EFFECT OF R1R2 OVER-EXPRESSION ON CARDIAC FUNCTION

R1R2 过度表达对心脏功能的影响

• 批准号: 8708949
• 负责人: MICHAEL REGNIER
• 金额: $ 57.26万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8708949
• 关键词: ATP Synthesis Pathway; ATP phosphohydrolase; Actins; Action Potentials; Acute; Adenine Nucleotides; Adenovirus Vector; Adrenergic Agents; Adult; Affect; Age; Animal Model; Animals; Anterior Descending Coronary Artery; Area; Behavior; Binding; Cardiac; Cardiac Myocytes; Chemosensitization; Chronic; Collaborations; Computer Simulation; Coronary Occlusions; Data; Depressed mood; Development; Dobutamine; Dose; Echocardiography; Effectiveness; Electrophysiology (science); Energy Metabolism; Exploratory/Developmental Grant for Diagnostic Cancer Imaging; Funding; Gene Delivery; Goals; Heart; Heart failure; Human; In Vitro; Infarction; Injection of therapeutic agent; Kinetics; Left; Left Ventricular Function; Ligation; Lung; Measurement; Measures; Mechanics; Mediating; Membrane Proteins; Metabolic; Metabolism; Microfilaments; Mitochondria; Modeling; Modification; Molecular; Mus; Myocardial; Myocardial Infarction; Myocardium; Myofibrils; Myosin ATPase; NMR Spectroscopy; Oxygen Consumption; Paper; Performance; Perfusion; Phosphorylation; Physiologic intraventricular pressure; Production; Property; Protein Isoforms; Proteins; Proteomics; Publishing; Pump; Rattus; Relaxation; Reporting; Research Personnel; Respiration; Ribonucleotide Reductase; Skeletal Muscle; Skin; Speed; Stress; Tail; Testing; Time; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; Veins; Ventricular; Viral; Viral Vector; Weight; Work; Workload; adeno-associated viral vector; adrenergic; editorial; experience; gene therapy; heart cell; heart function; improved; in vivo; inorganic phosphate; insight; loss of function; novel; novel strategies; prevent; promoter; research study; translational approach; vector

DESCRIPTION (provided by applicant): The goal of this project is to determine whether increased cardiomyocyte 2-deoxy ATP content [dATP], via over-expression of Ribonucleotide Reductase (R1R2), can be beneficial in potentiating cardiac function and treating heart failure. A multi-disciplinary team of investigators with considerable experience in the areas of cardiac contractile function, metabolism, electrophysiology and viral-mediated gene delivery has been assembled. Dr. Regnier (PI) previously demonstrated that dATP enhances contraction in demembranated cardiac muscle by increasing myosin binding to actin (crossbridge formation) and crossbridge cycling. In a recent paper (2011, JMCC. 51:894-901; Appendix 1), we demonstrated that increasing [dATP] in cultured adult rat cardiomyocytes by over-expression of R1R2 enhances contraction, speeds relaxation and has no effect on intracellular Ca2+ transient amplitude but speeds it's decay. In the same JMCC issue an editorial (2011, JMCC 51;883-4) urges that this novel approach be tested in animals to determine its potential for treating heart failure. This is the purpose of our proposal. Importantly, we present preliminary data demonstrating increasing [dATP] rescues depressed contractility and Ca2+ transients of cardiomyocytes from infarcted hearts. Additionally, we demonstrate that transgenic R1R2 over-expression mice (TG-R1R2) have elevated left ventricular (LV) function, measured by echocardiography and Langendorff perfusion. Here we propose a translational approach, i.e. delivery of an adeno-associated viral vector with a cardiac specific promoter (AAV6- R1R2cTnT455). We demonstrate that it results in R1R2 over-expression in the heart, but not skeletal muscle or lung, and that the AAV6 vector has sustained activity for at least 20 months in mice. We also report that a ~10x dose range of AAV6-R1R2cTnT455 injection is effective in increasing LV function (out to 6 weeks thus far). We will study AAV6-R1R2cTnT455 injected mice and TG-R1R2 mice under normal conditions (Aim 1) and in an acute (Aim 2) and chronic (Aim 3) infarct model. In vivo and in vitro whole heart studies will be complimented by trabeculae, intact cardiomyocyte and myofibril mechanical assessments during Ca2+ activated contraction. Because dATP increases crossbridge cycling and may affect other cellular ATPases, we will measure cardiac ATP synthesis and mitochondrial respiration, as well as high energy phosphate utilization, energetic reserve and oxygen consumption under basal conditions and with ¿-adrenergic stress using NMR spectroscopy. We will also study action potential and Ca2+ transient behavior and assess hearts and mice for potential pathological condition. Mechanistic interpretations will be aided by proteomic analysis of myofilament and membrane proteins to assess isoform and phosphorylation profiles. We will also use computational models (in collaboration with Dr. Andrew McCulloch, UCSD) to integrate the multi-scale data and provide mechanistic insight. Results from these studies will provide valuable insight on whether sustained enhancement of myofilament contractility (with dATP) has potential for treatment of heart failure in animal models and humans.

描述（由申请人提供）：该项目的目的是确定通过核糖核苷酸还原酶（R1R2）的过度表达增加心肌细胞2-脱氧ATP含量[dATP]是否有利于增强心功能和治疗心力衰竭。一个在心脏收缩功能、代谢、电生理和病毒介导的基因传递领域具有丰富经验的多学科研究团队已经组建。Regnier博士（PI）先前证明，dATP通过增加肌凝蛋白与肌动蛋白的结合（交叉桥形成）和交叉桥循环来增强脱膜心肌的收缩。在最近的一篇论文（2011年）中，JMCC。51:894 - 901;附录1)，我们证明了通过过度表达R1R2增加培养的成年大鼠心肌细胞的[dATP]增强收缩，加速松弛，对细胞内Ca2+瞬态振幅没有影响，但加速了它的衰减。在同一篇JMCC的社论（2011年，JMCC 51;883-4）中，敦促对这种新方法进行动物试验，以确定其治疗心力衰竭的潜力。这就是我们提议的目的。重要的是，我们提供的初步数据表明，增加[dATP]可以拯救梗死心脏心肌细胞的收缩力和Ca2+瞬态。此外，我们证明转基因R1R2过表达小鼠（TG-R1R2）具有升高的左心室（LV）功能，通过超声心动图和朗根多夫灌注测量。在这里，我们提出了一种翻译方法，即递送带有心脏特异性启动子（AAV6- R1R2cTnT455）的腺相关病毒载体。我们证明，它导致R1R2在心脏中过度表达，而不是在骨骼肌或肺中过度表达，并且AAV6载体在小鼠中持续活性至少20个月。我们还报道了~10倍剂量范围内的AAV6-R1R2cTnT455注射可有效提高左室功能（迄今为止持续6周）。我们将研究AAV6-R1R2cTnT455注射小鼠和TG-R1R2小鼠在正常情况下（Aim 1）和急性（Aim 2）和慢性（Aim 3）梗死模型。体内和体外全心研究将辅以小梁、完整心肌细胞和肌原纤维在Ca2+激活收缩期间的力学评估。由于dATP增加了跨桥循环并可能影响其他细胞ATP酶，我们将在基础条件下和在肾上腺素能应激下使用核磁共振光谱测量心脏ATP合成和线粒体呼吸，以及高能磷酸盐利用，能量储备和氧气消耗。我们还将研究动作电位和Ca2+瞬态行为，并评估心脏和小鼠的潜在病理状况。机制解释将通过肌丝和膜蛋白的蛋白质组学分析来评估异构体和磷酸化谱。我们还将使用计算模型（与UCSD的Andrew McCulloch博士合作）来整合多尺度数据并提供机制洞察力。这些研究的结果将为持续增强肌丝收缩性（使用dATP）是否具有治疗动物模型和人类心力衰竭的潜力提供有价值的见解。