OXYGEN RADICALS AND CARDIAC ADAPTATIONS TO ISCHEMIA

氧自由基和心脏对缺血的适应

基本信息

批准号：
7269782
负责人：
Roberto Bolli
金额：
$ 53.54万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-05-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7269782
关键词：
Ablation Acute Animals Arginine Atrial Natriuretic Factor Binding Binding Sites Biochemical Biochemistry Biological Assay CREB1 gene Cardiac Cellular biology Chloride Ion Chlorides Complex Coronary Arteriosclerosis Coronary Occlusions Coupled Cyclic AMP-Responsive DNA-Binding Protein Data Development Diethyldithiocarbamate Disruption Doctor of Medicine Dominant-Negative Mutation EGF gene EMSA Electrophoretic Mobility Shift Assay Electrospray Ionization Elements Engineering Epidermal Growth Factor Event Exercise Exposure to FOS gene Family Gene Targeting Gene Transfer Techniques Genes Genetic Genetic Techniques Genetic Transcription Genetically Engineered Mouse Glyceraldehyde-3-Phosphate Dehydrogenases Heart Heat shock proteins High Pressure Liquid Chromatography Hypoxia-Responsive Elements IRF1 gene IRF2 gene Immunoprecipitation Individual Interferon Regulatory Factor 2 Interferon Type II Interferons Interleukin-2 Interleukin-6 Investigation Ischemia Ischemic Preconditioning Isoenzymes Janus kinase Knockout Mice Laboratories Lavendustin A Left Lymphocyte-Specific p56LCK Tyrosine Protein Kinase MAP Kinase Gene Manganese Superoxide Dismutase Mass Spectrum Analysis Measures Mediating Mediator of activation protein Mitogens Modeling Molecular Molecular Biology Molecular Genetics Mus Mutation Myocardial Infarction NOS1 protein, human Nitrates Nitric Oxide Synthase Nitrites Nitroglycerin Nuclear Numbers Patients Peroxonitrite Phosphorylation Site Phosphotransferases Physiological Physiological reperfusion Physiology Play Positioning Attribute Protein Chemistry Protein Isoforms Protein Kinase Protein Kinase C Protein Tyrosine Kinase Proteins Pyrazolones Reactive Oxygen Species Recruitment Activity Reperfusion Injury Reperfusion Therapy Resistance Response Elements Role S-nitro-N-acetylpenicillamine STAT1 gene Signal Pathway Signal Transduction Site Spectrometry, Mass, Electrospray Ionization Stimulus Stress Structure Techniques Therapeutic Thiopronine Time Transcription Factor AP-1 Transducers Transgenic Mice Transgenic Organisms Tumor Necrosis Factor-alpha Tyrosine Tyrosine Kinase Inhibitor Tyrosine Phosphorylation Up-Regulation Ventricular Western Blotting clinically relevant day diethylenetriamine human NOS3 protein hypoxia inducible factor 1 inhibitor/antagonist insight interdisciplinary approach interest knockout gene nitrate novel preconditioning programs promoter protein kinase C epsilon protein structure protein-tyrosine kinase c-src pyrazolone transcription factor

项目摘要

ecent evidence demonstrates that exercise induces delayed cardioprotection similar to that of ischemic preconditioning (PC), ia mechanisms that are presently unknown. Unlike ischemic or pharmacologic PC, exercise PC is triggered by a physiologic itimulus and thus would appear to be a natural means for achieving cardioprotection. The overall objective of this proposal is .0 elucidate the molecular mechanisms .underlying the newly-discovered phenomenon of exercise PC. Our fundamental lypothesis is that exercise-induced release of NO (via eNOS) activates a signal transduction cascade that includes PKCe, Src/Lck, and multiple transcription factors and culminates in the upregulation of iNOS, which then mediates the protection, either singularly or in conjunction with eNOS. A broad multidisciplinary approach will be used that will combine diverse echniques (integrative physiology, protein chemistry, mass spectrometry, biochemistry, cell biology, molecular biology, gene .argeting, and transgenesis) and will integrate genetic information at the molecular level with biochemical information at the jrotein structure level and physiological information at the whole animal level. Unequivocal evidence for or against an Dbligatory role of three specific kinases (PKCe, Src,.Lck) in exercise PC will be provided by the use of a novel dominant negative PKCe transgenic mouse line and Src and Lck knockout mice, this will enable us to achieve, for the first time, (inase-specific modulation of PKCe. Src.and Lck during exercise. The role of PKCs in initiating exercise PC will be onclusivelv established by determining the effects of specific transgenic inhibition of this isozyme. The kinase-specific activity of all seven Src PTKs expressed in the mouse heart (Fyn, Fgr, Yes, Src, Lyn, Lck, and Blk) will be directly measured at serial times after exercise PC. The role of Src PTKs in triggering versus mediating exercise PC'will be discerned by comparing inhibition of these kinases on days 1 and 2 (during the exercise stimulus) versus day 3 (during coronary occlusion). Targeted disruption of the Src and Lck gene will be employed to conclusively establish the specific function of individual PTKs in the PC protection. The transcription factors responsible for exercise PC will be systematically interrogated by using mice with targeted genetic ablation of each of the main factors known to bind to the iNOS gene (IRF-1, TNF-a, STAT1, CREB, AP-1, IL-2, and IL-6). The specific NOS isoforms responsible for initiating as well as mediating exercise PC will be conclusively identified by targeted gene disruption of eNOS, iNOS, and nNOS. The post-translational modulation of iNOS 24 h after exercise will be elucidated by identifying the precise phosphorylation site(s) on iNOS with HPLC coupled- electrospray ionization mass spectrometry. Finally, the role of Src PTKs in the post-translational modulation of iNOS will be established by measuring iNOS activity and tyrosine phosphorylation in the absence and presence of Src PTK inhibitors. This proposal should produce important new insights into the molecular mechanisms whereby the heart adapts not only to physical stress but also to stress in general. Elucidation of the mechanism of exercise PC may have important therapeutic implications.

最近的证据表明，运动会导致延迟心脏保护，类似于缺血预适应（PC），目前未知的ia机制。与缺血性或药物性 PC 不同，运动性 PC 是由生理性 PC 触发的。因此，itimulus 似乎是实现心脏保护的自然手段。该提案的总体目标是 .0 阐明新发现的运动 PC 现象背后的分子机制。我们的根本推测是运动诱导的 NO 释放（通过 eNOS）激活信号转导级联，其中包括 PKCe、 Src/Lck 和多种转录因子，最终导致 iNOS 上调，从而介导保护，单独使用或与 eNOS 结合使用。将采用广泛的多学科方法，将不同的技术（综合生理学、蛋白质化学、质谱、生物化学、细胞生物学、分子生物学、基因 .靶向和转基因）并将分子水平的遗传信息与生物化学信息整合整个动物水平的蛋白结构水平和生理信息。支持或反对的明确证据三种特定激酶（PKCe、Src、.Lck）在运动 PC 中的必然作用将通过使用新型显性激酶来提供 PKCe 阴性转基因小鼠系以及 Src 和 Lck 敲除小鼠，这将使我们首次实现，（运动过程中 PKCe、Src 和 Lck 的酶特异性调节。PKC 在启动运动 PC 中的作用将是通过确定该同工酶的特异性转基因抑制的效果来确定结论。激酶特异性将直接测量小鼠心脏中表达的所有七个 Src PTK（Fyn、Fgr、Yes、Src、Lyn、Lck 和 Blk）的活性锻炼电脑后连续几次。 Src PTK 在触发与调节 PC 运动中的作用将通过以下方式来识别比较这些激酶在第 1 天和第 2 天（运动刺激期间）与第 3 天（冠状动脉闭塞期间）的抑制情况。 Src 和 Lck 基因的定向破坏将用于最终确定个体的特定功能 PC 保护中的 PTK。负责运动 PC 的转录因子将通过使用系统地进行询问对已知与 iNOS 基因结合的每个主要因子（IRF-1、TNF-a、STAT1、CREB、 AP-1、IL-2 和 IL-6）。负责启动和调节 PC 运动的特定 NOS 异构体是通过 eNOS、iNOS 和 nNOS 的靶向基因破坏最终确定。翻译后调制运动后 24 小时的 iNOS 将通过 HPLC 联用鉴定 iNOS 上的精确磷酸化位点来阐明 - 电喷雾电离质谱法。最后，Src PTKs 在 iNOS 翻译后调节中的作用将是通过在不存在和存在 Src PTK 抑制剂的情况下测量 iNOS 活性和酪氨酸磷酸化来建立。这该提案应该对心脏不仅适应身体的分子机制产生重要的新见解压力，而且还包括一般压力。阐明运动 PC 的机制可能具有重要的治疗意义。