The rate and rhythm of heart muscle contractions are coordinated by the cardiac conduction system (CCS), a generic term for a collection of different specialized muscular tissues within the heart. The CCS components initiate the electrical impulse at the sinoatrial node, propagate it from atria to ventricles via the atrioventricular node and bundle branches, and distribute it to the ventricular muscle mass via the Purkinje fibre network. The CCS thereby controls the rate and rhythm of alternating contractions of the atria and ventricles. CCS function is well conserved across vertebrates from fish to mammals, although particular specialized aspects of CCS function are found only in endotherms (mammals and birds). The development and homeostasis of the CCS involves transcriptional and regulatory networks that act in an embryonic-stage-dependent, tissue-dependent, and dose-dependent manner. This Review describes emerging data from animal studies, stem cell models, and genome-wide association studies that have provided novel insights into the transcriptional networks underlying CCS formation and function. How these insights can be applied to develop disease models and therapies is also discussed.

The rate of contraction of the heart relies on proper development and function of the sinoatrial node, which consists of a small heterogeneous cell population, including Tbx3 + pacemaker cells. Here, we have isolated and characterized the Tbx3 + cells from Tbx3 +/Venus knock-in mice. We studied electrophysiological parameters during development and found that Venus-labeled cells are genuine Tbx3 + pacemaker cells. We analyzed the transcriptomes of late fetal FACS-purified Tbx3 + sinoatrial nodal cells and Nppb-Katushka + atrial and ventricular chamber cardiomyocytes, and identified a sinoatrial node-enriched gene program, including key nodal transcription factors, BMP signaling and Smoc2, the disruption of which in mice did not affect heart rhythm. We also obtained the transcriptomes of the sinoatrial node region, including pacemaker and other cell types, and right atrium of human fetuses, and found a gene program including TBX3, SHOX2, ISL1 and HOX family members, and BMP and NOTCH signaling components conserved between human and mouse. We conclude that a conserved gene program characterizes the sinoatrial node region and that the Tbx3 +/Venus allele provides a reliable tool for visualizing the sinoatrial node, and studying its development and function.

Rationale: 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, ISL1 and SHOX2. Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. Objective: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. Methods and Results: We performed ATAC-seq on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative regulatory DNA elements specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse orthologue of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. 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. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. Conclusions: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3 and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3. exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.