IN VITRO MODEL OF PRECONDITIONING WITH ISOLATED MYOCYTES

分离的心肌细胞预处理的体外模型

• 批准号: 6043816
• 负责人: CHARLES E GANOTE
• 金额: $ 17.67万
• 依托单位: EAST TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6043816
• 关键词: adenosine triphosphate; calcium flux; cardiac myocytes; chemoprevention; cytoprotection; glycolysis; heart metabolism; laboratory rabbit; myocardial ischemia /hypoxia; phosphatase inhibitor; phosphorylation; potassium channel; species difference; swine; tissue /cell culture

DESCRIPTION: (Adapted from the Investigator's Abstract) This is a proposal to study mechanisms of ischemic preconditioning using isolated cardiac myocytes from rabbits, as an example of an animal with a fast heart rate, and from pigs, as an example of an animal with a slow heart rate. Preliminary data show 1) that rabbits precondition by mechanisms independent of K-ATP channels and ATP conservation, while both of these factors are required for protection of pig cardiomyocytes. 2) Ca+2-free ischemia protects in the pig but not in the rabbit. 3) Fostriecin, a water soluble, non-toxic protein phosphatase 2A-selective inhibitor, mimics ischemic preconditioning in both species, but protection can be blocked by K-ATP channel inhibitors in pig but not rabbit. Fostriecin is prototypic of ischemia-selective protein phosphatase inhibitors that could protect by either a) maintaining the phosphorylated state of K-ATP channels which would keep these channels open at higher ATP concentration or b) maintaining the phosphorylated state of cytoskeletal proteins during ischemia. 4) Protein phosphatase inhibitors protect rabbit cells even when added during ischemia, after the onset of rigor. This type of protection which occurs after the myocytes are already severely ATP depleted is new and of potential clinical importance. The basic hypothesis to be tested is that the fundamental protective effect of preconditioning is due to better maintenance of phosphorylation of critical proteins, and ultimately to phosphorylation dependent cytoskeletal integrity. In the rabbit, the hypothesis is that protection is due to a direct effect on cytoskeletal proteins whereas in the pig, the protective effect is an energy sparing effect which indirectly preserves cytoskeletal phosphorylation. The first Specific Aim is to compare the effects of protein phosphatase inhibitors and preconditioning in rabbit and pig cardiac myocytes. The applicant presents preliminary data that preconditioning does not affect protein phosphatase activity per se but if the endpoint of preconditioning is increased phosphorylation of critical proteins, then there should be considerable similarity between the two interventions, and where differences appear, they should be related to differences in the ultimate level of specific protein phosphorylation. The second specific aim is to compare the response of rabbit and pig myocytes to ATP-sparing interventions. The rationale is that ATP seems to be irrelevant in the rabbit myocytes whereas it seems to be very important in the pig myocytes. In the rabbit myocytes, a similar protective effect is obtained with preconditioning and with addition of protein phosphatase inhibitors after 75 minutes of ischemia, at which time the ATP is extremely low. In the pig myocytes, the hypothesis is that preconditioning is protective because of phosphorylation of the K-ATP channel which has an ATP-sparing effect, and that ultimately, the pig myocytes differ from rabbit in that ATP is necessary to maintain cytoskeletal protein phosphorylation. The proposal culminates with the third specific aim which is to show how specific cytoskeletal protein phosphorylation relates to lethal injury. Ultimately, if the hypothesis is correct, protection should be related to enhanced phosphorylation of specific proteins, regardless of whether the intervention is preconditioning or addition of a phosphatase inhibitor, and since he will be using phosphatase inhibitors with different specificities, some of which are protective and some of which are not, he has the opportunity to separate which protein phosphorylation is most important.

描述：（根据调查员的摘要改编）这是一个建议 使用孤立的心脏研究缺血性预处理的机制 来自兔子的心肌细胞，作为快速心率的动物的一个例子， 从猪起，作为心率缓慢的动物的一个例子。 初步数据显示1）独立于机制的兔子先决条件 K-ATP渠道和ATP保护的，而这两个因素均为 保护猪心肌细胞所需。 2）CA+2-无缺血 在猪中保护，但不能在兔子中保护。 3）福斯特环，水溶性， 非毒性蛋白磷酸酶2a选择性抑制剂，模仿缺血性 在这两个物种中进行预处理，但可以通过K-ATP阻止保护 在猪而不是兔子中引导抑制剂。 Fostriecin是原型的 缺血选择性蛋白磷酸酶抑制剂，可以通过 a）维持K-ATP通道的磷酸化状态 保持这些通道以较高的ATP浓度或b）维护 缺血期间细胞骨架蛋白的磷酸化状态。 4）蛋白质 磷酸酶抑制剂即使在缺血期间加入，也可以保护兔细胞 严格发作后。 这种类型的保护是在 心肌细胞已经严重耗尽了，这是新的，潜在的临床 重要性。 要测试的基本假设是基本 预处理的保护作用是由于更好地维护 临界蛋白的磷酸化，最终达到磷酸化 依赖性细胞骨架完整性。 在兔子中，假设是 保护是由于对细胞骨架蛋白的直接影响，而在 猪，保护效果是一种间接的能量支撑效应 保存细胞骨架磷酸化。 第一个具体目的是 比较蛋白质磷酸酶抑制剂的作用和预处理 兔子和猪心肌细胞。 申请人提供初步数据 该预处理本身不会影响蛋白质磷酸酶活性，而是 如果预处理的终点增加了关键的磷酸化 蛋白质，然后两者之间应该有很大的相似性 干预措施以及出现差异的地方，应与 特异性蛋白质磷酸化的最终水平的差异。 这 第二个具体目的是将兔和猪心肌的反应与 ATP比较干预措施。 理由是ATP似乎无关紧要 在兔心肌细胞中，而在猪中似乎很重要 心肌细胞。 在兔心肌细胞中，获得了类似的保护作用 通过预处理并添加蛋白质磷酸酶抑制剂 缺血75分钟后，此时ATP极低。 在 猪肌细胞，假设是预处理是保护性的 由于具有ATP的K-ATP通道的磷酸化 效果，最终，猪肌细胞与该ATP中的兔子不同 维持细胞骨架蛋白磷酸化是必要的。 提案 最终以第三个特定目的表明特定目标 细胞骨架蛋白磷酸化与致命损伤有关。 最终，如果假设正确，保护应与 不管是否是否 干预是预处理或添加磷酸酶抑制剂，并且 由于他将使用具有不同特异性的磷酸酶抑制剂 其中一些是保护性的，其中一些不是，他有 分离哪种蛋白质磷酸化的机会是最重要的。