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

1. 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 degreesC 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. 2. Relative to control conditions, application of 6 muM 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 (pCa(50)) increased from pCa 5.85 +/- 0.05 to 5.95 +/- 0.04 (change in pCa(50) (Delta pCa(50)) = 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 P-o (maximum Ca2+-activated force), i.e. the Hill coefficient (n(2)) decreased from 4.72 +/- 0.38 to 1.54 +/- 0.07. 3. In the absence of NEM-S1, the rate of force redevelopment (k(tr)) 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 k(tr) by eliciting maximal values at low levels of Ca2+, i.e. k(tr) was 9.38 +/- 0.30 s(-1) at pCa 6.6 compared to 9.23 +/- 0.27 s(-1) at pCa 4.5. At intermediate levels of Ca2+, k(tr) was less than maximal but was still greater than values obtained at the same pCa in the absence of NEM-S1. 4. 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. 5. 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 crossbridge attachment, potentiation of force at low levels of free Ca2+, and slowed rates of relaxation.