Impaired Proteostasis, not Protein Synthesis, Limits Recovery of Aged Skeletal Muscle After Disuse Atrophy

Abstract

Older individuals undergo more frequent inpatient hospitalizations and bedrest compared to younger persons, causing rapid losses of muscle mass and strength. Evidence suggests that only aged muscle fails to completely recover following disuse despite aged and adult muscle having similar levels of muscle loss. Current efforts to improve muscle recovery in older individuals are commonly aimed at increasing myofibrillar protein synthesis via mTOR stimulation despite recent evidence demonstrating that old muscle has chronically elevated levels of mTORC1 activity. We hypothesized that old muscle fails to fully recover muscle mass due to impaired proteostasis and not due limitations in protein synthesis.MethodsAdult (10 month) and old (30 month) F344BN rats were hindlimb unloaded (HU) for 14‐days to induce atrophy, followed by subsequent reloading (RE) to study muscle regrowth. During RE the rats were labeled with deuterium oxide (D2O) for 7, 15, 30, 45 or 60 days (n=4‐6 per timepoint per group) to determine bulk and individual protein synthesis and RNA synthesis. We also assessed muscle mass, fiber size by immunohistochemistry, mTORC1 signaling by western blot, and aggregate formation by tissue fractionation.ResultsAdult and old gastrocnemius (GA) muscle had significant losses of muscle mass (359 mg ±55 and 724 mg ±60 loss respectively,p<0.05) and fiber size (1296 um2±202 and 1480 um2 ±142 loss respectively, p<0.01) with HU. While adult muscle fully recovered GA mass and fiber size by day 15, old GA muscle did not fully recover during the entire 60 days of RE (18% CSA and 11% mass loss still present). Despite limited mass and fiber size recovery in old muscle, GA myofibrillar protein synthesis rates (1.45 ±0.18 vs 2.28 ±0.24, FSR%, p<0.05) and mTORC1‐related signaling were higher in old muscle compared to adult. Additionally, GA RNA synthesis rates (2.79 ±0.139 vs 5.18 ±0.684, p<0.05) and RNA concentration (183 ng ±3.6 vs 249 ng ±13.9, p<0.05), which are markers of translational capacity, were also elevated in old muscle relative to adult during RE. Old muscle had higher levels of insoluble protein aggregates, a marker of impaired proteostasis, during RE compared to adult (1.2 ±0.09 vs 1.5 ±012 fold increase, p<0.05). Lastly, we assessed individual protein synthesis rates of the whole muscle proteome and discovered that old GA muscle had a larger number of proteins with increased synthesis rates compared to adult. In conclusion, these data strongly suggest that limitations in old muscle to recover after disuse are not due to limitations in protein synthesis but are instead due to impaired proteostasis with age. Therefore, understanding how proteostasis responds during these periods surrounding unloading in old muscle are critical to improve muscle recovery after disuse.