The long term goal of this project is to understand the regulation of cardiac muscle contraction, particularly E-C coupling and the regulation of Ca fluxes. Isolated cardiac myocytes will be used with whole cell voltage clamp and the intracellular Ca indicator, indo-1. Experiments are also planned using multicellular preparations and isolated membrane vesicles. The specific goals for the next period will be to: a) Critically evaluate intracellular calibrations of indo-1 fluorescence signals rather than rely on in vitro calibrations which are likely to be inaccurate. This will allow several important quantitative issues to be addressed (e.g. direct assessment of the pCai vs tension relation in intact cardiac muscle). b) Measure intracellular Ca buffering by measuring Ca entry, Ca extrusion and [Ca]i, providing direct information about the overall Cai required for cardiac muscle activation. c) Estimate """"""""normal"""""""" and maximal SR Ca content in different species & conditions (e.g. in """"""""Ca overload""""""""). d) Determine the fraction of SR Ca released during normal and modified contractions. e) Evaluate if Em itself can alter SR Ca release at constant SR Ca load and constant ICa """"""""trigger"""""""". f) Evaluate whether Ca entry via Na/Ca exchange can induce SR Ca release. These two studies (e & f) are important in the evaluation of postulated mechanisms of E-C coupling. g) Determine the influence of Nao on Cai transients and assess the competition between the SR and Na/Ca exchange during relaxation (and [Ca]i decline). h) Characterize Em-dependent Ca binding at the inner sarcolemmal surface which could be involved in E-C coupling since depolarization may release Ca bound to the inner sarcolemmal surface. i) Evaluate if indo-1 alters sarcolemmal Na/Ca exchange or SR Ca-ATPase complicating the use of indo-1. These studies are focused on providing important new fundamental information of a quantitative nature about the regulation of intracellular Ca and the mechanism of E-C coupling in cardiac muscle cells (e.g. force vs pCai, Cai-buffering, SR Ca content & fractional release, role of Em, Na/Ca exchange and SR in E-C coupling and relaxation). They will also provide useful new information about the use of indo-1 in cardiac muscle. Cellular Ca fluxes are in a dynamic, yet delicate balance, which will be studied in detail. The consequences of disturbing this balance can include contractile failure, spontaneous contractions (i.e. arrhythmogenic) and impair relaxation. Any of these dysfunctions will compromise the ability of the heart to function effectively as a pump. Thus, comprehensive understanding of Ca regulation in the heart is crucial.
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