Myocardial ischemia-reperfusion injury (MIRI) is featured by post-ischemic cardiomyocyte death and reperfusion myocardial damage, which is an unresolved fatal complication in acute myocardial infarction (AMI) treatment. Recent literature has indicated that glutaminase 2 (GLS2) is involved in promoting the ferroptosis of cardiomyocytes, but whether it plays a role in MIRI is still unknown. This research aims to explore the role and mechanism of GLS2 in the development of MIRI. Cell viability and apoptosis were analyzed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry. Bcl-2 related X protein (Bax), Cleaved-caspase-3, Specificity protein 1 (SP1), Glutathione Peroxidase 4 (GPX4), GLS2, p-AMPK, AMPK, p-mTOR, and mTOR protein abundances were determined using Western blot. Interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) levels were analyzed using ELISA. The Fe2+ level in AC16 cells was assessed using the Iron assay kit. Glutathione (GSH), malondialdehyde (MDA), and reactive oxygen species (ROS) products were examined using special assay kits. After JASPAR prediction, the binding between SP1 and GLS2 promoter was verified using Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. The effect of GLS2 on myocardial injury was detected using a mouse MI model. Ischemia/Reperfusion treatment repressed AC16 cell proliferation and induced cell apoptosis, inflammatory response, and ferroptosis. Moreover, GLS2 knockdown relieved hypoxia/reoxygenation (H/R)-triggered AC16 cell injury and ferroptosis. In the mechanism, SP1 was a transcription factor of GLS2 and upregulated the transcription of GLS2 via binding to its promoter region. Silencing of SP1 activated the AMPK/mTOR pathway by inhibiting GLS2. Meanwhile, AMPK inhibitor Dorsomorphin overturned the protective effect of GLS2 downregulation on myocardial cells. GLS2 silencing repressed myocardial damage in vivo. SP1-activated GLS2 could aggravate H/R-induced cardiomyocyte injury and ferroptosis by inactivating the AMPK/mTOR pathway, providing a promising therapeutic target for MIRI treatment in the future.
Keywords: AMPK/mTOR; GLS2; SP1; ferroptosis; hypoxia/reoxygenation.
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