Comprehensive analysis of aberrant alternative splicing and RNA binding proteins regulators associated with myocardial ischemia reperfusion injury in mice

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Myocardial ischemia-reperfusion injury (MIRI) involves complex molecular mechanisms. However, the roles of alternative splicing (AS) and RNA-binding proteins (RBPs) in its pathogenesis remain largely elusive. In this study, we conducted an integrated analysis of the public RNA sequencing dataset GSE214122 to identify regulated alternative splicing events (RASEs) and differentially expressed genes (DEGs) in a murine MIRI model. We identified 1262 DEGs (883 upregulated and 379 downregulated), among which 232 were RBPs. Notably, 42 RASE-related genes overlapped with the DEGs. Functional enrichment analysis revealed that aberrantly spliced genes were primarily involved in critical signaling pathways, including mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK). Key genes identified within the mTOR pathway included Eif4e2, Atp6v1h, and Insr, while Traf6, Map4k4, and Nr4a1 were prominent in the MAPK pathway. Gene Ontology (GO) analysis further highlighted biological processes closely associated with MIRI, such as angiogenesis and cellular response to hypoxia. Co-expression network analysis demonstrated that the differentially expressed RBP LMNA was highly correlated with an alternative 5’ splice site (alt5p) event in Atp6v1h (clualt5p2389), the splicing ratio of which was significantly elevated in the MIRI group. Independent experimental validation confirmed the significant upregulation of splice isoforms for Eif4e2, Traf6, Insr, and Nr4a1. Furthermore, mRNA levels of seven RBPs (Anxa2, Fn1, Hyou1, Hif1a, Lmna, Myh9, and Stmn1) were significantly upregulated, whereas Crebrf was significantly downregulated. The Western blot results showed that the protein levels of HIF1A, FN1, LMNA, and EIF4E2 were increased in the MIRI group, while the expression of CREBRF protein was decreased. In conclusion, this study provides a systematic landscape of AS and RBP dysregulation in MIRI. We report for the first time that Lmna-regulated AS of Atp6v1h may participate in the hypoxic response and mTOR pathway modulation. These candidate RBPs and their associated AS events offer novel insights into the molecular mechanisms of MIRI and represent potential therapeutic targets.

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