Mechanisms of Cardiac Ryanodine Receptor Modulation by Phosphorylation The cardiac ryanodine receptor (RyR2) constitutes the main pathway for Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling of the heart. The force of cardiac contraction is greatly dependent on the magnitude of Ca2+ release by RyR2 channels: during periods of stress or in the setting of exercise, β-adrenergic stimulation of ventricular myocytes increases Ca2+ release and thus force of contraction. In isolated, beating hearts stimulated with β-adrenergic agonists, RyR2 channels are among the first proteins to undergo metabolic phosphorylation. This suggests that RyR2 channels are an integral part of the concert of events by which β-adrenergic receptors produce inotropic and lusitropic effects on the heart. Yet, the functional effect of RyR2 phosphorylation remains elusive. Wide ranges of importance have been attributed to RyR2 phosphorylation, from functionally inconsequential to a central mechanism in several cardiomyopathies. This proposal aims to elucidate the mechanisms underlying regulation of RyR2 by phosphorylation. We have gathered evidence to postulate that the present model of RyR2 phosphorylation, where one kinase (PKA or CaMKII) phosphorylates one RyR2 phosphoepitope (Ser2808 or Ser2814, respectively) and produces one discrete effect, is overly simplified and leads to erroneous assumptions. Instead, we propose a multi-site, bimodal and interactive model of RyR2 phosphorylation where Ser2808 and Ser2814 (and possibly Ser2811) all form part of a single signaling node (“phosphorylation hotspot”) that works in concert to modulate RyR2 gating in a graded manner. Our model also considers the emergent role of Ser2030, a PKA site, in the control of RyR2 modulation by phosphorylation, and the active contribution of protein phosphatases PP1 and PP2a to this process. We have generated key experimental animals and toolkits that will allow us to test with unprecedented integrative level the physiological and pathophysiological role of RyR2 phopshorylation and dephosphorylation in molecular, cellular, whole heart and intact animal settings. These studies are therefore likely to provide fresh, novel insight into the mechanisms that control RyR2 phosphorylation and the functional consequences of this process for the Ca2+ homeostasis of cardiac cells.