The long term goal of the project is to elucidate the mechanism(s) underlying the length dependence of force production and Ca2+ sensitivity in cardiac muscle, the Frank-Starling relationship.
Specific aim #1 is to determine whether recruitment of the cross bridges is a principal mechanism of enhanced force generation and/or Ca2+-sensitivity at increased sarcomere length (SL) or whether a modulation in their cycling rate also contributes significantly. This will be achieved by a combined fluorescence and flash photolysis study of skinned trabeculae muscle. Using a novel fluorescent MgATP analog demonstrating about 14 fold change in fluorescence between its bound and free forms as a substrate for contraction, an excess of non fluorescent ADP will be photogenerated in the muscle to displace fluorescent ADP bound to cross bridges in AM*ADP state(s). The amplitude and the kinetics of the fluorescent transient due to displacement of fluorescent ADP related directly to, respectively, the number of cross bridges in the force generating state (AM*ADP) and the rate of ADP release (determinant of cross bridge cycling rate) will be determined and compared in the same cardiac muscle activated by maximal and submaximal (about EC50) [Ca2+] at different SL. Contribution of each parameter (amplitude or rate) will be determined both for the enhanced force production (at maximal [Ca2+]) and Ca2+-sensitivity (at submaximal [Ca2+]) at increased SL.
Specific aim #2 is to determine whether the interfilament distance (di) is the major determinant of the length dependence offeree production and/or Ca2+-sensitivity in the stretched cardiac muscle. The changes in the cross bridge parameters of force production (determined as in Specific aim#1) at maximal and submaximal (about EC50) [Ca2+] will be determined in muscle having osmotically reduced di at constant SL and compared with corresponding changes obtained at di decreased to the same extent by lengthening of SL. If the effects of decreasing di on the cross bridge parameters achieved by the two independent methods will be significantly different at each [Ca2+], this will strongly indicate that the decrease in di is not a sole determinant of the length dependence neither of force production nor enhanced Ca2+ sensitivity in cardiac muscle. These findings will provide new insights into fundamental mechanisms underlying the Frank-Starling relationship in normal and pathological states, such as in left ventricular diastolic disfunction. ? ? ? ?
