LRP6调控心肌线粒体自噬在心力衰竭发生发展中的作用及机制

LRP6, an essential coreceptor for wnt ligands, regulates embryonic development in various systems and differentiation process from stem cells towards the different committed cell types. However, few studies report the role of LRP6 in adult cardiac muscle. During preliminary experiment, we observed that LRP6 was downregulated in myocardium of mice with heart failure induced by pressure overload, and cardiomyocyte-conditional knockout of LRP6 in mice induced cardiac dysfunction, severe myocardial mitophagy, and translocation of Drp1 to mitochondria. It suggests that the downregulation of LRP6 in myocardium may be involved in the development of heart failure by inducing excessive myocardial mitophagy. To explore the potential mechanisms, we will use mice with cardiomyocyte-conditional knockout of LRP6 or cultured human cardiomyocytes AC16 transfected with shRNA-LRP6, treated with PKA inhibitor or Drp1 inhibitor, to observe the production of ROS, the expression of PKA and Drp1, mitophagy, fatty acid metabolism and cardiac function. On the basis of it, we will build pressure overload in mice with cardiomyocyte-specific overexpression of LRP6 or provide mechanical stretch to LRP6-overexpressed-AC16 cells to observe the cardiac protective role of LRP6 against mitochondria injury and cardiac dysfunction. This study will firstly clarify the molecular mechanism of LRP6 in mitophagy regulation that is involved in the development of heart failure. This study will be helpful for finding a novel intervention strategy for heart failure induced by pressure overload.

Wnt受体LRP6参与干细胞和胚胎各器官的发育和分化，但其是否调控成年心肌功能目前还未见相关研究。我们在前期研究中观察到压力负荷小鼠模型心衰期LRP6出现下调以及心肌LRP6特异性敲除的小鼠出现心力衰竭、线粒体过度自噬、Drp1线粒体转位，提示LRP6下调可能是介导线粒体功能失调导致心衰发展的重要因素之一。为进一步探讨其具体机制，本项目拟通过LRP6心肌特异性敲除小鼠以及慢病毒干扰人心肌细胞系AC16的LRP6表达研究LRP6下调后如何调控Drp1的线粒体转位介导线粒体过度自噬引起脂肪酸代谢障碍从而参与心衰的发展的具体机制，并进一步通过LRP6心肌过表达小鼠及AC16心肌细胞建立压力负荷及机械牵张模型观察其是否改善线粒体自噬延缓或防治心衰的发展。本研究将阐明LRP6调控心肌线粒体自噬介导脂肪酸代谢障碍参与心衰发展的新机制，拓展人们对LRP6功能和机制的认识，为心衰防治提供新思路。

Wnt受体LRP6参与干细胞和胚胎各器官的发育和分化，但其是否调控心力衰竭的发生发展，研究甚少。在该项目研究中，我们观察到LRP6在人扩心病的心肌组织显著降低。我们成功构建了他莫昔芬诱导型LRP6心肌特异性敲除小鼠，发现LRP6敲除后，小鼠心功能失调，生存率下降，线粒体自噬小体增多及功能下降，心肌脂滴蓄积。蛋白组学发现差异性蛋白富集PPARs相关脂肪酸代谢通路。进一步研究发现心肌LRP6敲除后，激活Drp1,引起心肌核内TFEB下降，从而抑制自噬降解相关蛋白LC3B表达,p62的降解以及脂肪酸代谢相关蛋白PPARα和δ等表达。激活Drp1还通过调控转录因子CTCF抑制CPT1b的表达抑制脂肪酸代谢。以上机制最终引起自噬降解障碍和脂肪酸代谢紊乱，从而介导心衰。此外我们还发现LRP6是心肌闰盘结构的重要组成蛋白，与闰盘其他标志蛋白Cx43，N-cadherin 等结合，LRP6敲除后介导闰盘结构紊乱，Cx43，N-cadherin等蛋白表达改变，更易出现心律失常。通过心肌特异性LRP6过表达小鼠建立压力负荷模型，发现LRP6过表达可保护心功能，延缓心衰的发展。本研究阐明了LRP6调控心肌线粒体自噬降解通路、脂肪酸代谢以及心肌闰盘结构等在心衰发展中的新机制，为心衰防治提供新靶点。