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Ann Thorac Surg 1997;64:1656-1659
© 1997 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Sodium Nitroprusside Exacerbates Myocardial Ischemia–Reperfusion Injury

Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health Sciences Center, Charlottesville, Virginia

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. The role of nitric oxide in myocardial ischemia–reperfusion is controversial. Although many studies claim that nitric oxide ameliorates reperfusion injury, others suggest that it exacerbates such injury, possibly through peroxynitrite production. These discordant results may be attributable to a dose-dependent phenomenon.

Methods. Isolated rabbit hearts sustained sequential periods of blood perfusion (20 minutes), warm ischemia (30 minutes), and reperfusion (20 minutes). During reperfusion, four groups underwent intracoronary infusion of saline solution (n = 6), or the nitric oxide donor sodium nitroprusside (100 nm/min [SNP100, n = 6], 1 nmol • L^-1/min^-1 [SNP1, n = 6], or 0.01 nmol • L^-1 • min^-1 [SNP0.01]). Left ventricular-developed pressure and oxygen consumption were measured after preischemic perfusion and reperfusion. Levels of myocardial nitrotyrosine, a marker for peroxynitrite, were measured after reperfusion with an immunoradiochemical assay.

Results. Postischemic-developed pressure and myocardial oxygen consumption were significantly higher in the saline group than all nitroprusside-reperfused groups (p < 0.01 for both parameters). However, there were no differences in either parameter between SNP100, SNP1, or SNP0.01. Nitrotyrosine levels were similar among the four groups (p = 0.43).

Conclusions. Nitroprusside exacerbates myocardial ischemia–reperfusion injury over a wide range of doses, although the mechanism does not appear to be mediated by peroxynitrite.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
See also page 1659.

The role of nitric oxide (NO) in myocardial ischemia–reperfusion (IR) injury remains controversial. Many previous studies have demonstrated that NO administered as either the endogenous precursor L-arginine or as an exogenous NO-donating agent mitigates damage incurred during reperfusion of ischemic myocardium [1–4]. Proposed mechanisms for the beneficial effects of this substance include decreased leukocyte accumulation [5], inhibition of platelet aggregation [6], and neutralization of superoxide radicals [7]. Conversely, a number of recent investigations have proposed that NO may actually be deleterious to the myocardium [8–14]. It has been postulated that production of peroxynitrite, the free radical product of a chemical reaction between NO and superoxide anion, may contribute to these adverse effects [8, 13, 14]. We developed the hypothesis that the discordant results of previous studies are attributable to a dose-dependent phenomenon in which physiologic intracoronary NO concentrations ameliorate myocardial IR injury, whereas higher concentrations exacerbate such injury by virtue of production of toxic peroxynitrite levels. Using a blood-perfused isolated rabbit heart model, we tested this hypothesis by studying the effects of different doses of the NO donor sodium nitroprusside (SNP) administered during reperfusion after global normothermic myocardial ischemia.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Adult New Zealand white rabbits were used for all protocols described herein. The Animal Review Committee of the University of Virginia reviewed and approved the protocols for this study. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 86-23, revised 1996).

Experimental Design
An isolated heart apparatus was used for ex vivo blood perfusion. As recently described in detail [15], this apparatus relied on a support rabbit for the continuous provision of fresh arterial blood perfusate. Under intramuscular xylazine and ketamine anesthesia, study rabbits (2.8 to 3.0 kg) underwent tracheostomy and volume ventilation (12 mL/kg) with 100% oxygen. A median sternotomy was performed, followed by isolation of the ascending aorta and systemic heparinization (2,000 units intravenously). A glass cannula filled with saline solution was secured into the ascending aorta, the heart was rapidly excised, and was blood-perfused immediately on the isolated heart apparatus as described previously [15]. All hearts sustained 20 minutes of baseline perfusion, after which preischemic left ventricular developed pressure (LVP), myocardial oxygen consumption (MVO₂), and coronary flow (CF) were measured exactly as described previously [15]. A 30-minute interval of global normothermic (37°C) ischemia ensued. This was followed by a 20-minute period of reperfusion.

Four groups of hearts were studied. Three groups underwent reperfusion in the presence of one of three different doses of SNP, and a fourth group was administered saline vehicle only. Commencing at the onset of reflow and terminating at the 20-minute mark of reperfusion, one of three different doses of SNP (Sigma, St. Louis, MO) was infused at a constant rate directly into the coronary circulation through a side port in the aortic cannula. Three successive doses of SNP were dissolved in saline solution to yield concentrations of 0.002, 2, and 200 nmol/L. Each of these solutions were administered at a rate of 0.5 mL/min to achieve final intracoronary SNP infusion rates of 0.01 (SNP0.01, n = 6), 1 (SNP1, n = 6), or 100 nmol • L^-1 • min^-1 (SNP100, n = 6). These infusion rates were based on a previous study in which the concentration of NO in the coronary circulation under physiologic conditions was estimated to be 0.1 to 1 nmol/L [16]. It is assumed that an infusion rate of 1 mole of SNP per minute produces 1 mole of NO per minute. Hence, assuming coronary endothelial cells produce negligible amounts of NO after 30 minutes of warm ischemia, an SNP infusion rate of 1 nmol • L^-1 • min^-1 was considered "physiologic," whereas rates of 0.01 and 100 nmol • L^-1 • min^-1 were considered to be "subphysiologic" and "supraphysiologic," respectively. As mentioned, a group of saline control hearts (SC, n = 6) received an intracoronary infusion of saline solution only (0.5 mL/min).

After 20 minutes of reperfusion, postischemic LVP, MVO₂, and CF were measured and compared with preischemic values. At the conclusion of the reperfusion period, a blood sample (5 mL) was withdrawn from the right atrium and submitted to an outside laboratory (Quest Diagnostics, San Diego, CA) for colorimetric analysis of coronary sinus thiocyanate levels. This was followed immediately by rapid excision of the atria and great vessels, and quick-freezing of the remaining ventricular tissue by immersion in liquid nitrogen. This tissue was then stored at -80°C for a subsequent molecular analysis.

Immunoradiochemical Assay for Nitrotyrosine
Nitrotyrosine, a molecule formed by peroxynitrite-mediated nitration of tyrosine residues in protein, has been used as a marker to quantitate levels of peroxynitrite in biologic tissues [17]. Frozen hearts were removed from storage at -80°C and broken into small pieces over dry ice with a mortar and pestel. Approximately 1 g of ventricular tissue was retained for performance of the assay and the remainder was returned to -80°C for storage. The tissue was homogenized for 40 seconds in 7 mL of phosphate-buffered saline solution, pH 7.4, containing 0.5 mmol/L phenylmethylsulfonyl fluoride (Sigma Chemical Co, St. Louis, MO) using a polytron homogenizer (Brinkman model PT2000). Nitrotyrosine levels were quantitated and reported as nanograms per milligram of protein using a solid phase immunoradiochemical assay as previously described [18].

Statistical Analyses
All results are expressed as the mean ± standard error of the mean. All functional, metabolic, and molecular data were analyzed for between-group differences using analysis of variance and the post-hoc test of Tukey's multiple comparisons. Significant differences were identified by a p value of less than 0.05. All analyses were performed using Statistica software (Statsoft, Tulsa, OK).

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Functional and Metabolic Data
Table 1 presents mean LVP data of isolated hearts after 20 minutes of baseline preischemic perfusion (preischemic LVP) and after 20 minutes of reperfusion with saline or one of the three doses of SNP (postischemic LVP). Also presented is the percent recovery of ventricular function (%LVP recovery) for each group, which was calculated as follows: %LVP recovery = postischemic LVP/preischemic LVP x 100%. Although the mean baseline LVP before the initiation of ischemia was similar between the four groups, postischemic LVP and %LVP recovery were significantly lower in the three groups undergoing reperfusion with SNP compared with SC (p < 0.01 for both parameters).

Nitrotyrosine Levels in Ischemic-Reperfused Ventricular Myocardium
Table 4 presents the results of an immunoradiochemical assay for nitrotyrosine in ventricular myocardium after 20 minutes of reperfusion in the presence of one of three doses of SNP or saline vehicle. As demonstrated, mean nitrotyrosine levels ranged from 23.7 to 38.0 ng/mg ventricular protein, but there were no statistically significant differences between the four groups (p = 0.43 by analysis of variance).

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

As mentioned previously, the three doses of SNP used in this study were selected as a result of the work of Kelm and Schrader [16], who estimated that the physiologic coronary concentration of NO is approximately 0.1 to 1 nmol/L. As it has been established that reperfusion injury reduces basal NO production by the endothelium [19], postischemic coronary NO levels would be expected to be less than 0.1 to 1 nmol/L in the absence of NO replacement (eg, in the form of L-arginine or an NO donor). Thus, for the purposes of the current study, an intracoronary SNP infusion rate of 1 nmol • L^-1 • min^-1 would place the postischemic coronary concentration of NO within the physiologic range, whereas 0.01 and 100 nmol • L^-1 • min^-1 would be expected to yield subphysiologic and supraphysiologic coronary NO concentrations, respectively. Examination of our postischemic CF data support this notion. For example, the mean postischemic CF in the group receiving an SNP infusion of 100 nmol • L^-1 • min^-1 was higher than SC and significantly elevated over SNP0.01 and SNP1, an expected finding in hearts receiving a supraphysiologic (ie, vasodilatory) dose of NO. We speculate that the reduced postischemic CF in SNP0.01 and SNP1 may be attributable to subvasodilatory doses of exogenous NO causing increased reperfusion injury to the coronary endothelium, with a resultant impairment in endogenous basal NO release.

The major conclusion of our study, that infusion of an NO donor compounds myocardial IR injury, is consistent with the results of previous investigations in which NO was found to be deleterious to postischemic myocardial recovery [8–10]. Although the exact mechanism underlying the detrimental effects of NO remains to be elucidated, Matheis and colleagues [8] have proposed that peroxynitrite formation may be involved. Peroxynitrite is a strong, long-lived oxidant produced by reaction of NO with superoxide radical. This free radical species has both direct and indirect cytotoxic properties, the latter by virtue of its decomposition into the potent hydroxyl radical [13, 14]. Clearly, our data do not support the contention that peroxynitrite toxicity is the mechanism through which NO exerts its deleterious effects on postischemic myocardium, as nitrotyrosine levels were comparable among all four study groups. Alternative explanations for the detrimental effects of NO on postischemic myocardial recovery may involve production of the negative inotrope guanosine 3`,5`-cyclic monophosphate [11, 12] or derangements in expression of the inducible or endothelial isoforms of nitric oxide synthase. The latter proposal is currently under investigation in our laboratory.

In summary, we have demonstrated that intracoronary infusion of the NO donor SNP exacerbates myocardial IR injury over a wide range of doses. The mechanism underlying the pathophysiologic role of NO in myocardial IR injury, such as increased cyclic guanosine monophosphate production or altered NO synthase metabolism, certainly merits further investigation.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We express our appreciation to Anthony J. Herring for his invaluable technical assistance. In addition, we owe a debt of gratitude to Dr Harry Ischiropoulos for his generous efforts in performing the nitrotyrosine assays.

This work was supported by a National Research Service Award (Fellowship No. 1 F32 HL09065-01A2) granted by the National Heart, Lung, and Blood Institute of the National Institutes of Health.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Presented at the Thirty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Feb 3–5, 1997.

Address reprint requests to Dr Kron, Department of Surgery, University of Virginia Health Sciences Center, Box 310, Charlottesville, VA 22908.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Curtis G. Tribble Irving L. Kron Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cope, J. T. Articles by Kron, I. L. Search for Related Content PubMed PubMed Citation Articles by Cope, J. T. Articles by Kron, I. L. Related Collections Related Article