The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Nevertheless, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. This study aimed to identify the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation.

Assay for Transposase-Accessible Chromatin was performed using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues, including the atrioventricular conduction system, lungs, and liver.

In conclusion, a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1. Thus, our data suggest that reduction of Tbx3 expression in mutants functionally affects both ANS and SAN pacemaker cells or their interaction.