Activation Kinetics of Skinned Cardiac Muscle by Laser Photolysis of Nitrophenyl-EGTA

The kinetics of Ca2+-induced contractions of chemically skinned guinea pig trabeculae was studied using laser photolysis of NP-EGTA. The amount of free Ca2+ released was altered by varying the output from a frequency-doubled ruby laser focused on the trabeculae, while maintaining constant total [NP-EGTA] and [Ca2+]. The time courses of the rise in stiffness and tension were biexponential at 23°C, Ph 7.1, and 200 Mm ionic strength. At full activation (pCa \u3c 5.0), the rates of the rapid phase of the stiffness and tension rise were 56 ± 7 s-1 (n = 7) and 48 ± 6 s-1 (n = 11) while the amplitudes were 21 ± 2 and 23 ± 3%, respectively. These rates had similar dependencies on final [Ca2+] achieved by photolysis: 43 and 50 s-1 per Pca unit, respectively, over a range of [Ca2+] producing from 15% to 90% of maximal isometric tension. At all [Ca2+], the rise in stiffness initially was faster than that of tension. The maximal rates for the slower components of the rise in stiffness and tension were 4.1 ± 0.8 and 6.2 ± 1.0 s-1. The rate of this slower phase exhibited significantly less Ca2+ sensitivity, 1 and 4 s-1 per Pca unit for stiffness and tension, respectively. These data, along with previous studies indicating that the force-generating step in the cross-bridge cycle of cardiac muscle is marginally sensitive to [Ca2+], suggest a mechanism of regulation in which Ca2+ controls the attachment step in the cross-bridge cycle via a rapid equilibrium with the thin filament activation state. Myosin kinetics sets the time course for the rise in stiffness and force generation with the biexponential nature of the mechanical responses to steps in [Ca2+] arising from a shift to slower cross-bridge kinetics as the number of strongly bound cross-bridges increases