基于SIRT3/AMPK通路探讨Apelin-13调控肥厚型心肌病心肌代谢重构的作用及机制研究

Hytertrophic cardiomyopathy（HCM）is a genetically inherited disease. Cardiac energy impairment leads to metabolic remodeling and precedes the development of hypertrophy. However, there are no effective therapy for the metabolic remodeling. Apelin-13, an endogenous ligand for the apelin(APJ) receptor has been recognized as a new adipokine that plays an important role in regulating energy metabolism. Sirtuin-3(SIRT3), a major mitochondrial deacetylase has been shown to activate AMP-activated protein kinase (AMPK) that participates in regulating energy metabolism. In our preliminary study, Apelin-13 has been shown to ameliorate myocardial fibrosis and improve the energy metabolism, but the mechanism and functional target is still unclear. It has been not reported that Apelin-13 attenuated myocardial remodeling through the regulation of metabolic remodeling, which was mediated through the activation of the SIRT3/AMPK pathway. In this study, we choose HCM primary myocardial cells to explore the functional role and mechanism that Apelin-13 regulates myocardial metabolic remodeling through SIRT3/AMPK pathway. Some experimental techniques such as western blot、qRT-PCR、siNRA、HPLC、fluorescent probe labeling as well as special inhibitor were used. Furthermore we use the HCM cTnIR145G transgenic mice to explore the effects of apelin-13 in regulating metabolic remodeling and myocardial remodeling by virtue of echocardiography、Clark electrode etc. This study is designed to validate the potential role of apelin-13 in improving the metabolic remodeling, which will provide new perspectives for the treatment of HCM.

心肌代谢重构是肥厚型心肌病（HCM）重要特征，促进了心肌肥大的进展。Apelin-13是一类新型脂肪因子，在调节能量代谢中发挥重要作用。Sirtuin-3(SIRT3) 蛋白是一种重要的线粒体去乙酰化酶，可以激活AMPK参与能量代谢的调节。前期研究揭示Apelin可以减轻HCM心肌纤维化，增加能量生成，但其作用机制尚不明确。Apelin13能否通过SIRT3/AMPK改善代谢重构相关研究未见报道。本研究借助于HCM原代心肌细胞，利用Western blot 、qRT-PCR、siRNA、探针标记等实验方法，深入探讨SIRT3/AMPK通路在介导Apelin-13改善HCM代谢重构中的作用及相关机制。其次用cTnIR145GHCM转基因小鼠，探讨 Apelin-13能否改善代谢重构及其机制，若能揭示Apelin-13通过SIRT3/AMPK通路改善代谢重构，将为临床治疗HCM提供新思路。

肥厚型心肌病（hypertrophic cardiomyopathy，HCM）是一种常染色遗传的心肌疾病，在人群中占的比例约为1/500，其临床特征为难以解释的左室肥厚、心肌纤维化及潜在的恶性心律失常，是青少年猝死的最常见的原因。目前尚未逆转左室肥厚的治疗途径，HCM由于肌小节基因突变引发的能量供应不足或糖脂代谢失衡，导致心肌出现代谢重构，进而诱发心室重构。Apelin作为新发现的分子肽类物质可改善线粒体形态及功能。 SIRT3/AMPK通路在调节底物代谢、增加线粒体氧化磷酸化、 降低ROS生成保护线粒体功能中发挥重要作用。调控SIRT3/AMPK通路能否改善HCM代谢重构，减缓心肌细胞肥大目前尚无研究。在本研究中。 我们假定Apelin-13可能通过活化SIRT3/AMPK通路，促进代谢底物葡萄糖和脂肪酸氧化，增加线粒体生物合成，抗心肌细胞凋亡进而改善心室重构。首先在细胞实验中，利用AngII诱导的肥大心肌细胞，通过Apelin-13进行干预，证实Aplein可以通过促进PGC-1α、FoxO3a蛋白表达增加线粒体ATP生成，降低ROS产生，增加脂肪酸氧化水平抵抗心肌细胞肥大，其机制是通过激活SRIT3/AMPK通路实现的；在动物实验中，采用Apelin-13进行干预HCM转基因小鼠，从影像学和分子细胞层面两部分探讨Apelin-13对HCM心室肥大和纤维化的影响，首先通过小鼠心脏超声方面证实了Aplein-13干预3个月后可以减轻左室肥大，改善HCM舒张功能障碍；在小鼠心脏病理切片包括HE染色及马松染色中，证实Apelin-13可以减轻心肌肥大，细胞排列紊乱，同时可以减轻心肌纤维化程度，其次从分子层面，利用Westernblot、RT-PCR方法提示Apelin-13可以上调SIRT3/AMPK信号通路中，SIRT3及AMPK、P-AMPK及其下游分子如PGC-1α、FoxO3a蛋白及其mRNA表达，增加心肌线粒体生成，降低心肌ROS的产生，抗心肌细胞凋亡而发挥抗心肌肥大及纤维化作用。