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

用于达农病和心力衰竭的人类 iPSC 衍生心肌细胞模型

• 批准号: 8396855
• 负责人: Cynthia Perry
• 金额: $ 4.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8396855
• 关键词: Adrenergic Agents; Animal Model; Apoptosis; Autophagocytosis; Autophagosome; Behavior; Biology; Biopsy; Calcium; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cardiovascular system; Cell Death; Cell Line; Cell physiology; Cellular biology; Characteristics; Childhood; Chloroquine; Cytoplasmic Organelle; Cytoskeletal Modeling; Disease; Electron Microscopy; Epinephrine; Failure; Fibroblasts; Functional disorder; Generations; Genetic; Glycogen Storage Disease Type IIb; Heart Diseases; Heart failure; Hip region structure; Homeostasis; Human; In Situ Nick-End Labeling; In Vitro; Left; Life; Light; Link; Lysosomal Storage Diseases; Lysosomes; Malignant Neoplasms; Mediating; Membrane Proteins; Messenger RNA; Methods; Microtubules; Modeling; Muscle Cells; Mutation; Myocardium; Neurodegenerative Disorders; Operative Surgical Procedures; Organelles; Oxidative Stress; Pathway interactions; Patients; Play; Process; Protein Isoforms; Proteins; Reactive Oxygen Species; Recycling; Role; Sarcomeres; Second Pregnancy Trimester; Sirolimus; Skeletal Muscle; Small Interfering RNA; Staining method; Stains; Starvation; Stress; Study models; Subfamily lentivirinae; Syndrome; System; Testing; Third Pregnancy Trimester; Time; Tissues; Up-Regulation; Vacuole; Western Blotting; adrenergic; cytokine; cytotoxicity; diacetyldichlorofluorescein; fluorescence imaging; human disease; implantation; in vitro Model; induced pluripotent stem cell; insight; novel; overexpression; patch clamp; response; ventricular assist device

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.

描述（由申请人提供）：本提案的总体目标是使用Danon综合征的新型人类模型来确定受损的自噬和心肌病之间的关系。尽管心血管生物学取得了进展，但由于缺乏合适的体外研究人类心肌细胞行为的模型，对人类心脏病的理解受到限制。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细胞分解代谢过程，与许多人类疾病包括神经退行性疾病、恶性肿瘤和心力衰竭有关。这一过程对于细胞内稳态至关重要，但其在心血管疾病中的作用却知之甚少。Danon病是一种X连锁的自噬性疾病，与严重的心脏和骨骼肌异常有关。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中死亡。这种疾病的标志是由于自噬体-溶酶体融合失败而导致的大的自噬空泡的积累。Danon病是由溶酶体膜蛋白2（LAMP 2）突变引起的，然而LAMP 2在自噬中的功能尚未完全表征。我们最近从患有Danon病的患者中创建了两个人诱导多能干细胞（hiPSC）系，这些患者在LAMP 2中具有不同的突变。我们的指导性假设是，LAMP 2的缺陷导致心肌细胞功能障碍，这是由于应激诱导的自噬空泡积累和氧化应激增加。为了检验我们的假设，我们提出：1）确定Danon患者iPSC衍生的心肌细胞是否在体外重演Danon病，2）确定在基础和应激条件下Danon患者hiPSC衍生的心肌细胞中对LAMP 2损失的细胞反应，和3）描绘自噬途径中LAMP 1和LAMP 2同种型的功能。心肌细胞将通过精氨酸介导的定向分化从我们的hiPSC系产生。hiPS衍生的心肌细胞和患者成纤维细胞都将作为Danon病的体外模型。这些模型将通过与Danon患者和野生型对照相比确定其独特的病理生理学特征来验证。LAMP 2的三种亚型和密切相关的蛋白质LAMP 1的作用将通过慢病毒过表达研究和在我们的体外模型中通过siRNA沉默LAMP 1并评估清除自噬空泡的能力来确定。细胞凋亡、氧化应激和细胞骨架完整性将在我们的Danon模型中在应激条件下进行评估，以确定LAMP 2丢失和细胞死亡之间的关系。了解LAMP 2功能障碍的机制将对Danon病的治疗以及与自噬受损相关的广泛疾病具有深远的意义。 公共卫生关系：尽管在心血管遗传学和病理生理学方面取得了进展，但由于缺乏合适的体外研究人类心肌细胞行为的模型，对人类心脏病的理解受到限制。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衍生的心肌细胞是研究人类细胞机制的独特体外方法，并且从Danon病患者产生hiPS细胞系不仅提供了溶酶体贮积病的第一个人类体外模型，而且还提供了对正常和病理性心脏病至关重要的非常重要的生物途径的洞察。