1.Ca～（2+）转运机制—Na～+/Ca～（2+）交换对狗心室肌细胞复极的影响 　2.动作电位参数作为区分狗心室肌心内膜、心外膜和心肌中层M细胞的可行性研究

【作者】 巩燕；

【导师】 何秉贤； Bela Szabo；

【作者基本信息】 新疆医科大学 ， 心血管内科， 2003， 博士

【摘要】 研究背景和目的：人类及哺乳类动物心脏的复极期极为重要，因为它不仅调节兴奋和传导，而且还调节收缩活动。复极的异常往往导致心律失常和/或泵功能异常。可能参与复极的离子流有瞬间外向钾离子流(I_to)，钙离子流(I_Ca)，快钾离子流(I_kr)，慢钾离子流(I_ks)，内向整流离子流（I_kl）)及钠—钙交换电流(I_NCX)等。目前，国内外已有很多有关I_to、I_Ca、I_kl、I_kr、I_ks和心室肌复极关系的研究报道。不同部位的心室肌细胞所含离子流的种类和数量不同，因而表现为不同的电活动特性以及对药物和病理学状态的不同反应（电异质性），所致的透壁性心室肌复极离散（transmural dispersion of repolarization，TDR）是致心律失常性单向阻滞和折返的重要机制。近年来的研究发现同一心肌细胞的动作电位时程（action potential duration，APD）的变异很可能为心律失常的另一机制。推测细胞膜上的钠-钙转运蛋白(Na⁺/Ca²⁺exchange，NCX)可能是影响APD变异的重要因素之一。目前，国外研究病理状态下的NCX电生理特性的报道较多。Studer 1994年首先报道在人类心衰时NCX的mRNA表达上调，在心肌肥厚和心衰的动物模型以及人类心衰终末期NCX的表达和/或活性增强。提示NCX活性改变在心肌肥厚和心衰中的重要性在于其对收缩功能的影响和心电紊乱事件的引发，NCX的非对称性离子转运(3个Na⁺交换1个Ca²⁺)实际上起生电效应，必然影响复极过程。NCX的顺向模式是在细胞松弛时将钙离子转运出细胞外。生理状态下的NCX逆向转运模式在复极中的意义还不甚明确。目前NCX的离子转运模式失调是否为引发心律失常的另一重要机制的研究，在国内外是一个前沿课题。本课题研究的目的在于探讨生理状态下NCX双向模式的离子转换对复极的影响，以及在转换失调时是否成为潜在的触发心律失常的机制。 方祛：单个心室肌细胞试验。动物来源20只成年雄性杂种狗心脏。用Langandorff冠状动脉左前降支（LAD）灌注法，灌注无钙液和低钙胶原酶液分离单个左心室肌细胞。被灌注的组织经切碎，过滤，冲铣后保存在 17℃，含抗菌素的生理液中待用 门～3 6h)。应用细胞膜钳片技术，在钳电流的模式下记录动作电位归ctionpotential，AP卜测定在生理状态下细胞的电生理特征，AP以及收缩的参数。并用统计学方法评价所测量的参数是否将心内膜，心外膜及M细胞有效地区分开。用光感边缘探测器测定细胞的短缩状况以代表细胞的收缩活动。在钳电压的模式下，I讹x应用钙装载程序（Cay一loading protocol）测定。将电压从“0mV钳至＋60my水平，持续600msec以充分激活NCX的逆向模式。进行钙的装载。再将膜电位钳回＊0inV，并持续600iiis盯。此时记录到的电流即INCX。继而将膜电位钳至omV，并持续500msec再将电压钳至＊0mV。膜电位为oniV时记录到的电流为IC。L。 结果：固定频率刺激APD的变化范围和频率改变时APD的变化范围均在一个相对固定的膜电位范围内 卜40～＋10mV)。用10mM EGTA缓冲细胞内游离钙后，APD的变异系数（coefficientvarianility，CV）明显减刁（2．3土0．8～1．3土0．3，pwto，of）。而废除SR功能及应用钙通道阻滞剂nifedipine并不改变APD的变异程度（P＞0．05）。说明 NCX模式转换的随机性造成细胞内游离钙的彼动，而细胞内游离钙的波动是APD变异的关键。另外我们在不同细胞观察到了因刺频率减慢（0．6HZ～1．OHZ）造成复极和细胞松弛的不匹配，从而导致后收缩（After七ontraction，A－CON）的出现 以及 APD的延长（278fsmsec～320igmsec MeanfSD，n—5，50 APs，Pwto．05）。其原因考虑为NCX逆向活性的加强引起的SR内钙的蓄积及钙的自发释放。因此又测定了在强化NCX逆向活性的条件下 （用 40mM Li”替换 40mM Na“以减低细胞外 Na‘浓度），穹隆膜 — —一电位显著降低＊．8士0．3—q 石士1卫mV，Prto刀5)，而APD显著延长＊30土 13～368土 14thSCC，P＜0．05)。结果还显示 10 11Mryanodine和 3 u M thapsigargin废除 SR功能时，细胞收缩幅度明显减J（2．1士0．2～二．2士0．l，P＜0．05）。10nM ISO虽可以增力收缩幅度（＊＊土0．2～3．8土0．4，P＜0．01），却不能增加收缩上升的速度（542士234～3 54士27mVlmsec，Pwto．05）。说明SR作为细胞内的贮钙机制，在其ryano山ne受体激活后，钙大量快速的释放对收缩的重要作用。 IsoproterenolO)模拟神经体液因素作用，本试验以其为工具研究对以上NCX参与的钙转运机制的影响以及诱发心律失常的机制。小剂量ISO＊)的作用下，细胞平均需要60个AP，l刀Hi刺激约1分钟的时间达到新的稳定状态，表现为收缩幅度的增加。但在较大剂量的uO作用下（10、20、50nM）细胞因出现不规律的延迟／早期后除极（delayed／early after－depolerization，DAD／EAD）及A－CON均未达到新的平衡。随ISO剂量的增大，EAD，DAD及A－CO更多还原

【Abstract】 Background and aims: Naturally occurring temporal variability of action potential duration (APD) in isolated myocytes has been noted. Most of the studies have been focusing on analyzes of the differences in ionic channels and currents among the epicardial-, mid-myocardial-(M) and endocardial myocytes, and the rate-dependent (adaptation) characteristics of APD. We have found that the change in APD during a change in frequency of stimulation mostly reflects a change in rate of repolarization at distinct membrane potential levels.We assumed that in the myocytes, there is balancing mechanism, which is constantly adjusting the various ionic currents accommodating to the changing conditions. This intrinsic ability of adaptation is important and may offer some of the consequences of the transmural heterogeneity in adaptation of APD. This adaptive behaviors maybe equally important in maintaining the normal electrophysiological properties and in induction of arrhythmia in a case of error in normal adaptation. Though most studies of Na+/Ca2+ exchange (NCX) has been emphasized on its reverse activaty during pathyological condition. Our hypothesis is that reverse activaty of NCX also plays an important role in adjusting the repolarization of AP during a physiological condition. A mismatch between action potential (AP) repolarization and relaxation of the contraction can be caused by intracellular Ca2+ transport abnormalities. Ca2+ influx via reverse activation of NCX can load the sarcoplasmic recticulum (SR), which has arrhythmogenic effect.Methods: We studied the single myocytes from the heart of adult mongrel dogs. During the cell separation, Ca2+ -free Tyrode, low Ca2+ (60#,M) BS containing collagenase was perfused through LAD by Langandorff system. The tissue perfused by the LAD was then minced into a suspension, filtered , washed and resuspended 5 times in BS. The myocytes were stored in a minimum essential medium with Hank’s salt at 17癈. Penicillin (100,000 u/L) inhibited bacterial growth in the storage medium. We determinded AP in current clamp mode, we verified the origin of each myocyte by examining the characteristics of the APs under baseline conditions. By this method we managed to separate the three types of myocytes very effectively. Myocyte contraction was imaged by a video camera, shortening of unloaded myocytes was detected by a video edgemotion detector, using changes in light intensity at the edges of themyocyte.INcx was recorded in voltage clamp mode and activated by Ca2+loading protocol..Re8uItst From 60 consecutive recorded APs at a constant 1.0 Hzstimulation under steady state conditions we found there is avariance in the repolarization between l0mV and -40mV. We alsofound the variance in the APD during the rate adaptation range ofrepolarization. Fluctuation in the transient may contribute to theAPD variability. To test this hypothesis we block the transient byintracellular dialysis with l0 mM EGTA(n=l9), this caused asignificant reduction in the coeffcient variability (CV=SD/meanAPD%) from 2.3t0.8 to l .3t0.3 p <0.0l. During a rate change ofthe stimulation from 0.6Hz to 1.0Hz. The AP duration increasedfrom 278l8msec to 320f9msec (MeanlSD, n=5, 50 APs, P<0.05),contraction is accompanied by an after-contraction(A-CON). There1axation of contraction precedes the repolarization of the AP. Weassumed that the enhancemcnt of repolarization and the productionof after-contraction can be possibly induced by reverse mode ofNCX. Reducing [Na+l. by substitution of 40mM Na+ with Li+ favorsNCX actiyating the reverse mode, which significantly decreased thedome of the AP from 4.8 i 0.3 to -- 10.6 i l .2mV(P < 0.05) andincreased the APD from 330 I l3 to 368 f l4msec(P < 0.05). Whenthe SR function was abolished by 10 u M ryanodine and 3 ll Mthapsigargin, contraction was significantly decreased (from 2.l l0.2 tol .2 l 0. l, P r 0.05) o 10nM isopretorenol (ISO) can onlyincrease the amplitude of the contraction (from 2.l I 0.2 .to 3 .8 i0.4, P wt 0.0l), but the rate of rise decreased (542 I 234 to 354更多还原