Molecular Mechanisms of Cardiomyopathy-Causing TnT Mutants Implicated in Interactions with Tropomyosin

Abstract

Point mutations in genes encoding sarcomeric proteins often cause primary cardiomyopathies via changes in protein-protein interactions and/or alterations in calcium sensitivity. Cardiac troponin T (TnT) interacts with troponin I and troponin C (TnI, Tnc) and binds to tropomyosin (Tm) to provide control of contractility by calcium. A helical region of TnT (residues 80-150) is known to interact with Tm. There is a direct interaction of Tm residues at position d, g, a and e of the heptad repeat with residues e, a, d and g of TnT respectively. We hypothesize that the TnT mutations seen at the above mentioned positions will severe biochemical and biophysical changes than the residues at most other positions because of their direct interaction with Tm. To test the hypothesis, we have studied multiple biochemical parameters of 5 hypertrophic (HCM) and 2 dilated (DCM) cardiomyopathy-causing mutants at representative positions of the heptad repeat. Microscale thermophoresis revealed changes in binding patterns of mutants with Tm relative to wild-type. Alterations in calcium sensitivity were observed by ATPase assays. Significant changes were observed in velocities with in vitro motility analysis. We have also used the data from these mutants to better model the interaction between TnT and Tm. Our data illustrate multiple mechanistic routes by which single-residue changes in TnT alter contractility.