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Cardiovascular Research 1998 40(1):113; doi:10.1016/S0008-6363(98)00146-1
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Intra-coronary administration of L-arginine aggravates myocardial stunning through production of peroxynitrite in dogs

Etsuo Mori, Nobuya Haramaki*, Hisao Ikeda and Tsutomu Imaizumi

The Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan

* Corresponding author: Tel.: +81-942-35-3311 (ext. 3805); Fax: +81-942-33-6509; E-mail: haramaki@med.kurume-u.ac.jp

Received 16 September 1997; accepted 16 March 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim of this study was to investigate how the enhanced nitric oxide (NO) production by intra-coronary infusion of L-arginine acts in myocardial stunning in dogs by focusing on the involvement of peroxynitrite, a reaction product of NO and superoxide anion. Methods and Results: Dogs were divided into six groups; a control non-treated group (CON, n=9), and NG-nitro L-arginine methyl ester (L-NAME, n=6), 1 mM L-arginine (L-ARG, n=8), D-arginine (D-ARG, n=6), L-arginine plus superoxide dismutase (L-ARG+SOD, n=6), and SOD alone (SOD, n=6) treated groups. L-NAME, or L- or D-arginine was continuously infused into the left anterior descending coronary artery (LAD) starting just prior to reperfusion, whereas SOD was intravenously injected before occlusion. During 120 min of reperfusion after 15 min occlusion of LAD, myocardial contractile function in the ischemic region gradually recovered and reached approximately 70% of the preischemic level in CON, D-ARG and SOD, but it remained dyskinetic (–46%) in L-ARG. On the other hand, it was improved in L-NAME (90%). Tissue malondialdehyde was elevated (p<0.005) after reperfusion, and myocardial NO metabolites measured by an intratissue-microdialyzer increased (approximately 150%, p<0.05) in the ischemic region during reperfusion in L-ARG but not in the CON, L-NAME, D-ARG or SOD groups. In the L-ARG+SOD group, L-arginine-induced contractile dysfunction and elevation of malondialdehyde were prevented, but the increase in NO metabolites remained. These results suggest that L-arginine aggravated myocardial stunning through oxidative stress and the cytotoxicity was caused by NO derivatives but not by NO itself. The formation of nitrotyrosine, a footprint of peroxynitrite, was immunohistochemically confirmed in the ischemic region of L-ARG. Conclusions: Our results demonstrate for the first time in vivo that NO has a detrimental role in myocardial stunning through the production of peroxynitrite.

KEYWORDS Dog; Reperfusion injury; Oxidative stress; L-Arginine; Microdialysis; Nitrotyrosine


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Nitric oxide (NO) is synthesized from L-arginine through the reaction catalyzed by NO synthase (NOS) [1–3]. NO plays several important physiological roles, including vasodilatation [4]and inhibition of platelet aggregation [5, 6]. The role of NO in myocardial ischemia–reperfusion has gained considerable attention because of the controversy as to whether it acts protectively or detrimentally. Due to its vasodilatory and platelet inhibitory effects, NO may ameliorate ischemic injury. In addition, NO suppresses myocardial contractility [7], which also ameliorates ischemic injury by decreasing myocardial energy requirements [8]. In fact, in several animal models of ischemia–reperfusion, administration of L-arginine, a precursor of NO biosynthesis, has been reported to reduce reperfusion-induced myocardial injury and to protect against endothelial dysfunction [9–13]. Furthermore, NO inhibitors enhanced myocardial injury during reperfusion, suggesting that endogenous NO has protective effects against ischemia–reperfusion injury [14, 15]. In contrast to those beneficial effects of NO, detrimental effects of NO have been reported as well [16–20]. Inhibition of NO synthesis improved post-ischemic dysfunction [13, 17]and reduced infarct size in animal models [18, 19]. Moreover, L-arginine aggravated post-ischemic myocardial dysfunction in the isolated rabbit heart [20].

NO is a free radical containing an unpaired outer shell electron. Despite its free radical nature, NO is not particularly reactive in the absence of other radicals. Even reactions with metal centers are of a relatively low rate constant. However, in the presence of other radicals, NO could undergo radical–radical reactions with much faster rates. One of the critical radical–radical reactions that involve NO is that with superoxide anion to yield peroxynitrite. Superoxide anion reacts with NO at almost diffusion-limited rates (6.7x109 mol–1s–1) [21]. This reaction is at least three-fold faster than the reaction of superoxide anion with superoxide dismutase (SOD) [22]and is a million-fold faster than the superoxide anion-driven Fenton reaction that generates hydroxyl radical, a well recognized powerful oxidant. Thus, the formation of peroxynitrite is considered to be a major pathway of NO reactivity. Although peroxynitrite is not a free radical, it is a powerful oxidant that effectively oxidizes sulfhydryls, lipids and proteins, and diffuses 10 000-fold further than hydroxyl radical [21]. Moreover, the protonated form of peroxynitrite, peroxynitrous acid (ONOOH), exhibits powerful reactivity, like hydroxyl radicals, via metal-independent mechanisms [22]. Therefore, NO may exert its detrimental effect through the formation of peroxynitrite in vivo.

Myocardial stunning, i.e. prolonged contractile dysfunction during reperfusion following a brief episode of myocardial ischemia, involves the generation of superoxide anion [23]. In myocardial stunning, NO may react with superoxide anion, resulting in the formation of peroxynitrite. However, the detrimental role of peroxynitrite in myocardial stunning remains unproved in vivo. To clarify this issue, we examined the effects of intra-coronary infusion of L-arginine on post-ischemic dysfunction (stunning) after 15 min of coronary occlusion in dogs. L-Arginine aggravated myocardial stunning associated with increased myocardial content of NO metabolites (NOx) and lipid peroxidation. We demonstrated, for the first time in vivo, by immunohistochemistry that peroxynitrite was responsible for NO-induced aggravation of myocardial stunning.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Chemicals
Bovine Cu,Zn-SOD, NG-nitro L-arginine methyl ester (L-NAME), deferoxamine mesylate, butylated hydroxytoluene, thiobarbituric acid and sodium dodecyl sulfate were purchased from Sigma (St. Louis, MO, USA). Polyclonal anti-nitrotyrosine antibody was purchased from Upstate Biotechnology (New York, NY, USA). Nitrotyrosine was purchased from Aldrich (Milwaukee, WI, USA). Non-immune rabbit IgG was purchased from Zymed Laboratories (San Francisco, CA, USA). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).

2.2 Surgical preparation
Investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985) and was approved by the Animal Research Committee of the Kurume University School of Medicine. Adult healthy mongrel dogs of either sex (n=67, Center of Animal Care, Fukuoka), weighing 18 to 32 kg, were anesthetized with an intravenous injection of sodium pentobarbital (30 mg/kg body weight). After tracheal intubation, the dogs were ventilated with a tidal volume of 15 ml/kg at a respiratory rate of 15–18 cycles/min using an oxygen-flushed anesthesia machine (Model K-1, Igarashi Ika, Kyoto, Japan) and a ventilator. Arterial blood gases and pH were analyzed every 5 min during 15 min of reperfusion and every 15 min thereafter, and maintained within normal physiologic ranges; the body temperature was kept constant using a heating pad. A thoracotomy was performed in the fifth left intercostal space of each dog under sterile conditions and the heart was suspended in a pericardial cradle. The experimental preparation is shown in Fig. 1. Heparinized polyethylene catheters were placed in the right atrium through the right atrial appendage for drug administration. Left ventricular (LV) pressure was measured with a microtip catheter (Mikro-Tip Catheter Transducer model MPC-500, Millar Instruments, Houston, TX, USA) inserted through the left atrial appendage into the LV cavity. Electrocardiographic wires were sutured onto the surface of the chest wall. A segment of the left anterior descending coronary artery (LAD) was gently and circumferentially dissected free from the surrounding tissue, and a balloon occluder and a pulsed Doppler flow velocity probe (20 MHz, HDP-20S, Crystal Biotech, Holliston, MA, USA) were placed around the LAD. A catheter was inserted into a diagonal branch of the LAD, proximal to the occluder, to infuse drugs. A Doppler ultrasonic wall thickening probe (10 MHz, DMT-10, Crystal Biotech) was positioned in the center of the region to be rendered ischemic. A microdialysis probe, and a 10-mm flexible cellulose membrane (outer diameter, 220 µm) with a molecular cut-off point of 50 kDa was inserted into the myocardium in the ischemic region, which is surrounded by at least two epicardial coronary arteries distal to the occluder. An integrated measuring system based on the pulsed Doppler (Model VF-1, Crystal Biotech) was used to signal myocardial thickening and the LAD blood flow velocity. The electrocardiogram, LV pressure and dP/dt, myocardial thickening and the LAD blood flow velocity were continuously recorded on an eight-channel recorder (Model RS-3800, Gould, Valley View, OH, USA).


Figure 1
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  		Fig. 1 Experimental preparation.

 
2.3 Experimental protocols
All dogs were allowed to stabilize for approximately 40 min following the surgical preparation, and baseline hemodynamics and wall thickening were recorded. Myocardial ischemia was produced by tightening the occluder. After 15 min of occlusion, the ischemic myocardium was reperfused for 120 min. Hemodynamics and wall thickening data were obtained 5 min before coronary occlusion, 10 min after occlusion, and 15, 30, 45, 60, 75, 90, 105 and 120 min after reperfusion. Dogs were randomly divided into six groups (Fig. 2); the control group, the L-NAME group, the L-arginine group, the D-arginine group, the L-arginine plus SOD group and the SOD group. For the control group, saline was infused into the LAD through the catheter placed in the diagonal branch proximal to the occluder at 0.15 ml/min starting just prior to the reperfusion. L-NAME (20 µg/kg/min) was infused at an infusion rate of 0.15 ml/min. For the L-arginine or D-arginine group, the concentration of drug was adjusted on the basis of the coronary flow during preischemia. The concentration of L- and D-arginine was 1 mM in the LAD at the infusion rate of 0.15 ml/min, i.e., it was 100 mM when the coronary flow was 15 ml/min. For the L-arginine plus SOD group and for the SOD group, bovine Cu,Zn-SOD (15 000 U/kg body weight) was administrated into the right atrium at 15 min before the occlusion, and the same protocols as for the L-arginine and control groups were conducted thereafter. At the end of the study, the dogs were given a lethal dose of potassium chloride. The heart was removed and cut into 0.5 cm-thick slices in the plane parallel to the atrioventricular groove. Each slice was then rinsed briefly in saline and incubated in a 1% solution of 2,3,5-triphenyltetrazolium chloride buffered in 0.2 M Tris buffer to pH 7.8 at approximately 37°C for 5 min [24]. As a result, the absence of any myocardial infarction was confirmed in all dogs. The rest of the removed myocardium in the middle of the ischemic and non-ischemic regions was excised (0.5 to 1.0 g), promptly washed in ice-cold saline, immediately freeze-clumped by liquid nitrogen and stored at –80°C.


Figure 2
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  		Fig. 2 Experimental protocols. Myocardial ischemia (15 min) and reperfusion (120 min) were produced by tightening and releasing, respectively, the occluder that was placed around the left anterior descending coronary artery (LAD). In the control group, saline was infused into the LAD through the catheter placed in the diagonal branch proximal to the occluder, starting just prior to the reperfusion. In the L-NAME, L-arginine and D-arginine groups, drugs were infused instead of saline. In the L-arginine plus SOD group and in the SOD group, SOD (15 000 U/kg body weight) was administered into the right atrium 15 min before the occlusion, and the same protocol as for the L-arginine group and the control group was followed thereafter.

 
2.4 Measurement of myocardial function
Regional myocardial function was assessed using a pulsed Doppler epicardial probe according to the method previously described by Zhu et al. [25]. Briefly, the pulsed Doppler technique with a single epicardial transducer was applied to determine systolic myocardial wall displacement by digitally integrating the velocity of the myocardial layers that pass through a range-gated sample volume. The beginning and end of systole were determined from the onset of the rapid upstroke of the LV pressure tracing and the peak negative LV dP/dt, respectively. Net systolic thickening was defined as the maximal systolic increase in the wall thickness from the end-diastolic value. When paradoxical wall thinning (dyskinesis) persisted for 50% or more of systole, the maximal extent of wall thinning was subtracted from wall thickening to give the net systolic thickening. Transmural myocardial function was expressed as the percent systolic thickening fraction=(transmural net systolic thickening/end-diastolic wall thickness)x100 (%). If paradoxical systolic thinning was not observed until 10 min of coronary occlusion, the dog was excluded from this study because collateral circulation prevented ischemia [26, 27]. Consequently, five dogs were excluded.

2.5 Lipid peroxidation of myocardium
The myocardial content of malondialdehyde (MDA), a secondary product of lipid peroxidation, was determined as thiobarbituric acid (TBA)-reactive substance by modifying a previously described method [28]. Briefly, after 120 min of reperfusion, the samples were homogenized using an ultraturrax homogenizer and sonicated using a tip-sonicator (Model ms-50, Heat System-Ultrasonics, Framingdale, NY, USA) in ice-cold nitrogen-gassed 50 mM potassium phosphate buffer (pH 7.4) containing 1 mM deferoxamine mesylate, 1 mM butylated hydroxytoluene and 1.6% ethanol. After centrifugation at 1000 rpm for 10 min, 0.2 ml of the supernatant was mixed with 1 ml of 2.2% glycine solution (pH 3.6) and with 0.5 ml aqueous solution of 1% TBA and 0.6% sodium dodecyl sulfate. The mixture was incubated at 95°C for 30 min. After cooling with ice-cold water, 0.6 ml of acetic acid and 1.4 ml of chloroform were added and the mixture was shaken vigorously before being centrifuged at 3000 r.p.m. for 10 min. Then, the upper layer was spectrophotometrically measured at 532.5 nm. As a standard, 1,1,2,2-tetramethoxypropane was used.

2.6 Myocardial microdialysis
A microdialysis probe inserted into the myocardium was perfused with Ringer solution (140 mM NaCl, 4 mM KCl, 1.26 mM CaCl2 and 1.15 mM MgCl2, pH 7.4) at a constant flow rate of 6 µl/min. An equilibration period of approximately 30 min was allowed before starting the collection of dialysates. The dialysates were collected every 5 min until 15 min of reperfusion and every 15 min thereafter.

2.7 Measurement of myocardial NO metabolites
Each 20 µl sample of collected dialysate was injected into the high-performance liquid chromatography (HPLC) system. Nitrate in the dialysates was first reduced to nitrite by passing it through a copper-plated cadmium column (NO-RED, Eicom, Japan), and the total nitrite, which represents NO metabolites (NOx; nitrite plus nitrate), was mixed with a Griess reagent to form a purple azo dye in a reaction coil [29]. The absorbance of the colored product dye at 540 nm was measured using a flow-through spectrophotometer (NOD-10, Eicom). The mobile phase, which was delivered by a pump at a rate of 0.33 ml/min, was 0.15 M NaCl–NH4Cl with 0.5 g/l 4Na–EDTA in 10% methanol. The Griess reagent, which was 1.25% HCl containing 30 mM sulfanilamide and 1 mM N-(1-naphthyl)ethylenediamine, was delivered at a rate of 0.1 ml/min. A standard curve was made at the beginning of every experiment using several concentrations (10–7 to 10–5 M) of NOx standard (NaNO2–NaNO3; 1:1, v/v) (r=0.998–0.999, p<0.001), and the stability of the peak area was confirmed by injecting 10–6 M NOx standard after every ten injections of dialysate. In addition, to determine the in vitro recovery of NOx across the dialysis probe, a microdialysis probe was immersed in the solution containing several concentrations (10–7 to 10–5 M) of NOx standard, and perfused with Ringer solution at a constant flow rate of 6 µl/min at room temperature. The level of NOx in dialysates correlated well with that in the solution outside the dialyzing probe (r=0.998, p<0.0001), indicating that the NOx concentration in the myocardial dialysate could represent that in the myocardium. According to these results, on the assumption that the in vivo recovery was equal to the in vitro recovery, the myocardial NOx level was estimated to be 4.62±0.21 µM during preischemia.

2.8 Nitrotyrosine immunohistochemistry
For immunohistochemistry, animals (n=3–4 for each group) were sacrificed after 15 min of reperfusion using a lethal dose of potassium chloride, and the removed myocardium from the ischemic and non-ischemic regions was embedded in optimal cutting temperature compound and frozen using liquid nitrogen. Serial 4-µm-thick frozen sections were adhered to poly-L-lysine-coated slides and then fixed in cold acetone for 10 min. The labeled streptavidin–biotin method was used for immunohistochemical staining, as described previously (Histofine SAB-PO kit, Nichirei, Japan) [30]. Briefly, the specimens were treated with 0.6% hydrogen peroxide for 10 min to inhibit endogenous peroxidase and then blocked with 10% goat serum. Subsequently, they were incubated with 2 µg/ml of polyclonal anti-nitrotyrosine antibody or with a similar amount of non-immune rabbit IgG for 3 h at room temperature. As another negative control, sections were incubated with the anti-nitrotyrosine antibody, however, the antibody was preincubated with 5 mM nitrotyrosine (antigen-competed antibody). After washing three times in phosphate-buffered saline (pH 7.4), biotinylated anti-rabbit IgG secondary antibody was applied, followed by peroxidase-labeled streptavidin. Peroxidase activity was visualized using 3-amino-9-ethylcarbazole, and the sections were faintly counterstained with Mayer's hematoxylin.

2.9 Statistical analysis
Values are presented as mean±SEM. Statistical comparisons were performed by one-way ANOVA followed by the Scheffé test for post hoc analysis. Differences were considered to be statistically significant when the probability was less than 0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Hemodynamics
No significant differences among the six groups were found for heart rate, LV systolic or end-diastolic pressure, or mean coronary blood flow (Table 1). Although LV end-diastolic pressure tended to increase during ischemia in all groups, it was not statistically significant. No coronary blood flow was observed during ischemia and the same magnitude of reactive hyperemia was observed during reperfusion among all groups.


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  		Table 1 Hemodynamic variables before and during ischemia and after reperfusion

 
3.2 Myocardial contractile function
The cardiac wall-thickening fraction, an indicator of myocardial contractile function in the ischemic region during preischemia and ischemia, was 27.3±1.4 and –20.2±2.1%, respectively, in the control group. No differences in wall thickening were observed among the six groups (Fig. 3) during ischemia. Thus, the myocardium became dyskinetic during ischemia in all groups. The wall contractile function gradually recovered during reperfusion and, at 120 min of reperfusion, the thickening fraction reached 19.0±1.5 and 19.6±1.2% (approximately 70% of the preischemic level) in the control group and the D-arginine group, respectively. The wall thickening fraction in the L-NAME group was higher than that in the control group between 75 and 120 min of reperfusion. In contrast, it remained dyskinetic (–12.7±1.6% at 120 min, –46% of preischemic level; p<0.001) in the L-arginine group. This aggravation of cardiac contractile dysfunction by L-arginine infusion was prevented by pretreatment with SOD (19.5±1.1% at 120 min). Treatment with SOD alone did not affect myocardial stunning (Fig. 3). In addition, when L-arginine was infused into the LAD of the non-ischemic heart as a preliminary experiment, the wall thickening fraction of the infused area was not affected (n=3).


Figure 3
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  		Fig. 3 Changes in the wall thickening fraction. The wall thickening fraction, an index of myocardial contractile function in the ischemic region, during preischemia and ischemia was 27.3±1.4 and –20.2±2.1%, respectively, in the control group, and no differences was observed among the six groups. The wall thickening fraction gradually recovered during reperfusion and, at 120 min of reperfusion, it reached 19.0±1.5, 19.6±1.2 and 21.3±0.6% in the control (Figure 3), D-arginine (Figure 3) and SOD (Figure 3) groups, respectively. The contractile function in the L-NAME group ({square}) was higher than that in the control group after 75 min of reperfusion. In contrast, it remained dyskinetic (–12.7±1.6% at 120 min) in the L-arginine group (Figure 3). This aggravation of cardiac contractile dysfunction by L-arginine infusion was prevented by pretreatment with SOD (Figure 3; 19.5±1.1% at 120 min). *p<0.05 compared with the preischemic level. {dagger}p<0.05 (§p=0.08, §§p=0.06) compared with the control group.

 
3.3 Lipid peroxidation
The cardiac tissue level of malondialdehyde, a marker of lipid peroxidation, was significantly higher in the ischemic region than in the non-ischemic region (0.991±0.090 vs. 0.576±0.057 nmol/mg protein; p<0.005) for the L-arginine group, while no such difference was found in the other groups (Table 2). Thus, intra-coronary administration of L-arginine aggravated post-ischemic contractile dysfunction associated with biochemical evidence of oxidative tissue injury. Pretreatment with SOD prevented L-arginine-induced myocardial lipid peroxidation.


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  		Table 2 Myocardial malondialdehyde levels (nmol/mg protein)

 
3.4 Measurement of myocardial NO metabolites
When the preischemic level of cardiac NOx content was expressed as 100%, the level gradually decreased by approximately 30% during ischemia in all groups and returned to the preischemic level during reperfusion in the control, D-arginine and SOD groups (Fig. 4). In contrast, transient increases in the NOx levels were observed soon after the onset of reperfusion in the L-arginine and L-arginine plus SOD groups (151±11 and 143±3% at peak, respectively). Thus, SOD did not prevent L-arginine-induced increases in NOx.


Figure 4
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  		Fig. 4 Changes in NOx levels in dialysate. The preischemic dialysate NOx level (2.26±0.09 µM) was expressed as 100%. The myocardial NOx level gradually decreased by approximately 30% during ischemia in all groups. In the control ({circ}), L-NAME ({triangleup}), D-arginine (bullet) and SOD ({blacktriangleup}) groups, the level returned to the preischemic level soon after reperfusion. Transient increases in the NOx level were observed at 10 and 15 min of reperfusion in both the L-arginine ({square}) and L-arginine plus SOD groups ({blacksquare}). *p<0.05 compared with the preischemic level. {dagger}p<0.05 compared with the control group.

 
3.5 Immunohistochemical staining of myocardium
The myocardium in the ischemic or non-ischemic region was immunohistochemically stained at 15 min of reperfusion, using a polyclonal antibody directed against nitrotyrosine. The myocardial section of the ischemic region of the L-arginine group exhibited more intense staining than the non-ischemic region from the same animal or the ischemic region of the control group (Fig. 5). The immunostaining in the ischemic region of the L-arginine group was reduced to the control level when the myocardial section was incubated with the antigen-competed nitrotyrosine antibody or when the primary antibody was replaced with an equal amount of non-immune rabbit IgG (data not shown). When the immunostaining was performed with the samples at 120 min of reperfusion, although it was not as strong as the staining at 15 min of reperfusion, the ischemic region was more densely stained than the non-ischemic region in the L-arginine group (data not shown).


Figure 5
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  		Fig. 5 Representative immunohistochemical staining of nitrotyrosine in canine myocardium. Myocardial sections from the ischemic region of the control group (A) and the L-arginine group (B) were incubated with 2 µg/ml of polyclonal anti-nitrotyrosine antibody for 3 h at room temperature. The myocardial section from the ischemic region of the L-arginine group incubated with the polyclonal anti-nitrotyrosine antibody exhibited more intense staining than that of the control group.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The major findings of this study were as follows: (i) intra-coronary administration of L-arginine but not D-arginine aggravated post-ischemic contractile dysfunction associated with the increased cardiac level of malondialdehyde (MDA), (ii) SOD pretreatment prevented L-arginine-induced aggravation of contractile dysfunction and lipid peroxidation in the ischemic region, but did not prevent the increase in cardiac NOx induced by L-arginine and (iii) nitrotyrosine, a footprint of peroxynitrite, was immunohistochemically stained in the ischemic region of the heart that was administered L-arginine. We demonstrated, for the first time in vivo, that NO plays a toxic role in myocardial stunning through the production of peroxynitrite.

In the dog, a coronary occlusion lasting less than 20 min does not result in any myocardial necrosis, nevertheless, it produces prolonged abnormalities of contractile performance and this phenomenon is called myocardial stunning [23]. In a previous report, it was shown that, in dogs, it could take as long as two days to achieve full recovery in the contractile function following 15 min of ischemia, and that the recovery was very gradual between 120 min and two days [26]. Therefore, 120 min during reperfusion after 15 min of ischemia was presumed to be an adequate period to observe stunning. In this experiment, myocardial contractile function in the control group gradually recovered during 120 min of reperfusion and no necrosis was histologically present, indicating that ours was a model of myocardial stunning. In this model, the causes of stunning have been demonstrated to involve the generation of reactive oxygen species, such as superoxide anion [31–35]. In the present study, the myocardial NOx level measured by a microdialysis technique was not elevated during reperfusion in the control group. Therefore, we administrated L-arginine, a precursor of NO biosynthesis, into this model, to examine the hypothesis that enhanced production of nitric oxide leads to a reaction with superoxide anion and produces peroxynitrite, which is the primary detrimental factor of NO in this model. There are other models, in which prolonged coronary occlusion for 30 min or longer has been applied, causing irreversible myocardial injury as well as myocardial necrosis [36]. In these models, evaluation of the role of nitric oxide may be difficult because nitric oxide may exert beneficial effects by reducing leukocyte adhesion [37, 38]and myocardial energy requirements [7, 8]and, on the other hand, it may exert harmful effects by producing a great amount of NO through inducible NOS (NOS-2) in leukocytes and the myocardium [39]. In addition, if an irreversible injury was caused by prolonged ischemia, it may be difficult to evaluate the role of NO. Thus, compared to the model of myocardial stunning used in this study, prolonged ischemia–reperfusion injury models have many variables that complicate the investigation of the role of NO.

There are three isoforms of NOS. Our study does not dissect out the contributions of these isoforms of NOS. However, the involvement of NOS-1, nNOS, is unlikely in this study because of the experimental design. Since leukocytes do not play a major role in the pathogenesis of myocardial stunning [23], and since reperfusion was observed for only 120 min (which is too short for the induction of NOS-2), it may be taken for granted that the involvement of NOS-2 is unlikely in the present study.

In the present study, we infused L-arginine on the assumption that infused L-arginine enhances the production of NO. Regarding the dose of L-arginine, 1 mM was chosen because previous investigators infused L-arginine at 1–10 mM in ischemia–reperfusion experiments [9–12, 20]. Intra-coronary infusion of L-arginine aggravated myocardial stunning but D-arginine did not. This detrimental effect of L-arginine was not caused by effects of L-arginine on hemodynamics, if any, because cardiac hemodynamics were not different among the control, D-arginine and L-arginine groups. These results suggest that NO or its derivatives may have aggravated myocardial stunning. Although several previous investigators examined the effects of the intra-coronary infusion of L-arginine on ischemia–reperfusion injury during longer coronary occlusions [9–11, 20], this is the first experiment which showed the detrimental effects of L-arginine on myocardial stunning in vivo. To investigate the biochemical evidence of tissue injury, we measured the myocardial MDA content in the ischemic myocardium, an index of lipid peroxidation, and this did not differ from that of non-ischemic myocardium in the control and D-arginine groups. In contrast, a significantly higher level of MDA was found in the ischemic region of the L-arginine group. Taken together, our results may indicate that the intra-coronary administration of L-arginine aggravated myocardial stunning associated with biochemical evidence of lipid peroxidation, which may have been caused by NO-derivative oxidants (NO itself does not cause lipid peroxidation [40]).

To further confirm that L-arginine-induced aggravation of myocardial stunning was caused by NO-derivative oxidants, we measured the myocardial NOx, metabolites of NO, by HPLC in the dialysate collected by microdialysis. The microdialysis technique, which dialyzes myocardial interstitial fluid, has been employed to monitor levels of various metabolites in the myocardium [41–43]. Measurement of the NO level was based on the Griess reaction, which is commonly used to detect metabolites of NO, nitrite and nitrate. Intra-coronary infusion of L-arginine, which aggravated myocardial stunning associated with increased myocardial MDA, locally increased the myocardial NOx level. D-Arginine did not have such an effect. The present findings support the theory that the detrimental effects of L-arginine on myocardial function and tissue lipid peroxidation were possibly mediated through the increased generation of NO. To further examine this issue, we pretreated dogs with SOD, a specific scavenger of superoxide anion. Pretreatment with SOD prevented L-arginine-induced aggravation of myocardial stunning and lipid peroxidation but did not attenuate the increase in myocardial NOx levels induced by L-arginine. The treatment with SOD alone did not affect myocardial stunning. Therefore, it is unlikely that improvement by SOD modified the effect on the deterioration of myocardial stunning by L-arginine. These results suggest that the detrimental effects of L-arginine were not caused by NO itself but by its derivatives.

From the above-mentioned discussion, it is likely that L-arginine infusion enhanced the generation of NO, which reacted with superoxide anion and produced peroxynitrite, a very potent cytotoxic oxidant. Recently, peroxynitrite has been demonstrated in vitro to cause lipid peroxidation [44]and to have detrimental effects on myocytes [45]. In addition, peroxynitrite is reported to impair cardiac contractile function by decreasing cardiac efficiency [46]. Therefore, it is likely that the production of peroxynitrite is responsible for the aggravated myocardial stunning and increased myocardial MDA in L-arginine-infused hearts. Peroxynitrite, but not NO or superoxide anion per se, facilitates tyrosine nitration and produces nitrotyrosine [21, 47]. This nitration of the ortho position of tyrosine is a major product of peroxynitrite's attack on proteins. Therefore, the presence of nitrotyrosine has been used as a marker of the formation of peroxynitrite in vivo [48–50]. In previous studies, increased nitrotyrosine production has been immunohistochemically demonstrated in various human diseases and animal models associated with oxidative stress, such as acute lung injury, adult respiratory distress syndrome [48, 49]and of atherosclerotic plaques of human coronary arteries [50]using a polyclonal anti-nitrotyrosine antibody. Using a polyclonal anti-nitrotyrosine antibody, clear staining was found in the myocardial section of the ischemic region of the L-arginine group. However, it was not observed in the control, or in the D-arginine or L-arginine plus SOD groups (data not shown). These findings indicate the presence of peroxynitrite in the myocardium of the ischemic region of the L-arginine group and suggest the detrimental role of peroxynitrite in myocardial stunning. Our results are consistent with those of previous studies that demonstrated the detrimental effects of NO on the ischemia–reperfused myocardium through the production of peroxynitrite, although none of those studies demonstrated the presence of peroxynitrite in vivo [13, 16, 17, 19, 51, 52].

Several limitations are considered. We assumed that nitrotyrosine acted as a footprint of peroxynitrite. This is not so because the reaction of nitrite with hypochlorous acid has recently been reported to be an additional mechanism of tyrosine nitration that is independent of peroxynitrite [53]. However, this mechanism is not likely because the formation of nitrotyrosine was prevented by pretreatment with SOD. Second, to investigate the oxidative modification of myocardium, myocardial MDA was determined as TBA-reactive substances. It is sometimes claimed that this assay has relatively low specificity against MDA because several compounds other than MDA give products that absorb at 532.5 nm on heating with TBA [54]. However, we observed that a significantly higher value was obtained only in the ischemic region of the L-arginine-administered heart and that pretreatment with the radical scavenger, SOD, prevented the increase in TBA-reactive substance levels, despite the administration of L-arginine. These results strongly suggest that the increased level of TBA-reactive substances reflects the increase in the oxidized product, most likely MDA, because NO reacts with superoxide anion to generate peroxynitrite, which, in turn, generates lipid peroxidation, which is evidenced as MDA. Third, an excess amount of nitric oxide was not generated during ischemia–reperfusion under the experimental conditions examined and, therefore, the infusion of L-arginine was required to enhance the generation of NO. Nonetheless, there have been a number of reports showing elevated NO generation during prolonged myocardial ischemia–reperfusion [51, 55, 56]. In the present study, when L-NAME was infused into the coronary artery, the recovery of the wall thickening fraction improved compared to the control. This result suggests the involvement of endogenously produced myocardial NO in myocardial stunning, although L-arginine infusion was required to prove the involvement of peroxynitrite in this study.

In conclusion, we have demonstrated, for the first time, that NO causes a peroxynitrite-mediated detrimental effect on myocardial stunning in vivo.

Time for primary review 24 days.


   Acknowledgements
 
We thank Ms. Kimiko Kimura and Aya Shimizu for their technical assistance. This study was supported in part by a grant-in-aid for scientific research (09770519) from the Ministry of Education, Science and Culture of Japan, Tokyo.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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