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

Ca(2+)-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 Ca(2+)-activated force (F(max)) 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 F(max) increased in a greater than linear manner with WT sTnC, and suggests that Ca(2+) binding to each functional Tn activates < 7 actins of a structural regulatory unit in cardiac muscle and > 7 actins in skeletal muscle. The Ca(2+) sensitivity of force and rate of force redevelopment (k(tr)) was leftward shifted by 0.1-0.2 -log [Ca(2+)] (pCa) units as WT cTn content was increased, but the slope of the force-pCa relation and maximal k(tr) 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 Ca(2+) 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, Ca(2+) binding to individual Tn complexes is insufficient to completely activate their corresponding RUs, making thin filament activation level more dependent on concomitant Ca(2+) binding at neighbouring Tn sites and/or crossbridge feedback effects on Ca(2+) binding affinity.