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Am J Physiol Endocrinol Metab 293: E355-E363, 2007. First published April 17, 2007; doi:10.1152/ajpendo.00632.2006
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Angiotensin II-mediated oxidative stress promotes myocardial tissue remodeling in the transgenic (mRen2) 27 Ren2 rat

Adam Whaley-Connell,1,2 Gurushankar Govindarajan,1,2 Javad Habibi,1,2 Melvin R. Hayden,1,2 Shawna A. Cooper,1,2 Yongzhong Wei,1,2 Lixin Ma,1,3 Mahnaz Qazi,1 Daniel Link,1,2 Poorna R. Karuparthi,1,2 Craig Stump,1,2,3,4 Carlos Ferrario,5 and James R. Sowers1,2,3

1School of Medicine and 2Diabetes and Cardiovascular Laboratory, University of Missouri, and 3Harry S. Truman Veterans Affairs Medical Center, Columbia, Missouri; 4The University of Arizona Diabetes Center, Tucson, Arizona; and 5Wake Forest University School of Medicine, Winston-Salem, North Carolina

Submitted 21 November 2006 ; accepted in final form 12 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Angiotensin II (ANG II) contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. ANG II stimulation of the ANG type 1 receptor (AT1R) generates reactive oxygen species via NADPH oxidase, which facilitates this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo AT1R blockade (AT1B) (valsartan) or superoxide dismutase/catalase mimetic (tempol) treatment in a rodent model of chronically elevated tissue levels of ANG II, the transgenic (mRen2) 27 rat (Ren2). Ren2 rats overexpress the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6–7 wk old) male Ren2 and age-matched Sprague-Dawley rats were treated with valsartan (30 mg/kg), tempol (1 mmol/l), or placebo for 3 wk. Heart tissue NADPH oxidase (NOX) activity and immunohistochemical analysis of subunits NOX2, Rac1, and p22phox, heart tissue malondialdehyde, and insulin-stimulated protein kinase B (Akt) activation were measured. Structural changes were assessed with cine MRI, transmission electron microscopy, and light microscopy. Increases in septal wall thickness and altered systolic function (cine MRI) were associated with perivascular fibrosis and increased mitochondria in Ren2 on light and transmission electron microscopy (P < 0.05). AT1B, but not tempol, reduced blood pressure (P < 0.05); significant improvements were seen with both AT1B and tempol on NOX activity, subunit expression, malondialdehyde, and insulin-mediated activation/phosphorylation of Akt (each P < 0.05). Collectively, these data suggest cardiac oxidative stress-induced structural and functional changes are driven, in part, by AT1R-mediated increases in NADPH oxidase activity.

reduced nicotinamide adenine dinucleotide phosphate oxidase; malondialdehyde; Akt

ANGIOTENSIN II (ANG II) is known to play an important role in the development of cardiac hypertrophy (3, 21, 24, 38, 39, 41, 47), interstitial cardiac fibrosis (13, 35, 40, 46, 48), and left ventricular (LV) dysfunction (10, 19, 27, 41). Components of the renin-angiotensin system (RAS), including ANG II, constitute an autocrine/paracrine cardiac tissue RAS (3, 13, 21, 35, 38–41, 47, 48). ANG II receptors have been characterized in cardiomyocytes and cardiac fibroblasts (13, 35, 48) and in coronary arteries (35, 51). Although both ANG type 1 and 2 receptors (AT1R and AT2R) are present in cardiac and coronary tissue, many of the adverse effects of ANG II on growth, myocardial fibrosis, and LV dysfunction are mediated through the AT1R (13, 17, 24, 29, 48, 51).

Accumulating evidence suggests that many of the adverse cardiac effects of ANG II are triggered by redox cycling of reactive oxygen species (ROS), generated in part by an NADPH oxidase-dependent pathway (5, 9, 15, 16, 24, 31, 35, 36, 43). In cardiomyocytes, ANG II stimulates a phagocytic-type NADPH oxidase, which is composed of a membrane-bound gp91phox (now known as NOX2) and the p22phox heterodimer and four regulatory subunits: p40phox, p47phox, p67phox, and Rac1 (32–35). ANG II activation of the NADPH oxidase enzyme affects cell signaling responses and facilitates remodeling and hypertrophy in the heart (5, 8, 16, 36). The role of ANG II-induced NADPH oxidase activation and generation of ROS in cardiac pathology is underscored by the fact that inhibitors of NADPH oxidase and free radical scavengers (45, 51) and AT1 receptor blockers (AT1B) (13, 35, 48, 51) attenuate these adverse effects of ANG II.

This investigation addressed the hypothesis that chronic ANG II overexpression in the heart is associated with structural and functional abnormalities that are driven by NADPH oxidase-mediated generation of ROS. Additionally, this study sought to test the notion that oxidative stress and structural and functional abnormalities could be attenuated by in vivo treatment with by AT1B (valsartan) and/or a superoxide dismutase (SOD)/catalase mimetic (tempol) in a rodent model of chronically elevated tissue levels of ANG II, the transgenic (mRen2) 27 rat (Ren2). This rodent model overexpresses the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular injury (6, 17, 37, 49, 50). Furthermore, it has been shown that insulin resistance (6) and glomerular injury (50) are related to AT1R-mediated increases in ROS in skeletal muscle and glomerular tissue, respectively. Our group (6) also showed that abrogation of enhanced oxidative stress corrected the impaired insulin-stimulated metabolic signaling [i.e., the protein kinase B (Akt) pathway and glucose transport in skeletal muscle]. In addition, we posited that enhanced AT1R-mediated oxidative stress would be associated with structural and functional changes in the heart, and abrogation of oxidative stress would then attenuate these abnormalities in the heart.

We utilized immunostaining to evaluate NADPH oxidase subunit expression, assessed NADPH oxidase activity via spectrophotometric analysis, and measured malondialdehyde (MDA) as a surrogate end product of oxidative stress. Structural changes were evaluated by in vivo cine MRI, transmission electron microscopy (TEM), and histological, immunohistochemical, and biochemical techniques. We have further evaluated the impact of chronic ANG II stimulation of NADPH oxidase on insulin signaling via Akt and the consequence of in vivo treatment with a SOD/catalase mimetic (tempol) and AT1B (valsartan).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and Treatments

All animal procedures were approved by the University of Missouri animal care and use committees and housed in accordance with National Institutes of Health guidelines. Male transgenic Ren2 rats and male Sprague-Dawley controls were kindly provided by Dr. Carlos M. Ferrrario, Wake Forest University School of Medicine (Winston-Salem, NC), through the Transgenic Core Facility (supported in part by National Heart, Lung, and Blood Institute Grant P01 HL-51952). Ren2 rats (n = 6; 6–7 wk of age) and age-matched Sprague-Dawley rats (n = 6) were randomly assigned to untreated [Ren-2 control (Ren2-C) and Sprague-Dawley control (SDC) groups], valsartan-treated (n = 6 each; Ren2-V and SDV, respectively), or tempol-treated (n = 6 each; Ren2-T and SDT, respectively) groups. Ren2-V and SDV animals received valsartan (30 mg·kg–1·day–1) in their drinking water for 21 days. Ren2-T and SDT animals received tempol (1 mM) in their drinking water for 21 days (6, 50). Animals were injected with 2 U of insulin 5 min before death to evaluate Akt signaling (7).

Systolic Blood Pressure

Systolic blood pressure (SBP) was determined at the end of treatment using the tail-cuff method (student oscillometric recorder; Harvard Systems) three times for 10–15 min after acclimatization and restraint conditioning for 48 h (6, 23, 50).

Measurement of NADPH Oxidase Activity

NADPH oxidase activity was determined in plasma membrane fractions as previously described (6). Aliquots of membrane and cytosolic fractions (12.5–100 µg proteins) were incubated with NADPH (100 µM) at 37°C. NADPH activity was determined by measuring the conversion of radical detector (Cayman Chemical) in the absence and presence of NADPH inhibitor diphenylene iodonium sulfate (DPI; 500 µM) using spectrophotometric (450 nm) techniques.

Lipid Peroxidation Measured in Myocardial Tissue

To measure products of lipid peroxidation, tissue MDA levels were determined (25, 50). Butylated hydroxytoluene (5 mM) was added to heart tissue (100 mg) to prevent new lipid peroxidation. Samples were then homogenized in a buffer solution on ice and centrifuged at 4°C at 15,000 rpm for 10 min. The supernatant was collected for MDA and O-phthaldialdehyde (OPA) protein assays. Supernatant (200 µl) was used to measure free MDA with the use of an MDA-586 spectrophotometric assay kit. Total protein was measured by OPA fluorometric assay.

Immunofluorescent Studies

Harvested LVs were immersed and fixed in 3% paraformaldehyde (50). After fixation, tissues were placed in histological cassettes and dehydrated with ethanol, infiltrated with low-melting (50°C) paraplast, and embedded in high-melting (56°C) paraplast. Blocks were sectioned, deparaffinized in CitriSolv, and rehydrated in ethanol and HEPES wash buffer. The first section was washed (3 x 15 min) with HEPES wash buffer and then mounted with Mowiol. The second section was washed and incubated with 1:100 primary antibodies in 10-fold diluted blocking agent, and third and fourth sections were washed and kept in the blocker. Over the course of 48 h, a fifth, sixth, and seventh section were incubated with 1:100 goat gp91phox (NOX2) (Santa Cruz Biotechnology, Santa Cruz, CA), 1:100 mouse Rac1 antibody (Upstate Cell Signaling), and 1:100 goat p22phox antibody (Santa Cruz Biotechnology), respectively, in 10-fold diluted blocker. After 24 h, the slides were washed (3 x 15 min), and a seventh section was mounted with Mowiol. Other sections were incubated with 1:300 Alexa fluor rabbit anti-goat 647 (Molecular Probes, Eugene, OR) in 10-fold diluted blocker except the sixth, which was stained with 1:300 of Alexa fluor goat anti-mouse 647. After 4 h, the slides were washed, mounted with Mowiol, and examined with a laser confocal scanning microscope; images were captured with the use of Laser-sharp software (Bio-Rad), and signal intensities were measured by MetaVue analysis (23, 50).

In Vivo Cine MRI

Scans were performed on Ren2 and Sprague-Dawley rats at 4–5 and 8–9 wk of age using a Varian 7T horizontal bore MRI (Varian, Palo Alto, CA) with ECG and respiratory gating (SA Instruments, Stony Brook, NY). Animals were anesthetized with 2–3.5% isoflurane with the use of a nose-cone nonrebreathing system supplying continuous oxygen. Wall thickness measurements were determined with Image-J (NIH, Bethesda, MD) on a single transverse axial image at 0-ms delay after the R wave and with averaging of five septal width measurements per rat. Ejection fraction was assessed on a single long-axis plane captured at 12 equally spaced time points of the cardiac cycle. LV volume was determined with a modified ellipsoid equation (26). The largest volume was considered end diastolic (EDV) and lowest as end systolic (ESV). Ejection fraction was calculated as (EDV – ESV/EDV) x 100.

Light Microscopy

Fixed paraffin sections of LV were evaluated with Verhoeff-van Gieson stain, which stains elastin (black), nuclei (blue black), collagen (red), and connective tissue (yellow), as previously described (23). Slides were analyzed with a Nikon50i microscope, and x4, x10, and x40 images were captured with a Cool Snapcf camera. Morphometric analysis was performed with MetaVue software. A total of 15 images of coronary artery cross sections where taken from each animal. In each image, the perimeter of the adventitia, media, and lumen were traced, and the percent area was then calculated by subtracting the combined areas for media and lumen from the total area and then dividing by the total area.

TEM Methods

LV heart tissue was thinly sliced and placed immediately in primary EM fixative (2% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cadodylate buffer, pH 7.35), as previously described (50). A Pelco 3440 laboratory microwave was utilized for secondary fixation, with acetone dehydration and Epon-Spurr's resin infiltration. Specimens were placed on a rocker overnight at room temperature, embedded the following morning, and polymerized at 60°C for 24 h. A Leica Ultracut UCT microtome with a 45° Diatome diamond knife was used to prepare 85-nm thin sections. The specimens were then stained with 5% uranyl acetate and Sato's triple lead stain. A JOEL 1200-EX transmission microscope was utilized to view all renal samples.

Three fields were randomly chosen per rat to obtain three x60,000 images/LV-containing cross-sectional views of the mitochondria. Mitochondria were visualized and identified based on the identification of cristae. Then three (450 nm x 450 nm) images were cropped out of each x60,000 image obtained. These cropped images were then analyzed with Image J (a public domain Java image processing program made possible by the NIH). By viewing the mitochondria in cross section in each cropped image, we calculated, measured, and recorded the number in a blinded fashion (25, 50).

Citrate Synthase Activity

Mitochondrial fractions of LV prepared during sucrose differential centrifugation were freeze thawed three times to ensure mitochondrial disruption and were diluted as necessary. Samples were analyzed in an incubation buffer consisting of Tris (pH 8.3), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and oxaloacetate, and the assay was initiated by the addition of acetyl-CoA. Samples were analyzed at 37°C using linear kinetics (405 nm). Amount of DTNB converted per minute was determined with the molar extinction coefficient for DTNB. Rates for each sample were normalized to mitochondrial fraction proteins determined by Bradford assay.

Western Blot Analysis

Hearts were homogenized with a glass-on-glass Dounce homogenizer in sucrose homogenization buffer and centrifuged at 1,000 g to remove connective tissue. Protein concentrations were measured by OPA as above. Supernatants were analyzed under denaturing conditions with SDS-PAGE using a mini-protean 2 electrophoresis system. Protein (20 µg) was mixed with 5 µl of loading buffer and incubated at 92°C for 5 min. Samples were then loaded into wells of 4–15% precast gels (Bio-Rad) and run at 180 V for 35 min. Proteins were transferred onto a polyvinylidene difluoride membrane in a wet transfer system (Bio-Rad) at 30 V overnight. Blots were blocked with 5% nonfat dry milk in Tris-buffered saline-Tween 20 (TBST) for 4 h. Membranes were incubated with primary antibodies, anti-Akt (rabbit polyclonal; 1:500) antibody, and anti-phosphorylated Akt (serine 473) (rabbit polyclonal; 1:1,000) antibody in 5% nonfat dry milk in TBST overnight. Membranes were washed and incubated for 60 min with secondary antibody anti-rabbit horseradish peroxidase linked at dilutions of 1:5,000 (Akt) and 1:2,000 (phosphorylated Akt) in 5% nonfat dry milk in TBST. Blots were incubated with enhanced chemiluminescence for 1 min, and the protein bands were quantified by Quanty One one-dimensional analysis software (Bio-Rad). Equal loading of total proteins in each lane was verified by amido-black staining using a densitometer.

Statistical Analysis

All values are expressed as means ± SE. Statistical analyses were performed with SPSS 13.0 (SPSS, Chicago, IL) using ANOVA with Fisher's least significant difference test as appropriate and Student's t-test for paired analysis. Phosphorylated Akt was plotted against NADPH oxidase activity and Rac for linear regression analysis. Significance was accepted as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Systolic Blood Pressure

At the end of the treatment period (9–10 wk of age), there were improvements in SBP with valsartan in both the Ren2-V and SDV groups (each P < 0.05) (Table 1), although not with tempol treatment.


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  		Table 1. Systolic blood pressure and oxidative stress parameters

 
Oxidative Stress in Ren2 Heart Tissue

NADPH oxidase activity. NADPH oxidase activity, as measured by radical detector conversion, was significantly higher in the Ren2-C than in SDC group and attenuated by valsartan (Ren2-V) and tempol (Ren2-T) treatments (P < 0.05). This activity was abrogated by DPI, a specific inhibitor of NADPH oxidase in all groups (P < 0.05), thus validating increased plasma membrane NADPH oxidase activity in the Ren2 heart.

End product of lipid peroxidation as measured by MDA levels. MDA levels were elevated in Ren2-C myocardial tissue compared with that shown in SDC (P < 0.05) (Table 1) and significantly decreased by both treatment paradigms (P < 0.05). Furthermore, MDA levels were lower in Ren2-T than in Ren2-V (P < 0.05).

NADPH oxidase subunits. Evaluation of NADPH subunits NOX2, Rac1, and p22phox via immunohistochemistry revealed increased expression of each subunit in Ren2-C, as measured by gray-scale intensities (40.2 ± 7.2, 38.8 ± 6.4, 23.6 ± 2.9, respectively), compared with that shown in SDC (26.7 ± 1.0, 21.7 ± 1.2, 16.5 ± 0.8, respectively) (P < 0.05) (Fig. 1). Reduced expression of these subunits, as observed in both Ren2-V and Ren2-T groups, corroborates reductions seen in NADPH oxidase activity (NOX2: 27.9 ± 3.2 and 19.0 ± 2.3, respectively; Rac1: 22.4 ± 3.0 and 18.6 ± 2.6, respectively; and p22phox: 18.4 ± 1.3 and 15.6 ± 0.2, respectively) (each P < 0.05).


Figure 1
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  		Fig. 1. A: immunohistochemical (IHC) staining of NADPH oxidase subunits NOX2, Rac1, and p22phox. B: gray-scale intensity measures of IHC in A. SDC, SDV, and SDT, control, valsartan-treated, and tempol-treated Sprague-Dawley rats, respectively. Ren2-C, Ren2-V, and Ren2-T, control, valsartan-treated, and tempol-treated Ren2 rats, respectively. *P < 0.05 compared with Sprague-Dawley control (SDC). **P < 0.05 compared with Ren2 control (Ren2-C) at 4 wk.

 
Myocardial Remodeling in the Ren2 Heart

Cine MRI. In vivo cine MRI was used to measure septal wall thickness and systolic ejection fraction (Fig. 2). There was no difference in wall thickness in the Ren2-C (1.25 ± 0.06 mm) compared with SDC (1.21 ± 0.03 mm; P > 0.05) at 4–5 wk. However, as expected, the systolic ejection fraction in Ren2-C animals was higher than in SDC (74.3 ± 5.1 vs. 52 ± 2.1%; P < 0.05). Septal wall hypertrophy was present in Ren2-C animals (1.65 ± 0.05 nm) compared with that shown in SDC (1.35 ± 0.07 nm; P < 0.05) (Fig. 2A). In contrast to SDC (63.2 ± 5.7%), there was a reduction in systolic ejection fraction in the Ren2-C (65.0 ± 3.4%; P < 0.05), with a change of –9.3 ± –8.4 compared with at 4 wk (Fig. 2B).


Figure 2
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  		Fig. 2. In vivo cine MRI measures of heart. A: septal wall thickness. B: systolic ejection fraction (EF) before initiation and end of treatment in the Ren2. A depicts the septal wall thickness in control animals at 4 wk (pretreatment) and 8 wk (posttreatment), and B depicts systolic ejection fraction before initiation and at end of treatment. *P < 0.05 compared with SDC at 4 wk (Ren2-C and SDC, respectively). **P < 0.05 compared with SDC at 8 wk.

 
Light microscopy and TEM. Perivascular fibrosis, as represented by percent area fibrosis on Verhoeff-van Gieson staining on light microscopy, was increased in Ren2-C animals (70 ± 3.2%) compared with SDC (57 ± 3.7%; P < 0.05) and improved in Ren2-V (53 ± 4.4%) compared with that shown in Ren2-C animals (P < 0.05) (Fig. 3, A and B). Furthermore, Ren2-T animals displayed a trend toward reduction of perivascular fibrosis (62.7 ± 0.03%).


Figure 3
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  		Fig. 3. Perivascular fibrosis in the Ren2 heart. A: light microscopy of structural changes consistent with perivascular fibrosis using Verhoeff-van Gieson staining. B: percent areas of perivascular fibrosis in SDC and in Ren2 groups. *P < 0.05 compared with SDC. **P < 0.05 compared with Ren2-C. C and D: transmission electron microscopy (TEM) images demonstrating pericapillary fibrosis and the increased number of mitochondria seen in the Ren2 (D) compared with SDC (C). In C, SDC is shown at x10,000 and x20,000 (boxed area), demonstrating a normal capillary. In D, Ren2-C is shown at x10,000 and x40,000 (boxed area), demonstrating pericapillary fibrosis, with asterisks outlining the area of fibrosis.

 
Ultrastructural analysis with TEM (Fig. 3, C and D) corroborated light microscopy findings (Fig. 3A). Pericapillary fibrosis emanated from the perivascular area to the interstitium of the endomysium between cardiomyocytes (Fig. 3D) compared with that shown in SDC animals (Fig. 3C), similar to that observed on light microscopy (Fig. 3A). Figure 3C displays a normal capillary without pericapillary fibrosis in the SDC at x10,000 and at x20,000 (boxed area), and Fig. 3B displays pericapillary fibrosis in the Ren2-C at x10,000 and at x40,000 (boxed area); asterisks outline the area of pericapillary fibrosis. Both longitudinal banded collagen and cross-sectional collagen are also shown.

Mitochondrial Content in Ren2 Heart

TEM images taken at x10,000 (Fig. 4) demonstrated striking changes in the mitochondria (Fig. 4A). There were increased numbers of mitochondria in Ren2-C (83.6 ± 7.6) compared with in SDC animals (67.2 ± 5.2; P < 0.05) (Fig. 4, A and B), with trends to improvement in the Ren2-V (68.2 ± 4.8) but not in the Ren2-T (80.1 ± 7.9). Increased mitochondria was confirmed with citrate synthase activity, an enzyme used to quantify mitochondrial content. Similar to TEM analysis, there were increases in Ren2 (536.1 ± 60.7) compared with SDC animals (398.2 ± 26.3; P < 0.05) (Fig. 4C), which demonstrates a trend to improvement in the Ren2-V (444.7 ± 48.6) but not in the Ren2-T (556.3 ± 73.1).


Figure 4
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  		Fig. 4. Mitochondrial content in the Ren2 heart. A: TEM images at x10,000 represent increased mitochondria content observed in Ren2 heart tissue compared with SDC with improvements in the Ren2-V group, as quantified in B. C: mitochondrial content quantified by citrate synthase activity. *P < 0.05 compared with SDC.

 
Akt Phosphorylation in Ren2 Heart Tissue

Animals were stimulated with 2 U of insulin before death to evaluate Akt signaling. The ratio of Western blot densities of phosphorylated Akt to total Akt in heart tissue of Ren2-C was less than that of the SDC (P < 0.05) (Fig. 5, A and B). Phosphorylated Akt was inversely correlated with NADPH oxidase activity (r2 = 0.39) and Rac1 protein expression by immunohistochemistry (r2 = 0.37) (Fig. 5C). Akt phosphorylation was improved in Ren2-V and Ren2-T animals (each P < 0.05). Furthermore, Akt phosphorylation was increased in the SDT compared with SDC and SDV animals (each P < 0.05).


Figure 5
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  		Fig. 5. A: Western blot analysis of phosphorylated Akt (pAkt) compared with total Akt (tAkt) in cytosolic fractions. B: densitometry analysis of pAkt/tAkt. *P < 0.05 compared with SDC and SDV. **P < 0.05 compared with Ren2-C and SDV. #P < 0.05 compared with both Ren2-C, Ren2-V, and SDT. C: linear regression analysis of NADPH oxidase activity vs. insulin-stimulated Akt in the Ren2 rat. D: linear regression analysis of Rac1 vs. insulin-stimulated Akt in the Ren2 rat.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The data from this investigation indicate that the hypertensive, insulin-resistant transgenic (mRen2) 27 (Ren2) rat manifests increased oxidative stress in concert with structural and functional changes in the heart. Alterations visible by light microscopy and TEM include perivascular fibrosis, as previously observed, and increased mitochondrial content (20, 23). AT1B of the Ren2 abrogated these structural abnormalities, suggesting that these changes are mediated by the AT1R. Electron microscopy revealed ultrastructural remodeling in the Ren2 heart compared with Sprague-Dawley rats. These changes included increased mitochondria, consistent with limited previous observations in hypertrophied hearts displaying increases in oxidative stress (45). This may represent an adaptive response to greater energy requirements due to increased afterload. These TEM changes are the first such observations made in a model of chronic ANG II overexpression. Furthermore, this is the first investigation demonstrating that AT1B lessens the abnormalities in concert with attenuation of oxidative stress. The cardiac structural changes observed in this model of tissue ANG II overexpression occurred in parallel with LV remodeling, as evidenced by increases in septal wall thickness and diminished systolic function. The loss of systolic contractility in Ren2-C suggests a diminution of systolic function that parallels the cardiac structural alterations.

In concert with these structural and functional abnormalities, the Ren2 rats manifested increased cardiac NADPH oxidase activity that was abrogated by DPI and further validated it as membrane NADPH oxidase. Furthermore, this increase in NADPH oxidase activity was abolished with AT1B or the SOD/catalase mimetic (6, 20).

Although treatment with AT1B was associated with a reduction in SBP, there was not a significant reduction in SBP after tempol treatment. Furthermore, that tempol treatment reduced NADPH oxidase activity but not structural abnormalities suggests that this ultrastructural remodeling was driven through ANG II stimulation of AT1R, independent of SBP and cardiac afterload. This is the first report to document that increased myocardial activity of NADPH oxidase can be abrogated in the absence of concurrent changes in blood pressure and consequent increases in cardiac afterload, in an in vivo model of enhanced tissue RAS activity.

In conjunction with enhanced NADPH oxidase activity, there was an increase in lipid peroxidase products in the Ren2, which was similarly reduced by treatment with either an AT1B or a SOD mimetic. Increased cardiac levels of oxidized products have not been previously observed in the Ren2 rat, but studies have demonstrated that ANG II increases ROS in cultured myocardial fibroblasts and in cardiomyocytes (4, 27, 30). The heart possesses a relatively low endogenous antioxidant capacity as contributed by both enzymatic and nonenzymatic free radical scavengers and antioxidants, thus rendering it susceptible to oxidative stress, with attendant structural and functional abnormalities (20). Indeed, increased oxidative stress in the heart has been linked to ventricular hypertrophy, systolic and diastolic functional abnormalities, and abnormal metabolic signaling (20, 33). The observation that in vivo treatment with AT1B or a SOD mimetic normalized NADPH oxidase and MDA products is the first report that these therapeutic approaches can comparably attenuate consequent pathological effects of chronic AT1R activation.

Immunohistochemical analysis of cardiac tissue NADPH oxidase subunits NOX2, p22phox, and Rac1 demonstrated an association with increased NADPH oxidase activity. Furthermore, either AT1B or a SOD mimetic significantly reduced the observed increases in all three NADPH oxidase subunits. ANG II stimulation of Rac1 expression has been previously observed in prior studies (11, 28). The small GTP binding protein Rac1 is critical for the assembly and function of the multicomponent NADPH oxidase complex (11, 27). Prior studies have shown that treatment of rat cardiomyocytes with ANG II induces hypertrophy, which is partly dependent on increased Rac1 translocation to cell membranes and consequent activation of NADPH oxidase (11, 14, 28). Recent studies suggest that ANG II-induced ROS production is dependent on Rac1 activation, which cascades into JNK and p38 signaling pathways that promote growth (32). In the present study, there was a relationship between Rac1 expression, increased NADPH oxidase activity, and diminished Akt activation in response to insulin. In hepatocytes, Akt signaling suppresses Rac-regulated ROS production (34), and in endothelial cells Rac1 regulates the production of the potent anti-oxidant manganese SOD (1). These data collectively suggest a negative feedback loop between Rac/ROS formation and Akt and the SOD systems in various tissues.

The changes in NADPH oxidase activity and subunits in this investigation were inversely related to insulin-stimulated Akt phosphorylation, an indicator of Akt2 activation (2, 12, 42). Akt is a highly conserved protein kinase with a pleckstrin homology domain, which is required for phosphorylation and/or activation by phosphoinositol 3-kinase (42). Activation of Akt involves translocation to the plasma membrane where activated phosphoinositide-dependent kinases 1 and 2 phosphorylate Akt on threonine 308 and serine 473, with both phosphorylations required for maximum Akt activation (42). Activated Akt then phosphorylates intracellular proteins that regulate growth, metabolism, survival, and cardiac function (2, 12, 18, 42). Akt activity in the rodent heart is regulated by insulin, nutritional status, pressure overload, and redox status (2, 12, 18, 42). Optimal Akt signaling, although it promotes physiological growth, inhibits pathological hypertrophy (2, 12, 18, 42). Our observations that Akt serine 473 phosphorylation in response to insulin is decreased in a rodent model of ANG II-stimulated oxidative stress, hypertrophy, and reduced myocardial contractility are consistent with these previous observations (2, 12, 18, 42). Indeed, there was an inverse relationship between Rac1 expression, NADPH oxidase activity, and Akt phosphorylation. These findings, in concert with several prior studies, support a reciprocal relationship between reduced insulin-mediated Akt signaling and oxidative stress in the heart (2, 12, 18, 42). Thus enhanced ANG II generation of ROS in the heart, as well as insulin-sensitive skeletal muscle (6), contributes to an insulin resistance state in these insulin-sensitive tissues. Insulin-stimulated Akt signaling is crucial for glucose transport in the heart and in skeletal muscle (44). Thus this ANG II-induced abnormality may contribute to altered cardiac mechanical-energy coupling in the Ren2 model of chronic ANG II overexpression.

Collectively, these data suggest that, in the Ren2 rat, there is a relative shift to heightened myocardial oxidative stress and remodeling, which are corrected by in vivo treatment with AT1B. In interpreting the results of this, or any study, that investigate blood pressure-mediated effects, measurement of SBP is susceptible to large variability. However, the overall consistent trends in our elevated SBP in the Ren2 that improved with AT1B are consistent with the literature and support our conclusions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grants R01 HL-073101-01A1 (J. R. Sowers) and P01 HL-51952 (C. Ferrario), the Veterans Affairs Merit System (0018) for J. R. Sowers and Advanced Research Career Development for C. Stump, and Novartis Pharmaceuticals. Male transgenic Ren2 rats and male Sprague-Dawley controls were kindly provided by Dr. Carlos M. Ferrrario, Wake Forest University School of Medicine (Winston-Salem, NC), through the Transgenic Core Facility supported in part by National Heart, Lung, and Blood Institute Grant P01 HL-51952.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the support provided by the Veterans Affairs Biomolecular Imaging Center at the Harry S. Truman Veterans Affairs Hospital and the University of Missouri-Columbia. The authors also acknowledge the electron microscope core facility for help and tissue preparation. Special thanks go to Allison Farris for help in preparation of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Whaley-Connell, Univ. of Missouri-Columbia School of Medicine, Division of Nephrology, MA436, DC043.0, One Hospital Dr., Columbia, MO 65212 (e-mail: whaleyconnella{at}health.missouri.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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