PKG Redox Modulation of Cardiac Function and Disease

描述(申请人提供)：在肥大或衰竭的心脏中，诱导和改善细胞和器官功能的不良适应变化的信号级联被共同激活。目前的临床治疗主要旨在阻断病理信号，但努力 增强内在适应途径也引起了人们的兴趣。环GMP-蛋白激酶G1a(PKG1a)通路就是后者的一个典型例子。最近的研究，其中许多来自我的实验室，揭示了抑制磷酸二酯酶5型刺激PKG是如何阻止病情发展的，甚至逆转了既定的心脏病。这导致了NIH赞助的两个主要的多中心PDE5抑制剂治疗心力衰竭的多中心试验，其射血分数要么减少，要么保留。这种翻译的核心是心肌中PKG1a的功能， 在新的数据中，我们第一次显示，这种酶在患病的左心室中被氧化， 降低其抵消病理的能力。这涉及到每个PKG1a单体中半胱氨酸-42(C42)之间的二硫化物，一个残基恰好位于对蛋白质相互作用至关重要的N末端对接结构域中。带有阻止C42氧化的敲入突变(C42S)的小鼠表现出改善的病理生理学，以维持压力超负荷。然而，他们也表明，PKG不再能被PDE5抑制激活。这表明，利用PKG激活的治疗方法，如利钠肽、一氧化氮供体或PDE5抑制剂，本身可能严重依赖于PKG氧化还原状态。在这个项目中，我们确定了PKG1a C42氧化/二聚化对患病心脏的影响，验证了氧化还原修饰改变其蛋白相互作用体从而改变其激酶信号的假设，以及通过细胞内cGMP池激活的假设。这是通过三个目标实现的。在目标1中，我们确定了PKG1a C42-二聚体的预防对承受压力超负荷或梗塞的心脏与生理(运动)应激的心脏的影响，确定了激酶靶向和病理生理调节方面的差异。我们还测试了PKG1a-氧化还原是否影响了临床方法刺激激酶的能力，并确定了这种变化的机制。在目标2中，我们评估了PKG1a-氧化还原如何影响肌细胞肌节功能的激酶修饰，确定了肌原纤维丰富和耗尽的磷酸蛋白质组的变化。在目标3中，我们研究了PKG1a-氧化还原对细胞内激酶定位和蛋白质相互作用的影响。确定了相互作用体，并确定了C42-二聚体影响蛋白质对接结构域的机制。总的来说，这些研究将确定PKG氧化还原调节的作用、机制和翻译含义，这些信息对于优化设计的治疗方案至关重要，这些疗法旨在利用其信号来治疗心脏病。

DESCRIPTION (provided by applicant): In the hypertrophied or failing heart, signaling cascades that both induce and ameliorate maladaptive changes in cell and organ function are co-activated. Current clinical therapy largely aims at blocking pathological signaling, but efforts to enhance intrinsic adaptive pathways are also gaining interest. The cyclic GMP-protein kinase G1a (PKG1a) pathway is a prime example of the latter. Recent studies, many from my laboratory, have revealed how inhibiting phosphodiesterase type 5 to stimulate PKG blocks the progression and even reverses established heart disease. This has led to two major NIH- sponsored multicenter trials of PDE5 inhibitors in heart failure with either reduced or preserved ejection fraction. At the core of this translation is the functionality of PKG1a in the myocardium, and in new data we show for the first time that the enzyme is oxidized in diseased left ventricles, reducing its capacity to offset pathology. This involves a di-sulfide between cysteine-42 (C42) in each PKG1a monomer, a residue lying just within an N-terminus docking domain critical for protein interactions. Mice with a knock-in mutation (C42S) that precludes C42 oxidation show improved pathophysiology to sustained pressure-overload. However, they also show that PKG can no longer be activated by PDE5-inhibition. This indicates that therapies leveraging PKG activation, such as natriuretic peptides, nitric oxide donors, or PDE5-inhibitors, may themselves critically depend on PKG redox state. In this project, we determine the impact of PKG1a C42 oxidation/dimerization on the diseased heart, testing the hypothesis that redox modification alters its protein-interactome and thus kinase signaling, as well as activation by intracellular cGMP pools. This is accomplished in three Aims. In aim 1, we determine how prevention of PKG1a C42-dimer impacts hearts subjected to pressure overload or infarction versus physiological (exercise) stress, identifying differences in kinase targeting and patho- physiological regulation. We also test if PKG1a-redox impacts the capacity of clinical methods to stimulate the kinase, and identify mechanisms for such changes. In Aim 2, we assess how PKG1a- redox impacts kinase modification of myocyte sarcomere function, identifying changes in the myofibrillar enriched and depleted phospho-proteome. In Aim 3, we examine the impact of PKG1a- redox on intracellular kinase localization and protein interactions. The interactome is identified, and mechanisms by which the C42-dimer impacts the protein docking-domain determined. Collectively, these studies will determine the role, mechanisms, and translational implications for PKG redox modulation, information central to optimizing therapies designed to leverage its signaling for the treatment of treat heart disease.