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

Myocardial ischemia-reperfusion injury (MIRI) is a pathological condition involving complex molecular mechanisms, including aberrant alternative splicing and dysregulated RNA-binding proteins (RBPs). To investigate the role of RBPs and alternative splicing events (ASEs) in MIRI, we designed an integrated analytical workflow. Using the dataset GSE214122, we systematically profiled differentially expressed genes (DEGs) and MIRI-related alternative splicing signatures in a mouse cardiac I/R model. We identified 1,262 DEGs (883 up-regulated and 379 down-regulated) and further screened 232 RBPs genes, among which 42 alternatively spliced genes overlapped with the DEGs. Functional enrichment analysis of these dysregulated splicing genes revealed significant involvement in signaling pathways such as mTOR and MAPK, which have established roles in MIRI. Key genes in the mTOR pathway included Eif4e2, Atp6v1h, and Insr, whereas Traf6, Map4k4, and Nr4a1 were highlighted in the MAPK pathway. Furthermore, we identified differentially expressed RBPs between the MIRI and sham-operated groups, suggesting their potential involvement in the post-transcriptional regulation of MIRI. Gene Ontology (GO) analysis uncovered biological processes closely linked to MIRI, such as angiogenesis and cellular response to hypoxia. Of particular significance was the identification of a hypoxia-responsive regulatory axis involving DERBP-Lmna mediated modulation of RAS-clualt5p2389: Atp6v1h within the mTOR signaling pathway. To experimentally validate these findings, we quantified four pivotal alternative splicing events (Eif4e2, Traf6, Insr, Nr4a1) and eight differentially expressed RBPs genes (Anxa2, CREBRF, FN1, HYOU1, HIF1A, LMNA, MYH9, STMN1) via qRT-PCR in a murine MIRI model.Results demonstrated significant upregulation of all examined ASE genes in MIRI subjects compared to controls. Concurrently, pronounced elevation was observed in RBPs genes (Anxa2, FN1, HYOU1, HIF1A, LMNA, MYH9, STMN1), whereas CREBRF expression was substantially suppressed. These experimental outcomes not only provide robust validation of our bioinformatic predictions but also yield novel mechanistic insights into MIRI pathogenesis. Our findings posit therapeutic targeting of RBPs-mediated splicing regulation as a promising avenue for myocardial ischemia-reperfusion injury intervention.