Role of the N-terminal negative charges of actin in force generation and cross-bridge kinetics in reconstituted bovine cardiac muscle fibres

摘要

Mutant yeast actins were used to determine the role of actin's N-terminal negative charges in force generation. The thin filament was selectively removed from bovine cardiac skinned muscle fibres by gelsolin, and the actin filament was reconstituted from purified G-actin. In this reconstitution, yeast wild-type actin (2Ac: two N-terminal negative charges), yeast mutant actins (3Ac and 4Ac), and rabbit skeletal muscle actin (MAc) were used. The effects of phosphate, ATP and ADP on force development were studied at 25°C. With MAc, isometric tension was 77% of the initial tension owing to the lack of a regulatory system. With 2Ac, isometric tension was 10% of the initial tension; with 3Ac, isometric tension was 23%; and with 4Ac, isometric tension was 44%. Stiffness followed a similar pattern (2Ac < 3Ac < 4Ac < MAc). A similar trend was observed during rigor induction and relaxation. Sinusoidal analysis was performed to obtain the kinetic constants of the cross-bridge cycle. The results showed that the variability of the kinetic constants was ≤ 2.5-fold among the 2Ac, 4Ac and MAc muscle models. When the cross-bridge distribution was examined, there was no significant reapportionment among these three models examined. These results indicate that force supported by each cross-bridge is modified by the N-terminal negative charges of actin, presumably via the actomyosin interface. We conclude that two N-terminal negative charges are not adequate, three negative charges are intermediate, and four negative charges are necessary to generate force.