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Effect of l -arginine on metabolic recovery of the ischemic myocardium
ORIGINAL
ARTICLES: CARDIOVASCULAR
Effect of L-Arginine on Metabolic Recovery of the Ischemic Myocardium Michel Carrier, MD, Ahmad Khalil, MD, Alain Tourigny, Eng, B. Charles Solymoss, MD, and L. Conrad Pelletier, MD Department of Surgery, Montreal Heart Institute, Montreal, Quebec, Canada
Background. The release of nitric oxide is decreased after myocardial ischemia and reperfusion. Whereas the precursor L-arginine can stimulate the release of nitric oxide, its effect on metabolic recovery after myocardial ischemia is u n k n o w n . Methods. To s t u d y the effect of L-arginine on metabolic recovery after m y o c a r d i a l ischemia, c a r d i o p l e g i a i n f u s i o n , a n d r e p e r f u s i o n , 33 dogs were p l a c e d on c a r d i o p u l m o n a r y b y p a s s a n d s u b j e c t e d to a s e q u e n c e of 30 m i n u t e s of n o r m o t h e r m i c g l o b a l i s c h e m i a , 30 m i n u t e s of w a r m b l o o d c a r d i o p l e g i c arrest, a n d 30 m i n u t e s of r e p e r f u s i o n . A p H p r o b e was i n s e r t e d in the a n t e r i o r w a l l of the left ventricle, and tissue p H was m e a s u r e d t h r o u g h o u t the e x p e r i m e n t . C o r o n a r y b l o o d flow in the left a n t e r i o r d e s c e n d i n g coronary artery and the circumflex c o r o n a r y artery was m e a s u r e d . Blood s a m p l e s from the c o r o n a r y s i n u s were t a k e n to measure b l o o d p H a n d levels of lactate, creatine k i n a s e , and t r o p o n i n T. Results. In the control group of 9 dogs, tissue p H averaged 6.4 ± 0.1, 6.5 --- 0.1, and 6.8 + 0.1 after the end of global ischemia, cardioplegia, and reperfusion, respectively.
T i s s u e p H a v e r a g e d 6.4 __. 0.1, 6.6 --- 0.1, a n d 6.9 --- 0.1, respectively, in the experimental group of 9 animals w i t h 2 mmol/L of L-arginine a d d e d to the cardioplegic solution. Tissue p H averaged 6.2 + 0.1, 6.7 -" 0.1, 7.1 + 0.1, respectively, in the third group of 9 animals that received an additional infusion of t-arginine (10 mg • kg -1 • min -1) d u r i n g reperfusion. Tissue p H recovered faster in groups with L-arginine (p = 0.00001). A hyperemic response of coronary b l o o d flow was s h o w n at reperfusion in animals in the control group only. In 6 dogs, L-NAME (N-nitroarginine m e t h y l ester), an i n h i b i t o r of nitric oxide synthesis, was injected and resulted in a slower p H recovery on reperfusion compared with that of animals that received r-arginine. Conclusions. The a d d i t i o n of L-arginine to the cardioplegic solution and the systemic circulation d u r i n g reperfusion resulted in a significant increase in coronary b l o o d flow d u r i n g cardioplegia infusion and in a faster recovery of myocardial tissue pH, p o s s i b l y b y increasing coronary b l o o d flow through the release of nitric oxide.
-Arginine is an amino acid precursor of nitric oxide, which has b e e n identified as the e n d o t h e l i a l - d e r i v e d relaxing factor. The latter causes substantial vasorelaxation [1], inhibits platelet a d h e s i o n [2] a n d aggregation [3], reduces neutrophil interaction with the e n d o t h e l i u m [4], a n d m a y neutralize superoxide radicals [5]. The former was shown to reduce infarct size, to lower myeloperoxidase activity in the ischemic region, and to preserve endothelial function in an experimental m o d e l of ischemia and reperfusion [61. By p r o m o t i n g endothelial synthesis of nitric oxide, L-arginine may be a simple a n d effective additive to cardioplegic solution that could be useful in clinical practice. The objective of the present study was to evaluate L-arginine a d d e d to w a r m blood cardioplegia in the metabolic recovery of the m y o c a r d i u m after global myocardial ischemia, cardioplegia infusion, and reperfusion. We h y p o t h e s i z e d that a d d i n g L-arginine to w a r m b l o o d cardioplegia m a y i m p r o v e metabolic recovery from isch-
emia by preserving endothelial cell function a n d by p r o m o t i n g the release of nitric oxide. In addition, the effect of infusing L-arginine during myocardial reperfusion was studied.
L
Presented at the Thirty-second Annual Meeting of The Society of Thoracic Surgeons, Orlando, FL, Jan 29-31, 1996. Address reprint requests to Dr Carrier, Montreal Heart Institute, 5000 Belanger St, Montreal, PQ H1T 1C8, Canada. © 1996 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1996;61:1651-7)
Material and Methods The study was p e r f o r m e d in 33 dogs weighing 25 to 30 kg. All animals received h u m a n care in compliance with the " G u i d e for the Care a n d Use of Laboratory A n i m a l s " p u b l i s h e d by the National Institutes of Health (NIH publication 85-23, revised 1985). The animals were anesthetized with s o d i u m p e n t o b a r bital (30 mg/kg) a n d ventilated using a H a r v a r d respirator (Harvard Apparatus, South Natick, MA). After a m e d i a n sternotomy and heparinization (3 mg/kg), the left femoral artery was c a n n u l a t e d for arterial inflow a n d the right atrium, for venous return. The cannulas were connected to a b u b b l e oxygenator (Baxter Healthcare Corp, Irvine, CA) p r i m e d with Ringer's lactate solution. The coronary sinus was c a n n u l a t e d through a transatrial a p p r o a c h for blood sampling, a n d the left ventricle was v e n t e d with a line through the apex. A cannula for cardioplegia delivery was placed in the ascending aorta. 0003-4975/96]$15.00 PII S0003-4975(96)00101-4
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CARRIER ET AL L-ARGININE AND MYOCARDIAL METABOLIC RECOVERY
The systemic temperature was maintained between 28 ° and 30°C throughout the procedure. In the first group of 9 dogs, the cardioplegic solution was made of oxygenated blood diluted 4:1 with a crystalloid solution containing 130 mmol of sodium, 135 mmol of chloride, 3 mmol of calcium, 28 mmol of lactate, 20 g of mannitol, 0.17 g of sodium bicarbonate, and 80 mEq (high concentration) or 34 mEq (low concentration) of KC1 per liter. The cardioplegic solution was maintained between 35 ° and 37°C and was administered at a rate of 150 ml/min. In the second group of 9 animals, 2 mmol of L-arginine was added to 1 L of crystalloid cardioplegic solution. In the third group of 9 dogs, L-arginine was added to the crystalloid solution (2 mmol/L), and an additional infusion of 10 m g . kg 1 . min 1 of r-arginine was injected into the systemic circulation during myocardial reperfusion. L-NAME (N-nitro-arginine methyl ester), a powerful inhibitor of nitric oxide synthesis from L-arginine, was added to the cardioplegic solution (2 mmol/L) and injected at 10 ~ g • kg -1 • rain i during reperfusion in another group of 6 animals. After the institution of cardiopulmonary bypass, global myocardial ischemia was obtained by clamping the ascending aorta for 30 minutes. After the period of global ischemia, the cardioplegic solution was infused over 30 minutes, after which the aorta was unclamped and the heart reperfused for 30 minutes. Interstitial pH was measured in the anterior myocardial wall throughout the periods of ischemia, cardioplegia infusion, and reperfusion. A tissue pH probe (Vascular Technology Inc, North Chelmsford, MA) was used, and a reference electrode was placed in the mouth of the animal to be in contact with the saliva. Both electrodes were connected to an electronic pH-meter unit. Data were computed and analyzed on a personal computer using an expert database program. A thermoneedle was implanted to monitor myocardial temperature, and that measurement was used to correct myocardial pH for temperature changes on the basis of the Nernst equation. Both pH and thermal probes were positioned in the anterior wall of the left ventricle. The pH electrode was calibrated before each experiment using a standard laboratory buffer solution with a pH of 7 at 35°C. Measures of interstitial pH were recorded every 2 seconds throughout the experiment, and averages were calculated for standardized periods of 100 seconds [7]. Blood flow in the left anterior descending coronary artery and in the circumflex coronary artery was m e a s u r e d with an electronic flowmeter (Transonic, Ithaca, NY). Blood samples were withdrawn from the coronary sinus for determination of coronary venous pH and levels of lactate, creatine kinase, and troponin T before and after each period of ischemia, cardioplegia, and reperfusion. Lactate production and creatine kinase release were measured using specific enzymatic methods. Troponin T was measured with a newly developed enzymatic-linked immunosorbent assay (Boehringer Mannheim, Mannhelm, Germany) [8]. The hemoglobin concentration, hematocrit values, and serum potassium levels in the blood cardioplegic solu-
A n n Thorac S u r g 1996;61:1651-7
tion were similar between the three groups. The hemoglobin level averaged 4 +_ 1 g/L, the hematocrit averaged 13% + 2%, and the serum potassium concentration averaged 11 + 2 mEq/L. The data are presented as the mean _+ the standard error. Differences between groups were analyzed using the repeated-measures analysis of variance for a threefactor design (Solo; BMDP Statistical Software Inc, Los Angeles, CA) and the Scheff6 test for intergroup comparisons. Differences between the three cardioplegia groups (cardioplegia effect), changes in variables occurring with time during cardioplegia infusion and reperfusion (time effect), and the interaction between cardioplegia effects and time effects were analyzed. The primary hypothesis tested in this study was that a significant interaction exists between the cardioplegia effect and the time effect, ie, changes in studied variables were not similar over time in the three cardioplegia groups [9[. The level of significance was established at 95% (p ~ 0.05). Results
Effect of Global Ischemia on Interstitial pH and Myocardial Metabolic Markers After 30 minutes of global myocardial ischemia created by cross-clamping the ascending aorta, interstitial myocardial pH was similar in all experimental groups. It averaged 6.4 + 0.1 in the control group, 6.4 + 0.1 in the group with L-arginine added to the cardioplegic solution, 6.2 -+ 0.1 in animals treated with L-arginine during cardioplegia and reperfusion, and 6.2 _~ 0.1 in animals with L-NAME infusion, differences that were not significant. Venous pH and lactate, troponin T, and creatine kinase levels in the coronary sinus blood were similar in all groups at the end of the period of global ischemia (Table 1).
Effect of L-Arginine on Coronary Blood Flow There was no significant difference in absolute value of coronary blood flow in the left anterior descending coronary artery between the control group and the two groups treated with L-arginine (cardioplegia effect, p = 0.06), although changes in blood flow with time throughout the periods of cardioplegia infusion and reperfusion were significant (time effect, p = 0.04). There was also a significant difference in the interaction between cardioplegia effect and time effect (p = 0.00001) between groups. The analysis suggests that changes in blood flow occurred during cardioplegia infusion and reperfusion and that these changes were different for the three cardioplegia groups. Whereas a hyperemic response of coronary blood flow was shown at reperfusion in the control group, blood flow remained stable during reperfusion in animals treated with L-arginine (Fig 1). There were no significant differences in absolute values of coronary blood flow (cardioplegia effect, p = 0.17) or in flow changes with time (time effect, p = 0.37) in the circumflex coronary artery throughout cardioplegia infusion and reperfusion. The interaction between the three cardioplegia groups was significant (interaction between
Ann Thorac Surg 1996;61:1651-7
CARRIER ET AL L-ARGININE AND MYOCARDIAL METABOLIC RECOVERY
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Table 1. Metabolic Markers During Ischemia, Cardioplegia, and Reperfusion" Venous pH
Lactate b (mmol/L)
Troponin T (~,g/L)
Creatine Kinase flU/L)
reperfusion)
7.36 _+ 0.04 7.36 _+ 0.03 7.36 + 0.02
6.6 _+ 0.3 4.6 +_ 0.5 4.8 + 0.7
1.12 ± 0.17 0.76 +_ 0.31 0.55 + 0.14
246 -+ 29 442 _+ 202 177 + 20
reperfusion)
6.98 _+ 0.09 6.93 ± 0.14 7.03 + 0.03
12.6 _+ 1.1 10.9 + 1.9 10.8 + 1.1
1.61 : 0.37 0.93 _+ 0.25 1.21 ± 0.27
309 _+ 41 421 + 176 194 _+ 35
reperfusion)
7.23 ± 0.04 7.21 + 0.02 7.13 ± 0.03
9.1 +_ 0.5 6.9 ÷ 0.5 8.3 + 0.7
1.74 _+ 0.32 1.37 + 0.17 1.46 ÷ 0.31
585 ± 117 504 ± 157 252 ± 28
reperfusion)
7.23 ± 0.04 7.21 _+ 0.03 7.18 +- 0.04
8.1 ± 0.7 5.8 ± 0.6 6.6 -- 0.5
2.14 +_ 0.29 2.1 _+ 0.36 2.2 -+ 0.39
836 + 106 800 -+ 122 561 -+ 90
Group Baseline Control L-Arginine (cardioplegia) L-Arginine (cardioplegia + After 30 minutes of ischemia Control L-Arginine (cardioplegia) L-Arginine (cardioplegia + After cardioplegia Control L-Arginine (cardioplegia) L-Arginine (cardioplegia + After reperfusion Control L-Arginine (cardioplegia) L-Arginine (cardioplegia ~
Data are shown as the mean -+ the standard error, of variance and Scheff6 test (p = 0.02).
b Control group differed significantly (p < 0.05) from groups treated with L-arginine by analysis
c a r d i o p l e g i a effect a n d t i m e effect, p = 0.00001), i n d i c a t i n g t h a t t h e c h a n g e s in c o r o n a r y b l o o d flow w e r e differe n t in e a c h g r o u p d u r i n g c a r d i o p l e g i a i n f u s i o n a n d r e p e r f u s i o n (Fig 2). C o r o n a r y b l o o d flow d u r i n g c a r d i o p l e g i a i n f u s i o n w a s h i g h e r in a n i m a l s t r e a t e d w i t h r arginine. However, control animals displayed a hyperemic response on reperfusion.
s i o n ( c a r d i o p l e g i a effect, p = 0.8). T h i s i n d i c a t e s t h a t interstitial tissue pH decreased significantly during ische m i a b u t r e c o v e r e d f a s t e r d u r i n g r e p e r f u s i o n in a n i m a l s treated with L-arginine compared with the control group (Fig 3).
Effect of Inhibition of Nitric Oxide Synthesis W i t h LNAME
Effect of L-Arginine on Myocardial Tissue p H
T h e effect of i n h i b i t i n g t h e s y n t h e s i s of nitric o x i d e w i t h L - N A M E (6 a n i m a l s ) w a s c o m p a r e d w i t h nitric o x i d e s t i m u l a t i o n w i t h L - a r g i n i n e (9 a n i m a l s ) d u r i n g c a r d i o p l e gia i n j e c t i o n a n d r e p e r f u s i o n . C h a n g e s i n t i s s u e p H w i t h t i m e w e r e s i g n i f i c a n t (p = 0.00001), as w a s t h e i n t e r a c t i o n between the two groups (interaction between cardioplegia effect a n d t i m e effect, p = 0.01), a l t h o u g h a v e r a g e p H values were not different between the two groups
C h a n g e s in t i s s u e p H w i t h t i m e w e r e s i g n i f i c a n t ( t i m e effect, p = 0.00001), as w a s t h e i n t e r a c t i o n b e t w e e n t h e control group and the two groups treated with L-arginine ( i n t e r a c t i o n b e t w e e n c a r d i o p l e g i a effect a n d t i m e effect, p = 0.00001), a l t h o u g h t h e a b s o l u t e p H v a l u e s w e r e n o t d i f f e r e n t in t h e t h r e e g r o u p s t h r o u g h o u t t h e v a r i o u s p e r i o d s of i s c h e m i a , c a r d i o p l e g i a i n f u s i o n , a n d r e p e r f u -
Fig 1. Changes in blood flow in left anterior descending coronary artery. A significant hyperemic response was shown at reperfusion in the control group. (Control = control animals; LArginine (cardio) = animals treated with rarginine during cardioplegia infusion; L-Arginine (cardio + rep) = animals treated with L-arginine during cardioplegia and reperfusion; arrow = myocardial reperfusion.)
cc/min
I00 ---o--- Control ----D--- L-Arginine(cardio)
80
L-Arginine (cardio + rep)
60
40
20
i
.
0 30 Ischemia
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34
.
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38
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42
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46
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50
Cardloplegia
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54
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62
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66
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82
Reperfusion (rain)
.
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86
,
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90
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CARRIER ET AL L-ARGININE A N D MYOCARDIAL METABOLIC RECOVERY
Fig 2. Changes in blood flow in circumflex coronary artery. A significant hyperemic response was shown at reperfusion in the control group. Blood flow was higher during cardioplegia infusion in groups with r-arginine. (arrow = myocardial reperfusion; abbreviations are the same as in Fig 1.)
A n n Thorac S u r g 1996;61:1651-7
cc/min 100
- - o - - - Control
so
----o---
L.Arginine
(cardio)
---a-----
L.Arginine
(cardio
+
rep)
1
60
40
20
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,
i
30
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t
34
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46
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54
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62
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66
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70
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78
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82
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86
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90
(rain)
Reperfusion
Cardioplegia
throughout the various periods of ischemia, cardioplegia, and reperfusion (cardioplegia effect, p = 0.63). This analysis and the data shown in Figure 4 suggest that changes in myocardial tissue pH were similar during the period of cardioplegia infusion but that tissue pH recovered faster during reperfusion in the group with L-arginine compared with animals receiving L-NAME.
According to Nakanishi and co-workers [15], reperfusion with cold blood cardioplegia after normothermic ischemia impairs endothelium-dependent coronary relaxation and is associated with anatomic damage to the endothelium. Thus, blood cardioplegia does not prevent reperfusion injury to the endothelium, a finding suggesting that adjunct therapy may be necessary to prevent damage and dysfunction, c-Arginine, the physiologic precursor of nitric oxide, and donors of nitric oxide were suggested as potential adjuncts to blood cardioplegia. Several studies [10-16] have shown that no or minimal damage to and dysfunction of the endothelium occur during cold and warm ischemic periods but that reperfusion is mainly responsible for structural and functional changes in the coronary endothelium [17]. Sellke and colleagues [18] found that cold potassium cardioplegia impairs endothelium-dependent microvascular relaxation. On the other h a n g Evora and coauthors [19] suggested that the cardioplegic solution itself is not responsible for endothelial damage because relaxation responses of the coronary arteries to acetylcholine re-
Comment Several authors [10-12] have shown that endothelial dysfunction occurs after myocardial ischemia and reperfusion and that the release of nitric oxide is significantly reduced in both in vitro and in vivo experimental models. Pearson and associates [13] reported that global myocardial ischemia and reperfusion impairs the endotheliumdependent relaxation response to aggregating platelets and that G proteins, which normally regulate intracellular endothelial-derived relaxing factor synthesis, are damaged and therefore are probably responsible for the decrease in nitric oxide release [14].
Fig 3. Changes in myocardial tissue pH. Changes were similar between the three groups during ischemia and infusion of cardioplegic solution. The pH recovered faster on reperfusion in L-arginine-treated groups (p ~ 0.00001). (arrow = beginning of cardioplegia infusion; double arrow myocardial reperfusion; abbreviations are the same as in Fig 1.)
i
42
pH
7.2 Control
7.0
---o--
~
L-Arginine
(cardio)
L-Arginine
(cardio
+
rep)
6.8
6.6
6.4
6.2
6.0
• •
t
6
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48
Cardioplegia
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54
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60
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66
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72
Reperfusion
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78
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84 (rain)
"
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90
A n n Thorac S u r g 1996;61:1651-7
CARRIER ET AL L-ARGININE A N D MYOCARDIAL METABOLIC RECOVERY
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Fig 4. Changes in myocardial tissue pH in hearts treated with L-arginine and L-NAME (N-nitro-arginine methyl ester) during cardioplegia infusion and reperfusion. Tissue pH recovered faster on reperfusion in the group treated with L-arginine than in that treated with L-NAME.
pH 7.4 L-NAME 7.2 7.0 6.8 6.6 6.4 6.2 6.0 0
6
12
18
Ischemia
24
30
36
42
48
Cardioplegia
54
60
66
72
78
84
90
Reperfusion (rain)
mained normal in epicardial coronary arteries. Moreover, G proteins responded normally to stimulation by promoting endothelial-derived relaxing factor release and coronary vasorelaxation. Finally, decreased microvascular blood flow and increased neutrophil accumulation in tissues after perfusion with hypothermic hyperkalemic crystalloid cardioplegic solutions and blood reperfusion were observed by Keller and associates [20]. Several groups have investigated the ability of Larginine to protect the coronary endothelium from ischemic and reperfusion damage. L-Arginine administered during ischemia and reperfusion decreased the area of myocardial necrosis and myeloperoxidase activity in the ischemic region, with preservation of the endotheliumdependent relaxation to acetylcholine, in a model of ischemia and myocardial reperfusion in the cat [6]. In the neonatal lamb, L-arginine-supplemented hearts had better recovery of left ventricular function and coronary flow after hypothermic ischemia [1]. In the dog, Sato and co-workers [21] showed that L-arginine used as an adjunct to blood cardioplegia reduced infarct size, restored postischemic systolic and diastolic function in the ischemic and reperfused segment, and may act by recruiting endogenous nitric oxide from the vascular endothelium. Nitric oxide donor agent SPM-5185 improves coronary endothelial and ventricular function after global ischemia, cardioplegia and reperfusion [22]. On the other hand, Matheis [23], Naseem [24], and their colleagues suggested that inhibition of nitric oxide synthesis may protect against myocardial reoxygenation injury. In the present study, blood cardioplegic solution with L-arginine administered after normothermic global ischemia resulted in a higher blood flow in the circumflex coronary artery during cardioplegia infusion and caused a loss of the initial hyperemic response on reperfusion. Myocardial tissue pH recovered faster during reperfusion in L-arginine-treated animals than in the control group and the animals treated with L-NAME. Although troponin T and serum creatine kinase levels were similar in all groups during ischemia, cardioplegia injection, and
reperfusion, serum lactate levels in the coronary sinus were significantly higher in control animals than in those treated with L-arginine. The beneficial effect of L-arginine supplementation and nitric oxide stimulation during cardioplegia infusion and myocardial reperfusion remains controversial. Although blood flow in the circumflex artery was higher in L-arginine-treated animals during cardioplegia infusion, a finding suggesting a higher local release of nitric oxide, blood flow in the left anterior descending coronary artery was not significantly altered. In the dog, basal coronary blood flow is lower in the left anterior descending coronary artery than in the circumflex coronary artery, and small changes may be more difficult to measure accurately. The initial hyperemic response on reperfusion found in control animals suggests that coronary endothelium still released nitric oxide and adenosine, a response that was absent in L-arginine-treated animals [25-28]. Successful repayment of blood flow debt during infusion of cardioplegic solution with c-arginine could explain the absence of a hyperemic reaction in these hearts. Because recovery of myocardial tissue pH was similar in all groups during cardioplegia infusion but faster during reperfusion in hearts supplemented with L-arginine, mechanisms other than increase in coronary blood flow could be involved in the response. It is known that nitric oxide inhibits platelet and neutrophil aggregation and adhesion to subendothelial extracellular matrix and postcapillary veins and that endothelium-derived nitric oxide decreases neutrophil interaction with the endothelium under whole-blood arterial flow conditions [4]. Thus, by synthesizing nitric oxide from the amino-acid precursor L-arginine, vascular endothelial cells play an important role not only in the relaxation of the underlying vascular smooth muscle but also in the modulation of circulating blood cell interaction with the vessel wall [2, 4]. Reperfusion with neutrophil-depleted blood is associated with improved postischemic function after cardioplegic arrest [29, 30], and a decrease in basal nitric oxide release after myocardial ischemia and reperfusion promotes neutro-
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CARRIERET AL L-ARGININEAND MYOCARDIALMETABOLICRECOVERY
Ann Thorac Surg 1996;61:1651-7
phil adhesion to the coronary e n d o t h e l i u m [31]. A d d i n g L-arginine to coronary artery blood significantly attenuates the increase in neutrophil adherence in these experiments [32]. Several other possible mechanisms, including depletion of L-arginine, blockade of recycling of L-citrulline to L-arginine, decreased enzyme activity in nitric oxide synthase, and inhibition of nitric oxide synthesis by oxygen-derived free radicals, can explain the decrease in nitric oxide release with ischemia and reperfusion [31]. A l t h o u g h several questions r e m a i n u n a n s w e r e d , Larginine appears to improve both coronary blood flow during cardioplegia infusion without a hyperemic response at reperfusion a n d recovery of interstitial tissue pH on reperfusion compared with the results in hearts without L-arginine or s u p p l e m e n t e d with the inhibitor of nitric oxide synthesis L-NAME. In conclusion, nitric oxide plays a major role in the regulation of coronary endothelial function a n d vessel wall function u n d e r normal conditions, a regulatory m e c h a n i s m that is lost after ischemia a n d reperfusion. Our data suggest that warm blood cardioplegic solution with L-arginine and systemic blood reperfusion supplem e n t e d with L-arginine improves metabolic recovery of the heart monitored by interstitial tissue pH after global myocardial ischemia.
lium-dependent relaxation to acetylcholine and augments contractile reactivity in vitro. J Clin Invest 1987;79:265-74. Engelman DT, Watanabe M, Engelman RM, et al. Constitutive nitric oxide release is impaired after ischemia and reperfusion. J Thorac Cardiovasc Surg 1995;110:1047-53. Pearson PJ, Pyng JL, Schaff HV. Global myocardial ischemia and reperfusion impair endothelium-dependent relaxation to aggregating platelets in the canine artery. J Thorac Cardiovasc Surg 1992;103:1147-54. Evora PRB, Pearson PJ, Schaff HV. Impaired endotheliumdependent relaxation after coronary reperfusion injury: evidence for G-protein dysfunction. Ann Thorac Surg 1994;57: 1550-6. Nakanishi K, Zhao Z-Q, Vinten-JohansenJ, Lewis JC, McGee DS, Hammon JW Jr. Coronary artery endothelial dysfunction after global ischemia, blood cardioplegia, and reperfusion. Ann Thorac Surg 1994;58:191-9. Dignan RJ, Dyke CM, Abd-Elfattah AS, et al. Coronary artery endothelial cell and smooth muscle dysfunction after global myocardial ischemia. Ann Thorac Surg 1992;53:311-7. Fullerton DA, Mitchell MB, McIntyre RC Jr, et al. Mechanisms of coronary vasomotor dysfunction in the transplanted heart. Ann Thorac Surg 1994;58:86-92. Sellke FW, Shafique T, Schoen FJ, Weintraub RM. Impaired endothelium-dependent coronary microvascular relaxation after cold potassium cardioplegia and reperfusion. J Thorac Cardiovasc Surg 1993;105:52-8. Evora PRB, Pearson PJ, Schaff HV. Crystalloid cardioplegia and hypothermia do not impair endothelium-dependent relaxation or damage vascular smooth muscle of epicardial coronary arteries. J Thorac Cardiovasc Surg 1992;104: 1365-74. Keller MW, Geddes L, Spotnitz W, Kaul S, Duling BR. Microcirculatory dysfunction following perfusion with hyperkalemic, hypothermic, cardioplegic solutions and blood reperfusion. Effects of adenosine. Circulation 1991;84: 2485-94. Sato H, Zhao ZQ, McGeer S, et al. Supplemental L-arginine during cardioplegic arrest and reperfusion avoids regional postischemic injury. J Thorac Cardiovasc Surg 1995;110: 302-14. Nakanishi K, Zhao ZQ, Vinten-Johansen J, Hudspeth DA, McGee DS, Hammon JW. Blood cardioplegia enhanced with nitric oxide donor SPM-5185 counteracts postischemic endothelial and ventricular dysfunction. J Thorac Cardiovasc Surg 1995;109:1146-54. Matheis G, Sherman MP, Buckberg GD, Haybron DM, Young HH, Ignarro J. Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury. Am J Physiol 1992; 262:H616-20. Naseem SA, Kontos MC, Rao PS, Jesse RL, Hess ML, Kukreja RC. Sustained inhibition of nitric oxide by N-nitro-Larginine improves myocardial function following ischemia/ reperfusion in isolated perfused rat heart. J Mol Cell Cardiol 1995;27:419-26. Parent R, Par6 R, Lavall6e M. Contribution of nitric oxide to the dilation of resistance coronary vessels in conscious dogs. Am J Physiol 1992;262:H10-6. Olsson RA, Snow JA, Gentry MK. Adenosine metabolism in canine myocardial reactive hyperemia. Circ Res 1978;42: 358-62. Kostic MM, Schrader J. Role of nitric oxide in reactive hyperemia of the guinea pig heart. Circ Res 1992;70:208-12. Yamabe H, Okumura K, Ishizaka H, Tsuchiya T, Yasue H. Role of endothelium-derived nitric oxide in myocardial reactive hyperemia. Am J Physiol 1992;263:H8-14. Kawata H, Sawatari K, Mayer JE. Evidence for the role of neutrophils in reperfusion injury after cold cardioplegic ischemia in neonatal lambs. J Thorac Cardiovasc Surg 1992; 103:908-18. Breda MA, Drinkwater DC, Laks H, et al. Prevention of reperfusion injury in the neonatal heart with leukocytedependent blood. J Thorac Cardiovasc Surg 1989;97:654-65.
12. 13.
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16. 17. 18.
19.
20.
References 1. Hiramatsu T, Forbess JM, Miura T, Mayer JE. Effects of L-arginine and r-nitro-arginine methyl ester on recovery of neonatal lamb hearts after cold ischemia. J Thorac Cardiovasc Surg 1995;109:81-7. 2. De Graaf JC, Banga JD, Moncada S, Palmer RMJ, de Groot PG, Sixma JJ. Nitric oxide function as an inhibitor of platelet adhesion under flow conditions. Circulation 1992;85:2284-90. 3. Furlong B, Henderson AH, Lewis MJ, Smith JA. Endothelium-derived relaxing factor inhibits in vitro platelet aggregation. Br J Pharmacol 1987;90:687-92. 4. Provost P, Lain JYT, Lacoste L, Merhi Y, Waters D. Endothelium-derived nitric oxide attenuates neutrophil adhesion to endothelium under arterial flow conditions. Arterioscler Thromb 1994;14:331-5. 5. 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. 6. 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. 7. Carrier M, Tourigny A, Thorib6 N, et al. Effects of cold and warm blood cardioplegia assessed by myocardial pH and release of metabolic markers. Ann Thorac Surg 1994;58: 764-7. 8. Katus HA, Schoeppenthau M, Tanzeem A, et al. Noninvasive assessment of perioperative myocardial cell damage by circulating cardiac troponin T. Br Heart J 1991;65: 259-64. 9. Ludbrook J. Repeated measurement and multiple comparisons in cardiovascular research. Cardiovasc Res 1994;28: 303-11. 10. Tsao PS, Aoki N, Lefer DJ, Johnson G, Lefer AM. Time course of endothelial dysfunction and myocardial injury, during myocardial ischemia and reperfusion in the cat. Circulation 1990;82:1402-12. 11. Van Benthuysen KM, McMurtry IF, Horowitz LD. Reperfusion after acute coronary occlusion in dogs impairs endothe-
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30.
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31. Ma X, 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.
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32. Seccombe JF, Pearson PJ, Schaff HV. Oxygen radicalmediated vascular injury selectively inhibits receptordependent release of nitric oxide from canine coronary arteries. J Thorac Cardiovasc Surg 1994;107:505-9.
DISCUSSION DR WILLIAM A. GAY, JR (St. Louis, MO): That was a very interesting study, and I had just one question. Some of your slides suggested that there was an additive effect w h e n you added L-arginine to the reperfusate. This would then suggest that there is a systemic deficit of L-arginine in the preparation. How do you explain the additive effect of L-arginine in the reperfusate? DR CARRIER: We do not know yet how we can explain that, but effectively in some of the slides, the difference between the two groups that received L-arginine was shown just by the end of the period of reperfusion. I am not sure exactly how to explain both, but in most of the experiments that use segmental ischemia, the investigator used to inject L-arginine during reperfusion to show that there is a difference. I do not have any explanation for that at that moment. DR ROBERT A. GUYTON (Atlanta, GA): I enjoyed the paper very much, and it was a very nice presentation. I presume that the total volume of cardioplegia infused was almost twice as high with L-arginine. If that is the case, have you looked at any nonspecific vasodilator during the cardioplegia infusion to determine whether a vasodilator that does not lead to nitric oxide production could have the same beneficial effects, because nitric oxide production can be a double-edged sword? DR CARRIER: No, we have not. The only other group we've used was the r-NAME, and that was supposed to block the nitric oxide synthesis, but we should try to use, for example, just nitroglycerin or other vasodilators. DR GERALD D. BUCKBERG (Los Angeles, CA): Do you think it was an increase in coronary blood flow during your ischemia to that collateral area, or do you think that the r-arginine allowed the endothelium to produce normal nitric oxide to prevent white cell aggregation to the endothelial wall with the subsequent oxygen radical productivity that may follow that? DR CARRIER: 1 think that you have expressed the two theories behind it. The first one is you vasodilate the vessels; the second one is you decrease the interaction between neutrophils and the
vessel wall itself. Some recent data in the literature seem to suggest that the second hypothesis is probably the most interesting. DR RALPH J. DAMIANO (Hershey, PA): I congratulate you. You know you have a good study when you raise more questions than you answer. You did not show the effect of r-NAME on cardioplegic flow. I am very concerned that differences in the amount of cardioplegia delivered may account for some of the differences you found. Could you enlighten us on the effect that r-NAME had on cardioplegic flow? One thing I noticed on your pH slide is that there was no indication that that difference in pH was significant between the L-arginine and L-NAME group. Was it? DR CARRIER: The difference was significant. We added the fourth group just again to show whether trying to block the nitric oxide will make a difference. The other way around would be to use D-arginine, which was not available at that time. I do not have the answer regarding coronary flow. We still have to study it. We just measure the pH in that last group to make sure that we are not in the wrong direction with the r-arginine. The only thing I can tell you is that when we injected the r-NAME during reperfusion we saw an increase in blood pressure of the animals, but I do not know about the blood flow of the coronary arteries; we will know in a couple of weeks or months. DR DAMIANO: We are all familiar with the we find a metabolic deficit after ischemia does not correlate at all with a functional wondering if you happened to measure ventricular function in these groups.
fact that often when and reperfusion, it abnormality. I was any indices of left
DR CARRIER: Yes, we measured in a small group of animals the systolic shortening of the wall itself by microcrystals, and it was preserved in the r-arginine group and decreased by about 40% in the control group. We still need to do left ventricular loops and make sure that the global function is improved, but the segmental function seems to be well protected with r-arginine.
ARTICLES: CARDIOVASCULAR
Effect of L-Arginine on Metabolic Recovery of the Ischemic Myocardium Michel Carrier, MD, Ahmad Khalil, MD, Alain Tourigny, Eng, B. Charles Solymoss, MD, and L. Conrad Pelletier, MD Department of Surgery, Montreal Heart Institute, Montreal, Quebec, Canada
Background. The release of nitric oxide is decreased after myocardial ischemia and reperfusion. Whereas the precursor L-arginine can stimulate the release of nitric oxide, its effect on metabolic recovery after myocardial ischemia is u n k n o w n . Methods. To s t u d y the effect of L-arginine on metabolic recovery after m y o c a r d i a l ischemia, c a r d i o p l e g i a i n f u s i o n , a n d r e p e r f u s i o n , 33 dogs were p l a c e d on c a r d i o p u l m o n a r y b y p a s s a n d s u b j e c t e d to a s e q u e n c e of 30 m i n u t e s of n o r m o t h e r m i c g l o b a l i s c h e m i a , 30 m i n u t e s of w a r m b l o o d c a r d i o p l e g i c arrest, a n d 30 m i n u t e s of r e p e r f u s i o n . A p H p r o b e was i n s e r t e d in the a n t e r i o r w a l l of the left ventricle, and tissue p H was m e a s u r e d t h r o u g h o u t the e x p e r i m e n t . C o r o n a r y b l o o d flow in the left a n t e r i o r d e s c e n d i n g coronary artery and the circumflex c o r o n a r y artery was m e a s u r e d . Blood s a m p l e s from the c o r o n a r y s i n u s were t a k e n to measure b l o o d p H a n d levels of lactate, creatine k i n a s e , and t r o p o n i n T. Results. In the control group of 9 dogs, tissue p H averaged 6.4 ± 0.1, 6.5 --- 0.1, and 6.8 + 0.1 after the end of global ischemia, cardioplegia, and reperfusion, respectively.
T i s s u e p H a v e r a g e d 6.4 __. 0.1, 6.6 --- 0.1, a n d 6.9 --- 0.1, respectively, in the experimental group of 9 animals w i t h 2 mmol/L of L-arginine a d d e d to the cardioplegic solution. Tissue p H averaged 6.2 + 0.1, 6.7 -" 0.1, 7.1 + 0.1, respectively, in the third group of 9 animals that received an additional infusion of t-arginine (10 mg • kg -1 • min -1) d u r i n g reperfusion. Tissue p H recovered faster in groups with L-arginine (p = 0.00001). A hyperemic response of coronary b l o o d flow was s h o w n at reperfusion in animals in the control group only. In 6 dogs, L-NAME (N-nitroarginine m e t h y l ester), an i n h i b i t o r of nitric oxide synthesis, was injected and resulted in a slower p H recovery on reperfusion compared with that of animals that received r-arginine. Conclusions. The a d d i t i o n of L-arginine to the cardioplegic solution and the systemic circulation d u r i n g reperfusion resulted in a significant increase in coronary b l o o d flow d u r i n g cardioplegia infusion and in a faster recovery of myocardial tissue pH, p o s s i b l y b y increasing coronary b l o o d flow through the release of nitric oxide.
-Arginine is an amino acid precursor of nitric oxide, which has b e e n identified as the e n d o t h e l i a l - d e r i v e d relaxing factor. The latter causes substantial vasorelaxation [1], inhibits platelet a d h e s i o n [2] a n d aggregation [3], reduces neutrophil interaction with the e n d o t h e l i u m [4], a n d m a y neutralize superoxide radicals [5]. The former was shown to reduce infarct size, to lower myeloperoxidase activity in the ischemic region, and to preserve endothelial function in an experimental m o d e l of ischemia and reperfusion [61. By p r o m o t i n g endothelial synthesis of nitric oxide, L-arginine may be a simple a n d effective additive to cardioplegic solution that could be useful in clinical practice. The objective of the present study was to evaluate L-arginine a d d e d to w a r m blood cardioplegia in the metabolic recovery of the m y o c a r d i u m after global myocardial ischemia, cardioplegia infusion, and reperfusion. We h y p o t h e s i z e d that a d d i n g L-arginine to w a r m b l o o d cardioplegia m a y i m p r o v e metabolic recovery from isch-
emia by preserving endothelial cell function a n d by p r o m o t i n g the release of nitric oxide. In addition, the effect of infusing L-arginine during myocardial reperfusion was studied.
L
Presented at the Thirty-second Annual Meeting of The Society of Thoracic Surgeons, Orlando, FL, Jan 29-31, 1996. Address reprint requests to Dr Carrier, Montreal Heart Institute, 5000 Belanger St, Montreal, PQ H1T 1C8, Canada. © 1996 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1996;61:1651-7)
Material and Methods The study was p e r f o r m e d in 33 dogs weighing 25 to 30 kg. All animals received h u m a n care in compliance with the " G u i d e for the Care a n d Use of Laboratory A n i m a l s " p u b l i s h e d by the National Institutes of Health (NIH publication 85-23, revised 1985). The animals were anesthetized with s o d i u m p e n t o b a r bital (30 mg/kg) a n d ventilated using a H a r v a r d respirator (Harvard Apparatus, South Natick, MA). After a m e d i a n sternotomy and heparinization (3 mg/kg), the left femoral artery was c a n n u l a t e d for arterial inflow a n d the right atrium, for venous return. The cannulas were connected to a b u b b l e oxygenator (Baxter Healthcare Corp, Irvine, CA) p r i m e d with Ringer's lactate solution. The coronary sinus was c a n n u l a t e d through a transatrial a p p r o a c h for blood sampling, a n d the left ventricle was v e n t e d with a line through the apex. A cannula for cardioplegia delivery was placed in the ascending aorta. 0003-4975/96]$15.00 PII S0003-4975(96)00101-4
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CARRIER ET AL L-ARGININE AND MYOCARDIAL METABOLIC RECOVERY
The systemic temperature was maintained between 28 ° and 30°C throughout the procedure. In the first group of 9 dogs, the cardioplegic solution was made of oxygenated blood diluted 4:1 with a crystalloid solution containing 130 mmol of sodium, 135 mmol of chloride, 3 mmol of calcium, 28 mmol of lactate, 20 g of mannitol, 0.17 g of sodium bicarbonate, and 80 mEq (high concentration) or 34 mEq (low concentration) of KC1 per liter. The cardioplegic solution was maintained between 35 ° and 37°C and was administered at a rate of 150 ml/min. In the second group of 9 animals, 2 mmol of L-arginine was added to 1 L of crystalloid cardioplegic solution. In the third group of 9 dogs, L-arginine was added to the crystalloid solution (2 mmol/L), and an additional infusion of 10 m g . kg 1 . min 1 of r-arginine was injected into the systemic circulation during myocardial reperfusion. L-NAME (N-nitro-arginine methyl ester), a powerful inhibitor of nitric oxide synthesis from L-arginine, was added to the cardioplegic solution (2 mmol/L) and injected at 10 ~ g • kg -1 • rain i during reperfusion in another group of 6 animals. After the institution of cardiopulmonary bypass, global myocardial ischemia was obtained by clamping the ascending aorta for 30 minutes. After the period of global ischemia, the cardioplegic solution was infused over 30 minutes, after which the aorta was unclamped and the heart reperfused for 30 minutes. Interstitial pH was measured in the anterior myocardial wall throughout the periods of ischemia, cardioplegia infusion, and reperfusion. A tissue pH probe (Vascular Technology Inc, North Chelmsford, MA) was used, and a reference electrode was placed in the mouth of the animal to be in contact with the saliva. Both electrodes were connected to an electronic pH-meter unit. Data were computed and analyzed on a personal computer using an expert database program. A thermoneedle was implanted to monitor myocardial temperature, and that measurement was used to correct myocardial pH for temperature changes on the basis of the Nernst equation. Both pH and thermal probes were positioned in the anterior wall of the left ventricle. The pH electrode was calibrated before each experiment using a standard laboratory buffer solution with a pH of 7 at 35°C. Measures of interstitial pH were recorded every 2 seconds throughout the experiment, and averages were calculated for standardized periods of 100 seconds [7]. Blood flow in the left anterior descending coronary artery and in the circumflex coronary artery was m e a s u r e d with an electronic flowmeter (Transonic, Ithaca, NY). Blood samples were withdrawn from the coronary sinus for determination of coronary venous pH and levels of lactate, creatine kinase, and troponin T before and after each period of ischemia, cardioplegia, and reperfusion. Lactate production and creatine kinase release were measured using specific enzymatic methods. Troponin T was measured with a newly developed enzymatic-linked immunosorbent assay (Boehringer Mannheim, Mannhelm, Germany) [8]. The hemoglobin concentration, hematocrit values, and serum potassium levels in the blood cardioplegic solu-
A n n Thorac S u r g 1996;61:1651-7
tion were similar between the three groups. The hemoglobin level averaged 4 +_ 1 g/L, the hematocrit averaged 13% + 2%, and the serum potassium concentration averaged 11 + 2 mEq/L. The data are presented as the mean _+ the standard error. Differences between groups were analyzed using the repeated-measures analysis of variance for a threefactor design (Solo; BMDP Statistical Software Inc, Los Angeles, CA) and the Scheff6 test for intergroup comparisons. Differences between the three cardioplegia groups (cardioplegia effect), changes in variables occurring with time during cardioplegia infusion and reperfusion (time effect), and the interaction between cardioplegia effects and time effects were analyzed. The primary hypothesis tested in this study was that a significant interaction exists between the cardioplegia effect and the time effect, ie, changes in studied variables were not similar over time in the three cardioplegia groups [9[. The level of significance was established at 95% (p ~ 0.05). Results
Effect of Global Ischemia on Interstitial pH and Myocardial Metabolic Markers After 30 minutes of global myocardial ischemia created by cross-clamping the ascending aorta, interstitial myocardial pH was similar in all experimental groups. It averaged 6.4 + 0.1 in the control group, 6.4 + 0.1 in the group with L-arginine added to the cardioplegic solution, 6.2 -+ 0.1 in animals treated with L-arginine during cardioplegia and reperfusion, and 6.2 _~ 0.1 in animals with L-NAME infusion, differences that were not significant. Venous pH and lactate, troponin T, and creatine kinase levels in the coronary sinus blood were similar in all groups at the end of the period of global ischemia (Table 1).
Effect of L-Arginine on Coronary Blood Flow There was no significant difference in absolute value of coronary blood flow in the left anterior descending coronary artery between the control group and the two groups treated with L-arginine (cardioplegia effect, p = 0.06), although changes in blood flow with time throughout the periods of cardioplegia infusion and reperfusion were significant (time effect, p = 0.04). There was also a significant difference in the interaction between cardioplegia effect and time effect (p = 0.00001) between groups. The analysis suggests that changes in blood flow occurred during cardioplegia infusion and reperfusion and that these changes were different for the three cardioplegia groups. Whereas a hyperemic response of coronary blood flow was shown at reperfusion in the control group, blood flow remained stable during reperfusion in animals treated with L-arginine (Fig 1). There were no significant differences in absolute values of coronary blood flow (cardioplegia effect, p = 0.17) or in flow changes with time (time effect, p = 0.37) in the circumflex coronary artery throughout cardioplegia infusion and reperfusion. The interaction between the three cardioplegia groups was significant (interaction between
Ann Thorac Surg 1996;61:1651-7
CARRIER ET AL L-ARGININE AND MYOCARDIAL METABOLIC RECOVERY
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Table 1. Metabolic Markers During Ischemia, Cardioplegia, and Reperfusion" Venous pH
Lactate b (mmol/L)
Troponin T (~,g/L)
Creatine Kinase flU/L)
reperfusion)
7.36 _+ 0.04 7.36 _+ 0.03 7.36 + 0.02
6.6 _+ 0.3 4.6 +_ 0.5 4.8 + 0.7
1.12 ± 0.17 0.76 +_ 0.31 0.55 + 0.14
246 -+ 29 442 _+ 202 177 + 20
reperfusion)
6.98 _+ 0.09 6.93 ± 0.14 7.03 + 0.03
12.6 _+ 1.1 10.9 + 1.9 10.8 + 1.1
1.61 : 0.37 0.93 _+ 0.25 1.21 ± 0.27
309 _+ 41 421 + 176 194 _+ 35
reperfusion)
7.23 ± 0.04 7.21 + 0.02 7.13 ± 0.03
9.1 +_ 0.5 6.9 ÷ 0.5 8.3 + 0.7
1.74 _+ 0.32 1.37 + 0.17 1.46 ÷ 0.31
585 ± 117 504 ± 157 252 ± 28
reperfusion)
7.23 ± 0.04 7.21 _+ 0.03 7.18 +- 0.04
8.1 ± 0.7 5.8 ± 0.6 6.6 -- 0.5
2.14 +_ 0.29 2.1 _+ 0.36 2.2 -+ 0.39
836 + 106 800 -+ 122 561 -+ 90
Group Baseline Control L-Arginine (cardioplegia) L-Arginine (cardioplegia + After 30 minutes of ischemia Control L-Arginine (cardioplegia) L-Arginine (cardioplegia + After cardioplegia Control L-Arginine (cardioplegia) L-Arginine (cardioplegia + After reperfusion Control L-Arginine (cardioplegia) L-Arginine (cardioplegia ~
Data are shown as the mean -+ the standard error, of variance and Scheff6 test (p = 0.02).
b Control group differed significantly (p < 0.05) from groups treated with L-arginine by analysis
c a r d i o p l e g i a effect a n d t i m e effect, p = 0.00001), i n d i c a t i n g t h a t t h e c h a n g e s in c o r o n a r y b l o o d flow w e r e differe n t in e a c h g r o u p d u r i n g c a r d i o p l e g i a i n f u s i o n a n d r e p e r f u s i o n (Fig 2). C o r o n a r y b l o o d flow d u r i n g c a r d i o p l e g i a i n f u s i o n w a s h i g h e r in a n i m a l s t r e a t e d w i t h r arginine. However, control animals displayed a hyperemic response on reperfusion.
s i o n ( c a r d i o p l e g i a effect, p = 0.8). T h i s i n d i c a t e s t h a t interstitial tissue pH decreased significantly during ische m i a b u t r e c o v e r e d f a s t e r d u r i n g r e p e r f u s i o n in a n i m a l s treated with L-arginine compared with the control group (Fig 3).
Effect of Inhibition of Nitric Oxide Synthesis W i t h LNAME
Effect of L-Arginine on Myocardial Tissue p H
T h e effect of i n h i b i t i n g t h e s y n t h e s i s of nitric o x i d e w i t h L - N A M E (6 a n i m a l s ) w a s c o m p a r e d w i t h nitric o x i d e s t i m u l a t i o n w i t h L - a r g i n i n e (9 a n i m a l s ) d u r i n g c a r d i o p l e gia i n j e c t i o n a n d r e p e r f u s i o n . C h a n g e s i n t i s s u e p H w i t h t i m e w e r e s i g n i f i c a n t (p = 0.00001), as w a s t h e i n t e r a c t i o n between the two groups (interaction between cardioplegia effect a n d t i m e effect, p = 0.01), a l t h o u g h a v e r a g e p H values were not different between the two groups
C h a n g e s in t i s s u e p H w i t h t i m e w e r e s i g n i f i c a n t ( t i m e effect, p = 0.00001), as w a s t h e i n t e r a c t i o n b e t w e e n t h e control group and the two groups treated with L-arginine ( i n t e r a c t i o n b e t w e e n c a r d i o p l e g i a effect a n d t i m e effect, p = 0.00001), a l t h o u g h t h e a b s o l u t e p H v a l u e s w e r e n o t d i f f e r e n t in t h e t h r e e g r o u p s t h r o u g h o u t t h e v a r i o u s p e r i o d s of i s c h e m i a , c a r d i o p l e g i a i n f u s i o n , a n d r e p e r f u -
Fig 1. Changes in blood flow in left anterior descending coronary artery. A significant hyperemic response was shown at reperfusion in the control group. (Control = control animals; LArginine (cardio) = animals treated with rarginine during cardioplegia infusion; L-Arginine (cardio + rep) = animals treated with L-arginine during cardioplegia and reperfusion; arrow = myocardial reperfusion.)
cc/min
I00 ---o--- Control ----D--- L-Arginine(cardio)
80
L-Arginine (cardio + rep)
60
40
20
i
.
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.
i
86
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CARRIER ET AL L-ARGININE A N D MYOCARDIAL METABOLIC RECOVERY
Fig 2. Changes in blood flow in circumflex coronary artery. A significant hyperemic response was shown at reperfusion in the control group. Blood flow was higher during cardioplegia infusion in groups with r-arginine. (arrow = myocardial reperfusion; abbreviations are the same as in Fig 1.)
A n n Thorac S u r g 1996;61:1651-7
cc/min 100
- - o - - - Control
so
----o---
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82
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throughout the various periods of ischemia, cardioplegia, and reperfusion (cardioplegia effect, p = 0.63). This analysis and the data shown in Figure 4 suggest that changes in myocardial tissue pH were similar during the period of cardioplegia infusion but that tissue pH recovered faster during reperfusion in the group with L-arginine compared with animals receiving L-NAME.
According to Nakanishi and co-workers [15], reperfusion with cold blood cardioplegia after normothermic ischemia impairs endothelium-dependent coronary relaxation and is associated with anatomic damage to the endothelium. Thus, blood cardioplegia does not prevent reperfusion injury to the endothelium, a finding suggesting that adjunct therapy may be necessary to prevent damage and dysfunction, c-Arginine, the physiologic precursor of nitric oxide, and donors of nitric oxide were suggested as potential adjuncts to blood cardioplegia. Several studies [10-16] have shown that no or minimal damage to and dysfunction of the endothelium occur during cold and warm ischemic periods but that reperfusion is mainly responsible for structural and functional changes in the coronary endothelium [17]. Sellke and colleagues [18] found that cold potassium cardioplegia impairs endothelium-dependent microvascular relaxation. On the other h a n g Evora and coauthors [19] suggested that the cardioplegic solution itself is not responsible for endothelial damage because relaxation responses of the coronary arteries to acetylcholine re-
Comment Several authors [10-12] have shown that endothelial dysfunction occurs after myocardial ischemia and reperfusion and that the release of nitric oxide is significantly reduced in both in vitro and in vivo experimental models. Pearson and associates [13] reported that global myocardial ischemia and reperfusion impairs the endotheliumdependent relaxation response to aggregating platelets and that G proteins, which normally regulate intracellular endothelial-derived relaxing factor synthesis, are damaged and therefore are probably responsible for the decrease in nitric oxide release [14].
Fig 3. Changes in myocardial tissue pH. Changes were similar between the three groups during ischemia and infusion of cardioplegic solution. The pH recovered faster on reperfusion in L-arginine-treated groups (p ~ 0.00001). (arrow = beginning of cardioplegia infusion; double arrow myocardial reperfusion; abbreviations are the same as in Fig 1.)
i
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A n n Thorac S u r g 1996;61:1651-7
CARRIER ET AL L-ARGININE A N D MYOCARDIAL METABOLIC RECOVERY
1655
Fig 4. Changes in myocardial tissue pH in hearts treated with L-arginine and L-NAME (N-nitro-arginine methyl ester) during cardioplegia infusion and reperfusion. Tissue pH recovered faster on reperfusion in the group treated with L-arginine than in that treated with L-NAME.
pH 7.4 L-NAME 7.2 7.0 6.8 6.6 6.4 6.2 6.0 0
6
12
18
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24
30
36
42
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Reperfusion (rain)
mained normal in epicardial coronary arteries. Moreover, G proteins responded normally to stimulation by promoting endothelial-derived relaxing factor release and coronary vasorelaxation. Finally, decreased microvascular blood flow and increased neutrophil accumulation in tissues after perfusion with hypothermic hyperkalemic crystalloid cardioplegic solutions and blood reperfusion were observed by Keller and associates [20]. Several groups have investigated the ability of Larginine to protect the coronary endothelium from ischemic and reperfusion damage. L-Arginine administered during ischemia and reperfusion decreased the area of myocardial necrosis and myeloperoxidase activity in the ischemic region, with preservation of the endotheliumdependent relaxation to acetylcholine, in a model of ischemia and myocardial reperfusion in the cat [6]. In the neonatal lamb, L-arginine-supplemented hearts had better recovery of left ventricular function and coronary flow after hypothermic ischemia [1]. In the dog, Sato and co-workers [21] showed that L-arginine used as an adjunct to blood cardioplegia reduced infarct size, restored postischemic systolic and diastolic function in the ischemic and reperfused segment, and may act by recruiting endogenous nitric oxide from the vascular endothelium. Nitric oxide donor agent SPM-5185 improves coronary endothelial and ventricular function after global ischemia, cardioplegia and reperfusion [22]. On the other hand, Matheis [23], Naseem [24], and their colleagues suggested that inhibition of nitric oxide synthesis may protect against myocardial reoxygenation injury. In the present study, blood cardioplegic solution with L-arginine administered after normothermic global ischemia resulted in a higher blood flow in the circumflex coronary artery during cardioplegia infusion and caused a loss of the initial hyperemic response on reperfusion. Myocardial tissue pH recovered faster during reperfusion in L-arginine-treated animals than in the control group and the animals treated with L-NAME. Although troponin T and serum creatine kinase levels were similar in all groups during ischemia, cardioplegia injection, and
reperfusion, serum lactate levels in the coronary sinus were significantly higher in control animals than in those treated with L-arginine. The beneficial effect of L-arginine supplementation and nitric oxide stimulation during cardioplegia infusion and myocardial reperfusion remains controversial. Although blood flow in the circumflex artery was higher in L-arginine-treated animals during cardioplegia infusion, a finding suggesting a higher local release of nitric oxide, blood flow in the left anterior descending coronary artery was not significantly altered. In the dog, basal coronary blood flow is lower in the left anterior descending coronary artery than in the circumflex coronary artery, and small changes may be more difficult to measure accurately. The initial hyperemic response on reperfusion found in control animals suggests that coronary endothelium still released nitric oxide and adenosine, a response that was absent in L-arginine-treated animals [25-28]. Successful repayment of blood flow debt during infusion of cardioplegic solution with c-arginine could explain the absence of a hyperemic reaction in these hearts. Because recovery of myocardial tissue pH was similar in all groups during cardioplegia infusion but faster during reperfusion in hearts supplemented with L-arginine, mechanisms other than increase in coronary blood flow could be involved in the response. It is known that nitric oxide inhibits platelet and neutrophil aggregation and adhesion to subendothelial extracellular matrix and postcapillary veins and that endothelium-derived nitric oxide decreases neutrophil interaction with the endothelium under whole-blood arterial flow conditions [4]. Thus, by synthesizing nitric oxide from the amino-acid precursor L-arginine, vascular endothelial cells play an important role not only in the relaxation of the underlying vascular smooth muscle but also in the modulation of circulating blood cell interaction with the vessel wall [2, 4]. Reperfusion with neutrophil-depleted blood is associated with improved postischemic function after cardioplegic arrest [29, 30], and a decrease in basal nitric oxide release after myocardial ischemia and reperfusion promotes neutro-
1656
CARRIERET AL L-ARGININEAND MYOCARDIALMETABOLICRECOVERY
Ann Thorac Surg 1996;61:1651-7
phil adhesion to the coronary e n d o t h e l i u m [31]. A d d i n g L-arginine to coronary artery blood significantly attenuates the increase in neutrophil adherence in these experiments [32]. Several other possible mechanisms, including depletion of L-arginine, blockade of recycling of L-citrulline to L-arginine, decreased enzyme activity in nitric oxide synthase, and inhibition of nitric oxide synthesis by oxygen-derived free radicals, can explain the decrease in nitric oxide release with ischemia and reperfusion [31]. A l t h o u g h several questions r e m a i n u n a n s w e r e d , Larginine appears to improve both coronary blood flow during cardioplegia infusion without a hyperemic response at reperfusion a n d recovery of interstitial tissue pH on reperfusion compared with the results in hearts without L-arginine or s u p p l e m e n t e d with the inhibitor of nitric oxide synthesis L-NAME. In conclusion, nitric oxide plays a major role in the regulation of coronary endothelial function a n d vessel wall function u n d e r normal conditions, a regulatory m e c h a n i s m that is lost after ischemia a n d reperfusion. Our data suggest that warm blood cardioplegic solution with L-arginine and systemic blood reperfusion supplem e n t e d with L-arginine improves metabolic recovery of the heart monitored by interstitial tissue pH after global myocardial ischemia.
lium-dependent relaxation to acetylcholine and augments contractile reactivity in vitro. J Clin Invest 1987;79:265-74. Engelman DT, Watanabe M, Engelman RM, et al. Constitutive nitric oxide release is impaired after ischemia and reperfusion. J Thorac Cardiovasc Surg 1995;110:1047-53. Pearson PJ, Pyng JL, Schaff HV. Global myocardial ischemia and reperfusion impair endothelium-dependent relaxation to aggregating platelets in the canine artery. J Thorac Cardiovasc Surg 1992;103:1147-54. Evora PRB, Pearson PJ, Schaff HV. Impaired endotheliumdependent relaxation after coronary reperfusion injury: evidence for G-protein dysfunction. Ann Thorac Surg 1994;57: 1550-6. Nakanishi K, Zhao Z-Q, Vinten-JohansenJ, Lewis JC, McGee DS, Hammon JW Jr. Coronary artery endothelial dysfunction after global ischemia, blood cardioplegia, and reperfusion. Ann Thorac Surg 1994;58:191-9. Dignan RJ, Dyke CM, Abd-Elfattah AS, et al. Coronary artery endothelial cell and smooth muscle dysfunction after global myocardial ischemia. Ann Thorac Surg 1992;53:311-7. Fullerton DA, Mitchell MB, McIntyre RC Jr, et al. Mechanisms of coronary vasomotor dysfunction in the transplanted heart. Ann Thorac Surg 1994;58:86-92. Sellke FW, Shafique T, Schoen FJ, Weintraub RM. Impaired endothelium-dependent coronary microvascular relaxation after cold potassium cardioplegia and reperfusion. J Thorac Cardiovasc Surg 1993;105:52-8. Evora PRB, Pearson PJ, Schaff HV. Crystalloid cardioplegia and hypothermia do not impair endothelium-dependent relaxation or damage vascular smooth muscle of epicardial coronary arteries. J Thorac Cardiovasc Surg 1992;104: 1365-74. Keller MW, Geddes L, Spotnitz W, Kaul S, Duling BR. Microcirculatory dysfunction following perfusion with hyperkalemic, hypothermic, cardioplegic solutions and blood reperfusion. Effects of adenosine. Circulation 1991;84: 2485-94. Sato H, Zhao ZQ, McGeer S, et al. Supplemental L-arginine during cardioplegic arrest and reperfusion avoids regional postischemic injury. J Thorac Cardiovasc Surg 1995;110: 302-14. Nakanishi K, Zhao ZQ, Vinten-Johansen J, Hudspeth DA, McGee DS, Hammon JW. Blood cardioplegia enhanced with nitric oxide donor SPM-5185 counteracts postischemic endothelial and ventricular dysfunction. J Thorac Cardiovasc Surg 1995;109:1146-54. Matheis G, Sherman MP, Buckberg GD, Haybron DM, Young HH, Ignarro J. Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury. Am J Physiol 1992; 262:H616-20. Naseem SA, Kontos MC, Rao PS, Jesse RL, Hess ML, Kukreja RC. Sustained inhibition of nitric oxide by N-nitro-Larginine improves myocardial function following ischemia/ reperfusion in isolated perfused rat heart. J Mol Cell Cardiol 1995;27:419-26. Parent R, Par6 R, Lavall6e M. Contribution of nitric oxide to the dilation of resistance coronary vessels in conscious dogs. Am J Physiol 1992;262:H10-6. Olsson RA, Snow JA, Gentry MK. Adenosine metabolism in canine myocardial reactive hyperemia. Circ Res 1978;42: 358-62. Kostic MM, Schrader J. Role of nitric oxide in reactive hyperemia of the guinea pig heart. Circ Res 1992;70:208-12. Yamabe H, Okumura K, Ishizaka H, Tsuchiya T, Yasue H. Role of endothelium-derived nitric oxide in myocardial reactive hyperemia. Am J Physiol 1992;263:H8-14. Kawata H, Sawatari K, Mayer JE. Evidence for the role of neutrophils in reperfusion injury after cold cardioplegic ischemia in neonatal lambs. J Thorac Cardiovasc Surg 1992; 103:908-18. Breda MA, Drinkwater DC, Laks H, et al. Prevention of reperfusion injury in the neonatal heart with leukocytedependent blood. J Thorac Cardiovasc Surg 1989;97:654-65.
12. 13.
14.
15.
16. 17. 18.
19.
20.
References 1. Hiramatsu T, Forbess JM, Miura T, Mayer JE. Effects of L-arginine and r-nitro-arginine methyl ester on recovery of neonatal lamb hearts after cold ischemia. J Thorac Cardiovasc Surg 1995;109:81-7. 2. De Graaf JC, Banga JD, Moncada S, Palmer RMJ, de Groot PG, Sixma JJ. Nitric oxide function as an inhibitor of platelet adhesion under flow conditions. Circulation 1992;85:2284-90. 3. Furlong B, Henderson AH, Lewis MJ, Smith JA. Endothelium-derived relaxing factor inhibits in vitro platelet aggregation. Br J Pharmacol 1987;90:687-92. 4. Provost P, Lain JYT, Lacoste L, Merhi Y, Waters D. Endothelium-derived nitric oxide attenuates neutrophil adhesion to endothelium under arterial flow conditions. Arterioscler Thromb 1994;14:331-5. 5. 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. 6. 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. 7. Carrier M, Tourigny A, Thorib6 N, et al. Effects of cold and warm blood cardioplegia assessed by myocardial pH and release of metabolic markers. Ann Thorac Surg 1994;58: 764-7. 8. Katus HA, Schoeppenthau M, Tanzeem A, et al. Noninvasive assessment of perioperative myocardial cell damage by circulating cardiac troponin T. Br Heart J 1991;65: 259-64. 9. Ludbrook J. Repeated measurement and multiple comparisons in cardiovascular research. Cardiovasc Res 1994;28: 303-11. 10. Tsao PS, Aoki N, Lefer DJ, Johnson G, Lefer AM. Time course of endothelial dysfunction and myocardial injury, during myocardial ischemia and reperfusion in the cat. Circulation 1990;82:1402-12. 11. Van Benthuysen KM, McMurtry IF, Horowitz LD. Reperfusion after acute coronary occlusion in dogs impairs endothe-
21.
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31. Ma X, 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.
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32. Seccombe JF, Pearson PJ, Schaff HV. Oxygen radicalmediated vascular injury selectively inhibits receptordependent release of nitric oxide from canine coronary arteries. J Thorac Cardiovasc Surg 1994;107:505-9.
DISCUSSION DR WILLIAM A. GAY, JR (St. Louis, MO): That was a very interesting study, and I had just one question. Some of your slides suggested that there was an additive effect w h e n you added L-arginine to the reperfusate. This would then suggest that there is a systemic deficit of L-arginine in the preparation. How do you explain the additive effect of L-arginine in the reperfusate? DR CARRIER: We do not know yet how we can explain that, but effectively in some of the slides, the difference between the two groups that received L-arginine was shown just by the end of the period of reperfusion. I am not sure exactly how to explain both, but in most of the experiments that use segmental ischemia, the investigator used to inject L-arginine during reperfusion to show that there is a difference. I do not have any explanation for that at that moment. DR ROBERT A. GUYTON (Atlanta, GA): I enjoyed the paper very much, and it was a very nice presentation. I presume that the total volume of cardioplegia infused was almost twice as high with L-arginine. If that is the case, have you looked at any nonspecific vasodilator during the cardioplegia infusion to determine whether a vasodilator that does not lead to nitric oxide production could have the same beneficial effects, because nitric oxide production can be a double-edged sword? DR CARRIER: No, we have not. The only other group we've used was the r-NAME, and that was supposed to block the nitric oxide synthesis, but we should try to use, for example, just nitroglycerin or other vasodilators. DR GERALD D. BUCKBERG (Los Angeles, CA): Do you think it was an increase in coronary blood flow during your ischemia to that collateral area, or do you think that the r-arginine allowed the endothelium to produce normal nitric oxide to prevent white cell aggregation to the endothelial wall with the subsequent oxygen radical productivity that may follow that? DR CARRIER: 1 think that you have expressed the two theories behind it. The first one is you vasodilate the vessels; the second one is you decrease the interaction between neutrophils and the
vessel wall itself. Some recent data in the literature seem to suggest that the second hypothesis is probably the most interesting. DR RALPH J. DAMIANO (Hershey, PA): I congratulate you. You know you have a good study when you raise more questions than you answer. You did not show the effect of r-NAME on cardioplegic flow. I am very concerned that differences in the amount of cardioplegia delivered may account for some of the differences you found. Could you enlighten us on the effect that r-NAME had on cardioplegic flow? One thing I noticed on your pH slide is that there was no indication that that difference in pH was significant between the L-arginine and L-NAME group. Was it? DR CARRIER: The difference was significant. We added the fourth group just again to show whether trying to block the nitric oxide will make a difference. The other way around would be to use D-arginine, which was not available at that time. I do not have the answer regarding coronary flow. We still have to study it. We just measure the pH in that last group to make sure that we are not in the wrong direction with the r-arginine. The only thing I can tell you is that when we injected the r-NAME during reperfusion we saw an increase in blood pressure of the animals, but I do not know about the blood flow of the coronary arteries; we will know in a couple of weeks or months. DR DAMIANO: We are all familiar with the we find a metabolic deficit after ischemia does not correlate at all with a functional wondering if you happened to measure ventricular function in these groups.
fact that often when and reperfusion, it abnormality. I was any indices of left
DR CARRIER: Yes, we measured in a small group of animals the systolic shortening of the wall itself by microcrystals, and it was preserved in the r-arginine group and decreased by about 40% in the control group. We still need to do left ventricular loops and make sure that the global function is improved, but the segmental function seems to be well protected with r-arginine.