CARDIOPROTECTIVE AGENTS IN ISCHEMIC MYOCYTES

缺血性心肌细胞中的心脏保护剂

• 批准号: 6183372
• 负责人: RICHARD E HONKANEN
• 金额: $ 20.6万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6183372
• 关键词: binding sites; cardiac myocytes; cardiovascular agents; chemoprevention; cytoprotection; drug screening /evaluation; heart pharmacology; laboratory rabbit; myocardial ischemia /hypoxia; pharmacokinetics; phosphatase inhibitor; phosphoprotein phosphatase; reperfusion

DESCRIPTION: (adapted from the applicant's abstract) This is an application to study agents which may be protective against myocardial ischemic injury. The applicants have found a novel compound, fostriecin, which has been purified from the fermentation broth of Streptomyces pulveraceus, and which has a pronounced cardioprotective effect in ischemic rabbit hearts. At concentrations that have no apparent toxicity, fostriecin reduces the size of a myocardial infarct induced by a controlled coronary occlusion from approximately 33% to approximately 8% of the risk zone. On the molecular level, fostriecin is a potent inhibitor of type 2A, a strong inhibitor of type 5, and a weak inhibitor of type 1 serine/threonine protein phosphatases, suggesting that the cardioprotective properties of fostriecin arise from its inhibitory activity against these or related phosphatases. The goal of the proposal is to test the hypothesis that the inhibition of certain serine/threonine protein phosphatases is the biochemical basis for the cardioprotective properties of fostriecin observed in ischemic heart tissue. The aims are 1) to characterize fostriecin-sensitive protein phosphatases in heart; 2) to characterize the high affinity fostriecin binding domain of PP2A and PP5; and 3) to determine the role of fostriecin sensitive protein phosphatases in ischemic cardiac tissue. Through a better understanding of the molecular events by which the inhibition of these enzymes limits myocardial tissue damage, insight into the mechanisms that produce protection will be gained. Such insight should aid in the development of new and improved methods for the medical management of an acute MI. 1) Significance: The proposal is novel. PKC activation is thought to play an important role in ischemic preconditioning, and it is logical to investigate the role of protein phosphatases in the protective effect of ischemic preconditioning since inhibition of protein phosphatases could modulate or amplify the effects to PKC stimulation. Previous work to understand the importance of protein phosphatases has been hampered by the lack of selective inhibitors; non-selective inhibition of protein phosphatases could have offsetting effects which would make it very difficult to draw definitive conclusions. For example, early work by Ganote and co-workers showed that the protein phosphatase inhibitor calyculin A had conflicting results on ischemic injury. It accelerated the onset of contracture but slowed the development of lethal injury. Calyculin is a non-selective protein phosphatase inhibitor and the results could reflect opposing effects of sustained phosphorylation of different proteins. The proposed approach to develop more selective inhibitors will help to resolve this issue. 2) Approach: The first specific aim is to characterize the fostriecin-sensitive PPs in heart. This involves purification followed by dose-inhibition studies with fostriecin. The applicant published a 1998 paper in JBC reporting the cloning, expression, and characterization of PP7 in human retina, and he has provided more detail of the proposed PP purification methods in the revised application. The second specific aim is to characterize the fostriecin binding domains of the relevant PPs. Preliminary data are presented on the generation of PP1/PP2A chimeras to identify PP inhibitor binding sites, and the data suggest that okadaic acid and fostriecin bind to different sites. This specific aim has been modified to include a collaboration to generate crystal structures of PP/fostriecin complexes. The third specific aim is to characterize the role of PPs in ischemic preconditioning. The PI and his collaborators have already looked to see if PP1 or PP2A activity is affected by ischemic preconditioning and has found that there was no difference between non-preconditioned and preconditioned myocardium at any time during a subsequent sustained period of ischemia, and therefore, he proposes to pursue the hypothesis that it could be PP5 activity which is decreased by preconditioning. This is a reasonable hypothesis that would explain the similarity between the protective effect of ischemic preconditioning and fostriecin. An alternative hypothesis to explain the similarity between the effects of preconditioning and fostriecin is that preconditioning could stimulate PKC and achieve a relatively high level of phosphorylation of a PKC substrate whereas fostriecin treatment results in a comparably high level of phosphorylation of the same substrate by slowing the dephosphorylation. The latter hypothesis will be tested by observing whether the protective effect of fostriecin is abolished in the presence of a PKC inhibitor. Likewise, it is possible that one of the stress-activated protein kinase pathways could be involved in phosphorylation of a substrate that could be dephosphorylated by a fostriecin-sensitive PP; this possibility will be tested by pretreating with various MAP kinase inhibitors. Finally, they will explore the possibility that HSP27 phosphorylation is involved in the protective effect of ischemic preconditioning and fostriecin by measuring the effect of fostriecin on HSP27 phosphorylation A minor weakness is that the proposed method for assessing changes in PP5 activity may not be reliable. Measurement of PP5 activity in the myocardium will be performed using a subtraction procedure, and if PP1 and PP2A activities are much higher than PP5 activity, it may be difficult to detect changes in PP5 activity by subtracting PP1 and PP2A activity from the total PP activity. 3) Innovation: The possibility that PPs are involved in ischemic hypothesis that is difficult to test because of the lack of specific PP inhibitors. Most of the currently available PP inhibitors are toxic and inhibit multiple PPs. Better understanding of the interaction between various inhibitors and different PPs may aid in the development of better inhibitors that are selective for only one class of PP. A recently developed PP inhibitor has been found to be remarkably protective in a model of ischemia/reperfusion injury. Further study of the effects of this PP inhibitor may lead to greater insight concerning downstream effectors of preconditioning.

描述：（根据申请人的摘要进行了改编）这是 可能可以防止心肌脑缺血损伤的研究剂。这 申请人发现了一种新颖的化合物Fostriecin，已被纯化 从链霉菌的发酵汤中，它具有 缺血性兔心脏中明显的心脏保护作用。浓度 没有明显的毒性，Fostriecin降低了心肌的大小 受控冠状动脉闭塞引起的梗塞从大约33％到 大约8％的风险区。在分子水平上，fostriecin是一个 2A型的有效抑制剂，5型抑制剂和弱抑制剂 1型丝氨酸/苏氨酸蛋白磷酸酶的 Fostriecin的心脏保护特性是由其抑制活性引起的 反对这些或相关的磷酸酶。该提议的目的是测试 假设抑制某些丝氨酸/苏氨酸蛋白磷酸酶 是Fostriecin的心脏保护特性的生化基础 在缺血性心脏组织中观察到。目的是1）表征 心脏中的fostriecin敏感蛋白磷酸酶； 2）表征高 PP2A和PP5的亲和力Fostriecin结合域； 3）确定 fostriecin敏感蛋白磷酸酶在缺血性心脏组织中的作用。 通过更好地理解抑制的分子事件 这些酶限制了心肌组织损害，洞察机制 将获得生产保护。这种见解应有助于 开发急性医疗管理的新方法和改进的方法 mi。 1）意义：该提议是新颖的。 PKC激活被认为可以发挥 在缺血性预处理中的重要作用，研究是合乎逻辑的 蛋白质磷酸酶在缺血的保护作用中的作用 由于抑制蛋白质磷酸酶可能会调节或 扩大对PKC刺激的影响。以前的工作以了解 缺乏选择性的蛋白质磷酸酶的重要性受到阻碍 抑制剂；蛋白质磷酸酶的非选择性抑制可能具有 抵消效果，这将使绘制确定性很难 结论。例如，Ganote和同事的早期工作表明 蛋白质磷酸酶抑制剂钙钙蛋白A A在缺血性上有冲突的结果 受伤。它加速了缔约的发作，但减慢了 致命伤害。钙钙蛋白是一种非选择性蛋白质磷酸酶抑制剂和 结果可能反映了持续磷酸化的相反影响 不同的蛋白质。提出的方法是开发更多选择性抑制剂 将有助于解决这个问题。 2）方法：第一个具体目的是表征fostriecin敏感的 pps心中。这涉及纯化，然后进行剂量抑制研究 与Fostriecin。申请人在JBC上发表了一篇1998年的论文 人类视网膜中PP7的克隆，表达和表征，他有 提供了修订的PP纯化方法的更多详细信息 应用。 第二个具体目的是表征Fostriecin结合域 相关的PPS。初步数据介绍了PP1/pp2a的生成 嵌合体鉴定PP抑制剂结合位点，数据表明 冈田酸和fostriecin与不同位点结合。这个具体目标是 修改以包括一个合作，以生成晶体结构 PP/Fostriecin复合物。 第三个具体目的是表征PPS在缺血性中的作用 预处理。 PI和他的合作者已经看过PP1是否存在 或PP2A活性受缺血预处理的影响，发现 非重构和预处理之间没有区别 在随后的持续缺血期间的任何时候，心肌， 因此，他提议追求可能是PP5活动的假设 通过预处理减少。这是一个合理的假设 会解释缺血的保护作用之间的相似性 预处理和fostriecin。一个解释的替代假设 预处理和fostriecin的效果之间的相似性是 预处理可以刺激PKC并达到相对较高的水平 PKC底物的磷酸化，而Fostriecin治疗导致A 同一底物的磷酸化水平相当高 去磷酸化。后一个假设将通过观察是否是否 在存在PKC的情况下，消除Fostriecin的保护作用 抑制剂。同样，有应力激活蛋白之一可能 激酶途径可能参与底物的磷酸化 被fostriecin敏感的PP脱磷酸化；这种可能性将是 通过对各种MAP激酶抑制剂进行预处理测试。最后，他们会的 探索Hsp27磷酸化参与的可能性 缺血性预处理和fostriecin的保护作用通过测量 Fostriecin对HSP27磷酸化的影响 一个较小的弱点是评估PP5变化的建议方法 活动可能不是可靠的。测量心肌中PP5活性 将使用减法程序以及PP1和PP2A活动进行 比PP5活动高得多，可能很难检测到PP5的变化 通过从总PP活性中减去PP1和PP2A活性的活性。 3）创新：PPS参与缺血的可能性 由于很难检验的假设 缺乏特定的PP抑制剂。大多数当前可用的PP抑制剂 有毒并抑制多个pps。更好地理解互动 在各种抑制剂和不同的PP之间可能有助于发展 仅选择一类PP的更好的抑制剂。最近 已经发现开发的PP抑制剂在一个模型中具有明显的保护性 缺血/再灌注损伤。进一步研究该PP抑制剂的作用 可能会导致有关预处理下游效应子的更多见解。