ADENOSINE AND PYRUVATE PROTECTION DURING HEART SURGERY

描述(改编自申请者摘要)：心肌顿抑和 心肌梗塞是心脏手术后发病率和死亡率的主要原因， 心脏移植和无创心肌血管重建术。这个 申请者的长期目标是开发新的治疗策略 靶向心脏内源性激素提高心肌对缺血的耐受性 对抗氧化应激的防御机制。这种相互竞争的延续 应用重点放在内源化合物的有益作用 腺苷和丙酮酸。重要的新发现表明，腺苷 受体激活和丙酮酸均可降低活性氧自由基 在缺血再灌流的心脏中形成和增加抗氧化防御。 拟议研究的具体目标是确定：1)角色，如果 肌细胞腺苷A3和A2受体在介导腺苷诱导中的作用 心肌保护；2)腺苷受体的作用机制(S) 活化和丙酮酸减少细胞和亚细胞ROS的形成 氧化应激；以及3)腺苷和丙酮酸在促进 内源性抗氧化防御机制。研究将在一个 猪体内局部缺血制备，大鼠离体心灌流， 和大鼠分离的心室肌细胞。在心肌细胞中，ROS的测量方法是 荧光探针二氯二乙酸荧光素和线粒体 氧化应激将通过监测线粒体黄素蛋白来评估 自体荧光。心脏微透析技术将用于测量 组织间液中ROS和脂质过氧化产物的水平 丙二醛在猪体内的研究。氧化应激与细胞死亡 (凋亡和坏死)将在提交给模拟的心肌细胞中测量 缺血再灌流制剂将用于估计间质液体 (ISF)羟基自由基和。抗氧化剂水平将由以下因素决定 测定还原型谷胱甘肽、氧化谷胱甘肽、NADPH/NADP比率，以及 抗氧化/抗凋亡蛋白Bc l-2的表达及其与细胞凋亡的关系 促氧化/促凋亡蛋白Bax。腺苷受体的作用 激动剂和丙酮酸，单独和联合，对氧化应激和 抗氧化状态将与心肌细胞抽动幅度和 缺氧-复氧后细胞凋亡和坏死的程度及 模拟缺血再灌流。ROS、丙二醛和 猪心肌中的抗氧化剂将与负荷不敏感相关 测量局部收缩能力以及细胞的凋亡性和坏死性。 拟议的实验旨在阐明细胞和亚细胞 腺苷受体激活及其保健作用的机制 丙酮酸，意在进一步提高其疗效。调查结果 应引导发展新的治疗策略，以提高 缺血性心脏病患者的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.