A Novel in vitro Model using Organotypic Cardiac Slices Reveals Transmural Heterogeneity in Arrhythmogenic Ca²⁺ Events after Cardiac Injury

Introduction: Spontaneous Ca2+ release events (SCRE) can trigger left ventricular (LV) arrhythmias during disease remodeling. Intrinsic transmural heterogeneities in Ca2+ handling may differentially modulate SCRE, leading to regional-dependent arrhythmia susceptibility. The geometry of the LV wall further complicates the transmural study of SCRE. Using an in vitro approach mimicking disease remodeling in intact cardiac tissue slices, we aimed to dissect transmural heterogeneities of SCRE. Methods: LV myocardial slices were prepared from adult rats (established protocol by our group). Cryoinjury was induced by applying a frozen probe on the surface of the tissue slice. After 24h-culture, SCRE were studied following pro-arrhythmia pacing using Fluo8AM and high-resolution imaging in regions adjacent to the cryo-injury in endocardial (ENDO, ≤600 µm endo-surface) and epicardial (EPI, ≤600 µm epi-surface) slices. Results: Cryo-injured slices showed local remodeling with reduced T-tubule density and Ca2+ transient amplitudes in the region adjacent to the cryo-injury. After pro-arrhythmic conditioning, injured ENDO slices showed more Ca2+ foci (ENDO: 4.54±0.40 vs. EPI: 3.29±0.29 #foci/mm²/s), which are more closely distributed (ENDO: 67.7±2.7 vs. EPI: 77.9±3.7 µm) with reduced latency times to induce SCRE (ENDO: 222±48 ms vs. EPI: 601±160 ms) compared to injured EPI slices. Inhibition of CaMKII using AIP reduced the number of Ca2+ foci (61% in ENDO vs. 29% in EPI) and increased the distance between Ca2+ foci (40% in ENDO vs. 20% in EPI) more effectively in injured ENDO vs. EPI slices, without affecting the Ca2+ transient amplitude. Conclusions: Our novel in vitro model allows for studies of transmural heterogeneity upon disease remodeling. The current data reveals increased arrhythmogenic susceptibility in the injured endocardium and highlight regional-specific targeting in therapies.