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重要的小分子之一。它在以下方面的角色： 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 在无数的临床试验中进行了测试。烟酰胺磷酸核糖转移酶(NAMPT)是限速酶 NAD挽救途径，并决定心脏中的NAD水平。NAD级别和NAMPT都有 心脏有强烈的昼夜节律波动。NAD昼夜节律调节的分子机制只是部分的 明白了。振荡NAD的生理意义还没有在临床前模型中直接测试。 因此，这一信息尚未被纳入任何现有的临床试验。 我们在这个应用中的目标是确定NAMPT昼夜节律调节的分子机制和 建立心脏振荡NAD的生理学意义。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优的 以一天中的时间依赖的方式上调NAMPT。这种协调的昼夜节律是 振荡心肌NAD、心脏代谢和I/R阻力。为了验证这一中心假设，我们设计了 两个互补的目的首次确定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。新奇素材剪辑 将使用技术来生成删除iPSC-CM和鼠标模型的顺式元件，除了传统的 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性腺相关病毒(AAV) 系统)，以测试NAMPT在心脏中的同相、反相和恒定表达的生理效应 代谢与抗缺血/再灌流损伤。 拟议工作的成功完成将使我们能够(1)建立精确的分子 心脏NAD的昼夜振荡调节机制，以及(2)了解NAD的功能意义 NAMPT在心脏中的振荡表达。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，目前还很难理解。我们建立了第一个心脏 “从”时钟(KLF15)，并在此首次提出了核心时钟--从时钟改进的染色质环路模型， 与线性核心时钟&gt；从时钟&gt；目标模型形成对比。除了概念上的新颖性，我们还产生了 新的主要编辑工具，允许在IPSC-CM中进行精确编辑，并以特殊的时间调节方式进行编辑 在活体内。我们还设计了新的方法来首次测试振荡表达的生理后果 而不是恒定的表情。 我们建议的工作直接响应了当前的资助机会PA19-0-49， 生物学，包括生物系统的动态平衡调节和复原力现象“， 生理功能、同步性和协调性影响的基本途径“。

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”.