RATIONALE 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. OBJECTIVE Elucidate the pathophysiology of cardiac dysfunction in Danon disease. METHODS AND RESULTS 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. CONCLUSIONS 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.

中文翻译：

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受损的线粒体促进达农病中的线粒体损伤。

原理 溶酶体相关膜蛋白 2 型 (LAMP-2) 是一种高度保守、普遍存在的蛋白质，对自噬通量至关重要。LAMP-2 基因功能缺失突变导致达农病，这是一种罕见的 X 连锁疾病，其特征是发育迟缓、骨骼肌无力和严重的心肌病。我们之前发现，来自 Danon 患者的人类诱导多能干细胞衍生心肌细胞 (hiPSC-CMs) 表现出显着的线粒体氧化应激和细胞凋亡。了解 LAMP-2 表达的缺失如何导致心肌细胞功能障碍和心力衰竭，对于治疗达农病以及与自噬受损相关的各种其他心脏疾病具有重要意义。目的阐明达农病心功能不全的病理生理。方法和结果 我们从两名达农病患者中创建了 hiPSC，并使用完善的方案将这些细胞分化为 hiPSC-CM。Danon hiPSC-CM 显示出受损线粒体的积累、线粒体通量中断、线粒体呼吸能力降低以及关键线粒体途径的异常基因表达。恢复心脏中最丰富的 LAMP-2 异构体 LAMP-2B 的表达，挽救了线粒体自噬通量以及线粒体健康和生物能量学。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，在 Lamp-2 KO 小鼠中注意到自噬通量受损，以及异常线粒体数量增加、线粒体自噬不完全的证据和线粒体呼吸受损。生理上，Lamp-2 KO 小鼠表现出收缩功能障碍的早期特征，而没有明显的心力衰竭，这表明与达农病相关的代谢异常先于终末期疾病的发展，而不仅仅是与心力衰竭相关的继发变化的一部分。结论 在达农病的体外和体内模型中都注意到不完全的线粒体自噬通量和线粒体功能障碍，并导致明显的心脏收缩功能障碍。这表明线粒体清除受损可能是疾病发病机制的核心，也是治疗干预的潜在目标。表明与达农病相关的代谢异常先于终末期疾病的发展，而不仅仅是与心力衰竭相关的继发变化的一部分。结论 在达农病的体外和体内模型中都注意到不完全的线粒体自噬通量和线粒体功能障碍，并导致明显的心脏收缩功能障碍。这表明线粒体清除受损可能是疾病发病机制的核心，也是治疗干预的潜在目标。表明与达农病相关的代谢异常先于终末期疾病的发展，而不仅仅是与心力衰竭相关的继发变化的一部分。结论 在达农病的体外和体内模型中都注意到不完全的线粒体自噬通量和线粒体功能障碍，并导致明显的心脏收缩功能障碍。这表明线粒体清除受损可能是疾病发病机制的核心，也是治疗干预的潜在目标。