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：
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
项目类别：
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

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（由适用提供）：该项目的目的是确定通过过度表达核糖核苷酸还原酶（R1R2），增加心肌细胞2-脱氧ATP含量[DATP]是否可以对潜在的心脏功能和治疗心力衰竭有益。一支在心脏收缩功能，代谢，电生理学和病毒介导的基因递送方面具有可观经验的研究人员的多学科团队。 Regnier博士（PI）先前证明，DATP通过增加与肌动蛋白（Crossbridge组）和Crossbridge Cycling的结合来增强心脏肌肉的收缩。在最近的一篇论文（2011年，JMCC 51：894-901;附录1）中，我们证明，通过过度表达R1R2，培养的成年大鼠心肌细胞在培养的成年大鼠心肌细胞中增加[DATP]会增强收缩的速度，速度放松，对细胞内CA2+瞬时放大器没有影响，但速度速度却加快了IT的延迟。在同一JMCC中，社论（2011年，JMCC 51; 883-4）敦促在动物中测试这种新颖的方法，以确定其治疗心力衰竭的潜力。这是我们建议的目的。重要的是，我们提供的初步数据表明[DATP]响应抑郁症的收缩性和梗塞心脏心肌细胞的Ca2+瞬变。此外，我们证明了转基因R1R2过表达小鼠（TG-R1R2）的左心室（LV）功能升高，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，即用心脏特异性启动子（AAV6- R1R2CTNT455）传递与腺相关的病毒载体的传递。我们证明，它导致心脏中的R1R2过表达，而不是骨骼肌肉或肺部，并且AAV6载体在小鼠中的活性至少20个月。我们还报告说，AAV6-R1R2CTNT455注射的约10倍范围可有效地提高LV功能（到目前为止迄今为止6周）。我们将在正常条件下（AIM 1）和急性（AIM 2）和慢性（AIM 3）梗塞模型研究AAV6-R1R2CTNT455注射小鼠。在Ca2+活化收缩期间，小梁，完整的心肌细胞和肌原纤维机械评估将补充体内和体外全心脏研究。由于DATP增加了Crossbridge循环并可能影响其他细胞ATPase，因此我们将测量心脏ATP合成和线粒体呼吸，以及在基本条件下以及使用NMR光谱术的肾上力抗应力的高能量磷酸盐利用，能量储备和氧气消耗。我们还将研究动作潜力以及CA2+瞬态行为以及评估心脏和小鼠的潜在病理状况。机械解释将通过对肌丝和膜蛋白的蛋白质组学分析来帮助评估同工型和磷酸化谱。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械洞察力。这些研究的结果将提供有价值的见解，了解是否持续增强肌丝收缩力（带DATP）有可能治疗动物模型和人类心力衰竭。