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.
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.