HCM-linked ∆160E cardiac troponin T mutation causes unique progressive structural and molecular ventricular remodeling in transgenic mice.

Hypertrophic cardiomyopathy (HCM) is a primary disease of the 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 protein abundance and phosphorylation state. Finally, studies of whole heart physiology in the isovolumic mode demonstrated dose-dependent differences in the degree of cardiac dysfunction. 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 dysregulation of Ca(2+) homeostasis at the cellular level that results in a unique and highly progressive pattern of ventricular remodeling.

Correlation of molecular and functional effects of mutations in cardiac troponin T linked to familial hypertrophic cardiomyopathy: an integrative in silico/in vitro approach.

Nearly 70% of all of the known cTnT mutations that cause familial hypertrophic cardiomyopathy fall within the TNT1 region that is critical to cTn-Tm binding. The high resolution structure of this domain has not been determined, and this lack of information has hindered structure-function analysis. In the current study, a coupled computational experimental approach was employed to correlate changes in cTnT dynamics to basic function using the regulated in vitro motility assay (R-IVM). An in silico approach to calculate forces in terms of a bending coordinate was used to precisely identify decreases in bending forces at residues 105 and 106 within the proposed cTnT "hinge" region. Significant functional changes were observed in multiple functional properties, including a decrease in the cooperativity of calcium activation, the calcium sensitivity of sliding speed, and maximum sliding speed. Correlation of the computational and experimental findings revealed an association between TNT1 flexibility and the cooperativity of thin filament calcium activation where an increase in flexibility led to a decrease in cooperativity. Further analysis of the primary sequence of the TNT1 region revealed a unique pattern of conserved charged TNT1 residues altered by the R92W and R92L mutations and may represent the underlying "structure" modulating this central functional domain. These data provide a framework for further integrated in silico/in vitro approaches that may be extended into a high-throughput predictive screen to overcome the current structural limitations in linking molecular phenotype to genotype in thin filament cardiomyopathies.

Temporal and mutation-specific alterations in Ca2+ homeostasis differentially determine the progression of cTnT-related cardiomyopathies in murine models.

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.

Computational Characterization of Mutations in Cardiac Troponin T Known to Cause Familial Hypertrophic Cardiomyopathy.

Cardiac Troponin T (cTnT) is a central modulator of thin filament regulation of myofilament activation. The lack of structural data for the TNT1 tail domain, a proposed α-helical region, makes the functional implications of the FHC mutations difficult to determine. Studies have suggested that flexibility of TNT1 is important in normal protein-protein interactions within the thin filament. Our groups have previously shown through Molecular Dynamics (MD) simulations that some FHC mutations, Arg92Leu(R92L) and Arg92Trp(R92W), result in increased flexibility at a critical hinge region 12 residues distant from the mutation. To explain this distant effect and its implications for FHC mutations, we characterized the dynamics of wild type and mutational segments of cTnT using MD. Our data shows an opening of the helix between residues 105-110 in mutants. Consequently, the dihedral angles of these residues correspond to non-α-helical regions on Ramachandran plots. We hypothesize the removal of a charged residue decreases electrostatic repulsion between the point mutation and surrounding residues resulting in local helical compaction. Constrained ends of the helix and localized compaction results in expansion within the nearest non-polar helical turn from the mutation site, residues 105-109.