Integrated bioinformatics analysis, machine learning, and experimental validation reveal that ACSL1 drives myocardial ischemia reperfusion injury via ferroptosis.

Myocardial ischemia-reperfusion injury (MIRI) is a significant factor in the development of cardiac dysfunction following an acute myocardial infarction (AMI). Ferroptosis, a type of regulated cell death driven by iron and marked by lipid peroxidation, has attracted increasing attention for its pivotal role in the pathogenesis of MIRI. It has been reported that acyl-CoA synthetase long chain family member 1 (ACSL1), a ferroptosis promoter, could mediate ferroptosis of myocardial cells during AMI. However, the specific function and mechanism of ACSL1 in AMI are still unclear. Differentially expressed genes in AMI were identified from the Gene Expression Omnibus (GEO) databases (GSE166780 and GSE97320), and ferroptosis-related genes were acquired from the PathCards database. Through integrated analysis of the two genomes, ferroptosis-related genes in AMI were identified. Then, the identified genes were subjected to cross-validation using three machine learning algorithms (LASSO, SVM-RFE, and RF), ultimately identifying characteristic genes. ACSL1, Forkhead box protein O4 (FOXO4), tripartite motif-containing protein 25 (TRIM25), and GPX4 protein levels were detected using western blot. Cell viability and apoptosis were Cell Counting Kit-8 (CCK-8) and flow cytometry. Interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) levels were analyzed using enzyme-linked immunosorbent assay (ELISA). Fe2+ level, lipid reactive oxygen species (ROS) level, and GSH level were examined using commercial kits. Flow cytometric analysis of mitochondrial membrane potential using JC-1. Binding between FOXO4 and ACSL1 promoter was predicted by JASPAR and verified using dual-luciferase reporter and ChIP assays. The stability of ACSL1 was assessed by CHX assay. Interaction between TRIM25 and ACSL1 was verified using Co-immunoprecipitation (CoIP) assay. The effect of ACSL1 on myocardial injury was detected using a mouse MIR model. After screening and identification, ferroptosis-related gene ACSL1 in AMI was selected for this study. ACSL1 expression was increased in AMI patients and Ischemia/Reperfusion (H/R)-treated AC16 cells. H/R-triggered AC16 cell viability inhibition, and apoptosis, inflammatory response, ferroptosis, and mitochondrial dysfunction promotion were partly abolished by ACSL1 silencing. Mechanistically, FOXO4 activated ACSL1 transcription by binding to its promoter region. TRIM25 facilitated ACSL1 ubiquitination and decreased its protein stability. ACSL1 downregulation could relieve myocardial damage in vivo. ACSL1 was identified as a key ferroptosis-related gene in AMI. Furthermore, FOXO4-activated transcription and TRIM25-mediated ubiquitination-dependent degradation of ACSL1 could affect H/R-induced cardiomyocyte damage and ferroptosis, providing a promising therapeutic target for MIRI treatment.