Aging-associated Loss Of Hypoxia Signaling Limits Skeletal Muscle Regeneration
Indranil Sinha, MD1, Dharaniya Sakthivel, MS1, Kristo Nuutila, PhD1, Shalender Bhasin, MD1, Amy Wagers, PhD2.
1Brigham and Women's Hospital, Boston, MA, USA, 2Harvard Stem Cell Institute, Cambridge, MA, USA.
Introduction: Skeletal muscle regeneration is required for the maintenance of muscle mass. Hypoxia signaling, including aryl hydrocarbon nuclear translocator (ARNT, also known as HIF-1β), is necessary to maintain regenerative potential. The present study evaluates whether loss of hypoxia signaling in aging directly limits skeletal muscle precursor (SMP) myogenic potential. Methods: Young (8-12 weeks old) and old (21-23 months old) mice were utilized for all aging studies. Regeneration was quantified by evaluating cross-sectional area (CSA) of regenerating fibers after cryoinjury. Whole muscle was utilized for immunoblotting, PCR analysis, and fluorescence activated cell-sorting for SMP purification. To create a model of decreased hypoxia signaling, floxed ARNT mice were crossed with human skeletal α-actin (HSA) Cre recombinase promoter mice to create mice with muscle specific loss of ARNT following activation. Results: SMP frequency and myogenic potential decreased dramatically with aging (p<0.001). CSA of regenerating fibers decreased by 40% in old compared to young mice following injury (p<0.001). In muscle, ARNT levels in old mice were 4.7-fold lower by PCR and 5-fold lower by immunoblotting compared to young mice (p<0.01). A PCR hypoxia array confirmed down-regulation in the majority of hypoxia response genes by at least 2-fold in old mice. Activated HSA CreER ARNTfl/fl mice exhibited an 80% decrease in muscle ARNT expression (p<0.01) and a 25% decrease in regenerating fiber CSA (p<0.01) compared to littermate controls. Conclusion: Hypoxia signaling declines with aging and contributes to loss of muscle regeneration. Restoration of this pathway may improve maintenance of skeletal muscle mass in the elderly.
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