Journal Logo

Original Article

Preconditioning Effects of Levosimendan in a Rabbit Cardiac Ischemia-Reperfusion Model

Leprán, István PhD, DSc*; Pollesello, Piero PhD; Vajda, Szilvia MSc*; Varró, András MD, PhD, DSc*; Papp, Julius Gy MD, PhD, DSc*‡

Author Information
Journal of Cardiovascular Pharmacology: October 2006 - Volume 48 - Issue 4 - p 148-152
doi: 10.1097/01.fjc.0000246151.39758.2a
  • Free

Abstract

Preconditioning is a well-known phenomenon that protects the myocardium against ischemic damage. The preconditioning stimulus that has been most frequently investigated is short-lasting, repeated, reversible myocardial ischemia, which reduces infarct size1 and protects against the development of reperfusion arrhythmias.2

A number of drugs were shown to induce preconditioning. Even limiting to the field of intensive care, the list is impressive: volatile anaesthetics,3 nitrates,4 opioids,5,6 and even catecolamines such as noradrenaline and isoprenaline7-9 improved postischemic recovery. The present consensus is that the action of these different drugs in triggering the preconditioning effect is regulated by few cellular mediators, among which the opening of mitochondrial adenosine triphosphate (ATP)-dependent potassium channels (mitoKATP channels) plays a key role.10,11

Levosimendan, a new inodilator used for the treatment of acute decompensated heart failure, has recently been shown to open mitoKATP channels in isolated cardiomyocytes.12 Levosimendan has also been shown to prevent the development of arrhythmias during acute myocardial infarction in rats13 and to decrease myocardial infarct size and produce cardioprotective effects while simultaneously enhancing ventricular contractile function.14 An anti-ischemic effect of the drug has also been observed.15 It has therefore been hypothesized that levosimendan may also exert a preconditioning effect,16 which would open new possibilities for the therapeutic use of the drug.

The aim of the present investigations was to ascertain whether levosimendan has a preconditioning effect. In order to distinguish this effect from the other hemodynamic characteristics of the drug (i.e., its vasodilator and positive inotropic effects), we studied the effects of repeated short-term pretreatment with levosimendan on functional parameters and infarct size following global ischemia-reperfusion injury in isolated Langendorff-perfused rabbit hearts, and compared them with the effects of ischemic preconditioning.

METHODS

Animals

Male albino rabbits (New Zealand white) weighing 2.2 to 2.8 kg were used. The animals were housed in an animal room (temperature: 20 ± 1°C, humidity: 40-70%; lighting: 12 hours per day) for at least 1 week before the experiments. Animals were fed commercial laboratory rabbit food pellet and allowed to drink tap water ad libitum before the experiments. The animals were kept and treated, and the experiments were carried out, under conditions delineated in the guidelines of the Committee of Animal Experiments, University of Szeged, Hungary.

Chemicals

Levosimendan, which is the (-)enantiomer of {[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]hydrazono}propanedinitrile [CAS registry number 141505-33-1], was synthesized at Orion Pharma, Espoo, Finland. Concentrated stock solutions of levosimendan were prepared with 50 μl of 1 M NaOH in 10 ml phosphate buffer (2.32% Na2HPO4 in distilled water). Then 100 μl of this stock solution was added to 1,000 ml modified Krebs-Henseleit solution for use in the perfused hearts.

Isolated-Perfused Heart Preparation

Animals were anesthetized using sodium pentobarbitone (30 mg/kg intravenous). Thoracotomy was performed after administration of heparin (2000 IU intravenous), and the heart was quickly removed and mounted on a nonrecirculating Langendorff apparatus.

The heart was perfused at a constant pressure of 75 mm Hg at 38°C, with a modified Krebs-Hensenleit solution containing: NaCl, 118.5 mM; KCl, 4.7 mM; KH2PO4, 1.2 mM; MgSO4, 1.2 mM; NaHCO3, 24.8 mM; CaCl2, 2.5 mM; and glucose, 10 mM. The solution was continuously oxygenated by 95% O2 and 5% CO2.

In order to measure intraventricular pressure changes, a fluid-filled balloon was inserted into the left ventricle of the heart via the left atrium and connected to a pressure transducer (P23ID, Gould). After analog-to-digital conversion, the pressure signal was recorded using a PowerLab data acquisition system (SP8, ADInstruments Ltd, UK). The initial end-diastolic pressure was set to <5 mm Hg. Left ventricular systolic pressure (LVSP), end-diastolic pressure (LVEDP), developed pressure (LVDP), and the indices of left ventricular contractile function during systole and diastole (i.e., +dP/dtmax, and −dP/dtmax) were calculated offline. Coronary flow was measured by timed collection of the coronary effluent in a graduated cylinder. Computer recording was performed continuously and measurements were repeated after each intervention and at timed intervals throughout the experiment. Right atrial pacing was performed at 200 bpm when the heart rate was lowered.

Experimental Protocol

After 10 minutes of equilibration, the hearts were randomly assigned to one of three groups (Fig. 1). In the control group, hearts were perfused for 20 minutes and then exposed to global ischemia for 30 minutes and reperfusion for 120 minutes. In the ischemic preconditioning group, hearts underwent two cycles of 5-minute global ischemia followed by 5-minute washout periods, before 30 minutes of global ischemia and a further 120 minutes of reperfusion. In the levosimendan pretreatment group, levosimendan (0.1 μmol/L) was included in the perfusion fluid for two cycles of 5 minutes, corresponding to episodes of ischemia in the precondition group and each followed by 5-minute washout periods, and then subjected to 30 minutes of global ischemia followed by 120 minutes of reperfusion using drug-free Krebs-Henseleit solution.

FIGURE 1
FIGURE 1:
Protocol of the experiment. IPC, ischemic preconditioning.

Determination of the Infarcted Area

The infarct area was determined as described previously by Tanno et al.17 At the end of the experiment, hearts were sliced into approximately 2-mm thick slices parallel to the atrioventricular groove. The slices were incubated with 0.1% nitroblue-tetrazolium in phosphate buffer for 10 min. After staining, the heart slices were digitalized using a scanner (HP ScanJet 4400C) and areas of infarction were measured using image analysing software (ImageJ, National Institutes of Health). Areas of infarct and the left ventricle were multiplied by the wet weight of individual slices to calculate the percentage of damage.

Statistical Analysis

Parameters were expressed as means ± standard error of the mean (SEM) and, after analysis of variance, compared by means of the modified t statistical method of Wallenstein et al.18

RESULTS

Effect of Levosimendan Pretreatment or Ischemic Preconditioning on Functional Heart Parameters

LVSP increased during the two short perfusions of levosimendan pretreatment (from the baseline 105 ± 5.9 mm Hg to 116 ± 6.1 mm Hg and 117 ± 8.3 mm Hg, respectively) but quickly returned to baseline during the washout periods (Fig. 2). By contrast, LVSP rapidly declined during the two ischemic preconditioning cycles and then recovered during reperfusion. LVSP did not differ significantly between the two groups either immediately before ischemia (Fig. 2) or during the postischemic reperfusion (Fig. 3).

FIGURE 2
FIGURE 2:
Effects of ischemic preconditioning (circles) and levosimendan pretreatment (squares) on LVSP and LVEDP during preischemia and ischemia (indicated by black bars on the time axis). Values are expressed as means ± SEM (n = 12).
FIGURE 3
FIGURE 3:
Effects of ischemic preconditioning and levosimendan pretreatment on the function of the heart after 90 minutes of reperfusion. The three groups are control (black), levosimendan (stripes), and ischemic preconditioned hearts (white). LVEDP is expressed as the absolute difference between its value after 90 minutes of reperfusion and the preischemic value (mm Hg). LVSP, LVDP, left ventricular isovolumic contraction and relaxation (+dP/dt and -dP/dt, respectively), and coronary flow (CF) are expressed as a percent of their preischemic values. Values are expressed as means ± SEM (n = 12). *P < 0.05 levosimendan versus controls.

LVEDP was elevated during the ischemic preconditioning cycles (from the baseline 1.5 ± 0.5 mm Hg to 3.5 ± 1.6 mm Hg and 5.8 ± 2.5 mm Hg), while exposure to levosimendan had no effect on this parameter during its administration (2.0 ± 0.7 mm Hg). Altogether the LVEDP was significantly increased before the 30-minute test ischemia in the ischemic preconditioning group (7.7 ± 3.1 mm Hg), compared to the levosimendan group (2.0 ± 1.1 mm Hg; Fig. 2). The latter did not differ from the control response (1.3 ± 0.9 mm Hg; not shown). During the 30-minute period of global myocardial ischemia, LVEDP was significantly elevated in all groups compared to the baseline values (P < 0.05) as a result of ischemic contraction, starting approximately 10 minutes after the period of ischemia.

Following reperfusion, LVEDP was elevated in all groups, but this effect was not so pronounced in the levosimendan hearts (Fig. 3). LVEDP 90 minutes after the beginning of reperfusion in the levosimendan hearts was 37 ± 5 mm Hg, significantly lower (P < 0.05) than that in the control hearts (56 ± 4 mm Hg) or in the ischemic preconditioned hearts (53 ± 10 mm Hg). As a result, the recovery in LVDP (i.e., the difference between systolic and diastolic pressure) at the end of the experiment was significantly improved by levosimendan pretreatment (Fig. 3). LVDP in the levosimendan group 90 minutes after reperfusion was 38 ± 6% of the baseline (preischemia) value, compared with 16 ± 5% in the control group (P < 0.05). LVSP is recovered 90 minutes after reperfusion in the ischemic preconditioned hearts; however, due to the scattering of individual data, this difference is not statistically significant (80 ± 5.9% vs. 64 ± 5.6% in controls, t = 1.931, 0.01 < P < 0.05).

The first derivative of left ventricular pressure change (±dP/dt) significantly increased during levosimendan pretreatment (data not shown) but returned to baseline during washout. The recovery of both inotropic (+dP/dt) and lusitropic (−dP/dt) parameters was better with levosimendan pretreatment than in either comparator groups (P < 0.05 vs. controls; Fig. 3).

Coronary flow during reperfusion was changed to a similar extent in all three groups (Fig. 3).

Effect of Preconditioning on the Infarct Size after Global Ischemia and Reperfusion

Infarct size was expressed as percentage of the wet weight of the left ventricle, including the interventricular septum. Because global ischemia had been applied, the entire myocardium was at risk. Global myocardial ischemia for 30 minutes followed by 120 minutes of reperfusion in control hearts resulted in spotty staining, due to the loss of enzyme from damaged myocytes (Fig. 4). Relative to controls (average end-of-study infarct volume 52 ± 2%), the volume of infarcted myocardium was significantly smaller in the ischemic preconditioned hearts (38 ± 2%) and in the levosimendan group (45 ± 2%; P < 0.05 for both comparisons vs. controls; Fig. 4).

FIGURE 4
FIGURE 4:
Effects of ischemic preconditioning and levosimendan pretreatment on infarct size after ischemia-reperfusion. Top, Representative picture of staining with nitroblue-tetrazolium of a control rabbit heart slices after 30 minutes of global myocardial ischemia and 120 minutes of reperfusion (A) and evaluation of the infarcted area of the same heart using ImageJ software (B). Dark area represents noninfarcted myocardium. Bottom, Values of the infarct size in the three groups are expressed as means ± SEM (n = 12). *P < 0.05 versus controls.

DISCUSSION

The results of the present study indicate that two pretreatment perfusions of 5-minutes duration with 0.1 μmol/L levosimendan significantly improved the mechanical function of isolated perfused rabbit hearts and decreased infarct size after global myocardial ischemia and reperfusion. Such a dose was chosen because it results in a plasma concentration of the drug pharmacokinetically consistent with the one reached in therapy.19

The positive inotropic effect of levosimendan was observed during its administration, but there was a rapid and complete recovery during the washout period. The rapid recovery of LVSP, +dP/dT, and -dP/dT to baseline values during the washout period suggests that the compound was completely washed out from the heart before ischemia began. The fact that levosimendan demonstrated no effect on coronary flow during reperfusion confirmed that the drug, which has been shown to have a vasodilating effect on the coronaries,20 is no longer present in the heart tissue.

Despite this, the recovery of contractile activity was more complete and ischemia was better tolerated in the levosimendan-pretreated hearts than in control or ischemic preconditioned hearts. Our data are thus in accordance with previous findings that levosimendan decreases infarct size after coronary artery ligation in dogs14 and rabbits.21 In those others studies, however, the actions of the drug as a vasodilator (by opening the KATP channels on the sarcolemma of vascular smooth muscle cells) and as a cardioprotector (by opening mitochondrial KATP channels in cardiomyocytes) could not be distinuished because the drug was administered throughout the ischemic-reperfusion event. The results of the current study suggest that short-term administration of levosimendan before an ischemic event could act as a cardioprotector by decreasing myocardial damage. Our data are also consistent with the recent demonstration of a cardioprotective (presumed preconditioning) effect of preoperative levosimendan in patients undergoing coronary artery bypass grafting (CABG).22

It would be of interest to investigate further whether levosimendan could improve the effectiveness of ischemic preconditioning, and whether a synergistic effect between the two could facilitate adequate myocardial protection with the use of fewer or shorter ischemic preconditioning cycles. It would also be of interest to investigate whether levosimendan could exert a possible postconditioning effect.23,24 Undoubtly, experiments in which the heart is perfused also with a mitochondrial KATP channel blocker such as glibenclamide could be useful to substantiate the role these channels have in this response.

CONCLUSIONS

Levosimendan preconditioning improves mechanical function and reduces the infarct volume in isolated perfused rabbit hearts during recovery from myocardial ischemia. These data support possible alternative uses of levosimendan in therapeutical fields in which potassium channel openers that can elicit a preconditioning effect could be beneficial, such as the reduction of pain related to the first intra-aortic balloon inflation in coronary angioplasty25,26 or the reduction of ischemic adverse events after coronary artery bypass grafting.27

REFERENCES

1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124-1136.
2. Vegh A, Komori S, Szekeres L, et al. Antiarrhythmic effects of preconditioning in anaesthetised dogs and rats. Cardiovasc Res. 1992;26:487-495.
3. Tanaka K, Ludwig LM, Kersten JR, et al. Mechanisms of cardioprotection by volatile anesthetics. Anesthesiology. 2004;100:707-721.
4. Bolli R. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol. 2001;33:1897-1918.
5. Peart JN, Gross ER, Gross GJ. Opioid-induced preconditioning: recent advances and future perspectives. Vascul Pharmacol. 2005;42:211-218.
6. Peart JN, Gross ER, Gross GJ. Effect of exogenous kappa-opioid receptor activation in rat model of myocardial infarction. J Cardiovasc Pharmacol. 2004;43:410-415.
7. Minatoguchi S, Uno Y, Kariya T, et al. Cross-talk among noradrenaline, adenosine and protein kinase C in the mechanisms of ischemic preconditioning in rabbits. J Cardiovasc Pharmacol. 2003;41:S39-S47.
8. Asimakis GK, Inners-McBride K, Conti VR, et al. Transient beta adrenergic stimulation can precondition the rat heart against postischaemic contractile dysfunction. Cardiovasc Res. 1994;28:1726-1734.
9. Hearse DJ, Sutherland FJ. Catecholamines and preconditioning: studies of contraction and function in isolated rat hearts. Am J Physiol. 1999;277:H136-H143.
10. Honda HM, Korge P, Weiss JN. Mitochondria and ischemia/reperfusion injury. Ann N Y Acad Sci. 2005;1047:248-258.
11. Geshi E, Ishioka H, Nomizo A, et al. The role of ATP-sensitive potassium channels in the mechanism of ischemic preconditioning. J Cardiovasc Pharmacol. 1999;34:446-453.
12. Kopustinskiene DM, Pollesello P, Saris NE. Potassium-specific effects of levosimendan on heart mitochondria. Biochem Pharmacol. 2004;68:807-812.
13. Lepran I, Papp JG. Effect of long-term oral pretreatment with levosimendan on cardiac arrhythmias during coronary artery occlusion in conscious rats. Eur J Pharmacol. 2003;464:171-176.
14. Kersten JR, Montgomery MW, Pagel PS, et al. Levosimendan, a new positive inotropic drug, decreases myocardial infarct size via activation of K(ATP) channels. Anesth Analg. 2000;90:5-11.
15. Du Toit EF, Muller CA, McCarthy J, et al. Levosimendan: effects of a calcium sensitizer on function and arrhythmias and cyclic nucleotide levels during ischemia/reperfusion in the Langendorff-perfused guinea pig heart. J Pharmacol Exp Ther. 1999;290:505-514.
16. Maytin M, Colucci WS. Cardioprotection: a new paradigm in the management of acute heart failure syndromes. Am J Cardiol. 2005;96:26G-31G.
17. Tanno M, Miura T, Tsuchida A, et al. Contribution of both the sarcolemmal K(ATP) and mitochondrial K(ATP) channels to infarct size limitation by K(ATP) channel openers: differences from preconditioning in the role of sarcolemmal K(ATP) channels. Naunyn Schmiedebergs Arch Pharmacol. 2001;364:226-232.
18. Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res. 1980;47:1-9.
19. Sandell EP, Hayha M, Antila S, et al. Pharmacokinetics of levosimendan in healthy volunteers and patients with congestive heart failure. J Cardiovasc Pharmacol. 1995;26:S57-S62.
20. Kaheinen P, Pollesello P, Levijoki J, et al. Levosimendan increases diastolic coronary flow in isolated guinea-pig heart by opening ATP-sensitive potassium channels. J Cardiovasc Pharmacol. 2001;37:367-374.
21. Rump AF, Acar D, Rosen R, et al. Functional and antiischaemic effects of the phosphodiesterase inhibitor levosimendan in isolated rabbit hearts. Pharmacol Toxicol. 1994;74:244-248.
22. Tritapepe L, De Santis V, Vitale D, et al. Preconditioning effects of levosimendan in coronary artery bypass grafting-a pilot study. Br J Anaesth. 2006;96:694-700.
23. Yellon DM, Opie LH. Postconditioning for protection of the infarcting heart. Lancet. 2006;367:456-458.
24. Vinten-Johansen J, Yellon DM, Opie LH. Postconditioning: a simple, clinically applicable procedure to improve revascularization in acute myocardial infarction. Circulation. 2005;112:2085-2088.
25. Leesar MA, Stoddard M, Ahmed M, et al. Preconditioning of human myocardium with adenosine during coronary angioplasty. Circulation. 1997;95:2500-2507.
26. Tomai F, Crea F, Gaspardone A, et al. Ischemic preconditioning during coronary angioplasty is prevented by glibenclamide, a selective ATP-sensitive K+ channel blocker. Circulation. 1994;90:700-705.
27. Laskey WK, Beach D. Frequency and clinical significance of ischemic preconditioning during percutaneous coronary intervention. J Am Coll Cardiol. 2003;42:998-1003.
Keywords:

preconditioning; levosimendan; ischemia; cardiac mechanical activity; infarct size

© 2006 Lippincott Williams & Wilkins, Inc.