A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

Abstract

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 ϳ23% 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.

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

“…Ͼϩ0.08 pCa units) net alteration in the overall Ca 2ϩ affinity. We are learning that a more complete contractile apparatus better recapitulates the cellular phenotype produced by mutations in regulatory proteins (11,20,32,34). As shown by Güth and Potter (18) and others (36,37), when ADP is added to the solution, the equilibrium shifts toward strongly bound crossbridge states, and the apparent Ca 2ϩ sensitivity increases.…”

“…After cTnC reconstitution, the Ca 2ϩ sensitivity and the maximal force recovery were evaluated. Recently our group has shown by Western blot analysis that 14.0 Ϯ 5.3% of the endogenous cTnC remains in the TF after CDTA extraction, and incubation of fibers with WT or mutant cTnC is able to restore the full complement of cTnC back into the muscle fibers (32).…”

Functional Characterization of TNNC1 Rare Variants Identified in Dilated Cardiomyopathy

“…Subsequently, this dysfunction causes the heart to lose its dynamic response to stress. In humans, it has been shown that DCM mutations involve primarily defective force transmission (33), which in functional studies is observed as decreased force recovery in skinned fibers with a corresponding reduction in ATPase activation (8,15,26,27,29). In vitro motility assays also indicated a decrease in Ca 2ϩ sensitivity and a loss of thin filament sliding velocity (10).…”

“…By NMR spectroscopy, it was determined that the C-terminal domain of cTnC containing the G159D mutation has lower affinity for the cTnI-(37-71) peptide (14). In addition, the combinatory purported mutations E59D and D75Y were identified in a patient with idiopathic DCM, and functional characterization also suggested their potential relevance (15,16).…”

Predicting Cardiomyopathic Phenotypes by Altering Ca2+ Affinity of Cardiac Troponin C

“…This shortened duration of the force transient would decrease the amount of force-generating cross-bridges that could be recruited during contraction, effectively lowering the amount of force generated (19,22), which results in systolic dysfunction in patients. The ventricular walls have thinned, and the cardiac output is significantly reduced (23).…”