This application is for renewal of a grant that has been focused during eight years on the Ca2+ induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) of skinned (sarcolemma free) single cardiac cells. The following new working hypothesis will be tested: the small but rapid increase of myoplasmic [free Ca2+] mostly caused by the initial spike of the transsarcolemmal current (ICa) would not activate the myofilaments directly but through the induction of a Ca2+ release from the SR. The large increase of myoplasmic [free Ca2+] resulting from this CICR would both inactivate the CICR (a negative feedback explaining that this process is not all or none) and partially inactivate ICa causing it to load the SR with an amount of Ca2+ available for release during subsequent beats. The methods include the use of aequorin bioluminescence to detect Ca2+ release and a microprocessor-controlled system of extremely rapid (less than or equal to 5 ms) changes of solutions by aspiration and injection. The major categories of goals are: (1) improvements to this system and study of the newly found binding of aequorin within the cell; (2) definition of the ionic factors controlling activation and inactivation of CICR by changing free concentrations of Ca2+, Mg2+ and H+ during the course of a Ca2+ transient; (3) definition of the trigger for CICR by quantifying the relationship between the amount of Ca2+ release and (a) Delta[free Ca2+], (b) Delta[free Ca2+]/Deltat; (4) interpretation of the earliest detectable event of CICR which is observed with potential-sensitive dyes; (5) definition of the relationships between Ca2+ accumulation in the SR and CICR by studying the effects of changes of temperature, pH and free Mg2+, and substitution of Sr2+ for Ca2+ which are known to affect Ca2+ accumulation (Are the two processes related or is CICR through a channel controlled by time- and Ca2+ dependent activation and inactivation?); (6) study of the interactions between CICR and Ca2+ binding and release from the myofilaments by extracting their contractile and regulatory proteins and studying some aspects of the length dependence of activation; (7) attempt to reproduce the force-frequency relationships by stimulating the skinned cardiac cell with microprocessor-controlled simulated transsarcolemmal Ca2+ movements; (8) extension of the rationale and methods to skinned skeletal muscle fibers.
