Effect of Amino Acid Changes in a Troponin I FHC Hotspot on Protein:Protein Binding and Calcium Sensitivity of Force Development

Abstract

Mutations in human cardiac troponin I (cTnI) have been associated with restrictive, dilated and hypertrophic cardiomyopathies. The most commonly occurring residue on cTnI that is associated with familial hypertrophic cardiomyopathy (FHC) is arginine, which is also the most common residue at which multiple mutations occur. Two FHC mutations are known to occur at arginine 204, R204C and R204H, and both are associated with poor clinical prognosis. To determine the effect of these mutations (R204C and R204H), as well as other cTnI mutations, R204P, R204Q, and R204W, calcium-force measurements and cTnI:troponin C (TnC) and cTnI:troponin T (TnT) interactions using the mammalian two-hybrid luciferase assays were utilized. All five mutations showed significant increases in calcium sensitivity of force development ranging from ΔpCa50 0.23 (R204W) to 0.35 (R204P). The mutations associated with FHC, R204C and R204H, had ΔpCa50 values of 0.28 and 0.29 respectively. The cTnI containing the R204P mutation showed the weakest interaction with TnT when compared to wild-type cTnI or the other mutants. The R204H mutation also showed significant impairment in its ability to interact with TnT, while the R204C mutation showed mild impairment when compared to wild-type cTnI. The R204C and R204P mutations showed the greatest impairment in binding to TnC. These results suggest that mutations at the same site on cTnI could affect thin filament interactions differentially, and that significant impairment in the interaction of cTnI with TnT or TnC may be enough to cause significant changes in calcium sensitivity. If the large increase in calcium sensitivity of force development observed with these mutations is associated with the poor prognosis then other R204 mutations are likely to have a poor prognosis. This research was supported by a Hellman Fellowship.