SCN5A基因突变/缺失致窦房结功能改变的研究
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摘要
第一部分SCN5A基因点突变致病态窦房结综合征分子机制的研究
目的SCN5A基因点突变E161K,T187I和L212P对Nav1.5通道的动力学以及通道蛋白转运的影响。
方法采用定点突变的方法,得到含有E161K,T187I和L212P的SCN5A突变质粒,转染人胚胎肾293细胞(HEK293细胞),用膜片钳记录Nav1.5激活,失活和恢复等通道动力学改变。在Nav1.5通道跨膜结构域Ⅰ的第一和第二个跨膜片段胞外连接处插入flag序列,与Nav1.5通道共表达,了解三个突变点对通道蛋白的转运是否有影响。
结果正常钠通道在-20mv时,钠通道的电流最大,峰电流密度约为800pA/pF,激活曲线V1/2为-33.8±0.7 mV,斜率K为6.7±0.2;失活曲线V1/2为-80.3±0.9 mV,斜率K为6.2±0.1。T187I峰电流值减少约100%;E161K峰电流值减少约70%,L212P峰电流基本不变。E161K激活曲线明显右移,激活曲线斜率K为8.8±0.1mV,V1/2为-14.9±0.6mV,激活速度减慢,P<0.01;稳态失活曲线与正常接近,k为-6.5±0.2mV,V1/2为-78.4±0.8mV,通道失活不变。L212P其激活曲线明显左移,激活加快,激活曲线斜率k为8.4±0.2mV,V1/2为-14.9±0.6mV,通道激活速度加快,P<0.01;稳态失活曲线明显左移,曲线斜率k为-5.2±0.1mV,V1/2为-90.7±1.1mV,失活加快,P<0.01。E161K和L212P突变对通道的恢复无明显影响。三种突变通道上的Flag均能正常的表达在细胞膜上。
结论T187I表现为Nav1.5通道失功能;E161K和L212P表现为Nav1.5通道功能减弱。三种突变Nav1.5通道均能被转运到细胞膜表面。
第二部分Scn5a基因敲除小鼠模型的建立及鉴定
目的建立Scn5a基因敲除杂合子(Scn5a +/-)小鼠模型。
方法采用基因打靶技术,DNA同源重组的方法,建立Scn5a +/-小鼠模型。选择一组5月龄的同期培育的Scn5a +/-小鼠和正常野生型小鼠,取鼠尾提取DNA行PCR鉴定。分离小鼠的心室肌细胞,应用抗α-actin抗体鉴定分离下来的心室肌细胞。利用膜片钳技术记录Nav1.5通道电流。比较Nav1.5通道动力学的变化。
结果PCR结果显示野生型小鼠在980Bp处出现一条带,而Scn5a +/-小鼠则在980Bp和650Bp处各出现一条条带。与正常野生型小鼠相比, Scn5a+/-小鼠心室肌细胞钠电流峰电流密度减少约45%(16±6PA/PF vs 34±8PA/PF,n=5,P <0.05);其激活,失活曲线的半数有效电压和斜率均没有改变,无统计学差异。
结论Scn5a +/-小鼠可以很好的被用来作为研究Nav1.5通道异常的疾病模型。
第三部分Scn5a基因敲除小鼠心脏传导系统功能障碍的电生理研究
目的探讨Scn5a基因敲除杂合子(Scn5a +/-)小鼠窦房结等传导系统功能障碍的原因。
方法记录小鼠离体心脏的心表心电图,观察心率和节律的改变。通过电mapping了解窦房结发放冲动传导至外周冠状窦的时间。膜片钳技术检测小鼠窦房结细胞Nav1.5通道电流和功能。局部组织冰冻切片免疫组织化学检测窦房结区Nav1.5通道蛋白的表达。
结果与野生型小鼠相比,Scn5a +/-小鼠的心率明显减慢(259±27 bpm VS 336±8 bpm,P<0.05,n=6),并且该组小鼠间的心率变异度也大,并且出现2:1的房室传导阻滞;心表心电图上显示为P波时限(12.91±4.17ms VS 22.35±6.44ms, P<0.05,n=6)和PR间期延长(44.22±5.98ms VS 66.81±8.24ms, P<0.01,n=6) ;电mapping显示由窦房结发出的冲动传导到冠状窦的时间延长(8.16±1.36ms vs 3.56±0.26ms P<0.05,n=6)。Scn5a +/-小鼠窦房结细胞Nav1.5通道最大电流电流密度减小(36±1PA/PF vs 57±4PA/PF, P<0.05,n=5),通道功能没有改变。免疫荧光的结果显示Scn5a +/-小鼠窦房结区Nav1.5通道蛋白表达减少。
结论Nav1.5通道蛋白运输和表达障碍是导致Scn5a +/-小鼠Nav1.5通道电流减小,产生病态窦房结综合征的主要原因。
PartⅠStudy of Molecular Mechanism of Sick Sinus Syndrome Caused by Point Mutation of SCN5A Gene
Objective: Point mutations of SCN5A gene(E161K,T187I and L212P) and their effects on kenetics of Nav1.5 channel and transportation of channel protein.
Method: Site-directed mutagenesis method was used to construct plasmids, then transfected into HEK-293 cell. Sodium channel kenetics were studied by patch clamp technique between the wild type channel and mutant channels. The flag gene was inserted into the genes encoding the ecto-linker between segament I and II in homologous domain I of Nav1.5 sodium channel and co-expressed with Nav1.5. Flag was detected by immunofluorescence.
Result: At -20mv, Normal wild type sodium channel showed maximal current, the peak current was 800pA/pF, V1/2 and K of activation curve were -33.8±0.7 mV and 6.7±0.2 mV respectively, V1/2 and K of inactivation curve were -80.3±0.9 mV and 6.2±0.1 mV respectively. The peak current of T187I was reduced 100%; The peak current of E161K was reduced to 70%, stable activation curve showed obvious right shift, V1/2 and K of activation curve were -14.9±0.6mV and 8.8±0.1mV respectively (P<0.01 ); The peak current of L212P had no change, but stable activation curve showed obvious left shift, V1/2 and K of activation curve were -14.9±0.6mV and 8.4±0.2mV respectively (P<0.01); stable inactivation curve showed obvious left shift, V1/2 mV and K of inactivation curve were -90.7±1.1mV and -5.2±0.1mV respectively (P<0.01). E161K and L212P mutations did not affect the recovery of the Nav1.5 channel. Flags in three mutational channels could express on the cell membrance normally.
Conclusion:T187I showed non-function of Nav1.5 channel; E161K and L212P showed loss of function. Three mutations did not affect the transport of the Nav1.5 channel.
PartⅡEstablishment and Identification of Mouse Model of Targeted Disruption of Gene Scn5a
Objective: To establish the mouse model of targeted disruption of gene Scn5a.
Method: Scn5a gene targeted disruption mouse model was generated by gene targeting and homologous recombination of DNA. One group of Scn5a +/- mice and one group of wild type normal mice as control with the same matched age of 5 months were chosen. The template of PCR was got from DNA extracted from fresh mice tails. Single mouse ventricular cell was enzymatically isolated to be identified by anti-αactin antibody. Sodium currents from two different groups were recorded by patch clamp technique. Kenetics of sodium channels between cells from two different groups were compared by activation and inactivation.
Results: PCR showed in wild type mouse group, there was a band at size of 980 base pair, however, in Scn5a +/- mouse group, there was a band at size of 980 and 650 base pair respectively. At -20mv, showed the maximum current density, compared with WT mice cells, the current density from heterozygous mice cells was reduced to 45%(16±6PA/PF vs 34±8PA/PF,n=5,P<0.05). But there was no significant difference in V1/2 and K of activation and inactivation between these two groups.
Conclusion:Scn5a +/- mouse is a good animal model to investigate the disease related to abnormalities of Nav1.5 channel.
PartⅢElectrophysiological study of cardiac conduction system of Scn5a +/- mouse
Objective: To explore the electrophysiological disturbance of cardiac conduction systems of Scn5a +/- mouse.
Method: ECG was recorded in the condition of langendorff perfusion system, heart rate and rhythm were observed. Impulse conduction time from sinus node to coronary sinus was measured by electrical mapping recording. The current and function of Nav1.5 channel of sinus node cell were detected by patch clamp thechique. The expression of Nav1.5 protein in SAN area was detected in tissue frozen section by immunoflurescence.
Result: Compared with WT mouse, heart rates in the Scn5a +/- mouse were slowed (259±27 bpm VS 336±8 bpm,P<0.05,n=6)and discrepancy accompanied with 2:1 atrial ventricle block; the durations of P wave (12.91±4.17ms VS 22.35±6.44ms, P<0.05,n=6) and PR intervals (44.22±5.98ms VS 66.81±8.24ms, P<0.01,n=6) in ECG were prolonged; the conduction time from SAN to cornarary sinus was also prolonged(8.16±1.36ms VS 3.56±0.26ms P<0.05, n=6). The maximal current density of Nav1.5 channel in SAN cells was reduced (36±1PA/PF VS 57±4PA/PF, P<0.05, n=5) but without functional change. Expression of Nav1.5 protein in SAN area was reduced.
Conclusion: The trafficking and expression abnormalities of Nav1.5 protein contribute predominantly to the decrease of Nav1.5 currents resulting in sick sinus syndrome.
目的SCN5A基因点突变E161K,T187I和L212P对Nav1.5通道的动力学以及通道蛋白转运的影响。
方法采用定点突变的方法,得到含有E161K,T187I和L212P的SCN5A突变质粒,转染人胚胎肾293细胞(HEK293细胞),用膜片钳记录Nav1.5激活,失活和恢复等通道动力学改变。在Nav1.5通道跨膜结构域Ⅰ的第一和第二个跨膜片段胞外连接处插入flag序列,与Nav1.5通道共表达,了解三个突变点对通道蛋白的转运是否有影响。
结果正常钠通道在-20mv时,钠通道的电流最大,峰电流密度约为800pA/pF,激活曲线V1/2为-33.8±0.7 mV,斜率K为6.7±0.2;失活曲线V1/2为-80.3±0.9 mV,斜率K为6.2±0.1。T187I峰电流值减少约100%;E161K峰电流值减少约70%,L212P峰电流基本不变。E161K激活曲线明显右移,激活曲线斜率K为8.8±0.1mV,V1/2为-14.9±0.6mV,激活速度减慢,P<0.01;稳态失活曲线与正常接近,k为-6.5±0.2mV,V1/2为-78.4±0.8mV,通道失活不变。L212P其激活曲线明显左移,激活加快,激活曲线斜率k为8.4±0.2mV,V1/2为-14.9±0.6mV,通道激活速度加快,P<0.01;稳态失活曲线明显左移,曲线斜率k为-5.2±0.1mV,V1/2为-90.7±1.1mV,失活加快,P<0.01。E161K和L212P突变对通道的恢复无明显影响。三种突变通道上的Flag均能正常的表达在细胞膜上。
结论T187I表现为Nav1.5通道失功能;E161K和L212P表现为Nav1.5通道功能减弱。三种突变Nav1.5通道均能被转运到细胞膜表面。
第二部分Scn5a基因敲除小鼠模型的建立及鉴定
目的建立Scn5a基因敲除杂合子(Scn5a +/-)小鼠模型。
方法采用基因打靶技术,DNA同源重组的方法,建立Scn5a +/-小鼠模型。选择一组5月龄的同期培育的Scn5a +/-小鼠和正常野生型小鼠,取鼠尾提取DNA行PCR鉴定。分离小鼠的心室肌细胞,应用抗α-actin抗体鉴定分离下来的心室肌细胞。利用膜片钳技术记录Nav1.5通道电流。比较Nav1.5通道动力学的变化。
结果PCR结果显示野生型小鼠在980Bp处出现一条带,而Scn5a +/-小鼠则在980Bp和650Bp处各出现一条条带。与正常野生型小鼠相比, Scn5a+/-小鼠心室肌细胞钠电流峰电流密度减少约45%(16±6PA/PF vs 34±8PA/PF,n=5,P <0.05);其激活,失活曲线的半数有效电压和斜率均没有改变,无统计学差异。
结论Scn5a +/-小鼠可以很好的被用来作为研究Nav1.5通道异常的疾病模型。
第三部分Scn5a基因敲除小鼠心脏传导系统功能障碍的电生理研究
目的探讨Scn5a基因敲除杂合子(Scn5a +/-)小鼠窦房结等传导系统功能障碍的原因。
方法记录小鼠离体心脏的心表心电图,观察心率和节律的改变。通过电mapping了解窦房结发放冲动传导至外周冠状窦的时间。膜片钳技术检测小鼠窦房结细胞Nav1.5通道电流和功能。局部组织冰冻切片免疫组织化学检测窦房结区Nav1.5通道蛋白的表达。
结果与野生型小鼠相比,Scn5a +/-小鼠的心率明显减慢(259±27 bpm VS 336±8 bpm,P<0.05,n=6),并且该组小鼠间的心率变异度也大,并且出现2:1的房室传导阻滞;心表心电图上显示为P波时限(12.91±4.17ms VS 22.35±6.44ms, P<0.05,n=6)和PR间期延长(44.22±5.98ms VS 66.81±8.24ms, P<0.01,n=6) ;电mapping显示由窦房结发出的冲动传导到冠状窦的时间延长(8.16±1.36ms vs 3.56±0.26ms P<0.05,n=6)。Scn5a +/-小鼠窦房结细胞Nav1.5通道最大电流电流密度减小(36±1PA/PF vs 57±4PA/PF, P<0.05,n=5),通道功能没有改变。免疫荧光的结果显示Scn5a +/-小鼠窦房结区Nav1.5通道蛋白表达减少。
结论Nav1.5通道蛋白运输和表达障碍是导致Scn5a +/-小鼠Nav1.5通道电流减小,产生病态窦房结综合征的主要原因。
PartⅠStudy of Molecular Mechanism of Sick Sinus Syndrome Caused by Point Mutation of SCN5A Gene
Objective: Point mutations of SCN5A gene(E161K,T187I and L212P) and their effects on kenetics of Nav1.5 channel and transportation of channel protein.
Method: Site-directed mutagenesis method was used to construct plasmids, then transfected into HEK-293 cell. Sodium channel kenetics were studied by patch clamp technique between the wild type channel and mutant channels. The flag gene was inserted into the genes encoding the ecto-linker between segament I and II in homologous domain I of Nav1.5 sodium channel and co-expressed with Nav1.5. Flag was detected by immunofluorescence.
Result: At -20mv, Normal wild type sodium channel showed maximal current, the peak current was 800pA/pF, V1/2 and K of activation curve were -33.8±0.7 mV and 6.7±0.2 mV respectively, V1/2 and K of inactivation curve were -80.3±0.9 mV and 6.2±0.1 mV respectively. The peak current of T187I was reduced 100%; The peak current of E161K was reduced to 70%, stable activation curve showed obvious right shift, V1/2 and K of activation curve were -14.9±0.6mV and 8.8±0.1mV respectively (P<0.01 ); The peak current of L212P had no change, but stable activation curve showed obvious left shift, V1/2 and K of activation curve were -14.9±0.6mV and 8.4±0.2mV respectively (P<0.01); stable inactivation curve showed obvious left shift, V1/2 mV and K of inactivation curve were -90.7±1.1mV and -5.2±0.1mV respectively (P<0.01). E161K and L212P mutations did not affect the recovery of the Nav1.5 channel. Flags in three mutational channels could express on the cell membrance normally.
Conclusion:T187I showed non-function of Nav1.5 channel; E161K and L212P showed loss of function. Three mutations did not affect the transport of the Nav1.5 channel.
PartⅡEstablishment and Identification of Mouse Model of Targeted Disruption of Gene Scn5a
Objective: To establish the mouse model of targeted disruption of gene Scn5a.
Method: Scn5a gene targeted disruption mouse model was generated by gene targeting and homologous recombination of DNA. One group of Scn5a +/- mice and one group of wild type normal mice as control with the same matched age of 5 months were chosen. The template of PCR was got from DNA extracted from fresh mice tails. Single mouse ventricular cell was enzymatically isolated to be identified by anti-αactin antibody. Sodium currents from two different groups were recorded by patch clamp technique. Kenetics of sodium channels between cells from two different groups were compared by activation and inactivation.
Results: PCR showed in wild type mouse group, there was a band at size of 980 base pair, however, in Scn5a +/- mouse group, there was a band at size of 980 and 650 base pair respectively. At -20mv, showed the maximum current density, compared with WT mice cells, the current density from heterozygous mice cells was reduced to 45%(16±6PA/PF vs 34±8PA/PF,n=5,P<0.05). But there was no significant difference in V1/2 and K of activation and inactivation between these two groups.
Conclusion:Scn5a +/- mouse is a good animal model to investigate the disease related to abnormalities of Nav1.5 channel.
PartⅢElectrophysiological study of cardiac conduction system of Scn5a +/- mouse
Objective: To explore the electrophysiological disturbance of cardiac conduction systems of Scn5a +/- mouse.
Method: ECG was recorded in the condition of langendorff perfusion system, heart rate and rhythm were observed. Impulse conduction time from sinus node to coronary sinus was measured by electrical mapping recording. The current and function of Nav1.5 channel of sinus node cell were detected by patch clamp thechique. The expression of Nav1.5 protein in SAN area was detected in tissue frozen section by immunoflurescence.
Result: Compared with WT mouse, heart rates in the Scn5a +/- mouse were slowed (259±27 bpm VS 336±8 bpm,P<0.05,n=6)and discrepancy accompanied with 2:1 atrial ventricle block; the durations of P wave (12.91±4.17ms VS 22.35±6.44ms, P<0.05,n=6) and PR intervals (44.22±5.98ms VS 66.81±8.24ms, P<0.01,n=6) in ECG were prolonged; the conduction time from SAN to cornarary sinus was also prolonged(8.16±1.36ms VS 3.56±0.26ms P<0.05, n=6). The maximal current density of Nav1.5 channel in SAN cells was reduced (36±1PA/PF VS 57±4PA/PF, P<0.05, n=5) but without functional change. Expression of Nav1.5 protein in SAN area was reduced.
Conclusion: The trafficking and expression abnormalities of Nav1.5 protein contribute predominantly to the decrease of Nav1.5 currents resulting in sick sinus syndrome.
引文
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2. Benson DW, Wang DW, Dyment M, et al. Congenital sick sinus syndrome caused by recessive mutations in the cardiac sodium channel gene (SCN5A). J Clin Invest.2003;112:1019-1028.
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5. Veldkamp MW, Wilders R, Baartscheer A, et al .Contribution of sodium channel mutations to bradycardia and sinus node dysfunction in LQT3 families. Circ Res. 2003;92: 976-983.
6. Lei M, Jones SA, Liu J,et al .Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J Physiol .2004;559: 835-848
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8. Tellez JO, Dobrzynski H, Greener ID, et al. Differential expression of ion channel transcripts in atrial muscle and sinoatrial node in rabbit. Circ Res. 2006;99:1384-1393.
9. Smits JP, Koopmann TT, Wilders R, et al .A mutation in the human cardiac sodium channel (E161K) contributes to sick sinus syndrome, conduction disease and Brugada syndrome in two families. J Mol Cell Cardiol .2005;38:969-981.
10. Makita N, Sasaki K, Groenewegen WA, et al. Congenital atrial standstill associated with coinheritance of a novel SCN5A mutation and connexin 40 polymorphisms. Heart Rhythm. 2005;2:1128-1134.
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12. Rudy Y, Shaw R M. Cardiac excitation: an interactive process of ion channels and gap junctions. Adv Exp Med Biol. 1997;430:269-279.
13. Remme CA, Verkerk AO, Nuyens D, et al. Overlap Syndrome of Cardiac Sodium Channel Disease in Mice Carrying the Equivalent Mutation of Human SCN5A-1795insD. Circulation. 2006;114:2584-2594
1. Marban E. Cardiac channelopaties. Nature.2002;415:213–218.
2. Bennett PB, Yazawa K, Makita N, et al. Molecular mechanism for an inherited cardiac arrhythmia. Nature. 1995;376:683– 685.
3. Tan HL, Bezzina CR, Smits JP, et al. Genetic control of sodium channel function. Cardiovasc Res. 2003;57:961–973.
4. Antzelevitch C, Brugada P, Brugada J, et al. Brugada syndrome: from cell to bedside. Curr Probl Cardiol. 2005;30:9-54.
5. Wang Q, Shen J, Splawski I, et al. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell.1995;80:805–811.
6. Akai J, Makita N, Sakurada H, et al. A novel SCN5A mutation associated with idiopathic ventricular fibrillation without typical ECG findings of Brugada syndrome. FEBS Lett. 2000;479:29-34.
7. 7. Benson DW, Wang DW, Dyment M, et al. Congenital sick sinus syndrome caused by recessive mutations in the cardiac sodium channel gene (SCN5A). J Clin Invest. 2003;112:1019-1028.
8. George AL, Varkony TA, Drabkin HA, et al.Assignment of the human heart tetrodotoxin-resistant voltage-gated Na+ channel alpha-subunit gene (SCN5A) to band 3p21. Cytogenet Cell Genet. 1995;68:67-70.
9. Tester DJ, Will ML, Haglund CM, et al. Compendium of cardiac channel mutations in 541 consecutive unrelated patients referred for long QT syndrome genetic testing. Heart Rhythm. 2005; 2:507–517.
10. Nuyens D, Stengl M, Dugarmaa S, et al. Abrupt rate accelerations or premature beats cause lifethreatening arrhythmias in mice with long-QT3 syndrome. Nat Med.2001;7:1021–1027.
11. Remme CA, Verkerk AO, Nuyens D, et al. Overlap syndrome of cardiac sodiumchannel disease in mice carrying the equivalent mutation of human SCN5A- 1795insD. Circulation.2006;114: 2584–2594.
12. Tian XL, Yong SL, Wan X, et al. Mechanisms by which SCN5A mutation N1325S causes cardiac arrhythmias and sudden death in vivo Cardiovasc Res. 2004;61:256-267.
13. Probst V, Hoorntje TM, Hulsbeek M, et al. Cardiac conduction defects associate with mutations in SCN5A. Nat Genet. 1999;23:20-21.
14. Papadatos GA, Wallerstein PM, Head CE, et al. Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a. Proc Natl Acad Sci U S A. 2002;99:6210-6215.
15. Laitinen-Forsblom PJ,Makynen P,Makynen H, et al. SCN5A mutation associated with cardiac conduction defect and atrial arrhythmias. J Cardiovasc Electrophysiol. 2006;17:480–485.
16. Chen LY, Ballew JD, Herron KJ, et al. A common polymorphism in SCN5A is associated with Lone Atrial Fibrillation. Clin Pharmacol Ther.2007;81:35–41.
17. Hesse M. Dilated Cardiomyopathy is Associated with Reduced Expression of the Cardiac Sodium Channel Scn5a. Cardiovasc Res.2007;75:498–509.
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