Abstract Body: Introduction
The sinoatrial node (SAN) intrinsically paces and drives the large electrical load of the atrial myocardium. This ability to overcome source-sink mismatch fails in sinus node dysfunction, a prevalent disease lacking effective treatment. We developed a human SAN organoid model that recapitulates the native pacemaker’s ability to pace and drive myocardium.

Hypothesis
We hypothesized that 1) integration of epicardial cells (Epi), key contributors to SAN development, is essential for generating functional human SAN organoids (SANOs); and 2) Epi-integrated SANOs (Epi-SANOs) pace and entrain surrounding neonatal rat ventricular myocyte (NRVM) monolayers in a source-sink mismatch model.

Approach
Human iPSCs were differentiated toward atrial or ventricular lineages. Epi-SANOs and Epi-ventricular organoids (Epi-VOs) were generated by integrating hiPSC-derived Epi with atrial lineage containing 10-15% pacemaker cells or ventricular lineage cells.

Results
Single-cell RNAseq showed that Epi-SANOs recapitulate key SAN cell populations, including head, tail, and transitional zone cells, whereas Epi-VOs showed few nodal cell types. Epi-SANOs exhibited faster beating rates and lower beat-to-beat variability than Epi-VOs (137.2 ± 15.8 vs. 27.0 ± 6.6 bpm; CV of RR intervals: 0.4 ± 0.1 vs. 2.3 ± 0.5; n ≥ 10 per group; p < 0.0001). After one week of NRVM co-culture, high-resolution optical mapping revealed action potentials originating from a discrete pacemaker site and propagating across the ventricular syncytium. Although both organoid types served as the leading pacemaker under basal conditions, only Epi-SANOs exhibited spontaneous diastolic depolarization with linear and exponential phases consistent with the dual-clock SAN automaticity. β-adrenergic stimulation (0.3 µM isoproterenol) caused failure of Epi-VO-driven pacing with ventricular monolayer-driven tachyarrhythmias. In contrast, Epi-SANOs maintained pacemaker dominance and increased pacing frequency, demonstrating robust rate-adaptation.

Conclusion
Epicardial-integrated human SAN organoids reproduce key structural and functional features of the native pacemaker and provide a human platform for studying the conduction system and modeling sinus node dysfunction.