Role of tropomyosin (Tm) isoforms in skeletal muscle thin filament regulation

We investigated the influence of recombinantly expressed aa-Tm vs. bb-Tm dimers on myofibril contraction and thin filament sliding in vitro motility assays. We previously described methods to remove endogenous Tm and troponin (Tn) from myofibrils and replacement with exogenous regulatory proteins (Scellini et al. Biophys J 96(3): 228a, 2009). Replacement, evaluated by SDS-PAGE, was 92 ± 2% for aa-Tm and 77 ± 2% for bb-Tm reconstitutions. Maximal isometric tension (P0), Ca2+ sensitivity of tension (pCa50) and cooperativity of activation (nH) did not differ between the two groups, but rates of activation (kACT) and tension redevelopment (kTR) were reduced by *30%. Maximal sliding speed (pCa 5) and fraction of moving thin filaments on rabbit skeletal heavy meromyosin surfaces was not affected by Tm composition, but pCa50 of speed was decreased with bb-Tm and regulation required *2-fold more aa-Tm vs. bb-Tm, suggesting impaired regulation. This idea was supported in myofibrils where bb-Tm reconstitution compromised regulation at pCa 9.0. Slack sarcomere length (s.l.) was significantly shorter (1.83 ± 0.04 lm) compared to aa-Tm (2.28 ± 0.02 lm). and the passive tension-s.l. relationship was left-shifted with bb-Tm vs. aa-Tm. Both of these mechanical features were greatly reduced by 10 mM BDM, suggesting high contents of bb-Tm may result in Ca2+ independent activation of reconstituted thin filaments. Telethon-Italy GGP07133, EU STREP P. BIG-HEART 241577 (CP), HL065497 (MR).