Cardiac hypertrophy is the main compensatory response to stress in heart disease and a leading risk for the development of heart failure. In the adult heart, the serum response factor (SRF) transcription factor is an important regulator of myocyte gene expression, directing the transcription of genes encoding cytoskeletal and contractile proteins, calcium-handling molecules and factors controlling myocyte phenotype. In this application, I examine a novel mechanism for the control of the ER-RSK3-SRF pathway by the protein scaffold muscle A-kinase anchoring protein ? (mAKAP?). The Kapiloff lab has shown that mAKAP? is required in vitro for adrenergic and cytokine-induced neonatal myocyte hypertrophy. Preliminary Data suggest that mAKAP? anchored RSK3 is important for the phosphorylation of SRF S103 and induction of myocyte hypertrophy. In this application, I test the hypothesis that hypertrophic gene expression regulated by SRF is dependent upon SRF association with mAKAP? complexes. I propose to examine how SRF binding to mAKAP? facilitates RSK3-catalyzed SRF phosphorylation and activity and to test in vivo whether the mAKAP? scaffold is in fact required for concentric cardiac hypertrophy.
Specific Aim 1 : mAKAP?-SRF Signalosomes. mAKAP? and SRF can be co-immunoprecipitated using adult heart extracts. I propose to map the domain on mAKAP? that binds SRF to determine whether their interaction is direct. The mapping experiments are designed to identify a small peptide that can compete endogenous mAKAP?-SRF binding in cells, as well as to allow the construction of a full-length mAKAP? SRF-binding mutant In addition, using RNA interference and a currently available peptide that competes RSK3 binding to mAKAP?, I will test whether mAKAP? expression and mAKAP?-RSK3 binding is important for ?-adrenergic- induced SRF DNA binding and transcriptional activity.
Specific Aim 2 : The requirement for mAKAP? in pathological remodeling in vivo. I propose to stress a new cardiac-specific mAKAP? knock-out mouse by transverse aortic constriction and phenylephrine/angiotensin infusion via osmotic pump. I predict that in response to both pressure overload and neuroendocrine over-stimulation that the mAKAP? knock-out mouse will exhibit less concentric cardiac myocyte hypertrophy and decreased overall cardiac remodeling. I also predict that SRF transcriptional activity will be reduced upon genetic deletion The first Specific Aim will provide novel, detailed mechanistic insights into the proposed ERK-RSK3- SRF pathway, including the specific association of these molecules with mAKAP?. The second Specific Aim will show the physiologic relevance of the related scaffold protein. Together, my experiments should reveal a novel therapeutic approach for the prevention or treatment of heart failure, the targeting of critical scaffold proteins that provide the architectral infrastructure for the hypertrophic signaling network.
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.