The circadian REV-ERB nuclear receptors are essential drivers of heart metabolism and function

The heart is a highly metabolic organ that uses multiple energy sources to meet its demand for ATP production. Diurnal feeding-fasting cycles result in substrate availability fluctuations which, together with increased energetic demand during the active period, impose a need for rhythmic cardiac metabolism. The nuclear receptors REV-ERBa and b are essential repressive components of the molecular circadian clock and major regulators of metabolism. To investigate their role in the heart, we generated mice with cardiomyocyte (CM)-specific deletion of both Rev-erbs, which died prematurely due to dilated cardiomyopathy. Loss of Rev-erbs markedly downregulated fatty acid oxidation genes prior to overt pathology, which was mediated by induction of the transcriptional repressor E4BP4, a direct target of cardiac REV-ERBs. E4BP4 directly controls circadian expression of Nampt and its biosynthetic product NAD+ via distal cis-regulatory elements. Thus, REV-ERB-mediated E4BP4 repression is required for Nampt expression and NAD+ production by the salvage pathway. Together, these results highlight the indispensable role of circadian REV-ERBs in cardiac gene expression, metabolic homeostasis and function. RNA-Sequencing was performed on 2-months-old control versus CM-RevDKO hearts, that were harvested at ZT10. H3K27ac Cut&Run was performed on isolated adult cardiomyocytes from control versus CM-RevDKO mice at ZT10. ChIP-Seq for HA-Rev-erba and E4BP4 was performed on biventricles of male mouse hearts (age 2 months), harvested at ZT10.心脏是一类高度代谢活跃的器官，依赖多种能量来源以满足ATP生成的需求。昼夜进食-禁食循环会引发底物可利用性的波动，再加上活动期能量需求的升高，使得心脏代谢必须具备节律性。核受体REV-ERBα与REV-ERBβ是分子生物钟的关键抑制性组分，同时也是代谢调控的核心因子。为探究二者在心脏中的功能，我们构建了心肌细胞（cardiomyocyte, CM）特异性双敲除Rev-erbs的小鼠模型，该模型小鼠因扩张型心肌病过早死亡。在出现明显病理表型之前，Rev-erbs的缺失就显著下调了脂肪酸氧化相关基因的表达，这一调控过程由转录抑制因子E4BP4的诱导所介导，而E4BP4正是心脏REV-ERBs的直接靶标。E4BP4通过远端顺式调控元件，直接调控Nampt的节律性表达及其生物合成产物烟酰胺腺嘌呤二核苷酸（NAD+）。由此可见，REV-ERB介导的E4BP4抑制对于补救合成途径中的Nampt表达与NAD+生成是必需的。综上，本研究结果凸显了节律性REV-ERBs在心脏基因表达、代谢稳态及心脏功能中不可或缺的作用。我们对2月龄对照小鼠与CM-RevDKO小鼠的心脏开展了RNA测序（RNA-Sequencing），样本均采集于ZT10时刻。我们从对照小鼠与CM-RevDKO小鼠体内分离的成年心肌细胞中进行了H3K27ac Cut&Run实验，样本同样采集于ZT10时刻。我们对2月龄雄性小鼠心脏的双心室组织开展了针对HA-Rev-erbα与E4BP4的染色质免疫沉淀测序（ChIP-Seq），样本采集于ZT10时刻。