A current challenge in cellular signaling is to decipher the complex intracellular spatiotemporal organization that any given cell type has developed to discriminate among different external stimuli acting via a common signaling pathway. This obviously applies to cAMP and cGMP signaling in the heart, where these cyclic nucleotides determine the regulation of cardiac function by many hormones and neuromediators. Recent studies have identified cyclic nucleotide phosphodiesterases as key actors in limiting the spread of cAMP and cGMP, and in shaping and organizing intracellular signaling microdomains. With this new role, phosphodiesterases have been promoted from the rank of a housekeeping attendant to that of an executive officer.

Insight into the mechanisms underlying congenital heart defects and the use of stem cells for cardiac repair are major research goals in cardiovascular biology. In the early embryo, progenitor cells in pharyngeal mesoderm contribute to the rapid growth of the heart tube during looping morphogenesis. These progenitor cells constitute the second heart field (SHF) and were first identified in 2001. Direct or indirect perturbation of SHF addition to the heart results in congenital heart defects, including arterial pole alignment defects. Over the last 3 years, a number of studies have identified key intercellular signaling pathways that control the proliferation and deployment of SHF progenitor cells. Here, we review data concerning Wnt, fibroblast growth factor, bone morphogenetic protein, Hedgehog, and retinoic acid signaling that have begun to identify the ligand sources and responding cell types controlling SHF development. These studies have revealed the importance of signals from pharyngeal mesoderm itself, as well as critical inputs from adjacent pharyngeal epithelia and neural crest cells. Proliferation is emerging as a central checkpoint in the regulation of SHF development. Together, these studies contribute to defining the niche of cardiac progenitor cells in the early embryo, and we discuss the implications of these findings for the regulation of resident stem cell populations in the fetal and postnatal heart. Characterization of signals that maintain, expand, and regulate the differentiation of cardiac progenitor cells is essential for understanding both the etiology of congenital heart defects and the biomedical application of stem cell populations for cardiac repair.

CAMP is one of the most important second messenger in the heart. The discovery of Epac as a guanine exchange factor (GEF), which is directly activated by cAMP, raises the question of the role of this protein in cardiac cells. Here we show that Epac activation leads to morphological changes and induces expression of cardiac hypertrophic markers. This process is associated with a Ca ²⁺ -dependent activation of the small GTPase, Rac. In addition, we found that Epac activates a prohypertrophic signaling pathway, which involves the Ca ²⁺ sensitive phosphatase, calcineurin, and its primary downstream effector, NFAT. Rac is involved in Epac-induced NFAT dependent cardiomyocyte hypertrophy. Blockade of either calcineurin or Rac activity blunts the hypertrophic response elicited by Epac indicating these signaling molecules coordinately regulate cardiac gene expression and cellular growth. Our results thus open new insights into the signaling pathways by which cAMP may mediate its biological effects and identify Epac as a new positive regulator of cardiac growth.

Compartmentation of cAMP is thought to generate the specificity of G _s -coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca ²⁺ current ( I _Ca,L ) on stimulation with β ₁ -adrenergic receptors (β ₁ -ARs), β ₂ -ARs, glucagon receptors (Glu-Rs) and prostaglandin E ₁ receptors (PGE ₁ -Rs). All receptors but PGE ₁ -R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide–gated (CNG) channels, stimulation of β ₁ -AR and Glu-R increased cAMP, whereas β ₂ -AR and PGE ₁ -R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated β ₁ -AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE ₁ -R and β ₂ -AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE ₁ -R, the measurements of I _Ca,L closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled β ₁ -AR and β ₂ -AR regulation of I _Ca,L , whereas only PDE4 controlled Glu-R regulation of I _Ca,L thus demonstrating that receptor–PDE coupling has functional implications downstream of cAMP. PGE ₁ had no effect on I _Ca,L even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE ₁ to diffuse to L-type Ca ²⁺ channels. These results identify specific functional coupling of individual PDE families to G _s -coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.

Intracardiac cAMP levels are modulated by hormones and neuromediators with specific effects on contractility and metabolism. To understand how the same second messenger conveys different information, mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit CNGA2, encoded into adenoviruses, were used to monitor cAMP in adult rat ventricular myocytes. CNGA2 was not found in native myocytes but was strongly expressed in infected cells. In whole cell patch-clamp experiments, the forskolin analogue L-858051 (L-85) elicited a non-selective, Mg2+ -sensitive current observed only in infected cells, which was thus identified as the CNG current (ICNG). The beta-adrenergic agonist isoprenaline (ISO) also activated ICNG, although the maximal efficiency was approximately 5 times lower than with L-85. However, ISO and L-85 exerted a similar maximal increase of the L-type Ca2+ current. The use of a CNGA2 mutant with a higher sensitivity for cAMP indicated that this difference is caused by the activation of a localized fraction of CNG channels by ISO. cAMP-dependent protein kinase (PKA) blockade with H89 or PKI, or phosphodiesterase (PDE) inhibition with IBMX, dramatically potentiated ISO- and L-85-stimulated ICNG. A similar potentiation of beta-adrenergic stimulation occurred when PDE4 was blocked, whereas PDE3 inhibition had a smaller effect (by 2-fold). ISO and L-85 increased total PDE3 and PDE4 activities in cardiomyocytes, although this effect was insensitive to H89. However, in the presence of IBMX, H89 had no effect on ISO stimulation of ICNG. This study demonstrates that subsarcolemmal cAMP levels are dynamically regulated by a negative feedback involving PKA stimulation of subsarcolemmal cAMP-PDE.

To analyze individual steps of G_S-linked signaling in intact cells, we used fluorescence resonance energy transfer (FRET)-based assays for receptor-G protein interaction, G protein activation, and cAMP effector activation. To do so, we developed a FRET-based sensor to directly monitor G_S activation in living cells. This was done by coexpressing a Gα_s mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gβγ subunits and appropriate receptors in HEK293 cells. Together with assays for receptor activation and receptor-G protein interaction, it is possible to characterize large parts of the G_S signaling cascade. When A_2A-adenosine or β₁-adrenergic receptors are coexpressed with G_S in HEK293T cells, the receptor-G_S interaction was on the same time scale as A_2A receptor activation with a time constant of <50 ms. G_S activation was markedly slower and around 450 ms with similar kinetics following activation of A_2A- or β₁-receptors. Taken together, our kinetic measurements demonstrate that the rate of G_S activation limits initiation of G_S-coupled receptor signaling.