The mechanism of excitation-contraction coupling in cardiac muscle is generally assumed to involve Ca2+-induced Ca2+ release, while that in skeletal muscle remains for the time being an enigma. Numerous mechanisms have been proposed to suggest how depolarization of the surface and transverse tubule membranes could cause the release of Ca from muscle's internal stores, the sarcoplasmic reticulum (SR). Certain of these mechanisms studied in some detail with isolated SR and with skinned muscle fibers have been discredited or dismissed, but none have been rigorously proven incorrect and certainly none have been conclusively implicated as being involved, except for Ca induced Ca release in cardiac muscle. The current proposal details a systematic interdisciplinary approach utilizing pharmacologic tools to determine which mechanisms account for both skeletal and cardiac muscle excitation- contraction coupling.

Previous work has determined conditions appropriate to reproduce in vitro a number of model mechanisms of Ca release from isolated so (those elicited in response to Ca elevations, SR "depolarization", alkalinization, and SH oxidation, among others). We have identified specific lnhibitors which can be used to distinguish between each of these forms of Ca release.

In the present application we intend to introduce these specific inhibitors inside voltage clamped cut skeletal muscle fibers and isolated cardiac myocytes in order to test their effects on excitation-contraction (E-C) coupling in situ. Substances that are demonstrated to enter the muscle fibers or myocytes but fail to affect E-C coupling will indicate that the in vitro mechanisms of SR Ca release they block are not involved physiologically in normal muscle contraction. Substances that do affect E-C coupling in the appropriate concentration range without effects on a postulated prior step in E-C coupling. Voltage dependent charge movement in the t-tubule membrane, will demonstrate that the mechanisms they block in vitro do participate in E-C coupling. These investigations, therefore, will extend previous studies identifying inhibitors of specific mechanisms of Ca release from isolated muscle SR to the definitive identification of the mechanisms involved in E-C coupling in skeletal and cardiac muscle.

R01HL042527

1989-05-01

1994-04-30