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...

Hypertrophic cardiomyopathy (HCM) is a primary disease of cardiac muscle, and one of the most common causes of sudden cardiac death (SCD) in young people. Many mutations in cardiac troponin T (cTnT) lead to a complex form of HCM with varying degrees of ventricular hypertrophy and ~65% of all cTnT mutations occur within or flanking the elongated N-terminal TNT1 domain. Biophysical studies have predicted that distal TNT1 mutations, including Δ160E, cause disease by a novel, yet unknown mechanism as compared to N-terminal mutations. To begin to address the specific effects of this commonly observed cTnT mutation we generated two independent transgenic mouse lines carrying variant doses of the mutant transgene. Hearts from the 30% and 70% cTnT Δ160E lines demonstrated a highly unique, dose-dependent disruption in cellular and sarcomeric architecture and a highly progressive pattern of ventricular remodeling. While adult ventricular myocytes isolated from Δ160E transgenic mice exhibited dosage-independent mechanical impairments, decreased sarcoplasmic reticulum calcium load and SERCA2a calcium uptake activity, the observed decreases in calcium transients were dosage-dependent. The latter findings were concordant with measures of calcium regulatory proteins abundance and phosphorylation state. Finally, studies of whole heart physiology in the isovolumic mode demonstrated dose-dependent differences in the degree of cardiac dysfuction. We conclude that the observed clinical severity of the cTnT Δ160E mutation is caused by a combination of direct sarcomeric disruption coupled to a profound disregulation of Ca2+ homeostasis at the cellular level that results in a unique and highly progressive pattern of ventricular remodeling.