Changes in the chemical and dynamic properties of cardiac troponin T cause discrete cardiomyopathies in transgenic mice

Abstract

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...

Cardiac hypertrophy: stressing out the heart

“…trophic phenotype (mild) from the cellular phenotype (moderate to severe) in iTAC mice was striking; not only does this finding support several recent studies in which the pathogenic cardiovascular response was separated from myocellular growth, it extends our understanding of the central role of the nature of the inciting stimulus (10)(11)(12). Thus, these results definitively show that while the duration of the discrete pathogenic stimulus may dictate the magnitude of ventricular hypertrophy, chronicity is not sufficient to cause hypertrophic heart disease - the nature of the inciting stimulus must be pathogenic.…”

“…Activation of myocellular remodeling via alterations in biomechanical stretch (mechanotransduction) plays an important role in many cardiomyopathies including those caused by postinfarction remodeling and mutations in cytoskeletal and sarcomeric proteins (17). Indeed, animal models of both dilated and hypertrophic cardiomyopathies (representing intrinsic mechanical stress) have demonstrated a similar dissociation between myocellular pathology and growth, as seen in the iTAC mice (12,18). Thus, both extrinsic and intrinsic pathologic stimuli may activate similar myocellular responses involving both G protein-dependent and -independent signaling pathways.…”

Atomic resolution probe for allostery in the regulatory thin filament

“…3) to study both structural and dynamic changes as a result of mutation. As has been shown in previous studies, mutations cause changes to both local and long-range interactions (17,18,20,21). The long-range effects are difficult to discern when such complex systems are not studied in atomistic detail.…”

Multi-scale computational models of familial hypertrophic cardiomyopathy: genotype to phenotype

“…MD uses protein structure data to predict the motion of atoms within molecules on picosecond time scales. In one case, MD has been used to predict motion of a critical region of the troponin T (TnT) molecule in the presence of FHC-linked mutations R92W and R92L [19]. Simulations showed that both mutations tended to destabilize helical structures in the protein, increasing flexibility of the molecule relative to the wild-type sequence.…”

“…In such a case, the initial challenge to relate a specific mutation to the type of cardiac remodelling becomes one of relating the mutation to Ca 2þ sensitivity. Knowing the amino acid sequence of normal and mutant proteins should provide important clues, and some progress is being made in this regard through the use of molecular dynamics (MD) simulations (see Ertz-Berger et al [19] and Lorenz & Holmes [20]). MD uses protein structure data to predict the motion of atoms within molecules on picosecond time scales.…”