RNF5-mediated ubiquitination of ACSL4 attenuates ferroptosis and confers cardioprotection against myocardial ischemia/reperfusion injury.

Myocardial ischemia/reperfusion (I/R) injury remains a major clinical challenge, with ferroptosis emerging as a critical contributor to cardiomyocyte death. Notably, ring-finger protein 5 (RNF5), which functions as an E3 ubiquitin ligase that is recognized for its crucial involvement in the modulation of endoplasmic reticulum stress, unfolded protein responses, and inflammatory processes, has not been systematically investigated in the process of the onset and evolutionary trajectory of myocardial ischemia/reperfusion (MI/R) injury.Male 7-8 week-oldC57BL/6 mice were used to establish a myocardial injury model of ischemia/reperfusion injury. Adeno-associated virus serotype 9 (AAV9) was used to regulate the expression of RNF5 in vivo and in vitro. Western blot analysis and immunohistochemistry (IHC) were employed to assess the expression levels of RNF5. A TUNEL assay was used to detect the apoptosis of myocardial cells. Immunoprecipitation-mass spectrometry (IP-MS) and coimmunoprecipitation (co-IP) were used to identify the proteins that interact with RNF5 to further explore the underlying mechanism involved.RNF5 was significantly downregulated in the myocardial tissues of MI/R mice. RNF5 overexpression alleviated myocardial tissue damage, reduced ferroptosis levels, and mitigated oxidative stress injury in MI/R model mice. Moreover, RNF5 overexpression markedly suppressed ROS levels, oxidative stress damage, and apoptosis in H9C2 cells subjected to H/R. Mechanistically, we found that RNF5 interacted with Acyl-coA synthetase long-chain family member 4(ACSL4) via its transmembrane region and mediated the degradation of ACSL4, thereby inhibiting the ferroptosis of cardiomyocytes.Our findings reveal a previously unrecognized ubiquitin-dependent mechanism controlling ferroptosis in the heart and position RNF5 as a promising therapeutic target for ischemic cardiomyopathy.