Heart Care Info - Heart Disease Prevention & Treatment

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

EP Manning, PJ Guinto... - Journal of Biological Chemistry, 2012 - ASBMBBackground: Cardiomyopathy-causing mutations in the N-terminal domain of c Tn T disrupt thin filament function. Results: Amino acid substitutions at c Tn T Residue 92 alter motility while computational analysis reveals significant changes in structural forces. Conclusion: ...

Nearly 70% of all of the known c Tn T mutations that cause Familial Hypertrophic Cardiomyopathy (FHC) fall within the TNT1 region that is critical to c Tn-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 c Tn T dynamics to basic function using the regulated in vitro motility assay (R- IVM). An in silica 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 c Tn T "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 Arg92 Trp (R92W) and Arg92 Leu (R92L) mutations and may represent the underlying "structure" modulating this central functional domain. These data provide a framework for further integrated in silicon 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.