Human Engineered Heart Tissues Reproduce Trigger‐ and Substrate‐Driven Mechanisms of Ventricular Tachycardia

ABSTRACT Human iPSC‑derived engineered heart tissues (EHTs) are used to model cardiac physiology and drug responses, yet whether they reproduce tissue‑scale ventricular arrhythmia mechanisms remains unclear. We combined modified milliPillar EHT fabrication with high‐resolution dual‐channel voltage/Ca 2+ optical mapping. Baseline electrophysiology showed action potential durations (APD) within the range reported for human ventricles and conduction velocities approximately half of human longitudinal values, with rate‑dependent restitution of APD and Ca 2+ transient duration, conduction slowing, and physiologic AP‐Ca 2+ latency. To interrogate a trigger arm of ventricular tachycardia (VT), we modeled acquired long‐QT syndrome (aLQTS; hERG blockade with hypokalemia/hypomagnesemia) in EHTs from two hiPSC lines. aLQTS EHTs developed VT‐like episodes with beat‑to‑beat instability. Early afterdepolarizations propagated heterogeneously, generating triggered activity, tissue acceleration, APD dispersion, wavebreak, and rotor formation. These were short and self‐terminating (median 0.4 s) and clustered at head/neck region curvatures. To test whether a fixed spatial substrate supports prolonged reentry, we developed a reproducible focal cryoinjury model. After injury, all tissues developed VT‐like episodes, and 70% anatomically anchored rotors at the border; three persisted beyond 10 s, a recording‐limited lower bound. Dual voltage/Ca 2+ mapping at this resolution establishes EHTs as a non‐animal platform for mechanistic study of both trigger‐ and substrate‐driven ventricular arrhythmias.