Background: Sarcopenia, the loss of skeletal muscle mass during aging, increases the risk for falls and dependency. Resistance exercise (RE) training is an effective treatment to improve muscle mass and strength in older adults, but aging is associated with a smaller amount of training-induced hypertrophy. This may be due in part to an inability to stimulate muscle-protein synthesis (MPS) after an acute bout of RE. We hypothesized that older adults would have impaired mammalian target of rapamycin complex (mTORC)1 signaling and MPS response compared with young adults after acute RE. Methods: We measured intracellular signaling and MPS in 16 older (mean 70 ± 2 years) and 16 younger (27 ± 2 years) subjects. Muscle biopsies were sampled at baseline and at 3, 6 and 24 hr after exercise. Phosphorylation of regulatory signaling proteins and MPS were determined on successive muscle biopsies by immunoblotting and stable isotopic tracer techniques, respectively. Results: Increased phosphorylation was seen only in the younger group (P< 0.05) for several key signaling proteins after exercise, including mammalian target of rapamycin (mTOR), ribosomal S6 kinase (S6K)1, eukaryotic initiation factor 4E-binding protein (4E-BP)1 and extracellular signal-regulated kinase (ERK)1/2, with no changes seen in the older group (P > 0.05). After exercise, MPS increased from baseline only in the younger group (P< 0.05), with MPS being significantly greater than that in the older group (P < 0.05). Conclusions: We conclude that aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis. These age-related differences may contribute to the blunted hypertrophic response seen after resistance-exercise training in older adults, and highlight the mTORC1 pathway as a key therapeutic target to prevent sarcopenia.

A common characteristic of aging is loss of skeletal muscle (sarcopenia), which can lead to falls and fractures. MicroRNAs (miRNAs) are novel posttranscriptional modulators of gene expression with potential roles as regulators of skeletal muscle mass and function. The purpose of this study was to profile miRNA expression patterns in aging human skeletal muscle with a miRNA array followed by in-depth functional and network analysis. Muscle biopsy samples from 36 men [young: 31 ± 2 (n = 19); older: 73 ± 3 (n = 17)] were 1) analyzed for expression of miRNAs with a miRNA array, 2) validated with TaqMan quantitative real-time PCR assays, and 3) identified (and later validated) for potential gene targets with the bioinformatics knowledge base software Ingenuity Pathways Analysis. Eighteen miRNAs were differentially expressed in older humans (P < 0.05 and >500 expression level). Let-7 family members Let-7b and Let-7e were significantly elevated and further validated in older subjects (P < 0.05). Functional and network analysis from Ingenuity determined that gene targets of the Let-7s were associated with molecular networks involved in cell cycle control such as cellular proliferation and differentiation. We confirmed with real-time PCR that mRNA expression of cell cycle regulators CDK6, CDC25A, and CDC34 were downregulated in older compared with young subjects (P < 0.05). In addition, PAX7 mRNA expression was lower in older subjects (P < 0.05). These data suggest that aging is characterized by a higher expression of Let-7 family members that may downregulate genes related to cellular proliferation. We propose that higher Let-7 expression may be an indicator of impaired cell cycle function possibly contributing to reduced muscle cell renewal and regeneration in older human muscle.

Sarcopenia, skeletal muscle loss during aging, is associated with increased falls, fractures, morbidity, and loss of independence. MicroRNAs (miRNAs) are novel posttranscriptional regulators. The role of miRNAs in cell size regulation after an anabolic stimulus in human skeletal muscle is unknown. We hypothesized that aging would be associated with a differential expression of skeletal muscle primary miRNA (pri-miRNA) and mature miRNA (miR). To test this hypothesis, we used real-time PCR and immunoblotting before and after an anabolic stimulus (resistance exercise + ingestion of a 20-g leucine-enriched essential amino acid solution) to measure the expression of muscle-specific miRNAs (miR-1, miR-133a, and miR-206), upstream regulators (MyoD and myogenin), and downstream targets [insulin-like growth factor-I, histone deacetylase-4, myocyte enhancing factor-2, and Ras homolog enriched in brain (Rheb)] in skeletal muscle of young and older men. Muscle biopsies were obtained at baseline and 3 and 6 h after exercise. At baseline, we found pri-miRNA-1-1, -1-2, -133a-1, and -133a-2 expression elevated in older compared with young men (P < 0.05). Pri-miRNA-1-2, -133a-1, and -133a-2 were reduced at 6 h after exercise only in the young men compared with baseline, whereas pri-miRNA-206 was elevated at different postexercise time points in older and young men (P < 0.05). Compared with baseline, miR-1 was reduced only in the young men, whereas Rheb protein was increased in both age groups after the anabolic stimulus (P < 0.05). We conclude that skeletal muscle primary and mature miRNA expression in young men is readily altered by an anabolic stimulus of resistance exercise + essential amino acid ingestion. However, aging is associated with higher basal skeletal muscle primary miRNA expression and a dysregulated miRNA response after the anabolic stimulus.

Skeletal muscle atrophy during bed rest is attributed, at least in part, to slower basal muscle protein synthesis (MPS). Essential amino acids (EAA) stimulate mammalian target of rapamycin (mTORC1) signaling, amino acid transporter expression, and MPS and are necessary for muscle mass maintenance, but there are no data on the effect of inactivity on this anabolic mechanism. We hypothesized that bed rest decreases muscle mass in older adults by blunting the EAA stimulation of MPS through reduced mTORC1 signaling and amino acid transporter expression in older adults. Six healthy older adults (67 ± 2 yr) participated in a 7-day bed rest study. We used stable isotope tracers, Western blotting, and real-time qPCR to determine the effect of bed rest on MPS, muscle mTORC1 signaling, and amino acid transporter expression and content in the postabsorptive state and after acute EAA ingestion. Bed rest decreased leg lean mass by ∼4% ( P < 0.05) and increased postabsorptive mTOR protein ( P < 0.05) levels while postabsorptive MPS was unchanged ( P > 0.05). Before bed rest acute EAA ingestion increased MPS, mTOR (Ser²⁴⁴⁸), S6 kinase 1 (Thr³⁸⁹, Thr⁴²¹/Ser⁴²⁴), and ribosomal protein S6 (Ser^240/244) phosphorylation, activating transcription factor 4, L-type amino acid transporter 1 and sodium-coupled amino acid transporter 2 protein content ( P < 0.05). However, bed rest blunted the EAA-induced increase in MPS, mTORC1 signaling, and amino acid transporter protein content. We conclude that bed rest in older adults significantly attenuated the EAA-induced increase in MPS with a mechanism involving reduced mTORC1 signaling and amino acid transporter protein content. Together, our data suggest that a blunted EAA stimulation of MPS may contribute to muscle loss with inactivity in older persons.

Reduced lean mass and physical function is a characteristic of frailty. However, it is currently unknown if proteolysis through the E3 ubiquitin ligases and the autophagic lysosomal pathway is dysregulated in inactive frail older women. The purpose of this study was to determine the expression of key markers of ubiquitin-mediated and autophagic lysosomal proteolysis in inactive (N = 7) compared with active (N = 7) older women. Strength, mobility, leg lean mass, and physical activity assessment were used to characterize activity levels and frailty in older women. Vastus lateralis biopsies were collected after an overnight fast and were assessed for gene and protein targets related to E3 ubiquitin ligases and the autophagic lysosomal system. We found that AMP-activated protein kinase alpha (Thr172) was increased (p = .045), and forkhead box O3A (FOXO3A) gene expression (p = .047) was lower in inactive frail older women. Foxo3a (Ser253), Beclin1 (Ser93/96), and class III phosphatidylinositol-3-kinase (VPS34) protein expression were not different between the groups (p > .05). Neural precursor cell-expressed developmentally downregulated protein 4, muscle ring finger 1, muscle atrophy F-box, and the autophagy/mitophagy gene expression markers, Beclin1, autophagy-related-7, BCL2/adenovirus E1B 19kDa interacting protein 3 (BNIP3), dynamin-related protein 1, and Parkinson protein 2 (PARKIN) were lower in inactive frail older women (p < .05). Autophagy/mitophagy markers were positively correlated with the 6-minute walk and leg lean mass (p < .05). We conclude that physical inactivity in frail older women is associated with a downregulation of ubiquitin-mediated and autophagic lysosomal skeletal muscle gene expression, perhaps related to low muscle mass and poor physical function.