Purkinje Origin of Ischemic Ventricular Tachycardia and Fibrillation

描述(由申请人提供)： 本项目的目的是继续对犬急性心肌缺血模型进行机制研究。这是一个非常稳定的诱导性室性心动过速(VT)和室颤(VF)模型；一半的实验是局灶性心内膜或浦肯野室速，另一半是心外膜折返。从心内膜病灶切除的组织在体外进行研究，起搏方案产生基于细胞钙的振荡，称为延迟后除极(DADS)，导致一种称为触发活动(TA)的心律失常。临床实验室通过记录急性心肌缺血患者室速和室颤起始处的心内膜信号，证实了局灶性室速。此外，点源心内膜消融术可以预防局灶性室速/室颤。因此，该模型是一种与人类缺血性室速相关的模型，它可能是局灶性的，也可能是折返性的。上一次资助期间的实验表明，局灶性心内膜室速也可以通过预防TA的药物来预防，包括HMG co-A还原酶抑制剂(他汀类药物)和PD123319(血管紧张素AT-2受体拮抗剂)。此外，他汀类药物的作用可能是由于抑制了活性氧物种(ROS)，因为一种名为TEMPO的ROS清除剂以类似的方式阻断VT和TA，同时减少组织中ROS的测量。ROS还可能导致氧化钙钙调蛋白激酶II(CaMKII)，这是VT/VF的一个基本因素，包括在小动物和大动物模型中的折返。总的目的是验证这一假说，即ROS与氧化的CaMKII相关联是导致临床相关的VT/VF犬模型中局灶性和折返性VT/VF的基本机制。调查集中在两个具体目标上。目的1：确定VT/VF局部是否存在最高水平的缺血组织ROS，以及是否通过清除剂Temol或通过抑制促进ROS的各种酶系统(包括NADH氧化酶、线粒体或黄嘌呤氧化酶)来降低ROS更有效地预防VT/VF。目的2：确定VT/VF局部是否存在最高水平的CaMKII氧化活性，以及KN-93或KN-92抑制CaMKII是否可以预防缺血局灶性VT/VF。在活体计算机激活标测的多个跨壁双极心电图结合浦肯野信号，从放置在冠状动脉前降支危险区域的16极针允许三维标测冠状动脉闭塞后额外刺激产生的VT/VF。用于阻断VT/VF的药物包括随机注射TEMPO(30 mg/kg)、罗布宁(4 mg/kg)、二苯碘(7.6 mg/kg)、鱼藤酮(9.5 mg/kg)、氧嘌呤(3.7 mg/kg)、KN-93(0.2 mg/kg)、KN-92(0.2 g/kg)。在激活标测后，VTVF机制起始处的组织将被切除以进行ROS、CaMKII、RyR2、磷蛋白、SERCA2a和NCX的测量和体外微电极测试。这些研究所需的所有技术都在进行这项初步工作的实验室中掌握。这些研究将利用菲舍尔精确检验和方差分析进行定量数据。标准的微电极技术和组织检测将实现这些目标。最终目标将是开发新的有效疗法，以预防冠心病患者，特别是退伍军人的缺血性室速/室颤。

DESCRIPTION (provided by applicant): The aim of this project is to continue mechanistic investigation of a canine model of acute myocardial ischemia. This is a remarkably stable model of inducible ventricular tachycardia (VT) and ventricular fibrillation (VF); focal endocardial or Purkinje VT results in half of the experiments and epicardial reentry in the other. Tissue excised from the endocardial focus is studied in vitro with pacing protocols generating cellular calcium based oscillations termed delayed afterdepolarization (DADs) giving rise to an arrhythmia called triggered activity (TA). Clinical laboratories have confirmed focal VT by recording endocardial signals at the origin of VT and VF in patients with acute myocardial ischemia. Moreover, prevention of focal VT/VF with point source endocardial ablation has been proven. Thus this model is a relevant model for human ischemic VT, which may be either focal or reentry. Experiments from the prior funding period have shown that focal endocardial VT is prevented with agents which also prevent TA, including HMG co-A reductase inhibitors (statins) and PD123319 (angiotensin AT-2 receptor antagonist.) Furthermore the effect of statins may result from inhibition of reactive oxygen species (ROS) since a ROS scavenger called TEMPO blocks both VT and TA in a similar manner while decreasing tissue measurements of ROS. ROS may also result in oxidized calcium calmodulin kinase II (CaMKII), a fundamental contributor to VT/VF including reentry in small and large animal models. The overall aim is to test the hypothesis that ROS linked to oxidized CaMKII are basic mechanisms resulting in focal and reentrant VT/VF in a clinically relevant canine model of VT/VF. Investigation centers on two specific aims. Aim 1: Determine whether the highest levels of the ischemic tissue ROS occur at foci of VT/VF and whether decreasing ROS by the scavenger TEMPOL or by inhibition of various enzyme systems that promote ROS including NADH oxidase, mitochondrial or xanthine oxidase more effectively prevents VT/VF. Aim 2: Determine whether the highest levels of oxidized CaMKII activity occur at the foci of VT/VF and if CaMKII inhibition by KN-93 or KN-92 can prevent ischemic focal VT/VF. In vivo computerized activation mapping of multiple transmural bipolar electrograms incorporating Purkinje signals taken from 16 pole needles placed in the risk zone of the anterior descending coronary artery allow 3-D maps of VT/VF produced by extra-stimuli after coronary occlusion. Drugs to be infused to block VT/VF include TEMPO (30 mg/kg,) apocynin (4 mg/kg,) diphenyleneiodine (7.6 mg/kg,) rotenone (9.5 mg/kg,) oxipurinol (3.7 mg/kg,) KN-93 (0.2 <g/kg,) KN-92 (0.2 <g/kg) administered randomly. After activation mapping, tissue at the mechanistic origin of VTVF will be excised for measurements of ROS, CaMKII, RyR2, phospholamban, SERCA2a, and NCX and in vitro microelectrode testing. All the techniques required for these studies are in hand in the laboratories where this preliminary work was done. These studies will utilize Fishers exact test and analysis of variance for quantitative data. Standard microelectrode technique and tissue assays will fulfill the aims. The ultimate goal will be to develop new, effective therapies to prevent ischemic VT/VF in patients with coronary disease, particularly in Veterans.