Single‐cell sequencing combined with transcriptomics and in vivo and in vitro analysis reveals the landscape of ferroptosis in myocardial ischemia–reperfusion injury

Abstract Myocardial ischemia‐reperfusion injury (MIRI) is a significant risk factor for acute myocardial infarction and is closely associated with ferroptosis. This study aimed to identify key ferroptosis‐related genes as potential diagnostic markers for MIRI and to explore their roles in immune infiltration and therapeutic targeting in myocardial tissue. We obtained single‐cell RNA sequencing (scRNA‐seq) and RNA‐seq data on MIRI from the GEO database, applied Seurat and UMAP for data processing and clustering, and analyzed ligand‐receptor interactions using CellPhoneDB. By scoring ferroptosis in cardiomyocytes, we identified differentially expressed genes and conducted GO and KEGG pathway analyses. A protein interaction network was then constructed using the STRING database, and seven key genes (Atp5h, Vdac2, Pkm, Cycs, Hspa8, Tpi1, Ldha) were identified through Lasso regression modeling, showing significant associations with immune responses. In vivo experiments in a mouse ischemia‐reperfusion model confirmed the roles of these seven genes in MIRI via RT‐qPCR. To further investigate the role of Hspa8 in ferroptosis and MIRI, siRNA knockdown experiments were performed in H9C2 rat cardiomyocytes, and its involvement in ferroptosis was validated by JC‐1 staining and PCR analysis. This study reveals the importance of ferroptosis‐related genes in MIRI through integrated bioinformatics and experimental approaches, offering new insights into diagnostic markers and immune‐related therapeutic targets for MIRI.