HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 92/1/147    most recent expphysiol.2006.035097v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Itoh, T. Articles by Abe, K. Search for Related Content PubMed PubMed Citation Articles by Itoh, T. Articles by Abe, K.

¹ Department of Drug Safety Evaluation, Developmental Research Laboratories, Shionogi & Co., Ltd, 3-1-1 Futaba-cho, Toyonaka, Osaka, 561-0825, Japan

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Using a working perfused heart model, we investigated the hypothesis that alterations in the NO–cGMP pathway may exacerbate postischaemic mechanical dysfunction in the hypertrophied heart. Ischaemia for 25 min followed by reperfusion for 30 min produced marked cardiac mechanical dysfunction in both stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY). Exogenous treatment with S-nitroso-N-acetyl-DL-penicillamine (SNAP), a NO donor, had beneficial effects on the cardiac dysfunction induced by ischaemia–reperfusion (I/R) in the WKY heart, but the cardioprotective effect of SNAP was eliminated by guanylyl cyclase inhibitor. Cardiac cGMP levels were increased by SNAP or ischaemia in WKY. In contrast, in SHRSP hearts, SNAP could not alleviate the cardiac dysfunction caused by I/R. Pre-ischaemia, the cardiac cGMP level was significantly higher in SHRSP than in WKY; however, no significant difference was found after SNAP and ischaemia. The myocardial Ca²⁺-dependent NO synthase (NOS) activity increased at the end of ischaemia in WKY. Conversely, the Ca²⁺-independent NOS activity and protein levels were upregulated by I/R in the SHRSP myocardium. In the SHRSP hearts, non-selective NOS and selective Ca²⁺-independent NOS inhibitors or antioxidant treatment alleviated cardiac dysfunction caused by I/R. Moreover, mRNA expression and Western blotting analysis of cGMP-dependent protein kinase type I showed more deterioration of SHRSP hearts compared with WKY. These results suggest that: (1) the NO-dependent cardioprotective effect is depressed; and (2) overproduction of NO derived from Ca²⁺-independent NOS contributes to postischaemic heart injury in the hypertrophied heart of hypertensive status.

(Received 7 July 2006; accepted after revision 5 October 2006; first published online 9 October 2006)
Corresponding author T. Itoh: Department of Drug Safety Evaluation, Developmental Research Laboratories, Shionogi & Co., Ltd, 3-1-1 Futaba-cho, Toyonaka, Osaka, 561-0825, Japan. Email: tetsuji.itoh{at}shionogi.co.jp

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Nitric oxide (NO) plays a pivotal role in the physiological modulation of myocardial function and coronary artery tone (Kelly et al. 1996). Both endogenously produced and exogenously supplied NO are known to be able to migrate rapidly to adjacent coronary arterial smooth muscles and cardiomyocytes and to have several direct effects on the heart, such as a vasodilator action and a positive or negative inotropic effect (Shah, 1996). Several other potentially beneficial effects of NO on postischaemic cardiac function have been demonstrated with various experimental preparations and in vivo species (Williams et al. 1995; Draper & Shah, 1997). These various cardiac actions of NO appear to activate soluble guanylyl cyclase, leading to the production of cGMP and activation of cGMP-dependent protein kinase (cGK; Shah & MacCarthy, 2000). Although NO can exert cGMP-independent effects, the NO–cGMP signalling pathway is deeply involved in the cardioprotective action in ischaemia–reperfusion (I/R) injury (MacCarthy & Shah, 2000; Hanafy et al. 2001). In contrast, some investigators have reported that inhibition of NO synthase (NOS) shows a protective effect on I/R injury, which suggests detrimental effects of NO during I/R (Yasmin et al. 1997; Kobara et al. 2003). The protective effect of NOS inhibition has been attributed to a reduced formation of oxygen-derived free radicals. The role of NO in cardiac I/R injury is still controversial.

Hypertrophy is one of the major risk factors for ischaemic heart disease, such as myocardial infarction and heart failure, in both target patients and experimental animal models (Levy et al. 1990; Schoemaker et al. 1994). Impairment of NO-dependent coronary vasodilatation has been reported in spontaneously hypertensive rats (SHR), which develop hypertrophy of the left ventricular myocardium. Crabos et al. (1997) suggested that a decrease in endothelial NOS (eNOS) expression in the coronary endothelium might be related to this event. In addition, the responsiveness to NO donor was downregulated in isolated hypertrophied myocytes of the pressure-overloaded hypertrophy rat model (Ito et al. 1997). In contrast, we previously reported a long-lasting increased activity of inducible NOS (iNOS) in postischaemic cardiac myocytes of stroke-prone SHR (SHRSP), as an animal model for hypertension-induced cardiac hypertrophy (Abe et al. 2001).

We hypothesized that the role of the NO–cGMP pathway would be altered in the hypertrophied heart and that differential regulation of this pathway might be involved in the susceptibility to cardiac dysfunction induced by I/R in cardiac hypertrophy. In the present study, we investigated the effects of NO donor, NOS inhibitor or antioxidants on I/R injury in hearts isolated from control normotensive Wistar Kyoto rats (WKY) and SHRSP using the working perfused heart model. We also examined the cardiac cGMP levels and NOS activities during the I/R treatment and analysed the cardiac NOS isoforms and cGK protein levels in both strains. Our focus was on the alteration of the NO–cGMP pathway in hypertrophic hearts and the involvement of this change in cardiac dysfunction after I/R.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals

Male SHRSP and WKY (13–14 weeks old; Japan SLC, Inc., Shizuoka, Japan) were used in the present experiments. Five animals were housed in each aluminium cage (width x depth x height, 400 mm x 500 mm x 200 mm) under the following conditions: temperature, 23 ± 2°C; relative humidity, 55 ± 15%; and ventilation frequency over 10 times h^–1 with 100% fresh air under a 12 h light–12 h dark schedule (lights turned on at 08.00 h and turned off at 20.00 h). They were allowed free access to water and food (solid chow; CA–1, Clea Japan Inc., Tokyo, Japan).

All animal experiments and procedures were approved and conducted in accordance with the Institutional Animal Care and Use Committee.

Isolated perfused working heart preparation and experimental protocols

Isolated working heart perfusion was performed by a previously described method (Itoh et al. 2004) with minor modification. Briefly, the hearts were quickly removed under anaesthesia induced with diethyl ether, placed in the perfusion chamber and connected to an aortic cannula. The hearts were perfused with an oxygenated Krebs–Henseleit bicarbonate buffer (mM: 119.8 NaCl, 4.7 KCl, 1.2 MgSO₄, 2.5 CaCl₂, 1.2 KH₂PO₄, 25.0 NaHCO₃ and 11.1 glucose, pH 7.4, maintained at 37°C) by the Langendorff perfusion method. The left atrium was cannulated through a pulmonary vein for the working heart mode preparation. After a 10 min Langendorff perfusion, the heart was subjected to working heart perfusion at a left atrial filling pressure (preload) of 11 mmHg using a heart-perfusion apparatus (IPH-W2, Labo-Support, Osaka, Japan). Since some cardiac function remained (cf. Table 2), the perfusion pressure in the aorta (afterload) was set at 60 mmHg for WKY or 80 mmHg for SHRSP hearts as previously described (Itoh et al. 2004), modified according to Chen et al. (2001). After 15 min of perfusion of the working heart, ischaemia was induced by lowering the afterload to about 0 mmHg (atmospheric pressure) for 25 min. The hearts were then reperfused by raising the afterload to the original pressure for 30 min. S-Nitroso-N-acetyl-DL-penicillamine (SNAP; Affinity BioReagents Golden, CO, USA), N-nitro-L-arginine (L-NNA; Cayman Chemical, Ann Arbor, MI, USA), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Tocris Cookson, Bristol, UK), 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; Tocris Cookson), vinyl-L-N-5-(1-imino-3-butenyl)-L-ornithine (L-VNIO; Alexis Corporation, Lausen, Switzerland), superoxide dismutase (SOD; Sigma-Aldrich, St Louis, MO, USA) and catalase (Sigma-Aldrich) were each dissolved in 0.1% dimethyl sulphoxide solution. They added to the perfusion solution from 10 min before the onset of ischaemia until the end of reperfusion. The dose levels of these drugs were determined based on our preliminary study and other reports (Du Toit et al. 1998, 2001; Mazzetti et al. 2001).

Assay of NOS activity

Nitric oxide synthase activity was measured by the conversion of L-[³H]arginine to L-[³H]citrulline in the presence of saturated concentrations of the enzyme's cofactors, as previously described (Abe et al. 2001) with minor modifications. After 25 min of ischaemia or at the end of reperfusion, the left ventricle was quickly removed and frozen with liquid nitrogen. The freeze-clamped samples were homogenized in ice-cold Tris buffer (50 mM Tris-HCl, pH 7.4) containing 250 mM sucrose, 0.1 mM EDTA, 0.1 mM EGTA, 1 µM leupeptin, 1 µM pepstatin A, 1 mM phenylmethylsulphonyl fluoride, 1 mM dithiothreitol and 500 kallikrein inhibitor units of aprotinin. The homogenates were centrifuged at 800g for 10 min at 4°C. The supernatant was centrifuged at 100 000g for 60 min at 4°C, and the resultant supernatant was used for Ca²⁺-independent NOS assay. The remaining pellet was suspended in the above Tris buffer containing 1 mM KCl and incubated for 10 min at 4°C. The pellet was then homogenized in the above Tris buffer containing 20 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) and incubated for 40 min at 4°C. The homogenates were centrifuged at 100 000g for 20 min at 4°C, and the final supernatant fraction was used for Ca²⁺-dependent NOS assay.

To measure Ca²⁺-independent NOS activity, the cytosolic sample (20 µl) was incubated in 80 µl of assay buffer containing 50 mM Hepes (pH 7.4), 1 mM EGTA, 1 mM NADPH, 1 mM flavin adenine donucleotide, 1 mM flavin mononucleotide, 0.1 mM tetrahydrobiopterin, 1 mM dithiothreitol and 250 nM L-[2,3,4-³H]arginine monohydrochloride (PerkinElmer, Wellesley, MA, USA) for 20 min at 30°C. The reaction was terminated by addition of ice-cold stop buffer [100 mM Hepes (pH 5.0) and 10 mM EDTA]. L-[³H]Citrulline was eluted on an AG 50W-X8 column (100–200 µm mesh, Na⁺ form, Bio-Rad Laboratories) that had been pre-equilibrated with the stop buffer, and quantified using a liquid scintillation counter (LSC2000CA, Packard, Groningen, The Netherlands). The Ca²⁺-dependent NOS sample was incubated in the assay buffer containing 1 mM CaCl₂ and 30 nM calmodulin instead of 1 mM EGTA, and examined according to the same procedure as the Ca²⁺-independent NOS assay. The level of L-[³H]citrulline is expressed as picomoles per milligram protein per minute.

Measurement of cGMP

The freeze-clamped myocardial tissues were homogenized in cold 6% trichloroacetic acid and centrifuged at 1000g for 15 min at 4°C. The extracted sample was washed three times with five volumes of water-saturated diethyl ether. Levels of cGMP were measured with commercially available radioimmunoassay kits (Yamasa, Tokyo, Japan) after succinylation of the sample. Protein concentrations were determined using a protein fixed quantity kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as a standard.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was isolated from each heart sample using TRIzol reagent (Gibco-BRL Life Technologies). For the RT-PCR analysis, first-strand cDNA was synthesized from 1 µg total RNA according to the standard protocols (Abe et al. 2001). The 1 µl cDNA sample was amplified in a reaction with 50 µl solution containing 2.5 U/µL Taq polymerase (TaKaRa Bio, Shiga, Japan), 10x PCR buffer, 1.5 mM MgCl₂, 0.25 mM dNTP mixture, and 0.5 µM of each of the following primers according to Gerzanich et al. (2003): for cGKI, forward primer, 5'-AAGACGGCAAGCATGAAGCT-3' and reverse primer, 5'-CCCTTCTGTCCCTGTAAAGGTTT-3'; and for ß-actin, forward primer, 5'-GCTCGTCGTCGACAACGGCTC-3' and reverse primer, 5'-CAAACATGATCTGGGTCATCTTCTC-3'. Reactions were initiated by incubation at 94°C for 3 min and PCRs (25–35 cycles of 94–95°C for 30 s, 56–60°C for 15–25 s, 72°C for 30 s) with a final extension at 72°C for 4 min. The PCR products were visualized in ethidium bromide-stained 2.0% agarose gels and quantified using image analysis software (Scion Image Beta 4.02 for Windows, Scion, Frederick, MD, USA).

Western blot analysis

Frozen heart samples were homogenized with ice-cold 50 mM Tris-HCl buffer (pH 7.4) containing 0.5 mM EDTA, 0.5 mM EGTA, 1 µM leupeptin, 1 µM pepstatin A, 1 mM phenylmethylsulphonyl fluoride, 1 mM dithiothreitol, 500 kallikrein inhibitor units aprotinin and 20 mM CHAPS, and subsequently centrifuged at 800g for 10 min at 4°C. The supernatant was centrifuged at 100 000g for 20 min at 4°C, and the protein concentration in the resultant supernatant was measured. The supernatant was diluted in loading buffer (6.4% sodium dodecyl sulphate (SDS), 10% glycerol, 0.16% Bromphenol Blue and 1.6 mM dithiothreitol in 0.1 M Tris-HCl, pH 6.8) and denatured by boiling for 3 min. Proteins (30 µg) were separated by electrophoresis on 7.5% polyacrylamide gel containing 0.1% SDS and transferred to polyvinylidene (PVDF) membrane (Bio-Rad Laboratories). The PVDF membrane was then saturated with 5% non-fat dry milk overnight and probed at 4°C for 2 h with the specific primary antibody, i.e. rabbit polyclonal anti-eNOS, anti-nNOS and anti-iNOS antibodies (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or rabbit polyclonal anti-cGMP-dependent protein kinase I (cGKI) antibody (1:1000 dilution; Calbiochem, San Diego, CA, USA). After washing with PBS–Tween (0.1%), the membrane was incubated with antirabbit secondary antibodies conjugated with horseradish peroxidase (GE Healthcare Bio-sciences Corp., Piscataway, NJ, USA). Subsequent detection of the specific proteins was done by enhanced chemiluminescence on X-ray film with measurement using Scion Image for Windows.

Data analysis

The results are expressed as means ± S.E.M. Statistical analysis was carried out using the SAS system (version 6.12; SAS Institute Inc., Tokyo, Japan) for Microsoft Windows. The statistical significance of differences for experiments on groups of two was determined using Student's unpaired t test. Experiments on groups of three or more were subjected to Dunnett's test following one-way analysis of variance (ANOVA). The P values of less than 0.05 were considered to be statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Blood pressure and cardiac hypertrophy of SHRSP

As expected, values of systolic and mean blood pressure in 13- to 14-week-old SHRSP were significantly higher than those in WKY. Moreover, left ventricular hypertrophy in SHRSP was manifested as a significant increase in left ventricular weight, left ventricular weight to body weight ratio and left ventricular wall thickness compared with those of WKY (Table 1).

First, we determined the afterload pressure that is necessary for optimal functioning of each isolated working heart preparation by changing the pressure in steps of 10 mmHg from 50 to 90 mmHg. Maximum values of PRP were observed in WKY and SHRSP when the afterloads were set at 60 and 80 mmHg, respectively. The coronary flow was also highest at each condition (Table 2). Therefore, in the following tests, afterload pressure was set at 60 and 80 mmHg in WKY and SHRSP, respectively. Other reports have also suggested that higher afterload pressure is needed in working hypertrophied rat heart preparations to reflect the higher peripheral resistance in hypertension (Chen et al. 2001; Labarthe et al. 2005).

Effect of NO donor on cardiac mechanical function during I/R in WKY and SHRSP

Figure 1 shows the effect of SNAP, a NO donor, on cardiac mechanical function during I/R in WKY and SHRSP. Pre-ischaemia, the PRP values were 28 395 ± 873 and 32 611 ± 1866 mmHg min^–1 in WKY and SHRSP hearts, respectively. Twenty-five minutes of ischaemia produced a marked cardiac mechanical dysfunction in both WKY and SHRSP hearts, and PRP recovered only to 4444 ± 899 mmHg min^–1 (15.7 ± 3.2% of pre-ischaemic value) in WKY and to 2728 ± 472 mmHg min^–1 (8.4 ± 1.4%) in SHRSP at 30 min of reperfusion following the ischaemia. SNAP at 10 µM led to PRP recovery to 13798 ± 1711 mmHg min^–1 (48.6 ± 6.2%) at the end of 30 min reperfusion in the WKY heart. In contrast, treatment with SNAP (1, 10 and 100 µM) in SHRSP hearts could not improve the deleterious effect of I/R on PRP. Although SNAP at 100 µM resulted in slight but not significant recovery of PRP in WKY rats, this high concentration of SNAP worsened PRP even during pre-ischaemia (that is, 10 min treatment with 100 µM SNAP) in both WKY and SHRSP hearts (19 930 ± 2331 and 21 633 ± 3169 mmHg min^–1, respectively).

View larger version (26K): [in this window] [in a new window]   Figure 1.  Effect of SNAP on the change of pressure–rate product during I/R in WKY and SHRSP Values are expressed as means ± S.E.M. of 6 experiments. Vehicle or SNAP was administered during a period beginning 10 min before the onset of ischaemia until the end of reperfusion. *P < 0.05 and **P < 0.01 versus each vehicle-treated group.

As shown above, 10 µM SNAP led to gradual PRP recovery during the reperfusion period in WKY. The beneficial effect of SNAP in WKY was completely abolished by combination with ODQ (30 µM), a soluble guanylyl cyclase inhibitor (at the end of reperfusion, PRP for SNAP was 15 660 ± 1676 mmHg min^–1 and for SNAP + ODQ was 6071 ± 1132 mmHg min^–1). The I/R-induced cardiac mechanical dysfunction was significantly aggravated by treatment with ODQ alone in WKY (vehicle, 4444 ± 899 mmHg min^–1 and ODQ, 1353 ± 558 mmHg min^–1). Neither SNAP nor ODQ affected PRP during I/R in SHRSP (Fig. 2).

View larger version (26K): [in this window] [in a new window]   Figure 2.  Effect of SNAP, ODQ or cotreatment with SNAP and ODQ (SNAP + ODQ) on the change of pressure–rate product during I/R in WKY and SHRSP Values are expressed as means ± S.E.M. of 6 experiments. SNAP (10 µM), ODQ (30 µM), SNAP + ODQ or vehicle was administered during a period beginning 10 min before the onset of ischaemia until the end of reperfusion. *P < 0.05 versus each vehicle-treated group. #P < 0.05 versus each SNAP-treated group.

In WKY, ischaemia induced a gradual increase of cardiac cGMP level (pre-ischaemia, 324.3 ± 29.3 fmol (mg protein)^–1; 25 min ischaemia, 483.4 ± 48.6 fmol (mg protein)^–1, P < 0.05), and the following reperfusion restored the cGMP level to a similar value to that pre-ischaemia. In SHRSP cardiomyocytes, the cGMP level was significantly higher than in WKY pre-ischaemia; however, no significant change was found during I/R. The cGMP level was greatly increased by 10 µM SNAP treatment in WKY, and this significant incremental effect was retained during the I/R period. By contrast, in SHRSP, SNAP failed to increase the cGMP level significantly compared with the vehicle-treated hearts during the pre- and post-I/R periods. Treatment with ODQ decreased the cGMP levels in both strains at all the time points studied. Furthermore, combination treatment with SNAP and ODQ suppressed the SNAP-induced cGMP elevation in WKY. With 25 min ischaemia and 30 min reperfusion in WKY, the combined treatment lowered cGMP levels to values similar to those in the vehicle-treated group (Table 3).

Figure 3 shows Ca²⁺-dependent and -independent NOS activities during I/R in WKY and SHRSP cardiomyocytes. Calcium-dependent NOS activity was increased during ischaemia in WKY, and a significant increase of ca twofold compared with the pre-ischaemia level was observed after 25 min ischaemia (pre-ischaemia, 1.78 ± 0.16 pmol min^–1 (mg protein)^–1; 25 min ischaemia, 3.43 ± 0.41 pmol min^–1 (mg protein)^–1). The increment was reversed to the pre-ischaemic level following the 30 min reperfusion. In SHRSP hearts, there was no effect on the Ca²⁺-dependent NOS activity by 25 min ischaemia. Furthermore, the Ca²⁺-dependent NOS activity in SHRSP hearts was markedly attenuated by 30 min reperfusion (pre-ischaemia, 2.10 ± 0.10 pmol min^–1 (mg protein)^–1; 30 min reperfusion, 1.11 ± 0.30 pmol min^–1 (mg protein)^–1).

View larger version (22K): [in this window] [in a new window]   Figure 3.  Ca2+-dependent (A) and Ca2+-independent NOS activity (B) in cardiomyocytes pre-ischaemia, after 25 min ischaemia and at the end of 30 min reperfusion in WKY and SHRSP Values are expressed as means ± S.E.M. of 6 hearts for each group. *P < 0.05 and **P < 0.01 versus each pre-ischaemia group.

Effect of antioxidants and NO synthase inhibitor on cardiac mechanical function during I/R in WKY and SHRSP

Antioxidants, in the form of SOD with catalase, significantly improved the deleterious effect of I/R on PRP in SHRSP (at the end of reperfusion, vehicle, 8.2 ± 1.3% and SOD + catalase, 38.4 ± 5.3% of baseline values). In WKY, SOD + catalase produced slight amelioration of the PRP depression induced by I/R, but no significant effect was observed during the study. The NO synthase inhibitor L-NNA, at 1 µM, significantly ameliorated the PRP in both WKY and SHRSP hearts (at the end of reperfusion, 37.0 ± 8.4 and 24.7 ± 3.2% of baseline values, respectively). Cotreatment using L-NNA with SOD + catalase also significantly increased PRP at 30 min reperfusion in both rat strains. However, the cotreatment did not significantly ameliorate the PRP recovery induced by L-NNA in WKY and SOD + catalase in SHRSP, respectively (Fig. 4).

View larger version (29K): [in this window] [in a new window]   Figure 4.  Effect of L-NNA and antioxidants on the change of pressure-rate product during I/R in WKY and SHRSP Values are expressed as means ± S.E.M. of 6 experiments. L-NNA (1 µM), SOD (100 i.u. ml–1) with catalase (500 i.u. ml–1), L-NNA with SOD and catalase (L-NNA + SOD + catalase) or vehicle was administered during a period beginning 10 min before the onset of ischaemia until the end of reperfusion. *P < 0.05 and **P < 0.01 versus each vehicle-treated group.

Western blot analysis of NOS isoforms in WKY and SHRSP cardiac myocytes

The protein expression of eNOS and nNOS was studied in extracts from the left ventricle of WKY and SHRSP pre-ischaemia and after I/R (Fig. 5A and B). Pre-ischaemia, although there was no difference in the eNOS protein expression between WKY and SHRSP, the level of nNOS protein in SHRSP was significantly higher than that in WKY. The eNOS and nNOS protein expression decreased after I/R in SHRSP but not in WKY hearts.

View larger version (29K): [in this window] [in a new window]   Figure 5.  Protein expression of NOS isoforms in WKY and SHRSP hearts Representative Western blot and densitometric data reflecting eNOS (A), nNOS (B) and iNOS protein expression (C) in left ventricle of WKY and SHRSP pre-ischaemia (Pre) and after 25 min ischaemia and 30 min reperfusion (I/R). The densitometric data for eNOS and nNOS protein are expressed as a percentage of WKY values pre-ischaemia. Since iNOS protein was hardly detectable in WKY hearts, the relative density of iNOS protein is presented as a percentage of that for SHRSP hearts pre-ischaemia. Values are the means ± S.E.M. of 5 or 6 experiments. *P < 0.05 versus WKY pre-ischaemia. #P < 0.05 and ##P < 0.01 versus SHRSP pre-ischaemia.

RT-PCR and Western blot analysis of type I cGK in WKY and SHRSP cardiac myocytes

The level of cGKI mRNA expression in the left ventricle was significantly decreased in SHRSP compared with that in WKY (Fig. 6A and B). Moreover, Western blot analysis revealed low expression of cGKI in SHRSP cardiomyocytes relative to that in WKY (Fig. 6C).

View larger version (28K): [in this window] [in a new window]   Figure 6.  Cardiac cGKI mRNA and expression levels in WKY and SHRSP A, representative PCR products for cGKI and ß-actin mRNA in WKY (W) and SHRSP left ventricular cardiomyocytes (S). B, PCR results for cGKI mRNA in WKY and SHRSP. Data are presented as means ± S.E.M. of 6 experiments as relative expressions of cGKI mRNA relative to ß-actin mRNA. *P < 0.05 versus WKY. C, Western blot analysis of cGKI levels in the left ventricles from WKY and SHRSP.

	Discussion

Top Abstract Introduction Methods Results Discussion References

The SHRSP strain has a cardiac hypertrophic character accompanied by hypertension and is widely used for investigating the mechanism of hypertension-induced cardiac hypertrophy. Previous studies have suggested that the hypertrophied hearts of SHRSP are susceptible to postischaemic injury (Chen et al. 2001; Itoh et al. 2004). In the present study, the exposure of isolated hearts to 25 min ischaemia followed by 30 min reperfusion caused great damage to cardiac function in both WKY and SHRSP. SNAP (10 µM) improved the cardiac dysfunction induced by I/R only in WKY hearts. Exogenous NO supplementation has been suggested to express its cardioprotective effects through accumulation of cardiac cGMP by activating soluble guanylyl cyclase (Engelman et al. 1996; Du Toit et al. 1998, 2001). Indeed, elevated cardiac cGMP levels were observed following the NO donor treatment in the I/R WKY hearts. The soluble guanylyl cyclase inhibitor ODQ (30 µM) decreased the cGMP levels and worsened PRP recovery in the I/R WKY hearts. Moreover, cotreatment with SNAP and ODQ overrode the PRP recovery, accompanied by suppression of the cGMP level elevation, in WKY hearts. These results indicate that the cardioprotective effect in WKY displayed by SNAP was mainly linked to cGMP level elevation. Several other potential mechanisms for the beneficial effect of NO on postischaemic myocardial function are: (1) improvement in coronary arterial flow; (2) attenuation of the elevation of intracellular Ca²⁺ concentrations; (3) reduction in myofilament responsiveness to Ca²⁺; (4) action of NO as a scavenger of reactive oxygen species; and (5) inhibition of platelet aggregation (MacCarthy & Shah, 2000; Ferdinandy & Schulz, 2003).

In contrast, in SHRSP hearts, none of the doses of SNAP was able to ameliorate the cardiac mechanical dysfunction induced by I/R. The basal cardiac cGMP levels in SHRSP hearts were higher than those in WKY, but no further significant increment of cGMP levels was observed on SNAP treatment as found in WKY hearts. Kojda et al. (1998) suggested that excess superoxide production in SHRSP may trigger desensitization of the vascular smooth muscle soluble guanylyl cyclase to the stimulating effects of SNAP. We have previously reported a decrease in superoxide dismutase in the myocardial mitochondria of SHRSP compared with that of WKY (Itoh et al. 2004). In contrast, Carlos et al. (1998) showed that the cardiac glutathione level increased 1.3-fold in SHRSP compared with that in WKY. They also found that the activity of xanthine oxidase increased 6.2-fold in SHRSP, leading to their suggestion that reactive oxygen radicals produced by xanthine oxidase caused the elevation of cardiac glutathione level in the SHRSP heart. Altered cellular production of the reactive oxygen species may affect NO responses by oxidizing sites in proteins with which NO reacts or which otherwise influence NO binding (Hare & Stamler, 2005). These data suggest that some alteration of the NO–cGMP and related signalling pathways in SHRSP hearts might be at the root of the attenuation of the cardioprotective effect by exogenous NO treatment.

With regard to endogenous NOS activity in the hearts subjected to I/R, Ca²⁺-dependent NOS activity was activated 25 min after the onset of ischaemia in WKY hearts. The Ca²⁺-dependent NOS activity reflects the sum activity of eNOS and nNOS, both of which have been identified in cardiomyocytes (Pabla & Curtis, 1996). Ischaemia decreases myocardial pH mainly as a result of lactate production in aerobic glycolysis and induces an elevation in intracellular Ca²⁺ levels through Na⁺–H⁺ and Na⁺–Ca²⁺ exchangers. Therefore, cardiac Ca²⁺-dependent NOS has been reported to be activated by the elevation in cellular Ca²⁺ levels induced by ischaemia (Kitakaze et al. 2001). Some reports showed an increase of Ca²⁺-dependent activity, observed after up to ca 20–30 min of ischaemia in normotensive isolated rat hearts, but this was compromised by further prolonged ischaemia or following reperfusion (Giraldez et al. 1997; Muscari et al. 2004). Indeed, the activity of Ca²⁺-dependent NOS and the expression of eNOS and nNOS protein levels in the WKY heart after the end of I/R were not significantly different from those of the heart pre-ischaemia. By contrast, in SHRSP hearts, although unchanged Ca²⁺-dependent NOS activity was observed after end of the ischaemia, a reduction in Ca²⁺-dependent NOS activity appeared after the end of reperfusion. In contrast, the Ca²⁺-independent NOS activity (reflective iNOS) increased during the I/R treatment in SHRSP. Accordingly, Western blot analysis demonstrated a correlation between the changes of these NOS activities and each of the NOS isoform protein levels in SHRSP hearts. High concentrations of NO have been reported to exhibit toxic actions, mainly as a result of the production of peroxynitrite (ONOO^–), the reaction product of NO with superoxide anion (O₂^–; Beckman & Koppenol, 1996; Ferdinandy & Schulz, 2003). Furthermore, it has been reported that the activity of superoxide dismutase is decreased and O₂^– generation is increased in the hypertrophic myocardium, indicating increased oxidant stress (Piech et al. 2003; Itoh et al. 2004). Also, NOS contributes to superoxide release and may play an important role in human atrial fibrillation, which is associated with increased oxidative stress (Kim et al. 2005). Our results demonstrate that the cardiac dysfunction after I/R was ameliorated in SHRSP by treatment with antioxidants (SOD + catalase). The cardioprotective effect was also observed in SHRSP hearts after treatment with an NOS inhibitor (L-NNA); NOS inhibitors have been reported to attenuate the generation or action of reactive oxygen species (Wang & Zweier, 1996; Yasmin et al. 1997; Xie et al. 1998). We previously reported that iNOS plays an important role in the development of postischaemic cardiac dysfunction, and its selective inhibitor can ameliorate the haemodynamic parameters and histopathological injuries following permanent occlusion of the coronary artery in SHRSP (Abe et al. 2001). In this study, AMT, an iNOS inhibitor, also significantly ameliorated the cardiac dysfunction induced by I/R only in SHRSP hearts. Thus, these findings may explain why the overproduction of NO derived from Ca²⁺-independent NOS contributes to myocardial dysfunction via reaction with reactive oxygen species in the I/R-treated SHRSP hearts. In normotensive rat hearts, Wang et al. (1997) reported that the cardiac Ca²⁺-independent NOS is not changed after I/R and is seldom involved in cardiac dysfunction. We also confirmed that Ca²⁺-independent NOS activity was not affected during I/R in WKY hearts, and almost no signal for iNOS protein was detectable in cardiac extracts of WKY both pre-ischaemia and in I/R. These findings indicate that there is no overproduction of NO by Ca²⁺-independent NOS activity in WKY, at least under our conditions. In WKY hearts during I/R, the cardioprotective effect was observed following L-VNIO, a specific nNOS inhibitor. Gorren et al. (1997) showed that the optimal pH for nNOS is between 7.0 and 7.5 and suggested that nNOS shifts from the production of NO to the cytotoxic compounds ONOO^– and O₂^– at lower values of pH. Since ischaemia is accompanied by a decrease in intracellular pH, production of the reactive oxygen species may also contribute to cardiac dysfunction induced by I/R in WKY hearts. Our results suggest that the production of reactive oxygen species in I/R heart is mainly involved with nNOS in WKY and with iNOS in SHRSP.

In addition to NO synthase, essential cofactors, such as tetrahydrobiopterin (BH₄), are deeply involved in the production and control of NO in vivo. Vasquez-Vivar et al. (2003) have demonstrated that oxidation of the key NOS cofactor BH₄ transforms NO synthases into superoxide-producing enzymes. Cosentino et al. (1998) reported that production of superoxide by stimulation of calcium ionophore was significantly higher in segments of the aorta from SHR than in those from WKY. The natural BH₄ levels were similar in both strains. Treatment of SHR with BH₄ decreased superoxide formation but did not increase the activity of constitutive NOS (cNOS, Ca²⁺-dependent NOS), while WKY increased cNOS activity. Cosentino et al. (1998) suggested that the cNOS with insufficient cofactor BH₄ may generate damaging superoxide in SHR. In our study, treatment with L-NNA or antioxidants led to PRP recovery and their ameliorating effects were of a similar degree. Since the main mechanism of both effects has been suggested to be a reduction of oxidant stress, these findings might explain the non-additive beneficial effects of cotreatment with NOS inhibitor and with antioxidants.

To determine NOS activities, we used the L-arginine to citrulline assay, which is a well-validated and widely used method for evaluating in vitro NOS activities. However, this assay does not necessarily reflect NO bioavailability under conditions of increased oxidative stress, since the essential enzyme cofactors are added at saturating concentrations. Nitric oxide synthases are uncoupled in the hypertrophic myocardium, predominantly as a result of increased oxidant stress and BH₄ oxidation (Takimoto et al. 2005). Although the assay could show which NOS isozyme had the potential for synthesizing NO in the ischaemia–reperfusion injured heart preparations, the cardiac BH₄ levels might affect the NO production and/or uncouple NO synthases. Further experiments are required to elucidate the involvement of the essential cofactors, especially BH₄, in the ischaemia–reperfusion injury of hypertrophic hearts.

While two isozymes of cGK, coding for type I and type II cGK, have been identified, cardiac myocytes have been reported to contain the mainly I (cGKI) isoform (Keilbach et al. 1992; Feil et al. 2003). However, we found that mRNA expression and Western blotting analysis of cGKI were lower in SHRSP hearts compared with WKY. Ecker et al. (1989) also found half the concentration of cGK in cardiac tissues of SHRSP and renovascular hypertensive rats in comparison with those of the respective normotensive animals. They suggested the low cGK content to be mainly a result of a low index of vascularization in SHRSP hearts. Cyclic GMP can also regulate the L-type calcium current through activation of cGK in rat ventricular myocytes (Mery et al. 1991). Mazzetti et al. (2001) indicated that the low expression of cGKI in SHR cardiomyocytes may determine the lack of NO–cGMP-dependent regulation of transient calcium levels. Therefore, the deterioration of cGKI in hypertrophic hearts might induce susceptibility to cardiac pathological conditions, e.g. postischaemic injury.

In conclusion, our results indicate that an increase of NO levels induced by treatment with an endogenous NO donor or activation of the Ca²⁺-dependent NOS activity owing to ischaemia are linked to an elevation of cGMP levels in WKY hearts. Endogenous or exogenous NO may not be sufficiently bioactive to stimulate the formation of cGMP and maintain adequate NO-dependent cardioprotective effects in SHRSP hearts. In contrast, the increased activity of myocardial Ca²⁺-independent NOS might encourage cardiac injury via production or action of reactive oxygen species after I/R in hypertrophic myocytes. The differential regulation of the NO–cGMP pathway may contribute to impairment of cardiac dysfunction after I/R in SHRSP hearts.

	References

Top Abstract Introduction Methods Results Discussion References

 
Abe K, Tokumura M, Itoh T, Murai T, Takashima A & Ibii N (2001). Involvement of iNOS in postischemic heart dysfunction of stroke-prone spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 280, H668–H673.[Abstract/Free Full Text]

Beckman JS & Koppenol WH (1996). Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 271, C1424–C1437.[Medline]

Carlos DM, Goto S, Urata Y, Iida T, Cho S, Niwa M, Tsuji Y & Kondo T (1998). Nicardipine normalizes elevated levels of antioxidant activity in response to xanthine oxidase-induced oxidative stress in hypertensive rat heart. Free Radic Res 29, 143–150.[Medline]

Chen H, Higashino H, Maeda K, Zhang Z, Ohta Y, Wang Z, Su DF & Yuan WJ (2001). Reduction of cardiac norepinephrine improves postischemic heart function in stroke-prone spontaneously hypertensive rats. J Cardiovasc Pharmacol 38, 821–832.[CrossRef][Medline]

Cosentino F, Patton S, d'Uscio LV, Werner ER, Werner-Felmayer G, Moreau P, Malinski T & Luscher TF (1998). Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats. J Clin Invest 101, 1530–1537.[Medline]

Crabos M, Coste P, Paccalin M, Tariosse L, Daret D, Besse P & Bonoron-Adele S (1997). Reduced basal NO-mediated dilation and decreased endothelial NO-synthase expression in coronary vessels of spontaneously hypertensive rats. J Mol Cell Cardiol 29, 55–65.[CrossRef][Medline]

Draper NJ & Shah AM (1997). Beneficial effects of a nitric oxide donor on recovery of contractile function following brief hypoxia in isolated rat heart. J Mol Cell Cardiol 29, 1195–1205.[CrossRef][Medline]

Du Toit EF, McCarthy J, Miyashiro J, Opie LH & Brunner F (1998). Effect of nitrovasodilators and inhibitors of nitric oxide synthase on ischaemic and reperfusion function of rat isolated hearts. Br J Pharmacol 123, 1159–1167.[CrossRef][Medline]

Du Toit EF, Meiring J & Opie LH (2001). Relation of cyclic nucleotide ratios to ischemic and reperfusion injury in nitric oxide-donor treated rat hearts. J Cardiovasc Pharmacol 38, 529–538.[CrossRef][Medline]

Ecker T, Gobel C, Hullin R, Rettig R, Seitz G & Hofmann F (1989). Decreased cardiac concentration of cGMP kinase in hypertensive animals. An index for cardiac vascularization? Circ Res 65, 1361–1369.[Abstract/Free Full Text]

Engelman DT, Watanabe M, Maulik N, Engelman RM, Rousou JA, Flack JE, Deaton DW & Das DK (1996). Critical timing of nitric oxide supplementation in cardioplegic arrest and reperfusion. Circulation 94 (Suppl. 9), II407–II411.[Medline]

Feil R, Lohmann SM, De Jonge H, Walter U & Hofmann F (2003). Cyclic GMP-dependent protein kinases and the cardiovascular system: insights from genetically modified mice. Circ Res 93, 907–916.[Abstract/Free Full Text]

Ferdinandy P & Schulz R (2003). Nitric oxide, superoxide, and peroxynitrite in myocardial ischaemia-reperfusion injury and preconditioning. Br J Pharmacol 138, 532–543.[CrossRef][Medline]

Gerzanich V, Ivanov A, Ivanova S, Yang JB, Zhou H, Dong Y & Simard JM (2003). Alternative splicing of cGMP-dependent protein kinase I in angiotensin-hypertension: novel mechanism for nitrate tolerance in vascular smooth muscle. Circ Res 93, 805–812.[Abstract/Free Full Text]

Giraldez RR, Panda A, Xia Y, Sanders SP & Zweier JL (1997). Decreased nitric-oxide synthase activity causes impaired endothelium-dependent relaxation in the postischemic heart. J Biol Chem 272, 21420–21426.[Abstract/Free Full Text]

Gorren AC, Schrammel A, Schmidt K & Mayer B (1998). Effects of pH on the structure and function of neuronal nitric oxide synthase. Biochem J 331, 801–807.[Medline]

Hanafy KA, Krumenacker JS & Murad F (2001). NO, nitrotyrosine, and cyclic GMP in signal transduction. Med Sci Monit 7, 801–819.[Medline]

Hare JM & Stamler JS (2005). NO/redox disequilibrium in the failing heart and cardiovascular system. J Clin Invest 115, 509–517.[CrossRef][Medline]

Ito N, Bartunek J, Spitzer KW & Lorell BH (1997). Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes. Circulation 95, 2303–2311.[Abstract/Free Full Text]

Itoh T, Abe K, Tokumura M, Hirono S, Haruna M & Ibii N (2004). Cardiac mechanical dysfunction induced by ischemia-reperfusion in perfused heart isolated from stroke-prone spontaneously hypertensive rats. Clin Exp Hypertens 26, 485–498.[CrossRef][Medline]

Keilbach A, Ruth P & Hofmann F (1992). Detection of cGMP dependent protein kinase isozymes by specific antibodies. Eur J Biochem 208, 467–473.[Medline]

Kelly R, Balligand JL & Smith TW (1996). Nitric oxide and cardiac function. Circ Res 79, 363–380.[Free Full Text]

Kim YM, Guzik TJ, Zhang YH, Zhang MH, Kattach H, Ratnatunga C, Pillai R, Channon KM & Casadei B (2005). A myocardial Nox2 containing NAD(P)H oxidase contributes to oxidative stress in human atrial fibrillation. Circ Res 97, 629–636.[Abstract/Free Full Text]

Kitakaze M, Node K, Takashima S, Asanuma H, Asakura M, Sanada S, Shinozaki Y, Mori H, Sato H, Kuzuya T & Hori M (2001). Role of cellular acidosis in production of nitric oxide in canine ischemic myocardium. J Mol Cell Cardiol 33, 1727–1737.[CrossRef][Medline]

Kobara M, Tatsumi T, Takeda M, Mano A, Yamanaka S, Shiraishi J, Keira N, Matoba S, Asayama J & Nakagawa M (2003). The dual effects of nitric oxide synthase inhibitors on ischemia-reperfusion injury in rat hearts. Basic Res Cardiol 98, 319–328.[CrossRef][Medline]

Kojda G, Kottenberg K, Hacker A & Noack E (1998). Alterations of the vascular and the myocardial guanylate cyclase/cGMP-system induced by long-term hypertension in rats. Pharm Acta Helv 73, 27–35.[CrossRef][Medline]

Labarthe F, Khairallah M, Bouchard B, Stanley WC & Des Rosiers C (2005). Fatty acid oxidation and its impact on response of spontaneously hypertensive rat hearts to an adrenergic stress: benefits of a medium-chain fatty acid. Am J Physiol Heart Circ Physiol 288, H1425–H1436.[Abstract/Free Full Text]

Levy D, Garrison RJ, Savage DD, Kannel WB & Castelli WP (1990). Prognostic implications of echocardiographically determined left ventricular mass in the Framingham heart study. N Engl J Med 322, 1561–1566.[Abstract]

MacCarthy PA & Shah AM (2000). The role of nitric oxide in cardiac ischemia-reperfusion. In Handbook of Experimental Pharmacology, vol. 143, Nitric Oxide, ed. Mayer B, pp. 545–570. Springer-Verlag, Berlin.

Mazzetti L, Ruocco C, Giovannelli L, Ciuffi M, Franchi-Micheli S, Marra F, Zilletti L & Failli P (2001). Guanosine 3': 5'-cyclic monophosphate-dependent pathway alterations in ventricular cardiomyocytes of spontaneously hypertensive rats. Br J Pharmacol 134, 596–602.[CrossRef][Medline]

Mery PF, Lohmann SM, Walter U & Fischmeister R (1991). Ca²⁺ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci U S A 88, 1197–1201.[Abstract/Free Full Text]

Muscari C, Bonafe F, Gamberini C, Giordano E, Tantini B, Fattori M, Guarnieri C & Caldarera CM (2004). Early preconditioning prevents the loss of endothelial nitric oxide synthase and enhances its activity in the ischemic/reperfused rat heart. Life Sci 74, 1127–1137.[CrossRef][Medline]

Pabla R & Curtis MJ (1996). Endogenous protection against reperfusion-induced ventricular fibrillation: role of neuronal versus non-neuronal sources of nitric oxide and species dependence in the rat versus rabbit isolated heart. J Mol Cell Cardiol 28, 2097–2110.[CrossRef][Medline]

Piech A, Dessy C, Havaux X, Feron O & Balligand JL (2003). Differential regulation of nitric oxide synthases and their allosteric regulators in heart and vessels of hypertensive rats. Cardiovasc Res 57, 456–467.[Abstract/Free Full Text]

Schoemaker RG, Leenen FH & Harmsen E (1994). Age-related increase in sensitivity for ischemic ATP breakdown in hypertrophic hearts of SHR normalized by enalapril. J Mol Cell Cardiol 26, 649–660.[CrossRef][Medline]

Shah AM (1996). Paracrine modulation of heart cell function by endothelial cells. Cardiovasc Res 31, 847–867.[CrossRef][Medline]

Shah AM & MacCarthy PA (2000). Paracrine and autocrine effects of nitric oxide on myocardial function. Pharmacol Ther 86, 49–86.[CrossRef][Medline]

Takimoto E, Champion HC, Li M, Ren S, Rodriguez ER, Tavazzi B, Lazzarino G, Paolocci N, Gabrielson KL, Wang Y & Kass DA (2005). Oxidant stress from nitric oxide synthase-3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load. J Clin Invest 115, 1221–1231.[CrossRef][Medline]

Vasquez-Vivar J, Kalyanaraman B & Martasek P (2003). The role of tetrahydrobiopterin in superoxide generation from eNOS: enzymology and physiological implications. Free Radic Res 37, 121–127.[CrossRef][Medline]

Wang P & Zweier JL (1996). Measurement of nitric oxide and peroxynitrite generation in the postischemic heart. Evidence for peroxynitrite-mediated reperfusion injury. J Biol Chem 271, 29223–29230.[Abstract/Free Full Text]

Wang QD, Morcos E, Wiklund P & Pernow J (1997). L-Arginine enhances functional recovery and Ca²⁺-dependent nitric oxide synthase activity after ischemia and reperfusion in the rat heart. J Cardiovasc Pharmacol 29, 291–296.[CrossRef][Medline]

Williams MW, Taft CS, Ramnauth S, Zhao ZQ & Vinten-Johansen J (1995). Endogenous nitric oxide (NO) protects against ischaemia-reperfusion injury in the rabbit. Cardiovasc Res 30, 79–86.[CrossRef][Medline]

Xie YW, Kaminski PM & Wolin MS (1998). Inhibition of rat cardiac muscle contraction and mitochondrial respiration by endogenous peroxynitrite formation during posthypoxic reoxygenation. Circ Res 82, 891–897.[Abstract/Free Full Text]

Yasmin W, Strynadka KD & Schulz R (1997). Generation of peroxynitrite contributes to ischemia-reperfusion injury in isolated rat hearts. Cardiovasc Res 33, 422–432.[Abstract/Free Full Text]

	Acknowledgements

 
We are grateful to Mr Kengo Nakagawa (Labo-Support, Osaka, Japan) for his competent technical assistance. There are no conflicts of interest for our work.

This Article Abstract Full Text (PDF) All Versions of this Article: 92/1/147    most recent expphysiol.2006.035097v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Itoh, T. Articles by Abe, K. Search for Related Content PubMed PubMed Citation Articles by Itoh, T. Articles by Abe, K.