EFHD1: A Potential Prognostic Biomarker Related to Mitochondrial Function and Aging in Atherosclerosis Plaque.

Introduction: Atherosclerosis (AS) is prevalent among the elderly population and poses a significant global health burden. However, the precise underlying mechanisms linking aging and mitochondrial dysfunction in AS remain unclear.

Methods: Through comprehensive utilization of databases including the Gene Expression Omnibus (GEO), MitoCarta, Molecular Signatures Database (MSigDB), and Human Aging Genomic Resources (HAGR), we employed various bioinformatics methods to explore the possible function of EF-hand domain family member D1 (EFHD1). This included the functional enrichment analysis, immune cell infiltration, and the lncRNA-miRNA-EFHD1 network. The validity of EFHD1 was confirmed using additional datasets and through Receiver Operating Characteristic (ROC) curve evaluation. Lastly, in vitro experiments were conducted using THP-1 cells treated with oxidized low-density lipoprotein (ox-LDL) to validate the expression and function of EFHD1 through Western Blot and real-time quantitative PCR analyses. Additionally, in vivo experiments were performed on ApoE-/- mice exhibiting atherosclerotic phenotypes, utilizing immunofluorescence staining.

Results: Totally seven genes associated with aging and mitochondrial function (ALDH3A2, UCP1, BCL2, EFHD1, AHCYL1, HTRA2, and ALDH9A1) were discovered in AS, with EFHD1 identified as the principal hub gene. Immune infiltration analysis indicated that EFHD1 was negatively associated with myeloid suppressor cells (MDSC), activated B cells, and natural killer cells. An evident decline in EFHD1 was noted in unstable or advanced plaques compared to stable or early plaques, accompanied by significant area under the ROC curve (AUC) values of 0.917 (GSE100927) and 0.933 (GSE41571). Moreover, we recorded a reduction in EFHD1 expression in AS tissues and macrophages treated with ox-LDL. Following the silencing of EFHD1, TNF-α and IL-1β decreased, while ALODA, PKM2, MMP-9, JAK2, and STAT3 levels were upregulated. Furthermore, levels of ATP and reactive oxygen species (ROS) were diminished, while calcium ions and mitochondria levels remained unchanged.

Discussion: To date, the common pathogenic genes associated with aging and mitochondrial dysfunction in atherosclerotic disease have been scarcely investigated. Using bioinformatics approaches, we identified seven hub genes (ALDH3A2, UCP1, BCL2, EFHD1, AHCYL1, HTRA2, and ALDH9A1) related to mitochondrial function and aging. Among these, EFHD1 was determined as the final hub gene. As a calcium sensor, EFHD1 plays a pivotal role in regulating mitochondrial metabolism and has been implicated in the prognosis of various tumors. Our findings demonstrated that EFHD1 knockdown decreased the levels of pro-inflammatory cytokines, such as IL-1β and TNF-α, increased JAK2 and STAT3 protein levels, and elevated MMP-9 levels, all of which may contribute to the vulnerability and progression of atherosclerotic plaques.

Conclusion: Our research revealed a reduction in EFHD1 expression within atherosclerotic tissues, suggesting its potential role in inflammation and mitochondrial energy metabolism as a key regulator of the calcium signaling pathway. This discovery offers possible advancements in the early diagnosis and treatment strategies for AS.