A8V Mutation of Cardiac Troponin C Enhances Troponin I Binding

Abstract

We find in our model that higher affinity for TnI is a potential mechanism by which the A8V mutation renders troponin-C (TnC) capable of causing hypertrophy in hearts carrying TnCA8V. At issue is to explain how muscle fibers reconstituted with TnCA8V are sensitized to Ca2+, i.e. requiring ∼2.3 fold less [Ca2+] to achieve 50% maximum tension compared with fibers reconstituted with wild type TnC (TnCWT). The N-helix of TnC, which is the location of alanine 8, is situated between the EF hand that binds Ca2+ and the hydrophobic patch that binds the switch peptide of troponin-I (TnIsp). Binding measurements rule out a significant change in Ca2+ affinity of TnCA8V, but TnCA8V binds TnIsp ∼1.6 fold more strongly than TnCWT. Hence, we focus on elements of the model in which the TnC-TnIsp interaction competes with the TnI-actin interaction. Tension data are well-fit by specific conditions of the model, namely, with the TnCA8V-TnIsp affinity adjusted ∼1.5 fold higher than TnCWT-TnIsp affinity for all Ca2+ concentrations. Myofibrillar ATPase assays confirm greater activation by TnCA8V at higher Ca2+ concentration, but are inconclusive at low Ca2+ concentration. To test for altered binding in low Ca2+, displacement of the environmentally sensitive fluorescence probe, bis-ANS, from TnI is monitored as a function of added TnC. Whereas Ca2+-bound TnCWT (Ca-TnCWT) displaces significantly more bis-ANS than Ca2+-free TnCWT (Mg-TnCWT) both Ca-TnCA8V and Mg-TnCA8V displace nearly the same amount of probe as Ca-TnCWT. Hence, the mutation predicted to increase hydrophobicity promotes a more intimate association with TnI independent of Ca2+, consistent with the observed increase in activation. Our work suggests that contractility is constantly above normal in hearts made hypertrophic by TnCA8V. Supported by NIH-HL103840 (JRP) and AHA-15GRNT25280004 (JRP).