Time-Stable Ferroptosis-Related Candidate Genes in Myocardial Ischemia/Reperfusion Injury Identified by Multi-Time-Point Transcriptomic Analysis: Prioritization of NCF2 Through Public Datasets and AC16 Cell Validation

Background: Ferroptosis contributes to myocardial ischemia/reperfusion injury (MIRI), but time-stable ferroptosis-related transcriptional features across reperfusion stages and their links to peripheral immune profiles remain unclear. This study aimed to identify time-stable ferroptosis-related candidates in MIRI using public datasets and AC16 hypoxia/reoxygenation experiments. Methods: GSE160516, containing reperfusion samples at 6 h, 24 h and 72 h, was used as the primary time-course dataset. Differential expression was analyzed with limma and intersected with FerrDb genes. Candidates were screened for directionally consistent changes, followed by three-time-point FDR filtering and deprioritization of broad inflammatory/tissue-injury-response genes. STRING was used for functional and interaction-network analyses. Directional support was assessed in GSE61592, and human PBMC relevance in GSE59867 with MCPcounter. AC16 cardiomyocytes underwent hypoxia/reoxygenation (H/R) with or without ferrostatin-1 (Fer-1) for ferroptosis-related phenotypic and molecular assessment. Results: Temporal screening yielded 149 candidates, of which 37 remained significant after FDR correction at all three reperfusion time points; deprioritization of seven broad inflammatory/tissue-injury-response genes yielded a 30-gene shortlist, from which ASNS, PSAT1, NCF2, PRKAA2, ULK1 and HIF1A were prioritized. Functional analyses implicated hypoxic response, oxidative stress, metabolic stress, autophagy and inflammation-related processes. In GSE59867 PBMCs, support varied across candidates, with NCF2 and ULK1 remaining significantly elevated in STEMI admission samples after BH correction, while MCPcounter suggested marked acute immune remodeling. In AC16 H/R cells, a ferroptosis-related injury phenotype was partially attenuated by Fer-1; NCF2 protein increased after H/R and was partially reduced by Fer-1. Conclusion: This study identified ferroptosis-related genes with sustained transcriptional changes during MIRI. NCF2 showed the most consistent support across datasets, sample types and AC16 H/R experiments, suggesting it as a prioritized candidate linking ferroptosis-related oxidative stress and myeloid immune activation. A causal role for NCF2 in ferroptosis remains to be established.