- Email: [email protected]
reperfusion
L-Arginine Reduces Endothelial Inflammation and Myocardial Stunning During Ischemia/Reperfusion Daniel T. Engelman, MD, Masazumi Watanabe, MD, Nilanjana Maulik, PhD, Gerald A. Cordis, MS, Richard M. Engelman, MD, John A. Rousou, MD, Joseph E. Flack III, MD, David W. Deaton, MD, and Dipak K. Das, PhD Department of Surgery, University of Connecticut School of Medicine, Farmington, Connecticut, and Baystate Medical Center, Springfield, Massachusetts
Background. This study evaluated whether the nitric oxide precursor L-arginine could reduce ischemia/reperfusion injury by preventing leukocyte-endothelial interactions. Methods. Normothermic regional ischemia was induced in the open-chest working pig heart for 30 minutes followed by 90 minutes of reperfusion. A preischemic 10-minute intravenous infusion of 4 mg- kg -1 • min -1 of L-arginine (n = 12) was compared with 12 control pigs. Nitric oxide release was measured from the coronary sinus using an amperometric probe. Left ventricular function, malonaldehyde, creatine kinase, myocardial oxygen extraction, and the soluble adhesion molecules (intracellular adhesion molecule-I, endothelial leukocyte adhesion molecule-I, and vascular cell adhesion molecule-I) were measured. Results. Nitric oxide release was significantly reduced from baseline throughout ischemia/reperfusion only in
the control group. Systolic and diastolic function, and myocardial oxygen extraction were also significantly decreased during early reperfusion in the control compared with the L-arginine group. Peak creatine kinase release was not significantly different between groups. The incidence of ventricular fibrillation, malonaldehyde release, and soluble intracellular adhesion molecule-I, endothelial leukocyte adhesion molecule-I, and vascular cell adhesion molecule-1 were each significantly decreased during reperfusion in the r-arginine group. Conclusions. t-Arginine reduced lipid peroxidation, plasma levels of soluble adhesion molecules, myocardial stunning, and arrhythmias. These results support an excessive endothelial injury/inflammatory response after regional ischemia/reperfusion that can be ameliorated through augmented nitric oxide.
eperfusion of ischemic myocardium has been reported to cause a rapid degeneration of endothelial function, characterized by a decreased release of nitric oxide (NO) in response to endothelium-dependent vasodilators [1]. Nitric oxide, synonymous with endotheliurnderived relaxing factor, is a naturally occurring, continuously released, vasoactive agent that controls coronary vascular tone [2]. It is synthesized by the vascular endothelium through the conversion of L-arginine to L-citrulline by the enzyme NO synthase [3]. Nitric oxide donors given during reperfusion have been shown to preserve coronary artery ring vasorelaxation (an indirect measure of NO synthesis) and reduce myocardial injury associated with ischemia and reperfusion [4]. In the vascular system, NO has been shown to be an endogenous inhibitor of leukocyte chemotaxis, adherence, and activation [5]. In addition, NO may inactivate superoxide free radicals generated by leukocytes [6]. The present study was designed to assess the role of endothelial function/activation after regional ischemia and
reperfusion in L-arginine-supplemented in situ bloodperfused porcine hearts using continuous measurements of myocardial function and coronary sinus NO release. To further delineate the effects of r-arginine on myocardial energetics and cellular necrosis, malonaldehyde release, an indirect marker of free radical-mediated lipid peroxidation, myocardial oxygen extraction, and creatine kinase release were measured during ischemia and reperfusion. In addition, the soluble endothelial "proadhesive'" molecules vascular cell adhesion molecule-1 (sVCAM-1), endothelial leukocyte adhesion molecule-1 (sE-selectin), and intercellular adhesion molecule-1 (sICAM-1) were each quantified from coronary sinus blood.
R
Accepted for publication June 13, 1995. Address reprint requests to Dr Daniel T. Engelman, Surgical Research Center, University of Connecticut School of Medicine, 263 Farmington Ave, Farmington,CT 06030-1110. © 1995 by The Society of Thoracic Surgeons
(Ann Thorac Surg 1995;60:1275-81)
Material and M e t h o d s 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 85-23, revised 1985). 0003-4975/95/$9.50 SSDI 0003-4975(95)00614-Q
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Surgical Preparation Yorkshire pigs of either sex weighing 20 to 25 kg were tranquilized with ketamine (50 mg/kg), anesthetized with sodium pentobarbital (25 mg/kg), and placed on a mechanical ventilator. The electrocardiogram was continuously recorded. Cannulas were placed in the femoral vein and artery. The chest was then opened with a median sternotomy and the pericardium was suspended in a pericardial cradle. The azygous and hemiazygos veins were ligated, and the inferior vena cava was snared with 3-ram-wide tape (Umbilical Tape; Ethicon, Inc, Somerville, NJ). Heparin sodium (250 U/kg) was then administered systematically. Sonometric dimension crystals (diameter, 6 ram), which were made of 3 MHz piezoelectric crystals (Triton Technologies, Inc, San Diego, CA) were placed at the endocardial surface across the anteroposterior minor axis, septal-free wall minor axis, and base-apex major axis of the left ventricle. The anteroposterior crystals were placed adjacent to the anterior and posterior descending coronary arteries. The septal-free crystals were located one-half of the distance from the apex to the base. The base crystal was placed into the left ventricle adjacent to the origin of the left circumflex coronary artery and the apex crystal was placed into the left ventricular apex. Techniques for crystal placement have been previously described in detail by Freeman [7]. A pair of sonometric dimension crystals (diameter, 2.5 mm), which were made of 5 MHz piezoelectric crystals (Triton Technologies) were placed 1 cm apart in the left anterior descending coronary artery (LAD) distribution epicardium of the left ventricle. A 5F micromanometer-tipped catheter (Millar Instruments, Inc, Houston, TX) was inserted through the left ventricular apex for pressure measurements. A catheter was then placed into the coronary sinus. This catheter drained coronary sinus blood past an in-line NO probe and into a 37°C chamber that was connected via a roller pump to the femoral vein catheter. The experimental preparation is illustrated in Figure 1. The heart rate was kept at 120 beats/min using an electronic pacer (Phipps & Bird, Inc, Richmond, VA) when necessary and continuous monitoring of the electrocardiogram. Bretylium tosylate (2 mg/kg) was given as a bolus injection for antiarrhythrnic prophylaxis before ischemia and reperfusion, and as required for ventricular ectopy. No other ionotropes or antiarrhythmics were administered during the experiment. A defibrillator (Mennen-Greatbatch, Clerance, NY) was used as necessary for ventricular fibrillation. Sedation was maintained with inhalational isoflurane (0.5% to 1.5%; Ohmeda, Liberty Corner, NJ). Arterial blood gases were monitored, and the ventilator was adjusted to maintain a pH of 7.35 to 7.45, an oxygen tension of more than 100 mm Hg, and a carbon dioxide tension of 35 to 50 mm Hg. Mean arterial pressure was kept above 65 mm Hg by infusion of lactated Ringer's solution. Postmortem examination verified crystal placement.
~d |
1"9 cer
Fig 1. Experimental heart preparation. ( A o aorta; I V C = inferior vena cava; L A = left atrium; P A = pulmonary artery; RA = right atrium; S V C = superior vena cava.)
Experimental Protocol Baseline measurements were made during steady-state contractions after instrumentation and stabilization. Control animals (CON; n = 12) then received an intravenous infusion of lactated Ringer's solution for 10 minutes. Treated animals (ARG; n = 12) received an intravenous infusion of L-arginine (4 mg" kg -1" min -1) (Sigma Chemical, St. Louis, MO) for 10 minutes. The LAD was then ligated for 30 minutes just proximal to the origin of the first diagonal branch. The hearts were then reperfused for 90 minutes. In 6 ARG pigs and 6 CON pigs, functional data was obtained before ischemia (baseline), after 15 and 30 minutes of ischemia, and after 10, 20, 30, 60, and 90 minutes of reperfusion. Nitric oxide release was measured continuously throughout the experimental protocol. Three-milliliter blood samples were taken from the coronary sinus for creatine kinase and malonaldehyde measurements at baseline, after 30 minutes of ischemia, and 3, 5, 15, 30, 60, and 90 minutes of reperfusion. Myocardial oxygen extraction was measured from simultaneously drawn coronary sinus and femoral artery blood samples at baseline, after 30 minutes of ischemia, and after 30 minutes of reperfusion. In a separate group of 6 ARG and 6 CON pigs, 3-mL blood samples were taken from the coronary sinus for soluble adhesion molecule measurements at baseline, after 30 minutes of ischemia, and at 3, 5, 15, 30, 60, and 90 minutes of reperfusion. All blood was immediately centrifuged at 1,000 g for 10 minutes and the plasma was stored in aliquots at -20°C for subsequent analysis.
Ann Thorac Surg 1995;60:1275-81
Measurement of Myocardial Function The hemodynamic variables continuously recorded were left ventricular pressure, left ventricular dimensions, and LAD regional segment length. These data were digitized and recorded in real-time with a 12-bit AD converter sampling at 200 Hz using the Cordat II Data Acquisition, Analysis, and Presentation System (Data Integrated Scientific Systems, Pinckney, MI; Triton Technologies, Inc, San Diego, CA). The digitized data was later analyzed using the CV AutoReport Cardiovascular Data Analysis Program (Scitelligence, Inc, Brighton, MI). Left ventricular volume (V) was modeled as two half-ellipsoids by the equation: V = ~r/6 × ASL, where A, S, and L are the anteroposterior, septal-free, and base-apex dimensions [8]. The first derivative of left ventricular pressure was calculated as a polynomial approximation from the digital left ventricular pressure signal. End-diastolic volume was defined as the left ventricular volume at the first positive derivative of left ventricular pressure. Left ventricular stroke work (SW) was defined as the integral of left ventricular pressure, P, and volume, V, of the cardiac cycle by the equation SW = fPdV. The linear regression analysis was performed on the stroke work-end-diastolic volume relationship, to generate the preload recruitable stroke work with slope, M**, and x-axis intercept, V o. Similar data was generated from regional segmentlength data by substituting end-diastolic LAD segmentlength for end-diastolic left ventricular volume. The "stiffness" coefficient (]3) was derived from exponential modeling of the end-diastolic pressure (EDP)end-diastolic volume (EDV) relationship by the equation EDP = a × e(~ x EDV). The stiffness coefficient is the inverse of compliance [9]. Similar data was generated from LAD regional segment-length data by substituting end-diastolic LAD segment-length for end-diastolic left ventricular volume. To obtain pressure-volume relations, preload was reduced by transient occlusion of the inferior vena cava to produce a 30 m m Hg reduction in maximal left ventricular pressure. At each sampling time, data was recorded over a 10-second period with the respirator off at endexpiration. The animal was then allowed to equilibrate and caval occlusion data were again obtained. Three sets of occlusion data were obtained at each time point. Global and regional functional data were analyzed during each preload reduction.
Measurement of Nitric Oxide Release Nitric oxide release was measured continuously from the coronary sinus using an amperometric sensor (ISO-NO, World Precision Instrument, Inc, Sarasota, FL). This probe measures the concentration of NO gas in aqueous solution [10]. Briefly, NO diffuses through a semipermeable m e m b r a n e and is then oxidized at a working platin u m electrode resulting in an electric current. This redox current is proportional to the concentration of NO at the m e m b r a n e ' s outer surface. Electrode calibration was performed daily before each experiment. A calibration curve was obtained by measuring the current generated by the addition of liquid nitrite (NaNO2; Curtin Matheson Sci-
ENGELMAN ET AL NITRIC OXIDE AND ISCHEMIA
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entific, Inc, Wilmington, MA) to a solution containing KI, H~SO4, and K2SO 4. This resulted in the immediate generation of NO by the equation: 2NaNO 2 + 2KI + 2H2SO 4 + 2 K 2 S O 4 --~ 2 N O + 12 + 2 H 2 0 -b 3 K 2 S O 4 + N a 2 S O 4.
Nitric oxide calibration was always linear (r ~ 0.99). After stabilization, the NO probe was placed into the coronary sinus catheter 10 cm from the point of blood drainage. As shown in Figure 1, NO was measured from the coronary effluent as it flowed past the probe and was recirculated through the femoral vein. The concentration of NO was electronically digitized and recorded in realtime along with the hemodynamic measurements using the data acquisition system. At the conclusion of the experiment all hearts were weighed. Nitric oxide release was calculated in nanomole per gram wet weight.
Measurement of Malonaldehyde Formation, Myocardial Oxygen Extraction, and Creatine Kinase Release Malonaldehyde was measured from 1.5 mL of plasma that was mixed with an equal volume of 20% trichloroacetic acid and 5.3 mmol/L sodium metabisulfite. Protein was precipitated on ice for 10 minutes, and the suspension centrifuged at 3,000 g for 10 minutes. Two milliliters of supernatant was derivatized with 2,4-dinitrophenylhydrazine and extracted with pentane. Malonaldehyde formation was then measured using high-performance liquid chromatography as previously described [11]. Myocardial oxygen extraction was determined by simultaneously sampling coronary venous and femoral arterial blood samples for oxygen content using a BGE IL 1400 blood gas analyzer (Instrumentation Laboratory, Lexington, MA). Myocardial oxygen extraction was calculated as arterial-venous oxygen content. Creatine kinase was quantified from 0.5 mL of plasma by the enzymatic assay method using a creatine kinase assay kit (Sigma Diagnostics, St. Louis, MO). The absorbance was read at 340 n m using a Beckman DU-8 spectrophotometer.
Measurement of Adhesion Molecules Plasma (100 A) from the coronary sinus was assayed in duplicate for sVCAM-1, sE-selectin, and sICAM-1 by enzyme-linked immunosorbent assay. All assays were performed with commercially available kits (R&D Systems, Inc, Minneapolis, MN).
Statistical Analysis All data are expressed as a mean + standard error of the mean. Data was analyzed by a two-way analysis of variance for repeated measures followed by a multiple comparison Scheff6's test to determine differences between groups. The paired Student's t test was used for within-group comparisons with baseline values. Significance was considered at a p value of less than 0.05.
Results
Myocardial Function The effect of ischemia/reperfusion on myocardial systolic function is shown in Figure 2A. Preload recruitable stroke
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Fig 2. (A) Left anterior descending coronary artery regional and global
O:ARG
preload recruitable stroke work (PRSW) (.4) and "stiffness coefficient" ((3) (B) during ischemia (ISC) and reperfusion in 6 control (CON) and 6 L-arginine-pretreated (ARG) pigs. Data are the means +_ standard error of the mean. (*p < 0.05 versus control group.)
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work was r e d u c e d after 30 minutes of ischemia a n d d u r i n g the first 20 minutes of reperfusion in the LAD r e g i o n a l model. G l o b a l left v e n t r i c u l a r p r e l o a d recruitable stroke w o r k was not significantly different between the groups. Diastolic function was similarly decreased in the C O N hearts (Fig 2B). During reperfusion, the "stiffness" coefficient (13) was significantly increased in CON hearts (indicating reduced diastolic function/ventricular compliance) in both the LAD regional and global left ventricular models. Diastolic function in the ARG hearts were not significantly different from baseline levels t h r o u g h o u t the ischemia/reperfusion protocol. All diastolic a n d systolic functional abnormalities r e t u r n e d to near-baseline levels b y 90 m i n u t e s reperfusion, with
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the exception of LAD regional ]3, which r e m a i n e d significantly increased. Ventricular fibrillation occurred during ischemia in 50% of C O N hearts versus 0% of the ARG hearts (p < 0.05). During reperfusion, ventricular fibrillation occurred in 83% of C O N hearts versus 8% of the ARG hearts (p < 0.05). Electrical defibrillation was successful in all cases.
Nitric Oxide Release Baseline levels of NO m e a s u r e d from the coronary sinus varied b r o a d l y with a m e a n value of 160.5 nmol/L. Therefore, results are r e p o r t e d as the change in NO release from baseline. Nitric oxide is r e p o r t e d as molarity p e r g r a m of left ventricular heart tissue m e a s u r e d at a set
Ann Thorac Surg 1995;60:1275-81
ENGELMAN ET AL NITRIC OXIDEAND 1SCHEMIA
t i m e - p o i n t in the coronary sinus catheter. The concentration of N O in the catheter t u b i n g is i n d e p e n d e n t of coronary flow as the volume of m e a s u r e d b l o o d is fixed. There were no differences in the postischemic left ventricular wet weights b e t w e e n groups a n d we have found that the myocardial water content increases by less than 3 % after 30 m i n u t e s of regional ischemia a n d reperfusion. The time-course of N O release is illustrated in Figure 3. Nitric oxide release in the A R G group was not significantly different from baseline levels t h r o u g h o u t the course of the experiment. In contrast, in the C O N hearts, N O release was significantly d e c r e a s e d d u r i n g ischemia, a n d c o n t i n u e d to decline during reperfusion.
25
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M a l o n a l d e h y d e , M y o c a r d i a l O x y g e n Extraction, and Creatine K i n a s e
M a l o n a l d e h y d e release is illustrated in Figure 4. Malona l d e h y d e levels were significantly increased in the C O N group at 3 a n d 5 m i n u t e s of reperfusion (162% baseline at 3 minutes) c o m p a r e d with the ARG group (108% baseline at 3 minutes). M a l o n a l d e h y d e levels r e t u r n e d to baseline levels by 15 m i n u t e s of reperfusion. Myocardial oxygen extraction was significantly d e c r e a s e d in the C O N comp a r e d with the ARG group at 30 minutes of ischemia (80% versus 119% baseline, respectively). Both groups r e t u r n e d to baseline values b y 30 m i n u t e s of reperfusion. Peak creatine kinase release was not significantly different b e t w e e n groups at any time point.
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30
3
5
15
30
60
90
minutes Fig 4. Malonaldehyde (MDA) release at baseline (B), during ischemia (ISC), and reperfusion in 6 control (CON) and 6 L-argininepretreated (ARG) pigs. Malonaldehyde was measured by high performance liquid chromatography as described in Material and Methods. Data are the means +_ standard error of the mean. (*p < 0.05 versus control; #p < 0.05 versus baseline values.)
Adhesion Molecules
The levels of sVCAM-1, sE-selectin, a n d sICAM-1 are illustrated in Figure 5. The sVCAM-1 level b e g a n to rise after 3 m i n u t e s of reperfusion in both groups, a n d con-
NO release nM/g LV wt.
tinued to rise thereafter. The sVCAM-1 level was significantly increased in the C O N c o m p a r e d with the ARG group after 30 m i n u t e s of reperfusion, sE-selectin a n d sICAM-1 were similarly increased in the C O N group but were u n c h a n g e d from baseline in the ARG group, sEselectin a n d sICAM-1 were both significantly increased in the C O N c o m p a r e d with the ARG groups after 5 m i n u t e s of reperfusion b u t were not different after 60 m i n u t e s of reperfusion in the sICAM-1 group.
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minutes Fig 3. Change in nitric oxide (NO) release from baseline during ischemia (ISC) and reperfusion in 6 control (CON) and 6 L-arginine-pretreated (ARG) pigs. Nitric oxide release from the coronary sinus was measured using an amperometric probe as described in Material and Methods. Data are the means + standard error of the mean. (*p < 0.05 versus control group.)
Comment The m a i n t e n a n c e of constitutive N O release a p p e a r s to be i m p o r t a n t in the recovery of function after an ischemic injury. Previously, we have d e m o n s t r a t e d r e d u c e d constitutive N O release after i s c h e m i a / r e p e r f u s i o n in a working Krebs p e r f u s e d rat heart [12]. The p r e s e n t study has d e m o n s t r a t e d , using direct methods, that in a b l o o d p e r f u s e d in vivo working pig heart, coronary sinus NO release is r e d u c e d within the first 15 minutes of regional ischemia a n d continues to decline t h r o u g h o u t reperfusion. In addition, we have shown that a preischemic administration of exogenous r - a r g i n i n e can sustain N O release at baseline levels, preserve systolic a n d diastolic left ventricular function, increase myocardial oxygen extraction, a n d reduce m a l o n a l d e h y d e and soluble a d h e sion molecule release. A l t h o u g h the beneficial effects of N O supplementation in the setting of ischemia/reperfusion injury have b e e n well characterized [1, 13], the u n d e r l y ing m e c h a n i s m s r e m a i n unknown.
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Fig 5. Soluble vascular cell adhesion molecule-1 (sVCAM-1), endothelial leukocyte adhesion molecule-1 (sE-selectin), and intercellular adhesion molecule-1 (sICAM-1) during ischemia (I) and reperfusion in 6 control (CON) and 6 L-arginine (ARG) pigs. All assays were run in duplicate from coronary sinus plasma by enzyme-linked immunosorbent assay as described in Material and Methods. Data are the means +_ standard error of the mean. (*p < 0.05 versus control group.)
It is well known that neutrophil-derived free radicals can exacerbate ischemia/reperfusion injury [14]. It has been suggested that NO synthesis can reduce oxidative stress, scavenge ambiently produced superoxide radicals [15], and terminate free-radical chain reactions within the lipid membrane [16], thereby reducing inflammatory mediators, adhesion molecule expression, and neutrophil-endothelial interactions [17]. The present study supports these findings by demonstrating in an in vivo system that NO supplementation reduced the release of malonaldehyde after ischemia/reperfusion. Lipid oxidation products, especially malonaldehyde, are a presumptive marker for lipid peroxidation, which is an indicator of free-radical production and oxidative stress. By binding and eliminating these toxic metabolites, NO may reduce lipid peroxidation and retard the damaging effects of reperfusion injury. The adhesion of leukocytes to endothelial cells requires the expression of leukocyte-specific adhesion proteins on the surface of the vascular endothelium [18]. Upon up-regulation, these adhesion molecules are capable of attachment to activated polymorphonuclear neutrophils, allowing transendothelial migration and cytotoxic damage [19]. To assess whether decreased NO production during ischemia/reperfusion promotes endothelial-leukocyte interactions by increasing expression of these mediators, we measured three soluble adhesion molecules constitutively expressed on the vascular endothelium. We demonstrated that postischemic increases in each of these soluble adhesion molecules could be significantly ameliorated with t-arginine-induced NO supplementation. Elevations of sICAM-1, sE-selectin, and sVCAM-1 definitively indicate activation or damage to the vascular endothelium [20]. Although the biologic significance of these circulating soluble adhesion molecules is unclear, it has been reported that sE-selectin can
up-regulate neutrophil integrin function, thereby acting as a physiologic proadhesive effector [21]. In addition, targeted reduction of adhesion molecule expression has been used to reduce ischemia/reperfusion injury. It has been demonstrated that a monoclonal antibody against ICAM-1 significantly attenuated the increase in polymorphonuclear neutrophil adherence to ischemic/reperfused coronary endothelium [5]. Regardless of the specific biologic roles for these soluble adhesion molecules, their up-regulation signifies an increased inflammatory endothelial response after ischemia/reperfusion injury. Although coronary flows were not measured, coronary vasodilatation was probably not a major component of this protection. Previously, we have demonstrated in an isolated rat heart model that coronary flow is not significantly increased after L-arginine pretreatment [12]. Other investigators have demonstrated in dogs [4], cats [1], and rabbits [22] that arterial tone or coronary flows were not significantly changed after L-arginine administration. In addition, coronary collateral flow in pigs is very small. Many of the proposed theories regarding the protective effects of NO suggest a free radical-mediated reperfusion phenomenon. However, we have demonstrated that significant functional improvement in regional systolic function and myocardial oxygen extraction were evident in the L-arginine-supplemented pigs even before reperfusion (after 30 minutes of ischemia). This may be evidence of a direct cytoprotective activity of NO. The functional impairment in control hearts was almost completely resolved after 90 minutes of reperfusion and there was no d i f f e r e n c e in creatine kinase release between the groups. Therefore, in this m o d e l L-arginine pretreatment appears to have reduced myocardial stunning, rather than infarction. L-Arginine also afforded a profound antiarrhythmic protection in this study. This may be a
Ann Thorac Surg 1995;60:1275-81
c o n s e q u e n c e of r e d u c e d free radical g e n e r a t i o n / i n creased b i n d i n g [23], increased cellular antioxidant (glutathione) levels in endothelial cells [24], or a direct, as yet uncharacterized, consequence of NO on cardiac musculature [25]. The systemic effects of L-arginine are probably not responsible for its cardioprotective activity. Arginine has b e e n shown to result in the release of active hormones, including insulin, glucagon, growth hormone, a n d prolactin [1]. However the reported inability of D-arginine to provide cardioprotection [12] suggests that systemic horm o n a l effects are not operative. In summary, s u p p l e m e n t a l L-arginine given before a regional ischemic insult, reduced free radical/neutrophilmediated lipid peroxidation, plasma levels of soluble adhesion molecules, myocardial stunning, and reperfusion arrhythmias. These results support an excessive endothelial injury/inflammatory response after regional ischemia a n d reperfusion. S u p p l e m e n t a t i o n of the NO pathway provides dramatic antiinflammatory endothelial protection and may suggest novel cardioprotective strategies to ameliorate clinical ischemia/reperfusion injury. This study was supported by grants HL 22559-15 and HL 34360-07 from the National Institutes of Health.
References 1. Weyrich AS, Ma X, Lefer AM. The role of L-arginine in ameliorating reperfusion injury, after myocardial ischemia in the cat. Circulation 1992;86:279-88. 2. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524-6. 3. Lhuscher TF. Endothelium-derived relaxing factor. In: Lhuscher TF, Vanhoutte PM, eds. The endothelium: modulator of cardiovascular function. Boca Raton: CRC Press, 1990. 4. Nakanishi K, Vinten-Johansen J, Lefer DJ, et al. Intracoronary L-arginine during reperfusion improves endothelial function and reduces infarct size. Am J Physiol 1992;263: H1650-8. 5. Ma X-l, Weyrich AS, Lefer DJ, Lefer AM. Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium. Circ Res 1993;72:403-12. 6. Rubanyi GM, Ho EH, Cantor EH, Lumma WC, ParkerBotelho LH. Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes. Biochem Biophys Res Commun 1991;181:1392-7. 7. Freeman GL. Effects of increased afterload on left ventricular function in closed-chest dogs. Am J Physiol 1990;259: H619-25.
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8. Shintani H, Glantz SA. Effect of disrupting the mitral valve apparatus on left ventricular function in dogs. Circulation 1993;87:2001-15. 9. Misare BD, Krukenkamp IB, Lazar ZP, Levitsky S. Retrograde is superior to antegrade continuous warm blood cardioplegia for acute cardiac ischemia. Circulation 1992;869 (Suppl 2):393-7. 10. Tsukahara H, Gordienko DV, Goligorsky MS. Continuous monitoring of nitric oxide release from human umbilical vein endothelial cells. Biochem Biophys Res Commun 1993; 193:722-9. 11. Cordis GA, Maulik N, Bagchi D, Engelman RM, Das DK. Estimation of the extent of lipid peroxidation in the ischemic and reperfused heart by monitoring lipid metabolic products with the aid of high-performance liquid chromatography. ] Chromatogr 1993;632:97-103. 12. Engelman DT, Watanabe M, Engelman RM, et al. Constitutive nitric oxide release is impaired following ischemia and reperfusion. J Thorac Cardiovasc Surg (in press). 13. Lefer DJ, Nakanishi K, Johnston WE, Vinten-Johansen J. Antineutrophil and myocardial protection actions of a novel nitric oxide donor after myocardial ischemia and reperfusion in dogs. Circulation 1993;88:2337-50. 14. Lucchesi BR. Modulation of leukocyte-mediated myocardial reperfusion injury. Annu Rev Physiol 1990;52:561-76. 15. Niu X-f, Smith CW, Kubes P. Intracellular oxidative stress induced by nitric oxide synthesis inhibition increases endothelial cell adhesion to neutrophils. Circ Res 1994;74:1133-40. 16. Wink DA, Hanbauer I, Krinshna MC, DeGraff W, Gamson J, Mitchell JB. Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc Nat Acad Sci 1993;90:13-7. 17. Kubes P, Suzuli M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci 1991;88:4651-5. 18. Godin C, Caprani A, Dufaux J, Flaud P. Interactions between neutrophils and endothelial cells. J Cell Sci 1993;106:441-52. 19. Menasch6 P, Peynet J, Lavivi6re J, et al. Does normothermia during cardiopulmonary bypass increase neutrophil-endothelium interactions? Circulation 1994;90:II-275-9. 20. Gearing AJH, Newman W. Circulating adhesion molecules in disease. Immunol Today 1993;14:506-12. 21. Lo SK, Lee S, Ramos RA, et al. Endothelial-leukocyte adhesion molecule I stimulates the adhesive activity of leukocyte integrin CR3 (CDllb/CD18, Mac-l, alpha m beta 2) on human neutrophils. J Exp Med 1991;173:1493-500. 22. Cooke JP, Andon NA, Girerd XJ, Hirsch AT, Creager MA. Arginine restores cholinergic relaxation of hypercholesterolemic rabbit thoracic aorta. Circulation 1991;83:1057-62. 23. Tosaki A, Braquet P. DMPO and reperfusion injury: arrhythmia, heart function, electron spin resonance studies in isolated working guinea pig hearts. Am Heart J 1990;120: 819 -30. 24. White AC, Maloney El<, Boustani MR, Fanburg BL. Nitric oxide increases cellular glutathione in fibroblasts, endothelial cells and smooth muscle cells [Abstract]. Circulation 1994;90:I-150. 25. Parratt J, Vegh A. Pronounced antiarrhythmic effects of ischemic preconditioning. Cardioscience 1994;5:9-18.
Background. This study evaluated whether the nitric oxide precursor L-arginine could reduce ischemia/reperfusion injury by preventing leukocyte-endothelial interactions. Methods. Normothermic regional ischemia was induced in the open-chest working pig heart for 30 minutes followed by 90 minutes of reperfusion. A preischemic 10-minute intravenous infusion of 4 mg- kg -1 • min -1 of L-arginine (n = 12) was compared with 12 control pigs. Nitric oxide release was measured from the coronary sinus using an amperometric probe. Left ventricular function, malonaldehyde, creatine kinase, myocardial oxygen extraction, and the soluble adhesion molecules (intracellular adhesion molecule-I, endothelial leukocyte adhesion molecule-I, and vascular cell adhesion molecule-I) were measured. Results. Nitric oxide release was significantly reduced from baseline throughout ischemia/reperfusion only in
the control group. Systolic and diastolic function, and myocardial oxygen extraction were also significantly decreased during early reperfusion in the control compared with the L-arginine group. Peak creatine kinase release was not significantly different between groups. The incidence of ventricular fibrillation, malonaldehyde release, and soluble intracellular adhesion molecule-I, endothelial leukocyte adhesion molecule-I, and vascular cell adhesion molecule-1 were each significantly decreased during reperfusion in the r-arginine group. Conclusions. t-Arginine reduced lipid peroxidation, plasma levels of soluble adhesion molecules, myocardial stunning, and arrhythmias. These results support an excessive endothelial injury/inflammatory response after regional ischemia/reperfusion that can be ameliorated through augmented nitric oxide.
eperfusion of ischemic myocardium has been reported to cause a rapid degeneration of endothelial function, characterized by a decreased release of nitric oxide (NO) in response to endothelium-dependent vasodilators [1]. Nitric oxide, synonymous with endotheliurnderived relaxing factor, is a naturally occurring, continuously released, vasoactive agent that controls coronary vascular tone [2]. It is synthesized by the vascular endothelium through the conversion of L-arginine to L-citrulline by the enzyme NO synthase [3]. Nitric oxide donors given during reperfusion have been shown to preserve coronary artery ring vasorelaxation (an indirect measure of NO synthesis) and reduce myocardial injury associated with ischemia and reperfusion [4]. In the vascular system, NO has been shown to be an endogenous inhibitor of leukocyte chemotaxis, adherence, and activation [5]. In addition, NO may inactivate superoxide free radicals generated by leukocytes [6]. The present study was designed to assess the role of endothelial function/activation after regional ischemia and
reperfusion in L-arginine-supplemented in situ bloodperfused porcine hearts using continuous measurements of myocardial function and coronary sinus NO release. To further delineate the effects of r-arginine on myocardial energetics and cellular necrosis, malonaldehyde release, an indirect marker of free radical-mediated lipid peroxidation, myocardial oxygen extraction, and creatine kinase release were measured during ischemia and reperfusion. In addition, the soluble endothelial "proadhesive'" molecules vascular cell adhesion molecule-1 (sVCAM-1), endothelial leukocyte adhesion molecule-1 (sE-selectin), and intercellular adhesion molecule-1 (sICAM-1) were each quantified from coronary sinus blood.
R
Accepted for publication June 13, 1995. Address reprint requests to Dr Daniel T. Engelman, Surgical Research Center, University of Connecticut School of Medicine, 263 Farmington Ave, Farmington,CT 06030-1110. © 1995 by The Society of Thoracic Surgeons
(Ann Thorac Surg 1995;60:1275-81)
Material and M e t h o d s 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 85-23, revised 1985). 0003-4975/95/$9.50 SSDI 0003-4975(95)00614-Q
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Surgical Preparation Yorkshire pigs of either sex weighing 20 to 25 kg were tranquilized with ketamine (50 mg/kg), anesthetized with sodium pentobarbital (25 mg/kg), and placed on a mechanical ventilator. The electrocardiogram was continuously recorded. Cannulas were placed in the femoral vein and artery. The chest was then opened with a median sternotomy and the pericardium was suspended in a pericardial cradle. The azygous and hemiazygos veins were ligated, and the inferior vena cava was snared with 3-ram-wide tape (Umbilical Tape; Ethicon, Inc, Somerville, NJ). Heparin sodium (250 U/kg) was then administered systematically. Sonometric dimension crystals (diameter, 6 ram), which were made of 3 MHz piezoelectric crystals (Triton Technologies, Inc, San Diego, CA) were placed at the endocardial surface across the anteroposterior minor axis, septal-free wall minor axis, and base-apex major axis of the left ventricle. The anteroposterior crystals were placed adjacent to the anterior and posterior descending coronary arteries. The septal-free crystals were located one-half of the distance from the apex to the base. The base crystal was placed into the left ventricle adjacent to the origin of the left circumflex coronary artery and the apex crystal was placed into the left ventricular apex. Techniques for crystal placement have been previously described in detail by Freeman [7]. A pair of sonometric dimension crystals (diameter, 2.5 mm), which were made of 5 MHz piezoelectric crystals (Triton Technologies) were placed 1 cm apart in the left anterior descending coronary artery (LAD) distribution epicardium of the left ventricle. A 5F micromanometer-tipped catheter (Millar Instruments, Inc, Houston, TX) was inserted through the left ventricular apex for pressure measurements. A catheter was then placed into the coronary sinus. This catheter drained coronary sinus blood past an in-line NO probe and into a 37°C chamber that was connected via a roller pump to the femoral vein catheter. The experimental preparation is illustrated in Figure 1. The heart rate was kept at 120 beats/min using an electronic pacer (Phipps & Bird, Inc, Richmond, VA) when necessary and continuous monitoring of the electrocardiogram. Bretylium tosylate (2 mg/kg) was given as a bolus injection for antiarrhythrnic prophylaxis before ischemia and reperfusion, and as required for ventricular ectopy. No other ionotropes or antiarrhythmics were administered during the experiment. A defibrillator (Mennen-Greatbatch, Clerance, NY) was used as necessary for ventricular fibrillation. Sedation was maintained with inhalational isoflurane (0.5% to 1.5%; Ohmeda, Liberty Corner, NJ). Arterial blood gases were monitored, and the ventilator was adjusted to maintain a pH of 7.35 to 7.45, an oxygen tension of more than 100 mm Hg, and a carbon dioxide tension of 35 to 50 mm Hg. Mean arterial pressure was kept above 65 mm Hg by infusion of lactated Ringer's solution. Postmortem examination verified crystal placement.
~d |
1"9 cer
Fig 1. Experimental heart preparation. ( A o aorta; I V C = inferior vena cava; L A = left atrium; P A = pulmonary artery; RA = right atrium; S V C = superior vena cava.)
Experimental Protocol Baseline measurements were made during steady-state contractions after instrumentation and stabilization. Control animals (CON; n = 12) then received an intravenous infusion of lactated Ringer's solution for 10 minutes. Treated animals (ARG; n = 12) received an intravenous infusion of L-arginine (4 mg" kg -1" min -1) (Sigma Chemical, St. Louis, MO) for 10 minutes. The LAD was then ligated for 30 minutes just proximal to the origin of the first diagonal branch. The hearts were then reperfused for 90 minutes. In 6 ARG pigs and 6 CON pigs, functional data was obtained before ischemia (baseline), after 15 and 30 minutes of ischemia, and after 10, 20, 30, 60, and 90 minutes of reperfusion. Nitric oxide release was measured continuously throughout the experimental protocol. Three-milliliter blood samples were taken from the coronary sinus for creatine kinase and malonaldehyde measurements at baseline, after 30 minutes of ischemia, and 3, 5, 15, 30, 60, and 90 minutes of reperfusion. Myocardial oxygen extraction was measured from simultaneously drawn coronary sinus and femoral artery blood samples at baseline, after 30 minutes of ischemia, and after 30 minutes of reperfusion. In a separate group of 6 ARG and 6 CON pigs, 3-mL blood samples were taken from the coronary sinus for soluble adhesion molecule measurements at baseline, after 30 minutes of ischemia, and at 3, 5, 15, 30, 60, and 90 minutes of reperfusion. All blood was immediately centrifuged at 1,000 g for 10 minutes and the plasma was stored in aliquots at -20°C for subsequent analysis.
Ann Thorac Surg 1995;60:1275-81
Measurement of Myocardial Function The hemodynamic variables continuously recorded were left ventricular pressure, left ventricular dimensions, and LAD regional segment length. These data were digitized and recorded in real-time with a 12-bit AD converter sampling at 200 Hz using the Cordat II Data Acquisition, Analysis, and Presentation System (Data Integrated Scientific Systems, Pinckney, MI; Triton Technologies, Inc, San Diego, CA). The digitized data was later analyzed using the CV AutoReport Cardiovascular Data Analysis Program (Scitelligence, Inc, Brighton, MI). Left ventricular volume (V) was modeled as two half-ellipsoids by the equation: V = ~r/6 × ASL, where A, S, and L are the anteroposterior, septal-free, and base-apex dimensions [8]. The first derivative of left ventricular pressure was calculated as a polynomial approximation from the digital left ventricular pressure signal. End-diastolic volume was defined as the left ventricular volume at the first positive derivative of left ventricular pressure. Left ventricular stroke work (SW) was defined as the integral of left ventricular pressure, P, and volume, V, of the cardiac cycle by the equation SW = fPdV. The linear regression analysis was performed on the stroke work-end-diastolic volume relationship, to generate the preload recruitable stroke work with slope, M**, and x-axis intercept, V o. Similar data was generated from regional segmentlength data by substituting end-diastolic LAD segmentlength for end-diastolic left ventricular volume. The "stiffness" coefficient (]3) was derived from exponential modeling of the end-diastolic pressure (EDP)end-diastolic volume (EDV) relationship by the equation EDP = a × e(~ x EDV). The stiffness coefficient is the inverse of compliance [9]. Similar data was generated from LAD regional segment-length data by substituting end-diastolic LAD segment-length for end-diastolic left ventricular volume. To obtain pressure-volume relations, preload was reduced by transient occlusion of the inferior vena cava to produce a 30 m m Hg reduction in maximal left ventricular pressure. At each sampling time, data was recorded over a 10-second period with the respirator off at endexpiration. The animal was then allowed to equilibrate and caval occlusion data were again obtained. Three sets of occlusion data were obtained at each time point. Global and regional functional data were analyzed during each preload reduction.
Measurement of Nitric Oxide Release Nitric oxide release was measured continuously from the coronary sinus using an amperometric sensor (ISO-NO, World Precision Instrument, Inc, Sarasota, FL). This probe measures the concentration of NO gas in aqueous solution [10]. Briefly, NO diffuses through a semipermeable m e m b r a n e and is then oxidized at a working platin u m electrode resulting in an electric current. This redox current is proportional to the concentration of NO at the m e m b r a n e ' s outer surface. Electrode calibration was performed daily before each experiment. A calibration curve was obtained by measuring the current generated by the addition of liquid nitrite (NaNO2; Curtin Matheson Sci-
ENGELMAN ET AL NITRIC OXIDE AND ISCHEMIA
1277
entific, Inc, Wilmington, MA) to a solution containing KI, H~SO4, and K2SO 4. This resulted in the immediate generation of NO by the equation: 2NaNO 2 + 2KI + 2H2SO 4 + 2 K 2 S O 4 --~ 2 N O + 12 + 2 H 2 0 -b 3 K 2 S O 4 + N a 2 S O 4.
Nitric oxide calibration was always linear (r ~ 0.99). After stabilization, the NO probe was placed into the coronary sinus catheter 10 cm from the point of blood drainage. As shown in Figure 1, NO was measured from the coronary effluent as it flowed past the probe and was recirculated through the femoral vein. The concentration of NO was electronically digitized and recorded in realtime along with the hemodynamic measurements using the data acquisition system. At the conclusion of the experiment all hearts were weighed. Nitric oxide release was calculated in nanomole per gram wet weight.
Measurement of Malonaldehyde Formation, Myocardial Oxygen Extraction, and Creatine Kinase Release Malonaldehyde was measured from 1.5 mL of plasma that was mixed with an equal volume of 20% trichloroacetic acid and 5.3 mmol/L sodium metabisulfite. Protein was precipitated on ice for 10 minutes, and the suspension centrifuged at 3,000 g for 10 minutes. Two milliliters of supernatant was derivatized with 2,4-dinitrophenylhydrazine and extracted with pentane. Malonaldehyde formation was then measured using high-performance liquid chromatography as previously described [11]. Myocardial oxygen extraction was determined by simultaneously sampling coronary venous and femoral arterial blood samples for oxygen content using a BGE IL 1400 blood gas analyzer (Instrumentation Laboratory, Lexington, MA). Myocardial oxygen extraction was calculated as arterial-venous oxygen content. Creatine kinase was quantified from 0.5 mL of plasma by the enzymatic assay method using a creatine kinase assay kit (Sigma Diagnostics, St. Louis, MO). The absorbance was read at 340 n m using a Beckman DU-8 spectrophotometer.
Measurement of Adhesion Molecules Plasma (100 A) from the coronary sinus was assayed in duplicate for sVCAM-1, sE-selectin, and sICAM-1 by enzyme-linked immunosorbent assay. All assays were performed with commercially available kits (R&D Systems, Inc, Minneapolis, MN).
Statistical Analysis All data are expressed as a mean + standard error of the mean. Data was analyzed by a two-way analysis of variance for repeated measures followed by a multiple comparison Scheff6's test to determine differences between groups. The paired Student's t test was used for within-group comparisons with baseline values. Significance was considered at a p value of less than 0.05.
Results
Myocardial Function The effect of ischemia/reperfusion on myocardial systolic function is shown in Figure 2A. Preload recruitable stroke
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Fig 2. (A) Left anterior descending coronary artery regional and global
O:ARG
preload recruitable stroke work (PRSW) (.4) and "stiffness coefficient" ((3) (B) during ischemia (ISC) and reperfusion in 6 control (CON) and 6 L-arginine-pretreated (ARG) pigs. Data are the means +_ standard error of the mean. (*p < 0.05 versus control group.)
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work was r e d u c e d after 30 minutes of ischemia a n d d u r i n g the first 20 minutes of reperfusion in the LAD r e g i o n a l model. G l o b a l left v e n t r i c u l a r p r e l o a d recruitable stroke w o r k was not significantly different between the groups. Diastolic function was similarly decreased in the C O N hearts (Fig 2B). During reperfusion, the "stiffness" coefficient (13) was significantly increased in CON hearts (indicating reduced diastolic function/ventricular compliance) in both the LAD regional and global left ventricular models. Diastolic function in the ARG hearts were not significantly different from baseline levels t h r o u g h o u t the ischemia/reperfusion protocol. All diastolic a n d systolic functional abnormalities r e t u r n e d to near-baseline levels b y 90 m i n u t e s reperfusion, with
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the exception of LAD regional ]3, which r e m a i n e d significantly increased. Ventricular fibrillation occurred during ischemia in 50% of C O N hearts versus 0% of the ARG hearts (p < 0.05). During reperfusion, ventricular fibrillation occurred in 83% of C O N hearts versus 8% of the ARG hearts (p < 0.05). Electrical defibrillation was successful in all cases.
Nitric Oxide Release Baseline levels of NO m e a s u r e d from the coronary sinus varied b r o a d l y with a m e a n value of 160.5 nmol/L. Therefore, results are r e p o r t e d as the change in NO release from baseline. Nitric oxide is r e p o r t e d as molarity p e r g r a m of left ventricular heart tissue m e a s u r e d at a set
Ann Thorac Surg 1995;60:1275-81
ENGELMAN ET AL NITRIC OXIDEAND 1SCHEMIA
t i m e - p o i n t in the coronary sinus catheter. The concentration of N O in the catheter t u b i n g is i n d e p e n d e n t of coronary flow as the volume of m e a s u r e d b l o o d is fixed. There were no differences in the postischemic left ventricular wet weights b e t w e e n groups a n d we have found that the myocardial water content increases by less than 3 % after 30 m i n u t e s of regional ischemia a n d reperfusion. The time-course of N O release is illustrated in Figure 3. Nitric oxide release in the A R G group was not significantly different from baseline levels t h r o u g h o u t the course of the experiment. In contrast, in the C O N hearts, N O release was significantly d e c r e a s e d d u r i n g ischemia, a n d c o n t i n u e d to decline during reperfusion.
25
20
10 O:CON
@:ARG
M a l o n a l d e h y d e , M y o c a r d i a l O x y g e n Extraction, and Creatine K i n a s e
M a l o n a l d e h y d e release is illustrated in Figure 4. Malona l d e h y d e levels were significantly increased in the C O N group at 3 a n d 5 m i n u t e s of reperfusion (162% baseline at 3 minutes) c o m p a r e d with the ARG group (108% baseline at 3 minutes). M a l o n a l d e h y d e levels r e t u r n e d to baseline levels by 15 m i n u t e s of reperfusion. Myocardial oxygen extraction was significantly d e c r e a s e d in the C O N comp a r e d with the ARG group at 30 minutes of ischemia (80% versus 119% baseline, respectively). Both groups r e t u r n e d to baseline values b y 30 m i n u t e s of reperfusion. Peak creatine kinase release was not significantly different b e t w e e n groups at any time point.
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30
3
5
15
30
60
90
minutes Fig 4. Malonaldehyde (MDA) release at baseline (B), during ischemia (ISC), and reperfusion in 6 control (CON) and 6 L-argininepretreated (ARG) pigs. Malonaldehyde was measured by high performance liquid chromatography as described in Material and Methods. Data are the means +_ standard error of the mean. (*p < 0.05 versus control; #p < 0.05 versus baseline values.)
Adhesion Molecules
The levels of sVCAM-1, sE-selectin, a n d sICAM-1 are illustrated in Figure 5. The sVCAM-1 level b e g a n to rise after 3 m i n u t e s of reperfusion in both groups, a n d con-
NO release nM/g LV wt.
tinued to rise thereafter. The sVCAM-1 level was significantly increased in the C O N c o m p a r e d with the ARG group after 30 m i n u t e s of reperfusion, sE-selectin a n d sICAM-1 were similarly increased in the C O N group but were u n c h a n g e d from baseline in the ARG group, sEselectin a n d sICAM-1 were both significantly increased in the C O N c o m p a r e d with the ARG groups after 5 m i n u t e s of reperfusion b u t were not different after 60 m i n u t e s of reperfusion in the sICAM-1 group.
°
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]
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I
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I
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minutes Fig 3. Change in nitric oxide (NO) release from baseline during ischemia (ISC) and reperfusion in 6 control (CON) and 6 L-arginine-pretreated (ARG) pigs. Nitric oxide release from the coronary sinus was measured using an amperometric probe as described in Material and Methods. Data are the means + standard error of the mean. (*p < 0.05 versus control group.)
Comment The m a i n t e n a n c e of constitutive N O release a p p e a r s to be i m p o r t a n t in the recovery of function after an ischemic injury. Previously, we have d e m o n s t r a t e d r e d u c e d constitutive N O release after i s c h e m i a / r e p e r f u s i o n in a working Krebs p e r f u s e d rat heart [12]. The p r e s e n t study has d e m o n s t r a t e d , using direct methods, that in a b l o o d p e r f u s e d in vivo working pig heart, coronary sinus NO release is r e d u c e d within the first 15 minutes of regional ischemia a n d continues to decline t h r o u g h o u t reperfusion. In addition, we have shown that a preischemic administration of exogenous r - a r g i n i n e can sustain N O release at baseline levels, preserve systolic a n d diastolic left ventricular function, increase myocardial oxygen extraction, a n d reduce m a l o n a l d e h y d e and soluble a d h e sion molecule release. A l t h o u g h the beneficial effects of N O supplementation in the setting of ischemia/reperfusion injury have b e e n well characterized [1, 13], the u n d e r l y ing m e c h a n i s m s r e m a i n unknown.
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A n n Thorac S u r g 1995;60:1275-81
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Fig 5. Soluble vascular cell adhesion molecule-1 (sVCAM-1), endothelial leukocyte adhesion molecule-1 (sE-selectin), and intercellular adhesion molecule-1 (sICAM-1) during ischemia (I) and reperfusion in 6 control (CON) and 6 L-arginine (ARG) pigs. All assays were run in duplicate from coronary sinus plasma by enzyme-linked immunosorbent assay as described in Material and Methods. Data are the means +_ standard error of the mean. (*p < 0.05 versus control group.)
It is well known that neutrophil-derived free radicals can exacerbate ischemia/reperfusion injury [14]. It has been suggested that NO synthesis can reduce oxidative stress, scavenge ambiently produced superoxide radicals [15], and terminate free-radical chain reactions within the lipid membrane [16], thereby reducing inflammatory mediators, adhesion molecule expression, and neutrophil-endothelial interactions [17]. The present study supports these findings by demonstrating in an in vivo system that NO supplementation reduced the release of malonaldehyde after ischemia/reperfusion. Lipid oxidation products, especially malonaldehyde, are a presumptive marker for lipid peroxidation, which is an indicator of free-radical production and oxidative stress. By binding and eliminating these toxic metabolites, NO may reduce lipid peroxidation and retard the damaging effects of reperfusion injury. The adhesion of leukocytes to endothelial cells requires the expression of leukocyte-specific adhesion proteins on the surface of the vascular endothelium [18]. Upon up-regulation, these adhesion molecules are capable of attachment to activated polymorphonuclear neutrophils, allowing transendothelial migration and cytotoxic damage [19]. To assess whether decreased NO production during ischemia/reperfusion promotes endothelial-leukocyte interactions by increasing expression of these mediators, we measured three soluble adhesion molecules constitutively expressed on the vascular endothelium. We demonstrated that postischemic increases in each of these soluble adhesion molecules could be significantly ameliorated with t-arginine-induced NO supplementation. Elevations of sICAM-1, sE-selectin, and sVCAM-1 definitively indicate activation or damage to the vascular endothelium [20]. Although the biologic significance of these circulating soluble adhesion molecules is unclear, it has been reported that sE-selectin can
up-regulate neutrophil integrin function, thereby acting as a physiologic proadhesive effector [21]. In addition, targeted reduction of adhesion molecule expression has been used to reduce ischemia/reperfusion injury. It has been demonstrated that a monoclonal antibody against ICAM-1 significantly attenuated the increase in polymorphonuclear neutrophil adherence to ischemic/reperfused coronary endothelium [5]. Regardless of the specific biologic roles for these soluble adhesion molecules, their up-regulation signifies an increased inflammatory endothelial response after ischemia/reperfusion injury. Although coronary flows were not measured, coronary vasodilatation was probably not a major component of this protection. Previously, we have demonstrated in an isolated rat heart model that coronary flow is not significantly increased after L-arginine pretreatment [12]. Other investigators have demonstrated in dogs [4], cats [1], and rabbits [22] that arterial tone or coronary flows were not significantly changed after L-arginine administration. In addition, coronary collateral flow in pigs is very small. Many of the proposed theories regarding the protective effects of NO suggest a free radical-mediated reperfusion phenomenon. However, we have demonstrated that significant functional improvement in regional systolic function and myocardial oxygen extraction were evident in the L-arginine-supplemented pigs even before reperfusion (after 30 minutes of ischemia). This may be evidence of a direct cytoprotective activity of NO. The functional impairment in control hearts was almost completely resolved after 90 minutes of reperfusion and there was no d i f f e r e n c e in creatine kinase release between the groups. Therefore, in this m o d e l L-arginine pretreatment appears to have reduced myocardial stunning, rather than infarction. L-Arginine also afforded a profound antiarrhythmic protection in this study. This may be a
Ann Thorac Surg 1995;60:1275-81
c o n s e q u e n c e of r e d u c e d free radical g e n e r a t i o n / i n creased b i n d i n g [23], increased cellular antioxidant (glutathione) levels in endothelial cells [24], or a direct, as yet uncharacterized, consequence of NO on cardiac musculature [25]. The systemic effects of L-arginine are probably not responsible for its cardioprotective activity. Arginine has b e e n shown to result in the release of active hormones, including insulin, glucagon, growth hormone, a n d prolactin [1]. However the reported inability of D-arginine to provide cardioprotection [12] suggests that systemic horm o n a l effects are not operative. In summary, s u p p l e m e n t a l L-arginine given before a regional ischemic insult, reduced free radical/neutrophilmediated lipid peroxidation, plasma levels of soluble adhesion molecules, myocardial stunning, and reperfusion arrhythmias. These results support an excessive endothelial injury/inflammatory response after regional ischemia a n d reperfusion. S u p p l e m e n t a t i o n of the NO pathway provides dramatic antiinflammatory endothelial protection and may suggest novel cardioprotective strategies to ameliorate clinical ischemia/reperfusion injury. This study was supported by grants HL 22559-15 and HL 34360-07 from the National Institutes of Health.
References 1. Weyrich AS, Ma X, Lefer AM. The role of L-arginine in ameliorating reperfusion injury, after myocardial ischemia in the cat. Circulation 1992;86:279-88. 2. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524-6. 3. Lhuscher TF. Endothelium-derived relaxing factor. In: Lhuscher TF, Vanhoutte PM, eds. The endothelium: modulator of cardiovascular function. Boca Raton: CRC Press, 1990. 4. Nakanishi K, Vinten-Johansen J, Lefer DJ, et al. Intracoronary L-arginine during reperfusion improves endothelial function and reduces infarct size. Am J Physiol 1992;263: H1650-8. 5. Ma X-l, Weyrich AS, Lefer DJ, Lefer AM. Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium. Circ Res 1993;72:403-12. 6. Rubanyi GM, Ho EH, Cantor EH, Lumma WC, ParkerBotelho LH. Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes. Biochem Biophys Res Commun 1991;181:1392-7. 7. Freeman GL. Effects of increased afterload on left ventricular function in closed-chest dogs. Am J Physiol 1990;259: H619-25.
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