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Cardiovascular Research 2004 62(3):568-577; doi:10.1016/j.cardiores.2004.01.025
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Critical timing of L-arginine treatment in post-ischemic myocardial apoptosis—role of NOS isoforms

Feng Lianga, Erhe Gaob, Ling Taoa, Huirong Liua, Yan Qua, Theodore A Christophera, Bernard L Lopeza and Xin L Ma*,a

aDepartment of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Thompson Building, Room 239, Philadelphia, PA 19107, USA
bCenter for Translational Medicine, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107, USA

* Corresponding author. Tel.: +1-215-955-4994; fax: +1-215-923-6225. Email address: xin.ma{at}jefferson.edu

Received 11 September 2003; revised 19 January 2004; accepted 20 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 
Background: The role of nitric oxide (NO) in apoptotic cell death has been extensively studied in recent years. However, reported results are inconsistent and often controversial, and the mechanisms underlying its diverse effects in apoptosis regulation remain unidentified. The present study attempted to determine whether in vivo administration of L-arginine, the substrate for NOS, at different time points during the course of myocardial ischemia and reperfusion may differentially regulate post-ischemic myocardial apoptosis, and if so, to investigate the mechanisms involved. Methods and results: Male adult rats were subjected to 30 min of myocardial ischemia followed by 5 h of reperfusion. L-Arginine was administered as a bolus at either 10 min before (early treatment) or 3 h after reperfusion (late treatment). There was no difference in myocardial eNOS expression between any groups studied. Myocardial iNOS expression was detected at 3 h after reperfusion but not at 1 h after reperfusion. Administration of L-arginine 10 min before reperfusion markedly decreased TUNEL-positive staining cardiomyocytes, reduced myocardial caspase-3 activity, inhibited iNOS expression, and reduced myocardial nitrotyrosine content. In strict contrast, administration of L-arginine 3 h after reperfusion, a time point when iNOS was expressed, resulted in a significant increase in myocardial NOx content, myocardial injury, and toxic peroxynitrite formation as measured by nitrotyrosine. Conclusion: Our results demonstrated for the first time that L-arginine administered at different time points during ischemia/reperfusion exerted different effects on post-ischemic myocardial injury, and suggests that stimulation of eNOS reduces nitrative stress and decreases apoptosis whereas stimulation of iNOS increases nitrative stress and enhances myocardial reperfusion injury.

KEYWORDS Nitric oxide; Apoptosis; Myocardial ischemia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 
Early reperfusion after coronary occlusion remains the most effective means of limiting ischemic myocardial injury. However, evidence from animal studies as well as clinical observations demonstrates that reperfusion itself may cause additional cell death, defined as "reperfusion injury". Traditionally, this reperfusion injury was thought to be caused exclusively by necrosis. However, accumulating evidence now indicates that apoptosis, a special form of cell death that differs from necrosis in many aspects, plays an essential role in cardiomyocyte death after ischemia and reperfusion [1,2]. Given that apoptosis is an active, gene-directed process, inhibition of apoptosis could be achieved more successfully than prevention of necrosis, a passive form of cell death that is inflicted by the acute stimulus [3].

Nitric oxide (NO), a free radical that is produced ubiquitously in all cell types, is synthesized from L-arginine by three isoforms of NO synthase (NOS). Two isoforms (eNOS and nNOS) are constitutively expressed and are acutely regulated by calcium/calmodulin and phosphorylation, while the third (iNOS) is induced during inflammation and produces higher levels of NO for a longer period [4]. Previous studies have demonstrated that supplementation of L-arginine during the early stages of reperfusion decreases myocardial infarction after ischemia/reperfusion [5,6]. However, the direct link between L-arginine-initiated cardioprotection and L-arginine-stimulated NO production has not been established. Moreover, the cellular pathways by which L-arginine exerts its cardioprotection remain largely unknown. Specifically, whether the supplementation of L-arginine may reduce post-ischemic myocardial apoptosis, a critical factor that determines the final myocardial infarct size [2], has not been previously investigated.

High concentrations of exogenous NO have been reported to induce apoptotic cell death in several cell types including macrophages, thymocytes, pancreatic islets, certain neurons, and tumor cells [7]. However, short-term exposure to high levels of NO may overwhelm natural protective pathways, leading to the activation of apoptotic signaling pathways. Such toxic levels of NO may have limited relevance to the in vivo situation. Whether or not in vivo stimulation of iNOS by L-arginine may increase NO production, thus resulting in apoptotic cell death, has not been previously studied.

Therefore, the purposes of the present study were (1) to determine whether early supplementation of L-arginine may reduce post-ischemic apoptosis, and if so, to clarify the relationship between NO production and L-arginine's anti-apoptotic effect, and to determine the enzymatic source that is responsible for NO production; and (2) to elucidate whether the supplementation of L-arginine during a late phase of reperfusion when iNOS is expressed may aggravate post-ischemic myocardial apoptosis, and if so, to identify the mechanisms involved.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Limitations
 References
 
2.1. Experimental protocol
The experiments were performed in adherence to National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Thomas Jefferson University Committee on Animal Care. Male Sprague–Dawley rats were anesthetized with 2% isoflurane and placed on a Temperature Therapy Pad (Harvard Apparatus) connected to a heat pump. Myocardial ischemia was produced by temporarily exteriorizing the heart (<1 min) via a left thoracic incision and placing a 6-0 silk suture slipknot around the left anterior descending coronary artery. Animals were recovered from anesthesia within 5 min after the completion of surgery. After 30 min of ischemia, the slipknot was released and the myocardium was reperfused for 1 h (to determine the effect of L-arginine supplementation before reperfusion on NO production during early reperfusion) or 5 h. During the most part of ischemia (>25 min) and during the entire reperfusion period, animals were not anesthetized. Rats were randomized to receive vehicle (0.9% NaCl, 1 ml/kg), L-arginine (100 mg/kg, i.p.) or D-arginine (100 mg/kg, i.p.) either at 10 min before reperfusion (early treatment) or 3 h after reperfusion (late treatment). Sham-operated control rats (Sham MI/R) underwent the same surgical procedures except that the suture that was passed under the left coronary artery was not tied. At the end of the 5-h reperfusion period (or 1 h for myocardial NO production during early reperfusion), the ligature around the coronary artery was retied and 1 ml of 2% Evans blue dye was injected into the left ventricular cavity. The dye was circulated and uniformly distributed except in that portion of the heart previously perfused by the occluded coronary artery (area-at-risk, AAR). The heart was quickly excised, the AAR was isolated and cardiac tissue was processed according to the procedures described below for immunohistological (n=5–6/group), biochemical (n=12–14/group), infarct size (n=10/group), and Western blot assays (n=5–6/group).

2.2. Determination of myocardial apoptotic death
Myocardial apoptosis was determined by DNA ladder formation and TUNEL staining as described previously [8]. In brief, at the end of experiment, ischemic/reperfused cardiac tissue was isolated, minced while thawing and homogenized. The tissue was digested with proteinase K at 56 °C overnight and then incubated with DNA-free RNase at 37 °C for an additional hour. Digested tissues were precipitated, centrifuged, and supernatants containing DNA were precipitated and centrifuged again. The resulting DNA pellets were washed and dissolved in DNA hydration solution. DNA electrophoresis was carried out at 60 V for 1–2 h. DNA ladder formation, a hallmark for tissue apoptosis, was visualized under ultraviolet light and photographed for permanent records.

To determine myocardial apoptosis in a quantitative manner, the hearts were perfused first with 0.9% NaCl for 5 min and then with 4% paraformadehyde in PBS (pH 7.4) for 20 min. Four longitudinal sections from ischemic regions were cut and further fixed in 4% paraformadehyde in PBS for 24 h at room temperature. Fixed tissues were embedded in a paraffin block and 2 slides at 4–5 µm thickness were cut from each tissue block. Immunohistochemical procedures for detecting apoptotic cardiomyocytes were performed by using an apoptosis detection kit (Boehringer Mannheim, Ridgefield, CT) according to the manufacturer's instructions. An additional staining was performed with monoclonal anti-{alpha}-sarcomeric actin. This staining enables the identification of myocytes and, therefore, a distinction between myocyte nuclei and nuclei of other cells in the cardiac tissue. After being rinsed with PBS, slides were coverslipped with mounting medium containing DAPI to permit total nuclei counting.

For each slide, 10 fields were randomly chosen, and using a defined rectangular field area (20 x objective), a total of 100 cells per field were counted. The index of apoptosis was determined (i.e., number of positively stained apoptotic myocytes/total number of myocytes counted x 100) from a total of 80 fields per heart (n=1, at least five hearts were studied in each group). Assays were performed in a blinded manner.

2.3. Determination of myocardial infarct size
At the end of the 24-h reperfusion period, the ligature around the coronary artery was retied and 1 ml of 2% Evans blue dye was injected into the left ventricular cavity. The dye was circulated and uniformly distributed except in that portion of the heart previously perfused by the occluded coronary artery (area-at-risk, AAR). The heart was excised, frozen in –20 °C, and sliced into 1 mm thick sections perpendicular to the long axis of the heart. Slices were incubated individually using a 24-well culture plate in 1% TTC in phosphate buffer at pH 7.4 at 37 °C for 10 min, and photographed with a digital camera. Evan's blue stained area (area-not-at-risk, ANAR), TTC stained area (red staining, ischemic but viable tissue), and TTC staining negative area (infarct myocardium) were digitally measured using SigmaScan. The myocardial infarct size was expressed as a percentage of infarct area over AAR.

2.4. Determination of myocardial caspase-3 activity
Cardiac capase-3 activity was performed by using a caspase-3 colorimetric assay kit (Chemicon, Temecula, CA) following the manufacturer's instructions. In brief, myocardial tissue was homogenized in ice-cold lysis buffer for 30 s. The homogenates were centrifuged, supernatants were collected, and protein concentrations were measured by bicinchoninic acid method. To each well of a 96-well plate, supernatant containing 200 µg of protein was loaded and incubated with 30 µg caspase-3 substrate N-acethyl-Asp-Glu-Val-Asp (DEVD)-p-nitroaniline at 37 °C for 1.5 h. The optical density was measured at 405 nm with a SpectraMax-Plus microplate spectrophotometer. The activity of Caspase-3 in tissue samples was calculated using a standard curve and expressed as µmol pNA/mg protein.

2.5 Determination of total NOx content in cardiac tissue
Cardiac tissue samples from AAR were rinsed, homogenized in deionized water (1:10, wt/vol), and centrifuged at 14,000 x g for 10 min. The tissue NO and its in vivo metabolic products (NO2 and NO3) in the supernatant, collectively known as NOx, were determined using a chemiluminescence NO detector (SIEVER 280i NO Analyzer) as described in our previous study [8].

2.6. Quantitation of tissue nitrotyrosine content
Nitrotyrosine content, a footprint of in vivo ONOO– formation, was determined using an ELISA method described in our recent publication [9]. The results were presented as nmol of nitrotyrosine/g protein.

2.7. Immunoblotting
Protein from tissue homogenate were separated on SDS-PAGE gels, transferred to nitrocellulose membranes, and Western blotted with monoclonal antibodies against eNOS or iNOS (Transduction Laboratories). Nitrocellulose membranes were then incubated with HRP-conjugated anti-mouse IgG antibody (1:2000, Cell Signaling) for 1 h and the blot was developed with a Supersignal chemiluminescence detection kit (Pierce). The immunoblotting was visualized with a Kodak Image Station 400 and the blot densities were analyzed with Kodak 1D software.

2.8. Statistical analysis
All values in the text and figures are presented as means±S.E.M. of n independent experiments. All data (except Western blot density) were subjected to ANOVA followed by Bonferroni correction for post hoc t-test. Western blot densities were analyzed with the Kruskal–Wallis test followed by Dunn's post test. 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
 5. Limitations
 References
 
3.1 Opposing effect of early vs. late L-arginine treatment on post-ischemic myocardial injury
To determine the time dependence of L-arginine supplementation on post-ischemic myocardial injury, L-arginine was administered either at 10 min before reperfusion (early treatment) or 3 h after reperfusion (delayed treatment). Consistent with previous reports, myocardial ischemia and reperfusion resulted in marked myocardial infarction (Fig. 1) and significant cardiomyocyte apoptosis (Figs. 2 and 3)Go. The administration of L-arginine 10 min before reperfusion markedly, but not completely, inhibited post-ischemic myocardial apoptosis and reduced myocardial infarct size (Figs. 1–3)GoGo. In contrast, administration of L-arginine 3 h after reperfusion not only failed to reduce post-ischemic myocardial injury, but also further worsened myocardial reperfusion injury as evidenced by a significant increase in TUNEL-positive staining and enlarged infarct size (Figs. 1–3)GoGo.


Figure 1
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  		Fig. 1 Effect of L-arginine treatment on myocardial infarct size after 30 min of ischemia and 5 h of reperfusion. *P<0.05; **P<0.01 vs. MI+vehicle (n=10–12 hearts/group).

 

Figure 2
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  		Fig. 2 Representative photograph of electrophoretic analysis of internucleosomal DNA extracted from sham-operated rat hearts (S, lane 2) or hearts exposed to 30 min ischemia followed by 5 h of reperfusion receiving vehicle (V, lane 3), early L-arginine treatment (E, lane 4) or late L-arginine treatment (L, lane 5). No DNA ladder was detected from tissue of sham-operated hearts, while clear DNA ladders were seen in tissue of vehicle-treated hearts; Treatments with L-arginine before reperfusion significantly reduced the intensity of DNA ladders, whereas treatment with L-arginine 3 h after reperfusion significantly enhanced the intensity of DNA ladders. M represents DNA size markers (lane 1).

 

Figure 3
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  		Fig. 3 (A) Representative photomicrographs of in situ detection of DNA fragments in heart tissue from rats subjected to sham ischemia or 30 min of ischemia and 5 h of reperfusion receiving vehicle or L-arginine. Cardiac myocytes are identified by anti-{alpha}-actinin antibody (red), total nuclei were labeled with DAPI (blue), and apoptotic nuclei were detected by TUNEL staining (green). (B) Summary of percent TUNEL-positive myocytes. MI+L-Arg(E): L-arginine administered 10 min before reperfusion; MI+L-Arg(L): L-arginine administered 3 h after reperfusion. ##P<0.01 vs. sham MI/R; *P<0.05; **P<0.01 vs. MI+vehicle (n=5–6 hearts/group).

 
To provide more evidence in a quantitative and specific manner that treatment with L-arginine at different times during the course of myocardial ischemia and reperfusion exerts different effects on post-ischemic myocardial apoptosis, myocardial caspase-3 activation, a final common pathway in caspase-dependent apoptosis, was determined. As summarized in Fig. 4, 30 min of ischemia followed by 5 h of reperfusion caused a 2.1-fold increase in caspase-3 activity when compared with sham operative controls. Early treatment with L-arginine (i.e., 10 min before reperfusion) resulted in a substantial reduction in caspase-3 activity, further suggesting that L-arginine treatment attenuated post-ischemic myocardial apoptosis. In contrast, when L-arginine treatment was delayed to 3 h after reperfusion, myocardial caspase-3 activity further increased to a level that was significantly higher than that seen in the vehicle group. Taken together, these results provided clear evidence that timing is critical in determining the effect of L-arginine supplementation on post-ischemic myocardial apoptosis.


Figure 4
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  		Fig. 4 Effect of L-arginine treatment on myocardial caspase-3 activity after 30 min of ischemia and 5 h of reperfusion. ##P<0.01 vs. sham MI/R; *P<0.05; **P<0.01 vs. MI+vehicle (n=12–14 hearts/group).

 
3.2 Effect of L-arginine treatment on myocardial total NO content
To establish a direct link between L-arginine's anti- and pro-apoptotic effects and its NO stimulatory effect, we directly measured myocardial total NO content in hearts with different treatments. Compared with sham myocardial ischemia/reperfusion hearts, myocardial total NO content was significantly increased in hearts subjected to myocardial ischemia and reperfusion. Interestingly, although the supplementation of L-arginine before reperfusion significantly increased myocardial total NO production when compared to sham ischemia/reperfused hearts, this treatment slightly decreased myocardial NO content when compared to vehicle-treated hearts. In contrast, administration of L-arginine 3 h after reperfusion almost doubled myocardial NO content when compared to vehicle-treated hearts (Fig. 5), indicating that a massive increase of NO production occurred in ischemia/reperfused hearts receiving L-arginine at this delayed time point.


Figure 5
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  		Fig. 5 Effect of L-arginine treatment on myocardial NOx content after 30 min of ischemia and 5 h of reperfusion. ##P<0.01 vs. sham MI/R; **P<0.01 vs. MI+vehicle (n=12–14 hearts/group).

 
3.3 Role of NO in L-arginine's anti-apoptotic effect (early treatment)
That early treatment with L-arginine significantly reduced post-ischemic myocardial apoptosis but failed to increase myocardial NO content when compared with vehicle was surprising and raised the possibility that L-arginine may reduce post-ischemic myocardial apoptosis in an NO-independent fashion. To further determine the role of NO in modulating post-ischemic myocardial apoptosis and to seek an explanation for these apparently controversial results, three additional experiments were performed. In the first experiment, rats receiving either vehicle (n=10) or early L-arginine treatment (i.e., 10 min before reperfusion) (n=10) were sacrificed 1 h after reperfusion (rather than 5 h after reperfusion) and myocardial total NOx content was measured. Interestingly, although myocardial total NOx measured at 5 h after reperfusion was slightly lower in hearts treated with L-arginine before reperfusion when compared to vehicle-treated hearts (Fig. 5), myocardial total NOx was markedly higher in L-arginine treated hearts when measured at 1 h reperfusion (1.4±0.09- vs. 1.05±0.07-fold over sham MI/R, P<0.01). This result suggested that (1) high levels of NOx in the vehicle group were accumulated at a later phase of reperfusion (not within the first h); and (2) treatment with L-arginine before reperfusion stimulated NO production at an early phase of reperfusion, and this increased NO may in turn, exert a negative feedback for subsequent excessive NO production, presumably from iNOS.

To obtain direct evidence that early treatment with L-arginine may negatively regulate iNOS expression, an additional six animals in each group were studied and myocardial iNOS expression was determined. As illustrated in Fig. 6, 30 min of ischemia and 5 h of reperfusion resulted in a significant upregulation of iNOS expression. Most interestingly, administration of L-arginine before reperfusion, but not at 3 h after reperfusion, markedly inhibited iNOS expression.


Figure 6
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  		Fig. 6 Effect of L-arginine treatment on myocardial iNOS expression after 30 min of ischemia and 5 h of reperfusion. Insert: representative blots. Data obtained from quantitative densitometry were presented as mean±S.E.M. of at least five independent experiments. ##P<0.01 vs. sham MI/R; *P<0.05 compared with vehicle.

 
To further demonstrate that supplementation of L-arginine before reperfusion inhibited post-ischemic myocardial apoptosis by increasing NO production, an additional group (n=10) of rats were treated with D-arginine (100 mg/kg, 10 min before reperfusion). This treatment had no protective effect against post-ischemic myocardial apoptosis (TUNEL-positive cells: 19.9±1.5% vs. 20.6±1.8% in vehicle group, P>0.1). Taken together, these results provided strong evidence that early supplementation of L-arginine stimulated NO production in the early phase of reperfusion and inhibited myocardial apoptosis.

3.4. Alteration of eNOS and iNOS expression during ischemia and reperfusion
To determine the potential enzymatic sources from which L-arginine may increase NO production when administered at different time points during ischemia and reperfusion, myocardial eNOS and iNOS expression were examined. As summarized in Fig. 7A, 30 min of ischemia followed by 1 or 3 h of reperfusion had no significant effect on eNOS expression. Significant iNOS expression was not detected at 1 h reperfusion. However, iNOS expression was markedly upregulated 3 h after reperfusion (Fig. 7B). It has been previously determined that the half-life of L-arginine after a single bolus administration is approximately 1 h [10]. Therefore, it is likely that L-arginine supplemented before reperfusion may have provided additional substrate for eNOS, but not iNOS, resulting in a moderate increase in NO production. In contrast, L-arginine administered 3 h after reperfusion when iNOS is expressed may have provided additional fuel for iNOS, resulting in pathological levels of NO production.


Figure 7
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  		Fig. 7 Time course of eNOS (A) and iNOS (B) expression in sham MI hearts and hearts subjected to 30 min of ischemia and 1 or 3 h of reperfusion. Insert: representative blots. Data obtained from quantitative densitometry were presented as mean±S.E.M. of at least five independent experiments. **P<0.01 compared with vehicle.

 
3.5 Mechanism underlying the anti- and pro-apoptotic effects of L-arginine treatment
Considerable evidence exists that NO itself has low reactivity with most biological molecules and is cytoprotective. In contrast, the secondary reaction products between NO and other reaction species are highly reactive and often cytotoxic. Specifically, peroxynitrite (ONOO–), the reaction product between NO and superoxide, reacts with a variety of biological molecules including DNA, proteins and lipids, and has been reported to cause apoptotic cell death in vitro and in vivo. To determine whether the anti- and pro-apoptotic properties of L-arginine supplementation were related to their effects on peroxynitrite generation, myocardial nitrotyrosine content, an in vivo foot marker of peroxynitrite formation, was determined. As illustrated in Fig. 8, 30 min of myocardial ischemia and 5 h of reperfusion resulted in a fourfold increase in nitrotyrosine formation, indicating a significant increase in ONOO– formation in ischemic/reperfused cardiac tissue. Consistent with their anti- or pro-apoptotic effects, early and late treatment with L-arginine exerted opposite effects on nitrotyrosine content (Fig. 8), suggesting that supplementation of L-arginine before reperfusion reduced ONOO– formation, whereas supplementation of L-arginine 3 h after reperfusion increased ONOO– formation.


Figure 8
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  		Fig. 8 Effect of L-arginine treatment on myocardial nitrotyrosine content after 30 min of ischemia and 5 h of reperfusion. ##P<0.01 vs. sham MI/R; *P<0.05, **P<0.01 vs. vehicle group (n=12–14 hearts/group).

 
3.6 Role of iNOS in the late L-arginine treatment-induced apoptotic cell death
Having demonstrated that significant iNOS expression was detected 3 h after reperfusion and supplementation of L-arginine at this time point increased ONOO– formation and apoptotic cell death, we performed another experiment and attempted to obtain more direct evidence that iNOS-derived NO and its secondary reaction products are responsible for the late L-arginine supplementation-induced myocardial apoptosis. A group of rats were treated with 1400 W, a highly selective iNOS inhibitor, 1 h before L-arginine administration (i.e., 2 h after reperfusion), and myocardial apoptotic cell death was determined 5 h after reperfusion. Interestingly, pretreatment with 1400 W completely blocked L-arginine-induced increase in myocardial NOx content (1.24±0.19-fold over sham MI), abolished the pro-apoptotic effect of late L-arginine treatment (12.2±3.4% vs. 29.4±2.5%, n=11, P<0.01), and significantly lowered the number of TUNEL-positive cells compared to vehicle (20.6±1.8% in vehicle, P<0.01).

To determine whether the supplementation of L-arginine 3 h after reperfusion (when iNOS is expressed in ischemic/reperfused myocardial tissue) may reduce systemic blood pressure and thereby decrease coronary perfusion, an additional experiment was performed. Rats were subjected to MI/R as described above except that they were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) to permit continuous monitoring of arterial blood pressure via a Millar Mikro-Tip catheter pressure transducer inserted into the left carotid artery. Three hours after reperfusion, rats were treated with either vehicle (n=5) or L-arginine (100 µg/kg, n=5), and the systemic blood pressure was measured at 20 min of interval. The data shown that treatment with L-arginine had no significant effect on blood pressure at any time points observed when compared with vehicle treated group (P>0.1). This result demonstrated that although supplementation of L-arginine stimulated local NO production (ischemic/reperfused cardiac tissue) from locally expressed iNOS (ischemic reperfused cardiac tissue), this local NO generation is not sufficient to cause any systemic changes. Therefore, any difference between vehicle and L-arginine treated groups cannot be attributed to hemodynamic alterations.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Limitations
 References
 
In the present study, we demonstrated for the first time that supplementation of L-arginine can either inhibit or promote post-ischemic myocardial injury. The enzymatic sources where NO is generated, the time frame when NO production is stimulated, and the concurrent formation of the toxic reaction product between NO and other reactive species are critical factors that determine the cytoprotection and cytotoxicity of L-arginine supplementation in post-ischemic myocardial injury.

4.1 Anti-apoptotic effect of early L-arginine supplementation—role of eNOS derived NO
Our present study demonstrated for the first time that supplementation of L-arginine before reperfusion markedly reduced post-ischemic myocardial apoptosis, likely via increasing NO production from eNOS. Two lines of evidence exist that support our conclusion. First, supplementation of L-arginine markedly increased myocardial NOx content when measured at 1 h after reperfusion, a time point when iNOS expression is undetectable; second, supplementation of D-arginine in an identical fashion failed to increase NO production and exerted no protection against post-ischemic myocardial apoptosis. Because there is no selective eNOS inhibitor available, the final conclusive evidence that early L-arginine supplementation exerts its anti-apoptosis effect via eNOS-derived NO pathway can only be obtained with eNSO knockout animals. This experiment has been planned.

The precise mechanisms responsible for inhibition of apoptosis by NO are not clear. Several possibilities exist that may explain the anti-apoptotic effects of NO. NO has been shown to increase Bcl-2, thioredoxin, and heat-shock protein-70 and -32 expression, thus inhibiting the release of mitochondrial cytochrome c and apoptosis inducing factor (AIF) [7]. The activation of cGMP and cGMP-dependent protein kinase (PKG) by NO increases phosphorylation of Akt, a major intracellular anti-apoptotic protein, both directly and indirectly [7]. Recent biological and in vitro cell culture studies have demonstrated that most caspases, including caspase-3 in its inactive (pro-caspase) and active forms, are inhibited by NO through cysteine S-nitrosylation (Cys163) [11–13]. Finally, increased NO production by L-arginine supplementation may inhibit neutrophil accumulation in ischemic/reperfused myocardial tissue, thus attenuating neutrophil-induced myocardial apoptotic death [14,15].

Additionally, our present experiment provides direct evidence that the early supplementation of L-arginine inhibited iNOS expression and its NO production and attenuated toxic ONOO– formation. Since high concentrations and/or high reactive RNS have been demonstrated to cause apoptotic cell death, the negative feedback of early, physiological amounts of NO production on subsequent toxic levels of NO production may contribute to the anti-apoptotic effect of early L-arginine supplementation as observed in the present study.

4.2 Pro-apoptotic effect of late L-arginine supplementation—role of iNOS derived NO and peroxynitrite formation
Previous studies have demonstrated that exposing cultured cells to high concentrations of NO induces apoptotic cell death [7]. Short-term exposure to high levels of NO may overwhelm natural protective pathways, leading to the activation of apoptotic signaling pathways. Such toxic levels of NO may have limited relevance to the in vivo situation. Therefore, the contribution of endogenously produced NO from iNOS to apoptotic ell death in vivo remains largely speculative. In our present experiment, L-arginine, the substrate for NOS, rather than an NO donor, was administrated 3 h after reperfusion. Our results demonstrated for the first time that supplementation of L-arginine at a time point when iNOS expression is detected markedly increased post-ischemic myocardial apoptosis as evidenced by increased TUNEL-positive staining and caspase-3 activity.

Multiple lines of evidence support the notion that delayed treatment with L-arginine increased myocardial apoptosis via stimulation of NO production from iNOS with subsequent increase of toxic ONOO– formation. First, strong iNOS expression was detected at 3 h after reperfusion, a time point when L-arginine was supplemented (Fig. 7). Second, administration of L-arginine resulted in a marked increase in myocardial NOx content (Fig. 5) and a significant increase in nitrotyrosine formation (Fig. 8). Finally and most importantly, pretreatment with 1400 W, a highly selective iNOS inhibitor, blocked late L-arginine treatment-induced NOx production, ONOO– formation, and myocardial apoptosis.

The mechanisms by which peroxynitrite causes myocardial apoptosis were not directly addressed in the present study. However, previous studies have demonstrated that peroxynitrite may increase apoptotic cell death in a variety of cell types. The pro-apoptotic mechanisms of ONOO– include protein and DNA oxidation [16–19], lipid peroxidation [20], protein nitration [21–23], and endoplasmic reticulum stress with the subsequent release of caspase-12 [24].

It should be indicated that there is a huge variation in the rate of post-ischemic myocardial apoptosis in the literature (ranging from 4% to 40%). Species and model (length of ischemia and reperfusion) differences are probably two of the most important contributors for the observed variation. For instance, an apoptotic rate of 28.6% [15] and 40% [25] has been reported in ischemic/reperfused rat heart. Moreover, a 26% of apoptosis has been reported in ischemic/reperfused dog hearts [26]. The percentage of TUNEL-positive staining in our vehicle-treated group (20.6%) thus falls in the range as reported by other investigators.

In summary, our results demonstrated for the first time that L-arginine administered at different time points during ischemia/reperfusion exerted different effects on post-ischemic myocardial apoptosis, and suggests that stimulation of eNOS may reduce nitrative stress and decreases myocardial reperfusion injury. Stimulation of iNOS, on the other hand, increases nitrative stress and enhances myocardial reperfusion injury. These results provide a likely explanation for previously published controversial results concerning the role of nitric oxide in apoptotic cell death. We now have demonstrated that the enzymatic sources where NO is generated, the time frame when NO production is stimulated, and the concurrent formation of the toxic reaction product between NO and other reactive species are critical factors that determine the cytoprotection and cytotoxicity of NO in apoptotic cell death. Our results are of clinical significance. Previous studies have demonstrated that supplementation of L-arginine before reperfusion decreases myocardial infarct size and suggested that L-arginine may have therapeutic value in patients with myocardial infarction. Our present results clearly indicated that the effects of L-arginine treatment is time-dependent, and supplementation of L-arginine when iNOS has been expressed will increase, not decrease, post-ischemic myocardial injury.


   5. Limitations
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
References
 
The infarct size in the vehicle group from the current study is relatively small compared with previously reported results, although even a smaller infarct size has been found in this model [27]. In most myocardial ischemia and reperfusion models used to date, animals are anesthetized during the entire ischemia/reperfusion period and the chest remains open. A significant hypothermia would occur in these animals if their body temperature is not carefully controlled. In the present study, animals were not under anesthesia for most of the ischemic (>25 min), and for the entire reperfusion (5 h), periods. Their natural temperature controlling system should not have been compromised as would have occurred in anesthetized animals. Therefore, it is unlikely that a significant hypothermia occurred in our model to account for the smaller infarct size observed in our study. Previous animal study [28] and clinical observation [29] have demonstrated that there is a clear seasonal variation in tissue infarct after ischemia, with the smallest infarct size occurs in the summer. Whether a relatively smaller infarct size observed in the present study is related to the seasonal variation needs to be directly determined in future study. However, it should be pointed that the major focus of the present study is to compare the effect of L-arginine administered at different time during ischemia/reperfusion on post-ischemic myocardial injury and investigate the mechanisms involved. Therefore, a relatively smaller infarct size in vehicle group compared to what has been published by other investigators should not change our conclusion that the effects of L-arginine treatment is time-dependent and that enzymatic sources where NO is generated and the concurrent formation of the toxic reaction product between NO and other reactive species are critical factors that determine the cytoprotection and cytotoxicity of L-arginine supplementation in post-ischemic myocardial injury.


   Acknowledgements
 
This research was supported in part by NIH grant HL-63828.


   Notes
 
Time for primary review 25 days


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

  1. Gottlieb R.A, Engler R.L. Apoptosis in myocardial ischemia–reperfusion. Ann. N. Y. Acad. Sci. (1999) 874:412–426.[Abstract/Free Full Text]
  2. Kajstura J, Cheng W, Reiss K, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab. Invest. (1996) 74:86–107.[ISI][Medline]
  3. Bialik S, Geenen D.L, Bennett M.R, et al. Cardiovascular Pharmacotherapeutics. Frishman W.H, Sonnenblick E.H, eds. (1997) New York: McGraw-Hill. 955–972.
  4. Paulus W.J. The role of nitric oxide in the failing heart. Heart Fail. Rev. (2001) 6:105–118.[CrossRef][Medline]
  5. Weyrich A.S, Ma X.L, Lefer A.M. The role of L-arginine in ameliorating reperfusion injury after myocardial ischemia in the cat. Circulation (1992) 86:279–288.[Abstract/Free Full Text]
  6. Pabla R, Buda A.J, Flynn D.M, et al. Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion. Circ. Res. (1996) 78:65–72.[Abstract/Free Full Text]
  7. Chung H.T, Pae H.O, Choi B.M, Billiar T.R, Kim Y.M. Nitric oxide as a bioregulator of apoptosis. Biochem. Biophys. Res. Commun. (2001) 282:1075–1079.[CrossRef][ISI][Medline]
  8. Gao F, Gao E, Yue T.L, et al. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia–reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation (2002) 105:1497–1502.[Abstract/Free Full Text]
  9. Ma X.L, Gao F, Nelson A.H, et al. Oxidative inactivation of nitric oxide and endothelial dysfunction in stroke-prone spontaneous hypertensive rats. J. Pharmacol. Exp. Ther. (2001) 298:879–885.[Abstract/Free Full Text]
  10. Bode-Boger S.M, Boger R.H, Galland A, Tsikas D, Frolich J.C. L-Arginine-induced vasodilation in healthy humans: pharmacokinetic–pharmacodynamic relationship. Br. J. Clin. Pharmacol. (1998) 46:489–497.[CrossRef][ISI][Medline]
  11. Boyd C.S, Cadenas E. Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis. Biol. Chem. (2002) 383:411–423.[CrossRef][ISI][Medline]
  12. Kim P.K.M, Kwon Y.G, Chung H.T, Kim Y.M. Regulation of caspases by nitric oxide. Ann. N. Y. Acad. Sci. (2002) 962:42–52.[Abstract/Free Full Text]
  13. Mannick J.B, Schonhoff C, Papeta N, et al. S-nitrosylation of mitochondrial caspases. J. Cell Biol. (2001) 154:1111–1116.[Abstract/Free Full Text]
  14. Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ. Res. (1996) 79:949–956.[Abstract/Free Full Text]
  15. Nakamura M, Wang N.P, Zhao Z.Q, et al. Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc. Res. (2000) 45:661–670.[Abstract/Free Full Text]
  16. Yamakura F, Taka H, Fujimura T, Murayama K. Inactivation of human manganese-superoxide dismutase by peroxynitrite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosine. J. Biol. Chem. (1998) 273:14085–14089.[Abstract/Free Full Text]
  17. Virág L, Marmer D.J, Szabó C. Crucial role of apopain in the peroxynitrite-induced apoptotic DNA fragmentation. Free Radic. Biol. Med. (1998) 25:1075–1082.[CrossRef][ISI][Medline]
  18. Yu S.W, Wang H, Poitras M.F, et al. Mediation of poly(ADP-Ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science (2002) 297:259–263.[Abstract/Free Full Text]
  19. Chiarugi A, Moskowitz M.A. Cell biology: PARP-1—a perpetrator of apoptotic cell death? Science (2002) 297:200–201.[Free Full Text]
  20. Ushmorov A, Ratter F, Lehmann V, et al. Nitric-oxide-induced apoptosis in human leukemic lines requires mitochondrial lipid degradation and cytochrome C release. Blood (1999) 93:2342–2352.[Abstract/Free Full Text]
  21. MacMillan-Crow L.A, Crow J.P, Thompson J.A. Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues. Biochemistry (1998) 37:1613–1622.[CrossRef][ISI][Medline]
  22. Francescutti D, Baldwin J, Lee L, Mutus B. Peroxynitrite modification of glutathione reductase: modeling studies and kinetic evidence suggest the modification of tyrosines at the glutathione disulfide binding site. Protein Eng. (1996) 9:189–194.[Abstract/Free Full Text]
  23. Zou M.H, Hou X.Y, Shi C.M, et al. Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent ser1179 phosphorylation of endothelial nitric oxide synthase. J. Biol. Chem. (2002) 277:32552–32557.[Abstract/Free Full Text]
  24. Oyadomari S, Takeda K, Takiguchi M, et al. Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway. Proc. Natl. Acad. Sci. U. S. A. (2001) 98:10845–10850.[Abstract/Free Full Text]
  25. Kato K, Yin H, Agata J, et al. Adrenomedullin gene delivery attenuates myocardial infarction and apoptosis after ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol. (2003) 285:H1506–H1514.[Abstract/Free Full Text]
  26. Zhao Z.Q, Velez D.A, Wang N.P, et al. Progressively developed myocardial apoptotic cell death during late phase of reperfusion. Apoptosis (2001) 6:279–290.[CrossRef][ISI][Medline]
  27. Hangaishi M, Nakajima H, Taguchi J, et al. Lecithinized Cu, Zn-superoxide dismutase limits the infarct size following ischemia–reperfusion injury in rat hearts in vivo. Biochem. Biophys. Res. Commun. (2001) 285:1220–1225.[CrossRef][ISI][Medline]
  28. Lee K.C, Hamel D.W, Kunbel S. Rodent model of renal ischemia and reperfusion injury: influence of body temperature, seasonal variation, tumor necrosis factor, endogenous and exogenous antioxidants. Methods Find Exp. Clin. Pharmacol. (1993) 15:153–159.[ISI][Medline]
  29. Kloner R.A, Das S, Poole W.K, et al. Seasonal variation of myocardial infarct size. Am. J. Cardiol. (2001) 88:1021–1024.[CrossRef][ISI][Medline]

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