急性机械牵张诱发心律失常的发生机制研究

Heart disease is becoming the first killer in the world. In USA, most of the sudden death is due to serious arrhythmias. It is well known that important feed-back mechanisms exist between the mechanical and electrical activity of the heart, modulated by stretch activated ion channels. Commotio cordis is sudden death due to low energy trauma to the chest wall has been described in young sports participants, but the mechanism is unknown. With Commotio cordis, there appears to be a vulnerable window for induction of ventricular fibrillation (15-30ms before the peak of the T-wave in the electrocardiogram). ..Recently, optical mapping of cardiac action potentials (APs) has become important tools for the cardiac electrophysiology. Optical mapping is a non-contact method that can employed at various levels of cardiac structure. At the tissue and whole organ level, optical mapping has been used to gain spatio-temporal information previously unobtainable by other techniques and has provided vital insight into the spread of cardiac excitation and the role played by electrophysiological heterogeneities in the maintenance of normal cardiac rhythm and the initiation and sustenance of arrhythmias. ..In this research, optical mapping is combined with Langendorff perfused rabbit heart to study the mechanism the ventricular fibrillation induction by acute global strain (ventricular volume pulse). Hearts were excitation-contraction uncoupled (10uM Blebbistatin) and subjected to global strain. Membrane pulse was optical mapped (10-20ul of 10mM di-4-ANEPPS; with green (535±12.5nm band-pass filter) LED excitation and 610 nm or 700 nm high pass filter collection, respectively) at 511HZ using a 128×128 16 bit EMCCD camera. ECG was monitored by spring-loaded contact electrodes and intraventricular pressure using a cling film. Gd3+ and Grammostola spatulata mechanotoxin 4 (GsMTx4) are used in this study to clarify the effects of stretch activated channel. ..In conclusion,the mechano-electric feed back is a new research field, also optical mapping is a new technique to clarify the mechanism of the ventricular fibrillation and cardiac heterogeneities. Therefore, this project will help to develop a new anti-arrhythmias medicine and supply a scientific base to terminate serious arrhythmias.

心脏病已经成为危害人类健康的头号杀手。在美国因为心脏病突发而猝死的人中，大多数人死于严重的心律失常。实验和理论都证明，机械电反馈是人体引发心律失常的重要原因。心脏震荡是急性机械牵张引起恶性心律失常的典型例子,目前，关于心室震荡的发生机制并不十分清楚。本项研究是将电压敏感染料膜电位光学标测技术与Langendorff灌流心脏相结合，通过对左心室在心动周期的舒张期和易损期施加压力脉冲的刺激，诱发室颤和期前收缩，用CCD相机在时间和空间上多位点同步记录细胞膜电活动，动态显示膜电位的传导过程包括传导方向、速度及整个心脏的电活动,从而阐明心律失常的发生机制。另外,通过灌流钆离子（Gd3+)和蜘蛛毒肽(GsMTx4)考察机械牵张激活的离子通道在急性牵张引起的心律失常干预作用。膜电位光学标测技术是近年来用于探讨机械性心律失常发生机制的新技术，在国内还处于刚刚起步阶段,因此本项目的开展具有很重要的意义。

心脏收缩活动是电活动结果，反过来，心脏应力对自身电活动有反作用，这种反作用称为机械-电反馈。心脏机械电反馈现象与机械牵张离子通道密切相关而且牵张激活离子通道在诱发心律不齐中有重要作用。本研究采用Langendorff灌流装置对心脏进行离体灌流，在舒张期对心脏左心室进行20%、40%及60%的机械牵张刺激，结果表明期前收缩发生率随着牵张程度加大而增加并且这个期前收缩发生率可以被机械牵张离子通道阻断剂钆离子所抑制，同时Bepridil，作为临床抗心律失常药物，也对该期前收缩率有抑制作用。本项目在离体灌注心脏中同时灌注膜电位染料RH237(激发波长540nm/发射波长585nm)和钙离子敏感指示剂Rhod 2AM（激发波长540nm/发射波长650nm），待2种荧光试剂均匀地分布在心肌表面后，再灌注Blebbistatin,以消除运动伪迹。在一定的波长照射下，用2个CMOS相机记录了正常窦性心律下的心肌表面膜活动电位和钙离子的传播。之后，对心脏进行不同频率电刺激并成功诱发室性心律失常、对心脏进行缺血-再灌注，并记录室性心律失常及缺血-再灌注时活动电位及钙瞬变。本项研究采用光学双标测技术，该方法可以多位点同步记录细胞膜电活动和钙离子在心肌表面传递，动态显示膜电位的传导方向和速度，两种荧光试剂并无交叉影响，在阐明室性心律失常发生机理方面发挥重要作用。本项目心房钠尿肽对心肌缺血再灌注损伤有保护作用，本研究采用H9c2细胞、四甲基罗丹明乙酯（TMRE）荧光染料及激光共聚焦显微镜成像技术测定心肌细胞线粒体膜通透性转换孔开放程度。研究结果表明心房钠尿肽通过调节糖原合成酶激酶3β活性来抑制心肌细胞mPTP的开放，从而保护H9c2心肌细胞的缺血再灌注损伤。本项目在离体家兔心房研究中发现，急性缺氧能够激发分泌内源性内皮素1和心房钠尿肽，内源性内皮素1可通过PI3K/Akt信号通路参与调节缺氧促进心房ANP分泌的过程。