Resolution and automated analysis of single-cell Ca²⁺ waves within living myocardial tissue slices

Current approaches for the study of arrhythmogenic Ca²⁺ waves in intact cardiac tissue have limited capacity to study the relationship between single-cell properties and the tissue environment. Particularly, wide-field Ca²⁺ 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 Ca²⁺ wave dynamics across large fields within the intact multicellular tissue. Living myocardial slices were prepared from left ventricular tissue from pig and human hearts. Ca²⁺ 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, Ca²⁺ waves were evident during the rest period. Images were recorded at 200 fps. After image processing, single Ca²⁺ waves were identified and propagation paths were tracked using TrackMate. From these tracks, Ca²⁺ 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 Ca²⁺ wave dynamics, detecting variability within the population and an absence of synchronization. The recording of Ca²⁺ 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 Ca²⁺ activity and the translation into arrhythmias. KEY POINTS: We developed an approach that allows high frame-rate imaging of living cardiac tissue, while retaining capacity to identify Ca²⁺ waves in single cells. In myocardial slice preparations from pig and human hearts, we recorded Ca²⁺ 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 Ca²⁺ 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.