CARDIAC OXIDANTS & APOPTOSIS: LESSONS OF PRECONDITIONING

描述：本提案代表了研究者之间的合作， 肺内科、重症监护和急诊科 新成立的紧急复苏研究中心， 芝加哥。建议进行研究，以检查 缺血/再灌注（I/R）诱导的心肌细胞凋亡，并检查 低氧预适应对细胞凋亡的保护作用。的 该建议的中心假设是，预处理降低了 凋亡性细胞死亡通过一些机制，阻止启动 和/或细胞凋亡的传播。为了验证这些假设，我们提出了三个 具体目标：（1）测试哪些活性氧（ROS）作为 细胞凋亡的细胞内激活剂。实验将（a）确定 包括线粒体膜破坏的凋亡事件序列 细胞色素c释放到胞质溶胶中， 半胱天冬酶-9作为“起始物”半胱天冬酶以及下游半胱天冬酶-3和其它; (b)使用特定的抑制治疗，评估改变的 超氧化物和/或H2 O2对这些凋亡事件的影响。(2)确定 预处理是否通过Bcl-2家族阻断某些凋亡事件 通过PKC介导的磷酸化调节。实验将（a）确定 缺血前和缺血后Bcl-2、Bcl-x和Bax的表达水平， 在预处理（PC）或非PC细胞中的I/R期间的心肌细胞;并确定 PC对Bcl-2蛋白丝氨酸磷酸化的影响（B） PKC、Bcl-2的过度表达和低表达/阻断以及 非磷酸化Bcl-2蛋白对氧化应激，一氧化氮合成， NF-κ B活化和凋亡事件。(3)确定关系 NF-κ B和一氧化氮（NO）合成之间的关系。（一） 测定PC对IkappaB磷酸化的影响， NF-κ B的活化和核转位;（B）测定表达 的即时早期基因，铝和chIAP，并检查其影响， 线粒体事件，ROS生成，氧化应激，NO生成，半胱天冬酶 活性和凋亡细胞死亡通过相关和直接的过度 表达和反义研究。(c)确定NO生成水平 在有或没有PC的再灌注过程中， 特异性阻断NO生成对caspase活性及后续细胞 每种情况下都会死亡。这些研究的结果有可能 改善心脏骤停和其他缺血性心脏病后复苏后护理 心肌梗塞和中风等疾病。

DESCRIPTION: This proposal represents collaboration between investigators in the Sections of Pulmonary Medicine & Critical Care and Emergency Medicine under the newly formed Emergency Resuscitation Research Center at the University of Chicago. Studies are proposed to examine the mechanisms and consequences of ischemia/reperfusion (I/R)-induced apoptosis in cardiomyocytes and to examine the protective effects of hypoxic preconditioning on this apoptosis. The central hypotheses of this proposal are that preconditioning decreases apoptotic cell death through a number of mechanisms that block the initiation and/or propagation of apoptosis. To test these hypotheses, we propose three specific aims: (1) Test which reactive oxygen species (ROS) act as intracellular activators of apoptosis. Experiments will (a) Determine the sequence of apoptotic events including disruption of mitochondria membrane potential, release of cytochrome c into the cytosol, and activation of caspase-9 as "initiator" caspase as well as downstream casases-3 and others; (b) Using specific inhibitory treatments, assess the effects of altered superoxide and/or H202 during (I/R) on these apoptotic events. (2) Determine whether preconditioning blocks certain apoptotic events via Bcl-2 family regulation through PKC-mediated phosphorylation. Experiments will (a) Determine pre- and post-ischemic levels of expression of Bcl-2, Bcl-x, and Bax in cardiomyocytes during I/R in preconditioned (PC) or non-PC cells; and determine the effects of PC on serine phopshorylation of Bcl-2 proteins (b) Assess the effects of over and underexpression/blockade of PKC, Bcl-2, and expression of a nonphosphorylatable Bcl-2 protein on oxidant stress, nitric oxide synthesis, NF-kappaB activation and apoptotic events. (3) Determine the relationship between NF-kappaB and nitric oxide (NO) synthesis in preventing apoptosis. (a) Determine the effect of PC on phosphorylation Of IkappaB and subsequent activation and nuclear translocation of NF-kappaB; (b) Determine the expression of immediate-early genes, Al and chIAP, and examine their effects on mitochondria events, ROS generation, oxidant stress, NO generation, caspase activity and apoptotic cell death through correlative and direct over expression and antisense studies. (c) Determine the levels of NO generation during reperfusion with or without PC and the direct effects of generated NO or specific blockade of NO generation on caspase activity and subsequent cell death in each condition. Results from these studies have the potential to improve post-resuscitation care following cardiac arrest and other ischemic diseases such as myocardial infarction and stroke.