Motor Denervation Activates Muscle Stem Cells and Leads to Muscle TGFbeta Expression
Alvin Wong, MD, Steven Garcia, BS, Solomon Lee, BA, Stanley Tamaki, PhD, Jason Pomerantz, MD.
University of California San Francisco, San Francisco, CA, USA.
The pathophysiology underlying the irreversibility of muscle atrophy and inability to regenerate following prolonged denervation is incompletely understood. Satellite cells (SCs) are the primary resident stem cells in muscle responsible for repair after injury, and are defined by expression of the transcription factor paired box protein 7 (Pax7). Previous studies reported SC depletion after long-term denervation, possibly explaining the irreversibility of denervation atrophy, but did not quantify Pax7+ cells nor perform functional studies. It is therefore unknown to what extent SC depletion occurs or limits recovery after delayed reinnervation. This study attempts to elucidate the effect of motor denervation on SC behavior and regenerative capacity. METHODS:
The sciatic nerve of C57Bl6 mice was transected. Three, six, and 12 months following denervation, 100ug EdU was injected intraperitoneally for three consecutive days. Mice were sacrificed on day four and the tibialis anterior muscles (TAs) were processed for immunohistochemical analysis. Flow cytometry was used to select for SCs from lower leg muscles. Mitotracker staining was performed to assess mitochondrial activity in SC. SCs from three month-denervated legs or minced muscle grafts from six and twelve month-denervated TAs were transplanted into the pre-irradiated left TAs of immune-deficient/dystrophin-deficient (NSG-mdx) mice. SCs were isolated from the atrophic denervated gluteus maximus muscle of human patients with complete spinal cord injuries via flow cytometry and transplanted into irradiated left TAs of NSG mice. RESULTS:
TAs weighed 11.9±0.6mg after 3-month denervation vs 56.6.5±5.5mg (p<0.05). No significant differences between three, six, and twelve-month denervated TA weights were noted. Cross-sectional fluorescent immunohistochemical staining demonstrated a significant increase in the total number of Pax7+ cells per muscle fiber in denervated TAs three and six months following denervation compared to non-denervated TAs (p<0.05). Twelve months following denervation, the number of Pax7+ cells in denervated TAs remains unchanged relative to non-denervated TAs. EdU uptake by SCs is significantly increased three months following denervation. SCs isolated from denervated legs were larger and had higher mitotracker uptake by flow cytometry compared to uninjured legs. After transplantation into the irradiated left TA of NSG-mdx mice, SCs from 3 month-denervated leg muscles were able to engraft and produce new muscle fiber. Minced muscle grafts from six and 12 month-denervated TAs also regenerated new muscle fibers after transplantation into NSG-mdx mice. SCs isolated from denervated human gluteus maximus and transplanted into NSG mice engrafted and made human dystrophin-producing muscle fibers. Following denervation, TGFbeta expression is increased in muscle, and downstream TGFbeta signaling is significantly increased in SCs as measured by Pax7-Smad2/3 co-staining. CONCLUSION:Contrary to previous reports, SC depletion does not occur following prolonged periods of denervation in either rodents or humans. The SC phenotype is altered from quiescence towards activation as demonstrated by their increase in number, size, EdU uptake, and mitochondrial activity post-denervation. SCs also retain robust intrinsic regenerative capacity when transplanted into a different host. The irreversibility of denervation atrophy cannot be explained by SC depletion or dysfunction. TGFbeta expression increases in muscle following denervation and may affect SC regenerative capacity.
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