Human iPSC-Derived Cardiomyocyte Model for Danon Disease and Heart Failure

描述(由申请人提供)：这项提案的总体目标是使用一种新的达农综合征人类模型来确定自噬受损和心肌病之间的关系。尽管心血管生物学取得了进展，但由于缺乏合适的模型来研究人类心肌细胞的体外行为，对人类心脏病的理解一直受到限制。因此，我们对心脏病细胞生物学的机械理解大多来自动物模型，这些模型具有固有的局限性，可能无法模拟人类疾病。自噬是一种普遍存在的分解代谢的细胞过程，与许多人类疾病有关，包括神经退行性疾病、恶性肿瘤和心力衰竭。这一过程对细胞内环境的稳定至关重要，但人们对其在心血管疾病中的作用知之甚少。达农病是一种最近被描述的X连锁自噬障碍，与严重的心肌和骨骼肌异常有关。绝大多数患者在童年时患上严重的心肌病，并在生命的第二个或第三个十年死亡。这种疾病的特点是由于自噬小体-溶酶体融合失败而积累大的自噬空泡。Danon病是由溶酶体膜蛋白2(LAMP2)基因突变引起的，但LAMP2在自噬中的功能尚未完全确定。我们最近从具有不同LAMP2突变的达农病患者中创建了两个人类诱导多能干细胞(HiPSC)系。我们的指导性假设是LAMP2的缺陷会导致心肌细胞功能障碍，这是由于应激导致自噬空泡的堆积和氧化应激的增加。为了验证我们的假设，我们建议：1)确定Danon患者IPSC来源的心肌细胞是否在体外重现Danon病；2)确定Danon患者HiPSC来源的心肌细胞在基础和应激条件下对LAMP2缺失的细胞反应；3)描述LAMP1和LAMP2亚型在自噬途径中的功能。我们的HiPSC系将通过细胞因子介导的定向分化产生心肌细胞。髋关节来源的心肌细胞和患者成纤维细胞都将作为达农病的体外模型。这些模型将通过确定它们与达农患者和野生型对照的不同病理生理学特征进行比较来验证。在我们的体外模型中，LAMP2的三种异构体和密切相关的蛋白LAMP1的作用将通过慢病毒的过度表达研究和siRNA对LAMP1的沉默以及对清除自噬空泡的能力的评估来确定。我们的Danon模型将在应激条件下评估细胞凋亡、氧化应激和细胞骨架的完整性，以确定LAMP2丢失和细胞死亡之间的关系。了解LAMP2功能障碍的机制将对治疗达农病以及与自噬受损相关的一系列疾病具有深远的意义。 公共卫生相关性：尽管心血管遗传学和病理生理学取得了进展，但由于缺乏合适的模型来研究人类心肌细胞的体外行为，对人类心脏病的理解一直受到限制。因此，我们对心脏病细胞生物学的机械理解大多来自动物模型，这些模型具有固有的局限性，可能无法模拟人类疾病。使用iPS来源的心肌细胞是研究人类细胞机制的一种独特的体外方法，而达农病患者的HIPS细胞系的产生不仅提供了第一个人类溶酶体储存性疾病的体外模型，而且还提供了对正常和病理心脏疾病都至关重要的非常重要的生物途径的洞察。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to use a novel human model of Danon syndrome to determine the relationship between impaired autophagy and cardiomyopathy. Despite advances in cardiovascular biology, the understanding of human heart disease has been limited by lack of a suitable model for studying human cardiomyocyte behavior in vitro. Consequently, much of our mechanistic understanding of the cellular biology of heart disease has come from animal models which have inherent limitations and may not mimic human disease. Autophagy is a ubiquitous catabolic cellular process that has been implicated in many human diseases including neurodegenerative disease, malignancy, and heart failure. This process is critical for cellular homeostasis; yet its role in cardiovascular disease is poorly understood. Danon Disease is a recently described X-linked disorder of autophagy associated with severe cardiac and skeletal muscle abnormalities. The vast majority of patients develop severe cardiomyopathy in childhood and die in the second or third decade of life. The hallmark of this disorder is the accumulation of large autophagic vacuoles due to the failure of autophagosome-lysosome fusion. Danon Disease is caused by mutations in the lysosomal membrane protein 2 (LAMP2), however the function of LAMP2 in autophagy has not been fully characterized. We have recently created two human induced pluripotent stem cell (hiPSC) lines from patients with Danon Disease who have different mutations in LAMP2. Our guiding hypothesis is that deficiencies in LAMP2 result in cardiomyocyte dysfunction due to stress-induced accumulation of autophagic vacuoles and increased oxidative stress. To test our hypothesis we propose: 1) To determine if Danon patient iPSC-derived cardiomyocytes recapitulate Danon disease in vitro, 2) To determine the cellular response to the loss of LAMP2 in Danon patient hiPSC-derived cardiomyocytes under basal and stressed conditions and 3) To delineate the function of LAMP1 and LAMP2 isoforms in the autophagic pathway. Cardiac myocytes will be generated from our hiPSC lines by cytokine-mediated directed differentiation. Both hiPS-derived cardiomyocytes and patient fibroblasts will serve as in vitro models of Danon disease. These models will be validated by determining their distinct pathophysiologic characteristics in comparison to those from both Danon patients and wild type controls. The roles of the three isoforms of LAMP2 and the closely related protein, LAMP1, will be determined by lentiviral overexpression studies and silencing of LAMP1 by siRNA in our in vitro models and evaluating the ability to clear autophagic vacuoles. Apoptosis, oxidative stress and cytoskeletal integrity will be evaluated in our Danon models under stress conditions to determine the relationship between the loss of LAMP2 and cell death. Understanding the mechanisms of LAMP2 dysfunction will have profound implications for the treatment of Danon Disease as well as a broad array of disorders associated with impaired autophagy. PUBLIC HEALTH RELEVANCE: Despite advances in cardiovascular genetics and pathophysiolgy, the understanding of human heart disease has been limited by lack of a suitable model for studying human cardiomyocyte behavior in vitro. Consequently, much of our mechanistic understanding of the cellular biology of heart disease has come from animal models which have inherent limitations and may not mimic human disease. The use of iPS- derived cardiomyocytes is a unique in vitro method of studying human cellular mechanisms and the generation of hiPS cell lines from patients with Danon Disease not only provides the first human in vitro model of a lysosomal storage disease, but also provides insight into a very important biologic pathway that is crucial for both normal and pathological cardiac conditions.