Integrated RNA sequencing and in vivo biosensor imaging define the early pathogenic cascade of Duchenne muscular dystrophy

: Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by loss of dystrophin, and characterized by progressive muscle wasting, with massive replacement of muscle fibers with adipose tissue. Yet, the early molecular events that initiate pathology remain poorly defined. Here, we combined longitudinal RNA sequencing of sapje dystrophic zebrafish (a single-mutation vertebrate model of human DMD characterized by a severe phenotype), transcriptomic profiling of human DMD myoblasts and myotubes, and functional in vivo imaging using pathway-specific zebrafish biosensors to reconstruct the cascade of events triggered by dystrophin deficiency. We observed that the earliest stages of disease are characterized by marked downregulation of genes controlling cytosolic Ca2+ homeostasis, mitochondrial function and organization, and Pax3/Mef2a/Srf-mediated transcriptional programs essential for satellite cell maintenance and muscle differentiation. These early deficits precede robust but ineffective regenerative and metabolic compensatory responses, accompanied by extracellular matrix remodeling and TGFβ activation. At advanced stages, both sapje zebrafish and human DMD myotubes converge on profound mitochondrial dysfunction, impaired cell-cycle control, and chronic inflammation signaling. Live imaging of sapje zebrafish biosensors validated these transcriptomic signatures, revealing reduced Notch, Bmp, Shh, Hif-1a and Wnt signaling, along with aberrant TGFβ activity and disrupted mitochondrial dynamics in vivo. Together, these findings identify a conserved temporal sequence linking early Ca2+ dysregulation to mitochondrial failure, satellite cell hyperactivation, and fibrotic remodeling, providing mechanistic insights and therapeutic targets for early intervention in DMD patients.