Abstract 4366628: 4D Cardiac Optogenetics Enable Complex Arrhythmia Modelling and Precise Interventional Simulation

Background: Cardiac arrhythmias are a major cause of morbidity and mortality. Yet, current in vitro arrhythmia models neither permit reproducible creation of complex arrhythmia substrates, nor allow reversible perturbations of the system, which are crucial for deciphering arrhythmia mechanisms and identifying the sites that are critical for their perpetuation. A high-resolution, fully reversible platform permitting targeted electrical silencing is therefore urgently needed to model arrhythmia substrates and accelerate therapeutic discovery. Hypothesis: The PAC-K construct, a light-gated hyperpolarizing potassium channel, can be applied to in vitro cardiac models to (i) generate temporary arrhythmogenic substrates, (ii) reproducibly induce and terminate complex arrhythmias, and (iii) evaluate the effects of different ablation-like interventions within those substrates. Methods: We developed a four-dimensional (3D space + time) optogenetic platform combining a stable hiPSC PAC-K line and an optical mapping setup. A digital micromirror device was used for targeted illumination, and duration of inhibition was controlled by modulating the light dose. Results: Using this platform, we induced arrhythmias in hiPSC-derived cardiac monolayers with targeted illumination. These arrhythmias were successfully terminated (100% success, n=26, p<0.0001) using diffuse 470 nm light (82 mW/cm 2 , 500 ms). Next, we demonstrated the ability to generate high-resolution conduction blocks using patterned illumination. This enabled the creation of clinically relevant arrhythmogenic substrates by applying illumination patterns mimicking the post-infarct border zone. We then induced arrhythmias within these substrates and applied targeted light pulses to functionally ablate different conduction pathways. This allowed us to investigate which arrhythmias are most likely to develop in each substrate and isolate the critical pathways necessary for their perpetuation. Conclusion: This study introduces a 4D optogenetic platform for precise, reversible, and high-throughput arrhythmia modeling and interventional simulation. By dynamically controlling spatial and temporal electrophysiology, this system advances arrhythmia research by providing a powerful tool for studying arrhythmogenesis and testing anti-arrhythmic strategies. The ability to induce intricate and reversible conduction blocks, which behave like pathological and ablative scars, offers exciting research and translational opportunities.