D28-17 Single-Nucleus Transcriptomic Atlas of Right Ventricle Reveals Cell-Type-Specific Remodeling in Pre-Clinical Models of Right Ventricular Failure Due to Pulmonary Hypertension

Introduction

Pulmonary hypertension (PH) is a progressive, fatal disease that manifests initially as chronically elevated pulmonary artery pressure that leads to right ventricular (RV) hypertrophy, failure, and sudden death. PH-induced RV failure (PH-RVF) is a significant prognostic determinant of morbidity and mortality and is characterized by cardiomyocyte hypertrophy, endothelial-to-mesenchymal transition (EndMT), fibrosis, and extracellular matrix (ECM) remodeling. Our first-ever single-nucleus profiling of the decompensated RV tissue from two severe rat models of PH-RVF presents a promising method for uncovering novel insights into the PH-RVF pathophysiology.

Methods

Single-nucleus RNA sequencing (snRNA-seq) was performed on decompensated RV free wall tissue from monocrotaline (MCT) and Sugen-hypoxia (SuHx) rat models of PH-RVF using the 10x Genomics Chromium Frozen RNA Profiling platform. Rigorous quality control included ambient RNA correction with CellBender and doublet removal using scDblFinder. Data normalization, dimensionality reduction, clustering, and integration were performed in Seurat with Harmony-based batch correction. Cell populations were annotated using Azimuth and cluster-specific marker genes. Differential gene expression analysis was conducted using MAST, and pathway enrichment was assessed using Gene Set Enrichment Analysis with Hallmark gene sets.

Results

RVs from 12 male Sprague-Dawley rats were profiled, including 4 MCT, 4 SuHx, and 4 age- and sex-matched controls. After quality filtering, a median of 5,981 nuclei per sample was retained, yielding 75,091 total nuclei. A median of 1,856 transcripts and 1,151 genes were detected per nucleus. Unsupervised clustering identified 20 transcriptionally distinct clusters corresponding to 16 major cell types spanning endothelial, mural, stromal, cardiomyocyte, neural, myeloid, and lymphoid lineages, including multiple specialized endothelial and immune subpopulations. Differential expression analysis revealed significant disease-associated transcriptional changes across all major cell populations in both MCT and SuHx RVs compared with controls (FDR < 0.05). Hallmark pathway analysis revealed EndMT and TGFβ signaling as the top shared upregulated pathways and interferon-γ and interferon-α responses as the top shared downregulated pathways in endothelial subpopulations of MCT and SuHx rat RVs compared to the control, indicating convergent inflammatory and vascular remodeling programs despite distinct disease etiologies.

Conclusions

Our first-ever integrated snRNA-seq analysis of MCT and SuHx rat decompensated RVs delineates the cellular landscape, cell-type-specific remodeling, molecular pathways, and signaling programs underlying PH-induced RV failure. Widespread, cell-type-specific gene and pathway dysregulation underscores disease complexity and provides a valuable resource for advancing mechanistic and therapeutic insights.

This abstract is funded by: NHLBI (National Heart, Lung, and Blood Institute)