时钟基因Per1介导的钙循环紊乱在糖尿病心肌缺血再灌注易损性增加中的机制研究

Diabetic myocardium are more susceptible to ischemia reperfusion injury (IRI). The latest studies suggest that circadian genes are essential mechanisms involved in the regulation of the myocardial IRI. Our preliminary study found that the expression of core circadian gene Per1 reached the peak value during the sleep-awake transition period (S-A period) in diabetic heart, and the peak value was significantly higher than non-diabetes. Per1 RNAi treatment alleviate hypoxia reoxygenation injury under high glucose condition, however, the underlying mechanism remains unclear. Orderly uptaking calcium ions from cytoplasm to sarcoplasmic reticulum by calcium pump SERCA2a is the basis of calcium cycling and myocardial relaxation. The calcium cycling disorder caused by diabetic myocardial calcium pump SERCA2a dysfunction aggravate IRI and may be related to circadian gene regulation. Our further preliminary study showed that SERCA2a activity in diabetic myocardium reached the bottom during the S-A period. While Per1 RNAi restored SERCA2a activity and improved sarcoplasmic reticulum calcium uptake ability. Accordingly, we speculate that circadian gene Per1 induces calcium cycling disorder by impairing SERCA2 plays a key role in increased vulnerability to myocardial IRI in diabetes. In this proposal, we will use in vivo and in vitro models to investigate the mechanism of Per1 mediated calcium cycling disorder in increased vulnerability to IRI in diabetic hearts and may help facilitate the development of potential and novel therapies or primary intervention in combating ischemic heart disease in diabetes.

糖尿病心肌更不易耐受缺血再灌注损伤(IRI)。新近发现，时钟基因是参与调控心肌IRI病程的重要机制。我们前期发现糖尿病心肌中核心时钟基因Per1在睡眠-觉醒过渡期(S-A期)达到昼夜峰值，且较非糖尿病显著升高；而沉默Per1可减轻高糖缺氧复氧损伤，但其机制未明。肌质网钙泵SERCA2a有序回收钙离子是心肌钙循环及舒缩的基础，糖尿病心肌SERCA2a功能障碍致钙循环紊乱是IRI加重的关键，并可能受时钟基因调控。预实验显示，糖尿病心肌SERCA2a活性在S-A期达到昼夜最低；而沉默Per1可恢复SERCA2a活性，提高肌质网钙回收能力。据此，我们推测：糖尿病心肌Per1调控SERCA2a节律异常，进而介导钙循环紊乱是IRI加重的关键机制之一。本项目通过糖尿病大鼠在体及离体实验，探讨Per1介导的钙循环紊乱在糖尿病心肌IRI易损性增加中的机制，为糖尿病心肌损伤的防治和干预时机提供新策略。

糖尿病心肌更不易耐受缺血再灌注损伤（IRI），糖尿病心肌SERCA2a功能障碍致钙循环紊乱是IRI加重的关键，并可能受时钟基因调控，但详细机制未明。我们的研究阐明了糖尿病心肌中，时钟基因Per1通过调控O-连接糖基化相关酶介导的SP1糖基化，转录层面抑制Serca2a的表达，是糖尿病心肌缺血再灌注易损性增加的新机制。我们的结果表明：（1）不同时间点抽提心脏组织 mRNA，高通量测序并分析，Per1 KO小鼠心脏多个时钟基因控制基因（CCGs）失去昼夜节律性；（2）在Per1 KO的糖尿病大鼠中实现心肌组织特异性过表达Per1，过表达Per1能够改善糖尿病小鼠的心功能，提高左心室的射血分数；（3）Per1可直接调节O-连接糖基化相关酶活性的昼夜节律，转录因子SP1糖基化能够负性调节Serca2a的表达，同时降低糖尿病心肌的心功能；（4）我们构建并转染Sp1糖基化位点的突变体，证实时钟基因Per1通过调节Sp1的糖基化，进而影响Serca2a的转录，是时钟基因介导的糖尿病心脏钙循环紊乱的机制之一。基于上述研究结果，以期为糖尿病心脏缺血/再灌注损伤的防治提供有效的治疗靶点。