Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA.
Department of Pharmacology, University of California San Diego, La Jolla, CA, USA.
J Mol Cell Cardiol. 2017 Jul;108:86-94. doi: 10.1016/j.yjmcc.2017.05.007. Epub 2017 May 16.
Lysosomal associated membrane protein type-2 (LAMP-2) is a highly conserved, ubiquitous protein that is critical for autophagic flux. Loss of function mutations in the LAMP-2 gene cause Danon disease, a rare X-linked disorder characterized by developmental delay, skeletal muscle weakness, and severe cardiomyopathy. We previously found that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from Danon patients exhibited significant mitochondrial oxidative stress and apoptosis. Understanding how loss of LAMP-2 expression leads to cardiomyocyte dysfunction and heart failure has important implications for the treatment of Danon disease as well as a variety of other cardiac disorders associated with impaired autophagy.
Elucidate the pathophysiology of cardiac dysfunction in Danon disease.
We created hiPSCs from two patients with Danon disease and differentiated those cells into hiPSC-CMs using well-established protocols. Danon hiPSC-CMs demonstrated an accumulation of damaged mitochondria, disrupted mitophagic flux, depressed mitochondrial respiratory capacity, and abnormal gene expression of key mitochondrial pathways. Restoring the expression of LAMP-2B, the most abundant LAMP-2 isoform in the heart, rescued mitophagic flux as well as mitochondrial health and bioenergetics. To confirm our findings in vivo, we evaluated Lamp-2 knockout (KO) mice. Impaired autophagic flux was noted in the Lamp-2 KO mice compared to WT reporter mice, as well as an increased number of abnormal mitochondria, evidence of incomplete mitophagy, and impaired mitochondrial respiration. Physiologically, Lamp-2 KO mice demonstrated early features of contractile dysfunction without overt heart failure, indicating that the metabolic abnormalities associated with Danon disease precede the development of end-stage disease and are not merely part of the secondary changes associated with heart failure.
Incomplete mitophagic flux and mitochondrial dysfunction are noted in both in vitro and in vivo models of Danon disease, and proceed overt cardiac contractile dysfunction. This suggests that impaired mitochondrial clearance may be central to the pathogenesis of disease and a potential target for therapeutic intervention.
溶酶体相关膜蛋白 2（LAMP-2）是一种高度保守、普遍存在的蛋白，对自噬流至关重要。LAMP-2 基因的功能丧失突变导致 Danon 病，这是一种罕见的 X 连锁疾病，其特征为发育迟缓、骨骼肌无力和严重的心肌病。我们之前发现，Danon 病患者的人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）表现出明显的线粒体氧化应激和细胞凋亡。了解 LAMP-2 表达缺失如何导致心肌细胞功能障碍和心力衰竭，对于 Danon 病以及各种其他与自噬受损相关的心脏疾病的治疗具有重要意义。
阐明 Danon 病中心肌功能障碍的病理生理学。
我们从两名 Danon 病患者中创建了 hiPSCs，并使用成熟的方案将这些细胞分化为 hiPSC-CMs。Danon hiPSC-CMs 表现出受损线粒体的积累、线粒体自噬流中断、线粒体呼吸能力下降以及关键线粒体途径的基因表达异常。恢复心脏中最丰富的 LAMP-2 同工型 LAMP-2B 的表达，可挽救自噬流以及线粒体健康和生物能量。为了在体内证实我们的发现，我们评估了 Lamp-2 敲除（KO）小鼠。与 WT 报告小鼠相比，Lamp-2 KO 小鼠的自噬流受损，异常线粒体数量增加，表明不完全的线粒体自噬以及线粒体呼吸受损。在生理上，Lamp-2 KO 小鼠表现出早期的收缩功能障碍特征，而没有明显的心力衰竭，这表明与 Danon 病相关的代谢异常先于终末期疾病的发展，而不仅仅是心力衰竭相关的继发性变化的一部分。
在 Danon 病的体外和体内模型中都观察到不完全的线粒体自噬流和功能障碍，并出现明显的心脏收缩功能障碍。这表明受损的线粒体清除可能是疾病发病机制的核心，也是治疗干预的潜在靶点。
