INTRODUCTION

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SEVERAL DRUGS EMPLOYING NOVEL and diverse mechanisms of action have been developed to improve outcome and symptoms of patients with heart failure. However, cardiac glycosides are the only inotropes without harmful effects on outcome in patients with heart failure. Ca²⁺ sensitizers are pharmacological agents that improve cardiac contractility without increasing the intracellular Ca²⁺ transient. Therefore, Ca²⁺ sensitizers may be particular useful for the treatment of human heart failure for the following reasons: 1) they do not increase the activation energy that is required for handling intracellular Ca²⁺ ions, 2) they may not be associated with induction of arrhythmias and cell injury because they avoid Ca²⁺ overloading of myocardial cells, and 3) they may reverse the myocardial dysfunction that is produced under pathophysiological conditions.

However, Ca²⁺ sensitizers may impair diastolic cardiac function as a result of increased Ca²⁺ sensitivity of the myofilaments. The Ca²⁺ sensitizer EMD-57033, for example, has a direct effect on cross-bridge interaction (24). The positive inotropic effect of EMD-57033 has been shown to occur independently from changes of the intracellular Ca²⁺ transient (5). However, EMD-57033 has been shown to increase time to 80% relaxation and diastolic force especially in failing human myocardium (15).

Levosimendan is a newly developed inotropic agent that is reported to increase Ca²⁺ sensitivity of the heart by a novel mechanism of myofibrillar Ca²⁺ sensitization, i.e., via Ca²⁺-dependent stabilization of the Ca²⁺-bound conformation of cardiac troponin C (14), without affecting the Ca²⁺ affinity of troponin C (8). It has been proposed that levosimendan binds to troponin C in a Ca²⁺-dependent manner, which means that levosimendan binds to troponin C at systolic intracellular Ca²⁺ concentrations ([Ca²⁺]_i) and detaches from troponin C at diastolic [Ca²⁺] (13). The binding site of levosimendan is located in the NH₂ terminal domain of cardiac troponin C. In addition to this new Ca²⁺-sensitizing effect, levosimendan was shown to inhibit phosphodiesterase III at high concentrations, which may result in an increased [Ca²⁺]_i (16, 17).

Because levosimendan is considered a potential candidate for treatment in patients with congestive heart failure, it was the aim of the present study to investigate the effects of levosimendan on isolated myocardium of human left ventricular failing myocardium [(dilated cardiomyopathy; New York Heart Association class IV (NYHA IV)] as well as in nonfailing myocardium. Because the frequency-dependent force generation is impaired in the failing myocardium, possibly due to a dysregulation of the intracellular Ca²⁺ homeostasis, the effect of levosimendan on the intracellular Ca²⁺ transient and the force-frequency relationship was studied as well.

	MATERIALS AND METHODS

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Myocardial tissue. Failing human myocardium was obtained during cardiac transplantation (n = 18, 8 female and 10 male patients, 46 ± 4 yr old). These patients suffered from heart failure clinically classified as NYHA IV on the basis of clinical symptoms and signs diagnosed by the attending cardiologist. Patients were on angiotensin-converting enzyme inhibitors, nitrates, diuretics, and cardiac glycosides. Within 48 h before transplantation, none of these patients was given -adrenoreceptor or Ca²⁺ antagonists. Nonfailing human left ventricular myocardium was obtained from donor hearts that could not be transplanted because of technical reasons (n = 6; 3 female and 3 male patients; age: 38 ± 4 yr). All patients or patient donor families gave written informed consent before surgery. The study was approved by the local ethics committee and conformed to the Declaration of Helsinki. Immediately after explantation, the myocardial tissues were placed in ice-cold aerated modified Tyrode's solution and delivered to the laboratory within 10 min.

Contraction experiments. Experiments were performed on isolated electrically driven muscle preparations. Muscle strips (0.2 mm thick and 6-9 mm long) with muscle fibers running approximately parallel to the length of the strips were carefully dissected under microscopic control in aerated bathing solution on ice. The preparations were attached to a bipolar platinum-stimulating electrode and suspended individually in 75-ml glass tissue chambers to record the isometric contractions. The bathing solution used was the modified Tyrode's solution that contained (in mmol/l) 119.8 NaCl, 5.4 KCl, 1.05 MgCl₂, 1.8 CaCl₂, 22.6 NaHCO₃, 0.42 NaH₂PO₄, 0.05 Na₂EDTA, 5.5 glucose, and 0.28 ascorbic acid. It was continuously gassed with 95% O₂-5% CO₂ and maintained at 37°C (pH 7.4). The experiments were performed as previously described (22).

Simultaneous measurement of the intracellular Ca²+ transient and force of contraction. Intracellular Ca²⁺ was measured by the fluorescence indicator fura 2 (10). To facilitate cell loading, fura 2 was used as acetoxymethyl (AM) ester as described previously (5). These AM esters passively cross the plasma membrane and, once inside the cell, are cleaved to cell-impermeant products by intracellular esterases.

Materials. Levosimendan was kindly provided by Orion Pharma (Finland). All other chemicals were of analytic grade or the best grade commercially available. For studies with isolated cardiac preparations, stock solutions were prepared and applied to the organ bath. Levosimendan was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the bathing solution never exceeded 0.05%. All other compounds were dissolved in twice-distilled water. Applied agents did not change the pH of the medium.

Statistics. The data shown are means ± SE. For comparison within one group, the paired t-test was applied. Otherwise, statistical significance was analyzed with Student's t-test for unpaired observations. A value of P < 0.05 was considered significant. In the experiments, the inotropic effect of the drug in each muscle strip was compared with the control, drug-free situation of the very same preparation. The statistical analysis was confirmed by the Institute of Medical Statistics of the University of Cologne.

	RESULTS

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Concentration-dependent effects of levosimendan on isometric contraction. Concentration-response curves of levosimendan (0.03-10 µmol/l) were determined in isolated, electrically stimulated (1 Hz), isometrically contracting papillary muscle strips of human left ventricular failing (n = 6) and nonfailing myocardium (n = 4). Representative original tracings of the force of contraction (FOC) are given in Fig. 1. Levosimendan concentration dependently increased FOC in human nonfailing left ventricular myocardium: the maximal positive inotropic effect achieved after application of 10 µmol/l levosimendan was +13.2 ± 5.6 mN/mm² and the concentration that induced a 50% increase of the maximal positive inotropic effect (EC₅₀ of levosimendan) was 0.38 µmol/l (confidence interval: 0.27-0.54 µmol/l). Levosimendan (10 µM) significantly reduced time to half peak relaxation (T1/2T). As time to peak tension and tension decline are dependent on the developed force, the rate of maximal tension rise (+dT/dt) and maximal tension decay (dT/dt) were normalized by FOC. With the use of this analysis, levosimendan did not change the kinetics of force development or decline relative to force (Table 1). Diastolic force was not influenced by levosimendan under basal conditions.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Original registrations of the concentration-dependent (0.03-10 µM) effects of levosimendan on isometric force of contraction (FOC) in human left ventricular failing (A) and nonfailing (B) myocardium (stimulation frequency: 1 Hz). NYHA IV, New York Heart Association class IV.

Role of cAMP-dependent and phosphodiesterase-inhibitory actions. There is an ongoing discussion as to whether or not cAMP is at least partially involved in the positive inotropic action of levosimendan (16, 17). In human failing myocardium, the intracellular cAMP concentration is reduced (7). To further investigate whether cAMP is involved in the pharmacodynamic principle of levosimendan, papillary muscle strips of human failing myocardium were preincubated with isoprenaline before the cumulative application of levosimendan. Figure 2A summarizes the results. Under control conditions, i.e., in the absence of cAMP-dependent stimulation, levosimendan had no influence on FOC in human failing myocardium (n = 6). Application of isoprenaline (0.1 µmol/l, n = 8) increased FOC from 5.6 ± 0.8 to 10.8 ± 1.6 mN/mm². After prestimulation with isoprenaline, levosimendan (0.03-10 µmol/l) also increased FOC in left ventricular papillary muscle strips obtained from patients suffering from dilated cardiomyopathy (EC₅₀ levosimendan: 0.062 µmol/l, confidence interval: 0.049-0.074 µmol/l). These results indicate that levosimendan may be able to potentiate cAMP-dependent positive inotropic effects in human myocardium or may be effective to stimulate force after Ca²⁺-dependent mechanisms because isoprenaline enhances force via increasing [Ca²⁺]_i significantly.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Influence of cAMP-dependent stimulation on the effect of levosimendan in human failing myocardium shown as FOC (A) and time to half peak relaxation (T1/2T) (B). Isolated muscle strips were stimulated with isoprenaline (Iso; 0.1 µmol/l) to raise the intracellular cAMP concentration. In human failing myocardium, a positive inotropic effect of levosimendan was only observed after Iso prestimulation. Under basal conditions, as well as after Iso prestimulation, application of levosimendan significantly shortened T1/2T.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   A-C: original registrations of the simultaneous measurements of the intracellular Ca2+ transient (top) and the isometric FOC (bottom) in human left ventricular failing myocardium; application of 0.3 µmol/l levosimendan (Levo) was performed after prestimulation with Iso (0.1 µmol/l). R340/380, fura 2 ratio, fluorescence signals measured at 340 and 380 nm.

Ca²+-dependent positive inotropic action of levosimendan in failing myocardium. In human failing myocardium, the systolic [Ca²⁺]_i is reduced compared with nonfailing myocardium (1). To investigate whether the inotropic action of levosimendan is dependent on the systolic [Ca²⁺]_i, application of levosimendan (0.3 µmol/l) was performed after elevated extracellular [Ca²⁺] ([Ca²⁺]_o) (3.2 mmol/l). Figure 4 shows an original tracing of the experiment. At 3.2 mmol/l, but not at 1.8 mmol/l [Ca²⁺]_o, levosimendan increased FOC in human failing myocardium. Diastolic force was not influenced by levosimendan at an increased [Ca²⁺]_o.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Positive inotropic effect of levosimendan after elevating the extracellular Ca2+ concentration from 1.8 to 3.2 mmol/l.

Force-frequency relationship. Ca²⁺ sensitizers may impair relaxation at higher heart rates. This might influence contractile behavior, especially when Ca²⁺ is increased intracellularly, i.e., when stimulation frequency is enhanced (6, 23). Levosimendan exerts not only a Ca²⁺-dependent but also a use-dependent influence on Ca²⁺-triggered cross-bridge interaction. This may be of special importance when treating patients with already compromised contractility due to altered intracellular Ca²⁺ handling, i.e., reduced peak systolic and enhanced diastolic Ca²⁺ levels. Therefore, the influence of levosimendan (0.5 µmol/l) was investigated at increasing stimulation frequencies (0.5-3.0 Hz) in human failing left ventricular myocardium. Without levosimendan, FOC declined with increasing stimulation frequencies (Fig. 5). Application of levosimendan (0.5 µmol/l) did not improve FOC at low stimulation frequencies, i.e., at 0.5 and 1.0 Hz; however, when stimulation frequency was further increased, FOC was significantly improved compared with control. This positive influence of levosimendan may be due to its positive lusitropic effect. Under basal conditions, T1/2T was significantly decreased by levosimendan [0.5 Hz, control: 149 ± 4 ms, +levosimendan (0.5 µmol/l) 138 ± 6 ms, P < 0.05]. This positive lusitropic effect of levosimendan is still working at high stimulation frequencies [3.0 Hz, control: 109 ± 2 ms, + levosimendan (0.5 µmol/l): 99 ± 3 ms, P < 0.05]. At increasing stimulation frequencies, levosimendan slightly, but not significantly, reduced the frequency-dependent increase in diastolic tension [3.0 Hz, control: +2.3 ± 0.3 mN, +levosimendan (0.5 µmol/l): 0.89 ± 0.3 mN].

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of levosimendan on the frequency-induced changes of FOC (top trace) in human left ventricular failing myocardium.

	DISCUSSION

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Several agents have been developed to treat patients with compromised cardiac function: some of them are beneficial to improve symptoms (cardiac glycosides and diuretics), but only a few of them improve outcome as well (-blockers, angiotensin-converting enzyme inhibitors, and vasodilators). Digitalis is the only inotropic compound that improves symptoms with no harm on outcome. However, cardiac glycosides as well as cAMP-dependent inotropes increase Ca²⁺ intracellularly and thus cause energy expenditure. Levosimendan is a newly developed inotropic agent that is reported to increase Ca²⁺ sensitivity of the heart via Ca²⁺-dependent stabilization of the Ca²⁺-bound conformation of cardiac troponin C (13, 14). Thus its effect may not be linked with increased energy consumption. Furthermore, this mode of action may lead to increased contractility when intracellular Ca²⁺ is high and may favor relaxation under low-Ca²⁺ conditions. This mode of action may be advantageous especially in human heart failure with already altered intracellular Ca²⁺ homeostasis (1, 22). Thus the present study aimed to investigate the mode of action of levosimendan in human failing and nonfailing myocardium.

The present study demonstrates that levosimendan exerts a concentration-dependent positive inotropic effect in human left ventricular nonfailing myocardium. Also, in studies (2, 13, 14, 17) with healthy animals, levosimendan increased force. However, this inotropic effect of levosimendan was lacking in human terminally failing myocardium. This may be due to changes in the contractile apparatus or due to changes in Ca²⁺-related systems in diseased human myocardium, e.g., the changes from high systolic to lower diastolic Ca²⁺ are significantly smaller in human failing compared with nonfailing human myocardium (1) and there may be an already increased Ca²⁺ sensitivity in diseased human myocardium (19, 25).

Interestingly, in human failing myocardium, in which the basal cAMP levels are decreased due to a downregulation of the ₁-adrenoceptors (3, 4, 7, 9, 18), levosimendan was effective to increase force in the presence of isoprenaline or elevated extracellular Ca²⁺, i.e., under inotropic stimulation. Furthermore, levosimendan improved contraction only after prestimulation with isoprenaline. Thus the present study may be indicative that levosimendan needs an increased systolic Ca²⁺ change, a less-sensitized contractile apparatus, or both. However, from the present study, it may be not ruled out whether or not levosimendan also acts as phosphodiesterase III inhibitor in human myocardium. A positive lusitropic effect of levosimendan was observed in human failing myocardium, which occurred despite an increase in FOC. cAMP-dependent inotropic effects of levosimendan have also been shown in rabbit myocardium (17) as well as guinea pig papillary muscle strips (2, 8). However, the present findings demonstrate that the positive inotropic effect of levosimendan after application of isoprenaline was not mediated by a significant increase of the systolic Ca²⁺ transient. Similar results were obtained in single ventricular cardiomyocytes of the rabbit using the Ca²⁺ indicator indo 1 (17) as well as in multicellular muscle strips of human myocardium using the bioluminescent indicator aequorin (16). Thus the positive inotropic action of levosimendan seems to be dependent to a certain degree on systolic Ca²⁺ but may not be effective as an inotope via a cAMP-mediated activation. In addition, as shown by the present study, the positive inotropic action of levosimendan seems to be dependent on the systolic [Ca²⁺]_i, because in failing human myocardium, levosimendan only increased FOC after elevating [Ca²⁺]_o. These findings may be in line with a Ca²⁺-triggered activation being involved in the mode of action of levosimendan.

Levosimendan decreased T1/2T even in failing human myocardium. In addition, the positive lusitropic effect of levosimendan was preserved when stimulation frequency was increased. This has been demonstrated in failing human myocardium as well (16) and may be also in line with cAMP-dependent moiety or may result from the novel molecular action of levosimendan.

Levosimendan improved the frequency-induced force generation in human failing myocardium, it even turned the negative force-frequency relationship into a positive one as shown by the present study. An improvement of the frequency-induced force generation by levosimendan was also found by Hasenfuss et al. (16). However, in their study, the effect of levosimendan was only obvious at lower stimulation rates (0.5 to 1.5 Hz), whereas in the present study, levosimendan was effective at stimulation rates of 1.5 Hz and above. This difference may be due to the preparations used or because in the present study levosimendan was used in a concentration, which produced no inotropic effect itself, whereas in the study by Hasenfuss and co-workers (16), levosimendan significantly increased FOC. These findings are in line with the effect of the -agonist isoprenaline on frequency-dependent force generation in human failing myocardium (21). After cAMP-dependent stimulation phospholamban may be phosphorylated and thereby increase sarco(endo)plasmic reticulum Ca²⁺-ATPase activity. Thus more Ca²⁺ can be stored into the sarcoplasmic reticulum, which may be released during the following depolarization, thus increasing force to a higher degree. Accordingly, it has been shown that the lusitropic response towards cAMP-dependent stimulation was closely related to the phosphorylation of phospholamban, whereas the cAMP-dependent inotropic response was directly related to the phosphorylation of Ca²⁺ channels in guinea pig atria. These findings may go in line with the altered effectiveness of levosimendan in failing compared with nonfailing human myocardium. However, because the Ca²⁺ transient increases with enhanced frequencies (6), these beneficial effects of levosimendan on the force-frequency relationship may result from its Ca²⁺-dependent action on the contractile machinery as well. Whether or not a phosphodiesterase-inhibitory effect (which has been shown in vitro at very high concentrations) may play a role in vivo may be answered by the use of data on circadian heart rate variability or by its influence on mortality. With the use of the present data, this mode of action cannot be ruled out completely.

In conclusion, the inotrope levosimendan increases force via a novel Ca²⁺-dependent Ca²⁺ sensitization of the contractile apparatus. This use dependency allows also a rapid relaxation. However, a phosphodiesterase-inhibitory moiety cannot be ruled out by the present data.