Background: Myocardial ischemia is a prevalent cardiovascular disorder associated with significant morbidity and mortality. While prompt restoration of blood flow is essential for improving patient outcomes, the subsequent reperfusion process can result in myocardial ischemia–reperfusion injury (MIRI). Mitophagy, a specialized autophagic mechanism, has consistently been implicated in various cardiovascular disorders. However, the specific connection between ischemia–reperfusion and mitophagy remains elusive. This study aims to elucidate and validate central mitophagy-related genes associated with MIRI through comprehensive bioinformatics analysis. Methods: We acquired the microarray expression profile dataset (GSE108940) from the Gene Expression Omnibus (GEO) and identified differentially expressed genes (DEGs) using GEO2R. Subsequently, these DEGs were cross-referenced with the mitophagy database, and differential nucleotide sequence analysis was performed through enrichment analysis. Protein–protein interaction (PPI) network analysis was employed to identify hub genes, followed by clustering of these hub genes using cytoHubba and MCODE within Cytoscape software. Gene set enrichment analysis (GSEA) was conducted on central genes. Additionally, Western blotting, immunofluorescence, and quantitative polymerase chain reaction (qPCR) analyses were conducted to validate the expression patterns of pivotal genes in MIRI rat model and H9C2 cardiomyocytes. Results: A total of 2719 DEGs and 61 mitophagy-DEGs were identified, followed by enrichment analyses and the construction of a PPI network. HSP90AA1, RPS27A, EEF2, EIF4A1, EIF2S1, HIF-1α, and BNIP3 emerged as the seven hub genes identified by cytoHubba and MCODE of Cytoscape software. Functional clustering analysis of HIF-1α and BNIP3 yielded a score of 9.647, as determined by Cytoscape (MCODE). In our MIRI rat model, Western blot and immunofluorescence analyses confirmed a significant elevation in the expression of HIF-1α and BNIP3, accompanied by a notable increase in the ratio of LC3II to LC3I. Subsequently, qPCR confirmed a significant upregulation of HIF-1α, BNIP3, and LC3 mRNA in the MIRI group. Activation of the HIF-1α/BNIP3 pathway mediates the regulation of the degree of Mitophagy, thereby effectively reducing apoptosis in rat H9C2 cardiomyocytes. Conclusions: This study has identified seven central genes among mitophagy-related DEGs that may play a pivotal role in MIRI, suggesting a correlation between the HIF-1α/BNIP3 pathway of mitophagy and the pathogenesis of MIRI. The findings highlight the potential importance of mitophagy in MIRI and provide valuable insights into underlying mechanisms and potential therapeutic targets for further exploration in future studies.

Ischemic heart disease remains the leading cause of global disease burden among cardiovascular disorders. In addition to cardiomyocyte injury, ischemia-reperfusion (I/R)-induced microvascular damage plays a crucial role in determining tissue dysfunction and overall prognosis. Mitochondria-associated endoplasmic reticulum membranes (MAMs), specialized contact sites between the ER and mitochondria, are now recognized as key regulators of cardiovascular pathophysiology. The present review summarized current knowledge of the structure of MAMs and their effects on endothelial cells under hypoxia/reoxygenation conditions. Particular attention was given to their role in regulating mitochondrial quality control processes, including fission, fusion, oxidative stress, mitophagy and Ca2+ homeostasis, within the context of cardiac microvascular I/R injury. Targeting MAMs may represent a promising strategy for microvascular protection in ischemic heart disease.

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.