PRKAG2基因G100S新突变对小鼠心肌细胞单磷酸腺苷活化蛋白激酶活性的影响
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摘要
目的探讨位于非胱硫醚β-合成酶(cystathionineβ-synthase,CBS)区域的PRKAG2基因G100S突变对小鼠心肌细胞单磷酸腺苷活化蛋白激酶(AMPK)活性的影响。方法建立人源PRKAG2(G100S)转基因小鼠模型,分别随机选取N4代4周龄、12周龄的转基因小鼠和同窝野生型小鼠各6只,用磷酸化检测试剂盒检测小鼠心肌细胞中AMPK活性,比较转基因小鼠与同窝野生型小鼠AMPK活性的差异,并观察随着周龄的增长转基因小鼠AMPK活性的变化。结果 4周龄和12周龄的转基因小鼠心肌细胞中AMPK活性均低于同窝野生型小鼠(0.042±0.013 vs 0.063±0.013,0.032±0.008 vs 0.062±0.018),差异均有统计学意义(P=0.019,P=0.004)。12周龄和4周龄的转基因小鼠心肌细胞中AMPK活性差异无统计学意义(P=0.135)。结论 PRKAG2基因G100S突变可导致转基因小鼠心肌细胞AMPK活性下降,而且AMPK活性并不随着转基因小鼠周龄的增长而变化。
Objective To explore the effect of PRKAG2 gene G100 S mutation in cystathionine β-synthase(CBS) region on adenosine monophosphate-activated protein kinase(AMPK) activity in cardiomyocytes of mice. Methods A human PRKAG2(G100S) transgenic mouse model was established. Four-week-old and 12-week-old transgenic mice, and 4-week-old and 12-weekold wildtype littermate were randomly selected from N4 generation mice(n=6). The activity of AMPK in mouse cardiomyocytes was detected by phosphorylation assay kit. The difference of AMPK activity was compared between transgenic mice and wildtype littermate, and the changes of the activity of AMPK with the increase of age were observed in transgenic mice. Results The AMPK activities in cardiomyocytes of 4-week-old and 12-week-old transgenic mice were significantly lower than those of the wildtype littermate(0.042±0.013 vs 0.063±0.013, and 0.032±0.008 vs 0.062±0.018), and the differences were significant(P=0.019, P=0.004). There was no significant difference in the AMPK activity of cardiomyocytes between 4-week-old and 12-weekold transgenic mice(P=0.135). Conclusion The PRKAG2 gene G100 S mutation can cause a reduction of AMPK activity in cardiomyocytes of transgenic mice, and AMPK activity does not significantly increase or decrease with the growth of the transgenic mice.
Objective To explore the effect of PRKAG2 gene G100 S mutation in cystathionine β-synthase(CBS) region on adenosine monophosphate-activated protein kinase(AMPK) activity in cardiomyocytes of mice. Methods A human PRKAG2(G100S) transgenic mouse model was established. Four-week-old and 12-week-old transgenic mice, and 4-week-old and 12-weekold wildtype littermate were randomly selected from N4 generation mice(n=6). The activity of AMPK in mouse cardiomyocytes was detected by phosphorylation assay kit. The difference of AMPK activity was compared between transgenic mice and wildtype littermate, and the changes of the activity of AMPK with the increase of age were observed in transgenic mice. Results The AMPK activities in cardiomyocytes of 4-week-old and 12-week-old transgenic mice were significantly lower than those of the wildtype littermate(0.042±0.013 vs 0.063±0.013, and 0.032±0.008 vs 0.062±0.018), and the differences were significant(P=0.019, P=0.004). There was no significant difference in the AMPK activity of cardiomyocytes between 4-week-old and 12-weekold transgenic mice(P=0.135). Conclusion The PRKAG2 gene G100 S mutation can cause a reduction of AMPK activity in cardiomyocytes of transgenic mice, and AMPK activity does not significantly increase or decrease with the growth of the transgenic mice.
引文
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[11]SIDHU J S,RAJAWAT Y S,RAMI T G,GOLLOB MH,WANG Z,YUAN R,et al.Transgenic mouse model of ventricular preexcitation and atrioventricular reentrant tachycardia induced by an AMP-activated protein kinase loss-of-function mutation responsible for Wolff-ParkinsonWhite syndrome[J].Circulation,2005,111:21-29.
[12]张静,郑兴,秦永文,周炳炎,吴弘,王洪如.家族性传导系统异常伴心室预激及心肌肥厚一家系调查分析[J].中华心血管病杂志,2007,35:258-259.
[13]陈挺,刘杰,肖良,鲍礼智,余云华,郑兴.心脏过表达人源PRKAG2(G100S)转基因小鼠模型的建立[J].第二军医大学学报,2016,37:273-278.CHEN T,LIU J,XIAO L,BAO L Z,YU Y H,ZHENGX.Establishment of transgenic mouse model with cardiac overexpression of PRKAG2-G100S[J].Acad J Sec Mil Med Univ,2016,37:273-278.
[14]LIGHT P E,WALLACE C H,DYCK J R.Constitutively active adenosine monophosphate-activated protein kinase regulates voltage-gated sodium channels in ventricular myocytes[J].Circulation,2003,107:1962-1965.
[15]YOSHIDA H,BAO L,KEFALOYIANNI E,TASKIN E,OKORIE U,HONG M,et al.AMP-activated protein kinase connects cellular energy metabolism to KATP channel function[J].J Mol Cell Cardiol,2012,52:410-418.
[16]SUKHODUB A,JOVANOVI?S,DU Q,BUDASG,CLELLAND AK,SHENM,et al.A M P-activated protein kinase mediates preconditioning in cardiomyocytes by regulating activity and trafficking of sarcolemmal ATP-sensitive K+channels[J].J Cell Physiol,2007,210:224-236.
[17]TURRELLHE,RODRIG C,NOR A N RI,DICKENS M,STANDEN N B.Phenylephrine preconditioning involves modulation of cardiac sarcolemmal KATP current by PKC delta,AMPK and p38MAPK[J].J Mol Cell Cardiol,2011,51:370-380.
[18]ALESUTAN I,MUNOZ C,SOPJANI M,D?RMAKU-SOPJANI M,MICHAEL D,FRASER S,et al.Inhibition of Kir2.1(KCNJ2)by the AMP-activated protein kinase[J].Biochem Biophys Res Commun,2011,408:505-510.
[19]DYCK J R,LOPASCHUK G D.AMPK alterations in cardiac physiology and pathology:enemy or ally?[J].JPhysiol,2006,574(Pt 1):95-112.
[20]CHEUNG P C,SALT I P,DAVIES S P,HARDIE D G,CARLING D.Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMPbinding[J].Biochem J,2000,346 Pt 3:659-669.
[21]DANIEL T,CARLING D.Functional analysis of mutations in theγ2 subunit of AMP-activated protein kinase associated with cardiac hypertrophy and WolffParkinson-White syndrome[J].J Biol Chem,2002,277:51017-51024.
[22]ZHANG B L,YE Z,XU R L,YOU X H,QIN Y W,WUH,et al.Overexpression of G100S mutation in PRKAG2causes Wolff-Parkinson-White syndrome in zebrafish[J].Clin Genet,2014,86:287-291.
[2]THEVENON J,LAURENT G,ADER F,LAFORêT P,KLUG D,DUVA PENTIAH A,et al.High prevalence of arrhythmic and myocardial complications in patients with cardiac glycogenosis due to PRKAG2 mutations[J].Europace,2017,19:651-659.
[3]GOLLOB M H,GREEN M S,TANG A S,GOLLOB T,KARIBE A,ALI HASSAN A S,et al.Identification of a gene responsible for familial Wolff-Parkinson-White syndrome[J].N Engl J Med,2001,344:1823-1831.
[4]GOLLOB M H,SEGER J J,GOLLOB T N,TAPSCOTTT,GONZALES O,BACHINSKI L,et al.Novel PRKAG2 mutation responsible for the genetic syndrome of ventricular preexcitation and conduction system disease with childhood onset and absence of cardiac hypertrophy[J].Circulation,2001,104:3030-3033.
[5]BAYRAK F,KOMURCU-BAYRAK E,MUTLU B,KAHVECI G,BASARAN Y,ERGINEL-UNALTUNAN.Ventricular pre-excitation and cardiac hypertrophy mimicking hypertrophic cardiomyopathy in a Turkish family with a novel PRKAG2 mutation[J].Eur J Heart Fail,2006,8:712-715.
[6]ARAD M,BENSON D W,PEREZ-ATAYDE A R,MCKENNA W J,SPARKS E A,KANTER R J,et al.Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy[J].J Clin Invest,2002,109:357-362.
[7]ARAD M,MOSKOWITZ I P,PATEL V V,AHMAD F,PEREZ-ATAYDE A R,SAWYER D B,et al.Transgenic mice overexpressing mutant PRKAG2 define the cause of Wolff-Parkinson-White syndrome in glycogen storage cardiomyopathy[J].Circulation,2003,107:2850-2856.
[8]SCOTT J W,HAWLEY S A,GREEN K A,ANIS M,STEWART G,SCULLION G A,et al.CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations[J].J Clin Invest,2004,113:274-284.
[9]DAVIES J K,WELLS D J,LIU K,WHITROW H R,DANIEL T D,GRIGNANI R,et al.Characterization of the role ofγ2 R531G mutation in AMP-activated protein kinase in cardiac hypertrophy and Wolff-Parkinson-White syndrome[J].Am J Physiol Heart Circ Physiol,2006,290:H1942-H1951.
[10]BANERJEE S K,RAMANI R,SABA S,RAGER J,TIAN R,MATHIER M A,et al.A PRKAG2 mutation causes biphasic changes in myocardial AMPK activity and does not protect against ischemia[J].Biochem Biophys Res Commun,2007,360:381-387.
[11]SIDHU J S,RAJAWAT Y S,RAMI T G,GOLLOB MH,WANG Z,YUAN R,et al.Transgenic mouse model of ventricular preexcitation and atrioventricular reentrant tachycardia induced by an AMP-activated protein kinase loss-of-function mutation responsible for Wolff-ParkinsonWhite syndrome[J].Circulation,2005,111:21-29.
[12]张静,郑兴,秦永文,周炳炎,吴弘,王洪如.家族性传导系统异常伴心室预激及心肌肥厚一家系调查分析[J].中华心血管病杂志,2007,35:258-259.
[13]陈挺,刘杰,肖良,鲍礼智,余云华,郑兴.心脏过表达人源PRKAG2(G100S)转基因小鼠模型的建立[J].第二军医大学学报,2016,37:273-278.CHEN T,LIU J,XIAO L,BAO L Z,YU Y H,ZHENGX.Establishment of transgenic mouse model with cardiac overexpression of PRKAG2-G100S[J].Acad J Sec Mil Med Univ,2016,37:273-278.
[14]LIGHT P E,WALLACE C H,DYCK J R.Constitutively active adenosine monophosphate-activated protein kinase regulates voltage-gated sodium channels in ventricular myocytes[J].Circulation,2003,107:1962-1965.
[15]YOSHIDA H,BAO L,KEFALOYIANNI E,TASKIN E,OKORIE U,HONG M,et al.AMP-activated protein kinase connects cellular energy metabolism to KATP channel function[J].J Mol Cell Cardiol,2012,52:410-418.
[16]SUKHODUB A,JOVANOVI?S,DU Q,BUDASG,CLELLAND AK,SHENM,et al.A M P-activated protein kinase mediates preconditioning in cardiomyocytes by regulating activity and trafficking of sarcolemmal ATP-sensitive K+channels[J].J Cell Physiol,2007,210:224-236.
[17]TURRELLHE,RODRIG C,NOR A N RI,DICKENS M,STANDEN N B.Phenylephrine preconditioning involves modulation of cardiac sarcolemmal KATP current by PKC delta,AMPK and p38MAPK[J].J Mol Cell Cardiol,2011,51:370-380.
[18]ALESUTAN I,MUNOZ C,SOPJANI M,D?RMAKU-SOPJANI M,MICHAEL D,FRASER S,et al.Inhibition of Kir2.1(KCNJ2)by the AMP-activated protein kinase[J].Biochem Biophys Res Commun,2011,408:505-510.
[19]DYCK J R,LOPASCHUK G D.AMPK alterations in cardiac physiology and pathology:enemy or ally?[J].JPhysiol,2006,574(Pt 1):95-112.
[20]CHEUNG P C,SALT I P,DAVIES S P,HARDIE D G,CARLING D.Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMPbinding[J].Biochem J,2000,346 Pt 3:659-669.
[21]DANIEL T,CARLING D.Functional analysis of mutations in theγ2 subunit of AMP-activated protein kinase associated with cardiac hypertrophy and WolffParkinson-White syndrome[J].J Biol Chem,2002,277:51017-51024.
[22]ZHANG B L,YE Z,XU R L,YOU X H,QIN Y W,WUH,et al.Overexpression of G100S mutation in PRKAG2causes Wolff-Parkinson-White syndrome in zebrafish[J].Clin Genet,2014,86:287-291.