The molecular basis of the steep force-calcium relation in heart muscle

Abstract

Contraction of heart muscle is regulated by binding of Ca(2+) ions to troponin in the muscle thin filaments, causing a change in filament structure that allows myosin binding and force generation. The steady-state relationship between force and Ca(2+) concentration in demembranated ventricular trabeculae is well described by the Hill equation, with parameters EC(50), the Ca(2+) concentration that gives half the maximum force, and n(H), the Hill coefficient describing the steepness of the Ca(2)(+) dependence. Although each troponin molecule has a single regulatory Ca(2+) site, n(H) is typically around 3, indicating co-operativity in the regulatory mechanism. This review focuses on the molecular basis of this co-operativity, and in particular on the popular hypothesis that force-generating myosin cross-bridges are responsible for the effect. Although cross-bridges can switch on thin filaments at low MgATP concentrations, we argue that the evidence from contracting heart muscle cells shows that this mechanism does not operate in more physiological conditions, and would not play a significant role in the intact heart. Interventions that alter maximum force and EC(50) do not in general produce a significant change in n(H). Complete abolition of force generation by myosin inhibitors does not affect the n(H) values for either Ca(2+) binding to the thin filaments or changes in troponin structure, and both values match that for force generation in the absence of inhibitors. These results provide strong evidence that the co-operative mechanism underlying the high value of n(H) is not due to force-generating cross-bridges but is rather an intrinsic property of the thin filaments.

Fig. 1

Steady-state force–calcium relation from demembranated trabeculae of rat ventricle; mean ± SE for 5 trabeculae, data from ‘Before reconstitution of TnC-BR_55–62’ in Table 1 of Sun et al. . The line is the Hill equation with pCa₅₀ = 5.60, n_H = 3.51.

Fig. 2

Four-state model for calcium regulation of contraction in heart muscle cells.

Fig. 3

Force (squares) and bound Ca (circles) as a function of pCa, replotted from Figs. 2A and 3A of Wang and Fuchs . The open circles denote control activations and the closed circles in the presence of 1 mM vanadate, which almost completely eliminated active force. The force data are fitted by a standard Hill curve with pCa₅₀ 5.8 and n_H 2.8. The bound Ca data are fitted by the sum of two Hill curves assuming a 2:1 ratio of high-affinity sites (pCa₅₀ 6.5 and n_H 0.7 in control; pCa₅₀ 6.7 and n_H 0.7 in 1 mM vanadate) to low-affinity sites (pCa₅₀ 5.8 and n_H 3.9 in control; pCa₅₀ 5.6 and n_H 3.1 in 1 mM vanadate).

Fig. 4

Force (squares) and the orientation of a bifunctional rhodamine probe on the C helix of TnC, θ (circles, triangles) as a function of pCa, replotted from Figs. 1B and 2A of Sun et al. . The circles denote control activations and the triangles in the presence of 25 μM blebbistatin, which almost completely eliminated active force. The Hill parameters for force (short dashed line) are pCa₅₀ 5.28, n_H 3.10; for control orientation (continuous line), pCa₅₀ 5.23, n_H 3.01; for blebbistatin (dashed line), pCa₅₀ 5.14, n_H 2.86.