Receptor-mediated changes in cAMP production play an essential role in hormone regulation of the electrical, mechanical, and metabolic activity of cardiac myocytes. However, responses to receptor activation cannot be easily ascribed to a uniform increase or decrease in cAMP activity throughout the entire cell. In the present study, we used a systems biology approach to investigate the role played by cAMP compartmentation in cardiac ventricular myocytes and the mechanisms underlying it.
In the first part of this study, we used a computational approach to test the hypothesis that in ventricular myocytes the effects of β1-adrenergic receptor (β1AR) and M2 muscarinic receptor (M2R) activation involve compartmentation of cAMP. Results obtained with the developed model indicate that (1) bulk basal cAMP can be high (~1 μM) and only modestly stimulated by β1AR activation (~2 μM), but caveolar cAMP varies in a range more appropriate for regulation of protein kinase A - PKA (~100 nM to ~2 μM); (2) M2R activation strongly reduces the β1AR-induced increases in caveolar cAMP, with less effect on bulk cAMP; and (3) during weak β1AR stimulation, M2R activation not only reduces caveolar cAMP, but also produces a rebound increase in caveolar cAMP.
The original model suggests that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the second part of this study we experimentally tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transfer (FRET)-based biosensor constructed from the type 2 exchange protein activated by cAMP. Our results support the conclusion that even though β1 and M2 receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell.
In the final part of the current study, we investigated the potential mechanisms responsible for cAMP compartmentation. We conclude that both limited cAMP diffusion between the submembrane and bulk compartments and an increased level of phosphodiesterase (PDE) activity in the membrane compartment are necessary for generating and maintaining the type of cAMP gradients previously characterized using intracellular FRET based cAMP probes.