Sex Hormones and Cardiac Arrhythmia in Transgenic LQT2 Rabbits

描述（由申请人提供）：心源性猝死（SCD）每年夺去大约35万美国人的生命。新出现的证据表明SCD遗传易感性的重要作用；然而，分子决定因素仍然难以捉摸。本提案的总体目标是通过应用创新技术和新的长QT综合征2 （LQT2）遗传模型来研究SCD的新机制。这种多管齐下的方法包括研究激素治疗的青春期前卵巢切除的LQT2兔，利用体外和硅实验方法探索性激素对心律失常影响的新机制范式，这些方法将整合新的分子、细胞、组织和理论模型。由于性别和交感神经系统在LQT2中触发SCD起着关键作用，我们将详细研究性激素和自主神经因素对SCD风险的影响。该提案由四个具体目标组成：目标1：利用遥测心电图监测和体内有创电生理研究，表征性激素诱导的心脏复极（QT持续时间和心脏不应期）、自发性TdP发生率和心源性猝死的变化；目的2：利用动作电位和钙瞬态的光学作图分析动作电位持续时间、复极化弥散、传导、触发活动以及电/机械恢复和传导阻滞的异质性。目的3：通过使用蛋白质表达研究、实时定量PCR和细胞电生理研究，分析再极化钾电流、钙电流和控制Ca++循环的蛋白质或亚基编码基因表达的性别或性激素相关差异，定义（基于光学研究）心脏再极化和兴奋中性别差异的分子决定因素；目的4：利用膜片钳和荧光指示剂技术，在对照条件下和自主受体刺激期间，对激素处理的LQT2兔分离的肌细胞的动作电位和钙处理特性进行比较分析。b)使用计算机建模来检查这些LQT2模型中实验定义的细胞/分子改变如何影响模拟心脏组织中的后去极化和致心律失常底物形成。这些研究将有助于理解激素治疗家兔心律失常的触发和维持机制，从而更好地理解长QT综合征中与性别相关的心律失常。公共卫生相关性：心律失常转基因兔模型的特性、性激素的影响，以及编码功能失调离子通道和危及生命的室性心动过速的突变LQTS基因之间关系的鉴定，应该为复极障碍中涉及的遗传和电生理因素提供更完整的知识。这些关于心室复极改变的研究将有助于对伴有缺血性和非缺血性心肌病的获得性心脏疾病以及qt延长药物相关的心源性猝死机制提供重要的新见解。这种知识的增强将导致更有效的策略，以预防广泛的遗传性和获得性心脏疾病中的猝死，并具有重大的公共卫生效益。

DESCRIPTION (provided by applicant): Sudden cardiac death (SCD) claims the lives of approximately 350,000 Americans each year. Emerging evidence indicates an important role for genetic predisposition to SCD; however, the molecular determinants have remained elusive. The overall goal of this proposal is to investigate new mechanisms that underlie SCD through the application of innovative technology and the novel use of new genetic models of long QT syndrome 2 (LQT2). This multi-pronged approach includes the investigation of hormone-treated prepubertal, ovariectomized LQT2 rabbits to explore new mechanistic paradigms that underlie the effects of sex hormones on cardiac arrhythmias using in vitro and in silico experimental approaches that will integrate novel molecular, cellular, tissue, and theoretical models. Because gender and the sympathetic nervous system plays a key role in triggering SCD in LQT2, the influence of sex hormones and the autonomic factors on SCD risk will be studied in detail. The proposal is composed of Four Specific Aims: Aim 1: To characterize of the sex hormone induced changes in cardiac repolarization (QT duration and cardiac refractory periods), incidence of spontaneous TdP and sudden cardiac death with the use of telemetric ECG monitoring and in vivo invasive electrophysiological studies; Aim 2: To analyze the action potential duration, dispersion of repolarization, conduction, triggered activity and the heterogeneities in electrical/mechanical restitution, and conduction block using optical mapping of action potential and calcium transients. Aim 3: To define (based on the optical studies) the molecular determinants of the gender differences in cardiac repolarization and excitation by analyzing the gender or sex hormone related differences in the expression of genes coding for the or subunits of the repolarizing potassium currents, calcium currents, and proteins that control Ca++ cycling, and the adrenergic receptors with the use of protein expression studies and quantitative real-time PCR, and cellular electrophysiological studies; and Aim 4: To perform a comparative analysis of action potential and calcium-handling properties in myocytes isolated from hormone-treated LQT2 rabbits under control conditions and during autonomic receptor stimulation, using patch-clamp and fluorescent-indicator techniques. b) To use computer modeling to examine how experimentally defined cellular/molecular alterations in these LQT2 models influence afterdepolarizations and arrhythmogenic substrate formation in simulated cardiac tissue. These studies will contribute to the understanding of the mechanisms that trigger and maintain arrhythmias in hormone-treated rabbits, and therefore lead to a better understanding of gender-related arrhythmias in long QT syndrome. PUBLIC HEALTH RELEVANCE: The characterization of transgenic rabbit models for cardiac arrhythmias, the effects of sex hormones, and the identification of the relationship between mutant LQTS genes that encode for dysfunctional ion channels and life-threatening ventricular tachyarrhythmias should provide more complete knowledge into the genetic and electrophysiologic factors involved in repolarization disorders. These studies into altered ventricular repolarization should contribute important new insights into sudden cardiac death mechanisms associated with acquired cardiac disorders that accompany ischemic and nonischemic cardiomyopathy and QT-prolonging drugs. This enhanced knowledge should lead to more effective strategies for prevention of sudden death in a broad spectrum of genetic and acquired cardiac disorders with meaningful public health benefits.