β-AR基因多态性参与代谢综合征合并心肌功能不全的机制研究

Gene polymorphism in β-adrenergic receptor (β-AR) is a susceptibility factor for metabolic syndrome (MS). It is also one of the important causes for diverse clinical manifestations and medication efficacy in cardiovascular disease. Our previous research identified the relationship between myocardial insufficiency and β1-/β2-AR in MS rats; but the detailed regulatory mechanisms are still unclear. Recently, we found that β2-AR stimulated downstream signaling pathway showed different activities between wide type and Gln27Glu、Thr164Ile mutants, respectively. Therefore, this project will continue to study the molecular mechanisms of β-AR gene polymorphism regulated downstream signaling pathway in myocardial insufficiency with MS. First, we will construct β-AR mutants to analyze the regulation of β-AR signaling pathway by different mutants through bioluminescence resonance energy transfer (BRET), fluorescent probe, Western Blot, etc. at cellular level. Then, we will evaluate the different activities under the influences of β-AR agonist / antagonist on the normal and mutant receptors. And then, we will use knock-in animals to observe the effects of mutant genotype on functions and β-AR signaling pathways in vivo (heart level) and in vitro (isolated myocardium and cardiomyocyte levels). Finally, we will confirm the relationship between gene polymorphism in β-AR and the changes of signaling pathway and myocardial functions in diseased animals. This project will provide theoretical support for the precise treatment of targeted β-AR drugs in myocardial insufficiency with MS.

β-肾上腺素受体(β-AR)基因多态性是代谢综合征(MS)的易感因素，也是心血管疾病临床表现与药物疗效多样性的重要原因之一。我们前期研究已证实MS大鼠心肌功能不全与β1-/β2-AR有关，但调控机制尚不清楚；近期又发现β2-AR野生型和Gln27Glu、Thr164Ile突变体激活的β-AR下游信号通路存在差异。因此，本项目将进一步研究β-AR基因多态性参与MS心肌功能不全的分子机制：首先构建β-AR突变体，利用生物发光共振能量转移(BRET)、荧光探针和Western Blot等方法在细胞水平检测突变体调节的β-AR信号通路，评估β-AR激动剂、阻滞剂作用下的激活差异；然后利用基因敲入动物观察突变体基因型对在体心脏、离体心肌和细胞功能及β-AR信号通路的影响；最后在疾病动物中验证β-AR基因多态性与信号通路及心肌功能改变的关联，为MS心肌功能不全中靶向β-AR药物的精准治疗提供理论支持。

β-肾上腺素受体(β-AR)基因多态性是代谢综合征(MS)的易感因素，会导致其携带者的心血管疾病临床表现和药物疗效具有明显个体差异。本项目研究了β-AR基因多态性参与MS合并心肌功能不全的分子机制。首先构建β-AR突变体，利用HEK-293工具细胞发现：非选择性β-AR激动剂Isoproterenol和选择性β2-AR激动剂Terbutaline在激活β2-AR下游cAMP、钙流或募集β-arrestin1/2的效应方面不受 SNPs 的影响；而使用选择性β2-AR激动剂Procaterol和Zinterol后，R16G和T164I突变体相比于野生型受体在激活钙流和/或募集β-arrestin1/2方面有显著的增强效应。β1-AR的突变体R389G和野生型类似，对β1-AR选择性抑制剂比索洛尔最为敏感，而对另一种选择性抑制剂美托洛尔最不敏感；对于突变体S49G，β1-AR非选择性抑制剂卡维地洛的抑制效果最好。基于上述结果，已构建ARDB2（G16R）和ARDB2（T164I）两个位点的点突变小鼠，目前正在繁育中；未来将进一步验证细胞层面β-AR下游信号通路的发现，并构建MS的基因敲入小鼠模型，研究其心肌功能和β-AR下游信号通路变化。我们同时构建了H9C2心肌细胞和大鼠的MS模型。H9C2细胞中加入棕榈酸（Palmitic acid, PA）可模拟MS造成的心肌损伤。在H9C2细胞MS模型中，心肌损伤标志物Myog和cTnT升高，细胞形态明显变化，脂质聚集明显增多，且β1/β2-AR的表达升高。采用高脂饮食喂养Wistar大鼠16w成功构建MS动物模型。超声心动图提示MS大鼠在体情况下，其正性肌力效应并未受损。左心室乳头肌肌力测定提示MS大鼠离体心肌对β-肾上腺素能刺激产生的正性肌力效应显著弱于Control组。MS组左室的β1/β2-AR表达增加。总之，该研究有望明确代谢综合征合并心肌功能不全中β-AR的信号机制及其基因多态性对下游信号通路的影响和不同配体激活的药理学差异，为MS并发的心血管疾病靶向药物的精准治疗提供理论依据。