Circadian regulation of NAMPT in the heart

心脏 NAMPT 的昼夜节律调节

• 批准号: 10509597
• 负责人: Lilei Zhang
• 金额: $ 20万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-22 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10509597
• 关键词: Binding; Biological Process; Biology; Brain; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular system; Cellular biology; Chromatin Loop; Chronotherapy; Clinical Trials; Complex; Data; Dependence; Dependovirus; Disease; Ectopic Expression; Elements; Enhancers; Enzymes; Fibroblasts; Foundations; Funding Opportunities; Gene Expression; Gene Expression Regulation; Genes; Genetic Enhancer Element; Genetic Transcription; Genus Hippocampus; Goals; Guide RNA; Heart; Homeostasis; Human; In Vitro; Introns; Ischemia; Knock-in; Knockout Mice; Life; Light; Liver; Longevity; Mammalian Cell; Measures; Metabolism; Methods; Modeling; Molecular; Mus; Muscle; Myocardial; Myocardial Ischemia; Niacinamide; Nicotinamide adenine dinucleotide; Pathway interactions; Periodicity; Phase; Physiological; Physiology; Play; Pre-Clinical Model; Predisposition; Proteins; Regulation; Reperfusion Injury; Reperfusion Therapy; Resistance; Role; Site; Slave; Supplementation; Technology; Testing; Tetracyclines; Therapeutic Uses; Time; Tissues; Up-Regulation; Work; Zinc Fingers; base; biological systems; cardiovascular health; chromosome conformation capture; circadian; circadian regulation; design; heart metabolism; in vivo; induced pluripotent stem cell; insight; mouse model; nicotinamide phosphoribosyltransferase; novel; prime editing; prime editor; promoter; resilience; response; small molecule; stem cell differentiation; therapeutic evaluation; tool; transcription factor

Project Summary Nicotinamide or NAD+ is one of the most essential small molecules in mammalian cells. Its roles in cardiovascular health and longevity are increasingly being appreciated. As a result, its therapeutic use is being tested in numerous clinical trials. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme of the NAD+ salvage pathway and determines NAD+ level in the heart. Both NAD+ levels and NAMPT have a strong circadian oscillation in the heart. The molecular mechanism for NAD+ circadian regulation is only partially understood. The physiological significance of oscillating NAD+ has not been directly tested in preclinical models. Thus, this information has not been incorporated in any of the existing clinical trials. Our goal in this application is to determine the molecular mechanism of NAMPT circadian regulation and establish the physiological significance of oscillatory NAD+ in the heart. Based on our preliminary data, we hypothesize that co-occupancy of BAML1 and KLF15 in a promoter-enhancer loop is required for optimal upregulation of Nampt in a time-of-a-day dependent fashion. This coordinated circadian regulation is key for oscillatory myocardial NAD+, cardiac metabolism and I/R resistance. To test this central hypothesis, we designed two complementary aims to first determine the molecular mechanism of NAMPT circadian regulation in both induced pluripotent stem cell differentiation cardiomyocytes (iPSC-CM) and mouse hearts. Novel prime editing technology will be used to generate cis-element deleting iPSC-CM and mouse models in addition to traditional cardiac KO mice. In the second aim, we will use our novel tetracycline inducible adeno-associated virus (AAV system) to test the physiological effect of in-phase, anti-phase and constant expression of NAMPT in cardiac metabolism and resistance to ischemia/reperfusion injury. Successful completion of the proposed work will allow us to (1) establish the precise molecular mechanism of circadian oscillatory regulation of cardiac NAD+, and (2) understand the functional significance of oscillatory Nampt expression in the heart. This will further shed light on the molecular mechanism of tissue specific circadian gene regulation, which is very poorly appreciated at this time. We established the first cardiac “slave” clock (KLF15) and here we first proposed the core clock-slave clock promoted chromatin looping model, in contrast to the linear core clock>slave clock>targets model. In addition to the conceptual novelty, we generated novel prime editing tools that allow precise editing in iPSC-CM as well as in a special-temporal regulated fashion in vivo. We also designed novel method to first time test the physiological consequence of oscillatory expression versus constant expression. Our proposed work directly responds to the current Funding Opportunity PA19-0-49, “studying normal biology including homeostatic regulation of biological systems and the phenomenon of resilience”, “defining the basic pathways that underlie effects of circadian function, synchronization and harmonization”.

项目摘要 烟酰胺或NAD+是哺乳动物细胞中最重要的小分子之一。它的角色 心血管健康和寿命越来越受到赞赏。结果，它的治疗用途是 在许多临床试验中进行了测试。烟酰胺磷脂基转移酶（NAMPT）是限速酶 NAD+打捞途径的确定，并确定心脏中的NAD+水平。 NAD+级别和NAMPT都有一个 心脏强烈的昼夜节律振荡。 NAD+昼夜节律调节的分子机制仅部分是 理解。振荡NAD+的物理意义尚未在临床前模型中直接测试。 这一点尚未纳入任何现有的临床试验中。 我们在此应用中的目标是确定NAMPT昼夜节律调节的分子机制和 在心脏中建立振荡性NAD+的物理意义。根据我们的初步数据，我们 假设BAML1和KLF15在启动子增强器环中的共占者是最佳的 以每天的依赖方式上调NAMPT。这项协调的昼夜节律是 振荡性心肌NAD+，心脏代谢和I/R抗性。为了检验这个中心假设，我们设计了 两个完整的目的是首先确定两者中NAMPT昼夜节律调节的分子机制 诱导多能干细胞分化心肌细胞（IPSC-CM）和小鼠心脏。新颖的Prime编辑 技术还将用于生成顺式元素删除IPSC-CM和鼠标模型。 心脏KO小鼠。在第二个目标中，我们将使用我们的新型四环素诱导腺相关病毒（AAV） 系统），以测试心脏内相，抗相常和恒定表达心脏中的物理作用 代谢和对缺血/再灌注损伤的抵抗力。 成功完成拟议的工作将使我们能够（1）建立精确的分子 心脏NAD+的昼夜节律调节机制，（2）了解功能意义 心脏中振荡的NAMPT表达。这将进一步阐明组织的分子机制 特定的昼夜节律调节，目前非常不欣赏。我们建立了第一个心脏 “从属”时钟（KLF15），在这里我们首先提出了核心时钟锁时钟促进染色质循环模型， 与线性核心时钟>从属时钟>目标模型相反。除了概念性的新颖性，我们还产生了 新颖的主要编辑工具，允许在IPSC-CM以及以特殊规范的方式中进行精确编辑 体内。我们还设计了新的方法来首次测试振荡表达的物理后果 相对于恒定表达。 我们提议的工作直接回应当前的资金机会PA19-0-49，“研究正常 生物学包括生物系统的稳态调节和弹性现象”，“定义 昼夜节律功能，同步和协调的影响的基本途径。”