犬右房不同部位Kv4.3、KchIP2、Cav1.2 mRNA的表达

目的研究犬右房不同部位短暂外向钾电流、L型钙电流亚单位mRNA的表达情况,探讨其在致房性心律失常中的意义。方法应用逆转录-聚合酶链反应半定量分析犬界嵴、梳状肌、右心耳的短暂外向钾电流α亚单位(Kv4.3)、β亚单位(KchIP2)及L型钙电流的α亚单位(Cav1.2)mRNA的表达量(以β-actin为内参照)。结果界嵴和梳状肌Kv4.3、KchIP2 mRNA高于右心耳(P<0.05或0.01);界嵴Cav1.2 mRNA高于梳状肌和右心耳(P均<0.05),而梳状肌和右心耳之间没有差异。结论Kv4.3、KchIP2、Cav1.2 mRNA在右房空间表达上的差异与其相应离子流在右房空间上的差异一致,可能是其离子流差异的分子基础。     

犬上腔静脉肌袖与右房心肌某些通道亚单位mRNA的表达

目的研究犬上腔静脉肌袖与右房游离壁快速激活延迟整流钾电流(IKr),L型钙电流(ICa-L),短暂外向钾电流(Ito)通道亚单位mRNA表达水平。方法8只健康杂种犬,取上腔静脉肌袖及右房游离壁,采用逆转录聚合酶链反应的方法测定IKrα亚单位ERG、ICa-Lα1亚单位CaV1.2、Itoα亚单位Kv4.3及β亚单位KChIP2mRNA表达水平并进行半定量分析。结果上腔静脉肌袖中ERG表达水平高于右房(P<0.05),而CaV1.2、Kv4.3、KChIP2的mRNA表达均低于右房(P<0.05)。结论上腔静脉肌袖与右房之间存在离子通道基因表达水平的差异。     

心力衰竭窦性心律患者右心耳肌细胞多种离子通道基因表达的变化

目的通过对心力衰竭(HF)窦性心律(简称窦律)患者右心耳肌细胞多种离子通道mRNA水平的测定,试图揭示HF窦律患者心房肌多种离子通道电流变化的分子机制,并进一步揭示HF患者易患心房颤动(AF)的可能机制。方法采集HF组患者(18例)和心功能正常组患者(18例)的右心耳组织,应用逆转录-聚合酶链反应技术(RT-PCR)以GAPDH为内参照,测定瞬时外向钾电流(Ito1)通道的决定基因Kv4.3αmRNA、慢速激活的延迟整流钾电流(Iks)通道的决定基因KvLQT1mRNA、超速激活的延迟整流钾电流(Ikur)通道的决定基因Kv1.5mRNA、L-型Ca2+通道基因L-Caα1cmRNA、钠钙交换(NCX)基因mRNA表达水平。结果与心功能正常组比较,HF组患者Kv4.3αmRNA、KvLQT1mRNA、L-型Ca2+通道基因L-Caα1cmRNA表达均降低(P均<0.01),Kv1.5mRNA表达无明显变化(P>0.05),NCX基因mRNA表达升高(P<0.01)。结论HF患者心房肌细胞离子通道基因表达的变化可能是其相应离子电流改变的分子基础。HF介导的心房肌细胞离子通道重构在形成AF电生理基质中可能起重要作用,可能是HF易患AF的机制之一。     

心力衰竭家兔左室短暂外向钾电流下调的分子基础

目的为探讨心力衰竭(简称心衰)兔左室短暂外向钾电流(Ito)下调的分子基础。方法采用结扎家兔冠状动脉左前降支的方法制备缺血性心衰模型。应用膜片钳全细胞记录方法记录左室心肌细胞Ito,描记电流-电压(I-V)曲线;应用半定量-聚合酶链式反应(RT-PCR)法检测电压依赖性Kv1.4和Kv4.3钾通道α亚单位mRNA表达,并以图象分析系统对其进行半定量分析。结果心衰组家兔左室心肌细胞Ito密度较对照组显著降低,I-V曲线明显下移;指令电压为+70mV时,心衰组Ito密度(9.73±0.94pA/pF,n=5)显著低于对照组(14.35±1.16pA/pF,n=4)(P<0.01)。Kv1.4和Kv4.3钾通道α亚单位mRNA表达心衰组(分别为0.66±0.05,0.21±0.02,n=5)也较对照组(分别为0.95±0.07,0.531±0.04,n=5)显著降低(P均<0.01)。结论心衰家兔左室Ito电流密度下调可能受转录水平调节。     

糖尿病对大鼠左室心外膜细胞短暂外向钾电流的影响及分子基础

目的研究大鼠左心室肌短暂外向钾电流(Ito)在糖尿病状态发生下降改变的分子机制。方法取体重150～200 g的雄性Sprague-Dawley大鼠,腹腔注射链脲菌素建立糖尿病大鼠模型,采用酶解法获得单个左室心外膜细胞,应用膜片钳全细胞方法记录Ito;用逆转录-聚合酶链式反应技术半定量编码该通道α亚单位mRNA的表达水平。结果与对照组相比,糖尿病大鼠左室心外膜细胞Ito密度显著降低,+60 mV时分别为27.38±1.16pA/pF(n=12)和16.85±2.31 pA/pF(n=25)(P<0.01);糖尿病大鼠左室心外膜肌细胞Ito通道α亚单位编码基因Kv4.2、Kv4.3 mRNA表达水平较对照组分别显著下调56.88%和46.57%;而Kv1.4 mRNA则较对照组上调约48.02%,三组基因表达水平的改变均具有显著性差异。结论糖尿病大鼠左心室肌外膜细胞Ito密度显著降低系编码该通道α亚单位的基因表达下调所致。     

钾流在糖尿病大鼠心室肌细胞的改变机制

目的 研究糖尿病对大鼠心室肌细胞瞬间外向钾流(Ito)的影响及其分子机制,探讨糖尿病引起的心脏损害与心律失常的关系.方法 取体质量150～200 g的雄性Sprague-Dawley大鼠,单次腹腔注射链脲菌素(STZ,65 mg/kg,pH=4.5)建立糖尿病大鼠模型,采用酶解法获得单个心室肌细胞,应用膜片钳全细胞方法记录Ito;并用反转录聚合酶链式反应技术进一步半定量编码该电流通道α亚单位基因(Kv4.2、Kv4.3和Kv1.4)mR-NA的表达水平.结果 与对照组比较, 70 mV时,糖尿病大鼠心室肌细胞Ito密度显著降低[对照组:(30.6±3.8)比糖尿病组:(18.9±3.3)pA/pF,P<0.01);半定量分析法显示糖尿病大鼠心室肌细胞Ito通道α亚单位编码基因Kv4.2、Kv4.3 mRNA表达水平分别下调56.9%和46.6%;而Kv1.4 mRNA表达则上调约48.0%,3组基因表达水平的改变差异均有统计学意义(P<0.05).结论 糖尿病大鼠心室肌细胞Ito密度显著降低主要与编码该通道α亚单位的基因表达下调有关.     

氯沙坦调节钾电流的分子基础

目的探讨氯沙坦对自发性高血压大鼠(SHR)心室肌细胞编码瞬间外向钾电流(Ito)、延迟整流性钾电流(Ik)、内向整流钾电流(Ik1)关键钾通道α和β亚基[Ito(Kv4.2、KChIP2)、IK(ERG、KvLQT1)、IK1(Kir2.1)]mRNA和蛋白水平的变化,研究氯沙坦抗室性心律失常效应的分子基础。方法SHR随机分成2组:对照组(n=12)和氯沙坦组[10mg/(kg.d),n=12,灌胃]。年龄、体质量匹配的WKY(n=12)作为对照。用药8周后采用膜片钳技术记录离体心脏、酶分解所得左室心肌细胞动作电位、Ito、Ik1、Ik,并采用逆转录聚合酶链反应(RT-PCR)及免疫印迹(Westernblot)方法测定Kv4.2、KChIP2、ERG、KvLQT1、Kir2.1的mRNA及蛋白水平。结果氯沙坦组心肌动作电位50%及90%复极化时程[(16.8±3.8)、(68.5±13.2)ms]短于对照组[(24.6±4.6)、(73.3±15.5)ms,均P<0.01]。氯沙坦组的Ito电流密度(从+40到+70mV)高于对照组(P<0.01)。氯沙坦组Kv4.2、Kir2.1mRNA及蛋白水平高于对照...     

兔肥厚心肌慢反应延迟整流钾通道表达的变化

目的:慢反应延迟整流钾通道(IKs)是心肌细胞复极的重要组成部分,本实验观察左心室肥厚对心外膜下心肌(Epi)和心内膜下心肌(Endo)IKs的mRNA表达水平变化。方法:家兔16只随机分为假手术组和心肌肥厚组。心肌肥厚组通过部分结扎腹主动脉的方法造成兔压力负荷性心肌肥厚模型,假手术组只暴露腹主动脉而不行缩窄术。应用RT-PCR技术检测IKs通道基因KvLQT1(α亚单位)和minK(β亚单位)的mRNA表达。结果:假手术组EpiIKs通道α亚单位基因KvLQT1为Endo的2.5倍,EpiIKs通道β亚单位基因minK为Endo的3.3倍。与假手术组相比,心肌肥厚组Epi和EndoIKs通道α亚单位基因KvLQT1表达分别降低40%和25%,心肌肥厚组Epi和EndoIKs通道β亚单位基因minK表达分别降低50%和33%。结论:IKs通道基因KvLQT1和minK的mRNA表达存在跨室壁的差异。心肌肥厚可造成Epi和EndoIKs的mRNA表达不均一的下降。     

氯沙坦调节瞬间外向钾电流的分子学研究

目的 观察氯沙坦对自发性高血压大鼠(SHR)心室肌细胞编码瞬间外向钾电流(Ito)关键钾通道α亚基(Kv4.2、Kv4.3)、β亚基(KChIP2)mRNA和蛋白水平变化的影响,探讨氯沙坦抗室性心律失常效应的分子基础.方法 SHR随机分成2组:氯沙坦组(10 mg·d-1·kg-1灌胃)和SHR对照组各12只大鼠.鼠龄、体质量匹配的WKY大鼠12只为WKY对照组.用药8周后采用膜片钳技术记录左心室心肌细胞动作电位、Ito,并采用反转录聚合酶链反应及免疫印迹反应(Western blot)方法测定Kv4.2、Kv4.3、KChIP2 mRNA及蛋白水平.结果 氯沙坦组左心室细胞的动作电位复极至50%及90%时程分别为(16.82±3.79)ms和(68.49±13.25)ms,短于SHR对照组的(24.56±4.59)ms和(73.26±15.47)ms,二者差异有统计学意义(均P<0.01).氯沙坦组的Ito电流密度高于SHR对照组(从+40 mV到+70 mV,均P<0.01).氯沙坦组Kv4.2、Kv4.3 mRNA及蛋白水平高于SHR对照组(均P<0.01).氯沙坦组KChIP2 mRNA及蛋白水平低于SHR对照组(均P<0.01).结论 氯沙坦慢性阻滞血管紧张素受体,逆转SHR左心室的电重构,缩短单个心肌细胞动作电位时程,增加Ito电流密度,这与Kv4.2、Kv4.3表达增加及KChIP2表达降低相关.     

快速心房起搏对家兔L-型钙通道和Kv4.3钾通道基因表达的影响

目的观察快速心房起搏对家兔心房L型钙通道亚单位和Kv4.3钾通道基因表达的影响。方法新西兰大耳白家兔36只,随机分成6组,经右颈外静脉穿刺置入电极于右房,分别给予0、3、6、12、24或48h快速心房起搏,停止起搏后取右房组织,应用半定量反转录聚合酶链式反应测定各时相点L型钙通道α1c,β1,α2亚单位,钾通道Kv4.3mRNA的表达水平。结果L型钙通道α1c、β1亚单位在快速起搏6h后表达水平下调,并随着起搏时间的延长进一步下调。α2亚单位的mRNA表达在各时相点无显著差异。Kv4.3的mRNA的表达在快速起搏的24h和48h下降分别达55.50%（P<0.01）和59.12%（P<0.01）。48h后下降达到一个平台期。结论快速心房起搏可导致L型钙通道亚单位和Kv4.3钾通道mRNA表达下调。     

Spatial alterations of Kv channels expression and K(+) currents in post-MI remodeled rat heart.

OBJECTIVE: The hypothesis being tested in the present study is that increased anisotropic properties occurs in the remodeled post-infarction heart due to spatial alterations in Kv channels expression and K(+) currents of the remodeled myocardium. METHODS: Three to 4 weeks post myocardial infarction (MI) in the rat, we measured the two components of the outward K(+) current, I(to-fast (f)) and I(to-slow(s)) in the epicardium (epi) and endocardium (endo) of noninfarcted remodeled left ventricle (LV) using patch clamp techniques. Alterations in mRNA and/or protein levels of potassium channel genes Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 were measured in epi, midmyocardium (mid), and endo regions of LV and in the right ventricle (RV). RESULTS: In sham operated rat heart, the density of I(to-f) was 2.3 times greater in epi compared to endo myocytes. In post-MI heart, the density of I(to-f) and I(to-s) decreased to a similar degree in LV epi and endo but the difference in I(to-f) density between epi and endo persisted. The mRNA and/or protein levels of Kv1.4, Kv2.1, Kv4.2 and Kv4.3 but not Kv1.5 decreased to a varying extent in different regions of LV but not in RV of post-MI heart. CONCLUSIONS: Our results suggest that regional downregulation of Kv channels expression and density of K(+) currents can be a significant determinant of increased spatial electrophysiological heterogeneity and contribute to increased electrical instability of the post-MI heart.     

Kv4.3 is not required for the generation of functional Ito,f channels in adult mouse ventricles

Accumulated evidence suggests that the heteromeric assembly of Kv4.2 and Kv4.3 α-subunits underlies the fast transient Kv current (I_to,f) in rodent ventricles. Recent studies, however, demonstrated that the targeted deletion of Kv4.2 results in the complete elimination of I_to,f in adult mouse ventricles, revealing an essential role for the Kv4.2 α-subunit in the generation of mouse ventricular I_to,f channels. The present study was undertaken to investigate directly the functional role of Kv4.3 by examining the effects of the targeted disruption of the KCND3 (Kv4.3) locus. Mice lacking Kv4.3 (Kv4.3−/−) appear indistinguishable from wild-type control animals, and no structural or functional abnormalities were evident in Kv4.3−/− hearts. Voltage-clamp recordings revealed that functional I_to,f channels are expressed in Kv4.3−/− ventricular myocytes, and that mean I_to,f densities are similar to those recorded from wild-type cells. In addition, I_to,f properties (inactivation rates, voltage dependences of inactivation and rates of recovery from inactivation) in Kv4.3−/− and wild-type mouse ventricular myocytes were indistinguishable. Quantitative RT-PCR and Western blot analyses did not reveal any measurable changes in the expression of Kv4.2 or the Kv channel interacting protein (KChIP2) in Kv4.3−/− ventricles. Taken together, the results presented here suggest that, in contrast with Kv4.2, Kv4.3 is not required for the generation of functional mouse ventricular I_to,f channels.     

Apico-basal inhomogeneity in distribution of ion channels in canine and human ventricular myocardium

OBJECTIVES: The aim of the present study was to compare the apico-basal distribution of ion currents and the underlying ion channel proteins in canine and human ventricular myocardium. METHODS: Ion currents and action potentials were recorded in canine cardiomyocytes, isolated from both apical and basal regions of the heart, using whole-cell voltage clamp techniques. Density of channel proteins in canine and human ventricular myocardium was determined by Western blotting. RESULTS: Action potential duration was shorter and the magnitude of phase-1 repolarization was significantly higher in apical than basal canine myocytes. No differences were observed in other parameters of the action potential or cell capacitance. Amplitude of the transient outward K(+) current (29.6+/-5.7 versus 16.5+/-4.4 pA/pF at +65 mV) and the slow component of the delayed rectifier K(+) current (5.61+/-0.43 versus 2.14+/-0.18 pA/pF at +50 mV) were significantly larger in apical than in basal myocytes. Densities of the inward rectifier K(+) current, rapid delayed rectifier K(+) current, and L-type Ca(2+) current were similar in myocytes of apical and basal origin. Apico-basal differences were found in the expression of only those channel proteins which are involved in mediation of the transient outward K(+) current and the slow delayed rectifier K(+) current: expression of Kv1.4, KChIP2, KvLQT1 and MinK was significantly higher in apical than in basal myocardium in both canine and human hearts. CONCLUSIONS: The results suggest that marked apico-basal electrical inhomogeneity exists in the canine-and probably in the human-ventricular myocardium, which may result in increased dispersion, and therefore, cannot be ignored when interpreting ECG recordings, pathological alterations, or drug effects.     

Expression of voltage-gated K<Superscript>+</Superscript> channels in human atrium

Voltage-gated K⁺ channels underlie repolarisation of the cardiac action potential and represent a potential therapeutic target in the treatment of cardiac dysrhythmias. However, very little is known about the relative expression of K⁺ channel subunits in the human myocardium. We used a semi-quantitative RT-PCR technique to examine the relative expression of mRNAs for the voltage-gated K⁺ channel subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, KvLQT1, HERG and IsK in samples of human atrial appendage. Data were expressed as a percentage expression density relative to an 18S ribosomal RNA internal standard. The most abundant K⁺ channel mRNAs were Kv4.3 (80.7 ± 10.1 %), Kv1.5 (69.7 ± 11.2 %) and HERG (55.9 ± 21.5 %). Significant expression of KvLQT1 (33.5 ± 5.5 %,) and Kv1.4 (26.7 ± 9.6 %) was also detected. Levels of mRNAs for Kv1.2 and IsK were very low and neither Kv2.1 nor Kv4.2 mRNA were detected in any experiments. Whole-cell patch-clamp techniques were used to examine the outward currents of isolated human atrial myocytes at 37 °C. These recordings demonstrated the existence of transient (I_to1) and sustained (I_so) outward currents in isolated human atrial myocytes. I_to1, and not I_so, showed voltage-dependent inactivation during 100 ms pre-pulses. Both I_to1 and I_so were inhibited by high concentrations (2 mM) of the K⁺ channel blocker, 4-aminopyridine (4-AP). However, lower concentrations of 4-AP (10 μM) inhibited I_so selectively. I_to1 recovered from inactivation relatively rapidly (t ∼21 ms). These data, with published information regarding the properties of expressed K⁺ channels, suggest that Kv4.3 represents the predominant K⁺ channel subunit underlying I_to1 with little contribution of Kv1.4. The sensitivity of Iso to very low concentrations of 4-aminopyridine and the relatively low expression of mRNA for Kv1.2 and Kv2.1 is consistent with the major contribution of Kv1.5 to this current. The physiological significance of the expression of KvLQT1 and Kv1.4 mRNA in the human atrium warrants further investigation. Received: 30 August 2000, Returned for 1. revision: 21 September 2000, 1. Revision received: 21 June 2002, Returned for 2. revision: 15 July 2002, 2. Revision received: 30 July 2002, Accepted: 31 July 2002 Correspondence to: Dr. A. F. James     

Functional protein expression of multiple sodium channel α- and β-subunit isoforms in neonatal cardiomyocytes

Voltage-gated sodium channels are composed of pore-forming α- and auxiliary β-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel α-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na_v1.5 sodium channel α-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel α- and β-subunits. α-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel α-subunit isoforms Na_v1.1, Na_v1.2, Na_v1.3, Na_v1.4 and Na_v1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac α-subunit isoform, Na_v1.5. Each of the β-subunit isoforms (β1-β4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the α-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel α-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na_v1.5 α-subunit and they contribute to the total sodium current.