Computational and Experimental Studies of Divergent Clinical Effects in Proximate Thin Filament Mutations

Abstract

The cardiac thin filament controls the contraction and relaxation of cardiac muscle. Mutations in this molecular machine have varying effects on the regulatory function of the cardiac sarcomere, and have been linked to both hypertrophic (HCM) and dilated cardiomyopathy (DCM). In the current study we describe both computational and experimental investigations of 2 cardiac Troponin T (cTnT) mutations that occur in similar regions of the thin filament but result in divergent clinical phenotypes. Both experimental (Time resolved FRET (TR-FRET)) and computational results show that the Δ160E mutation repositions the flexible linker, moving it closer to the C-terminus of tropomyosin. This in turn alters weak electrostatic binding that decreases linker flexibility, ultimately leading to HCM. By contrast, Arg173Trp (R173W) also lies within the linker domain on cTnT but results in a dilated cardiomyopathy (DCM). Computation shows both average structure and time dependent dynamics are differentially modified in this mutational state. Specifically, the in silico average structure of the thin filament shows an increased distance between the linker region and the C-terminus of tropomyosin due to the R173W mutation. Differential scanning calorimetry on reconstituted thin filaments both with and without the R173W mutation suggest an impact on thermal stability and thus the thermodynamics of protein interaction. Our simulations and TR-FRET results illuminate the resultant perturbations to the WT structure and dynamics caused by the R173W mutation, and we contrast these effects to those found in the Δ160E mutation. Our findings provide a new framework for understanding the varying pathogenic mechanisms that drive clinical phenotypes in HCM and DCM in proximate thin filament mutations.