EFFECT OF R1R2 OVER-EXPRESSION ON CARDIAC FUNCTION

描述(由申请人提供)：该项目的目标是确定通过过表达核糖核苷酸还原酶(R1R2)而增加的心肌细胞2-脱氧ATP含量[dATP]是否有助于增强心功能和治疗心力衰竭。一支在心脏收缩功能、新陈代谢、电生理学和病毒介导的基因传递领域具有相当经验的多学科研究团队已经组建完成。Regnier博士(PI)此前证明，dATP通过增加肌球蛋白与肌动蛋白的结合(交叉桥的形成)和交叉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)，我们证明了通过过度表达R1R2而增加培养的成年大鼠心肌细胞的[dATP]增强收缩，加快松弛，对细胞内钙瞬变幅度没有影响，但加速了它的衰变。在同一期《美国医学会杂志》上，一篇社论(2011，JMCC 51；883-4)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。这就是我们建议的目的。重要的是，我们提供的初步数据表明，增加[dATP]可以挽救梗塞心脏心肌细胞受抑制的收缩能力和钙瞬变。此外，我们证明了转基因R1R2过表达小鼠(TG-R1R2)通过超声心动图和朗宁多夫血流灌注测量左心(LV)功能。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的腺相关病毒载体(AAV6-R1R2cTnT455)。我们证明，它导致R1R2在心脏过度表达，但不是骨骼肌或肺，并且AAV6载体在小鼠身上至少保持了20个月的活性。我们还报道了~10倍剂量范围的AAV6-R1R2cTnT455注射可以有效地改善左心功能(到目前为止已达6周)。我们将研究AAV6-R1R2cTnT455注射小鼠和TG-R1R2小鼠在正常条件下(目标1)以及在急性(目标2)和慢性(目标3)脑梗塞模型中。体内和体外的全心脏研究将得到小梁、完整的心肌细胞和肌原纤维在钙离子激活的收缩过程中的力学评估的支持。由于dATP增加了交叉桥循环，并可能影响其他细胞ATPase，我们将使用核磁共振波谱测量心脏ATP合成和线粒体呼吸，以及基础条件下和肾上腺素能应激条件下的高能磷酸盐利用、能量储备和氧气消耗。我们还将研究动作电位和钙瞬变行为，并评估心脏和小鼠的潜在病理状况。肌丝和膜蛋白的蛋白质组学分析将有助于机制的解释，以评估异构体和磷酸化特征。我们还将使用计算模型(与加州大学安德鲁·麦卡洛克博士合作)来整合多尺度数据并提供机械洞察力。这些研究的结果将为持续增强肌丝收缩能力(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。

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.