Abstract 11735: Single-Nucleus RNA Sequencing Analysis Reveals Subpopulation of Endothelial Cells Activation in Hearts of Porcine Double Injury Model

Abstract

Pigs underwent a double injury model: neonatal day 1 apical resection (AR P1 ) and postnatal day 28 LAD ligation (MI P28 ) can remuscularize the AMI by postnatal day 56 (P56) with little evidence of myocardial scarring, which is accompanied by increased cardiomyocytes (CMs) cell-cycle activity. The vascularization that occurred to support remuscularization has not been studied. Therefore, we analyze the single-nucleus RNA sequencing (snRNA-seq) data collected from the MI-border zone of AR P1 -MI P28 , MI P28 (without AR P1 ), naïve, and fetal pig endothelial cells (ECs) using the Artificial Intelligence autoencoder and cluster analyses. Three clusters of EC were identified. Among them, vascular and lymphatic EC clusters (EC1 and EC2) were presented in all postnatal hearts. Besides expressing vascular EC gene signatures, the remaining cluster, named ‘EC3’, also upregulated Transforming grow factor-beta (TGFβ) and Bone Morphogenetic Proteins (BMP) signaling markers. The signaling indicated EC3 was still under development. EC3 strongly presented in the fetal heart (31% of ECs); then, EC3 proportions decreased as the pig matured and virtually dismissed (<2%) in the naïve heart from P28 to later. Up to 1-week following MI P28 injury, EC3 proportions remained low (<2%) in MI P28 -only hearts; meanwhile, EC3 proportions significantly increased (>7%) in the regenerative AR P1 -MI P28 hearts. Thus, in the AR P1 -MI P28 injured hearts, the subpopulation of EC3 is activated to support remuscularization. Figure. SnRNA-seq analysis identifies a cluster of under-development endothelial cells (EC3). A) UMAP: three EC clusters. B) UMAP: vascular EC marker PLVAP expressesed (calculated using Seurat) in EC1 and EC3. C) UMAP: lymphatic EC marker CCL21 expressesed in EC2. D) Heatmap: TGFβ and BMP signaling upregulated in EC3. C) Bargraph: proportions of EC3 in naïve and injured hearts on postnatal days P28, P30, and P35; P-value and Fold-change (FC) were calculated using Fisher’s exact test.