Chemogenetic approaches to define the roles of redox dysfunction in the cardiomyopathy of aging

此R21应用程序提出了将开发和验证新模型的实验， 通过使用化学发生方法产生氧化还原应激， 心脏和其他组织。“年龄相关性心肌病”的发展与 心脏收缩和舒张功能障碍，氧化还原应激，以及高死亡率- 已经被广泛地记录下来。我们最近开发了一种新的化学基因 直接和特异性地导致心肌病发展的方法 这是由心脏氧化还原应激增加引起的。在建议的研究中，我们会 在衰老的背景下检查这种新的心肌病模型。我们用了强心剂 腺相关病毒血清型9（AAV 9）以表达酵母D-氨基酸氧化酶（DAAO） 在体内选择性地在心肌细胞中产生ROS，引起心脏的氧化还原应激 导致扩张型心肌病DAAO是一种立体特异性酶， 过氧化氢（H2 O2）仅当D-氨基酸作为底物时。由于大多数 哺乳动物组织仅含有L-氨基酸，酵母DAAO是无活性的，直到D-氨基酸 提供酸。我们将重组DAAO-AAV 9病毒注射到小鼠（或大鼠）中，并使用 细胞成像方法显示H2 O2仅在心肌细胞中产生 在向肌细胞中加入D-丙氨酸后。然后，我们开发了一种体内化学发生模型， 在心脏中产生慢性氧化还原应激：我们将D-丙氨酸添加到小鼠的饮用水中， 感染了重组DAAO-AAV 9。与对照动物相比， DAAO-AAV 9感染小鼠在D-丙氨酸治疗3周内出现扩张性心肌病 治疗我们建议扩展这些方法，以研究氧化还原应激在 年龄相关性心肌病的研究 重组DAAO-AAV 9，然后分析生理、代谢、生化， 转录组学和蛋白质组学特征， 接近。这些实验可能会发现新的治疗靶点，以防止或 治疗老年性心肌病我们还建议开发DAAO-TGLoxP 转基因小鼠品系，将允许分析衰老中的组织特异性氧化还原应激 使用Cre-Lox方法在心脏和其他组织中表达DAAO。我们 预期DAAO的组织特异性转基因表达将提供新的 对广泛的疾病状态的机械见解，其中年龄相关的 组织病理学与氧化还原功能障碍有关。

This R21 application proposes experiments that will develop and validate new models of aging-related cardiac pathology by using chemogenetic approaches to generate redox stress in the heart and other tissues. The development of an “age-related cardiomyopathy”– associated with both systolic and diastolic cardiac dysfunction, redox stress, and a high mortality rate– has been extensively documented. We have recently developed a new chemogenetic approach that leads to the development of cardiomyopathy that is directly and specifically caused by inducing an increase in redox stress in the heart. In the proposed studies, we will examine this new cardiomyopathy model in the context of aging. We used a cardiotropic adeno-associated virus serotype 9 (AAV9) to express a yeast D-amino acid oxidase (DAAO) that selectively generates ROS in cardiac myocytes in vivo, causing redox stress in the heart and leading to dilated cardiomyopathy. DAAO is a stereospecific enzyme that generates hydrogen peroxide (H2O2) only when D-amino acids are available as substrate. Since most mammalian tissues contain only L-amino acids, the yeast DAAO is inactive until D-amino acids are provided. We injected recombinant DAAO-AAV9 virus into mice (or rats), and used cellular imaging methods to show that H2O2 is robustly generated in cardiac myocytes only after adding D-alanine to myocytes. We then developed an in vivo chemogenetic model to generate chronic redox stress in the heart: we added D-alanine to the drinking water of mice that had been infected with recombinant DAAO-AAV9. Compared to control animals, the DAAO-AAV9-infected mice develop a dilated cardiomyopathy within 3 weeks of D-alanine treatment. We propose to extend these approaches to study the roles of redox stress in age-associated cardiomyopathy by infecting middle-aged and elderly mice with recombinant DAAO-AAV9, and then analyzing physiological, metabolic, biochemical, transcriptomic, and proteomic characteristics using both in vivo and ex vivo approaches. These experiments are likely to identify new therapeutic targets to prevent or treat the age-associated cardiomyopathy. We also propose to develop DAAO-TGLoxP transgenic mouse lines that will permit analyses of tissue-specific redox stress in aging using Cre-Lox methodologies to express DAAO in the heart and other tissues. We anticipate that tissue-specific transgenic expression of DAAO will provide new mechanistic insights into the broad range of disease states in which age-associated tissue pathology is associated with redox dysfunction.