Year: 2007
Type: article
Abstract: Many hormones mediate their effects on the heart by triggering cyclic adenosine monophosphate (cAMP) signaling pathways in cardiomyocytes.However, not all the hormone-receptors that activate cAMP signaling pathways produce the same responses.While β 1 adrenergic receptors trigger a stimulatory cAMP signaling pathway, muscarinic receptor activation inhibits and stimulates cAMP-dependent effects.In another example, while prostaglandin PGE1 also enhances cAMP levels, it does not result in the functional responses associated with β 1 AR activation.To study the spatial and temporal regulation of cAMP in cardiomyocytes that could account for such disparate behavior, it is important to monitor real-time changes in cAMP and PKA activity in live cardiomyocytes.Recently developed genetically encoded cAMP biosensors based on fluorescence resonance energy transfer (FRET) have enabled such studies.These sensors consist of cAMP effectors, PKA and exchange protein activated by cAMP (Epac), tagged with fluorescent proteins CFP and YFP.Binding to cAMP affects the conformation of these recombinant proteins and are detected as changes in FRET.In this project, we examine the spatiotemporal nature of cAMP changes responsible for the disparate effects discussed above using x cardiac myocytes expressing PKA and Epac-based biosensors.Whole-cell Ltype calcium current was monitored as a functional response.First, we use the PKA-based biosensor to directly demonstrate the temporal pattern of cAMP changes produced by muscarinic receptor activation in cardiomyocytes.The biosensor was sensitive to physiological levels of β 1 AR signaling and had the same localization pattern as endogenous PKA which is targeted by binding to A kinase anchoring proteins (AKAPs).Thereafter, using the freely diffusible Epac-based biosensor or the AKAP-tethered PKA biosensor, we demonstrate that the difference in signaling between β 1 AR and PGE1 receptors is due to β1ARs stimulating cAMP production in cytosolic and caveolar compartments of intact cardiomyocytes, while PGE1 only stimulates cAMP production in the cytosolic compartment.Compartmentation of PGE1 responses was not due to activation of a G i -dependent pathway or phosphodiesterases.Instead it appears to be due to transient stimulation of cAMP in a microdomain that can communicate directly with the bulk cytosolic compartment but not the compartment associated with βAR regulation of L-type Ca 2+ channels.
Author: Sunita Warrier
Institution: Case Western Reserve University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W51290497