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(Circulation. 1997;96:1616-1623.)
© 1997 American Heart Association, Inc.

Articles

Selective Modulation of Inducible Nitric Oxide Synthase Isozyme in Myocardial Infarction

Stephen M. Wildhirt, MD; Hiroshi Suzuki, MD; Damian Horstman; Susanne Weismüller, MD; Ryszard R. Dudek, MD, PhD; Kazuhide Akiyama, MD; ; Bruno Reichart, MD

From the Department of Cardiac Surgery, Ludwig-Maximilians University, Munich, Germany (S.M.W., S.W., W.P.W., B.R.), and the Department of Experimental Cardiology, Huntington Medical Research Institutes, Pasadena, Calif (H.S., D.H., R.R.D., K.A.).

Correspondence to Stephen M. Wildhirt, MD, Department of Cardiac Surgery, Ludwig-Maximilians University, Marchioninistr 15, 81377 Munich, Germany. E-mail wildhirt{at}hch.med.uni-muenchen.de

	Abstract

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Background Inducible nitric oxide synthase (iNOS) is activated in cardiac disorders. We investigated the contribution of increased iNOS activity to the development of left ventricular dysfunction after myocardial infarction by selective inhibition of the isozyme.

Methods and Results Male New Zealand rabbits were subjected to myocardial infarction. Animals were treated with either saline, S-methylisothiourea sulfate (SMT) (a selective iNOS inhibitor), or N-nitro-L-arginine (L-NNA) (a nonselective NOS inhibitor). Inducible and constitutive NOS (cNOS) activity, plasma NO_x, cGMP, hemodynamics, and myocardial blood flow were measured before and 5, 24, and 72 hours after coronary occlusion. Infarction 72 hours after occlusion resulted in increased myocardial iNOS activity, increased cardiac NO_x production, and elevated cGMP levels. cNOS remained unchanged. Infarction increased left ventricular end-diastolic pressure (LVEDP) and decreased maximum +dP/dt and -dP/dt. L-NNA inhibited iNOS and cNOS activities and plasma NO_x levels. L-NNA further increased LVEDP and reduced myocardial blood flow. Administration of SMT 72 hours after infarction significantly inhibited iNOS and cardiac NO_x production but had no effects on cNOS. SMT improved left ventricular maximum +dP/dt and -dP/dt and decreased LVEDP. Myocardial blood flow in the remote myocardium increased.

Conclusions These findings suggest that induction of iNOS activity 72 hours after infarction exerts negative inotropic effects and contributes to the development of myocardial dysfunction; selective modulation of increased iNOS activity by SMT improves cardiac performance, enhances myocardial blood flow, and may be beneficial in the treatment of acute myocardial infarction.

Key Words: myocardial infarction • regional blood flow • hemodynamics • ischemia

	Introduction

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Induction of iNOS has been reported in cardiac disorders involving inflammatory processes, such as dilated cardiomyopathy, acute allograft rejection, or MI.^{1 2 3 4 5} Various studies showed that infiltrating macrophages and cardiomyocytes are major cellular sources of enhanced iNOS activation under these conditions.^{3 5 6} Increased production of NO derived from macrophage iNOS has been shown to depress the contractile response of ventricular cardiomyocytes to ß-adrenergic agonists.^{7 8 9 10}

Non–isoform-selective inhibition of NOS has been investigated as a potential therapeutic strategy in several experimental settings. However, nonselective inhibition of NOS isozymes has been shown to affect endothelial cNOS; it was deleterious because of marked vasoconstriction and resulted in reduction of cardiac output and increased mortality, as shown in endotoxemic rats.¹¹ Similar harmful effects on the regulation of vasomotor tone and blood pressure were reported by others.^{12 13 14}

Thus, selective modulation of the inducible isozyme, while preserving endothelial cNOS activity, could avoid these complications and may provide a novel therapeutic modality in cardiac disorders associated with induction of NOS. SMT, a non–amino acid analogue of L-arginine, and other isothioureas are potent and selective inhibitors of human and rat iNOS.^{15 16 17} Selective modulation of increased iNOS activation by SMT exerted beneficial effects and improved survival in a rodent model of septic shock.¹⁸ The concept of preferential inhibition of iNOS activity was also investigated by Worral et al² ; they reported prolonged cardiac allograft survival and improvement of graft contractile function after administration of aminoguanidine in rats.

MI is associated with increased iNOS activity, left ventricular and endothelial dysfunction, and depressed coronary flow and flow reserve in the surviving myocardium and may lead to the development of heart failure.^{19 20 21} The functional significance of increased iNOS activation after acute MI has not been studied to date. We therefore investigated the differential hemodynamic effects of SMT, a selective iNOS inhibitor, and L-NNA, a non–isoform-selective NOS inhibitor, in a time course of iNOS induction in rabbits with postinfarction left ventricular dysfunction.

	Methods

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Experimental Protocol
The experimental protocol was approved by the institutional Animal Care and Use Committee. Animals were randomly assigned to one of the four groups: A, sham (n=6); B, treatment with saline (vehicle; 3.0 mL) before and B1, 5 hours (n=5), B2, 24 hours (n=5), or B3, 72 hours (n=6) after coronary occlusion; C, treatment with L-NNA (10 mg/kg IV) before and C1, 5 hours (n=5), C2, 24 hours (n=5), or C3, 72 hours (n=6) after coronary occlusion; and D, administration of SMT (3 mg/kg IV) before and D1, 5 hours (n=5), D2, 24 (n=5) hours, or D3, 72 hours (n=6) after coronary occlusion. An additional 21 animals were used for dose-response measurements and determination of plasma nitrite levels. In the present setting, only acute effects of the inhibitors were assessed, and no consideration was given to continuous inhibition of NOS activity. The time interval chosen was based on the time course of iNOS induction in this model. iNOS peaked 72 to 96 hours after coronary occlusion and subsequently decreased over time until day 14, when levels comparable to those of sham animals were observed (Fig 1A). We hypothesized that administration of the inhibitors at peak iNOS activity exerted maximal effects on hemodynamics and changes in MBF.

View larger version (27K): [in this window] [in a new window]   Figure 1. A, iNOS activity (fmol · µg-1 · min-1) expressed as production of L-citrulline in left ventricular myocardium. Significant increase is noted in infarcted regions, with peak activity occurring 72 hours after coronary occlusion. Significant differences vs sham (0.26±0.08) are noted at 48 (0.71±0.12*), 72 (1.24±0.16*), and 96 hours (1.09±0.11*) and on postoperative day (POD) 7 (0.85±0.13*). No significant increase is noted in noninfarcted regions and 5 and 24 hours after coronary occlusion. On POD 14, iNOS activity has declined to levels determined in sham animals. Data are mean±SD. *P<.05 vs sham. B, Effects of various concentrations of L-NNA (NOLA) and SMT (2.5, 10, and 25 mmol/L) on cNOS and iNOS activity (in % of total enzyme activity). Both inhibitors act in a dose-dependent manner. L-NNA inhibits both cNOS and iNOS but exerts greater effects on cNOS. In contrast, SMT has nearly no effects on cNOS activity but potently inhibits iNOS activity. Data are mean±SD. n=4 left ventricular myocardial preparations from infarcted regions and noninfarcted control regions per measurement point. C, Inhibitory effects of in vivo administration of L-NNA (10 mg/kg) and SMT (3 mg/kg) on cNOS and iNOS activity (fmol · µg-1 · min-1) in infarcted regions and noninfarcted control regions of the left ventricle 72 hours after coronary occlusion. As shown, L-NNA exerts potent inhibitory effects on cNOS activity in both infarcted and noninfarcted regions. However, SMT shows only effects on increased iNOS activity in infarcted regions and is significantly more potent than L-NNA in inhibiting iNOS activity (infarct+saline, 1.24±0.09; infarct+L-NNA, 0.78±0.1*; infarct+SMT, 0.43±0.06*). Values are mean±SD. *P<.05 vs sham-operated animals. P<.05 vs infarct+L-NNA.

Heart rate, LVESP and LVEDP, and maximum positive and negative dP/dt (+dP/dt, -dP/dt) as an index of the inotropic state were monitored and recorded continuously with a Simultrace recorder (Honeywell) and a heart performance analyzer (HPA-100, Micro-Med). Rate-pressure product, an index of cardiac work, was calculated from arterial systolic pressure and heart rate. CVR was estimated from the ratio of mean arterial blood pressure (mm Hg), recorded just before each microsphere injection, and the corresponding MBF (mL · g^-1 · min^-1).

RMBF was measured by four different colors of fluorescent microspheres injected in random order at definite time points: (1) when animals had returned to stable hemodynamic conditions (baseline); (2) 5 minutes after administration of drugs (SMT, L-NNA, saline); (3) at definite time points after coronary occlusion (5, 24, and 72 hours); and (4) at definite time points after coronary occlusion (5, 24, and 72 hours) 5 minutes after administration of drugs (saline, L-NNA, SMT).

Surgical Preparation
Anesthesia was maintained with pentobarbital as described.²² An 18-gauge catheter was inserted into the left common carotid artery and advanced into the aortic root for measurement of hemodynamics and withdrawal of reference blood samples. After a left thoracotomy was performed in the fourth intercostal space, an 18-gauge catheter was inserted into the left ventricle via the left ventricular apex for continuous measurement of hemodynamics. A 20-gauge catheter was inserted into the left atrium for injection of microspheres. Coronary sinus blood was drawn through a 25-gauge winged infusion set. Catheters were primed with heparinized saline. After completion of catheterization, animals were allowed to stabilize for 30 minutes before the experimental protocol was started.

To produce MI, the anterolateral branch of the circumflex coronary artery was occluded midway between the left atrial appendage and apex with a 4-0 prolene suture. In sham-operated animals, the suture was kept in place without ligation. Appearance of cyanosis and bulging of the anterolateral aspect of the left ventricle documented successful coronary occlusion. At the end of the experiment, the chest was closed in layers, and animals were inspected daily until their scheduled reoperation. All animals received a nitrate/nitrite-poor diet.

Determination of Infarct Size
After euthanasia with sodium pentobarbital, hearts were removed, mounted on a Langendorff apparatus, and perfused with saline for 1 minute. In sham animals, the suture was ligated before perfusion. The infarcted region was determined as described previously with triphenyltetrazolium chloride and Evans blue dye.²³ Left ventricular rings were placed between clear overlays, and regions were traced on paper. Images were scanned on an Apple Macintosh computer, and areas were planimetered with appropriate software. Infarct size was expressed as percentage of total left ventricular myocardium.

NOS Assay
The general method of Bredt and Snyder²⁴ was used to measure the conversion of L-[¹⁴C]arginine to [¹⁴C]citrulline. In brief, 40.0 mg tissue from the infarcted and noninfarcted regions was homogenized in 1.0 mL Tris buffer (0.05 mol/L, pH 7.4). Supernatants were adjusted to a protein content of 2.0 mg/mL with protein assay kit P5656 (Sigma) with BSA as standard and were used for the enzyme activity assay immediately. L-[¹⁴C]Arginine was purified by column cation exchange chromatography. cNOS activity was measured in the presence of NADPH (1 mmol/L), calmodulin (30 nmol/L), Ca²⁺ (2 mmol/L), and tetrahydrobiopterin (5 µmol/L) (all from Sigma). iNOS was determined in the presence of the above factors and EGTA (5 mmol/L) but without Ca²⁺. NOS activity (fmol · µg^-1 · min^-1) was linear with respect to time for at least 30 minutes.

Determination of Plasma NO_x^- by Chemiluminescence
Blood samples from peripheral vein, coronary sinus, and carotid artery catheter (ascending aortic position) were withdrawn before coronary occlusion and at definite time points (5, 24, and 72 hours) after coronary occlusion. Plasma was deproteinized by ultrafiltration (Centrifree micropartition system, Amicon). The nitrate content of the sample was reduced to nitrite by incubation of 100 µL deproteinized plasma for 1 hour at 37°C with nitrate reductase (20 µL), FAD (6.0 mmol/L), NADPH (5.0 mmol/L), and phosphate buffer (1.2 mmol/L) in appropriate dilutions to give a final sample volume of 150 µL. Ten microliters of the reduced sample was injected into the reaction vessel containing reducing solution (30.0 mg of 1,1'-dimethylferrocene in 3.0 mL acetonitrile, acidified with 49.0 µL 70% perchloric acid). Under these conditions, nitrite was reduced to NO and detected by chemiluminescence with an NO analyzer (207B, Sievers Research Inc). Standard curves were performed in each experiment with a solution of NaNO₂ (20 to 80 µmol/L). Correlation coefficients of all standard curves were r>.98.

Determination of Myocardial cGMP Levels
cGMP levels were determined as described previously.²⁵ In brief, left ventricular tissue samples from infarcted and noninfarcted regions were analyzed in duplicate with a radioimmunoassay kit (TRK 500, Amersham Corp). Frozen myocardial samples were homogenized with 0.5 mL of 6% trichloroacetic acid at 4°C in a glass tissue grinder. Homogenates were centrifuged at 4°C (10 000g; 10 minutes). The supernatant was removed and extracted four times with 5 mL water-saturated diethyl ether. The aqueous phase was lyophilized, and the residue was dissolved in 0.3 mL assay buffer. The protein pellet was solubilized by addition of 1.5 mL of 1 mol/L NaOH for 24 hours. cGMP concentration was expressed as pmol/mg protein.

Measurement of MBF
For determination of RMBF, the method described previously was followed, with slight modification.²⁶ At each time point, 250 000 fluorescent microspheres (15 µm in diameter; blue, blue-green, yellow-green, orange, or red; Triton Technology) were vortexed, sonicated, and injected into the left atrial catheter, followed by a flush of heparinized, prewarmed saline (3.0 mL; 37°C). Reference blood samples were withdrawn from the carotid artery catheter at a rate of 1.36 mL/min (model 600, Harvard Apparatus) starting 20 seconds before injection of microspheres, for a total of 120 seconds.

Myocardial samples were taken from the infarcted and noninfarcted regions of the left ventricle. In sham-operated animals, samples were taken from corresponding regions. Tissue samples were processed as described previously. During microsphere filtration, lipids, triphenyltetrazolium chloride, and Evans blue dye were removed by rinsing of filters with a solution consisting of 2% Tween 80 in distilled water (60%) and ethyl alcohol (40%). Emission peaks were measured with a fluorescence spectrophotometer (F-4500, Hitachi Instruments). Preliminary experiments showed absence of autofluorescent activities for triphenyltetrazolium chloride, Evans blue dye, and tissue blanks (no microspheres added) within the spectral range of the different colors.

Statistical Analysis
Mean values are shown with their SDs. Paired t test was used to assess differences in hemodynamics and blood flow within each individual animal. ANOVA was used to assess differences between groups. Correlation coefficient was determined by simple regression analysis. Values of P<.05 were considered statistically significant.

	Results

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Histopathology
Infarcts at 5, 24, and 72 hours after coronary occlusion were verified by histopathological examination. All hearts showed transmural infarction, which consisted of hemorrhage, myocyte coagulation necrosis (5, 24, and 72 hours), infiltrating mononuclear cells (72 hours), and some neutrophils and lymphocytes (24 and 72 hours) (hematoxylin-eosin staining) (data not shown). These findings did not differ between groups.

Myocardial Infarct Size
Mean infarct size as a percentage of the total left ventricular myocardium did not differ significantly among groups, indicating that infarct size per se had no effect on differences in hemodynamics and MBF between groups (5 hours: saline, 22.5±4.8% versus L-NNA, 22.1±5.6%, P=.98; SMT, 21.3±4.2%, P=.87 versus saline; 24 hours: saline, 25.6±6.3% versus L-NNA, 24.9±3.7%, P=.83; SMT, 25.2±3.6%, P=.91 versus saline; 72 hours: saline, 24.89±5.2% versus L-NNA, 23.44±6.8%, P=.94; SMT, 23.10±4.5%, P=.91 versus saline).

Left Ventricular NOS Activity
Fig 1A shows the time course of iNOS activation in infarcted animals. iNOS activity peaked 72 to 96 hours after coronary occlusion. No significant increase was determined 5 and 24 hours after coronary occlusion.

Fig 1B illustrates inhibitory effects of various concentrations of L-NNA and SMT regarding cNOS and iNOS activity.

iNOS activity in noninfarcted regions did not differ significantly compared with sham-operated animals (Fig 1C). Seventy-two hours after coronary occlusion, iNOS activity increased significantly in infarcted regions of saline-treated animals compared with sham animals (1.24±0.16 versus 0.26±0.08, P<.05), respectively. Administration of L-NNA significantly inhibited iNOS activity compared with saline-treated animals (iNOS, 0.78±0.17 versus 1.24±0.16 fmol · µg^-1 · min^-1, P=.02). SMT was more potent than L-NNA in inhibiting iNOS activity compared with saline-treated animals (0.43±0.08 versus 1.24±0.16 fmol · µg^-1 · min^-1, P=.002), respectively (Fig 1C).

cNOS activity in infarcted animals did not change significantly over time (Fig 1A). Interestingly, there were still considerable levels of cNOS activity (fmol · µg^-1 · min^-1) within tissues obtained from infarcted regions (Fig 1C). Administration of L-NNA almost completely inhibited cNOS activity compared with sham (sham, 0.19±0.03; infarct+saline, 0.14±0.05; infarct+L-NNA, 0.03±0.04; P=.004) (Fig 1C). Inhibitory effects of SMT on cNOS were noted in some animals; however, the effect was not statistically significant (sham, 0.19±0.03; infarct+saline, 0.14±0.05; infarct+SMT, 0.12±0.09; P=NS).

Plasma NO_x and Myocardial cGMP Levels
Standard curves revealed a linear relationship for known amounts of nitrite (NO₂) and nitrate (NO₃) in plasma. All results represent total plasma NO_x levels (µmol/L) (as the sum of plasma nitrite and nitrate). Fig 2A shows the time course of plasma NO_x (µmol/L) in peripheral venous blood. NO_x levels determined before coronary occlusion were comparable to those of sham animals (29.12±1.6 versus sham, 31.52±1.03, P=NS). No significant increase versus sham was noted 5 hours (30.45±1.3) and 24 hours (32.16±1.2) after coronary occlusion. However, a marked and significant increase was observed in all three parts of the vasculature 72 hours after infarction, and a significantly higher NO_x level is observed in coronary sinus blood compared with aortic values, indicating cardiac release of NO_x 72 hours after infarction (sham, 31.52±1.03; peripheral vein, 52.42±1.5; aorta, 52.7±1.6; coronary sinus, 58.9±2.1). The increase in plasma NO_x correlates with peak iNOS activity. Both L-NNA and SMT significantly inhibited plasma NO_x concentration. However, cardiac NO_x release (difference between aortic and coronary sinus levels) was abolished by administration of SMT (aorta, 37.7±2.3 versus coronary sinus, 37.02±1.7) but not by L-NNA (aorta, 44.8±2.2 versus coronary sinus, 49.5±1.9; P<.05).

View larger version (20K): [in this window] [in a new window]   Figure 2. A, Plasma NOx concentration in peripheral vein, aorta, and coronary sinus at various time points. No significant increase vs sham is noted 5 and 24 hours after coronary occlusion. However, a marked and significant increase is observed in all three parts of the vasculature 72 hours after infarction, and a significantly higher NOx level is observed in coronary sinus blood compared with aortic values, indicating cardiac release of NOx 72 hours after infarction (sham, 31.52±1.03; peripheral vein, 52.42±1.5*; aorta, 52.7±1.6*; coronary sinus, 58.9±2.1*). Increase in plasma NOx correlates with peak iNOS activity. Both L-NNA (NOLA) and SMT significantly inhibit plasma NOx concentration. However, cardiac NOx release (difference between aortic and coronary sinus levels) is abolished by administration of SMT (aorta, 37.7±2.3 vs coronary sinus, 37.02±1.7) but not by L-NNA (aorta, 44.8±2.2 vs coronary sinus, 49.5±1.9; P<.05). Data are mean±SD. B, Simple regression analysis reveals a highly significant positive correlation (r=.973, P=.001) between myocardial iNOS activity of infarcted left ventricular regions and total plasma nitrite/nitrate (NOx) levels obtained from coronary sinus at various time points (5, 24, and 72 hours) after coronary occlusion. n=19 animals. C, cGMP levels in infarcted and noninfarcted regions of left ventricular myocardium. Significant increase of cGMP is noted in infarcted regions of saline-treated animals vs sham 72 hours after coronary occlusion (1.27±0.3 vs sham, 0.49±0.11; *P=.001).

Simple regression analysis revealed a significant positive correlation between iNOS activity and cardiac NO_x production (r=.97, n=19, P<.001) (Fig 2B).

Left ventricular cGMP levels did not differ significantly between noninfarcted regions of animals in the saline group and sham-operated animals 72 hours after occlusion (0.63±0.24 versus 0.49±0.11 pmol/mg protein, P=.24). A significant increase of myocardial cGMP levels was measured in infarcted tissues 72 hours after coronary occlusion compared with sham (1.27±0.3 versus 0.49±0.11 pmol/mg protein, P=.001), respectively (Fig 2C).

Hemodynamics
Hemodynamic changes in response to administration of various concentrations of both inhibitors are shown in Fig 3A and 3B. No significant changes in LVESP response are noted with administration of SMT before occlusion as well as 5 and 24 hours after infarction. However, SMT exerts significant pressure response 72 hours after coronary occlusion (in the presence of increased iNOS activity). In contrast, L-NNA exerts significant pressure response before coronary occlusion as well as 5, 24, and 72 hours after coronary occlusion (Fig 3B).

View larger version (35K): [in this window] [in a new window]   Figure 3. A and B, Effects of SMT (1 to 10 mg/kg IV) (A) and L-NNA (NOLA; 1 to 30 mg/kg IV) (B) on LVESP response before and 5, 24, and 72 hours after coronary occlusion. L-NNA exerts pressure response at all time points. In contrast, no significant effects are noted with SMT before or 5 or 24 hours after occlusion. Additional administration of excess L-arginine (300 mg/kg IV) reverses effects observed by both inhibitors. Data are mean±SD. *P<.05 vs levels before occlusion and 5 and 24 hours after MI. P<.05 vs 72 hours after MI for 3.0, 5.0, and 10.0 mg/kg of SMT (A). **P<.05 vs pressure response observed with 30.0 mg/kg L-NNA at all time points. C and D, Effects of saline, SMT, and L-NNA on LVEDP. MI causes significant increase in LVEDP in all infarcted groups. Administration of L-NNA further increases LVEDP at all time points. SMT does not affect LVEDP 5 and 24 hours after infarction. However, a significant reduction of LVEDP is noted after administration of SMT 72 hours after coronary occlusion (3.1±0.8 vs 5.3±0.7, respectively) (presence of increased iNOS activity). Effects of saline, SMT, and L-NNA on maximum +dP/dt. No significant changes are noted in all groups before coronary occlusion. Infarction results in significant reduction of maximum +dP/dt (*P<.05 vs sham) after 5, 24, and 72 hours. Administration of SMT 72 hours after infarction exerts a significant improvement of maximum +dP/dt (2267±112 vs 1694±398, P<.05, respectively). In contrast, L-NNA has no beneficial effects on maximum +dP/dt at any time point. *P<.05 vs sham; P<.05 vs infarcted animals treated with saline 72 hours after infarction.

Hemodynamic data of sham-operated animals determined on the first day of surgery did not differ significantly from measurements after 5, 24, and 72 hours, indicating that the surgical procedure per se did not affect the experimental protocol. In all groups, infarcted animals developed left ventricular dysfunction (Table). At 5, 24, and 72 hours after coronary occlusion, LVEDP was increased; maximum +dP/dt and -dP/dt and LVESP were significantly reduced compared with levels obtained before coronary occlusion (Table).

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Effects of Saline (3.0 mL), L-NNA (10 mg/kg), and SMT (3 mg/kg) Before Coronary Occlusion and 5, 24, and 72 Hours After MI

Administration of saline did not alter baseline and postinfarction cardiovascular hemodynamics (Table). Injection of L-NNA (10 mg/kg IV) on day 1 before coronary occlusion significantly increased LVESP and LVEDP; it had no significant effects on maximum +dP/dt and -dP/dt. Administration of L-NNA 5, 24, and 72 hours after coronary occlusion resulted in effects comparable to those observed before occlusion, although peak LVESP response appeared to be attenuated after 72 hours (Table).

Administration of SMT (3 mg/kg IV) before coronary occlusion as well as 5 and 24 hours after coronary occlusion had no significant effects on maximum +dP/dt and -dP/dt, LVESP, or LVEDP (Fig 3C and 3D; Table). In contrast, administration of SMT 72 hours after coronary occlusion resulted in a prompt and significant increase in maximum +dP/dt (2267±112 versus 1694±398, P<.05) and -dP/dt (1871±201 versus 1569±131, P<.05) and LVESP (72.6±3.1 versus 53.9±4.7 mm Hg, P<.05). Moreover, SMT significantly decreased LVEDP (3.1±0.8 versus 5.3±0.7 mm Hg, P<.05), respectively (Fig 3C; Table).

Neither L-NNA nor SMT had significant effects on rate-pressure product before and 72 hours after coronary occlusion (Table).

Effects of L-NNA and SMT on RMBF
Coronary occlusion resulted in transmural infarction. RMBF within the infarcted region was reduced by >90% in all groups. This is because rabbits do not possess sufficient collateral blood flow within the myocardium.

RMBF before coronary occlusion averaged 1.05±0.11 mL · min^-1 · g^-1. Administration of saline (3.0 mL) at any time point had no significant effect on RMBF in noninfarcted regions (Fig 4A and 4B). L-NNA given before coronary occlusion significantly reduced MBF (0.65±0.13 versus 1.01±0.2 mL · min^-1 · g^-1; P=.03), most likely because of inhibition of endothelial cNOS (Figs 1B, 1C, and 4). SMT did not significantly change RMBF before coronary occlusion (1.08±0.18 versus 1.06±0.1 mL · min^-1 · g^-1, P=.33) (Fig 4A).

View larger version (23K): [in this window] [in a new window]   Figure 4. A, MBF in surviving left ventricular myocardium in response to saline (3.0 mL), SMT (3 mg/kg), and L-NNA (NOLA; 10 mg/kg) before coronary occlusion. Neither saline nor SMT has significant effects on MBF. However, administration of L-NNA results in a significant reduction of MBF, most likely a result of vasoconstrictive effects due to inhibition of endothelial cNOS. *P<.05 vs baseline levels without inhibitor. n=7 blood flow measurements for MI+saline. n=6 blood flow measurements for MI+L-NNA and n=6 for MI+SMT. B, MBF measurements obtained in noninfarcted left ventricular regions 72 hours after coronary occlusion. Although no effects are noted with saline administration, a significant reduction in MBF is observed with L-NNA. In contrast, SMT enhances MBF in surviving myocardium 72 hours after infarction (presence of increased iNOS activity). This effect was not observed 5 and 24 hours after coronary occlusion. *P<.05 vs measurements obtained 72 hours after infarction without inhibitor. n=7 blood flow measurements for MI+saline. n=6 blood flow measurements for MI+L-NNA and n=6 for MI+SMT.

At 5, 24, and 72 hours after coronary occlusion, no significant changes of blood flow measurements (mL · min^-1 · g^-1) in noninfarcted regions in response to saline (3.0 mL) were noted (5 hours, 1.03±0.12 versus 1.02±0.08; 24 hours, 1.05±0.10 versus 1.04±0.11; 72 hours, 0.94±0.07 versus 0.96±0.09; P=.4).

RMBF (mL · min^-1 · g^-1) in noninfarcted regions was significantly reduced in rabbits given L-NNA (5 hours, 0.58±0.14; 24 hours, 0.53±0.09; 72 hours, 0.55±0.18 versus 1.03±0.23; P<.05 versus baseline [Fig 4B]).

SMT did not have significant effects on blood flow 5 hours after MI (1.07±0.09 versus 1.08±0.12, P=NS). After 24 hours, a slight increase in blood flow was observed; however, these changes were not significant (1.03±0.13 versus 1.09±0.11, P=NS). In contrast, administration of SMT 72 hours after MI significantly increased MBF in noninfarcted regions (1.24±0.15 versus 1.04±0.11 mL · min^-1 · g^-1, P=.04) (Fig 4B).

As shown in the Table, injection of L-NNA significantly increased CVR; it is consistent with the potent inhibition of myocardial cNOS activity by L-NNA as shown in Fig 1C. This was not observed in animals treated with either saline or SMT.

	Discussion

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The major finding in the present study is a significant increase of iNOS activity associated with increased cardiac NO_x production 72 hours after acute MI. During the time course of iNOS activation, the cNOS activity remains at levels comparable to baseline values. The present data indicate that increased iNOS activation contributes to depressed left ventricular performance and reduced MBF in the remote myocardium. Isoform-selective modulation of iNOS by SMT inhibits cardiac NO_x release, improves the cardiac contractile state of infarcted hearts, and enhances RMBF in the surviving myocardium.

Effects of L-NNA
In the present study, administration of L-NNA, a non–isoform-selective NOS inhibitor, exerts inhibitory effects on both cNOS and iNOS (Fig 1B and 1C) associated with decreased plasma NO_x levels (Fig 2A). With respect to hemodynamics, L-NNA increases LVESP but significantly reduces maximum +dP/dt and -dP/dt, important determinants of cardiac contractile function. In addition, L-NNA further increases the already elevated LVEDP (Table). RMBF in the noninfarcted (surviving) myocardium is significantly reduced and CVR is significantly increased by L-NNA, which is most likely mediated by inhibition of the endothelial cNOS (Fig 1C, Table).

Similar findings were shown in several other experimental settings in which non–isoform-selective NOS inhibitors were used.^{27 28} Cobb et al²⁹ reported increased systemic vascular resistance and a decreased cardiac index after administration of N-monomethyl-L-arginine in endotoxemic beagles. Moreover, a recent report by Petros et al¹² showed a dose-dependent reduction in cardiac output after administration of N^G-monomethyl-L-arginine, possibly because of coronary vasoconstriction and subsequent reduction of MBF.³⁰ These data suggest that non–isoform-selective inhibition of NOS exaggerates myocardial dysfunction because of reduction of regional blood flow in the surviving myocardium and illustrates the necessity of preserved endothelial cNOS activity in maintaining adequate coronary perfusion. Elevation of blood pressure by L-NNA is most likely due to increased systemic vascular resistance at the expense of reduced cardiac output.³⁰

Effects of SMT
SMT, a competitive NOS inhibitor with selectivity toward the inducible isoform, significantly inhibits iNOS activity without significant effects on cNOS (Fig 1B and 1C); it is associated with a significant reduction in cardiac NO_x production (Fig 2A). SMT causes a prompt restoration of maximum +dP/dt, -dP/dt, and LVESP and reduces LVEDP. In addition, with SMT, a significant increase in MBF to the surviving myocardium is noted (Fig 4B).

The inhibitory effect of SMT on iNOS activity is likely to be the major underlying reason for improvement of left ventricular performance. First, heart rate was not significantly different between untreated and SMT-treated infarcted animals, indicating that peripheral vascular resistance was not decreased after SMT. Second, increased preload was probably not the reason for improvement in cardiac performance, because LVEDP decreased after treatment with SMT. Third, the hemodynamic effects of SMT observed 72 hours after coronary occlusion (in the presence of increased iNOS activity) were not observed before coronary occlusion (absence of iNOS activity). Evidence supporting the contention that afterload did not decrease in infarcted animals is that a significant increase of LVESP was observed in this postinfarction group after administration of SMT. Moreover, CVR was not significantly altered by SMT, most likely because of preservation of endothelial cNOS.

In addition, the pressure response to SMT (10 mg/kg IV) observed 72 hours after infarction is attenuated by excess L-arginine (300 mg/kg IV), suggesting that SMT competitively and reversibly inhibits iNOS activity at the L-arginine site in vivo (Fig 3A). Finally, Szabo et al¹⁸ showed that the effects of SMT are selective for NOS, because it does not inhibit the activities of other enzymes, including xanthine oxidase, diaphorase, lactate dehydrogenase, monoamine oxidase, catalase, cytochrome P450, or superoxide dismutase.

The increase in RMBF in the surviving myocardium observed after administration of SMT is likely to be due to reduction of LVEDP and improvement of blood pressure, thereby reducing wall stress and increasing the coronary driving pressure. In addition, in the present study, no significant effects on cNOS were noted; preserving cNOS while inhibiting iNOS may be one key mechanism for improvement of cardiac performance. This is supported by others who showed that inhibition of enhanced NO production by SMT while endothelial cNOS is preserved significantly increased renal perfusion pressure and blood flow in septic rats.¹⁸

What are the potential underlying mechanisms by which iNOS mediates left ventricular dysfunction? Increased cardiac cGMP formation may be one explanation of NO-mediated contractile dysfunction, as previously reported.^{9 31 32} As shown in Fig 2C, increased iNOS activity in MI is associated with enhanced production of intracellular cGMP, a second messenger that mediates cardiac cell and smooth muscle cell relaxation, possibly by activation of cGMP kinase, subsequently decreasing intracellular Ca²⁺ levels.³³ Induction of the NO/cGMP pathway is known to attenuate the response of vascular smooth muscle cells and cardiomyocytes to - and ß-adrenergic stimuli.³⁴ Inhibition of iNOS by SMT may therefore enhance the cellular response to adrenergic stimuli, leading to improvement of vasomotor tone and the contractile state of the preserved myocardium.^{18 35}

Other NO-mediated factors might also contribute to left ventricular dysfunction, such as inhibition of mitochondrial respiration through covalent modification of iron sulfur–centered respiratory enzymes and thus reduction of ATP synthesis.³⁶ Moreover, reaction of NO with superoxide anion at diffusion-limited rates leads to the formation of peroxynitrite, which is potentially injurious to myocardial tissue after its rapid protonation and decomposition to highly oxidant species.^{37 38}

Involvement of increased iNOS formation in pathophysiological processes has been shown in several cardiac disorders.^{1 4 6} Worral et al² reported elevated serum NO_x levels and the presence of iNOS messenger RNA in cardiac transplant rejection in rats. Selective inhibition of iNOS production by aminoguanidine prolonged graft survival and improved graft contractile function. This is supported by Russel et al,³ who recently reported that modulation of increased iNOS activity in rat cardiac allografts inhibited the inflammatory response and significantly decreased intimal thickening, suggesting an important role for macrophage-derived iNOS in mediating transplant arteriosclerosis.

In conclusion, induction of NOS in MI contributes, at least in part, to the development of left ventricular dysfunction; the findings reported here support the view that selective modulation of the high-output NO pathway regulated by iNOS while endothelial cNOS is preserved may be an additional therapeutic approach in the treatment of certain cardiac disorders, including MI.

	Selected Abbreviations and Acronyms

 

cNOS = constitutive nitric oxide synthase CVR = coronary vascular resistance iNOS = inducible nitric oxide synthase L-NNA = N-nitro-L-arginine LVEDP = left ventricular end-diastolic pressure LVESP = left ventricular end-systolic pressure MBF = myocardial blood flow MI = myocardial infarction NOS = nitric oxide synthase RMBF = regional myocardial blood flow SMT = S-methylisothiourea sulfate

	Footnotes

 
Preliminary data from this study were presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl I]:I-773).

Received November 4, 1996; revision received March 4, 1997; accepted March 7, 1997.

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