Microdystrophins Partially Rescue Duchenne Muscular Dystrophy Deficits in iPSC-Cardiomyocytes

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Dilated cardiomyopathy is the leading cause of death in DMD patients. Smaller variants of dystrophin, called microdystrophins, amenable to packaging into adeno-associated virus (AAV), have shown to be effective in improving skeletal muscle function in animal models; however, the functional benefit of these microdystrophins in the DMD heart remains unclear. To determine the efficacy of microdystrophin gene therapy, we compared three microdystrophin variants in DMD cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs). We used three DMD lines of different genetic backgrounds and benchmarked against healthy isogenic controls expressing full-length dystrophin. DMD iPSC-cardiomyocytes exhibit hallmarks of disease, including arrhythmia and poor viability compared to healthy isogenic controls. In parallel, we tested a dystrophin variant, called minidystrophin, which is larger than the microdystrophins. We validated dystrophin expression and compared transcriptional profiles by bulk RNA sequencing. For morphological analysis, we performed immunohistochemistry of cardiac troponin on fixed cells. To test the functional benefit of the transgenes, we performed calcium imaging with a ratiometric calcium indicator as well as viability assays. Our results show that the microdystrophins partially rescue disease phenotypes; however, the results are variable among genetic backgrounds. None of the gene therapies altered the global transcriptional profile of the DMD iPSC-CMs to resemble a healthy profile. Minidystrophin significantly improved cell viability of DMD iPSC-CMs in two of three lines of different genetic backgrounds. Our findings suggest that microdystrophins may have limited efficacy in DMD cardiomyocytes and that the delivery of larger dystrophin variants may be necessary to delay the onset of cardiac complications.