Circadian regulation of NAMPT in the heart

项目摘要 烟酰胺或NAD+是哺乳动物细胞中最必需的小分子之一。其在以下方面的作用： 心血管健康和长寿越来越受到重视。因此，它的治疗用途正在被 在许多临床试验中测试。烟酰胺磷酸核糖基转移酶（NAMPT）是限速酶 NAD+补救途径，并确定心脏中的NAD+水平。NAD+水平和NAMPT均具有 心脏中强烈的昼夜节律振荡。NAD+昼夜节律调节的分子机制只是部分的 明白振荡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临床试验。 我们在本申请中的目标是确定NAMPT昼夜节律调节的分子机制， 建立振荡NAD+在心脏中的生理意义。根据初步数据，我们 假设启动子-增强子环中BAML 1和KLF 15共占据是最佳 Nampt以一天中的时间依赖性方式上调。这种协调的昼夜节律调节是 振荡心肌NAD+、心脏代谢和I/R阻力。为了验证这一核心假设，我们设计了 两个互补的目的是首先确定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。小说精华剪辑 技术将用于产生除了传统的外，顺式元件缺失的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）检测NAMPT在心脏中的同相、反相和恒定表达的生理效应。 代谢和抗缺血/再灌注损伤。 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 特定的昼夜节律基因调控，这是非常不好的赞赏，在这个时候。我们建立了第一个心脏 “从”时钟（KLF 15），在这里我们首次提出了核心时钟-从时钟促进的染色质循环模型， 与线性核心时钟>从时钟>目标模型相反。除了概念上的新奇，我们还生成了 新的主要编辑工具，允许在iPSC-CM中以及以特殊的时间调节方式进行精确编辑 in vivo.我们还设计了一种新的方法，首次测试了振荡表达的生理后果 相对于常量表达式。 我们建议的工作直接响应当前的资助机会PA 19 -0-49，“学习正常 生物学，包括生物系统的稳态调节和弹性现象”，“定义 生理机能、同步和协调作用的基本途径”。

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”.