Dissecting the molecular pathways controlling human pacemaker development

About This Grant

Abstract The human sinoatrial node (SAN) is the heart’s natural pacemaker, initiating electrical impulses that maintain rhythmic contraction and coordinate blood flow. While its role is essential, our understanding of SAN development—particularly in humans—remains limited. Sinus node dysfunction (SND), often caused by SAN cell loss or fibrosis, is a major cause of arrhythmia and the primary reason for up to 50% of artificial pacemaker implants in the US. There are no approved cellular or pharmacological treatments. Compared to chamber muscle formation, the mechanisms underlying SAN lineage specification and subpopulation diversity are poorly defined, representing a critical gap in knowledge. In preliminary studies, we used single-nucleus multiomics and spatial transcriptomics to analyze the human fetal SAN and identified candidate regulatory networks. We also developed a dual knock-in human pluripotent stem cell (hPSC) reporter line (SHOX2:eGFP; MYH6:mCherry) and established protocols to generate functional pacemaker cells, SAN organoids (Sinoids) and SAN-paced cardioids (SAN-PCOs). These tools enable real-time visualization, isolation, and functional analysis of human pacemaker cells and tissues. Three Aims are proposed to 1) identify key transcriptional regulators of SAN differentiation using inducible CRISPR interference (CRISPRi) and activation (CRISPRa) in hPSCs. Validated genes will be tested in SAN-PCO models using molecular and electrophysiological assays to assess their role in pacemaker function; 2) dissect the molecular programs guiding SAN subpopulation specification, including head, tail, and transitional zone domains. Using CRISPR-based perturbation and single-cell RNA sequencing, we will evaluate how candidate regulators shape subpopulation identity and influence SAN-cardiomyocyte interactions; and 3) assess how genetic variants associated with SND affect SAN development and function. We will generate isogenic SNP knock-in and gene knockout hPSC lines based on top candidate GWAS hits and evaluate the variant–gene–cell axis using multiomics and functional assays. By integrating single-cell genomics, gene editing, and human stem cell-derived SAN models, this study will define the molecular architecture of human pacemaker development and identify genetic mechanisms underlying SND. These findings will advance our understanding of cardiac conduction system biology and support future therapeutic strategies.