Reversible Covalent Binding to Cardiac Troponin C by the Ca²⁺-Sensitizer Levosimendan

The binding of Ca²⁺ to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca²⁺, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca²⁺ (Ca²⁺-sensitizers) have therapeutic promise. The only Ca²⁺-sensitizer used regularly in clinical settings is levosimendan. While the primary target of levosimendan is thought to be cTnC, the molecular details of this interaction are not well understood. In this study, we used mass spectrometry, computational chemistry, and nuclear magnetic resonance spectroscopy to demonstrate that levosimendan reacts specifically with cysteine 84 of cTnC to form a reversible thioimidate bond. We also showed that levosimendan only reacts with the active, Ca²⁺-bound conformation of cTnC. Finally, we propose a structural model of levosimendan bound to cTnC, which suggests that the Ca²⁺-sensitizing function of levosimendan is due to stabilization of the Ca²⁺-bound conformation of cTnC.