Abstract 4144825: 4-Hydroxy-2-Nonenal Alters Alternative Polyadenylation to Regulate mRNA Isoform Diversity in the Transition from Human Cardiac Fibroblasts to Myofibroblasts

Background: Cardiac fibrosis involves excessive deposition of extracellular matrix (ECM) proteins, leading to myocardial stiffness and remodeling, exacerbating diastolic and systolic dysfunction. Notably, cardiac fibroblast (CF) differentiation into myofibroblasts drives ECM deposition, contributing to adverse remodeling. Lipid peroxidation by 4-hydroxynonenal (4HNE) is crucial in fibrosis initiation. Our research explores the role of alternative polyadenylation (APA) in ECM production specifically the impact of 4HNE on APA-mediated modifications in CF activation. Objective: To investigate global APA events during the transition of human CFs to myofibroblasts induced by 4HNE, using the PolyA miner algorithm. Methods: Human CFs were treated with 1 or 2 µM 4HNE or vehicle control to investigate 4HNE induced heterogeneity in 3’UTR lengths. Poly(A)-click RNA sequencing and PolyA-miner analysis were used to examine 3’UTR length changes in genes related to myofibroblast activation. Results: Our analysis revealed cells treated with 1 µM 4HNE altered 236 differential APA genes (DAGs), of which 169 genes exhibited PolyAIndex and among them, 77 underwent 3’ UTR lengthening, and 92 genes underwent 3’UTR shortening. Whereas cells treated with 2µM 4HNE altered 461 DAGs of which 345 genes exhibited PolyAIndex, with 127 genes exhibiting elongation and 218 genes showing shortening. This shows, 4HNE altered 3’UTR lengths in dose dependent manner. Importantly, pathway analysis unveiled that the shortened 3’UTR lengths of genes in 4HNE treated fibroblasts were associated with TGF-β, hippo, and focal adhesion signaling pathways implicated in myofibroblast activation, whereas genes with altered elongated 3’UTR lengths were mostly linked to hedgehog signaling. Furthermore, 4HNE promoted differential APA of fibrotic genes, including fibronectin, collagen 1 α, and α-smooth muscle actin, correlating with increased protein expression. Altogether, our results identify 4HNE as an APA regulator during cardiac myofibroblast activation, though the exact mechanism remains unknown. Conclusion: This study provides novel insights into the molecular mechanisms by which 4HNE governs the transition of human CF to myofibroblasts through regulation of 3'UTR length of fibrotic genes and subsequent translational control. Understanding 4HNE-mediated APA-mediated changes in CFs may pave the way for the development of targeted therapeutic strategies to mitigate adverse outcomes in cardiac fibrosis.