Age-related increase of early afterdepolarization in calsequestrin-2 knock-in mouse cardiomycyte

Objective To characterize early afterdepolarizations （EADs） caused triggered activity （TA） among calsequestrin-2 （CASQ2） knock-in （CASQ2 KI） mice and its relationship with aging. Methods Electrophysiological properties of ventricular myocytes from 3- month （mo, young）, 9-mo （adult-l） and 12-too （adult-2） in wild-type （WT） and CASQ2 KI mice were investigated with patch-clamp technique. Results The incidences of EADs and TA in CASQ2 KI cardiomyocytes increased with increasing age. In contrast, WT mice cardiomyocytes showed no significant change in matched-age groups. Compared with that in 3-mo CASQ2 KI mice, the 50% repolarization of action potential （APD50） showed prolongation in both 9-mo and 12-mo ones （9.2±0.9 ms of 9-mo and 10.3 ± 1.2 ms of 12- mo vs. 5.6± 0.3 ms of 3-mo）, while the 90 % repolarization of action potential （APD90） was similar among 3 age groups. Compared with 3-mo mice, the 9-mo and 12-mo CASQ2 KI mice showed markedly reduced transient outward potassium current （Ito） densities but increased L-type calcium current （ICa-L） densities. Conlcusion This study suggested that events of EADs and TA in CASQ2 KI mice increased with increasing age, It might be associated partly with the augment of cellular calcium concentration and the prolongation of APD50 induced by decrease of Ito and increase of ICa-L in adult CASQ2 KI mice     

Effect of pacing and mexiletine on dispersion of repolarisation and arrhythmias in DeltaKPQ SCN5A (long QT3) mice

OBJECTIVE: It has been suggested that both pacing and treatment with mexiletine may reduce torsade de pointes (TdP) arrhythmias in patients with long QT syndrome 3 (LQT3), but it is not fully understood how these interventions could prevent TdP. We therefore studied the effects of pacing and mexiletine in mice with a heterozygous knock-in DeltaKPQ SCN5A(Delta/+) deletion (SCN5A-Tg), a murine LQT3 model. METHODS: Three right and left ventricular monophasic action potentials (MAPs) were simultaneously recorded in Langendorff-perfused hearts of SCN5A-Tg and wild type (WT) littermates. AV block was induced, and pacing was performed at baseline and during mexiletine infusion (4 microg/ml). MAP recordings were analysed for action potential duration (APD), APD dispersion, and early afterdepolarisations (EADs) and related to spontaneous arrhythmias. RESULTS: After inducing AV block, SCN5A-Tg hearts were bradycardic [SCN5A-Tg 532+/-60 vs. WT 284+/-48 ms cycle length (CL, mean+/-S.E.M., P<0.05(*))]. EADs occurred in 16/18, and polymorphic ventricular tachycardia (pVT) in 11/18 SCN5A-Tg but not in 19 WT. SCN5A-Tg had longer APD than WT hearts*. At CL of 200 ms and longer, APD dispersion was higher in SCN5A-Tg [dispersion (APD70): 12+/-3 ms vs. 5+/-2 ms at CL=200 ms*], and increased to 35+/-4 ms* directly prior to pVT episodes. Sudden rate accelerations initially increased APD dispersion due to EADs and APD alternans in SCN5A-Tg, but pacing then reduced APD dispersion. Pacing suppressed (n=9/9) and prevented (n=49/50) pVT. Mexiletine shortened APD at long CL*, and suppressed pVT (n=4/5*), but did not prevent pVT during normal rhythm. CONCLUSIONS: Bradycardia, increased dispersion of APD and EADs provoke ventricular ectopy and pVT in SCN5A-Tg hearts. Ventricular pacing reduces APD dispersion, suppresses EADs and prevents pVT in SCN5A-Tg hearts. These effects provide a pathophysiological rationale for pacing in LQT3.     

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）.     

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。     

新生大鼠心肌细胞短暂外向钾电流下降与心肌肥大的相关性研究

在某些心力衰竭患者中常观察到心肌细胞短暂外向钾电流 (Ito)下降和动作电位时程 (APD)延长 ,笔者主要探讨Ito下降与心肌肥大的关系及内在机制。用Ito阻断剂 ,4 氨基吡啶 (4 AP) ,处理新生大鼠心室肌细胞 ,观察作为心肌肥大指标的细胞膜电容和3 H 亮氨酸 (3 H Leu)掺入量 ,同时测Ito振幅和APD。结果 :Ito振幅下降近 5 0 %(1 5 0 .3± 1 8.6pA ,n =7vs 74 .0± 1 1 .5pA ,n =1 1 ,P <0 .0 5 )。APD50 (5 0 %复极 )显著的延长 (75 .8± 1 4 .1ms,n =7vs2 0 1 .7± 2 3.5ms,n =1 1 ,P <0 .0 5 )。膜电容和3 H Leu掺入量分别增加 4 7%和 31 % (P均 <0 .0 5 )。L 型钙通道阻断剂维拉帕米 ,能抑制 4 AP诱导的APD延长以及膜电容和3 H 亮氨酸掺入量的增加。环孢素A(CsA)也可抑制 4 AP诱导的膜电容和3 H Leu掺入量的增加 ,但对APD影响不明显。结论 :Ito下降通过延长APD ,致细胞内钙增加 ,激活钙调神经磷酸酶反应途径 ,可能引起心肌肥大     

Pause induced early afterdepolarizations in the long QT syndrome: a simulation study.

OBJECTIVE: The long QT syndrome (LQTS) is characterized by prolonged repolarization and propensity to syncope and sudden death due to polymorphic ventricular tachycardias such as torsade de pointes (TdP). The exact mechanism of TdP is unclear, but pause-induced early afterdepolarizations (EADs) have been implicated in its initiation. In this study we investigate the mechanism of pause-induced EADs following pacing at clinically relevant rates and characterize the sensitivity of different cell types (epicardial, midmyocardial, and endocardial) to EAD development. METHODS: Simulations were conducted using the Luo-Rudy (LRd) model of the mamalian ventricular action potential (AP). Three cell types--epicardial, midmyocardial (M), and enocardial--are represented by altering the channel density of the slow delayed rectifier current, IKs. LQTS is modelled by enhanced late sodium current (LQT3), or reduced density of functional channels that conduct IKr (LQT2) and IKs (LQT1). The cell is paced 40 times at a constant Basic Cycle Length (BCL) of 500 ms. Following a 1500 ms pause, an additional single stimulus is applied. RESULTS: Our results demonstrate that pause-induced EADs develop preferentially in M cells under conditions of prolonged repolarization. These EADs develop at plateau potentials ('plateau EADs'). Mechanistic investigation shows that prolongation of the plateau phase of the post-pause AP due to a smaller delayed rectifier potassium current, IKs' and enhancement of the sodium-calcium exchange current, INaCa, allows for the reactivation of the L-type calcium current, ICa(L), which depolarizes the membrane to generate the EAD. CONCLUSIONS: APD is a very important determinant of arrhythmogenesis and its prolongation, either due to acquired or congenital LQTS, can result in the appearance of EADs. The formation of pause-induced EADs preferentially in M cells suggests a possible role for these cells in the generation of arrhythmias that are associated with abnormalities of repolarization (e.g., TdP). The ionic mechanism of pause-induced EADs involves reactivation of the L-type calcium current during the prolonged plateau of the post-pause AP.     

L-type calcium current recovery versus ventricular repolarization: preserved membrane-stabilizing mechanism for different QT intervals across species

BACKGROUND: Long QT syndrome is associated with early after-depolarization (EAD) that may result in torsade de pointes (TdP). Interestingly, the corrected QT interval seems to be proportional to body mass across species under physiologic conditions. OBJECTIVE: The purpose of this study was to test whether recovery of L-type calcium current (I(Ca,L)), the primary charge carrier for EADs, from its inactivated state matches ventricular repolarization time and whether impairment of this relationship leads to development of EAD and TdP. METHODS: Transmembrane action potentials from the epicardium, endocardium, or subendocardium were recorded simultaneously with a transmural ECG in arterially perfused left ventricular wedges isolated from cow, dog, rabbit, and guinea pig hearts. I(Ca,L) recovery was examined using action potential stimulation in isolated left ventricular myocytes. RESULTS: The ventricular repolarization time (action potential duration at 90% repolarization [APD(90)]), ranging from 194.7 +/- 1.8 ms in guinea pig to 370.2 +/- 9.9 ms in cows, was linearly related to the thickness of the left ventricular wall among the species studied. The time constants (tau) of I(Ca,L) recovery were proportional to APD(90), making the ratios of tau to APD(90) fall into a relatively narrow range among these species despite markedly different ventricular repolarization time. Drugs with risk for TdP in humans were shown to impair this intrinsic balance by either prolongation of the repolarization time and/or acceleration of I(Ca,L) recovery, leading to the appearance of EADs capable of initiating TdP. CONCLUSION: An adequate balance between I(Ca,L) recovery and ventricular repolarization serves as a "physiologic stabilizer" of ventricular action potentials in repolarization phases.     

Reduced repolarization reserve in ventricular myocytes from female mice

OBJECTIVE: Cardiac repolarization is prolonged and repolarization reserve (RR) is diminished in female rabbits and humans, compared to males. Reduced RR is evidenced by the relatively greater increase in ventricular action potential duration (APD) in myocytes from females in response to drugs that block repolarizing K(+) currents. Mice are an increasingly important experimental model animal for cardiovascular research, but gender-dependent differences have not been reported for repolarization in murine ventricular myocytes. METHODS: APD and repolarizing K(+) currents were measured in isolated ventricular myocytes from adult littermate male and female mice. Repolarizing K(+) currents were dissected into transient (I(to)) and sustained (I(sus)) components and the selective I(sus) antagonist FK506 was used to probe for differences in RR. RESULTS: Under control conditions APD at 50% (APD(50)) and at 90% (APD(90)) repolarization was significantly longer in females (APD(50)=15 +/- 3 ms, n=6 and APD(90)=63 +/- 6 ms, n=6) compared to males (APD(50)=8 +/- 2 ms, n=7 and APD(90)=42 +/- 9 ms, n=7) at 1.0 Hz. At 0.3 Hz stimulation frequency APD(90), but not APD(50), was significantly longer in females (APD(50)=12 +/- 2 ms and APD(90)=54 +/- 5 ms, n=10) compared to males (APD(50)=11 =/- 2 ms and APD(90)=47 +/- 7 ms, n=10). FK506 treatment (25 microM) selectively and equally inhibited I(sus) in all cells, and significantly increased APD(50) and APD(90) in males and females at 0.3 and 1.0 Hz. However, increases in APD(50) and APD(90) (0.3 and 1.0 Hz) in response to FK506 were significantly greater in myocytes from females compared to males. Voltage clamp measurement of I(to) and I(sus) revealed that males had a relatively more prominent I(to) while females exhibited a more prominent I(sus). CONCLUSIONS: Ventricular action potential repolarization is prolonged in myocytes from female compared to male mice. Female mice have reduced RR that is unmasked by FK506. These findings suggest that gender is an important variable for cardiovascular studies using mice.     

Lidocaine and Nisoldipine Attenuate Almokalant-Induced Dispersion of Repolarization and Early Afterdepolarizations In Vitro

Attenuation of Almokalant-Induced Proarrhythmias In Vitro. Introduction: Treatment with Class III antiarrhythmic agents may lead to increased dispersion or repolarization and early afterdepolarizations (EADs), which are both likely substrates for torsades de pointes. Recent studies in vivo have shown that the prevalence of proarrhythmias induced by Class III agents may be reduced by Na⁺ or Ca²⁺-blocking agents. In the present study, tentative mechanisms for this protective effect were investigated in vitro. Methods and Results: Transmembrane action potentials were recorded simultaneously from rabbit isolated ventricular muscle (VM) and Purkinje fibers (PF). At a basic cycle length (BCL) of 500 msec, the Class III agent almokalant (0.1 μM) increased the dispersion by prolonging the action potential duration (APD) significantly more in the PF (33%± 4.2%, n = 18) than in the VM (17%± 5.9%, n = 18. P < 0.05). In six of the preparations, addition of 1, 5, and 25 μM lidocaine reduced the almokalant-induced prolongation in a concentration-dependent manner mainly in the PF, thereby decreasing the dispersion. At 5 μM lidocaine, the remaining prolongation was 7%± 12.2% (P < 0.05 vs time controls) in the PF and 14%± 6.4% in the VM, respectively. In six other preparations, the addition of 0.01, 0.05, and 0.25 μM nisoldipine did not reduce the almokalant-induced prolongation in the PF and VM, hut attenuated the spike-and-dome appearance of the action potential in the PF. In separate experiments performed at a BCL of 1000 msec, EADs developed in 2 of 6 and 5 of 6 PF during superfusion with almokalant (0.3 and 1 μM, respectively) at an API) of 828 ± 41.4 msec. In six separate preparations pretreated with lidocaine (5 μ) the almokalant-induced prolongation in the PF was less pronounced and EADs were not observed. Pretreatment with nisoldipine (0.05 μM) did not influence the response to almokalant, and in 4 of 6 preparations the APD exceeded 1000 msec. Despite this extensive prolongation, EADs did not appear. Conclusion: At concentrations that did not affect the APD in the VM hut reduced the APD in the PE. lidocaine suppressed almokalant-induced dispersion and the development of EADs. Nisoldipine, (m the other hand, inhibited almokalant-induced EADs directly. Hence, (he primary APD-prolonging effect of a Class III agent may he preserved, but the risk of proarrhythmary reduced, during concomitant treatment with low concentrations of a Na⁺- or Ca²⁺ blocking agent.     

Prominent I(Ks) in epicardium and endocardium contributes to development of transmural dispersion of repolarization but protects against development of early afterdepolarizations

INTRODUCTION: Previous studies from our laboratory demonstrated (1) a much larger I(Ks) and (2) inability to induce early afterdepolarization (EAD) activity in epicardial and endocardial cells versus M cells. This study tests the hypothesis that these two characteristics are interrelated. METHODS AND RESULTS: Standard and floating microelectrode techniques were used to record transmembrane activity from the canine left ventricular epicardial, M, and endocardial regions in isolated tissue slices and arterially perfused wedge preparations. The I(Kr) blocker E-4031 (1 to 10 microM) caused prominent prolongation of action potential duration (APD) and induced EADs in tissues isolated from the M region, but not those from epicardium or endocardium, causing a large transmural dispersion of APD. In contrast, the I(Ks) blocker chromanol 293B (10 to 30 microM) produced moderate prolongation of APD without EADs in all three tissue types. The combination of E-4031 (1 microM) and chromanol 293B (30 microM) resulted in profound prolongation of APD and the development of EADs in all three tissue types. In the perfused wedge, neither E-4031 nor chromanol 293B alone could induce EADs. In combination, the two drugs caused significant prolongation of APD and EADs in all three transmural regions. CONCLUSION: Our results support the hypothesis that a prominent I(Ks) is responsible for the ability of epicardium and endocardium to resist some but not all of the arrhythmogenic effects of I(Kr) block. The data highlight the critical importance of I(Ks) in the canine heart and the significant role of electrotonic interactions in minimizing the development of an arrhythmogenic substrate when repolarization reserve is reduced.     

Developmental changes in IKr and IKs contribute to age-related expression of dofetilide effects on repolarization and proarrhythmia

OBJECTIVE: Clinical and experimental studies suggest that immature hearts are as or more sensitive than adult hearts to adverse effects of I(Kr) blocking drugs. We hypothesized that age-dependent changes in I(Kr) and I(Ks) contribute to the different repolarization reserves and proarrhythmic effects of I(Kr) blockers in the young and adult heart. METHODS: Dogs aged 1-150 days and adults were used to study (1) proarrhythmic effects in situ of the I(Kr) blocker dofetilide; (2) dofetilide effects on action potential duration (APD) recorded with microelectrodes from left ventricular (LV) slabs; (3) I(Kr) and I(Ks) in single LV myocytes using whole-cell voltage clamp. RESULTS: In situ, dofetilide-induced proarrhythmia occurred in 40% of adults, 86% of young (20-150 day) dogs and 0% of neonatal (1-19 day) dogs (P<0.05). Isolated tissue experiments showed no transmural gradient for repolarization from neonate through 3 months of age, after which the gradient increased through adulthood. In the presence of dofetilide, the greatest APD prolongation occurred in neonates. Yet, transmural dispersion did not increase in neonates but significantly increased in young and adults. Dofetilide-induced early after depolarization (EAD) incidence was 23% in adults, 59% in young and 8% in neonates (P<0.05). I(Kr) but not I(Ks) was expressed at <30 days, whereas both currents were present in adult myocardium. CONCLUSIONS: Our data suggest that a lack of I(Ks) results in a greater dependence on I(Kr) for repolarization in neonates and is associated with exaggerated effects of I(Kr)-blockade on APD. However, APD prolongation alone is insufficient for expression of proarrhythmia, which also requires transmural dispersion of repolarization and EADs. The extent to which APD prolongation, transmural dispersion and EADs are manifested at various ages in the absence and presence of I(Kr) blocking drugs appears to be the ultimate determinant of proarrhythmia.     

LQT2模型室性心律失常电生理机制研究

为探讨心室肌跨壁复极离散度 (TDR)和心脏兴奋的恢复性质在长QT综合征 (LQTS)室性心律失常发生过程中的作用 ,应用冠状小动脉灌流的兔左室肌楔形组织块标本 ,分模型组和对照组 ,采用浮置玻璃微电极法同步记录心室肌内、外膜心肌细胞动作电位和跨壁心电图。模型组以 30 μmol/L的d sotalol台氏液灌流 ,制备LQT2模型。对照组以标准台氏液灌流。结果 :模型组和对照组比较TDR有显著性差异 (83.6± 14 .0msvs 4 8.6± 5 .3ms,P <0 .0 1,n =10 )。模型组内、外膜动作电位时程 (APD)恢复曲线最大斜率均大于 1,而对照组均小于 1,两组间APD恢复曲线最大斜率比较有显著性差异 (P <0 .0 1,n =2 0 )。模型组在S1S2 程序刺激下尖端扭转型室性心动过速的发生率为70 %。对照组无 1例发生室性心律失常。结论 :心脏兴奋的恢复性质和心室肌TDR均参与了LQT2室性心律失常的发生。     

Two-Dimensional Vector Analysis of Beat-to-Beat Dynamics of Ventricular Repolarization

Objective: Duration of ventricular repolarization is a result of complex interaction between autonomic modulation and heart rate (HR). Methods: To study the dynamics of ventricular repolarization of the human right ventricle, beat-to-beat variability of ventricular repolarization was measured by plotting the duration of each monophasic action potential at the 90% phase of repolarization (APD₉₀) as a function of the previous APD90 both in sinus rhythm and during steady-state atrial pacing (cycle length of 600 rns) in 12 subjects without structural heart disease. Results: Quantitative analyses of APD₉₀ and RR interval variability in sinus rhythm showed that the total standard deviation (SD) of APD90 was only 8% of the SD of all RR intervals. Both longterm, continuous variability (SD2) of APD90 (3.9 ± 1.5 ms) and instantaneous beat-to-beat variability (SD1) of APD₉₀ (1.2 ± 0.3 ms) were smaller than the SD2 and SD1 of RR intervals (46 ± 17 ms and 15 ± 9 ms, respectively) (P < 0.001 for both), but the shapes of the APD₉₀ and RR interval plots and the SD1/SD2 ratio did not differ. SD1 of APD90 correlated well with the SD1 of RR intervals (r = 0.64, P < 0.05) but no significant correlation was observed between the SD2 of APD₉₀ and SD2 of RR intervals (r = 0.32, NS). During steady-state atrial pacing, only minimal instantaneous beat-to-beat variability in APD₉₀ (0.9 ± 0.3 rns) was observed but the SD2 was larger than the SD2 of RR intervals (2.3 ± 1.0 ms vs. 1.6 ± 0.8 ms, P < 0.001). Conclusion: The results suggest that instantaneous beat-to-beat variability of ventricular repolarization is mainly due to beat-to-beat fluctuation of HR, but the long-term dynamics of repolarization are partly influenced by other factors than the HR variability.     

Block of I(Ks) does not induce early afterdepolarization activity but promotes beta-adrenergic agonist-induced delayed afterdepolarization activity

INTRODUCTION: An early afterdepolarization (EAD)-induced triggered beat is thought to precipitate torsade de pointes (TdP) in the long QT syndrome (LQTS). Previous studies demonstrated the development of EAD activity and dispersion of repolarization under LQT2 (reduced I(Kr)) and LQT3 (augmented late I(Na)), but not LQT1 (reduced I(Ks)), conditions. The present study examines these electrophysiologic characteristics during I(Ks) block. METHODS AND RESULTS: Canine epicardial (Epi), M, and endocardial (Endo) tissues and Purkinje fibers isolated from the canine left ventricle were studied using standard microelectrode recording techniques. The I(Ks) blocker chromanol 293B (293B, 30 microM), produced a homogeneous rate-independent prolongation of action potential duration (APD) in Epi, M, and Endo, but little to no APD prolongation in Purkinje. Chromanol 293B 1 to 30 microM failed to induce EADs or delayed afterdepolarizations (DADs) in any of the four tissue types. Isoproterenol (ISO, 0.1 to 1.0 microM) in the presence of 293B 30 microM significantly prolonged the APD of the M cell (basic cycle length > or = 1 sec), abbreviated that of Purkinje, and caused little change in that of Epi and Endo. The combination of 293B 30 microM and ISO 0.2 microM did not induce EADs in any of the four tissue types, but produced DAD activity in 4 of 8 Epi, 7 of 10 M cells, and 3 of 8 Endo. CONCLUSION: Our results indicate that I(Ks) block alone or in combination with beta-adrenergic stimulation does not induce EADs in any of the four canine ventricular tissue types, but that the combination of the two induces DADs as well as accentuated dispersion of repolarization.     

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.     

Species plays an important role in drug-induced prolongation of action potential duration and early afterdepolarizations in isolated Purkinje fibers

INTRODUCTION: Although isolated Purkinje fibers (PFs) often are used to evaluate the electrophysiologic effects of new drugs in terms of prolongation of action potential duration (APD) and induction of early afterdepolarizations (EADs), species differences in this respect remain elusive. We evaluated potential species-specific differences in drug-induced prolongation of APD and EADs in isolated PF from various species. METHODS AND RESULTS: Using a microelectrode technique, PFs (n = 7 to 11 per species) were isolated from hearts of rabbits, guinea pigs, dogs, swine, goats, or sheep, superperfused in Tyrode's solution with dofetilide (1 x 10(-8) M) or quinidine (1 x 10(-5) M) for 25 minutes, and stimulated at 1 Hz for 20 minutes and at 0.2 Hz for another 5 minutes. Dofetilide increased APD at 90% repolarization (APD90) at 1 Hz by 83% (rabbit), 24% (guinea pig), 65% (dogs), 18% (swine), 61% (goat), and 30% (sheep), and prolonged APD90 at 0.2 Hz by 187% (rabbit), 31% (guinea pig), 154% (dog), 17% (swine), 61% (goat), and 8% (sheep). Similarly, quinidine changed APD90 by 93% (rabbit), 0% (guinea pig), 16% (dog), -3% (swine), 0% (goat), and -24% (sheep) at 1 Hz, and by 124% (rabbit), 15% (guinea pig), 53% (dog), 17% (swine), 11% (goat), and -39% (sheep) at 0.2 Hz in PF. During superfusion of dofetilide or quinidine, EADs occurred in most preparations in rabbit PFs at 0.2 Hz, but not in any of the PFs from other species at 0.2 Hz. CONCLUSION: Our study demonstrates that species plays an important role in the response of PF to drug-induced prolongation of APD and EADs. Rabbit PFs constitute the most sensitive model for detecting drug-induced, potential long APD and proarrhythmogenic effects in vitro.     

Calmodulin antagonist W-7 prevents sparfloxacin-induced early afterdepolarizations (EADs) in isolated rabbit purkinje fibers: importance of beat-to-beat instability of the repolarization

INTRODUCTION: The occurrence of early afterdepolarizations (EADs) has been related to the incidence of torsades de pointes in drug-induced long QT (LQT). The generation of EADs may be facilitated by Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase). METHODS AND RESULTS: In the present study, we investigated a possible involvement of Ca(2+)/Calmodulin dependent protein kinase in the generation of sparfloxacin-induced EADs in isolated rabbit Purkinje fibers by means of a calmodulin antagonist W-7. EADs were evident in 8 of the 10 preparations perfused with sparfloxacin at 1 x 10(-4) M and stimulated at 0.2 Hz. The induction of EADs by sparfloxacin was associated with a large prolongation of the duration of the action potential (APD), an increase in the triangulation, and the short-term instability of the repolarization. CaM kinase blockade with the calmodulin antagonist W-7 inhibited sparfloxacin-induced EADs in a concentration-dependent manner (EADs were induced in 3 of 10, 1 of 10, and 0 of 8 preparations in the presence of W-7 at 5 x 10(-7) M, 5 x 10(-6) M, and 5 x 10(-5) M, respectively; P < 0.01 at 5 x 10(-6) M and 5 x 10(-5) M). The inhibition of sparfloxacin-induced EADs by W-7 at 5 x 10(-7) M and 5 x 10(-6) M was associated with a significant decrease in the beat-to-beat instability but not associated with a significant shortening of the APD and reduction of V(max). CONCLUSION: The present findings support the hypothesis that CaM kinase may be a proarrhythmic signaling molecule and demonstrate that CaM kinase may be involved in the generation of EADs in drug-induced LQT and enhanced beat-to-beat instability of repolarization is essential for the genesis of EADs in rabbit in vitro.     

Effects of Alpha Adrenergic Stimulation and Blockade on Early Afterdepolarizations Induced by Cesium in Canine Cardiac Purkinje Fibers

Alpha Adrenergic Stimulation and EADs. The effects of alpha adrenergic stimulation and three alpha adrenoceptor blockers on early afterdepolarizations (EADs) were examined in canine card diac Purkinje fibers. In the first group of 18 preparations, EADs were induced by superfusion with 7.5 mM cesium (Cs) dissolved in low potassium (2.7 mM KCl) Tyrode's solution. During alpha adrenoceptor stimulation (norepinephrine 1 μM and propranolol 1 μM) to enhance EADs, the effects of phentotamine and two new alpha 1 adrenoceptor antagonists, benoxathian and WB 4101 (1, 3, and 10 μM), were examined. WB 4101 (1 μM) suppressed EADs in all six preparations. Benoxathian required 3 μM in five preparations and 10 μM in one to suppress EADs. Phentolamine (10 μM) suppressed EADs in two of six preparations. In the second group of 21 preparations, the effects on cesium-induced EADs of the three alpha adrenoceptor hlockers (10 μM) were examined without alpha adrenoceptor stimulation. WB 4101 (10 μM) suppressed EADs in seven of seven preparations, while benoxathian (10 μM) suppressed EADs in five of seven. Phentolamine (10 μM) enhanced EADs in six of seven preparations. In the third group of 24 Purkinje fibers, the direct effects (without alpha adrenoceptor stimulation) of these three antagonists on the characteristics of normal Purkinje fiber action potentials superfused with normal Tyrode's solution were examined. Phentolamine (1, 3, and 10 μM, n = 8) prolonged action potential duration at 90% repolarization (APD90) by 5.3%± 1.3%, 10.0%± 1.8%, and 14.3%± 2.7%, respectively. Benoxathian (n = 8) and WB 4101 (n = 8) shortened APD90 equally: 6.8%± 1.1% vs 7.7%± 1.3% at 1 μM, 13.6%± 1.0% vs 14.5%± 1.6% at 3 μM, and 21.1%± 1.2% vs 20.8%± 2.1% at 10 μM, respectively. We conclude that in cesium treated Purkinje fibers: (1) Alpha adrenoceptor stimulation enhanced EADs; (2) Phentolamine was least effective in suppressing EADs, probably because of a direct effect that prolonged MM)90,: and (3) Although benoxathian and WB 4101 had similar etTects on APD90, WB 4101 was more effective in suppressing EADs at lower concentrations than henoxathian.     

Abnormal cardiac Na(+) channel properties and QT heart rate adaptation in neonatal ankyrin(B) knockout mice

The cytoskeleton of the cardiomyocyte has been shown to modulate ion channel function. Cytoskeletal disruption in vitro alters Na(+) channel kinetics, producing a late Na(+) current that can prolong repolarization. This study describes the properties of the cardiac Na(+) channel and cardiac repolarization in neonatal mice lacking ankyrin(B), a cytoskeletal "adaptor" protein. Using whole-cell voltage clamp techniques, I(Na) density was lower in ankyrin(B)(-/-) ventricular myocytes than in wild-type (WT) myocytes (-307+/-26 versus -444+/-39 pA/pF, P<0.01). Ankyrin(B)(-/-) myocytes exhibited a hyperpolarizing shift in activation and inactivation kinetics compared with WT. Slower recovery from inactivation contributed to the negative shift in steady-state inactivation in ankyrin(B)(-/-). Single Na(+) channel mean open time was longer in ankyrin(B)(-/-) versus WT at test potentials (V(t)) of -40 mV (1.0+/-0.1 versus 0. 61+/-0.04 ms, P<0.05) and -50 mV (0.8+/-0.1 versus 0.39+/-0.05 ms, P<0.05). Ankyrin(B)(-/-) exhibited late single-channel openings at V(t) -40 and -50 mV, which were not seen in WT. Late I(Na) contributed to longer action potential durations measured at 90% repolarization (APD(90)) at 1 Hz stimulation in ankyrin(B)(-/-) compared with WT (354+/-26 versus 274+/-22 ms, P<0.05). From ECG recordings of neonatal mice, heart rates were slower in ankyrin(B)(-/-) than in WT (380+/-14 versus 434+/-13 bpm, P<0.01). Although the QT interval was similar in ankyrin(B)(-/-) and WT at physiological heart rates, QT-interval prolongation in response to heart rate deceleration was greater in ankyrin(B)(-/-). In conclusion, Na(+) channels in ankyrin(B)(-/-) display reduced I(Na) density and abnormal kinetics at the whole-cell and single-channel level that contribute to prolonged APD(90) and abnormal QT-rate adaptation.     

氯沙坦调节瞬间外向钾电流的分子学研究

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