哌唑嗪(PRA)拮抗去甲肾上腺素(NE)诱导人类胚胎干细胞来源的心肌细胞(hESCs-CMs)肥大
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摘要
目的在人类胚胎干细胞来源的心肌细胞(human embryonic stem cells-cardiomyocytes,hESCs-CMs)上,研究α受体拮抗剂-哌唑嗪(prazosin,PRA)对去甲肾上腺素(norepinephrine,NE)诱导的心肌肥大效果的影响。方法通过体外二维培养分化的方法将hESCs-H7细胞系定向诱导分化为CMs,并通过imageJ软件计算免疫荧光染色图片来检测CMs面积的改变,qRT-PCR方法检测CMs肥大相关基因mRNA表达的改变,通过Leica显微镜FelixGX细胞动缘检测系统来测定CM收缩力及跳动情况的改变。结果 20μmol/L NE处理可使hESCs-CMs面积明显增大,肥大相关基因mRNA水平表达上调,收缩力及跳动频率增加;15μmol/L PRA处理后hESCs-CMs面积相较NE组有所降低,肥大相关基因BNP、TNNT2 mRNA表达明显降低,CM收缩力及跳动频率明显减弱。结论α受体拮抗剂PRA在hESCs-CMs上通过抑制NE诱导的细胞面积增大、肥大相关基因表达上调、收缩力增强以及跳动频率增加,从而改善NE诱导的心肌肥大。
Objective To investigate the effect of prazosin(PRA),α receptor antagonist,on cardiac hypertrophy induced by norepinephrine(NE) in human embryonic stem cells-cardiomyocytes-cardiomyocytes(hESCs-CMs). Methods Human embryonic stem cells(H7) were differentiated into CMs through 2D culture in vitro.Immunofluorescence staining images were calculated by software imageJ to analyze the cell size changes of CMs.The expression level of hypertrophy associated genes in CMs were analyzed by real time PCR.The contractility and of Leica beating rates of CMs were examined by FelixGX detection system. Results NE(20 μmol/L) significantly induced the increase of cell size,the expression of hypertrophy-associated genes,contraction force and beating rates of hESCs-CMs.PRA(15 μmol/L) treatment significantly inhibited NE-induced increase of cell size,mRNA expressions of BNP and TNNT2,contraction force and beating rates of hESCs-CMs. Conclusions PRA can alleviate NE-induced cardiac hypertrophy in hESCs-CMs by reducing cell size,down-regulatingthe expression of hypertrophy-associated genes,reducing contractility and beating frequency.
Objective To investigate the effect of prazosin(PRA),α receptor antagonist,on cardiac hypertrophy induced by norepinephrine(NE) in human embryonic stem cells-cardiomyocytes-cardiomyocytes(hESCs-CMs). Methods Human embryonic stem cells(H7) were differentiated into CMs through 2D culture in vitro.Immunofluorescence staining images were calculated by software imageJ to analyze the cell size changes of CMs.The expression level of hypertrophy associated genes in CMs were analyzed by real time PCR.The contractility and of Leica beating rates of CMs were examined by FelixGX detection system. Results NE(20 μmol/L) significantly induced the increase of cell size,the expression of hypertrophy-associated genes,contraction force and beating rates of hESCs-CMs.PRA(15 μmol/L) treatment significantly inhibited NE-induced increase of cell size,mRNA expressions of BNP and TNNT2,contraction force and beating rates of hESCs-CMs. Conclusions PRA can alleviate NE-induced cardiac hypertrophy in hESCs-CMs by reducing cell size,down-regulatingthe expression of hypertrophy-associated genes,reducing contractility and beating frequency.
引文
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[2] HUGHES SE.The pathology of hypertrophic cardiomyopathy[J].Histopathology,2004,44(5):412-427.
[3] MARON BJ,MARON MS.Hypertrophic cardiomyopathy[J].Lancet,2013,381(9862):242-255.
[4] CHEN YZ,QIAO SB,HU FH,et al.Left ventricular remodeling and fibrosis:sex differences and relationship with diastolic function in hypertrophic cardiomyopathy[J].Eur J Radiol,2015,84(8):1487-1492.
[5] XIAO D,DASGUPTA C,CHEN M,et al.Inhibition of DNA methylation reverses norepinephrine-induced cardiac hypertrophy in rats[J].Cardiovasc Res,2014,101(3):373-382.
[6] BRIEST W,HOMAGK L,RABLER B,et al.Norepinephrine-induced changes in cardiac transforming growth factor-β isoform expression pattern of female and male rats[J].Hypertension,2004,44(4):410-418.
[7] LUO JD,XIE F,ZHANG WW,et al.Simvastatin inhibits noradrenaline-induced hypertrophy of cultured neonatal rat cardiomyocytes[J].Br J Pharmacol,2001,132(1):159-164.
[8] AMIN JK,XIAO L,PIMENTAL DR,et al.Reactive oxygen species mediate alpha-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes[J].J Mol Cell Cardiology,2001,33(1):131-139.
[9] MORISCO C,ZEBROWSKI D,CONDORELLI G,et al.The Akt-glycogen synthase kinase 3beta pathway regulates transcription of atrial natriuretic factor induced by beta-adrenergic receptor stimulation in cardiac myocytes[J].J Biol Chem,2000,275(19):14466-14475.
[10] KEHAT I,DAVIS J,TIBURCY M,et al.Extracellular signal-regulated kinases 1 and 2 regulate the balance between eccentric and concentric cardiac growth[J].Circ Res,2011,108(2):176-183.
[11] WANG X,FINEGAN KG,ROBINSON AC,et al.Activation of extracellular signal-regulated protein kinase 5 downregulates FasL upon osmotic stress[J].Cell Death Differ,2006,13(12):2099-2108.
[12] MENASCHE P,VANNEAUX V,FABREGUETTES JR,et al.Towards a clinical use of human embryonic stem cell-derived cardiac progenitors:a translational experience[J].Eur Heart J,2015,36(12):743-750.
[13] CHONG JJ,YANG X,DON CW,et al.Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts[J].Nature,2014,510(7504):273-277.
[14] FAGARD RH.Impact of different sports and training on cardiac structure and function[J].Cardiol Clin,1997,15(3):397-412.
[15] ZANATI BS,BORGES VM,MARTIN LC,et al.Disproportionate pregnancy-induced myocardial hypertrophy in women with essential hypertension[J].Am J Hypertens,2013,26(6):816-821.
[16] SEGERS VF,De KEULENAER GW.Pathophysiology of diastolic dysfunction in chronic heart failure[J].Future Cardiol,2013,9(5):711-720.
[17] YOU J,WU J,JIANG G,et al.Olmesartan attenuates cardiac remodeling through DLL4/Notch1 pathway activation in pressure overload mice[J].J Cardiovasc Pharmacol,2013,61(2):142-151.
[18] DUAN Q,NI L,WANG P,et al.Deregulation of XBP1 expression contributes to myocardial vascular endothelial growth factor-a expression and angiogenesis during cardiac hypertrophy in vivo[J].Aging Cell,2016,15(4):625-633.
[19] HUNTER JJ,CHIEN KR.Signaling pathways for cardiac hypertrophy and failure[J].N Engl J Med,1999,341(17):1276-1283.
[20] PELLIEUX C,SAUTHIER T,AUBERT JF,et al.Angiotensin Ⅱ-induced cardiac hypertrophy is associated with different mitogen-activated protein kinase activation in normotensive and hypertensive mice[J].J Hypertens,2000,18(9):1307-1317.
[21] YANG F,LIU Z,WANG Y,et al.Hydrogen sulfide endothelin-induced myocardial hypertrophy in rats and the mechanism involved[J].Cell Biochem Biophys,2014,70(3):1683-1686.
[22] PASQUALE F,SYRRIS P,KASKI JP,et al.Long-term outcomes in hypertrophic cardiomyopathy caused by mutations in the cardiac troponin T gene[J].Circ Cardiovasc Genet,2012,5(1):10-17.
[23] 徐旖旎,王羿,柏帅,等.环维黄杨星D对高交感活性诱导大鼠实验性心肌损伤氧化应激及能量代谢的影响[J].中药材,2014,37(7):1213-1217.
[24] BISHOPRIC NH,SIMPSON PC,ORDAHL CP.Induction of the skeletal alpha-actin gene in alpha 1-adrenoceptor-mediated hypertrophy of rat cardiac myocytes[J].J Clin Invest,1987,80(4):1194-1199.
[25] LIAN X,BAO X,ZILBERTER M,et al.Chemically defined,albumin-free human cardiomyocyte generation[J].Nat Methods,2015,12(7):595-596.
[26] SHIBA Y,FERNANDES S,ZHU WZ,et al.Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts[J].Nature,2012,489(7415):322-325.
[27] LUNDY SD,ZHU WZ,REGNIER M,et al.Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells[J].Stem Cells Dev,2013,22(14):1991-2002.
[28] BLAZESKI A,ZHU R,HUNTER DW,et al.Electrophysiological and contractile function of cardiomyocytes derived from human embryonic stem cells[J].Prog Biophys Mol Biol,2012,110(2-3):178-195.
[29] COTECCHIA S,DEL VC,COLELLA M,et al.The alpha1-adrenergic receptors in cardiac hypertrophy:signaling mechanisms and functional implications[J].Cell Signal,2015,27(10):1984-1993.
[30] PAPAY RS,SHI T,PIASCIK MT,et al.alpha(1)A-adrenergic receptors regulate cardiac hypertrophy in vivo through interleukin-6 secretion[J].Mol Pharmacol,2013,83(5):939-948.
[31] VIDAL M,WIELAND T,LOHSE MJ,et al.beta-Adrenergic receptor stimulation causes cardiac hypertrophy via a Gbetagamma/Erk-dependent pathway[J].Cardiovasc Res,2012,96(2):255-264.
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