Clinically Divergent Mutation Effects on the Structure and Function of the Human Cardiac Tropomyosin Overlap

Abstract

The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm–Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.

Combinatorial genetic replenishments in myocardial and outflow tract tissues restore heart function in tnnt2 mutant zebrafish

“…Besides, mutations in a single sarcomere gene like cardiac troponin T ( TNNT2 ) are sufficient to cause cardiomyopathy, and TNNT2 mutations are the most common ‘drivers’ of thin filament deficiency in both DCM and HCM (Hershberger et al, 2013; Veselka et al, 2017). Most human TNNT2 mutations are located in central and C-terminal domains of cardiac troponin T and are responsible for both familial cardiomyopathy and sporadic cardiomyopathy (Forissier et al, 1996; Van Driest, 2003; McConnell et al, 2017).…”

Mechanism of Cardiac Tropomyosin Transitions on Filamentous Actin As Revealed by All-Atom Steered Molecular Dynamics Simulations

“…By comparing the wild-type and the mutated models, we develop an understanding of how single-point mutations can alter the function of the complex molecular machinery of the CTF. 1,12–16 In this Letter, we face a far more basic problem. While the basic outline for Tm control of actin binding is accepted, the physical nature of this mechanism remains opaque because no currently available experimental technique is able to elucidate the transition mechanism.…”

Proof of Principle that Molecular Modeling Followed by a Biophysical Experiment Can Develop Small Molecules that Restore Function to the Cardiac Thin Filament in the Presence of Cardiomyopathic Mutations

“…As reported previously, this corresponds to the average distance between residues 94–133 of cTnT and the center of the TM coiled coil. 7 A candidate drug is thus identified as one that restores the overlap distance in a mutant to be similar to that of the WT. This winnowed the number of tested ligands to three.…”

“…7,10,11 A detailed description of the TM D230N mutation from molecular dynamics studies and experiments has been previously presented. 7,10 (We note that the computational and experimental model are both chosen to have mutant amino acid residues in both chains of the TM dimer. This is done to create interpretable experimental results and then to match the calculations to the experimental model).…”