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.

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。 “年龄相关性心肌病”的发展——相关 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—— 已被广泛记录。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 由诱导心脏氧化还原应激增加引起。在拟议的研究中，我们将 在衰老的背景下研究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 (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 并导致扩张型心肌病。 DAAO 是一种立体特异性酶，可产生 仅当 D-氨基酸作为底物时才产生过氧化氢 (H2O2)。由于大多数 哺乳动物组织仅含有 L-氨基酸，酵母 DAAO 在 D-氨基酸之前不活跃 提供酸。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 向肌细胞添加 D-丙氨酸后。然后我们开发了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 已被重组 DAAO-AAV9 感染。与对照动物相比， 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 治疗。我们建议扩展这些方法来研究氧化还原应激在 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 接近。这些实验可能会确定新的治疗靶点来预防或 治疗与年龄相关的心肌病。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。我们 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。