Work proposed in this application addresses specific hypotheses regarding the fall-off of tension in myocardium at short lengths. Length-dependence of isometric tension will be assessed in single skinned myocytes from rat ventricles by measuring tension- pCa relationships as a function of sarcomere length. Further experiments will test the hypothesis that the kinetics of force-generating transitions in the cross-bridge interaction cycle are slowed at short lengths, which would contribute to reduced twitch tension in living myocardium. Kinetics of force development will be assessed by measuring the rate of tension development under conditions of steady activation and by photolyzing caged Pi in order to probe specific force-generating steps in the cross-bridge cycle. Osmotic compression of myocytes will determine whether length-dependent changes in tension or the rate of rise of tension are due to changes in lateral separation of thick and thin filaments. Experiments will also test the hypothesis that changes in length and filament lattice spacing affect activation of mechanical properties by modulating the probability of strong binding of cross-bridges to the thin filament, thereby modulating cooperative activation of additional binding and the kinetics of cross- bridge binding. These studies will measure mechanical properties, first in the absence and then in the presence of N-ethylmaleimide-conjugated myosin sub-fragment-1, a derivative that forms long-lived, strongly- bound complexes with the thin filament. Since Ca2+-sensitivity of tension may vary less with length in skeletal muscle, comparative measurements on single skinned fibers from skeletal muscles will provide interpretive insight into underlying mechanisms of length-dependent activation.
