Cyclic AMP (cAMP) is a familiar second messenger that controls essential functions (including gene expression) in all cells. In the cardiovascular system, cAMP regulates many physiological processes such as contractility and relaxation. Congestive heart failure is associated with aberrations in cAMP/PKA signaling. Therapeutic treatments using beta-adrenergic receptor agonists (e.g. Xamoterol) or phosphodiesterase inhibitors (e.g. Milrinone, Rolipram) that increase intracellular cAMP are effective in increasing myocardial contractility in the short-term, but their chronic use has many serious adverse effects. These poorly understood long-term actions might ultimately be explained by compensatory alterations in cAMP/PKA-dependent gene expression. It has now become clear that cAMP metabolism and PKA-dependent phosphorylation can be regulated independently in different compartments of the cell. One such cAMP signaling microdomain is possibly represented by the nucleus, which recently was shown to contain all of the components of the cAMP signaling pathway (a soluble adenylyl cyclase, PKA holoenzyme and the transcription factor CREB). Here we hypothesize that nuclear cAMP signals are linked to the control of particular programs of gene expression. Long-term treatment with cAMP elevating agents may result in alteration of this signaling microdomain, ultimately leading to attenuation of the CREB-mediated gene transcription. In order to determine the functions and specific effectors of the nuclear cAMP signal, we propose here to use a novel genetically encoded cAMP 'buffer' to perturb the nuclear cAMP microdomain in HEK293 cells and primary neonatal cardiac myocytes. The effects of the buffer on free cAMP levels, PKA-dependent phosphorylation, and CREB activation will be monitored in real-time in single living cells using a panel of FRET-based biosensors. We will integrate these observations with RT-PCR on cell populations, in order to understand the effects of nuclear cAMP signals on the transcriptional regulation of the cAMP regulated genes. These proposed studies will allow us to determine whether the nuclear cAMP microdomain is subject to independent and unique modes of regulation. This might potentially explain the paradoxical responses to cAMP-elevating drugs during congestive heart failure.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||07/01/2008 → 06/30/2010|