Abstract 18853: Rescue of Pulmonary Hypertension-Induced Right Ventricular Failure by Silencing Snai1 Induces Alterations in Methylation Status of Critical Genes Regulating Endothelial-to-Mesenchymal-Transition and Fibrosis

Abstract

Introduction: Pulmonary hypertension (PH)-induced RV failure (PH-RVF), a significant prognostic determinant, is characterized by RV endothelial-to-mesenchymal transition (EndMT), fibrosis, and remodeling. However, the regulatory control of RV EndMT and fibrosis remains unclear. Hypothesis: TGFβ1-Snai1-LOXL2 axis regulates RV EndMT and fibrosis in PH-RVF. Key gatekeeper transcription factor (TF) Snai1 mediates RV EndMT and fibrosis via differential methylation changes in EndMT and fibrotic genes and may serve as a novel therapeutic target. Methods: Male rats received s.c. Monocrotaline (MCT, 60mg/kg, n=9; 30-days), Sugen (20mg/kg, n=9; 3-wk hypoxia+2-wk normoxia; SuHx) or PBS (CTRL, n=9). For Snai1-knockdown (KD), MCT rats received siSnai1 (n=6; 5nM every 3-4d, IV) or scramble (n=7) from day 14-30. Echo, cath, RV-RNASeq, qPCR, WB, IF validation, and RV-DNA methylome RRBS-Seq were performed. Differentially methylated sites (DMS) and regions (DMR) were assessed. Secondary RNASeq analysis was performed on human decompensated-RVF vs Control (dRVF; GSE198618) and human RVs were assessed for EndMT, fibrosis, myofibroblast (MFB) transition, and Snai1+LOXL2. Snai1-KD was performed on human coronary artery endothelial cells (HCAECs) and human cardiac fibroblasts (HCFs) under hypoxia+TGFβ1. Results: MCT and SuHx had increased RVSP and Fulton index and decreased RVFAC (all p<0.001). RNASeq showed EndMT and Snai1 as the top-upregulated pathway and TF respectively in MCT and SuHx and human dRVF. RV EndMT, fibrosis, MFB transition, TGFβ1-Snai1-LOXL2, and peri-nuclear colocalization of Snai1+LOXL2 were increased in MCT, SuHx and dRVF patients. Snai1-KD rescued PH-RVF via significantly reducing Snai1+LOXL2. RV-RNASeq demonstrated EndMT as the top-downregulated pathway and decreased fibrotic DEGs. RV RRBS-Seq showed significant DMS and DMR remodeling of Dnmt3a, Kdm1a, Snai1, TGFβ1/2, and other critical EndMT and fibrotic genes. Snai1-KD inhibited hypoxia+TGFβ1-induced EndMT in HCAEC and MFB transition in HCF by decreasing Snai1+LOXL2 expression and reversing EndMT and fibrotic DEGs respectively. Conclusions: Rescue of PH-RVF by silencing Snai1 induces alterations in methylation status of critical genes regulating EndMT and fibrosis.