Department of Cardiology,the First Affiliated Hospital of Nanjing Medical University
The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)
目的：探究生物钟基因Bmal1(brain and muscle ARNT-like protein-1,Bmal1)调控T型钙离子通道(T-type calcium channel, TTCC)对慢性心衰(chronic heart failure,CHF)室性心律失常(ventricular arrhythmia,VA)昼夜节律的影响及可能的调控机制。方法：采用主动脉缩窄术(TAC)构建C57BL/6J小鼠慢性心力衰竭模型(CHF),设定假手术对照组(CON)及TTCC抑制剂米贝拉地尔(MIB)治疗组(CHF+MIB)。使用心脏超声评估各组小鼠心功能,免疫组织化学染色(IHC)及蛋白免疫印迹法(WB)测定心肌TTCC表达。使用程序性电刺激(PES)诱发室性心律失常(VA),采用WB及实时定量逆转录聚合酶链反应(qRT-PCR)检测心肌组织Bmal1、TTCC昼夜节律变化及关系。使用染色质免疫共沉淀技术(ChIP)揭示Bmal1调控TTCC的分子机制。结果：与CON组相比,CHF小鼠心脏收缩功能显著降低,心腔扩大且伴有明显心肌肥厚,心肌中TTCC表达量明显增加,行PES检测发现CHF小鼠VA发作率明显增加,且有昼夜节律差异,使用MIB可一定程度降低CHF小鼠VA的发生。WB及qRT-PCR结果提示CHF小鼠心肌中Bmal1及TTCC表达呈昼夜节律性变化,ChIP检测提示Bmal1可直接结合到TTCC启动子区域的E-box位点调控TTCC的转录。结论：CHF小鼠中,TTCC通道重新高表达且呈昼夜节律性,使用MIB可降低CHF小鼠VA发作,Bmal1可通过直接结合TTCC启动子区域的E-box位点调控TTCC的转录。
Objective: Explore the effect of circadian clock gene brain and muscle ARNT-like protein-1(Bmal1) regulated T-type calcium channel (TTCC) on circadian rhythm of chronic heart failure (CHF) ventricular arrhythmia (VA) and its possible mechanisms. Methods: The C57BL/6J mice chronic heart failure (CHF) model was established by transverse aortic constriction (TAC) and mice in control group (CON) were treated with sham operation. Some TAC mice were randomly assigned to receive TTCC inhibitor mibefradil (MIB) for 2 weeks (CHF+MIB). Cardiac function was assessed by echocardiographic and the expression level of TTCC was determined by immune histochemical (IHC) staining and western blot (WB). Ventricular arrhythmia (VA) was induced through programmed electrical stimulation (PES) in each group. The circadian rhythms of Bmal1 and TTCC were detected by WB and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the molecular mechanism by which Bmal1 regulated TTCC was determined by chromatin immunoprecipitation (ChIP). Results: Compared with CON group, CHF mice had significantly reduced cardiac systolic function, increased internal diameter accompanied by obvious cardiac hypertrophy. Significantly increased TTCC re-expression level in CHF mice myocardium was detected by IHC staining and WB. PES indicated that VA occurrence was significantly increased when comparing CHF mice with CON mice. To a certain extent, using MIB could reduce the occurrence of VA in CHF mice. In addition, VA occurrence appeared circadian rhythm in all three groups (CON, CHF and CHF+MIB). WB and qRT?PCR results suggested that the expressions of Bmal1 and TTCC in the myocardium of CHF mice exhibited circadian rhythm and ChIP suggested that Bmal1 could directly bind to the E-box site of TTCC promoter to regulate TTCC. Conclusion: In CHF mice, TTCC channel are highly re-expressed and show circadian rhythm, and MIB can reduce the occurrence of VA in CHF mice. Bmal1 can directly bind to the E-box site of TTCC promoter region to regulate the transcription of TTCC.