Molecular and Cellular Characterization of Core Genes Associated with Endoplasmic Reticulum Stress in Myocardial Ischemia-Reperfusion Injury

Background: Myocardial ischemia-reperfusion injury (MIRI) contributes to myocardial injury following reperfusion and adversely affects clinical outcomes. Endoplasmic reticulum stress (ERS), as a crucial cellular stress response mechanism, plays a central regulatory role in the development of MIRI. Currently, the molecular pathways and cellular phenotypic characteristics of ERS-related core genes regulating MIRI remain incompletely elucidated. Targeted research in this area could provide a solid theoretical foundation for developing novel myocardial protection strategies. Methods: In the present study, two microarray gene expression datasets (GSE100499 and GSE108940) were obtained from the Gene Expression Omnibus (GEO) database. Simultaneously, ERS-related genes were collected from the GeneCards and Molecular Signatures Database (MSigDB) databases. Differentially expressed genes (DEGs) were identified using the limma package, and key module genes were selected through weighted gene co-expression network analysis (WGCNA). An intersection analysis was performed among DEGs, genes from the selected WGCNA module, and ERS-related genes. Three machine learning algorithms [least absolute shrinkage and selection operator (LASSO), Random Forest (RF) and Support Vector Machine (SVM)] were employed to identify core ERS genes; functional association analysis was performed to explore potential relationships among the core genes. Concurrently, single-nucleus RNA sequencing analysis was performed to investigate the functional mechanisms of core genes at the single-cell level. Furthermore, the pathological roles of these core genes in MIRI were validated by constructing rat MIRI models and H9c2 cell hypoxia-reoxygenation models, where 4-phenylbutyrate was used to inhibit ERS. Results: ERS-related DEGs were predominantly enriched in tumor necrosis factor (TNF) signaling pathway, lipid metabolism and atherosclerosis, and leukocyte transendothelial migration pathways, which are all critical pathological pathways in the progression of MIRI. Four ERS-related genes (Bcl2, Thbs1, Cebpb and Calr) performed best in Lasso, RF and SVM analyses. All four genes showed significant interactions with pathways associated with MIRI and ERS, including ERS activation, inflammatory response, cardiomyocyte apoptosis and inflammatory cell migration. In the single-cell transcriptome of MIRI cardiac tissue, among the 12 cell types, the aforementioned four genes were primarily upregulated in cardiomyocytes, vascular endothelial cells and cardiac macrophages. Notably, cardiomyocytes and cardiac macrophages exhibited a pronounced pro-inflammatory state and the highest levels of pyroptosis. This suggests that they may be the primary effectors of the four key genes mediating MIRI-related injury. Conclusions: Collectively, Bcl2, Thbs1, Cebpb, and Calr may serve as potential ERS-associated biomarkers and therapeutic targets for MIRI, providing novel insights into ERS-mediated molecular mechanisms underlying myocardial injury.