Optical Mapping Could Reveal Two-Dimensional Morphology of Atrioventricular Electrical Insulation in the Foetal Avian Heart

Veronika Olejnickova1, Oto Janousek2, Marina Ronzhina2, Jakub Hejc3, Alena Kvasilova4, Eva Zabrodska4, David Sedmera4
11First Faculty of Medicine Charles University, Prague, Czech Republic, 2Department of Biomedical Engineering, Brno University of Technology, Brno, Czech Republic, 3Department of Biomedical Engineering, Brno University of Technology, 4First Faculty of Medicine, Charles University, Prague, Czech Republic


Abstract

Formation of the atrioventricular (AV) fibrotic skeleton with the sole electrical connection through the AV bundle is one of the crucial steps in cardiac development. Inappropriate electrical insulation between atria and ventricles results in syndromes of pre-excitation or AV re-entry. However, this electrical remodelling process remains poorly understood, mainly due to limited possibility to examine spontaneous electrical activity of foetal hearts and its visualisation in 2D. Here we demonstrate optical mapping of the developing avian heart as a tool for tracing of AV junction functional remodelling during embryonic and foetal stages. Embryonic hearts (day 4 and 7) were stained with voltage sensitive dye di-4-ANEPPS for 5 or 10 minutes, respectively (0.05 mg/ml in Tyrode´s solution – TS on ice). We introduced retrograde perfusion with oxygenated TS for foetal hearts staining (day 13 and 18) to achieve appropriate dye loading and oxygenation. Dye perfusion (37°C; 10.0µl of 1 mg/ml; 5-7 minutes) was followed by 5 minutes of stabilization. The spread of depolarization across the myocardium was monitored in superfused embryonic or retrogradely perfused foetal hearts (both at 37°C, oxygenated TS) by changes in the fluorescence signals of di-4-ANEPPS using a ULTIMA-L high-speed camera at 2 kHz. Blebbistatin was administered before recording to limit motion artefacts. Observed changes in ventricular activation pattern in embryonic day 7 hearts (50% both branches, 25% right bundle branch, and 25% primitive pattern) correspond to functional connections of the conduction system in the ventricular septum and AV electrical insulation. However, in post-septated hearts (day 13), persistence of incomplete AV electrical separation as accessory pathways was present in 40% of hearts (central anterior or left side posterior). The optical mapping proved its ability to visualize the process of AV junction insulation with exact 2D location of accessory AV pathways for whole embryonic and foetal hearts during avian development.