Strong cross-bridges potentiate the Ca²⁺ affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: A fast kinetic approach

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca²⁺ dissociation (koff) 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 Ca²⁺ 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 Ca²⁺-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.