The Effect of Increased HSF1-HMOX1 Expression in H9C2 Cardiomyocytes Following Hypoxia/Reoxygenation on Myocardial Ferroptosis and M1 Polarization of Macrophages

Background: Myocardial ischemia/reperfusion injury (MIRI) remains a leading cause of morbidity and mortality in patients with cardiovascular disease. Ferroptosis, an iron-dependent form of regulated cell death, has been increasingly implicated in cardiomyocyte damage during MIRI. Heat shock factor 1 (HSF1) and heme oxygenase-1 (HMOX1) are stress-responsive proteins, but their interplay in regulating ferroptosis during myocardial injury has not been fully elucidated. This study aimed to investigate the role of the HSF1–HMOX1 axis in modulating ferroptosis and myocardial injury after ischemia/reperfusion (I/R). Methods: An I/R rat model was established by transient ligation of the left anterior descending coronary artery, with sham-operated rats serving as controls. H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R) and co-cultured with RAW 264.7 macrophages were used for in vitro experiments. HSF1 overexpression and knockdown, as well as HMOX1 knockdown via siRNA, were performed. Myocardial injury was assessed by measurement of serum creatine kinase–myocardial band (CK-MB) and cardiac troponin I (cTn-I), as well as histologic and immunohistochemical means. Ferroptosis was evaluated using cell viability assays, reactive oxygen species (ROS) detection, and protein detection of glutathione peroxidase 4 (GPX4), SLC7A11, and ACSL4 by means of Western blotting. Inflammatory responses and macrophage polarization were analyzed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Results: HSF1 and HMOX1 expression were transiently upregulated in myocardial tissues during early I/R but decreased at 24 h. HSF1 overexpression further increased ROS accumulation and exacerbated ferroptosis, as reflected by GPX4 downregulation and ACSL4 upregulation. Conversely, HSF1 knockdown attenuated ferroptosis and injury. HMOX1 knockdown reversed the pro-ferroptotic effects of HSF1 overexpression, indicating that HMOX1 mediates HSF1-induced ferroptosis. Furthermore, the HSF1–HMOX1 axis promoted macrophage polarization toward the pro-inflammatory M1 phenotype, enhancing tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 secretion. Conclusions: The HSF1–HMOX1 axis promotes ferroptosis and exacerbates myocardial damage in ischemia/reperfusion injury by integrating stress response pathways and inflammatory regulation. Inhibiting this axis may represent a promising therapeutic strategy for reducing MIRI and improving cardiac outcomes.