Adenosine and oxidative stress in myocardial tissue

本项目的目标是开发新的治疗策略，通过针对心脏的内源性防御机制来提高心肌对缺血的耐受性。拟议研究的具体目的是确定腺苷和腺苷受体激活减少细胞和亚细胞活性氧（RO2）形成和氧化应激的机制；腺苷在增强内源性抗氧化防御机制中的作用。研究将在分离的大鼠心室肌细胞和猪体内局部缺血制备中进行。在猪体内研究中，心脏微透析技术将用于测量间质液中的活性氧水平。收缩功能障碍将通过区域收缩力的负荷不敏感措施来评估。在肌细胞中，RO2将用荧光探针测量二乙酸二氯荧光素的收缩性。在肌细胞中，ROS将用荧光探针二氯荧光素二醋酸酯测量，线粒体氧化应激将通过监测线粒体黄蛋白自身荧光来估计。氧化应激和细胞功能将测量肌细胞提交刺激缺血和再灌注。抗氧化水平将通过测定还原型谷胱甘肽、氧化谷胱甘肽和NADPH/NADP+比值来确定。腺苷受体激动剂的作用，单独或联合氧化应激和抗氧化状态将与心肌细胞抽搐幅度和休克程度以及缺氧-再氧化后的细胞功能相关。这一发现可能会导致新的治疗策略的发展，以改善缺血性心脏病患者的血流动力学表现和生存。

The goal of this project is to develop new therapeutic strategies to improve myocardial tolerance to ischemia by targeting the heart's endogenous defense mechanisms against oxidative stress. The specific aim of the proposed research is to determine the mechanism(s) whereby adenosine and adenosine receptor activation reduce cellular and subcellular reactive oxygen species (RO2) formation and oxidative stress; and the role adenosine plays in enhancing endogenous antioxidant defense mechanisms. Studies will be performed in isolated rat ventricular myocytes and a porcine in vivo regional ischemia preparation. In the in vivo porcine studies, the cardiac microdialysis technique will be used to measure interstitial fluid levels of ROS. Contractile dysfunction will be evaluated by load insensitive measures of regional contractility. In myocytes, RO2 will be measures with the fluorescent probe dichlorofluorescein diacetate, contractility. In myocytes, ROS will be measured with the fluorescent probe dichlorofluorescein diacetate, and mitochondrial oxidative stress will be estimated by monitoring mitochondrial flavoprotein autofluorescence. Oxidative stress and cell function will be measured in myocytes submitted to stimulated ischemia and reperfusion. Antioxidant levels will be determined by measuring reduced glutathione, oxidized glutathione and the NADPH/NADP+ ratio. The effects of adenosine receptor agonists, alone and in combination of oxidative stress and antioxidant status will be correlated with myocyte twitch amplitude and the extent of stunning and cell function following hypoxia-reoxygenation. The findings could lead to the development of new therapeutic strategies to improve hemodynamic performance and survival in patients with ischemic heart disease.