MYOCARDIAL ISCHEMIC INJURY--MODIFICATION BY DRUG THERAPY

该建议的总体目标是阐明早期和晚期预处理对抗致死性缺血的机制。我们的主要假设是，在实验和药物干预的影响下，细胞内Ca++的轻微增加是预处理的强烈触发因素，蛋白激酶C是导致保护的信号级联的主要参与者。线粒体KATP通道的打开被认为是保护的关键，它依赖于细胞内[ca++]i增加对PKC的激活。线粒体占细胞总体积的三分之一，具有多因子功能，在应激和缺血条件下的肌细胞存活中起着重要作用。在即时应激条件下，线粒体KATP通道的打开通过调节Ca++稳态和增加ATP合成来维持细胞完整性，而延迟保护是通过增强抗氧化剂合成来介导的。细胞培养和完整的心脏都将被使用。具体目的是：确定氧化应激引起的[Ca++]i波动是否引起早期和晚期保护；[ca++]i介导的PKC激活是否与早期和晚期PC有关；线粒体KATP通道的开放是否在晚期PC的保护中起重要作用；线粒体KATP通道的打开是否依赖于PKC的激活及其在PC早期和晚期向线粒体的易位；[Ca++]i通过调节通透性转变抑制细胞凋亡，从而减轻细胞损伤；如果晚期PC由[Ca++]，这是由于合成抗氧化剂，抑制细胞凋亡。广泛的多学科技术，包括生物化学、细胞生物学、分子生物学、电子显微镜、免疫细胞化学和药理学方法，将被用于整合细胞水平的信息和器官水平的功能。这项研究将对预适应的分子机制产生重要的新见解，并将对基于心肌独特的内源性适应性反应设计治疗干预具有重要意义。

The overall objective of this proposal is to elucidate the mechanism of early and late preconditioning against lethal ischemia. Our major hypothesis is that a mild increase in intracellular Ca++ effected by experimental and pharmacological interventions is a strong trigger for preconditioning and protein kinase C is a major player in the signaling cascade leading to protection. The opening of mitochondrial KATP channel which is believed to be pivotal in protection is dependent on the activation of PKC by intracellular [Ca++]i increase. Mitochondria which occupy a third of total cellular volume and perform multifactorial functions play a significant role in the survival of myocytes in stress and ischemic conditions. Under immediate stress conditions, the opening of mitochondrial KATP channel maintains the cell integrity by regulation of Ca++ homeostasis and increased ATP synthesis while the delayed protection is mediated by augmented synthesis of antioxidants. Both cell cultures and intact hearts will be used. The specific aims are to: determine whether [Ca++]i fluctuations by oxidative stress elicit both early and late protection; whether [Ca++]i mediated activation of PKC is responsible for early and late PC; whether opening of mitochondrial KATP channel is important in protection in late PC; whether opening of mitochondrial KATP channel is dependent on PKC activation and its translocation to mitochondria during early and late PC; if early and late PC by [Ca++]i reduces cell injury by inhibiting apoptosis by regulating permeability transition; if late PC by [Ca++]i is due to synthesis of antioxidants which inhibits apoptosis. A wide range of multidisciplinary techniques, including biochemistry, cell biology, molecular biology, electron microscopy, immunocytochemistry and pharmacological approaches will be used to integrate the information at the cell level with the function at the organ level. This investigation will produce important new insights into the molecular mechanisms of preconditioning and will have important implications for designing therapeutic interventions based on the unique endogenous adaptive responses by the myocardium.