The Muscle Stem Cell Mediates Remodeling of Skeletal Muscle Mitochondrial Networks

Skeletal muscle regeneration is an energy‐demanding biological process that relies on mitochondria to generate ATP. While interfibrillar mitochondrial networks in healthy skeletal muscle fibers exhibit an organized architecture segregated into columns along the Z‐line of the myofiber, regenerating fibers demonstrate altered mitochondrial networks that span several sarcomeres. Although it’s been established that skeletal muscle regeneration parallels altered mitochondrial dynamics and biogenesis, the cells that mediate the remodeling of mitochondrial networks have not been thoroughly investigated. Because muscle stem cells (MuSCs) play a critical role in muscle regeneration, we explored the relationship between MuSCs and mitochondria in regenerating muscle fibers. In order to induce muscle regeneration, we employed 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 mark MuSCs, track nuclei, and label mitochondria, respectively. Fluorescently‐labelled 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. Following injury, there was a dramatic increase in the number of endogenous MuSCs 7 days post‐injury compared to contralateral control while there was a significant increase in fusion of the accreted MuSCs into the myofiber at days 14 and 28. Interestingly, all fused myofibers at days 14 and 28 were regenerating, demarcated by central nucleation, suggesting that fusion of the MuSC is critical for myofiber regeneration. To determine the origin of the centrally located myonuclei, primary MuSCs from H2B‐EGFP reporter mice were isolated and transplanted into injured muscle. For myofibers in which the transplanted MuSCs fused into, centrally located myonuclei were GFP+, implying that these myonuclei were derived from MuSCs. Surprisingly, regenerating muscle fibers isolated from the mitoDendra2 mouse model exhibited high densities of mitochondrial content adjacent to the centrally located myonuclei. In order to test whether MuSCs are responsible for producing these central myonuclei near mitochondria, primary MuSCs isolated from mitoDendra2 reporter mice were transplanted into mdx mice and fused myofibers indeed exhibited high Dendra2 + mitochondrial density between central nuclei as well as 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. Overall, these data indicate that following skeletal muscle injury, MuSC‐derived myonuclei synthesize new mitochondria to generate the bioenergetics for myofiber regeneration. This relationship between MuSCs and myofiber mitochondria provide a basis for MuSC‐derived mitochondrial transplantation as a therapy for muscular diseases that are concomitant with mitochondrial dysfunction. Support or Funding Information NIH, REMMitochondrial network in isolated myofibers from healthy, hindlimb ischemia, and mdx mice.MuSC‐derived mitochondrial network in regenerating myofibers following MuSC transplantation.