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.