The development of pathological cardiac hypertrophy requires the stimulation of gene transcription activated by Ca2+-dependent signaling pathways. However, targeting specifically these Ca2+-dependent pathways is difficult due to the multiple functions of Ca2+ in myocyte physiology. Recent evidence suggests that formation of distinct Ca2+ microdomains provide the molecular mechanism that allows for specificity in Ca2+ signaling. Therefore, understanding the components and regulation of each microdomain is key for the development of novel therapeutics for the prevention and treatment of pathological hypertrophy. We have previously demonstrated that binding of the Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN) to the muscle-specific A Kinase Anchoring Protein mAKAPβ mediates the induction of myocyte hypertrophy. New data show that the mAKAPβ signalosome is also required for a perinuclear Ca2+ transient required for the activation of transcription factors responsible for myocyte hypertrophy. We hypothesize that a pool of perinuclear RyR2 localized to the mAKAPβ signalosome is responsible for this Ca2+ microdomain, and importantly, that these perinuclear RyR2 are segregated from those involved in excitation-contraction coupling (E-C coupling). The central hypothesis of this proposal is that RyR2 localized to mAKAPb signalosomes induces perinuclear Ca2+ transients required for CaN-dependent gene expression that are independent of the canonical function of RyR2 in E-C coupling. Aim 1: Perinuclear RyR2 associated with mAKAPβ signalosomes are within an independent Ca2+ signaling compartment that regulates myocyte hypertrophy. The goal of Aim 1 is to demonstrate that mAKAPb- signalosome associated RyR2 is responsible for bAR-stimulated perinuclear Ca2+ transients that induce myocyte hypertrophy and that this pool of RyR2 is regulated independently from RyR2 involved in E-C coupling. Using novel, targeted activators and inhibitors of the perinuclear RyR2, we will demonstrate the importance of perinuclear RyR2 for the regulation of pathological gene transcription, and show that modulation of perinuclear RyR2 does not impact contractility. Furthermore, the importance of PKA-mediated phosphorylation of RyR2 at several sites will be investigated. Aim 2: The dimensions of the mAKAPβ Ca2+/CaN compartment. Aim 2 will map the perinuclear Ca2+ domain that is specified by the mAKAPb signalosome and demonstrate that this perinuclear compartment does not affect cytosolic CaN activity, but functions to maintain perinuclear CaN activity. Aim 3: Requirement of perinuclear RyR2 signaling for pathological remodeling in vivo. The therapeutic potential of targeting perinuclear RyR2 in mouse models of cardiac hypertrophy will be investigated in Aim 3. Through these Aims, this proposal will define a novel signaling compartment orchestrated by mAKAPb that is required for pathological gene transcription and induction of cardiac disease, but does not affect E-C co...