HIF-1信号介导LAMP2缺失所致肥厚型心肌病的机制研究

Danon disease, a hypertrophic cardiomyopathy (HCM) subtype in children, is a genetic disease caused by mutations in the gene coding lysosomal associated membrane protein 2 (LAMP2). Autophagy dysregulation due to LAMP2 deficiency is considered as the critical pathogenesis of Danon disease, but it is still unclear how the LAMP2 deficiency-induced autophagy dysregulation leads to hypertrophic myocardium. Previously, several studies have shown that LAMP2 interacts with hypoxia-inducible factor-1(HIF-1) and mediates its degradation, and that the overexpression of HIF-1α can cause myocardial hypertrophy. Our preliminary data also found an abnormal elevation of HIF-1α level in LAMP2-deficient cardiomyocytes. Therefore, we hypothesize that the abnormal overactivation of HIF-1 pathway is the essential mechanism in the pathogenesis of LAMP2 deficiency-induced HCM. In this project, using cardiomyocytes differentiated from Danon disease patient-derived iPS cells and Lamp2-deficient mice, we plan to study if the LAMP2-induced HCM is dependent on the HIF-1 pathway by combined approaches such as molecular genetics, etc. Finally, we will test if HIF-1α modulation can alleviate the disease-related symptoms in these disease models. The study will shed light on the molecular mechanism of Danon disease, providing novel therapeutic strategies to treat this devastating disorder.

Danon病是溶酶体相关膜蛋白2（LAMP2）基因突变所致的遗传性疾病，是引起儿童肥厚型心肌病（HCM）的重要病因之一。自噬障碍是Danon病的重要发病机制，但自噬异常导致该病患者心肌肥厚的机制尚不清楚。既往研究表明，LAMP2与缺氧诱导因子-1（HIF-1）存在相互作用并介导后者的降解，而HIF-1的活性亚基HIF-1α过表达则可导致心肌肥厚。我们的预实验也发现LAMP2缺失心肌细胞中HIF-1α水平异常升高。因此推测HIF-1信号异常升高是Danon病发生心肌肥厚的一个关键机制。本项目拟以Danon病患者来源iPS细胞定向分化的心肌细胞及Lamp2敲除小鼠为研究模型，运用分子遗传等方法研究HIF-1通路在LAMP2缺失所致HCM中的作用及机制，并通过改变HIF-1α水平来减轻LAMP2缺失导致的心肌肥厚。本项目研究有助于阐明Danon病心肌肥厚的发生机制，并为HCM的治疗提供新的靶点。

Danon病是一种由溶酶体相关膜蛋白2（LAMP2）基因突变所致的X连锁显性遗传病，并伴发儿童肥厚型心肌病。作为溶酶体跨膜蛋白，LAMP2功能丧失会引起细胞自噬功能障碍及细胞代谢稳态失衡。本项目采用Danon病患者来源iPS细胞诱导分化的心肌细胞（hiPSC-CMs)及Lamp2敲除小鼠，通过免疫荧光观察和心肌细胞转录组（RNA-seq）分析，发现LAMP2缺失延迟了hiPSC-CMs的成熟；心脏超声心动图显示20周龄LAMP2-/y小鼠的心脏体重比（HW/BW）明显增加和心肌舒张功能障碍；IonOptix测量系统表明，成年LAMP2-/y小鼠原代心肌细胞存在收缩和舒张功能障碍及兴奋收缩偶联异常；透射电镜和免疫印迹结果表明LAMP2-/y小鼠心肌细胞存在自噬体与溶酶体融合障碍及线粒体形态和数目异常；四甲基罗丹明甲酯（TMRM）染色结果显示，LAMP2-/y小鼠心肌细胞线粒体膜电位下降；荧光定量PCR结果表明LAMP2-/y小鼠心肌细胞线粒体DNA（mtDNA）拷贝数代偿性增加；RNA-seq数据表明LAMP2-/y小鼠原代心肌细胞线粒体三羧酸循环过程相关基因表达下调而糖酵解途径相关基因表达上调；通过进一步的非靶向代谢组学，证实了心肌细胞上述途径中关键代谢物的改变；转录组学和非靶向代谢组学联合分析提示LAMP2缺失导致与氨基酸生物合成相关的基因上调，并在该模型中发现精氨酸含量显著升高；免疫印迹结果也证实mTOR通路激活。上述实验结果表明LAMP2缺失会导致心肌细胞线粒体结构和功能异常及代谢重编程的发生，并可能通过影响心肌细胞精氨酸代谢及激活mTOR信号通路引起心肌细胞体积增大。因此，阐明LAMP2对心肌细胞自噬、能量代谢以及氨基酸合成的影响，有望揭示Danon病患者心肌病的发生机制，为Danon病治疗提供新策略。