PKG Redox Modulation of Cardiac Function and Disease

PKG 氧化还原对心脏功能和疾病的调节

• 批准号: 8530799
• 负责人: David Alan Kass
• 金额: $ 38.56万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8530799
• 关键词: Acute; Address; Adrenergic Agents; Adverse effects; Affect; Antioxidants; Binding; Blood Vessels; Cardiac; Cardiac Myocytes; Cell membrane; Cells; Chronic; Clinical; Clinical Treatment; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cysteine; Cytosol; Data; Depressed mood; Dimerization; Disease; Docking; Doxorubicin; EFRAC; Enzymes; Exercise; Functional disorder; Goals; Heart; Heart Diseases; Heart failure; Homo; Hypertrophy; Infarction; Intracellular translocation; Knock-in Mouse; Laboratories; Left ventricular structure; Leucine Zippers; Link; Mechanics; Mediating; Methods; Modification; Molecular; Multicenter Trials; Mus; Muscle Cells; Mutation; Myocardial; Myocardium; Natriuretic Peptides; Nitric Oxide; Nitric Oxide Donors; Nuclear; Organ; Oxidation-Reduction; Oxidative Stress; Pathology; Pathway interactions; Phosphotransferases; Physiological; Prevention; Protein Kinase; Proteins; Proteome; Regulation; Role; Sarcomeres; Side; Signal Transduction; Stimulus; Stress; Study models; Sulfides; Testing; Time; Transcript; Translations; Troponin I; Troponin T; United States National Institutes of Health; Vasodilation; War; Work; X-Ray Crystallography; adrenergic; biological adaptation to stress; clinically relevant; connectin; dimer; disulfide bond; drug efficacy; drug testing; heart function; improved; in vivo; inhibitor/antagonist; insight; interest; loss of function; monomer; novel; oxidation; phospholamban; phosphoric diester hydrolase; pressure; protein transport; public health relevance; therapeutic effectiveness; therapy design; trafficking

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.

描述（由申请人提供）：在肥大或衰竭的心脏中，诱导和改善细胞和器官功能适应不良变化的信号级联被共激活。目前的临床治疗主要旨在阻断病理信号传导，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G1 a（PKG 1a）途径是后者的一个主要例子。最近的研究，许多来自我的实验室，已经揭示了如何抑制磷酸二酯酶5型刺激PKG阻止进展，甚至逆转已建立的心脏病。这导致了两个主要的NIH赞助的多中心试验的PDE 5抑制剂在心力衰竭与减少或保留射血分数。这种翻译的核心是PKG 1a在心肌中的功能， 在新的数据中，我们首次表明这种酶在病变的左心室中被氧化， 降低其抵消病理的能力。这涉及每个PKG 1a单体中半胱氨酸-42（C42）之间的二硫键，该残基恰好位于对蛋白质相互作用至关重要的N端对接结构域内。具有排除C42氧化的敲入突变（C42 S）的小鼠显示出对持续压力超负荷的改善的病理生理学。然而，他们也表明PKG不能再被PDE 5抑制激活。这表明利用PKG激活的疗法，如利钠肽、一氧化氮供体或PDE 5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PKG 1a C42氧化/二聚化对患病心脏的影响，测试氧化还原修饰改变其蛋白质相互作用组的假设，从而激酶信号，以及细胞内cGMP池的激活。这是通过三个目标实现的。在目标1中，我们确定PKG 1a C42-二聚体的预防如何影响压力超负荷或梗死与生理（运动）应激的心脏，确定激酶靶向和病理生理调节的差异。我们还测试PKG 1a-氧化还原是否影响临床方法刺激激酶的能力，并确定这种变化的机制。在目标2中，我们评估PKG 1a-氧化还原如何影响肌细胞肌节功能的激酶修饰，确定肌原纤维富集和耗尽磷酸化蛋白质组的变化。在目标3中，我们研究PKG 1a-氧化还原对细胞内激酶定位和蛋白质相互作用的影响。确定了相互作用组，并确定了C42-二聚体影响蛋白质停靠结构域的机制。总的来说，这些研究将确定PKG氧化还原调节的作用、机制和翻译意义，这是优化旨在利用其信号传导治疗心脏病的治疗方法的核心信息。