OXYGEN RADICALS AND CARDIAC ADAPTATIONS TO ISCHEMIA

近年来的研究表明，运动诱导的延迟性心肌保护作用与缺血预处理（PC）相似， 这是目前未知的机制。与缺血性或药物性PC不同，运动PC是由生理性 因此似乎是实现心脏保护的天然手段。本建议的总体目标是 .0阐明运动PC这一新发现现象的分子机制。我们的根本 假设是运动诱导的NO释放（通过eNOS）激活了包括PKCe的信号转导级联， Src/Lck和多种转录因子，并最终上调iNOS，然后介导保护， 单独或与eNOS结合。将采用广泛的多学科方法，将联合收割机 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 转基因），并将分子水平上的遗传信息与生物化学水平上的生物化学信息相结合。 蛋白质结构水平和整体动物水平的生理信息。明确的证据支持或反对 三种特异性激酶（PKCe，Src，Lck）在运动PC中的拮抗作用将通过使用一种新的优势激酶来提供。 阴性PKCe转基因小鼠系和Src和Lck敲除小鼠，这将使我们能够实现，首次， （PKCe的蛋白酶特异性调节。Src.和Lck在运动期间。PKC在启动运动PC中的作用将是 结论通过测定特异性转基因抑制该同工酶的作用而建立。激酶特异性 将直接测量小鼠心脏中表达的所有七种Src PTK（Fyn、Fgr、Yes、Src、林恩、Lck和Blk）的活性 在运动PC后的连续时间。Src PTK在触发与介导运动PC'中的作用将通过以下方式来辨别： 比较第1天和第2天（在运动刺激期间）与第3天（在冠状动脉闭塞期间）这些激酶的抑制。 Src和Lck基因的靶向破坏将用于最终确定个体的特异性功能， PC保护中的PTK。负责运动PC的转录因子将通过使用 对已知与iNOS基因结合的每种主要因子（IRF-1，TNF-α，STAT 1，CREB， AP-1、IL-2和IL-6）。负责启动和介导运动PC的特定NOS亚型将被 通过eNOS、iNOS和nNOS的靶向基因破坏最终鉴定。翻译后调节 将通过用HPLC偶联的方法鉴定iNOS上的精确磷酸化位点来阐明运动后24小时的iNOS。 电喷雾离子化质谱最后，Src PTKs在iNOS翻译后调节中的作用将被进一步研究。 通过在Src PTK抑制剂存在和不存在下测量iNOS活性和酪氨酸磷酸化来建立。这 这项提案应该产生重要的新见解，使心脏不仅适应物理机制， 强调，也强调一般。阐明运动PC的机制可能具有重要的治疗意义。

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.