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）是收缩性心力衰竭的最常见形式，预后不良 也没有针对潜在收缩缺陷的特殊治疗。除了收缩力的丧失， 弗兰克-斯塔林关系也有损害，这是一个适应性过程，被描述为 响应于增加的前负荷而增加收缩力。我们打算调查一部小说 治疗DCM，解决收缩力丧失和Frank-Starling关系受损。 这种新的治疗方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（R1 R2）的表达， 新dNTP生物合成。心肌细胞内dATP水平的增加通过以下方式增加收缩： 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，增加dATP水平也增强了收缩。 弗兰克·史达琳的关系。我将提出具体的目标，以评估增加dATP对弗兰克的影响。 两种扩张型心肌病动物模型的Starling关系和收缩。在第一个目标中，我将使用一种遗传学方法， 在α-原肌球蛋白中具有D230 N突变（D230 N Tm）的DCM模型。使用重组D230 N Tm， 我将检验这一假设，即这种突变减少了细丝的激活，因此， 收缩激活和舒张的动力学。然后我将确定增加dATP含量的效果 这些赤字。使用转基因D230 N Tm小鼠的组织，我将检验这种突变 降低力和长度依赖性激活的钙敏感性。我将纠正这些功能 增加dATP含量。使用AAV 6-cR 1 R2，一种腺相关病毒载体， R1 R2在心肌细胞中的过表达，我将检验增加dATP水平改善心肌细胞的假设。 收缩性的分离的心肌细胞，并改善收缩功能，在成人和年轻的D230 N Tm 小鼠同样，我将使用大鼠心肌梗死后DCM模型来评估跨桥激活的效果 长度依赖性激活（LDA）-在肌节水平上的Frank Starling关系。接下来我会 使用替代药物增加（右旋糖酐，EMD 50733）或减少（BDM，氟化铍，高 无机磷酸盐）跨桥募集，并评估它们对脱膜后 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， 增加DCM患者的人心肌中的LDA。 扩展项目：在D230 N Tm DCM啮齿动物模型中使用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。

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.