Functional and Structural Changes Induced By cTNT-Related FHC Mutations in TNT1 Alter Actomyosin Binding Interactions

Abstract

Familial Hypertrophic Cardiomyopathy (FHC) is a primary disease of the cardiac sarcomere. Many FHC mutations in hcTnT are found within the TNT1 domain, with a mutational hotpot at residues 160-163. These residues fall within a highly charged region (158-RREEEENRR-166), which may create a flexible hinge necessary for function, the structure and function of which is affected by FHC mutations. We are investigating the effects of these hotspot mutations using in vitro motility (IVM) assays, SDSL-EPR, and transgenic mouse models. IVM data indicate that mutations Δ160E and E163R disrupt actin binding to heavy meromyosin under standard assay conditions. By reducing the ionic strength of the motility solutions, thin filament binding and sliding are restored suggesting that mutations in this region cause disease by disrupting the weak electrostatic interactions between the thin filament and myosin necessary for crossbridge formation. CW-EPR spectra show an increase in spin label isotropic rotational rate at hcTnT residue 153 (upstream of the putative hinge region) between Troponin ternary complexes containing Δ160E verses WT hcTnT, suggesting an increase in flexibility due to backbone changes induced by the mutation. We are expanding our SDSL-EPR experiments with additional cysteine substitutions superimposed onto 160-163 mutant proteins to provide further data regarding secondary structural changes imposed by these mutations. These results correspond with our Δ160E mouse model showing dose dependant myofilament disarray. Preliminary observations of an E163R model suggest that this mutant is less severely affected, tolerating a higher transgene dose. The structural and functional changes observed in vitro may contribute to the structural impairment seen in vivo. By correlating our IVM and SDSL-EPR findings with in vivo data generated from the Δ160E and E163R models, a mechanism of disease for these hotspot mutations can be determined.