Involvement of Inducible Nitric Oxide Synthase in - CiteSeerX

AJP-Heart Articles in PresS. Published on February 14, 2002 as DOI 10.1152/ajpheart.00872.2001

Involvement of Inducible Nitric Oxide Synthase in Cardiac Dysfunction With Tumor Necrosis Factor-α α

Hajime Funakoshi, MD 1; Toru Kubota, MD, PhD 1; Yoji Machida, MD 1; Natsumi Kawamura, MD 1; Arthur M. Feldman, MD, PhD 2; Hiroyuki Tsutsui, MD, PhD 1; Hiroaki Shimokawa, MD, PhD 1; Akira Takeshita, MD, PhD 1

Department of Cardiovascular Medicine, Kyushu University Garaduate School of Medical Sciences, Fukuoka, Japan 1, and the Cardiovascular Institute, University of Pittsburgh Medical Center, Pittsburgh, PA 2

Running head:

Funakoshi et al.

iNOS in TNF Cardiomyopathy

Correspondence:

Toru Kubota, MD, PhD. Department of Cardiovascular Medicine Kyushu University Graduate School of Medical Sciences 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan 812-8582 Tel: +81-92-642-5360

Fax: +81-92-642-5375

e-mail: [email protected]

Copyright 2002 by the American Physiological Society.

Funakoshi et al. Manuscript #00872-2001

ABSTRACT Transgenic mice with cardiac-specific overexpression of tumor necrosis factor (TNF)-α (TG) develop dilated cardiomyopathy with myocardial inflammation. The purpose of this study was to investigate the role of nitric oxide (NO) in this mouse model of cardiomyopathy. Female TG and wild-type mice (WT) at the age of 10 weeks were studied. Expression and activity of inducible nitric oxide synthase (iNOS) were significantly increased in the TG myocardium, while those of endothelial NOS were not altered. Majority of the iNOS protein was isolated in the interstitial cells. A selective iNOS inhibitor ONO-1714 was used to examine the effects of iNOS induction on myocardial contractility. Echocardiography and left ventricular pressure measurements were performed. Both fractional shortening and +dP/dtmax were significantly suppressed in TG. Although ONO-1714 did not change hemodynamic parameters or contractility at baseline, it significantly improved β-adrenergic inotropic responsiveness in TG. These results indicate that induction of iNOS may play an important role in the pathogenesis of cardiac dysfunction in this mouse model of cytokine-induced cardiomyopathy.

KEYWORDS cytokine; heart failure; transgenic mice

Funakoshi et al. Manuscript #00872-2001 1

Tumor necrosis factor (TNF)-α is a proinflammatory cytokine that is involved in a variety of cardiovascular diseases, including endotoxin shock, acute myocarditis, cardiac allograft rejection, myocardial infarction, and congestive heart failure (8,23). Recent studies indicate that the heart itself is a source of TNF-α in these disorders (11,21,30,31). To investigate the pathophysiological role of myocardial production of TNF-α, we made transgenic mice that overexpress TNF-α specifically in the heart under the control of α-myosin heavy chain promoter (20). These mice developed ventricular hypertrophy, ventricular dilatation, interstitial infiltrates, interstitial fibrosis, attenuation of adrenergic inotropic responsiveness, and re-expression of atrial natriuretic factor in the ventricle. Furthermore, the mice that died spontaneously demonstrated exceptional dilatation of the heart, organized atrial thrombus, and massive pleural effusion, suggesting that they died of congestive heart failure. Several aspects of these results have since been confirmed by another laboratory (4). These results indicate that myocardial production of TNF-α may play an important role in the pathogenesis of cardiac dysfunction. However, the mechanisms by which TNF-α damages the myocardium remain undefined. Recent basic and clinical studies have shown that nitric oxide (NO) exerts versatile effects on cardiovascular function (16) (1,18). NO is a free radical gas synthesized from L-arginine by a family of nitric oxide synthases (NOS), including neuronal (nNOS), inducible (iNOS), and endothelial isoforms (eNOS). Both nNOS and eNOS are constitutively expressed, whereas iNOS is induced by inflammation, allograft rejection, and cytokine activation (16). Recent studies have indicated that iNOS is increased in the failing human heart (6,11,30). A small amount of NO produced by nNOS and eNOS seems cardioprotective by improving myocardial perfusion and inhibiting apoptosis (17). In contrast, a large amount of NO produced by iNOS may be cardiotoxic by suppressing myocardial contractility (18) and promoting apoptosis (17). Since TNF-α is a potent inducer of iNOS (16), the negative


inotropic effect of TNF-α may be mediated by the enhanced production of NO in the myocardium. However, conflicting results have been reported regarding the effects of NOS inhibition on cytokine-induced cardiac dysfunction: some investigators reported that negative inotropic effects of cytokines were ameliorated by NOS inhibition (3,9,10,27), while others did not (25,33). The present study was thus designed to investigate the role of NO in our mouse model of cardiomyopathy caused by cardiac-specific overexpression of TNF-α. The results indicated that iNOS was highly induced in the myocardium, playing an important role in the pathogenesis of cardiac dysfunction caused by cardiac-specific overexpression of TNF-α.


METHODS Animal Model. Transgenic mice with cardiac-specific overexpression of TNF-α (TG) (19,20,22) and wild-type littermates (WT) were studied. All the mice were 10-week-old female unless otherwise mentioned. We did not use TG males because they did not tolerate physiological experiments due to the premature development of congestive heart failure (15). This experiment was reviewed by the Committee of the Ethics on Animal Experiment, Kyushu University Graduate School of Medical Sciences and carried out under the control of the Guideline for Animal Experiment, Kyushu University and the Law (No.105) and Notification (No.6) of the Government.

Northern Blot Analysis. Total RNA was extracted from the left ventricle by using an acid guanidium thiocyanate-phenol chloroform method. RNA samples (10 µg) were electrophoresed in a formaldehyde-agarose gel, and transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech, INC). The membrane was then hybridized with 32

P-labeled probes listed as follows: nNOS, nucleotides 2582 to 3169 (GenBank, D14552);

iNOS, 235 to 744 (GenBank, U43428); eNOS, 2882 to 3287 (GenBank, U53142); and 18S ribosomal RNA (19,20). The radioactivity of hybridized bands was quantified by a MacBAS Bioimage Analyzer (Fuji Film, Tokyo, Japan). Results of the cDNA hybridization were normalized to that of the 18S probe to correct for differences in RNA mass and efficiency of transfer. Data were in turn normalized to the mean of the WT samples, arbitrarily set at 1.

Western Blot Analysis.

Tissue samples from the left ventricle were homogenized in

Tris-buffer containing proteinase inhibitors. The protein samples were then separated in a 7.5% resolving gel, blotted onto a nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad Lab), and blocked with blocking buffer (Block Ace, Dainippon-Pharm) at room


temperature for 60 minutes. Immunoblotting was performed using a rabbit polyclonal antibody to murine iNOS (1:500 dilution, M-19, Santa Cruz), or a rabbit polyclonal antibody to human eNOS (1:500 dilution, N30030/Lot6, Transduction Laboratories). Immunodetection was accomplished using a horseradish anti-rabbit secondary antibody (1:2000 dilution, Amersham) with an enhanced chemiluminescence kit (Amersham). The data were quantified by the densitometry.

Measurement of NOS Activity. NOS activity was evaluated by monitoring the conversion of [3H]L-arginine to [3H]L-citrulline as previously reported (32). Briefly, the tissue was homogenized in ice-cold buffer containing 50 mmol/L Tris/HCl (pH 7.5), 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DL-dithiothreitol, 10 µg/mL antipain and leupeptin, and 0.1 mg/mL phenylmethylsulfonyl fluoride. After centrifugation, the supernatant was incubated in the presence of L-arginine/[3H]L-arginine, 1 mmol/L NADPH, 10 µg/mL calmodulin, 5 µmol/L tetrahydrobiopterin, and 1 mmol/L CaCl2 in Tris/HCl buffer for 30 minutes at 37°C. The reaction was stopped with the addition of HEPES buffer (pH 5.5) containing 10 mmol/L EDTA. The reaction mixture was applied to Dowex 50W-X8 columns, and the eluted [3H]L-citrulline was measured by scintillation counting for total NOS activity. Parallel experiments in the absence of CaCl2 and the presence of EGTA (1 mmol/L) determined iNOS activity. Protein concentration was measured by use of the bicinchoninic acid assay (Pierce) with bovine serum albumin as standard. NOS activity was expressed as picomoles of citrulline formed per milligram of protein per 30 minutes.

Immunohistochemistry.

Frozen sections of 4 µm thick were thawed on slides, air dried,

and fixed in acetone for 10 minutes at −20°C. Endogenous peroxidase activity was quenched by incubating sections in 5 mM periodic acid for 10 minutes. Nonspecific binding sites were


blocked by incubation with 20% normal goat serum for 15 minutes at room temperature before the addition of a rabbit polyclonal antibody to murine iNOS (1:100 dilution, M-19, Santa Cruz). The sections were then incubated for overnight at 4°C, washed three times, and incubated with affinity-purified, biotinylated rabbit anti-rabbit IgG for 1 hour at room temperature. They were washed again and overlaid with streptoavidin/biotin/peroxidase complex for 1 hour at room temperature (Nichirei, Tokyo, Japan). After a final wash, the labeling was visualized with 3,3'-diaminobenzidine and 0.05% H2O2 in acetate buffer (50 mM Tris-HCl pH7.6). Counterstaining was then performed with Mayer's hematoxylin.

Plasma Levels of Nitrate + Nitrite (NOx). Plasma levels of NOx were measured by a modified chemiluminescence method of Radomski et al (29) with a NO analyzer (model 270B, SIEVERS) according to the manufacturer's instruction.

Echocardiography. Echocardiographic studies were performed using an ultrasonographic system (ALOKA SSD-5500, Tokyo, Japan) as previously reported (15,19). After anesthetization with 2.5% Avertin (14 µl/g body weight, IP, Aldrich Chemical Co), mice were placed in a supine position. A 7.5-MHz transducer (ALOKA, Tokyo, Japan) was applied to the left hemithorax. Two-dimensional targeted M-mode imaging was obtained from the short-axis view at the level of the greatest left ventricular dimension at baseline and 2 minutes after low and high doses of isoproterenol (0.02 µg and 0.5 µg, IP). M-mode measurements of left ventricular end-diastolic and end-systolic diameter and left ventricular anterior- and posterior-wall thickness were made using the leading-edge convention of the American Society of Echocardiography. End diastole was determined at the maximal left ventricular diastolic dimension, and end systole was taken at the peak of posterior-wall motion. The percentage of left ventricular fractional shortening (LVFS) was calculated as: LVFS (%) =


(LVEDD – LVESD)/LVEDD x 100, where LVEDD and LVESD indicate left ventricular end-diastolic and end-systolic diameter, respectively.

Left Ventricular Pressure Measurement. After anesthetization with 2.5% Avertin (14 µl/g body weight, IP, Aldrich Chemical Co), mice were placed in a supine position. A 1.4 F micromanometer catheter (Millar Instruments) was inserted into the left ventricle through the right carotid artery. Left ventricular pressure was then recorded at baseline and 2 minutes after low and high doses of isoproterenol (0.02 µg and 0.5 µg, IP).

iNOS Inhibition With ONO-1714.

A novel cyclic amidine analogue, (1S,5S,6R,7R)-7-

Chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714, Ono Pharmaceutical Company, Osaka, Japan) (26), was used to inhibit iNOS activity. ONO-1714 was found to be 10-fold selective for human iNOS (Ki = 1.88 nM) over human eNOS (Ki = 18.8 nM). The inhibitory effect of ONO-1714 on iNOS was found to be 451-fold and >20,000-fold more potent than L-NMMA and aminoguanidine, respectively (26). In terms of the selectivity for human iNOS, ONO-1714 was approximately 34- and 2-fold more selective for iNOS than L-NMMA and aminoguanidine, respectively. ONO-1714 (Ono Pharmaceutical Company, Osaka, Japan) was used to inhibit iNOS activity (26). Echocardiography and left ventricular pressure measurement were performed 20 minutes after intravenous injections of ONO-1714 (0.1 mg/kg). Phosphate buffered saline was used as vehicle.

Statistical Analysis.

The results are presented as mean ± SD. Student's t test was used to

compare each variable between TG and WT in Figure 1. One-way ANOVA with Student-Newman-Keuls test was used in Figure 3. Two-way ANOVA with repeated measures was used in Figure 4 and 5. Two-way ANOVA without repeated measures was used in Table


1 and 2. Differences were considered to be statistically significant at P