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.

Abstract

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. 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 subjected 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. 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 conditions 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. 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.