Irisin Mitigates Myocardial Hypoxia/Reoxygenation Injury by Preserving Mitochondrial Redox Homeostasis via the UCP2-SOD2 Axis

Abstract

Introduction: Mitochondrial redox homeostasis is of utmost significance in myocardial ischemia-reperfusion (I/R) injury. Irisin, a myokine, has drawn extensive attention in research regarding the protection against cardiovascular diseases.

Methods: This study utilized in vitro Hypoxia/Reoxygenation (H/R) models in H9c2 cardiomyocytes to simulate I/R injury. Cells were pretreated with irisin (20 ng/mL) prior to reoxygenation. UCP2 knockdown was achieved via siRNA/shRNA transfection. Cell viability and apoptosis were assessed using CCK-8 and flow cytometry (Annexin V-FITC/PI staining), respectively. Intracellular calcium dynamics were monitored by Fluo-3/AM confocal imaging, while ROS levels were quantified via DCFH-DA flow cytometry. Key oxidative stress markers (LDH, MDA, GSH-Px, and CAT) and protein expression (ASC, NLRP3, SIRT1, UCP2, and SOD2) were evaluated using commercial kits and Western blotting. Protein interactions were analyzed by coimmunoprecipitation, and ubiquitination levels were measured under proteasomal/lysosomal inhibition (MG132/Leupeptin).

Results: Irisin attenuated H/R injury in cardiomyocytes by suppressing apoptosis, calcium/ROS overload, and NLRP3 activation through a UCP2-dependent pathway. UCP2 knockdown significantly attenuated irisin's protection and reduced SOD2 protein stability. Mechanistically, UCP2 bound SOD2 and inhibited its ubiquitin-proteasomal degradation.

Discussion: This study reveals a novel mechanism where irisin enhances mitochondrial redox homeostasis by promoting UCP2's function, which stabilizes SOD2 against ubiquitin-proteasomal degradation. This UCP2-SOD2 axis attenuates oxidative stress and inhibits NLRP3 inflammasome activation during cardiac injury, offering a promising dual-targeted therapeutic strategy for I/R injury.

Conclusion: Irisin protects cardiomyocytes against H/R injury primarily via a novel UCP2-SOD2 axis.

Keywords: Irisin; SOD2; UCP2; hypoxia/reoxygenation (H/R); oxidative stress; ubiquitylation..