Odz4 upregulates SAN-specific genes to promote differentiation into cardiac pacemaker-like cells

Cardiac arrhythmias stemming from abnormal sinoatrial node (SAN) function can lead to sudden death. Developing a biological pacemaker device for treating sick sinus syndrome (SSS) could offer a potential cure. Understanding SAN differentiation is crucial, yet its regulatory mechanism remains unclear. We reanalyzed published RNA-seq data and identified Odz4 as a SAN-specific candidate. In situ hybridization revealed Odz4 expression in the cardiac crescent and throughout the cardiac conduction system (CCS). To assess role of Odz4 in CCS differentiation, we utilized a Tet-Off inducible system for its intracellular domain (ICD). Odz4-ICD exogenously expressing embryonic bodies (EBs) exhibited an increased propensity to develop into pacemaker-like cells with enhanced automaticity and upregulated expression of SAN-specific genes. CellChat and GO analyses unveiled SAN-specific enrichment of ligand-receptor sets, especially Ptn-Ncl, and extracellular matrix components in the Odz4-ICD exogenously expressing group. Our findings underscore the significance of Odz4 in SAN development and offer fresh insights into biological pacemaker establishment Overall design: EBRTcH3 cells were employed to establish the Odz4-ICD inducible cell line, and the expression was controlled by using Tet-Off system. After forming embryonic bodies for differentiation using the hanging drop method, EBs cultured with or without Tet from Day2, 2+3, 3+5, and 2+7 were dissociated for single cell sequencing.