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.     

Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action

The mechanism by which isoproterenol (ISO) prevents the prolongation of action potential duration (APD) and refractory period (RP) by the class III antiarrhythmic agent E-4031 was studied. E-4031 (1 microM) increased RP by 50% with no effect on contractile force in papillary muscles isolated from guinea pig heart. ISO (1 microM) increased force of contraction more than fivefold and decreased RP by 25%. The prolongation of RP by E-4031 was prevented by pretreatment of muscles with ISO. The prolongation of APD in isolated guinea pig ventricular myocytes by 5 microM E-4031 also was antagonized by prior exposure of the cells to 1 microM ISO. Instantaneous currents and delayed rectifier K+ currents, IK, were measured in isolated myocytes using the suction microelectrode voltage-clamp technique. Currents were measured in response to 225-msec depolarizing pulses from a holding potential of -40 mV. Previous studies have demonstrated that IK in these cells results from activation of two distinct outward K+ currents, IKs and IKr (specifically blocked by E-4031). ISO doubled the magnitude of IKs without significant effect on IKr. The instantaneous current, putatively identified as a Cl- current, also was doubled by ISO but was unaffected by E-4031. The augmented conductance of IKs and instantaneous current by ISO results in a decrease in RP. The small effect of E-4031 on APD and RP in the presence of ISO results from the smaller contribution of IKr relative to the augmented repolarizing currents.     

Block of delayed rectifier potassium current, IK, by flecainide and E-4031 in cat ventricular myocytes

Block of the delayed rectifier potassium current, IK, by the class IC antiarrhythmic agent, flecainide, and by the novel selective class III antiarrhythmic agent, E-4031, were compared in isolated cat ventricular myocytes using the single suction-pipette, voltage-clamp technique. Flecainide (10 microM) markedly reduced IK elicited on depolarization steps to plateau voltages (+10 mV) and nearly completely blocked the "tail currents" elicited on repolarization to -40 mV (93 +/- 4% block at +40 mV, n = 3). E-4031 (1 microM) produced similar effects (96 +/- 3% block at +40 mV, n = 3). Slow voltage ramps from -100 to +40 mV confirmed inward rectifying properties of IK and showed that flecainide and E-4031 have no effects on the background potassium current, IK1. Thus, the results demonstrate that block of IK is a common feature of flecainide and E-4031. IK block by E-4031 most likely underlies the drug's potent class III antiarrhythmic properties. On the other hand, flecainide block of IK during an action potential would tend to prolong repolarization, but this effect may be obscured by concomitant block of plateau Na+ channels to produce little or no change in action potential duration, consistent with its class IC classification.     

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）.     

芍药苷对心脏钾通道电生理功能的影响

目的研究芍药苷对内向整流钾电流(IK1)、瞬时外向钾电流(Ito)以及延迟整流钾电流(IKs和IKr)的作用。方法用全细胞膜片钳技术记录大鼠心室肌细胞的Ito和IK1电流。而IKs和IKr电流在转染相应质粒的HEK293细胞上记录。对比芍药苷使用前后的电流图,观察芍药苷对各种离子通道电流的影响。结果在-100mV测试电压下,100μmol/L的芍药苷能使IK1峰值密度从(-25.26±8.21)pA/pF降至(-17.65±6.52)pA/pF,平均抑制率为30.13%(n=6,P<0.05),但对其反转电位以及内向整流特性无影响。此外,100μmol/L芍药苷对Ito、IKs和IKr电流无明显作用。结论芍药苷对IK1电流具有明显的抑制作用,而对Ito、IKs及IKr无明显作用。     

Repolarizing K+ currents ITO1 and IKs are larger in right than left canine ventricular midmyocardium

BACKGROUND: The ventricular action potential exhibits regional heterogeneity in configuration and duration (APD). Across the left ventricular (LV) free wall, this is explained by differences in repolarizing K+ currents. However, the ionic basis of electrical nonuniformity in the right ventricle (RV) versus the LV is poorly investigated. We examined transient outward (ITO1), delayed (IKs and IKr), and inward rectifier K+ currents (IK1) in relation to action potential characteristics of RV and LV midmyocardial (M) cells of the same adult canine hearts. METHODS AND RESULTS: Single RV and LV M cells were used for microelectrode recordings and whole-cell voltage clamping. Action potentials showed deeper notches, shorter APDs at 50% and 95% of repolarization, and less prolongation on slowing of the pacing rate in RV than LV. ITO1 density was significantly larger in RV than LV, whereas steady-state inactivation and rate of recovery were similar. IKs tail currents, measured at -25 mV and insensitive to almokalant (2 micromol/L), were considerably larger in RV than LV. IKr, measured as almokalant-sensitive tail currents at -50 mV, and IK1 were not different in the 2 ventricles. CONCLUSIONS: Differences in K+ currents may well explain the interventricular heterogeneity of action potentials in M layers of the canine heart. These results contribute to a further phenotyping of the ventricular action potential under physiological conditions.     

Differential effects of the new class III antiarrhythmic agents almokalant, E-4031 and D-sotalol, and of quinidine, on delayed rectifier currents in guinea pig ventricular myocytes.

OBJECTIVES: The effects of almokalant (4-[3-ethyl[3-(propylsulphinyl)propyl]-amino]-2-hydroxy-propoxy]- benzonitrile), E-4031 (1-[2-(6-methyl-2-pyridyl)-ethyl]-4-(4-methylsulphonyl-amino- benzoyl)piperidine), d-sotalol, and quinidine were investigated on the delayed K+ rectifier current IK. The aim of the study was to compare the drug action on the two components of this current. METHODS: Membrane currents were measured in ventricular myocytes from guinea pig hearts with the whole cell voltage clamp technique. IK was activated during clamp steps from a holding potential of -40 mV to test potentials -30 and +50 mV. The tail current Itail was measured upon stepping back to holding potential. RESULTS: In control experiments. IK and Itail declined spontaneously ("run down"). With 300 ms long test pulses to +50 mV, only d-sotalol (10(-4) M) caused a significant further decrease in IK, whereas all four agents significantly reduced Itail (almokalant 10(-6) M, E-4031 10(-7) M, quinidine 10(-5) M). When tested with 1 s long clamp steps at various potentials almokalant (3 x 10(-6) M), E-4031 (10(-6) M), quinidine (10(-5) M), and d-sotalol (10(-4) M) reduced IK in the potential range between -20 and +40 mV, yielding a bell shaped inward rectifying drug sensitive current. Itail was reduced by almokalant and E-4031 over the whole voltage range with saturation of block positive to +20 mV. Similar reductions with quinidine but not with d-sotalol were also significant. With rest pulses to +50 mV of increasing duration (25 ms-4000 ms), Itail developed with a faster time course than IK and therefore the ratio of Itail/IK declined with pulse duration. With almokalant and E-4031, this ratio became independent of test pulse duration. For 250 ms pulses, Itail/IK was also significantly reduced by d-sotalol and quinidine. CONCLUSION: Inhibition of the rapidly activating inwardly rectifying component of IK is prominent with almokalant and E-4031 and less pronounced with d-sotalol and quinidine. Since inhibition of this component prolongs the cardiac action potential, it should contribute to the antiarrhythmic properties of the agents.     

Contribution of IKr to rate-dependent action potential dynamics in canine endocardium

Previous modeling studies have suggested that the rapid component of the delayed rectifier (I(Kr)) may contribute importantly to action potential dynamics during tachycardia. To test this idea experimentally, I(Kr) was measured as the E-4031-sensitive current in isolated canine endocardial myocytes at 37 degrees C using the perforated patch-clamp technique. Command potentials were trains of action potential waveforms recorded at cycle lengths (CLs) of 1000, 500, 320, 170, and 120 ms. Action potential duration (APD) alternans occurred at CLs of 170 and 120 ms. During an action potential, I(Kr) increased gradually to a maximum at -55 to -60 mV. Peak I(Kr) increased initially as CL was shortened from 1000 to 500 ms (from 0.55+/-0.03 to 0.57+/-0.03 pA/pF), but decreased progressively as CL was shortened further (to 0.45+/-0.03 pA/pF at CL=120 ms). Baseline I(Kr) was negligible at CLs of 1000 to 320 ms, but increased to 0.12+/-0.01 pA/pF at a CL of 120 ms. During APD alternans, peak I(Kr) was larger for the short than for the long action potential (0.48+/-0.03 versus 0.46+/-0.03 pA/pF). A computer model of I(Kr) based on these data indicated that increasing I(Kr) suppressed alternans and decreasing I(Kr) increased alternans. In support of the latter result, inhibition of I(Kr) by E-4031 increased the maximal amplitude of alternans. These results indicate that I(Kr) contributes importantly to rate-related alterations of repolarization, including APD alternans. Modifying I(Kr) may be a promising approach to suppressing alternans and thereby preventing ventricular tachyarrhythmias.     

Pacing-induced heart failure causes a reduction of delayed rectifier potassium currents along with decreases in calcium and transient outward currents in rabbit ventricle

OBJECTIVE: Heart failure in patients and in animal models is associated with action potential prolongation of the ventricular myocytes. Changes in several membrane currents have been already demonstrated to underlie this prolongation. However, information on the two components (I(Kr) and I(Ks)) of the delayed rectifier potassium current (I(K)) in rapid pacing induced heart failure is lacking. METHODS AND RESULTS: Action potentials and whole-cell currents, I(K), I(to1), I(K1), and I(Ca-L) were recorded in apical myocytes of left ventricle from 10 rabbits subjected to left ventricular pacing at 350-380 beats/min for 3-4 weeks and 10 controls with sham operation. Action potential duration at 90% repolarization (APD(90)) was prolonged in myocytes from failing hearts compared to controls at both cycle lengths of 333 and 1000 ms. Both E-4031-sensitive and -resistant components of I(K) (I(Kr), I(Ks)) in myocytes from failing hearts were significantly less than those of control hearts; tail current densities of I(Kr) and I(Ks) following depolarization to +50 mV were 0.62+/-0.05 vs. 0.96+/-0.12 pA/pF (P<0.05), and 0.27+/-0.08 vs. 0.52+/-0.08 pA/pF (P<0.05), respectively. There was no significant difference between control and failing myocytes in the voltage- and time-dependence of activation of total I(K), I(Kr) and I(Ks). The peak of L-type Ca(2+) current (I(Ca-L)) was significantly reduced in myocytes from failing hearts (at +10 mV, -9.29+/-0.52 vs. -12.28+/-1.63 pA/pF, P<0.05), as was the Ca(2+)-independent transient outward current (I(to1); at +40 mV, 4.8+/-0.9 vs. 9.6+/-1.3 pA/pF, P<0.05). Steady state I-V curve for I(K1) was similar in myocytes from failing and control hearts. CONCLUSIONS: Decrease of I(K) (both I(Kr) and I(Ks)) in addition to reduced I(to1), may underly action potential prolongation at physiological cycle length and thereby contribute to arrhythmogenesis in heart failure.     

Open-state unblock characterizes acute inhibition of I potassium current by amiodarone in guinea pig ventricular myocytes

INTRODUCTION: The aim of the present study was to investigate the acute action of amiodarone on the slow component of delayed rectifier K+ current (IKs) under basal conditions and during beta-adrenoceptor stimulation in guinea pig ventricular myocytes. METHODS AND RESULTS: Using the whole-cell patch-clamp method, IKs was evoked by depolarizing voltage-clamp steps, during superfusion with the Na+-, K+-, and Ca2+-free solution supplemented with 0.4 microM nisoldipine and 5 microM E-4031. The acute effect of amiodarone was evaluated, within approximately 10 minutes after starting the bath application, by the amplitude of deactivating tail currents at -50 mV. Amiodarone concentration dependently blocked I(Ks) and exerted a more potent effect on IKs when activated by shorter pulse durations; the degree of block by 30 microM amiodarone on IKs activated by 200 ms, 500 ms, and 2000 ms depolarizing pulses to +30 mV was 55.9 +/- 5.8%, 38.6 +/- 6.0%, and 27.1 +/- 4.0% (n = 5 each), respectively. An envelope of tails test conducted at +10, +30, and +60 mV demonstrated that the degree of IKs block by amiodarone was gradually attenuated during membrane depolarization, which can be described by a monoexponential function, thus supporting the presence of open channel unblock. Amiodarone also blocked IKs maximally stimulated by 1 microM isoprenaline, to an extent similar to control, when IKs was activated by pulse durations of < or =2000 ms. CONCLUSION: We propose that amiodarone acutely blocks native IKs with characteristics associated with open channel unblock, and that the protein kinase A-mediated phosphorylation of channel proteins only minimally affects the amiodarone block.     

慢性压力超负荷左室电重构的异质性及离子基础

目的研究慢性压力超负荷左室电重构的异质性及离子基础。方法新西兰兔通过肾上腹主动脉次全结扎诱发左室慢性压力超负荷。采用全细胞膜片钳技术分别记录对照组及手术组左室内膜、中层及外膜细胞的动作电位,慢激活的延迟整流钾电流(IKs)及快激活的延迟整流钾电流(IKr)等。结果与对照组比较,基础周长为2s时,手术组左室内膜、中层及外膜细胞的动作电位复极90%的时程(APD90)分别延长27.7%、27.2%(P<0.05或0.01)、19.6%(P>0.05);对照组中层细胞的APD90较外膜细胞长50.8%,而手术组为60.4%;在测试电位为+50mV时,手术组左室内膜、中层及外膜细胞IKs尾电流密度分别减小26.1%、36.3%(P均<0.05)、23.0%(P>0.05);IKr尾电流密度分别减小31.7%、30.5%、30.0%(P<0.01或0.05)。对照组外膜细胞的IKs尾电流密度较中层细胞大49.1%,而手术组为77.6%;两组三层细胞之间IKr尾电流密度均无差别。结论正常兔左室存在跨壁复极异质性,心肌肥厚时进一步扩大,IKs分布及下调的不均一性是其离子流基础。     

Prolonged repolarization and triggered activity induced by adenoviral expression of HERG N629D in cardiomyocytes derived from stem cells

OBJECTIVE: The long QT syndrome, N629D HERG mutation, alters the pore selectivity signature sequence, GFGN to GFGD. Heterologous co-expression of N629D and the wildtype HERG resulted in a relative loss of the selectivity of K+ over Na+, but its physiologic relevance has not been assessed in cardiac myocytes. METHODS AND RESULTS: Accordingly, N629D was overexpressed, via adenoviral gene transfer, in cardiomyocytes derived from mouse stem cells. Three IKr phenotypes were observed: (1) the wildtype-like IKr showed inward rectification and a positive tail current; (2) the N629D-like IKr showed outward rectification and an inward tail current; and (3) intermediate IKr showed a small outward tail current. Action potentials (AP) were paired with the IKr measurements in each cell. Resting membrane potential (RMP) was critically dependent on the IKr phenotype. The resting membrane potential of the cells was -61 +/- 5 mV (n=40) in wildtype, -63 +/- 3 mV (n=18) in wildtype-like IKr phenotype, -30 +/- 2 mV (n=12) in N629D-like and -47 +/- 2 mV (n=24) in intermediate phenotype (p<0.00001). Triggered action potential durations (APD) were: 62 +/- 12 ms (n=6) in wildtype, 65 +/- 11 ms (n=6) in wildtype-like IKr phenotypes and 106 +/- 10 ms (n=6) (p<0.01) in intermediate IKr phenotypes. Lowering [K+]o hyperpolarized wildtype cells and cells with a wildtype-like IKr phenotype, but depolarized those with intermediate phenotype (from -45 +/- 1 to -35 +/- 0.5 mV (n=12), p<0.01). In 6 of 12 cells, with intermediate phenotype, the hypokalemia-induced depolarization resulted in triggered activity. TTX suppressed this triggered activity. CONCLUSION: Overexpression of N629D in cardiomyocytes derived from stem cells results in phenotypic variability in IKr, which was the critical determinant of the resting membrane potential, action potential duration and arrhythmogenic response to low [K+]o.     

Chronic angiotensin II stimulation in the heart produces an acquired long QT syndrome associated with IK1 potassium current downregulation

Cardiac hypertrophy is an independent predictor of cardiovascular morbidity and mortality. It predisposes patients to heart failure, QT interval prolongation and ventricular arrhythmias. Angiotensin II (Ang II) exerts direct actions on cardiac tissue inducing cardiomyocyte hypertrophy and electro-mechanical dysfunction. However, a direct association between Ang II and cardiomyocyte electrical remodeling has yet to be demonstrated. Transgenic TG1306/1R (TG) mice with cardiac-specific Ang II overproduction demonstrate blood pressure-independent cardiac hypertrophy and exhibit significant increase in sudden death associated with mechanical dysfunction. The present study makes use of TG mice to evaluate the direct effects of high levels of intracardiac Ang II on cardiac electrophysiology. Surface-limb ECG measurements were recorded on 50- to 60-week-old TG and wild-type (WT) mice. QT interval was significantly prolonged (+20%) in TG mice relative to WT. TG mice also showed an increased incidence of ventricular arrhythmias. QT prolongation was associated with prolongation of cardiomyocyte action potential at 90% repolarization (APD90). The change in APD90 correlated with a reduction in IK1 potassium current density in TG vs. WT cardiomyocytes (at -70 mV: 0.3+/-0.1 pA/pF vs. 0.8+/-0.2 pA/pF, P<0.05). In TG mice, reduction in IK1 was associated with a significant reduction (-50%) of the mRNA encoding Kir2.1 and Kir2.2 subunits of IK1-related KCNJ2 and KCNJ12 potassium channels. These data suggest that cardiac Ang II overproduction leads to the emergence of a long QT syndrome resulting from an IK1-dependent prolongation of the action potential duration through modulation of channel subunit expression.     

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。     

Characterisation of the Na, K pump current in atrial cells from patients with and without chronic atrial fibrillation

OBJECTIVE: To assess the contribution of the Na, K pump current (I(p)) to the action potential duration (APD) and effective refractory period (ERP) in human atrial cells, and to investigate whether I(p) contributes to the changes in APD and ERP associated with chronic atrial fibrillation (AF). METHODS: Action potentials and ion currents were recorded by whole-cell patch clamp in atrial myocytes isolated from consenting patients undergoing cardiac surgery, who were in sinus rhythm (SR) or AF (>3 months). RESULTS: In cells from patients in SR, the I(p) blocker, ouabain (10 microM) significantly depolarised the membrane potential, V(m), from -80+/-2 (mean+/-S.E.) to -73+/-2 mV, and lengthened both the APD (174+/-17 vs. 197+/-23 ms at 90% repolarisation) and ERP (198+/-22 vs. 266+/-14 ms; P<0.05 for each, Student's t-test, n=7 cells, 5 patients). With an elevated pipette [Na(+)] of 30 mM, I(p) was measured by increasing extracellular [K(+)] ([K(+)](o)) from 0 to 5.4 mM. This produced an outward shift in holding current at -40 mV, abolished by 10 microM ouabain. K(+)- and ouabain-sensitive current densities were similar, at 0.99+/-0.13 and 1.12+/-0.11 pA/pF, respectively (P>0.05; n=9 cells), confirming the K(+)-induced current as I(p). I(p) increased linearly with increasing V(m) between -120 and +60 mV (n=25 cells). Stepwise increments in [K(+)](o) (between 0 and 10 mM) increased I(p) in a concentration-dependent manner (maximum response, E(max)=1.19+/-0.09 pA/pF; EC(50)=1.71+/-0.15 mM; n=27 cells, 9 patients). In cells from patients in AF, the sensitivity of I(p) to both V(m) and [K(+)](o) (E(max)=1.02+/-0.05 pA/pF, EC(50)=1.54+/-0.11 mM; n=44 cells, 9 patients) was not significantly different from that in cells from patients in SR. Within the group of patients in AF, long-term digoxin therapy (n=5 patients) was associated with a small, but significant, reduction in E(max) (0.92+/-0.07 pA/pF) and EC(50) (1.35+/-0.15 mM) compared with non-treatment (E(max)=1.13+/-0.08 pA/pF, EC(50)=1.76+/-0.14 mM; P<0.05 for each, n=4 patients). In cells from non-digoxin-treated patients in AF, the voltage- and [K(+)](o)-sensitivity (E(max) and EC(50)) were similar to those in cells from patients in SR. CONCLUSIONS: The Na, K pump current contributes to the human atrial cell V(m), action potential shape and ERP. However, the similarity in I(p) sensitivity to both [K(+)](o) and V(m) between atrial cells from patients with and without chronic AF indicates that I(p) is not involved in AF-induced electrophysiological remodelling in patients.     

兔界嵴细胞离子通道特性研究

目的研究生理状态下及异丙肾上腺素灌流对兔界嵴(CT)与梳状肌(PM)细胞动作电位(AP)及钠电流(INa)、短暂外向钾电流(Ito)、L型钙电流(ICa-L)、延迟整流钾电流(IK)及内向整流性钾电流(IK1)的影响,探讨CT与房性心律失常的关系。方法酶解法分离兔CT及PM细胞,利用全细胞膜片钳技术,记录生理状态下及异丙肾上腺素灌流后CT与PM细胞AP及INa、Ito、ICa-L、IK及IK1的变化。结果①生理状态下,CT细胞动作电位时程(APD)较长,可见明显的平台期;PM细胞AP形态与普通心房肌细胞相似,1期复极迅速,平台期短,类似三角形。②生理状态下,CT细胞Ito电流密度比PM细胞明显降低(7.13±0.38 pA/pF vs 10.70±0.62 pA/pF,n=9,P<0.01),而INa、Ito、ICa-L、IK及IK1则无明显差别。③异丙肾上腺素灌流时CT与PM细胞APD20、APD50、APD90均延长(n=8,P<0.01);指令电位+50 mV时,CT与PM细胞Ito电流密度均减少(n=9,P<0.01)而IK均增加(n=8,P<0.05);指令电位+10 mV时,CT与PM细胞ICa-L电流密度均增加(n=9,P<0.01);IK1在两种心肌细胞均无明显差异。结论 CT与PM细胞AP差异与Ito有关。异丙肾上腺素灌流时ICa-L与IK增强,Ito抑制使CT与PM细胞APD延长,触发机制可能是CT参与房性心律失常的机制之一。     

Potassium rectifier currents differ in myocytes of endocardial and epicardial origin.

Whole-cell voltage-clamp experiments and single-channel current recordings in cell-attached patch mode were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to compare the characteristics of inward rectifier K+ current (IK1) and delayed rectifier K+ current (IK) between endocardial and epicardial cells and to test the hypothesis that the differential characteristics of IK1 and/or IK are responsible for the differences in action potential configuration between the two cell types. IK1 in endocardial cells displayed a distinct N-shaped current-voltage (I-V) relation, with a prominent outward current at potentials between -80 and -30 mV. In epicardial cells, an outward current region was much smaller, and the I-V relation demonstrated a blunted N-shaped I-V relation. In single-channel current recordings in cell-attached patch mode, neither unitary current amplitude of IK1 nor probability of channel opening was different between endocardial and epicardial cells, suggesting that the difference in the number of functional channels might be responsible for the differential IK1 I-V relations. The characteristics of IK also differed between endocardial and epicardial cells. The time course of growth of tail current of IK (IK,tail) (activation of IK) was significantly enhanced and that of IK,tail deactivation was delayed in epicardial cells compared with endocardial cells. The time constant of the slow component of IK activation at +20 mV was 3,950 +/- 787 msec in endocardial cells and 2,746 +/- 689 msec in epicardial cells (p less than 0.05); the corresponding values for IK deactivation at -50 mV were 1,041 +/- 387 msec and 1,959 +/- 551 msec, respectively (p less than 0.01). The voltage dependence of steady-state activation of IK,tail was similar between endocardial and epicardial cells, suggesting that the probability of channel opening at any potential was not different in the two cell types. The amplitude and density of fully activated IK (IK,full) were significantly greater in epicardial cells than in endocardial cells. At repolarization to -20 mV, IK,full amplitude was 452 +/- 113 pA in endocardial cells and 578 +/- 135 pA in epicardial cells (p less than 0.05), and the corresponding values for IK,full density were 2.86 +/- 0.73 and 4.21 +/- 0.83 microA/cm2, respectively (p less than 0.05). A nonstationary fluctuation analysis revealed that the amplitude of IK unitary current was similar between endocardial and epicardial cells (0.23 +/- 0.07 versus 0.22 +/- 0.03 pA, p = NS).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

A novel mutation in KCNQ1 associated with a potent dominant negative effect as the basis for the LQT1 form of the long QT syndrome

Introduction: Long QT Syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and life-threatening polymorphic ventricular tachyarrhythmias. LQT1 caused by KCNQ1 mutations is the most common form of LQTS.
Methods and Results: Patients diagnosed with LQTS were screened for disease-associated mutations in KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2, and SCN5A . A novel mutation was identified in KCNQ1 caused by a three-base deletion at the position 824–826, predicting a deletion of phenylalanine at codon 275 in segment 5 of KCNQ1 (ΔF275). Wild-type (WT) and ΔF275- KCNQ1 constructs were generated and transiently transfected together with a KCNE1 construct in CHO-K1 cells to characterize the properties of the slowly activating delayed rectifier current (IKs) using conventional whole-cell patch–clamp techniques. Cells transfected with WT- KCNQ1 and KCNE1 (1:1.3 molar ratio) produced slowly activating outward current with the characteristics of IKs. Tail current density measured at −40 mV following a two-second step to +60 mV was 381.3 ± 62.6 pA/pF (n = 11). Cells transfected with ΔF275- KCNQ1 and KCNE1 exhibited essentially no current. (Tail current density: 0.8 ± 2.1 pA/pF, n = 11, P = 0.00001 vs WT). Cotransfection of WT- and ΔF275- KCNQ1 (50/50), along with KCNE1, produced little to no current (tail current density: 10.3 ± 3.5 pA/pF, n = 11, P = 0.00001 vs WT alone), suggesting a potent dominant negative effect. Immunohistochemistry showed normal membrane trafficking of ΔF275- KCNQ1 .
Conclusion: Our data suggest that a ΔF275 mutation in KCNQ1 is associated with a very potent dominant negative effect leading to an almost complete loss of function of IKs and that this defect underlies a LQT1 form of LQTS.     

Ionic basis of ventricular arrhythmias in remodeled rat heart during long-term myocardial infarction.

OBJECTIVE: Deleterious electrical abnormalities evolve during myocardial infarction. The goal of this study was to analyse current changes during the late decompensated phase of heart disease induced by coronary ligation and to compare them in various heart regions. METHODS: Young rats were submitted to left coronary ligature. After 4-6 months, cells were enzymatically dissociated and isolated from the upper part basal region of the left ventricle, as well as from the septum, apex and the right ventricle before being studied under whole-cell patch-clamp. RESULTS: Basal L-type Ca2+ current, ICaL elicited at +10 mV did not exhibit regional dependence neither in control nor after post-myocardial infarction (PMI). ICaL showed both a significantly reduced peak amplitude (17.1 +/- 2.8 pA/pF versus 9.9 +/- 1.4 pA/pF in seven control and seven PMI hearts, n = 32 and 40, respectively) and a slower inactivation, such that the amount of inward charges during a 200 ms-depolarizing pulse was nearly unchanged. beta-Adrenergic stimulation was less effective in increasing ICaL in PMI cells but it slowed inactivation further. Significant differences in the K+ currents were observed. A regional distribution was seen for Ito only, with the largest amplitude in the right ventricle (in pA/pF: 23.1 +/- 2.4, 18.2 +/- 3.9, 14.8 +/- 2.4, 8.3 +/- 1.7 in the right ventricle, apex, septum and left ventricle, respectively n = 8, 7, 8 and 9). This was also true in failing heart cells despite Ito being halved in each of the four regions (in pA/pF: 12.2 +/- 2.5, 11.2 +/- 1.9, 5.1 +/- 1.0 and 4.8 +/- 1.0, respectively n = 12, 12, 11 and 13). IK1 was also significantly reduced by 20% in the PMI cells. Two-way analyses of variance demonstrated the absence of interaction between the topographical origin of the cells and the physiological state of the rats. The alpha 1-adrenergic agonist, methoxamine significantly reduced Ito and IK1 to the same extent in both sham and PMI cells, by about 35% and 20% respectively. CONCLUSIONS: Long-term left coronary occlusion induces significant alterations in both Ca2+ and K+ currents that occur with similar amplitude in both ventricles. They include a marked reduction in Ito amplitude as well as a slowing of ICaL inactivation. Both factors could contribute to the disturbances in cellular electrical behaviour and the occurrence of arrhythmias in the post-myocardial infarcted heart.