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

• 批准号: 10391887
• 负责人: Farid Moussavi-Harami
• 金额: $ 4.46万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2022-01-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10391887
• 关键词: Address; Adult; Anabolism; Animal Model; Beryllium; Binding; Calcium; Cardiac; Cardiac Myocytes; Data; Deoxyadenosines; Dextrans; Dilated Cardiomyopathy; Enzymes; Epidemic; Fluorides; Genetic Models; Heart; Heart failure; Human; Impairment; Infarction; Kinetics; Length; Medical; Modeling; Mus; Mutation; Myocardium; Patients; Process; Prognosis; Rattus; Recombinants; Relaxation; Ribonucleotide Reductase; Rodent Model; Sarcomeres; Sturnus vulgaris; Systolic heart failure; Testing; Thin Filament; Tissues; Transgenic Organisms; Workload; adeno-associated viral vector; alpha Tropomyosin; improved; inorganic phosphate; machine learning method; mortality; novel; novel therapeutics; overexpression; recruit; response; tripolyphosphate

Abstract 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. In addition to the loss of contractility, there is also impairment in the Frank-Starling 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. 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 (D230N Tm). 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 tissue 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-cR1R2, 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 both adult and young D230N Tm mice. Similarly, I will use post-infarct model of DCM in rats to evaluate the effect of cross-bridge activation on length dependent activation (LDA)-the Frank Starling relationship at the sarcomere level. 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. Extension Project: Use data from above studies in D230N Tm DCM rodent model to simulate realistic cardiac twitches and use machine-learning methods for parameter optimization.

抽象的 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。除了收缩力丧失之外， 弗兰克-斯塔林关系也受到损害，这是一种适应性过程，被描述为 收缩力随着预紧力的增加而增加。我们提议调查一部小说 治疗扩张型心肌病（DCM）的方法，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心肌细胞内 2-脱氧 ATP (dATP) 的水平来实现的 通过增加核糖核苷酸还原酶 (R1R2) 的表达，这是 de 的限速步骤 新的 dNTP 生物合成。心肌细胞内 dATP 水平增加可增强收缩力 增强跨桥结合和循环动力学并改善收缩的变构激活。 最近的数据表明，除了改善收缩外，增加 dATP 水平还可以增强 弗兰克·史达林的关系。我将提出具体目标来评估增加 dATP 对 Frank 的影响 两种 DCM 动物模型中八哥的关系和收缩。在第一个目标中，我将使用基因 α-原肌球蛋白 (D230N Tm) 具有 D230N 突变的 DCM 模型。使用重组D230N Tm， 我将测试这一假设，即这种突变会减少细丝激活，因此， 收缩激活和松弛的动力学。然后我将确定增加 dATP 含量的效果 关于这些赤字。使用来自转基因 D230N Tm 小鼠的组织，我将检验这种突变的假设 降低钙对力和长度依赖性激活的敏感性。我会纠正这些功能 通过增加 dATP 含量来弥补缺陷。使用AAV6-cR1R2，一种腺相关病毒载体，限制 R1R2 在心肌细胞中过度表达，我将检验增加 dATP 水平可以改善的假设 分离心肌细胞的收缩力，并改善成人和年轻 D230N Tm 的收缩功能 老鼠。同样，我将使用大鼠DCM梗死后模型来评估跨桥激活的效果 长度依赖性激活（LDA）-肌节水平的 Frank Starling 关系。接下来，我将 使用替代药物来增加（葡聚糖、EMD 50733）或减少（BDM、氟化铍、高 无机磷酸盐）跨桥募集并评估其对脱膜后LDA的影响 梗死 DCM 大鼠小梁。我将通过测试跨桥激活的假设来完成这个目标 增强 DCM 患者心肌中的 LDA。 扩展项目：使用 D230N Tm DCM 啮齿动物模型中上述研究的数据来模拟现实 心脏抽搐并使用机器学习方法进行参数优化。