Altered cardiac progenitor transcriptional program accompanies NOS-associated DNA damage in a human Duchenne muscular dystrophy organoid model

Duchenne muscular dystrophy (DMD), a rare X-linked disorder caused by dystrophin mutations, leads to progressive muscle degeneration and cardiomyopathy. We previously showed elevated DNA damage and mutagenesis in DMD pluripotent stem cells, due to nitric oxide synthase (NOS) dysregulation and reactive species. Here we examined how these disruptions influence cardiac tissue development. In developing cardiac organoids, the DMD cardiac progenitor (CP) population shows a premature, elevated activation of CP markers, followed by early maturation gene expression and reduced proliferation. DMD organoids exhibit elevated DNA damage in CPs, persistent pro-inflammatory signaling, and increased expression of inducible NOS. These early molecular disruptions lead to an impaired cardiac differentiation with a lower fraction of spontaneously contracting organoids. DMD organoids show higher cardiac troponin release and accelerated collagen accumulation, mirroring heart failure pathology in mdx mice and DMD patients. Inducing DNA damage in early-stage wild-type organoids reproduced part of the altered gene expression seen in DMD. NOS inhibition in DMD organoids reduced DNA damage and improved organoid contraction, though it did not prevent CM death or collagen accumulation or restore normal gene expression. Together, these findings position NO/NOS modulation as relevant to early cardiac differentiation defects, with isoform balance likely shifting across developmental stages.