犬右心室心外膜下细胞瞬间外向钾电流特性的研究

目的　探讨犬右心室心外膜下 (Epi)细胞复极 1期瞬间外向钾电流 (Ito1)的电生理特性。方法　应用全细胞钳制技术 ,对右心室Epi细胞复极 1期Ito1的电流强度、动力学过程和动作电位切迹进行定量观察。结果　 (1)犬右心室Epi细胞具有强大的Ito1,其激活过程呈明显的电压依赖性 ,在37℃、0 2Hz和去极化试验电压为 +70mV时 ,Epi细胞的峰值Ito1强度和密度分别达 (4 15 0± 1780 )pA和 (31± 11 4)pA/pF ,其激活和失活动力学过程符合Boltzmann分布 ;(2 )犬右心室Epi细胞Ito1具有明显的频率依赖性 ,即在基础刺激周长增加时 ,Ito1明显增大 ,并和动作电位的“尖峰 穹隆”幅度的增加相对应 ;(3)温度对Ito1强度的影响明显 ,在去极化试验电压为 +70mV、0 2Hz和温度为 2 2℃、37℃条件下 ,右心室Epi细胞的Ito1强度和密度分别为 (2 380± 10 5 0 )pA与 (18± 7 6 )pA/pF、(4 15 0± 1780 )pA与 (31± 11 4)pA/pF ,差异明显。结论　犬右心室Epi细胞存在强大的Ito1,这种具有明显电压依赖性、频率依赖性和温度依赖性的Ito1及其参与介导的“尖峰 穹隆”状动作电位图形是右心室Epi细胞复极1期一个突出的电生理特点。     

人右心室瞬间外向钾电流电不均一性的初步研究

目的探讨人右心室复极1相瞬间外向钾电流(Ito)的跨壁电不均一性。方法应用全细胞钳制技术,对人右心室心外膜下(Epi)细胞、中间层(M)细胞和心内膜下(Endo)细胞复极1相Ito离子流的电流强度、密度和动力学过程进行研究。结果人右心室三层细胞之间存在明显的Ito离子流电不均一性,在37℃、0·2Hz和除极化试验电压为+70mV时,Epi细胞和M细胞峰值Ito离子流的密度分别为(13·2±2·6)pA/pF,和(8·9±3·1)pA/pF,远大于Endo细胞(2·7±0·35)pA/pF,(P<0·01);在37℃条件下,人右心室Epi细胞和M细胞Ito离子流的激活和失活过程符合Boltzmann分布,两组细胞的激活和失活V1/2、斜率因子K等均差异无统计学意义(P>0·05)。结论在人右心室跨越室壁的3层细胞间,特别是Epi细胞与Endo细胞之间、M细胞与Endo细胞之间,复极1相存在明显的Ito离子流强度差异和强大的Ito离子流梯度。     

犬右室瞬间外向钾电流异质性的研究

应用全细胞钳制技术对犬右室心外膜下 (epi)细胞、中层 (M)细胞和心内膜下 (endo)细胞复极 1期瞬间外向钾电流 (Ito1)的强度、密度和动力学过程进行系统定量研究 ,以期从复极 1期主要离子流的角度 ,探讨右室复极 1期跨越室壁的电异质性。结果发现 :犬右室epi细胞和M细胞存在强大的Ito1离子流 ,在刺激频率为 0 .2Hz、37℃和去极化试验电压为 +70mV时 ,epi细胞和M细胞峰值Ito1离子流的强度分别为 46 5 0± 176 0 ,36 2 0± 1880pA ,其激活和失活动力学过程符合Boltzmann分布。与epi细胞和M细胞相比 ,endo细胞Ito1离子流微小 ,同样条件下其平均峰值Ito1离子流仅为 480± 130pA。表明在右室跨越室壁的三层细胞间 ,特别是epi细胞与endo细胞之间、M细胞与endo细胞之间 ,复极 1期存在明显的Ito1离子流强度差异和强大的Ito1离子流梯度 ,此为右室电异质性的一种突出表现 ,它可能是Brugada综合征等疾病所致恶性心律失常的重要离子基础之一     

自发性高血压大鼠左心室肌细胞动作电位延长的离子机制

目的:研究自发性高血压大鼠(SHR)左心室肌细胞动作电位时程延长的膜离子流基础.方法:应用酶解方法分离获得正常血压Wistar大鼠和SHR的左心室肌细胞,采用玻璃微电极技术记录动作电位,膜片钳全细胞记录膜离子流,对比正常心室肌细胞和肥大心室肌细胞间动作电位及膜离子流差别.结果:(1)SHR和Wistar大鼠的心脏/体重比分别为5.66±0.46 mg/g和3.7±0.29 mg/g (P<0.001) ,细胞平均膜电容分别为280.68±67.98 pF 和189.94±56.59 pF(P<0.05).提示SHR 心脏肥厚、心肌细胞增大;(2)SHR动作电位APD50和APD90较Wistar大鼠明显延长(21.33±1.56 ms vs 14.91±2.95 ms,P<0.001; 164.6±74 ms vs 93.27±10.59 ms,P<0.00 1),说明SHR心室肌细胞存在复极延迟;(3)SHR的平均ICa-L幅值显著大于Wistar大鼠,分别为1944±466.8 pA和1136±33.3 pA(P<0.001),电流密度二者间无差异(6.932±1.7 1 pA/pF vs 6.19±2.85 pA/pF) ,但SHR的慢失活时间常数明显延长(56.01±13.36 ms vs 43.63±17.89 ms,P<0.001);(4)S HR的Ik1内向电流密度显著小于Wistar大鼠(11.3±2.26 pA/pF vs 14.33 pA/pF,P<0.05),外向电流密度二者间差异无显著性(2.36±0.86 pA/pF vs 2.96±1.27 pA/pF);(5)SHR的Ik密度与Wista r大鼠间无差别(12.38±5.46 pA/pF vs 11.86±3.59 pA/pF);(6)SHR的Ito密度显著地低于Wistar 大鼠(+70 mV时, 8.21±6.64 pA/pF vs 19.16±6.17 pA/pF, P<0.001).但通道的激活和失活时间常数二者无差异,提示Ito的降低可能仅是通道数减少所致.结论:SHR左心室肌细胞动作电位时程延长系外向复极钾流(Ito、Ik1)减小和慢钙通道失活时间常数延长所致.     

血管紧张素Ⅱ对人心房肌细胞膜钾钙离子电流的作用

观察血管紧张素Ⅱ对人心房肌细胞膜主要离子流的作用,揭示其参与房性心律失常的细胞电生理机制。急性分离单个人心房肌细胞,采用全细胞膜片钳方法记录细胞膜短暂外向钾电流(Ito)、内向整流钾电流(Ik1)和L型钙电流(ICaL)。结果:0.1μmol/LAngⅡ使人心房肌细胞膜Ito峰值电流密度明显下降6.54±0.49pA/pFvs12.65±0.86pA/pF(P<0.05),在-100mV电压下使IK1峰值电流密度显著升高-8.93±1.12pA/pFvs-5.23±0.95pA/pF,(P<0.05),并明显促进人心房肌细胞膜ICaL-12.72±1.69pA/pFvs-5.79±0.84pA/pF(P<0.05)。结论:AngⅡ可促进人心房肌细胞膜IK1及ICaL,抑制人心房肌细胞膜Ito。     

奎尼丁对正常大鼠心室肌细胞瞬间外向钾电流的影响

研究大鼠心室肌细胞瞬间外向钾电流(Ito1)的特性及奎尼丁对其的影响,从而从细胞离子的层面去探讨奎尼丁作为治疗Brugada综合征的侯选药物的机制。用酶解法分离大鼠单个左室细胞,应用膜片钳全细胞方法记录Ito1,观察不同浓度的奎尼丁对心室肌细胞Ito1的作用。结果:①大鼠心室肌细胞具有强大的Ito1,在0.2Hz,+70mV和32℃条件下,其平均峰值Ito1强度和密度分别为1940±440pA和12.9±2.6pA/pF;②在奎尼丁1,2.5,5,7.5,10μmol/L不同的浓度下,奎尼丁抑制Ito1程度越明显(自身对照P<0.05),其作用呈浓度依赖性。结论:奎尼丁可以通过抑制Ito1,延长动作电位的复极时程,此很有可能是其作为治疗Brugada综合征的侯选药物的机制之一。     

心房颤动心房肌细胞短暂外向钾电流的特点

观察心房颤动 (AF)心房肌细胞的电生理特点 ,探讨短暂外向钾电流 (Ito)与AF电重构的关系。采用组织块酶消化法分离 7例AF(AF组 )和 14例非AF(非AF组 )患者的右心耳心房肌细胞 ,利用全细胞记录技术观察单个心房肌细胞的Ito。在各钳制电位下 ,AF组的Ito值均明显低于对照组。钳制电位在 + 60mV时AF组的Ito值 ( 14 2 .87±4 2 .2 5pA)与非AF组 ( 4 80 .12± 3 9.2 4pA)相比 ,减少 70 .2 4 %(P <0 .0 0 1)。在各钳制电位下 ,AF组的稳态电流 (Isus)均高于非AF组。钳制电位在 + 60mV时AF组的Isus值 ( 83 1.88± 72 .90pA)与非AF组 ( 5 4 3 .93± 65 .3 4pA)相比 ,增加 5 2 .94 %(P <0 .0 1)。随着刺激频率的增加 ,AF与非AF患者心房肌细胞在钳制电位 + 60mV时的Ito强度逐渐降低 ,与非AF组不同的是 ,AF组心房肌细胞的Ito在刺激频率 1～ 2Hz间电流改变的差异具有显著性。结论 :AF心房肌不应期的缩短与Ito的降低有关 ,Ito可能是AF电重构的离子机制 ,Isus可能也参与了电重构的发生。     

犬Marshall韧带心肌细胞短暂外向钾电流的特性

目的　探讨Marshall束 (Marshallbundle ,MB)内心肌细胞短暂外向钾电流 (transientout wardpotassiumcurrent,Ito)的离子通道特性。方法　运用组织块酶解法分离犬MB内单个心肌细胞 ,在倒置显微镜下直接比较细胞形态 ;采用全细胞膜片钳技术记录MB内单个心肌细胞Ito电流密度及动力学特性。在细胞外液中加入 1μM异丙肾上腺素 ,比较加药前后Ito电流密度及动力学特性的变化。结果　MB内有两种形态迥异的心肌细胞 :一种为短矩形 ,短而宽 ,呈典型的矩形或略呈锥型 ;另一种为长杆型 ,长而窄。短矩形细胞数量明显多于长杆型细胞。短矩形细胞和长杆型细胞的长宽比分别为2 99± 0 95和 12 0 5± 2 4 1(P <0 0 1)。两种心肌细胞Ito的动力学无明显差别 ,但短矩形细胞的Ito电流密度明显小于长杆型细胞 ,8 77pA/pF (n =8)对 14 95 pA/pF (n =8) ,P <0 0 1。加用异丙肾上腺素后 ,在 70mV ,长杆型细胞Ito峰值减小到 7 96pA/pF (n =8) ;短矩形细胞减小到 3 89pA/pF(n =8) ;两种细胞加药后Ito分别减小 (5 0 4± 0 32 ) pA/pF和 (6 86± 0 4 9) pA/pF (P <0 0 5 ) ;稳态激活曲线均右移 ,短矩形细胞与长杆型细胞的V1/ 2 分别从 (- 7 1± 0 8)mV和 (- 6 8± 0 7)mV变为 (-1 8± 0 2 )mV和 (- 1 6± 0 4 )mV     

西洛他唑对大鼠右心室肌细胞瞬间外向钾电流的影响

目的 :观察西洛他唑对大鼠右心室肌细胞瞬间外向钾电流 (Ito1)的影响 ,探讨西洛他唑抗心律失常作用的确切机制。方法 :酶解法分离SD大鼠右心室肌细胞 ,采用全细胞膜片钳技术记录右心室肌细胞Ito1。结果 :5 0 μmol/L西洛他唑显著降低Ito1,使Ito1幅值由加药前 (12 .7± 0 .5 ) pA/ pF降至 (7.6± 0 .4 ) pA/ pF ,降低 (4 0 .1± 3.0 ) % (P <0 .0 1,n =9)。在 1～ 5 0 μmol/L范围内 ,西洛他唑的作用呈浓度依赖性 ,半抑制浓度为 (17.2±2 .2 ) μmol/L。此外 ,该药对Ito1电压依赖性的激活和失活曲线无显著影响。结论 :西洛他唑浓度依赖性地阻滞大鼠右心室肌细胞的Ito1。     

增强型绿色荧光蛋白转染小鼠心脏后瞬时外向钾电流及钠钙交换电流的变化

目的探讨绿色荧光蛋白(GFP)转染对心脏瞬时外向钾电流(Ito)及钠钙交换电流(INCX)的影响。方法20只雄性小鼠等量随机分为对照组和增强型GFP(EGFP)组。EGFP组小鼠采用8点注射法均匀注射100μl腺病毒于左室游离壁上,对照组注射等量无菌生理盐水。一周后分离单个心室肌细胞,用膜片钳记录Ito及INCX。结果与对照组相比,EGFP组几乎所有测定电压下,Ito电流密度显著减小[如+60 mV时为8.40±1.55 pA/pF(n=9)vs 36.77±8.12 pA/pF(n=11),P<0.05]。INCX的前向模式不因转染EGFP变化[如-80 mV时为-0.35±0.05 pA/pF(n=8)vs-0.42±0.08 pA/pF(n=10),P>0.05],但反向模式电流显著增大[如+80 mV时为1.47±0.10 pA/pF(n=8)vs 0.72±0.05 pA/pF(n=10),P<0.05]。结论 EGFP转染可使心脏Ito显著减小,而INCX仅反向模式电流增大,其综合效应可能导致细胞内钙增加。     

乙酰胆碱对犬右室三层心肌细胞L型钙电流不均一性的影响

目的测定犬右室三层心肌细胞上的L型钙电流(ICa,L),并研究其对自主神经递质乙酰胆碱的反应。方法经酶解法分离获得犬右室三层心肌细胞,应用全细胞膜片钳技术,记录并比较三层心肌细胞的ICa,L,以及应用2μmol/L乙酰胆碱前后电流-电压曲线的差异。结果ICa,L的峰值电流密度外膜下大于M细胞,而M细胞又大于内膜下心肌细胞,分别为-4.896±1.907pA/pF(n=31),-3.406±0.904pA/pF(n=37),-2.788±0.756pA/pF(n=33)(P<0.05)。使用乙酰胆碱后,右室外膜下及M细胞的峰值电流密度减小[-4.921±1.023pA/pF vs -3.462±0.997pA/pF(n=12);-3.803±1.115pA/pF vs -2.959±0.883pA/pF(n=13),P均<0.05]。心内膜下心肌细胞用药前后无差异(P>0.05)。结论ICa,L在犬右室三层心肌细胞存在不均一性,乙酰胆碱可以减小心外膜下、M细胞的ICa,L,对内膜下心肌细胞的ICa,L无影响。     

胺碘酮对模拟缺氧状态下大鼠心室肌细胞瞬时外向和内向整流钾电流的影响

目的通过观察胺碘酮对模拟缺氧状态下急性分离的大鼠心室肌单细胞复极相中瞬时外向钾电流(Ito)和内向整流钾电流(IK1)通道的影响,探讨其在该条件下抗心律失常的作用机制。方法使用酶解法分离获取大鼠单个心室肌细胞,通过持续通以模拟缺氧细胞外液建立体外模拟缺氧模型,采用全细胞膜片钳实验技术研究胺碘酮对该条件下Ito和IK1的作用。结果胺碘酮呈剂量依赖性降低Ito和IK1电流幅值,对Ito抑制效应的起始浓度为1μmol/L,100μmol/L时抑制作用达最大,最大抑制幅度为56.78%±4.27%(23.98±2.18pA/pFvs10.38±4.27pA/pF;测试电压为+70mV;P<0.01;n=5),IC50(半数抑制浓度)为74.35μmol/L,但Ito的I-V曲线趋势并没有发生变化,稳态激活和失活曲线几乎不发生移动。胺碘酮对IK1内向电流部分抑制起始浓度为1μmol/L,外向电流部分抑制效应的起始浓度为2μmol/L,其最大抑制幅度分别为58.77%±10.76%(56.32±7.24pA/pFvs23.22±7.30pA/pF;测试电压为-150mV;P<0.01)和33.29%±2.15%(6.70±0.89pA/pFvs4.46±0.93pA/pF;测试电压为+40mV;P<0.01;n=5)。对内向电流成分的IC50为63.75μmol/L,IK1通道的稳态激活曲线无明显改变。结论在大鼠离体心室肌单细胞模拟缺氧条件下,胺碘酮对Ito和IK1电流幅度呈剂量依赖性抑制,有对抗缺氧本身造成的动作电位时程缩短效应;对I内向电流成分的敏感性高于外向成分。     

正常大鼠心室肌细胞动作电位和瞬时外向钾离子流的特点

目的研究正常Spraque-Dawley大鼠外层、中层和内层心室肌细胞动作电位(AP)和瞬时外向钾离子流(Ito)的特点。方法采用酶消化法获得大鼠外层、中层和内层心室肌细胞,以全细胞膜片钳技术记录心室肌细胞AP和Ito。结果成功记录到大鼠心室肌细胞外、中和内层心肌细胞AP和Ito。外层至内层心室肌细胞动作电位时程(APD)逐渐延长(P<0.05)。在+70mV刺激时外层至内层心室肌细胞Ito电流密度逐渐减小,分别为59.50±15.99,29.15±5.53和12.29±3.62pA/pF(P<0.05)。三层心室肌细胞曲线半激活电压、半失活电压及失活后恢复时间均无差异(P>0.05)。结论大鼠三层心室肌细胞AP形态和Ito大小存在分层差别。     

Properties of the transient outward current in rabbit sino-atrial node cells

The electrophysiological properties of the transient outward current were investigated in voltage-clamped single cells from the rabbit sino-atrial node. To make a regional comparison, some experiments were repeated in atrial myocytes. The current-voltage relationship showed a characteristic outward rectification with an activation threshold of -30 mV. External 4-aminopyridine (0.01-5 mM) inhibited this current in a dose-dependent manner (IC50 = 0.28 mM, Hill coefficient = 1.38). The steady-state inactivation exhibited a half-maximum voltage of -35 mV and a slope factor of -.4 mV. The current density of the transient outward current was 6.3 +/- 0.5 pA/pF in sino-atrial node cells and 12.3 +/- 1.2 pA/pF in atrial cells. The inactivation time constant was faster in sino-atrial node cells (time constants 4.2 +/- 0.5 and 26.0 +/- 0.6 ms, respectively, for the fast and slow components) than in atrial cells (9.7 +/- 1.2 and 44.8 +/- 3.2 ms, respectively). Recovery from inactivation was much faster in sino-atrial node cells (time constants 44.7 +/- 9.0 ms) than in atrial cells (time constants 1.39 +/- 0.32 and 6.70 +/- 0.1 s, respectively, for the fast and slow components). These results suggest that the kinetic properties, as well as the current density, of the transient outward current differs between sino-atrial node and atrial cells. Taking the current density of Ito at +10 mV as 2.5 +/- 0.3 pA/pF gives a total Ito of approximately 100 pA at the peak of the action potential in rabbit sino-atrial node cells. The action potential duration was increased by 24.8 +/- 1.3% by 0.5 mM 4-AP. Thus, Ito may contribute significantly to the repolarization phase in mammalian sino-atrial node cells.     

Repolarizing currents in ventricular myocytes from young patients with tetralogy of Fallot.

OBJECTIVE: It was the aim of our study to describe repolarizing currents in ventricular myocytes isolated from children with tetralogy of Fallot. This is the first report on outward currents in ventricular myocytes from children. METHODS: Ventricular myocytes were isolated from tissue samples of the outflow tract of the right ventricle which were obtained during corrective surgery of tetralogy of Fallot. Action potentials and whole-cell currents were recorded with the patch clamp technique at a temperature of 36-37 degrees C. RESULTS: The mean resting potential was -71.7 +/- 1.92 mV, action potential amplitude was 110 +/- 2.96 mV and action potential duration at 90% repolarization was 794 +/- 99.5 ms (n = 12). In four out of 12 myocytes early afterdepolarizations (EADs) were observed. Upon hyperpolarization Ba(2+)-sensitive inward currents similar to the inward rectifier current (IKl) could be observed. The current density at -120 mV was -22.8 +/- 2.47 pA/pF (n = 14). A transient outward current (Itol) could be recorded in all myocytes studied, the current density varied from 0.3 to 8.6 pA/pF with a mean of 3.77 +/- 0.47 pA/pF at +40 mV (n = 38). Recovery of Itol from inactivation was fast (70% recovery within 100 ms), rate-dependent reduction amounted to 38.2% at 4 Hz. A delayed rectifier current was seen in only two out of 38 myocytes (rapid component IKr). CONCLUSIONS: The electrophysiological characteristics of right ventricular myocytes isolated from children with tetralogy of Fallot resemble in most cases subendocardial myocytes from adults. The most prominent difference is a fast recovery from inactivation as well as a small rate dependent reduction of Itol. The observed EADs may have clinical implications.     

The ionic mechanisms of long QT interval in diabetic rabbits

Objective Abnormal QT prolongation associated with arrhythmias is considered the major cardiac electrical disorder and a significant predictor of mortality in diabetic patients. The precise ionic mechanisms for diabetic QT prolongation remained unclear. The present study was designed to analyze the changes of ventricular repolarization and the underlying ionic mechanisms in diabetic rabbit hearts. Methods Diabetes was induced by a single injection ofalloxan （145mg/kg, Lv. ）. After the development of diabetes （10 weeks）, ECG was measured. Whole-cell patch-clamp technique was applied to record the action potential duration （APD50, APD90）, slowly activating outward rectifying potassium current （IKs）, L-type calcium current （ICa-L） and inward rectifying potassium current （IK1）. Results The action potential duration （APD50 and APD90） of ventricular myocytes was obviously prolonged from 271.5＋32.3 ms and 347.8＋36.3 ms to 556.6~72.5 ms and 647.9~72.2 ms respectively （P〈 0.05）. Meanwhile the normalized peak current densities of IKs in ventricular myocytes investigated by whole-cell patch clamp was smaller in diabetic rabbits than that in control group at test potential of＋50mV （1.27~0.20 pA/pF vs 3.08~0.67 pA/pF, P〈0.05）. And the density of the ICa-L was increased apparently at the test potential of 10 mV （-2.67~0.41 pA/pF vs -5.404-1.08 pA/pF, P〈0.05）. Conclusion Ventricular repolarization was prolonged in diabetic rabbits, it may be partly due to the increased L-type calcium current and reduced slow delayed rectifier K＋ current （IKs） （J Geriatr Cardio12010; 7：25-29）.     

Nav1.1抗体对豚鼠心室肌细胞钠电流的影响

目的探讨Nav1.1抗体对豚鼠心室肌细胞钠电流(INa)的影响。方法采用全细胞膜片钳技术,观察急性分离的豚鼠心室肌细胞在应用Nav1.1抗体后心肌INa的变化。结果在Nav1.1抗体作用下,钠离子通道电流密度呈浓度依赖性减小。用药前与三个浓度抗体组(1∶100,1∶50,1∶25)的电流密度分别为34.22±6.22,32.13±3.45,25.49±7.4,19.23±1.69pA/pF。其中1∶25Nav1.1抗体明显降低钠电流密度(P<0.05),并且,1∶25Nav1.1抗体使INa电流-电压曲线明显向正电位方向移动,但是其稳态失活曲线和恢复曲线没有显著性改变。结论Nav1.1可能对INa有影响。