Abstract
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 helixϵ of the N-terminal 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.
| Original language | English |
|---|---|
| Pages (from-to) | 479-491 |
| Journal | Journal of Molecular and Cellular Cardiology |
| Volume | 32 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2000 |
| MoE publication type | A1 Journal article-refereed |
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Further evidence for the cardiac troponin C mediated calcium sensitization by levosimendan : Structure-response and binding analysis with analogs of levosimendan. / Levijoki, Jouko; Pollesello, Piero; Kaivola, Juha; Tilgmann, Carola; Sorsa, Tia; Annila, Arto; Kilpeläinen, Ilkka; Haikala, Heimo (Corresponding Author).
In: Journal of Molecular and Cellular Cardiology, Vol. 32, No. 3, 2000, p. 479-491.Research output: Contribution to journal › Article › Scientific › peer-review
TY - JOUR
T1 - Further evidence for the cardiac troponin C mediated calcium sensitization by levosimendan
T2 - Structure-response and binding analysis with analogs of levosimendan
AU - Levijoki, Jouko
AU - Pollesello, Piero
AU - Kaivola, Juha
AU - Tilgmann, Carola
AU - Sorsa, Tia
AU - Annila, Arto
AU - Kilpeläinen, Ilkka
AU - Haikala, Heimo
PY - 2000
Y1 - 2000
N2 - 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 helixϵ of the N-terminal 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.
AB - 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 helixϵ of the N-terminal 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.
U2 - 10.1006/jmcc.1999.1093
DO - 10.1006/jmcc.1999.1093
M3 - Article
VL - 32
SP - 479
EP - 491
JO - Journal of Molecular and Cellular Cardiology
JF - Journal of Molecular and Cellular Cardiology
SN - 0022-2828
IS - 3
ER -