肺静脉肌袖心肌细胞内向整流性钾电流特性研究

心房颤动（房颤）是临床上最常见的一种快速性心律失常,严重影响患者的生活质量,因其机制复杂,至今仍缺乏理想的治疗方法.近年来一些学者发现,大多数阵发性房颤存在异位的局灶兴奋点,对该部位行射频消融可以有效地终止或减少房颤的发生.而90%以上的异常兴奋灶集中在肺静脉口及其附近,推测这些兴奋灶以某种机制触发房颤,可能与触发活动、自律性增高或微折返有关.     

咪达普利对家兔肥厚心肌内向整流钾电流跨室壁异质性的作用

目的探讨咪达普利（IMI）对家兔左室肥厚心肌（LVH）内向整流钾电流（IK1）的跨室壁不均一性的影响。方法用腹主动脉缩窄法制备家兔LVH模型,并口服IMI[0.625mg/（kg·d）]连续8周进行干预。取心脏分离左室游离壁3层心肌细胞,用全细胞膜片钳技术记录IK1。结果因LVH模型心肌细胞膜电容增加所致IK1密度明显减少,心外膜下心肌、中层心肌和心内膜下心肌分别从（5.9±0.7）pA/pF、（6.1±0.5）pA/pF和（4.9±0.3）pA/pF降低为（4.4±0.5）pA/pF、（4.5±0.4）pA/pF和（2.1±0.2）pA/pF,各层细胞间电流密度差异加大。IMI处理后,可逆转心肌的病变,IK1的密度明显高于IMI未干预的LVH组（P<0.05）,心外膜下心肌、中层心肌和心内膜下心肌分别为（5.4±0.8）pA/pF、（5.8±0.6）pA/pF和（4.3±0.5）pA/pF,使3层细胞间电流密度的差异减小。结论IMI可逆转LVH后心肌细胞IK1的改变,减少跨室壁差异,提示可能是其减少LVH后发生快速心律失常的机制之一。     

夹竹桃麻素对兔心房肌IK1氧化损伤的保护作用

目的 探讨夹竹桃麻素(APO)对兔左心房肌细胞内向整流钾电流(I_K1)的保护作用。 方法 运用低浓度(50 μmol/L)的过氧化氢(H₂O₂)建立氧化应激模型。应用膜片钳全细胞技术,探讨APO(100 μmol/L)对兔左心房肌细胞I_K1及动作电位时程(APD)氧化应激损伤的保护作用。Western blot检测各组兔左心房中Kir2.1蛋白的表达。反转录聚合酶联反应(RT-PCR)检测兔左心房中的KCNJ2 mRNA表达。 结果 与对照组比较,低浓度H₂O₂(50 μmol/L)组I_K1峰值从(?182.2±15.6) pA/pF 下降到(?119.3±8.9)pA/pF (P<0.05),APO(100 μmol/L)组I_K1峰值为(?175.3±15.2)pA/pF无差异;与H₂O₂组比较,H₂O₂(50 μmol/L)+APO(100 μmol/L)组I_K1峰值恢复到(?160.5±13.5)pA/pF (P<0.05);APO使由于H₂O₂处理后减小的静息膜电位(RMP)绝对值及缩短的90%的APD(APD₉₀)得以恢复;与对照组相比,H₂O₂组Kir2.1蛋白表达下降(P<0.05);与H₂O₂组相比,H₂O₂+APO组Kir2.1蛋白表达明显恢复(P<0.05);与对照组相比,H₂O₂组KCNJ2 mRNA表达下降(P<0.05);与H₂O₂组相比APO+H₂O₂组KCNJ2 mRNA表达恢复(P<0.05) 结论 APO对兔左心房肌细胞I_K1具有保护作用。     

急性心肌梗死对心室肌细胞钾电流的影响

目的 :研究急性心肌梗死 （AMI）心室肌细胞瞬时外向钾电流 （Ito）和内向整流性钾电流 （IK1 ）的变化。方法 :采用结扎兔冠状动脉左前降支的方法建立 AMI动物模型 ,应用膜片钳全细胞记录方法 ,记录比较 AMI后 1周心外膜梗死区心肌细胞 Ito和 IK1 的变化。结果 :心梗组 Ito明显下降 ,I- V曲线明显下移。指令电位为 +60 m V时 ,Ito在心梗组为 1.0 8± 0 .2 4n A（n=12 ） ,与对照组 （2 .0 9± 0 .3 9n A ,n=16）相比 ,显著下降 ,P<0 .0 1;心梗组 IK1 与对照组比较 ,明显下降 ,特别在超极化时。指令电位为 - 12 0 m V时 ,心梗组 IK1 为 3 .0 1± 0 .49n A （n=11） ,对照组为 4.12±0 .5 1n A（n=10 ,P<0 .0 5 ）。结论 :AMI可引起心室肌细胞 Ito和 IK1 的下降 ,从而导致动作电位平台期延长、复极异常 ,这可能是导致 AMI后出现折返性室性心律失常的原因     

Delayed rectifier potassium current in undiseased human ventricular myocytes

OBJECTIVE: The purpose of the study was to investigate the properties of the delayed rectifier potassium current (IK) in myocytes isolated from undiseased human left ventricles. METHODS: The whole-cell configuration of the patch-clamp technique was applied in 28 left ventricular myocytes from 13 hearts at 35 degrees C. RESULTS: An E-4031 sensitive tail current identified the rapid component of IK (IKr) in the myocytes, but there was no evidence for an E-4031 insensitive slow component of IK (IKs). When nifedipine (5 microM) was used to block the inward calcium current (ICa), IKr activation was fast (tau = 31.0 +/- 7.4 ms, at +30 mV, n = 5) and deactivation kinetics were biexponential and relatively slow (tau 1 = 600.0 +/- 53.9 ms and tau 2 = 6792.2 +/- 875.7 ms, at -40 mV, n = 7). Application of CdCl2 (250 microM) to block ICa altered the voltage dependence of the IKr considerably, slowing its activation (tau = 657.1 +/- 109.1 ms, at +30 mV, n = 5) and accelerating its deactivation (tau = 104.0 +/- 18.5 ms, at -40 mV, n = 8). CONCLUSIONS: In undiseased human ventricle at 35 degrees C IKr exists having fast activation and slow deactivation kinetics; however, there was no evidence found for an expressed IKs. IKr probably plays an important role in the frequency dependent modulation of repolarization in undiseased human ventricle, and is a target for many Class III antiarrhythmic drugs.     

Calcium-activated chloride current in rabbit ventricular myocytes

We have used the whole-cell patch-clamp technique to examine the ionic basis for a transient outward current in rabbit ventricular myocytes. High concentrations of intracellular calcium buffer prevented the current, isoproterenol increased it, and cadmium, nisoldipine, ryanodine, or caffeine blocked it. These data are consistent with a current that is calcium activated, by the calcium transient that causes contraction. The current was not blocked by external 4-aminopyridine or tetraethylammonium, and it was still present if external potassium was omitted and internal potassium was replaced by cesium. The current was absent when intracellular and extracellular chloride concentrations were drastically reduced, even when intracellular and extracellular potassium concentrations were normal. The current was blocked by the anion transport blockers 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and responded to extracellular chloride changes as expected for a chloride current. We used SITS and DIDS to define the voltage dependence of the transient outward current. The current first appeared at voltages positive to the threshold of the calcium current and declined as voltage approached the calcium reversal potential. Tail-current experiments suggested that the current rectified strongly in the outward direction. We propose that the 4-aminopyridine-resistant transient outward current of rabbit ventricular myocytes is a calcium-activated chloride current.     

Mechanisms of closure of cardiac sodium channels in rabbit ventricular myocytes: single-channel analysis

We have examined the kinetics of closure of sodium channels using single-channel recordings in cell-attached and excised membrane patches of rabbit ventricular myocytes. Sodium-channel closure was dependent on membrane potential. The closing rate initially decreased with depolarization. The rate then passed through a minimum and increased at strongly depolarized potentials. We attempted to determine the separate voltage dependence of the deactivation and inactivation rate constants using the method of Aldrich, Corey, and Stevens. In a majority of experiments, the method did not give internally consistent results. As an alternative approach, batrachotoxin was used to remove inactivation and determine the voltage dependence of deactivation rate. The deactivation rate decreased with depolarization. To account for the increase in the closing rate at strongly depolarized test potentials, one must postulate voltage dependence of inactivation. The ensemble average current relaxed with a time course that was usually best described by the sum of two exponentials. The larger of the two rate constants that described the relaxation was strongly voltage-dependent, increasing with depolarization. The larger rate constant may reflect voltage-dependent inactivation. We found evidence of two possible mechanisms for the slow component of relaxation: 1) cardiac sodium channels may open repetitively during a given depolarizing epoch, and 2) channels may return from the inactivated state with low probability and burst for as much as 200 msec with open times that are longer than those during usual gating. The slow component appears to be more prominent in cardiac muscle than in nerve and may play an important role in the control of the action potential duration and the inotropic state of the heart.     

Changes in morphology and inward rectifier currents in human atrial myocytes depend on culture conditions

Human atrial myocytes were cultured under systematically varied conditions in order to obtain stable cells for future gene manipulation. Transient (I_to) and sustained outward current (I_so), and voltage- and muscarinic receptor-activated inward rectifier K⁺ currents (I_K1, I_K,ACh) were measured in freshly isolated cells and after 5 days in culture. Myocytes were grown on polylysin or laminin in medium with or without 10 % serum (medium+S, medium-S). Cultured myocytes dedifferentiated to a greater extent in medium+S than medium-S, but independent of the chemical nature of the adherence surface. Apparent surface area increased in medium+S, whereas membrane capacitance declined under all culture conditions. I_to of myocytes cultured in medium-S was increased. Myocytes grown on polylysin and laminin exhibited reduced I_K1 current density. Under all culture conditions, I_K,ACh was attenuated with carbachol but hardly affected with sphingosine-1-phosphate as agonists. In conclusion, morphological and electrophysiological changes depended on serum in the culture medium rather than on adherence surface being coated with laminin or polylysin. Received: 4 October 2001, Returned for revision: 25 October 2001, Revision received: 4 July 2002, Accepted: 25 July 2002 H. M. H. and M. P. contributed equally to this work. Correspondence to: U. Ravens     

Unblock of the slow inward current induces the arrhythmogenic transient inward current in isolated guinea-pig myocytes.

This study investigated whether abrupt changes in extracellular Ca2+ concentration or washout of the Ca2+ antagonists Mn2+ or verapamil, could induce transient inward current (ITI) in enzymatically disaggregated guinea-pig myocytes. Single electrode voltage-clamp techniques were used. ITI was elicited upon repolarization to various voltage steps from an activating step to +20 mV. The holding potential was -80 mV. Slow inward current (ICa) was induced by steps to -10 mV. Continuous exposure to either 2.5 or 6.0 mM Ca2+ did not induce ITI; however, following exposure of cells to 0.5 mM Ca2+ for 20 min which decreased ICa, return to 2.5 or 6.0 mM Ca2+ induced ITI. ITI could be observed for 10 to 20 min following sudden elevations of Ca2+. Similar effects also were seen when Ca2+ was increased from 2.5 to 6.0 mM. Exposure to 2.0 mM Mn2+ or 2.0 microM verapamil blocked ICa. Washout of either blocker induced ITI, particularly in 6.0 mM Ca2+. Peak ITI occurred upon repolarization at c. -70 mV; a reversal potential could not be demonstrated. Thus, abrupt changes in Ca2+ influx, produced either by sudden changes in external Ca2+ or by washout of Ca2+ antagonists, induced ITI with characteristics similar to those described for ITI induced by toxic concentrations of cardiac glycosides.     

The slow component of the delayed rectifier potassium current in undiseased human ventricular myocytes

OBJECTIVE: The purpose of this study was to investigate the properties of the slow component of the delayed rectifier potassium current (I(Ks)) in myocytes isolated from undiseased human left ventricles. METHODS: The whole-cell configuration of the patch-clamp technique was applied in 58 left ventricular myocytes from 15 hearts at 37 degrees C. Nisoldipine (1 microM) was used to block inward calcium current (I(Ca)) and E-4031 (1-5 microM) was applied to inhibit the rapid component of the delayed rectifier potassium current (I(Kr)). RESULTS: In 31 myocytes, an E-4031 insensitive, but L-735,821 and chromanol 293B sensitive, tail current was identified which was attributed to the slow component of I(K) (I(Ks)). Activation of I(Ks) was slow (tau=903+/-101 ms at 50 mV, n=14), but deactivation of the current was relatively rapid (tau=122.4+/-11.7 ms at -40 mV, n=19). The activation of I(Ks) was voltage independent but its deactivation showed clear voltage dependence. The deactivation was faster at negative voltages (about 100 ms at -50 mV) and slower at depolarized potentials (about 300 ms at 0 mV). In six cells, the reversal potential was -81.6+/-2.8 mV on an average which is close to the K(+) equilibrium potential suggesting K(+) as the main charge carrier. CONCLUSION: In undiseased human ventricular myocytes, I(Ks) exhibits slow activation and fast deactivation kinetics. Therefore, in humans I(Ks) differs from that reported in guinea pig, and it best resembles I(Ks) described in dog and rabbit ventricular myocytes.     

兔急性心肌梗死后心室肌细胞L-钙离子通道电流的变化

目的 :研究急性心肌梗死 （AMI）后心室肌细胞钙离子通道电流的变化。方法 :采用结扎兔冠状动脉左前降支的方法建立 AMI动物模型 ,应用膜片钳全细胞记录方法 ,观察 AMI后 1周及 2月心外膜梗死区心肌细胞 L -钙通道电流 （ICa- L）的变化。结果 :1梗死后 1周组、2月组与对照组相比 ,I- V曲线上移。ICa- L电流密度峰值 （0 m V时 ）的比较显示 :对照组为 5 .6± 1.5 p A/ pf（n=10 ） ;梗死后 1周组为 3.5± 0 .9p A / pf（n=6 ） ,较对照组显著减小 ,P<0 .0 5 ;梗死后 2月组为 4 .8± 1.5 p A/ pf（n=11） ,较对照组减小 ,较梗死后 1周组增大 ,但均无统计学差异 ,P>0 .0 5。 2梗死后 1周组、梗死后 2月组与对照组相比 ,失活曲线明显左移 （即向超极化方向移动 ） ,以梗死后 1周组左移更加明显。梗死后 1周组半数失活电压 （V0 .5）为 - 2 6± 7m V（n=6 ） ,与对照组 （- 13± 4 m V ,n=8）比较相差显著 ,P<0 .0 5。梗死后 2月组半数失活电压 V0 .5为 - 2 1± 6 m V（n=8） ,与对照组比较无统计学差异 ,P>0 .0 5。结论 :AMI后1周梗死区心室肌细胞 ICa- L下降、钙通道动力学发生变化 ,在 AMI后 2月这种电生理异常有恢复趋势     

兔左室三层心肌细胞非特异性阳离子电流的特性

目的研究兔左室除钾电流外是否还存在其它复极外向电流,并进一步研究其在三层心肌细胞上的分布。方法采用胶原酶二步消化法分离兔心肌细胞,用锐利眼科剪分离左室游离壁内、中、外三层心肌,改变灌流液的成份,采用全细胞膜片钳记录离子电流。结果在兔左室肌细胞记录到一种新的电压依赖性、非特异性阳离子电流。该离子通道对Na+、K+、Li+、Cs+通透,对Cl-不通透,能够被Gd3+和La3+阻断。该通道在三层心肌细胞的密度分布不同,中层心肌细胞的电流密度明显小于内层和外层心肌细胞。结论非特异性阳离子电流参与了兔左室心肌细胞的电生理异质性的形成。     

Effect of the angiotensin-converting enzyme inhibitor,perindoprilat, and of angiotensin-II on the transient inward current of rabbit ventricular myocytes

Summary Angiotensin-converting enzyme (ACE) inhibitors protect the myocardium from experimental lethal ventricular arrhythmias induced by ischemia or reperfusion. Hypothetically, such arrhythmias may result from the calcium-dependent transient inward current I_ti. It is already known that perindoprilat decreased the transient inward current in guinea-pig myocytes [1]. In the same preparation, however, angiotensin-IIdecreased the transient inward current, an effect opposite to that required to prove that the ACE inhibitor exerted its beneficial effects on I_ti by lessening the action of angiotensin-II. We, therefore, selected another species, the rabbit, in which angiotensin-II was known to have a positive inotropic effect. Perindoprilat (1 µM but not 0.01 µM) decreased the transient inward current from –8.93±0.80 µA/cm² to –5.33±0.74 µA/cm² (p<0.05). Perindoprilat (1 µM) also protected from the effects of angiotensin-II (0.01 and 0.1 µM), which on its own increased the amplitude of the transient inward current. Based on our results, we conclude that perindoprilat (1 µM) prevents the effect of angiotensin-II in promoting the transient inward current in the rabbit. Hence our data support the hypothesis that the ACE inhibitor, perindoprilat, might in relatively high concentrations have an antiarrhythmic effect, at least in part through inhibition of angiotensin-II-evoked calcium-dependent I_ti.This article was evaluated by the San Francisco Editorial Office by two anonymous referees.     

Characterization of functional capacity of adult ventricular myocytes in long-term culture

Background

Functional properties of freshly isolated adult ventricular myocytes (AVMs) or those of AVMs during first few weeks in culture were well described. However, the functional capacity of these AVMs such as regenerative potential remains unknown, in part, due to the short lifespan of AVMs in culture. This study modified culture conditions that extended the lifespan of AVMs, isolated from adult rat hearts, longer than 6 months.

Methods

Temporal changes in the morphology of individual AVMs, cell–cell interaction, formation of myofibers, self-repair capacity after injury, expression of senescence biomarkers, and contractile function of AVMs over 5 weeks (defined as long-term culture) were chronologically characterized and quantified with live-cell video and fluorescence microscopy, and immunocytochemistry.

Results

Cell growth in size reached a plateau after 4 weeks in culture concomitantly with continuous increase in structural remodeling in long-term culture. Dynamic remodeling of AVMs promoted self-contact of filopodia and cell–cell contact where these contained abundant myofilaments, connexin 43 proteins, and high density and high integrity of mitochondria. Such high capacity also enabled self-repair of AVMs after injury, cytokinesis, and formation of myofibers. AVMs in long-term culture displayed spontaneous contraction and importantly were responsive to electrical stimulation. Moreover, AVMs expressed senescence-associated β-galactosidase, p16, and stress-associated atrial natriuretic peptides that resulted likely from cellular modeling.

Conclusions

Prolonged longevity of AVMs in culture with characteristics of high functional capacity of organelle regeneration and contraction makes them invaluable for further longitudinal mechanistic studies in cardiac (patho)physiology (e.g., hypertrophy and aging), single-cell analysis (e.g., function of hetero-phenotypes) and drug discovery.     

伊贝沙坦对兔心室肌细胞L-型钙通道电流的影响

目的 研究伊贝沙坦(irbesartan)对正常家兔心室肌细胞L-型钙电流(ICa-L)、内向整流性钾电流(IK1)、快钠流(INa)以及跨膜动作电位的影响。方法 应用膜片钳全细胞记录方法记录各项离子流和跨膜动作电位。结果 (1)伊贝沙坦呈浓度依赖性阻断ICa-L；(2)伊贝沙坦可使ICa-L I—V曲线上移，但不改变其激活电位、峰值电位和反转电位；(3)伊贝沙坦呈使用依赖性阻滞ICa-L；(4)伊贝沙坦对ICa-L激活曲线无明显影响，但ICa-L失活后再激活的恢复时间常数(τ)明显延长；(6)伊贝沙坦对IKl和INa无明显影响；(7)伊贝沙坦(100nM)可使单个心室肌细胞动作电位时限缩短，对静息电位(RP)、动作电位幅度(APA)无影响。结论 伊贝沙坦作用于L-型钙通道的失活态从而阻断ICa-L。     

U69 593对家兔心室肌细胞L型Ca2+电流的影响

目的 :研究 U695 93对家兔心室肌细胞 L 型 Ca2 +电流 （ ICa· L）的影响。方法 :采用全细胞膜片钳技术记录家兔心室肌细胞 IC a· L。结果 :在 -2 0～ +4 0 m V测试电压范围内 ,10 0 nmol/ L U695 93显著增加 IC a· L,以 +10 m V为最大激活电压 ,其峰值 ICa· L由加药前 -7.1± 0 .4p A/ p F增至 -8.3± 0 .5 p A/ p F （ P<0 .0 1,n=8）。 10 0 nmol/ LU 695 93不影响 IC a· L电压依赖性激活与失活特性。结论 :U695 93可增加家兔心室肌细胞 ICa· L。     

SERCA2A overexpression decreases the incidence of aftercontractions in adult rabbit ventricular myocytes

K. Davia, E. Bernobich, H. K. Ranu, F. del Monte, C. M. N. Terracciano, K. T. MacLeod, D. L. Adamson, B. Chaudhri, R. J. Hajjar and S. E. Harding. SERCA2a Overexpression Decreases the Incidence of Aftercontractions in Adult Rabbit Ventricular Myocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 1005-1015. Slow relaxation and poor contractile response to increasing stimulation frequency in failing human heart have been strongly linked to a decrease in the activity of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a). Restoration of SERCA2a levels using gene transfer has beneficial effects on contractile function but, like beta -adrenoceptor stimulation, could potentially produce excess SR Ca(2+), arrhythmias and cell death. We have examined the effects of SERCA2a overexpression in adult rabbit cardiac myocytes, and compared changes in relaxation with those following beta -adrenoceptor stimulation. Myocytes were infected with an adenovirus carrying both SERCA2a and green fluorescent protein (GFP) for positive identification of infected cells. Myocyte survival was significantly enhanced in the infected cultures. There was a reduction in both time-to-peak contraction and time-to-50% relaxation (R50) 48 h after infection. Time-to-90% relaxation (R90) was particularly improved (non-infected 516+/-41 ms, AD.SERCA2a-GFP 230+/-23 ms, n=7 preparations, P<0.001). There was also a decreased incidence of aftercontractions in Ad.SERCA2a-GFP infected myocytes (21+/-5%v 41+/-4% in controls, P<0.01). This contrasts with beta -adrenoceptor stimulation, which reduced R50 but prolonged R90 by 158+/-76 ms (P<0.02, n=16). At higher stimulation frequencies (2-3 Hz) contraction amplitude and SR calcium content were increased and diastolic contracture was reduced following SERCA2a overexpression. Overall, increasing levels of SERCA2a resulted in an improvement in systolic and diastolic function and a reduction in cell death and arrhythmic aftercontractions. SERCA2a overexpression therefore lacks the detrimental effects associated with some other inotropic interventions.