Strong Cross-bridges Potentiate the Ca2+ Affinity Changes Produced by Hypertrophic Cardiomyopathy Cardiac Troponin C Mutants in Myofilaments

Abstract

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca 2؉ affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca 2؉ affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP 2؊ , the condition conducive to rigor cross-bridge formation, further increased the apparent Ca 2؉ affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca 2؉ dissociation (k off ) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF ؉ S1), an increase in the Ca 2؉ affinity and decrease in k off was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca 2؉ off-rate is most likely to be affected rather than the Ca 2؉ on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca 2؉ -sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k off from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.

Enhanced troponin I binding explains the functional changes produced by the hypertrophic cardiomyopathy mutation A8V of cardiac troponin C

“…Human TnC WT , TnC A8V , and TnI were recombinantly expressed and purified according to established protocols (25,28). …”

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility

“…Pinto et al . reported that cTnC A8V and cTnC D145E significantly slowed Ca 2+ dissociation kinetics ( k off ) in solution, suggesting both mutations may alter muscle relaxation properties [178]. Considering that most of the findings of these HCM mutations were focused on the Ca 2+ sensitivity of ATPase activity and/or force development, it is of importance to also investigate how they influence the muscle relaxation.…”

A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation

“…This gives an overall stoichiometry between the thin filament and S1 as 1:1.58, respectively. We have previously determined that this is the ideal concentration/ratio of S1 that caused the half-maximal change in fluorescence from thin filaments (24). The change in the fluorescence spectra was recorded during the titration of microliter amounts of CaCl 2 .…”