PARP in Cardiac Arrest

PARP 在心脏骤停中的应用

• 批准号: 6825318
• 负责人: RICHARD J TRAYSTMAN
• 金额: $ 24.73万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6825318
• 关键词: ADP ribosylation; SDS polyacrylamide gel electrophoresis; brain injury; cardiopulmonary resuscitation; cerebral ischemia /hypoxia; genetically modified animals; heart arrest; histopathology; immunocytochemistry; laboratory mouse; molecular genetics; neuroprotectants; nicotinamide adenine dinucleotide; nitric oxide; nitric oxide synthase; transfection /expression vector; western blottings

Despite four decades of research concerning cardiac arrest/cardiopulmonary resuscitation (CPR), clinical outcome remains poor. Much of this research has taken the form of developing new methodologic or pharmacologic approaches to CPR. However, now, novel basic science and molecular genetic approaches to CPR are needed to improve our understanding of critical mechanisms of injury during cardiac arrest/CPR to develop new therapeutic interventions. Poly (ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme which helps maintain genomic integrity in neurons and numerous other cell types. Inhibition of PARP with pharmacologic agents or PARP deficiency via PARP knockout animals reduces injury after focal cerebral ischemia, but there is little information concerning PARP and cardiac arrest/CPR. Our preliminary studies show that cell injury after cardiac arrest/CPR in PARP null mutants (PARP-/-) is markedly reduced relative to wild type mice. Aim 1 will investigate this apparent neuroprotection observed in PARP deficient mice, characterizing extent and longevity of protection in intact animals over days of the evolving injury. We will use a combination of functional behavioral evaluation over time and terminal histopathology. Aim 2 will establish the importance of nitric oxide generation to PARP activation in vivo and subsequent injury during cardiac arrest/CPR. The relative consequences of inhibiting PARP vs neuronal or inducible nitric oxide synthase will be examined in the cardiac arrest/CPR mouse model. Lastly, using a strategy of transfecting virus carrying PARP mutant at two distinct catalytic and cleavage sites, we will begin to examine the molecular mechanism of PARP's importance in cardiac arrest/CPR. The role of the C-terminal NAD binding domain in PARP-mediated neuroprotection will be determined by comparison of cardiac arrest/CPR outcome in PARP-/- with or without transfection of a mutant form of PARP with inactive catalytic domain. Also, we will examine cysteine aspartase (CASPASE)-PARP interactions by comparison of cardiac arrest/CPR outcome in PARP-/- with and without transfection of a mutant form of PARP lacking in CASPASE 3 cleavage site (D214G mutant). These experiments will further our understanding of PARP-mediated mechanisms of in vivo ischemic brain injury and may provide a rational basis for development of effective, new therapeutic strategies to preserve neurological function atter cardiac arrest/CPR.

尽管关于心脏骤停/心肺复苏（CPR）的研究已经进行了四十年，但临床结果仍然很差。这些研究的大部分采取了开发新的方法学或药理学方法的形式。然而，现在，需要新的基础科学和分子遗传学方法来改善我们对心脏骤停/CPR期间损伤的关键机制的理解，以开发新的治疗干预措施。聚腺苷二磷酸核糖聚合酶（PARP）是一种丰富的核酶，有助于维持 神经元和许多其他细胞类型的基因组完整性。用药理学试剂抑制PARP或通过PARP敲除动物的PARP缺陷减少局灶性脑缺血后的损伤，但关于PARP和心脏骤停/CPR的信息很少。我们的初步研究表明，心脏骤停/心肺复苏后，在PARP无效突变体（PARP-/-）的细胞损伤显着减少相对于野生型小鼠。目的1将研究在PARP缺陷小鼠中观察到的这种明显的神经保护作用，表征在不断发展的损伤的数天内完整动物中的保护程度和寿命。我们将使用随时间推移的功能行为评价和终末组织病理学相结合的方法。目的2将确定一氧化氮的产生对体内PARP激活和随后的心脏骤停/CPR损伤的重要性。将在心脏骤停/CPR小鼠模型中检查抑制PARP与神经元或诱导型一氧化氮合酶的相对后果。最后，采用在两个不同的催化和切割位点上转染携带PARP突变体的病毒的策略，我们将 开始检查PARP在心脏骤停/CPR中重要性的分子机制。C-末端NAD结合结构域在PARP介导的神经保护中的作用将通过比较在有或没有转染具有无活性催化结构域的PARP突变形式的PARP-/-中的心脏骤停/CPR结果来确定。此外，我们还将通过比较转染和不转染缺少半胱氨酸天冬氨酸蛋白酶3切割位点（D214 G）的突变型PARP的PARP-/-中的心脏骤停/CPR结果来检查半胱氨酸天冬氨酸蛋白酶（CASPASE）-PARP相互作用 突变体）。这些实验将进一步加深我们对PARP介导的体内缺血性脑损伤机制的了解，并可能为开发有效的新治疗策略以在心脏骤停/心肺复苏后保护神经功能提供合理的基础。