E/C COUPLING AND CALCIUM REGULATION IN ATRIAL MYOCYTES

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The overall goal of the proposed study is to characterize [Ca2+]i regulation and sarcoplasmic reticulum (SR) Ca2+ release mechanisms during excitation-contraction (e-c) coupling in mammalian atrial muscle. We will test the hypothesis that in atrial cells normal e-c coupling involves Ca2+ release from both junctional (j-SR) and non-junctional SR (nj-SR). However only release from j-SR is directly dependent on membrane voltage (i.e. via Ca2+ entering through voltage-gated Ca2+ channels triggering Ca 2+-induced Ca2+-release (CICR)), whereas release from nj-SR is triggered solely by diffusion of Ca2+ and CICR (by a mechanism similar to cardiac [Ca 2+]~ wave propagation). In atrial muscle the model of 'local control' of e-c coupling strictly applies only to release from j-SR. SR Ca2+ release is tightly regulated by a phosphorylation-dephosphorylation cycle for which ATP is produced glycolytically in the microdomain of the SR release channel. Metabolically-induced electromechanical and [Ca2+]1 transient alternans, a major risk factor for atrial arrhythmias, will provide a model system to study the dynamic regulation of e- c coupling by compartmentalized glycolytic ATP formation. The five major specific aims of the proposed research are: 1. Characterize the spatio-temporal properties of whole-cell [Ca2+]1-transients in mammalian atrial cells triggered by action potentials. 2. Test the validity of the local control model for e-c coupling in atrial cells and characterize the mechanisms of e-c coupling and Ca2+ release from j-SR and nj-SR. 3. Define quantitatively the properties of elementary events of Ca2+ release (Ca2+ sparks) from j-SR and nj-SR in atrial cells. 4. Characterize metabolically-induced [Ca2+]i alternans and its cellular mechanisms. 5. Define the role of compartmentalized glycolytic ATP production in modulating SR Ca2+ release and CICR. To achieve these aims a multitude of experimental techniques will be used, including high resolution [Ca2+]i imaging by laser scanning confocal microscopy in single atrial myocytes, whole- cell voltage clamp techniques to study membrane currents, single channel recordings through cardiac SR Ca2+ release channels reconstituted into planar lipid bilayers, subcellular photolysis of caged Ca2+ by 2-photon excitation, and pharmacological manipulation of Ca2+ entry, release and uptake. The proposed research will provide fundamental new information on atrial e-c coupling and Ca2+ release under normal and altered conditions relevant to atrial arrhythmias.