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

 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，没有特异性治疗来解决潜在的收缩缺陷。DCM有多种原因，包括冠状动脉疾病、毒素、代谢物和基因突变，所有这些都导致扩张和收缩力降低的共同途径。除了收缩力的丧失之外，Frank-Staring关系也有损伤，这是一种适应性过程，被描述为响应于增加的前负荷而增加收缩力。我们建议研究一种治疗扩张型心肌病的新疗法，既能治疗收缩力丧失，又能治疗Frank-Starling关系受损。这种新疗法是通过增加核糖核苷酸还原酶（R1 R2）的表达来增加心肌细胞中2-脱氧ATP（dATP）的细胞内水平来实现的，核糖核苷酸还原酶是从头dNTP生物合成中的限速步骤。我们以前发表的工作表明，增加心肌细胞内dATP水平增加收缩通过增强跨桥结合和循环动力学和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水平也增强了Frank Starling关系。我将提出具体的目标，以评估增加dATP的影响，对弗兰克斯塔林的关系和收缩的两种动物模型的DCM。 在第一个目标中，我将使用在α-原肌球蛋白（Tm）中具有D230 N突变的DCM遗传模型。这使我们能够测试增加跨桥结合是否改善了由细丝突变引起的收缩缺陷，并表明我们的疗法可以用于各种各样的疾病。使用重组D230 N Tm，我将测试的假设，这种突变减少细丝激活，因此，收缩激活和松弛的动力学。然后，我将确定增加dATP含量对这些缺陷的影响。使用转基因D230 N Tm小鼠的去膜小梁，我将测试的假设，这种突变降低钙敏感性的力量和长度依赖性激活。我将通过增加dATP含量来纠正这些功能缺陷。使用AAV 6-R1 R2 cTnT 455（一种限制R1 R2过度表达于心肌细胞的腺相关病毒载体），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，并改善成年D230 N Tm小鼠的收缩功能，并延缓或预防患有DCM的年轻小鼠心力衰竭的进展。 在第一个目标中，我将使用大鼠心肌梗死后DCM模型以及D230 N Tm小鼠来评估跨桥激活对肌节水平上的长度依赖性激活（LDA）-Frank Starling关系的影响。我将首先测试dATP在DCM的两个模型中增强LDA的假设。接下来，我将使用替代药物来增加（右旋糖酐，EMD 50733）或减少（BDM，氟化铍，高无机磷酸盐）跨桥募集，并评估其对去膜梗死后DCM大鼠小梁中LDA的影响。我将通过验证跨桥激活增加DCM患者心肌中LDA的假设来完成这一目标。 该项目的长期目标是为DCM提供安全有效的治疗方法，无论其原因如何。我建议进行必要的临床前实验，以将这种新的治疗方法应用于临床，从而改善DCM的发病率和死亡率。

 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.