Cardiovascular Research

Cardiovascular Research 1997 36(2):195-204; doi:10.1016/S0008-6363(97)00179-X
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Peroxynitrite aggravates myocardial reperfusion injury in the isolated perfused rat heart

Xin L Ma^a,*, Bernard L Lopez^a, Gao-Lin Liu^a, Theodore A Christopher^a and Harry Ischiropoulos^b

^aDivision of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA 19107-5004, USA
^bInstitute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA 19104-6068, USA

^* Corresponding author. Tel. (+1-215) 9556844; Fax (+1-215) 9236225.

Received 20 January 1997; accepted 12 June 1997

	Abstract

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Objective: This study examined the effects of peroxynitrite (ONOO^–) on cardiac function and cellular injury following ischemia (30 min) and reperfusion (60 min) in isolated perfused rat hearts. Methods: 3-Morpholinosydnonimine (SIN-1, 0.1 mM), an ONOO^– donor, was administered alone or combined with superoxide dismutase (SOD, 300 U/ml) or glutathione (GSH, 1 mM) at the time of reperfusion. Results: Administration of SIN-1 alone significantly aggravated post-ischemic myocardial injury characterized by depressed cardiac function recovery (p<0.05 vs. vehicle), increased lactic dehydrogenase (LDH) and creatine kinase (CK) release (p<0.01 vs. vehicle), and enlarged necrotic size (p<0.01 vs. vehicle). The co-administration of either SOD to decrease the formation of ONOO^–, or GSH to increase the detoxification of ONOO^–, completely blocked the detrimental effects of SIN-1 and exerted significant cardioprotective effects against reperfusion injury. Conclusion: These results suggest that ONOO^– may play a significant role in postischemic myocardial injury.

KEYWORDS Peroxynitrite; Nitric oxide; Reperfusion injury

	1 Introduction

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Early reperfusion after myocardial ischemia is the most effective means of limiting myocardial injury. However, abundant evidence suggests that reperfusion may initiate an inflammatory response and cause additional cell death. The cause of reperfusion-induced cardiac injury is apparently multifactorial. Numerous experiments, however, have demonstrated that reactive oxygen species (ROS), including superoxide (·O₂^–), hydrogen peroxide (H₂O₂) and hydroxyl radical (·OH), play a pivotal role in post-ischemic myocardial injury [1, 2].

Nitric oxide (·NO), a simple molecule produced primarily by endothelial cells (ECs) in the cardiovascular system, has been shown to attenuate myocardial reperfusion injury [3]. In addition to its well-demonstrated vasorelaxation effects, ·NO has also been shown to possess significant anti-neutrophil properties and thereby attenuate neutrophil-induced myocardial injury following reperfusion [4]. Moreover, ·NO reacts with ·O₂^– in a nearly diffusion-limited speed (6.7x10⁹ M^–1 s^–1) [5]. This reaction can scavenge ·O₂^– and can therefore prevent further reactions that lead to the biosynthesis of more potent oxidants such as ·OH. Since ·OH is believed to be the major mediator for reperfusion injury [1, 2], this ·NO/·O₂^– reaction has been thought to be a major mechanism of ·NO protection in myocardial reperfusion injury [6]. Recent biochemical studies, however, have shown that peroxynitrite (ONOO^–), the reacting product of ·NO and ·O₂^–, is a potent cytotoxic agent per se. It is highly reactive with a wide variety of molecules, including deoxyribose, cellular lipids, and protein sulfhydryls and results in an oxidative tissue damage apparently similar to that caused by ·OH in vitro [7]. It is well recognized that ischemia-reperfused endothelial cells and activated leukocytes can generate ·O₂^– and ·NO simultaneously, suggesting that ONOO^– might be formed in post-ischemic myocardial tissue and thus may play a significant role in myocardial reperfusion injury. To date, however, the role of ONOO^– in post-ischemic myocardial injury has not been fully evaluated at the organ level or in vivo. Therefore, the objective of this study was to (1) determine the effects of exogenous ONOO^– on cardiac function and myocardial cellular injury following ischemia and reperfusion in an isolated, cell-free medium-perfused heart model; and (2) elucidate the factors that may attenuate ONOO^–-induced myocardial injury.

	2 Materials and methods

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
2.1 Heart preparation and measurement of functional parameters
Adult male Sprague–Dawley rats (250–300 g) were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal) and heparinized with sodium heparin (1000 U/kg, i.v. via penis vein). Five minutes after heparin injection, a midsternal thoracotomy was performed, and the heart was rapidly excised and placed into ice-cold Krebs–Henseleit (KH) buffer solution consisting of (in mM): NaCl, 118; KCl, 4.75; KH₂PO₄, 1.19; MgSO₄·7H₂O, 1.19; CaCl₂·2H₂O, 2.54; NaHCO₃, 25; EDTA, 0.5; and glucose, 11 [8, 9]. Within 30 seconds, the heart was mounted onto a non-recirculation Langendorff heart perfusion apparatus (Radnoti Glass Technology, Inc. Monrovia, CA). The hearts were perfused in a retrograde fashion via the aorta at a constant pressure of 60 mm Hg with KH solution oxygenated with 95% O₂+5% CO₂ to maintain pH 7.4 at 37°C [10]. An epicardial electrocardiogram (ECG) was recorded using two stainless steel electrodes, one attached to the apex of the heart and the other to the aortic cannula [11]. Reperfusion-induced ventricular fibrillation (VF) was defined according to the Lambeth Conventions [12]and the incidence of VF was compared among the groups.

To assess contractile function, a latex balloon was inserted into the left ventricular cavity through the mitral orifice and connected to a pressure transducer (Cobe CDXIII, Lakewood, CO). The balloon was initially inflated with water to produce an end-diastolic pressure of 8 to 10 mm Hg, which is on the plateau of the Starling curve for this preparation. During the 30 minute ischemic period, the balloon was deflated using a gas-tight microsyringe to minimize balloon-induced myocardial injury. At three minutes of reperfusion, the same volume of saline was injected slowly back to the balloon. The ECG and left ventricular pressure (LVP) signals were continually monitored and acquired over five seconds by using a PC-based data acquisition system (Data Translation, Marlboro, MA) at the following time points: immediately before global ischemia (0 min), 5, 10, 20, 40 and 60 minutes after reperfusion. The left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), heart rate (HR), and pressure-rate product (PRP=HRxLVDP) were obtained using computer algorithms and an interactive videographics program (Bowman Gray School of Medicine, Winston-Salem, NC) [13]. Coronary flow was measured via an in-line flow probe connected to an ultrasonic flow meter (Transonic Systems, Inc, Ithaca, NY). In our preliminary experiments we observed that this preparation is functionally stable for at least two hours. Spontaneous decline of left ventricular developed pressure (LVDP=LVSP–LVEDP) is typically <7% per hour.

2.2 Experimental protocol
After a 20 minute equilibration time, hearts were subjected to complete global ischemia for 30 minutes by turning off the perfusion system. After 30 minutes of ischemia, the perfusion system was restarted, and the hearts were reperfused for an additional 60 minutes. At the time of reperfusion, hearts were randomized to receive one of the following treatments: (1) vehicle (50 mM phosphate buffer, pH 5.0); (2) SIN-1 (3-morpholinosydnonimine, final concentration 0.1 mM); (3) superoxide dismutase (Cu,Zn-SOD, 300 U/ml); (4) glutathione monoester (GSH, 1 mM final concentration); (5) SIN-1+SOD; and (6) SIN-1+GSH. Drugs were infused into the heart via a sidearm in the perfusion line located just proximal to the heart cannula for 60 minutes. The rate of infusion was adjusted based on the coronary flow rate so that the desirable final concentration was obtained. Sham ischemic-reperfusion hearts were perfused with KH solution for two hours.

For exposure to ONOO^–, we used SIN-1, a compound that simultaneously generates ·NO and ·O₂^–. SIN-1 is stable at pH 5.0. At pH 7.4, it undergoes a base-catalyzed ring opening to form SIN-1A. During the ring opening, oxygen is univalently reduced to form ·O₂^–. SIN-1A then releases ·NO and is converted to the stable metabolite SIN-1C. ·O₂^– reacts with ·NO at a near diffusion-limited rate to form ONOO^– (rate constant=6.7x10⁹ M^–1 s^–1). It has been previously determined that the rate of ONOO^– formation (µM/min) from SIN-1 in buffer solution at 37°C is approximately 1% of the initial SIN-1 concentration [14]. Addition of 0.1 mM SIN-1 thus will generate ONOO^– at a rate of 1 µM/min [15]. In preliminary experiments, we determined that 0.1 mM SIN-1 was the minimal concentration that resulted in significant additional tissue injury in our ischemia-reperfusion model. This concentration (i.e., 0.1 mM) was thus used in this study. Stock solutions of SIN-1 were dissolved in 50 mM phosphate buffer, pH 5.0. The formation of ONOO^– from the decomposition of SIN-1 was confirmed by oxidation of dihydrorhodamine 123 (DHR 123) to rhodamine. Previous studies have shown that DHR 123 is oxidized by ONOO^– but not by ·O₂^– or ·NO alone [14]. The yield of rhodamine formation after 60 minute incubation at 37°C was determined using the extinction coefficient=78 000 M^–1 cm^–1 at 500 nm. The release of ·NO from the decomposition of SIN-1 in KH solution at 37°C in the presence of SOD was detected by an ·NO-selective electrochemical detector (World Precision Instruments, Sarasota, FL). The release of ·NO was followed continuously for 30 minutes and, at the end of the measurement, 5 µM oxyhemoglobin was added to quench ·NO. The addition of oxyhemoglobin resulted in the rapid loss of the ·NO signal, indicating the selectivity of ·NO detection.

2.3 Biochemical and morphological assessment of injury
The coronary effluent during reperfusion was collected at three consecutive time segments: 0 to 5 minutes, 5 to 30 minutes and 30 to 60 minutes. Lactic dehydrogenase (LDH) was measured spectrophotometrically using a microplate reader (Molecular Devices, Sunnyvale, CA). In brief, stock solutions of sterile 100 mM potassium phosphate buffer (pH 7.4) and 27.2 mM sodium pyruvate in potassium phosphate buffer were prepared in advance and stored at 4°C until use. A solution of NADH (3 mg/10 ml phosphate buffer) was prepared prior to use. 25 µl of coronary effluent was placed in a 96-well assay plate and 125 µl phosphate buffer and 100 µl NADH solution were then added to each well. After a 10-minute incubation at room temperature, 25 µl of pyruvate solution was added to each well, and the absorbance of the reaction mixture at 340 nm was determined at six-second intervals for two minutes. LDH concentrations in coronary effluent (U/l) were determined from a standard curve obtained from sequential dilutions of LDH enzyme solution (Sigma enzyme control 2-E). Total LDH release per gram of heart was calculated as follows: LDH release (U/g)=LDH concentration in coronary effluent (U/l)xvolume of coronary effluent (l)/heart weight (g) [16, 17].

Our preliminary study showed that 120 minutes of reperfusion were required in order to obtain a clear and consistent necrotic injury in vehicle-treated hearts. We thus extended the reperfusion period for an additional 60 minutes after stoppage of drug infusion. The ventricles were separated into two parts. One part was homogenized in cold 0.25 M sucrose (1:10, w/v) containing 1 mM EDTA and 0.1 mM mercaptoethanol using a PRO 200 homogenizer (PRO Scientific Inc, Monroe, CT). Homogenates were centrifuged at 36 000 g at 4°C for 30 minutes. The supernatants were decanted and analyzed spectrophotometrically for myocardial creatine kinase (CK) activity as reported previously [18]. Protein concentration was determined by the BCA method (Pierce). The ischemia-reperfusion induced CK loss was calculated by subtracting CK activity of ischemic-reperfused hearts from that of sham ischemia-reperfusion hearts. The results were expressed in international unites per 100 mg of protein. Another part of ventricle was sliced into approximately 2 mm thick slices and incubated in 0.1% nitroblue tetrazolium (NBT) in phosphate buffer at pH=7.4 and 37°C for 15 minutes. The unstained portion (which is the irreversibly injured, necrotic region) was then separated from the stained (non-necrotic) portion. Both sections were weighed and the results were expressed as a percentage of necrotic over total ventricular mass.

2.4 Materials
3-Morpholinosydnonimine (SIN-1) was obtained from Molecular Probes, Inc. (Eugene, OR). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

2.5 Statistical analysis
All values in the text and figures are presented as means±standard errors of the mean of n independent experiments. Incidence of VF among the groups was compared by ² analysis. All other data were analyzed with repeated measures analysis of variance (ANOVA). Differences between groups were further assessed with the Bonferroni correction for post-hoc t-test comparison. Probabilities of 0.05 or less were considered to be statistically significant.

	3 Results

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
3.1 Formation of peroxynitrite after decomposition of SIN-1
Consistent with previous reports [19], we have found that ·NO is not detectable by an electrochemical sensor unless SOD is present to dismutate ·O₂^–. As shown in Fig. 1A, no ·NO was detected from the decomposition of 100 µM SIN-1 in KH buffer. However, when 300 U/ml of SOD was added with SIN-1, a significant ·NO release occurred. The release of ·NO increased in a linear fashion for the first five minutes and reached a plateau that persisted for the next 20 minutes. The formation of ONOO^– from SIN-1 was confirmed by the oxidation of DHR 123 to rhodamine. As shown in Fig. 1B, the production of rhodamine was proportional to the amount of SIN-1 added to the reaction mixture. The yield of rhodamine formed over 60 minutes at 37°C by 10 and 100 µM SIN-1 corresponded approximately to the yield of rhodamine formed by a bolus addition of 24 and 162 µM chemically synthesized ONOO^–, respectively. These results confirmed that SIN-1 functioned as an ONOO^– donor, rather than an ·NO donor, under these conditions.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 (A) Representative tracing of nitric oxide detection from 100 µM SIN-1 at the presence and absence of 300 U/ml SOD. SIN-1 was dissolved in 2 ml KH solution, pH 7.4 at 37°C, and nitric oxide was detected by a nitric oxide-selective electrochemical sensor. (B) Concentration-response relationship of SIN-1 on DHR 123 (300 µM) oxidation. The yield of rhodamine was determined after a 60 min incubation in EBSS, pH 7.4 at 37°C. Data are presented as the mean values of three determinations±SEM.

 
3.2 Incidence of ventricular fibrillation after reperfusion
Global ischemia followed by reperfusion frequently resulted in the induction of episodes of ventricular tachyarrhythmias which occurred mostly in the first five minutes of reperfusion. However, most hearts reverted to a stable sinus rhythm within 15 minutes of reperfusion. In 14 hearts receiving vehicle, only 1 heart developed VF which reverted to normal sinus rhythm within one minute by a gentle tap with a small forceps handle. Our preliminary study demonstrated that this defibrillation technique did not significantly alter myocardial CK loss or necrotic size. In the SOD or GSH treated group, no VF was observed during the entire reperfusion period. In 13 hearts treated with SIN-1 alone, 3 hearts developed VF which was successfully reverted to normal sinus rhythm using the defibrillation technique described above. When either SOD or GSH was used in combination with SIN-1, no VF occurred in the reperfusion period. However, the difference in the incidence of VF was not statistically significant among any groups.

3.3 Effects of peroxynitrite on cardiac contractile function changes following ischemia and reperfusion
To determine the effects of ONOO^– on cardiac functional injury following reperfusion, the LVDP and HR were measured and percent recovery of PRP after reperfusion was calculated. Thirty minutes of global ischemia followed by reperfusion caused a severe cardiac dysfunction manifested by elevated LVEDP, decreased LVSP, and reduced HR (Table 1). At 60 minutes of reperfusion, PRP recovered only to 23.8±1.4% of the control value (before ischemia). Administration of 0.1 mM SIN-1 significantly aggravated cardiac function (PRP recovered to 16.5±2.1% of control value, p<0.05 vs. vehicle). However, the co-administration of either SOD, an enzyme that scavenges ·O₂^– released from SIN-1 and thus inhibits ONOO^– formation, or GSH, a molecule that has been reported to react with ONOO^– and decrease its reactivity, completely reversed the effects of SIN-1 on cardiac function (Fig. 2). Thus, after 60 minutes of reperfusion, PRP recovered to 42.9±3.8% in the SOD plus SIN-1 group (p<0.05, vs. vehicle, p<0.01 vs. SIN-1 alone) and 47.9±4.1% in the GSH plus SIN-1 group (p<0.01 vs. vehicle and SIN-1 alone) (Fig. 2).

View this table: [in this window] [in a new window]   Table 1 The effects of different treatments on left ventricular developed pressure (LVDP) and heart rate (HR) changes following reperfusion

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effects of different treatments on percent recovery of pressure-rate-product (PRP) after reperfusion. Each point represents the mean of 12 to 14 hearts with SEM shown by vertical bars. *p<0.05, **p<0.01 vs. vehicle; p<0.005 vs. SIN-1.

 
3.4 Effects of peroxynitrite on cardiac cellular injury following ischemia and reperfusion
To examine the effects of ONOO^– on cellular injury, we measured the release of LDH to the effluent, myocardial CK loss, and tissue necrosis. As summarized in Table 2 and Fig. 3, global ischemia and reperfusion resulted in marked LDH release and myocardial CK loss, and caused a significant necrotic injury. In the sham ischemic group, only 1.1±0.09 U/g LDH was released during the entire time-matched 60 minute reperfusion period and no necrotic tissue was found after 120 minutes of normal perfusion, indicating that perfusion with KH solution under the conditions employed in the present experiment did not cause significant cellular injury. However, 24.1±1.82 U/g LDH was released during reperfusion, and 45±3.2% of the ventricular mass became necrotic in ischemia-reperfused hearts which received vehicle. Moreover, the myocardial CK content was significantly decreased in ischemia-reperfusion hearts. Consistent with the results reported previously by other investigators [20, 21], the administration of either SOD or GSH at the time of reperfusion significantly attenuated myocardial injury in our study. In contrast, the administration of ONOO^– markedly exaggerated cellular injury as evidenced by the higher LDH release (30.2±1.94 U/g, p<0.01 vs. vehicle), larger necrosis (59±3.5%, p<0.05 vs. vehicle) and more significant CK loss. The combinations of either SOD or GSH with SIN-1, however, not only completely blocked the cardiotoxic effects of SIN-1, but also exerted more cardioprotective effects than either SOD or GSH when used alone (Table 2 and Fig. 3).

View this table: [in this window] [in a new window]   Table 2 The effects of different treatments on lactate dehydrogenase release to coronary effluent during reperfusion after 30 min of global ischemia (U/g)

 

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effects of different treatments on myocardial creatine kinase (CK) loss (left) and tissue necrotic injury after 120 minutes of reperfusion. Bar heights are means and brackets indicate±SEM. Numbers at the bottom of the bars represent number of hearts studied. *p<0.05, **p<0.01 vs. vehicle; p<0.005 vs. SIN-1.

 
To further ascertain whether or not the detrimental effects of SIN-1 in myocardial reperfusion injury was related to its generation of ONOO^–, the effects of a stable metabolite of SIN-1, SIN-1C, on post-ischemic cardiac function recovery and cellular injury were studied. SIN-1 was stirred for 12 hours in phosphate buffer pH 8.0 and followed by another 12 hours in phosphate buffer pH 7.4. During this time the loss of absorbance at 290 nm was followed and a shift in absorbance indicated base hydrolysis of SIN-1 first to SIN-1A and then to SIN-1C. The SIN-1C prepared by this method neither released NO in the presence of SOD, nor oxidized DHR123. In seven separate rat hearts, 0.1 mM of SIN-1C (final concentration) was continually infused into the hearts beginning at the onset of reperfusion. The results showed that SIN-1C had no significant effects on cardiac function recovery (PRP recovery to 26.3±3.1% at 60 minute reperfusion, p>0.1 vs. vehicle). Interestingly, administration of SIN-1C not only failed to aggravate the cellular injury like SIN-1 did, it slightly (although not statistically significant) reduced LDH release (total LDH release, 20.1±1.58 U/g) and decreased necrotic size (39±3.1%). This was likely due to direct effects of SIN-1C on reoxygenation-induced cardiomyocyte injury [22]. These results indicated that the detrimental effects of SIN-1 on reperfusion injury were not attributable to its stable metabolite, SIN-1C.

3.5 Effects of peroxynitrite on coronary flow recovery following reperfusion
To clarify whether the deleterious effects of SIN-1 on reperfusion injury were due to its vasoactive effects on the coronary vasculature, coronary flow was measured before and after SIN-1 administration. As summarized in Table 3, administration of SIN-1 at 100 µM did not impair coronary flow recovery after reperfusion. In contrast, coronary flow was slightly, although not significantly, increased in this SIN-1 treatment group. Thus, the detrimental effects of SIN-1 on cardiac function and cellular injury observed in this model could not be attributed to the effects of SIN-1 on coronary circulation.

View this table: [in this window] [in a new window]   Table 3 The effects of different treatments on coronary flow rate recovery following reperfusion (ml/min)

 

	4 Discussion

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Timely reperfusion with intravenous thrombolytic agents has been shown to reduce mortality in patients with acute myocardial infarction. However, the magnitude of improvement in left ventricular function has always been less than expected. The introduction of oxygen into previously ischemic tissue has been shown to initiate a particular form of inflammation and tissue damage that has been termed ‘reperfusion injury’ [23]. Substantial evidence indicates that ROS plays a major role in the pathogenesis of reperfusion injury [1]. Initial evidence was indirectly based on the beneficial effects of free radical scavengers administered exogenously at the time of reperfusion [20, 24]. Recent electron paramagnetic resonance (EPR) spectroscopy studies have directly demonstrated that there is a burst of superoxide anion generation during the first 60 seconds of reflow. Administration of either a free radical scavenger, such as SOD, or an iron chelator, such as deferoxamine, prevents ·O₂^–, burst [25, 26].

Recent biochemical evidence suggests that the formation of ONOO^– by the reaction of ·NO and ·O₂^– may also play an important role in ·O₂^–-initiated cellular injury [7]. ONOO^– is highly reactive with a wide variety of compounds, including deoxyribose, cellular lipids, and protein sulfhydryls, and this reaction can result in an oxidative tissue damage similar as that caused by ·OH [27]. It is well demonstrated that vascular ECs generate ·NO constitutively and produce a burst of ·O₂^– production upon reperfusion, suggesting that endothelial cells may generate ONOO^– following ischemia and reperfusion and thus result in cell injury. In this connection, Wang and Zweier [28]have recently showed that ONOO^– formation can be detected in the cell-free crystalloid solution of perfused hearts in the early period of reperfusion. Inhibiting ONOO^– formation by blocking ·NO production with L-NAME, or scavenging ONOO^– by administration of uric acid, significantly improved post-ischemic cardiac function recovery. These results provided direct evidence that ONOO^– can be formed by the structure cells of hearts, e.g. endothelial cells and cardiomyocytes, and contributes significantly to the post-ischemic myocardial injury.

Recent studies have demonstrated that activated leukocytes generate a large amount of ·NO and ·O₂^– simultaneously and yield ONOO^– [29–32]. It is well documented that there is a substantial leukocyte accumulation in the ischemia-reperfused myocardium in vivo. It is therefore likely that, in addition to ischemia-reperfused endothelial cells and myocytes, leukocytes that have migrated to the tissue may be a significant source of ONOO^– following reperfusion. To determine the effects of this ‘foreign-cell’-derived ONOO^– on post-ischemic myocardial injury, we administered exogenous ONOO^– to crystalloid-perfused hearts. SIN-1, an ONOO^– donor, was added to the perfusion system at the time of reperfusion. Although SIN-1 has been used as an ·NO donor in some early studies and has been shown to be protective in myocardial ischemia and reperfusion in vivo [33], recent biochemical studies using direct ·NO measurement has revealed that SIN-1 does not release measurable ·NO in physiological solutions, but rather, it releases ONOO^– [19, 34]. Based on these observations, SIN-1 has been used as an ONOO^– donor in recent studies [35, 36]. Our present study clearly demonstrated that SIN-1, when administered alone, exerted significant detrimental effects on reperfusion injury in crystalloid-perfused hearts. In contrast, when given in combination with SOD or GSH, SIN-1 significantly attenuated myocardial injury. However, it should be indicated that the final effects of ONOO^– on reperfusion injury in vivo are likely much more complex and may critically depend on time, sources, concentrations and the interactions between this oxidant and other mediators that play important roles in reperfusion injury [37–39].

As has been previously reported by other investigators [19], we observed that adding SOD with SIN-1 to physiologic solution leads to a significant ·NO release. Although we did not directly observe the effects of ·NO on myocardial injury in our present experiment, other studies have previously demonstrated that the administration of ·NO donors in the isolated perfused heart model exerts significant cardioprotective effects [40–42]. Therefore, the likely explanation for the elimination of the detrimental effects of SIN-1 by co-administration of SOD is that SOD can scavenge ·O₂^– generated by SIN-1. This reaction not only prevents the formation of cytotoxic ONOO^–, but also leads to a release of cytoprotective ·NO from SIN-1. This ·NO may exert cardioprotective effects additively or synergistically with SOD, thus providing more significant protection against reperfusion injury.

The mechanisms for SIN-1's conversion from cytotoxicity to cytoprotection by GSH is perhaps more complicated and could not be answered precisely by the present study. Since the combination of GSH and SIN-1 exerted even more (although not statistically significant) protective effects than GSH alone, the protective effects of this combination cannot be explained by the notion that GSH simply masked the cytotoxic effects of SIN-1-derived ONOO^–. Moreover, the rate constant for the interaction of ·O₂^– and ·NO to form ONOO^– is larger by six to seven orders of magnitude than the corresponding value for the reaction of ·O₂^– and GSH (10²–10³ M^–1 s^–1) [43], it seems unlikely that GSH could directly inhibit the formation of ONOO^–, as does SOD. Several recent studies have shown that GSH may act as an ONOO^– scavenger. Using isolated canine coronary vessels, Liu et al. [44]has shown that ONOO^–-induces a ·NO-like vasorelaxation. Further studies by Wu et al. [45]and Moro et al. [37]have demonstrated that ONOO^– reacts with GSH to form an S-nitrothiol compound to regenerate ·NO over a prolonged period. Moreover, more recent studies have demonstrated that ONOO^– stimulates guanylyl cyclase in a GSH-dependent manner and induces cyclic GMP accumulation in endothelial and smooth muscle cells [46, 47]. Therefore, co-administration of GSH with SIN-1 may convert a cytotoxic agent, ONOO^–, to a cytoprotective agent, ·NO. ·NO, in turn, can attenuate reperfusion injury. Another possible explanation is that GSH can act as a substrate for glutathione peroxidase in removing H₂O₂, thus preventing ·OH formation and attenuating ROS-induced cell injury.

Administration of SIN-1 at the time of reperfusion slightly increased the incidence of VF (3/13 vs. 1/14 in vehicle group). This difference, however, was not statistically significant. In the global ischemia model that we used in the present study, it is known that the overall incidence of VF is low [10]. Future studies using a regional ischemia model, which has a higher incidence of ventricular arrhythmias [48–50], may provide more direct evidence of the effects of ONOO^– on cardiac electrophysiologic changes after reperfusion.

In summary, our results demonstrate that, in the cell-free crystalloid perfused heart, administration of additional ONOO^– aggravated reperfusion tissue injury. It has been well demonstrated that there is a significant leukocyte accumulation in the ischemic-reperfused myocardial tissue, and that activated leukocytes generate significant ONOO^–. Our present study suggests that ONOO^– generated from accumulated leukocytes in the myocardium under in vivo conditions, which was mimicked by administration of exogenous ONOO^– to cell-free crystalloid solution perfused hearts in the present study, may contribute significantly to tissue injury associated with ischemia and reperfusion. Co-administration of GSH with SIN-1 converted the cytotoxicity of SIN-1 to cytoprotection. This anti-ONOO^– effect of GSH may represent a novel mechanism of protection by this molecule in the treatment of myocardial reperfusion injury.

Time for primary review 23 days.

	Acknowledgements

 
We gratefully acknowledge Ms. Ya-Ping Guo for her excellent technical assistance in the biochemical analyses reported in this study.

	References

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 

1. Kilgore K.S., Lucchesi B.R. Reperfusion injury after myocardial infarction: The role of free radicals and the inflammatory response. Clin Biochem (1993) 26:359–370.[CrossRef][Web of Science][Medline]
2. Granger D.N., Hollwarth M.E., Parks D.A. Ischemia-reperfusion injury: role of oxygen-derived free radicals. Acta Physiol Scand (1986) 548:47–63.
3. Vinten-Johansen J., Sato H., Zhao Z.Q. The role of nitric oxide and NO-donor agents in myocardial protection from surgical ischemic-reperfusion injury. Int J Cardiol (1995) 50:273–281.[CrossRef][Web of Science][Medline]
4. Egdell R.M., Siminiak T., Sheridan D.J. Modulation of neutrophil activity by nitric oxide during acute myocardial ischaemia and reperfusion. Basic Res Cardiol (1994) 89:499–509.[CrossRef][Web of Science][Medline]
5. Huie R.E., Padmaja S. The reaction of NO with superoxide. Free Rad Res Commun (1993) 18:195–199.[Web of Science][Medline]
6. Rubanyi G.M., Ho E.H., Cantor E.H., Lumma W.C., Botelho L.H. Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes. Biochem Bioph Res Co (1991) 181:1392–1397.[CrossRef][Web of Science][Medline]
7. Beckman J.S. Peroxynitrite versus hydroxyl radical: The role of nitric oxide in superoxide-dependent cerebral injury. Ann NY Acad Sci (1994) 738:69–75.[Web of Science][Medline]
8. Lefer D.J., Shandelya S.M.L., Serrano C.V. Jr., Becker L.C., Kuppusamy P., Zweier J.L. Cardioprotective actions of a monoclonal antibody against CD-18 in myocardial ischemia-reperfusion injury. Circulation (1993) 88:1779–1787.[Abstract/Free Full Text]
9. Neely J.R., Liebermeister H., Battersby E.J., Morgan H.E. Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol (1967) 212:804–814.[Free Full Text]
10. Shimada Y., Hearse D.J., Avkiran M. Impact of extracellular buffer composition on cardioprotective efficacy of Na⁺/H⁺ exchanger inhibitors. Am J Physiol (1996) 270:H692–H700.[Medline]
11. Pabla R., Bland-Ward P., Moore P.K., Curtis M.J. An endogenous protectant effect of cardiac cyclic GMP against reperfusion-induced ventricular fibrillation in the rat heart. Brit J Pharmacol (1995) 116:2923–2930.[Web of Science][Medline]
12. Walker M.J., Curtis M.J., Hearse D.J., Campbell R.W., Janse M.J., Yellon D.M., Coker S.J., Harness J.B., Harron D.W., et al. The Lambeth Conventions: guidelines for the study of arrhythmias in ischaemia infarction, and reperfusion. Cardiovasc Res (1988) 22:447–455.[Abstract/Free Full Text]
13. Sato H., Zhao Z.Q., McGee D.S., Williams M.W., Hammon J.W. Jr., Vinten-Johansen J. Supplemental L-arginine during cardioplegic arrest and reperfusion avoids regional postischemic injury. J Thorac Cardiovasc Surg (1995) 110:302–314.[Abstract/Free Full Text]
14. Kooy N.W., Royall J.A., Ischiropoulos H., Beckman J.S. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radic Biol Med (1994) 16:149–156.[CrossRef][Web of Science][Medline]
15. Brunelli L., Crow J.P., Beckman J.S. The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch Biochem Biophys (1995) 316:327–334.[CrossRef][Web of Science][Medline]
16. Verbunt R.J.A.M., Van Dockum W.G., Bastiaanse E.M.L., Egas J.M., Van der Laarse A. Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts. Mol Cell Biochem (1995) 144:85–93.[CrossRef][Web of Science][Medline]
17. Engelman D.T., Watanabe M., Engelman R.M., Rousou J.A., Kisin E., Kagan V.E., Das D.K. Hypoxic preconditioning preserves antioxidant reserve in the working rat heart. Cardiovasc Res (1995) 29:133–140.[Abstract/Free Full Text]
18. Lefer A.M., Tsao P.S., Ma X.L., Sampath T.K. Anti-ischaemic and endothelial protective actions of recombinant human osteogenic protein (hOP-1). J Mol Cell Cardiol (1992) 24:585–593.[CrossRef][Web of Science][Medline]
19. Ischiropoulos H., Duran D., Horwitz J. Peroxynitrite-mediated inhibition of DOPA synthesis in PC12 cells. J Neurochem (1995) 65:2366–2372.[Web of Science][Medline]
20. Ambrosio G., Weisfeldt M.L., Jacobus W.E., Flaherty J.T. Evidence of a reversible oxygen radical mediated component of reperfusion injury: Reduction by recombinant human superoxide dismutase administered at the time of reflow. Circulation (1987) 75:282–291.[Abstract/Free Full Text]
21. Liu P., Fisher M.A., Farhood A., Smith C.W., Jaeschke H. Beneficial effects of extracellular glutathione against endotoxin-induced liver injury during ischemia and reperfusion. Circ Shock (1994) 43:64–70.[Web of Science][Medline]
22. Schluter K.D., Weber M., Schraven E., Piper H.M. NO donor SIN-1 protects against reoxygenation-induced cardiomyocyte injury by a dual action. Am J Physiol (1994) 267:H1461–H1466.[Web of Science][Medline]
23. Zimmerman B.J., Granger D.N. Mechanisms of reperfusion injury. Am J Med Sci (1994) 307:284–292.[Web of Science][Medline]
24. Ambrosio G., Zweier J.L., Jacobus W.E., Weisfeldt M.L., Flaherty J.T. Improvement of post-ischemic myocardial function and metabolism by administration of deferoxamine at the time of reflow: The role of iron in the pathogenesis of reperfusion injury. Circulation (1987) 76:906–915.[Abstract/Free Full Text]
25. Zweier J.L., Rayburn B.K., Flaherty J.T., Weisfeldt M.L. Recombinant superoxide dismutase reduces oxygen free radical concentrations in reperfused myocardium. J Clin Invest (1987) 80:1728–1734.[Web of Science][Medline]
26. Bolli R., Jeroudi M.O., Patel B.S., Aruoma O.I., Halliwell B., Lai E.K., McCay P.B. Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion. Circ Res (1989) 65:607–622.[Abstract/Free Full Text]
27. Pryor W.A., Squadrito G.L. The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide. Am J Physiol (1995) 268:L699–L722.[Web of Science][Medline]
28. Wang P.H., Zweier J.L. Measurement of nitric oxide and peroxynitrite generation in the postischemic heart — Evidence for peroxynitrite-mediated reperfusion injury. J Biol Chem (1996) 271:29223–29230.[Abstract/Free Full Text]
29. Carreras M.C., Pargament G.A., Catz S.D., Poderoso J.J., Boveris A. Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils. FEBS Lett (1994) 341:65–68.[CrossRef][Web of Science][Medline]
30. Rodenas J., Mitjavila M.T., Carbonell T. Simultaneous generation of nitric oxide and superoxide by inflammatory cells in rats. Free Rad Biol Med (1995) 18:869–875.[CrossRef][Web of Science][Medline]
31. Ischiropoulos H., Zhu L., Beckman J.S. Peroxynitrate formation from macrophage-derived nitric oxide. Arch Biochem Bioph (1992) 298:446–451.[CrossRef][Web of Science][Medline]
32. Evans T.J., Buttery L.D.K., Carpenter A., Springall D.R., Polak J.M., Cohen J. Cytokine-treated human neutrophils contain inducible nitric oxide synthase that produces nitration of ingested bacteria. Proc Natl Acad Sci USA (1996) 93:9553–9558.[Abstract/Free Full Text]
33. Siegfried M.R., Erhardt J., Rider T., Ma X.L., Lefer A.M. Cardioprotection and attenuation of endothelial dysfunction by organic nitric oxide donors in myocardial ischemia-reperfusion. J Pharmacol Exp Ther (1992) 260:668–675.[Abstract/Free Full Text]
34. Haddad I.Y., Crow J.P., Hu P., Ye Y., Beckman J., Matalon S. Concurrent generation of nitric oxide and superoxide damages surfactant protein A. Am J Physiol (1994) 267:L242–L249.[Web of Science][Medline]
35. Darley-Usmar V.M., Hogg N., O'leary V.J., Wilson M.T., Moncada S. The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Free Rad Res Comms (1992) 17:9–20.[Web of Science][Medline]
36. Szabo C., Salzman A.L. Endogenous peroxynitrite is involved in the inhibition of mitochondrial respiration in immuno-stimulated J774.2 macrophages. Biochem Biophys Res Commun (1995) 209:739–743.[CrossRef][Web of Science][Medline]
37. Moro M.A., Darley-Usmar V.M., Goodwin D.A., Read N.G., Zamora-Pino R., Feelisch M., Radomski M.W., Moncada S. Paradoxical fate and biological action of peroxynitrite on human platelets. Proc Natl Acad Sci USA (1994) 91:6702–6706.[Abstract/Free Full Text]
38. Lopez B.L., Christopher T.A., Ma X.L. The effects of peroxynitrite on the myocardium during ischemia-reperfusion is dependent upon the biological environment. Endothelium (1995) 3:s76.
39. Lefer D.J., Scalia R., Campbell B., Nossuli T., Hayward R., Salamon M., Grayson J., Lefer A.M. Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats. J Clin Invest (1997) 99:684–691.[Web of Science][Medline]
40. Beresewicz A., Karwatowska-Prokopczuk E., Lewartowski B., Cedro-Ceremuzynska K. A protective role of nitric oxide in isolated ischaemic reperfused rat heart. Cardiovasc Res (1995) 30:1001–1008.[CrossRef][Web of Science][Medline]
41. Li X.S., Uriuda Y., Wang Q.D., Nordlander R., Sjöquist P.O., Pernow J. Role of L-arginine in preventing myocardial and endothelial injury following ischaemia/reperfusion in the rat isolated heart. Acta Physiol Scand (1996) 156:37–44.[CrossRef][Web of Science][Medline]
42. Massini E., Bianchi S., Mugnai L., Gambassi F., Lupini M., Pistelli A., Mannaioni P.F. The effect of nitric oxide generators on ischemia reperfusion injury and histamine release in isolated perfused guinea-pig heart. Agents Actions (1991) 33:53–56.[CrossRef][Web of Science][Medline]
43. Winterbourn C.C., Metodiewa D. The reaction of superoxide with reduced glutathione. Arch Biochem Biophys (1994) 314:284–290.[CrossRef][Web of Science][Medline]
44. Liu S., Beckman J.S., Ku D.D. Peroxynitrite, a product of superoxide and nitric oxide, produces coronary vasorelaxation in dogs. J Pharmacol Exp Ther (1994) 268:1114–1121.[Abstract/Free Full Text]
45. Wu M., Pritchard K.A. Jr., Kaminski P.M., Fayngersh R.P., Hintze T.H., Wolin M.S. Involvement of nitric oxide and nitrosothiols in relaxation of pulmonary arteries to peroxynitrite. Am J Physiol (1994) 266:H2108–H2113.[Web of Science][Medline]
46. Tarpey M.M., Beckman J.S., Ischiropoulos H., Gore J.Z., Brock T.A. Peroxynitrite stimulates vascular smooth muscle cell cyclic GMP synthesis. FEBS Lett (1995) 364:314–318.[CrossRef][Web of Science][Medline]
47. Mayer B., Schrammel A., Klatt P., Koesling D., Schmidt K. Peroxynitrite-induced accumulation of cyclic GMP in endothelial cells and stimulation of purified soluble guanylyl cyclase. Dependence on glutathione and possible role of S-nitrosation. J Biol Chem (1995) 270:17355–17360.[Abstract/Free Full Text]
48. Connaughton M., Kelly F.J., Haddock P.S., Hearse D.J., Shattock M.J. Ventricular arrhythmias induced by ischaemia-reperfusion are unaffected by myocardial glutathione depletion. J Mol Cell Cardiol (1996) 28:679–688.[CrossRef][Web of Science][Medline]
49. Pabla R., Curtis M.J. Effects of NO modulation on cardiac arrhythmias in the rat isolated heart. Cir Res (1995) 77:984–992.[Abstract/Free Full Text]
50. Coronel R., Wilms-Schopman F.J., Dekker L.R., Janse M.J. Heterogeneities in [K⁺]₀ and TQ potential and the inducibility of ventricular fibrillation during acute regional ischemia in the isolated perfused porcine heart. Circulation (1995) 92:120–129.[Abstract/Free Full Text]

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