Drug binding to cardiac troponin C

Compounds that sensitize cardiac muscle to Ca2+ by intervening at the level of regulatory thin filament proteins would have potential therapeutic bene fit in the treatment of myocardial infarctions. Two putative Ca2+ sensitize rs, EMD 57033 and levosimendan, are reported to bind to cardiac troponin C (cTnC). In this study, we use heteronuclear NMR techniques to study drug bi nding to [methyl-C-13]methionine-labeled cTnC when free or when complexed w ith cardiac troponin I (cTnI). In the absence of Ca2+, neither drug interac ted with cTnC. In the presence of Ca2+, one molecule of EMD 57033 bound spe cifically to the C-terminal domain of free cTnC. NMR and equilibrium dialys is failed to demonstrate binding of levosimendan to free cTnC, and the pres ence of levosimendan had no apparent effect on the Ca2+ binding affinity of cTnC. Changes in the N-terminal methionine methyl chemical shifts in cTnC upon association with cTnI suggest that cTnI associates with the A-B helica l interface and the N terminus of the central helix in cTnC. NMR experiment s failed to show evidence of binding of levosimendan to the cTnC cTnI compl ex. However, levosimendan covalently bound to a small percentage of free cT nC after prolonged incubation with the protein. These findings suggest that levosimendan exerts its positive inotropic effect by mechanisms that do no t involve binding to cTnC.