腺苷A2A受体在心肌肥厚中的作用及机制研究

Myocardial hypertrophy is recognized as an adaptive process of the myocardium to increased workload, characterized by increase of myocardial mass. Although initially a beneficial adaptive response, prolonged hypertrophy is a leading predictor of arrhythmias and sudden death as well as heart failure. But the precise mechanisms remain unclear. Adenosine is an endogenous purine nucleoside that plays an important role in cardioprotection. The A2AR is expressed on the cardiomyocytes and has a high affinity for adenosine.Our pilot study revealed that the A2AR knockout（A2AR KO）mice exhibited markedly increased myocardial hypertrophy and expression of fetal genes after 4 weeks of transverse aortic constriction(TAC). And the NF-κB signaling pathway was activated in vitro study. Therefore we hypothesized that A2AR KO exacerbates pressure overload-induced hypertrophy in mice via the NF-κB signaling pathway. Based on our hypothesis, in the present study, we examined the effect of A2AR KO mice on TAC-induced myocardial hypertrophy in vivo and extended our examination to the roles of A2AR in modulating hypertrophy in cultured neonatal mice cardiomyocytes, free from hemodynamic and neurohormonal factors that can influence the in vivo heart. To elucidate the underling mechnisms, the cultured myocytes will be treated with A2AR antagonist and A2AR agonist. The protein synthesis rate was measured by [3H]-leucine incorporation. And Western-blot analysis will be used to evaluated the expression of ANP、BNP. EMSA was used to analysis the NF-κB signaling pathway. These meaningful results will enlarge our scope about the mechanism of cardiac hypertrophy, and may shed light on the prevention and treatment of myocardial hypertrophy.

心肌肥厚是心肌长期压力、容量超负荷引起的一种适应性改变。腺苷对心肌肥厚有保护作用，但机制尚未阐明。我们的预实验结果发现：在主动脉缩窄术诱导的心肌肥厚模型，腺苷A2A受体(A2AR)敲除小鼠心肌肥厚较野生型小鼠明显加重，心肌组织ANP、ß-MHC的mRNA表达增加；体外研究发现心肌肥厚时NF-κB通路被激活。因此提出假说：腺苷A2AR通过激活NF-κB信号通路，抑制心肌肥厚的进展。本研究采用A2AR敲除小鼠，建立心肌肥厚小鼠模型，超声评价小鼠心脏结构与功能，免疫组化检测心肌细胞肥大，RT-PCR、Western blot检测心肌肥大标志物的mRNA和蛋白表达水平。体外培养心肌细胞，AngⅡ诱导心肌细胞肥大，给予A2AR激活剂、抑制剂及NF-κB抑制剂干预，用分子生物学方法进一步验证我们的假说。本课题的完成将有助于阐明A2AR在心肌肥厚中的作用机制，为心肌肥厚的防治提供新的理论依据和药物靶点。

【背景和目的】心肌肥厚是心脏对压力、容量超负荷等应激反应的一种长期、慢性的代偿机制，由多种神经体液因素所介导，是心衰的前期病变。腺苷是体内重要的神经调质，在减轻心肌缺血再灌注损伤、拮抗心肌肥厚、改善心肌重构和心功能等多方面均起重要保护作用。目前，腺苷A2AR在心肌肥厚的确切机制尚未完全阐明。【主要研究内容】：建立TAC法的心肌肥厚动物模型，取野生型（WT）和A2AR KO 小鼠各40只，予以分组：WT 假手术组（WT+Sham）、WT 主动脉缩窄术组(WT+TAC)、A2AR KO 假手术组（A2ARKO+Sham）、A2AR KO 主动脉缩窄术组（A2AR KO+TAC），采用主动脉缩窄法（TAC）制作心肌肥厚动物模型。超声评价小鼠心脏结构与功能，免疫组化检测心肌细胞肥大，RT-PCR、Western blot检测心肌细胞肥大标志物的mRNA和蛋白表达水平。体外培养心肌细胞，AngⅡ诱导心肌细胞肥大，给予A2AR激活剂、抑制剂及NF-κB抑制剂干预，探讨腺苷A2A受体保护心肌肥厚机制。【主要结果】：成功建立了野生型（WT）和A2AR KO 小鼠的心肌肥厚动物模型，在TAC诱导的心肌肥厚模型，腺苷A2AR KO小鼠心肌肥厚较WT小鼠明显加重，WT+TAC组较WT+Sham组小鼠的LVEDD、LVESD显著增加，FS则显著减少（各组P<0.05）,而且，A2AR KO+TAC较WT+TAC组小鼠的小鼠的LVEDD、LVESD显著增加，FS则显著减少（各组P<0.05）心肌细胞胚胎型基因ANP、ß-MHC表达显著增加；体外研究发现，在Ang II诱导的心肌细胞肥厚中，pP65在心肌细胞的蛋白表达水平显著升高，而A2AR和NF-κB选择性抑制剂均可抑制pP65的表达。【科学意义】本课题的完成将有助于阐明A2AR在心肌肥厚中的作用机制，为心肌肥厚的防治提供新的理论依据和药物靶点。