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

• 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
• 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

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.

此R21应用程序建议进行实验，以开发和验证新的 使用化学发生方法产生氧化还原应激的衰老相关心脏病理 心脏和其他组织。与年龄相关的心肌病的发展 有收缩和舒张期心脏功能障碍，氧化还原压力，和高死亡率- 已经被广泛记录在案。我们最近开发了一种新的化学遗传学 导致心肌病发展的直接和特异的方法 引起心脏氧化还原应激增加所致。在建议的研究中，我们会 在老龄化的背景下研究这一新的心肌病模型。我们用了一种强心剂 表达酵母D-氨基酸氧化酶的腺相关病毒血清9型(AAV9) 在活体心肌细胞中选择性地产生ROS，在心脏引起氧化还原应激 并导致扩张型心肌病。DAAO是一种立体专一性酶，它能产生 过氧化氢(H_2O_2)只有在有D-氨基酸作为底物时才能产生。因为大多数人 哺乳动物组织只含有L氨基酸，酵母菌只有在D-氨基之前是不起作用的 提供了酸。我们将重组DAAO-AAV9病毒注射到小鼠(或大鼠)体内，并使用 显示过氧化氢仅在心肌细胞中大量产生的细胞成像方法 在心肌细胞中加入D-丙氨酸后。然后我们开发了一个体内化学发生模型来 在心脏产生慢性氧化还原压力：我们在小鼠的饮用水中添加D-丙氨酸 已经感染了重组DAAO-AAV9的人。与控制动物相比， DAAO-AAV9感染的小鼠在服用D-丙氨酸3周内出现扩张性心肌病 治疗。我们建议将这些方法扩展到研究氧化还原应激在 感染中老年小鼠的增龄性心肌病 重组DAAO-AAV9，然后分析生理、代谢、生化、 利用体内和体外转录和蛋白质组学特征 接近了。这些实验可能会确定新的治疗靶点，以预防或 治疗年龄相关性心肌病。我们还建议开发DAAO-TGLoxP 能够分析衰老过程中组织特异性氧化还原应激的转基因小鼠品系 使用Cre-Lox方法在心脏和其他组织中表达DAAO。我们 预计DAAO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械论洞察 组织病理学与氧化还原功能障碍有关。