Transplantation of healthy muscle stem cells improves the bioenergetic function of dystrophic myofibers

Introduction: Skeletal muscle regeneration is an energy-demanding process that relies on mitochondria to generate ATP. While healthy skeletal muscle fibers have organized interfibrillar mitochondrial networks organizing architecture segregated into columns along the Z-line of the myofiber, regenerating fibers exhibits altered mitochondrial networks that span several sarcomeres. Despite links between muscle regeneration and altered mitochondrial dynamics and biogenesis, the role of specific cells in remodeling these networks remains unclear. Because muscle stem cells (MuSCs) play a critical role in muscle regeneration, we explored the relationship between MuSCs and mitochondria in regenerating muscle fibers. Methods: To induce muscle regeneration, we used the hindlimb ischemia mouse model of peripheral artery disease (PAD) and the mdx mouse model of Duchenne’s muscular dystrophy (DMD). Furthermore, transgenic reporter mice such as Pax7-tdTomato, H2B-EGFP, and mitoDendra2 were used to mark MuSCs, track nuclei, and label mitochondria, respectively. Fluorescently labeled MuSCs were also transplanted into BaCl 2 -injured muscle to elucidate their behavior and interactions throughout muscle regeneration. Immunofluorescence, single fiber staining, and biochemical assays from harvested muscles were performed for data analysis. Results: Following injury, a significant increase in the number of endogenous MuSCs was observed 7 days post-injury compared to contralateral control, while a dramatic increase in the fusion of MuSCs into myofibers was noted at days 14 and 28. Interestingly, all fused myofibers at days 14 and 28 displayed central nucleation, a hallmark of regeneration that fusion of the MuSC is critical for myofiber regeneration. Primary MuSCs from H2B-EGFP reporter mice were isolated and transplanted into the injured muscle to determine the origin of the centrally located myonuclei. In myofibers where the transplanted MuSCs had fused, centrally located myonuclei were GFP + , indicating their origin from MuSCs. Surprisingly, regenerating muscle fibers isolated from the mitoDendra2 mouse model exhibited high densities of mitochondrial content near the centrally located myonuclei. To evaluate whether MuSCs contribute to producing these central myonuclei near mitochondria, primary MuSCs isolated from mitoDendra2 reporter mice were transplanted into mdx mice. Fused myofibers indeed exhibited high Dendra2 + mitochondrial density between central nuclei along with a restoration of the mitochondrial network. Finally, we also observed a change in myogenic potential and mitochondrial respiration after transplanting MuSCs that have been preconditioned to enhance mitochondrial biogenesis. SIGNIFICANCE RELEVANCE: Overall, these data indicate that MuSC-derived myonuclei synthesize new mitochondria to generate the bioenergetics for myofiber regeneration after muscle injury. This relationship between MuSCs and myofiber mitochondria provides a basis for MuSC-derived mitochondrial transplantation as a therapeutic approach for muscular diseases associated with mitochondrial dysfunction. Acknowledgements: This work was supported by the NIH R01AR080169, R01AG072309, VA Merit Award I02RX005009 (to YCJ) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.