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）：该项目的目标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] 是否有利于增强心脏功能和治疗心力衰竭。 Regnier 博士（PI）之前已经组建了一个由在心脏收缩功能、代谢、电生理学和病毒介导的基因传递领域拥有丰富经验的多学科研究人员组成的团队。 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，JMCC. 51：894-901；附录 1）中，我们证明了培养成体中 [dATP] 的增加。大鼠心肌细胞通过过度表达 R1R2 增强收缩，加速舒张，对细胞内 Ca2+ 瞬态振幅没有影响，但加速其收缩在同一期 JMCC 中，一篇社论（2011，JMCC 51；883-4）敦促在动物身上测试这种新方法，以确定其治疗心力衰竭的潜力。重要的是，我们提出了初步建议。数据表明，增加 [dATP] 可挽救梗死心脏中心肌细胞的收缩力下降和 Ca2+ 瞬变。此外，我们还证明转基因 R1R2 过表达小鼠 (TG-R1R2) 的心肌细胞收缩力和 Ca2+ 瞬变有所提高。左心室 (LV) 功能，通过超声心动图和 Langendorff 灌注测量。在这里，我们提出了一种转化方法，即递送带有心脏特异性启动子 (AAV6-R1R2cTnT455) 的腺相关病毒载体，我们证明它会导致 R1R2 过度。 AAV6 载体在心脏中表达，但在骨骼肌或肺中不表达，并且 AAV6 载体在小鼠中具有至少 20 个月的持续活性。约 10 倍剂量范围的 AAV6-R1R2cTnT455 注射可有效增强左心室功能（迄今为止长达 6 周）我们将在正常条件下（目标 1）和急性条件下（目标 1）研究注射 AAV6-R1R2cTnT455 的小鼠和 TG-R1R2 小鼠。 2）和慢性（目标 3）梗塞模型将得到体内和体外全心脏研究的补充。 Ca2+ 激活收缩过程中小梁、完整心肌细胞和肌原纤维的机械评估由于 dATP 增加横桥循环并可能影响其他细胞 ATP 酶，因此我们将测量心脏 ATP 合成和线粒体呼吸，以及基础下的高能磷酸盐利用率、能量储备和氧消耗。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。我们还将通过肌丝和膜蛋白的蛋白质组学分析来辅助解释，以评估同种型和磷酸化谱。我们还将使用计算模型（与加州大学圣地亚哥分校的 Andrew McCulloch 博士合作）来整合多尺度数据并提供机制见解。这些研究将为了解持续增强肌丝收缩力（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。