In this study we explore the mechanisms by which a double mutation (E59D/D75Y) in cardiac
troponin C (CTnC) associated with
dilated cardiomyopathy reduces the Ca(2+)-activated maximal tension of cardiac muscle. Studying the single mutants (i.e. E59D or D75Y) indicates that D75Y, but not E59D, causes a reduction in the
calcium affinity of CTnC in
troponin complex, regulated thin filaments (RTF), and the Ca(2+) sensitivity of contraction and
ATPase in cardiac muscle preparations. However, both D75Y and E59D are required to reduce the
actomyosin ATPase activity and maximal force in muscle fibers, indicating that E59D enhances the effects of D75Y. Part of the reduction in force/
ATPase may be due to a defect in the interactions between CTnC and cardiac
troponin T, which are known to be necessary for
ATPase activation. An additional mechanism for the reduction in force/
ATPase comes from measurements of the binding stoichiometry of
myosin subfragment-1 (S-1) to the RTF. Using wild type RTFs, 4.8 mol S-1 was bound per mol filament (seven
actins), whereas with E59D/D75Y RTFs, the number of binding sites was reduced by approximately 23% to 3.7. Altogether, these results suggest that the reduction in force and
ATPase activation is possibly due to a thin filament conformation that promotes fewer accessible S-1-binding sites. In the absence of any family segregation data, the functional results presented here support the concept that this is likely a
dilated cardiomyopathy-causing mutation.