Abstract Body (Do not enter title and authors here): Introduction
The sinoatrial node (SAN) is heterogeneous and comprises pacemaker cells (PCs), atrial cardiomyocytes (ACMs), transitional zone (TZ) cells, and stromal cells. Epicardial cells (Epics) serve as a major source of nonmyocytes, contributing to sinoatrial node (SAN) development and function.

Hypothesis
Integrating pacemaker organoids with Epics (Epi-SANOs) recapitulate heterogeneity of SAN and enable stable pacing with proper response to autonomic stimuli.

Approach
Epi-SANOs were generated by combining ACMs and PCs with WTC11-derived Epics at a 1:1 ratio at d15. Control SANOs were generated from WTC11-derived ACMs and PCs. Single-cell RNA sequencing was performed at d20, d25, and d30. Beating period and variability were analyzed using microelectrode array. The chronotropic response to autonomic stimuli was measured by optical mapping.

Results
The content of PCs marked by SHOX2+/cTNT+ cells was higher in Epi-SANOs (20%) than in control SANOs (16%) at d30. Fibroblast (FB) population was higher in Epi-SANOs than in control SANOs at d20 and d30, suggesting commitment of Epics to PCs and FBs. Native SAN is delineated into head, tail, and TZ cells. Compared to the native SAN single-cell transcriptome, most PCs in control SANOs were mapped to TZ cells. In contrast, PCs in Epi-SANOs were identified as head and tail cells. To understand whether heterogenous SAN cells mimic SAN-like pacing, we assessed the beat to beat periods of Epi-SANOs and control SANOs; 389±24ms and 422±25ms, respectively (p=0.36, n=9). Analysis of beat to beat variability using Poincaré plots revealed that the SD1 (short term variability) was smaller in Epi-SANOs than in control-SANOs (9.0±2.6 23.5±7.5ms, p=0.09, n=9). This result showed that Epics did not affect the mean pacing frequencies, however, Epi-SANOs tended to have more regular beat to beat automaticity compared to control SANOs. Following isoproterenol (sympathetic agonist) treatment, the beating rates of Epi-SANOs were increased (136±6.6bpm to 162±8.0bpm, p=0.02, n=16), and acetylcholine (parasympathetic agonist) decreased their beating rates (159±8.6bpm to 135bpm±5.4bpm, p=0.03, n=15). This data showed Epi-SANOs exhibit the ability to rate-adapt to physiological stimuli.

Conclusion
Epics contribute to the heterogeneity of PCs in the hiPSC-derived pacemaker organoids and stabilize beat to beat regularity. Our data support the potential of Epi-SANOs as organoid models of the native SAN.