J Mol Cell Cardiol 32, 479–491 (2000) doi:10.1006/jmcc.1999.1093, available online at http://www.idealibrary.com on
Further Evidence for the Cardiac Troponin C Mediated Calcium Sensitization by Levosimendan: Structure-response and Binding Analysis with Analogs of Levosimendan Jouko Levijoki1, Piero Pollesello1, Juha Kaivola1, Carola Tilgmann1, Tia Sorsa2, Arto Annila3, Ilkka Kilpela¨inen2 and Heimo Haikala1 1
Orion Pharma, Preclinical Research, Department of Drug Discovery & Pharmacology, PO Box 65, FIN-02101 Espoo, Finland; 2Institute of Biotechnology, University of Helsinki, PO Box 17, FIN00014 Helsinki, Finland; 3VTT, Chemical Technology, PO Box 1401, FIN-02044 VTT, Finland (Received 13 July 1999, accepted in revised form 31 December 1999) J. L, P. P, J. K, C. T, T. S, A. A, I. K¨ H. H. Further Evidence for the Cardiac Troponin C Mediated Calcium Sensitization by Levosimendan: Structure-response and Binding Analysis with Analogs of Levosimendan. Journal of Molecular and Cellular Cardiology (2000) 32, 479–491. Levosimendan, an inodilatory drug discovered using troponin C as a target protein, has a cardiac effect deriving from the calcium sensitization of contractile proteins. The aim of this study was to give further evidence that levosimendan binds to cardiac troponin C and that the binding involves amino acid residues on helix e of the Nterminal domain of this calcium-binding protein. Nine organic molecules, obtained by chemical modification of levosimendan, were tested both for their calcium-dependent binding to troponin C and troponin complex affinity HPLC columns, and for their ability to increase the calcium sensitivity of myofilaments in cardiac skinned fibers. A good correlation between the calcium sensitization and the calcium-dependent binding to troponin complex (r=0.90) and to cardiac troponin C (r=0.91) for the analogs of levosimendan was shown. In addition, the effect of levosimendan on the calcium-induced conformational changes in native and point-mutated cTnC was studied. Cys84→Ser, Asp87→Lys and Asp88→Ala point-mutated cTnC were shown to maintain a high affinity to calcium, but their Ca2+ titration curves were not influenced by levosimendan as for the native protein. Finally, it was demonstrated that the NMR chemical shifts of the terminal methyl groups of Met47, Met81, and Met85 on calcium-saturated cTnC were changed after addition of levosimendan in water solution at pH 7.4. This effect was not seen when adding an analog of levosimendan, which did not bind to the troponin C affinity HPLC column and did not increase the calcium-induced tension in cardiac skinned fibers. 2000 Academic Press K W: cTnC; Troponin complex; Affinity HPLC; Dansylated troponin; Calcium binding; NMR; Calcium sensitizer; Skinned fibers.
Introduction Levosimendan is an inodilatory drug intended for treating congestive heart failure. The main mech-
anism of action of this drug is the calcium sensitization of contractile proteins in cardiac muscle, as was directly demonstrated in cardiac muscle skinned fibers.1,2 In this model, where small
Please address all correspondence to: Heimo Haikala, Orion Pharma, Preclinical Research, Department of Drug Discovery & Pharmacology, PO Box 65, FIN-02101 Espoo, Finland. Tel: +358-9-429-2897; Fax: +358-9-429-2924; E-mail:
[email protected]
0022–2828/00/030479+13 $35.00/0
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molecules and ions can freely penetrate into the cell and intracellular calcium stores are destroyed,3 the fiber produces tension only as a function of the calcium concentration in the medium, and the calcium-sensitizing effect of a compound can be directly analysed. Levosimendan increases the tension of the fibers in a concentration-dependent manner,2,4 and its effect is reversible, i.e. when levosimendan is washed away, the tension rapidly returns to the level observed before its administration. It was also shown that levosimendan binds to a troponin C affinity HPLC column in a calciumdependent manner, which supports the hypothesis that the calcium-sensitizing effect of levosimendan is mediated by the calcium-dependent binding of the drug to troponin C.1 Levosimendan was in fact discovered using troponin C as a target protein. However, due to the lack of X-ray crystallographic and NMR data on the structure of cardiac troponin C (cTnC)∗ in the past, the open structure of the Ca2+-saturated skeletal TnC (sTnC)5 was used as a model for the drug discovery.6 Afterwards, evidence was given that levosimendan does bind to the N-terminal domain of human recombinant cTnC.7 However, the use of sTnC as a model was later questioned when the structure of Ca2+-saturated cTnC was determined,8 and it was proposed that its calciumsaturated form does not assume an open conformation, as in the case of the skeletal protein. Only recently, it was demonstrated that an open and closed form of calcium-saturated cTnC are in equilibrium,9 and that this equilibrium is influenced by cTnI fragments.10,11 The aim of this study was to demonstrate that the calcium-sensitizing effect of levosimendan is closely related to its binding to cardiac troponin C and that the binding involves amino acid residues on helix e of cTnC. In this study, three different approaches were used. (a) In a structure-to-activity relationship (SAR) study, a family of organic molecules were compared, obtained by chemical modification of levosimendan, both for their calcium-dependent binding to cTnC and troponin complex affinity HPLC columns, and for their ability to increase the calcium-induced tension in cardiac skinned fibers. A good correlation between these test models would be expected if a molecule is a calcium sensitizer. (b) In addition, the effect of levosimendan on the
calcium-induced conformational changes in native and point-mutated cTnC was compared. Cys84→Ser, Asp87→Lys and Asp88→Ala point mutations were selected in order to study how those residues on the helix e of the regulatory N-terminal of cTnC influence the binding of levosimendan. (c) Finally, the NMR spectra of calcium-saturated cTnC was analysed after addition of levosimendan (or some of its analogs) in water solution at pH 7.4, and changes in the chemical shifts of the terminal methyl groups of the methionines were looked for. It was expected that levosimendan and its active analogs would affect the chemical shifts of some methionine residues located in the proximity of the putative binding site, while no effects would be seen when adding the analogs of levosimendan which do not bind to cTnC affinity HPLC columns and do not increase the calcium-induced tension in cardiac skinned fibers.
Materials and Methods Chemicals Levosimendan, (R)-{[4-(1,4,5,6-tetrahydro-4-methyl6-oxo-3-pyridazinyl)phenyl]hydraz-ono}propanedinitrile, CAS registry number [141505-33-1] as well as its analogs II, III, IV, V, VI, VII, VIII and IX (see Table 1), were synthesized at Orion-Pharma (Espoo, Finland). Trifluoperazine, dansyl chloride, saponin and troponin complex from porcine slow skeletal muscle were purchased from Sigma Chemical Company (St. Louis, MO, USA).
Cloning and expression of recombinant human cTnC, its amino terminal fragment, and point mutations The coding sequence of human cTnC was cloned by using the human heart poly A+ RNA (purchased from Clontech, Palo Alto, CA, USA) as template for the cDNA synthesis as described previously.7 The amplified DNA fragment was digested with BamHI and EcoRI, purified and subcloned to the pGEM3-vector (Promega).12 For protein expression, the cTnC-insert from pGEM3 was isolated and ligated to BamHI-EcoRI-digested GST-fusion protein-vector pGEX-2T (Pharmacia PL-Biochemicals, Milwaukee, WI, USA) to yield the clone pOGL501 as described.7
∗ Abbreviations used in the text: cTnC, cardiac troponin C; sTnC, skeletal troponin C; cTnI, cardiac troponin I; cTnT, cardiac troponin T; SAR, structure-to-activity relationship; MOPS, 3-[N-morpholino] propanesulphonic acid; CT-13C HSQC, constant time heteronuclear single quantum coherence.
Binding of Levosimendan to Cardiac Troponin C Table 1 Levosimendan (I) and its analogs
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Amino acid substitutions Cys84→Ser, Asp87→Lys and Asp88→Ala were carried out by applying the “megaprimer” technique,13 by using mutant oligonucleotides. The accuracy of the mutant clones was verified by DNA sequencing. The expression construct was introduced into E. coli BL21 (DE3) or DH-5 cells (Promega) which were grown and induced with isopropyl-b-D-galactopyranoside (0.5 m final concentration) as previously described.14 The expression construct coding for the amino terminal fragment of cTnC (amino acids 1-91; pOGL522) was made by PCR using as the 3′ primer an oligonucleotide (5′-AGCTGAATTCTCACCCTTTGCTGTCGTCCT-3′) that incorporates the translation stop-codon after the sequence coding for amino acid 91. The recombinant Troponin C proteins were purified by Glutathione-Sepharose 4B-affinity chromatography (Pharmacia) as described elsewhere.15 Recombinant cTnC protein was cleaved from the fusion protein with thrombin and eluted from the column with the cleavage buffer. Aliquots from the eluted fractions were analysed by SDSPAGE (15% acrylamide)16 and Coomassie Brilliant Blue staining. The fractions containing recombinant cTnC protein were pooled, concentrated, and the buffer changed by ultrafiltration (Omega Filter NMWL 3K, Filtron). Further purification was performed on an anion exchange Mono-Q chromatography column (HR 5/5, Pharmacia). The proteins were finally analysed by reversed-phase chromatography.17
Binding on cTnC- and troponin complex-affinity HPLC The cTnC-affinity HPLC column was prepared as previously described,1 and the cardiac troponin complex HPLA column was constructed in a similar way. Briefly, Tn complex (50 mg) or recombinant human TnC (27 mg) were dissolved in 50 ml of a coupling buffer containing (in m): MOPS (3-[Nmorpholino] propanesulfonic acid), 10; MgCl2, 3; CaCl2, 2; KCl, 100; at pH 7.5. This solution was then run through an activated tresyl HPLAC column (SelectiSpher-10TM, 10 cm×5 mm ID., Pierce Chemical Company, Rockford, IL, USA). After coupling, the column was washed and then the residual tresyl groups were deactivated with 0.1 of TrisHCl, pH 8.0. The UV-absorption of Tn solutions was measured before and after the coupling to calculate the amount of Tn or cTnC on the column. Temperature of the solutions was 20–22°C, and the columns were stored at 4–8°C while not in use. The
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Figure 1 pCa/force relationship in guinea pig skinned fibers prepared with the sonication/saponin treatment. Hill plot of the pCa/force relationship. h is the tension (expressed as percentage of the maximum contraction) obtained in the skinned fiber at a certain [Ca2+]. For every [Ca2+], 4–8 individual experiments were done. The Hill parameters calculated after fitting the single data on a line are pK=6.28±0.1 and nH=4.2±0.3. Table 2 Retention times of levosimendan and its analogs in cTnC- and Tn complexHPLAC columns. The compounds were run through the columns in mobile phases containing Ca2+ or EGTA (pH 6.9, ionic strength 0.12). The conditions are described in the Materials and Methods section Compound Ca2+ min I (Levosimendan) II III IV V VI VII VIII IX
10.3 7.4 32.5 22.4 10.3 7.3 4.9 5.4 5.6
cTnC-HPLAC EGTA Ca2+/ EGTA min ratio 6.6 4.5 19.4 13.6 6.5 5.7 4.7 5.3 5.7
methods were validated as published previously.1 Levosimendan and its analogs were dissolved in DMSO, diluted in 100 ll of distilled water and then injected into the column. EGTA buffer or calciumcontaining buffer was used as the mobile phase. The EGTA buffer contained (in m): MOPS, 10; EGTA, 2; and KCl, 87 (pH 6.9), while the calcium buffer contained (in m): Ca2+, 30; MOPS, 10 (pH 6.9). The ionic strength (I) of both buffers was adjusted to 0.12 by KCl. A flow rate of 0.5 ml/min was used. The HPLAC columns were connected to a Spectroflow 400 pump (Applied Biosystem, USA), and the compounds were detected by a Spectroflow 757 absorbance detector (Kratos Analytical, USA) at
1.56 1.64 1.68 1.65 1.58 1.28 1.04 1.02 0.98
Tn complex-HPLAC Ca2+ EGTA Ca2+/ EGTA min min ratio 9.4 3.2 10.5 4.8 3.3 2.8 2.4 1.8 1.5
5.6 1.8 4.8 2.1 2.1 2.4 2.4 1.7 1.7
1.68 1.78 2.19 2.28 1.57 1.17 1.00 1.06 0.88
their UV-absorption maximum. The actual retention times of the compounds were calculated by subtracting the time interval between injection and the appearance of the void peak of a DMSO blank run from the measured retention times. The ratio of the retention times in Ca2+ buffer to those in EGTA buffer showed the Ca2+-dependent binding to the cTnC- and Tn complex-HPLAC column.
Skinned fibers Adult guinea-pigs of either sex (Duntley Hartley, Mollegaard Breeding Center Ltd, Denmark), weighing 300–350 g were used. Guinea-pigs were killed,
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Figure 2 Correlation between the calcium-dependent binding to cTnC-HPLAC and Tn complex-HPLAC columns for levosimendan and its analogs. The ratios of retention times in Ca2+ over EGTA buffer in cTnC- and Tn complex-HPLAC are plotted for the nine molecules studied. The correlation index (r) is 0.92.
Table 3 Calcium-sensitizing effects of levosimendan and its analogs in skinned fibers. Differences in calcium-induced tension (at pCa 6.5) ±... from controls, expressed as percentage (n=7) Compound
I (Levosimendan) II III IV V VI VII VIII IX
0.3 l Mean 16.3 14.8 21.8 12.9 12.4 0.3 −2.0 −2.2 0.5
...
1 l Mean
Ca2+ Sensitization 3 l ... Mean
...
10 l Mean
...
2.9 2.5 7.9 3.5 4.9 2.2 1.8 1.5 0.3
41.0 34.0 55.6 44.0 29.4 1.2 −2.3 −2.9 0.7
7.8 9.1 20.0 9.5 10.8 0.9 2.2 1.3 0.5
11.8 12.0 50.1 15.7 17.6 2.8 3.6 1.0 0.9
152.1 101.9 46.5 105.9 94.8 0.1 2.5 1.8 4.1
30.0 22.1 6.6 24.9 37.4 4.1 3.1 3.0 2.2
and the heart was excised. Right ventricular papillary muscle was dissected and rinsed in ice-cold Tyrode solution. Thereafter, the papillary muscle was immersed in a solution containing (in m): K+-acetate, 75; EGTA-Na2, 10; MgSO4, 5.4; ATPNa2, 4; DTT, 2; MOPS, 20 (pH 7.0). Subsequently, the papillary muscle was sonicated at 10 W for 60 s. The fibers (
