Single-Cell WGCNA Combined with Transcriptome Sequencing to Study the Molecular Mechanisms of Inflammation-Related Ferroptosis in Myocardial Ischemia-Reperfusion Injury

Zhuohua Zhang,1,2 Yan Liu,2 Da Huang,2 Zhaohe Huang1,3,4 1Department of Cardiology, First Affiliated Hospital of Jinan University, Guangzhou, 510630, People’s Republic of China; 2Department of Cardiology, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, People’s Republic of China; 3Affiliated Southwest Hospital, Youjiang Medical University for Nationalities, Baise, 533000, People’s Republic of China; 4Graduate School, Youjiang Medical University for Nationalities, Baise, 533000, People’s Republic of ChinaCorrespondence: Zhaohe Huang, Department of Cardiology, First Affiliated Hospital of Jinan University, Guangzhou, 510630, People’s Republic of China, Tel +86 13977691455, Fax +86 776-2853272, Email [email protected]: Myocardial ischemia-reperfusion injury (MIRI) is characterized by inflammation and ferroptosis, but the precise mechanisms remain unknown. This study used single-cell transcriptomics technology to investigate the changes in various cell subtypes during MIRI and the regulatory network of ferroptosis-related genes and immune infiltration.Methods: Datasets GSE146285, GSE83472, GSE61592, and GSE160516 were obtained from Gene Expression Omnibus. Each cell subtype in the tissue samples was documented. The Seurat package was used for data preprocessing, standardization, and clustering. Cellphonedb was used to investigate the ligand-receptor interactions between cells. The hdWGCNA analysis was used to create a gene co-expression network. GSVA and GSEA were combined to perform functional enrichment and pathway analysis on the gene set. Furthermore, characteristic genes of the disease were identified using Lasso regression and SVM algorithms. Immune cell infiltration analysis was also performed. MIRI rat models were created, and samples were taken for RT-qPCR and Western blot validation.Results: The proportion of MIRI samples in the C2, C6, and C11 subtypes was significantly higher than that of control samples. Three genes associated with ferroptosis (CD44, Cfl1, and Zfp36) were identified as MIRI core genes. The expression of these core genes was significantly correlated with mast cells and monocyte immune infiltrating cells. The experimental validation confirmed the upregulation of Cd44 and Zfp36 expression levels in MIRI, consistent with current study trends.Conclusion: This study used single-cell transcriptomics technology to investigate the molecular mechanisms underpinning MIRI. Numerous important cell subtypes, gene regulatory networks, and disease-associated immune infiltration were also discovered. These findings provide new information and potential therapeutic targets for MIRI diagnosis and treatment.Keywords: myocardial ischemia-reperfusion injury, ferroptosis, immune cell infiltration, single-cell transcriptome sequencing, WGCNA