Antioxidant transgene in diabetic cardiomyopathy

描述（申请人提供）：许多糖尿病患者在没有血管疾病的情况下也患有严重的心肌病。来自动物模型和临床样本的证据表明活性氧（ROS）与糖尿病性心肌病的发展有关。我们的研究结果证实了活性氧的重要性。在I型糖尿病的OVE26模型中，我们发现心脏氧化损伤增加，糖尿病心肌细胞ROS的产生增加，抗氧化转基因的保护作用增强。针对心肌细胞的两种不同的抗氧化蛋白，金属硫蛋白（MT）和过氧化氢酶阻止了糖尿病性心肌病的大多数主要特征，包括收缩性降低、钙稳态受损和形态破坏。我们最近的数据表明线粒体电子传递和NADPH氧化酶在糖尿病心肌细胞氧化损伤的产生中起作用。本项目将确定糖尿病心脏中ROS生成增加的细胞来源，确定哪些蛋白质是ROS修饰的靶点，并测试ROS在I型糖尿病中的重要性是否也适用于II型糖尿病的心肌病。ROS的来源将通过测量糖尿病心肌细胞中ROS的产生作为基因敲除或药物抑制潜在来源的功能来测试。培育糖尿病小鼠以敲除小鼠的NADPH氧化酶将确定该酶是否为糖尿病心肌病的发展所必需。利用蛋白质组学分析结合抗氧化修饰氨基酸的抗体，鉴定整个心脏、线粒体和肌浆网中ROS修饰的蛋白靶点。为了分析ROS在II型糖尿病心肌病中的作用，我们将在II型糖尿病agouti模型中表征心脏形态、ROS产生、心功能和氧化损伤。将Agouti小鼠培育成心脏特异性抗氧化转基因保护的小鼠，以确定氧化损伤是否是该模型心肌病的必要条件。

DESCRIPTION (provided by applicant): Many diabetics suffer from severe cardiomyopathy even in the absence of vascular disease. Evidence from animal models and clinical samples implicate reactive oxygen species (ROS) in the development of diabetic cardiomyopathy. Our results confirm the importance of ROS. In the OVE26 model of Type I diabetes we find increased cardiac oxidative damage, increased production of ROS by diabetic cardiomyocytes and protection by antioxidant transgenes. Two different antioxidant proteins targeted to the cardiomyocyte, metallothionein (MT) and catalase prevented most of the primary characteristics of diabetic cardiomyopathy, including reduced contractility, impaired calcium homeostasis and disrupted morphology. Our most recent data implicate mitochondrial electron transport and the enzyme NADPH oxidase in the production of oxidative damage in diabetic cardiomyocytes. This project will identify the cellular source of increased ROS production in diabetic hearts, determine which proteins are the targets of ROS modification and test whether the importance of ROS in Type I diabetes also applies to cardiomyopathy in Type II diabetes. Sources of ROS will be tested by measuring ROS production in diabetic cardiomyocytes as a function of genetic knockout or drug inhibition of potential sources. Breeding diabetic mice to knockout mice for NADPH oxidase will determine if this enzyme is required for the development of diabetic cardiomyopathy. Protein targets of ROS modification in whole heart, mitochondria and sarcoplasmic reticulum will be identified by using proteomic analysis combined with antibodies against oxidatively modified amino acids. To analyze the role of ROS in Type II diabetic cardiomyopathy we will characterize cardiac morphology, ROS production, cardiac function and oxidative damage in the agouti model of Type II diabetes. Agouti mice will be bred to mice protected by cardiac specific antioxidant transgenes to determine if oxidative damage is a requirement for cardiomyopathy in this model.