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J Thorac Cardiovasc Surg 1995;110:1054-1062
© 1995 Mosby, Inc.
CARDIOPULMONARY BYPASS, |
Oakland, Calif.
Supported in part by a grant from the East-Bay Neonatology Foundation
Received for publication Dec. 9, 1994. Accepted for publication March 15, 1995. Address for reprints: William M. DeCampli, MD, PhD, Division of Cardiothoracic Surgery, Children'sHospital Oakland, 747 52nd St., Oakland, CA 94609
Abstract
The effects of aspirin and L-arginine (biological precursor of nitric oxide) on the production of hydroxyl radicals, cyclic guanosine monophosphate levels, vascular tone, and the recovery of the ischemic myocardium were investigated in isolated rat hearts subjected to ischemia and reperfusion. After 30 minutes of perfusion, hearts were arrested with St. Thomas' Hospital cardioplegic solution, global ischemia was induced at 37° C for 45 minutes, and the hearts were then reperfused at 37° C for 30 minutes. The percent change in recovery of pulse pressure and maximal change of this pressure with time were better in the group perfused with Krebs-Henseleit solution containing aspirin plus L-arginine (17%±23%, p = 0.001, and 10%±25%, p = 0.002, respectively) compared with these values in the control group perfused with Krebs-Henseleit solution alone (-7%±14% and -11%±16%, respectively). Coronary vascular resistance before and after ischemia were lower in the aspirin plus L-arginine group (0.19±0.03 dynes sec/cm 5, p = 0.001, and 0.23±0.04 dynes sec/cm 5, p = 0.0, respectively) compared with those of the control group (0.24±0.02 and 0.28±0.07 dynes 
sec/cm 5, respectively). Cyclicguanosine monophosphate levels increased from 22.5±6 pmol/100 mg of tissue in the control group to 37.1±8.9 pmol/100 mg (p = 0.002) in the aspirin plus L-arginine group. Adding N
-nitro-L-arginine methyl ester to the perfusion medium caused a deterioration in pulse pressure and maximal change of this pressure with time, a decrease in cyclic guanosine monophosphate, and a rise in coronary vascular resistance. The addition of L-arginine to the solution in the Krebs-Henseleit solution plus aspirin group increased the production of hydroxyl radicals from 0.32±0.18 nmol/gm per 3 minutes to 0.75±0.33 nmol/gm per 3 minutes (p = 0.03). Despite the association of nitric oxide with increased hydroxyl radical production, it appears that nitric oxide has an overall beneficial effect on the recovery of the ischemic myocardium. The synergism between aspirin and arginine may be caused in part by the scavenging of hydroxyl radicals. Alternatively, by inhibiting the prostaglandin pathway, aspirin may reduce the generation of superoxide anion, a free radical that inactivates nitric oxide. The prolonged half-life of nitric oxide may explain the increased levels of cyclic guanosine monophosphate seen in the group perfused with Krebs-Henseleit solution plus aspirin plus L-arginine. Aspirin and L-arginine, both readily available, may be useful adjuncts to clinical cardioplegia strategy. (J THORAC CARDIOVASC SURG 1995;110:1054-62)
A growing number of studies have implicated oxygen-derived free radicals as important in ischemia-induced myocardial reperfusion injury. 
1,2 Produced atreperfusion by a variety of mechanisms, 3-8 oxygen free radicals depress myocardial function and cause lipid peroxidation of membranes and scission of deoxyribonucleic acid and polypeptide chains both in vivo and in vitro. 9 Additionally, they have been implicated as contributors to the phenomenon of stunned myocardium. 2
Since the discovery of endogenous nitric oxide by Furchgott and Zawadski 10 in 1980, there have been conflicting reports as to the effect of nitric oxide on the recovery of ischemic myocardium. Some animal studies have shown a beneficial effect of nitric oxide through its regulation of coronary vascular tone. 11,12 However, other investigators have recently suggested that nitric oxide (NO) may cause an adverse effect on myocardial recovery by increasing the production of hydroxyl radicals (OH) via the following pathway 13,14:
Direct cellular, tissue, or whole organ 15 measurements of hydroxyl radical production by nitric oxide degeneration have heretofore not been made. Detection of oxygen free radicals is complicated by their highly reactive and transient nature. Recently, however, acetylsalicylic acid (aspirin) has been used as a "trap molecule" to detect and quantify hydroxyl radical production in biochemical reactions. 16 Given this function, we thought that aspirin might therefore also serve as a scavenger of hydroxyl radical in biologic systems. These complementary properties of aspirin suggest that it might be used to help uncover the effect that nitric oxide has on myocardial recovery after ischemia.
In the present study, we designed a whole-organ (isolated rat heart) model that allows us to investigate the following hypotheses: (1) exogenously administered nitric oxide increases the production of hydroxyl radicals in the ischemic myocardium, (2) despite this increased production of hydroxyl, nitric oxide does not have a significantly deleterious effect during reperfusion after ischemia, and (3) aspirin acts synergistically with nitric oxide to substantially improve myocardial recovery after ischemia.
MATERIALS AND METHODS
Heart preparation
Male Sprague-Dawley rats (250 to 300 gm) were given anticoagulation treatment by injection of 1000 USP units of heparin sodium per kilogram and anesthetized by intraperitoneal injection of a 30 mg/kg dose of pentobarbital. The hearts were rapidly excised and immersed in ice-cold perfusion buffer. Within 1 minute of excision, the hearts were perfused in a retrograde fashion through the aorta according to the Langendorff method with a modified Krebs-Henseleit solution (composition of the Krebs-Henseleit solution in millimoles per liter: NaCl 118.5, NaHCO3 25.0, KCl 4.9, KH2PO4 1.2, MgSO4 1.2, CaCl2, 1.4, and glucose 11.1; pH 7.4). All solutions, including the cardioplegic solution were prepared fresh daily, gassed with 95% O2 plus 5% CO2, warmed to 37° C, and passed through a 0.45 µm filter before use. Each heart beat spontaneously and none was paced.
All animals in this study were treated humanely in accordance 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 No. 86-23, revised 1985), as well as the requirements of the Research Animal Review Committee of the Children's Hospital Oakland Research Institute, which reviewed and approved the experimental protocol.
Experimental protocol
L-Arginine (biologic precursor of nitric oxide) was used to stimulate the production of nitric oxide in the myocardium. N-nitro-L-arginine methyl ester (L-NAME) was used to inhibit basal nitric oxide production. Aspirin served as a scavenger and a trap molecule for hydroxyl radical production. By design, the experimental protocol was set to simulate conditions encountered in a typical cardiac operation. Therefore our hearts were subjected to conditions that inflicted on them a minimal to moderate amount of damage. Hearts were perfused at 37° C for a 35-minute period at a pressure of about 70 mm Hg with one of the five perfusion media listed in Table I. The perfusion was then stopped, and St. Thomas' Hospital cardioplegic solution (composition of St. Thomas' Hospital solution in millimoles per liter: NaCl 110.0, NaHCO3 10.0, KCl 16.0, CaCl2 1.2, MgCl2 16.0, and glucose 11.1; pH 7.8) was infused into the aortic root at 45 mm Hg for 1 minute. The hearts were kept arrested for 45 minutes, a period simulating the typical duration of cardioplegic arrest during heart operations. During this time the hearts were immersed in St. Thomas' Hospital solution (at 37° C) to prevent oxidative reactions from taking place on the surface of the hearts. Rat hearts are known to recover well from a 45-minute period of hypothermic (5° to 15° C) ischemia. To inflict measurable damage during the ischemic period, therefore, the hearts were maintained at 37° C. This was followed by another 35 minutes of aerobic reperfusion.
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Mechanical function quantities were expressed as percent fractional change (%
), whereby % of a variable X = ([value of X after ischemia minus value of X before ischemia]/value of X before ischemia) x100.
Measurement of hydroxyl radical
Aspirin is known to react with hydroxyl radical to form catechol and 2,3- and 2,5-dihydroxybenzoic acid (DHBA) as shown in Fig. 2. These reaction products were extracted from the coronary sinus effluent and quantified with the use of high-performance liquid chromatography (HPLC) as described by Floyd and Wong 16 and modified by Onodera and Ashraf. 17
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In brief, 2 ml of the total coronary sinus effluent volume collected in the first 3 minutes after reperfusion was treated with 50 µl of 100 µmol/L 2,4-DHBA (an internal standard) and 100 µl of 1N HCl. The solution was then extracted with 6 ml of HPLC-grade diethyl ether, centrifuged at room temperature. The extraction and centrifugation steps were repeated three times. The diethyl ether layers were combined and evaporated to dryness in a water bath at 45° C. The residue was then dissolved in 60 µl of 1N HCl and 40 µl of mobile phase described later. Forty microliters of this solution was injected into the HPLC apparatus. Separation was made on a Bio-sil C18 HL90-5S column, 250 x 4.6, from Bio-Rad Laboratories (Richmond, Calif.), with an attached precolumn. The effluent was 80% 0.03 mol/L citric acid, 0.03 mol/L acetic acid buffer, pH 3.6, and 20% methanol at a flow rate of 1.0 ml/min. The ultraviolet detection was set at a wavelength of 315 nm.
Cyclic guanosine monophosphate assay
The concentration of cyclic guanosine monophosphate (cGMP) was assayed as an indicator of nitric oxide activation of guanylate cyclase. 18,19 Frozen heart samples were weighted and homogenized in cold 6% (weight/volume) trichloroacetic acid at 2° to 8° C to give a 10% (weight/volume) homogenate. After centrifugation at 2000 g for 15 minutes at 4° C, the supernatant was collected and washed four times with four volumes of water-saturated diethyl ether. The aqueous extract remaining was then lyophilized. cGMP contents of the extracted supernatant were determined with the use of Amersham enzyme immunoassay kits (Arlington Heights, Ill.). Concentration of cGMP was expressed as picomoles per 100 mg of tissue.
Chemicals
All chemicals were obtained from Sigma Chemical Co., St. Louis, Missouri, or VWR, Boston, Massachusetts. Diethyl ether and methanol were of HPLC grade. Distilled deionized water was used throughout.
Statistical analysis
All data are expressed as mean plus or minus the standard deviation. Statistical analysis was done comparing results in control versus intervention groups by analysis of variance followed by the Student's t test with the Bonferroni correction. The p value is listed for each measurement.
RESULTS
Mechanical function
Hearts in the control group, perfused with Krebs-Henseleit solution without any additional agent, had values of % (P) and % (dP/dt) of -7 ± 14 and -11 ± 16, respectively. The addition of aspirin or L-arginine alone to the perfusion medium improved the values of % (P) and % (dP/dt) modestly but not to statistically significant levels. When the perfusion medium was changed to Krebs-Henseleit solution plus aspirin plus L-arginine, % (P) and % (dP/dt) values improved significantly to +17 ± 23 (p = 0.001) and +10 ± 25 (p= 0.002), respectively, as shown in Fig. 3. Conversely, the addition of L-NAME to the Krebs-Henseleit solution caused a significant decrease in both P and dP/dt values before and after ischemia when compared with values in control hearts.
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Coronary vascular resistance
Ischemia caused an increase in the resistance in all groups studied (Fig. 4). Control values of coronary vascular resistance before and after-ischemia were 0.24 ± 0.02 and 0.28 ± 0.07 dynes sec/cm 5 , respectively. In the groups perfused with L-arginine or aspirin alone coronary vascular resistance was modestly lower than that in control hearts but not to statistically significant levels. In the group perfused with Krebs-Henseleit solution plus aspirin plus L-arginine, coronary vascular resistance values before and after ischemia were 0.19 ± 0.03 (p = 0.001) and 0.23 ± 0.04 dynes sec/cm 5 (p = 0.01), respectively (Fig. 4). As expected, by decreasing basal nitric oxide concentration, L-NAME caused a dramatic increase in both preischemic and postischemic coronary vascular resistance to 0.39 ± 0.03 (p = 0.3 x 10 -5 ) and 0.42 ± 0.07dynes sec/cm 5 (p = 0.002), respectively, when compared with values in control hearts (Fig. 4).
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Nitric oxide is a labile molecule synthesized from the amino acid L-arginine via the enzyme nitric oxide synthase and is equivalent to endothelium-derived relaxing factor). 20 Once released, nitric oxide is believed to carry on intracellular messenger functions leading to the activation of guanylate cyclase by a heme-dependent mechanism. Activated guanylate cyclase causes an increase in cGMP concentration. 18,19 Increased levels of cGMP lead, in turn, to vasorelaxation and better tissue perfusion. 11,12 Nitric oxide is continuously released from the endothelium 21 and appears to play other important roles, such as mediation of inflammation 22 and thrombosis 23 and inhibition of platelet adhesion and aggregation. 24 In addition to its vasodilatory properties, nitric oxide has been implicated as an independent source for the production of the deleterious hydroxyl radicals in the ischemic myocardium. 13,14
Hydroxyl is a short-lived radical, produced in the myocardium during different pathologic conditions, including ischemia/reperfusion. Although other radicals are also born in the ischemia/reperfusion process (notably superoxide anion and hydrogen peroxide) these are not considered to be as injurious to the myocardium. For example, superoxide anion reacts slowly under physiologic conditions and hydrogen peroxide is a relatively weak oxidant. 25,26 Hydroxyl radical, on the other hand, is highly reactive and has been shown to cause membrane damage, lipid peroxidation, and deoxyribonucleic acid breakage. 9 Studies have demonstrated that by preventing the formation of hydroxyl radicals with the use of allopurinol, an inhibitor of xanthine oxidase, 27 or deferoxamine, an inhibitor of iron-catalyzed hydroxyl radical generation, 28,29 one can improve myocardial recovery after ischemia. Because of its short half-life, the hydroxyl radical is extremely difficult to measure in biologic systems.
In an attempt to investigate whether nitric oxide represents a significant source of these hydroxyl radicals in the myocardium, we used a novel and specific chromatographic technique (HPLC) developed by Floyd and Wong 16 and modified by Onodera and Ashraf, 17 which permitted us to compare the quantities of hydroxyl radical released into the coronary effluent in the presence or absence of L-arginine. Our study has provided the first direct measurement of increased hydroxyl radical production, in an isolated perfused heart, as a result of enhancing nitric oxide production in the myocardium with the use of L-arginine. These findings are complementary to the work of Beckman and Freeman, 13 who found that nitric oxide leads to increased production of hydroxyl radical in chemical reactions. Hydroxyl radical release peaked in the first 3 minutes after reperfusion. This time course has been reported in other studies that investigated other radical species and used other techniques. 30,31 By restricting our perfusion media in this study to crystalloid, we eliminated the contribution of polymorphonuclear leukocytes, which can produce a variety of toxic species such as NH2Cl, HOCl, and hydroxyl radical. This was done to isolate the contribution of the endothelial cells and myocytes to oxidative damage.
The current experiment demonstrates a modest improvement of P and dP/dt and a slight decrease in coronary vascular resistance in an isolated rat heart perfused with L-arginine. One possible interpretation of these findings is that nitric oxide does not affect myocardial function. We tested this hypothesis by perfusing hearts with a nitric oxide inhibitor, L-NAME. We noted a significant increase in coronary vascular resistance and a dramatic decrease in mechanical function, thus implying that the presence of nitric oxide, at least at a baseline level, is important for the maintenance of myocardial function. This finding is also supported by Amrani and Yacoub, 32 who found that inhibition of the release of basal nitric oxide was associated with a significant decrease in cardiac output and that L-NAME itself was not toxic to myocardial tissue.
Another possible interpretation of our results is that the addition of exogenous nitric oxide to the perfusate results in both deleterious and beneficial effects in roughly comparable proportions. Thus the harmful effect of the increase in production of hydroxyl radicals might be roughly balanced by the salutory effects of the decrease in coronary vascular resistance and presumed improvement in tissue perfusion. To test this supposition, we studied myocardial recovery in the presence of both exogenous nitric oxide and a known free radical scavenger, aspirin. Both substances added simultaneously to the perfusate resulted in a significant increase in postischemic recovery of myocardial mechanical function and a decrease in coronary vascular resistance. By scavenging some of the hydroxyl radicals, aspirin may have unmasked the salutory effect of nitric oxide on vascular tone and allowed better tissue perfusion and, eventually, better myocardial recovery after ischemia. The importance of the presence of nitric oxide in this synergism is further demonstrated by the fact that improvement in function was only modest when aspirin alone was added to the perfusate, that is, hydroxyl radicals were scavenged without the additional benefit of enhanced nitric oxide.
Although the direct free radical scavenging activity of aspirin may well have played a significant role in the synergism displayed in this study between aspirin and L-arginine, there is another possible mechanism by which aspirin might have acted. By inhibiting the cyclooxygenase pathway, aspirin may affect tissue levels of nitric oxide. Cyclooxygenase catalyzes the oxygenation of arachidonic acid to prostaglandin G2 and hydroperoxidase converts this to prostaglandin H2. The peroxidase-catalyzed conversion of prostaglandin G2 to prostaglandin H2 yields superoxide anion, 33,34 a well-known scavenger of nitricoxide. 35,36 It is, therefore, possible that aspirin, by inhibiting the cyclooxygenase pathway, reduces tissue production of .O2- , leading to an increased half-life of nitric oxide (Fig. 7). The increase half-life of nitric oxide will subsequently increase cGMP production by activating guanylate cyclase. 18,19 Indeed, we found augmented levels of cGMP in the group perfused with aspirin plus L-arginine, whereas L-NAME, by inhibiting basal nitric oxide production, significantly decreased the levels of this cyclic nucleotide in tissue. Thus the cGMP data lend support to the proposed mechanism of interaction between the cyclooxygenase and nitric oxide pathways. This mechanism is further supported by the work of Johnson and Lefer, 37 who found that nitric oxide acted synergistically with a scavenger of superoxide anion, superoxide dismutase, to confer significant protection on the myocardium subjected to ischemia and reperfusion injury.
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381O2 has been shown to cause amino acid photooxidation, damage cardiac sarcoplasmic reticulum function, 39 and cause arrythmias. 40 Therelative role that .OH and 1O2 play in ischemic/reperfusion injury remains unclear. Yet another limitation of this study is the addition of nitric oxide and aspirin to the perfusion in the preischemic and the reperfusion intervals. This design does not permit one to tell specifically what, if any, preconditioning effect these agents might have had to explain the postischemic improvement in recovery. Additionally, the clinical use of aspirin might be limited by its tendency to increase bleeding because of platelet dysfunction. This problem could be obviated, however, by isolating the coronary perfusion circuit from the systemic circuit during early reperfusion, a method that is already used often in the clinical setting.
In conclusion, we have demonstrated that nitric oxide leads to an increased generation of hydroxyl radicals immediately after the start of reperfusion in the isolated perfused rat heart model. Despite increasing the production of hydroxyl radicals, exogenous nitric oxide does not appear to be detrimental to myocardial recovery after ischemia. Furthermore, we found a significant synergism between nitric oxide and aspirin in terms of improvement of coronary flow and myocardial recovery after ischemia/reperfusion injury. Aspirin appears to have acted in part as a scavenger of hydroxyl radicals. Moreover, aspirin, by inhibiting the endoperoxide (prostaglandin) pathway, reduces .O2- production, decreases theproduction of nitric oxide–derived hydroxyl radicals, and prolongs the half-life of nitric oxide. This study provides a biochemical basis for the possible utility of two readily available agents in clinical myocardial protection strategies.
Footnotes
From the Division of Cardiothorac Surgery, Children's Hospital Oakland, c Children's Hospital Oakland Research Institute, a and the Department of General Surgery, University of California, Davis-East Bay, b Oakland, Calf. 
References
This article has been cited by other articles:
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 R. Schulz, M. Kelm, and G. Heusch Nitric oxide in myocardial ischemia/reperfusion injury Cardiovasc Res, February 15, 2004; 61(3): 402 - 413. [Abstract] [Full Text] [PDF]
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J. E. Jordan, Z.-Q. Zhao, and J. Vinten-Johansen The role of neutrophils in myocardial ischemia-reperfusion injury Cardiovasc Res, September 1, 1999; 43(4): 860 - 878. [Abstract] [Full Text] [PDF]
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p < 0.3 x 10-5; 
