Explore novel molecular mechanisms of FNDC5 in ischemia-reperfusion (I/ R) injury by analyzing transcriptome changes in mouse model of skeletal muscle I/R injury with FNDC5 knockout

Background: Irisin, a myokine derived from proteolytic cleavage of the fibronectin type III domain-containing protein 5 (FNDC5) protein, is crucial in protecting tissues and organs from ischemia-reperfusion (I/R) injury. However, the underlying mechanism of its action remains elusive. In this study, we investigated the expression patterns of genes associated with FNDC5 knockout to gain insights into its molecular functions.Methods: We employed a mouse model of skeletal muscle I/R injury with FNDC5 knockout to examine the transcriptional profiles using RNA sequencing. Differentially expressed genes (DEGs) were identified and sub-jected to further analyses, including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein-protein interaction (PPI) network analysis, and miRNA-transcription factor network analysis. The bioinformatics findings were validated using qRT-PCR and Western blotting.Results: Comparative analysis of skeletal muscle transcriptomes between wild-type (WT; C57BL/6), WT-I/R, FNDC5 knockout (KO), and KO-I/R mice highlighted the significance of FNDC5 in both physiological condi-tions and I/R injury. Through PPI network analysis, we identified seven key genes (Col6a2, Acta2, Col4a5, Fap, Enpep, Mmp11, and Fosl1), which facilitated the construction of a TF-hub genes-miRNA regulatory network. Additionally, our results suggested that the PI3K-Akt pathway is predominantly involved in FNDC5 deletion-mediated I/R injury in skeletal muscle. Animal studies revealed reduced FNDC5 expression in skeletal muscle following I/R injury, and the gastrocnemius muscle with FNDC5 knockout exhibited larger infarct size and more severe tissue damage after I/R. Moreover, Western blot analysis confirmed the upregulation of Col6a2, Enpep, and Mmp11 protein levels following I/R, particularly in the KO-I/R group. Furthermore, FNDC5 deletion inhibited the PI3K-Akt signaling pathway.Conclusion: This study demonstrates that FNDC5 deletion exacerbates skeletal muscle I/R injury, potentially involving the upregulation of Col6a2, Enpep, and Mmp11. Additionally, the findings suggest the involvement of the PI3K-Akt pathway in FNDC5 deletion-mediated skeletal muscle I/R injury, providing novel insights into the molecular mechanisms underlying FNDC5's role in this pathological process.