DSG2~(F536C)点突变基因敲入小鼠心脏表型的观察研究
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摘要
目的观察桥粒芯糖蛋白2(DSG2)点突变(DSG2~(F536C))基因敲入小鼠心脏结构、功能、病理学改变,并初步探讨其纤维化的发生机制。方法构建DSG2~(F536C)突变基因敲入小鼠模型,分为纯合子突变组(DSG2~(mt/mt))、杂合子突变组(DSG2~(mt/wt))和野生型组(DSG2~(wt/wt))。分别行超声心动图检查小鼠心脏结构和功能,Masson三色染色和HE染色观察心肌组织病理特点,并用Western blot法测定心肌组织转化生长因子-β1(TGF-β1)的蛋白表达。结果与DSG2~(wt/wt)组小鼠相比,DSG2~(mt/wt)组小鼠左室射血分数和缩短分数显著降低,DSG2~(mt/mt)组小鼠则较DSG2~(mt/wt)组进一步降低,并伴有室壁明显变薄和心腔显著扩大。DSG2~(mt/mt)组小鼠的右心室出现心肌排列紊乱、纤维化和炎性反应,约1/3的小鼠同时累及左心室,而DSG2~(mt/wt)组小鼠仅见心肌排列紊乱。与DSG2~(wt/wt)组小鼠相比,DSG2~(mt/wt)组和DSG2~(mt/mt)组小鼠心肌组织TGF-β_1蛋白表达明显升高。结论 DSG2~(F536C)小鼠表现出心室扩大、室壁变薄、心功能下降、炎性反应和心室纤维化浸润和TGF-β_1升高等表征,类似于临床致心律失常性右室心肌病(ARVC)患者的表型,提示其为致病性突变。此外,DSG2~(F536C)基因敲入小鼠为深入研究DSG2突变导致ARVC的发病机制提供了良好的动物模型。
Objective To investigate the structural,functional and pathological characteristics of DSG2~(F536C) mutation knock-in mice heart and the preliminary pathogenesis of cardiac fibrosis. Methods DSG2~(F536C) mutation knock-in mice were generated and assigned to three groups,i. e. homozygous mice group( DSG2~(mt/mt)),heterozygous mice group( DSG2~(mt/wt)) and wild type mice group( DSG2~(wt/wt)). Structural and functional changes of mice heart were evaluated by echocardiography. Pathological characteristics of the myocardium were assessed by Masson' s trichrome staining and HE staining. Expression level of Transforming growth factor β1( TGF-β1) was measured by Western blot. All studies were performed in mice at ten weeks of age. Results Compared with DSG2~(wt/wt) mice,DSG2~(mt/wt) ones hadsignificantly decreased left ventricular ejection fraction( LVEF) and fractional shortening( LVFS),while DSG2~(mt/mt) mice showed further dramatically decrease in LVEF and LVFS. Meanwhile,left ventricular diameter and volume were significantly increased,but left ventricular wall thickness was significantly decreased in both DSG2~(mt/wt) and DSG2~(mt/mt)mice. Moreover,DSG2~(mt/mt) mice were observed to have disordered cardiac muscle,fibrosis and inflammatory cell infiltrates of cardiac myocytes in the right ventricle,and left ventricle was affected in 1/3 of them. DSG2~(mt/wt)mice were noted to have disordered cardiac muscle only.Compared with DSG2~(wt/wt) mice,both DSG2~(mt/wt) and DSG2~(mt/mt)ones showedsignificantly increased expression of cardiac TGF-β_1. Conclusions DSG2~(F536C) knock-in mice show phenotype similar to those of ARVC patients,including enlarged ventricles,thinned ventricular wall,decreased heart function,inflammatory response,ventricular fibrotic infiltration and increased TGF-β_1,which indicate that the mutation might be a pathogenic cause of ARVC. In addition,DSG2~(F536C) knock-in mice could be a useful model for the study of the pathogenesis of ARVC.
Objective To investigate the structural,functional and pathological characteristics of DSG2~(F536C) mutation knock-in mice heart and the preliminary pathogenesis of cardiac fibrosis. Methods DSG2~(F536C) mutation knock-in mice were generated and assigned to three groups,i. e. homozygous mice group( DSG2~(mt/mt)),heterozygous mice group( DSG2~(mt/wt)) and wild type mice group( DSG2~(wt/wt)). Structural and functional changes of mice heart were evaluated by echocardiography. Pathological characteristics of the myocardium were assessed by Masson' s trichrome staining and HE staining. Expression level of Transforming growth factor β1( TGF-β1) was measured by Western blot. All studies were performed in mice at ten weeks of age. Results Compared with DSG2~(wt/wt) mice,DSG2~(mt/wt) ones hadsignificantly decreased left ventricular ejection fraction( LVEF) and fractional shortening( LVFS),while DSG2~(mt/mt) mice showed further dramatically decrease in LVEF and LVFS. Meanwhile,left ventricular diameter and volume were significantly increased,but left ventricular wall thickness was significantly decreased in both DSG2~(mt/wt) and DSG2~(mt/mt)mice. Moreover,DSG2~(mt/mt) mice were observed to have disordered cardiac muscle,fibrosis and inflammatory cell infiltrates of cardiac myocytes in the right ventricle,and left ventricle was affected in 1/3 of them. DSG2~(mt/wt)mice were noted to have disordered cardiac muscle only.Compared with DSG2~(wt/wt) mice,both DSG2~(mt/wt) and DSG2~(mt/mt)ones showedsignificantly increased expression of cardiac TGF-β_1. Conclusions DSG2~(F536C) knock-in mice show phenotype similar to those of ARVC patients,including enlarged ventricles,thinned ventricular wall,decreased heart function,inflammatory response,ventricular fibrotic infiltration and increased TGF-β_1,which indicate that the mutation might be a pathogenic cause of ARVC. In addition,DSG2~(F536C) knock-in mice could be a useful model for the study of the pathogenesis of ARVC.
引文
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[14]YU CC,YU CH,HSUEH CH,et al.Arrhythmogenic right ventricular dysplasia:clinical characteristics and identification of novel desmosome gene mutations[J].J Formos Med Assoc,2008,107(7):548-558.
[15]RASMUSSEN TB,PALMFELDT J,NISSEN PH,et al.Mutated desmoglein-2 proteins are incorporated into desmosomes and exhibit dominant-negative effects in arrhythmogenic right ventricular cardiomyopathy[J].Hum Mutat,2013,34(5):697-705.
[16]BIERNACKA A,DOBACZEWSKI M,FRANGOGIANNIS NG.TGF-βsignaling in fibrosis[J].Growth Factors,2011,29(5):196-202.
[2]ROMERO J,MEJIA-LOPEZ E,MANRIQUE C,et al.Arrhythmogenic Right ventricular cardiomyopathy(ARVC/D):A systematic literature review[J].Clin Med Insights Cardiol,2013,7:97-114.
[3]BASSO C,BAUCE B,CORRADO D,et al.Pathophysiology of arrhythmogenic cardiomyopathy[J].Nat Rev Cardiol,2011,9(4):223-233.
[4]LEASK A.TGFbeta,cardiac fibroblasts,and the fibrotic response[J].Cardiovasc Res,2007,74(2):207-212.
[5]DOBACZEWSKI M,CHEN W,FRANGOGIANNIS NG.Transforming growth factor(TGF)-beta signaling in cardiac remodeling[J].J Mol Cell Cardiol,2011,51(4):600-606.
[6]LAZZARINI E,JONGBLOED JD,PILICHOU K,et al.The ARVD/C genetic variants database:2014 update[J].Hum Mutat,2015,36(4):403-410.
[7]KANT S,HOLTHFER B,MAGIN TM,et al.Desmoglein 2-dependent arrhythmogenic cardiomyopathy is caused by a loss of adhesive function[J].Circ Cardiovasc Genet,2015,8(4):553-563.
[8]PILICHOU K,REMME CA,BASSO C,et al.Myocyte necrosis underlies progressive myocardial dystrophy in mouse dsg2-related arrhythmogenic right ventricular cardiomyopathy[J].J Exp Med,2009,206(8):1787-1802.
[9]ZHOU X,CHEN M,SONG H,et al.Comprehensive analysis of desmosomal gene mutations in Han Chinese patients with arrhythmogenic right ventricular cardiomyopathy[J].Eur J Med Genet,2015,58(4):258-265.
[10]THOMASON H,SCOTHERN A,MCHARG S,et al.Desmosomes:adhesive strength and signalling in health and disease[J].Biochem J,2010,429(3):419-433.
[11]RIZZO S,LODDER EM,VERKERK AO,et al.Intercalated disc abnormalities,reduced Na+current density,and conduction slowing in desmoglein-2 mutant mice prior to cardiomyopathic changes[J].Cardiovasc Res,2012,95(4):409-418.
[12]KRUSCHE CA,HOLTHFER B,HOFE V,et al.Desmoglein 2mutant mice develop cardiac fibrosis and dilation[J].Basic Res Cardiol,2011,106(4):617-633.
[13]DUBASH AD,KAM CY,AGUADO BA,et al.Plakophilin-2 loss promotes TGF-β1/p38 MAPK-dependent fibrotic gene expression in cardiomyocytes[J].J Cell Biol,2016,212(4):425-438.
[14]YU CC,YU CH,HSUEH CH,et al.Arrhythmogenic right ventricular dysplasia:clinical characteristics and identification of novel desmosome gene mutations[J].J Formos Med Assoc,2008,107(7):548-558.
[15]RASMUSSEN TB,PALMFELDT J,NISSEN PH,et al.Mutated desmoglein-2 proteins are incorporated into desmosomes and exhibit dominant-negative effects in arrhythmogenic right ventricular cardiomyopathy[J].Hum Mutat,2013,34(5):697-705.
[16]BIERNACKA A,DOBACZEWSKI M,FRANGOGIANNIS NG.TGF-βsignaling in fibrosis[J].Growth Factors,2011,29(5):196-202.