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Ann Thorac Surg 1995;60:1187-1192
© 1995 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Effect of L-Arginine Cardioplegia on Recovery of Neonatal Lamb Hearts After 2 Hours of Cold Ischemia

Department of Cardiac Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. Despite hypothermia and cardioplegia, myocardial ischemia followed by reperfusion results in both ventricular and endothelial dysfunction. The endothelial dysfunction is characterized by a reduced response to acetylcholine, which implies a reduced ability of the endothelium to release nitric oxide after hypothermic ischemia and reperfusion. We have previously demonstrated that infusion of the nitric oxide precursor L-arginine only during reperfusion after hypothermic ischemia significantly improves the recovery of ventricular function and results in an increased vasodilator response to the infusion of acetylcholine. In contrast, other investigators have found that nitric oxide has deleterious effects during postischemic reperfusion.

Methods. In the current experiments we have further examined the role of endothelial production of nitric oxide by adding 10 mmol/L L-arginine to cardioplegia in isolated, blood-perfused neonatal lamb hearts having 2 hours of cold cardioplegic ischemia. In another group 10 mmol/L D-arginine, an inactive enantiomer of L-arginine, was added to the cardioplegia. Controls received only cardioplegia (dextrose-potassium).

Results. At 30 minutes of reperfusion, the L-arginine group showed a significantly improved recovery in left ventricular systolic function (maximum developed pressure, developed pressure at a constant balloon volume [V10] resulting in an end-diastolic pressure of 10 mm Hg before ischemia, positive maximum dP/dt, and dP/dt at V10), diastolic function (negative maximum dP/dt and end-diastolic pressure at V10), coronary blood flow, endothelial function (assessed by the coronary vascular resistance response to acetylcholine), and myocardial oxygen consumption compared with the control group (p < 0.05). There were no significant differences in the recovery of any variables between the D-arginine and control groups.

Conclusions. These results suggest that provision of more substrate for the endothelial production of nitric oxide during ischemia has an important salutary effect on the recovery of postischemic myocardial and endothelial function and provide further evidence for an important role for the endothelial production of nitric oxide in the response to hypothermic ischemia and reperfusion in the neonatal lamb heart.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
See also page 1192.

Myocardial ischemia and reperfusion result in both ventricular and endothelial dysfunction despite the use of hypothermia and cardioplegia [1]. We have found that the endothelial dysfunction is characterized by reduced vasodilator response to intraarterial administration of acetylcholine, implying a reduced release of nitric oxide (NO) by the endothelium [2]. Recent investigations have characterized endothelium-derived relaxing factor as NO [3, 4], which is produced from the semiessential amino acid L-arginine as it is converted to L-citrulline by NO synthase in endothelial cells [5, 6]. In the vascular system, NO released from endothelial cells causes vasorelaxation [7, 8] but also inhibits leukocyte adhesion [9] and platelet aggregation [10]. Moreover, NO is a primary radical species and is inactivated by superoxide radicals [11, 12], but conversely NO may neutralize superoxide radicals [13]. Despite these potential benefits, NO production has been shown to be deleterious by some authors [14, 15] in crystalloid-perfused models of ischemia and reperfusion. We have previously demonstrated that infusion of L-arginine during the early phase of reperfusion significantly improved endothelial dysfunction and recovery of left ventricular (LV) function after hypothermic ischemia and reperfusion in a blood-perfused model [16]. This study was designed to investigate the effects of L-arginine administered during ischemia as a cardioplegia additive on the recovery of both myocardial and endothelial function after hypothermic ischemia.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Experimental Preparation
A previously described [1, 2, 16] isolated blood-perfused heart model was used to study 24 hearts from neonatal lambs (2.3 to 5.4 kg; 2 to 5 days old). Coronary perfusion was established with a roller pump and oxygenator system before isolation of the heart. Heparinized fresh homologous blood was used as the perfusate. Arterial pH was kept at 7.4 (corrected to perfusate temperature). Myocardial temperature was monitored by thermal probes, and the perfusate temperature was controlled at 37°C except during the hypothermic phase. Coronary perfusion pressure was maintained at 60 mm Hg except during the hypothermic and reperfusion phases. A latex balloon that contained a pressure transducer was placed inside the LV through the apex to measure LV function.

Measurements
Left ventricular function was measured during isovolumic contraction by inflating the intraventricular balloon as described previously [1, 2, 16]. The recovery of systolic function was evaluated by measuring the maximum developed pressure, positive maximum rate of change in LV pressure (dP/dt), peak developed pressure at a constant balloon volume to produce an end-diastolic pressure of 10 mm Hg during baseline measurement (V10), and peak dP/dt at V10. To assess the diastolic function, negative maximum dP/dt and end-diastolic pressure at V10 were measured. Coronary blood flow was assessed by an electromagnetic flow meter, which was connected to the venous cannula. Coronary endothelial function was assessed by the coronary vascular resistance (CVR) response to acetylcholine (10^-7 mol/L) infusion [1, 2, 16]. Maximum decrease in CVR during the acetylcholine infusion, divided by baseline CVR, was defined as the CVR response. To assess endothelium-independent vasodilator capacity, trinitroglycerin (3 x 10^-5 mol/L) was infused in the same way and the CVR response was measured. Arterial and venous blood were collected, and myocardial oxygen consumption was calculated from the hemoglobin concentration, the oxygen content, and saturation [1, 2, 16].

Experimental Protocol
Baseline measurements were made after a 20-minute equilibrium period. Then the perfusate was cooled to 15°C. After 10 minutes of cooling (myocardial temperature = 15°C), coronary perfusion was stopped and 20 mL/kg of cardioplegic solution was given followed by topical cooling (myocardial temperature was kept at 10°C). A second dose of 10 mL/kg was given after 60 minutes. The cardioplegic solution was 0.45% sodium chloride and 2.5% dextrose solution with 20 mEq/L of potassium chloride and 6 mEq/L of sodium bicarbonate (pH 7.4 at 37°C, osmolarity = 360 mOsm/L). After 2 hours of ischemia, reperfusion was begun with the perfusate at room temperature (25°C) and the heart was rewarmed to normothermia over 25 minutes. Mean coronary perfusion pressure was maintained at 20 mm Hg for the first 5 minutes, raised to 40 mm Hg for the second 5 minutes, and then kept at 60 mm Hg until the end of experiment [1, 2]. High oxygen concentration (95% O₂, 5% CO₂) was used during the cooling phase and the first 15 minutes of reperfusion. Thereafter the gas was changed to a low-oxygen mixture (20% O₂, 5% CO₂, 75% N₂).

Experimental Groups
The hearts were divided into three groups: (1) In the control group (n = 8), the crystalloid cardioplegic solution was given. (2) In the L-arginine group (n = 8), the NO precursor L-arginine was added to the cardioplegia to achieve a concentration of 10 mmol/L. (3) In D-arginine group (n = 8), D-arginine, an inactive enantiomer of L-arginine, was added to the cardioplegia at a concentration of 10 mmol/L.

Animals in this study received humane care in compliance with ``Principles of Laboratory Animal Care'' formulated by the National Society for Medical Research and ``Guide for the Care and Use of Laboratory Animals'' prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23, revised in 1985).

Statistics
All values are expressed as mean ± standard deviation and analyzed by a statistical analysis system. One-way analysis of variance and repeated-measures two-way analysis of variance were used to compare the differences in recovery between groups. Data were compared using the Student-Newman-Keuls test if the result of the analysis of variance was significant. A p value less than 0.05 was considered to be significant.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Baseline Measurement
There was no significant difference among the three groups in baseline data (Table 1).

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

A number of recent studies are pertinent to the results of the current study. Normothermic ischemia and reperfusion have been shown to result in diminished NO release from endothelial cells [17], and attempts have been made to supplement the reduced NO production and to thereby attenuate the myocardial injury associated with ischemia/reperfusion injury. The administration of an NO donor [18], NO itself [19], or L-arginine ameliorates reperfusion injury after normothermic ischemia in cats [20] and dogs [21]. A recent study in patients with microvascular angina showed that L-arginine improved exercise-induced myocardial perfusion abnormalities [22]. We have previously demonstrated that infusion of L-arginine only during the early phases of reperfusion significantly improved endothelial dysfunction and functional recovery of LV after hypothermic ischemia/reperfusion in neonatal lamb hearts [16]. The results of the present study suggest that supplying more substrate for NO production during ischemia by adding L-arginine to the cardioplegic solution resulted in increased NO production during reperfusion, because the response to intraarterial administration of acetylcholine was better preserved in the L-arginine–treated hearts. It is possible that the L-arginine in the cardioplegia ``loads'' the endothelial cells with this NO substrate so that NO production during reperfusion is enhanced. We were not able to measure endothelial production of NO in our model due to its extremely short half-life, and we have not found any reports in which L-arginine levels in endothelial cells have been measured during ischemia. However, the current experiment combined with prior experiments [2, 16, 21] seems to imply that the ability of endothelial cells to release NO is reduced after ischemia and that enhanced NO production is beneficial during reperfusion after hypothermic ischemia.

The exact mechanism by which L-arginine has a beneficial effect still remains speculative, but one possible mechanism would be through increased coronary blood flow [7, 8]. The significantly higher levels of coronary blood flow after ischemia in the L-arginine group would support this hypothesis. Our laboratory has previously found that administration of trinitroglycerin (which is generally thought to act as an NO donor) offset the deleterious effects of high-pressure reperfusion [23] and that trinitroglycerin enhanced the recovery of mechanical function in the postischemic period after cold cardioplegic ischemia [24]. Increases in coronary blood flow may lead to improved ventricular function through the Gregg or ``garden hose'' effect [25]. However, we have shown that an infusion of L-arginine without ischemia at normothermia does not increase coronary blood flow or improve ventricular function [16]. Moreover, prior experiments from our laboratory have also shown that postischemic infusion of theophylline (an adenosine-receptor antagonist) caused increased coronary blood flow and lowered coronary resistance, but was associated with worse recovery of ventricular function [26]. These experiments have implied to us that coronary vasodilation alone during reperfusion is not sufficient to improve the recovery of contractile function after hypothermic ischemia or to have a Gregg effect that outweights the depression of ventricular function produced by ischemia and reperfusion. We think it is reasonably likely that L-arginine has other cardioprotective mechanisms beyond coronary vasodilation that affect the response to ischemia/reperfusion.

If NO release is maintained close to the site of injury, it could have cytoprotective effects by inhibiting neutrophil aggregation and adherence. Recently, NO has been shown to be an endogenous inhibitor of leukocyte chemotaxis [27], adherence [28], and activation [29]. Because normal endothelial cells release NO basally, and because this NO may prevent leukocytes from adhering to endothelial cells, decreased basal release of NO after myocardial ischemia and reperfusion may lead to enhanced adherence of leukocytes to the coronary endothelium, which could enhance leukocyte-induced myocardial injury [30].

Increased endothelial production of NO with L-arginine infusion also may be cardioprotective through the action of directly quenching superoxide free radicals. Nitric oxide is a primary radical species and is inactivated by superoxide radicals [11, 12], but NO also neutralizes superoxide radicals [13]. Conditions associated with enhanced production of superoxide have been shown to increase neutrophil adherence, and therefore administration of NO could prevent endothelial injury resulting from superoxide radicals.

In contrast with our results, it should be noted that others have reported that augmentation of NO by L-arginine increases postischemic injury [14, 15]. It has been hypothesized that the mechanism by which NO has deleterious effects is through the formation of peroxynitrite and hydroxyl radical formation from NO [23]. However, these observed deleterious effects may be model-dependent. Takeuchi and associates [14] reported a negative inotropic effect of L-arginine in isolated rabbit hearts, but they used a crystalloid perfusate, which is likely to induce maximum vasodilation under baseline conditions and which lacks circulating neutrophils. In the report by Matheis and colleagues [15], the model was normothermic hypoxia and reoxygenation, which clearly differs from hypothermic ischemia and reperfusion. Despite the cytotoxic effects of NO observed in these studies, the beneficial effects of NO that have been outlined above seem to play much more important roles in the in vivo environment after hypothermic ischemia and reperfusion, which was present in our model.

There are several limitations to the current studies. First, the model that was used is an isolated, blood-perfused heart system, which involves extensive surfaces for blood contact and neutrophil activation. The advantages and disadvantages of this model for the assessment of cardiac function after ischemia have been previously discussed [1, 2]. We have continued to use this model because of the elimination of the influence of adrenergic, neural, and anesthetic variations on ventricular function, and because the model provides coronary blood flow independent of the mechanical function of the heart. In addition, the presence of an extracorporeal circuit and large surface area for neutrophil activation are very similar to those encountered in the clinical situation in which ischemia/reperfusion is induced as part of cardiac surgical procedures. The use of high oxygen tensions during early reperfusion also mimics the conditions of reperfusion during cardiac operations. The oxygen tension is reduced in the late stage of reperfusion to allow the evaluation of the coronary response to acetylcholine. In prior experiments, we have found it difficult to induce coronary relaxation with acetylcholine under hyperoxic conditions. When room air is supplied to the oxygenator, coronary sinus oxygen saturation data do not suggest global myocardial ischemia. However, repeat experiments in the whole animal would seem appropriate before making a firm recommendation that L-arginine be included in cardioplegia solutions.

In summary, the current experiments provide additional evidence to suggest an important role for the endothelial production of NO in the recovery of neonatal lamb hearts after hypothermic ischemia and reperfusion. The mechanism of these beneficial effects of L-arginine seems likely to involve enhancement of endothelial cell NO production by supplying more substrate for NO production, although the precise mechanisms by which increased NO production has this beneficial effect remain incompletely defined. Experiments to further investigate the mechanism for the beneficial effect of NO will involve attempts at independent manipulation of neutrophil function and vascular tone.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We sincerely thank Mark A. Cioffi, MAT, for his technical assistance.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Presented at the Forty-first Annual Meeting of the Southern Thoracic Surgical Association, Marco Island, FL, Nov 10–12, 1994.

Address reprint requests to Dr Mayer, Department of Cardiac Surgery, Children's Hospital, 300 Longwood Ave, Boston, MA 02115.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Joseph M. Forbess Takuya Miura John E. Mayer, Jr Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hiramatsu, T. Articles by Mayer, J. E. Search for Related Content PubMed PubMed Citation Articles by Hiramatsu, T. Articles by Mayer, J. E., Jr Related Collections Related Article