Abstract Body: Introduction
Human induced pluripotent stem cells (hiPSCs) can be differentiated into ventricular and atrial cardiomyocytes (vCMs and aCMs). aCMs arise from the posterior second heart field, a retinoic acid-patterned region that also generates sinoatrial node (SAN) pacemaker cells. Although hiPSC-aCM cultures are largely atrial, whether a minor nodal population influences emergent tissue electrophysiology remains unresolved.

Hypothesis
We hypothesize that retinoic acid-directed hiPSC-aCM monolayers harbor functional pacemaker cells with I(f)-dependent automaticity, and that this minor cell population drives tissue-level electrophysiology toward a pacemaker.

Methods
Single cell RNAseq was performed on d15 hiPSC-aCM and vCM monolayers. Microelectrode array recordings and voltage-sensitive dye optical mapping quantified spontaneous activity, beat variability and action potential dynamics.

Results
Single cell transcriptomics identified a distinct SAN pacemaker-like population (13%) expressing SHOX2, ISL1, and TNNT2 in aCM cultures but rarely in vCM cultures (0.7%). Cardiomyocyte content was lower in aCMs (64%) than in vCM cultures (87%), consistent with flow cytometry data. Despite predominantly atrial transcriptional identity, aCM monolayers displayed markedly faster spontaneous beating rates (mean beat rate 127 ± 34 vs. 34 ± 12 bpm) and lower beat-to-beat variability (coefficient of variation of the beat period 0.76 ± 0.01 vs. 22.6 ± 5.1 %, P<0.05, n=24 wells/group) than vCMs. Ivabradine, an I(f) inhibitor, dose-dependently slowed beating rates in aCM monolayers but minimally affected vCMs. Optical mapping revealed steeper diastolic depolarization slopes in aCMs and a shift in dominant pacemaker frequency from 3.3 Hz to 2.1 Hz after 10 uM ivabradine, whereas vCM was unchanged. Chronotropic response to isoproterenol and carbachol were greater in aCMs than in vCMs, indicating SAN-like autonomic regulation.

Conclusion
Although hiPSC-aCM cultures are transcriptionally atrial, a small pacemaker cell population dominates tissue-level electrophysiology and confers SAN-like automaticity. These findings suggest that rare nodal cells can disproportionately shape emergent electrical behavior in engineered cardiac tissues.