The formation of multimolecular complexes called """"""""signalosomes"""""""" by A-kinase anchoring proteins (AKAPs) contributes to the spatial and temporal restriction of intracellular signaling by the second messenger cAMP. Targeting unique protein-protein interactions present within individual signalosomes may constitute a novel approach to drug discovery, yielding a new class of selective cardiac therapies displaying minimal off- target side-effects. One such signalosome is organized by mAKAPb, a scaffold protein that binds adenylyl cyclase 5, the cAMP-dependent enzymes protein kinase A and Epac1, and the cAMP-specific phosphodiesterase PDE4D3. By including all of the enzymes necessary for cAMP synthesis, degradation, and function, mAKAPb complexes may autonomously regulate and respond to locally controlled cAMP levels. mAKAPb signalosomes also contain ERK5 mitogen-activated protein kinase and the Ca2???? dependent phosphatase calcineurin Ab. Accordingly, the induction of neonatal myocyte hypertrophy in vitro by adrenergic and gp130 cytokine/leukemia inhibitory factor receptors is impaired by RNAi of mAKAPb expression. This application has three Specific Aims that address two central hypotheses: (1) that mAKAPb plays a critical role in the regulation of cardiac remodeling in vivo, and (2) that the mAKAPb signalosome forms an autonomous cAMP signaling compartment whose disruption will result in changes both in local cAMP levels and overall myocyte phenotype.
Specific Aim 1 : The role of mAKAPb in cardiac remodeling. The in vivo relevance of the mAKAPb scaffold to pathologic remodeling will be tested in mice using a new """"""""floxed"""""""" mAKAP allele. The mAKAP gene will be deleted using a cardiac-specific cre transgene, and both unstressed, aging mice and mice subjected to chronic isoproterenol infusion, transverse aortic constriction and myocardial infarction will be studied.
Specific Aim 2 : Regulation of AC5 by mAKAPb Complexes. AC5 directly binds to a N-terminal domain in mAKAPb. How AC5 activity is regulated by binding mAKAPb will be investigated in vitro and in vivo using a novel transgenic mouse in which an AC5-binding peptide is conditionally expressed in the heart.
Specific Aim 3 : Control of local cAMP levels by the mAKAPb signalosome. mAKAPb signalosome regulation of local cAMP levels in living cells will be investigated by the expression in cultured adult and neonatal cardiac myocytes of cAMP FRET sensors fused to mAKAPb. Signals obtained with a wildtype mAKAPb fusion sensor will be compared to that obtained using mAKAPb sensors lacking binding sites for individual binding partners, thereby revealing how the disruption of an individual scaffold protein complex affects intracellular signaling. Data obtained by these Specific Aims should establish the mAKAPb signalosome as an important node in the hypertrophic signaling network and as a candidate target for specific drug therapy for maladaptive remodeling and the prevention of heart failure.
Heart failure is a syndrome of major public heath significance accountable for nearly 300,000 deaths each year. It is estimated that 5.7 million US citizens suffer from heart failure, with nearly 670,000 new cases diagnosed annually. A better understanding of the cellular mechanisms that control cardiac remodeling, including myocyte hypertrophy, may yield better therapeutic regimens with decreased mortality.
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