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

摘要 扩张型心肌病(DCM)是最常见的收缩性心力衰竭，预后差 也没有具体的治疗方法来解决潜在的收缩缺陷。除了收缩能力的丧失外， 弗兰克-斯塔林的关系也有损害，这是一种被描述为 随着预紧力的增加而增加的收缩力。我们提议调查一部小说 治疗扩张型心肌病，同时解决了Frank-Starling关系中的收缩能力丧失和损害。 这种新的治疗方法是通过增加心肌细胞内2-脱氧三磷酸腺苷(DATP)的水平来实现的 通过增加核糖核酸还原酶(R1R2)的表达，在DEC中的限速步骤 Novo dNTP生物合成。心肌细胞内dATP水平升高通过以下途径增加收缩 增强跨桥结合和循环动力学，改善收缩的变构激活。 最近的数据表明，除了改善收缩外，dATP水平的增加也增强了 弗兰克·斯塔林的关系。我将提出具体目标来评估dATP增加对Frank的影响 两种扩张型心肌病动物模型的启动关系和收缩。在第一个目标中，我将使用基因 α-原肌球蛋白D230N突变的扩张性心肌病模型使用重组D230N TM， 我将测试这一假设，即这种突变会降低细丝的活性，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 在这些赤字上。使用转基因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的激活的敏感性。我将更正这些功能 通过增加dATP含量来减少赤字。使用腺相关病毒载体AAV6-cR1R2 R1R2过度表达到心肌细胞，我将检验增加dATP水平改善的假设 分离的心肌细胞的收缩能力，并改善成年和年轻D230N TM的收缩功能 老鼠。同样，我将使用大鼠心肌梗死后模型来评估跨桥激活的效果。 依赖长度的激活(LDA)--肌节水平的Frank Starling关系。接下来，我会 使用替代试剂来增加(葡聚糖、EMD 50733)或减少(bdm、氟化铍、高 无机磷)跨桥募集并评价其对去膜后大鼠LDA的影响 梗死型DCM大鼠骨小梁。我将通过测试跨桥激活的假设来完成这一目标 增强扩张型心肌病患者心肌中的LDA。 扩展项目：在D230N TM DCM啮齿动物模型中使用上述研究的数据来模拟逼真 心脏抽动，并使用机器学习方法进行参数优化。

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.