Regulated HSPG Signaling Directs Epicardial Behavior to Support Cardiac Formation.

Abstract

Pathways controlling cardiac cell behavior share a common dependency on heparan sulfate proteoglycans (HSPGs), which tightly regulate signaling at extracellular locations. This signaling is essential for cardiac development, yet how HSPGs are regulated in the forming heart is unknown. The epicardium is a rich source of HSPG-dependent signaling and cellular progenitors. We hypothesized that extracellular heparan sulfate modifiers, 6-<i>O</i>-endosulfatases, orchestrate progenitor cell behavior to support cardiogenesis. We used single-cell RNA sequencing, microscopy, and flow cytometry-based single-molecule RNA ISH to systematically profile 6-<i>O</i>-endosulfatases and target HSPGs in the embryonic mouse heart. Subsequently, we utilized knockout and knockdown models that identified gene associations and a role for the main epicardial 6-<i>O</i>-endosulfatase isoform, <i>Sulf1</i>. Transcriptional regulation of <i>Sulf1</i> was assessed using ATAC and CUT&RUN sequencing, luciferase assays, and siRNA, and the impact on epicardial cell behavior was confirmed in vivo and using in vitro functional assays. Despite identical function, we find that <i>Sulf1</i> is expressed in the embryonic epicardium, while <i>Sulf2</i> is expressed broadly throughout the myocardium. We show that epicardial SULF1 dynamically regulates HSPG sulfation to fine-tune the magnitude and duration of signaling to impact cell fate and cardiac morphogenesis. Single-cell genomics and lineage tracing studies reveal <i>Sulf1</i> to be strongly coexpressed with key transcriptional regulator <i>Wt1</i> (Wilms tumor 1) in the epicardium, with reduction of both coinciding with epithelial-to-mesenchymal transition and quiescence. CUT&RUN-seq revealed transcriptional control of <i>Sulf1</i> by WT1, which directly impacts essential HSPG-dependent downstream signaling. Ligand-receptor interaction predictions and functional assays indicated that FGF (fibroblast growth factor)-2 and TGF-β (transforming growth factor β)-driven processes were governed by this regulatory interaction. Our study highlights, for the first time, essential fine-tuning of HSPG-dependent signaling to modulate key processes in heart formation, offering potential insights for therapeutically targeting congenital heart disease and enhancing epicardial proregenerative behaviors.