Interaction between PEVK-titin and actin filaments - Origin of a viscous force component in cardiac myofibrils

The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of r elaxed cardiomyocytes. Here, human N2-B-cardiac PEVK was expressed in Esche richia coli and tested-along with recombinant cardiac titin constructs cont aining immunoglobulin-like or fibronectin-like domains-for a possible inter action with actin filaments. In the actomyosin in vitro motility assay, onl y the PEVK construct inhibited actin filament sliding over myosin. The slow down occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca2+] reversed PEVK effect. PEVK concentrations greater than or equal to 10 mug/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescenc e binding assays without myosin at physiological ionic strength. In cosedim entation assays, PEVK-titin interacted weakly with actin at 0 degreesC, but more strongly at 30 degreesC, suggesting involvement of hydrophobic intera ctions. To probe the interaction in a more physiological environment, nonac tivated cardiac myofibrils were stretched quickly, and force was measured d uring the subsequent hold period. The observed force decline could be fit w ith a three-order exponential-decay function, which revealed an initial rap id-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the w hole decay amplitude. The rapid, viscous decay component, but not the slowe r decay components, decreased greatly and immediately on actin extraction w ith Ca2+-independent gelsolin fragment, both at physiological sarcomere len gths and beyond actin-myosin overlap. Steady-state passive force dropped on ly after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag dur ing diastolic stretch of cardiac myofibrils. The interaction could also opp ose shortening during contraction.