NCoR1-HDAC3/BMAL1介导的线粒体自噬节律紊乱在糖尿病心肌再灌注易损性增加中的作用及机制

Mitophagy dysfunction is a major why hearts from diabetic subjects are more vulnerable to ischemia reperfusion (IR) injury. Our recent study showed that mitophagy dysfunction was characterized as dysrhythmia, but the underlying mechanisms are still unclear. Mitophagy rhythm depends on clock gene oscillation.Rev-erbα and BMAL1 are crucial for the stability of clock gene oscillation. Recent study indicated that NcoR1-HDAC3 were key factors to regulate the function of clock gene and glycometabolism. Our preliminary study showed that during myocardial IRI, higher HDAC activity and lower BMAL1 expression were detected in diabetic heart than in non-diabetic control, that was accompanied with BMAL1 oscillation disorder. Accordingly, we speculate that abnormal regulation of mitophagy rhythmia by NCoR1-HDAC3 mediated BMAL1 oscillation disorder plays a key role in increasing diabetic heart vulnerability to IR injury. In this proposal, we will use specific gene knockout mice and primarily cultured cardiomyocytes to establish in vivo and in vitro models of ischemia/reperfusion or hypoxia/reoxygenation to investigate the roles of Rev-erbα/BMAL1 oscillation and its regulation by NCoR1-HDAC3 in mediating mitophagy dysrhythmia in in vitro and in vivo study, and to observe its effects on increased vulnerability of IR injury in diabetic hearts. This proposed studies may help facilitate the development of novel and optimal therapies or intervention interval in combating ischemic heart disease in diabetes .

线粒体自噬功能障碍是糖尿病心肌缺血再灌注(IR)易损性增加的重要机制。我们前期研究发现，线粒体自噬功能异常的特征为节律紊乱，但其调控机制未明。研究表明，线粒体自噬节律依赖于时钟基因振荡，Rev-erbα、BMAL1是维持时钟基因振荡稳定性的基础；新近发现，NCoR1-HDAC3 是调控时钟基因功能和糖代谢的关键因子。我们预实验表明，糖尿病心肌IR期间HDAC活性增高，BMAL1表达降低且振荡失调。据此，我们推测NCoR1-HDAC3调控BMAL1基因振荡异常，进而介导线粒体自噬节律紊乱是糖尿病心肌IR易损性的关键机制之一。本项目拟用特异性基因敲除小鼠在体、离体心脏及原代心肌细胞为研究对象，探讨NCoR1-HDAC3调控的Rev-erbα/BMAL1基因振荡在线粒体自噬节律紊乱中的作用，并观察其对糖尿病心肌IR易损性的影响及机制，为糖尿病心肌IR损伤的药物防治和干预时机提供新靶点和新策略。

线粒体自噬功能障碍是糖尿病心肌缺血再灌注(IR)易损性增加的重要机制。我们前期研究发现，线粒体自噬功能异常的特征为节律紊乱，但其调控机制未明。NCoR1-HDAC3调控Rev-erbα/BMAL1基因振荡异常，进而介导线粒体自噬节律紊乱可能是糖尿病心肌IR易损性的关键机制。我们的研究表明：（1）非糖尿病大鼠时钟基因Rev-erbα，BMAL1和CEBP/β的mRNA表达具有显着的昼夜节律性，而糖尿病大鼠心肌Rev-erbα，BMAL1和CEBP/β的mRNA表达无明显昼夜节律；（2）非糖尿病和糖尿病大鼠在ZT0，ZT6，ZT12，ZT18时间点行心肌IR后，心肌损伤程度具有节律性，且ZT12时损伤最重，自噬水平最低；糖尿病心肌损伤更加严重，且不同时间点心肌损伤和自噬水平无明显节律性；（3）ZT12时进行IR处理后，糖尿病心肌组织中HDAC3和Rev-erbα水平升高、BMAL1表达降低，IR损伤加重且心肌自噬水平显著下调；(4) HBAAV9-r-HDAC3敲低糖尿病大鼠心肌HDAC3表达后，HDAC3和Rev-erbα表达降低、BMAL1水平显著上调，线粒体自噬水平明显增加，IR损伤显著减轻；（5）高糖条件下，原代心肌细胞HDAC3和Rev-erbα表达增高，BMAL1表达下调且自噬水平降低；（6）高糖缺氧复氧刺激后，HDAC3和Rev-erbα表达增高，BMAL1表达下降，线粒体损伤加重，细胞自噬水平显著降低；（7）慢病毒过表达BMAL1基因后，细胞损伤明显下降；而BMAL1沉默后，细胞损伤明显增加；（8）siRNA沉默HDAC3和Rev-erbα基因显著降低高糖缺氧复氧损伤，同时上调BMAL1表达水平和线粒体自噬水平，减轻心肌细胞损伤。基于我们的上述研究，以期待为糖尿病心肌IR损伤的药物防治和干预时机提供新靶点和新策略。