Truncation of the Mobile Domain of Cardiac Troponin I Results in Biphasic Calcium-Dependent Thin Filament Activation

Abstract

Cardiac troponin I (cTnI) contains a ∼40 residue C-terminal mobile domain (cTnI-Md, residues 168-211 in Rattus norvegicus) that is known to promote the blocked state of thin filament regulation. Because it has also been shown to stabilize the closed-state position of tropomyosin, we hypothesized that cTnI-Md is involved in the cooperativity of thin filament activation. To test this hypothesis, we generated the truncation mutant cTnI(1-167) from R.norvegicus wherein the entire cTnI-Md had been removed. We used passive exchange to incorporate cTnI(1-167) and FRET labeled cTnC(T13C/N51C)AEDENS-DDPM into left ventricular detergent skinned myocardial fibers. SDS-PAGE and Western blotting demonstrated that cTnC(T13C/N51C)AEDENS-DDPM and cTnI(1-167) were efficiently exchanged into skinned fibers. Intriguingly, incorporation of cTnI(1-167) resulted in biphasic Ca2+ dependent thin filament activation as indicated by the tightly coupled force−Ca2+ and N-cTnC-opening-Ca2+ relationships. Simultaneous force and FRET measurements showed that treatment with 1 mM orthovanadate inhibited force, reduced ensemble-averaged N-cTnC opening, and decreased the Ca2+-sensitivity of activation, but did not affect the cooperativity underlying the biphasic response of N-cTnC opening to increasing Ca2+. Akaike information criteria indicated that a weighted sum of two Hill equations was >109-fold superior in describing biphasic activation than a single Hill equation. Interestingly, a steady-state cooperativity model based on the concept of tropomyosin being pinned by cTnI (J. Mol. Biol., vol. 340, pp. 295-305) was 41-fold superior to the weighted sum of two Hill equations and suggested that blocked-state allosteric communication is severely disrupted by removal of cTnI-Md. We concluded that cTnI-Md helps facilitate allosteric communication between thin filament regulatory units in the blocked state and is therefore essential to achieving a proper contractile response to sarcomeric Ca2+ signals during early systole.