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”.

项目概要 烟酰胺或 NAD+ 是哺乳动物细胞中最重要的小分子之一。它的角色在 心血管健康和长寿越来越受到重视。因此，其治疗用途正在 经过大量临床试验的测试。烟酰胺磷酸核糖转移酶 (NAMPT) 是限速酶 NAD+ 挽救途径并确定心脏中的 NAD+ 水平。 NAD+ 水平和 NAMPT 都有 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阐明 明白了。振荡 NAD+ 的生理意义尚未在临床前模型中直接测试。 因此，该信息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 确定心脏中振荡 NAD+ 的生理意义。根据我们的初步数据，我们 假设 BAML1 和 KLF15 在启动子-增强子环中的共同占据是最佳效果所必需的 Nampt 以一天中时间依赖性方式上调。这种协调的昼夜节律调节是关键 振荡心肌 NAD+、心脏代谢和 I/R 抵抗。为了检验这个中心假设，我们设计了 两个互补的目标首先确定 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。小说素素编辑 除了传统的技术外，技术还将用于生成顺式元件删除 iPSC-CM 和小鼠模型 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型腺相关病毒（AAV 系统）测试NAMPT同相、反相和恒定表达对心脏的生理效应 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+ 的昼夜节律振荡调节机制，以及 (2) 了解 心脏中出现振荡的Nampt表达。这将进一步揭示组织的分子机制 特定的昼夜节律基因调控，目前人们对此知之甚少。我们建立了第一个心脏 “从”时钟（KLF15），在这里我们首先提出了核心时钟-从时钟促进染色质循环模型， 与线性核心时钟>从时钟>目标模型相反。除了概念上的新颖性之外，我们还生成了 新颖的主要编辑工具，允许在 iPSC-CM 中以及以特殊时间调节方式进行精确编辑 体内。我们还设计了新方法来首次测试振荡表达的生理后果 与恒定表达。 我们提出的工作直接响应当前的资助机会 PA19-0-49，“研究正常 生物学，包括生物系统的稳态调节和复原力现象”，“定义 昼夜节律功能、同步化和协调性影响的基本途径”。