Effects of quinidine on action potentials and ionic currents in isolated canine ventricular myocytes

We examined the effects of quinidine (5-20 microM) on transmembrane action potentials and ionic currents of isolated canine ventricular myocytes. Collagenase treatment of canine ventricular tissue produced a yield of 40-60% healthy cells. Myocytes had normal resting and action potentials as measured using conventional microelectrodes. Quinidine decreased Vmax, amplitude, overshoot, and the duration of action potentials stimulated by passage of brief current pulses through the recording pipette. Recovery was complete after washout except that action potential duration was prolonged compared with control. A discontinuous single microelectrode voltage ("switch") clamp was used to measure ionic currents. Quinidine irreversibly reduced steady-state outward current as measured with three different voltage clamp protocols. Quinidine reversibly decreased peak calcium current as well as the slowly inactivating and/or steady-state inward currents in the plateau voltage range, presumably both "late" sodium (tetrodotoxin-sensitive) and calcium (tetrodotoxin-insensitive) currents. The effect on calcium current showed both tonic and use-dependent block. Thus, quinidine has a multitude of actions on both inward and outward currents, which combine to produce the net effect of quinidine on action potential configuration.     

Effects of trimetazidine on action potentials and membrane currents of guinea-pig ventricular myocytes

The effects of trimetazidine on membrane potentials and membrane currents of enzymatically isolated guinea-pig ventricular cells were studied with the use of giga-seal suction pipettes for patch clamp. Trimetazidine (3 X 10(-5) M) decreased the action potential duration from 433 +/- 179 ms (mean and S.D., n = 9) to 319 +/- 156 ms within 8 mins. In voltage clamp experiment, trimetazidine at a concentration of 1.5 X 10(-4) M decreased the peak amplitude of calcium current by 40% (0.92 +/- 0.46 nA to 0.55 +/- 0.19 nA, mean +/- S.D., n = 5). The effect on calcium current was rate-dependent, e.g., at 1 Hz, trimetazidine blocked a larger fraction of the calcium current than at 0.2 Hz. The drug decreased the conductance of potassium current which flows via inward rectifier potassium channel from 28 +/- 11 nS to 19 +/- 10 nS, n = 5, P less than 0.05). Trimetazidine shifted the steady state current-voltage relationship outward at potentials positive to -20 mV. This shift was not due to the enhanced time- and voltage-dependent outward current (Ik). From these findings, it was concluded that trimetazidine shortens action potential duration by blocking the calcium channels with increases in steady state outward current or a possible blockade of non-inactivated component of the calcium current, at the plateau potentials. The reduction of calcium current and of inward rectifier potassium current may protect the cardiac cells from accumulation of calcium ions and from loss of potassium ions, in the presence of ischemia.     

Anoxia depresses sodium-calcium exchange currents in guinea-pig ventricular myocytes.

Cardiac Na-Ca exchange is related to the intracellular calcium overload that occurs during ischemia and reperfusion. However, direct observation of the membrane current through Na-Ca exchange during ischemia has not been performed. The purpose of this study was to clarify the effect of simulated ischemia (substrate-free anoxia) and intracellular acidification on the Na-Ca exchange current. The electrogenic Na-Ca exchange current was recorded from isolated guinea-pig ventricular myocytes by using patch-clamp techniques. Exposure to anoxia significantly decreased both the inward and outward directed Na-Ca exchange currents (from -1.21+/-0. 18 to -0.04+/-0.32 pA/pF at -80 mV; from 6.58+/-1.06 to 3.14+/-1.06 pA/pF at +40 mV). The reversal potential of Na-Ca exchange current shifted to negative direction during anoxia. Subsequent reoxygenation rapidly restored the amplitude of exchange currents and the reversal potential. These anoxia/reoxygenation-induced changes were completely inhibited when the intracellular pH was clamped at 7.3 by using 20 m m HEPES-buffer. Furthermore, the anoxia-induced changes of Na-Ca exchange current were mimicked by the intracellular acidosis induced by a brief exposure to ammonium chloride in normoxic conditions. We conclude that the Cardiac Na-Ca exchange is suppressed by anoxia secondary to intracellular acidosis, and that these changes were reversed by reoxygenation.     

Effects of quinidine on ventricular repolarization

The possibility that an asynchronous increase in the ventricularmonophasic action potential duration is the basis of the quinidine-inducedtorsade de pointes, has led us to study the electrophysiologicaleffects of increasing doses of intravenous quinidine. We measuredthe monophasic action potential duration and the ventriculareffective refractory period at several right ventricular myocardialsites in the anaesthetized dog.Our results showed that quinidineinduces a dose-dependent prolongation in ventricular effectiverefractory period and in ventricular monophasic action potentialduration. These increases were uniform throughout the rightventricle. No variations in repolarization or in refractorinesswere observed between the four ventricular sites studied.Theresults suggest that quinidine does not have a direct effecton dispersion of repolarization, and that mechanisms other thanits direct electrophysiological action are involved in the developmentof torsade de pointes.     

Effects of quinidine on ventricular repolarization

The possibility that an asynchronous increase in the ventricularmonophasic action potential duration is the basis of the quinidine-inducedtorsade de pointes, has led us to study the electrophysiologicaleffects of increasing doses of intravenous quinidine. We measuredthe monophasic action potential duration and the ventriculareffective refractory period at several right ventricular myocardialsites in the anaesthetized dog.Our results showed that quinidineinduces a dose-dependent prolongation in ventricular effectiverefractory period and in ventricular monophasic action potentialduration. These increases were uniform throughout the rightventricle. No variations in repolarization or in refractorinesswere observed between the four ventricular sites studied.Theresults suggest that quinidine does not have a direct effecton dispersion of repolarization, and that mechanisms other thanits direct electrophysiological action are involved in the developmentof torsade de pointes.     

阿魏酸钠对家兔心室肌细胞钾通道电流的影响

目的:探讨阿魏酸钠对家兔心室肌细胞膜延迟整流钾电流快速与缓慢激活成分(IKr、IKs)、内向整流钾电流(IK1)、瞬时外向钾电流(Ito)的影响.方法:酶解法分离单个家兔心室肌细胞,以经典的Ⅲ类药胺碘酮为对照,采用全细胞膜片钳技术记录浓度为3.0、10.0、30.0,100.0 μmol/L的阿魏酸钠对IKr,IKs、IK1、Ito的作用.结果:阿魏酸钠的作用弱于胺碘酮,二者均可浓度依赖性抑制IKr、ILs时间依赖性外向电流及尾电流(IKr,tail、IKs,tail).不同浓度的阿魏酸钠对IKr,tail的抑制率为:(12.1±2.5)%、(24.1±3.0)%、(47.0±5.8)%及(58.5±8.3)%(n=5,P＜0.05);对IKs,tail的抑制率为:(15.6±6.4)%、(27.1±6.5)%、(45.6±5.8)%及(51.8±6.6)%(n=5,P＜0.05),其对IKr,tail及IKs,tail的半数抑制浓度(IC50)均大于胺碘酮(43.6:3.48 μmol/L,44.9:5.11 μmol/L).30.0、100.0 μmol/L阿魏酸钠及10.0、30.0 μmol/L胺碘酮可使IK1的I-V曲线左移,在-100 mV及-20 mV测试电压下,阿魏酸钠对IK1内向、外向电流抑制率小于胺碘酮(n=5,P＜0.05).阿魏酸钠与胺碘酮均不影响Ito及其I-V曲线.结论:阿魏酸钠复合阻滞复极期多种钾电流,可能是其抗心律失常作用的电生理机制之一.     

Effects of ambasilide, quinidine, flecainide and verapamil on ultra-rapid delayed rectifier potassium currents in canine atrial myocytes

OBJECTIVE: A dog atrial ultra-rapid delayed rectifier current (I(Kur. d)) is involved in canine atrial repolarization and shares similarities with the human atrial ultra-rapid delayed rectifier (I(Kur)). Almost no information is available about the actions of antiarrhythmic drugs on I(Kur.d). This study evaluated effects of ambasilide, quinidine, flecainide and verapamil on I(Kur.d) in isolated canine atrial myocytes. METHODS: Standard whole-cell patch clamp techniques were used to study the effects of multiple concentrations of each drug. RESULTS: All drugs produced reversible concentration-, voltage- and time-dependent I(Kur.d) inhibition. Significant effects of quinidine, flecainide and ambasilide were noted at atrial-effective antiarrhythmic concentrations in the dog. Upon the onset of a depolarizing pulse, block developed exponentially in relation to time, with the blocking rate-constant increasing with drug concentration, consistent with open-channel blockade and permitting the calculation of forward and reverse rate-constants. For all drugs, the 50% blocking concentration (EC(50)) showed significant voltage-dependence, decreasing at more positive potentials. The magnitude of voltage-dependent block was directly related to the degree of drug-induced shift in the voltage dependence of activation (r=0.97), pointing to open-channel block as a mechanism for voltage-dependent action. An additional component of voltage-dependence suggested that blocking sites were subjected to 17-21% of the transmembrane voltage field. CONCLUSIONS: Ambasilide, quinidine, flecainide and verapamil inhibit I(Kur.d), with preferential action on the open state. I(Kur.d) inhibition may play a role in antiarrhythmic effects in canine atrial arrhythmia models. Comparisons between the effects of these drugs on I(Kur.d) and previously studied effects on I(Kur) suggest potential opportunities for investigating the molecular structural determinants of drug-blocking action on atrial-specific ultrarapid delayed rectifiers.     

Sarcolemmal hydraulic conductivity of guinea-pig and rat ventricular myocytes

OBJECTIVE: Osmotic gradient-induced volume change and sarcolemmal water permeability of cardiac myocytes were evaluated to characterize the mechanism of water flux across the plasma membranes. METHODS: Cell surface dimensions were measured from isolated guinea-pig and rat ventricular myocytes by digital videomicroscopy, and membrane hydraulic conductivity (L(p)) was obtained by analyzing the time course of cell swelling and shrinkage in response to osmotic gradients. RESULTS: Superfusion with anisosmotic solution (0.5-4 times normal osmolality) caused a rapid (<3 min to steady states) and reversible myocyte swelling or shrinkage. L(p) was approximately 1.9 x 10(-10) l N(-1) s(-1) for guinea-pig myocytes and approximately 1.7 x 10(-10) l N(-1) s(-1) for rat myocytes at 35 degrees C. Arrhenius activation energy (E(a)), a measure of the energy barrier to water flux, was approximately 3.7 (guinea-pig) and approximately 3.6 kcal mol(-1) (rat) between 11 and 35 degrees C; these values are equivalent to E(a) of self-diffusion of water in bulk solution ( approximately 4 kcal mol(-1)). Treatment with 0.1 mM Hg(2+), a sulfhydryl-oxidizing reagent that blocks membrane water channels, reduced L(p) by approximately 80%, and the sulfhydryl-reducing reagent dithiothreitol (10 mM) antagonized the inhibitory action of Hg(2+). Inhibition of the volume-sensitive cation (30 microM Gd3+) and anion (1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate) channels and Na+-K+ pump (10 microM ouabain) modified the size of osmotic swelling but had little effect on L(p). CONCLUSIONS: Although the observed L(p) is relatively small in magnitude, the low E(a) and the sulfhydryl reagent-induced modification of L(p) are characteristic of channel-mediated water transport. These data suggest that water flux across the sarcolemma of guinea-pig and rat heart cells occurs through parallel pathways, i.e., the majority passing through water channels and the remainder penetrating the lipid bilayers.     

牵拉刺激对豚鼠心室肌动作电位时程的影响及其机制

目的本实验观察了牵拉刺激对离体豚鼠左心室乳头肌动作电位的影响,为探讨心肌牵张激活离子通道电流的特点提供实验依据。方法实验采用标准微电极细胞内记录技术引导豚鼠心室乳头肌细胞动作电位,用微机化生理信号采集分析系统(NSA-Ⅲ)记录并处理信号。结果①牵拉心肌可加快整个复极过程,APD20、APD50和APD90均有缩短(P<0.05)。牵拉作用呈心肌长度依赖性。牵拉刺激对RP和APA无显著性影响(P>0.05)。②内向整流钾通道(IK1)阻断剂BaCl2(100μmol/L)可明显减弱因牵拉刺激导致的动作电位时程缩短。应用格列本脲、奎尼丁和氯化钆与未用药组比较无显著差异(P>0.05)。结论①在豚鼠心室肌存在牵拉刺激激活的外向电流,这种电流加快复极过程,使动作电位时程明显缩短。②该电流可能与内向整流钾通道(IK1)有关,但是与ATP敏感性钾通道和IKr无关。     

1-磷酸鞘氨醇对心肌细胞延迟整流钾电流2种成分的作用

目的:研究1-磷酸鞘氨醇(S1P)对豚鼠心室肌细胞延迟整流钾电流的2种成分快速激活整流钾电流(IKr)和缓慢激活整流钾电流(IKs)的作用。方法:用胶原酶酶解法急性分离豚鼠心室肌细胞,随机分为正常对照组、S1P(1.1μmol/L)组、S1P(1.1μmol/L)加苏拉明(Suramin)(200μmol/L)组。利用全细胞膜片钳的方法记录心室肌细胞IKr和IKs及其尾电流。结果:①对照组IKr和IKr的尾电流分别为(0.85±0.53)nA和(0.65±0.40)nA。加入S1P后,IKr和IKr的尾电流受到明显抑制,下降到(0.63±0.37)nA和(0.56±0.29)nA(P<0.05,n=6)。而加入S1P加Suramin后,抑制作用消失,IKr和IKr的尾电流为(0.85±0.41)nA和(0.71±0.43)nA,与对照组相比差异无统计学意义(P>0.05,n=6)。②对照组IKs和IKs的尾电流分别为(1.53±0.61)nA和(0.82±0.34)nA。加入S1P后,下降到(1.47±0.46)nA和(0.79±0.41)nA,但差异无统计学意义(P>0.05,n=6)。结论:S1P可降低豚鼠心室肌细胞IKr的幅值,并且是通过其特异性的G蛋白耦联S1P受体介导而产生这些作用。S1P对豚鼠心室肌细胞IKs没有作用。     

Effects of simvastatin on ion channel currents in ventricular myocytes from rabbit with acute myocardial infarction

Objective To investigate the effects of simvastatin on membrane ionic currents in left ventricular myocytes after acute myocardial infarction (AMI,so as to explore the ionic mechanism of statin treatment for antiarrhythmia.Methods Fourty-five New Zeland rabbits were randomly divided into three groups:AMI group,simvastatin intervention group （statin group） and sham-operated control group （CON）.Rabbits were infarcted by ligation of the left anterior descending coronary artery after administration of oral simvastatin 5 mg·kg^-1·d^-1 （Statin group） or placebo （AMI group）for 3 days.Twenty-four hours later,single ventricular myocytes were isolated enzymatically from the epicardial zone of the infracted region.Whole cell patch clamp technique was used to record membrane ionic currents,including sodium current (I_Na),L-type calcium current (I_Ca-L) and transient outward potassium current (I_to).Results①There was no significant difference in serum cholesterol concentration among three groups.②The peak I_Na current density （at-30 mV） was significantly decreased in AMI group （-23.26±5.18） compared with CON （-42.78±5.48,P<0.05）,while it was significantly increased in Statin group （-39.23±5.45） compared with AMI group （P<0.01）;The peak I_Ca-L current density （at 0 mV） was significantly decreased in AMI group （-3.23±0.91） compared with CON （-4.56±1.01,P<0.05）,while it was significantly increased in Statin group （- 4.18±0.95） compared with AMI group （P<0.05）;The I_to current density（at +60 mV） was significantly decreased in AMI group（10.41±1.93）compared with CON （17.41±3.13,P<0.01）,while it was significantly increased in Statin group（16.21±2.42）compared with AMI group （P<0.01）.Conclusions AMI induces significant down-regulation of I_Na,I_Ca-L and I_to.Pretreatment with simvastatin could attenuate this change wit     

Effects of sea anemone toxin anthopleurin-Q on potassium currents in rats and guinea pig ventricular myocytes

To investigate the effect of sea anemone toxin anthopleurin-Q (AP-Q) on potassium currents in isolated rats and guinea pig ventricular myocytes.Methods The ventricular cells of guinea pigs and SD rats were obtained by enzymatic dissociation method.Whole cell patch clamp technique was used to record potassium currents (Ito,IK,and IK1).Results AP-Q 3-100 nmol/L increased Ito in a concentration-dependent manner,with an EC50 value of 12.7 nmol/L.At a potential of +50mV,AP-Q 10nmol/L increased Ito from (13.3±3.4) pA pF-1 to (19.46±4.3) pA pF-1.AP-Q 0.1-100 nmol/L increased IK and IK tail in a concentration-dependent manner with EC50 values of 4.7 nmol/L and 5.0 nmol/L,respectively.AP-Q 1 pmol/L-100 nmol/L increased IK1 in dose-dependent manner,with an EC50 of 0.2 nmol/L.Conclusions The effect of AP-Q on Ito,IK and IK1 may partly explain its mechanism in shortening APD and increasing RP.(J Geriatr Cardiol 2008;5:243-247)     

Effects of neuropeptide Y on cell length and membrane currents in isolated guinea pig ventricular myocytes

Direct effects of neuropeptide Y were studied in left ventricular myocytes isolated from guinea pigs. Contraction was measured as the change in unloaded cell length using a photodiode array. Action potentials were elicited at 1 Hz in current-clamp mode, and membrane currents were measured using a switch-clamp amplifier with 2 M-KCl microelectrodes. At concentrations of 10(-6) M and above, neuropeptide Y reduced contraction in a concentration-dependent fashion. The reduction in contraction by the peptide was proportionately greater in the presence of isoproterenol, and the increase in contraction caused by isoproterenol was completely inhibited by 10(-6) M neuropeptide Y. In response to neuropeptide Y, action potential duration was shortened, and the time course of the shortening was similar to that of the reduction in contraction. Under voltage clamp, 1 x 10(-5) M neuropeptide Y reduced peak L-type calcium current by 32% and shifted the myocyte current-voltage relation during a slow ramp in a manner that suggested a reduction in the background rectifier K+ current. The effects of the peptide on membrane currents were greatly attenuated by preincubation of the cells with pertussis toxin (100 ng/ml). We conclude that neuropeptide Y reduces developed shortening, action potential duration, L-type calcium current, and background rectifier current in single guinea pig ventricular myocytes and that these effects are mediated, at least in part, via membrane G proteins.     

乙酰胆碱对离体豚鼠心室肌的直接作用机制探讨

目的 :研究乙酰胆碱 （ACh）对离体豚鼠心室肌的直接负性作用及机制。方法 :采用标准玻璃微电极细胞内记录技术记录动作电位 （AP）及肌力换能器记录心肌收缩力 （FC）的方法观察 ACh对离体豚鼠心室肌的作用 ,并观察几种受体或通道水平的阻断剂阿托品、氯化铯 （Cs Cl）、氯化镉 （Cd Cl2 ）对 ACh直接作用的影响。结果 :10 - 5 mol/LACh对心室肌动作电位持续时间 （APD）及 FC的抑制率分别为 7.31%和 37.5 7% （P<0 .0 5 ） ,10 - 5 mol/L阿托品和 2 0 m mol/L Cs Cl可阻断该作用 ,0 .1m mol/L Cd Cl2 对该作用无影响。结论 :10 - 5 m ol/L ACh对离体豚鼠心室肌有直接负性作用 ,ACh的作用与毒蕈碱型胆碱受体及 K+电流有关 ,而与 Ca2 +电流的关系可能不大。     

Effects of atorvastatin and simvastatin on atrial plateau currents

Recent evidence has shown that the inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) might exert antiarrhythmic effects both in experimental models and in humans. In this study we analyzed the effects of atorvastatin and simvastatin acid (SVA) on the currents responsible for the duration of the plateau of human atrial action potentials: hKv1.5, Kv4.3, and L-type Ca(2+) (I(Ca,L)). hKv1.5 and Kv4.3 currents were recorded in transfected Ltk(-) and Chinese hamster ovary cells, respectively, and I(Ca,L) in mouse ventricular myocytes, using whole-cell patch-clamp. Atorvastatin and SVA produced a concentration-dependent block of hKv1.5 channels (IC(50)=4.5+/-1.7 microM and 5.7+/-0.03 microM, respectively) and shifted the midpoint of the activation and inactivation curves to more negative potentials. Importantly, atorvastatin- and SVA-induced block was added to that produced by quinidine, a drug that blocks hKv1.5 channels by binding to their pore cavity. Atorvastatin and SVA blocked Kv4.3 channels in a concentration-dependent manner (IC(50)=13.9+/-3.6 nM and 7.0+/-0.8 microM, respectively). Both drugs accelerated the inactivation kinetics and shifted the inactivation curve to more negative potentials. SVA (10 nM), but not atorvastatin, also blocked I(Ca,L) producing a frequency-dependent block that, at 2 Hz, reached a 50.2+/-1.5%. As a consequence of these effects, at nanomolar concentrations, atorvastatin lengthened, whereas SVA shortened, the duration of mouse atrial action potentials. The results suggest that atorvastatin and SVA alter Kv1.5 and Kv4.3 channel activity following a complex mechanism that does not imply the binding of the drug to the channel pore.     

Effects of chronic oral quinidine on left ventricular performance

We studied the effects of 10 to 14 days of oral quinidine administration (200 mg every 8 hrs) on left ventricular (LV) $\text{dP}\text{dt}$ max and shortening fraction (%ΔD) in seve preinstrumented consclous dogs in the resting state, during atrial pacing at 120 bpm, and during an acute pressure load produced by intravenous phenylephrine. Dogs were studied before, during, and after oral quinidine administration with control measurements varying by < 10%. In the resting state, heart rate (85 ± 6 SEM vs 88 ± 7 bpm), LV end-diastolic pressure (7.2 ± 1.4 vs 6.7 ± 1.1 mm Hg), LV end-diastolic diameter (39.6 ± 3.2 vs 38.9 ± 2.7) did not differ (p > 0.05) before or during quinidine, respectively. During atrial pacing LV $\text{dP}\text{dt}$ increased similarly during the control and quinidine periods (+ 13 and + 11%), and %ΔD decreased equally (?26% and ?21%) during phenylephrine infusion off and on quinidine. Thus chronic oral quinidine administration in clinically therapeutic doses (serum quinidine levels 2.3 to 7.5 μg/ml) produced no depression in LV performance at rest or during an acute pressure load.     

Characterisation of the function of adult guinea-pig ventricular myocytes following co-culture with neonatal rat myocytes

Adult guinea-pig myocytes were co-cultured with a layer of spontaneously contracting neonatal rat myocytes based on a method described by Weisensee D. (In Vitro Cell Dev Biol 31: 190–195, 1995). Contractile studies were performed on freshly isolated, 24 and 48 h co-cultured adult guinea-pig myocytes to investigate whether alterations in contractile function had occurred. No difference was found between freshly isolated and 24 h co-cultured adult guinea-pig myocytes in terms of sensitivity to calcium, isoprenaline, frequency response and beat duration. After 48 h, the frequency response was depressed (P<0.02) and the beat was prolonged (P<0.05) when compared to that of freshly isolated myocytes. In the presence of the SR Ca²⁺ ATPase inhibitor, thapsigargin, the beat was significantly prolonged (P=0.003) in 24 h co-cultured myocytes but not in freshly isolated myocytes. These findings show that adult guinea-pig myocytes can be maintained in co-culture with neonatal rat myocytes with little change in contractile function for 24 h but after this time contractile function begins to deteriorate. Received: 3 March 1998, Returned for 1. revision: 25 March 1998, 1. Revision received: 26 May 1998, Returned for 2. revision: 18 June 1998, 2. Revision received: 23 July 1998, Accepted: 23 July 1998     

急性低氧对家兔心室肌细胞钙、钾通道电流的影响

目的:研究急性低氧对单个家兔心室肌细胞L-型钙通道电流(L-Ica)、ATP敏感性钾通道电流(Ik(ATP)以及短暂外向钾电流(Ito)的影响,以探讨急性低氧导致动作电位时程(APD)缩短的机制。方法:应用膜片钳全细胞记录方法。结果:低氧15分钟后心室肌细胞APD明显缩短(由491±57ms降至287±53ms,P<0.01);L-Ica峰值降低(由1.57±0.29 nA降至0.83士0.15 nA,P<0.01),电流-电压曲线上移;IK(ATP)通道开放,短暂外向钾电流(Ito)峰值增大(由4.76士0.43nA升至5.41±0.53hA,P<0.05);复氧2分钟后,IK(ATP)受到抑制(由2.98±0.37nA降至610.9±42.IPA,P<0.01)。结论:急性低氧时APD的缩短是L-Ica、IK(ATP)以及Ito变化综合作用的结果。