Effect of elevated dATP on contractile function and the Frank-Starling relationship in models of dilated cardiomyopathy

dATP 升高对扩张型心肌病模型收缩功能和 Frank-Starling 关系的影响

基本信息

批准号：
9272928
负责人：
Farid Moussavi-Harami
金额：
$ 16.12万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-15 至 2021-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9272928
关键词：
Actins Address Adult Affinity Anabolism Animal Model Beryllium Binding Calcium Cardiac Cardiac Myocytes Clinic Coronary Arteriosclerosis DNA Sequence Alteration Data Deoxyadenosines Dextrans Dilated Cardiomyopathy Disease model Electrostatics Enzymes Epidemic Etiology Family suidae Fluorides Genetic Genetic Models Goals Heart Heart Transplantation Heart failure Human Impairment In Vitro Infarction Kinetics Laboratories Length Mechanics Mediating Medical Methods Modeling Molecular Morbidity - disease rate Mus Muscle Cells Mutation Myocardium Myofibrils Myosin ATPase Nucleotides Pathway interactions Patients Power stroke Process Protocols documentation Publishing Rattus Recombinants Recruitment Activity Relaxation Ribonucleotide Reductase Sampling Sarcomeres Stretching Striated Muscles Sturnus vulgaris Systolic heart failure Testing Therapeutic Intervention Thin Filament Toxin Training Transgenic Organisms Translational Research Tropomyosin Work Workload X ray diffraction analysis X-Ray Diffraction adeno-associated viral vector adenoviral-mediated alpha Tropomyosin base effective therapy efficacy testing experimental study improved in vivo inorganic phosphate insight mortality novel novel strategies novel therapeutics outcome forecast overexpression pre-clinical prevent public health relevance response tool tripolyphosphate

项目摘要

DESCRIPTION (provided by applicant): Dilated cardiomyopathy (DCM) is the most common form of systolic heart failure with poor prognosis and no specific treatment to address the underlying contractile deficit. DCM has various causes including coronary artery disease, toxins, metabolites and genetic mutations with all leading to a common pathway of dilation and reduced contractility. In addition to the loss of contractility, there is also impairment in the Frank-Staring relationship, an adaptive process that is described as the increase in contractile force in response to increased preload. We are proposing to investigate a novel therapy for DCM that addresses both loss of contractility and impairment in the Frank-Starling relationship. This novel therapy is achieved by increasing intracellular levels of 2-deoxy ATP (dATP) in cardiomyocytes via increasing the expression of the enzyme ribonucleotide reductase (R1R2), the rate-limiting step in de novo dNTP biosynthesis. Our previous published work has shown that that increased intracellular levels dATP in cardiomyocytes increases contraction by enhancing cross-bridge binding and cycling kinetics and improving allosteric activation of contraction. Recent data suggests that in addition to improved contraction, increased dATP level also enhances the Frank Starling relationship. I will propose specific aims to assess the effect of increased dATP on the Frank Starling relationship and contraction in two animal models of DCM. In the first aim, I will use a genetic model of DCM with a D230N mutation in alpha-tropomyosin (Tm). This allows us to test whether augmenting cross-bridge binding improves the contractile deficit caused by a thin filament mutation and suggest that our therapy can be used a wide variety of conditions. Using recombinant D230N Tm, I will test the hypothesis that this mutation reduces thin filament activation and, as a consequence, the kinetics of contractile activation and relaxation. I will then determine the effect of increasing dATP content on these deficits. Using demembranated trabecula from transgenic D230N Tm mice, I will test the hypothesis that this mutation decreases calcium sensitivity of force and length-dependent activation. I will correct these functional deficits by increasing dATP content. Using AAV6-R1R2cTnT455, an adeno-associated viral vector that restricts R1R2 over-expression to cardiac myocytes, I will test the hypothesis that increasing dATP levels improves contractility of isolated cardiomyocytes, and improves systolic function in adult D230N Tm mice and retard or prevent progression of heart failure in young mice with DCM. In the first aim, I will use post-infarct model of DCM in rats in addition to the D230N Tm mice to evaluate the effect of cross-bridge activation on length dependent activation (LDA)-the Frank Starling relationship at the sarcomere level. I will first tes the hypothesis that dATP enhances LDA in the two models of DCM. Next, I will use alternative agents to increase (dextran, EMD 50733) or decrease (BDM, beryllium fluoride, high inorganic phosphate) cross-bridge recruitment and evaluate their effect on LDA in demembranated post-infarct DCM rat trabecula. I will complete this aim by testing the hypothesis that cross-bridge activation augments LDA in human myocardium from patients with DCM. The long-term goal of this project is provide a safe and effective treatment for DCM regardless of its cause. I am proposing the necessary preclinical experiments necessary to take this novel therapy to the clinic to improve morbidity and mortality in DCM.

描述（由申请人提供）：扩张型心肌病（DCM）是收缩性心力衰竭的最常见形式，预后不良，并且没有特定的治疗方法来解决潜在的收缩缺陷。DCM 的病因多种多样，包括冠状动脉疾病、毒素、代谢物和基因突变。所有这些都会导致扩张和收缩力降低的共同途径除了收缩力丧失之外，弗兰克-凝视关系也会受到损害，这是一种适应性过程，被描述为收缩力的增加。我们提议研究一种针对 DCM 的新疗法，该疗法可解决收缩性丧失和 Frank-Starling 关系受损的问题。这种新疗法是通过增加细胞内 2-脱氧 ATP (dATP) 水平来实现的。在心肌细胞中，通过增加核糖核苷酸还原酶 (R1R2) 的表达，这是从头 dNTP 生物合成的限速步骤，表明这会增加细胞内水平。心肌细胞中的 dATP 通过增强跨桥结合和循环动力学以及改善收缩的变构激活来增加收缩。最近的数据表明，除了改善收缩之外，增加的 dATP 水平还可以增强 Frank Starling 关系，我将提出评估效果的具体目标。 dATP 增加对两种 DCM 动物模型中 Frank Starling 关系和收缩的影响在第一个目标中，我将使用 α-原肌球蛋白中存在 D230N 突变的 DCM 遗传模型。 (Tm)。这使我们能够测试增强跨桥结合是否可以改善细丝突变引起的收缩缺陷，并表明我们的疗法可以用于多种条件，使用重组 D230N Tm，我将测试以下假设。这种突变减少了细丝激活，因此，我将使用去膜小梁确定增加 dATP 含量对这些缺陷的影响。从转基因 D230N Tm 小鼠中，我将测试这种突变降低钙敏感性和长度依赖性激活的假设，我将通过增加 dATP 含量（一种限制 R1R2 的腺相关病毒载体）来纠正这些功能缺陷。心肌细胞过度表达，我将检验以下假设：增加 dATP 水平可改善分离心肌细胞的收缩性，并改善成人的收缩功能D230N Tm 小鼠并延缓或预防患有 DCM 的年轻小鼠的心力衰竭进展在第一个目标中，除 D230N Tm 小鼠外，我还将使用大鼠的梗死后模型来评估跨桥激活对 DCM 的影响。长度依赖性激活 (LDA) - 肌节水平的 Frank Starling 关系我将首先测试 dATP 在两种 DCM 模型中增强 LDA 的假设。增加（右旋糖酐，EMD 50733）或减少（BDM，氟化铍，高无机磷酸盐）跨桥募集并评估其对去膜性梗死后 DCM 大鼠小梁中 LDA 的影响，我将通过测试交叉假设来完成此目标。 -桥激活增强 DCM 患者心肌中的 LDA 该项目的长期目标是为 DCM 提供安全有效的治疗，无论其类型如何。我建议进行必要的临床前实验，将这种新疗法应用于临床，以改善 DCM 的发病率和死亡率。