Background: Mitochondrial metabolism and immune inflammation play pivotal roles in the MI pathogenesis of myocardial infarction (MI); however, their interplay remains unclear. This study aimed to clarify the roles of mitochondrial metabolism and immune infiltration in MI, using a combination of bioinformatics analyses and experimental validation.

Methods: MI chip data (GSE96561, GSE181872, and GSE183272) were obtained from the Gene Expression Omnibus (GEO) database, and mitochondrial gene data were sourced from the MitoCarta3.0 database. Differentially expressed genes (DEGs) were identified and subjected to functional enrichment analyses. Mitochondria-related DEGs (mitoDEGs) were determined by intersecting DEGs with mitochondrial genes and associated Gene Ontology (GO) terms were analyzed using the Metascape database. A protein-protein interaction (PPI) network of mitoDEGs was constructed, and hub mitoDEGs associated with MI were identified using CytoHubba and molecular complex detection (MCODE) algorithms. Transcription factor (TF) and microRNA (miRNA) targets of hub mitoDEGs were predicted using iRegulon and miRWalk plug-ins, respectively, and a regulatory network involving TFs, hub mitoDEGs, and miRNA was established. Immune infiltration in MI was analyzed using ImmuCellAI, and the relationship between hub mitoDEGs and immune infiltration abundance was assessed using the Spearman method. Experimental validation of hub mitoDEGs, immune cell markers (F4/80, CD163 and CD86), and apoptosis-related proteins (BAX/BCL-2 and cleaved caspase-3) was conducted in MI mice, and the association with cardiac function was explored.

Results: MitoDEGs in the MI group were significantly enriched in pathways related to mitochondrial transport and gene expression. Nine hub mitoDEGs closely associated with MI were identified. Immune analysis revealed increased infiltration of mast and plasma cells infiltration and decreased CD4 T cell infiltration in the MI immune microenvironment. Spearman analysis showed positive correlations between hub mitoDEGs and M1 macrophages, Th2 Cells, and monocytes and negative correlations with eosinophils and activated T cells. In MI mice, expression trends of four hub MitoDEGs (Cox5b, Ndufa2, Ndufs6, and Uqcr11) were consistent with the bioinformatics results, and their downregulation was associated with reduced cardiac function. CD86 and apoptosis-related proteins (BAX/BCL-2 and cleaved caspase-3) were markedly elevated in MI groups.

Conclusion: These findings suggest that Cox5b, Ndufa2, Ndufs6, and Uqcr11 act as core regulatory molecules in immunometabolism during MI, providing new insights into its pathogenesis and diagnosis.

Keywords: bioinformatics analysis; differentially expressed genes; immune infiltration; immunohistochemistry; immunometabolism; ischemia; mitochondrial metabolism; myocardial infarction.

Copyright © 2025 Hou, Wang, Zhu and Li.

背景： 线粒体代谢和免疫炎症在心肌梗死（MI）的发病机制中起着关键作用，但它们之间的相互作用仍然不清楚。本研究旨在通过结合生物信息学分析和实验验证来阐明线粒体代谢和免疫浸润在 MI 中的作用。

方法： 从 Gene Expression Omnibus (GEO) 数据库获取 MI 芯片数据（GSE96561，GSE181872 和 GSE183272），并从 MitoCarta3.0 数据库中获得线粒体基因数据。识别差异表达基因 (DEGs)，并对它们进行功能富集分析。通过将 DEGs 与线粒体基因相交来确定线粒体相关 DEGs（mitoDEGs），使用 Metascape 数据库对相关的 Gene Ontology (GO) 术语进行分析。利用 CytoHubba 和分子复合物检测 (MCODE) 算法构建 mitoDEGs 的蛋白质-蛋白质相互作用 (PPI) 网络，并识别与 MI 相关的中心 mitoDEGs。使用 iRegulon 插件预测 hub mitoDEGs 的转录因子 (TF) 和 microRNA (miRNA) 目标，分别使用 miRWalk 插件进行，从而建立涉及 TF、hub mitoDEGs 和 miRNA 的调控网络。使用 ImmuCellAI 分析 MI 中的免疫浸润，并利用 Spearman 方法评估 hub mitoDEGs 与免疫浸润丰度之间的关系。在 MI 小鼠中对 hub mitoDEGs、免疫细胞标记物（F4/80，CD163 和 CD86）和凋亡相关蛋白（BAX/BCL-2 和裂解的 caspase-3）进行实验验证，并探讨其与心脏功能的关系。

结果： MI 组中的 mitoDEGs 在线粒体转运和基因表达相关的通路中显著富集。确定了九个与 MI 密切相关的 hub mitoDEGs。免疫分析显示，MI 免疫微环境中肥大细胞和平滑肌成纤维细胞浸润增加而 CD4 T 细胞浸润减少。Spearman 分析表明，hub mitoDEGs 与 M1 巨噬细胞、Th2 细胞和单核细胞呈正相关，而与嗜酸性粒细胞和活化的 T 细胞呈负相关。在 MI 小鼠中，四个 hub MitoDEGs（Cox5b、Ndufa2、Ndufs6 和 Uqcr11）的表达趋势与生物信息学结果一致，并且它们下调与心脏功能下降有关。MI 组中的 CD86 以及凋亡相关蛋白 (BAX/BCL-2 和裂解的 caspase-3) 显著升高。

结论： 这些发现表明，Cox5b、Ndufa2、Ndufs6 和 Uqcr11 在 MI 中作为免疫代谢的核心调控分子发挥作用，为该病的发病机制和诊断提供了新的见解。

关键词： 生物信息学分析；差异表达基因；免疫浸润；免疫组织化学；免疫代谢；缺血；线粒体代谢；心肌梗死。

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