MYOCARDIAL ISCHEMIC INJURY--MODIFICATION BY DRUG THERAPY

本研究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，实验和药理学干预影响细胞内Ca++的轻度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中的主要参与者。 线粒体KATP通道的开放依赖于细胞内[Ca++]i升高激活PKC，而KATP通道的开放被认为是保护心肌的关键。线粒体占据了总细胞体积的三分之一，并执行多因素功能，在应激和缺血条件下的心肌细胞存活中起着重要作用。 在即刻应激条件下，线粒体KATP通道的开放通过调节Ca++稳态和增加ATP合成来维持细胞的完整性，而延迟保护则通过增加抗氧化剂的合成来介导。将使用细胞培养物和完整心脏。 具体目标是：确定氧化应激引起的[Ca++]i波动是否会引起早期和晚期保护作用;[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的保护中发挥重要作用;线粒体KATP通道的开放是否依赖于PKC激活及其在早期和晚期PC期间向线粒体的移位;如果早期和晚期PC由[Ca++]i通过调节通透性转换抑制细胞凋亡而减轻细胞损伤，如果晚期PC由[Ca++]i是由于合成抑制细胞凋亡的抗氧化剂。 将使用多种多学科技术，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，将细胞水平的信息与器官水平的功能结合起来。 这项研究将产生重要的新的见解预处理的分子机制，并将有重要的意义，设计治疗干预措施的基础上独特的内源性适应性反应的心肌。

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.