Background-The developmental mechanisms underlying the persistence of myocardial accessory atrioventricular pathways (APs) that bypass the annulus fibrosis are mainly unknown. In the present study, we investigated the role of epicardium-derived cells (EPDCs) in annulus fibrosis formation and the occurrence of APs. Methods and Results-EPDC migration was mechanically inhibited by in ovo microsurgery in quail embryos. In ovoECGs were recorded in wild-type (nϭ12) and EPDC-inhibited (nϭ12) hearts at Hamburger-Hamilton (HH) stages 38 to 42. Subsequently, in these EPDC-inhibited hearts (nϭ12) and in additional wild-type hearts (nϭ45; HH 38 -42), ex ovo extracellular electrograms were recorded. Electrophysiological data were correlated with differentiation markers for cardiomyocytes (MLC2a) and fibroblasts (periostin). In ovo ECGs showed significantly shorter PR intervals in EPDC-inhibited hearts (45Ϯ10 ms) than in wild-type hearts (55Ϯ8 ms, 95% CI 50 to 60 ms, Pϭ0.030), whereas the QRS durations were significantly longer in EPDC-inhibited hearts (29Ϯ14 versus 19Ϯ2 ms, 95% CI 18 to 21 ms, Pϭ0.011). Furthermore, ex ovo extracellular electrograms (HH 38 -42) displayed base-first ventricular activation in 44% (20/45) of wild-type hearts, whereas in all EPDC-inhibited hearts (100%, 12/12), the ventricular base was activated first (PϽ0.001). Small periostin-and MLC2a-positive APs were found mainly in the posteroseptal region of both wild-type and EPDC-inhibited hearts. Interestingly, in all (nϭ10) EPDC-inhibited hearts, additional large periostinnegative and MLC2a-positive APs were found in the right and left lateral free wall coursing through marked isolation defects in the annulus fibrosis until the last stages of embryonic development. Conclusions-EPDCs

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