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

Abstract

The binding of Ca to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca (Ca-sensitizers) have therapeutic promise. The only Ca-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, Ca-bound conformation of cTnC. Finally, we propose a structural model of levosimendan bound to cTnC, which suggests that the Ca-sensitizing function of levosimendan is due to stabilization of the Ca-bound conformation of cTnC.

Dynamic Equilibrium of Cardiac Troponin C’s Hydrophobic Cleft and Its Modulation by Ca2+ Sensitizers and a Ca2+ Sensitivity Blunting Phosphomimic, cTnT(T204E)

“…These results imply that levosimendan is unable to substantially bind with cTnC in the absence of Ca 2+ and cTnI at the concentrations tested, likely because the probability of hydrophobic cleft opening is too low. This agrees with an NMR study that showed the presence of Ca 2+ was necessary for levosimendan to substantially bind with cTnC; the authors suggested that insufficient opening of the hydrophobic cleft limits levosimendan’s ability to bind . Accordingly, we propose that when the probability of hydrophobic cleft opening is increased by cTnC binding with either Ca 2+ or cTnI, levosimendan is able to induce a greater effect on the dynamic equilibrium in cTnC, as was observed in the present study.…”

“…This agrees with an NMR study which showed the presence of Ca 2+ was necessary for levosimendan to substantially bind with cTnC, the authors suggested that insufficient opening of the hydrophobic cleft limits levosimendan’s ability to bind. 21 Accordingly, we propose that when the probability of hydrophobic cleft opening is increased, either by cTnC binding with Ca 2+ or cTnI, levosimendan is able to induce a greater effect on the dynamic equilibrium in cTnC, as was observed in the present study. Thus, levosimendan appears to amplify the extent of cTnC opening induced by Ca 2+ or cTnI binding.…”

Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery

“…It plays a critical role in regulating sensitivity to Ca 2+ . Levosimendan is a positive inotrope that modulates troponin-C, 1 protein of the trimer and increases its sensitivity to regulation by Ca 2+ (Pollesello et al , 1994; Robertson et al , 2016). Treatment of ECTs with levosimendan induced a maximal 3.3 ± 0.4-fold increase in contractile force at a concentration of 2 μM with an EC 50 of 234 nM (Figure 4C).…”

Structural Changes Induced by the Binding of the Calcium Desensitizer W7 to Cardiac Troponin

“…Physiological studies have shown greatly enhanced contractility of cardiac trabeculae when cTnC–i9 was exchanged in the fibers, proving that targeting cTnC can be effective . It has also been demonstrated that levosimendan can make a reversible covalent thioimidate bond in situ with cTnC . Recent computational studies have discovered three novel compounds, NSC147866, NSC600285, and NSC611817, that exhibit positive switch peptide–drug binding cooperativity. , …”

“…16 It has also been demonstrated that levosimendan can make a reversible covalent thioimidate bond in situ with cTnC. 17 Recent computational studies have discovered three novel compounds, NSC147866, NSC600285, and NSC611817, that exhibit positive switch peptide−drug binding cooperativity. 18,19 The calmodulin antagonist W7 has been used to explore a wide range of physiological processes involving calcium signaling.…”