Changes in Diaphragm Collagen Proteostasis With Aging, Exercise, and Rapamycin Treatment

The diaphragm is the primary inspiratory muscle. Like limb muscles, the diaphragm shows decreases in mass and function with aging, which has been associated with the age-related increased susceptibility to respiratory complications. Increased content of extracellular matrix proteins has been proposed to contribute to increased stiffness and decreased contractile function of the diaphragm with age. However, the age-related changes in diaphragm proteostatic mechanisms with aging remain largely unknown. We hypothesized that aged diaphragm would have increased total and insoluble collagen content and impaired collagen turnover. Further, we hypothesized that two different interventions: 1) long-term voluntary running; and 2) rapamycin treatment at 42 ppm, would improve collagen proteostasis in aged diaphragm. C57BL/6 mice were assigned to four groups: adult sedentary (adult, 7-9 months-old, n=27), old sedentary (old-sed, 22-24 months-old, n=27), old exercise (old-ex, 22-24 months-old, n=18), old rapamycin (old-rapa, 22-24 months-old, n=13). We used long-term (0, 4, 8, 15, 30, 45, and 60 days) deuterium oxide (D 2 O) labeling (3 mice/time-point) to determine the fraction of the collagen pool that is actively turning over. The exercise and rapamycin interventions were started at the same time and lasted for the duration of D 2 O labeling. In addition, we used a hydroxyproline assay in 6 mice/group to determine the total, soluble, and insoluble collagen contents in the adult and old-sed groups. Aged diaphragm had greater total (+53%, p=0.016) and insoluble (+54%, p=0.017) collagen content compared to adult diaphragm. Although not statistically significant, aged diaphragm had a lower proportion of the collagen fraction that turns over compared to adult (adult: 39%; old-sed: 19%; p=0.200), indicating a greater fraction of the pool that is fibrotic and resistant to turnover. Neither exercise (old-ex: 21%) nor rapamycin treatment (old-rapa: 26%) improved the fraction of collagen turning over. Our results suggest that there is a loss of collagen turnover in the diaphragm with aging, which may contribute to an accumulation of insoluble collagen content. Further, neither voluntary running nor rapamycin treatment changed collagen turnover in the aged diaphragm. NIH R01 AG074551, American Lung Association Catalyst Award. 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.