Kinetics of ADP Release From Cycling Cross Bridges In Contracting Skinned Cardiac Muscle Monitored With A Fluorescent Probe

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Kinetics of ADP release from cycling cross bridges were studied in Ca activated skinned papillary muscle by displacement of fluorescent ADP bound to the cross bridges in AM*ADP state(s) by non-fluorescent ADP photogenerated from caged ADP. A strip of glycerinated papillary muscle (100um, 2mm) from guinea pig left ventricular in Ca-free solution was loaded with a mixture of non-fluorescent ATP (1mM), fluorescent 3′-amino-deoxy ATP (aminoATP) (50μM) and 5 mM caged ADP in the presence of an ATP back-up system. At the plateau of force at pCa5.8 the muscle was rapidly transferred into the photolysis trough filled with silicone oil and irradiated by a 437 nm laser pulse. Alternatively, a muscle loaded with only fluorescent amino ATP (50μM) was allowed to contract at pCa4.5 in oil and develop rigor with amino ADP bound to the cross bridges. Following photolysis of caged ADP the kinetics of force and fluorescent transients were found markedly different in contracting and rigor muscles. In contracting muscle the force and fluorescence both increased following caged ADP photolysis, while in rigor muscle the photolysis induced an increase in fluorescence, but decrease in force. Kinetics of ADP release estimated by the rate of fluorescence increase was significantly slower in contracting muscle than that in rigor: 2-4 s-1 vs 18-20s-1, suggesting that at least two different AM*ADP states exist during ATP hydrolysis by cycling cross bridge in contracting papillary muscle. Supported by NIH grant R03 AR05 2885 for A.K.