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

 描述(申请人提供)：扩张型心肌病(DCM)是最常见的收缩性心力衰竭形式，预后很差，没有特殊的治疗方法来解决潜在的收缩功能缺陷。扩张型心肌病有多种原因，包括冠状动脉疾病、毒素、代谢物和基因突变，所有这些都会导致共同的扩张和收缩能力下降。除了收缩能力的丧失外，Frank-Starting关系也有损害，这是一种适应性过程，被描述为对增加的前负荷做出反应的收缩力量的增加。我们建议研究一种新的治疗扩张型心肌病的方法，以解决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核糖核苷酸还原酶(R1R2)的表达来增加心肌细胞内2-脱氧ATP(DATP)的水平实现的，R1R2是dNTP生物合成的限速步骤。我们以前发表的工作表明，心肌细胞内dATP水平的增加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，dATP水平的增加也加强了Frank Starling的关系。我将提出具体的目标来评估dATP增加对两种DCM动物模型Frank Starling关系和收缩的影响。在第一个目标中，我将使用在α-原肌球蛋白(TM)中存在D230N突变的扩张性心肌病的遗传模型。这使我们能够测试增强跨桥结合是否改善了由细丝突变引起的收缩缺陷，并表明我们的治疗方法可以用于各种条件。利用重组的D230N TM，我将检验这样的假设，即该突变降低了细丝的激活，从而降低了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D230N TM小鼠的去膜小梁，我将检验这种突变降低钙对力和长度依赖的激活敏感性的假设。我会通过增加dATP含量来纠正这些功能缺陷。利用AAV6-R1R2cTnT455，一种可限制R1R2在心肌细胞中过度表达的腺相关病毒载体，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能力，改善成年D230N TM小鼠的收缩功能，并延缓或防止DCM幼鼠心力衰竭的进展。在第一个目标中，我将使用D230N TM小鼠和大鼠的DCM梗死后模型来评估跨桥激活对长度依赖激活(LDA)的影响-在肌节水平上的Frank Starling关系。我将首先提出假设，在两种DCM模型中，dATP增强LDA。接下来，我将使用替代药物增加(葡聚糖，EMD 50733)或减少(BDM，氟化铍，高无机磷)跨桥募集，并评估它们对去膜后心肌梗死后弥漫性心肌梗死大鼠小梁LDA的影响。我将通过验证跨桥激活增加扩张型心肌病患者心肌LDA的假设来完成这一目标。该项目的长期目标是为DCM提供一种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，将这种新疗法应用于临床，以改善扩张型心肌炎的发病率和死亡率。

 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.