Age-related loss of muscle mass occurs to varying degrees in all individuals and has a detrimental effect on morbidity and mortality. Muscle RING Finger 1 (MuRF1), a muscle-specific E3 ubiquitin ligase, is believed to mediate muscle atrophy through the ubiquitin proteasome system (UPS). Deletion of MuRF1 (KO) in mice attenuates the loss of muscle mass following denervation, disuse, and glucocorticoid treatment; however, its role in age-related muscle loss is unknown. In this study, skeletal muscle from male wild-type (WT) and MuRF1 KO mice was studied up to the age of 24 months. Muscle mass and fiber cross-sectional area decreased significantly with age in WT, but not in KO mice. In aged WT muscle, significant decreases in proteasome activities, especially 20S and 26S β5 (20-40% decrease), were measured and were associated with significant increases in the maladaptive endoplasmic reticulum (ER) stress marker, CHOP. Conversely, in aged MuRF1 KO mice, 20S or 26S β5 proteasome activity was maintained or decreased to a lesser extent than in WT mice, and no increase in CHOP expression was measured. Examination of the growth response of older (18 months) mice to functional overload revealed that old WT mice had significantly less growth relative to young mice (1.37- vs. 1.83-fold), whereas old MuRF1 KO mice had a normal growth response (1.74- vs. 1.90-fold). These data collectively suggest that with age, MuRF1 plays an important role in the control of skeletal muscle mass and growth capacity through the regulation of cellular stress.

Development of osteoarthritis commonly involves degeneration of epiphyseal trabecular bone. In previous studies, we observed 30-44% loss of epiphyseal trabecular bone (BV/TV) from the distal femur within 1 week following non-invasive knee injury in mice. Mechanical unloading (disuse) may contribute to this bone loss; however, it is unclear to what extent the injured limb is unloaded following injury, and whether disuse can fully account for the observed magnitude of bone loss. In this study, we investigated the contribution of mechanical unloading to trabecular bone changes observed following non-invasive knee injury in mice (female C57BL/6N). We investigated changes in gait during treadmill walking, and changes in voluntary activity level using Open Field analysis at 4, 14, 28, and 42 days post-injury. We also quantified epiphyseal trabecular bone using μCT and weighed lower-limb muscles to quantify atrophy following knee injury in both ground control and hindlimb unloaded (HLU) mice. Gait analysis revealed a slightly altered stride pattern in the injured limb, with a decreased stance phase and increased swing phase. However, Open Field analysis revealed no differences in voluntary movement between injured and sham mice at any time point. Both knee injury and HLU resulted in comparable magnitudes of trabecular bone loss; however, HLU resulted in considerably more muscle loss than knee injury, suggesting another mechanism contributing to bone loss following injury. Altogether, these data suggest that mechanical unloading likely contributes to trabecular bone loss following non-invasive knee injury, but the magnitude of this bone loss cannot be fully explained by disuse. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1680-1687, 2016.

Testosterone is considered a potent anabolic agent in skeletal muscle with a well-established role in adolescent growth and development in males. However, the role of testosterone in the regulation of skeletal muscle mass and function throughout the lifespan has yet to be fully established. While some studies suggest that testosterone is important for the maintenance of skeletal muscle mass, an understanding of the role this hormone plays in young, adult, and old males with normal and low serum testosterone levels is lacking. We investigated the role testosterone plays in the maintenance of muscle mass by examining the effect of orchiectomy-induced testosterone depletion in C57Bl6 male mice at ages ranging from early postnatal through old age (1.5-, 5-, 12-, and 24-month old mice). Following 28 days of testosterone depletion, we assessed mass and fiber cross-sectional-area (CSA) of the tibialis anterior, gastrocnemius, and quadriceps muscles. In addition, we measured global rates of protein synthesis and degradation using the SuNSET method, western blots, and enzyme activity assays. Twenty-eight days of testosterone depletion resulted in reduced muscle mass in the two youngest cohorts, but had no effect in the two oldest cohorts. Mean CSA decreased only in the youngest cohort and only in the tibialis anterior muscle. Testosterone depletion resulted in a general increase in proteasome activity at all ages. No change in protein synthesis was detected at the terminal time point. These data suggest that within physiological serum concentrations, testosterone may not be critical for the maintenance of muscle mass in mature male mice; however, in young mice testosterone is crucial for normal growth.

Age-related loss of muscle mass and strength can be accelerated by impaired recovery of muscle mass following a transient atrophic stimulus. The aim of this study was to identify the mechanisms underlying the attenuated recovery of muscle mass and strength in old rats following disuse-induced atrophy. Adult (9 month) and old (29 month) male F344BN rats underwent hindlimb unloading (HU) followed by reloading. HU induced significant atrophy of the hindlimb muscles in both adult (17-38%) and old (8-29%) rats, but only the adult rats exhibited full recovery of muscle mass and strength upon reloading. Upon reloading, total RNA and protein synthesis increased to a similar extent in adult and old muscles. At baseline and upon reloading, however, proteasome-mediated degradation was suppressed leading to an accumulation of ubiquitin-tagged proteins and p62. Further, ER stress, as measured by CHOP expression, was elevated at baseline and upon reloading in old rats. Analysis of mRNA expression revealed increases in HDAC4, Runx1, myogenin, Gadd45a, and the AChRs in old rats, suggesting neuromuscular junction instability/denervation. Collectively, our data suggests that with aging, impaired neuromuscular transmission and deficits in the proteostasis network contribute to defects in muscle fiber remodeling and functional recovery of muscle mass and strength.

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

Rotator cuff tears (RCTs) are among the most common injuries seen in orthopedic patients. Chronic tears can result in the development of muscular atrophy and fatty infiltration. Despite the prevalence of RCTs, little is known about the underlying molecular pathways that produce these changes. Recently, we have shown that mammalian target of rapamycin (mTOR) signaling plays an important role in muscle atrophy that results from massive RCTs in a rat model. The purpose of this study was therefore to extend our understanding of mTOR signaling and evaluate its role in fatty infiltration after a combined tendon transection and suprascapular nerve denervation surgery. Akt/mTOR signaling was significantly increased and resulted in the up-regulation of two transcription factors: SREBP-1 and PPARγ. We also saw an increase in expression of adipogenic markers: C/EBP-α and FASN. Upon treatment with rapamycin, an inhibitor of mTOR, we observed a decrease in mTOR signaling, activity of transcription factors, and reduction in fatty infiltration. Therefore, our study suggests that mTOR signaling mediates rotator cuff fatty infiltration via SREBP-1 and PPARγ. Clinically, our finding may alter current treatment methods to address rotator cuff fatty infiltration.

Background

Bone structure and strength are rapidly lost during conditions of decreased mechanical loading, and aged bones have a diminished ability to adapt to increased mechanical loading. This is a concern for older patients that experience periods of limited mobility or bed rest, but the acute effects of disuse on the bones of aged patients have not been thoroughly described. Previous animal studies have primarily examined the effect of mechanical unloading on young animals. Those that have studied aged animals have exclusively focused on bone loss during unloading and not bone recovery during subsequent reloading. In this study, we investigated the effect of decreased mechanical loading and subsequent reloading on bone using a hindlimb unloading model in Adult (9 month old) and Aged (28 month old) male rats.

Methods

Animals from both age groups were subjected to 14 days of hindlimb unloading followed by up to 7 days of reloading. Additional Aged rats were subjected to 7 days of forced treadmill exercise during reloading or a total of 28 days of reloading. Trabecular and cortical bone structure of the femur were quantified using ex vivo micro-computed tomography (μCT), and mechanical properties were quantified with mechanical testing.

Results

We found that Adult rats had substantially decreased trabecular bone volume fraction (BV/TV) following unloading (- 27%) while Aged animals did not exhibit significant bone loss following unloading. However, Aged animals had lower trabecular BV/TV after 3 days of reloading (- 20% compared to baseline), while trabecular BV/TV of Adult rats was not different from baseline values after 3 days of reloading. Trabecular BV/TV of Aged animals remained lower than control animals even with exercise during 7 days of reloading and after 28 days of reloading.

Conclusions

These data suggest that aged bone is less responsive to both increased and decreased mechanical loading, and that acute periods of disuse may leave older subjects with a long-term deficit in trabecular bone mass. These finding indicate the need for therapeutic strategies to improve the skeletal health of elderly patients during periods of disuse.

Anabolic resistance to feeding in aged muscle is well-characterized; however, whether old skeletal muscle is intrinsically resistant to acute mechanical loading is less clear. The aim of this study was to determine the impact of aging on muscle protein synthesis (MPS), ribosome biogenesis, and protein breakdown in skeletal muscle following a single bout of resistance exercise. Adult male F344/BN rats aged 10 (Adult) and 30 (Old) months underwent unilateral maximal eccentric contractions of the hindlimb. Precursor rRNA increased early post-exercise (6-18 h), preceding elevations in ribosomal mass at 48 h in Adult and Old; there were no age-related differences in these responses. MPS increased early post-exercise in both Adult and Old; however, at 48 h of recovery, MPS returned to baseline in Old but not Adult. This abbreviated protein synthesis response in Old was associated with decreased levels of IRS1 protein and increased BiP, CHOP and eIF2α levels. Other than these responses, anabolic signaling was similar in Adult and Old muscle in the acute recovery phase. Basal proteasome activity was lower in Old, and resistance exercise did not increase the activity of either the ATP-dependent or independent proteasome, or autophagy (Cathepsin L activity) in either Adult or Old muscle. We conclude that MPS and ribosome biogenesis in response to maximal resistance exercise in old skeletal muscle are initially intact; however, the MPS response is abbreviated in Old, which may be the result of ER stress and/or blunted exercise-induced potentiation of the MPS response to feeding.

The loss of muscle strength with age has been studied from the perspective of a decline in muscle mass and neuromuscular junction (NMJ) stability. A third potential factor is force transmission. The purpose of this study was to determine the changes in the force transfer apparatus within aging muscle and the impact on membrane integrity and NMJ stability. We measured an age-related loss of dystrophin protein that was greatest in the flexor muscles. The loss of dystrophin protein occurred despite a twofold increase in dystrophin mRNA. Importantly, this disparity could be explained by the four- to fivefold upregulation of the dystromir miR-31. To compensate for the loss of dystrophin protein, aged muscle contained increased α-sarcoglycan, syntrophin, sarcospan, laminin, β1-integrin, desmuslin, and the Z-line proteins α-actinin and desmin. In spite of the adaptive increase in other force transfer proteins, over the 48 hours following lengthening contractions, the old muscles showed more signs of impaired membrane integrity (fourfold increase in immunoglobulin G-positive fibers and 70% greater dysferlin mRNA) and NMJ instability (14- to 96-fold increases in Runx1, AchRδ, and myogenin mRNA). Overall, these data suggest that age-dependent alterations in dystrophin leave the muscle membrane and NMJ more susceptible to contraction-induced damage even before changes in muscle mass are obvious.

Key points

Ribosome biogenesis is the primary determinant of translational capacity, but its regulation in skeletal muscle following acute resistance exercise is poorly understood. Resistance exercise increases muscle protein synthesis acutely, and muscle mass with training, but the role of translational capacity in these processes is unclear. Here, we show that acute resistance exercise activated pathways controlling translational activity and capacity through both rapamycin-sensitive and -insensitive mechanisms. Transcription factor c-Myc and its downstream targets, which are known to regulate ribosome biogenesis in other cell types, were upregulated after resistance exercise in a rapamycin-independent manner and may play a role in determining translational capacity in skeletal muscle. Local inhibition of myostatin was also not affected by rapamycin and may contribute to the rapamycin-independent effects of resistance exercise.

Abstract

This study aimed to determine (1) the effect of acute resistance exercise on mechanisms of ribosome biogenesis, and (2) the impact of mammalian target of rapamycin on ribosome biogenesis, and muscle protein synthesis (MPS) and degradation. Female F344BN rats underwent unilateral electrical stimulation of the sciatic nerve to mimic resistance exercise in the tibialis anterior (TA) muscle. TA muscles were collected at intervals over the 36 h of exercise recovery (REx); separate groups of animals were administered rapamycin pre-exercise (REx+Rapamycin). Resistance exercise led to a prolonged (6-36 h) elevation (30-50%) of MPS that was fully blocked by rapamycin at 6 h but only partially at 18 h. REx also altered pathways that regulate protein homeostasis and mRNA translation in a manner that was both rapamycin-sensitive (proteasome activity; phosphorylation of S6K1 and rpS6) and rapamycin-insensitive (phosphorylation of eEF2, ERK1/2 and UBF; gene expression of the myostatin target Mighty as well as c-Myc and its targets involved in ribosome biogenesis). The role of c-Myc was tested in vitro using the inhibitor 10058-F4, which, over time, decreased basal RNA and MPS in a dose-dependent manner (correlation of RNA and MPS, r(2) = 0.98), even though it had no effect on the acute stimulation of protein synthesis. In conclusion, acute resistance exercise stimulated rapamycin-sensitive and -insensitive mechanisms that regulate translation activity and capacity.