The Ca2+/calmodulin-dependent phosphatase calcineurin (CaN, PP2B) is a pleiotropic signaling enzyme important for the regulation of cardiac hypertrophy. While CaN inhibition will attenuate pathological cardiac remodeling, therapeutic targeting of CaN is problematic as clinically established CaN inhibitors are immunosuppressant and as CaN targeting can worsen myocardial injury due to ischemia/reperfusion. In theory, however, if CaN signaling pathways mediating pathological cardiac hypertrophy could be targeted in isolation, a new therapeutic paradigm could be established. Two isoforms of CaNA, α and β, are expressed as proteins equally in the heart, and yet genetic targeting of CaNAβ (PPP3cb) is sufficient to blunt pathological hypertrophy in mice. We recently discovered that CaNAβ2 is targeted through its unique N-terminal polyproline (PP) domain to a myocyte compartment organized by the scaffold protein Cdc4-Interacting Protein 4 (CIP4, TRIP10). Imaging using Forster Resonance Energy Transfer (FRET) reporters revealed that CIP4-bound CaNAβ2 is activated by G-protein coupled receptor signaling, including angiotensin II, α- and β-adrenergic receptors, but not by pacing that induces myocyte contraction. Notably, both CIP4 gene targeting and adeno-associated virus-mediated PP- domain anchoring disruption inhibited cardiac remodeling and improved cardiac function in response to pressure overload. Thus, CaNAβ PP-dependent anchoring constitutes a novel mechanism for specification of CaN function, providing an explanation for the specific role of CaNAβ in the cardiac myocyte, as well as for the important question why hypertrophic CaN signaling is not active in normal contracting myocytes. In this project, we will test the novel hypothesis that in the cardiac myocyte PP-domain anchoring and CIP4 compartmentation confer CaNAβ2 action selectively promoting pathological cardiac hypertrophy, thereby comprising a new therapeutic target for the prevention and/or treatment of heart failure. Specific Aim 1: Localization and function of the CIP4 signaling compartment. This Aim will define CIP4 localization in the myocyte, study the relevance of the various CIP4 domains for myocyte hypertrophy, and explore whether CIP4-associated CaNAβ2 acts locally or distally from CIP4 complexes to promote hypertrophy. Specific Aim 2: Regulation of Ca2+ in the CIP4-CaNAβ2 compartment. Functional independence of a Ca2+ signaling compartment requires mechanisms for both local influx of Ca2+ and for insulating the compartment against elevations in Ca2+ associated with other cellular functions. In this Aim, we will define the source of Ca2+ and how this compartment is insulated using live myocyte imaging. Specific Aim 3: PP-anchored CaNAβ and ischemic heart disease. This aim will test whether targeting of PP-anchored CaNAβ can attenuate the development of heart failure without worsening myocyte survival in ischemic heart disease, These Aims will elucidate how PP-domain-mediated anch...