Anesth Analg 2006;103:1094-1098
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000244326.38206.a0
CARDIOVASCULAR ANESTHESIA
The Vasodilatory Effects of Levosimendan on the Human Internal Mammary Artery
Félix R. Montes, MD*,
Darío Echeverri, MD
,
Lorena Buitrago, MB
,
Isabel Ramírez, IE
,
Juan C. Giraldo, MD*,
Javier D. Maldonado, MD||, and
Juan P. Umaña, MD||
From the Departments of *Anesthesiology, Cardiology, ||Cardiovascular Surgery, Laboratory of Vascular Function, Fundación CardioInfantil, Instituto de Cardiología; and Facultad de Ingeniería Industrial, Universidad de Los Andes, Bogota, Colombia, South America.
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Abstract
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BACKGROUND: Levosimendan, an inotropic drug that enhances myocardial contractility through myofilment calcium sensitazion, induces peripheral vasodilation via opening ATP-dependent K+ channels. It is unknown whether this drug can be used for the treatment of perioperative vasospasm of arterial conduits used for coronary artery bypass grafting.
METHODS: We investigated the effects of levosimendan on human internal mammary artery (IMA) specimens taken from patients undergoing coronary artery bypass surgery. The rings were carefully prepared and placed between two wire hooks in organ bath chambers and then constricted submaximally with norepinephrine and thromboxane A2 analog (U46619). Nitroglycerin, milrinone, and levosimendan were separately added in a cumulative fashion and concentration response curves for relaxation were constructed. In parallel experiments, the response to levosimendan was evaluated on rings with and without functional endothelium. Levosimendan prevention of norepinephrine-induced contraction was also estimated.
RESULTS: Nitroglycerin, milrinone, and levosimendan completely reversed the contraction of the IMA segments induced by U46619 and norepinephrine. Levosimendan produced a potent, concentration-dependent preventive effect on the norepinephrine-induced contraction of IMA. The responses to levosimendan were similar in preparations with or without endothelium.
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Introduction
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Levosimendan is a pyridazinone-dinitrile derivate that increases myocardial contractility by stabilizing the calcium bound conformation of troponin C. In addition to its cardiac effects, levosimendan is also a pulmonary and systemic vasodilator (1). This combination of positive inotropic and vasodilator activity has been shown to be beneficial in increasing cardiac output and decreasing left ventricular end-diastolic pressure, pulmonary wedge pressure, right atrial pressure, and systemic vascular resistance in patients with congestive heart failure (2). Levosimendan has also been used successfully to treat low cardiac output states post cardiopulmonary bypass surgery (3,4).
The internal mammary artery (IMA) is commonly used as a coronary artery bypass graft for myocardial revascularization because of its good short- and long-term patency (5). However, spasm of the IMA may contribute to early myocardial ischemia, increasing perioperative morbidity and mortality (6). The mechanism for IMA spasm includes injury to the endothelium during the surgical procedure, release of endogenous mediators, or is a result of vasoconstrictive drugs administered systemically to support the circulation (7). Prevention and treatment of IMA spasm with vasodilator drugs is thus often an important management goal during coronary artery bypass grafting to prevent myocardial ischemic injury. The purpose of this study was to investigate the vasodilator effects of levosimendan in an in vitro model of human IMA vasospasm.
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METHODS
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Vessel Preparation
After receiving approval from the IRB and written informed consent from all subjects, 186 segments of the right or left IMA were collected from 75 patients undergoing coronary artery bypass surgery. The normally discarded distal end of the artery was removed carefully and placed in chilled modified Krebs-Henseleit solution of the following composition (in mmol/L): NaCl 118, KCl 4.69, CaCl2 3.35, MgSO4 1.04, NaHCO3 25, and d-glucose 11.1, pH 7.40 ± 0.05. The vessels were transferred to the laboratory and then cleaned of adherent connective tissue and cut into 3-mm ring segments. One to six rings were obtained from each vessel. The time delay between the harvest and the preparation of the vessels was <15 min.
Organ-Chamber Experiments
The rings were suspended between two wire hooks in an organ bath chamber filled with 25 mL Krebs-Henseleit solution (37°C, pH 7.40) aerated with 95% O2/5% CO2. The upper hook was connected to a force transducer (Kent-Scientific Corporation, Litchfield, CT), and changes in isometric force were recorded (PowerLab® System, ADI Instruments; Mountain View, CA). A resting tension of 2 g initially defined by preliminary studies was progressively applied, and the rings were allowed to stabilize for 60 min. In the first series of experiments, 122 IMA segments resected from 59 patients were precontracted with a thromboxane A2 analog (U46619, 10–8 mol/L, Cayman Chemical, Ann Arbor, MI) or norepinephrine (3.16 x 10–6 mol/L, Sterop Laboratories, Belgium) to reach optimal constriction. The concentrations were determined from cumulative concentration-response curves to achieve 50%–80% of the maximum contraction. After a second equilibration period of 15 minutes (time needed for the development of a stable contraction) the rings were randomly selected to be exposed to levosimendan (10–8 to 10–4 mol/L, Abbott Laboratories, Chicago, IL), milrinone (10–8 to 10–4 mol/L, Sanofi Winthrop Industries, France), or nitroglycerin (10–11 to 10–6 mol/L, Luitpold Pharmaceuticals, USA). The concentrations of levosimendan, milrinone and nitroglycerin were within the range of plasma concentrations likely to be achieved clinically. Relaxant drugs were applied in a cumulative manner in 0.5-log unit steps. The relaxation response to each dose was allowed to develop for 10–15 min, until a stable baseline was obtained. Each IMA ring was exposed to only one drug.
In a separate set of experiments, four rings were taken from six patients. One of the rings was used as a control, and the other three were pretreated for 15 min with one of three increasing concentrations (10–7, 10–6, or 10–5 mol/L) of levosimendan. A cumulative concentration-contraction curve was then constructed for norepinephrine using similar concentrations as in the earlier experiments.
To assess whether endothelium mediates levosimendan-induced vasorelaxation, the endothelium was removed by inserting a microforceps into the ring lumen and gently rubbing the luminal surface in half of 40 IMA segments taken from 10 different patients. The presence or absence of functional endothelium was tested by the response to acetylcholine (3 x 10–6 mol/L, Sigma Chemical, St. Louis, MO) in rings precontracted with norepinephrine (3.16 x 10–6 mol/L). Rings that exhibited >50% relaxation response to acetylcholine were presumed to be endothelium positive, while rings demonstrating absence of relaxation were categorized to be endothelium negative. The rings were washed twice with fresh buffer solution, and then precontracted with norepinephrine (3.16 x 10–6 mol/L). Concentration-relaxation curves to levosimendan were then constructed in both endothelium positive and endothelium negative rings. All experiments were performed in the presence of indomethacin (10–5 mol/L, Sigma Chemical) to prevent the synthesis of vascular prostaglandins. The thromboxane A2 analog was diluted in ethanol (95%) to 0.05 M and then serially diluted in distilled water. All other drugs were serially diluted in distilled water before each experiment. The concentration of the drugs is expressed as final concentration in the bath solution.
Data and Statistical Analysis
Relaxation responses were calculated as a percentage of norepinephrine or the thromboxane A2 analog-induced contraction; the resulting value was used for the analysis. Data were averaged for each patient in all experiments and n equaled the number of patients from whom the IMA segments were obtained. The effective concentration of study drug to cause 50% of maximal relaxation (EC50) was determined for each ring by the logistic curve fitting equation:
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where E is response, M is maximal relaxation, A is concentration, K is EC50 and p is the slope parameter. To compare the maximal contraction values between the groups, one-way analysis of variance followed by Scheffé test was used. A t-test for unpaired comparison was used to compare the response to levosimendan in rings with and without endothelium. A nonparametric test for unpaired comparison (Mann-Whitney U-test) was used to compare the EC50 values of the vasodilators, according to the vasoconstrictor drug. For the entire study, a probability value of <0.05 was considered significant. Data are presented as mean ± sd.
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RESULTS
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Comparison of Levosimendan with Other Vasodilators
Levosimendan as well as nitroglycerin and milrinone resulted in 80%–100% maximal relaxation of the IMA segments contracted with norepinephrine (Fig. 1) or the thromboxane A2 analog (Fig. 2). In vessels precontracted with norepinephrine, the relaxing effect of nitroglycerin was greater (EC50, 2.7 ± 2.4 x 10–8) when compared with levosimendan (EC50, 7.07 ± 5.8 x 10–6, P < 0.001) and milrinone (EC50, 4.89 ± 3.6 x 10–6, P < 0.001). Also, in vessels precontracted with thromboxane A2 analog, nitroglycerin demonstrated greater potency (EC50, 1.49 ± 1.3 x 10–8) than levosimendan (EC50, 4.89 ± 7.6 x 10–6, P < 0.001) and milrinone(EC50, 2.06 ± 1.7 x 10–6, P < 0.001). There was no difference between the EC50 for levosimendan and milrinone in vessels precontracted with norepinephrine (P = 0.25) or the thromboxane A2 analog (P = 0.87). The relaxation response obtained with levosimendan was similar in vessels contracted with the thromboxane A2 analog and in vessels contracted with norepinephrine (EC50 4.89 ± 7.6 x 10–6 vs 7.07 ± 5.8 x 10–6, P < 0.12).
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Figure 1. Concentration-response curves for nitroglycerin (n = 10), milrinone (n = 11), and levosimendan (n = 10) in human internal mammary artery contracted with norepinephrine (3.16 x 10–6 mol/L). Data are expressed as mean ± sd.
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Figure 2. Concentration-response curves for nitroglycerin (n = 11), milrinone (n = 10), and levosimendan (n = 10) in human internal mammary artery contracted with tromboxane A2 analog (U46619, 10–8 mol/L). Data are expressed as mean ± sd.
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Prevention of IMA Contraction
Pretreatment of the IMA rings for 15 min with increasing concentrations (10–7 to 10–5 mol/L) of levosimendan significantly prevented norepinephrine-induced contraction in a concentration-dependent manner (Fig. 3). Values of maximal contraction were as follows: 99.9% ± 10.4% for the control group and 38.0% ± 26.4%, 21.0% ± 16.9%, and 10.6% ± 8.5% for the groups treated with 10–7, 10–6, and 10–5 mol/L of levosimendan, respectively.
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Figure 3. Preventive effect of levosimendan on contraction of human human internal mammary artery induced by norepinephrine. Data are expressed as mean ± sd, n = 6 for each dose of levosimendan. (*P < 0.05 when compared with control. **P < 0.01 when compared with control).
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Relaxing Effect of Levosimendan on Rings with or Without Endothelium
The efficacy and potency of levosimendan in relaxing the IMA segments were similar in rings with and without endothelium (P > 0.05) (Fig. 4).
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Figure 4. Graph illustrating responses to increasing concentrations (3.16 x 10–9 mol/L to 1 x 10–4 mol/L) of levosimendan in human internal mammary artery contracted with norepinephrine (3.16 x 10–6 mol/L). There was no significant difference in the relaxing effect of levosimendan between rings with and without endothelium.
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DISCUSSION
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The current study shows that levosimendan, a myofilament calcium sensitizer, effectively reversed thromboxane A2 analog and norepinephrine-induced contraction of IMA segments in vitro. The vasodilator effect is not dependent on the presence of endothelium. Levosimendan also exerts a potent, concentration-dependent, inhibitory effect on IMA norepinephrine-mediated contraction.
Levosimendan, nitroglycerin, and milrinone reversed both norepinephrine- and thromboxane A2 analog-induced contractions. Judging from their EC50 values, levosimendan and milrinone appeared to have comparable potency for this effect. Based on similar comparisons, nitroglycerin was significantly more potent in reversing norepinephrine and thromboxane A2 vasoconstriction. The EC50 for vasorelaxation of preconstricted IMA rings for levosimendan in our study (4.89 to 7.07 x 10–6 mol/L) was higher that the therapeutic level reported in patients (1.24 x 10–7 mol/L) (8). This value corresponds to a relaxation of approximately 40%–50% in our study.
Our results are in agreement with other's studies which have reported that nitroglycerin is more potent than other drugs for reverting precontracted IMA (9,10). The mechanism of action of nitroglycerin is because of the release of nitric oxide resulting in activation of guanylate cyclase and increased formation of cyclic guanosine monophosphte leading to smooth muscle relaxation (11). The EC50 for this effect of nitroglycerin found in the present study is in the therapeutic range (10–9 to 10–8 mol/L) reported for this drug (12) and comparable to the EC50 for nitroglycerin reported in other studies (9,10). A clinical limitation with nitroglycerin, though, is the development of tachyphylaxis, limiting its usefulness for long durations or for patients chronically treated with this drug before surgery. Consequently, different drugs have been evaluated as an alternative to nitroglycerin (10).
Milrinone is a bypyridine derivative that selectively inhibits phosphodiesterase type III and prevents the degradation of cyclic adenosine monophosphate. Liu et al. (13) investigated the effects of milrinone on IMA segments and established that it produces a potent endothelium-independent relaxation on the IMA. The data of the present study show an EC50 in the range of 2.1 to 4.9 x 10–6 mol/L; similar to the optimal therapeutic plasma concentration clinically reached (14) and similar to that previously demonstrated in in vitro vascular ring studies (10,13). Despite the beneficial clinical myocardial inotropic and vasodilatory effects on the pulmonary and systemic vasculature that both enhance right and left ventricular stroke volume, a rate-limiting factor for milrinone administration can increase myocardial oxygen demand with consequent risk for ischemia and arrhythmia (15,16).
Levosimendan enhances the contractile force of myocardium by binding to troponin C without increasing the intracellular calcium concentration at therapeutic doses. In another cellular action, levosimendan promotes vasodilation by opening the ATP-sensitive K+ channels (17). De Witt et al. (18) showed that responses to levosimendan are reduced, yet not completely blocked, by K+ATP-channel blockers; this observation suggests that additional mechanisms may be involved in mediating the smooth muscle response to levosimendan. In vitro levosimendan has been found to be a highly selective inhibitor of phosphodiesterase; however, at concentrations exceeding the pharmacologically relevant concentrations for inducing positive inotropic effects (19). After levosimendan infusion, the resultant venous and arterial dilation reduces cardiac preload and postload, improves oxygen supply to the myocardium, and enhances the renal blood flow (1–3). Such vasodilation by levosimendan is also thought to underlie the reductions in infarct size and myocardial ischemia as well as afford anti-myocardial stunning benefits (20). The results of this study suggest that levosimendan might have other beneficial clinical effects for preventing or treating IMA vasospam.
There are promising human studies and clinical experience with levosimendan in cardiac surgery (3,4). The preliminary data in these relatively small studies suggest that levosimendan may be beneficial in low cardiac output states after cardiopulmonary bypass. In both studies, levosimendan increased cardiac output, heart rate, and decreased systemic and pulmonary vascular resistance. Despite improved cardiac performance, levosimendan did not increase myocardial oxygen consumption or change myocardial metabolism. Potential disadvantages of levosimendan include its association with arterial hypotension and its relatively high cost.
Some of the factors contributing to the development of IMA spasm include endogenous and exogenous catecholamines acting via 
-adrenoreceptors and other mediators, such as endothelin and thromboxane A2 (21,22). In this study, the selection of two receptor agonists, norepinephrine and thromboxane A2 analog, as contracting agents was made because they are likely to play an important role in producing IMA-graft spasm in patients undergoing myocardial revascularization surgery. Plasma levels of those two vasoconstrictors are increased during and after conventional coronary artery bypass surgery and they may act synergistically in producing perioperative vasospasm (7).
Our findings might have relevance to the care of patients who receive arterial grafts and have suspected vasospasm. However, as shown in the figures, there is a considerable variability in vasodilator effects among the vessel segments. Consequently, the EC50 value might not be correlated with the dose leading to an expected vasodilator effect in vivo. At present, there are little quantitative clinical data clearly supporting the use of a specific drug as a preventive strategy for postoperative IMA spasm. Nitroglycerin, although effective in reversing established spasm, may be much less effective if it is given before the constrictor stimulus (21). In our study, when added as pretreatment, levosimendan antagonized norepinephrine-induced vasoconstriction. This suggests a prophylactic role for calcium sensitizers, such as that has been reported for drugs acting through receptor-operated calcium channels, such as nifedipine, and the phosphodiesterase III inhibitor milrinone (13,23).
Although effective for reversing or preventing vasoconstriction of IMA segments, these effects might not be observed in other arterial bypass graft conduits, such as the radial artery. Differences between the radial artery and IMA to a contractile agonist have been reported, with the radial artery showing a stronger contractile response than the IMA (24). In addition, there may be differences between the two arteries in the mechanisms underlying vasodilation (25). Thus, the optimal treatment to attenuate vasospasm may not be identical for the two vessels. Postoperative saphenous venous spasm is a rarity, and can be readily reversed by nitroglycerin. However, spasm of the saphenous vein during harvesting is a common phenomenon that can be minimized by careful surgical technique. Future studies are necessary in order to investigate the vasodilator effects of levosimendan on other arterial conduits and the saphenous vein.
In conclusion, our results indicate that levosimendan is a potent, endothelium-independent vasodilator of human IMA. In light of its positive inotropic and vasodilator properties, levosimendan might be beneficial for the perioperative treatment of patients undergoing coronary artery bypass grafting.
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge the advice and suggestions of Fania Szlam, MMS, Department of Anesthesiology, Emory University.
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Footnotes
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Accepted for publication July 18, 2006.
Presented, in part, at the annual meeting of the American Society of Anesthesiologists, Las Vegas, Nevada, October 24–27, 2004.
Supported by Abbott Laboratories de Colombia.
Author correspondence and reprint requests to Félix R. Montes, MD, TRANEXCO 1194tp6co, P.O. Box 025512, Miami, Florida 33102. Address e-mail to felixmontes{at}etb.net.co.
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Abstract
Introduction
