Journal of Shandong University (Health Sciences) ›› 2026, Vol. 64 ›› Issue (8): 48-55.doi: 10.6040/j.issn.1671-7554.0.2025.0847

• Preclinical Medicine • Previous Articles    

N-glycoproteomic analysis of AC16 cardiomyocytes after hypoxia/reoxygenation injury

REN Yanxin1,2, HUANG Qingwen2, JIA Wenjuan2, YANG Jun1,2   

  1. 1. Second Clinical Medical College of Binzhou Medical University, Yantai 264003, Shandong, China;
    2. Department of Cardiology, Yantai Yuhuangding Hospital, Yantai 264001, Shandong, China
  • Published:2026-08-14

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Abstract: Objective To profile the quantitative N-glycoproteome of AC16 cardiomyocytes under normoxia and following hypoxia/reoxygenation(H/R)via mass spectrometry-based glycoproteomics, aiming to identify novel therapeutic targets for myocardial ischemia-reperfusion injury(MIRI). Methods After subjecting the cells to hypoxia/reoxygenation treatment, quantitative N-glycoproteomics mass spectrometry was employed to identify differentially expressed glycoproteins between the control group and the H/R group. Bioinformatics analysis was performed to screen for key regulatory molecules. The expression profiles of core candidate targets were subsequently validated by Western blotting analysis. Results Proteomics identified 698 proteins, including 66 upregulated(42 differentially glycosylated, 186 modification sites)and 29 downregulated(7 differentially glycosylated, 27 sites). Pathway analysis showed these proteins were mainly involved in the PI3K/AKT signaling pathway(P<0.05)and ECM-receptor interaction(P<0.05). Mechanistic studies found cluster of differentiation 44(CD44)and hypoxia up-regulated 1(HYOU1)to be significantly upregulated in the H/R model(P<0.05). Conclusion The study offers a systems-level view of altered N-glycoproteins and glycosylation sites in AC16 cells after H/R injury. It discovered a series of glycoproteins with abnormal N-glycosylation levels related to MIRI, such as CD44 and HYOU1. These findings may offer novel treatment strategies for targeted glycotope intervention and myocardial protection.

Key words: AC16 cardiomyocyte, Myocardial ischemia-reperfusion injury, Proteomics, N-glycosylation, Mass spectrometry analysis

CLC Number: 

  • R33
[1] Mensah G A, Fuster V, Roth G A. A heart-healthy and stroke-free world: using data to inform global action[J]. J Am Coll Cardiol, 2023, 82(25): 2343-2349.
[2] Banharak S, Threeranut A, Metprommarat A. Effects of health education applying role play on decision making in acute myocardial infarction situation among older adults in community[J]. Sci Rep, 2024, 14(1): 19627. DOI:10.1038/s41598-024-70747-2
[3] Salari N, Morddarvanjoghi F, Abdolmaleki A, et al. The global prevalence of myocardial infarction: a systematic review and meta-analysis[J]. BMC Cardiovasc Disord, 2023, 23(1): 206. DOI:10.1186/s12872-023-03231-w
[4] Huang Z Y, Lai P F, Cocker A T H, et al. Roles of N-linked glycosylation and glycan-binding proteins in placentation: trophoblast infiltration, immunomodulation, angiogenesis, and pathophysiology[J]. Biochem Soc Trans, 2023, 51(2): 639-653.
[5] Chan M Y, Efthymios M, Tan S H, et al. Prioritizing candidates of post-myocardial infarction heart failure using plasma proteomics and single-cell transcriptomics[J]. Circulation, 2020, 142(15): 1408-1421.
[6] Nurmohamed N S, Kraaijenhof J M, Mayr M, et al. Proteomics and lipidomics in atherosclerotic cardiovascular disease risk prediction[J]. Eur Heart J, 2023, 44(18): 1594-1607.
[7] 王根弟, 田心, 张军茹.心肌缺血再灌注损伤的代谢组学研究进展[J]. 宁夏医科大学学报, 2023, 45(1): 99-105. Wang Gendi, Tian Xin, Zhang Junru. Progress in metabonomics of myocardial ischemia-reperfusion injury[J]. Journal of Ningxia Medical University, 2023, 45(1): 99-105.
[8] Schjoldager K T, Narimatsu Y, Joshi H J, et al. Global view of human protein glycosylation pathways and functions[J]. Nat Rev Mol Cell Biol, 2020, 21(12): 729-749.
[9] Esmail S, Manolson M F. Advances in understanding N-glycosylation structure, function, and regulation in health and disease[J]. Eur J Cell Biol, 2021, 100(7/8): 151186. DOI:10.1016/j.ejcb.2021.151186
[10] Ramakrishnan K, Johnson R L, Winter S D, et al. Glycosylation increases active site rigidity leading to improved enzyme stability and turnover[J]. FEBS J, 2023, 290(15): 3812-3827.
[11] Ji Y, Li B Z, Qiao M, et al. Advances on the in vivo and in vitro glycosylations of flavonoids[J]. Appl Microbiol Biotechnol, 2020, 104(15): 6587-6600.
[12] Hansen A L, Reily C, Novak J, et al. Immunoglobulin a glycosylation and its role in disease[J]. Exp Suppl, 2021, 112: 433-477. DOI:10.1007/978-3-030-76912-3_14
[13] He X F, Hu X L, Wen G J, et al. O-GlcNAcylation in cancer development and immunotherapy[J]. Cancer Lett, 2023, 566: 216258. DOI:10.1016/j.canlet.2023.216258
[14] Yang X Y, Qian K. Protein O-GlcNAcylation: emerging mechanisms and functions[J]. Nat Rev Mol Cell Biol, 2017, 18(7): 452-465.
[15] Li S T, Meng J J, Lv Y Z, et al. Changes in serum IgG glycosylation patterns for abdominal aortic aneurysm patients[J]. J Cardiovasc Dev Dis, 2022, 9(9): 291. DOI:10.3390/jcdd9090291
[16] Pinho S S, Reis C A. Glycosylation in cancer: mechanisms and clinical implications[J]. Nat Rev Cancer, 2015, 15(9): 540-555.
[17] Cho S H, Park J Y, Kim C H. Systemic lectin-glycan interaction of pathogenic enteric bacteria in the gastrointestinal tract[J]. Int J Mol Sci, 2022, 23(3): 1451. DOI:10.3390/ijms23031451
[18] 茹凝玉, 陈果, 余志斌. CD147糖基化与心血管疾病[J]. 心脏杂志, 2020, 32(1): 82-87. Ru Ningyu, Chen Guo, Yu Zhibin. Glycosylation of CD147 and cardiovascular diseases[J]. Chinese Heart Journal, 2020, 32(1): 82-87.
[19] Chatham J C, Patel R P. Protein glycosylation in cardiovascular health and disease[J]. Nat Rev Cardiol, 2024, 21(8): 525-544.
[20] Radovani B, Gudelj I. N-glycosylation and inflammation; the not-so-sweet relation[J]. Front Immunol, 2022, 13: 893365. DOI:10.3389/fimmu.2022.893365
[21] 王思敏, 燕银芳, 纪玉强.蛋白质N-糖基化在心血管疾病中的研究进展[J]. 中国动脉硬化杂志, 2023, 31(12): 1091-1097. Wang Simin, Yan Yinfang, Ji Yuqiang. Progression of research on protein N-glycosylation in cardiovascular diseases[J]. Chinese Journal of Arteriosclerosis, 2023, 31(12): 1091-1097.
[22] Zhong L, Zhao J Q, Huang L, et al. Runx2 activates hepatic stellate cells to promote liver fibrosis via transcriptionally regulating Itgav expression[J]. Clin Transl Med, 2023, 13(7): e1316. DOI:10.1002/ctm2.1316
[23] 周晨明, 崔芳, 石葛明, 等. HYOU1在糖尿病施万细胞凋亡中的作用及对JAK-STAT通路的影响研究[J]. 河北医科大学学报, 2024, 45(12): 1374-1379. Zhou Chenming, Cui Fang, Shi Geming, et al. Effect of HYOU1 on apoptosis of diabetic Schwann cells and its effect on JAK-STAT pathway[J]. Journal of Hebei Medical University, 2024, 45(12): 1374-1379.
[24] Ge C W, Peng Y Q, Li J C, et al. Hydroxysafflor yellow a alleviates acute myocardial ischemia/reperfusion injury in mice by inhibiting ferroptosis via the activation of the HIF-1α/SLC7A11/GPX4 signaling pathway[J]. Nutrients, 2023, 15(15): 3411. DOI:10.3390/nu15153411
[25] Han X, Jiang Z C, Hou Y F, et al. Myocardial ischemia-reperfusion injury upregulates nucleostemin expression via HIF-1α and c-Jun pathways and alleviates apoptosis by promoting autophagy[J]. Cell Death Discov, 2024, 10(1): 461. DOI:10.1038/s41420-024-02221-x
[26] Zheng J, Chen P E, Zhong J F, et al. HIF-1α in myocardial ischemia-reperfusion injury(Review)[J]. Mol Med Rep, 2021, 23(5): 352. DOI:10.3892/mmr.2021.11991
[27] Wang Z, Tan C, Duan C H, et al. FUT2-dependent fucosylation of HYOU1 protects intestinal stem cells against inflammatory injury by regulating unfolded protein response[J]. Redox Biol, 2023, 60: 102618. DOI:10.1016/j.redox.2023.102618
[28] Rao S, Oyang L, Liang J X, et al. Biological function of HYOU1 in tumors and other diseases[J]. Onco Targets Ther, 2021, 14: 1727-1735. DOI:10.2147/OTT.S297332
[29] Vuorio J, Škerlová J, Fábry M, et al. N-Glycosylation can selectively block or foster different receptor-ligand binding modes[J]. Sci Rep, 2021, 11(1): 5239. DOI:10.1038/s41598-021-84569-z
[30] Kashyap T, Pramanik K K, Nath N, et al. Crosstalk between Raf-MEK-ERK and PI3K-Akt-GSK3β signaling networks promotes chemoresistance, invasion/migration and stemness via expression of CD44 variants(v4 and v6)in oral cancer[J]. Oral Oncol, 2018, 86: 234-243. DOI:10.1016/j.oraloncology.2018.09.028
[31] Zhang Y F, Wang X C, Ji Y W, et al. All-trans retinoic acid pretreatment of mesenchymal stem cells enhances the therapeutic effect on acute kidney injury[J]. Cell Commun Signal, 2024, 22(1): 291. DOI:10.1186/s12964-024-01671-1
[32] Walkowski B, Kleibert M, Majka M, et al. Insight into the role of the PI3K/Akt pathway in ischemic injury and post-infarct left ventricular remodeling in normal and diabetic heart[J]. Cells, 2022, 11(9): 1553. DOI:10.3390/cells11091553
[33] Ghafouri-Fard S, Khanbabapour Sasi A, Hussen B M, et al. Interplay between PI3K/AKT pathway and heart disorders[J]. Mol Biol Rep, 2022, 49(10): 9767-9781.
[34] Yao Z Y, Wu J H, Fang Y. Moderate constraint facilitates association and force-dependent dissociation of HA-CD44 complex[J]. Int J Mol Sci, 2023, 24(3): 2243. DOI:10.3390/ijms24032243
[35] Suleiman M, Abdulrahman N, Yalcin H, et al. The role of CD44, hyaluronan and NHE1 in cardiac remodeling[J]. Life Sci, 2018, 209: 197-201. DOI:10.1016/j.lfs.2018.08.009
[36] Guo N N, Wang X, Xu M R, et al. PI3K/AKT signaling pathway: Molecular mechanisms and therapeutic potential in depression[J]. Pharmacol Res, 2024, 206: 107300. DOI:10.1016/j.phrs.2024.107300
[37] Wei X H, Chen J, Wu X F, et al. Salvianolic acid B alleviated myocardial ischemia-reperfusion injury via modulating SIRT3-mediated crosstalk between mitochondrial ROS and NLRP3[J]. Phytomedicine, 2025, 136: 156260. DOI:10.1016/j.phymed.2024.156260
[38] Huang Q W, Dong H B, Jia W J, et al. Regulation of N-glycosylation of CDNF on its protein stability and function in hypoxia/reoxygenation model of H9C2 cells[J]. Cell Biol Int, 2025, 49(5): 472-483.
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