Receptor-mediated changes in cAMP production play an essential role in sympathetic and parasympathetic 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 this study, we used a computational approach to test the hypothesis that in cardiac ventricular myocytes the effects of beta(1)-adrenergic receptor (beta(1)AR) and M(2) muscarinic receptor (M(2)R) activation involve compartmentation of cAMP. A model consisting of two submembrane (caveolar and extracaveolar) microdomains and one bulk cytosolic domain was created using published information on the location of beta(1)ARs and M(2)Rs, as well as the location of stimulatory (G(s)) and inhibitory (G(i)) G-proteins, adenylyl cyclase isoforms inhibited (AC5/6) and stimulated (AC4/7) by G(i), and multiple phosphodiesterase isoforms (PDE2, PDE3, and PDE4). Results obtained with the model indicate that: 1), bulk basal cAMP can be high ( approximately 1 microM) and only modestly stimulated by beta(1)AR activation ( approximately 2 microM), but caveolar cAMP varies in a range more appropriate for regulation of protein kinase A ( approximately 100 nM to approximately 2 microM); 2), M(2)R activation strongly reduces the beta(1)AR-induced increases in caveolar cAMP, with less effect on bulk cAMP; and 3), during weak beta(1)AR stimulation, M(2)R activation not only reduces caveolar cAMP, but also produces a rebound increase in caveolar cAMP following termination of M(2)R activity. We conclude that compartmentation of cAMP can provide a quantitative explanation for several aspects of cardiac signaling.

.-In cardiac myocytes there is evidence that activation of some receptors can regulate protein kinase A (PKA)-dependent responses by stimulating cAMP production that is limited to discrete intracellular domains. We previously developed a computational model of compartmentalized cAMP signaling to investigate the feasibility of this idea. The model was able to reproduce experimental results demonstrating that both ␤ 1-adrenergic and M2 muscarinic receptor-mediated cAMP changes occur in microdomains associated with PKA signaling. However, the model also suggested that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the present study we tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transferbased biosensor constructed from the type 2 exchange protein activated by cAMP (Epac2-camps). It was determined that in adult ventricular myocytes the basal cAMP concentration detected by the probe is ϳ1.2 M, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both ␤1 and M2 receptor activation. Modeling suggests that responses detected by Epac2-camps mainly reflect what is happening in a bulk cytosolic compartment with little contribution from microdomains where PKA signaling occurs. These results support the conclusion that even though ␤1 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. This allows receptor stimulation to regulate cAMP activity over concentration ranges appropriate for modulating both higher (e.g., PKA) and lower affinity (e.g., Epac) effectors.␤-adrenergic receptor signaling; muscarinic receptor signaling; live cell imaging; fluorescence resonance energy transfer; biosensors MANY DIFFERENT NEUROTRANSMITTERS and hormones control a wide range of cellular processes by regulating the production of a common second messenger, cAMP (32). This signaling pathway plays a particularly important role in sympathetic regulation of cardiac function, where ␤ 1 -adrenergic receptor (␤ 1 AR) activation generates significant changes in the electrical, mechanical, and metabolic properties of the heart by stimulating the production of cAMP and activating protein kinase A (PKA) (4). Although other G protein-coupled receptors are also able to affect cAMP production in the heart, they do not all produce the same responses. This has led to the hypothesis that cAMP production is compartmentalized and that different receptors can regulate cAMP production in specific microdomains (31).The recent development of several different biosensors capable of monitoring cAMP activity in intact living cells has provided a means of obtaining more direct proof that compartmentation occurs (20,33,37). Some of these sensors are targeted to specific subcellular locations. This includes the fluorescence resonance energ...

1. beta(1)-Adrenoceptor and M(2) muscarinic receptor regulation of cAMP production plays a pivotal role in autonomic regulation of cardiac myocyte function. However, not all responses are easily explained by a uniform increase or decrease in cAMP activity throughout the entire cell. 2. Adenovirus expression of fluorescence resonance energy transfer (FRET)-based biosensors can be used to monitor cAMP activity in protein kinase A (PKA) signalling domains, as well as the bulk cytoplasmic domain of intact adult cardiac myocytes. 3. Data obtained using FRET-based biosensors expressed in different cellular microdomains have been used to develop a computational model of compartmentalized cAMP signalling. 4. A systems biology approach that uses quantitative computational modelling together with experimental data obtained using FRET-based biosensors has been used to provide evidence for the idea that compartmentation of cAMP signalling is necessary to explain the stimulatory responses to beta(1)-adrenoceptor activation as well as the complex temporal responses to M(2) muscarinic receptor activation.

phthalate (DEHP) is a widely used plasticizer found in a variety of polyvinyl chloride (PVC) medical products. The results of studies in experimental animals suggest that DEHP leached from flexible PVC tubing may cause health problems in some patient populations. While the cancerogenic and reproductive effects of DEHP are well recognized, little is known about the potential adverse impact of phthalates on the heart. This study used preparations of confluent, synchronously beating cultures of neonatal rat cardiomyocytes to examine possible adverse effects of clinically relevant concentration of DEHP on cardiac tissue. Seventy two hour-long exposure to 50 mg/ml DEHP led to a marked decrease in conduction velocity and asynchronous cell beating in DEHP-treated samples but not in time-matched controls. The mechanism behind DEHP-induced changes was a loss of junctional connexin-43, documented using western blot analysis, dye-transfer assay and immunofluorescence. Use of organelle-specific connexin-43 antibodies, IF1 and CT1, allowed for further analysis of changes in intracellular distribution of connexin-43. In DEHPtreated samples the amount of gap-junctional connexin-43 (IF1-sensitive) was significantly decreased as compared to the controls. In contrast, Golgi and perinuclear (CT1-sensitive) staining was more pronounced. The data suggests that DEHP modifies connexin-43 trafficking and protein assembly into functional gap junctions, which impairs the electrical behavior of a cardiac cell network. Applicability of these findings to human patients remains to be established.