Abstract 17079: In Vitro Modeling of Duchenne Muscular Dystrophy (DMD) Cardiomyopathy Using Human Induced Pluripotent Stem Cells (hiPSC)
Introduction: Duchenne Muscular Dystrophy (DMD) is the most common and debilitating X-linked muscular dystrophy characterized by absence of dystrophin. A significant cardiomyopathic phenotype is seen in DMD and has emerged as a leading cause of death in DMD. There is limited knowledge of pathophysiology of DMD cardiomyopathy due to the limited availability of patient tissues and no human cardiomyocyte model of DMD exists. We hypothesized the pathophysiology of DMD cardiomyopathy can be modeled by utilizing DMD hiPSC CM and interrogating them at the cellular and molecular level.
Methods: Dermal fibroblasts were obtained from patients with DMD cardiomyopathy (dystrophin deletion of exon 4-43) and normal healthy controls. The fibroblasts were reprogrammed to hiPSC using retroviral vectors containing the human transcription factors, OCT4, SOX2, KLF4 and C-MYC. The DMD and control hiPSC lines were differentiated to CM using a directed differentiation protocol. Molecular and physiologic assays were performed at day 30 of differentiation.
Results: The newly-derived DMD and control hiPSC lines expressed pluripotent markers as detected by qRT-PCR and immunostaining, formed teratomas, and were karyotypically normal. The DMD hiPSC showed deletion of DYSTROPHIN gene exons 4-43. Both DMD and control hiPSC differentiated to beating, electrically coupled CM sheets. Western blot of DMD hiPSC CM lack dystrophin immunoreactivity compared to dystrophin band at 427kDa in all control hiPSC CM. Dystrophin is associated with the dystroglycan complex in all control hiPSC CM and absent in DMD hiPSC CM as assayed by wheat germ agglutinin co-immunoprecipitation of beta-dystroglycan and dystrophin at d30. Increased membrane fragility, a hallmark feature of DMD, was observed by increased release of CK-MB in DMD hiPSC CM in response to both osmotic and dobutamine stress.
Conclusion: Our results suggest that DMD hiPSC can be differentiated to cardiomyocytes and have a unique phenotype for disease investigation. Further evaluation of the molecular and physiologic phenotype of DMD hiPSC cardiomyocytes will serve as a platform for developing novel therapeutic regimens for patients with DMD cardiomyopathy.
- © 2013 by American Heart Association, Inc.