Transcriptional Regulation of Sinoatrial Node Development

PROJECT SUMMARY The sinoatrial node (SAN) is a tiny structure consisting of a precise arrangement of specialized pacemaker cardiomyocytes (PCs) that trigger each heartbeat. Sinus node dysfunction (SND), resulting from loss or malfunction of PCs, is a common and morbid disease that is not well understood. There is currently no treatment that can delay or prevent SND, so sufferers must undergo permanent pacemaker implantation. This proposal will leverage novel tools and experimental approaches to determine how a multipotent population of SAN progenitor cells are allocated to different fates within the SAN, and to define how transcriptional hierarchies govern gene expression programs and spatial organization in the SAN. Ultimately, we hope that our findings will lead to new approaches to prevent or reverse SND by targeting the biological pathways that control SAN formation and maintenance. Previous efforts in this area have been hampered by the lack of specific genetic tools to mark the SAN progenitor population and the lack of an in vitro system that can model SAN development. In the past, our group has co- discovered a key activating role for the transcription factor Isl1 in SAN development and we have identified an Isl1 SAN Enhancer (ISE) that is specifically active in the SAN and its progenitor population. We have used this enhancer to generate a new mouse line, ISE-CreERT2, that allows conditional genetic modification of the SAN progenitor population. Clonal fate mapping with this mouse line and single cell sequencing analysis have defined the dynamics of the SAN progenitor population and allowed us to develop a system to explore how SAN progenitors are allocated among several possible fates. To gain further insight into how this fate allocation occurs, we developed a novel in vitro protocol using hiPSCs that recapitulates key aspects of SAN differentiation, including diversification of SAN progenitors into anatomically and functionally distinct pacemaker cardiomyocyte subtypes. Using this model, with in vivo studies as validation, the present work explores the hypothesis that SAN progenitor allocation depends upon Isl1, activator protein-1 (AP-1), and Nuclear Factor I (NFI) transcriptional programs that guide cells in the SAN progenitor field to different fates. In Aim 1, we will determine how Isl1 shifts from playing a role in regulating cardiac progenitor cell proliferation to playing a key role in activating the PC-specific gene expression in a cellular subtype-specific manner. In Aim 2 we will determine how AP-1 and NFI regulate progenitor cell allocation and adoption of cell type-specific gene programs in the SAN. Aim 3 will determine how these pathways regulate the spatial architecture of the SAN using spatial transcriptomics in WT and genetically modified mice. Taken together, the work proposed will establish new mechanistic paradigms for the how the mammalian cardiac pacemaker is formed and could set the stage for novel approaches to treating and preventing SND.