and hair loss.5,6 Thus, a further understanding of Ang II/AT1R downstream signalling, which could facilitate the development of novel therapeutic approaches for cardiovascular disease with the overactivation of Ang II signalling, is of clinical significance.

Indeed, Ang II signalling in the heart has been extensively investigated for several decades.7 One of the notable findings is that the Ang II-induced formation of reactive oxygen species (ROS) causes oxidative stress leading to cardiac hypertrophy and heart failure.79 An NADPH oxidase-dependent ROS formation and the subsequent activation of p38 mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK), and nuclear factor (NF)-κB have been identified as important mechanisms by which Ang II induces cardiomyocyte hypertrophy and cardiac dysfunction. On the other hand, up-regulation of antioxidant enzymes, including haeme oxygenase-1 (HO-1) and thioredoxin 2 (Txn-2), has been demonstrated to inhibit Ang II-induced oxidative stress and cardiac hypertrophy,10,11 suggesting an important role of endogenous antioxidant defences in the control of Ang II-mediated redox signalling in the heart. Thus, it is likely that myocardial antioxidant systems scavenge excess ROS in the heart, thereby suppressing Ang II-induced myocardial oxidative stress and subsequent maladaptive cardiac remodelling and dysfunction. Nevertheless, the precise interplay between Ang II and antioxidant defence systems in the pathogenesis of cardiac disease remains unclear. Of note, the failure of large clinical trials using ROS scavengers of antioxidant vitamins for the treatment of cardiovascular disease suggests that non-selective scavenging of ROS is ineffective or even harmful.1215 These results highlight a unique regulatory role of endogenous antioxidant defences in the control of redox signalling for cardiovascular homeostasis. Accordingly, studies with a focus on the interaction between Ang II and intrinsic antioxidant pathways in the heart will not only extend our understanding of myocardial oxidative stress but also potentially lead to therapeutic interventions for the prevention of pathologic cardiac hypertrophy and dysfunction.

Nuclear factor erythroid-2-related factor 2 (Nrf2) belongs to the Cap ‘n’ Collar family of basic leucine zipper transcription factors, which include NF-E2, Nrf1-3, and Bach1-2.16 Nrf2 is a pleiotropic protein that binds to a cis-acting enhancer sequence known as the antioxidant response element with a core nucleotide sequence of 5′-RTGACNNNGC-3′ to control the basal and inducible expression of a battery of antioxidant genes and other cytoprotective phase II detoxifying enzymes, such as HO-1, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferases, NAD(P)H:quinone oxidoreductase (NQO1), NQO2, γ-glutamylcysteine synthase , and glucuronosyltransferase. In addition to the wealth of evidence showing Nrf2 to be a major regulator of cellular defences against various pathological stresses in diverse organs, such as lung and kidney, we have demonstrated that it is a critical endogenous inhibitor of maladaptive cardiac remodelling and dysfunction through the co-ordination of a group of its downstream antioxidant genes, including GPx, HO-1, NQO-1, Txn-1, thioredoxin reductase-1 (Txnrd-1), SOD-2, and SOD-3, to suppress myocardial oxidative stress.17 Moreover, recent studies have revealed that Nrf2 is critical for the cellular defence against high-glucose-induced oxidative damage in cardiomyocytes18 and the proteosome-inhibitor-mediated cardioprotection in vivo.19 These results underscore a central role for Nrf2 in orchestrating antioxidant defences of the heart. Furthermore, these findings also raise an intriguing issue whether or not Nrf2 plays a role in Ang II-induced oxidative stress and the subsequent hypertrophic remodelling in the heart.

In the present study, we demonstrate that Nrf2 is a negative regulator of Ang II-induced cardiac hypertrophy, independent of changes in blood pressure. At a molecular level, we provide for the first time evidence that activation of Nrf2 serves as a feedback mechanism against an axis of Ang II-induced oxidative stress and the subsequent down-regulation of p27kip1 leading to cardiac hypertrophy.

2. Methods

2.1. Animals

Littermate wild-type (WT; Nrf2+/+) and Nrf2 knockout (Nrf2−/−) mice were generated by breeding heterozygous Nrf2 (Nrf2+/−) mice as previously described.17 Male mice at the age of 8 weeks were sham-operated or subcutaneously infused with Ang II (Sigma-Aldrich) at a rate of 1.4 μg/kg/min for 2 weeks by osmotic mini-pumps (Alzet model 1002, Alza Corp.). All of the animal procedures were conducted in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at University of South Carolina, USA.

2.2. Echocardiographic and haemodynamic analysis

Blood pressures were measured in conditioned, unanaesthetized mice using the tail-cuff method (Hatteras Instruments, MC4000 Blood Pressure Analysis System).17 Echocardiography was performed on anaesthetized (isoflurane) mice, using the Vevo 770 High-Resolution Imaging System (VisualSonics Inc.) with a 37.5 MHz high-frequency linear transducer, as previously described.17

2.3. Histological and immunochemical analysis

Hearts were cannulated via the left ventricular apex, cleared by perfusion with phosphate buffered saline at 90 mmHg, arrested in diastole with 60 mM KCl, fixed by perfusion with 10% formalin, and embedded in paraffin. Paraffin sections were prepared (5 μm, Leica RM2030, rotary microtome) and stored at room temperature until staining. Histology and immunohistochemistry were performed as previously described.17 Primary antibodies included a rabbit Nrf2 polyclonal antibody (H-300, sc-13032, Santa Cruz Biotechnology, Inc.), a mouse 4-hydroxy-2-nonenal (4-HNE) antibody (ab48506, Abcam Inc.), a mouse 8-hydroxydeoxyguanosine (8-OHdG) antibody (sc-660369, Santa Cruz Biotechnology, Inc.), and a rabbit polyclonal p27kip1 antibody (sc-528, Santa Cruz Biotechnology, Inc.), respectively. Cardiomyocytes were stained with a rabbit tropomyosin I antibody (ab55915, Abcam Inc.) or a mouse monoclonal cardiac myosin heavy chain antibody (ab15, Abcam Inc.). Nuclei were labelled with 4′, 6-diamidino-2-phenylindole (DAPI; Cat. No. D9542, Sigma-Aldrich).

2.4. Cell culture and adenovirus infection

Rat neonatal cardiac myocytes were isolated and cultured, and treated as previously described.17 Nrf2 gain- or loss-of-function approaches were achieved by infecting the cells with adenovirus of green fluorescent protein (GFP), murine Nrf2, scramble shRNA, or rat Nrf2 shRNA at a dose of 20 MOI in serum-free PC-1 media (Lonza Walkersville, Inc., Walkersville, MD, USA) for 48 h. For double infection experiments, each adenovirus was used at a dose of 10 MOI. Adenovirus of p27kip1 was purchased from Cell Biolabs, Inc.

2.5. [3H]Leucine incorporation and intercellular ROS detection

Rat neonatal cardiac myocytes were infected with adenovirus and treated as indicated. [3H]leucine incorporation was determined as described previously.17 Generation of superoxide (O2), hydrogen peroxide (H2O2), and peroxynitrite (ONOO•) was measured by using the fluorescent probes dihydroethidium (DHE), 5-(and-6)-carboxy-2'7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA), and dihydrorhodamine (DHR)-123,2022 respectively. DHE, a non-fluorescent membrane-permeant probe, interacts with O2, leading to the liberation of membrane-impermeant ethidium cations that fluoresce on intercalating with nuclear DNA. Carboxy-H2DCFDA diffuses through the cell membrane and is hydrolysed by intracellular esterases to non-fluorescent dichlorofluorescin (carboxy-DCFH) that is rapidly oxidized to highly fluorescent dichlorofluorescein by ROS such as H2O2, hydroxyl radical (OH), and hydroperoxides. DHR-123 is oxidized by ONOO to the highly fluorescent product rhodamine in vitro. Rat neonatal cardiomyocytes were cultured in serum-free PC-1 media for 24 h, pre-treated with or without Tiron (10 μM) for 30 min, and then treated with or without Ang II (1 μM) and/or Tiron (10 μM) in Hank's buffered salt solution for 1 h. The fluorescent probes including DHE (10 µM), H2DCFDA (10 µM), and DHR-123 (10 µM) were added to the media at the same time as Ang II. The cells were observed under Nikon E600 fluorescent microscope and photographed. ROS levels in the cardiomyocytes were semi-quantified by measuring integrated optical density (IOD) per field as previously.17

2.6. Western blot analysis

Cell lysates were subjected to western blot analysis using an anti-p27kip1 antibody (sc-528, Santa Cruz Biotechnology, Inc.), anti-Txnrd-1 (ab16840, Abcam Inc.), anti-NQO-1 (ab34173, Abcam Inc.), anti-HO-1 (SPA-896, Stressgen Biotechnologies), and anti-Txn-1 (ab26320, Abcam Inc.) as described previously.17

2.7. Reverse transcription-PCR and quantitative real-time PCR

The genotypes of mice were verified by examining the size of the polymerase chain reaction (PCR) products: Nrf2+/+ (700 bp), Nrf2−/− (400 bp), and Nrf2+/− (700 and 400 bp). Expression levels of target genes were normalized by concurrent measurement of glyceraldehyde-3-phosphate dehydrogenase mRNA levels as previously described.17 Primers for genotypes: 5′-TGGACGGGACTATTGAAGGCTG-3′ (sense for Nrf2+/+ and Nrf2−/−), 5′-CGCCTTTTCAGTAGATGGAGG-3′ (antisense for Nrf2+/+), and 5′-GCGGATTGACCGTAATGGGATAGG-3′ (antisense for LacZ). Primers that were used for quantitative (Q-PCR) are summarized in Supplementary material online, Table SI.

2.8. Statistical analysis

Data are shown as mean ± SEM. Results were compared by ANOVA, followed by Bonferroni test for multiple comparisons. Differences were considered significant at P < 0.05.

3. Results

3.1. Nrf2 deficiency exaggerates Ang II-induced cardiac hypertrophy

To determine a role of Nrf2 in Ang II-induced cardiac hypertrophy, we analysed the myocardial hypertrophic responses elicited by chronic Ang II infusion in WT and Nrf2−/− mice. Ang II infusion for 2 weeks increased blood pressure similarly in WT and Nrf2−/− mice (Table 1) and Supplementary material online, Figure S1. Ang II infusion led to concentric cardiac hypertrophy, including an increased heart weight to body weight ratio (HW/BW); increased cardiomyocyte cross sectional area; increased thickening of the diastolic left ventricle posterior wall (LVPWd) and the diastolic interventricular septum (IVSd); and decreased diastolic left ventricular internal dimension (LVIDd), whereas left ventricular (LV) systolic function was preserved (Table 1). In addition, there was an up-regulation of atrial natriuretic factor (ANF), brain natriuretic factor (BNP), and beta-myosin heavy chain (β-MHC; Table 1). However, the Ang II-induced hypertrophic changes were exaggerated in Nrf2−/− mice (Table 1).

View this table:
Table 1

Body weight, blood pressure, heart rate, echocardiographic results, and pathology of WT and Nrf2−/− mice 2 weeks after Ang II infusion

WTNrf2−/−
Ang II (−)Ang II (+)P-valueAng II (−)Ang II (+)P-value
 BW (g)37.9 ± 1.15 (13)30.2 ± 1.25 (19)A38.3 ± 2.04 (12)28.1 ± 2.36 (21)B
 BP systolic (mmHg)115.0 ± 4.24 (11)159.8 ± 6.70 (12)A118.4 ± 1.88 (11)143.6 ± 6.99 (12)B
 BP diastolic (mmHg)92.6 ± 3.73 (11)133.8 ± 7.11 (12)A89.1 ± 2.89 (11)133.1 ± 7.14 (12)B
 BP MAP (mmHg)99.7 ± 3.72 (11)141.4 ± 6.88 (12)A98.8 ± 2.31 (11)138.3 ± 3.45 (12)B
 HR (b.p.m.)516.6 ± 15.7 (11)597.1 ± 32. 8 (12)571.8 ± 23.9 (11)621.5 ± 22.4 (12)
Echocardiography(13)(17)(12)(16)
 IVSd (mm)0.73 ± 0.020.96 ± 0.04A0.76 ± 0.021.04 ± 0.03B C
 LVIDd (mm)4.44 ± 0. 073.72 ± 0.10A4.57 ± 0.053.66 ± 0.11B
 LVIDs (mm)3.30 ± 0.402.65 ± 0.52A3.35 ± 0.372.65 ± 0.44B
 LVPWd (mm)0.84 ± 0.020.94 ± 0.03A0.85 ± 0.021.10 ± 0.04B C
 FS (%)26.1 ± 1.3029.1 ± 2.5526.9 ± 1.5527.9 ± 1.70
Pathology
 HW/BW4.44 ± 0.21 (6)5.69 ± 0.12 (10)A4.09 ± 0.08 (6)6.46 ± 0.09 (11)B C
 MC CSA (µm2)152 ± 3.85 (6)210 ± 14.62 (7)A148 ± 8.21 (6)271 ± 17.07 (8)B C
 Lung wt/BW6.96 ± 0.41 (6)8.11 ± 0.60 (10)7.33 ± 0.61 (6)9.89 ± 0.68 (11)B C
 mRNAs(7)(9)(6)(10)
  ANF0.97 ± 0.252.60 ± 5.33A0.72 ± 1.614.63 ± 6.67B C
  BNP0.98 ± 0.143.08 ± 0.42A0.54 ± 0.122.40 ± 0.39B
  α-MHC1.02 ± 0.090.72 ± 0.061.03 ± 0.050.86 ± 0.08
  β-MHC1.00 ± 0.132.63 ± 0.60A0.68 ± 0.073.87 ± 0.66B C
  SERCA1.05 ± 0.100.91 ± 0.161.00 ± 0.110.93 ± 0.12
  • IVSd, interventricular septum diastolic; LVIDd, left ventricular internal dimension diastolic; LVIDs, left ventricular internal dimension systolic; LVPWd, left ventricular posterior wall diastolic; FS, fractional shortening; BW, body weight; BP, blood pressure; MAP, mean artery pressure; HR, heart rate; b.p.m., beat per minute; HW/BW, heart weight/body weight ratio; MC CSA, left ventricular myocyte cross-sectional area; Lung wt/BW, lung weight/body weight ratio; ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; α-MHC, alpha-myosin heavy chain; β-MHC, beta-myosin heavy chain; SERCA, sarcoplasmic reticulum calcium ATPase2a. A and B, P < 0.05, Ang II (+) vs. Ang II (−); C, P < 0.05, Nrf2−/− Ang II (+) vs. WT Ang II (+).

3.2. Nrf2 deficiency impairs endogenous antioxidant defence in Ang II-induced hypertrophic heart

In view of the anti-oxidative defence role of Nrf2 in the heart,17 we investigated the role of Nrf2 in regulating Ang II-induced myocardial oxidative stress. Ang II infusion increased myocardial levels of 4-HNE—a marker of lipid peroxidation—and 8-OHdG—a marker of DNA oxidation, indicating elevated levels of oxidative stress in the heart (Figure 1). The Ang II-induced oxidative stress levels in the heart were markedly enhanced as a result of Nrf2 deficiency (Figure 1). In response to the sustained Ang II stimulation, several Nrf2 downstream genes, including HO-1, Txn-1, and Txnrd-1, in WT but not in Nrf2−/− hearts were up-regulated at both mRNA and protein levels (Figure 2 and Supplementary material online, Figure S2). In addition, the chronic Ang II infusion-enhanced Nrf2 nuclear translocation in WT myocardium (data not shown).

Figure 1

Oxidative stress in the hearts of WT and Nrf2−/− mice following 2 weeks of Ang II infusion. Upper panel: representative confocal microscopic images of left ventricular (LV) 4-HNE staining and 8-OHdG staining. Areas positive for 4-HNE and 8-OHdG are shown in red. Nuclei (blue) were labelled with DAPI. Cardiomyocytes (green) were marked using anti-Tropomyosin I antibody against cardiac myocyte tropomyosin. Lower panel: Levels of 4-HNE and 8-OHdG were semi-quantified by measuring IOD of eight randomly chosen fields in each myocardial tissue section. Number of hearts (n) are indicated.

Figure 2

Effect of Ang II on Nrf2 downstream gene expression in the heart. Male littermate WT and Nrf2−/− mice at 8 weeks of age were infused with Ang II (1.4 µg/kg/min) for 2 weeks. The hearts were harvested for Q-PCR analysis of HO-1, NQO-1, Txn-1, and Txnrd-1 as described in ‘Methods’.

3.3. Nrf2 acts as a negative regulator of Ang II-induced ROS formation and hypertrophic growth in cardiomyocytes

We have demonstrated that Nrf2 is an essential regulator of ROS formation in cardiomyocytes.17 On the other hand, it is well established that Ang II induces cardiomyocyte hypertrophy at least partly via increasing ROS production.23,24 Thus, we determined the role of Nrf2 in regulating Ang II-operated ROS formation and hypertrophic growth in isolated cardiomyocytes using adenoviral Nrf2 gain- and loss-of-function techniques as previously described.11 A forced activation of Nrf2 via adenoviral overexpression of Nrf217,25 suppressed basal ROS formation in a primary culture of rat neonatal cardiomyocytes, whereas suppressing Nrf2 activity via adenoviral Nrf2 knockdown exerted an opposite effect (Figure 3A). While we observed that Ang II (1 h stimulation)-induced ROS formation was suppressed by the activation of Nrf2 in rat neonatal cardiomyocytes, we did not observe any significant enhancement of the Ang II-induced ROS formation by the suppression of Nrf2 activity (Figure 3A). Moreover, Nrf2 overexpression suppressed and Nrf2 knockdown increased the basal leucine uptake in rat neonatal cardiomyocytes; however, these effects were negated by N-acetyl-l-cysteine (NAC), an ROS scavenger (Figure 3B). Ang II-induced leucine update was inhibited by Nrf2 overexpression and enhanced by Nrf2 knockdown in rat neonatal cardiomyocytes (Figure 3B). On the other hand, Ang II (6 h stimulation)-activated Nrf2-driven transcriptional activity in rat neonatal cardiomyocytes (data not shown).

Figure 3

Effect of Nrf2 overexpression and knockdown on Ang II-induced ROS production and hypertrophic growth in rat neonatal cardiomyocytes. (A): Representative images (upper panel) and quantitative analysis (lower panel) of intracellular ROS production in vehicle and Ang II (1 µmol/L)-treated cardiac myocytes that were infected with 20 MOI of Ad-βGal, Ad-Nrf2, Ad-cont shRNA, or Ad-Nrf2 shRNA. Measurements of IOD are representative of three separate experiments (n = 4). Ad, adenovirus; βGal, beta galactosidase; cont, control. (B): Cells were infected with 20 MOI of Ad-GFP, Ad-Nrf2, Ad-cont shRNA, or Ad-Nrf2 shRNA for 2 days, and then stimulated with vehicle, NAC (300 µmol/L), or Ang II (0.1 µmol/L) for 1 day. NAC was added 30 min before Ang II stimulation. [3H]Leucine uptake was measured as described in ‘Methods’. *P < 0.05 vs. Ad-GFP vehicle; P < 0.05 vs. Ad-cont shRNA vehicle; #P < 0.05 vs. Ad-Nrf2 shRNA vehicle. Results are representative of three separate experiments (n = 4).

3.4. Nrf2 negatively regulates Ang II/ROS formation/p27kip1 protein degradation pathway in the heart

It has recently been demonstrated that down-regulation of p27kip1 expression is critical for Ang II-induced cardiac hypertrophy.26 Therefore, we investigated the possible role of Nrf2 in Ang II-mediated down-regulation of p27kip1 expression in the heart. Consistent with the previous report,26 we observed that Ang II infusion down-regulated p27kip1 protein expression without affecting its mRNA expression (Figure 4 and Supplementary material online, Figure S3). Importantly, we found that Nrf2 deficiency enhanced the Ang II-induced down-regulation of myocardial p27kip1 protein expression alone (Figure 4 and Supplementary material online, Figure S3).

Figure 4

Expression of p27kip1 in the hearts of WT and Nrf2−/− mice 2 weeks after Ang II infusion. (A) Upper panel: representative confocal microscopic images of p27kip1 staining in the left ventricle (LV) of WT and Nrf2−/− mice that were infused with vehicle or Ang II for 2 weeks. p27kip1 protein (red) was stained with a rabbit polyclonal p27kip1 antibody. Cardiomyocytes (green) were stained with mouse anti-cardiac myosin heavy chain antibody. Nuclei were labelled with DAPI. Lower panel, semi-quantitative measurements of IOD were performed as described in Figure 1. Numbers of hearts are indicated. (B) Western blot analysis of p27kip1 expression. LV lysates of WT and Nrf2−/− mice that were infused with vehicle or Ang II for 2 weeks were subjected to western blot analysis of p27kip1 as described in ‘Methods’. Results are representatives of four separated experiments.

To further explore the role of Nrf2 in regulating the Ang II/p27kip1 pathway in the heart, we determined the effects of forced activation or inactivation of Nrf2 on p27kip1 expression in isolated cardiomyocytes. Expression of p27kip1 protein was up-regulated by Nrf2 overexpression in a primary culture of rat neonatal cardiomyocytes (Supplementary material online, Figure S4); however, expression of p27kip1 mRNA levels was only minimally affected by either Nrf2 overexpression or Nrf2 knockdown (data not shown). Notably, overexpression of Nrf2 reversed the Ang II-induced down-regulation of p27kip1 protein expression in rat neonatal cardiomyocytes (Figure 5A).

Figure 5

Role of Nrf2 in Ang II-mediated down-regulation of p27kip1 in cardiomyocytes. (A) Rat neonatal cardiomyocyte infected with Ad-GFP (20 MOI) or Ad-Nrf2 (20 MOI) for 2 days and then stimulated with Ang II (0.1 µM) for additional 16 h. p27kip1 staining and IOD measurement were performed as described above. *P < 0.05 vs. Ad-GFP Ang II (−), n = 4. Results are representative of two separated experiments. (B) Effect of Tiron on Ang II-induced p27kip1 protein expression in rat neonatal cardiomyocytes. Rat neonatal cardiomyocytes were pre-treated with or without Tiron (10 µM) for 30 min, and then stimulated with or without Ang II (0.1 µM) for 16 h. Expression of p27kip1 protein was measured by immunochemical staining as described in ‘Methods’. For each sample, IOD was quantified in eight randomly chosen fields. *P < 0.01 vs. Ang II (−), n = 4. (C) Rat neonatal cardiomyocytes were serum starved for 24 h, and then stimulated with Ang II (0.1 µM), PDGF (20 ng/mL), and Tiron (10 µM) as indicated for 16 h. Tiron was added 30 min before the administration of Ang II and PDGF. p27kip1 protein levels were determined by western blot. Results are representative of four separate experiments.

It has been documented that ROS is linked to either up- or down-regulation of p27kip1 expression depending on cell types.2729 However, the role of ROS in the Ang II-mediated down-regulation of p27kip1 in cardiomyocytes remains to be determined. Thus, we examined the effect of Tiron, a ROS scavenger, on Ang II-induced ROS formation as well as on p27kip1 down-regulation in a primary culture of rat neonatal cardiomyocytes. Firstly, we established the ability of Tiron to scavenge Ang II-induced ROS formation in rat neonatal cardiomyocytes. We observed that a 30 min pre-treatment with Tiron (10 µM) prevented the Ang II-induced generation of superoxide (O2), ONOO•, and H2O2 in the cells (Supplementary material online, Figure S5). Secondly, we examined the effect of Tiron pre-treatment on Ang II-induced down-regulation of p27kip1 protein expression in rat neonatal cardiomyocytes with a positive control of the down-regulation of p27kip1 protein expression by platelet-derived growth factor (PDGF).30 Of significance, both western blot analysis and immunochemical staining revealed that the Ang II-induced down-regulation of p27kip1 expression was completely recovered by Tiron pre-treatment (Figure 5B and C). Moreover, Ang II-induced down-regulation of p27kip1 protein expression was also reversed by NAC (Supplementary material online, Figure S6).

3.5. Nrf2 inhibits Ang II-induced cardiomyocyte hypertrophy via up-regulating p27kip1

To determine the role of p27kip1 in Nrf2-mediated inhibition of cardiomyocyte hypertrophy, we examined the effect of overexpression of p27kip1 on the Nrf2 deficiency-enhanced hypertrophic growth in rat neonatal cardiomyocytes. Since we demonstrated that adenovirus of Nrf2 shRNA (10 MOI) effectively knocked down Nrf2 expression in rat neonatal cardiomyocytes17 and adenovirus of p27kip1 (10 MOI) induced dramatic p27kip1 protein expression in the cardiomyocytes (Figure 6), we applied a double infection of adenovirus of control, p27kip1, and Nrf2 shRNA with a non-toxic total dose of 20 MOI. As we demonstrated in Figure 3B, Nrf2 deficiency enhanced both basal and Ang II-induced [3H]leucine uptake in cardiomyocytes (Figure 6). However, overexpression of p27kip1 inhibited the Ang II-induced [3H]leucine uptake in both control and Nrf2 deficient cells (Figure 6).

Figure 6

Effect of p27kip1 overexpression on Ang II-induced hypertrophic growth in cardiomyocytes with or without Nrf2 deficiency. (A) (left): Rat neonatal cardiomyocytes were infected with 10 MOI of Ad-GFP, Ad-p27kip1, Ad-cont shRNA, and Ad-Nrf2 shRNA for 2 days, and then stimulated with Ang II (0.1 µM) for 1 day. [3H]Leucine uptake was performed as described in ‘Method’. *P < 0.05 vs. Ad-GFP + Ad-cont shRNA (−); ns, non-significant, n = 4. (A) (right): Representative analysis of three western blot analysis of p27kip1 protein expression.

4. Discussion

In the present study, there are several novel findings regarding Ang II signalling and the regulation of cardiac hypertrophy as follows: (i) Ang II down-regulates p27kip1 protein expression through increased ROS formation in cardiomyocytes thereby contributing to cardiac hypertrophy; (ii) activation of Nrf2 suppresses whereas inactivation of Nrf2 enhances the axis of Ang II-oxidative stress-p27kip1 inhibition-cardiomyocyte hypertrophy, and (iii) Nrf2 plays a minimal role in regulating Ang II-induced increase in blood pressure.

It has been firmly established that sustained Ang II infusion increases blood pressure and induces cardiac hypertrophy.31,32 At the molecular level, Ang II activates NADPH oxidase with increased ROS formation to induce cardiac hypertrophy, including cardiomyocyte hypertrophy, myocardial fibrosis, and apoptosis, leading to heart failure.79 Considering a critical role of Nrf2 in antioxidant defences,16 it was not surprising to find that Nrf2 knockout enhances Ang II-induced cardiac hypertrophy by further increasing oxidative stress in the heart. Recently, we have demonstrated that Nrf2 is a negative regulator of oxidative stress in cardiomyocytes.17 We further observed that Ang II activated Nrf2 in the heart and Nrf2 deficiency exaggerated Ang II-induced cardiac hypertrophy. A robust increase in ROS formation appeared prior to Nrf2 activation in Ang II-stimulated cardiomyocytes in vitro. Thus, it is conceivable that the increased Nrf2 activity in the myocardium is secondary to the Ang II-induced oxidative stress. Additionally, Nrf2 overexpression suppressed basal ROS formation and blocked Ang II-induced ROS production in cultured cardiomyocytes, whereas Nrf2 knockdown dramatically enhanced basal ROS formation without further increases in Ang II-stimulated ROS production. These results demonstrate that Nrf2 not only plays a pivotal role in the control of basal ROS levels but also serves as a negative regulator of Ang II-induced oxidative stress in cardiomyocytes. However, the lack of enhancement of Ang II-induced ROS production in Nrf2-deficient cardiomyocytes is intriguing. Although not conclusive at this point, a plausible explanation is likely that Ang II-induced ROS production is masked by the dramatic increases in ROS levels due to Nrf2 deficiency in cultured cardiomyocytes. Accordingly, we observed that NAC, a ROS scavenger, hardly affects the Nrf2 overexpression-suppressed but reversed the Nrf2 knockdown-enhanced basal leucine uptake, while Nrf2 overexpression blocked and Nrf2 knockdown-enhanced Ang II-induced leucine uptake. These results clearly demonstrate that Nrf2 is a negative regulator of Ang II-induced cardiomyocyte hypertrophy via suppressing oxidative stress. On the other hand, the inhibitory role of Nrf2 in regulating myocardial fibrosis and apoptosis could also be postulated from the recent findings of Nrf2-mediated anti-fibrotic and anti-apoptotic actions in cardiac fibroblasts and cardiomyocytes.17,18,33 However, the observation that Nrf2 deficiency had no impact on Ang II-induced increase in arterial blood pressure was unexpected, because we have predicted that loss of Nrf2 will exaggerate Ang II-induced oxidative stress in vasculature leading to hypertension.34 Probably, Nrf2 exerts tissue-specific actions, or Nrf2 deficiency could be compensated by enhancing other endogenous antioxidant defence systems in vasculature. Nevertheless, this finding suggests a major role for Nrf2 in cardiac hypertrophy in Ang II-dependent hypertension. In addition, we observed that, following 4 weeks of Ang II infusion, cardiac diastolic function was impaired in Nrf2−/− mice but not in WT mice (data not shown). The early onset of cardiac diastolic dysfunction in Nrf2−/− mice suggests that Nrf2 is one of the critical inhibitors of the transition from physiological to Ang II-induced pathological myocardial hypertrophy and cardiac dysfunction. Therefore, our results support the notion that decreased antioxidant capacity in association with increased oxidative stress in the myocardium contributes to the transition from compensated hypertrophy to overt heart failure.35 Importantly, these results suggest that Nrf2 activation provides a negative feedback for maladaptive cardiac hypertrophy and heart failure rather than increase in blood pressure in the setting of sustained Ang II stimulation. Taken together, these results demonstrate that Nrf2 is a novel negative regulator of Ang II-mediated cardiomyocyte hypertrophy and maladaptive cardiac hypertrophic remodelling at least partly via the suppression of oxidative stress, independent of changes in blood pressure.

Mechanistically, the downstream events of Ang II-induced oxidative stress contributing to pathological cardiac hypertrophy remain unclear. Although a critical role of down-regulation of p27kip1 expression for Ang II-induced cardiac hypertrophy has been recently demonstrated,26 whether or not Ang II-induced oxidative stress contributes to p27kip1 down-regulation in the heart is unclear. In this context, we demonstrated that Nrf2 deficiency exaggerated Ang II-induced myocardial oxidative stress and p27kip1 down-regulation, and cardiac hypertrophy, indicating a novel axis of Ang II-oxidative stress-p27kip1 down-regulation-cardiomyocyte hypertrophy with Nrf2 as a negative regulator. Nrf2 gain- and loss-of-function approaches demonstrated that Nrf2 is an endogenous inhibitor of the Ang II-mediated down-regulation of p27kip1 via a post-transcriptional mechanism in the heart. On the other hand, we found that scavenging ROS reversed Ang II-induced down-regulation of p27kip1 protein expression in isolated cardiomyocytes, suggesting that Ang II-induced oxidative stress down-regulates p27kip1 protein expression in the heart. It is worthy to note that Ang II down-regulates p27kip1 protein expression predominantly via enhancing its proteosomal degradation in cardiomyocytes.26 Collectively, these results indicates that Ang II triggers post-transcriptional p27kip1 degradation via increasing ROS formation in the heart and Nrf2 activation inhibits the Ang II-induced p27kip1 protein degradation via suppressing ROS formation. Of importance, we observed that overexpression of p27kip1 blocked Ang II-induced hypertrophic growth in Nrf2 deficient cardiomyocytes, suggesting that p27kip1 is a downstream effector of Nrf2 in the suppressing of Ang II-induced cardiomyocyte hypertrophy, providing critical evidence to support the notion that Nrf2 acts a negative regulator for the axis of Ang II/oxidative stress/p27kip1 down-regulation/cardiomyocyte hypertrophy. In contrast, the increased leucine incorporation in non-stimulated Nrf2 deficient cardiomyocytes could not explain the lack of cardiac hypertrophy in the heart of Nrf2−/− mice at basal conditions. A precise reason for the discrepancy is unknown. Perhaps it is because of multiple and complex compensatory regulations in the setting of Nrf2 deficiency in the heart, which is absent in isolated cardiomyocytes or maybe because of a critical role of Nrf2 in regulating basal protein turnover via an as yet-unknown mechanism independent of ROS-mediated cardiomyocyte hypertrophy. These possibilities still need further investigation.

Clinically, our results highlight a novel therapeutic potential by targeting Nrf2 against Ang II-mediated cardiac hypertrophy and heart failure. Consistent with our finding that Ang II-induced hypertrophic signalling was inhibited by the forced activation of Nrf2, recent studies have documented that sulphoraphane and triterpenoid 2-cyano-3, 12-dioxooleana-1, 9 (11)-dien-28-oic acid (CDDO)-imidazolide (two established Nrf2 activators) protect the heart from ischaemic injury in rats36 and cigarette-smoke-induced cardiac dysfunction in mice,37 respectively. We have also demonstrated that dihydro-CDDO-trifluoroethyl amide (dh404) is a novel potent Nrf2 activator and suppresses Ang II-induced oxidative stress in cardiomyocytes38. Thus, the impact of Nrf2 activation by either genetic or pharmacological approaches on Ang II-induced cardiac hypertrophy and dysfunction in vivo needs to be delineated.

It should be noted that the exact molecular mechanisms of the Ang II-mediated Nrf2 activation in the heart have not been fully dissected in the present study. However, several hypotheses could be postulated from the known mechanisms of Nrf2 activation and from the established redox signalling of Ang II in the cardiovascular system.79,16 Firstly, Ang II-induced oxidative stress leads to direct modification of cysteine residues of Keap1 thereby decreasing the Keap1-mediated ubiquinitation and degradation of Nrf2. Accordingly, Nrf2 activity is enhanced. Secondly, Ang II-induced oxidative stress results in the modulation of cysteine residues in Nrf2 per se that regulates its nuclear translocation. Thirdly, Ang II-activated kinases phosphorylates Nrf2, thereby increasing its nuclear translocation. Moreover, it has been reported that G-alpha12 and G-alpha13 transmit a JNK-dependent signal for Nrf2 ubiquitination and degradation, whereas G-alpha13 regulates Nrf2 phosphorylation, which is negatively balanced by G-alpha12.39 Since G-alpha 12/13-mediated ROS production is essential for Ang II-induced JNK activation in cardiomyocytes,40 the possibility that G-alpha 12 and 13 proteins serve as switches for the Ang II-induced Nrf2 activation in the heart remains to be addressed.

In conclusion, our present study demonstrates that Nrf2 is a critical regulator for maintaining structural and functional integrity of the heart in the setting of sustained Ang II stimulation. Mechanistically, it is likely that Nrf2 protects against maladaptive cardiac remodelling and dysfunction by repressing ROS production and up-regulating p27kip1 expression. A further dissection of molecular mechanisms by which Ang II regulates Nrf2 activity in the heart will provide not only novel insights into Ang II-operated redox signalling but also new perspectives for the rational design and development of a novel class of Nrf2 activators for the treatment of cardiovascular disease, especially Ang II-induced pathogenesis.

Funding

This work was supported by a grant from the American Heart Association (0865101E).

Acknowledgements

We thank Xiaoyu Dong for her invaluable technical expertise in conducting these experiments.

Conflict of interest: none declared.

References

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