Metformin Attenuates Myocardial Ischemia-Reperfusion Injury through the AMPK-HMGCR-ROS Signaling Axis

Objective. To explore the role and mechanism of metformin (MET) in regulating myocardial injury caused by cardiac ischemia-reperfusion.
Material and methods. A rat model of myocardial ischemia-reperfusion injury was established by ligation of the anterior descending branch of the left coronary artery. The myocardial area at risk and the infarction size were measured by Evans blue and 2,3,5‑triphenyltetrazole chloride (TTC) staining, respectively. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) staining was used to detect apoptosis of cardiomyocytes. The expression of 4‑hydroxynonenal (4‑HNE) was detected by immunohistochemical staining. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot were used to detect mRNA and expression of the Adenosine 5‘-monophosphate-activated protein kinase (AMPK) – 3‑hydroxy-3‑methylglutaryl-CoA reductase (HMGCR) signaling pathway, respectively.
Results. MET treatment decreased the infarct size and the activity of the myocardial enzyme profile, thus demonstrating protection of ischemic myocardium. The number of TUNEL positive cells significantly decreased. Immunohistochemical results showed that MET decreased the expression of 4‑HNE in myocardial tissue and the content of malondialdehyde (MDA) in myocardial cells. Further experimental results showed that MET decreased HMGCR transcription and protein expression, and increased AMPK phosphorylation. In the model of hypoxia and reoxygenation injury of cardiomyocytes, MET increased the viability of cardiomyocytes, decreased the activity of lactic dehydrogenase (LDH), decreased malondialdehyde content and intracellular reactive oxygen species (ROS) concentrations, and regulate the AMPK-HMGCR signaling pathway through coenzyme C (ComC).
Conclusion. MET inhibits the expression of HMGCR by activating AMPK, reduces oxidative damage and apoptosis of cardiomyocytes, and alleviates myocardial ischemia-reperfusion injury.

1. Duggan JP, Peters AS, Trachiotis GD, Antevil JL. Epidemiology of Coronary Artery Disease. Surgical Clinics of North America. 2022;102(3):499–516. DOI: 10.1016/j.suc.2022.01.007

2. Algoet M, Janssens S, Himmelreich U, Gsell W, Pusovnik M, Van Den Eynde J et al. Myocardial ischemia-reperfusion injury and the influence of inflammation. Trends in Cardiovascular Medicine. 2023;33(6):357–66. DOI: 10.1016/j.tcm.2022.02.005

3. Wang K, Li Y, Qiang T, Chen J, Wang X. Role of epigenetic regulation in myocardial ischemia/reperfusion injury. Pharmacological Research. 2021;170:105743. DOI: 10.1016/j.phrs.2021.105743

4. Belhadj Slimen I, Najar T, Ghram A, Dabbebi H, Ben Mrad M, Abdrabbah M. Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. A review. International Journal of Hyperthermia. 2014;30(7):513–23. DOI: 10.3109/02656736.2014.971446

5. Xu H, Shen Y, Liang C, Wang H, Huang J, Xue P et al. Inhibition of the mevalonate pathway improves myocardial fibrosis. Experimental and Therapeutic Medicine. 2021;21(3):224. DOI: 10.3892/etm.2021.9655

6. Zhang Y, Wang Y, Xu J, Tian F, Hu S, Chen Y et al. Melatonin attenuates myocardial ischemia-reperfusion injury via improving mitochondrial fusion/mitophagy and activating the AMPK-OPA1 signaling pathways. Journal of Pineal Research. 2019;66(2):e12542. DOI: 10.1111/jpi.12542

7. Sanchez-Rangel E, Inzucchi SE. Metformin: clinical use in type 2 diabetes. Diabetologia. 2017;60(9):1586–93. DOI: 10.1007/s00125-017-4336-x

8. Ritsinger V, Lagerqvist B, Lundman P, Hagström E, Norhammar A. Diabetes, metformin and glucose lowering therapies after myocardial infarction: Insights from the SWEDEHEART registry. Diabetes and Vascular Disease Research. 2020;17(6):1479164120973676. DOI: 10.1177/1479164120973676

9. Bu Y, Peng M, Tang X, Xu X, Wu Y, Chen AF et al. Protective effects of metformin in various cardiovascular diseases: Clinical evidence and AMPK-dependent mechanisms. Journal of Cellular and Molecular Medicine. 2022;26(19):4886–903. DOI: 10.1111/jcmm.17519

10. Fei Q, Ma H, Zou J, Wang W, Zhu L, Deng H et al. Metformin protects against ischaemic myocardial injury by alleviating autophagy-ROS-NLRP3-mediated inflammatory response in macrophages. Journal of Molecular and Cellular Cardiology. 2020;145:1–13. DOI: 10.1016/j.yjmcc.2020.05.016

11. Loi H, Boal F, Tronchere H, Cinato M, Kramar S, Oleshchuk O et al. Metformin Protects the Heart Against Hypertrophic and Apoptotic Remodeling After Myocardial Infarction. Frontiers in Pharmacology. 2019;10:154. DOI: 10.3389/fphar.2019.00154

12. Yang F, Qin Y, Wang Y, Meng S, Xian H, Che H et al. Metformin Inhibits the NLRP3 Inflammasome via AMPK/mTOR-dependent Effects in Diabetic Cardiomyopathy. International Journal of Biological Sciences. 2019;15(5):1010–9. DOI: 10.7150/ijbs.29680

13. Kuburas R, Gharanei M, Haussmann I, Maddock H, Sandhu H. Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK. Journal of Cardiovascular Pharmacology. 2022;79(6):799–807. DOI: 10.1097/FJC.0000000000001256

14. Al-Ani B, Alzamil NM, Hewett PW, Al-Hashem F, Bin-Jaliah I, Shatoor AS et al. Metformin ameliorates ROS-p53-collagen axis of fibrosis and dyslipidemia in type 2 diabetes mellitus-induced left ventricular injury. Archives of Physiology and Biochemistry. 2023;129(3):734–40. DOI: 10.1080/13813455.2020.1869265

15. Choi R, Ham JR, Lee H, Cho HW, Choi M, Park S et al. Scopoletin Supplementation Ameliorates Steatosis and Inflammation in Diabetic Mice. Phytotherapy Research. 2017;31(11):1795–804. DOI: 10.1002/ptr.5925

16. Poornima MS, Sindhu G, Billu A, Sruthi CR, Nisha P, Gogoi P et al. Pretreatment of hydroethanolic extract of Dillenia indica L. attenuates oleic acid induced NAFLD in HepG2 cells via modulating SIRT-1/p-LKB-1/AMPK, HMGCR & PPAR-α signaling pathways. Journal of Ethnopharmacology. 2022;292:115237. DOI: 10.1016/j.jep.2022.115237

17. Wang H, Lin C, Yao J, Shi H, Zhang C, Wei Q et al. Deletion of OSB-PL2 in auditory cells increases cholesterol biosynthesis and drives reactive oxygen species production by inhibiting AMPK activity. Cell Death & Disease. 2019;10(9):627. DOI: 10.1038/s41419-019-1858-9

18. Apaijai N, Inthachai T, Lekawanvijit S, Chattipakorn SC, Chattipakorn N. Effects of dipeptidyl peptidase-4 inhibitor in insulin-resistant rats with myocardial infarction. Journal of Endocrinology. 2016;229(3):245–58. DOI: 10.1530/JOE-16-0096

19. Zeng B, Liu L, Liao X, Zhang C. Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling. Journal of Cellular and Molecular Medicine. 2021;25(7):3205–15. DOI: 10.1111/jcmm.16389

20. Solskov L, Løfgren B, Kristiansen SB, Jessen N, Pold R, Nielsen TT et al. Metformin Induces Cardioprotection against Ischaemia/Reperfusion Injury in the Rat Heart 24 Hours after Administration. Basic Clinical Pharmacology Toxicology. 2008;103(1):82–7. DOI: 10.1111/j.1742-7843.2008.00234.x

21. Mongkolpathumrat P, Kijtawornrat A, Prompunt E, Panya A, Chattipakorn N, Barrère-Lemaire S et al. Post-Ischemic Treatment of Recombinant Human Secretory Leukocyte Protease Inhibitor (rhSL-PI) Reduced Myocardial Ischemia/Reperfusion Injury. Biomedicines. 2021;9(4):422. DOI: 10.3390/biomedicines9040422

22. Patel AMR, Apaijai N, Chattipakorn N, Chattipakorn SC. The Protective and Reparative Role of Colony-Stimulating Factors in the Brain with Cerebral Ischemia/Reperfusion Injury. Neuroendocrinology. 2021;111(11):1029–65. DOI: 10.1159/000512367

23. Oesterle A, Laufs U, Liao JK. Pleiotropic Effects of Statins on the Cardiovascular System. Circulation Research. 2017;120(1):229–43. DOI: 10.1161/CIRCRESAHA.116.308537

24. Shen C, Tan S, Yang J. Effects of continuous use of metformin on cardiovascular outcomes in patients with type 2 diabetes after acute myocardial infarction: A protocol for systematic review and meta-analysis. Medicine. 2021;100(15):e25353. DOI: 10.1097/MD.0000000000025353

25. Rosenstock J, Kahn SE, Johansen OE, Zinman B, Espeland MA, Woerle HJ et al. Effect of Linagliptin vs Glimepiride on Major Adverse Cardiovascular Outcomes in Patients With Type 2 Diabetes: The CAROLINA Randomized Clinical Trial. JAMA. 2019;322(12):1155–66. DOI: 10.1001/jama.2019.13772

26. Liu C, Li Z, Li B, Liu W, Zhang S, Qiu K et al. Relationship between ferroptosis and mitophagy in cardiac ischemia reperfusion injury: a mini-review. PeerJ. 2023;11:e14952. DOI: 10.7717/peerj.14952

27. Zheng D, Chen L, Wei Q, Zhu Z, Liu Z, Jin L et al. Fucoxanthin regulates Nrf2/Keap1 signaling to alleviate myocardial hypertrophy in diabetic rats. Nan Fang Yi Ke Da Xue Xue Bao = Journal of Southern Medical University. 2022;42(5):752–9. DOI: 10.12122/j.issn.1673-4254.2022.05.18

28. Li Y, Yang H, Nong H, Wang F, Wang Y, Xu Y et al. 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) protects hair cells from cisplatin-induced ototoxicity in vitro: possible relation to the activities of p38 MAPK signaling pathway. Archives of Toxicology. 2023;97(11):2955–67. DOI: 10.1007/s00204-023-03588-z

29. Xing H, Liang C, Wang C, Xu X, Hu Y, Qiu B. Metformin mitigates cholesterol accumulation via the AMPK/SIRT1 pathway to protect osteoarthritis chondrocytes. Biochemical and Biophysical Research Communications. 2022;632:113–21. DOI: 10.1016/j.bbrc.2022.09.074

30. Pokhrel RH, Acharya S, Ahn J-H, Gu Y, Pandit M, Kim J-O et al. AMPK promotes antitumor immunity by downregulating PD-1 in regulatory T cells via the HMGCR/p38 signaling pathway. Molecular Cancer. 2021;20(1):133. DOI: 10.1186/s12943-021-01420-9

31. Feng Y, Zhang Y, Xiao H. AMPK and cardiac remodelling. Science China Life Sciences. 2018;61(1):14–23. DOI: 10.1007/s11427-017-9197-5