Aging-associated Loss Of Hypoxia Signaling Limits Skeletal Muscle Regeneration
Kodi Udeh, MS1,2, Bin Li, MD2, Adriana Panayi, MD2, Ronald Neppl, PhD3, Amy Wagers, PhD4, Indranil Sinha, MD2,4.
1University of Connecticut School of Medicine, Farmington, CT, USA, 2Divison of Plastic Surgery, Brigham and Women's Hospital, Boston, MA, USA, 3Divison of Orthopedic Surgery, Brigham and Women's Hospital, Boston, MA, USA, 4Harvard Stem Cell Institute, Cambridge, MA, USA.
PURPOSE: Growing evidence suggests that the loss of skeletal muscle precursor (SMP) function, which are required for muscle regeneration, is partially responsible for sarcopenia, the age-associated loss of skeletal muscle mass and function. Hypoxia signaling, including aryl hydrocarbon nuclear translocator (ARNT), is necessary to maintain regenerative potential. The present study evaluates whether loss of hypoxia signaling in aging directly limits skeletal muscle precursor (SMP) regenerative potential.
METHODS: Young (Y, 8-12 weeks) and old (O, 21-23 months) mice were utilized to determine changes in regenerative potential and muscle hypoxia signaling that occur with aging. Regeneration was quantified using cross-sectional area (CSA) of regenerating fibers following cryoinjury. Whole muscle was utilized for ELISA, PCR, FACS sorting for skeletal muscle precursors (SMPs) and immunohistochemistry. Mice containing the human skeletal α-actin (HSA) Cre recombinase promoter crossed with a homozygous ARNTfl/fl allele were created to assess regeneration in the setting of skeletal muscle specific loss of ARNT following tamoxifen activation. Experimentation regarding these mice were completed at 8-12 weeks of age. ML228, a pharmacologic mimetic of ARNT, or dimethyl sulfoxide (vehicle control) was injected by intraperitoneal injection for 5 consecutive days in aged mice to determine if muscle regenerative capacity could be restored.
RESULTS: SMP frequency and myogenic potential decrease dramatically in aging (p<0.01). CSA of regenerating fibers decreases by 40% in O mice as compared to Y following injury at 5 (p<0.01) and 10 (p<0.01) days post-injury. In hind-limb skeletal muscle, ARNT levels are 4.7-fold lower by PCR (p<0.01) and 5-fold lower by immunoblotting in O versus Y mice (p<0.01). Using a focused PCR array, we demonstrated that the majority of hypoxia response genes were significantly down-regulated with aging. Young, tamoxifen-activated HSA-Cre ER ARNTfl/fl mice, created to mimic the loss of hypoxia signaling in old mice, exhibit an 80%, skeletal muscle specific decrease in ARNT expression (p<0.01) and a 30% decrease in regenerating muscle fiber CSA at 5 (p<0.01) and 10 (p<0.01) days post-injury, as compared to controls. ML228 administration resulted in a 30% increase in regenerating fiber CSA in O mice at day 5 (p<0.01) versus O mice treated with vehicle.
CONCLUSION: Hypoxia signaling declines with aging and contributes to loss of skeletal muscle regeneration. Restoring the hypoxia pathway may promote regeneration and prevent muscle loss in aging.
Back to 2019 Posters