Background: Phosphodiesterases (PDE) critically regulate myocardial cAMP and cGMP levels. PDE2 is stimulated by cGMP to hydrolyze cAMP, mediating a negative crosstalk between both pathways. PDE2 upregulation in heart failure contributes to desensitization to β-adrenergic overstimulation. After isoprenaline (ISO) injections, PDE2 overexpressing mice (PDE2 OE) were protected against ventricular arrhythmia. Here, we investigate the mechanisms underlying the effects of PDE2 OE on susceptibility to arrhythmias. Methods: Cellular arrhythmia, ion currents, and Ca2+-sparks were assessed in ventricular cardiomyocytes from PDE2 OE and WT littermates. Results: Under basal conditions, action potential (AP) morphology were similar in PDE2 OE and WT. ISO stimulation significantly increased the incidence of afterdepolarizations and spontaneous APs in WT, which was markedly reduced in PDE2 OE. The ISO-induced increase in ICaL seen in WT was prevented in PDE2 OE. Moreover, the ISO-induced, Epac- and CaMKII-dependent increase in INaL and Ca2+-spark frequency was blunted in PDE2 OE, while the effect of direct Epac activation was similar in both groups. Finally, PDE2 inhibition facilitated arrhythmic events in ex vivo perfused WT hearts after reperfusion injury. Conclusion: Higher PDE2 abundance protects against ISO-induced cardiac arrhythmia by preventing the Epac- and CaMKII-mediated increases of cellular triggers. Thus, activating myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.

Rationale: Fibrosis promotes the maintenance of atrial fibrillation (AF), making it resistant to therapy. Improved understanding of the molecular mechanisms leading to atrial fibrosis will open new pathways towards effective antifibrotic therapies. Objective: This study aims to decipher the mechanistic interplay between polo-like kinase 2 (PLK2) and the pro-fibrotic cytokine osteopontin (OPN) in the pathogenesis of atrial fibrosis and atrial fibrillation. Methods and Results: Atrial PLK2 mRNA expression was 10-fold higher in human fibroblasts than in cardiomyocytes. Compared to sinus rhythm (SR), right atrial appendages and isolated right atrial fibroblasts from AF patients showed downregulation of PLK2 mRNA and protein, along with increased PLK2 promotor methylation. Genetic deletion as well as pharmacological inhibition of PLK2 induced pro-fibrotic phenotype conversion in cardiac fibroblasts and led to a striking de novo secretion of OPN. Accordingly, PLK2-deficient (PLK2 KO) mice showed cardiac fibrosis and were prone to experimentally induced AF. In line with these findings, OPN plasma levels were significantly higher only in AF patients with atrial low-voltage zones (surrogates of fibrosis) compared to SR controls. Mechanistically, we identified ERK1/2 as the relevant downstream mediator of PLK2 leading to increased OPN expression. Finally, oral treatment with the clinically-available drug mesalazine, known to inhibit ERK1/2, prevented cardiac OPN overexpression and reversed the pathological PLK2 KO phenotype in PLK2 KO-mice. Conclusions: In summary, abnormal PLK2/ERK1/2/OPN axis function critically contributes to AF-related atrial fibrosis, suggesting reinforcing PLK2 activity and/or OPN inhibition as innovative targets to prevent fibrosis progression in AF. Mesalazine derivatives may be used as lead compounds for the development of novel anti-AF agents targeting fibrosis.

A novel series of dimeric melatonin analogues obtained by connecting two melatonin molecules through N1 with spacers of 15-24 atoms was synthesized and characterized in 2-[ 125 -I]-iodomelatonin binding and bioluminescence resonance energy transfer (BRET) experiments at MT 1 and MT 2 receptors. Compounds 4 (16 atoms spacer) and 13 (24 atoms spacer) are among the ligands inducing the maximal BRET at MT 2homodimers as well as at both types of MT 1 /MT 2 heterodimers. Notably, ligand-induced BRET changes observed for compounds linked through spacers of 22-24 atoms could be attributed to ligand-induced conformational changes between the two protomers of MT 1 and MT 2 homo-and heterodimers providing evidence for the binding of both pharmacophores of dimeric melatonin analogues to the two protomers of receptor dimers.

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