Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury GEORG MATHEIS, MICHAEL P. SHERMAN, GERALD D. BUCKBERG, DAVID M. HAYBRON, HELEN H. YOUNG, AND LOUIS J. IGNARRO Departments of Cardiothoracic Surgery, Pediatrics, and Pharmacology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024 Matheis, Georg, Michael P. Sherman, Gerald D. Buckberg, David M. Haybron, Helen H. Young, and Louis J. Ignarro. Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H616-H620,1992.-In view of the recent findings that NO reacts with superoxide anion to generate hydroxyl radical, the present study was conducted to ascertain the role of endogenous NO in mediating myocardial reoxygenation injury in the hypoxic piglet on cardiopulmonary bypass. Anesthetized piglets were made hypoxic (PaO, = 20-30 mmHg) for up to 120 min, followed by reoxygenation on cardiopulmonary bypass for 30 min. Reoxygenation caused rapidly developing myocardial injury characterized by decreased contractility (expressed as endsystolic elastance) and increased lipid peroxidation (measured as conjugated dienes). Systemic venous and coronary sinus blood content of NO decreased significantly during hypoxia and increased substantially above prehypoxic levels during reoxygenation on cardiopulmonary bypass. Administration of either the antioxidants mercaptopropionyl glycine and catalase or the NO synthase inhibitor, NG-nitro-L-arginine methyl ester, to the extracorporeal circuit afforded similar and nearly complete protection against myocardial reoxygenation injury. The protective effects of NG-nitro-L-arginine methyl ester were nullified by adding an excess of L-arginine to the pump circuit, suggesting that the L-arginine-NO pathway is involved in myocardial reoxygenation injury. superoxide anion; peroxynitrite anion; hydroxyl radical; nitro-L-arginine methyl ester; catalase; mercaptopropionyl tine
N”gly-
have increased the use of cardiopulmonary bypass (CPB) and extracorporeal membrane oxygenation (ECMO) during infancy and childhood (2, 13, 20). Clinical reports have described myocardial dysfunction either after correction of congenital heart disease on CPB (15) or during the treatment of pulmonary failure on ECMO (18). This study tests the hypothesis that the institution of either CPB or ECMO causes an immediate reoxygenation injury to the infantile, hypoxic heart and subsequent alterations in cardiac contractility. The hypothesis is supported by the observations of de1 Nido et al. (8) that cyanotic infants undergoing reoxygenation on CPB had an increased content of conjugated dienes in their myocardial biopsies and supported by a report of reduced systolic function in hypoxemic newborns placed on ECMO (18). To investigate the biochemical mechanisms responsible for the injury to the hypoxic heart during reoxygenation, we used an in vivo piglet
TECHNICAL
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model of acute hypoxia, induced by lowering the fractional concentration of O2 in inspired gas (FIN ) on the ventilator. The hypoxic period was followed by reoxygenation on CPB and a recovery phase. Although de1 Nido and associates (8) found evidence of myocardial lipid peroxidation in hypoxic hearts placed on CPB, there are no studies that indicate reoxygenation injury of the hypoxic, beating myocardium is due to reactive oxygen intermediates (ROI). This assumption must be derived from investigations using the ischemic and reperfused myocardium. Bolli and associates (4) utilized an in vivo canine model of regional ischemia and reperfusion to produce myocardial contractile dysfunction that was associated with immediate detection of ROI in coronary venous blood. Their use of the hydroxyl radical (HO.) scavenger mercaptopropionyl glycine (MPG) attenuated “myocardial stunning” and suppressed ROI generation. It is generally accepted that highly reactive HO. is responsible for this oxidant injury to the myocardium, and HO* formation occurs via the iron-catalyzed Haber- Weiss (Fenton) reaction (12, 19). Beckman and colleagues have suggested that generation of HO. via the Haber-Weiss pathway (3) may be limited in vivo, and they have proposed that NO reacts with 0; in pathological states to produce cytotoxic species via the following biochemical pathway NO + 0, s ONOO- + H+ c ONOOH r HO. + NO 2 c
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ONOOONOOH HO. + NO2 NO, + H+
This chemical reaction may be viewed as an alternate pathway of HO* generation during tissue reoxygenation injury and is consistent with the known protective effects of superoxide dismutase (1, 16). This investigation tests the hypothesis that in vivo reoxygenation injury of the infantile, hypoxic heart may be mediated, at least in part, by the L-arginine-NO pathway leading to the formation of peroxynitrite anion (ONOO-) and HO.. METHODS Twenty-five premeditation
Yorkshire Duroc piglets (4-6 kg, 3 wk) received (0.5 mg/kg diazepam im), were anesthetized (30
Copyright 0 1992 the American Physiological Society
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mg/kg pentobarbital ip initially, followed by 5 mg/kg iv per hour), and underwent tracheotomy for intermittent mandatory ventilation. Transducer-tipped catheters were placed into the left ventricle, left atrium, pulmonary artery, abdominal aorta, and right atrium for continuous pressure monitoring. A singlestage right atria1 cannula and an ascending aortic cannula were inserted for initiation of CPB. The ductus arteriosus was always ligated with a surgical clip via a left fourth interspace thoracotomy. Piezoelectric sonomicrometer crystals were placed along the anterior-posterior axis for continuous measurement of left ventricular dimension, and cardiac output was determined by thermodilution. Intravascular pressures and crystal signals were amplified and digitalized for inscribing computer-analyzed pressure-dimension loops. A sonomicrometer (Triton Technology, model 210, San Diego, CA) converted transit time of the ultrasonic impulse between anterior and posterior wall into intracavitary distance. A series of pressure-dimension loops under variable loading conditions was generated by rapid transient occlusion of the inferior vena cava during a 12-s period of apnea (17). The end-systolic pressure-dimension point of each loop was determined and left ventricular performance was described as ventricular elastance (26). End-systolic elastance, as a measurement of systolic performance, is relatively independent of loading conditions (25). Five normoxic control animals were anesthetized, instrumented, and observed over 5 h before functional and biochemical assessments. In the 20 animals undergoing studies of reoxygenation, hypoxia was produced by lowering inspired FIN, ( 400 mmHg). The criterion for instituting reoxygenation before 120 min was a mean arterial pressure of ~30 mmHg. Arterial pH was maintained above 7.3 by periodic NaHCOs infusions. The extracorporeal circuit prime was isosmotic and included packed red blood cells, hetastarch (Hespan, DuPont, Wilmington, DE), and Plasma-Lyte electrolyte solution (Baxter Healthcare, Deerfield, IL). Five animals undergoing reoxygenation had no additives in the extracorporeal fluid, whereas the pump fluid was supplemented with either 1) MPG (20 mg/kg) plus catalase (5 mg/kg), or 2) NG-nitro-L-arginine methyl ester (4 mg/kg), or 3) NG-nitro-L-arginine methyl ester (L-NAME, 4 mg/kg) plus L-arginine (20 mg/kg) in the remaining three groups of five animals each. All reagents were obtained from Sigma (St. Louis, MO). A 30-min period of reoxygenation on CPB was followed by an additional 30-min interval of observation before final functional assessments were performed and specimens for biochemical analyses were obtained. At the end of each experiment, a visual inspection of brain, thoracic, and intra-abdominal organs was performed, and the patency of the foramen ovale was assessed. Myocardial conjugated dienes were measured as a marker of lipid peroxidation. Endocardial biopsy specimens were immediately frozen and stored in liquid nitrogen, and tissue levels of hydroxyconjugated dienes were determined using modifications of previously described methods (1, 16). With the use of nitrogen-equilibrated solutions at 4°C 200 mg of endocardium was homogenized in ice-cold chloroform:methanol (2:l, vol/vol). After being centrifuged at 1,000 g at 4°C for 10 min, 5 ml of the organic layer was washed with 2.0 ml of 0.003 M HCl. Two millilters of the organic layer was dried under 100% N, with an Alltech oxygen trap and resuspended in 3.0 ml hexane. A Beckman DU 70 spectrophotometer with capture and curvefitting software (Beckman Instruments, Irvine, CA) and interfaced with an IBM computer, was used to determine absorbance (A) at 233 nm by performing a wavelength scan between 200 and 310 nm. Hexane was used to blank the instrument. The
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content of conjugated dienes was expressed as AZri:, nanometers per milligrams lipid. Total myocardial lipid was measured by the spectrophotometric method of Chiang et al. (6). Direct measurements of NO cannot be made because of its short half-life. Thus NO was determined in plasma as its spontaneous oxidation product, nitrite, which was reconverted to NO and quantitated with a sensitive chemiluminescence assay (22) using a nitrogen oxides analyzer (DASIBI Environmental, model 2108, NO, analyzer, Glendale, CA). The method was modified to increase the sensitivity of the detector to 0.8 parts/billion of NO (1 pmol/O.l ml of test sample). Plasma samples (0.1 ml) were injected into 75 ml of refluxing glacial acetic acid containing 1% potassium iodide in a nitrogen atmosphere. Systemic venous and coronary sinus plasma samples were used to assess whole body and myocardial NO production, respectively. Plasma was obtained before hypoxia, immediately before reoxygenation, and 30 min after reoxygenation. Plasma concentrations taken during CPB were adjusted for dilutional effects of the volume in the extracorporeal circuit. Statistical analysis. Data were analyzed with StatView V2.0 on an Apple Macintosh IICi. Analysis of variance was employed for comparisons between groups (10). Differences were considered significant at a probability level of P < 0.05. Group data are expressed as means * SE. RESULTS Hypoxia
initially increased the cardiac index, followed decline over an average tolerated hypoxic of 91 t 4 min. There was no difference in the
by a gradual period
mean duration of hypoxia among the four experimental groups. The changes in systemic vascular resistance observed during hypoxia, reoxygenation, and the postbypass period are shown in Fig. 1. The presence of LNAME in the CPB circuit during reoxygenation caused an increase in systemic vascular resistance to 167% of prehypoxic values, a phenomenon that was largely reSVRS
L-NAME (n=5) L-NAME + L-Arginine no Rx (n=5) M PG/Catalase (n=4)
--&-
--&__c_ w
1200-
(n=5) I*
I-
TA
off Bypass
I
1
lOOO-
800-
600-
400-
1
200!
I
e
I
.
I
+~Hou~s++--------------
.
I
-
I
-
I
-’
I
30min
-
I
-
I
.
1
-
1
Time
P
HYPOXIA REOXYGENATION Fig. 1. Effect of hypoxia and reoxygenation on systemic vascular resistance index (SVRI) and modulating influence of MPG/catalase, L-NAME, or L-NAME + L-arginine on SVRI. SVRI was calculated as follows: [(mean arterial pressure - central venous pressure)/ cardiac output] X weight Data are presented as means t SE. x-axis shows con dition and time intervals. L-NAME, Nc’-nitroL-arginine methyl ester (4 mg/kg); L-arginine (20 mg/kg); no R,, reoxygenation without any CPB additives; MPG, mercaptopropionyl glycine (20 mg/kg); and catalase (5 mg/kg). *P c 0.05 vs. no R, and MPG/catalase; tP < 0.05 vs. L-NAME + L-arginine.
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H618
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versed by adding L-arginine to the circuit with L-NAME. Reoxygenation also caused myocardial dysfunction characterized by markedly depressed contractility as expressed by end-systolic elastance (62% decrease vs. prehypoxic control values) and injury characterized by increased endocardial conjugated dienes (89% increase above normoxic, instrumented piglets) (Figs. 2 and 3). The addition of either L-NAME or MPG/catalase to the extracorporeal circuit resulted in near complete recovery of cardiac contractility to 98 and 84% of control, respectively (Fig. 2), and maintenance of endocardial conjugated diene concentrations to within 16% of the control (Fig. 3). Inclusion of L-arginine (20 mg/kg) in the extracorporeal circuit completely prevented the functional and biochemical protection afforded by L-NAME (Figs. 2 and 3) . Average prehypoxic NO levels in systemic venous and coronary sinus blood were 185 and 235 pmol/O.l ml plasma, respectively (Fig. 4), corresponding to mean plasma NO concentrations of 1.85 and 2.35 PM, respectively. The concentration of NO in coronary sinus blood, taken during either prehypoxia or hypoxia, was significantly higher (>27%) than that in systemic venous blood. Hypoxia caused more than a 21% decline in plasma NO levels in systemic and coronary sinus blood. During reoxygenation, the systemic and coronary sinus venous NO content of reoxygenated (untreated) and L-NAME + L-arginine-treated animals rose well above either prehypoxic or hypoxic values (>44%), whereas the plasma content of NO in L-NAME-treated piglets was equivalent to their prehypoxic concentrations. Pancreatic damage ranging from petechiae to frank hemorrhage was found in all L-NAME-treated piglets, but evidence of macroscopic injury to other organs was not seen. The foramen ovale was always anatomically closed.
REOXYGENATION
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1.
52 .--cl 2 0. +
E" ‘00. 2 < 0. 0.
•m Control
n
(n=5)
no Rx (n=5)
q
MPG/Catalase
0
L-NAME
(n=5)
q
L-NAME
+
(n=5)
L-Arg
(n=5)
Fig. 3. Conjugated diene content in reoxygenated endocardium and effect of adding either MPG/catalase, L-NAME, or L-NAME + Larginine to CPB circuit on this measure of lipid peroxidation. Vertically lined bar (designated control) shows range of endocardial values & SE (Azs3/mg total lipid) from normoxic instrumented piglets. Data for 4 experimental groups are presented as means t SE. We used capture software to do curve overlays, thereby determining the difference between experimental samples and a reference standard prepared by averaging the results of lipid extracts from 5 normoxic hearts. Specific absorption peak of conjugated dienes at A,,, appeared as a shoulder on a broader peak of unsaturated lipids. Contribution of other unsaturated lipids to absorbance was eliminated by differential spectroscopy (7). This procedure also showed a significant increase in conjugated dienes in no-treatment and L-NAME + L-arginine hearts compared with values obtained for MPG/catalaseand L-NAME-treated piglets. See Fig. 1 for abbreviations and doses. 600
KI
Systemic Venous oronary Sinus * p< 0.05 vs. Systemic
Ek
Venous
“p p< 0.05 vs. Control § p< 0.05 vs. Hypoxia ,400 l-d . 5 E a
loo-
P 2 200 2 a
.0 5 67
*:..._:.:. :.:._.; n_. 0
-6 20 5
H
no Rx (n&)
q
MPCXataiase
(n=5)
0
L-NAME
(n=5)
q
L-NAME
+ L-Arg (n=5)
Fig. 2. Reoxygenation-induced impairment of myocardial contractility, its prevention by MPG/catalase or L-NAME, and the nullifying effect of adding L-arginine along with L-NAME to CPB circuit. Values are changes in left ventricular end-systolic elastance after discontinuation of CPB, expressed as percent of individual prehypoxic control values. Data are presented as means of: SE. See Fig. 1 for abbreviations. *P < 0.05 vs. control; ‘fP < 0.05 vs. no R, and L-NAME + L-arginine.
CONTROL
HYPOXIA
(n=15)
(n=15)
(n=5)
EOXYGENAT L-NAME (n=5)
‘ION L-NAM
E/L-Arg (n=5)
Fig. 4. Plasma levels of NO during prehypoxia, hypoxia, and reoxygenation, and modulating effect of L-NAME or L-NAME + L-arginine on plasma NO content during reoxygenation. Values are expressed as pmol/O.l ml of plasma. Data are presented as means & SE. See Fig. 1 for abbreviations and doses.
DISCUSSION
The present study provides the first demonstration that the endogenous L-arginine-NO pathway is causally involved in myocardial reoxygenation injury. NG-nitroL-arginine methyl ester (L-NAME), a potent NO syn-
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thase inhibitor (11), afforded nearly complete protection against myocardial reoxygenation injury during reoxygenation on CPB and was as effective as the antioxidants, MPG and catalase. This suggests that NO formation contributes to reoxygenation injury of the hypoxic piglet heart by interacting with 02 to form ONOO-, which then decays homolytically after protonation to HO. and nitrogen dioxide radical (3). The protective action of L-NAME was completely prevented by the concomitant administration of a fivefold excess of Larginine, the proposed principal substrate for NO synthase (21). This effect of L-arginine is attributed to its capacity to compete with and override the inhibitory action of L-NAME on NO synthase. Our conclusion concerning the L-arginine-NO pathway is strengthened by the internal consistency of the present findings. The reoxygenation-only group and the LNAME + L-arginine group had significantly increased NO content in coronary sinus blood at the termination of CPB (Fig. 4), and this observation coincided with a marked decline in end-systolic elastance (Fig. 2) and a substantial rise in endocardial conjugated dienes during the recovery period (Fig. 3). In contrast, the groups that received either L-NAME or MPG and catalase in the extracorporeal circuit during reoxygenation on CPB had a near normal recovery of contractile function and minimal evidence of lipid peroxidation of the heart. The protective effect of MPG and catalase during myocardial reoxygenation can be attributed to scavenging of HO. and H202, respectively (4,5). MPG is neither a scavenger of 0; nor H202 (4). Recent reports also suggest that MPG, a thiol, may prevent HO. formation via direct interaction with NO and ONOO- (23). By adding catalase to the CPB circuit, we attempted to avoid the direct cytotoxic effects of HzOz on the heart. By reducing the availability of H202, we may also have prevented its interaction with 02 in an iron-catalyzed Haber-Weiss reaction to form HO. (12, 19). This leaves MPG as the sole scavenger of HO* generated via the homolytic decay of ONOO-. If this assumption is correct concerning the relative actions of MPG and catalase, it explains the equal efficacy of L-NAME and MPG + catalase in ameliorating reoxygenation injury. Direct limitation of ONOO- reaction with membrane lipids (24) may be an additional or alternative explanation for the limitation of lipid peroxidation achieved by either MPG and catalase or L-NAME. The average prehypoxia NO levels in coronary sinus blood were 27% higher than that observed in systemic venous blood, indicating that the heart generates significant quantities of NO under basal conditions. These in vivo findings concur with findings seen in isolated perfused hearts (14). Hypoxia decreased plasma NO levels, perhaps reflecting the oxygen requirement of NO synthase in the catalysis of L-arginine to NO and L-citrulline. After hypoxia, reoxygenation with a high intraarterial oxygen tension resulted in an elevated NO content in both the systemic and coronary venous blood compared with baseline (prehypoxia) levels. This suggests that intra-arterial oxygen tension may influence NO generation and raises the possibility of controlling NO production by modifying how CPB is initiated.
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The increased systemic vascular resistance during the administration of L-NAME (Fig. 1) suggests that although inhibition of NO formation may protect against myocardial reoxygenation injury, it may superimpose ischemic injury on other previously hypoxic organs. Macroscopic evidence of pancreatic damage ranging from petechiae to frank hemorrhage was limited to L-NAMEtreated piglets. This would not have been detected during in vitro studies of isolated hearts and indicates the importance of in vivo models that simulate clinical conditions. Pancreatic injury is a complication of CPB in human subjects, and its pathogenesis seems partially related to ischemia (9). These data emphasize the salutary effects of either free radical scavenger supplementation or NO inhibition during reoxygenation. The protective effect of a L-arginine analogue in alleviating myocardial reoxygenation injury should serve to stimulate the search for other compounds or strategies that regulate NO generation so that the potential benefits of NO, such as vasodilation, inhibition of platelet aggregation and adhesion, and inhibition of neutrophil adherence can be maintained. Finally, our observations underscore the importance of adding chemical agents to the extracorporeal circuit that limit the generation of reactive oxygen or nitrogen intermediates if myocardial reoxygenation injury is to be minimized during repair of cyanotic congenital heart defects or the treatment of infantile respiratory failure. The authors thank Russell Byrns, Garland Hodges, Nanci Stellino, and Paul Reed for excellent technical assistance, and Judith Becker for word processing assistance. This research was supported by National Heart, Lung, and Blood Institute Grants HL-40675 and HL-40922, the Laubisch Fund for Cardiovascular Research, and the University of California TobaccoRelated Disease Research program. G. Matheis is a recipient of a Research Fellowship of the Deutsche Herzstiftung. M. P. Sherman is an Established Investigator of the American Lung Association of California. Address for reprint requests: G. D. Buckberg, Dept. of Cardiothoracic Surgery, UCLA School of Medicine, Los Angeles, CA 90024. Received 20 September 1991; accepted in final form 8 November 1991. REFERENCES 1. Ambrosio,
G., J. T. Flaherty, C. Duillo, I. Tritto, M. Condorelli, and M. Chiariello.
G. Santoro,
Oxygen radicals generated at reflow induce peroxidation of membrane lipids in reperfused hearts. J. Clin. Inuest. 87: 2056-2066, 1991. P. P. Elia,
2. Anderson,
H. L., III, R. J. Attori, J. R. Custer, R. A. ChapR. H. Bartlett. Extracorporeal membrane oxygenation pediatric cardiopulmonary failure. J. Thorac. Cardiouusc. Surg.
man,
and
for 99:1011-1021,199o. 3. Beckman,
J. S., T. W. Beckman, B. A. Freeman. Apparent
J. Chen,
P. A. Marshall,
hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Nutl. Acad. Sci. USA 87: 1620-1624, 1990. and
4. Bolli,
R.,
M. 0. Jeroudi, E. K. Lai, and
B. S. Patel, P. B. McCay.
0.
I. Aruoma,
B.
Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion. Evidence that myocardial “stunning” is a manifestation of reperfusion injury. Circ. Res. 65: 607-622,1989. Halliwell,
5. Brown,
J. M., M. A. Grosso, L. S. Terada, A. Banerjee, C. W. White, A. H. Harken,
G. J. Whitman, and J. E. Repine.
Endotoxin pretreatment increases endogenous myocardial catalase activity and decreases ischemia-reperfusion injury of isolated rat hearts. Proc. NutZ. Acud. Sci. USA. 86: 2516-2520, 1989.
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6. Chiang, S. P., C. F. Gessert, and 0. H. Lowry. CoZorimetric Determination of Extracted Lipids. School of Aviation Medicine, USAF, Randolph AFB, TX, 1957 (No. 56-113). 7. Corongiu, F. P., and A. Milia. An improved and simple method for determining diene conjugation in autoxidized polyunsaturated fatty acids. Chem. Biol. Interact. 44: 289-297, 1983 8. Del Nido, P. J., D. A. G. Mickle, G. J. Wilson, L. N. Benson, J. G. Coles, G. A. Trusler, and W. G. Williams. Evidence of myocardial free radical injury during elective repair of tetralogy of Fallot. Circulation 76, Suppl. V: V-174-V-179, 1987. 9. Fernandez-de1 Castillo, C., W. Harringer, A. L, Warshaw, G. J. Vlahakes, G. Koski, A. M. Zaslavsky, and D. W. Rattner. Risk factors for pancreatic cellular injury after cardiopulmonary bypass. N. Eng. J. Med. 325: 382-387, 1991. 10. Godfrey, K. Comparing the means of several groups. N. Eng. J. Med. 313: 1450-1460,1985. 11. Gross, S. S., D. J. Stuehr, K. Aisaka, E. A. Jaffe, R. Levi, and 0. W. Griffith. Macrophage and endothelial cell nitric oxide synthesis: cell-type selective inhibition by p-aminoarginine, pnitroarginine and p-methylarginine. Biochem. Biophys. Res. Commun. 170: 96-103, 1990. 12. Halliwell, B. Oxidants and human disease: some new concepts. FASEB J . 1: 358-364,1987. M. N. Current status of surgery for congenital heart 13. Ilbawi, disease. Clin. Perinatol. 16: 157-176, 1989. 14. Kelm, M., and J. Schrader. Control of coronary vascular tone by nitric oxide. Circ. Res. 66: 1561-1575, 1990. 15. Kirklin, J. K., E. H. Blackstone, J. W. Kirklin, R. McKay, A. D. Pacifico, and L. J. Bargeron, Jr. Intracardiac surgery in infants under age 3 months: incremental risk factors for hospital mortality. Am. J. Cardiol. 48: 500-506, 1981. 16. Lesnefsky, E. J., P. M. Fennessey, K. M. Van Benthuysen, I. F. McMurtry, V. L. Travis, and L. D. Horwitz. Superoxide dismutase decreases early reperfusion release of conjugated dienes following regional canine ischemia. Basic Res. CardioZ. 84: 191-196, 1989.
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17. Little, W. C., C. P. Cheng, M. Mumma, Y. Igarashi, J. Vinten-Johansen, and W. E. Johnston. Comparison of measures of left ventricular contractile performance derived from pressure-volume loops in conscious dogs. CircuZation 80: 1378-1387, 1989. 18. Martin, G. R,, and B. L. Short. Doppler echocardiographic evaluation of cardiac performance in infants on prolonged extracorporeal membrane oxygenation. Am. J. Cardiol. 62: 929-934, 1988. P., C. Grousset, Y. Gauduel, C. Mouas, and A. lg* Menasche, Piwnica. Prevention of hydroxyl radical formation: a critical concept for improving cardioplegia. Protective effects of deferoxamine. Circulation 76, Suppl. V: V-180-V-185, 1987. P. P., and R. K. Crone. Pediatric applications of 20. O’Rourke, extracorporeal membrane oxygenation. J. Pediatr. 116: 393-394, 1990. 21. Palmer, R. M. J., D. S. Ashton, and S. Moncada. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature Lond. 333: 664-666,1988. 22. Palmer, R. M. J., A. G. Ferrige, and S. Moncada. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature Lond. 327: 524-526, 1987. 23 . Radi, R., J. S. Beckman, K. M. Bush, and B. A. Freeman. Peroxynitrite oxidation of sulfhydrils. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266: 4244-4250, 1991. 24. Radi, R., J. S. Beckman, K. M. Bush, and B. A. Freeman. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch. Biochem. Biophys. 288: 481-487,199l. K. The ventricular pressure-volume diagram revisited. 25* Sagawa, Circ. Res. 43: 677-687, 1978. K., W. L. Manghan, H. Suga, and K. Sunagawa. 26. Sagawa, Cardiac contraction and pressure-volume relationship. New York: Oxford University, 1988, p. 56-61 and 110-119.
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N”gly-
have increased the use of cardiopulmonary bypass (CPB) and extracorporeal membrane oxygenation (ECMO) during infancy and childhood (2, 13, 20). Clinical reports have described myocardial dysfunction either after correction of congenital heart disease on CPB (15) or during the treatment of pulmonary failure on ECMO (18). This study tests the hypothesis that the institution of either CPB or ECMO causes an immediate reoxygenation injury to the infantile, hypoxic heart and subsequent alterations in cardiac contractility. The hypothesis is supported by the observations of de1 Nido et al. (8) that cyanotic infants undergoing reoxygenation on CPB had an increased content of conjugated dienes in their myocardial biopsies and supported by a report of reduced systolic function in hypoxemic newborns placed on ECMO (18). To investigate the biochemical mechanisms responsible for the injury to the hypoxic heart during reoxygenation, we used an in vivo piglet
TECHNICAL
H616
model of acute hypoxia, induced by lowering the fractional concentration of O2 in inspired gas (FIN ) on the ventilator. The hypoxic period was followed by reoxygenation on CPB and a recovery phase. Although de1 Nido and associates (8) found evidence of myocardial lipid peroxidation in hypoxic hearts placed on CPB, there are no studies that indicate reoxygenation injury of the hypoxic, beating myocardium is due to reactive oxygen intermediates (ROI). This assumption must be derived from investigations using the ischemic and reperfused myocardium. Bolli and associates (4) utilized an in vivo canine model of regional ischemia and reperfusion to produce myocardial contractile dysfunction that was associated with immediate detection of ROI in coronary venous blood. Their use of the hydroxyl radical (HO.) scavenger mercaptopropionyl glycine (MPG) attenuated “myocardial stunning” and suppressed ROI generation. It is generally accepted that highly reactive HO. is responsible for this oxidant injury to the myocardium, and HO* formation occurs via the iron-catalyzed Haber- Weiss (Fenton) reaction (12, 19). Beckman and colleagues have suggested that generation of HO. via the Haber-Weiss pathway (3) may be limited in vivo, and they have proposed that NO reacts with 0; in pathological states to produce cytotoxic species via the following biochemical pathway NO + 0, s ONOO- + H+ c ONOOH r HO. + NO 2 c
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ONOOONOOH HO. + NO2 NO, + H+
This chemical reaction may be viewed as an alternate pathway of HO* generation during tissue reoxygenation injury and is consistent with the known protective effects of superoxide dismutase (1, 16). This investigation tests the hypothesis that in vivo reoxygenation injury of the infantile, hypoxic heart may be mediated, at least in part, by the L-arginine-NO pathway leading to the formation of peroxynitrite anion (ONOO-) and HO.. METHODS Twenty-five premeditation
Yorkshire Duroc piglets (4-6 kg, 3 wk) received (0.5 mg/kg diazepam im), were anesthetized (30
Copyright 0 1992 the American Physiological Society
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mg/kg pentobarbital ip initially, followed by 5 mg/kg iv per hour), and underwent tracheotomy for intermittent mandatory ventilation. Transducer-tipped catheters were placed into the left ventricle, left atrium, pulmonary artery, abdominal aorta, and right atrium for continuous pressure monitoring. A singlestage right atria1 cannula and an ascending aortic cannula were inserted for initiation of CPB. The ductus arteriosus was always ligated with a surgical clip via a left fourth interspace thoracotomy. Piezoelectric sonomicrometer crystals were placed along the anterior-posterior axis for continuous measurement of left ventricular dimension, and cardiac output was determined by thermodilution. Intravascular pressures and crystal signals were amplified and digitalized for inscribing computer-analyzed pressure-dimension loops. A sonomicrometer (Triton Technology, model 210, San Diego, CA) converted transit time of the ultrasonic impulse between anterior and posterior wall into intracavitary distance. A series of pressure-dimension loops under variable loading conditions was generated by rapid transient occlusion of the inferior vena cava during a 12-s period of apnea (17). The end-systolic pressure-dimension point of each loop was determined and left ventricular performance was described as ventricular elastance (26). End-systolic elastance, as a measurement of systolic performance, is relatively independent of loading conditions (25). Five normoxic control animals were anesthetized, instrumented, and observed over 5 h before functional and biochemical assessments. In the 20 animals undergoing studies of reoxygenation, hypoxia was produced by lowering inspired FIN, ( 400 mmHg). The criterion for instituting reoxygenation before 120 min was a mean arterial pressure of ~30 mmHg. Arterial pH was maintained above 7.3 by periodic NaHCOs infusions. The extracorporeal circuit prime was isosmotic and included packed red blood cells, hetastarch (Hespan, DuPont, Wilmington, DE), and Plasma-Lyte electrolyte solution (Baxter Healthcare, Deerfield, IL). Five animals undergoing reoxygenation had no additives in the extracorporeal fluid, whereas the pump fluid was supplemented with either 1) MPG (20 mg/kg) plus catalase (5 mg/kg), or 2) NG-nitro-L-arginine methyl ester (4 mg/kg), or 3) NG-nitro-L-arginine methyl ester (L-NAME, 4 mg/kg) plus L-arginine (20 mg/kg) in the remaining three groups of five animals each. All reagents were obtained from Sigma (St. Louis, MO). A 30-min period of reoxygenation on CPB was followed by an additional 30-min interval of observation before final functional assessments were performed and specimens for biochemical analyses were obtained. At the end of each experiment, a visual inspection of brain, thoracic, and intra-abdominal organs was performed, and the patency of the foramen ovale was assessed. Myocardial conjugated dienes were measured as a marker of lipid peroxidation. Endocardial biopsy specimens were immediately frozen and stored in liquid nitrogen, and tissue levels of hydroxyconjugated dienes were determined using modifications of previously described methods (1, 16). With the use of nitrogen-equilibrated solutions at 4°C 200 mg of endocardium was homogenized in ice-cold chloroform:methanol (2:l, vol/vol). After being centrifuged at 1,000 g at 4°C for 10 min, 5 ml of the organic layer was washed with 2.0 ml of 0.003 M HCl. Two millilters of the organic layer was dried under 100% N, with an Alltech oxygen trap and resuspended in 3.0 ml hexane. A Beckman DU 70 spectrophotometer with capture and curvefitting software (Beckman Instruments, Irvine, CA) and interfaced with an IBM computer, was used to determine absorbance (A) at 233 nm by performing a wavelength scan between 200 and 310 nm. Hexane was used to blank the instrument. The
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content of conjugated dienes was expressed as AZri:, nanometers per milligrams lipid. Total myocardial lipid was measured by the spectrophotometric method of Chiang et al. (6). Direct measurements of NO cannot be made because of its short half-life. Thus NO was determined in plasma as its spontaneous oxidation product, nitrite, which was reconverted to NO and quantitated with a sensitive chemiluminescence assay (22) using a nitrogen oxides analyzer (DASIBI Environmental, model 2108, NO, analyzer, Glendale, CA). The method was modified to increase the sensitivity of the detector to 0.8 parts/billion of NO (1 pmol/O.l ml of test sample). Plasma samples (0.1 ml) were injected into 75 ml of refluxing glacial acetic acid containing 1% potassium iodide in a nitrogen atmosphere. Systemic venous and coronary sinus plasma samples were used to assess whole body and myocardial NO production, respectively. Plasma was obtained before hypoxia, immediately before reoxygenation, and 30 min after reoxygenation. Plasma concentrations taken during CPB were adjusted for dilutional effects of the volume in the extracorporeal circuit. Statistical analysis. Data were analyzed with StatView V2.0 on an Apple Macintosh IICi. Analysis of variance was employed for comparisons between groups (10). Differences were considered significant at a probability level of P < 0.05. Group data are expressed as means * SE. RESULTS Hypoxia
initially increased the cardiac index, followed decline over an average tolerated hypoxic of 91 t 4 min. There was no difference in the
by a gradual period
mean duration of hypoxia among the four experimental groups. The changes in systemic vascular resistance observed during hypoxia, reoxygenation, and the postbypass period are shown in Fig. 1. The presence of LNAME in the CPB circuit during reoxygenation caused an increase in systemic vascular resistance to 167% of prehypoxic values, a phenomenon that was largely reSVRS
L-NAME (n=5) L-NAME + L-Arginine no Rx (n=5) M PG/Catalase (n=4)
--&-
--&__c_ w
1200-
(n=5) I*
I-
TA
off Bypass
I
1
lOOO-
800-
600-
400-
1
200!
I
e
I
.
I
+~Hou~s++--------------
.
I
-
I
-
I
-’
I
30min
-
I
-
I
.
1
-
1
Time
P
HYPOXIA REOXYGENATION Fig. 1. Effect of hypoxia and reoxygenation on systemic vascular resistance index (SVRI) and modulating influence of MPG/catalase, L-NAME, or L-NAME + L-arginine on SVRI. SVRI was calculated as follows: [(mean arterial pressure - central venous pressure)/ cardiac output] X weight Data are presented as means t SE. x-axis shows con dition and time intervals. L-NAME, Nc’-nitroL-arginine methyl ester (4 mg/kg); L-arginine (20 mg/kg); no R,, reoxygenation without any CPB additives; MPG, mercaptopropionyl glycine (20 mg/kg); and catalase (5 mg/kg). *P c 0.05 vs. no R, and MPG/catalase; tP < 0.05 vs. L-NAME + L-arginine.
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versed by adding L-arginine to the circuit with L-NAME. Reoxygenation also caused myocardial dysfunction characterized by markedly depressed contractility as expressed by end-systolic elastance (62% decrease vs. prehypoxic control values) and injury characterized by increased endocardial conjugated dienes (89% increase above normoxic, instrumented piglets) (Figs. 2 and 3). The addition of either L-NAME or MPG/catalase to the extracorporeal circuit resulted in near complete recovery of cardiac contractility to 98 and 84% of control, respectively (Fig. 2), and maintenance of endocardial conjugated diene concentrations to within 16% of the control (Fig. 3). Inclusion of L-arginine (20 mg/kg) in the extracorporeal circuit completely prevented the functional and biochemical protection afforded by L-NAME (Figs. 2 and 3) . Average prehypoxic NO levels in systemic venous and coronary sinus blood were 185 and 235 pmol/O.l ml plasma, respectively (Fig. 4), corresponding to mean plasma NO concentrations of 1.85 and 2.35 PM, respectively. The concentration of NO in coronary sinus blood, taken during either prehypoxia or hypoxia, was significantly higher (>27%) than that in systemic venous blood. Hypoxia caused more than a 21% decline in plasma NO levels in systemic and coronary sinus blood. During reoxygenation, the systemic and coronary sinus venous NO content of reoxygenated (untreated) and L-NAME + L-arginine-treated animals rose well above either prehypoxic or hypoxic values (>44%), whereas the plasma content of NO in L-NAME-treated piglets was equivalent to their prehypoxic concentrations. Pancreatic damage ranging from petechiae to frank hemorrhage was found in all L-NAME-treated piglets, but evidence of macroscopic injury to other organs was not seen. The foramen ovale was always anatomically closed.
REOXYGENATION
INJURY
1.
52 .--cl 2 0. +
E" ‘00. 2 < 0. 0.
•m Control
n
(n=5)
no Rx (n=5)
q
MPG/Catalase
0
L-NAME
(n=5)
q
L-NAME
+
(n=5)
L-Arg
(n=5)
Fig. 3. Conjugated diene content in reoxygenated endocardium and effect of adding either MPG/catalase, L-NAME, or L-NAME + Larginine to CPB circuit on this measure of lipid peroxidation. Vertically lined bar (designated control) shows range of endocardial values & SE (Azs3/mg total lipid) from normoxic instrumented piglets. Data for 4 experimental groups are presented as means t SE. We used capture software to do curve overlays, thereby determining the difference between experimental samples and a reference standard prepared by averaging the results of lipid extracts from 5 normoxic hearts. Specific absorption peak of conjugated dienes at A,,, appeared as a shoulder on a broader peak of unsaturated lipids. Contribution of other unsaturated lipids to absorbance was eliminated by differential spectroscopy (7). This procedure also showed a significant increase in conjugated dienes in no-treatment and L-NAME + L-arginine hearts compared with values obtained for MPG/catalaseand L-NAME-treated piglets. See Fig. 1 for abbreviations and doses. 600
KI
Systemic Venous oronary Sinus * p< 0.05 vs. Systemic
Ek
Venous
“p p< 0.05 vs. Control § p< 0.05 vs. Hypoxia ,400 l-d . 5 E a
loo-
P 2 200 2 a
.0 5 67
*:..._:.:. :.:._.; n_. 0
-6 20 5
H
no Rx (n&)
q
MPCXataiase
(n=5)
0
L-NAME
(n=5)
q
L-NAME
+ L-Arg (n=5)
Fig. 2. Reoxygenation-induced impairment of myocardial contractility, its prevention by MPG/catalase or L-NAME, and the nullifying effect of adding L-arginine along with L-NAME to CPB circuit. Values are changes in left ventricular end-systolic elastance after discontinuation of CPB, expressed as percent of individual prehypoxic control values. Data are presented as means of: SE. See Fig. 1 for abbreviations. *P < 0.05 vs. control; ‘fP < 0.05 vs. no R, and L-NAME + L-arginine.
CONTROL
HYPOXIA
(n=15)
(n=15)
(n=5)
EOXYGENAT L-NAME (n=5)
‘ION L-NAM
E/L-Arg (n=5)
Fig. 4. Plasma levels of NO during prehypoxia, hypoxia, and reoxygenation, and modulating effect of L-NAME or L-NAME + L-arginine on plasma NO content during reoxygenation. Values are expressed as pmol/O.l ml of plasma. Data are presented as means & SE. See Fig. 1 for abbreviations and doses.
DISCUSSION
The present study provides the first demonstration that the endogenous L-arginine-NO pathway is causally involved in myocardial reoxygenation injury. NG-nitroL-arginine methyl ester (L-NAME), a potent NO syn-
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AND MYOCARDIAL
thase inhibitor (11), afforded nearly complete protection against myocardial reoxygenation injury during reoxygenation on CPB and was as effective as the antioxidants, MPG and catalase. This suggests that NO formation contributes to reoxygenation injury of the hypoxic piglet heart by interacting with 02 to form ONOO-, which then decays homolytically after protonation to HO. and nitrogen dioxide radical (3). The protective action of L-NAME was completely prevented by the concomitant administration of a fivefold excess of Larginine, the proposed principal substrate for NO synthase (21). This effect of L-arginine is attributed to its capacity to compete with and override the inhibitory action of L-NAME on NO synthase. Our conclusion concerning the L-arginine-NO pathway is strengthened by the internal consistency of the present findings. The reoxygenation-only group and the LNAME + L-arginine group had significantly increased NO content in coronary sinus blood at the termination of CPB (Fig. 4), and this observation coincided with a marked decline in end-systolic elastance (Fig. 2) and a substantial rise in endocardial conjugated dienes during the recovery period (Fig. 3). In contrast, the groups that received either L-NAME or MPG and catalase in the extracorporeal circuit during reoxygenation on CPB had a near normal recovery of contractile function and minimal evidence of lipid peroxidation of the heart. The protective effect of MPG and catalase during myocardial reoxygenation can be attributed to scavenging of HO. and H202, respectively (4,5). MPG is neither a scavenger of 0; nor H202 (4). Recent reports also suggest that MPG, a thiol, may prevent HO. formation via direct interaction with NO and ONOO- (23). By adding catalase to the CPB circuit, we attempted to avoid the direct cytotoxic effects of HzOz on the heart. By reducing the availability of H202, we may also have prevented its interaction with 02 in an iron-catalyzed Haber-Weiss reaction to form HO. (12, 19). This leaves MPG as the sole scavenger of HO* generated via the homolytic decay of ONOO-. If this assumption is correct concerning the relative actions of MPG and catalase, it explains the equal efficacy of L-NAME and MPG + catalase in ameliorating reoxygenation injury. Direct limitation of ONOO- reaction with membrane lipids (24) may be an additional or alternative explanation for the limitation of lipid peroxidation achieved by either MPG and catalase or L-NAME. The average prehypoxia NO levels in coronary sinus blood were 27% higher than that observed in systemic venous blood, indicating that the heart generates significant quantities of NO under basal conditions. These in vivo findings concur with findings seen in isolated perfused hearts (14). Hypoxia decreased plasma NO levels, perhaps reflecting the oxygen requirement of NO synthase in the catalysis of L-arginine to NO and L-citrulline. After hypoxia, reoxygenation with a high intraarterial oxygen tension resulted in an elevated NO content in both the systemic and coronary venous blood compared with baseline (prehypoxia) levels. This suggests that intra-arterial oxygen tension may influence NO generation and raises the possibility of controlling NO production by modifying how CPB is initiated.
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The increased systemic vascular resistance during the administration of L-NAME (Fig. 1) suggests that although inhibition of NO formation may protect against myocardial reoxygenation injury, it may superimpose ischemic injury on other previously hypoxic organs. Macroscopic evidence of pancreatic damage ranging from petechiae to frank hemorrhage was limited to L-NAMEtreated piglets. This would not have been detected during in vitro studies of isolated hearts and indicates the importance of in vivo models that simulate clinical conditions. Pancreatic injury is a complication of CPB in human subjects, and its pathogenesis seems partially related to ischemia (9). These data emphasize the salutary effects of either free radical scavenger supplementation or NO inhibition during reoxygenation. The protective effect of a L-arginine analogue in alleviating myocardial reoxygenation injury should serve to stimulate the search for other compounds or strategies that regulate NO generation so that the potential benefits of NO, such as vasodilation, inhibition of platelet aggregation and adhesion, and inhibition of neutrophil adherence can be maintained. Finally, our observations underscore the importance of adding chemical agents to the extracorporeal circuit that limit the generation of reactive oxygen or nitrogen intermediates if myocardial reoxygenation injury is to be minimized during repair of cyanotic congenital heart defects or the treatment of infantile respiratory failure. The authors thank Russell Byrns, Garland Hodges, Nanci Stellino, and Paul Reed for excellent technical assistance, and Judith Becker for word processing assistance. This research was supported by National Heart, Lung, and Blood Institute Grants HL-40675 and HL-40922, the Laubisch Fund for Cardiovascular Research, and the University of California TobaccoRelated Disease Research program. G. Matheis is a recipient of a Research Fellowship of the Deutsche Herzstiftung. M. P. Sherman is an Established Investigator of the American Lung Association of California. Address for reprint requests: G. D. Buckberg, Dept. of Cardiothoracic Surgery, UCLA School of Medicine, Los Angeles, CA 90024. Received 20 September 1991; accepted in final form 8 November 1991. REFERENCES 1. Ambrosio,
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17. Little, W. C., C. P. Cheng, M. Mumma, Y. Igarashi, J. Vinten-Johansen, and W. E. Johnston. Comparison of measures of left ventricular contractile performance derived from pressure-volume loops in conscious dogs. CircuZation 80: 1378-1387, 1989. 18. Martin, G. R,, and B. L. Short. Doppler echocardiographic evaluation of cardiac performance in infants on prolonged extracorporeal membrane oxygenation. Am. J. Cardiol. 62: 929-934, 1988. P., C. Grousset, Y. Gauduel, C. Mouas, and A. lg* Menasche, Piwnica. Prevention of hydroxyl radical formation: a critical concept for improving cardioplegia. Protective effects of deferoxamine. Circulation 76, Suppl. V: V-180-V-185, 1987. P. P., and R. K. Crone. Pediatric applications of 20. O’Rourke, extracorporeal membrane oxygenation. J. Pediatr. 116: 393-394, 1990. 21. Palmer, R. M. J., D. S. Ashton, and S. Moncada. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature Lond. 333: 664-666,1988. 22. Palmer, R. M. J., A. G. Ferrige, and S. Moncada. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature Lond. 327: 524-526, 1987. 23 . Radi, R., J. S. Beckman, K. M. Bush, and B. A. Freeman. Peroxynitrite oxidation of sulfhydrils. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266: 4244-4250, 1991. 24. Radi, R., J. S. Beckman, K. M. Bush, and B. A. Freeman. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch. Biochem. Biophys. 288: 481-487,199l. K. The ventricular pressure-volume diagram revisited. 25* Sagawa, Circ. Res. 43: 677-687, 1978. K., W. L. Manghan, H. Suga, and K. Sunagawa. 26. Sagawa, Cardiac contraction and pressure-volume relationship. New York: Oxford University, 1988, p. 56-61 and 110-119.
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