大鼠急性低氧运动对心肌细胞瞬时外向钾电流的影响

目的 通过建立大鼠急性低氧运动模型,观察大鼠单个心室肌细胞瞬时外向钾电流(I_to)的改变,在细胞水平探究模拟高原低氧条件下力竭运动对心脏电生理的影响。 方法 将40只健康雄性清洁级SD大鼠随机分为低氧运动组、低氧安静组、常氧运动组和常氧安静组,每组10只。利用小动物低压氧舱和常氧状态下进行力竭运动试验。取出各组大鼠心脏,利用灌流酶解法分离大鼠单个心室肌细胞,采用全细胞膜片钳技术记录大鼠单个心室肌细胞的瞬时外向钾电流。采用SPSS 20.0统计软件进行数据处理,多组间比较采用ANOVA方差分析,组间两两比较采用SNK-q检验。 结果 与常氧安静组比较,+40mV时,低氧运动组的I_to电流密度显著降低,且低于低氧安静组及常氧运动组,此效应呈现电压依赖性。门控机制研究显示,低氧运动时大鼠心肌细胞I_to稳态激活曲线失活向超极化方向移动, 而稳态失活曲线则向去极化方向移动,二者综合效应使I_to电流显著降低。 结论 急性低氧运动可通过改变钾通道稳态激活与稳态失活过程,降低大鼠心室肌细胞I_to,这可能是急性低氧运动导致心律失常的主要原因之一。     

Effects of allitridum on the transient outward potassium current in rats with heart failure

Objective To study the effect of allitridum on the transient outward potassium current (I_to) of ventricular myocytes in heart failure (HF). Methods The dual enzymatic method was used to separate single ventricular myocytes from Sprague Dawley rats. Patch-clamping was used to record I_to and analyze the effect of allitridum on the current. Results The I_to current had a significant decrease in the HF group, compared with the control group. The density of I_to in the HF group was increased after treatment of allitridum (30 μmol/L). The peak current densities of I_to were enhanced in the HF group from 6.01 ± 0.30 pA/pF to 8.41 ± 0.54 pA/pF (P < 0.01) at +50 mV after treatment with allitridum (30 μmol/L). We also determined the effect of allitridum on the gating mechanism of the I_to in the HF group. Conclusions We found that allitridum increased the I_to by accelerating the activation of channels and shortened the time constants of inactivation, and allitridum decreased the remodeling of I_to in ventricular myocytes of rats with HF.     

胺碘酮及Ⅲ类AAD的电药理

Ⅲ类抗心律失常药物(Ⅲ类AAD)为钾通道阻滞剂，自CAST试验以来是发展较快，受人关注的抗心律失常药物，胺碘酮属其中之一，应用至今已有30年历史，是较为成熟的Ⅲ类AAD。Ⅲ类AAD共同的特点为延长动作电位时程(APD)，心电图上表现QT间期延长，使心肌细胞有效不应期(ERP)延长，达到抗心律失常目的。ERP延长使折返波长延长，所以能有效地终止各种折返性心动过速。APD延长有利于诱发早后除极电位(EAD)，导致尖端扭转性室速(TdP)，此为Ⅲ类AAD促心律失常特征。     

醛固酮对心室肌细胞动作电位及L型钙通道的影响

目的研究醛固酮对心室肌细胞动作电位及L型Ca2+通道的影响,探讨其致心律失常的机制。方法分离Wister大鼠心室肌细胞,随机分为对照组和Ald组,采用全细胞膜片钳记录方法记录动作电位时程(APD)、L型钙电流(ICa-L)。结果 Ald组APD较对照组显著延长(P<0.05)。Ald组ICa-L电流密度峰值较对照组显著增大[-(9.73±0.90)pA/pF vs-(7.07±0.83)pA/pF,P<0.01]。Ald组与对照组比较,I-V曲线显著下移。结论醛固酮可能通过增加L型Ca2+通道的电流密度,延长心肌细胞APD,参与醛固酮的致心律失常作用。     

源于右室流出道的室性心律失常的电生理机制研究

目的探讨右室流出道(RVOT)室性心律失常的发生机制。方法采用全细胞膜片钳技术记录RVOT和右室(RV)心肌细胞的动作电位及离子流,对比分析动作电位及离子流的特性。结果RVOT心肌细胞动作电位表现出复极离散度较RV大。在单细胞电流的记录中,RVOT心肌细胞的瞬时外向钾电流离散度较RV心肌细胞大。在稳态电流中,RVOT心肌细胞的非特异性阳离子流(NSCC)小于RV心肌细胞的NSCC,甚至某些RVOT心肌细胞缺乏NSCC,与之对应的是心肌细胞较长的动作电位时程(APD)并记录到早期后除极(EAD)。当激活NSCC后,可使RVOT心肌细胞较长的APD缩短。激活的NSCC可以消除在RVOT记录到的EAD。结论RVOT心肌细胞的复极离散度和APD的离散大,此易导致折返性心律失常的发生。NSCC较小或缺如是RVOT心肌细胞APD延长,并且产生EAD的原因。     

兔右室流出道心肌细胞动作电位及钠钙交换尾电流特性

目的研究兔右室流出道(RVOT)心肌细胞动作电位及钠钙交换尾电流(INCX,tail)相关特性,探讨源于RVOT室性心律失常的发生机制。方法采用全细胞膜片钳技术记录兔右室(RV)游离壁和RVOT心肌细胞的动作电位,在不更换细胞及电极内液情况下连续记录INCX,tail,对比分析两者动作电位和INCX,tail特性。结果兔RVOT心室肌细胞动作电位复极时程(APD)的变异程度大于RV游离壁心肌细胞。在RVOT心肌细胞记录到早期后除极及显著延长的APD。动作电位显著延长及后除极的RVOT心肌细胞所对应的INCX,tail到达峰值时程较动作电位正常的细胞延迟,并且电流强度大于RV游离壁对照组心肌细胞(P<0.05)。结论 RVOT心肌细胞APD变异程度大,而且APD显著延长的RVOT细胞INCX,tail到达峰值时程延迟及相应电流显著增大,这是RVOT部位好发触发活动的重要机制。     

糖尿病心肌钾离子通道改变研究进展

心肌细胞短暂外向钾电流(Ito)、延迟整流性外向钾电流(IK)、超快速激活型钾电流(IKur)、ATP敏感性钾通道(KATP)等钾离子通道在糖尿病(DM)模型中均发生异常改变,可引起动作电位(AP)、动作电位时程(APD)、QT间期、有效不应期(ERP)改变,以及折返、触发等活动,导致心律失常发生。DM心肌电生理改变可能与体内糖代谢紊乱造成心肌细胞葡萄糖利用障碍、ATP供给下降有关,这些改变可导致细胞骨架稳定性的破坏、基因转录的抑制和蛋白翻译水平的下降。但机制远不止这些,还需要大量的研究工作来探索心肌电生理发生的改变及其机制。     

电压依赖型钾通道的失活及其分子机制

心肌细胞中存在多种钾通道，主要作用于动作电位的平台期。通道的激活和复活对心肌细胞的复极有较大影响。钾通道分为电压依赖性、受体依赖性、ATP依赖性、Ca‘“依赖性等类型，其中电压依赖性钾通道占重要地位。随着分子生物学技术的发展，已克隆出多种钾通道基因，从而有可能从基因结构上了解钾通道的功能，以及基因结构与通道动力学、通道调节之间的关系，更进一步了解心律失常的发生及药物的作用机制。本文着重从分子结构＊阐述通道的失活及其调节机制。！电压依范性钾通过的基本结构离子通道实际上是一类跨膜糖蛋白，是由多个亚基构…     

从索他洛尔电生理作用评价其抗心律失常作用

从索他洛尔 (sotalol)电药理和电生理作用探索其抗心律失常机制。方法 :①采用经典的微电极方法和膜片钳技术观察sotalol对豚鼠心肌细胞动作电位时程 (APD)和膜离子流的作用 ;②运用心内膜单相动作电位 (MAP)技术观察sotalol对狗APD、有效不应期 (ERP)的影响 ;③监测药物浓度与QT间期关系。结果 :①索他洛尔对内向整流性钾流、钠内流、慢钙内流无影响 ,仅对延迟整流性钾流 (IK)有抑制作用 ;②索他洛尔可延长APD ,呈反转使用依赖 ;③静脉注射索他洛尔后 ,QTc ,APD90 和心室ERP均延长 ,缺血区心肌与非缺血区心肌对索他洛尔的反应一致 ;④QT间期随血药浓度升高而延长 ,血药浓度随剂量增大而上升。结论 :索他洛尔阻滞IK 外流、延长APD ,呈反转使用依赖。索他洛尔延长QTc与血药浓度呈正相关。因此 ,如能掌握索他洛尔特性 ,本药还不失为一安全有效的Ⅲ类抗心律失常药物。     

活性氧与ATP敏感性钾通道的相互作用参与高糖对

目的　研究活性氧（ROS）和ATP敏感性钾通道（K_ATP通道）的相互作用在高糖（HG）引起的心肌细胞损伤中的作用。方法　应用Western　blot检测心肌细胞K_ATP通道蛋白、Cleaved　Caspase-3的表达水平;双氯荧光素染色荧光显微镜照相检测胞内ROS水平;细胞计数盒测定心肌细胞存活率;Hoechst　33258核染色荧光显微镜照相测定凋亡细胞数量的变化;JC-1染色法测定线粒体膜电位。结果　应用高糖（35　mmol/L葡萄糖）处理H9c2心肌细胞24　h能明显下调K_ATP通道蛋白的表达水平,1000　μmol/L　N-乙酰半胱氨酸（ROS清除剂）预处理心肌细胞60　min可阻断HG对心肌细胞K_ATP通道蛋白表达的下调作用。100　μmol/L二氮嗪（线粒体K_ATP通道开放剂）和50　μmol/L吡拉地尔（非选择性K_ATP通道开放剂）预处理均显著抑制HG引起的心肌细胞ROS的堆积。1000　μmol/L　N-乙酰半胱氨酸、100　μmol/L二氮嗪和50　μmol/L吡拉地尔均能抑制HG引起的心肌细胞损伤,使细胞存活率升高,凋亡细胞数量、Cleaved　Caspase-3表达及线粒体膜电位丢失减少。结论　在HG状态下,心肌细胞的ROS和K_ATP通道存在相互作用,两者在HG引起的心肌细胞损伤中发挥重要作用。     

Stretch Current‐Induced Abnormal Impulses in CaMKIIδ Knockout Mouse Ventricular Myocytes

CaMKII and Arrhythmias. Background: CaMKII activation is proarrhythmic in heart failure where myocardium is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle has not been well understood. Objective: We tested abnormal impulse inducibility by stretch current in myocytes isolated from CaMKIIδ knockout (KO) mouse left ventricle (LV) where CaMKII activity is reduced by ≈ 62%. Methods and Results: Action potentials were recorded by whole‐cell patch clamp, and abnormal impulses were induced in LV myocytes by a simulation of stretch‐activated channel (SAC) current. SAC activation failed to induce abnormal impulses in wild type (WT) myocytes but steadily produced early after‐depolarizations and automaticity in KO myocytes in which an increase in L‐type calcium channel (LTCC) current (I_Ca) and a reduction of sarcoplasmic reticulum Ca²⁺ leak and action potential duration (APD) were observed. The abnormal impulses were not suppressed by CaMKII inhibitor AIP whereas a low concentration of nifedipine eliminated abnormal impulses without shortening APD, implicating I_Ca in promoting stretch‐induced abnormal impulses. In addition, APD prolongation by LTCC opener S(‐)Bay K 8644 or isoproterenol facilitated abnormal impulse induction in WT ventricular myocytes even in the presence of CaMKII inhibitor AIP, whereas APD prolongation by K⁺ channel blocker 4‐aminopyridine promoted abnormal impulses in KO myocytes but not in WT myocytes. Conclusion: I_Ca activation plays a central role in stretch‐induced abnormal impulses and APD prolongation is arrhythmogenic only when I_Ca is highly activated. At increased I_Ca activation, CaMKII inhibition cannot suppress abnormal impulse induction. (J Cardiovasc Electrophysiol, Vol. 24, pp. 457‐463, April 2013)     

Effects of nerve growth factor on the action potential duration and repolariz-ing currents in a rabbit model of myocardial infarction

Objective To investigate the effect of nerve growth factor (NGF) on the action potential and potassium currents of non-infarcted myocardium in the myocardial infarcted rabbit model. Methods Rabbits with occlusion of the left anterior descending coronary artery were prepared and allowed to recover for eight weeks (healed myocardial infarction, HMI). During ligation surgery of the left coronary artery, a polyethylene tube was placed near the left stellate ganglion in the subcutis of the neck for the purpose of administering NGF 400 U/d for eight weeks (HMI + NGF group). Cardiomyocytes were isolated from regions of the non-infarcted left ventricular wall and the action potentials and ion currents in these cells were recorded using whole-cell patch clamps. Results Compared with HMI and control cardiomyocytes, significant prolongation of APD₅₀ or APD₉₀ (Action potential duration (APD) measured at 50% and 90% of repolarization) in HMI + NGF cardiomyocytes was found. The results showed that the 4-aminopyridine sensitive transient outward potas?sium current (I_to), the rapidly activated omponent of delayed rectifier potassium current (I_Kr), the slowly activated component of delayed rectifier potassium current (I_Ks), and the L-type calcium current (I_CaL) were significantly altered in NGF + HMI cardiomyocytes compared with HMI and control cells. Conclusions Our results suggest that NGF treatment significantly prolongs APD in HMI cardiomyocytes and that a decrease in outward potassium currents and an increase of inward Ca²⁺ current are likely the underlying mechanism of action.     

Effects of allocryptopine on outward potassium current and slow delayed rec-tifier potassium current in rabbit myocardium

Objective Allocryptopine (ALL) is an effective alkaloid of Corydalis decumbens (Thunb.) Pers. Papaveraceae and has proved to be anti-arrhythmic. The purpose of our study is to investigate the effects of ALL on transmural repolarizing ionic ingredients of outward potassium current (I_to) and slow delayed rectifier potassium current (I_Ks). Methods The monophasic action potential (MAP) technique was used to record the MAP duration of the epicardium (Epi), myocardium (M) and endocardium (Endo) of the rabbit heart and the whole cell patch clamp was used to record I_to and I_Ks in cardiomyocytes of Epi, M and Endo layers that were isolated from rabbit ventricles. Results The effects of ALL on MAP of Epi, M and Endo layers were disequilibrium. ALL could effectively reduce the transmural dispersion of repolarization (TDR) in rabbit transmural ventricular wall. ALL decreased the current densities of I_to and I_Ks in a voltage and concentration dependent way and narrowed the repolarizing differences among three layers. The analysis of gating kinetics showed ALL accelerated the channel activation of I_to in M layers and partly inhibit the channel openings of I_to in Epi, M and Endo cells. On the other hand, ALL mainly slowed channel deactivation of I_Ks channel in Epi and Endo layers without affecting its activation. Conclusions Our study gives partially explanation about the mechanisms of transmural inhibition of I_to and I_Ks channels by ALL in rabbit myocardium. These findings provide novel perspective regarding the anti-arrhythmogenesis application of ALL in clinical settings.     

Modeling Reentry in the Short QT Syndrome With Human-Induced Pluripotent Stem Cell–Derived Cardiac Cell Sheets

BackgroundThe short QT syndrome (SQTS) is an inherited arrhythmogenic syndrome characterized by abnormal ion channel function, life-threatening arrhythmias, and sudden cardiac death.ObjectivesThe purpose of this study was to establish a patient-specific human-induced pluripotent stem cell (hiPSC) model of the SQTS, and to provide mechanistic insights into its pathophysiology and therapy.MethodsPatient-specific hiPSCs were generated from a symptomatic SQTS patient carrying the N588K mutation in the KCNH2 gene, differentiated into cardiomyocytes, and compared with healthy and isogenic (established by CRISPR/Cas9-based mutation correction) control hiPSC-derived cardiomyocytes (hiPSC-CMs). Patch-clamp was used to evaluate action-potential (AP) and I_Kr current properties at the cellular level. Conduction and arrhythmogenesis were studied at the tissue level using confluent 2-dimensional hiPSC-derived cardiac cell sheets (hiPSC-CCSs) and optical mapping.ResultsIntracellular recordings demonstrated shortened action-potential duration (APD) and abbreviated refractory period in the SQTS-hiPSC-CMs. Similarly, voltage- and AP-clamp recordings revealed increased I_Kr current density due to attenuated inactivation, primarily in the AP plateau phase. Optical mapping of the SQTS-hiPSC-CCSs revealed shortened APD, impaired APD-rate adaptation, abbreviated wavelength of excitation, and increased inducibility of sustained spiral waves. Phase-mapping analysis revealed accelerated and stabilized rotors manifested by increased rotor rotation frequency, increased rotor curvature, decreased core meandering, and increased rotor complexity. Application of quinidine and disopyramide, but not sotalol, normalized APD and suppressed arrhythmia induction.ConclusionsA novel hiPSC-based model of the SQTS was established at both the cellular and tissue levels. This model recapitulated the disease phenotype in the culture dish and provided important mechanistic insights into arrhythmia mechanisms in the SQTS and its treatment.     

Impaired IKs channel activation by Ca2 +-dependent PKC shows correlation with emotion/arousal-triggered events in LQT1

BackgroundThe most common inherited cardiac arrhythmia, LQT1, is due to I_Ks potassium channel mutations and is linked to high risk of adrenergic-triggered cardiac events. We recently showed that although exercise-triggered events are very well treated by ß-blockers for these patients, acute arousal-triggered event rate were not significantly reduced after beta-blocker treatment, suggesting that the mechanisms underlying arousal-triggered arrhythmias may be different from those during exercise. I_Ks is strongly regulated by β-adrenergic receptor (β-AR) signaling, but little is known about the role of α1-AR-mediated regulation.Methods and resultsHere we show, using a combination of cellular electrophysiology and computational modeling, that I_Ks phosphorylation and α1-AR regulation via activation of calcium-dependent PKC isoforms (cPKC) may be a key mechanism to control channel voltage-dependent activation and consequently action potential duration (APD) in response to adrenergic-stimulus. We show that simulated mutation-specific combined adrenergic effects (β + α) on APD were strongly correlated to acute stress-triggered cardiac event rate for patients while β-AR effects alone were not.ConclusionWe were able to show that calcium-dependent PKC signaling is key to normal QT shortening during acute arousal and when impaired, correlates with increased rate of sudden arousal-triggered cardiac events. Our study suggests that the acute α1-AR-cPKC regulation of I_Ks is important for QT shortening in “fight-or-flight” response and is linked to decreased risk of sudden emotion/arousal-triggered cardiac events in LQT1 patients.     

Bone marrow mesenchymal stem cells upregulate transient outward potassium currents in postnatal rat ventricular myocytes

Bone marrow mesenchymal stem cell (BMSC) transplantation has been shown to effectively improve cardiac function in experimental animals and patients with myocardial infarction and heart hypertrophy. BMSCs exert potent effects on cardiomyocytes through the inhibition of cardiac apoptosis, the attenuation of cardiac inflammation, etc. However, novel biological actions of BMSCs on cardiomyocytes remain to be explored. The present study was designed to investigate whether BMSCs affect electrophysiological features of neonatal rat ventricular myocytes (NRVMs). BMSCs and NRVMs were indirectly co-cultured at a ratio of 1:10 with a semi-permeable membrane. We found that compared with mono-cultured NRVMs, co-cultured NRVMs exhibited an obvious increase of transient outward potassium current (I_to), accompanied by significant changes in activation, inactivation and recovery of I_to. Meanwhile, K_V4.2 mRNA which encodes the channel carrying I_to was more abundant in co-cultured NRVMs than mono-cultured NRVMs. The increases in basic fibroblast growth factor (bFGF) and insulin growth factor-1 (IGF-1) levels were observed in culture medium of BMSCs. bFGF but not IGF-1 upregulated the K_V4.2 mRNA expression and enhanced I_to currents. Taken together, we conclude that BMSCs upregulate I_to of NRVMs, at least partially, by secreting bFGF that in turn upregulates K_V4.2 expression and alters the kinetics of I_to.     

High-mobility group box 1 (HMGB1) downregulates cardiac transient outward potassium current (Ito) through downregulation of Kv4.2 and Kv4.3 channel transcripts and proteins

Transient outward potassium currents (I_to) are major early repolarization currents in shaping cardiac action potential (AP). Downregulation of I_to contributes to AP configuration alteration in myocardial infarction (MI) and numerous other heart diseases. High-mobility group box 1 (HMGB1), a proinflammatory cytokine, has been reported to increase dramatically in the serum of patients with MI, participating in ischemia-reperfusion injury and recovery of post-infarction failing heart. This study investigated the possible role of HMGB1 in regulating cardiac I_to and electrical stability. HMGB1 treatment for 24 h significantly inhibited the current densities of heterologously expressed Kv4.3 and Kv4.2 in COS-7 cells and native I_to in neonatal rat ventricular myocytes (NRVMs) in a dose-dependent manner. HMGB1 decreased the mRNA and protein levels of the I_to α subunits Kv4.2 and Kv4.3 channels, but not the β subunit KChIP2 and KCNE2 in NRVMs. The receptor binding domain (150-186 amino acid residues) responsible for receptor of advanced glycation end product (RAGE) binding similarly inhibited I_to, while treatment with soluble RAGE that blocks binding of ligands to cell-surface RAGE partially restored I_to current density and Kv4 protein expressions. Box A which possesses no proinflammatory activity of HMGB1 still remained part of the I_to suppression effect. In addition to downregulating I_to, HMGB1 modestly inhibited L-type Ca²⁺ current, but not I_K1. The AP duration (APD) was slightly prolonged by HMGB1 treatment. These results collectively establish HMGB1 as a novel pathological factor downregulating I_to partially through HMGB1-RAGE interaction, providing new insights into the potential molecular mechanisms underlying the electrical remodeling in MI.     

KChIP2 attenuates cardiac hypertrophy through regulation of Ito and intracellular calcium signaling

Recent evidence shows that the auxiliary subunit KChIP2, which assembles with pore-forming Kv4-subunits, represents a new potential regulator of the cardiac calcium-independent transient outward potassium current (I_to) density. In hypertrophy and heart failure, KChIP2 expression has been found to be significantly decreased. Our aim was to examine the role of KChIP2 in cardiac hypertrophy and the effect of restoring its expression on electrical remodeling and cardiac mechanical function using a combination of molecular, biochemical and gene targeting approaches. KChIP2 overexpression through gene transfer of Ad.KChIP2 in neonatal cardiomyocytes resulted in a significant increase in I_to-channel forming Kv4.2 and Kv4.3 protein levels. In vivo gene transfer of KChIP2 in aortic banded adult rats showed that, compared to sham-operated or Ad.β-gal-transduced hearts, KChIP2 significantly attenuated the developed left ventricular hypertrophy, robustly increased I_to densities, shortened action potential duration, and significantly altered myocyte mechanics by shortening contraction amplitudes and maximal rates of contraction and relaxation velocities and decreasing Ca²⁺ transients. Interestingly, blocking I_to with 4-aminopyridine in KChIP2-overexpressing adult cardiomyocytes significantly increased the Ca²⁺ transients to control levels. One-day-old rat pups intracardially transduced with KChIP2 for two months then subjected to aortic banding for 6–8 weeks (to induce hypertrophy) showed similar echocardiographic, electrical and mechanical remodeling parameters. In addition, in cultured adult cardiomyocytes, KChIP2 overexpression increased the expression of Ca²⁺-ATPase (SERCA2a) and sodium calcium exchanger but had no effect on ryanodine receptor 2 or phospholamban expression. In neonatal myocytes, KChIP2 notably reversed Ang II-induced hypertrophic changes in protein synthesis and MAP-kinase activation. It also significantly decreased calcineurin expression, NFATc1 expression and nuclear translocation and its downstream target, MCiP1.4. Altogether, these data show that KChIP2 can attenuate cardiac hypertrophy possibly through modulation of intracellular calcium concentration and calcineurin/NFAT pathway.