Mfn2 gene transfer inhibites neonatal rat cardiomyocytes apoptosis induced by ischemia/reperfusion via activation of the Ras-PI3K-Akt signaling pathway

Abstract

Abstract: AIM: To investigate the effect of Mfn2 gene transfer on inhibiting the apoptosis of cardiomyocytes induced by ischemia/reperfusion. METHODS: Treating neonatal rat cardiomyocytes by hypoxia/reoxygenation to simulating myocardial ischemia/reperfusion injury. And then the cardiomyocytes were infected by adenovirus-mediated Mfn2 (Adv-Mfn2). TUNEL staining, cell death ELISA and FACS analysis were used to investigate the role of Adv-Mfn2 gene transfer on neonatal rat cardiomyocytes apoptosis. Western blot was used to analyze the protein expression of Bcl-2, Bax, cleaved caspase-9 and p-Akt. RESULTS: TUNEL staining evidenced that there were less positive-apoptoic cardiomyocytes in Adv-Mfn2 group than in H/Re group and Adv-LacZ group (P<0.01). ELISA and FACS results showed that cardiomyocytes apoptosis in Adv-Mfn2 group was significantly reduced compared with H/Re group and Adv-LacZ group, and this effect was time-dependent (P<0.01). The protein expression of Bcl-2 and phosphorylated Akt were significantly upregulated in Adv-Mfn2 group (P<0.05), whereas Bax and cleaved caspase-9 protein expressions were significantly downregulated in Adv-Mfn2 group (P<0.05). CONCLUSION: Mfn2 gene promotes the protein expression of Bcl-2 and phosphorylated Akt, supppres the protein expression of Bax and cleaved caspase-9 via activation of the Ras-PI3K-Akt signaling pathway to inhibite neonatal rat cardiomyocytes apoptosis induced by ischemia/reperfusion.

Key words: Mfn2, adenovirus, cell apoptosis, ischemia/reperfusion injury

CLC Number:

R965.2

ZHOU Jie, YAN Ling, HUANG Duo-xin. Mfn2 gene transfer inhibites neonatal rat cardiomyocytes apoptosis induced by ischemia/reperfusion via activation of the Ras-PI3K-Akt signaling pathway[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2014, 19(9): 973-978.

References 26

[1]	Colucci WS.Apoptosis in the heart[J]. N Engl J Med, 1996, 335(16): 1224-1226.
[2]	Nakamura T, Mizuno S, Matsumoto K, et al.Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF[J]. J Clin Invest, 2000, 106(12): 1511-1519.
[3]	Chen KH LN, Zhou AR.The role of hypertension-related gene in aortic vascular smooth muscle cells from mice and rats[J]. Chin Med J (Engl), 2001, 114(8): 833-836.
[4]	Eura Y, Ishihara N, Yokota S, et al.Two mitofusin proteins, mammalian homologues of FZO, with distinct functions are both required for mitochondrial fusion[J]. J Biochem, 2003, 134(3): 333-344.
[5]	Ishihara N, Eura Y, Mihara K.Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity[J]. J Cell Sci, 2004, 117(Pt 26): 6535-6546.
[6]	Tanner FC, Boehm M, Akyurek LM, et al.Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation[J]. Circulation, 2000, 101(17): 2022-2025.
[7]	Harbour JW, Dean DC.Rb function in cell-cycle regulation and apoptosis[J]. Nat Cell Biol, 2000, 2(4): E65-E67.
[8]	Miele A, Braastad CD, Holmes WF, et al.HiNF-P directly links the cyclin E/CDK2/p220NPAT pathway to histone H4 gene regulation at the G1/S phase cell cycle transition[J]. Mol Cell Biol, 2005, 25(14): 6140-6153.
[9]	康品方, 高琴, 王晓梅. 线粒体融合素2在糖尿病大鼠心肌损伤中的变化[J]. 中国临床药理学与治疗学, 2012, 17(4): 398-402.
[10]	Zhang C, Meng X, Zhu Z, et al.Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro[J]. Cell Biol Int, 2004, 28(12): 863-873.
[11]	Koyama T, Temma K, Akera T.Reperfusion-induced contracture develops with a decreasing [Ca2+]i in single heart cells[J]. Am J Physiol, 1991, 261(4 Pt 2): H1115-1122.
[12]	Peyton KJ, Reyna SV, Chapman GB, et al.Heme oxygenase-1-derived carbon monoxide is an autocrine inhibitor of vascular smooth muscle cell growth[J]. Blood, 2002, 99(12): 4443-4448.
[13]	Gottlieb RA, Burleson KO, Kloner RA, et al.Reperfusion injury induces apoptosis in rabbit cardiomyocytes[J]. J Clin Invest, 1994, 94(4): 1621-1628.
[14]	Fliss H, Gattinger D.Apoptosis in ischemic and reperfused rat myocardium[J]. Circ Res, 1996, 79(5): 949-956.
[15]	Bialik S, Geenen DL, Sasson IE, et al.Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53[J]. J Clin Invest, 1997, 100(6): 1363-1372.
[16]	Mattson MP, Kroemer G.Mitochondria in cell death: novel targets for neuroprotection and cardioprotection[J]. Trends Mol Med, 2003, 9(5): 196-205.
[17]	Datta SR, Brunet A, Greenberg ME.Cellular survival: a play in three Akts[J]. Genes Dev, 1999, 13(22): 2905-2927.
[18]	Datta SR, Dudek H, Tao X, et al.Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery[J]. Cell, 1997, 91(2): 231-241.
[19]	Cantley LC.The phosphoinositide 3-kinase pathway[J]. Science, 2002, 296(5573): 1655-1657.
[20]	Nagata S.DNA degradation in development and programmed cell death[J]. Annu Rev Immunol, 2005, 23: 853-875.
[21]	Wajant H.The Fas signaling pathway: more than a paradigm[J]. Science, 2002, 296(5573): 1635-1636.
[22]	Jiang X, Wang X.Cytochrome C-mediated apoptosis[J]. Annu Rev Biochem, 2004, 73: 87-106.
[23]	Ong SB, Subrayan S, Lim SY, et al.Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury[J]. Circulation, 2010, 121(18): 2012-2022.
[24]	Zhou L, Ma W, Yang Z, et al.VEGF165 and angiopoietin-1 decreased myocardium infarct size through phosphatidylinositol-3 kinase and Bcl-2 pathways[J]. Gene Ther, 2005, 12(3): 196-202.
[25]	Renatus M, Stennicke HR, Scott FL, et al.Dimer formation drives the activation of the cell death protease caspase 9[J]. Proc Natl Acad Sci USA, 2001, 98(25): 14250-14255.
[26]	Acehan D, Jiang X, Morgan DG, et al.Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation[J]. Mol Cell, 2002, 9(2): 423-432.