What does this research mean for the field?

Dysregulation of alternative splicing and RNA-binding proteins, specifically Lmna-regulated alternative splicing of Atp6v1h, plays a critical role in the pathogenesis of myocardial ischemia-reperfusion injury by modulating the hypoxic response and mTOR signaling pathway. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

What question did this study set out to answer?

The research aims to explore the roles of alternative splicing and RNA-binding proteins in myocardial ischemia-reperfusion injury.

March 29, 2026Open Access

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

Key Points

The research aims to explore the roles of alternative splicing and RNA-binding proteins in myocardial ischemia-reperfusion injury.
Conducted an integrated analysis using public RNA sequencing dataset GSE214122.
Identified regulated alternative splicing events and differentially expressed genes in a murine MIRI model.
Performed functional enrichment analysis and co-expression network analysis.
Validated findings through independent experiments including western blotting.
Identified 1262 differentially expressed genes, with 232 classified as RNA-binding proteins.
Discovered 42 regulated alternative splicing-related genes among the differentially expressed genes.
Key genes in the mTOR pathway included Eif4e2, Atp6v1h, and Insr, with Traf6, Map4k4, and Nr4a1 prominent in MAPK.
Significantly elevated mRNA and protein levels of several candidates, including LMNA and HIF1A, in the MIRI group.

Abstract

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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