Role of the C-terminus mobile domain of cardiac troponin I in the regulation of thin filament activation in skinned papillary muscle strips.

The C-terminus mobile domain of cTnI (cTnI-MD) is a highly conserved region which stabilizes the actin-cTnI interaction during the diastole. Upon Ca2+-binding to cTnC, cTnI-MD participates in a regulatory switching that involves cTnI to switch from interacting with actin toward interacting with the Ca2+-regulatory domain of cTnC. Despite many studies targeting the cTnI-MD, the role of this region in the length-dependent activation of cardiac contractility is yet to be determined. The present study investigated the functional consequences of losing the entire cTnI-MD in cTnI(1-167) truncation mutant, as it was exchanged for endogenous cTnI in skinned rat papillary muscle fibers. The influence of cTnI-MD truncation on the extent of the N-domain of cTnC hydrophobic cleft opening and the steady-state force as a function of sarcomere length (SL), cross-bridge state, and [Ca2+] was assessed using the simultaneous in situ time-resolved FRET and force measurements at short (1.8 µm) and long (2.2 µm) SLs. Our results show the significant role of cTnI-MD in the length dependent thin filament activation and the coupling between thin and thick filament regulations affected by SL. Our results also suggest that cTnI-MD transmits the effects of SL change to the core of troponin complex.

Functional significance of C-terminal mobile domain of cardiac troponin I.

Ca2+-regulation of cardiac contractility is mediated through the troponin complex, which comprises three subunits: cTnC, cTnI, and cTnT. As intracellular [Ca2+] increases, cTnI reduces its binding interactions with actin to primarily interact with cTnC, thereby enabling contraction. A portion of this regulatory switching involves the mobile domain of cTnI (cTnI-MD), the role of which in muscle contractility is still elusive. To study the functional significance of cTnI-MD, we engineered two cTnI constructs in which the MD was truncated to various extents: cTnI(1-167) and cTnI(1-193). These truncations were exchanged for endogenous cTnI in skinned rat papillary muscle fibers, and their influence on Ca2+-activated contraction and cross-bridge cycling kinetics was assessed at short (1.9 µm) and long (2.2 µm) sarcomere lengths (SLs). Our results show that the cTnI(1-167) truncation diminished the SL-induced increase in Ca2+-sensitivity of contraction, but not the SL-dependent increase in maximal tension, suggesting an uncoupling between the thin and thick filament contributions to length dependent activation. Compared to cTnI(WT), both truncations displayed greater Ca2+-sensitivity and faster cross-bridge attachment rates at both SLs. Furthermore, cTnI(1-167) slowed MgADP release rate and enhanced cross-bridge binding. Our findings imply that cTnI-MD truncations affect the blocked-to closed-state transition(s) and destabilize the closed-state position of tropomyosin.