CBL knockdown protects cardiomyocytes against hypoxia‑reoxygenation injury by downregulating GRB2 expression

Lv,Zhengbing; Luo,Xiaojia; Hong,Biying; Ye,Qiran; Liu,Jianxiong; Hu,Yongmei

doi:10.3892/etm.2022.11111

CBL knockdown protects cardiomyocytes against hypoxia‑reoxygenation injury by downregulating GRB2 expression

Authors:
- Zhengbing Lv
- Xiaojia Luo
- Biying Hong
- Qiran Ye
- Jianxiong Liu
- Yongmei Hu
View Affiliations

Affiliations: Department of Cardiology, The Second People's Hospital of Chengdu, Chengdu, Sichuan 610017, P.R. China, Department of Biotechnology, College of Life Science Sichuan University, Chengdu, Sichuan 610000, P.R. China
Published online on: January 3, 2022 https://doi.org/10.3892/etm.2022.11111
Article Number: 188
Copyright: © Lv et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Myocardial ischemia‑reperfusion injury (MIRI) is an event that follows a myocardial infarction. As such, close observation and appropriate patient management is paramount in the treatment process of interventional surgery. The pathogenesis of MIRI has not been fully elucidated. Therefore, the aim of the present study was to explore of novel targets for MIRI treatment whilst also determining their possible underlying mechanism of action. The plasma samples used in the present study were collected from 30 patients with ischemic cardiomyopathy and 30 healthy volunteers. H9c2 rat cardiomyoblasts were subjected to hypoxia and reoxygenation (H/R) modeling to establish an in vitro MIRI model. Initially, the expression levels of Cbl proto‑oncogene (CBL) in ICM heart tissue, normal heart tissue, H/R‑induced H9c2 cells and normal H9c2 cells were detected using quantitative PCR and western blotting. With the application of Cell Counting Kit‑8, western blotting and Tunnel assay, the proliferation, oxidative stress and apoptosis of H/R‑induced cells were assessed. Moreover, co‑IP assay was employed to testify the interaction between CBL and GRB2. The present study revealed that CBL expression was upregulated in patients with ischemic cardiomyopathy and H/R‑induced H9c2 cells in comparison with that in normal heart tissue and normal H9c2 cells, respectively. The genetic silence of CBL using small interfering RNA promoted the proliferation and oxidative stress of H/R‑induced cells but repressed the apoptosis. The full‑length wild‑type of growth factor receptor‑bound protein 2 (GRB2) was ligated into pcDNA3.1 to achieve GRB2 overexpression, which revealed that GRB2 overexpression reversed the effects of CBL knockdown on cells, suggesting that it may mediate these processes downstream. In conclusion, under hypoxic conditions, CBL knockdown promoted the proliferation and antioxidant capacity of cardiomyocytes whilst inhibiting apoptosis, by downregulating GRB2 expression. These findings revealed the underlying mechanism of action of this pathway, which can be exploited for the prevention or treatment of MIRI.

View Figures

View References

1	Brueck M, Koerholz D, Nuernberger W, Juergens H, Goebel U and Wahn V: Elimination of l-asparaginase in children treated for acute lymphoblastic leukemia. Dev Pharmacol Ther. 12:200–204. 1989.PubMed/NCBI
2	Roth GA, Mensah GA, Johnson CO, Addolorato G, Ammirati E, Baddour LM, Barengo NC, Beaton AZ, Benjamin EJ, Benziger CP, et al: Global burden of cardiovascular diseases and risk factors, 1990-2019: Update from the GBD 2019 study. J Am Coll Cardiol. 76:2982–3021. 2020.PubMed/NCBI View Article : Google Scholar
3	Dang H, Ye Y, Zhao X and Zeng Y: Identification of candidate genes in ischemic cardiomyopathy by gene expression omnibus database. BMC Cardiovasc Disord. 20(320)2020.PubMed/NCBI View Article : Google Scholar
4	Dixon SR, Henriques JP, Mauri L, Sjauw K, Civitello A, Kar B, Loyalka P, Resnic FS, Teirstein P, Makkar R, et al: A prospective feasibility trial investigating the use of the Impella 2.5 system in patients undergoing high-risk percutaneous coronary intervention (The PROTECT I Trial): Initial U.S. experience. JACC Cardiovasc Interv. 2:91–96. 2009.PubMed/NCBI View Article : Google Scholar
5	Henriques JP, Remmelink M, Baan J Jr, van der Schaaf RJ, Vis MM, Koch KT, Scholten EW, de Mol BA, Tijssen JG, Piek JJ and de Winter RJ: Safety and feasibility of elective high-risk percutaneous coronary intervention procedures with left ventricular support of the Impella recover LP 2.5. Am J Cardiol. 97:990–992. 2006.PubMed/NCBI View Article : Google Scholar
6	Kiyooka T and Satoh Y: Mid-ventricular obstructive hypertrophic cardiomyopathy with an apical aneurysm caused by vasospastic angina. Tokai J Exp Clin Med. 39:29–33. 2014.PubMed/NCBI
7	Fanari Z, Abraham N, Hammami S and Qureshi WA: High-risk acute coronary syndrome in a patient with coronary subclavian steal syndrome secondary to critical subclavian artery stenosis. Case Rep Cardiol. 2014(175235)2014.PubMed/NCBI View Article : Google Scholar
8	Wang X, Chen J and Huang X: Rosuvastatin attenuates myocardial ischemia-reperfusion injury via upregulating miR-17-3p-mediated autophagy. Cell Reprogram. 21:323–330. 2019.PubMed/NCBI View Article : Google Scholar
9	Bulluck H and Hausenloy DJ: Ischaemic conditioning: Are we there yet? Heart. 101:1067–1077. 2015.PubMed/NCBI View Article : Google Scholar
10	Hausenloy DJ and Yellon DM: Myocardial ischemia-reperfusion injury: A neglected therapeutic target. J Clin Invest. 123:92–100. 2013.PubMed/NCBI View Article : Google Scholar
11	Wu MY, Yiang GT, Liao WT, Tsai AP, Cheng YL, Cheng PW, Li CY and Li CJ: Current mechanistic concepts in ischemia and reperfusion injury. Cell Physiol Biochem. 46:1650–1667. 2018.PubMed/NCBI View Article : Google Scholar
12	Ziegler M, Wang X and Peter K: Platelets in cardiac ischaemia/reperfusion injury: A promising therapeutic target. Cardiovasc Res. 115:1178–1188. 2019.PubMed/NCBI View Article : Google Scholar
13	Fanari Z, Weiss S and Weintraub WS: Cost effectiveness of antiplatelet and antithrombotic therapy in the setting of acute coronary syndrome: Current perspective and literature review. Am J Cardiovasc Drugs. 15:415–427. 2015.PubMed/NCBI View Article : Google Scholar
14	World Health Organization. The top 10 causes of death. Available from: http://www.who.int/mediacentre/factsheets/fs310/en/. WHO website, 2018.
15	Huang ZQ, Xu W, Wu JL, Lu X and Chen XM: MicroRNA-374a protects against myocardial ischemia-reperfusion injury in mice by targeting the MAPK6 pathway. Life Sci. 232(116619)2019.PubMed/NCBI View Article : Google Scholar
16	Xie B, Liu X, Yang J, Cheng J, Gu J and Xue S: PIAS1 protects against myocardial ischemia-reperfusion injury by stimulating PPARγ SUMOylation. BMC Cell Biol. 19(24)2018.PubMed/NCBI View Article : Google Scholar
17	Liang S, Ping Z and Ge J: Coenzyme Q10 regulates antioxidative stress and autophagy in acute myocardial ischemia-reperfusion injury. Oxid Med Cell Longev. 2017(9863181)2017.PubMed/NCBI View Article : Google Scholar
18	Chen S, Zhu Q, Ju H, Hao J, Lai Z, Zou C, Zhang W, Zhao S, Chen X, Zhang H, et al: The role of oxygen free radicals in myocardial ischemia/reperfusion injury. Chin Med Sci J. 6:127–131. 1991.PubMed/NCBI
19	Tian L, Cao W, Yue R, Yuan Y, Guo X, Qin D, Xing J and Wang X: Pretreatment with Tilianin improves mitochondrial energy metabolism and oxidative stress in rats with myocardial ischemia/reperfusion injury via AMPK/SIRT1/PGC-1 alpha signaling pathway. J Pharmacol Sci. 139:352–360. 2019.PubMed/NCBI View Article : Google Scholar
20	Grøvdal LM, Stang E, Sorkin A and Madshus IH: Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation. Exp Cell Res. 300:388–395. 2004.PubMed/NCBI View Article : Google Scholar
21	Schmidt MHH and Dikic I: The Cbl interactome and its functions. Nat Rev Mol Cell Biol. 6:907–918. 2005.PubMed/NCBI View Article : Google Scholar
22	Wu WJ, Tu S and Cerione RA: Activated Cdc42 sequesters c-Cbl and prevents EGF receptor degradation. Cell. 114:715–725. 2003.PubMed/NCBI View Article : Google Scholar
23	Swaminathan G and Tsygankov AY: The Cbl family proteins: Ring leaders in regulation of cell signaling. J Cell Physiol. 209:21–43. 2006.PubMed/NCBI View Article : Google Scholar
24	Rafiq K, Kolpakov MA, Seqqat R, Guo J, Guo X, Qi Z, Yu D, Mohapatra B, Zutshi N, An W, et al: c-Cbl inhibition improves cardiac function and survival in response to myocardial ischemia. Circulation. 129:2031–2043. 2014.PubMed/NCBI View Article : Google Scholar
25	Yu SY, Dong B, Fang ZF, Hu XQ, Tang L and Zhou SH: Knockdown of lncRNA AK139328 alleviates myocardial ischaemia/reperfusion injury in diabetic mice via modulating miR-204-3p and inhibiting autophagy. J Cell Mol Med. 22:4886–4898. 2018.PubMed/NCBI View Article : Google Scholar
26	Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O'Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I, et al: Report of the 1995 World Health Organization/international society and federation of cardiology task force on the definition and classification of cardiomyopathies. Circulation. 93:841–842. 1996.PubMed/NCBI View Article : Google Scholar
27	Liu D: Effects of Nicorandil combined with Danhong injection on SOD and MDA content in patients with myocardial ischemia-reperfusion injury. Mod J Integr Tradit Chin West Med. 1:78–80. 2016.
28	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar
29	Szklarczyk D, Gable AL, Nastou KC, Lyon D, Kirsch R, Pyysalo S, Doncheva NT, Legeay M, Fang T, Bork P, et al: The STRING database in 2021: Customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res. 49:D605–D612. 2021.PubMed/NCBI View Article : Google Scholar
30	Hausenloy DJ: Conditioning the heart to prevent myocardial reperfusion injury during PPCI. Eur Heart J Acute Cardiovasc Care. 1:13–32. 2012.PubMed/NCBI View Article : Google Scholar
31	Hausenloy DJ, Candilio L, Evans R, Ariti C, Jenkins DP, Kolvekar S, Knight R, Kunst G, Laing C, Nicholas J, et al: Remote ischemic preconditioning and outcomes of cardiac surgery. N Engl J Med. 373:1408–1417. 2015.PubMed/NCBI View Article : Google Scholar
32	Miao W, Yan Y, Bao TH, Jia WJ, Yang F, Wang Y, Zhu YH, Yin M and Han JH: Ischemic postconditioning exerts neuroprotective effect through negatively regulating PI3K/Akt2 signaling pathway by microRNA-124. Biomed Pharmacother. 126(109786)2020.PubMed/NCBI View Article : Google Scholar
33	Heusch G, Bøtker HE, Przyklenk K, Redington A and Yellon D: Remote ischemic conditioning. J Am Coll Cardiol. 65:177–195. 2015.PubMed/NCBI View Article : Google Scholar
34	Soares ROS, Losada DM, Jordani MC, Évora P and Castro-E-Silva O: Ischemia/reperfusion injury revisited: An overview of the latest pharmacological strategies. Int J Mol Sci. 20(5034)2019.PubMed/NCBI View Article : Google Scholar
35	Ivary SHA, Jajarmy N, Shahri MK, Shokoohi M, Shoorei H, Ebadi A, Moghimian M and Sigaroodi F: Effect of fish and flaxseed oil supplementation on isoprenaline-induced myocardial infarction in rats: Inhibition of mitochondrial permeability transition pore opening. Crescent J Med Biol Sci. 6:158–163. 2019.
36	Zheng Y, Shi B, Ma M, Wu X and Lin X: The novel relationship between Sirt3 and autophagy in myocardial ischemia-reperfusion. J Cell Physiol. 234:5488–5495. 2019.PubMed/NCBI View Article : Google Scholar
37	Han D, Wang Y, Chen J, Zhang J, Yu P, Zhang R, Li S, Tao B, Wang Y, Qiu Y, et al: Activation of melatonin receptor 2 but not melatonin receptor 1 mediates melatonin-conferred cardioprotection against myocardial ischemia/reperfusion injury. J Pineal Res. 67(e12571)2019.PubMed/NCBI View Article : Google Scholar
38	Li J, Zheng X, Ma X, Xu X, Du Y, Lv Q, Li X, Wu Y, Sun H, Yu L and Zhang Z: Melatonin protects against chromium(VI)-induced cardiac injury via activating the AMPK/Nrf2 pathway. J Inorg Biochem. 197(110698)2019.PubMed/NCBI View Article : Google Scholar
39	Yang D, Yang Q, Fu N, Li S, Han B, Liu Y, Tang Y, Guo X, Lv Z and Zhang Z: Hexavalent chromium induced heart dysfunction via Sesn2-mediated impairment of mitochondrial function and energy supply. Chemosphere. 264(128547)2021.PubMed/NCBI View Article : Google Scholar
40	Bouayed J and Bohn T: Exogenous antioxidants-double-edged swords in cellular redox state: Health beneficial effects at physiologic doses versus deleterious effects at high doses. Oxid Med Cell Longev. 3:228–237. 2010.PubMed/NCBI View Article : Google Scholar
41	Puente BN, Kimura W, Muralidhar SA, Moon J, Amatruda JF, Phelps KL, Grinsfelder D, Rothermel BA, Chen R, Garcia JA, et al: The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response. Cell. 157:565–579. 2014.PubMed/NCBI View Article : Google Scholar
42	Sun F, Zhuang Y, Zhu H, Wu H, Li D, Zhan L, Yang W, Yuan Y, Xie Y, Yang S, et al: LncRNA PCFL promotes cardiac fibrosis via miR-378/GRB2 pathway following myocardial infarction. J Mol Cell Cardiol. 133:188–198. 2019.PubMed/NCBI View Article : Google Scholar
43	Peng X, Lin L, Zhou X, Yang D, Cao Y, Yin T and Liu Y: miR-133b inhibits myocardial ischemia-reperfusion-induced cardiomyocyte apoptosis and accumulation of reactive oxygen species in rats by targeting YES1. Nan Fang Yi Ke Da Xue Xue Bao. 40:1390–1398. 2020.PubMed/NCBI View Article : Google Scholar : (In Chinese).
44	Su C, Fan X, Xu F, Wang J and Chen Y: Prostaglandin E1 attenuates post-cardiac arrest myocardial dysfunction through inhibition of mitochondria-mediated cardiomyocyte apoptosis. Mol Med Rep. 23(110)2021.PubMed/NCBI View Article : Google Scholar
45	Chuang GC, Xia H, Mahne SE and Varner KJ: Environmentally persistent free radicals cause apoptosis in HL-1 cardiomyocytes. Cardiovasc Toxicol. 17:140–149. 2017.PubMed/NCBI View Article : Google Scholar
46	Pirocanac EC, Nassirpour R, Yang M, Wang J, Nardin SR, Gu J, Fang B, Moossa AR, Hoffman RM and Bouvet M: Bax-induction gene therapy of pancreatic cancer. J Surg Res. 106:346–351. 2002.PubMed/NCBI View Article : Google Scholar
47	Dejean LM, Martinez-Caballero S, Guo L, Hughes C, Teijido O, Ducret T, Ichas F, Korsmeyer SJ, Antonsson B, Jonas EA and Kinnally KW: Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel. Mol Biol Cell. 16:2424–2432. 2005.PubMed/NCBI View Article : Google Scholar
48	Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP and Wang X: Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked. Science. 275:1129–1132. 1997.PubMed/NCBI View Article : Google Scholar