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Eur J Cardiothorac Surg 1998;14:70-75
© 1998 Elsevier Science NL

Calcium sensitization as new principle of inotropic therapy in end-stage heart failure?¹

^a Klinik für Thorax- und Kardiovaskuläre Chirurgie, Heinrich Heine-Universität, Moorenstrasse 5, D-40225 Düsseldorf, Germany
^b Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität, Domagkstrasse 12, D-48129 Münster, Germany
^c Ciba-Geigy Limited, Basle, Pharmaceuticals Division, Research and Development, CH-4002 Basle, Switzerland
^d Abteilung Allgemeine Pharmakologie, Universitäts-Krankenhaus Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany

Received 29 September 1997; received in revised form 15 April 1998; accepted 15 April 1998.

^* Corresponding author. Tel.:+49 211 8118331; fax: +49 211 8118333.

	Abstract

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Objective: Due to shortage of donor hearts and increasing waiting-lists of patients with end-stage heart disease, new pharmacological principles for bridging therapies are necessary. The positive inotropic effects of cAMP-increasing drugs (e.g. catecholamines, phosphodiesterase-inhibitors) are diminished in the failing myocardium. Hence, we investigated the usefulness and mechanism of the two calcium sensitizers, levosimendan and CGP 48506 in preparations from end-stage failing human hearts since the exact mechanism of the positive inotropic effects is not yet clearly understood. Methods: Failing human hearts which required orthotopic heart transplantation due to idiopathic dilated cardiomyopathy were investigated. Contraction experiments were performed using muscle strips of ventricles. Calcium sensitization was investigated in skinned fibers and phosphodiesterase activity was measured in ventricular homogenate. In addition, cAMP levels were quantified in myocytes from guinea-pig hearts. Results: In muscle stripes from failing human hearts levosimendan (10 µmol/l) increased the force of contraction only to 112.8±6.7% of predrug values. In contrast, CGP 48506 increased the force of contraction to 311±59% of predrug values at 100 µmol/l. The time to peak tension and time of relaxation were increased to 175±4% and 205±15% of control levels at 100 µmol/l. Skinned fibers from failing human hearts were sensitized to calcium with an EC₅₀ of 10 µmol/l. Other mechanisms of action were excluded since CGP 48506 affected neither the activity of phosphodiesterase isoenzymes I–IV in failing human hearts, nor cAMP levels in guinea-pig cardiomyocytes. On the other hand, levosimendan (1 µmol/l) increased cAMP content from 6.3±0.3 to 8.1±0.3 pmol/mg protein. Conclusion: CGP 48506 is an inotropic agent with calcium-sensitizing properties in the human heart, that is devoid of inhibitory activity on human cardiac phosphodiesterase isoenzymes. It offers, therefore, a new form of positive inotropic therapy that can be useful for the bridging treatment of heart failure before transplantation. On the other hand, levosimendan is a calcium sensitizer showing less-effective inotropic effects accompanied by increased cAMP levels.

Key Words: Calcium sensitizer • CGP 48506 • Levosimendan • End-stage heart failure

	Introduction

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There are two main indications to support the myocardium pharmacologically in cardiac surgery: first, myocardial dysfunction due to brief periods of ischemia (`stunning') after open-heart surgery and second, treatment of end-stage heart failure in patients waiting for transplantation.

Myocardial stunning is characterized by reversibly-impaired postischemic systolic and diastolic function [1]. The mechanisms responsible for myocardial stunning are not fully understood yet, but a decrease of myofibrillar calcium sensitivity seems to be an important factor [2]. Hence, myocardial dysfunction after cardiac surgery could be treated causally by increasing calcium sensitivity of myofilaments.

As the number of patients with end-stage heart disease waiting for transplantation increases more and more, attempts are made to develop new and better bridging therapies. The positive inotropic effect of drugs such as catecholamines (ß-adrenoceptor agonists) that increase intracellular cAMP and calcium levels is markedly reduced in human end-stage heart failure. Unfortunately, increased calcium levels may potentially be harmful and lead to unwanted side effects associated with calcium overload, like cardiac necrosis, tachycardia and arrhythmia. Moreover, these kinds of inotropic drugs increase the oxygen requirements of the myocardium due to both the greater force-production and the extra energy required for pumping increased systolic calcium concentrations back to the diastolic level [3].

Therefore, the clinical use of the available positive inotropic agents, especially during episodes with reduced calcium sensitivity (stunning) or in chronic treatment before heart transplantation, is limited and attempts have to be made to find new cardiotonic drugs capable of increasing myocardial contractility by a different mechanism. One possibility is to enhance the sensitivity of myofilaments to calcium, which means to increase tension at a given intracellular calcium level by calcium sensitizers. Up to recently all pharmacological agents described with noteworthy calcium-sensitizing properties (e.g. EMD 57033, pimobendan) exhibited additional phosphodiesterase inhibitory activity [4] [5]. In the present work, we report positive inotropic effects of different calcium sensitizers: levosimendan which acts via troponin C [6] in comparison to investigations of CGP 48506 which affects crossbridges [7] [8]. These investigations have in part been incorporated into a parallel publication [9].

	Materials and methods

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Failing human hearts were obtained from patients undergoing orthotopic heart transplantation as a result of end-stage (NYHA IV) heart failure. After explantation, contraction experiments were started immediately. Ventricular tissue samples were frozen in liquid nitrogen and stored at minus80°C for biochemical experiments.

These procedures complied with the Helsinki Declaration and permission for the experiments was approved by the local Ethics Committee.

Contraction experiments
Trabeculae carneae were isolated from ventricles of failing human hearts (diameter less than 1 mm, length 5–8 mm) which were dissected in gassed bathing solution at 4°C. The bathing solution contained (in mmol/l) NaCl 119.8, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.05, NaH₂PO₄ 0.42, NaHCO₃ 22.6, Na₂EDTA 0.05, ascorbic acid 0.28, glucose 5.05, continuously gassed with 95% O₂ and 5% CO₂ and maintained at 37°C and pH 7.4. Preparations were attached to a bipolar stimulating electrode and suspended individually in 10-ml glass tissue chambers for recording isometric contractions. Force of contraction was measured with inductive force transducers (W. Fleck, Mainz, Germany) connected to a Hellige Helco Scriptor recorder (Hellige, Freiburg, Germany). Each muscle was stretched to the length of maximal force of contraction. Resting force (about 5 mN) was kept constant throughout the experiment. Trabeculae carneae were electrically stimulated at 0.5 Hz with rectangular pulses of 5 ms duration (Grass stimulator SD9; Grass, Quincy, MA), the voltage was about 10–20% greater than threshold. All preparations equilibrated in bathing solution until complete stabilization.

Concentration–response curves were obtained cumulatively. Levosimendan, CGP 48506, CaCl₂ or the solvent DMSO (dimethyl sulfoxide) were added. The time-course of contraction was evaluated using twitches recorded at high chart speed.

Measurement of calcium sensitization (skinned fiber experiments)
From the ventricular free wall of failing human hearts, parallel to the length axis of the cardiomyocytes, subendocardial fiber bundles about 5–10 mm in length and less than 200 µm in diameter were dissected and subjected to a detergent/glycerol extraction using triton extraction solution (containing glycerol 50 vol.%, histidine HCl 20 mmol/l, EGTA 5 mmol/l, NaN₃ 10 mmol/l, DDT 1 mmol/l, Triton X-100 0.1% w/v, pH 7.3 at 4°C). By this extraction procedure, sarcolemma and sarcoplasmic reticulum were destroyed, mitochondria and soluble cellular components were washed out, leaving behind isolated contractile structures, the myofibrils of the fiber bundle. Using a fast-setting glue (cellulose nitrate dissolved in acetone), the preparations were attached to a mechanical apparatus designed for the measurement of isometric force. This consisted of an AE 801 force transducer element (Aksjeselskapet Mikroelektronikk, Horten, Norway), an amplifier and a 2-channel recorder.

In the presence of exogenous Mg-ATP, isometric contractions were elicited by incubation of the preparations in solutions with various EGTA-buffered calcium concentrations i.e. mixtures of activating solution (containing ATP 10 mmol/l, MgCl₂ 12 mmol/l, EGTA 5 mmol/l, CaCl₂ 5 mmol/l, imidazole HCl 20 mmol/l, NaN₃ 5 mmol/l, phosphoenol pyruvate 5 mmol/l, pyruvate kinase 20 U/ml) and relaxing solution (identical in composition to activating solution with the exception of no added CaCl₂). The sensitivity of the force generation of the contractile structures in response to calcium was measured by the calcium concentration at which half of the maximum force (EC₅₀) was generated.

CGP 48506 was added to the incubation media from 1000-fold concentrated stock solutions in DMSO. Control incubation media contained 0.1% DMSO.

PDE activity
The different forms of PDE were separated by diethyl aminoethanol (DEAE) sepharose anion exchange liquid chromatography as described by Reeves et al. [10].

PDE activity was determined in a two-step procedure according to the method of Thompson and Appleman [11].

Concentrations of drugs producing 50% of the maximal effect (EC₅₀) or reducing the basal activity of PDE to 50% (IC₅₀) were determined graphically. IC₅₀ values are given as geometric means with 95% confidence limits.

Determination of cAMP content
For determination of cAMP content, cardiomyocytes of guinea-pig hearts were isolated as described by Neumann et al. [12]. Cardiomyocytes were incubated with CGP 48506. At the indicated periods of time, reaction was stopped by adding 150 µl of 0.1 mol/l HCl. Samples were heat-treated for 10 min at 95°C and then centrifuged.

cAMP was measured by radioimmunoassay as published by Neumann et al. [12]. Protein was determined according to Bradford [13].

Statistics
Values are means±SEM. Statistical significance was estimated using Student's t-test for paired or unpaired observations; P<0.05 was considered significant.

Drugs
Levosimendan was provided by Dr. H. Haikala (Orion Farmos Pharmaceuticals, Espoo, Finland). CGP 48506 was obtained from Ciba Geigy, Basle, Switzerland.

All chemicals were of analytical or best commercial grade available. Deionized and twice-distilled water was used throughout.

	Results

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Effects on force of contraction
In isolated trabeculae from failing human myocardium, levosimendan (10 µmol/l) increased the force of contraction only to 112.8±6.7% of predrug value (n=8; see Fig. 1 ). In contrast, the positive inotropic effect of CGP 48506 was 311±59% of the predrug value at 100 µmol/l (n=6). The effect of CGP 48506 on force of contraction was reversible within 30 min on washout in drug-free bathing solution. A functional way to investigate the participation of cAMP in the positive inotropic effect of CGP 48506 is to study the antagonism between CGP 48506 and carbachol. Carbachol (10 µmol/l) did not reduce the positive inotropic effect of 100 µmol/l CGP 48506 which is indirect functional evidence that the positive inotropic effect is not mediated by an increase in cAMP. For comparison, contraction experiments were additionally performed using CaCl₂, since the positive inotropic effect of Ca²⁺ does not differ significantly between failing and non-failing human hearts. The force of contraction was increased concentration-dependently to 440±29% at 12.6 mmol/l CaCl₂ (n=6).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Effects of levosimendan (10 µmol/l, n=8), CGP 48506 (100 µmol/l, n=6) and CaCl2 (12.6 mmol/l, n=6) on force of contraction (% of predrug value) in trabeculae carneae from failing human hearts at the highest concentrations investigated.

Effects on calcium sensitivity
Human ventricular skinned fibers were isometrically activated at EGTA buffered calcium concentrations. CGP 48506 concentration dependently shifted the calcium activation curve to the left, to lower calcium concentrations (calcium-sensitizing effect). In concentrations of up to 100 µmol/l, CGP 48506 did not impair skinned fiber relaxation at low free calcium (pCa 8), nor did it, in contrast to the known `PDE-inhibitor type' calcium sensitizers like EMD 53998 [9], increase maximum calcium activated force F_max. 1 µmol/l was a threshold concentration for the calcium-sensitizing effect of CGP 48506 and 100 µmol/l reduced the calcium requirement for skinned fiber activation by a factor of about ten.

At a constant, buffered calcium concentration of 0.912 µmol/l (pCa 6.02), CGP 48506 concentration dependently increased force at pCa 6.02 up to 191% of the predrug value, the EC₅₀ for this effect being 10 µmol/l.

Effects on PDE activity
In the subsequent study, PDE isoenzymes were purified and characterized by their elution from a DEAE-sepharose column.

PDE I, II, III and IV activities were not affected by CGP 48506 up to 300 µmol/l (IC₅₀ values>1000 µmol/l). Only at 1000 µmol/l CGP 48506, were PDE III and IV slightly inhibited.

For reasons of comparison, the well-characterized PDE-inhibitors 3-isobutyl-1-methyl-xanthine (IBMX) and milrinone were investigated. IBMX inhibited unselectively all types of PDE (IC₅₀ in µmol/l; PDE I: 18.0, PDE II: 34.5, PDE III: 4.74 and PDE IV: 7.66, n=3), whereas milrinone showed the typical properties of a specific PDE III inhibitor (IC₅₀ in µmol/l; PDE I: 269.8, PDE II: 301.0, PDE III: 1.03 and PDE IV: 3.04, n=3).

Effects on cAMP content
In addition, cAMP content was measured in guinea-pig ventricular myocytes incubated with levosimendan or CGP 48506. CGP 48506 (100 µmol/l) did not affect cAMP levels (2.28±0.29 pmol/mg vs. control value of 2.23±0.26 pmol/mg, n=4), whereas levosimendan (1 µmol/l) increased cAMP content from 6.3±0.3 pmol/mg protein to 8.1±0.3 pmol/mg protein (P<0.05, n=10).

	Discussion

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The presented data clearly indicate that both calcium sensitizers, levosimendan and CGP 48506, show positive inotropic effects in failing human myocardium, but the latter agent is much more effective.

CGP 48506 sensitizes myofilaments to calcium, accompanied by a distinct positive inotropic response. Other mechanisms of action were excluded since CGP 48506 affected neither the activity of phosphodiesterase isoenzymes I–IV in failing human hearts, nor cAMP levels in guinea-pig cardiomyocytes. In accordance, the positive inotropic effect of CGP 48506 in failing human hearts was not sensitive to 10 µmol/l of the cAMP effect antagonizing carbachol. In contrast, levosimendan increased cAMP levels in accordance to its PDE inhibitory properties [14]. It has been reported that CGP 48506 can increase whole-cell L-type calcium current in cardiomyocytes [15]. It is unclear whether these effects are functionally relevant, since it has been shown that CGP 48506 does not increase free calcium levels in cardiac myocytes [16]. Moreover, there are no effects of CGP 48506 on sarcolemmal calcium channels or on ₁, ₂, ß₁ and histamine₁ receptors [7].

For comparison, values for maximal positive inotropic effects in failing human myocardium, EC₅₀ values for calcium sensitization and IC₅₀ values for PDE III inhibition of CGP 48506, levosimendan and two other agents with calcium sensitizing properties (EMD 57033 and pimobendan) are summarized in Table 1 [17] [18] [19]. As judged from the EC₅₀ values, levosimendan and EMD 57033 increase force of contraction more potently than CGP 48506 and pimobendan. On the other hand, CGP 48506 appears to be the more effective positive inotropic drug and is even more effective in failing human hearts than isoprenaline (maximal positive inotropic effect about 200% of predrug value) but less effective than calcium. Moreover, one should note that – in contrast to all other agents – the positive inotropic effect of calcium does not differ significantly between failing and non-failing human hearts [6].

Since the main cause of heart failure is myocardial damage, it seems reasonable to enhance the contractility of the residual myocardium in order to produce beneficial effects on symptoms, which are mainly the result of reduced cardiac output and secondary compensation. Hence, we investigated the effects of cardiotropic agents in an isolated organ bath in vitro using preparations of failing human hearts. Those in vitro experiments are well established and reliable. The results of an isolated preparation, however, do not allow characterization of hemodynamic action or clinical significance of drugs. These kinds of data have to be evaluated in clinical trials. Although short-term trials using levosimendan in patients with left-ventricular dysfunctions were performed [23], long-term data are lacking and it is impossible to draw conclusions on the effect of calcium sensitizers on patient survival. As far as we know, studies in patients using CGP 48506 have not yet been started. No data on either toxicology or clinical pharmacology are currently available in the published literature, and animal studies on the pharmacokinetics and toxicology of CGP 48506 have not yet been published [24].

Will calcium sensitizers play a role in the treatment of heart failure? A number of new cardiovascular agents have appeared that act by calcium sensitization. The simple observation, first made by Ringer in 1895, that calcium is essential for cardiac contraction, now turns out to be infinitely more complex. Calcium sensitization is a promising new approach to the treatment of heart failure [25] [26]. Drugs that sensitize the contractile proteins to calcium, increase cardiac contractility without changes in the intracellular calcium concentration. This concept appears to be associated with fewer adverse effects, a lower arrhythmogenic potential, and a favorable effect on myocardial oxygen consumption compared with established inotropic agents. A possible adverse effect of such agents is slowing of cardiac relaxation. Compared to inotropic agents that increase intracellular calcium, calcium sensitizers probably represent a more economic way of increasing force development. Clinical data on calcium sensitizers are yet very sparse but further clinical studies will pronounce the final judgment of clinical usefulness.

	Footnotes

 
¹ Presented at the 11th Annual Meeting of the European Association for Cardio-thoracic Surgery, Copenhagen, Denmark, September 28 – October 1, 1997.

² Present address: Department of Pharmacology and Physiology, Aga Khan University, P.O. Box 3500, Karachi 74800, Pakistan.

	Appendix A. Conference discussion

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Dr V. Vaage (Stockholm, Sweden): I am quite sure that there are still some people in the audience that think, why should we use a calcium sensitizer, why not just dobutamine? Maybe you should elaborate a bit more why should they use a calcium sensitizer. What are the advantages?

Dr Zimmermann: It's important to mention that all positive inotropic drugs used in the clinic increase intracellular calcium levels. For example, digitalis inhibits the sodium–potassium ATPase, hence, intracellular sodium levels are elevated, and activation of the sodium–potassium exchanger leads to an increase in intracellular calcium.

Second, catecholamines lead via GTP-binding stimulatory proteins to enhanced activity of adenylyl cyclase. The enzyme converts ATP to cAMP; cAMP binds to and activates the cAMP-dependent kinase (protein kinase A, PKA). PKA catalyzes the phosphorylation of numerous target proteins, for example, troponin inhibitor, calcium channels or phospholamban.

This pathway is also used by phosphodiesterase inhibitors. These compounds increase the level of intracellular cAMP via blocking its degradation by phosphodiesterases. Thus phosphodiesterase inhibitors and ß-adrenoceptor agonists share some of the same pathway involving cAMP-generation and increase of intracellular calcium.

In normal contractions of healthy hearts, intracellular calcium rises to a level at which approximately half maximum force is produced. Drugs that increase intracellular calcium call upon the reserve and may be harmful to the myocardium due to calcium overload.

Hence, calcium sensitization might be a new approach to the development of positive inotropic agents for chronic heart failure.

Dr T. Ferguson (St. Louis, MO, USA): The objective, as you outlined in the first paragraph of your abstract, is to use this technique for prolongation of bridging activities. I did not hear anything that tells me these effects would be continued over time. In other words, what is the time-frame relationship, or have you looked at that?

Dr Zimmermann: We performed these contraction experiments in an isolated organ bath and there were no problems to keep force of contraction elevated during a period of 3 or 4 h. But we did not investigate long-term effects of CGP 48506. To my knowledge, no data on either toxicology or clinical pharmacology of CGP 48506 are currently available in the published literature, and animal studies on the pharmacokinetics and toxicology have not been performed yet.

	References

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