Investigation of thin filament near‐neighbour regulatory unit interactions during force development in skinned cardiac and skeletal muscle

Ca2+-dependent activation of striated muscle involves cooperative interactions of cross-bridges and thin filament regulatory proteins. We investigated how interactions between individual structural regulatory units (RUs; 1 tropomyosin, 1 troponin, 7 actins) influence the level and rate of demembranated (skinned) cardiac muscle force development by exchanging native cardiac troponin (cTn) with different ratio mixtures of wild-type (WT) cTn and cTn containing WT cardiac troponin T/I + cardiac troponin C (cTnC) D65A (a site II inactive cTnC mutant). Maximal Ca2+-activated force (Fmax) increased in less than a linear manner with WT cTn. This contrasts with results we obtained previously in skeletal fibres (using sTnC D28A, D65A) where Fmax increased in a greater than linear manner with WT sTnC, and suggests that Ca2+ binding to each functional Tn activates 7 actins in skeletal muscle. The Ca2+ sensitivity of force and rate of force redevelopment (ktr) was leftward shifted by 0.1–0.2 −log [Ca2+] (pCa) units as WT cTn content was increased, but the slope of the force–pCa relation and maximal ktr were unaffected by loss of near-neighbour RU interactions. Cross-bridge inhibition (with butanedione monoxime) or augmentation (with 2 deoxy-ATP) had no greater effect in cardiac muscle with disruption of near-neighbour RU interactions, in contrast to skeletal muscle fibres where the effect was enhanced. The rate of Ca2+ dissociation was found to be > 2-fold faster from whole cardiac Tn compared with skeletal Tn. Together the data suggest that in cardiac (as opposed to skeletal) muscle, Ca2+ binding to individual Tn complexes is insufficient to completely activate their corresponding RUs, making thin filament activation level more dependent on concomitant Ca2+ binding at neighbouring Tn sites and/or crossbridge feedback effects on Ca2+ binding affinity.