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EFFECT OF R1R2 OVER-EXPRESSION ON CARDIAC FUNCTION

R1R2 过度表达对心脏功能的影响

基本信息

批准号：
8891479
负责人：
MICHAEL REGNIER
金额：
$ 57.55万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-15 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8891479
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

项目摘要

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）：本项目的目的是确定通过核糖核苷酸还原酶（R1 R2）的过表达增加心肌细胞2-脱氧ATP含量[dATP]是否有利于增强心脏功能和治疗心力衰竭。一个多学科的研究小组，在心脏收缩功能，代谢，电生理学和病毒介导的基因传递领域具有相当丰富的经验已经组装。Regnier博士（PI）先前证明，dATP通过增加肌球蛋白与肌动蛋白的结合（横桥形成）和横桥循环来增强去膜心肌的收缩。在最近的一篇论文（2011年，JMCC。51：894-901;附录1），我们证明了在培养的成年大鼠心肌细胞中通过R1 R2的过度表达增加[dATP]增强收缩，加速舒张，对细胞内Ca 2+瞬时振幅没有影响，但加速其衰减。在同一期JMCC中，一篇社论（2011，JMCC 51;883-4）敦促在动物中测试这种新方法，以确定其治疗心力衰竭的潜力。这就是我们提出建议的目的。重要的是，我们目前的初步数据表明，增加[dATP]救援抑郁的收缩性和Ca 2+瞬变的心肌细胞从梗死的心脏。此外，我们证明，转基因R1 R2过表达小鼠（TG-R1 R2）具有升高的左心室（LV）功能，通过超声心动图和Langendorff灌注测量。在这里，我们提出了一种翻译方法，即递送具有心脏特异性启动子的腺相关病毒载体（AAV 6-R1 R2 cTnT 455）。我们证明了它导致R1 R2在心脏中过表达，但不是骨骼肌或肺，并且AAV 6载体在小鼠中具有持续至少20个月的活性。我们还报道了约10倍剂量范围的AAV 6-R1 R2 cTnT 455注射在增加LV功能方面是有效的（迄今为止长达6周）。我们将研究在正常条件下（Aim 1）以及在急性（Aim 2）和慢性（Aim 3）梗塞模型中注射AAV 6-R1 R2 cTnT 455的小鼠和TG-R1 R2小鼠。在体内和体外全心脏研究将补充小梁，完整的心肌细胞和肌原纤维的机械评估在钙激活收缩。由于dATP增加了跨桥循环，并可能影响其他细胞ATP酶，我们将测量心脏ATP合成和线粒体呼吸，以及高能磷酸盐利用率，能量储备和氧消耗在基础条件下，并与β-肾上腺素能应激使用NMR光谱。我们还将研究动作电位和Ca 2+瞬态行为，并评估心脏和小鼠的潜在病理状况。机制解释将有助于肌丝和膜蛋白的蛋白质组学分析，以评估亚型和磷酸化概况。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械见解。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。