Resolution and automated analysis of single‐cell Ca 2+ waves within living myocardial tissue slices

Abstract Current approaches for the study of arrhythmogenic Ca 2+ waves in intact cardiac tissue have limited capacity to study the relationship between single‐cell properties and the tissue environment. Particularly, wide‐field Ca 2+ imaging captures large areas but merges signals from multiple cells, while confocal imaging provides (sub)cellular resolution but is limited to a small number of cells. The aim of this study was to develop an imaging and analysis pipeline capable of extracting single‐cell Ca 2+ wave dynamics across large fields within the intact multicellular tissue. Living myocardial slices were prepared from left ventricular tissue from pig and human hearts. Ca 2+ transients and waves reported by Fluo8 were imaged in regions of 20–100 cells at the slice surface. Following a 2 min conditioning period of 2 Hz pacing under adrenergic stimulation, Ca 2+ waves were evident during the rest period. Images were recorded at 200 fps. After image processing, single Ca 2+ waves were identified and propagation paths were tracked using TrackMate. From these tracks, Ca 2+ wave parameters (number of tracks, coordinates and kinetics) were extracted for quantitative analysis and assigned to individual cardiomyocytes using a maximum fluorescence intensity mask to identify cellular borders. In healthy pig cardiac tissue, we captured and quantified single‐cell Ca 2+ wave dynamics, detecting variability within the population and an absence of synchronization. The recording of Ca 2+ dynamics across a large cell population and their cell–cell interactions bridges the gap between cell‐ and tissue‐level observations. Future studies of diseased tissue will offer new insights into mechanisms driving aberrant cellular Ca 2+ activity and the translation into arrhythmias. image Key points We developed an approach that allows high frame‐rate imaging of living cardiac tissue, while retaining capacity to identify Ca 2+ waves in single cells. In myocardial slice preparations from pig and human hearts, we recorded Ca 2+ waves and, using image analysis tools, tracked their paths and properties simultaneously within individual cells and across a large cell population (20–100 cells) within a region of interest. This approach bridges a key gap between single‐cell observations and whole‐tissue behaviour, and can offer deeper insights into how cellular Ca 2+ waves could expand and propagate in the heart. Our approach provides a new methodology to study cellular mechanisms that lead to arrhythmias in diseased cardiac tissue.