Titin-actin interaction in mouse myocardium: Passive tension modulation and its regulation by calcium/S100A1

Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the sub domains of the extensible region of cardiac N2B titin (tandem-ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected fo r the other fragments. Comparison with a skeletal muscle PEVK fragment reve aled that only the cardiac PEVK binds actin at physiological ionic strength s. The significance of PEVK-actin interaction was investigated using in vit ro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F -actin retarded filament sliding. Myocyte results suggest that a similar in teraction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK-actin interaction. Although calc ium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK-actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 boun d the PEVK region in vitro in a calcium-dependent manner, and S100A1 bindin g was observed at several sites along titin's extensible region in situ, in cluding the PEVK domain. In vitro motility results indicate that S100A1-PEV K interaction reduces the force that arises as F-actin slides relative to t he PEVK domain, and we speculate that S100A1 may provide a mechanism to fre e the thin filament from titin and reduce titin-based tension before active contraction.