ADENOSINE AND PYRUVATE PROTECTION DURING HEART SURGERY

描述（改编自申请人摘要）：心肌顿抑和 梗塞是心脏手术后发病率和死亡率主要原因， 心脏移植和非侵入性心肌血管重建。 的 申请人的长期目标是开发新的治疗策略， 通过靶向心脏内源性 对氧化应激的防御机制。 这种相互竞争的延续 应用集中于内源性化合物的有益效果 腺苷和丙酮酸。 重要的新发现表明腺苷 受体激活和丙酮酸都可以减少活性氧（ROS） 形成和增加抗氧化剂防御缺血再灌注心脏。 拟议研究的具体目标是确定：1）如果 任何一种肌细胞腺苷A3和A2受体都在介导腺苷诱导的 心肌保护; 2）腺苷受体 活化和丙酮酸减少细胞和亚细胞ROS形成， 氧化应激;以及，3）腺苷和丙酮酸在增强 内源性抗氧化防御机制。 研究将在 猪体内局部缺血制备，离体灌注大鼠心脏， 和大鼠离体心室肌细胞。 在肌细胞中，ROS将用 荧光探针二氯荧光素二乙酸酯和线粒体 氧化应激将通过监测线粒体黄素蛋白来估计 自发荧光 心脏微透析技术将用于测量 组织液中ROS和脂质过氧化产物的水平 体内猪研究中的丙二醛。 氧化应激和细胞死亡 （凋亡和坏死）将在提交给模拟的心肌细胞中测量。 将使用缺血再灌注制备物来估计间质液 (ISF)羟基自由基水平。 抗氧化剂水平将通过以下方法确定： 测量还原型谷胱甘肽、氧化型谷胱甘肽、NADPH/NADP比率，以及 抗氧化/抗凋亡蛋白Bcl-2的表达和 促氧化/促凋亡蛋白Bax。 腺苷受体的作用 激动剂和丙酮酸盐单独和组合对氧化应激的作用， 抗氧化状态将与肌细胞抽搐幅度相关， 缺氧-复氧后凋亡和坏死细胞死亡的程度， 模拟缺血再灌注。 ROS、丙二醛和 猪心肌中的抗氧化剂将与负荷不敏感性相关 局部收缩性和凋亡及坏死细胞死亡的测量。 所提出的实验旨在阐明细胞和亚细胞 腺苷受体激活的有益作用的机制， 丙酮酸盐，目的是进一步增强它们的功效。 这些发现 应该导致新的治疗策略的发展，以改善 缺血性心脏病患者血流动力学表现和存活率。

Description (Adapted From The Applicant's Abstract): Myocardial stunning and infarction are a major cause of morbidity and mortality after heart surgery, heart transplantation, and noninvasive myocardial revascularization. The long-term goal of the applicant is to develop new therapeutic strategies to improve myocardial tolerance to ischemia by targeting the heart's endogenous defense mechanisms against oxidative stress. This competing continuation application focuses on the beneficial effects of the endogenous compounds adenosine and pyruvate. Significant new findings indicate that adenosine receptor activation and pyruvate may both reduce reactive oxygen species (ROS) formation and increase antioxidant defenses in the ischemic-reperfused heart. The specific aims of the proposed research are to determine: 1) the role, if any, myocyte adenosine A3 and A2, receptors play in mediating adenosine-induced myocardial protection; 2) the mechanism(s) whereby adenosine receptor activation and pyruvate reduce cellular and subcellular ROS formation and oxidative stress; and, 3) the role adenosine and pyruvate play in enhancing endogenous antioxidant defense mechanisms. Studies will be performed in a porcine in vivo regional ischemia preparation, isolated perfused rat hearts, and rat isolated ventricu1ar myocytes. In myocytes ROS will be measured with the fluorescent probe dichlorofluorescein diacetate, and mitochondrial oxidative stress will be estimated by monitoring mitochondrial flavoprotein autofluorescence. The cardiac microdialysis technique will be used to measure interstitial fluid levels of ROS and the lipid peroxidation product malonaldehyde in the in vivo porcine studies. Oxidative stress and cell death (apoptosis and necrosis) will be measured in myocytes submitted to simulated ischemia reperfusion preparation will be used t o estimate interstitial fluid (ISF) levels of hydroxyl radical and. Antioxidant levels will be determined by measuring reduced glutathione, oxidized glutathione, the NADPH/NADP ratio, and expression of the antioxidant/anti-apoptotic protein Bcl-2 and the pro-oxidant/pro-apoptotic protein Bax. The effects of adenosine receptor agonists and pyruvate, alone and in combination, on oxidative stress and antioxidant status will be correlated with myocyte twitch amplitude and the extent of apoptotic and necrotic cell death following hypoxia-reoxygenation and simulated ischemia-reperfusion. The levels of ROS, malonaldehyde, and antioxidants in porcine myocardium will be correlated with load insensitive measurements of regional contractility and apoptotic and necrotic cell death. The proposed experiments are designed to elucidate the cellular and subcellular mechanisms of the salutary effects of adenosine receptor activation and pyruvate with the intent to further enhance their efficacy. The findings should lead to the development of new therapeutic strategies to improve hemodynamic performance and survival in patients with ischemic heart disease.