Dysregulation of Hypoxia Signaling Limits Skeletal Muscle Regeneration Following Injury in Aging
Yori Endo1, Yuteng Zhang1, Bin Li1, Kodi Baldino2, Dharaniya Sakthivel3, Adriana Panayi1, Amy Wagers4, Indranil Sinha1.
1Harvard Medical School, Brigham and Women's Hospital, Boston, MA, USA, 2University of Connecticut School of Medicine, Farmington, CT, USA, 3Baylor College of Medicine, Houston, TX, USA, 4Stem Cell and Regenerative Biology, Harvard Medical School, Boston, MA, USA.
Background: The ability of skeletal muscle to regenerate declines significantly with aging. This leads to clinical problems that manifest as sarcopenia and incomplete healing of muscular injuries. In the present study, we examined the role of the hypoxia pathway and ARNT in the age-related decline in regenerative response of skeletal muscle following injury, and explored the effect of ML228, a hypoxia signaling activator, in the restoration of such response in aged muscle. Methods: Young (2-3 months old) and old (23-25 months old) mice were compared in their hypoxia-related gene expression. A mouse model of inducible skeletal-muscle specific ARNT knockout was created (ARNT mKO) to study the effect of suppressing the HIF-induced hypoxia-related gene expression in mature muscle fibers. Injured skeletal muscle as well as muscle stem cells were harvested from young, old, ARNT WT and ARNT mKO mice treated with or without ML228, a hypoxia pathway activator, for histological as well as biochemical analysis. Blood flow at mid-thigh level was assessed using doppler ultrasound, and capillary density in skeletal muscle was analyzed to study the potential effects of the loss of ARNT in blood flow and angiogenesis, respectively.Results: The RNA and protein expression of aryl hydrocarbon receptor translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2-3 months old) mice. Furthermore, Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle-specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), rescued skeletal muscle regeneration in both old and ARNT-deleted mice. While the blood flow at mid-thigh was lower in aged muscle compared to young, ARNT deletion
did not affect the blood flow in young mice. The capillary density in skeletal muscle was found to be similar between young and old mice, and was unaffected by skeletal muscle-specific ARNT deletion or ML228 treatment. Conclusion: The loss of ARNT in the aged skeletal muscle contributes to diminished myogenic potential with associated decline in the Notch signaling activation, and activation of hypoxia signaling holds promise for rescuing the regenerative activity in aged muscle.
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