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Nicorandil but not ISDN Upregulates Endothelial Nitric Oxide Synthase Expression, Preventing Left Ventricular Remodeling and Degradation of Cardiac Function in Dahl Salt-sensitive Hypertensive Rats With Congestive Heart Failure

Horinaka, Shigeo PhD, MD; Kobayashi, Naohiko PhD, MD; Yagi, Hiroshi PhD, MD; Mori, Yousuke PhD, MD; Matsuoka, Hiroaki PhD, MD

Journal of Cardiovascular Pharmacology: May 2006 - Volume 47 - Issue 5 - pp 629-635
doi: 10.1097/01.fjc.0000211741.47960.c2
Original Articles

Cardiac endothelial nitric oxide synthase (ecNOS) was suppressed and inducible NOS (iNOS) enhanced at the decompensated heart failure stage in 18-week-old Dahl salt-sensitive (DS) hypertensive rats to which a high-salt diet had been administered from the age of 6 weeks. Nicorandil (NIC) enhanced ecNOS by activating Adenosine triphosphate-sensitive potassium channels (K-ATP channels) in the normal rat left ventricle. In this study, left ventricular hypertrophy, remodeling, function, cardiac ecNOS, and iNOS were compared between NIC and isosorbide dinitrate (ISDN) treatments in DS hypertensive rats with congestive heart failure. We examined DS hypertensive rats of 18 weeks of age to which 8% NaCl had been administered from the age of 6 weeks, and to which subdepressor doses of NIC (6 mg/kg/d), ISDN (6 mg/kg/d), and vehicle (CON) were administered from the age of 11 weeks. Contractility (Ees), stiffness (Eed), left ventricular end-diastolic volume, and left ventricular end-systolic volume were measured by conductance catheter and micromanometer on the basis of the pressure-volume relationship, and mRNA and protein levels of ecNOS and iNOS in the left ventricle were measured by reverse transcription-polymerase chain reaction and Western blot analysis at 18 weeks. LV mass index and LV dimensions were smaller in the NIC and ISDN groups than in the CON group (P<0.01), and the first parameter was lower in the NIC than in the ISDN group (P<0.01). Ees was also better maintained in the NIC and ISDN groups than in the CON group (NIC: 3349±649; ISDN: 2950±577, P<0.05 vs. NIC; CON: 1424±375 mL/mmHg, P<0.01 vs. treatments). Eed was exacerbated only in the ISDN group. NIC enhanced whereas ISDN suppressed ecNOS mRNA and protein levels (NIC 2.0-fold and 1.8-fold, ISDN 0.70-fold and 0.8-fold vs. CON; P<0.01, respectively). However, no intragroup differences in iNOS mRNA or protein levels were observed for the 3 groups. More significant improvements in cardiac function and LV hypertrophy regression were observed in an NIC group than in an ISDN group of DS hypertensive rats. Activation of the K-ATP channel seems to induce this beneficial effect, which may be mediated in part by enhanced ecNOS expression in the heart in DS hypertensive congestive heart failure rat model.

Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi, 321-0293, Japan

Reprints: Shigeo Horinaka, MD, Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, Japan (e-mail:

Received for publication August 9, 2005; accepted November 28, 2005

Cardiac endothelial nitric oxide synthase (ecNOS) is structurally present in vascular endothelial cells as an important enzyme in the production of nitric oxide (NO).1 NO production is blunted in the microvasculature of the failing myocardium, contributing to the development of endothelial dysfunction in heart failure.2,3 In recent years, NO has been demonstrated to promote antihypertrophy and inhibit cardiac remodeling.4–6

Recent reports indicate impaired endothelium-dependent relaxation and downregulation of aortic ecNOS activity at the decompensated heart failure stage in 18-week-old hypertensive Dahl salt-sensitive (DS) rats administered high-salt diets from the age of 8 weeks.7 Previously, we reported that cardiac ecNOS was suppressed and inducible NOS (iNOS) enhanced in DS rats during this stage.8,9 We have also demonstrated that nicorandil (NIC) enhances cardiac ecNOS expression by activating ATP-sensitive K channels in normal and infarct left ventricles of Sprague-Dawley rats.10,11 In contrast, Vaziri et al12 report that isosorbide dinitrate (ISDN), a donor of NO, suppresses aortic ecNOS expression through cGMP-mediated negative feedback regulation by NO in Sprague-Dawley rats.

The recent introduction of miniature conductance catheters enables analysis of cardiac function in small animals.9,13,14 Researchers over the past decade have examined the characteristics of left ventricular contraction using the LV end-systolic pressure-volume relationship (ESPVR), recently recognized as useful for studying cardiac hemodynamics. The end-systolic volume elastance (Ees), which is the slope of the ESPVR, is relatively independent of preload and afterload and serves as a reliable index of LV systolic function (contractility) within the physiologically normal range of LV pressures.15–18 This index may be suitable for assessing left ventricular function. Comparisons of control and treatment groups with respect to values of this index may permit assessment of the protection from impaired cardiac function provided in congestive heart failure (CHF) cases.

The present study sought to test the hypothesis that compared with ISDN, NIC-induced enhancement of ecNOS and activation of K-ATP channel resulted in improved cardiac function and remodeling in a DS hypertensive CHF rat model.

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All experimental procedures and protocols used in this study met the guidelines of the Dokkyo University School of Medicine and of the NIH Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication No 85-23, revised 1985). Thirty male DS rats (Eisai Co, Tokyo, Japan) were fed 0.3% NaCl (low-salt) chow from birth until the age of 6 weeks, then switched to 8% NaCl (high-salt) chow. The special chow and tap water were given ad libitum throughout the experiment. At 11 weeks of age, the rats were divided into 3 groups. Before treatment, hypertension was confirmed in each rat by tail cuff plethysmography. Treatment with NIC (6 mg/kg), ISDN (6 mg/kg), or vehicle (CON) was initiated at 11 weeks of age, with twice-daily administrations by gastric gavage for 8 weeks. The doses selected for NIC and ISDN were based on preliminary studies, suggesting that these doses would not change arterial pressure in this strain of rats and previous report.19 At the conclusion of this treatment period, all rats were subjected to hemodynamic examination. On the day of euthanasia, the blood pressure and heart rate of each rat were measured by tail cuff plethysmography. Rats were anesthetized by intraperitoneal administration of pentobarbital (20 mg/kg), and the trachea was intubated with PE-240 tubing connected to a small animal ventilator (Harvard respirator, Harvard Apparatus, South Natick, MA) to permit controlled ventilation. After mid-sternal incision, the positive-end expiratory pressure was held constant by submerging the outflow tube tip of the respirator in water to a depth of 10 mm to prevent pulmonary atelectasis.

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The LV ESPVR was determined by the conductance catheter technique, as previously described in detail.9 Briefly, the conductance catheter was inserted into the left ventricle through the apex and pushed until the distal tip was positioned in the ascending aorta along the longitudinal axis of the left ventricle. A 2.5 Fr catheter-tip micromanometer (SPR-524, Millar Instruments, Houston, TX) was inserted into the left ventricle through the apex to permit continuous measurement of LV pressure. To change the preload, a snare was placed around the inferior vena cava. We recorded conductance volume and LV pressure simultaneously during gradual inferior vena cava occlusion. Electrical signals from the conductance-measuring and pressure-measuring equipment (VPR 1002, Unique Medical, Tokyo, Japan) were digitized by an analog-to-digital converter (AD12-8, Contec, Tokyo, Japan) at a sampling frequency of 1 kHz at 12-bit resolution and saved to a personal computer (Dynabook SS 330, Toshiba, Tokyo, Japan). The points of the ESPVR and end-diastolic pressure-volume relationship (EDPVR) were determined by the previously reported iterative technique using Integra 3 software (Unique Medical, Tokyo, Japan). To avoid respiratory drift, all data were obtained during a 10-second period during which the respirator was turned off in the expiration phase. Finally, parallel conductance volume was measured by the hypertonic saline-dilution method. All rats were decapitated after the experiment and the heart immediately excised. The left ventricle was carefully separated from the atria and right ventricle, weighed, immediately frozen in liquid nitrogen, and stored at −80°C until extraction of total RNA and protein measurement. After an interval of several days, the RNA and protein samples were pooled and their expression simultaneously analyzed.

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Reverse Transcription-Polymerase Chain Reaction Analysis

Total RNA was extracted from frozen specimens as described elsewhere,20 and reverse transcription-polymerase chain reaction (RT-PCR) was allowed to proceed by the standard methods, with 1 and 3 μg of total RNA for ecNOS and iNOS analysis, respectively.21 First-strand cDNA was synthesized using random primers and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). Sequences were amplified by PCR using synthetic gene-specific primers for ecNOS (upstream primer, 5′-TCCAGTAACACAGACAGTGCA-3′; downstream primer, 5′-CAGGAAGTAAGTGAGAGC-3′),22 iNOS (descending 5′-AGAATGGAGATAGGACGT-3′; ascending 5′-GAGATCAATGCAGCTGTG-3′),23 and a DNA PCR kit (PerkinElmer, Norwalk, CT) over 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 1 minute. Rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was amplified in parallel using primers, as described.24 Reaction conditions were optimized to obtain reproducible and reliable amplification within the logarithmic phase of the reaction, as determined by preliminary experiments. Ethidium bromide detection indicated a linear reaction for up to 35 cycles. PCR products separated by electrophoresis on 2% agarose gels containing ethidium bromide were visualized by ultraviolet-induced fluorescence. The intensity of each band was scanned using an Epson GT-8500 Scanner and quantified by ZERO-Dscan software (Scanalytics Inc, Fairfax, VA). The resulting densities of the ecNOS and iNOS bands are expressed relative to the corresponding densities of the GAPDH bands from the same RNA sample.20,25

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Western Blot Analysis

The left ventricle from each rat was homogenized (25% wt/vol) with a Polytron homogenizer (Kinematica, Littau, Switzerland) in 10 mmol/L HEPES buffer, pH 7.4, containing 320 mmol/L sucrose, 1 mmol/L ethylendiaminetetraacetic acid, 1 mmol/L dithiothreitol, 10 mg/mL leupeptin, and 2 μg/mL aprotinin at 0°C to 4°C. The homogenate was then centrifuged at 1000g at 4°C for 5 minutes and the resulting supernatant used as the postnuclear fraction. Protein concentrations were determined by the Bradford method.26 The postnuclear fraction (ecNOS and iNOS: 200 μg of protein) was subjected to SDS-PAGE through 10% gels.27 The proteins on the gels were transferred electrophoretically to Polyvinylidine difloride sheets for 1 hour at 2 mA/cm2, as described previously.28 The sheets were immunoblotted with the anti-ecNOS and anti-iNOS antibodies (Transduction Laboratories, Lexington, KY) in a buffer containing 10 mmol/L Tris/HCl, pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20, and 5% skim milk, followed by peroxidase-conjugated goat antimouse IgG (Amersham Life Science Inc, Piscataway, NJ).28 eNOS and iNOS proteins on the sheets were detected with the enhanced chemiluminescence immunoblotting detection system (Amersham Life Science Inc). The developed images were scanned using an Epson GT-8500 Scanner and quantified by ZERO-Dscan software (Scanalytics Inc). The amount of each protein was quantified using a densitometer in a linear range and expressed as a percentage relative to that in control rats.8,9

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Statistical Analysis

All calculated data are expressed as means±standard deviation of the mean. A 1-way analysis of variance with Fisher test was used to determine the statistical significance of differences. Survival curves were determined by Kaplan-Meier analysis, whereas the statistical significance of differences was determined by the log-rank test. Statistical significance was accepted at the level of P<0.05.

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Cumulative Survival Rate

Two of the CON rats died at 16 weeks of age. One and 2 of the ISDN-treated rats died at 15 and 17 weeks of age, respectively. All NIC-treated rats survived until 18 weeks of age. Figure 1 shows the cumulative survival curves for CON, NIC, and ISDN rats. Survival rates were 100% in NIC-treated rats, 70% in ISDN-treated rats, and 80% in CON rats at 18 weeks of age. NIC rats demonstrated higher survival rates than ISDN rats, although this difference was not statistically significant.

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LV Weight and Hemodynamics

The body weight of NIC-treated and ISDN-treated rats was significantly greater than that of CON rats (P<0.01). However, we found no significant differences in the body weight of NIC-treated and ISDN-treated rats. The LV mass of NIC-treated and ISDN-treated rats was significantly lower than that of CON rats (P<0.01, respectively), whereas that of NIC-treated rats was significant lower than that of ISDN-treated rats (P<0.05) (Table 1). Although rats were conscious, we found no significant differences in systolic arterial pressure among NIC, ISDN, and CON rats. The heart rates of NIC-treated and ISDN-treated rats tended to be lower than that of CON rats (Table 2). Upon anesthesia, systolic LV pressure in each group decreased by a mean of 40%, whereas heart rates decreased by about 20%. We observed no differences in LV systolic pressure or heart rates among the 3 groups. The LV end-diastolic pressure was significantly greater in CON rats than in the NIC-treated and ISDN-treated rats (P<0.01, respectively).

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Left Ventricular Geometry and Cardiac Function

The LV end-systolic volume and end-diastolic volume were significantly smaller in NIC-treated and ISDN-treated rats than in CON rats (P<0.01, respectively), whereas LV end-diastolic volumes were significantly smaller in NIC-treated rats than in ISDN-treated rats (P<0.05) (Table 2). Thus, NIC rats demonstrated the greatest suppression of LV geometric remodeling. Figure 2 depicts the pressure-volume relationships recorded for a CON rat, an NIC-treated rat, and an ISDN-treated rat. The ESPVR and EDPVR in the NIC-treated and ISDN-treated rats consistently showed an upward shift compared with those in CON rats. As shown in Table 2, Ees was significantly greater in NIC-treated and ISDN-treated rats than in CON rats (P<0.01, respectively). The V0 of the NIC-treated rats was significantly smaller than that of CON rats (P<0.01) and that of ISDN rats (P<0.05).These data suggest that NIC treatment generated the most marked improvements in LV contractility and LV geometric remodeling. Eed was significantly greater in ISDN-treated rats than in CON and NIC rats (P<0.01, respectively), indicating stiffer LV diastolic chambers in ISDN-treated rats than in CON and NIC rats (Table 2).

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NOS mRNA and Protein Levels

Figure 3 presents an RT-PCR analysis of rat cardiac ecNOS and GAPDH as an internal control to correct for differences in RNA loading in CON (n=8), NIC (n=10), and ISDN (n=7) rats. ecNOS mRNA levels were more significantly elevated in NIC-treated rats than in CON rats, but ecNOS mRNA levels were lower in ISDN-treated rats than in CON rats. Figure 4 shows the densitometric quantification of these results. ecNOS mRNA showed a 2.0-fold increase in the NIC group (P<0.01 vs. CON) and a 0.7-fold decrease in the ISDN group (P<0.01 vs. CON). Significant differences were observed in ecNOS mRNA levels between NIC-treated and ISDN-treated rats (P<0.01). Figure 5 presents a Western blot analysis of rat cardiac ecNOS protein levels in CON (n=8), NIC (n=10), and ISDN (n=7) rats. NIC increased ecNOS protein levels by 76% (P<0.01 vs. CON and ISDN). In contrast, ISDN reduced ecNOS protein levels by 21% (P<0.01 vs. CON and NIC) (Fig. 6). No differences in iNOS mRNA levels were observed between CON rats and treated rats. iNOS protein levels also remained constant in treatments (data not shown).

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Our study is the first demonstration of the beneficial effects of NIC on salt-induced hypertensive LV remodeling and impaired LV function through the activation of K-ATP channels in DS hypertensive rats with heart failure.

NIC has a potassium channel-opening effect that activates K-ATP channels and induces NO in the same way as nitrates such as ISDN, resulting in increased intracellular cyclic GMP.29,30 This different pathophysiologic action may be induced by the activation of K-ATP channels.

In this study, NIC showed greater regression of LV hypertrophy, inhibition of LV remodeling, and improvement in cardiac function, effects presumably mediated by the K-ATP channel, because ISDN, a NO donor, demonstrates only modest effects.

Although this study did not demonstrate a precise underlying mechanism, 2 hypotheses have been advanced to account for NIC's stronger suppression of cardiac remodeling and improved cardiac function compared with ISDN.

First, NIC activates cardiac K-ATP channels. Activation of K-ATP channels by NIC-attenuated cardiac remodeling by inhibiting 70-kd S6 kinase in long-term inhibition of NOS with -nitro-L-arginine methyl ester rats.31 Other reports indicate that NIC treatment improves LV remodeling.32,33

K-ATP channel openers may maintain cardiac contractility by inhibiting voltage-dependent Ca2+ channels and Na+/Ca2+ exchange, thereby reducing Ca2+ overload in myocytes29 and suppressing microvascular injury caused by neutrophils,34 as demonstrated in this study in DS hypertensive rats with heart failure.

Endocardial ischemia may be found in cases involving severe LV hypertrophy. K-ATP channel openers have been observed to provide cardioprotective effects in the ischemic myocardium, mimicking ischemic preconditioning.35–37 In addition, microcirculatory dysfunction may also be present in cases of hypertensive heart failure. NIC, but not ISDN, dilated coronary artery microvessels (vessel size <100 μm).38 This ischemic protection of the endocardium and improvement in microvascular dysfunction may also maintain cardiac contractility.

Second, NIC enhances cardiac ecNOS by activating K-ATP channels. In contrast, ISDN suppresses ecNOS. Compared with Dahl salt-resistant rats, DS rats with heart failure exhibited suppressed levels of ecNOS in the LV, whereas subdepressor doses of imidapril, an angiotensin-converting enzyme inhibitor, increased ecNOS, with improved CHF and left ventricular remodeling.8 It remains unknown whether the enhanced production of endogenous NO resulting from ecNOS activation after NIC administration provides greater benefits for the heart in treatment by exogenous NO donors such as ISDN, which release NO at a rate about 2.5-fold greater than NIC.39 Nevertheless, endogenous NO may have positive inotropic effects.40,41 Several mechanisms have been suggested to explain the positive inotropic effects of NO at the cardiomyocyte.42–47

Reports also indicate that overproduction of ecNOS in ecNOS transgenic mice inhibits cardiac hypertrophy induced by chronic isoproterenol infusion.48 In mice, ecNOS deletion leads to left ventricular hypertrophy with systemic hypertension.49 Thus, NIC produces exogenous NO from the action of the NO donor itself and endogenous NO via activation of ecNOS, which may increase potency with respect to regression of LV hypertrophy.

Ikeda et al50 report that the occurrence of cardiomyocyte apoptosis contributes to ventricular eccentric dilatation in 18-week-old DS hypertension rats with heart failure to whom 8% NaCl had been administered from the age of 6 weeks. Endogenous endothelial NO production may inhibit myocardial caspase 3 activity that promotes apoptosis in heart failure.51 Nagata et al52 demonstrated that NIC inhibited apoptosis in rat neonatal cultured cardiac myocytes through a NO/c-GMP-dependent mechanism by activating K-ATP channels and by inhibiting caspase 3 activities directly. These mechanisms are also likely to be involved in this study's finding of stronger improvements in cardiac remodeling and function for NIC.

Vaziri et al12 report that ISDN, an NO donor, inhibits the expression of aortic ecNOS via cGMP in Sprague-Dawley rats. In contrast, our observations indicate that NIC enhances the expression of cardiac ecNOS by activating K-ATP channels in Sprague-Dawley rats; a mechanism posited in a previous report.10 This study demonstrates for the first time that similar contrasting effects on cardiac ecNOS are observed in DS hypertensive heart failure rats.

Furthermore, the enhanced expression of cardiac ecNOS observed with NIC but not with ISDN may prove crucial when the heart is subjected to acute stress, such as acute CHF and myocardial ischemia. NIC has been reported to improve outcomes by reducing major coronary events in patients with stable angina.53 We believe that both NIC's K-ATP channel activation and its enhancement of cardiac ecNOS expression are involved in the improved outcomes observed for coronary events in the IONA study. However, the survival rates in this study for NIC-treated rats failed to show statistically significant improvement, a result that needs to be confirmed through further study.

In conclusion, NIC was associated with greater LV regression and improved LV function than ISDN treatments. NIC, but not ISDN, enhanced cardiac ecNOS expression in DS hypertensive rats with heart failure. In addition to activation of K-ATP channels, the improvements in LV remodeling and function observed with NIC may be attributable to ecNOS enhancement via activated K-ATP channels.

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nicorandil; isosorbide dinitrate; endothelial nitric oxide synthase; left ventricular remodeling; cardiac function; congestive heart failure; ATP-sensitive potassium channel

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