The Mechanism of HCM-Related Mutation R21C on the Modulation of C-I Interactions and Contractile Kinetics

Abstract

The R21C mutation in cTnI, associated with hypertrophic cardiomyopathy (HCM), is the only known mutation within N-terminus of cTnI. The mechanisms by which this mutation leads to heart failure, and how this mutation affects PKA regulation of cTn function and myofibril relaxation still remain elusive. Currently, we are studying this mutation using an integrated experimental-computational approach. We generated triple-mutated cTnI (cTnI-R21C/S2324D) to mimic the phosphorylation. From steady-state fluorescence measurement, which using a fluorescent probe (IANBD) coupled to cTnCC35S, C-I binding affinity (KC-I) and calcium binding affinity (KCa) to whole cTn were monitored. cTnIR21C increased both KC-I and KCa compared to cTnIWT. Phosphorylation of cTnI reduced KCa for both complexes. However, while PKA treatment weakened KC-I for cTnIWT, this effect was not absent in cTnIR21C. To study myofibril contraction/relaxation mechanics and kinetics, endogenous cTn was exchanged with either WT or R21C recombinant cTn, and mechanics were monitored at maximum and submaximal Ca2+ conditions. The kinetics of activation (KACT) is slower at submaximal vs maximal Ca2+ conditions, and not affected by PKA treatment of myofibrils containing either cTnIWT or cTnIR21C. However, PKA treatment of cTn with cTnIWT significantly affected slow phase relaxation, increasing slow phase rate (KREL,slow) and decreasing slow phase duration (tREL, slow), and these effects are more clear at submaximal calcium levels. Interestingly, these PKA mediated effects were blunted in cTnIR21C. Molecular dynamics (MD) simulations of whole cTn with R21C and R21C/S23DS24D substitution are ongoing. Interactions between cNTnC-cNTnI, cNTnC-switch peptide of cTnI, and cTnC-inhibitory peptide of cTnI will be monitored, and calcium binding sites will be studied. Computational results will provide the structural basis at molecular level of how R21C influence C-I binding and relaxation of cardiac muscles. HL65497, HL11197 (MR), 8P41GM103426 (AM & AM).