Loss of Muscle Fiber Hypoxia Signaling Impairs Skeletal Muscle Adaptability to Exercise with Aging

Background: Exercise remains beneficial at any age, but the physiological response to aerobic exercise declines with aging. This study examines differentially regulated pathways in aging skeletal muscle that may be responsible for this decreased adaptation to aerobic exercise. Methods: To assess differential effects of aerobic exercise with age, Young (Y, 10-12 weeks) and Old (O, 23-25 months) mice were subjected to 8 weeks of treadmill running at similar relative workloads (70% VO 2max ) or no regimented exercise. All mice were maintained in reverse light/dark cycles with exercise performed at similar times following acclimatization. Mice were then evaluated for maximal running speed, distance, and fatiguability with repeated tetanic stimulation. Gastrocnemius (G) muscle was then harvested for single nuclei (sn)RNA sequencing (Azenta) and other G muscle was assessed for fiber type by immunofluorescence. To assess the importance of hypoxia signaling in exercise response with aging, we generated mice with inducible, skeletal muscle fiber-specific knock-in of PHD2 (muscle (m)PHD2), which is a critical upstream inhibitor of hypoxia signaling. mPHD2 and control mice were similarly exercised or underwent no regimented exercise and then similarly evaluated. IOX2, a PHD2 inhibitor, or vehicle control was administered to Y and O mice in a separate experiment during the training period and these mice were similarly evaluated. Results: Y, trained mice experienced a significant increase in maximal distance running, decreased fatigue with repeated tetanic stimulation, and a fiber type transition to oxidative fibers in comparison to age-matched, untrained controls. O mice improved to a lesser degree in running distance and speed but did not improve significantly in fatiguability or fiber type transition. snRNA sequencing analysis of G from Y and O mice, both with and without exercise training, demonstrated many of the most differentially regulated genes with exercise in young mice, which were not similarly regulated in old mice, were hypoxia signaling factors and produced by myofibers. Using our genetically modified mice, immunoblotting demonstrated prolyl hydroxylase domain 2 (PHD2), which limits hypoxia signaling, was 5-fold higher in skeletal muscle at baseline and following exercise in O in contrast to Y mice. Similar to O mice, mPHD2 mice, which exhibit 6-fold higher levels of PHD2 in skeletal muscle versus controls, did not improve maximal distance running, fatiguability, or exhibit a fiber type transition to oxidative fibers whereas littermate control mice did. Administration of IOX2, a PHD2 inhibitor, increased maximal running distance and fiber type adaptation in response to exercise training in O mice. Conclusion: Loss of skeletal muscle hypoxia signaling due to increased PHD2 may be responsible for diminished response to endurance exercise in aging. Activation of hypoxia signaling by inhibition of PHD2 holds promise for rescuing exercise adaptability in old muscle. IS is supported by National Institute of Aging R01 AG078307 and P30AG031679. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.