Abstract
Aim:
CYP2J3 in myocardium metabolizes arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs), which have diverse biological activities in rat heart. In this study we examined whether CYP2J3 was involved in cardioprotective effects of ophiopogonin D (OPD), a steroidal glycoside isolated from Chinese herb Radix ophiopogonis.
Methods:
Rat cardiomyoblast cell line (H9c2 cells) was tested. Intracellular Ca2+ concentrations ([Ca2+]i) were measured using Fluo-4/AM. The expression of calcium-regulating molecules and ER stress signaling molecules was measured with qRT-PCR and Western blot analyses. Cell apoptosis was quantified with Hoechst 33258 staining and TUNEL assay. The level of 14,15-DHET, a stable metabolite of 14,15-EET, was assessed with ELISA.
Results:
Angiotensin II (10−6 mol/L) significantly decreased the expression of calcium-regulating molecules (SERCA2a, PLB, RyR2 and FKBP12.6), and elevated [Ca2+]i in H9c2 cells. Furthermore, angiotensin II markedly increased the expression of ER stress signaling molecules (GRP78, CHOP, p-JNK and cleaved caspase-12) and ER stress-mediated apoptosis. OPD (100, 250 and 500 nmol/L) dose-dependently increased CYP2J3 expression and 14,15-DHET levels in normal H9c2 cells. Pretreatment of H9c2 cells with OPD suppressed angiotensin II-induced abnormalities in Ca2+ homeostasis, ER stress responses and apoptosis. Overexpression of CYP2J3 or addition of exogenous 14,15-EET also prevented angiotensin II-induced abnormalities in Ca2+ homeostasis, whereas transfection with CYP2J3 siRNA diminished the effects of OPD on Ca2+ homeostasis. Furthermore, the intracellular Ca2+ chelator BAPTA suppressed angiotensin II-induced ER stress responses and apoptosis in H9c2 cells.
Conclusion:
OPD is a novel CYP2J3 inducer that may offer a therapeutic benefit in treatment of cardiovascular diseases related to disturbance of Ca2+ homeostasis and ER stress.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Change history
26 May 2020
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
References
Clapham DE . Calcium signaling. Cell 2007; 131: 1047–58.
Wang W, Barnabei MS, Asp ML, Heinis FI, Arden E, Davis J, et al. Noncanonical EF-hand motif strategically delays Ca2+ buffering to enhance cardiac performance. Nat Med 2013; 19: 305–12.
Frey N, McKinsey TA, Olson EN . Decoding calcium signals involved in cardiac growth and function. Nat Med 2000; 6: 1221–7.
Gianni D, Chan J, Gwathmey JK, del Monte F, Hajjar RJ . SERCA2a in heart failure: role and therapeutic prospects. J Bioenerget Biomembr 2005; 37: 375–80.
Camors E, Valdivia HH . CaMKII regulation of cardiac ryanodine receptors and inositol triphosphate receptors. Frontiers Pharmacol 2014; 5: 101.
Liu Y, Huang H, Xia W, Tang Y, Li H, Huang C . NADPH oxidase inhibition ameliorates cardiac dysfunction in rabbits with heart failure. Mol Cell Biochem 2010; 343: 143–53.
Kawase Y, Ladage D, Hajjar RJ . Rescuing the failing heart by targeted gene transfer. J Am Coll Cardiol 2011; 57: 1169–80.
Engin F, Hotamisligil GS . Restoring endoplasmic reticulum function by chemical chaperones: an emerging therapeutic approach for metabolic diseases. Diabetes Obesity Metab 2010; 12 Suppl 2: 108–15.
Ron D . Translational control in the endoplasmic reticulum stress response. J Clin Invest 2002; 110: 1383–8.
Michalak M, Robert Parker JM, Opas M . Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum. Cell Calcium 2002; 32: 269–78.
Verkhartsky A . Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiol Rev 2005; 85: 201–79.
Sozen E, Karademir B, Ozer NK . Basic mechanisms in endoplasmic reticulum stress and relation to cardiovascular diseases. Free Radical Biol Med 2015; 78: 30–41.
Groenendyk J, Sreenivasaiah PK, Kim do H, Agellon LB, Michalak M . Biology of endoplasmic reticulum stress in the heart. Circ Res 2010; 107: 1185–97.
Askari A, Thomson SJ, Edin ML, Zeldin DC, Bishop-Bailey D . Roles of the epoxygenase CYP2J2 in the endothelium. Prostaglandins Other Lipid Mediat 2013; 107: 56–63.
Medhora M, Narayanan J, Harder D, Maier KG . Identifying endothelium-derived hyperpolarizing factor: recent approaches to assay the role of epoxyeicosatrienoic acids. Jap J Pharmacol 2001; 86: 369–75.
Westphal C, Spallek B, Konkel A, Marko L, Qadri F, DeGraff LM, et al. CYP2J2 overexpression protects against arrhythmia susceptibility in cardiac hypertrophy. PloS One 2013; 8: e73490.
Campbell WB . New role for epoxyeicosatrienoic acids as anti-inflammatory mediators. Trends Pharmacol Sci. 2000; 21: 125–7.
Wang HX, Zhang DM, Zeng XJ, Mu J, Yang H, Lu LQ, et al. Upregulation of cytochrome P450 2J3/11,12-epoxyeicosatrienoic acid inhibits apoptosis in neonatal rat cardiomyocytes by a caspase-dependent pathway. Cytokine 2012; 60: 360–8.
Liu L, Chen C, Gong W, Li Y, Edin ML, Zeldin DC, et al. Epoxyeicosatrienoic acids attenuate reactive oxygen species level, mitochondrial dysfunction, caspase activation, and apoptosis in carcinoma cells treated with arsenic trioxide. J Pharmacol Exp Ther 2011; 339: 451–63.
Wang X, Ni L, Yang L, Duan Q, Chen C, Edin ML, et al. CYP2J2-derived epoxyeicosatrienoic acids suppress endoplasmic reticulum stress in heart failure. Mol Pharmacol 2014; 85: 105–15.
Minamisawa S, Hoshijima M, Chu G, Ward CA, Frank K, Gu Y, et al. Chronic phospholamban–sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell 1999; 99: 313–22.
White DC, Hata JA, Shah AS, Glower DD, Lefkowitz RJ, Koch WJ . Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction. Proc Natl Acad Sci U S A 2000; 97: 5428–33.
Sikkel MB, Hayward C, MacLeod KT, Harding SE, Lyon AR . SERCA2a gene therapy in heart failure: an anti-arrhythmic positive inotrope. Br J Pharmacol 2014; 171: 38–54.
Wehrens XH, Lehnart SE, Reiken S, Vest JA, Wronska A, Marks AR . Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression. Proc Natl Acad Sci U S A 2006; 103: 511–8.
Kou J, Tian Y, Tang Y, Yan J, Yu B . Antithrombotic activities of aqueous extract from Radix Ophiopogon japonicus and its two constituents. Biol Pharm Bull 2006; 29: 1267–70.
Lin Y, Zhu D, Qi J, Qin M, Yu B . Characterization of homoisoflavonoids in different cultivation regions of Ophiopogon japonicus and related antioxidant activity. J Pharm Biomed Anal 2010; 52: 757–62.
Lu LY, Zheng GQ, Wang Y . An overview of systematic reviews of shenmai injection for health care. Evidence-based Complement Alter Med 2014; 2014: 840650.
Qian J, Jiang F, Wang B, Yu Y, Zhang X, Yin Z, et al. Ophiopogonin D prevents H2O2-induced injury in primary human umbilical vein endothelial cells. J Ethnopharmacol 2010; 128: 438–45.
Zhang YY, Meng C, Zhang XM, Yuan CH, Wen MD, Chen Z, et al. Ophiopogonin D attenuates doxorubicin-induced autophagic cell death by relieving mitochondrial damage in vitro and in vivo. J Pharmacol Exp Ther 2015; 352: 166–74.
Wang YG, Zhou JM, Ma ZC, Li H, Liang QD, Tan HL, et al. Pregnane X receptor mediated-transcription regulation of CYP3A by glycyrrhizin: a possible mechanism for its hepatoprotective property against lithocholic acid-induced injury. Chem Biol Interact 2012; 200: 11–20.
Zhou T, Wang YG, Ma ZC, Xiao Y, Tang XL, Tan HL, et al. Ginkgolide B induces CYP3A4 expression through activation of human pregnane X receptor. Chin Pharmacol Bull 2014; 30: 931–6.
Zhao L, Wu J, Zhang X, Kuang H, Guo Y, Ma L . The effect of Shenmai injection on the proliferation of rat airway smooth muscle cells in asthma and underlying mechanism. BMC Complement Altern Med 2013; 13: 221.
Jiang JG, Chen CL, Card JW, Yang S, Chen JX, Fu XN, et al. Cytochrome P450 2J2 promotes the neoplastic phenotype of carcinoma cells and is up-regulated in human tumors. Cancer Res 2005; 65: 4707–15.
Samokhvalov V, Alsaleh N, El-Sikhry HE, Jamieson KL, Chen CB, Lopaschuk DG, et al. Epoxyeicosatrienoic acids protect cardiac cells during starvation by modulating an autophagic response. Cell Death Disease 2013; 4: e885.
Deniaud A, Sharaf el dein O, Maillier E, Poncet D, Kroemer G, Lemaire C, et al. Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene 2008; 27: 285–99.
Xiao B, Li X, Yan J, Yu X, Yang G, Xiao X, et al. Overexpression of cytochrome P450 epoxygenases prevents development of hypertension in spontaneously hypertensive rats by enhancing atrial natriuretic peptide. J Pharmacol Exp Ther 2010; 334: 784–94.
Feng Y, Tang XY, Dai DZ, Dai Y . Reversal of isoproterenol-induced downregulation of phospholamban and FKBP12.6 by CPU0213-mediated antagonism of endothelin receptors. Acta Pharmacol Sin 2007; 28: 1746–54.
Saliaris AP, Amado LC, Minhas KM, Schuleri KH, Lehrke S, St John M, et al. Chronic allopurinol administration ameliorates maladaptive alterations in Ca2+ cycling proteins and beta-adrenergic hyporesponsiveness in heart failure. Am J Physiol Heart Circ Physiol 2007; 292: 1328–35.
Mattiazzi A, Kranias EG . The role of CaMKII regulation of phospho-lamban activity in heart disease. Frontiers Pharmacol 2014; 5: 5.
Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 2000; 101: 365–76.
Jiang MT, Lokuta AJ, Farrell EF, Wolff MR, Haworth RA, Valdivia HH . Abnormal Ca2+ release, but normal ryanodine receptors, in canine and human heart failure. Circ Res 2002; 91: 1015–22.
Li J, Imtiaz MS, Beard NA, Dulhunty AF, Thorne R, vanHelden DF, et al. ss-Adrenergic stimulation increases RyR2 activity via intracellular Ca2+ and Mg2+ regulation. PloS One 2013; 8: e58334.
Clapham DE . Calcium signaling. Cell 2007; 131: 1047–58.
Jing G, Wang JJ, Zhang SX . ER stress and apoptosis: a new mechanism for retinal cell death. Exp Diabetes Res 2012; 10: 1155.
Shen M, Wang L, Wang B, Wang T, Yang G, Shen L, et al. Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt. Cell Death Disease 2014; 5: e1528.
Zhu YR, Xu Y, Fang JF, Zhou F, Deng XW, Zhang YQ . Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation. Biochem Biophys Res Commun 2014; 451: 112–8.
Domenighetti AA, Wang Q, Egger M, Richards SM, Pedrazzini T, Delbridge LM . Angiotensin II-mediated phenotypic cardiomyocyte remodeling leads to age-dependent cardiac dysfunction and failure. Hypertension 2005; 46: 426–32.
Kajstura J, Cigola E, Malhotra A, Li P, Cheng W, Meggs LG, et al. Angiotensin II induces apoptosis of adult ventricular myocytes in vitro. J Mol Cell Cardiol 1997; 29: 859–70.
Hajjar RJ, Zsebo K, Deckelbaum L, Thompson C, Rudy J, Yaroshinsky A, et al. Design of a phase 1/2 trial of intracoronary administration of AAV1/SERCA2a in patients with heart failure. J Cardiac Failure 2008; 14: 355–67.
Lee AC, Murray M . Up-regulation of human CYP2J2 in HepG2 cells by butylated hydroxyanisole is mediated by c-Jun and Nrf2. Mol Pharmacol 2010; 77: 987–94.
Acknowledgements
This work was supported by the National Basic Research Program of China (“973 Program”) (No 2011CB505304 and 2012CB518402), and the Scientific and Technological Major Special Project Major Creation of New Drugs (No 2009ZX09501-304).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Supplementary information is available at the Acta Pharmacologica Sinica's website.
Supplementary information
Supplemental Table 1
Primer sequences used for qRT-PCR (DOC 31 kb)
Supplemental Figure 1
Stable H9c2 cell lines with CYP2J3 overexpression. (DOC 46 kb)
Supplemental Figure 2
11,12-EET and 14,15-EET restored SERCA2a expression in cardiomyocytes treated with AngII (DOC 58 kb)
Supplemental Figure 3
14,15-EET restored calcium-regulating molecules expression in cardiomyocytes treated with AngII (DOC 131 kb)
Supplemental Figure 4
Knocking down CYP2J3 expression using its specific siRNA (DOC 48 kb)
Rights and permissions
About this article
Cite this article
You, Wt., Zhou, T., Ma, Zc. et al. Ophiopogonin D maintains Ca2+ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/EETs and suppressing ER stress. Acta Pharmacol Sin 37, 368–381 (2016). https://doi.org/10.1038/aps.2015.146
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/aps.2015.146
Keywords
This article is cited by
-
TPPU inhibits inflammation-induced excessive autophagy to restore the osteogenic differentiation potential of stem cells and improves alveolar ridge preservation
Scientific Reports (2023)
-
Global metabolomic and lipidomic analysis reveals the potential mechanisms of hemolysis effect of Ophiopogonin D and Ophiopogonin D' in vivo
Chinese Medicine (2021)
-
FAM134B Attenuates Seizure-Induced Apoptosis and Endoplasmic Reticulum Stress in Hippocampal Neurons by Promoting Autophagy
Cellular and Molecular Neurobiology (2020)
-
Soluble Epoxide Hydrolase Regulation of Lipid Mediators Limits Pain
Neurotherapeutics (2020)
-
Low-Dose Radiation Prevents Chemotherapy-Induced Cardiotoxicity
Current Stem Cell Reports (2019)
