Ca2+-Regulatory Function of the Inhibitory Peptide Region of Cardiac Troponin I is Aided by the C-Terminus of Cardiac Troponin T: Effects of FHC Mutations Ctni R145G and Ctnt R278C, Alone and in Combination, on Filament Sliding

Abstract

Investigations of cardiomyopathy mutations in Ca2+ regulatory proteins troponin and tropomyosin provide crucial information about cardiac disease mechanisms, and provide insights into functional domains in the affected polypeptides. Hypertrophic cardiomyopathy-associated mutations TnI R145G, located within the inhibitory peptide (Ip) of human cardiac troponin I (hcTnI), and TnT R278C, located immediately C-terminal to the IT arm in human cardiac troponin T (hcTnT), share remarkable features: structurally, biochemically, and pathologically. Using bioinformatics, we find compelling evidence that affected regions of hcTnI and hcTnT, may be related_not just structurally_but also evolutionarily. Because alignment of TnI and TnT coincides with the known structure of the IT-arm, and both Arg mutations are located close to the C-terminal end of the IT-arm, we investigated functional relationships between hcTnI R145G and hcTnT R278C. We hypothesized that if the mutations affected function independently, then their effects would be additive in a double mutant complex. We characterized Tn complexes containing either mutation alone, or both mutations simultaneously, using in vitro motility assays run with varying [Ca2+], temperature, or HMM density. Our most significant findings show that TnT R278C rescued some deleterious effects of TnI R145G at high Ca2+, but exacerbated the loss of function (i.e., switching off the actomyosin interaction) at low Ca2+. Taken together, our results raise the likelihood that cTnI's Ip sequence might share a common evolutionary origin with, and thus be structurally and functionally related to, the C-terminus of cTnT. In accord with this prediction, our experimental results suggest that the C-terminus of cTnT aids Ca2+-regulatory function of cTnI Ip within the troponin complex.