Abstract Body: Background: The sinoatrial node (SAN) is the natural pacemaker structure of the heart. Pacemaker cells (PCs) are specialized cardiomyocytes residing in the SAN that initiate and maintain rhythmic electrical signals. PC deficiency leads to SAN dysfunction (SND), which causes arrhythmias and even sudden death. Aging is a major risk factor for SND, yet its underlying mechanisms remain poorly defined. Currently, there are no human in vitro models that faithfully recapitulate aging-associated SND.

Hypothesis: D-galactose-treated human pluripotent stem cell (hPSC)-derived pacemaker cells (hPCs) partially recapitulate molecular and functional features of the aging mouse SAN.

Methods: hPCs derived from a dual SHOX2-GFP; MYH6-mCherry reporter line were validated by PC-marker enrichment and distinct molecular identity relative to hPSC-derived cardiomyocytes, then treated with D-galactose and analyzed by senescence assays, immunoblotting, immunostaining, optical electrophysiology, calcium imaging, and bulk RNA-seq. Young and aged mouse SAN was analyzed by immunostaining, telemetry, and bulk RNA-seq. RNA-seq used n=3 per group in human cells and n=8 per group in mouse SAN; telemetry used n=4 per group.

Results: D-galactose induced a senescence phenotype in hPCs, with increased senescence-associated beta-galactosidase activity, p53-dependent p21 upregulation, and reduced Lamin A/C, which were also observed in aged mouse SAN. Senescent hPCs further exhibited electrophysiologic and calcium-handling remodeling, including shortened action potential durations, accelerated calcium transient kinetics, and reduced contractility. Consistent with this, aged mice showed increased bradycardia and a greater pause burden. Mouse and human transcriptomics revealed conserved findings, including activation of senescence and direct p53-target programs, repression of electrophysiology, ion-channel, and calcium-handling pathways, and enrichment of conduction- and arrhythmia-related disease signatures.

Conclusions: D-galactose-treated hPCs recapitulate key cellular, transcriptomic, and functional features of aging in vivo and define a conserved aging phenotype centered on senescence activation and impaired SAN functional integrity.