Circadian regulation of NAMPT in the heart

点击翻译按钮获取中文摘要

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”.