A structural and functional perspective into the mechanism of Ca2+-sensitizers that target the cardiac troponin complex

Abstract

The Ca 2+ dependant interaction between troponin I (cTnI) and troponin C (cTnC) triggers contraction in heart muscle. Heart failure is characterized by a decrease in cardiac output, and compounds that increase the sensitivity of cardiac muscle to Ca 2+ have therapeutic potential. The Ca 2+ -sensitizer, levosimendan, targets cTnC; however, detailed understanding of its mechanism has been obscured by its instability. In order to understand how this class of positive inotropes function, we investigated the mode of action of two fluorine containing novel analogues of levosimendan; 2' ,4'-difluoro(1,1'-biphenyl)-4-yloxy acetic acid (dfbp-o) and 2' ,4'-difluoro(1,1'-biphenyl)-4-yl acetic acid (dfbp). The affinities of dfbp and dfbp-o for the regulatory domain of cTnC were measured in the absence and presence of cTnI by NMR spectroscopy, and dfbp-o was found to bind more strongly than dfbp. Dfbp-o also increased the affinity of cTnI for cTnC. Dfbp-o increased the Ca 2+ -sensitivity of demembranated cardiac trabeculae in a manner similar to levosimendan. The high resolution NMR solution structure of the cTnC-cTnI-dfbp-o ternary complex showed that dfbp-o bound at the hydrophobic interface formed by cTnC and cTnI making critical interactions with residues such as Arg147 of cTnI. In the absence of cTnI, docking localized dfbp-o to the same position in the hydrophobic groove of cTnC. The structural and functional data reveal that the levosimendan class of Ca 2+ -sensitizers work by binding to the regulatory domain of cTnC and stabilizing the pivotal cTnC-cTnI regulatory unit via a network of hydrophobic and electrostatic interactions, in contrast to the destabilizing effects of antagonists such as W7 at the same interface.

A Novel Calcium Binding Site in the Slow Vacuolar Cation Channel TPC1 Senses Luminal Calcium Levels

“…To model the luminal calcium sensor in TPC1, the sequence from Ile-445 to Ile-474, located between the transmembrane helices 7 and 8, were manually aligned onto the sequence of the EF-hand of Troponin C (PDB entry 2L1R; (Robertson et al, 2010) with Glu-450 aligning onto the first Asp residue of the EF-hand occupying the +X position Asp-65. With this placement, Asp-454 of TPC1 would then be positioned at position 5 and coordinating the Ca 2+ ion in the +Z position.…”

Sarcomere neutralization in inherited cardiomyopathy: small-molecule proof-of-concept to correct hyper-Ca2+-sensitive myofilaments

“…W7 is an excellent molecule to begin these investigations because of the wealth of biochemical data demonstrating W7 directly binds to cTnC to alter its Ca 2ϩ -binding properties (23). Specifically, W7 binds to the NH 2 -terminal regulatory domain of cTnC, alters Ca 2ϩ affinity, and decreases cTnI switch peptide affinity for cTnC's hydrophobic patch (23,29). Mechanistically, W7's positive charged tail (Fig.…”

“…Mechanistically, W7's positive charged tail (Fig. 1A) is modeled to repel two arginines at the beginning of the cardiac TnI switch peptide domain, thus decreasing TnI's affinity for cTnC (29). Studies using membrane-permeabilized isolated myocytes show that W7 decreases the Ca 2ϩ sensitivity of force development (1).…”

The Evaluation of the Ameliorative Effect of Montelukast Against Arsenic Trioxide-Induced Cardiotoxicity in Rats

“…It is measured in the blood to diagnose various heart disorders, including unstable angina, myocardial infarction, and acute coronary syndrome (21,22). The quantitative determination of cardiac troponin-I in the serum was performed using an immunodiagnostic kit, purchased from Monobind, Inc. Lake Forest, CA (92630), USA.…”