Ann Thorac Surg 2006;81:1715-1719
© 2006 The Society of Thoracic Surgeons
Original article: Cardiovascular
Potassium Channel-Related Relaxation by Levosimendan in the Human Internal Mammary Artery
Oguzhan Yildiz, MD, PhD
a
,
b
,
c
,
*
,
Melik Seyrek, MD
a
,
b
,
c
,
Vedat Yildirim, MD
a
,
b
,
c
,
Ufuk Demirkilic, MD
a
,
b
,
c
,
Cahit Nacitarhan, MD, PhD
a
,
b
,
c
a Department of Pharmacology, Gulhane School of Medicine, Ankara, Turkey
b Department of Cardiovascular Surgery, Gulhane School of Medicine, Ankara, Turkey
c Department of Pharmacology, Faculty of Medicine, Akdeniz University, Antalya, Turkey
Accepted for publication December 8, 2005.
* Address correspondence to Dr Yildiz, Gulhane School of Medicine, Department of Pharmacology, Etlik, 06018, Ankara, Turkey (Email: oyildiz{at}gata.edu.tr).
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Abstract
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BACKGROUND: Levosimendan is a potent inotropic and vasodilator drug used in the treatment of decompensated heart failure. There is no study on in vitro effects of levosimendan in human isolated arteries.
METHODS: We investigated the effect of levosimendan on contractile tone of human isolated internal mammary artery (IMA). The responses in IMA were recorded isometrically by a force-displacement transducer in isolated organ baths. Levosimendan was added to organ baths either at rest or after precontraction with phenylephrine (1 µmol/L). Levosimendan-induced relaxations were tested in the presence of cyclooxygenase inhibitor indomethacin (10 µmol/L), nitric oxide synthase inhibitor N
122-nitro-L-arginine methyl ester (100 µmol/L), large-conductance calcium-activated potassium-channel inhibitor tetraethylammonium (1 mmol/L), adenosine triphosphate–sensitive potassium-channel inhibitor glibenclamide (10 µmol/L), and voltage-sensitive potassium-channel inhibitor 4-aminopyridine (1 mmol/L).
RESULTS: Levosimendan (10 nmol/L to 3 µmol/L) produced potent relaxation in human IMA (maximal effect, 75.3% ± 4.9% of phenylephrine maximum contraction, 6.8 ± 0.1, n = 15; –log10 of 50% effective concentration). Vehicle had no significant relaxant effect. The relaxation to levosimendan is not affected by either potassium-channel inhibitors (tetraethylammonium and 4-aminopyridine) or cyclooxygenase and nitric oxide synthase inhibitors. Glibenclamide (10 µmol/L) inhibited levosimendan-induced relaxation significantly (p < 0.01).
CONCLUSIONS: Levosimendan effectively and directly decreases the tone of IMA. The mechanism of levosimendan-induced relaxation in IMA appears in part to be adenosine triphosphate–sensitive potassium-channel opening action. Levosimendan may be a cardiovascular protective agent by its relaxing action on the major arterial graft, IMA.
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Introduction
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Levosimendan is a potent inotropic and vasodilator drug under current investigation in the treatment of decompensated heart failure. It increases the contractile force of the heart by sensitizing cardiac troponin C to calcium during cardiac systole [1, 2]. In addition to its cardiac effects, levosimendan is also a vasodilator both in vitro [3, 4], and in vivo [5, 6]. Animal studies have demonstrated that the vasodilation by levosimendan may be partially related to a lowering of intracellular free calcium ([Ca2+]i) through potential inhibition of phosphodiesterase III [1, 7], calcium desensitization [8], or opening of adenosine triphosphate (ATP)-sensitive potassium channels [9, 10].
It has been proposed that levosimendan produces vasorelaxation through activation of the various types of potassium channels: ATP-sensitive, voltage-sensitive, and calcium-activated types [11]. Recently, ATP-sensitive potassium channel–mediated vasodilator effect of levosimendan in humans has been reported in human portal and saphenous veins [12, 13]. No studies have, however, examined the vasorelaxant action of levosimendan in human conduit arteries. The human conduit arteries such as the internal mammary artery (IMA) are commonly used as coronary grafts in coronary artery bypass graft surgery. After coronary artery bypass graft surgery, the graft becomes a part of the new vascular system to supply blood to the heart. The effect of levosimendan on the graft may therefore affect the blood flow to the heart. It is obviously important to know the effect of levosimendan on the conduit arteries used as coronary grafts. Furthermore, it is possible that levosimendan may be effective as a vasodilatory agent against vasospasm during or after coronary artery bypass grafting, which is one of the major complications of the coronary artery bypass graft surgery using arterial grafts. There are several studies on the effects of vasoactive substances on the IMA [14–16]. However, there is no study on the effects of levosimendan. As mentioned above, there are several proposed mechanisms for the vasodilatory effect of levosimendan. The present study was designed to investigate the involvement of potassium channels in the vascular effect of levosimendan on the IMA in vitro.
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Material and Methods
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Tissue Preparation
Eighty-seven IMA segments were collected from 21 patients undergoing coronary artery bypass graft surgery. There were 18 men and 3 women with a age range between 49 and 73 years. Patients had New York Heart Association (NYHA) classification of II through IV. Patients took a nitrate (91%), and some patients also took a ß-blocker, diuretic, and angiotensin-converting enzyme inhibitor. Approval to use discarded IMA tissue was granted by the ethics committee of Gulhane Faculty of Medicine, and this investigation conforms with the principles outlined in the Declaration of Helsinki. The arteries were placed in oxygenated Krebs–Henseleit solution (in mmol/L: NaCl, 118; KCl, 4.7; CaC12, 2.5; KH2PO4, 1.2; MgSO4, 1.2; glucose, 10; and NaHCO3, 25; pH 7.4) at room temperature and transferred immediately to the laboratory. The arteries were dissected from adhering fat and connective tissue then cut into 3- to 4-mm length rings. The rings were mounted in an organ bath, containing 10 mL of Krebs–Henseleit solution, on an L-shaped brace for tension measurement along the former circumferential axis. The solution was gassed with 95% O2 and 5% CO2 at 37°C. Changes in arterial tensions were recorded isometrically by a force-displacement transducer (model FT03; Grass Instruments, Astro-Med Inc, West Warwick, RI) and recorded continuously on a multichannel recorder polygraph (model P122; Grass Instruments, Astro-Med Inc) by using computer software (Polyview, version 2.0; Grass Instruments, Astro-Med Inc). The segments were allowed to equilibrate under final resting force of 2 g for at least 1.5 hours, and they were washed every 20 minutes before one of the following protocols was undertaken.
Experiments with levosimendan
After the equilibration period, arterial rings were challenged with phenylephrine (PE; 1 µmol/L) to test their viability. Rings that were contracted more than 1 g were included in the experimental protocol. The tissues were washed every 10 minutes during a 30-minute additional waiting period. After precontraction with PE (1 µmol/L) reached a plateau, cumulative relaxations to levosimendan (10 nmol/L to 3 µmol/L) were recorded. In some PE- precontracted rings, levosimendan was not added, and a time control of the arterial tension was recorded in parallel.
Effect of endothelial denudation on levosimendan responses
In this group of experiments, endothelial layers of some IMA segments were removed by mechanical rubbing. After arterial rings were precontracted with PE (1 µmol/L), the effect of levosimendan (10 nmol/L to 3 µmol/L) was recorded. Then, the tissues were washed every 10 minutes during a 30-minute additional waiting period. After a third precontraction with PE (1 µmol/L) reached a plateau, relaxation to acetylcholine (10 µmol/L) was used to test the success of endothelial denudation.
Experiments with inhibitors of nitric oxide synthase, cyclooxygenase, and potassium channels
In another set of experiments, after the equilibration period arterial rings were challenged with PE (1 µmol/L) to test their viability. Rings that were contracted more than 1 g were included in the experimental protocol. The tissues were washed every 10 minutes during a 30-minute additional waiting period. Before precontraction with PE (1 µmol/L), the rings were incubated with the cyclooxygenase inhibitor indomethacin (10 µmol/L), nitric oxide synthase inhibitor N
-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L), large-conductance calciun-activated potassium-channel inhibitor tetraethylammonium (TEA; 1 mmol/L), ATP-sensitive potassium-channel inhibitor glibenclamide (10 µmol/L), and voltage-sensitive potassium-channel inhibitor 4-aminopyridine (4-AP; 1 mmol/L) for 30 minutes; cumulative relaxations to levosimendan (10 nmol/L to 3 µmol/L) were recorded. The concentration of these inhibitors were chosen according to our previous studies with IMA [17–19]. Only one concentration–response curve to levosimendan was obtained in each preparation, and experiments were performed in parallel, control versus inhibitor, in adjacent segments from the same patient (paired design). Each preparation was used in only one experimental protocol, and only one ring in each group came from the same patient. The number of segments (patients) is represented by n.
Drugs and Chemicals
Tetraethylammonium chloride was purchased from Merck Company (Darmstadt, Germany). Glibenclamide, indomethacin, and L-NAME were purchased from Sigma Chemical Company (St. Louis, MO). 4-Aminopyridine was purchased from Acros Organics (Morris Plains, NJ). Levosimendan was obtained from Orion-Pharma (Espoo, Finland). Glibenclamide was dissolved in dimethyl sulfoxide, indomethacin was dissolved in ethanol, and all other inhibitors were dissolved in saline; no effects of vehicle were noted if total vehicle was 0.3% or less.
Statistical Evaluations
Levosimendan-evoked responses are expressed as arithmetic mean ± standard error of the mean of the percentage of the maximum PE response in each corresponding tissue. The concentrations of levosimendan required to produce 50% of the calculated maximum response (EC50) will be used to determine pEC50 values (negative log10 of the EC50 value). Data analysis was made by Student's paired t test for paired data. A value of p less than 0.05 was considered statistically significant.
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Results
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Cumulative concentrations of levosimendan (10 nmol/L to 3 µmol/L) elicited concentration–dependent relaxation of PE (1 µmol/L) -induced active tone in human IMA (pEC50, 6.8 ± 0.1; maximal effect (Emax), 75.3% ± 4.9% of PE maximum contraction; n = 15). However, in another group of experiments, levosimendan-induced relaxation in tissues precontracted with KCl (40 mmol/L) was significantly lower than that of PE-precontracted rings (24.1% ± 4.8%; n = 3; p < 0.001). Vehicle had no significant relaxant effect (<7%; n = 6). There was no spontaneous relaxation in PE-precontracted rings in time control experiments (data not shown). A representative tracing of the relaxant effect of levosimendan on the steady-state active tone elicited by PE (1 µmol/L) is shown in Figure 1.
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Fig 1. Original tracing of phenylephrine (PE; 1 µmol/L) -precontracted human internal mammary artery ring showing relaxation to levosimendan (10 nmol/L to 3 µmol/L). The concentration was changed every 5 minutes after a plateau was reached.
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Mechanical rubbing of the endothelium did not significantly modify relaxation induced by levosimendan (10 nmol/L to 3 µmol/L) in human IMA (n = 6). In contrast, relaxation induced by 10 µmol/L acetylcholine in endothelium-intact rings (72.4% ± 6.7% of PE maximum contraction) was significantly reduced in endothelium-free IMA (12.1% ± 5.6% of PE maximum contraction; p < 0.01; n = 5; Fig 2). Incubation of IMA with the nitric oxide synthase inhibitor L-NAME (100 µmol/L; n = 6) and cyclooxygenase inhibitor indomethacin (10 µmol/L; n = 6) did not significantly modify relaxation to levosimendan (10 nmol/L to 10 µmol/L; Fig 3).
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Fig 2. Original tracing of phenylephrine (PE; 1 µmol/L)-precontracted human internal mammary artery ring showing no relaxation to acetylcholine (ACh; 10 µmol/L), which was used to test the success of endothelial denudation.
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Fig 3. Cumulative relaxation to levosimendan in human internal mammary artery rings precontracted with phenylephrine (PE; 1 µmol/L) in the presence of (A) nitric oxide synthase inhibitor N
-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L; n = 6) and (B) cyclooxygenase inhibitor indomethacin (10 µmol/L; n = 6). Data are expressed as percentage of phenylephrine-induced maximum contraction. Vertical lines represent standard error of the mean.
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Relaxation to levosimedan (10 nmol/L to 3 µmol/L) was not significantly modified by incubation of IMA with the selective large-conductance calcium-activated potassium-channel inhibitor TEA (1 mmol/L) and voltage-sensitive potassium-channel inhibitor 4-AP (1 mmol/L; Fig 4A, 4C). On the other hand, the selective ATP-sensitive potassium-channel blocker glibenclamide (10 µmol/L) inhibited levosimendan-induced relaxation significantly (Fig 4B). None of the antagonists altered resting tension.
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Fig 4. Cumulative relaxation to levosimendan in human internal mammary artery rings precontracted with phenylephrine (PE; 1 µmol/L) in the presence of (A) 4-aminopyridine, a voltage-sensitive potassium-channel inhibitor (n = 8); (B) glibenclamide, an adenosine triphosphate–sensitive potassium-channel inhibitor (n = 10); and (C) tetraethylammonium chloride, a large-conductance calcium-activated potassium-channel inhibitor (n = 9). Data are expressed as percentage of phenylephrine-induced maximum contraction. Vertical lines represent standard error of the mean. *p < 0.05 and **p < 0.01 versus levosimendan control by paired Student's t test.
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Comment
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We demonstrated that levosimendan was able to relax PE-contracted human IMA. The drug caused vasodilation in concentrations that are attained in the clinical therapy of heart failure (approximately 0.2 µmol/L) [20].
In the present study, significant reduction in the efficacy of levosimedan in KCl-precontracted arteries demonstrated that levosimendan may be acting to open potassium channels, as has been previously suggested [21]. In this study, the vasodilating effect of levosimendan was inhibited in part by glibenclamide, reflecting some role of the hyperpolarizing ATP-sensitive potassium channels in the mechanism of action of the drug. Previously, a similar vasodilating potency of levosimendan against norepinephrine-induced contractions in human isolated portal vein was reported [12]. Recently, levosimendan was found to decrease the 5-hydroxytryptamine–induced tone of human isolated saphenous vein [13]. This suggests a receptor-independent vasodilation induced by the drug, as it was also found in isolated porcine coronary artery [4]. The ATP-sensitive potassium channel has recently been proposed as a target for levosimendan in isolated mesenteric artery myocytes [9], small coronary arteries of the dog [10], and human portal and saphenous veins [12, 13]. However, other potassium channels also serve as targets for the drug in large epicardial coronary arteries of the pig [9, 21, 22]. In addition to potassium channels, other proposed mechanisms of vasodilation induced by levosimendan involve changes in smooth muscle membrane potential [9], desensitization of intracellular calcium-binding proteins [8], elevation of adenosine 3',5'-cyclic monophosphate concentration in smooth muscle cells, and antagonism of endothelin-1–induced vasoconstriction [4]. The site of action through which levosimendan decreases the tone of a smooth muscle appears to depend on the concentration of the drug, the species, and probably the type of blood vessel.
Vasodilators are effective in heart failure because they provide a reduction in preload (through venodilation) or reduction in afterload (through arteriolar dilation), or both [23]. Previously, levosimendan has been shown to decrease the preload of the heart in intact conscious animals [24] and in experimental heart failure [5]. A recent report has shown the direct effect of levosimendan on the tone of isolated human saphenous vein [13], which is a human capacitance blood vessel. In the present study, we demonstrated a direct vasodilatory effect of levosimendan in human IMA, a conduit artery used to replace diseased coronary vessels.
A recent study by Michaels and colleagues [25] has provided evidence that levosimendan given intravenously exerts vasodilator effects on human coronary conductance and resistance arteries. Michaels and coworkers [25] have also demonstrated that intravenous levosimendan improves left ventricular systolic function and decreases myocardial oxygen extraction. These findings together suggest that levosimendan has the potential to produce favorable effects on systemic and coronary hemodynamics and to improve myocardial metabolic function.
In light of the present findings, we conclude that levosimendan effectively and directly decreases the tone of IMA. It appears that the mechanism of vasodilating action of levosimendan depends, at least in part, on ATP-sensitive potassium channels. In combination with the aforementioned, the present study has shown that levosimendan may be a cardiovascular protective agent by its relaxing action on the major arterial graft, the IMA.
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Abstract
Introduction
