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

Preparations

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.

Ca²⁺-Binding Measurements

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 (V_max) 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.

Data Analysis

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.

Materials

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.

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Figure 1.

The chemical structure of (R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl] hydrazono]propanedinitrile (Levosimendan).

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.

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Figure 2.

Recordings showing effects of Levosimendan concentration on force developed by saponin-treated bundles of cardiac muscle fibers. A, Increased Ca²⁺ responsiveness at pCa 6.0 and 22°C. B, Increased Ca²⁺ responsiveness at pCa 6.0 and 37°C. C, Results demonstrating a lack of effect of Levosimendan at pCa 4.5 and 22°C, levels at which the preparations developed maximum force. There was also no effect at 37°C. Measurements were made as described in “Materials and Methods.”

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Figure 3.

Graphs summarizing the dependence of force developed by saponin-treated cardiac muscle preparations on the concentration of Levosimendan. A, Measurements were made at pCa 6.0 and 22°C as described in “Materials and Methods.” B, Measurements were made at pCa 6.0 and 37°C. Each value is expressed as the percentage of maximum force (% of Max.) obtained at pCa 4.5 and represents the mean±SD of six preparations from different hearts. *P<.05; **P<.001.

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Figure 4.

Graph comparing the relation between pCa and force developed by cardiac muscle preparations made permeable with saponin under control conditions (•) and in the presence of 10 μmol/L Levosimendan (▪). Measurements were made at 22±1°C under conditions described in “Materials and Methods.” Each value represents the mean±SD of four preparations from different hearts. *P<.05.

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.

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Figure 5.

Graph showing the lack of effect of 10 μmol/L Levosimendan on the relation between pCa and Ca²⁺ binding to troponin C in myofilaments. Each value represents the mean±SEM for four measurements at each pCa value. There were no significant differences between the two curves.

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 (V_max) 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 (V_max) 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%).

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Figure 7.

Graph showing dose-response relation of the effect of Levosimendan on heart rate (•), −dP/dt (▴), and +dP/dt (▪) values. Guinea pig hearts were perfused with various concentrations of Levosimendan for 6 minutes as described in “Materials and Methods.” Results are expressed as percentage of control (0-minute) values. Each value represents the mean±SD of at least five different hearts. *P<.05 compared with 0-minute values. Zero-minute values for heart rate, −dP/dt, and +dP/dt were 175±10 beats per minute, 164±12 kPa/s, and 160 kPa/s, respectively.

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Figure 6.

A, Graph showing the direct effect of Levosimendan on Ca²⁺ dependence of sarcoplasmic reticulum (SR) Ca²⁺ uptake from cardiac homogenates. The initial rates of SR Ca²⁺ uptake were measured from control (•) and Levosimendan-incubated (▪) samples at various free Ca²⁺ concentrations, as described in “Materials and Methods.” The synthetic inhibitor of the cAMP-dependent protein kinase catalytic subunit was included in the uptake buffer at a final concentration of 2 μg/mL. Rates of Ca²⁺ uptake are expressed as percentage of the maximal rates obtained at pCa 5.5. Each value represents the mean±SD of four different hearts, each assayed in triplicate. B, Graph showing the effects of Levosimendan on Ca²⁺ dependence of SR Ca²⁺ uptake from cardiac homogenates under conditions optimal for cAMP-mediated protein phosphorylation. Cardiac homogenate samples were preincubated with 2 μmol/L cAMP and 0.1 μmol/L okadaic acid in the presence (▪) and absence (•) of 0.3 μmol/L Levosimendan, and SR Ca²⁺ uptake was measured at various free Ca²⁺ concentrations, as described in “Materials and Methods.” Each value represents the mean±SD of four different hearts, each assayed in triplicate. See text for analysis of kinetic parameters.

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.