Biophysical and physiologic characterization of cardiac troponin T mutations in the TNT1 domain that cause FHC

Abstract

Cardiac troponin T (cTnT) is a central modulator of thin filament regulation of myofilament activation. Mutations found in cTnT are associated with Familial Hypertrophic Cardiomyopathy, a primary cardiac muscle disorder that is one of the most common causes of sudden death in young people in the field. Cardiac TnT residue 92, flanking the TNT1 N-terminal tail domain, has been shown to be a mutational "hotspot" resulting in variable complex, cardiovascular phenotypes. The lack of structural data for the TNT1 tail domain, a proposed alpha-helical region, makes functional implications of FHC mutations difficult to determine. Studies have suggested that flexibility of TNT1 is important in normal protein-protein interactions within the thin filament. Through Molecular Dynamics (MD) simulations, we showed that R92 and R94 FHC mutations cause local alpha-helical structural alterations, changes in local forces, and increased flexibility at a critical hinge region 18 Angstroms distant from the mutation. We hypothesize that these local structural alterations in mutational cTnT segments lead to electrostatic perturbations, possibly interfering with cTnT-TM complex formation and thin filament function. In vitro motility assays with wildtype-cTnT and hotspot FHC-cTnT mutants support this hypothesis, whereby local structural alterations correlated with global changes in the cooperativity of thin filament regulatory function. Moreover, to determine the mechanistic links between the primary mutational effects on FHC-cTnT structure/function and resultant cardiovascular phenotypes, we characterized the myocellular response of several transgenic mouse models carrying cTnT mutations corresponding to the peptides studied via MD (R92L and R92W). Results showed that independent cTnT mutations in the TNT1 domain resulted in primary mutation-specific effects and differential temporal onset of altered myocellular mechanics, Ca2+ kinetics, and Ca 2+ homeostasis. For R92L, control of Ca2+ handling and homeostasis suggested unique pathogenic mechanisms at the level of the myofilament taking precedence, while R92W pathogenesis invoked both Ca 2+ handling and myofilament level mechanisms. Additionally, the beta-adrenergic response of the heart plays a role in the differential disease progression of the R92 FHC mutations. Together with the primary perturbations on structure and function, these downstream myocellular responses reveal complex mechanisms, which may contribute to the clinical variability in cTnT-related FHC mutations.