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Original Articles – Critical Care

Influence of temperature on the positive inotropic effect of levosimendan, dobutamine and milrinone

Rieg, Annette Da; Schroth, Sylvia Ca; Grottke, Olivera,b; Hein, Marca; Ackermann, Dianac; Rossaint, Rolfa; Schälte, Gereona

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European Journal of Anaesthesiology: November 2009 - Volume 26 - Issue 11 - p 946-953
doi: 10.1097/EJA.0b013e328330e9a0
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Abstract

Introduction

Heart failure is a common problem after myocardial infarction and cardiopulmonary bypass. Both pharmacological interventions and mechanical assistance [intraaortic cannula pump (IACP and assist device)] may help to overcome this clinical situation. Cardiotonic agents, such as digitalis, adrenaline, dobutamine (Sigma Chemical Co., St. Louis, Missouri, USA) and the phosphodiesterase-III inhibitors (PDE-III inhibitors), all lead, although by different mechanisms, to an increase in intracellular Ca2+ concentration. Thus, they act as Ca2+ mobilizers via their upstream mechanism and so improve cardiac contractility. However, dependent on elevated Ca2+ levels, myocardial oxygen cost (MVO2) and occurrence of dysrhythmias are also increased [1–3]. In contrast, levosimendan (Orion Corporation, Espoo, Finland) acts, because of its Ca2+-sensitizing effect, via central and downstream mechanisms [4–6]. At higher dosages, it also exerts a PDE-III-inhibiting effect [7,8]. Finally, levosimendan-mediated inotropy does not first cause a significant increase in intracellular Ca2+ levels. Because of this, it does not impair diastolic relaxation [9,10] and does not alter MVO2[11–13]. Antiischaemic and cardioprotective properties have also been proven for levosimendan, owing to its ability to open ventricular K+ATP channels [14–17]. In addition, levosimendan acts to open K+ATP channels in vascular smooth muscles and, therefore, leads to a decrease in pulmonary, coronary artery and systemic resistance [18–20]. In the case of septic cardiomyopathy, in which myofilament properties are altered due to increased levels of troponin I phosphorylation, levosimendan still improves inotropy, possibly by acting as a Ca2+ resensitizer [21–24]. Although a large body of work is devoted to studies of the different mechanisms of levosimendan, dobutamine and milrinone (Sanofi-Winthrop, Collegeville, Pennsylvania, USA), there are only a few studies that focus on their inotropic effect under hypothermic and hyperthermic conditions [25–27]. However, this is a situation of great interest because patients depending on inotropic support are often hypothermic because of extracorporal bypass or hyperthermic because of an inflammatory response. It was our intention to determine whether levosimendan, with its preferable pharmacological profile, improves contractility independent of temperature. For this reason, we investigated the positive inotropic effect of levosimendan under varied temperature conditions. We also compared it with milrinone and dobutamine, which have both been used for a significant amount of time in clinical practice.

Methods

The study was performed in accordance with the guidelines of the Animal Care Committee of the University Hospital Aachen (RWTH Aachen, Aachen, Germany) and German law concerning the care and use of animals.

Female guinea pigs (n = 32, tribe pbw) were killed by a blow to the head, followed by the opening of both carotid arteries. The hearts were excised and placed in oxygenated 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) buffer solution (NaCl 136 mmol l−1, KCl 3.3 mmol l−1, KH2PO4 1.2 mmol l−1, MgSO4 1.1 mmol l−1, CaCl2 2.5 mmol l−1, glucose 10 mmol l−1 and HEPES 10 mmol l−1). The pH of the buffer was adjusted to 7.40 at 37°C. During preparation of the ventricular trabeculae, the heart was set in a HEPES buffer bath, which was supplemented with 2,3-butanedione-monoxime (BDM; Sigma Chemical Co.). BDM was shown to have a reversible negative inotropic effect, which protects the myocardium from cutting injury [28]. The size of the chosen ventricular trabeculae ranged from 0.5 to 1.0 mm (mean 0.73 mm). After preparation of the ventricular trabeculae and rinsing of the BDM, the muscle strings were fixed with two special clips, one of which was connected to a precalibrated force transducer (Scientific Instruments, Heidelberg, Germany) and placed in the HEPES buffer bath (37°C), which was continuously bubbled with 100% oxygen. During stimulation (1.3 Hz and a current of 8 mA), the muscle strings were first allowed to reach a steady state in force contraction and then prestretched until a maximum force of contraction was achieved. Muscle strings that did not reach a stable steady state of contraction or that developed dysrhythmias during prestretching were excluded from the experiments. Subsequently, the target temperature in the bath was changed to 31°C, 34°C or 40°C (Fig. 1a and b), and the maximum developed force of contraction (corresponds to baseline 100%) was again recorded. According to the protocol, levosimendan, dobutamine or milrinone was increased stepwise (from 10−9 to 10−5 mol l−1, milrinone up to 10−4 mol l−1) until maximum inotropy was achieved and no induction of dysrhythmias occurred. In addition to the temperature, the following parameters were continuously recorded (Software: Twitch; Scientific Instruments): maximum developed force (in μN); time to peak tension (TPT in ms), which is defined as the time until maximum systolic force is reached; Tsystolic50% (in ms), which is part of the systolic period until 50% of systolic force has developed; and Tdiastolic50% (in ms), which is defined as the period until 50% of systolic muscle tension remains. At the end of each experiment, the trabeculae were tested for functional integrity by adding calcium (final concentration 10 mmol l−1). In total, 120 muscle strings were tested for levosimendan, dobutamine and milrinone, and 10 ventricular trabeculae for each temperature and each substance.

Fig. 1
Fig. 1

Statistical analysis

Statistical analyses were conducted using SAS software version 9.1 (SAS Institute, Cary, North Carolina, USA). The absolute values varied markedly throughout the population of muscle strings. Therefore, the force of contraction was normalized at each temperature to baseline 100%, before adding any cardiotonic agent. Values are expressed as mean ± SD. The influence of temperature and concentration of the tested drug on the maximum developed force, TPT, Tsystolic50% and Tdiastolic50% was investigated using a two-way (dose vs. temperature) repeated measures analysis of variance (ANOVA). A statistically significant influence was identified if the resulting F test showed a P value lower than 0.05.

Changes in contractility induced by temperature change were compared by the exact Wilcoxon test (paired samples). Paired samples were as follows: 37°C vs. 31°C, 37°C vs. 34°C and 37°C vs. 40°C. The comparison of effectiveness of the three different inotropic substances at the respective optimal concentration was evaluated by the exact Mann–Whitney U test, separately for every temperature. P values lower than 0.05 were considered to be statistically significant.

Results

Temperature influences force and time of contraction of untreated ventricular trabeculae

Both the inotropic force of contraction and the time of contraction were temperature dependent (Fig. 1a and b). The force of contraction (Fig. 1a) was significantly influenced by temperature (P < 0.0001), leading to higher values under hypothermic conditions, with a maximum at 31°C in comparison with normothermia. Even moderate hypothermia (34°C) caused accelerated inotropy (P = 0.0012). In contrast, induction of hyperthermia (40°C) resulted in significantly reduced contractility (P < 0.0001). A temperature decrease from 37°C to 34°C or to 31°C led to the prolongation of TPT, Tsystolic50% and Tdiastolic50% (P < 0.0001 at 31°C and 34°C, for all three times) and was most distinctive at 31°C (Fig. 1b). In contrast, induction of hyperthermia (40°C) led to significant shortening of all three times (P < 0.0001). Duration of the systolic period was prolonged under hypothermic conditions and reduced in the case of hyperthermia, in general.

Increase of inotropic force by levosimendan, dobutamine and milrinone

Levosimendan exerted a significant dose-dependent positive inotropic effect (P < 0.0001). Maximum achieved increase of inotropy (Fig. 2a) did not differ significantly in the various temperature ranges (P = 0.0643). Also, no interaction of temperature and concentration could be seen (P = 0.1886). The maximum of inotropy was reached under concentrations of 10−6 mol l−1. Higher concentrations (≥10−5 mol l−1) of levosimendan did not lead to any further increase in inotropy. No concentration of levosimendan provoked the occurrence of dysrhythmias. Thus, levosimendan increased inotropy in a dose-dependent, but temperature-independent, manner.

Fig. 2
Fig. 2

Dobutamine (Fig. 2b) showed a significant, dose-dependent, positive inotropic effect (P < 0.0001). Maximum contractility was reached if it was added at a concentration of 10−5 mol l−1. Further increases up to 10−4 mol l−1 led to the occurrence of dysrhythmias. At 31°C, the inotropic effect of dobutamine was nearly abolished. At 34°C and 37°C, dobutamine caused an enhancement of inotropy, and at 40°C, its inotropic effect was at a maximum. The experimental setting gave rise to the suspicion that dobutamine-related inotropy was temperature dependent. However, the statistical evaluation did not show a significant influence of temperature (P = 0.0624) on the positive inotropic effect of dobutamine. There existed a significant interaction between temperature and concentration (P = 0.0001). Development of dobutamine-induced contractility differed in the various temperature ranges. Maximum achieved force was reached at 31°C if a concentration of 10−6 mol l−1 was used. Within the other temperature ranges, maximum achieved force was reached if dobutamine was used at a concentration of 10−5 mol l−1.

Milrinone (Fig. 2c) increased inotropy in a dose-dependent manner (P < 0.0001). The maximum effect was reached if it was used at a dosage of 10−4 mol l−1, with higher concentrations leading to the occurrence of dysrhythmias. A maximum increase of inotropy was reached under normothermic conditions (37°C). At 31°C and 34°C, its inotropic effect was abolished. At 40°C, contractility was enhanced, although the effect was less distinctive than at 37°C. Thus, milrinone acts in a temperature-dependent manner (P < 0.0001). The interaction of temperature and concentration of milrinone had a significant influence (P < 0.0001) on the development of force over the various temperatures. At 31°C, maximum force was reached if milrinone was used at a concentration of 10−7 mol l−1; at 34°C at a concentration of 10−5 mol l−1; and at 37°C and 40°C at a concentration of 10−4 mol l−1.

A statistical comparison (Mann–Whitney U test) was carried out for the three agents at their optimal concentrations for maximum inotropic effect for each temperature. At 31°C (Fig. 3a), levosimendan was superior to dobutamine (P = 0.0185) and to milrinone (P = 0.0266). A significant difference did not exist between dobutamine and milrinone (P = 0.4598).

Fig. 3
Fig. 3

At 34°C (Fig. 3b), levosimendan exerted a superior inotropic effect in comparison with milrinone (P = 0.0143). Although dobutamine exerted a higher increase in force than levosimendan, the forces did not differ statistically (P = 0.6305). This lack of significance might be explained by a high SD in the dobutamine group. Milrinone and dobutamine did not differ significantly (P = 0.1839) in this temperature range.

At 37°C (Fig. 3c), milrinone influenced inotropy more strongly than levosimendan (P = 0.002). No significant difference was found for dobutamine and levosimendan (P = 0.1145) or for milrinone and dobutamine (P = 0.061).

At 40°C (Fig. 3d), dobutamine and milrinone exerted a superior inotropic effect compared with levosimendan (both P = 0.0185). Milrinone and dobutamine did not differ statistically (P = 0.2475).

Influence of levosimendan, dobutamine and milrinone on duration of contraction

Levosimendan shortened TPT and Tsystolic50% (P < 0.0001 for both) in a dose-dependent fashion. Reduction of both periods was most pronounced if the levosimendan concentration was 10−5 mol l−1. Levosimendan shortens both times independently of temperature (P = 0.2637 for TPT and P = 0.4423 for Tsystolic50%). No interaction of temperature and concentration existed (P = 0.9636 for TPT and P = 0.8683 for Tsystolic50%). Levosimendan reduced Tdiastolic50% dose-dependently at 31°C, 37°C and 40°C if a high concentration (10−6 and 10−5 mol l−1) was used. In contrast, at 34°C, levosimendan prolonged Tdiastolic50%. Low concentrations extended Tdiastolic50%. Statistical evaluation showed a concentration (P = 0.0423) and temperature-dependent (P = 0.0315) effect, as well as an interaction of both (P = 0.0262).

Dobutamine reduced TPT, Tsystolic50% and Tdiastolic50%. Reduction of these times depended significantly on the concentration of dobutamine (P < 0.0001 for all). The time-reducing effect was most pronounced if dobutamine was added at a concentration of 10−5 mol l−1. Statistical evaluation of dobutamine-induced dose-dependent reduction for any time during the systolic period showed no influence by the experimental temperature (P = 0.0595 for TPT, P = 0.4288 for Tsystolic50% and P = 0.2699 for Tdiastolic50%). No interaction existed between the temperature and concentration of dobutamine (P = 0.2395 for TPT, P = 0.2326 for Tsystolic50% and P = 0.3703 for Tdiastolic50%).

Milrinone had a statistically significant effect on TPT and Tsystolic50% (P < 0.0001 for both). It also reduced both times in a dose-dependent manner at each temperature level, even at 31°C and 34°C, in which milrinone-related inotropy was suppressed. Statistical evaluation showed independence of the experimental temperatures (P = 0.3384 for TPT and P = 0.1185 for Tsystolic50%). For TPT, there was no interaction between temperature and concentration (P = 0.1519) in contrast to Tsystolic50% (P < 0.0001). The concentration of milrinone did not influence Tdiastolic50% (P = 0.8704) nor did the temperature (P = 0.7528) or the interaction between temperature and concentration (P = 0.6078).

Discussion

To the best of our knowledge, this is the first study investigating and comparing the inotropic effect of levosimendan, dobutamine and milrinone at various temperature levels. Our study reveals a dose-dependent inotropic effect of levosimendan, dobutamine and milrinone and is in accordance with other studies [22,29,30] that have been performed in distinct species (rabbit, guinea pig and humans). Moreover, we demonstrated that levosimendan increases inotropy independent of the temperature. Dobutamine-related inotropy exhibited a clear trend towards temperature dependence, although statistical evaluation failed to show a statistically significant relationship. In contrast, milrinone acts as a positive inotrope that is dependent on the experimental temperature. At 31°C, levosimendan was the most effective inotropic agent, and at 34°C, it was definitely superior to milrinone. In the case of normothermia, milrinone proved to be the most effective substance, and at 40°C, dobutamine accelerated contractility best of all three agents. Apart from their different optimum temperature range, all three cardiotonic agents reduced the duration of the systolic period in a dose-dependent fashion but independent of temperature. Shortening of the systolic period occurred independent of their inotropic effect, shown for dobutamine at 31°C, for milrinone at 31°C and 34°C and for levosimendan at a concentration of 10−5 mol l−1 at each temperature. Thus, these inotropic agents abbreviate even hypothermia-induced prolongation or hyperthermia-provoked shortening of the systolic period.

It has long been known that myocardial contractility and the duration of contraction are both temperature dependent [31–40]. Nevertheless, the underlying mechanisms of hypothermia-induced positive inotropy have still not been resolved. Kaufmann and Fleckenstein [41] postulated that the effect was due to longer opening times of Ca2+ channels, because of a prolonged duration of action potential and consecutive accelerated intracellular Ca2+ influx and Ca2+ content. This has been confirmed by later studies [42]. In contrast, Weisser et al. [34] showed hypothermia-induced inotropy to be independent of elevated intracellular Ca2+ content or Ca2+ transients. They assumed that hypothermia-related inotropism was due to enhanced Ca2+ responsiveness. This theory is supported by Churcott et al. [38] and Fabiato and Fabioto [43], who demonstrated improved Ca2+ responsiveness by cooling-induced intracellular alkalosis. Hypothermia-induced reduction of Na+–K+-ATPase activity and consecutive enhanced Na+/Ca2+ exchange also contribute to hypothermia-related increased intracellular Ca2+ levels and hypothermia-related increased inotropy [44]. These conflicting results may be explained, in part, by the use of different species and various hypothermic temperature ranges. These findings suggest that both modulation of intracellular Ca2+ homeostasis and Ca2+ responsiveness play a central role, or possibly act synergistically, in temperature-altered inotropy.

Milrinone has never been investigated and compared under hypothermic conditions, whereas for levosimendan and dobutamine, only a few studies exist. Kaheinen et al. [27] studied the inotropic effect of levosimendan in papillary muscles of the guinea pig at 30°C and demonstrated a preserved inotropic effect. In a similar model, Melnikov et al. [32] investigated cyclic AMP (cAMP)-dependent isoproterenol under hypothermic conditions and proved an abolished inotropic effect at 28°C, although its effect remained at 35°C. In cat papillary muscle, Sys et al. [45] showed that the inotropic effect of the PDE-III inhibitor amrinone was suppressed at 29°C relative to its effect at 37°C. On the contrary, apparently contradictory results exist. Riishede and Nielsen-Kudsk [25] demonstrated a manifest inotropic effect of dobutamine at 22°C in the isolated perfused rabbit heart. Oung et al. [26] demonstrated dobutamine-induced inotropy even at 30°C in the pig. Lochner et al. [46] proved a clear positive inotropic effect of levosimendan after normothermic cardiac arrest in the isolated perfused guinea pig heart; in contrast, after hypothermic (20°C) cardiac arrest, contractility was not improved. In contradiction to this, Nijhawan et al. [47] showed that cardiac performance was improved in patients after hypothermic cardiac arrest (28°–30°C) in comparison with control patients treated with placebo. Our own clinical observations support the results of Nijhawan et al. [47]. Clinical studies in patients focusing on the efficacy of different cardiotonic agents under hypothermia and hyperthermia have not been performed until now.

What might be the reason for suppression of the inotropic effect of dobutamine and milrinone under hypothermia? Both agents improve contractility through elevation of intracellular cAMP levels and Ca2+ levels [48,49]. Additionally, hypothermia leads to highly increased intracellular Ca2+ levels [42]. It is possible that the addition of pharmacological agents, such as Ca2+ mobilizers, may not lead to a further increase in intracellular Ca2+ content because maximum levels have already been reached. Therefore, treatment with Ca2+ mobilizers might fail to enhance inotropy further under hypothermic conditions. Further, the enzymes adenylate cyclase and PDE-III might be inhibited under low temperature conditions. On the contrary, Ca2+ mobilizers exert a clear increase in contractility under normothermia and hyperthermia. During normothermia or hyperthermia, Ca2+ levels are normal or at least able to be increased. However, dobutamine and milrinone differ in regard to their optimal temperature range. Milrinone exerts its maximum effect at 37°C, indicating that PDE-III acts most effectively at 37°C. Dobutamine acts well in the extended temperature field between 34°C and 40°C. The best effect is achieved at 40°C. A possible explanation might be reduced sensitivity to temperature for adenylate cyclase relative to PDE-III.

Although the inotropic effect of dobutamine and milrinone is suppressed at 31°C, and that of milrinone at 34°C, they both counteract the contraction-prolonging effect of hypothermia. These results are in line with those of Endoh et al. [48]. Therefore, the diastolic relaxation time is prolonged, as is the perfusion time in the left ventricle; together with improved filling conditions, this contributes to improved haemodynamics.

Levosimendan, which has a Ca2+-sensitizing effect and, at concentrations more than 10−7 mol l−1, also has a PDE-III-inhibiting effect [6], improves inotropy between 31°C and 40°C. It is known that the usage of levosimendan up to a concentration of 10−7 mol l−1 increases inotropy without affecting Ca2+ transients or sarcoplasmic reticulum Ca2+ content [8]. Thus, a preserved inotropic effect under hypothermia is not surprising. Hypothermia-related increases in intracellular Ca2+ content may act in synergy with levosimendan's Ca2+-sensitizing effect. Assuming this, levosimendan is suspected to exert reduced inotropic effects between 37°C and 40°C. This is in contradiction to our results. An explanation can be found in its additional function as a PDE-III inhibitor.

The findings of this study suggest that, depending on the experimental temperature, either the Ca2+-sensitizing effect or the PDE-III-inhibiting effect of levosimendan predominates, which results in a preserved inotropic effect independent of temperature. This hypothesis is supported by Haikala et al. [50], who could demonstrate no cAMP-dependent component of levosimendan (3 × 10−7 mol l−1) at 22°C, which is in contrast to Edes et al. [6], who proved that levosimendan (10−7 mol l−1) induced a significant increase in cAMP at 37°C. In clinical practice, plasma concentrations of levosimendan of at least 10−7 mol l−1 are usually reached. Jonsson et al. [51] measured plasma concentrations of 100 ng ml−1 in humans, which is equal to 3.5 × 10−7 mol l−1, after a levosimendan bolus of 6–24 μg kg−1 and a continuous levosimendan infusion of 0.05–0.2 μg kg−1 min−1. McGough et al. [52] measured a maximal plasma concentration of 120 ng ml−1 in conscious dogs, which corresponds to 4.2 × 10−7 mol l−1, after a levosimendan bolus of 24 μg kg−1 followed by a continuous application of 0.4 μg kg−1 min−1. Both results are similar to those of Nijhawan et al. [47], who demonstrated, in humans, a plasma concentration of 8.5 × 10−7 mol l−1 after a high bolus of 36 μg levosimendan followed by a continuous infusion of 0.3 μg kg−1 min−1. Therefore, the PDE-III-inhibiting effect of levosimendan contributes to improved cardiac performance after application of levosimendan.

Our results provide indirect evidence that temperature-altered myocardial contractility is more probably generated by intracellular modulation of Ca2+ content than by Ca2+ responsiveness of myofibrils. If temperature-altered inotropism was primarily due to changed Ca2+ responsiveness, we would expect the strongest inotropic effect of a Ca2+ sensitizer under hyperthermia, when Ca2+ responsiveness of myofibrils is decreased due to acidotic intracellular milieu [53].

Levosimendan reduces the duration of contraction between 31°C and 40°C, even if used at a concentration of 10−5 mol l−1, which does not further improve inotropy. This is in line with Yokoshiki et al. [54], who proved that levosimendan (at a concentration of 10−5 mol l−1) reduces the duration of action potentials by a mechanism dependent on its ability to open KATP channels in ventricular myocytes.

Our model has some limitations. For solving the underlying mechanism of the temperature-independent inotropic action of levosimendan and the temperature-dependent inotropic actions of dobutamine and milrinone, investigations of intracellular Ca2+ levels and Ca2+ transients under various temperature ranges are necessary. Further in-vivo studies and clinical studies are warranted to determine the haemodynamic benefit of levosimendan under hypothermic conditions.

Conclusion

Our data suggest no modulation of the positive inotropic effect of levosimendan by the experimental temperature chosen in vitro. Acting as a Ca2+ sensitizer, in contrast to the cAMP-coupled dobutamine and milrinone, levosimendan might be the most advantageous positive inotropic substance for patients with a hypothermic body temperature. Particularly during reperfusion after cardiopulmonary bypass, additional intracellular Ca2+ overload can be prevented by the application of levosimendan instead of Ca2+ mobilizers. Another favourable use might be the inotropic support during neuroprotective hypothermia following successful cardiopulmonary resuscitation.

Acknowledgements

Funding was received for this work from the Department of Anaesthesiology, University Hospital Aachen of the RWTH Aachen.

We acknowledge Professor René Tolba, MD (Institute for Laboratory Animal Science, Pauwelsstrasse, Aachen, Germany) and his team for their support and the opportunity to carry out this study in his laboratory.

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Keywords:

cardiotonic agents; dobutamine; hyperthermia; hypothermia; levosimendan; milrinone; ventricular trabeculae

© 2009 European Society of Anaesthesiology