Cardiovascular disease is the major cause of death in the United States. Regardless of the etiology the heart compensates mainly through myocyte hypertrophy in adaptation to chronic stress. Cardiac hypertrophy is induced by signaling through intracellular pathways composed of diffusable second messenger molecules such as cAMP and Ca2+, soluble enzymes, and anchored signaling complexes. The mAKAP signaling complex contains protein kinase A, a phosphodiesterase, a protein phosphatase, and the ryanodine receptor Ca2+-activated, Ca2+ channel. In this application we show that mAKAP also binds in a regulated manner the transcription factor NFATc1. Further, new data reveals that displacement of the mAKAP complex from its normal location at the nuclear envelope and inhibition of ryanodine receptors will block the induction of myocyte hypertrophy by cytokine agonists. We, therefore, propose a model in which upon agonist stimulation, ryanodine receptors in the mAKAP complex contribute to the de-phosphorylation and activation of NFATc1 by releasing Ca2+ that will activate the phosphatase calcineurin. De-phosphorylated NFATc factors will translocate to the nucleus and transactivate genes involved in hypertrophy. Upon calcineurin activation, NFATc1 will, in addition, be recruited into the mAKAP complex, where it may be rephosphorylated by protein kinase A in the complex. NFATc1 association with mAKAP may constitute a mechanism by which the induction of hypertrophy can be attenuated, preventing unrestrained hypertrophy. In this application three Specific Aims are proposed that test elements of this model and investigate the possible negative regulation of NFATc1 by the mAKAP complex.
In Specific Aim #1 the functional importance of protein kinase A, phosphodiesterase 4D3, ryanodine receptor and NFATc1 binding to the mAKAP complex is examined in primary myocyte cultures by expression of mAKAP forms that lack binding sites for individual components, by RNA interference, and by assessing the induction of myocyte growth, hypertrophic gene expression, and NFATc1 activity.
In Specific Aim #2, the composition of the NFATc1 bound mAKAP complex will be established and the binding domains permitting the mAKAP and NFATc1 interaction mapped.
In Specific Aim #3 we propose to examine whether mAKAP-associated NFATc1 is dephosphorylated and whether NFATc1 is a substrate for protein kinase A in the complex.
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