SIRT3调控VLCAD乙酰化修饰在脂肪酸代谢异常介导房颤的机制研究

Disordered myocardial energy metabolism is a critical mechanism of the incidence of atrial fibrillation (AF). Our preliminary study presented that the very long-chain specific acyl-CoA dehydrogenase (VLCAD), one rate-limiting enzyme in fatty acid oxidation, was differently expressed in atrial tissues of AF patients, and the level of VLCAD acetylation was also significantly increased. Fatty acid oxidation is known as the most important energy source for myocardial cells. It has also been identified that acetylation could affect the biological activity of proteins and silent information regulator of transcription 3 (SIRT3) plays a key role in mitochondrial proteins acetylation. Thus, we suppose that differential expression of VLCAD, whose activity is influenced by acetylation, could affect the incidence and development of AF through regulating myocardial fatty acid metabolism and energy production, and VLCAD acetylation could be controlled by SIRT3. In the present project, we will construct wild-type and acetylation-mutated VLCAD viruses, SIRT3 gene overexpression and lentiviral vector to investigate (1) the effect of differential expression and acetylation of VLCAD on fatty acid metabolism and AF, and (2) the regulatory site of VLCAD acetylation and the effect of SIRT3 on the acetylation level of VLCAD during AF in both canine atrial fibrillation model and the primary myocardial cells. Our project is likely to illustrate the influence of VLCAD acetylation on regulating energy metabolism during AF, and the effect of SIRT3 on VLCAD acetylation, which may provide new targets for the prevention and treatment of AF.

心肌能量代谢失衡是心房颤动（房颤）发生机制之一。我们前期研究发现脂肪酸β氧化限速酶超长链脂酰辅酶A脱氢酶（VLCAD）在房颤患者心肌中表达异常，其乙酰化水平显著上调；已知脂肪酸氧化为心肌最重要供能途径，而乙酰化修饰显著影响蛋白质活性，且沉默信息调节因子3（SIRT3）是调控线粒体蛋白乙酰化修饰的关键因子。据此我们科学假说：VLCAD受乙酰化修饰调控，通过差异性表达调节心肌脂肪酸代谢与心肌供能影响房颤的发生发展；而VLCAD乙酰化程度受SIRT3调控。本研究拟构建VLCAD野生型/乙酰化位点突变型病毒及SIRT3过表达/低表达病毒，在犬房颤模型和原代心肌细胞上研究：①VLCAD的表达及乙酰化修饰对脂肪酸代谢及房颤的影响，②房颤时VLCAD乙酰化调节位点及SIRT3对其乙酰化水平的影响，以期阐明SIRT3调控VLCAD乙酰化修饰影响房颤心肌能量代谢受损的机制，为防治房颤寻找新的干预靶点。

研究背景：课题组前期研究发现房颤与脂肪酸代谢改变密切相关，在2015年国家自然科学基金工作中发现乙酰化翻译后修饰对心肌能量代谢有重要影响。前期研究发现，脂肪酸代谢相关分子，尤其是极长链酰基辅酶脱氢酶(VLCAD)在房颤的发生发展中起重要作用，且VLCAD的活性受乙酰化调控，但VLCAD的乙酰化如何介导房颤脂肪酸代谢异常的具体机制尚未阐明。.研究内容：收集左心耳临床标本并进行蛋白组学研究；建立慢性犬房颤模型，分为假手术组，快速心房起搏组，快速心房起搏+二甲双胍组，分别检测其脂肪酸代谢指标，有氧糖酵解指标，房颤易感性指标，心房纤维化标志物等；心外膜脂肪对房颤的发生发展起重要影响，分别在犬房颤模型和HL-1心房肌细胞与3T3-L1脂肪细胞共培养建立相互作用模型，探讨心外膜脂肪在房颤中的作用及机制。.研究结果及关键数据：首先，我们通过蛋白质组学研究发现房颤与AMP依赖的蛋白激酶（AMPK）信号通路受损关系十分密切。此后课题组进一步研究发现AMPK的激动剂二甲双胍能够通过影响脂肪酸代谢相关过程进而逆转房颤的心房重构作用。在上述研究过程中，我们还发现二甲双胍可减轻房颤诱导的左心耳组织脂质沉积。同时，二甲双胍也能改变脂肪酸代谢关键因子的蛋白表达，包括脂肪酸转运体(FAT/CD36)、肉碱棕榈酰转移酶-1(CPT-1)和VLCAD。其机制为在房颤病理过程中，二甲双胍通过AMPK/PGC-1α/PPARα通路上调这些因子的表达来发挥重要调节作用。正常情况下，心房肌细胞以脂肪酸代谢为主，但是在房颤过程中会转变为以糖酵解为主，即Warburg效应，而二甲双胍能逆转Warburg效应，恢复心房肌正常代谢状态。此外，我们还发现房颤的发生发展与心外膜脂肪重构密切相关，二甲双胍能通过ROS/NF-κB和PPARγ/APN/adipoR1通路改善心房和心外膜脂肪重构。.科学意义：本研究发现房颤病程中，脂肪酸代谢关键分子VLCAD表达水平下调，心房代谢由脂肪酸代谢为主转变为糖酵解代谢为主，心外膜脂肪组织重构导致房颤“恶性循环”，二甲双胍能逆转上述现象。