C-Terminal Truncation of Troponin I and Substitutions of Non- Canonical Residues in the Central Part of Tropomyosin 1.1 Disrupt Thin Filament Switched Off State in Rabbit Psoas Myofibrils

The Ca2+-dependent and highly cooperative activation of striated muscle contraction critically depends on the factors affecting Tpm-Tn position on actin filament. Based on previously developed methods to remove/reconstitute striated muscle myofibrils with exogenous regulatory proteins [1], we investigate here the functional impact of the exchange of: (i) native fast skeletal Tn for human recombinant cTn containing either full length cTnI (cTnIFL) or truncated cTnI (cTnI1- 192); (ii) native Tpm-Tn with purified skeletal Tn and recombinant Tpm1.1 either wild type (WT) or carrying one (D137L) or two (D137L/G126R) stabilizing substitutions in the central part of the molecule, decreasing its flexibility [2]. In myofibrils replaced with cTnI1-192 or D137L/G126R Tpm (15C) force relaxation from maximal activation (pCa 4.5) to relaxing solution (pCa 9.0), was similarly prolonged (increased duration of slow phase, decreased rate of fast phase). Effects on maximal isometric tension and on rates of force activation (kACT) and redevelopment (kTR) were small (cTnI1-192) or absent (D137L or D137L/G126R Tpm). Both cTnI1- 192 and Tpm substitutions strongly and additively decreased slack sarcomere length (sl) at sub-maximal activating [Ca2+] and increased the steepness of the sl-passive tension relation. These effects were reversed by 10 mM BDM, suggesting that both cTnI1-192 and Tpm substitutions compromise the full inhibition of acto-myosin interactions in the absence of Ca2+. This hypothesis is further supported by the significant increase of ATPase activity in relaxing solution of D137L and D137L/G126R Tpm myofibrils compared to WT Tpm. Data support the hypothesis that flexibility of the Tpm coiled-coiled structure critically modulates the turning off of the thin filament system and muscle relaxation dynamics, likely in interaction with the C-term of TnI.