Phosphodiesterase 8 governs cAMP/PKA-dependent reduction of L-type calcium current in human atrial fibrillation: a novel arrhythmogenic mechanism.

AIMS: Atrial fibrillation (AF) is associated with altered cAMP/PKA signaling and an AF-promoting reduction of L-type Ca2+-current (ICa,L), the mechanisms of which are poorly understood. Cyclic-nucleotide phosphodiesterases (PDEs) degrade cAMP and regulate PKA-dependent phosphorylation of key calcium-handling proteins, including the ICa,L-carrying Cav1.2α1C subunit. The aim was to assess whether altered function of PDE type-8 (PDE8) isoforms contributes to the reduction of ICa,L in persistent (chronic) AF (cAF) patients. METHODS AND RESULTS: mRNA, protein levels, and localization of PDE8A and PDE8B isoforms were measured by RT-qPCR, western blot, co-immunoprecipitation and immunofluorescence. PDE8 function was assessed by FRET, patch-clamp and sharp-electrode recordings. PDE8A gene and protein levels were higher in paroxysmal AF (pAF) vs. sinus rhythm (SR) patients, whereas PDE8B was upregulated in cAF only. Cytosolic abundance of PDE8A was higher in atrial pAF myocytes, whereas PDE8B tended to be more abundant at the plasmalemma in cAF myocytes. In co-immunoprecipitation, only PDE8B2 showed binding to Cav1.2α1C subunit which was strongly increased in cAF. Accordingly, Cav1.2α1C showed a lower phosphorylation at Ser1928 in association with decreased ICa,L in cAF. Selective PDE8 inhibition increased Ser1928 phosphorylation of Cav1.2α1C, enhanced cAMP at the subsarcolemma and rescued the lower ICa,L in cAF, which was accompanied by a prolongation of action potential duration at 50% of repolarization. CONCLUSION: Both PDE8A and PDE8B are expressed in human heart. Upregulation of PDE8B isoforms in cAF reduces ICa,L via direct interaction of PDE8B2 with the Cav1.2α1C subunit. Thus, upregulated PDE8B2 might serve as a novel molecular mechanism of the proarrhythmic reduction of ICa,L in cAF.

Impact of phosphodiesterases PDE3 and PDE4 on 5-hydroxytryptamine receptor4-mediated increase of cAMP in human atrial fibrillation.

Atrial fibrillation (AF)-associated remodeling includes contractile dysfunction whose reasons are only partially resolved. Serotonin (5-HT) increases contractile force and causes arrhythmias in atrial trabeculae from patients in sinus rhythm (SR). In persistent atrial fibrillation (peAF), the force responses to 5-HT are blunted and arrhythmic effects are abolished. Since force but not arrhythmic responses to 5-HT in peAF could be restored by PDE3 + PDE4 inhibition, we sought to perform real-time measurements of cAMP to understand whether peAF alters PDE3 + PDE4-mediated compartmentation of 5-HT4 receptor-cAMP responses. Isolated human atrial myocytes from patients in SR, with paroxysmal AF (paAF) or peAF, were adenovirally transduced to express the FRET-based cAMP sensor Epac1-camps. Forty-eight hours later, cAMP responses to 5-HT (100 µM) were measured in the absence or concomitant presence of the PDE3 inhibitor cilostamide (0.3 µM) and the PDE4 inhibitor rolipram (1 µM). We successfully established real-time cAMP imaging in AF myocytes. 5-HT increased cAMP in SR, paAF, and peAF, but in line with previous findings on contractility, this increase was considerably smaller in peAF than in SR or paAF. The maximal cAMP response to forskolin (10 µM) was preserved in all groups. The diminished cAMP response to 5-HT in peAF was recovered by preincubation with cilostamide + rolipram. We uncovered a significantly diminished cAMP response to 5-HT4 receptor stimulation which may explain the blunted 5-HT inotropic responses observed in peAF. Since both cAMP and force responses but not arrhythmic responses were recovered after concomitant inhibition of PDE3 + PDE4, they might be regulated in different subcellular microdomains.