Objective
Levosimendan, a compound that exerts effects on calcium sensitivity and intracellular free calcium, in addition to opening ATP-sensitive K-channels, is widely used in the treatment of heart failure. Because of its dual mechanism of action, we hypothesized that it would modulate human uterine contractility.
Study Design
Biopsies of human myometrium were obtained at elective cesarean section (n = 16). Dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of 1 nmol/L to 100 mmol/L. In separate experiments, the effects of prior exposure to the K-ATP antagonist glibenclmide (100 mmol) on the effects of levosimendan on myometrial contractility were evaluated. Simultaneous controls were performed for all experiments.
Results
Levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, when compared with control strips. The mean maximal inhibition (MMI) values were as follows: 45.34% ± 5.92% for spontaneous contractions (n = 6; P < .05), and 41.88% ± 5.40% for oxytocin-induced contractions (n = 6; P < .05). The inhibitory effect of levosimendan was significantly antagonized by glibenclamide, resulting in the mean maximal inhibition for levosimendan reduced to 19.04% ± 3.61% for spontaneous contractions (n = 6; P < .05), and 16.53% ± 4.08% for oxytocin induced contractions (n = 6; P < .05).
Conclusion
Levosimendan exerted a potent relaxant effect on spontaneous and agonist-induced human uterine contractility in vitro. This effect was reduced in the presence of K-ATP blockade. Because of the putative role of levosimendan in inflammatory conditions, the findings here may have implications for its future use as therapy for preterm labor.
Levosimendan is an unusual compound in that it has a dual mechanism of action. It is a pyridazinone-dinitrile derivative, which is largely classified as being a calcium sensitizer, albeit it has varying effects related to intracellular calcium in both cardiac and vascular smooth muscle. Although it appears to predominantly increase the sensitivity of contractile proteins to calcium, it may also reduce intracellular calcium, and reduce the calcium sensitivity of contractile proteins in vascular smooth muscle particularly. In addition to these contradictory effects in terms of intracellular calcium, it has also been shown to activate K+ channels, mainly of the ATP-dependent type (K-ATP). Because of its combined effect on intracellular calcium, which in cardiac smooth muscle results in an inotropic effect, and the potent vasodilatory effect elicited by K+ channel activation, it has been under intense investigation for its benefits for acute and chronic heart failure in recent years. The resultant physiologic effects of its use in cardiac smooth muscle have been that of positive inotropy and vasodilatation, although the relative contributions of each of these mechanisms in terms of its vasorelaxation are unclear. Second, by presumably separate mechanisms, levosimendan appears to exert an immunomodulatory effect resulting in a reduction of circulating proinflammatory cytokines and soluble apoptosis mediators, in patients with decompensated heart failure.
There are no data to our knowledge outlining the effects of levosimendan on nonvascular smooth muscle. Because it could potentially increase or reduce sensitivity to intracellular calcium in uterine smooth muscle, and, in addition, activate K-ATP channels, we hypothesized that it would modulate human uterine contractility. The aim of this study was to evaluate the effects of levosimendan on uterine contractility in isolated preparations obtained during human pregnancy, in relation to spontaneous and agonist-induced contractility. The secondary aim was to evaluate the effects of levosimendan in the absence and presence of K-ATP blockade using glibenclamide.
Materials and Methods
Tissue collection
Biopsies of human myometrial tissue were obtained at elective cesarean section in the third trimester of pregnancy in the Department of Obstetrics and Gynecology, University College Hospital, Galway, Ireland. Ethical committee approval for tissue collection was obtained from the Research Ethics Committee at University College Hospital Galway and recruitment of patients was by informed written consent. The biopsies were excised from the upper portion of the lower segment of the uterus. Once collected, all tissue biopsies were placed in Krebs-Henseleit physiologic salt solution (PSS) at pH 7.4 containing the following: 4.7 mmol/L potassium chloride, 118 mmol/L sodium chloride, 1.2 mmol/L magnesium sulphate, 1.2 mmol/L calcium chloride, 1.2 mmol/L potassium phosphate, 25 mmol/L sodium bicarbonate, and 11 mmol/L glucose (Sigma-Aldrich, Dublin, Ireland). Tissues were stored at 4°C and used within 12 hours of collection.
Tissue bath experiments
Longitudinal myometrial strips (measuring approximately 2 × 2 × 10 mm) were dissected free of uterine decidua and serosa and mounted for isometric recording under 2 g of tension in organ baths as previously described. The tissue baths contained 10 mL Krebs-Henseleit PSS maintained at 37°C, pH 7.4, and were gassed continuously with a mixture of 95% oxygen/5% carbon dioxide. Myometrial strips were allowed to equilibrate for a period of at least 1 hour, during which time the Krebs-Henseleit PSS was changed every 20 minutes. After equilibration, a 30-minute period was allowed to achieve spontaneous phasic contractions, or, alternatively, contractions were stimulated by bath exposure of the strips to oxytocin (0.5 nmol/L). Levosimendan was then added to the tissue bath in a cumulative manner at bath concentrations of 1 nmol/L, 10 nmol/L, 100 nmol/L, 1 μmol/L, and 10 μmol/L at 20-minute intervals. There were 2 sets of control experiments performed as follows: control 1, strips exposed to either PSS only (spontaneous contractions) or PSS and 0.5 nmol/L oxytocin (oxytocin-induced contractions); and control 2, strips exposed to PSS and vehicle for levosimendan (spontaneous contractions) or PSS, 0.5 nmol/L oxytocin and vehicle for levosimendan (oxytocin-induced contractions). The effects of levosimendan and the respective controls were assessed by calculation of the integral from a minimum of selected areas for each 20-minute intervals and expressed as a percentage of the integral obtained in the 20-minute period before any levosimendan addition using the PowerLab hardware unit and Chart v4.0 software (AD Instruments, Hastings, UK). The inhibitory effect of levosimendan was corrected for the reduction in the contractile activity observed in the vehicle control (control 2), and the effects of levosimendan were interpreted as the final additional relaxant effect. This value, when subtracted from 100% represents the mean maximum inhibition (MMI) of levosimendan on myometrial contractility. The MMI provided, therefore, represents the net final inhibition resulting from exposure to levosimendan, that is, after subtraction of any alteration observed in vehicle control (control 2) experiments.
Separate experiments were performed to evaluate the effects of levosimendan on uterine contractility in the presence of the ATP-dependant potassium channel (K-ATP) blocker, glibenclamide. After the period of equilibration, strips were allowed to contract spontaneously, or were exposed to oxytocin, as outlined previously, for a 30-minute period. Glibenclamide 10 μM was then added to the tissue bath for a 30-minute period, before exposure to levosimendan cumulatively, exactly as outlined previously. Simultaneous control experiments involving exposure to 10 μM glibenclamide alone were also performed. The MMI calculated represents the net final inhibition resulting from exposure to glibenclamide and levosimendan, that is, after subtraction of any alteration observed in the glibenclamide-only control experiments.
Drugs and solutions
Levosimendan was kindly provided by Abbott Pharmaceuticals, Abbott Park, IL. A stock solution (100 μmol/L) was made in 70% EtOH. Serial dilutions were made in deionized water on the day of experimentation. Fresh Krebs-Henseleit solution was made daily. A stock solution of oxytocin (1 mmol/L; Sigma-Aldrich) was prepared using deionized water. Serial dilutions were prepared in deionized water on the day experiments were carried out. Glibenclamide was purchased from Sigma-Aldrich.
Statistical analysis
Comparisons of contractile effect, for each bath concentration of levosimendan were performed using analysis of variance, followed by Tukey HSD post hoc testing to determine significant differences among data groups. A P value of < .05 was considered to be statistically significant. A paired sample t test was used to compare spontaneous and oxytocin-induced vehicle controls with spontaneous and oxytocin-induced controls, respectively. The statistical package SPSS for Windows version 11.0 (SPSS Inc, Chicago, IL) was used for these statistical calculations.
Results
Levosimendan exerted an inhibitory effect on both spontaneous and oxytocin-induced contractions in human myometrium in vitro, in all strips, in comparison to control measurements. A representative recording of spontaneous myometrial contractions, as shown in Figure 1 , A (control 1) and Figure 1 , B, demonstrates a recording of spontaneous myometrial contractions treated with increasing concentrations of vehicle for levosimendan (1 nmol/L, 10 nmol/L, 100 nmol/L, 1 μmol/L, and 10 μmol/L) (control 2). At maximal concentration, the integrals of contractile activity measured for spontaneous control 1 experiments (n = 6), and those for control 2 experiments (n = 6), were compared, and no significant difference was found. It was, therefore, concluded that the vehicle for levosimendan exerted no significant effect on myometrial contractions. Figure 1 , C shows a representative recording of spontaneous myometrial contractions treated with levosimendan and demonstrates the relaxant effect of levosimendan on contractility. The reduction in contractility for spontaneous contractions was cumulative and attained statistical significance at a bath levosimendan concentration of 10 nmol/L, 100 nmol/L, 1 μmol/L, and 10 μmol/L ( P < .05) in comparison to control strips (with vehicle). This is demonstrated in Figure 2 , where a graphical representation of the effect of increasing concentrations of levosimendan on spontaneous myometrial contractions is shown.
