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

描述（由申请人提供）：本提案的总体目标是使用一种新的Danon综合征人类模型来确定自噬受损与心肌病之间的关系。尽管心血管生物学取得了进展，但由于缺乏合适的模型来体外研究人类心肌细胞的行为，对人类心脏病的了解受到限制。因此，我们对心脏病细胞生物学机制的理解大多来自动物模型，这些模型具有固有的局限性，可能无法模仿人类疾病。自噬是一种普遍存在的细胞分解代谢过程，涉及许多人类疾病，包括神经退行性疾病、恶性肿瘤和心力衰竭。这个过程对细胞内稳态至关重要；然而，人们对其在心血管疾病中的作用知之甚少。Danon病是最近发现的一种与严重心脏和骨骼肌异常相关的自噬x连锁疾病。绝大多数患者在儿童时期发展为严重的心肌病，并在生命的第二或第三个十年死亡。这种疾病的标志是由于自噬体-溶酶体融合失败导致大的自噬液泡的积累。Danon病是由溶酶体膜蛋白2 （LAMP2）突变引起的，但LAMP2在自噬中的功能尚未得到充分表征。我们最近从具有不同LAMP2突变的Danon病患者身上创建了两种人类诱导多能干细胞（hiPSC）系。我们的指导假设是，由于应激诱导的自噬空泡积累和氧化应激增加，LAMP2的缺乏导致心肌细胞功能障碍。为了验证我们的假设，我们提出：1)确定Danon患者ipsc衍生的心肌细胞是否在体外重现Danon病，2)确定在基础和应激条件下Danon患者hipsc衍生的心肌细胞中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。通过细胞因子介导的定向分化，我们的hiPSC细胞系将产生心肌细胞。hips衍生的心肌细胞和患者成纤维细胞将作为Danon病的体外模型。这些模型将通过确定其与Danon患者和野生型对照的不同病理生理特征来验证。在我们的体外模型中，我们将通过慢病毒过表达研究和siRNA沉默LAMP1，以及评估清除自噬空泡的能力来确定LAMP2的三种亚型和密切相关的蛋白LAMP1的作用。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完整性，以确定LAMP2缺失与细胞死亡之间的关系。了解LAMP2功能障碍的机制将对Danon病以及与自噬受损相关的一系列疾病的治疗产生深远的影响。

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.