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

化学遗传学方法确定氧化还原功能障碍在衰老心肌病中的作用

基本信息

批准号：
9922852
负责人：
Thomas Michel
金额：
$ 21.66万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-15 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9922852
关键词：
Age Aging Alanine Amino Acids Animal Model Biochemical Biological Cardiac Cardiac Myocytes Cardiomyopathies Cells Characteristics Chronic Code Coin Control Animal Country Cre-LoxP D-Amino Acid Dehydrogenase Dependovirus Development Dilated Cardiomyopathy Disease EFRAC Elderly Enterobacteria phage P1 Cre recombinase Enzymes Functional disorder Future Heart Heart failure Hydrogen Peroxide LoxP-flanked allele Metabolic Methodology Modeling Molecular Morbidity - disease rate Mus Muscle Cells Myocardial dysfunction Oxidation-Reduction Pathologic Pathology Patients Phenotype Physiological Population Prevalence Proteins Proteome Proteomics Public Health Rattus Recombinants Research Role Serotyping Signal Pathway Site Stimulus Stress Sulfhydryl Compounds System Terminator Codon Tissues Transgenic Mice Transgenic Organisms Virus Yeasts age related aged cellular imaging drinking water experimental study feeding hemodynamics imaging modality in vivo information model insight mature animal middle age mortality new therapeutic target novel therapeutic intervention novel therapeutics optogenetics partial recovery prevent programs promoter response transcriptome transcriptomics transgene expression young adult

项目摘要

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.

该 R21 应用提出了开发和验证新模型的实验通过使用化学遗传学方法产生氧化还原应激来研究与衰老相关的心脏病理学心脏和其他组织的“年龄相关性心肌病”的发展。具有收缩性和舒张性心功能障碍、氧化还原应激和高死亡率—— 我们最近开发了一种新的化学遗传学。直接且特异性地导致心肌病发展的方法在拟议的研究中，我们将通过诱导心脏氧化还原应激增加来引起。我们在衰老的背景下研究了这种新的心肌病模型。腺相关病毒血清型 9 (AAV9) 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，引起心脏氧化还原应激 DAAO 是一种立体特异性酶，可产生扩张型心肌病。过氧化氢 (H2O2) 仅当 D-氨基酸可用作底物时才产生。哺乳动物组织仅含有 L-氨基酸，酵母 DAAO 在 D-氨基酸之前没有活性我们将重组DAAO-AAV9病毒注射到小鼠（或大鼠）中，并使用。细胞成像方法表明 H2O2 仅在心肌细胞中大量产生在向肌细胞添加 D-丙氨酸后，我们开发了一种体内化学遗传学模型在心脏中产生慢性氧化还原应激：我们在小鼠的饮用水中添加了 D-丙氨酸与对照动物相比，感染了重组 DAAO-AAV9 的动物。感染 DAAO-AAV9 的小鼠在服用 D-丙氨酸后 3 周内出现扩张型心肌病我们建议扩展这些方法来研究氧化还原应激在治疗中的作用。通过感染中年和老年小鼠来治疗与年龄相关的心肌病重组DAAO-AAV9，然后分析生理、代谢、生化、使用体内和离体的转录组和蛋白质组特征这些实验可能会确定新的治疗靶点来预防或治疗。我们还建议开发 DAAO-TGLoxP。转基因小鼠品系将允许分析衰老过程中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。预计 DAAO 的组织特异性转基因表达将提供新的对与年龄相关的广泛疾病状态的机制见解组织病理学与氧化还原功能障碍有关。