Biophysical and Physiologic Characterization of Cardiac Troponin T Mutations in the TNT1 Domain that Cause FHC

Abstract

Familial Hypertrophic Cardiomyopathy (FHC) is one of the most frequently occurring cardiac genetic disorders and a common cause of sudden cardiac death in young people. The majority of FHC mutations in the thin filament regulatory protein cTnT are found within the TNT I domain, with a mutational hotpot occurring at residues 160/163 (Delta160E, E163K, E163R). These residues fall within a highly charged region (158-RREEEENRR-166), which we hypothesize creates a flexible hinge necessary for function, the structure and function of which is affected by FHC mutations. We investigated the effects of these hotspot mutations using regulated in vitro motility (R-IVM) assays and transgenic mouse models.;R-IVM data indicate that 160/163 mutations disrupt weak electrostatic actomyosin binding interactions. By altering the conditions of the motility assay to facilitate weak binding, function is fully rescued. These weak electrostatic interactions are crucial for the recruitment of the strong actomyosin crossbridges required for muscle contraction. This is the first observation of FHC mutations in cTnT disrupting weak actomyosin binding. Additionally, the R-IVM experiments reveal an increase in ATPase activity for the 160/163 hotspot mutations, suggesting these mutations alter actomyosin-ATP kinetics.;In order to link the in vitro findings to an in vivo system, transgenic mouse models of Delta160E and E163R were generated. Ultrastructural analysis shows profound sarcomeric disruption and myofilament disarray. Impairment in weak actomyosin binding may cause a destabilization of the sarcomere, leading to progressive myofibrillar disarray. By correlating our in vitro data with in vivo findings, a novel mechanism of disease pathogenesis for these hotspot mutations is determined.;R-IVM data reveal impaired weak actomyosin binding to be the primary molecular mechanism of disease for these hotspot mutations. Nevertheless, it is critical to determine the downstream effects of these changes, as they represent important potential therapeutic targets. In order to study these downstream effects, transgenic mouse models of Deltat160E expressing different levels of mutant protein (35% and 70%) were characterized. In addition to ultrastructural changes, these animals showed dose-dependent, progressive remodeling of the heart. This is consistent with the mutant protein acting as a "poison polypeptide" that destabilizes the myofilament and leads to pathologic changes in whole heart morphology. These animals dose showed dose-dependent changes in systolic and diastolic function. Ex vivo measurements of myocellular mechanics reveal significant impairments in unloaded myocellular contraction and relaxation, and measurements of calcium transient kinetics showed significant, dose-dependent alterations. In contrast with the dose dependent changes observed in calcium transients, SR calcium load is impaired independently of dose, suggesting that compensatory mechanisms are in play at higher transgene doses.;The TNT1 domain is an important tropomyosin-binding domain. However, the FHC mutations at the C-terminal TNT I hotspot have previously been shown to have no effect on the tropomyosin-dependent functions of TNT1, and have been posited to cause disease by "some other mechanism", which we have elucidated with our R-IVM data. Mutations at cTnT residues 160/163 may alter the flexibility of a putative hinge region, causing a disruption in weak actomyosin binding interactions. This impairs the transition from weak to strong actomyosin crossbridge formation, altering regulation of the crossbridge cycle and impairing sarcomere function. The presence of these mutant proteins destabilizes the myofilaments, leading to sarcomeric disarray. The downstream effects of this are whole-heart remodeling and changes in myocellular mechanics and calcium homeostasis. The determination of the elusive genotype-to-phenotype link for these FHC mutations by understanding the molecular disease mechanisms and downstream physiologic consequences reveal potential mutation-specific therapeutic targets for this disease.