Duchenne Muscular Dystrophy Cardiac Exosomes Contribute to the Pathogenesis of Dystrophin-deficient Cardiomyopathy

Introduction: Cardiomyopathy is a devastating consequence of Duchenne muscular dystrophy (DMD) and its pathogenesis is not well known. Exosomes (exos) are small, secreted vesicles with the ability to exert paracrine effects in target cells by transfer of their molecular cargo. This cargo can be dysregulated in disease and may contribute to pathogenesis. Duchenne muscular dystrophy exos in skeletal muscle promoted pathology, but whether exos are pathological in the DMD heart is unclear. We hypothesize that exposure to DMD cardiac exos promotes pathogenesis and their inhibition will protect against cardiomyocyte injury. To investigate, we used DMD iPSC-derived cardiomyocytes (iCM) and mdx mouse models of DMD. Methods/Results: In vitro, 2 non-affected (N), 2 patient-derived DMD and 1 gene-edited DMD iCMs were exposed to either exos isolated from N- or DMD-iCMs for 48 hr, or to GW4869, an exo inhibitor for 24 hr, followed by 1 hr stress (100 μM H 2 O 2 in 10 mM deoxyglucose in RPMI - glucose) and 4 hr recovery (RPMI + glucose) and stained with either DHE, TMRE and propidium iodide to assess ROS levels, mitochondrial membrane potential (Δψm) and cell death, respectively. Stress increased ROS levels in all 3 DMD-iCMs from 413-1102AU to 955-2008AU, which was diminished with N-exo (346-1061AU) but not DMD-exo (882-1995AU). Stress caused Δψm loss in DMD-iCM from 4049-4082AU to 439-1315AU, and N-exo (1917-2867AU) but not DMD-exo (870-1586AU) were protective. Stress increased cell death in DMD-iCM from 3-10% to 30-45% which was decreased with N-exo (10-21%) but not DMD-exo (26-46%). GW4869 decreased ROS levels from 955-2008AU to 344-528AU , cell death from 30-45% to 6-28% and partially rescued Δψm from 439-1315AU to 1947-3842AU in DMD-iCM. In mdx mice, GW4869 reduced isoproterenol induced cardiac injury ( GW+Iso: 3±2% vs. Veh+Iso: 13±3% ). Conclusions: In vitro , unlike N-exo, DMD-exo exposure failed to protect DMD-iCM from stress. Inhibiting DMD-exo with GW4869 protected DMD-iCM against cell stress and reduced mdx cardiac isoproterenol-induced injury. Together, these data suggest that DMD-exo may dysregulate the DMD cardiomyocyte response to stress thereby contributing to injury. Furthermore, exosome reduction may be a strategy for the treatment of DMD cardiomyopathy .