Effects of Levosimendan, a Cardiotonic Agent Targeted to Troponin C, on Cardiac Function and on Phosphorylation and Ca2+ Sensitivity of Cardiac Myofibrils and Sarcoplasmic Reticulum in Guinea Pig Heart
Abstract A new cardiotonic agent, (R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl] hydrazono]propanedinitrile (Levosimendan), has been developed and screened for its ability to bind to cardiac troponin C. In perfused hearts, low concentrations of 0.03 or 0.1 μmol/L Levosimendan increased +dP/dt, but did not affect the speed of relaxation and produced only a slight increase in spontaneous heart rate in the hearts perfused with 0.1 μmol/L of the drug. In these same hearts, perfusion with 0.03 μmol/L Levosimendan did not alter the 32P incorporation into troponin I or C protein, whereas a slight but significant increase was noted for phospholamban, with no detectable change in tissue cAMP levels. Administration of 0.1 or 0.3 μmol/L Levosimendan significantly increased myocardial cAMP levels as well as the phosphorylation of phospholamban, troponin I, and C protein. Levosimendan (0.03 to 10 μmol/L) reversibly increased force generated by detergent-extracted fiber bundles over a range of submaximally activating free Ca2+ concentrations with no significant effect on maximum force or on Ca2+ binding to myofilament troponin C. There was no direct effect of Levosimendan on Ca2+ uptake by vesicles of sarcoplasmic reticulum (SR). In contrast, under conditions optimal for cAMP-dependent phosphorylation, Levosimendan slightly but significantly lowered the concentration of Ca2+, yielding half-maximal uptake rates by the SR vesicles. Our results indicate that at low concentrations Levosimendan acts preferably as a Ca2+ sensitizer, whereas at higher concentrations its action as a phosphodiesterase inhibitor contributes to the positive inotropic effect.
A new and promising approach to pharmacological stimulation of cardiac contractility in the failing heart is the development of agents, so- called Ca2+ sensitizers, that increase the response of the myofilaments to Ca2+.1 An advantage of these agents is that increases in contractility may occur with little or no change in the Ca2+ transient. This avoids a property associated with administration of other cardiotonic agents, such as digitalis, which increase the Ca2+ transient and may thus lead to arrhythmias. Moreover, it would be expected that such agents may reduce the energy cost of contractility by reducing the energy cost of Ca2+ transport. Unique among the Ca2+-sensitizing agents is the compound (R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl] hydrazono]propanedinitrile (Levosimendan), which has been synthesized with a rationale based on its ability to bind to cardiac troponin C (cTnC),2 3 the myofilament Ca2+-receptor protein that triggers activation of the contractile machinery.1
Although Levosimendan has been targeted to cTnC, this may not be its only mechanism of action. An interesting, but poorly understood, feature of agents with Ca2+-sensitizing activity is that some also have activity as phosphodiesterase (PDE) inhibitors.1 4 5 Therefore, in intact cardiac preparations, the mechanism of the positive inotropic action may be related to both cAMP-mediated protein phosphorylation as well as an altered response of the myofilaments to Ca2+. Cardiac proteins important as determinants of the inotropic state and potentially phosphorylated in association with PDE inhibition include phospholamban,6 which regulates activity of the sarcoplasmic reticulum (SR) Ca2+ pump, Ca2+ channel subunits,7 and myofilament proteins.8
In the present study, we have used an integrative approach to characterize Levosimendan in guinea pig hearts. We tested its effects on (1) Ca2+ responsiveness and troponin C (TnC)–Ca2+ binding of myofilaments, (2) Ca2+ uptake of SR vesicles, (3) left ventricular function in perfused hearts, (4) phosphorylation of phospholamban, troponin I (TnI), and C protein in intact hearts, and (5) myocardial cAMP and cGMP levels. Our results indicate that Levosimendan is a relatively potent myofilament Ca2+ sensitizer acting through TnC and that this action is the basis for the inotropic effect at low concentrations. The activity of Levosimendan as a PDE inhibitor was apparent only at the higher concentrations.
Materials and Methods
Hearts were rapidly excised from Hartley guinea pigs anesthetized with 30 mg/kg sodium pentobarbital and heparinized with 500 U/kg. The hearts were immediately cannulated through the aorta and prepared for retrograde perfusion with modified Krebs buffer containing (mmol/L) NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, Na2EDTA 0.5, KH2PO4 0.23, and glucose 5.5. The buffer solution was saturated with 95% O2/5% CO2, pH 7.4, at 37°C. The hearts were initially perfused at a constant aortic pressure (5 kPa) for 20 to 25 minutes in a drip-through mode. The perfusion circuit was then switched to a recirculating system containing 200 cpm/pmol [32P]orthophosphate for 30 minutes. After this labeling period, the circuit was returned to the drip-through mode with nonradioactive buffer for 1 minute. The drugs of interest (Levosimendan or isoproterenol) were administered into the buffer flow line. Six minutes after the administration of various concentrations of Levosimendan (0.03, 0.1, and 0.3 μmol/L), the hearts were freeze-clamped with a precooled Wollenberger clamp, powdered, and stored under liquid nitrogen, as previously described.9 10 11 Hearts perfused with 0.1 μmol/L isoproterenol served as internal controls for protein phosphorylation and were stimulated for only 2 minutes. Heart rate and left ventricular pressure were continuously monitored. Derivatives of the mechanical function (dP/dt) were electronically derived and stored in a personal computer. Microsomes and myofibrils were prepared from perfused hearts as previously described.9 11
Gel Electrophoresis and Autoradiography
Polyacrylamide gel electrophoresis of 32P-labeled protein was performed according to the method of Laemmli12 by using 10% to 18% and 5% to 20% gradient slab gels. After electrophoresis, the gels were fixed, stained with Coomassie blue, destained, and placed in sealed plastic bags into Kodak Lanex cassettes loaded with Kodak Ortho-G films for 48 to 72 hours. The radioactive bands corresponding to phospholamban, TnI, C protein, and myosin P light chain were identified and cut from the gel for counting in scintillation fluid. For the identification of TnI, C protein, and P light chain, partially purified standards were used on the same gel. Phospholamban was identified on the basis of its characteristic molecular weight shift upon boiling before electrophoresis. Phosphate incorporation was quantified by dividing the 32P incorporation into the phosphoproteins by the specific activity of [γ-32P]ATP determined for each heart and expressed as picomoles phosphate per milligram protein loaded onto the gel.
Force Measurements on Skinned Fiber Bundles
Papillary muscles were isolated from hearts of male guinea pigs (200 to 300 g) that had been deeply anesthetized with pentobarbital and killed by decapitation. Thin strips, 100 to 150 μm in diameter and 1.5 to 2.0 mm in length, were dissected. These fiber bundles were treated according to the procedure previously described,13 in which the fibers are soaked for 30 minutes in a relaxing solution described below containing 250 μg/mL saponin. This treatment increases the permeability of SR membranes as well as the surface membranes. The fiber bundles were attached to a strain gauge arranged in a micromanipulator for determination of force as a function of pCa (−log [Ca2+]). Relaxing solution contained (mmol/L) KCl 79.3, MgCl2 6.5, Na2ATP 5.4, EGTA 0.1 or 10, MOPS 20, and creatine phosphate 12 (ionic strength, 160), along with creatine phosphokinase (10 U/mL). The sarcomere length was measured and set at 2.0 to 2.1 μm in relaxing solution by using a laser diffraction pattern as described by Hibberd and Jewell.14 Maximum force at this sarcomere length was computed to be 45 to 50 mNmm2. Measurements were made at 22±1°C and at 37°C. Various pCa values were determined by using a computer program15 and achieved by varying the total CaCl2 while maintaining ionic strength and pH.
Myofilaments were subjected to centrifugation by using a sucrose gradient as described by Muir et al16 to remove mitochondria and SR and subsequently extracted with saponin as described above. The myofilaments were incubated in a solution containing (mmol/L) MgATP2− 5, free Mg2+ 2, imidazole 60, creatine phosphate 12, d-glucose 1, and sodium azide 0.4. The solution also contained 1.0 U/mL creatine kinase, 0.3 μCi/mL 45Ca, and 0.3 μCi/mL [3H]d-glucose. The pH was set at 7.0, and the ionic strength of the solution was adjusted with KCl to 0.15 mol/L. After equilibrium binding was achieved as previously described,4 radioactivity in the fibers was eluted in a solution containing (mmol/L) EGTA 10, KCl 50, MgCl2 2, d-glucose 1, and imidazole 60 (pH 7.0). Samples were assayed for 45Ca and 3H, and bound Ca2+ was computed from the ratio of 45Ca and 3H in the binding solution and the elution solution. Protein concentration was determined as previously described.4
Ca2+ Transport by SR Vesicles
Rates of Ca2+ uptake by the SR vesicles were determined in homogenates at 37°C in the presence or absence of 0.3 μmol/L Levosimendan and/or 2 μmol/L cAMP as described in the figure legends. Uptake was measured with the aid of 45CaCl2 by using a Millipore filtration assay as previously described.17 Incubation was carried out in 1.5-mL baths containing 2 mg/mL homogenate protein. Other conditions were as follows (mmol/L): imidazole 40 (pH 7.0), KCl 100, potassium oxalate 5, MgCl2 5, ATP 5, ruthenium red 0.005, EGTA 0.5, and sodium azide 5. Total CaCl2 was varied to achieve various free concentrations of CaCl2 in a final volume of 1.5 mL. Free Ca2+ concentrations were calculated by a computer program.15 In some samples the synthetic inhibitor of the cAMP-dependent protein kinase catalytic subunit was also included in the uptake buffer in a final concentration of 2 μg/mL. This was done to permit examination of the direct effect (ie, not protein phosphorylation–mediated) of Levosimendan on SR Ca2+ transport. When Levosimendan was included, the samples were preincubated with the compound for 3 minutes, and the Ca2+ transport was initiated by the addition of ATP.
The effect of Levosimendan on SR Ca2+ transport was also examined under conditions optimal for cAMP-dependent protein phosphorylation. Homogenate protein (2 mg/mL) was added to the reaction mixture (1.5 mL) containing (mmol/L) imidazole 40 (pH 7.0), EGTA 0.5, sodium azide 5, and MgCl2 5, along with 2 μmol/L cAMP and 0.1 μmol/L okadaic acid, and incubated in the presence or absence of 0.3 μmol/L Levosimendan. Reactions were initiated by the addition of 0.1 mmol/L ATP (final concentration). After 3 minutes of incubation at 37°C, the tubes were placed at 0°C, and samples were then taken for determination of Ca2+ uptake. The rates of SR Ca2+ uptake were calculated by the least-squares linear regression analyses of the 30-, 60-, and 90-second values of Ca2+ uptake. The initial rates of SR Ca2+ uptake were linear, with cardiac homogenate protein concentration up to 150 μg. The concentrations of Ca2+ yielding half-maximal rates (EC50) and the highest rates of Ca2+ uptake (Vmax) by the cardiac SR were calculated by using a curve-fitting computer program (MicroCal Origin).
Analysis of Nucleotides
The specific radioactivity of [γ-32P]ATP was determined from the specific activity of [32P]phosphocreatine at the end of perfusion.18 Tissue levels of cAMP and cGMP were determined from identically treated nonradioactive hearts with specific assay kits ([3H]cAMP assay kit and [3H]cGMP assay kit, Amersham). Results were corrected for recoveries, which were monitored with [3H]cAMP and [3H]cGMP included in separate portions of the cardiac homogenates. The protein content was determined by the method of Peterson,19 with bovine serum albumin used as standard.
The mean±SD was obtained for each parameter determined. Statistical analysis was performed by using Student’s t test for unpaired observations and ANOVA. When ANOVA was used, comparisons were made by Scheffé’s test, and values with P<.05 were regarded as statistically significant.
Levosimendan (Fig 1⇓) was synthesized by Orion-Farmos Pharmaceuticals. [32P]Orthophosphate and [45Ca]Cl2 were obtained from Amersham and DuPont NEN, respectively. All other chemicals, including a synthetic inhibitor of protein kinase A (PKA, rabbit sequence), were analytical grade and purchased from Sigma Chemical Co.
Studies With Skinned Fiber Bundles
Fig 2⇓ shows tracings of the effects of Levosimendan concentration on force generated by skinned fiber bundles. In one series of experiments, the measurements were made at 25°C at a submaximally activating pCa value of 6.0 (Fig 2A⇓) and at a maximally activating pCa value of 4.5 (Fig 2C⇓). These results demonstrate the ability of Levosimendan at pCa 6.0 to increase force by a direct effect on the myofilaments in a concentration-dependent manner. However, as demonstrated by the data in Fig 2C⇓, Levosimendan had no effect on force at pCa 4.5, where force was maximally activated by Ca2+. Fig 3⇓ summarizes the dependence of force (expressed as percent maximal force) on the concentration of Levosimendan at 25°C. Fig 4⇓, which demonstrates the relation between pCa and force, summarizes the results of experiments on the effect of 10 μmol/L Levosimendan over a range of pCa values at 25°C. In a second series of experiments, effects of Levosimendan on the force generated by the skinned fiber bundles was measured at 37°C. The results, which are shown as force recordings in Fig 2B⇓ and as summarized data in Fig 3B⇓, indicate that the myofilaments were more sensitive to Levosimendan at the warmer temperature. The EC50 was ≈1.0 μmol/L at 25°C and ≈0.1 μmol/L at 37°C. At 37°C there was no effect of Levosimendan on maximum force at pCa 4.5.
To investigate the mechanism for the Ca2+-sensitizing effect of Levosimendan on cardiac myofilaments, we measured Ca2+ binding to myofilament TnC in control conditions and in the presence of 10 μmol/L Levosimendan. As shown in Fig 5⇓, there was no significant change in the titration of myofilament TnC with Ca2+ in the presence of 10 μmol/L Levosimendan.
Effects of Levosimendan on Ca2+ Uptake by SR Vesicles
Initial rates of ATP-dependent oxalate-facilitated Ca2+ uptake were obtained in cardiac homogenates at various Ca2+ concentrations in the presence and absence of Levosimendan (0.3 μmol/L). The incubation conditions under which Ca2+ uptake is restricted to SR vesicles in the homogenate have been defined, and the validity and advantages of this approach have been previously reported.11 20 To test for effects of Levosimendan independent of PDE inhibition, we did one series of measurements under conditions in which cAMP-dependent protein kinase (PKA) activity was inhibited by a synthetic inhibitor (Fig 6A⇓). Results of these experiments indicated that Levosimendan (0.3 μmol/L) did not have any direct effect on the SR Ca2+ transport. Under conditions in which the phosphorylation of proteins was prevented by the PKA inhibitor, the EC50 (Vmax) values were 0.31±0.03 μmol/L Ca2+ (85.9±2.9 nmol Ca2+ per milligram protein per minute) for control preparations and 0.31±0.05 μmol/L Ca2+ (83.9±6.0 nmol Ca2+ per milligram protein per minute) for preparations treated with Levosimendan. By contrast, as shown in Fig 6B⇓, where the possibility of phosphorylation of phospholamban was optimized by inclusion of cAMP and okadaic acid and exclusion of the cAMP-dependent protein kinase inhibitor, Levosimendan (0.3 μmol/L) slightly but significantly lowered the concentration of Ca2+, yielding half-maximal uptake rates by the SR vesicles. The calculated EC50 (Vmax) values in the Levosimendan (0.3 μmol/L)–treated preparations were 0.25±0.02 μmol/L Ca2+ (87.0±4.4 nmol Ca2+ per milligram protein per minute) and in control preparations were 0.34±0.03 μmol/L (80.0±6.5 nmol Ca2+ per milligram protein per minute). When cardiac homogenates were incubated in the presence of 100 μmol/L [γ-32P]ATP, 2 μmol/L cAMP, and 0.3 μmol/L Levosimendan and the membrane vesicle fraction was purified and analyzed by autoradiography of SDS-polyacrylamide gels, the 32P label associated with phospholamban was increased by 1.8-fold compared with identically treated control preparations (data not shown). Thus, it appears that 0.3 μmol/L Levosimendan resulted in partial phosphorylation of phospholamban at levels comparable to those achieved in situ upon stimulation with isoproterenol (Table 1⇓).
Effects of Levosimendan on Left Ventricular Function
Function of the left ventricle, as measured by +dP/dt (contractility), −dP/dt (relaxation), and spontaneous heart rate, was determined in the same hearts in which the degree of phosphorylation of SR vesicles and myofibrils was assessed (Fig 7⇓). Perfusion of guinea pig hearts with various concentrations of Levosimendan resulted in a significant increase in the contractility (+dP/dt), even at a concentration of 0.03 μmol/L (Fig 7⇓). In contrast, the speed of relaxation (−dP/dt) in the presence of 0.03 and 0.1 μmol/L Levosimendan did not change. In hearts perfused with higher (0.3 μmol/L) concentrations of Levosimendan (Fig 7⇓), there was a slight but significant elevation in −dP/dt. However, even at this concentration of the drug, the increase in −dP/dt was less pronounced than the increase in +dP/dt. Levosimendan at a relatively low concentration (0.03 μmol/L) did not significantly change the spontaneous heart rate, whereas administration of higher doses (0.1 and 0.3 μmol/L) of the drug stimulated the heart rate in a time-dependent manner (data not shown). As a positive control and for purposes of comparison in our preparations, we also measured the effect of isoproterenol in these guinea pig hearts. Stimulation of the guinea pig hearts by the β-adrenoceptor agonist isoproterenol (0.1 μmol/L) elicited strong positive inotropic, lusitropic, and chronotropic responses, producing a large elevation in +dP/dt (69% increase), −dP/dt (51%), and heart rate (39%).
Effects of Levosimendan and Isoproterenol on In Situ Protein Phosphorylation in Perfused Hearts
Using 32P-labeled guinea pig hearts, we assayed the ability of Levosimendan to increase in situ 32P incorporation into phospholamban in SR membranes and into TnI, C protein, and myosin P light chains in myofibrils. As summarized in Table 1⇑, exposure of the perfused beating hearts to 0.03 μmol/L Levosimendan slightly but significantly increased 32P incorporation into phospholamban but did not alter phosphorylation of TnI, C protein, or myosin P light chain. Administration of higher concentrations of Levosimendan (0.1 or 0.3 μmol/L) was associated with significant increases in the phosphorylation of both TnI and C protein and phospholamban compared with the levels observed with 0.03 μmol/L Levosimendan (Table 1⇑). As a positive control in parallel experiments, we perfused hearts with isoproterenol under conditions identical to those used with Levosimendan. Compared with hearts perfused with Levosimendan, hearts perfused with isoproterenol (0.1 μmol/L) demonstrated much higher 32P incorporation into phospholamban, TnI, and C protein (Table 1⇑).
Effects of Levosimendan and Isoproterenol on Myocardial cAMP and cGMP Content
The content of cAMP was measured in hearts treated under conditions identical to those described above but in the absence of radioactivity. At the low concentration (0.03 μmol/L) of Levosimendan, there was no significant change in myocardial cAMP levels compared with those of control hearts (Table 2⇓). However, perfusion of guinea pig hearts with 0.1 and 0.3 μmol/L Levosimendan was associated with significant elevations in the myocardial cAMP levels. No significant change was observed for the tissue cGMP levels in the same hearts. In parallel experiments, perfusion of hearts with isoproterenol (0.1 μmol/L) resulted in higher increases in cAMP levels than did perfusion of hearts with 0.3 μmol/L Levosimendan.
Our experiments are the first to use an integrated approach to study the mechansim of the cardiotonic actions of Levosimendan, which was rationally designed as a TnC-binding molecule.2 The docking site of this unique compound is at the amino-terminal region of TnC near the regulatory Ca2+-binding domain.2 Binding of Levosimendan to this site has been hypothesized to stabilize the Ca2+-bound conformation2 and thereby increase the level of thin-filament activation with no change in TnC-Ca2+ binding. Our data fit this hypothesis. We found that at relatively high pCa values, in which activation of the myofilaments is limited by Ca2+, there was a concentration-dependent Ca2+-sensitizing effect of Levosimendan with no significant change in TnC-bound Ca2+. However, at relatively low pCa values, where the effect of Ca2+-TnC on the thin filament is fully elaborated, there was no effect of Levosimendan on skinned fiber force. Moreover, the lack of effect of Levosimendan at maximally activating pCa values indicates that Levosimendan most likely does not directly affect the actin-myosin interaction.
These effects of Levosimendan are most likely due to a direct action on the myofilaments rather than an indirect effect, such as through inhibition of PDE. No exogenous cAMP was added, and we would expect all endogenous cAMP to be washed out in the preparation. Moreover, potent PDE inhibitors, such as milrinone, have no Ca2+-sensitizing effect.4 In any case, an increase in cAMP-dependent phosphorylation of the myofilaments would decrease the Ca2+-sensitivity8 and thus could not account for the increase in Ca2+ sensitivity we report in the present study.
An important question is whether the Ca2+-sensitizing action of Levosimendan is important in situ. To address this question, we tested inotropic effects of Levosimendan on perfused hearts, with an emphasis on the possibility that some actions of Levosimendan may be due to PDE inhibition. Our results on Langendorff-perfused guinea pig hearts demonstrated that low concentrations of Levosimendan (0.03 μmol/L) were able to increase the inotropy of the myocardium without affecting the spontaneous heart rate and relaxation. This same concentration of Levosimendan was able to significantly increase myofilament force in skinned fiber bundles when the measurements were made at 37°C. Examination of the 32P label associated with the major cardiac phosphoproteins in these hearts revealed that only phospholamban was partially phosphorylated, whereas the phosphorylation levels of TnI, C protein, and the P light chain of myosin did not change significantly. The lack of increases in total tissue cAMP levels and 32P incorporation into TnI and C protein suggests that under these conditions the compound primarily acts as a Ca2+ sensitizer. The PDE-inhibitory potential of Levosimendan may be more pronounced on the SR compartment, and the observed slight increases in the phosphorylation status of phospholamban may be sufficient to override potential impairment on relaxation by Ca2+ sensitization of the myofibrils in these hearts.
In the perfused hearts, at the highest concentrations of Levosimendan studied (0.3 μmol/L), there were effects on heart rate, inotropy, lusitropy, and protein phosphorylation, suggesting that PDE inhibition was the predominant mechanism for the effects on cardiac function. Phosphorylation of all proteins studied was significantly increased in these hearts, as were total tissue cAMP levels. Moreover, our in vitro studies on SR Ca2+ transport in the presence of Levosimendan and cAMP indicated a potential for an effect of PDE inhibition of the Ca2+ uptake rate of the SR at the higher (0.3 μmol/L) concentrations of Levosimendan. Consequently both −dP/dt and +dP/dt were significantly increased. Inasmuch as the phosphorylation of cardiac myofilaments by PKA was reported to desensitize the myofibrils to Ca2+ and shift the pCa-force relation to the right,11 21 it is possible that the Ca2+-sensitizing effect of Levosimendan at concentrations at which it has strong PDE inhibition may be blunted, at least in part.
In summary, it appears that low concentrations of Levosimendan preferably act by increasing the response of the myofilaments to Ca2+, but at higher concentrations, its activity as a PDE inhibitor strongly contributes to the positive inotropic effect. Our results suggest that directing the actions of pharmacological agents to specific sites on Ca2+-binding proteins may be a useful approach not only in the cardiac myofilaments but also in other systems. The intriguing overlap of this activity with inhibition of PDE activity requires further study.
This study was supported in part by National Institutes of Health grants R01 HL-22231 (Dr Solaro), R01 HL-26057 (Dr Kranias), and T32 HL-07692 (Dr Powers); by the Hungarian Academy of Sciences (grant OTKA 2717); and by the USA-Hungarian Science and Technology Joint Fund, in cooperation with the Department of Health and Human Services and the Hungarian Ministry of Social Welfare, under Project No. 203/91b.
- Received January 18, 1995.
- Accepted March 14, 1995.
- © 1995 American Heart Association, Inc.
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