beta-adrenergic modulation of cardiac ryanodine receptor

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DESCRIPTION (provided by applicant): In the heart, a small influx of Ca2+ through L-type Ca2+ channels (DHPRs) triggers the opening of ryanodine receptors (RyRs), which in turn release massive amounts of Ca2+ from the sarcoplasmic reticulum (SR) by the process of Ca2+-induced Ca2+ release (CICR). The force of heart contraction is thus greatly dependent on the magnitude of Ca2+ release by RyRs; during periods of stress or in the setting of exercise, beta-adrenergic stimulation of ventricular myocytes increases Ca2+ release and thus force of contraction. Beta-adrenergic stimulation increases Ca2+ release by activation of PKA, which phosphorylates phospholamban, thereby relieving its inhibition on the SR Ca 2 pump (SERCA) and increasing SR Ca2+ load. Higher SR Ca2+ load then leads to higher Ca2+ release. Substantial phosphorylation of RyRs is also seen upon beta-adrenergic stimulation, but the role of RyR phosphorylation in normal and diseased states remains controversial. This proposal seeks to establish the physiological impact of RyR phosphorylation, the molecular players involved in this reaction, and its implications for heart failure. Using a combination of phospho-specific antibodies against RyRs, beta-adrenergic stimulation of whole hearts, cellular Ca2+ imaging, and recording of single RyR activity under quasi-physiological conditions, we propose: 1) To identify the protein kinase(s) activated by beta-adrenergic stimulation that phosphorylate RyRs, their specific phosphorylation site(s) in the RyR sequence, and their extent of participation under physiological conditions. 2) To determine the functional output of specific phosphorylation sites in the RyR, by directly recording wild type and mutant RyR channel activity under quasi-physiological conditions. 3) To establish the role of RyR phosphorylation and their counterpart reaction, RyR dephosphorylation, as a pathogenic mechanism for the blunted Ca2+ release characteristic of heart failure. These studies are likely to provide fresh, novel insight into the mechanisms that control RyR phosphorylation and the functional consequences of this process for the Ca2+ homeostasis of cardiac cells. Thus, they may help resolve the controversial role of RyR phosphorylation in the pathogenesis of heart failure.