Cardiac troponin T (cTnT) is a central component of the regulatory thin filament. Mutations in cTnT have been linked to severe forms of familial hypertrophic cardiomyopathy. A mutational ''hotspot'' that leads to distinct clinical phenotypes has been identified at codon 92. Although the basic functional and structural roles of cTnT in modulating contractility are relatively well understood, the mechanisms that link point mutations in cTnT to the development of this complex cardiomyopathy are unknown. To address this question, we have taken a highly interdisciplinary approach by first determining the effects of the residue 92 mutations on the molecular flexibility and stability of cTnT by means of molecular dynamics simulations. To test whether the predicted alterations in thin filament structure could lead to distinct cardiomyopathies in vivo, we developed transgenic mouse models expressing either the Arg-92-Trp or Arg-92-Leu cTnT proteins in the heart. Characterization of these models at the cellular and whole-heart levels has revealed mutation-specific early alterations in transcriptional activation that result in distinct pathways of ventricular remodeling and contractile performance. Thus, our computational and experimental results show that changes in thin filament structure caused by single amino acid substitutions lead to differences in the biophysical properties of cTnT and alter disease pathogenesis.contractility ͉ molecular dynamics ͉ thin filament ͉ familial hypertrophic cardiomyopathy T he regulatory function of the cardiac sarcomere resides in the thin filament. Muscle contraction depends on the access of the myosin head to the actin filament, which is regulated by a cascade of allosteric changes in the interactions of the proteins within the troponin [cardiac troponin T (cTnT), cTnI, and cTnC] and tropomyosin-actin complexes upon the binding of Ca 2ϩ (1, 2). Disruption of these important protein-protein interactions by many naturally occurring thin filament mutations is poorly tolerated. Many mutations in cTnT result in a severe form of genetic cardiomyopathy, familial hypertrophic cardiomyopathy (FHC). FHC caused by cTnT mutations is characterized by a high frequency of early sudden cardiac death, often in the absence of overt ventricular hypertrophy (3). The direct link between mutations in the structural components of the cardiac sarcomere and the resultant complex clinical phenotype remains unknown.cTnT is a highly elongated protein that interacts with all other components of the thin filament and has been described as the ''glue'' of the contractile regulatory system (1). Codon 92 in cTnT is a mutational ''hotspot,'' and patients carrying each of the three predicted missense mutations have been identified and exhibit distinct clinical phenotypes (4-6). Patients carrying the Arg-92-Trp (R92W) missense mutation in cTnT develop mild or no ventricular hypertrophy, yet they experience a high frequency of early cardiac sudden death (7). In contrast, although carriers of the Arg-92-Leu (R92L) mutatio...

Naturally occurring mutations in cardiac troponin T (cTnT) result in a clinical subset of familial hypertrophic cardiomyopathy. To determine the mechanistic links between thin-filament mutations and cardiovascular phenotypes, we have generated and characterized several transgenic mouse models carrying cTnT mutations. We address two central questions regarding the previously observed changes in myocellular mechanics and Ca(2+) homeostasis: 1) are they characteristic of all severe cTnT mutations, and 2) are they primary (early) or secondary (late) components of the myocellular response? Adult left ventricular myocytes were isolated from 2- and 6-mo-old transgenic mice carrying missense mutations at residue 92, flanking the TNT1 NH(2)-terminal tail domain. Results from R92L and R92W myocytes showed mutation-specific alterations in contraction and relaxation indexes at 2 mo with improvements by 6 mo. Alterations in Ca(2+) kinetics remained consistent with mechanical data in which R92L and R92W exhibited severe diastolic impairments at the early time point that improved with increasing age. A normal regulation of Ca(2+) kinetics in the context of an altered baseline cTnI phosphorylation suggested a pathogenic mechanism at the myofilament level taking precedence for R92L. The quantitation of Ca(2+)-handling proteins in R92W mice revealed a synergistic compensatory mechanism involving an increased Ser16 and Thr17 phosphorylation of phospholamban, contributing to the temporal onset of improved cellular mechanics and Ca(2+) homeostasis. Therefore, independent cTnT mutations in the TNT1 domain result in primary mutation-specific effects and a differential temporal onset of altered myocellular mechanics, Ca(2+) kinetics, and Ca(2+) homeostasis, complex mechanisms which may contribute to the clinical variability in cTnT-related familial hypertrophic cardiomyopathy mutations.

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