The effect of tropomyosin on force and elementary steps of the cross-bridge cycle in reconstituted bovine myocardium

The role of tropomyosin (Tm) in the elementary steps of the cross-bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed using gelsolin (thin filament severing protein), and the actin filament was reconstituted from G-actin. Tm was further reconstituted without troponin (Tn), and the kinetic constants of the elementary steps of the cross-bridge cycle were deduced using sinusoidal analysis at pCa ≤ 4.66, pH 7.00, and 25°C. The association constant of MgATP to cross-bridges (K₁) after reconstitution of Tm was 20.7 ± 2.3 mM^-1, which was about 2 × the control (untreated) myocardium (9.1 ± 1.3 mM^-1). Following reconstitution of Tm, the equilibrium constant of the cross-bridge detachment step (K₂), the phosphate (P_i) association constant (K₅) and the equilibrium constant of the force-generation step (K₄), which significantly changed in the actin filament-reconstituted myocardium, recovered to those of the control myocardium. Active tension after reconstitution of Tm was 0.69 × the control myocardium, a value between the control (1.00 ×) and the actin filament-reconstituted myocardium (0.59 ×). Tm-reconstituted myocardium was further reconstituted with Tn, and the effect of MgATP on the rate constants (K₁, K₂) was studied. Following reconstitution with Tn, the myocardium regained the Ca²⁺ sensitivity and the active tension became 0.83 × the control myocardium. In addition, K₁ recovered to the value of the control myocardium with Tn reconstitution. These results indicate that both Tm and Tn enhance the force generated by each cross-bridge, and that Tm is primarily responsible for the change in the kinetic constants of the elementary steps of the cross-bridge cycle.