cAMP and cGMP signalling pathways are common targets in the pharmacological treatment of heart failure, and often drugs that modulate the level of these second messengers are simultaneously administered to patients. cGMP can potentially affect cAMP levels by modulating the activity of PDEs (phosphodiesterases), the enzymes that degrade cyclic nucleotides. This biochemical cross-talk provides the means for drugs that increase cGMP to concomitantly affect cAMP signals. Recent studies using FRET (fluorescence resonance energy transfer) reporters and real-time imaging show that, in cardiac myocytes, the interplay between cGMP and cAMP has different outcomes depending on the specific location where the cross-modulation occurs. cGMP can either increase or decrease the cAMP response to catecholamines, based on the cyclase that generates it and on the PDEs associated with each subcellular compartment. cGMP-mediated modulation of cAMP signals has functional relevance as it affects protein phosphorylation downstream of protein kinase A and myocyte contractility. The physical separation of positive and negative modulation of cAMP levels by cGMP offers the previously unrecognized possibility to selectively modulate local cAMP signals to improve the efficacy of therapy.
cAMP and cGMP are intracellular second messengers that mediate the activity of several neurotransmitters, hormones and NO (nitric oxide). cAMP is synthesized by adenylate cyclases upon activation of GPCRs (G-protein-coupled receptors). cGMP is generated by the stimulation of the soluble (s) and the particulate (p) GC (guanylate cyclase) by NO and natriuretic peptides respectively. These cyclic nucleotides control key physiological processes in the cardiovascular system. cAMP regulates the force and frequency of cardiac contraction via activation of PKA (protein kinase A), whereas cGMP promotes vasorelaxation and reduces cardiac contractility (inotropy) via mechanisms classically attributed to the activation of PKG (protein kinase G) . Key regulators of the physiological response to cAMP and cGMP signalling are PDEs (phosphodiesterases), the enzymes that degrade cyclic nucleotides. PDEs belong to a large superfamily of proteins encoded by 21 genes and including more than 80 transcription variants grouped in 11 families (PDE1–PDE11) . PDE isoforms are characterized by different intracellular localization and distinctive regulatory mechanisms . Cardiac myocytes express several PDE isoforms: PDEs 1, 2, 3, 4, 5, 8 and 9 . Of these, PDEs 1, 2 and 3 can degrade both cAMP and cGMP and are regulated by cGMP . The interplay between cAMP and cGMP is of particular interest in the cardiovascular system, where cAMP and cGMP signals are concomitantly generated to regulate cardiac function. The cross-talk between these two signalling pathways has important implications for the therapeutic effects of common treatments of cardiac disease. For example, a combination of inotropic drugs that increase cAMP levels and nitrates and vasodilators that increase cGMP levels may be simultaneously administered to patients suffering from heart failure. Understanding how these two signalling pathways are intertwined and how the interplay between cAMP and cGMP may affect cardiac pathophysiology is therefore of critical importance.
PDE2 and PDE3: integration of cAMP and cGMP signals
The dual-specificity enzymes PDE2 and PDE3 degrade both cAMP and cGMP, albeit with different affinity and catalytic rate [5,6]. Their hydrolytic activity in living cells is dictated by the physiological concentrations of cAMP and cGMP. The enzymatic activity of PDE2 is regulated by the GAF-B domain (GAF domains are found in mammalian cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases and Escherichia coliformate hydrogen lyase transcription activator FhlA), an allosteric binding site for cGMP located at the N-terminus of the protein. Binding of cGMP to the GAF-B domain of PDE2 increases its cAMP-degrading activity approximately 10-fold . Therefore cGMP-elevating agents can oppose the physiological effects of cAMP via activation of PDE2. Modulation of cAMP signals by cGMP via this mechanism has been shown both in rabbit atrioventricular nodal cells, where the activation of PDE2 via NO–cGMP mediates the muscarinic cholinergic attenuation of L-type Ca2+ current induced by isoprenaline (isoproterenol) , and in rat neonatal ventriculocytes, where the stimulation of sGC with NO donor has been shown to attenuate β-adrenergic stimulation via PDE2 activation . In marked contrast with the effect of cGMP on PDE2 activity, cGMP acts as a competitive inhibitor for PDE3. PDE3 has similar affinity for cAMP and cGMP, but, because of the higher catalytic rate for cAMP than for cGMP, PDE3 is inhibited by cGMP . Through this mechanism, signals that increase cGMP levels can increase cAMP levels. For instance, in cardiac sinoatrial cells, the inhibition of PDE3 potentiates the delayed rectifier K+ currents via activation of the cAMP–PKA system  and in frog ventricular myocytes, L-type Ca2+ channel currents are potentiated via a mechanism that involves sGC-mediated cGMP generation and subsequent inhibition of PDE3 .
Interestingly, the impact of cGMP signals on cAMP appears to be dependent on the intracellular concentrations of cGMP. At low concentrations (less than 50 nM), cGMP exclusively inhibits PDE3, whereas at higher concentrations (between 200 and 500 nM), cGMP also activates PDE2 . The biphasic effect of cGMP on cAMP levels was shown in human atrial myocytes, where low concentrations of cGMP potentiates the L-type Ca2+ channel currents via inhibition of PDE3, whereas higher concentrations of cGMP reduce such currents via PDE2 activation .
Although evidence suggested that cGMP concentration can dictate, to an extent, the amplitude of cAMP signals, how the opposing effects of cGMP on PDE2 and PDE3 are balanced in the cell was not known. Recent findings now provide new insights and show that the interplay between cGMP and cAMP is subtly regulated at the subcellular level and that the location where the cross-regulation takes place plays a critical role in determining the functional outcome .
A FRET (fluorescence resonance energy transfer)-based approach to study cAMP compartmentalization
cAMP and cGMP are ubiquitous signalling molecules that regulate a plethora of cellular events via the activation of an intricate network of signalling pathways operating through the activity of their two main effectors, PKA and PKG respectively. These cyclic nucleotides are small and hydrophilic molecules which, in principle, can freely diffuse in the cytosol, raising the question of how they can mediate so many different functional effects in a specific manner. To attain specificity of response, mechanisms must be in place that allow for individual pathways to be activated selectively in response to a given stimulus. The recent development of approaches to directly visualize cAMP and cGMP in living cells has provided a better understanding of the mechanisms that allow parallel and spatially segregated cyclic nucleotide signalling pathways to coexist within the cell. By using genetically encoded FRET-based sensors for cAMP  and cGMP , it is possible to monitor cyclic nucleotide signals with high spatial and temporal resolution and to capture this information in the exact cellular environment in which these signalling events occur [17,18]. Using these tools, it was demonstrated that, in neonatal cardiac myocytes, cAMP is compartmentalized and that activation of β-adrenergic receptors increases cAMP in discrete subcellular locations , whereas prostaglandin receptor activation increases cAMP levels in different subcellular compartments . Importantly, individual microdomains of cAMP are spatially restricted by the activity of localized PDEs [19,21]. The use of FRET reporters targeted to specific subcellular compartments increased the spatial resolution of this approach, making it possible to establish functional coupling of individual PDE isoforms with selected cAMP pools [20,22].
cAMP–cGMP interplay in subcellular compartments
The main effector of cAMP, PKA, is a protein that exists as a tetramer composed of two catalytic (C) and two regulatory (R) subunits. Two distinct PKA isoforms are expressed in cardiac myocytes, PKA-RI and PKA-RII, which differ in their R subunits. These two isoforms possess different physical and biological properties and are spatially confined to specific subcellular domains via binding to AKAPs (A-kinase-anchoring proteins) [23,24], a family of functionally related proteins that tether PKA in proximity to its phosphorylation targets .
FRET-based sensors for cAMP targeted to either RI- or RII-selective AKAPs have been developed to monitor cAMP dynamics at the level of the subcellular domains where PKA-RI and PKA-RII normally reside . Using this approach, it was possible to show that, in rat neonatal cardiac myocytes, the two PKA isoforms are activated by cAMP pools generated by different GPCRs . β-Adrenergic receptors mainly activate PKA-RII and trigger phosphorylation of TnI (troponin I), PLB (phospholamban) and the β2-adrenoreceptor, whereas stimulation of the prostaglandin receptor activates PKA-RI and does not affect the phosphorylation of the same targets .
More recently, by using cGMP sensors that had also been targeted to RI- and RII-selective AKAPs, the cross-talk between cAMP and cGMP signalling was investigated . These studies demonstrated that cGMP profoundly modulates the cAMP pools that selectively activate PKA-RI and PKA-RII, albeit with different effects. The cGMP-mediated modulation of cAMP, in fact, depends both on the cyclase that generates cGMP and on the PDEs that are associated with each compartment. In catecholamine-stimulated cardiac myocytes, PDE2 activity appears to be mainly coupled to the PKA-RII compartment, whereas PDE3 activity is coupled to the PKA-RI compartment. When the impact of the activation of sGC and pGC on the cAMP generated by isoprenaline was examined, different outcomes were observed. Activation of sGC exerts opposite effects on cAMP signals in the PKA-RI and PKA-RII compartments. In the presence of cGMP, the cAMP pool that activates PKA-RI is increased via PDE3 inhibition, whereas the cAMP pool that activates PKA-RII is decreased via PDE2 activation. On the other hand, activation of pGC by atrial natriuretic peptide A shows a selective effect on the cAMP pool that activates the PKA-RII compartment, where the rise in cGMP blunts the response to catecholamine via activation of PDE2, whereas the cAMP response in the PKA-I compartment is not affected. In these experiments, the effects of cGMP on the PKA-RII compartment were abolished by displacement of the endogenous PDE2 by overexpression of a catalytically inactive version of the enzyme, suggesting that the specific intracellular localization of PDE2 is important to effect the cGMP–cAMP cross-talk. Modulation of cAMP levels by cGMP was also found to propagate downstream of the second messenger. FRET-based PKA activity reporters targeted to RI and RII compartments were used to measure the impact of cGMP signals on local PKA activity and it emerged that stimulation of sGC reduced PKA activity in the PKA-RII compartment while increasing PKA activity in the PKA-RI compartment. In contrast, activation of pGC selectively reduced PKA activity in the PKA-RII compartment without affecting the PKA-RI compartment. Importantly, activation of sGC also resulted in reduced phosphorylation of PLB and TnI and reduced myocyte contractility, effects that were shown to be PDE2-dependent.
Implications for heart pathophysiology
Heart failure is a severe and complex pathology that remains a leading cause of mortality in the Western world. Heart failure is characterized by reduced cardiac contractility and inability of the heart to sustain adequate organ perfusion and to provide oxygen and substrates to all tissues. Treatment is aimed at improving overall cardiac function and, depending on the type and gravity of the symptoms, relies on inotropes that raise cAMP levels and on nitrates and vasodilators that increase cGMP levels. β-Agonists and PDE3 inhibitors significantly improve the haemodynamic and clinical status of patients with decompensated hearts. However, it is clear that inotropic therapy in the long term has a negative impact on patients' survival. Paradoxically, long-term treatment with β-blockers was shown to be beneficial in chronic heart failure [26–28]. Whether an increase in cAMP is beneficial or detrimental in the failing heart remains to be established; however, it appears that any manipulation intended to alter cardiac contractility must be carefully tuned. It is possible to speculate that the positive or negative effects of cAMP very much depend on the compartments in which it is raised and that generating a global increase of cAMP may not be an appropriate therapeutic approach. Another interesting consideration is that nitrates and natriuretic peptides have been shown to blunt cardiac contractility [29–31], a side effect that might worsen the already serious conditions of a decompensated heart. Although such an effect has been normally ascribed to cGMP-mediated activation of PKG [32,33], an alternative explanation could be that the anti-inotropic effect of cGMP is secondary to cGMP-mediated activation of PDE2. This is an interesting possibility as pharmacological inhibition of PDE2 may be a way to achieve selective manipulation of cAMP levels only in those compartments coupled to positive inotropy without affecting other pathways that may lead to deleterious effects.
Treatment of heart failure remains a challenge in the clinic, and how to improve cardiac output without further complicating the clinical picture of the patient is a critical question. cAMP and cGMP signalling pathways are a common target in the treatment of this condition; however, their manipulation is often associated with unwanted side effects. The appreciation that positive and negative modulation of cAMP levels by cGMP occurs in physically distinct compartments and is effected by distinct PDEs may offer the possibility to develop novel strategies for a tailored and more effective therapy.
Signalling 2011: a Biochemical Society Centenary Celebration: A Biochemical Society Focused Meeting held at the University of Edinburgh, U.K., 8–10 June 2011. Organized and Edited by Nicholas Brindle (Leicester, U.K.), Simon Cook (The Babraham Institute, U.K.), Jeff McIlhinney (Oxford, U.K.), Simon Morley (University of Sussex, U.K.), Sandip Patel (University College London, U.K.), Susan Pyne (University of Strathclyde, U.K.), Colin Taylor (Cambridge, U.K.), Alan Wallace (AstraZeneca, U.K.) and Stephen Yarwood (Glasgow, U.K.).
The work described in the present paper was supported by the Fondation Leducq [grant number O6 CVD 02], the British Heart Foundation [grant number PG/07/091/23698] and the National Science Foundation/National Institutes of Health CRCNS (Collaborative Research in Computational Neuroscience) programme [grant number NIH R01 AA18060].