(*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). Compared with the WT group, the SI/R group exhibited an upregulation in O-GlcNAcylation levels Nifuroxazide (P<0.01) (Fig.4c,d), but SIRT3 activity showed a slight decrease (P<0.01) (Fig.4e). while mitochondrial reactive oxygen species (MROS) levels and cellular apoptosis were assessed using immunofluorescence. We have identified an interaction between SIRT3 and OGT, where SIRT3 undergoes dynamic O-GlcNAcylation at the S190 site, facilitating SIRT3 deacetylase activity. S5mt During SI/R, elevated levels of O-GlcNAcylation activate SOD2 by promoting SIRT3 enzyme activity, thereby inhibiting excessive MROS production. This significantly mitigates the occurrence of malignant autophagy in myocardial cells during reperfusion, promoting their survival. Conversely, blocking SIRT3 O-GlcNAcylation at the S190 site exacerbates SI/R injury. We demonstrate that O-GlcNAcylation is a crucial post-translational modification (PTM) of SIRT3 during SI/R, shedding light on a promising mechanism for future therapeutic approaches. Keywords:SI/R, SIRT3, O-GlcNAcylation Subject terms:Glycosylation, Myocardial infarction == Introduction == Acute myocardial infarction (AMI) is a serious cardiovascular disease that poses a significant threat to human health1,2. Reperfusion therapy is currently the most effective treatment method for reducing ischemic damage3. Unfortunately, myocardial ischemia-reperfusion (MI/R) can lead to additional myocardial injury4,5. Therapeutic strategies aimed at reducing MIRI have been found to be significantly limited in their effectiveness6,7. Targeted reperfusion therapy has been a focal point and bottleneck of research. Autophagy is believed to play a crucial role in MIRI8. Autophagy acts as a protective mechanism during cardiac ischemia by rapidly releasing energy substrates into the cytoplasm, generating ATP through the tricarboxylic acid cycle, thereby reducing cellular metabolic burden and providing energy9. Many studies have demonstrated the protective role of autophagy during the ischemic process10,11. However, continuous expression of autophagy during ischemic organ reperfusion may cause damage or even cell death1214. Extensive research has been conducted on the molecular mechanisms of Nifuroxazide excessive autophagy during myocardial reperfusion. Some studies suggest that excessive MROS generated during reperfusion can significantly induce autophagy and exacerbate myocardial injury1517. MROS are primarily degraded by SOD2, whose activity is mainly influenced by its acetylation level18. SIRT3 is a member of the class III histone deacetylases (HDACs) and is the main deacetylase of mitochondria. SIRT3 regulates the antioxidant enzyme activity of SOD2 through deacetylation19. Studies have shown that SIRT3 plays a crucial role in cardiac ROS regulation and can thereby modulate autophagy levels during reperfusion, alleviating reperfusion injury2022. Therefore, elucidating the activity regulation mechanism of SIRT3 during MI/R is crucial, yet relevant research remains scarce. In recent years, O-GlcNAcylation has emerged as a PTM that links changes in cellular metabolism with protein function, becoming a regulatory factor in cardiovascular homeostasis23. Protein O-GlcNAcylation is a highly dynamic and reversible nutrient-driven process24, controlled by OGT and O-GlcNAcase (OGA), Nifuroxazide where OGT catalyzes the attachment of a single Nifuroxazide sugar to target proteins, while OGA reverses this process25. One of the earliest cellular responses to various stresses is a rapid increase in O-GlcNAcylation rates26. Numerous studies have shown that this adaptive increase contributes to enhanced cell survival during MI/R, exerting a protective effect2729. However, the specific mechanisms remain unclear. The overlapping roles of SIRT3 and O-GlcNAcylation in MIRI protection have led us to consider the possibility of O-GlcNAcylation modifying SIRT3. In recent years, several members of the Sirtuins family have been found to possess O-GlcNAcylation sites, significantly regulating their activity and involvement in physiological and pathological processes. For instance, SIRT1 is O-GlcNAcylated at Ser 549 at its carboxyl terminus, enhancing its deacetylase activity and protecting cells from stress-induced apoptosis30. Similarly, O-GlcNAcylation modification at Ser 136 of SIRT7 is crucial for maintaining its protein stability and deacetylase ability, and blocking SIRT7 O-GlcNAcylation can delay tumor progression31. Therefore, we speculate that SIRT3 may also possess O-GlcNAcylation sites and significantly regulate its deacetylation ability. In this study, we established an SI/R model to confirm that O-GlcNAcylation of SIRT3 enhances its deacetylase activity, thereby activating SOD2 activity, promoting the degradation of MROS during SI/R, and inhibiting maladaptive autophagy. During SI/R, the loss of SIRT3 O-GlcNAcylation suppresses the SIRT3-SOD2-MROS axis, exacerbating cell apoptosis. These data elucidate a novel molecular mechanism of SIRT3, which may offer new insights and approaches for the clinical treatment of myocardial infarction in the future. == Methods == == Cell culture == The cell lines H9C2 were obtained from Procell and cultured in H9C2-specific growth medium (Procell, CM-0089) at 37 C with 5% carbon dioxide in a cell culture incubator (Thermo). The medium was changed based on the cell growth and adherence status. During medium change, the old medium was first discarded, followed by washing with PBS (Sevenbiotech, SC106) for 23 times, and then fresh complete medium was added. If drug stimulation was applied, the drug was added after medium change. == PUGNAC drug intervention == When H9C2 cells reach 80% confluence in the culture flask, PUGNAC (Santa Cruz.