Hepatic BMAL1 and HIF1a regulate a time-dependent hypoxic response and their absence leads to Hepatopulmonary Syndrome II

The transcriptional response to hypoxia is temporally regulated yet the molecular underpinnings and physiological implications are unknown. We examined herein the roles of hepatic Bmal1 and Hif1a in the circadian response to hypoxia in mice. We found that the majority of the transcriptional response to hypoxia is dependent on Bmal1 or Hif1a, through shared and distinct roles that are daytime determined. We further show that HIF1a accumulation upon hypoxia is temporally regulated and Bmal1-dependent. Unexpectedly, mice lacking both hepatic Bmal1 and Hif1a are hypoxemic and exhibit increased mortality upon hypoxic exposure in a daytime-dependent manner. These mice display mild liver dysfunction with pulmonary vasodilation likely due to ERK activation, endothelial nitric oxide synthase and nitric oxide accumulation in lungs, suggestive of hepatopulmonary syndrome. Our findings indicate that hepatic BMAL1 and HIF1a are key time-dependent regulators of the response to hypoxia and provide molecular insight on the pathophysiology of hepatopulmonary syndrome. RNA-sequencing on livers of AlbCRE, HLKO or BLKO mice exposed to 4 hour normoxia (21% oxygen) or hypoxia (6% hypoxia) at CT4-8 or CT16-20. Lights were turned off at the transition of dark to light regiment on the day of the experiment, following regular LD regimen.机体对低氧的转录应答呈时序调控特征，但其分子基础与生理意义迄今尚未阐明。本研究以小鼠为模型，探讨了肝脏Bmal1（Bmal1）与Hif1a（Hif1a）在低氧昼夜节律应答中的作用。研究发现，绝大多数低氧诱导的转录应答依赖于Bmal1或Hif1a，二者通过具有昼夜依赖性的共有与独特功能发挥调控作用。本研究进一步证实，低氧诱导的HIF1a蛋白蓄积呈时序调控特性，且该过程依赖于Bmal1。出乎意料的是，同时缺失肝脏Bmal1与Hif1a的小鼠会出现低氧血症，且在低氧暴露下的死亡率呈昼夜依赖性升高。此类小鼠表现出轻度肝功能异常，并伴随肺血管舒张，该现象可能与肺组织中细胞外调节蛋白激酶（ERK）激活、内皮型一氧化氮合酶（endothelial nitric oxide synthase）及一氧化氮蓄积相关，提示存在肝肺综合征（hepatopulmonary syndrome）。本研究结果表明，肝脏BMAL1与HIF1a是低氧应答的关键时序依赖性调控因子，并为肝肺综合征的病理生理学机制提供了分子层面的新见解。实验对AlbCRE、HLKO或BLKO小鼠的肝脏组织开展了RNA测序（RNA-sequencing），这些小鼠分别在昼夜节律时间点CT4-8与CT16-20接受了4小时常氧（21%氧气）或低氧（6%氧气）处理。实验当日遵循常规光暗（LD）周期，于暗转亮的节点关闭照明光源。