昼夜节律基因Clock/Bmal1在拟胆碱药调控心力衰竭钙紊乱中的作用机制研究

Circadian rhythm disorders can induce cardiovascular events. Autonomic imbalance is an important pathological basis for cardiovascular diseases such as heart failure. Our previous studies have shown that vagal nerve activation ameliorates abnormal calcium handling in myocardium and blood vessels. However，the intrinsic relations of vagal regulation, circadian rhythm, and calcium dyshomeostasis in heart failure are not yet clear. In the present project, we will use the heart failure models combined with disruption of circadian clocks, and investigate the molecular mechanisms underlying cholinergic drugs regulating circadian rhythm and calcium homeostasis. We will 1) detect the changes of cardiac circadian gene expression rhythm by RT-qPCR, in order to determine the involvement of the disturbances of cardiac circadian clock system in heart failure; explore the effect of the disrupted core circadian clock genes Clock/Bmal1 on store-operated calcium channels and receptor-operated calcium channels and related calcium responses by application of laser confocal microscopy and siRNA, in order to clarify the calcium regulation mechanism by circadian rhythm genes; 2) explore the effect of cholinergic drugs on Clock/Bmal1 regulation by applying ChIP, in order to disclose the signal pathway of choline regulating circadian gene and ameliorating calcium dyshomostasis. This study may help to provide a new way of autonomic intervention and pharmacological therapeutic targets for the regulation of circadian rhythm in the prevention and treatment of heart failure.

昼夜节律紊乱诱使心血管事件的发生，自主神经失衡是心力衰竭等心血管疾病的重要病理基础。前期研究发现活化迷走可改善心血管钙调控异常，但迷走神经调节、昼夜节律、心衰钙稳态失衡三者的内在联系尚不清楚。本项目拟建立心衰模型结合生物钟紊乱，研究拟胆碱药对生物钟基因和钙稳态调控的分子机制：1）采用实时定量PCR检测心脏生物钟基因表达变化，明确生物钟系统异常参与心衰发病机制；应用激光共聚焦显微镜技术和siRNA研究生物钟核心基因Clock/Bmal1对钙通道SOCC/ROCC及其介导钙反应的作用，探讨昼夜节律基因与钙离子稳态间的内在联系；2）运用染色质免疫共沉淀技术探讨拟胆碱药对Clock/Bmal1的调控机制，阐明拟胆碱药调控心衰生物钟基因节律异常及钙紊乱的信号通路。本研究有望为调控昼夜节律防治心衰提供自主神经干预的新途径和药理学治疗靶点。

昼夜节律紊乱诱使心血管事件的发生，自主神经失衡是心力衰竭等心血管疾病的重要病理基础。前期研究发现活化迷走可改善心血管钙调控异常，但迷走神经调节、昼夜节律、心衰钙稳态失衡三者的内在联系尚不清楚。本项目拟建立心衰模型，研究拟胆碱药对生物钟基因和钙稳态调控的分子机制：（1）通过SD大鼠腹主动脉缩窄（AAC）建立压力超负荷诱导的心肌重构模型，结果表明AAC改变了大鼠心脏中七个主要时钟基因（Bmal1、Clock、Rev-erbα、Per1/2、Cry1/2）转录水平的昼夜节律。AAC还上调了大鼠心脏中SOCC/ROCC（STIM1、Orai1和TRPC6）的蛋白水平。胆碱改善AAC大鼠的昼夜节律紊乱，降低上调的STIM1、Orai1和TRPC6蛋白水平，并减轻心功能障碍和重构（降低心肌肥厚、纤维化和凋亡）。（2）应用血管紧张素II（AngⅡ）刺激建立原代乳鼠心肌细胞肥大模型。体外实验显示在AngII干预的心肌细胞中，胆碱减轻钙超载和STIM1、Orai1、TRPC6的上调和抑制TG诱导的SOCE和OAG诱导的ROCE。表明昼夜节律紊乱和胞内钙稳态失衡的减轻可能是胆碱保护作用的重要机制。（3）胆碱通过激活M3型毒蕈碱乙酰胆碱受体（M3AChR）和Nrf2信号通路，改善了AngII/AAC诱导的血管平滑肌细胞表型转化和血管重构。综上，胆碱可减弱AAC诱导的心脏重构和心功能障碍，这与减轻昼夜节律紊乱和SOCC/ROCC介导的细胞内钙稳态失衡有关。AAC诱导的心脏生物钟的改变可能会破坏外界刺激和系统反应性之间的同步性，从而促进组织重构，最终发展为心力衰竭。以昼夜节律和钙稳态为靶点，通过给予胆碱调节迷走神经活性具有治疗心脏病的前景。