犬左室三层细胞的分离和电生理记录
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摘要
目的探讨分离耐钙犬左室三层细胞的方法,并利用全细胞膜片钳技术进行电生理记录。方法采用改良的Langendorff灌流装置,经主动脉左冠窦插管灌流带有左室前降支的犬心肌组织块,分离出单个耐钙心室肌细胞。先根据解剖部位将心室肌细胞分为内、中、外三层;再采用全细胞膜片钳技术,在电流钳模式下,各层随机选取8个细胞记录动作电位(AP)、快激活延迟整流钾电流(I_(Kr))、慢激活延迟整流钾电流(I_(Ks))、晚钠电流(I_(Na-L)),分析三层的心室肌细胞的电生理特性。结果分离状态良好的单个心室肌细胞率为90%以上,呈杆状,表面光滑,横纹清晰,边缘锐利,静止不动;内层(Endo)心室肌细胞没有尖峰-圆顶样形态,动作电位复极90%的时程(APD_(90))最短;中层(Mid)心室肌细胞也有尖峰-圆顶样形态,但不如外层(Epi)细胞明显,APD_(90)最长;Epi心室肌细胞有明显的尖峰-圆顶样形态,APD_(90)介于Mid与Endo之间。在-30 mV电压下,Mid细胞I_(Na-L)电流密度较Endo、Epi的电流密度稍大,但无统计学差异;三层细胞I_(Kr)的电流密度无明显差异;Mid细胞的I_(Ks)的电流密度在三层心肌细胞中最小,Endo、Epi中无明显差异。结论该方法更加易于操作,节约实验成本和时间,分离的细胞数量多、活性高;结合解剖部位和电生理特点,能有效鉴别各层心室肌细胞;成功分离出细胞,可用于离子通道特性的研究。
Objective To explore an efficient method to isolate calcium-tolerant cells in different layers of canine left ventricular myocytes, and to use the whole-cell patch clamp technique for electrophysiological recording. Method The modified Langendorff perfusion apparatus was used in the experiments. Connecting aortic left sinus to the cannula of the perfusion apparatus, a single calcium-tolerant ventricular myocyte was obtained from the canine myocardium of left ventricular anterior descending artery. According to the anatomical position, the ventricular myocardium were divided into three layers: endocardial(Endo), midmyocardium(Mid) and epicardium(Epi). Then 8 cells of each layer were selected randomly. Their action potential, late sodium current(I_(Na-L)), the delayed rectifier potassium current, including I_(Kr)(rapid) and I_(Ks)(slow), were recorded with the whole-cell patch clamp technique and to be analysis later. Results Specific ventricular myocytes, which identified as rod-shaped, smooth surface, clear stripes, sharp edges and quiescent with normal physiological properties, accounted for more than 90% of isolated cells.Endocardial myocytes had no spike-dome-like morphology, APD_(90) was the shortest; Mid cells also had a spike-dome-like morphology, but not as obvious as Epi cells, and APD_(90) was the longest; Epi myocytes had a distinct spike-dome-like morphology with APD_(90) between the Mid cells′ and Endo cells′. At-30 mV, the current density of I_(Na-L) of Mid cells was slightly larger than that of Endo and Epi, but there was no statistical difference.The current density of I_Kr among Mid,Endo and Epi cells was not significantly different.The current density of IKsof Mid cells was the smallest among the three layers of cardiomyocytes,and there was no significant statistically difference between Endo and Epi. Conclusion This method is more easy to operate and can save experimental cost and time.The isolated cells are high quantity and activity.Combining with the anatomical position and electrophysiological characteristics,it can effectively identify the ventricular myocytes of each layer.The isolated cells can be used to study the characteristics of ion channels.[Chinese Journal of Cardiac Pacing and Electrophysiology,2019,33(4):333-338]
Objective To explore an efficient method to isolate calcium-tolerant cells in different layers of canine left ventricular myocytes, and to use the whole-cell patch clamp technique for electrophysiological recording. Method The modified Langendorff perfusion apparatus was used in the experiments. Connecting aortic left sinus to the cannula of the perfusion apparatus, a single calcium-tolerant ventricular myocyte was obtained from the canine myocardium of left ventricular anterior descending artery. According to the anatomical position, the ventricular myocardium were divided into three layers: endocardial(Endo), midmyocardium(Mid) and epicardium(Epi). Then 8 cells of each layer were selected randomly. Their action potential, late sodium current(I_(Na-L)), the delayed rectifier potassium current, including I_(Kr)(rapid) and I_(Ks)(slow), were recorded with the whole-cell patch clamp technique and to be analysis later. Results Specific ventricular myocytes, which identified as rod-shaped, smooth surface, clear stripes, sharp edges and quiescent with normal physiological properties, accounted for more than 90% of isolated cells.Endocardial myocytes had no spike-dome-like morphology, APD_(90) was the shortest; Mid cells also had a spike-dome-like morphology, but not as obvious as Epi cells, and APD_(90) was the longest; Epi myocytes had a distinct spike-dome-like morphology with APD_(90) between the Mid cells′ and Endo cells′. At-30 mV, the current density of I_(Na-L) of Mid cells was slightly larger than that of Endo and Epi, but there was no statistical difference.The current density of I_Kr among Mid,Endo and Epi cells was not significantly different.The current density of IKsof Mid cells was the smallest among the three layers of cardiomyocytes,and there was no significant statistically difference between Endo and Epi. Conclusion This method is more easy to operate and can save experimental cost and time.The isolated cells are high quantity and activity.Combining with the anatomical position and electrophysiological characteristics,it can effectively identify the ventricular myocytes of each layer.The isolated cells can be used to study the characteristics of ion channels.[Chinese Journal of Cardiac Pacing and Electrophysiology,2019,33(4):333-338]
引文
1 郭玉君,侯月梅.适用于膜片钳实验的犬心房肌细胞的分离方法分析研究[J].新疆医科大学学报,2010(08):904
2 于涛,吴若彬,邓春玉,等.犬左心房心肌细胞的分离方法及电生理记录[J].广东医学,2010(24):3 157
3 刘颖,王雷,彭珍,等.犬心肌细胞分离方法的改良[J].现代生物医学进展,2013(08):1 412
4 郭凯,黄从新,吴攀,等.犬三层心室肌细胞的分离和鉴别[J].中国心脏起搏与心电生理杂志,2008,22(03):252
5 Tadevosyan A,Allen BG,Nattel S.Isolation and study of cardiac nuclei from canine myocardium and adult ventricular myocytes[J].Methods Mol Biol,2015.1234:69
6 Hammill OP,Marty A,Neher E,et al.Improved patch-clamp techniques for high-resolution current recording from cells and cell-freemembrane patches[J].Pflugers Arch,1981,391(2):85
7 王如兴,蒋文平.正常耐钙Spraque-Dawley大鼠心室肌细胞分离方法及体会 [J].中国心脏起搏与心电生理杂志,2007,21(1):59
8 王焱,巩燕,黄卫斌,等.动作电位参数作为区分狗心室肌三种细胞的可行性研究[J].中国心脏起搏与心电生理杂志,2005,19:472
9 Zygmunt AC,Eddlestone GT,Thomas GP,et al.Larger late sodium conductance in M cells contributes to electrical heterogeneity in canine ventricle[J].Am J Physiol Heart Circ Physiol,2001,281(2):H689
10 Xiao L,Zhang LM,Han W,et al.Sex-based transmural differences in cardiac repolarization and ionic-current properties in canine left ventricles[J].Am J Physiol Heart Circ Physio,2006,291:H570
11 Liu DW,Antzelevitch C.Characteristics of the delayed rectifier current(IKr and IKs) in canine ventricular epicardial,midmyocardial,and endocardial myocytes.A weaker IKs contributes to the longer action potential of the M cell[J].Circ Res,1995,76(3):351
12 Viswanathan PC,Shaw RM,Rudy Y.Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence:a simulation study[J].Circulation,1999,99:2 466
13 Suthedand FJ,Shattock MJ,Baker KE,et al.Mouse isolated perfused heart:characteristics and cautions[J].Clin Exper Pharm Physiol,2003,30:867
14 李泱,马杰,肖建民,等.兔左室壁三层心肌细胞的分离及动作电位、钙和钾电流分布的异质性[J].生理学报,2002(05):369
15 Wasserstrom JA,Salata JJ.Basis for tetrodotoxin and lidocaine effects on action potentials in dog ventricular myocytes[J].Am J Physiol Heart Circ Physiol,1988,254:H1 157
2 于涛,吴若彬,邓春玉,等.犬左心房心肌细胞的分离方法及电生理记录[J].广东医学,2010(24):3 157
3 刘颖,王雷,彭珍,等.犬心肌细胞分离方法的改良[J].现代生物医学进展,2013(08):1 412
4 郭凯,黄从新,吴攀,等.犬三层心室肌细胞的分离和鉴别[J].中国心脏起搏与心电生理杂志,2008,22(03):252
5 Tadevosyan A,Allen BG,Nattel S.Isolation and study of cardiac nuclei from canine myocardium and adult ventricular myocytes[J].Methods Mol Biol,2015.1234:69
6 Hammill OP,Marty A,Neher E,et al.Improved patch-clamp techniques for high-resolution current recording from cells and cell-freemembrane patches[J].Pflugers Arch,1981,391(2):85
7 王如兴,蒋文平.正常耐钙Spraque-Dawley大鼠心室肌细胞分离方法及体会 [J].中国心脏起搏与心电生理杂志,2007,21(1):59
8 王焱,巩燕,黄卫斌,等.动作电位参数作为区分狗心室肌三种细胞的可行性研究[J].中国心脏起搏与心电生理杂志,2005,19:472
9 Zygmunt AC,Eddlestone GT,Thomas GP,et al.Larger late sodium conductance in M cells contributes to electrical heterogeneity in canine ventricle[J].Am J Physiol Heart Circ Physiol,2001,281(2):H689
10 Xiao L,Zhang LM,Han W,et al.Sex-based transmural differences in cardiac repolarization and ionic-current properties in canine left ventricles[J].Am J Physiol Heart Circ Physio,2006,291:H570
11 Liu DW,Antzelevitch C.Characteristics of the delayed rectifier current(IKr and IKs) in canine ventricular epicardial,midmyocardial,and endocardial myocytes.A weaker IKs contributes to the longer action potential of the M cell[J].Circ Res,1995,76(3):351
12 Viswanathan PC,Shaw RM,Rudy Y.Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence:a simulation study[J].Circulation,1999,99:2 466
13 Suthedand FJ,Shattock MJ,Baker KE,et al.Mouse isolated perfused heart:characteristics and cautions[J].Clin Exper Pharm Physiol,2003,30:867
14 李泱,马杰,肖建民,等.兔左室壁三层心肌细胞的分离及动作电位、钙和钾电流分布的异质性[J].生理学报,2002(05):369
15 Wasserstrom JA,Salata JJ.Basis for tetrodotoxin and lidocaine effects on action potentials in dog ventricular myocytes[J].Am J Physiol Heart Circ Physiol,1988,254:H1 157
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