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

Figures

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  Fig. 1. Increase of cAMP levels after stimulation of 5-HT receptors in human atrial myocytes. a Schematics of the Epac1-camps sensor containing yellow (YFP) and cyan fluorescent proteins (CFP) as donor and acceptor fluorophores linked to a cyclic nucleotide-binding domain (CNBD) of Epac1. Binding of cAMP to this domain leads to a conformational change resulting in a decrease of FRET (Nikolaev et. al 2004; Nikolaev and Lohse 2006). b Representative image of human AF atrial myocyte (HAM) after 48 h of culture and transfected with the cytosolic cAMP-sensor Epac1-camps (top, transmitted light image; bottom, fluorescence image in the CFP channel). c Real-time measurement of cAMP levels in this HAM. The graph shows the time course of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) emissions in response to 5-HT (100 μM) and forskolin (+FSK, 10 μM). Increased cAMP binding to the sensor induces a decrease in fluorescence resonance energy transfer between CFP (excited at 440 ± 20 nm) and YFP visualized by a simultaneous increase in donor emission (CFP; blue squares) and a decrease in acceptor emission (YFP; yellow squares). Acquisitions were performed every 5 s. The…

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  Fig. 2. cAMP responses to 5-HT but not to FSK are reduced in peAF. Time courses of representative FRET signals indicating changes in cytosolic cAMP in 3 HAMs from patients with SR (a), paAF (b), and peAF (c) exposed to 100 μM 5-HT and in the continuous presence of 5-HT to 10 μM forskolin (FSK). d Summary of the results from panels a–c. Depicted are FRET responses to both 5-HT and forskolin in the presence of 5-HT (+FSK) in individual myocytes. Mean values ± SEM are indicated by the circles. n = number of myocytes/number of patients. *p < 0.05 vs. 5-HT in SR (one-way ANOVA; Tukey’s multiple comparisons test, based on myocytes); #p < 0.05 FSK vs. 5-HT (paired t test). Values for FSK were not significantly different between groups (one-way ANOVA; Tukey’s multiple comparisons test)

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  Fig. 3. Inhibition of PDE3 and PDE4 restores cAMP response to 5-HT in peAF. Time courses of the representative cAMP responses in 3 HAMs from patients in SR (a), paAF (b), and in peAF (c) exposed to 0.3 μM cilostamide and 1 μM rolipram (Cil + Rol). Summary of the results is in panel. d Depicted are FRET responses to Cil + Rol, to 100 μM 5-HT and to 10 μM forskolin in the presence of Cil + Rol and 100 μM 5-HT (+FSK) in individual myocytes. Mean values ± SEM are indicated by the circles. n = number of myocytes/number of patients. #p < 0.05 vs. the corresponding values for Cil + Rol alone. Values for FSK in the presence of Cil + Rol and 5-HT were not significantly different between groups (one-way ANOVA followed by Tukey’s multiple comparisons test)

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  Fig. 4. Inhibition of PDE3 and PDE4 increases cAMP levels upon 5-HT stimulation in peAF. Analysis of 5-HT responses in the absence (−PDEi) and in the presence of PDE inhibitors (0.3 μM cilostamide and 1 μM rolipram, +PDEi), indicating single-value cells based and mean values ± SEM patients based. n = number of myocytes/number of patients. *p < 0.05 vs. in the absence of PDE(−PDEi) in SR or paAF (one-way ANOVA, followed by Tukey’s multiple comparisons test); #p < 0.05 vs. respective values for −PDEi (non-parametric Mann-Whitney test)