Background: Cardiac arrhythmias, such as atrial fibrillation (AF), are often related to remodeling of membrane receptors and alterations in cAMP-dependent regulation of Ca²⁺ handling mechanisms. For instance, decreased L-type calcium current density but upregulated RyR2 are major hallmarks of AF. These inhomogeneous AF-associated changes of protein phosphorylation point to a local regulation of PKA activity within these intracellular compartments. Previous studies have shown varying physiological and cAMP-dependent effects upon stimulation of different G-protein-coupled receptors (GPCRs). However, up to date no study has elucidated the reason. The aim of this study was to investigate whether stimulation of different GPCRs in human atrial myocytes from patients in sinus rhythm (SR) leads to different cAMP signaling into different cellular compartments and how AF affects these compartmentalized effects.

Methods: Atrial myocytes were isolated from tissues of 130 patients in SR or with AF. Cells were then transfected with adenovirus (Epac1-camps, pm-Epac1-camps and Epac1-JNC) in order to express the (cytosolic, membrane or RyR2 respectively) FRET-based cAMP sensor and cultured for 48 hours. Förster-resonance energy transfer (FRET) was then used to measure cAMP in 464 isolated human atrial myocytes. Stimulation with β-adrenergic agonist Isoprenaline (ISO, 100 µM) was used and compared with Serotonin (Ser, 100mM) and A2ARs (with CGS, 30mM) stimulation.

Results: In SR ISO stimulation promotes a bigger increase in cytosolic cAMP than Ser and CGS. AF promotes cytosolic desensitization to β-adrenergic and serotonin stimulation and promotes dysregulation on the control of the cAMP levels in a compartment-dependent manner. However, while the cAMP increase is smaller in AF than in SR upon Ser stimulation in the sarcolemma, ISO effects are bigger in AF in this compartment. Furthermore, although CGS promoted no changes on sarcolemmal cAMP levels, it significantly increases cAMP levels in RyR2 microdomain, especially in AF. Nevertheless, the effect of ISO and Ser into this compartment was smaller in AF than in SR.

Conclusions: Collectively, our data show that cAMP levels are highly compartmentalized and differentially regulated by different GPCRs. Furthermore, the β-adrenergic pathway suffers a profound remodeling in myocytes from patients with atrial fibrillation. In AF stimulation with Isoprenaline seems to have a decreased effect on cytosolic cAMP and the RyR2 microdomains, contrary to the larger effect observed in cAMP levels on the LTCC. By contrast, in AF stimulation with CGS increased cAMP levels more than in SR in the RyR2 microdomain.