In Vitro Modeling of Duchenne Muscular Dystrophy (DMD) Cardiomyopathy Using Human Induced Pluripotent Stem Cells (hiPSC)

Abstract

Purpose DMD is the most common an debilitating X-linked muscular dystrophy that leads to devastating skeletal and cardiac muscle damage. DMD cardiomyopathy has now emerged as the leading cause of death in DMD, however there is limited knowledge of pathophysiology of DMD cardiomyopathy. The purpose of this is to utilize patient-specific hiPSC derived cardiomyocytes (CM) to model DMD cardiomyopathy. Methods and Materials Dermal fibroblasts were obtained from a DMD patient with severe cardiomyopathy and a normal healthy control. The fibroblasts were reprogrammed to hiPSC with retrovirus containing the human transcription factors, Oct4, Sox2, Klf4, and c-Myc. The DMD and control hiPSC lines were differentiated to CM using a monolayer, extra cellular matrix, growth factor directed differentiation. Molecular and physiologic assays were performed at day 30 of differentiation. Results The DMD and control hiPSC lines were fully characterized and demonstrated pluripotent markers with qRT-PCR and immunostaining, formed teratomas with all three germ layers, and were karyotypically normal. DMD hiPSC were shown to have absence of dystrophin exons 4-43, consistent with the patient’s mutation. DMD hiPSC differentiated to beating CM sheets and initiation of beating was observed between days 10-14 in both DMD and control hiPSC CM. Increased membrane fragility was seen in d30 DMD hiPSC CM compared to control CM as assayed by hypo-osmotic stress and LDH release. Initial gene expression, demonstrated significantly lower dystrophin expression in DMD hiPSC CM as compared to control hiPSC CM, while there was significantly higher induction of beta-1-adrenergic receptor gene expression in DMD hiPSC CM. Conclusions Our results suggest that DMD hiPSC CM can be differentiated to cardiomyocytes and have a unique phenotype for disease investigation. Further evaluation of the molecular and physiologic phenotype of DMD hiPSC CM will serve as a platform for developing novel therapeutic regimens for patients with muscular dystrophy cardiomyopathies.