Further evidence for the cardiac troponin C mediated calcium sensitizationby levosimendan: Structure-response and binding analysis with analogs of levosimendan

Levosimendan, an inodilatory drug discovered using troponin C as a target p rotein. has a cardiac effect deriving from the calcium sensitization of con tractile proteins. The aim of this study was to give further evidence that levosimendan binds to cardiac troponin C and that the binding involves amin o acid residues on helix epsilon of the N-terminal domain of this calcium-b inding protein, Nine organic molecules, obtained by chemical modification o f levosimendan, were tested both for their calcium-dependent binding to tro ponin C and troponin complex affinity HPLC columns, and for their ability t o increase the calcium sensitivity of myofilaments in cardiac skinned fiber s. A good correlation between the calcium sensitization and the calcium-dep endent 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 l evosimendan on the calcium-induced conformational changes in native and poi nt-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. T his effect was not seen when adding an analog of levosimendan, which did no t bind to the troponin C affinity HPLC column and did not increase the calc ium-induced tension in cardiac skinned fibers. (C) 2000 Academic Press.