Cross-bridge interaction kinetics in rat myocardium are accelerated by strong binding of myosin to the thin filament

Abstract

To determine the ability of strong-binding myosin cross-bridges to activate the myocardial thin filament, we examined the Ca2+ dependence of force and cross-bridge interaction kinetics at 15 degrees C in the absence and presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1) in chemically skinned myocardium from adult rats. Relative to control conditions, application of 6 microM NEM-S1 significantly increased Ca2+-independent tension, measured at pCa 9.0, from 0.8 +/- 0.3 to 3.7 +/- 0.8 mN mm-2. Furthermore, NEM-S1 potentiated submaximal Ca2+-activated forces and thereby increased the Ca2+ sensitivity of force, i.e. the [Ca2+] required for half-maximal activation (pCa50) increased from pCa 5.85 +/- 0.05 to 5.95 +/- 0.04 (change in pCa50 (dpCa50) = 0.11 +/- 0.02). The augmentation of submaximal force by NEM-S1 was accompanied by a marked reduction in the steepness of the force-pCa relationship for forces less than 0.50 Po (maximum Ca2+-activated force), i.e. the Hill coefficient (n2) decreased from 4.72 +/- 0.38 to 1.54 +/- 0.07. In the absence of NEM-S1, the rate of force redevelopment (ktr) was found to increase from 1.11 +/- 0.21 s-1 at submaximal [Ca2+] (pCa 6.0) to 9.28 +/- 0.41 s-1 during maximal Ca2+ activation (pCa 4.5). Addition of NEM-S1 reduced the Ca2+ dependence of ktr by eliciting maximal values at low levels of Ca2+, i.e. ktr was 9.38 +/- 0.30 s-1 at pCa 6.6 compared to 9.23 +/- 0.27 s-1 at pCa 4. At intermediate levels of Ca2+, ktr was less than maximal but was still greater than values obtained at the same pCa in the absence of NEM-S1. NEM-S1 dramatically reduced both the extent and rate of relaxation from steady-state submaximal force following flash photolysis of the caged Ca2+ chelator diazo-2. These data demonstrate that strongly bound myosin cross-bridges increase the level of thin filament activation in myocardium, which is manifested by an increase in the rate of cross-bridge attachment, potentiation of force at low levels of free Ca2+, and slowed rates of relaxation.

Figure 6. NEM-S1 markedly slows the rate of force relaxation

Myocardial preparations were bathed for 15 min in a pCa 9.0 solution in the absence or presence of 6 μm NEM-S1 and subsequently incubated for 2 min in loading solution containing (mm): Bes, 100; potassium propionate, 40.9; creatine phosphate, 15; dithiothreitol, 5; MgATP, 4; free Mg²⁺, 1; CaCl₂, 0.12; and diazo-2, 0.25; pH 7.0 at 15°C. Force relaxation following flash photolysis of diazo-2 was recorded either in the absence (trace a) or in the presence of 6 μm NEM-S1 (trace b), where F refers to the time point at which the flash lamp was triggered. Each of the myocardial preparations generated ≈0.70 P_o in the loading solution prior to photolysis of diazo-2.

Figure 1. Photomicrographs of rat skinned myocardium

Representative skinned myocardial preparation (dimensions: 430 μm w 174 μm) while relaxed (A, pCa 9.0) and during maximal activation (B, pCa 4.5). Sarcomere length in pCa 9.0 and 4.5 was 2.35 and 2.27 μm, respectively. Scale bar represents 50 μm.

Figure 3. Effects of 6 μm NEM-S1 on force-pCa relationships in skinned myocardium

Force-pCa relationships were determined from skinned ventricular myocardium (n = 7) in the absence (0) and presence (1) of NEM-S1. Smooth lines were generated by fitting the mean data with the equation: P_rel=[Ca²⁺]ⁿ/(kⁿ+[Ca²⁺]ⁿ), where P_rel is force as a fraction of maximal Ca²⁺-activated force (P_o) or maximal total force (P_Tot), n is the Hill coefficient, and k is the [Ca²⁺] required for half-maximal activation (i.e. pCa₅₀). Data points are means and the error bars are s.e.m. A, Ca²⁺-activated force-pCa relationship in the absence (pCa₅₀= 5.85 ± 0.05, n₂= 4.72 ± 0.38) and presence of NEM-S1 (pCa₅₀= 5.95 ± 0.04, n₂= 1.54 ± 0.07). B, total force-pCa relationship in the absence (pCa₅₀= 5.86 ± 0.05, n₂= 4.50 ± 0.69) and presence of NEM-S1 (pCa₅₀= 6.09 ± 0.05, n₂= 0.93 ± 0.22). Total force is the sum of Ca²⁺-independent and Ca²⁺-activated forces at each pCa.

Figure 2. NEM-S1 increases Ca²⁺-independent force

A mechanical release-restretch manoeuvre was used to measure the rate of force redevelopment in a solution of pCa 9.0 in the absence and presence of 6 μm NEM-S1. This skinned myocardial preparation generated Ca²⁺-independent force of 0.05 P_o in the absence of NEM-S1 in pCa 9.0 (trace a), whereas Ca²⁺-independent force increased to 0.22 P_o following NEM-S1 treatment (trace b).

Figure 4. NEM-S1 potentiates submaximal force and the rate of force redevelopment

A, force records showing redevelopment of submaximal force developed by a skinned ventricular preparation at pCa 5.8 both in the absence (trace b) and in the presence of 6 μm NEM-S1 (trace c). Force in each case is scaled to the maximum force (P_o) generated by the same preparation when exposed to a solution of pCa 4.5 (trace a). The upper panel shows the mechanical release-restretch manoeuvre used to measure k_tr. B, to illustrate differences in the rate of force redevelopment, the force records in A are re-plotted with the peak force at each pCa scaled to 1.0.

Figure 5. NEM-S1 nearly eliminates the activation dependence of the rate of force redevelopment

Force redevelopment following rapid release and restretch was measured in rat skinned myocardium (n = 7) in the absence (0) and presence (1) of NEM-S1. Data points are means and the error bars are the s.e.m. A, the Ca²⁺ dependence of the rate constant of force redevelopment, k_tr. B, the rate constant of force redevelopment (k_tr) as a function of force (as a fraction of total force) measured at each pCa. Total force is the sum of Ca²⁺-independent and Ca²⁺-activated forces at each pCa.