TRIM28 Is an E3 Ligase of IRP2 Suppressing Ischemia/Reperfusion–Induced Myocardial Ferroptosis

BACKGROUND: Myocardial ischemia/reperfusion (I/R) injury is a common and severe clinical complication in patients with ischemic heart disease after reperfusion therapy. Effective therapeutic strategies for myocardial I/R injury remain limited. Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation. However, the mechanisms underlying ferroptosis in myocardial I/R injury are not fully understood. METHODS: Transcriptomic data from patients with heart failure and cardiomyocytes undergoing ferroptosis were analyzed. Based on the screening results, TRIM28 (tripartite motif-containing 28) expression was evaluated in ferroptotic cardiomyocytes. Cardiac I/R injury models in mice and hypoxia/reoxygenation injury models in neonatal rat ventricular myocytes were established. To explore the function of TRIM28, we used adeno-associated virus serotype 9 to achieve cardiomyocyte-specific overexpression and generated tamoxifen-inducible cardiomyocyte-specific TRIM28 knockout mice. RNA sequencing, coimmunoprecipitation coupled with mass spectrometry, and ubiquitinome profiling were applied to elucidate the underlying mechanisms. Human heart samples from patients with ischemic heart disease were used to evaluate the expression of TRIM28 and its related signaling molecules. The Connectivity Map database was used to screen potential activators of TRIM28. RESULTS: We found that TRIM28 expression was downregulated in ferroptosis inducer-treated and hypoxia/reoxygenation-injured cardiomyocytes, as well as in I/R-injured mouse hearts. Cardiomyocyte-specific overexpression of TRIM28 protected the heart against I/R-induced ferroptosis, whereas its deficiency exacerbated myocardial I/R-induced ferroptotic injury. Mechanistically, TRIM28 functioned as an E3 ubiquitin ligase that directly bound to IRP2 (iron regulatory protein 2) and promoted K48-linked ubiquitination at the K877 site, leading to the downregulation of IRP2 and TFR1 (transferrin receptor 1), suppression of intracellular iron uptake, and consequent attenuation of cardiomyocyte ferroptosis. Furthermore, p55γ interacted with and upregulated TRIM28, thereby mitigating I/R-induced myocardial ferroptosis. Consistent with these findings, protein levels of TRIM28 and p55γ were decreased in heart samples from patients with ischemic heart disease, whereas IRP2 and TFR1 were increased. Last, we demonstrated that perhexiline inhibited I/R-induced myocardial ferroptosis by upregulating p55γ and TRIM28. CONCLUSIONS: Our study identifies TRIM28 as an essential E3 ubiquitin ligase of IRP2 and delineates that TRIM28-mediated inhibition of myocardial ferroptosis by targeting IRP2-TFR1 signaling protects the heart against I/R injury, indicating that targeting TRIM28 represents a promising therapeutic strategy for suppressing cardiomyocyte ferroptosis and I/R injury.