Calcineurin Aβ-Specific Anchoring Confers Isoform-Specific Compartmentation and Function in Pathological Cardiac Myocyte Hypertrophy.

Background: The Ca2+ /calmodulin-dependent phosphatase calcineurin is a key regulator of cardiac myocyte hypertrophy in disease. An unexplained paradox is how the Aβ isoform of calcineurin (CaNAβ) is required for induction of pathological myocyte hypertrophy, despite calcineurin Aα expression in the same cells. In addition, it is unclear how the pleiotropic second messenger Ca2+ drives excitation-contraction coupling, while not stimulating hypertrophy via calcineurin in the normal heart. Elucidation of the mechanisms conferring this selectively in calcineurin signaling should reveal new strategies for targeting the phosphatase in disease. Methods: Primary adult rat ventricular myocytes were studied for morphology and intracellular signaling. New Forster Resonance Energy Transfer (FRET) reporters were used to assay Ca2+ and calcineurin activity in living cells. Conditional gene deletion and adeno-associated virus (AAV)-mediated gene delivery in the mouse were used to study calcineurin signaling following transverse aortic constriction in vivo . Results: Cdc42-interacting protein (CIP4/TRIP10) was identified as a new polyproline domain-dependent scaffold for CaNAβ2 by yeast-2-hybrid screen. Cardiac myocyte-specific CIP4 gene deletion in mice attenuated pressure overload-induced pathological cardiac remodeling and heart failure. Accordingly, blockade of CaNAβ polyproline-dependent anchoring using a competing peptide inhibited concentric hypertrophy in cultured myocytes, while disruption of anchoring in vivo using an AAV gene therapy vector inhibited cardiac hypertrophy and improved systolic function after pressure overload. Live cell FRET biosensor imaging of cultured myocytes revealed that Ca2+ levels and calcineurin activity associated with the CIP4 compartment were increased by neurohormonal stimulation, but minimally by pacing. Conversely, Ca2+ levels and calcineurin activity detected by non-localized FRET sensors were induced by pacing and minimally by neurohormonal stimulation, providing functional evidence for differential intracellular compartmentation of Ca2+ and calcineurin signal transduction. Conclusions: These results support a structural model for Ca2+ and CaNAβ compartmentation in cells based upon an isoform-specific mechanism for calcineurin protein-protein interaction and localization. This mechanism provides an explanation for the specific role of CaNAβ in hypertrophy and its selective activation under conditions of pathologic stress. Disruption of CaNAβ polyproline-dependent anchoring constitutes a rational strategy for therapeutic targeting of CaNAβ-specific signaling responsible for pathological cardiac remodeling in cardiovascular disease deserving of further pre-clinical investigation.