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

Abstract

Several studies have suggested that conformational dynamics are important in cardiac troponin C’s (cTnC’s) regulation of thin filament activation, however little direct evidence has been offered to support these claims. In this study, a dye homodimerization approach is developed and implemented which allows for determination of the dynamic equilibrium between open and closed conformations in cTnC’s hydrophobic cleft. Modulation of this equilibrium by Ca2+, cardiac troponin I (cTnI), cardiac troponin T (cTnT), Ca2+-sensitizers, and a Ca2+ desensitizing phosphomimic of cTnT (cTnT(T204E) is characterized. Isolated cTnC contained a small open conformation population in the absence of Ca2+, which increased significantly upon addition of saturating levels of Ca2+. This suggests the Ca2+ induced activation of thin filament arises from an increase in the probability of hydrophobic cleft opening. Inclusion of cTnI increased the population of open cTnC while inclusion of cTnT had the opposite effect. Samples containing Ca2+-desensitizing cTnT(T204E) showed a slight but insignificant decrease in open conformation probability compared to samples with cTnT(wt), while Ca2+-sensitizer treated samples generally increased open conformation probability. These findings show that an equilibrium between open and closed conformations of cTnC’s hydrophobic cleft are play a significant role in tuning the Ca2+ sensitivity of the heart.

Development of a Novel Sensor System Based on Magnetic Microspheres to Detect Cardiac Troponin T

“…It acts as a regulatory protein in myocardial contraction and is released into the bloodstream when the heart is damaged. In addition, the detection of cTn has high sensitivity and specificity and may substitute traditional cardiac enzymes as a diagnostic indicator of AMI [10][11][12][13][14]. Therefore, the fluctuation of cTnT content is regarded as an important indicator of AMI diagnosis by the European and American College of Cardiology [15].…”