Wang Xiaoping, Jiang Yanyan, Zhang Yawen, Sun Qianbin, Ling Guanjing, Jiang Jinchi, Li Weili, Tian Xue, Jiang Qianqian, Lu Linghui, Wang Yong
School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China.
School of Life Science, Beijing University of Chinese Medicine, Beijing 100029, China.
Phytomedicine. 2022 May;99:154009. doi: 10.1016/j.phymed.2022.154009. Epub 2022 Feb 18.
Mitophagy can regulate mitochondrial homeostasis, preserve energy metabolism and cardiomyocytes survival effectively to restrain the development of heart failure (HF). Danqi Pill (DQP), composed of the dry roots of Salvia miltiorrhiza Bunge and Panax notoginseng, is included in the 2015 national pharmacopeia and effective in the clinical treatment of coronary heart diseases. Our previous studies have approved that DQP exerted remarkable cardioprotective effects on HF. However, the effect and mechanism of DQP on mitophagy have not been proved yet.
HYPOTHESIS/PURPOSE: We aim to explore whether DQP regulates mitophagy to protect against HF and to elucidate the in-depth mechanism.
The HF rat model for evaluating DQP's efficacy was established with left anterior descending coronary artery ligation. The oxygen-glucose deprivation-reperfusion-induced cardiomyocyte model was conducted to clarify the potential mechanism of DQP.
The mitochondria-targeted fluorescent protein Keima (mt-Keima) was applied for detecting mitophagy flux. Co-immunofluorescence and co-immunoprecipitation were performed to detect protein co-localization. Flow cytometry for JC-1 and Annexin-FITC/PI staining was utilized for assessing mitochondrial activity and function.
In vivo, medium dose of DQP (1.5 g/kg) notably improved cardiac function and inhibited cardiac apoptosis in HF rats. Co-immunofluorescent staining of LC3B and TOM20 showed that DQP restored mitophagy. Further co-immunoprecipitation demonstrated that DQP increased the co-localization of FUNDC1 with either ULK1 or PGAM5. In vitro, DQP markedly protected mitochondrial membrane potential damage, reduced cardiomyocytes apoptosis, decreased the level of mitochondrial ROS, and increased the ATP level. Parallel with the in vitro results, DQP increased the interaction of FUNDC1 and LC3B, while knockdown of FUNDC1 diminished the interaction. Besides, Mt-Keima signaling detection further confirmed that DQP significantly promoted mitophagy. Intriguingly, knockdown of ULK1 or PGAM5 separately weakened rather than eliminated these effects of DQP on FUNDC1-mediated mitophagy, mitochondrial homeostasis and energy metabolism.
Our results demonstrated that DQP protected against HF by improving FUNDC1-mediated mitophagy to perverse energy metabolism through the coordinated regulation of ULK1 and PGAM5.
线粒体自噬可调节线粒体稳态，有效维持能量代谢和心肌细胞存活，从而抑制心力衰竭（HF）的发展。丹芪滴丸（DQP）由丹参和三七的干燥根组成，被收录于2015年国家药典，在冠心病的临床治疗中有效。我们之前的研究已证实DQP对HF具有显著的心脏保护作用。然而，DQP对线粒体自噬的作用及机制尚未得到证实。
假设/目的：我们旨在探讨DQP是否通过调节线粒体自噬来预防HF，并阐明其深层机制。
通过结扎左冠状动脉前降支建立评估DQP疗效的HF大鼠模型。采用氧糖剥夺-再灌注诱导的心肌细胞模型来阐明DQP的潜在机制。
应用线粒体靶向荧光蛋白Keima（mt-Keima）检测线粒体自噬通量。进行共免疫荧光和共免疫沉淀以检测蛋白共定位。利用流式细胞术检测JC-1以及Annexin-FITC/PI染色来评估线粒体活性和功能。
在体内，中剂量的DQP（1.5 g/kg）显著改善了HF大鼠的心脏功能并抑制了心脏细胞凋亡。LC3B和TOM20的共免疫荧光染色显示DQP恢复了线粒体自噬。进一步的共免疫沉淀表明DQP增加了FUNDC1与ULK1或PGAM5的共定位。在体外，DQP显著保护线粒体膜电位损伤，减少心肌细胞凋亡，降低线粒体ROS水平，并提高ATP水平。与体外结果一致，DQP增加了FUNDC1与LC3B的相互作用，而敲低FUNDC1则减少了这种相互作用。此外，Mt-Keima信号检测进一步证实DQP显著促进了线粒体自噬。有趣的是，分别敲低ULK1或PGAM5会削弱而非消除DQP对FUNDC1介导的线粒体自噬、线粒体稳态和能量代谢的这些作用。
我们的结果表明，DQP通过改善FUNDC1介导的线粒体自噬，通过ULK1和PGAM5的协同调节来维持能量代谢，从而预防HF。
