It is reported that LGR4 (leucine-rich repeat domain containing G protein-coupled receptor 4) plays a crucial role in the physiological function of many organs. However, few data are available on the function and mechanism of LGR4 in myocardial ischemia–reperfusion (I/R) injury. The aim of this study was to explore the function and mechanism of LGR4 in I/R injury. We incubated H9c2 cells in simulating ischemia buffer and then re-incubated them in normal culture medium to establish a model of I/R injury in vitro. The expression of LGR4 was evaluated by RT-PCR and western blot. Besides, the cell apoptosis was evaluated by flow cytometric analysis and the content of ROS, SOD, MDA, LDH, CK, ATP, cyt c were detected by special commercial kits. The expression of mitochondrial function-related proteins were detected by western blot. Then, the roles of ERK signaling pathway was determined with TBHQ (ERK activator) treatment. Our data have demonstrated that I/R boosted the expression of LGR4 in H9c2 cells. Knockdown of LGR4 increased the apoptosis rate of H9c2 cells and led to excessed oxidant stress and impaired mitochondrial function by increasing the levels of ROS, MDA, LDH, CK and cyt c and inhibiting SOD activity, ATP production. In addition, LGR4 silence inhibited the activation of ERK pathway. And TBHQ partially reversed the effects of LGR4 knockdown on H9c2 cells. To conclude, our study indicated that LGR4 regulated mitochondrial dysfunction and oxidative stress by ERK signaling pathways, which provides a potential cardiac protective target against I/R.
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Leucine-rich repeat domain containing G protein-coupled receptor 4
Dulbecco’s modified Eagle’s medium
Fetal bovine serum
Ischemic heart disease
Reactive oxygen species
Mitogen-activated protein kinases
Extracellular regulated protein kinases
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Aikawa R et al (1997) Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest 100(7):1813–1821
Boengler K, Lochnit G, Schulz R (2018) Mitochondria “THE” target of myocardial conditioning. Am J Physiol Heart Circ Physiol 315(5):H1215–H1231
Borutaite V (2003) Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia. J Mol Cell Cardiol 35(4):357–366
Carmon KS et al (2011) R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling. Proc Natl Acad Sci 108(28):11452–11457
Chen WR et al (2015) Effects of liraglutide on left ventricular function in patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention. Am Heart J 170(5):845–854
Dabkowski ER et al (2010) Mitochondrial dysfunction in the type 2 diabetic heart is associated with alterations in spatially distinct mitochondrial proteomes. Am J Physiol Heart Circ Physiol 299(2):H529–H540
Du B et al (2013) Lgr4/Gpr48 negatively regulates TLR2/4-associated pattern recognition and innate immunity by targeting CD14 expression. J Biol Chem 288(21):15131–15141
Fryer RM et al (2001) Differential activation of extracellular signal regulated kinase isoforms in preconditioning and opioid-induced cardioprotection. J Pharmacol Exp Ther 296(2):642–649
Giordano FJ (2005) Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Investig 115(3):500–508
Hausenloy DJ, Yellon DM (2004) New directions for protecting the heart against ischaemia–reperfusion injury: targeting the reperfusion injury salvage kinase (RISK)-pathway. Cardiovasc Res 61(3):448–460
Hausenloy DJ et al (2005) Ischemic preconditioning protects by activating prosurvival kinases at reperfusion. Am J Physiol Heart Circ Physiol 288(2):H971–H976
Heusch G (2016) Myocardial ischemia: lack of coronary blood flow or myocardial oxygen supply/demand imbalance? Circ Res 119(2):194–196
Heusch G (2020) Myocardial ischaemia-reperfusion injury and cardioprotection in perspective. Nat Rev Cardiol 17:773–789
Hu X et al (2007) Stromal cell derived factor-1 alpha confers protection against myocardial ischemia/reperfusion injury: role of the cardiac stromal cell derived factor-1 alpha CXCR4 axis. Circulation 116(6):654–663
Kalogeris T et al (2012) Cell biology of ischemia/reperfusion injury. Int Rev Cell Mol Biol 298:229–317
Kato S et al (2006) Leucine-rich repeat-containing G protein-coupled receptor-4 (LGR4, Gpr48) is essential for renal development in mice. Nephron Exp Nephrol 104(2):e63-75
Ke Z et al (2017) Crude terpene glycoside component from Radix paeoniae rubra protects against isoproterenol-induced myocardial ischemic injury via activation of the PI3K/AKT/mTOR signaling pathway. J Ethnopharmacol 206:160–169
Khan M et al (2006) C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling. Am J Physiol-Heart Circ Physiol 290(5):H2136–H2145
Lesnefsky EJ et al (2017) Mitochondrial dysfunction and myocardial ischemia-reperfusion: implications for novel therapies. Annu Rev Pharmacol Toxicol 57:535–565
Li Z et al (2019) LGR4 protects hepatocytes from injury in mouse. Am J Physiol Gastrointest Liver Physiol 316(1):G123–G131
Liang F et al (2020) Ablation of LGR4 signaling enhances radiation sensitivity of prostate cancer cells. Life Sci 265:118737
Liu S et al (2013) Lgr4 gene deficiency increases susceptibility and severity of dextran sodium sulfate-induced inflammatory bowel disease in mice. J Biol Chem 288(13):8794–8803
Liu Y et al (2015) RNA-Seq identifies novel myocardial gene expression signatures of heart failure. Genomics 105(2):83–89
Liu S et al (2018) R-spondin3-LGR4 signaling protects hepatocytes against DMOG-induced hypoxia/reoxygenation injury through activating beta-catenin. Biochem Biophys Res Commun 499(1):59–65
Loh E et al (2000) Chromosomal localization1 of GPR48, a novel glycoprotein hormone receptor like GPCR, in human and mouse with radiation hybrid and interspecific backcross mapping. Cytogenet Genome Res 89(1–2):2–5
Maack C et al (2009) Endogenous activation of mitochondrial KATP channels protects human failing myocardium from hydroxyl radical–induced stunning. Circ Res 105(8):811–817
Mazerbourg S et al (2004) Leucine-rich repeat-containing, G protein-coupled receptor 4 null mice exhibit intrauterine growth retardation associated with embryonic and perinatal lethality. Mol Endocrinol 18(9):2241–2254
Padanilam BJ (2003) Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am J Physiol Renal Physiol 284(4):F608–F627
Pan H et al (2014) Lgr4 gene regulates corpus luteum maturation through modulation of the WNT-mediated EGFR-ERK signaling pathway. Endocrinology 155(9):3624–3637
Rink L, Hebel T, Fukumot J (2015) Sepsis and signal transduction pathway: cross-talk TLR4/MyD88/TRIF. Am J BioMed 3(4):150–163
Sanada S, Komuro I, Kitakaze M (2011) Pathophysiology of myocardial reperfusion injury: preconditioning, postconditioning, and translational aspects of protective measures. Am J Physiol Heart Circ Physiol 301(5):H1723–H1741
Styrkarsdottir U et al (2013) Nonsense mutation in the LGR4 gene is associated with several human diseases and other traits. Nature 497(7450):517–520
Turer AT, Hill JA (2010) Pathogenesis of myocardial ischemia-reperfusion injury and rationale for therapy. Am J Cardiol 106(3):360–368
Vickers NJ (2017) Animal Communication: When I’m Calling You, Will You Answer Too? Curr Biol 27(14):R713–R715
Wang J, Zhou H (2020) Mitochondrial quality control mechanisms as molecular targets in cardiac ischemia-reperfusion injury. Acta Pharm Sin B 10(10):1866–1879
Wang Z et al (2010) GPR48-Induced keratinocyte proliferation occurs through HB-EGF mediated EGFR transactivation. FEBS Lett 584(18):4057–4062
Wang F et al (2014) LGR4 acts as a link between the peripheral circadian clock and lipid metabolism in liver. J Mol Endocrinol 52(2):133–143
Xu R et al (2016) Ascending aortic adventitial remodeling and fibrosis are ameliorated with Apelin-13 in rats after TAC via suppression of the miRNA-122 and LGR4-beta-catenin signaling. Peptides 86:85–94
Yang Y et al (2013) JAK 2/STAT 3 activation by melatonin attenuates the mitochondrial oxidative damage induced by myocardial ischemia/reperfusion injury. J Pineal Res 55(3):275–286
Yu L et al (2015) Protective effect of berberine against myocardial ischemia reperfusion injury: role of Notch1/Hes1-PTEN/Akt signaling. Apoptosis 20(6):796–810
Yue T-L et al (2000) Inhibition of extracellular signal–regulated kinase enhances ischemia/reoxygenation–induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res 86(6):692–699
Zhang J et al (2016) Lgr4 promotes prostate tumorigenesis through the Jmjd2a/AR signaling pathway. Exp Cell Res 349(1):77–84
Zhang M et al (2017) RSPO3-LGR4 regulates osteogenic differentiation of human adipose-derived stem cells via ERK/FGF signalling. Sci Rep 7(1):1–15
Zhu J et al (2015) Targeted deletion of the murine Lgr4 gene decreases lens epithelial cell resistance to oxidative stress and induces age-related cataract formation. PLoS ONE. https://doi.org/10.1371/journal.pone.0119599
Zorov DB et al (2000) Reactive oxygen species (Ros-Induced) Ros release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J Exp Med 192(7):1001–1014
This study was supported by the National Natural Science Foundation of China(81870330).
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Chen, T., Qiao, X., Cheng, L. et al. LGR4 silence aggravates ischemic injury by modulating mitochondrial function and oxidative stress via ERK signaling pathway in H9c2 cells. J Mol Histol (2021). https://doi.org/10.1007/s10735-021-09957-1
- Ischemic myocardial injury
- Mitochondrial function
- Oxidative stress
- ERK signaling pathway