Near N-terminal region of troponin I is critical in modifying feedback effects of cross-bridge activation

Force generating, strong cross-bridges are required to fully activate cardiac thin filaments, but the signaling mechanisms for this mode of activation remains unclear. Based on studies comparing effects of cardiac (cTnI) and slow skeletal (ssTnI) troponin I, our hypothesis is that cross-bridge binding activates the thin filament through a signaling pathway that involves a critical near N-terminal region of cTnI surrounding Ala 66. To probe cross-bridge dependent activation, we measured Ca2+ activated tension at pH 7.0 and pH 6.5 before and after NEM-S1 treatment of detergent-extracted cardiac fibers in which endogenous Tn complexes were exchanged with those containing either cTnI, cTnI(A66H), ssTnI or ssTnI(H34A). Cross-bridge dependent activation was amplified at pH 6.5 in myofilaments containing cTnI than in those containing ssTnI. When cTnI(A66H) replaced cTnI, this effect of strong cross-bridges was blunted, whereas replacement of ssTnI with ssTnI(H34A) had little effect. Moreover, force-pCa relations demonstrated that myofilaments containing cTnI(A66H) were desensitized to Ca2+ at both pH 7.0 and pH 6.5 with no changes in Ca2+ dissociation rates from cTnC in the Tn complex. Studies demonstrating altered antibody binding to an epitope at the C-terminus of cTnI indicated a long range structural effect of the A66H mutation. A solid-phase protein binding assay indicated cTnT had lower affinity for GST-cTnI(A66H) than for GST-cTnI only at pH 6.5; there was no difference between cTnT binding to GST-ssTnI and GST-ssTnI(H34A) at either pH 7.0 or pH 6.5. Our data indicate that the near N-terminal region of cTnI surrounding Ala 66 plays a special role in cross-bridge dependent activation by signaling involving both cTnI and cTnT interactions and possible modifications of cTnI interactions with actin.