Ann Thorac Surg 1996;61:36-40
© 1996 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Effects of Endothelin-1 and L-Arginine After Cold Ischemia in Lamb Hearts
Takeshi Hiramatsu, MD,
Joseph M. Forbess, MD,
Takuya Miura, MD,
Stephen J. Roth, MD,
Mark A. Cioffi,
Mat,
John E. Mayer, Jr, MD
Department of Cardiac Surgery and Cardiology, Children's Hospital and Harvard Medical School, Boston, Massachusetts
 |
Abstract
|
|---|
Background. Prior studies from our laboratory have suggested an important role for the coronary endothelium in the injury resulting from hypothermic ischemia and reperfusion. A decreased endothelial response to intraarterial acetylcholine occurs after ischemia/reperfusion, implying a reduced release of the vasodilator nitric oxide by endothelial cells, but the role of endothelial-derived vasoconstrictor endothelin-1 in ischemia/reperfusion and interactions between endothelin-1 and nitric oxide in ischemia/reperfusion are still unclear.
Methods. We examined the effects of endothelin-1 and L-arginine, the precursor for nitric oxide, on functional recovery of isolated, blood-perfused neonatal lamb hearts undergoing 2 hours of ischemia at 10°C. One group (n = 8) received 10 pmol/L endothelin-1 before reperfusion, and a second group (n = 8) received a continuous infusion of 3 mmol/L L-arginine during the initial 20 minutes of reperfusion. The third group (n = 8) received both endothelin-1 and L-arginine in the same way as in the endothelin-1 and L-arginine groups. The fourth group underwent the same period of hypothermic ischemia without interventions during reperfusion.
Results. After 30 minutes of reperfusion, the endothelin-1-treated hearts showed significantly reduced recovery of left ventricular systolic function (positive maximum dP/dt and volume normalized [V10] dP/dt) and diastolic function (negative maximum dP/dt), coronary blood flow, and myocardial oxygen consumption compared with the control group (p < 0.05). These effects of endothelin-1 were offset to equal the values observed in controls having unmodified reperfusion by adding L-arginine. The L-arginine group had significantly greater recovery of left ventricular systolic function (positive maximum dP/dt, maximum developed pressure, dP/dt at V10, and developed pressure at V10) and diastolic function (negative maximum dP/dt), coronary blood flow, and myocardial oxygen consumption compared with the control group (p < 0.05).
Conclusion. These results, combined with our previous observations that endothelin-1 levels are unchanged by hypothermic ischemia and reperfusion, suggest that there is an imbalance between the endothelial production of endothelin-1 and nitric oxide, which affects postischemic coronary blood flow and the recovery of ventricular function. Interventions that modify this imbalance of endothelially derived substances could favorably influence the outcome after a period of hypothermic ischemia and reperfusion.
|
Introduction
|
|---|
See also page 40.
Prior studies from our laboratory have suggested that the coronary endothelium has an important role in the functional outcome after a period of hypothermic ischemia and reperfusion [1–4]. The endothelium is the source of both vasodilator (eg, nitric oxide, prostacyclin) and vasoconstrictor (eg, endothelin) substances [5, 6]. Lüscher and colleagues [6] have proposed that interactions between these endothelium-derived factors contribute both to the normal regulation of blood flow and to the pathogenesis of certain cardiovascular disorders. We have previously provided evidence to suggest that decreased release of nitric oxide occurs after hypothermic ischemia and reperfusion [2], and we have also found that infusion of the nitric oxide precursor L-arginine during reperfusion improves the recovery of left ventricular and coronary endothelial function after hypothermic ischemia [3]. More recently, we have presented preliminary data suggesting that exogenous endothelin administered at the start of reperfusion worsens recovery of ventricular function after hypothermic ischemia [4]. The present study attempts to determine how interactions between endothelin-1 (ET-1) and nitric oxide influence the recovery of postischemic ventricular function by administering ET-1 and L-arginine alone and in combination to isolated, blood-perfused neonatal lamb hearts subjected to hypothermic ischemia.
|
Material and Methods
|
|---|
Experimental Preparation
An isolated blood-perfused heart model described previously [1–31-3] was used to study 32 hearts from neonatal lambs (2.3 to 5.9 kg; 2 to 7 days old). Briefly, coronary perfusion was established by a cannula placed into the ascending aorta using a roller pump and oxygenator system before isolation of the heart. Coronary venous blood was returned to the pump using a right ventricular cannula, which was inserted once the heart was isolated and perfused. A sampling catheter was placed in the coronary sinus through the hemiazygous vein for coronary venous blood analysis. 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 was maintained 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 left ventricle (LV) through the apex to measure LV function.
Measurements
Left ventricular function was assessed during isovolumic contraction by incrementally inflating the intraventricular balloon, as described previously [1, 3, 4]. The recovery of systolic function was evaluated by measuring the maximum (max) developed pressure (DP), positive maximum LV dP/dt, peak DP at a constant balloon volume (V10), and peak dP/dt at V10; V10 is the balloon volume to produce an end-diastolic pressure of 10 mm Hg during the preischemic baseline measurements. To assess diastolic function, we measured negative max dP/dt and end-diastolic pressure at V10. Coronary blood flow was assessed by an electromagnetic flowmeter connected to the venous cannula. Arterial and venous blood was collected, and myocardial oxygen consumption (MVO2) was calculated from the hemoglobin concentration, the oxygen content, and saturation [1, 3, 4].
Experimental Protocol
Baseline measurements were made after a 20-minute equilibrium period. The perfusate was then 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. Total ischemic time was 2 hours. 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). Reperfusion was begun with the perfusate at room temperature (25°C) and then rewarmed to normothermic levels over 25 minutes. Mean coronary perfusion pressure was maintained at 20 mm Hg for the first 5 minutes and raised to 40 mm Hg for the second 5 minutes, then kept at 60 mm Hg until the end of the experiment [1, 3, 4]. High oxygen (95% O2, 5% CO2) was used during the cooling phase and the first 15 minutes of reperfusion. Thereafter, the gas was changed to low oxygen (20% O2, 5% CO2, 75% N2).
Experimental Groups
The hearts were divided into four groups, all of which underwent 2 hours of cold ischemia: (1) The control group (n = 8) had no intervention during reperfusion; (2) in the ET-1 group (n = 8), ET-1 (Bachem, Philadelphia, PA) was given just before reperfusion at a concentration of 10 pmol/L (this group was previously reported in abstract form [4]); (3) in the L-arginine group (n = 8), L-arginine (Sigma Chemicals, St. Louis, MO) was infused during the initial 20 minutes of reperfusion at a concentration of 3 mmol/L (this group was previously reported [3] but is included for comparison purposes); and (4) the ET-1 + L-arginine group (n = 8) received both ET-1 and L-arginine in the same way as the groups receiving ET-1 or L-arginine alone.
Animals in this study received humane care in compliance with ``Principles of Laboratory Animal Care'' formulated by the National Society for Medical Research and the ``Guide for the Care and Use of Laboratory Animals'' prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Statistics
All values are expressed as mean ± standard deviation and were 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 analysis of variance was significant. A p value less than 0.05 was considered significant.
|
Results
|
|---|
Baseline Measurements
There were no significant differences among the four groups in baseline data (Table 1
).
Left Ventricular Function
The ET-1-treated hearts achieved significantly reduced recovery of LV systolic function (positive max dP/dt, dP/dt at V10) and diastolic function indices (negative max dP/dt) at 30 minutes of reperfusion compared with the control group (Table 2). The L-arginine group had significantly greater recovery of LV systolic function (max DP, DP at V10, positive max dP/dt, dP/dt at V10) and diastolic function (negative max dP/dt) than the control group. The ET-1 + L-arginine group showed no significant difference in LV functional recovery compared with the control group.
Coronary Blood Flow
Hearts in the ET-1 group had significantly lower coronary blood flow than the control group at 25 and 30 minutes of reperfusion (Table 3). The L-arginine group had significantly higher flow than the control group from 10 to 30 minutes of reperfusion. The ET-1 + L-arginine group showed no significant difference in coronary blood flow compared with the control group.
Oxygen Consumption
At 30 minutes of reperfusion, the ET-1 hearts showed significantly lower MVO2 than the control group (Table 4). The L-arginine group had significantly higher MVO2 than the control group from 15 to 30 minutes of reperfusion. The ET-1 + L-arginine group showed no significant difference in MVO2 compared with the control group.
Effects of Endothelin-1 at Normothermia Without Ischemia
To assess the effect of ET-1 at normothermia without ischemia, we infused a bolus of ET-1 calculated to achieve a concentration of 10 pmol/L in the same isolated heart models (Table 5). We assessed changes in LV function, coronary blood flow, and MVO2 30 minutes later. Endothelin-1 was infused just after baseline measurements. Data were compared with an additional control group perfused at normothermia without ischemia (n = 4). The group receiving ET-1 at normothermia (n = 4) showed no significant difference in LV function indices except for negative max dP/dt and nonsignificantly (p > 0.05) reduced coronary blood flow and MVO2.
View this table:
[in this window]
[in a new window]
|
Table 5. . Percent Recovery of Left Ventricular Function, Coronary Blood Flow, and Myocardial Oxygen Consumption at Normothermia Without Ischemia in the Endothelin-1-Treated Heartsa
|
|
|
Comment
|
|---|
It is now clear that the endothelium is an important source of vasoactive agents, including both ET-1 and nitric oxide [5, 6]. Endothelin-1 is a peptide with 21 amino acid residues that has multiple biologic actions [7], but it is an extremely potent vasoconstrictor in the coronary, renal, and systemic vascular beds [8]. Nitric oxide is produced in endothelial cells as L-arginine and is converted to L-citrulline. Nitric oxide has now been shown to be the endothelium-derived relaxing factor [5]. Its vasodilator effects are limited to the immediately adjacent smooth muscle, as the half-life of this compound is extremely short [5]. The balance of the effects of nitric oxide and ET-1 seems to play a major role in modulation of vascular tone in the circulation [9].
The mechanisms by which ET-1 and nitric oxide are involved in the response to ischemia and reperfusion remain incompletely defined, but several studies from our own laboratory and others have suggested at least two possibilities. One such mechanism is a direct effect on coronary blood flow. In previous studies, we have found correlations between the postischemic recovery of coronary blood flow and the recovery of ventricular function [1, 10, 11], although vasodilation induced by theophylline administration during reperfusion was associated with reduced recovery of ventricular function [10]. In the current experiments, ET-1 administration at the initiation of reperfusion resulted in significantly reduced coronary blood flow throughout the reperfusion period and reduced recovery of ventricular function. In studies in which no exogenous ET-1 was added, we have found that ET-1 levels remained constant during hypothermic ischemia and that administration of an endothelin receptor antagonist was associated with improved postischemic recovery of coronary blood flow and ventricular function [4]. Other investigators have reported that ET-2 had 30 times greater vasoconstrictive potency after ischemia compared with before ischemia [12], and Nayler [13] reported up-regulation of endothelin receptors after ischemic or hypoxic insults. Previous studies from our laboratory suggested that there is a reduced capacity to release nitric oxide after hypothermic ischemia [1, 2], and in the current studies, we found that the hearts treated with the nitric oxide precursor L-arginine had significantly higher coronary blood flow during reperfusion and improved ventricular function compared with both controls and ET-1-treated hearts. Furthermore, infusion of L-arginine during reperfusion after exogenous ET-1 administration offset the effects of exogenous ET-1 and resulted in a recovery of coronary blood flow and ventricular function equaling that found in the control hearts, which underwent only hypothermic ischemia with cardioplegia. These findings suggest that during reperfusion after hypothermic ischemia, there is an alteration in the balance between endothelially derived vasodilator (nitric oxide) and vasoconstrictor (endothelin) substances which leads to lower coronary blood flow. The precise mechanisms underlying the observed relation between reduced coronary blood flow and worse recovery of ventricular function are unclear, but it is intuitively attractive to hypothesize that reduced coronary blood flow could be the cause of the reduced ventricular function observed after hypothermic ischemia and reperfusion. However, these studies do not prove that this is the mechanism responsible for the reduced ventricular function observed after ischemia.
A second potential mechanism by which ET-1 and nitric oxide could interact to influence the outcome of a period of ischemia and reperfusion is through their contrasting effects on leukocytes. Nitric oxide has been shown to be an inhibitor of leukocyte chemotaxis [14], adherence [15, 16], and activation [17]; reduced endothelial production of nitric oxide therefore could be associated with an increase in leukocyte-mediated injury after ischemia. In contrast, ET-1 has been shown to increase neutrophil adhesion to endothelial cells by inducing leukocyte adhesion molecule (integrin) expression [18]. Nitric oxide has also been shown to reduce the release of ET-1 from endothelial cells [19, 20]. Nitric oxide can neutralize superoxide radicals and therefore could be expected to reduce the injury due to oxygen radicals produced by neutrophils [21]. Thus, reduced production of nitric oxide combined with continued production of ET-1 could increase ischemia-reperfusion injury through a leukocyte-mediated mechanism as well as by modulation of vascular tone.
Although the precise mechanisms are incompletely defined, the present studies do show that in the isolated, blood-perfused neonatal lamb heart subjected to hypothermic ischemia with cardioplegia, there are likely to be important interactions between ET-1 and nitric oxide production that have a substantial impact on postischemic recovery. The current studies show that L-arginine administration offsets the effects of exogenous ET-1. When these results are considered with the findings of a detrimental effect of ET-1 and a beneficial effect of L-arginine when each was administered alone, it seems likely that in the postischemic coronary circulation, there is reduced nitric oxide production with continued ET-1 production, with a resulting imbalance between these endothelially produced substances. We hypothesize that this imbalance results in the reduced coronary blood flow (which may be due to vasoconstriction or leukocyte-endothelial interactions) and that the decreased flow is likely related to the reduced recovery of ventricular function. Interventions that alter this imbalance may be useful in the postischemic state to reduce the deleterious effects of ischemia and reperfusion.
These conclusions are limited in that the experiments were performed in an isolated, blood-perfused heart model and not a whole animal. The advantages and disadvantages of this model for the assessment of cardiac function after ischemia have been discussed [1, 3]. We continue to use this model because it precludes the influences of adrenergic, neural, and anesthetic variations on ventricular function and provides coronary blood flow independent of mechanical function of the heart. The use of cold crystalloid cardioplegia may also influence these results, although our previous experiments comparing crystalloid with blood cardioplegia in the isolated neonatal lamb heart model did not demonstrate significant differences in recovery after hypothermic ischemia [22]. Experiments are in progress to examine these concepts further regarding the role of the endothelium in the whole-animal response to hypothermic ischemia and reperfusion.
|
Acknowledgments
|
|---|
We sincerely thank Pascal Gebeyan, Michael E. Roy, Tara E. Hamond, and Sigrid H. Wolfram, BS, for their technical assistance.
|
Footnotes
|
|---|
Presented at the Thirty-first Annual Meeting of The Society of Thoracic Surgeons, Palm Springs, CA, Jan 30-Feb 1, 1995.
Address reprint requests to Dr Mayer, Department of Cardiac Surgery, Children's Hospital, 300 Longwood Ave, Boston, MA 02115.
|
References
|
|---|
- Kawata H, Sawatari K, Mayer JE Jr. Evidence for the role of neutrophils in reperfusion injury after cold cardioplegic ischemia in neonatal lambs. J Thorac Cardiovasc Surg 1992;103:908–18.[Abstract]
- Sawatari K, Kadoba K, Berger KA, Daich JA, Mayer JE Jr. Influence of initial reperfusion pressure after hypothermic cardioplegia on endothelial modulation of coronary tone in neonatal lambs: impaired coronary vasodilation response to acetylcholine. J Thorac Cardiovasc Surg 1991;101:777–82.[Abstract]
- Hiramatsu T, Forbess JM, Miura T, Mayer JE Jr. Effects of L-arginine and L-NAME on recovery of neonatal lamb hearts after cold cardioplegic ischemia: evidence for an important role for endothelial production of nitric oxide. J Thorac Cardiovasc Surg 1995;109:81–7.[Abstract/Free Full Text]
- Hiramatsu T, Forbess JM, Miura T, Roth SJ, Cioffi MA, Mayer JE Jr. Effects of endothelin-1 and endothelin-A receptor antagonist on recovery after hypothermic cardioplegic ischemia in neonatal lamb hearts [Abstract]. Circulation 1994;90(Suppl 1):422.
- Luscher TF, Vanhoutte PM. The endothelium: modulator of cardiovascular function. Boca Raton, FL: CRC Press, 1990:1-6.
- Lüscher TF, Boulanger CM, Yang Z, Noll G, Dohi Y. Interactions between endothelium-derived relaxing and contracting factors in health and cardiovascular disease. Circulation 1993;87(Suppl 5):36–43.
- Yanagisawa M, Kurihara H, Tomobe Y, et al. Novel potent vasoconstrictor peptide produced by vascular endothelial cell. Nature 1988;3321:411–5.
- Goetz KL, Wang BC, Nadwed JB, Zhu JL, Leadley RJ Jr. Cardiovascular, renal, and endocrine responses to intravenous endothelin in conscious dogs. Am J Physiol 1988;255:R1064–8.[Abstract/Free Full Text]
- Lüscher TF, Yang Z, Tschudi M, et al. Interactions between endothelin-1 and endothelium-derived relaxing factor in human arteries and veins. Circ Res 1990;66:1088–94.[Abstract/Free Full Text]
- Nomura F, Aoki M, Mayer JE Jr. Effects of adenosine infusion during reperfusion after cold cardioplegic ischemia in neonatal lambs. Circulation 1993;88(Suppl 2):380–6.
- Kawata H, Aoki M, Mayer JE Jr. Nitroglycerin improves functional recovery of neonatal lamb hearts after 2 hours of cold ischemia. Circulation 1993;88(Suppl 2):366–71.
- Liu J, Casley DJ, Nayler WG. Ischemia causes externalization of endothelin-1 binding sites in rat cardiac membranes. Biochem Biophys Res Commun 1989;164:1220–5.[Medline]
- Nayler WG. The endothelins. Berlin: Springer Verlag, 1990:1-188.
- Bath PMW, MacGregor GA. Spontaneous nitric oxide donors inhibit monocyte chemotaxis and increase intracellular cGMP concentration [Abstract]. J Vasc Res 1992;29:170.
- Kubes P, Suzuki M, Granger N. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991;88:4651–5.[Abstract/Free Full Text]
- McCall T, White BJR, Boughton-Smith NK, Moncada S. Inhibition of FMLP-induced aggregation of rabbit neutrophils by nitric oxide. Br J Pharmacol 1988;85:517P.
- Moilanen E, Vuorinen P, Kankaarenta H, Metsaketela T, Vapaatalo H. NO donors inhibit human neutrophil activation in vivo [Abstract]. J Vasc Res 1992;29:170.
- Lopez-Farro A, Riesco A, Espinosa G, et al. Effects of endothelin-1 on neutrophil adhesion to endothelial cells and perfused heart. Circulation 1993;88:1166–71.[Abstract/Free Full Text]
- DeNucci G, Thomas R, D'Orleans-Juste P, Antunes E, Walder TD, Vane JR. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci USA 1988;85:9797–800.[Abstract/Free Full Text]
- Boulanger C, Luscher TF. Release of endothelin from porcine aorta inhibition by endothelium-derived nitric oxide. J Clin Invest 1990;85:587–90.
- Rubanyi GM, Ho EH, Cantor EH, Lumma WC, Parker-Botelho LH. Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes. Biochem Biophys Res Commun 1991;181:1392–7.[Medline]
- Fujiwara T, Heinle J, Britton LW, Mayer JE. Myocardial preservation in neonatal lambs: comparison of hypothermia with crystalloid and blood cardioplegia. J Thorac Cardiovasc Surg 1991;101:703–12.[Abstract]
Related Article
-
Discussion
Ann. Thorac. Surg. 1996 61: 40-41.
[Extract]
[Full Text]
This article has been cited by other articles:
|
|
|
|
 
M. M. Multani, J. S. Ikonomidis, P. Y. Kim, E. A. Miller, K. J. Payne, R. Mukherjee, B. H. Dorman, and F. G. Spinale
Dynamic and differential changes in myocardial and plasma endothelin in patients undergoing cardiopulmonary bypass
J. Thorac. Cardiovasc. Surg.,
March 1, 2005;
129(3):
584 - 590.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
