Impaired proteostatic mechanisms other than decreased protein synthesis limit old skeletal muscle recovery after disuse atrophy

Abstract

Background

Skeletal muscle mass and strength diminish during periods of disuse but recover upon return to weight bearing in healthy adults but are incomplete in old muscle. Efforts to improve muscle recovery in older individuals commonly aim at increasing myofibrillar protein synthesis via mammalian target of rapamycin (mTOR) stimulation despite evidence demonstrating that old muscle has chronically elevated levels of mammalian target of rapamycin complex 1 (mTORC1) activity. We hypothesized that protein synthesis is higher in old muscle than adult muscle, which contributes to a proteostatic stress that impairs recovery.

Methods

We unloaded hindlimbs of adult (10‐month) and old (28‐month) F344BN rats for 14 days to induce atrophy, followed by reloading up to 60 days with deuterium oxide (D₂O) labelling to study muscle regrowth and proteostasis.

Results

We found that old muscle has limited recovery of muscle mass during reloading despite having higher translational capacity and myofibrillar protein synthesis (0.029 k/day ± 0.002 vs. 0.039 k/day ± 0.002, P < 0.0001) than adult muscle. We showed that collagen protein synthesis was not different (0.005 k (1/day) ± 0.0005 vs. 0.004 k (1/day) ± 0.0005, P = 0.15) in old compared to adult, but old muscle had higher collagen concentration (4.5 μg/mg ± 1.2 vs. 9.8 μg/mg ± 0.96, P < 0.01), implying that collagen breakdown was slower in old muscle than adult muscle. This finding was supported by old muscle having more insoluble collagen (4.0 ± 1.1 vs. 9.2 ± 0.9, P < 0.01) and an accumulation of advanced glycation end products (1.0 ± 0.06 vs. 1.5 ± 0.08, P < 0.001) than adult muscle during reloading. Limited recovery of muscle mass during reloading is in part due to higher protein degradation (0.017 1/t ± 0.002 vs. 0.028 1/t ± 0.004, P < 0.05) and/or compromised proteostasis as evidenced by accumulation of ubiquitinated insoluble proteins (1.02 ± 0.06 vs. 1.22 ± 0.06, P < 0.05). Last, we showed that synthesis of individual proteins related to protein folding/refolding, protein degradation and neural‐related biological processes was higher in old muscle during reloading than adult muscle.

Conclusions

Our data suggest that the failure of old muscle to recover after disuse is not due to limitations in the ability to synthesize myofibrillar proteins but because of other impaired proteostatic mechanisms (e.g., protein folding and degradation). These data provide novel information on individual proteins that accumulate in protein aggregates after disuse and certain biological processes such as protein folding and degradation that likely play a role in impaired recovery. Therefore, interventions to enhance regrowth of old muscle after disuse should be directed towards the identified impaired proteostatic mechanisms and not aimed at increasing protein synthesis.

Introduction

Acute periods of muscle loss with failed recovery in the elderly accelerate the progression towards sarcopenia and the associated health risks such as decreased mobility, ¹ , ² development of metabolic disorders, ³ , ⁴ increased risk of falls, ² and mortality. ² Skeletal muscle mass and strength diminish during periods of disuse. Healthy adult muscle recovers upon return to weight bearing (WB), but old muscle does not. ⁵ , ⁶ , ⁷ In a 2‐week immobilization study, old men had diminished restoration of muscle volume and attenuated recovery of muscle fibre size compared to adult men during 4 weeks of recovery and exercise training. ⁷ Similarly, when adult and old rats were hindlimb unloaded for 2 weeks followed by 14 days of normal ambulation, old rats had attenuated recovery of muscle mass and strength while adults had full recovery. ⁸ There are currently no effective interventions that completely restore muscle mass and strength in older individuals, despite ongoing research into the underlying mechanisms of recovery after disuse.

Efforts to improve the restoration of muscle mass after periods of disuse often target stimulation of mammalian target of rapamycin (mTOR) activity to promote myofibrillar protein synthesis. ⁹ , ¹⁰ However, when we used the stable isotope deuterium oxide (D₂O) to assess protein synthesis rates in adult (24‐month) and older (28‐month) rats during recovery from hindlimb unloading (HU), we found that the older rats had higher protein synthesis and ribosomal biogenesis rates in soleus (SOL) muscle and no differences in tibialis anterior (TA) and extensor digitorum longus (EDL) muscles. ⁸ Interestingly, the lack of increase in TA and EDL muscles was because myofibrillar protein synthesis rates were already higher in older rats than younger rats during WB conditions. Together, these data demonstrate that myofibrillar protein synthesis is not impaired in old muscle and is even greater in some muscles. ⁸ It is however unclear whether the age‐related difference in protein synthesis occurs in all proteins or a subset and how that influences muscle recovery.

Previous studies investigating recovery of muscle after disuse atrophy have mainly focused on a single time point of reloading. We have developed an approach that uses D₂O labelling over an extended period with multiple sampling points, which allows us to determine the size of protein pool that becomes resistant to turnover. ¹¹ Our time‐course approach allows us to determine the size of the pool of proteins that become resistant to turnover by determining the value at which the fraction of new proteins plateaus. ¹¹ Further, we showed that some protein pools, such as collagen, reach a plateau early and at a surprisingly low fraction. Therefore, any labelling that goes beyond that point artificially decreases fractional synthesis rates (FSRs) or results in values that appear to not be different even though significant differences exist. For the current study, we took advantage of a time‐course approach and labelled over 60 days of recovery to better understand proteostatic decline in several protein pools, including collagen.

Proteostasis is maintained through multiple proteostatic mechanisms including protein synthesis, breakdown and folding. Protein aggregation is a hallmark of compromised proteostasis, which can arise from impaired protein folding, ¹² , ¹³ insufficient catabolic clearance of misfolded or aggregated proteins, ¹⁴ , ¹⁵ or aberrant protein synthesis leading to improperly translated proteins. ¹⁶ , ¹⁷ Although protein aggregates receive much attention in tissues like the brain, there are few studies that measure aggregates in muscle. In this study, we sought to determine if older rats had increased protein aggregates in muscle compared to adult rats during a period of reloading after disuse.

The goal of this study was to determine factors that contribute to impaired recovery from disuse in old skeletal muscle. To do so, we subjected adult (10‐month) and old (28‐month) rats to a period of disuse followed by normal ambulation for recovery and a time‐course approach of D₂O labelling up to 60 days. We hypothesized that an inability to synthesize myofibrillar proteins is not the primary cause of reduced recovery in old muscle after disuse. Instead, we hypothesized that protein synthesis would be higher in old muscle than adult muscle and this would contribute to a proteostatic stress or would be matched by higher rates of protein breakdown, thus impairing recovery. To further understand the nature of the proteostatic stress, we assessed several protein pools and individual proteins in both soluble and insoluble fractions of muscle.

Methods

Methods are provided in brief with full details available in the Methods section in the supporting information.

Ethical approval

We performed all animal procedures in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Kentucky and the guidelines provided by the National Research Council's Guide for the Care and Use of Laboratory Animals: Eighth Edition.

Animals and study design

Adult (10‐month) and old (30‐month) male Fisher 344/BN rats had free access to water and were fed ad libitum. We kept rats on a 14:10‐h light/dark cycle in a temperature‐controlled (27°C) and humidity‐controlled facility according to procedures described in our previous studies. ¹⁸ Rats were housed two per cage during a 1‐week acclimatization period (WB) after which they were transferred to custom‐built single‐house cages for HU. Following 14 days of HU, we reloaded (RE) the rats and allowed them to ambulate freely for a period of up to 60 days (Figure 1 A ). Following an intraperitoneal (i.p.) injection of deuterium oxide (D₂O), we provided D₂O‐enriched drinking water (8%) to the rats during RE for 0, 7, 15, 30, 45, or 60 days to measure protein synthesis and breakdown. ¹⁸ At designated time points, we euthanized rats by i.p. injection of sodium pentobarbital (150 mg/kg; Euthanasia III Solution, Covetrus, Portland, ME) followed by exsanguination through cardiac puncture. Serum from the cardiac puncture was stored at −80°C for body water enrichment determination. We dissected and weighed gastrocnemius (GA), SOL and TA muscles. Muscles were placed on cards at resting length, frozen in liquid nitrogen and stored at −80°C. Investigators were blinded to group assignment of the animals.

Figure 1.

Muscle from adult, but not old rats, fully recovers mass and myofibre size after disuse. (A) Study design for adult (10‐month) and old (28‐month) F344BN male rats. Blue arrow indicates D₂O start. Harvest days indicate endpoints for each group from the end of hindlimb unloading (HU) to 60 days of reloading (RE). (B) Body mass collected at weight bearing (WB), HU and RE Day 15 and 60 periods (n = 3–8). (C) Wet muscle mass and (D) myofibre cross‐sectional area (CSA) of soleus (SOL) and gastrocnemius (GA) muscles (n = 3–8). (E) Representative images of GA and SOL muscles following immunofluorescence staining. (F) SOL and GA muscle myofibre CSA frequency distribution within WB, HU and RE periods (n = 3–8). Data are means ± SEM. *P < 0.05, ^** P < 0.01, ^*** P < 0.001 and ^**** P < 0.0001.

Cross‐sectional area

We determined mean fibre cross‐sectional area (CSA) for GA and SOL muscles on 7‐μm sections as previously described. ¹⁸ We took a total of five regionally representative images of the medial and lateral heads of the GA muscle with an average of 500 muscles fibres represented per muscle or three images of SOL muscle with a minimum of 300 muscle fibres. Image capture was performed by a technician blinded to experimental conditions and CSA quantified using MyoVision. ¹⁹

Isolation of detergent‐soluble and detergent‐insoluble proteins

We fractionated 20 mg of GA muscle into soluble and insoluble (aggregate‐rich fraction) ²⁰ proteins as previously described ²¹ and detailed in the Methods section in the supporting information. For total protein analysis, we stained and imaged gels with GelCode Blue Stain (aka Coomassie) Reagent (24590, Thermo Fisher Scientific, Waltham, MA). Actin and myosin were present in the insoluble fraction, which is common. ²²

Pepsin collagen solubility and hydroxyproline assay

To quantify the proportion of mature cross‐linked collagen from non‐cross‐linked and immature cross‐linked collagen in GA muscles, we treated ∼30 mg of tissue with pepsin as previously described. ¹¹ We determined the collagen concentration in the pepsin‐soluble and pepsin‐insoluble fractions using a hydroxyproline (OHP) assay as we previously described. ¹¹

Protein and RNA turnover, proteomics and bioinformatics

We determined deuterium incorporation into protein of GA, TA and SOL muscles as previously described. ¹¹ , ²³ , ²⁴ To determine ribosomal biogenesis, we assessed RNA synthesis using isolated RNA as previously described. ⁸ , ²⁵ Derivatized samples of alanine and RNA were run on an Agilent 7890A GC coupled to an Agilent 5975C MS. ²³ , ²⁴ Body water enrichment was determined on serum using a Liquid Water Isotope Analyzer (LWIA‐45‐EP, Los Gatos Research, San Jose, CA). ²⁶

For protein and RNA turnover, we calculated the rise to plateau of fraction new over the time‐course according to our previously published study. ¹¹ This analysis calculates the synthesis rate (k) over the 60‐day period using a one‐phase association. This approach provides a plateau value that shows the fraction of a protein pool that is resistant to renewal as an indicator of proteostatic decline. ¹¹ We then used our previously published non‐steady‐state equations to account for changes in protein mass. ²⁵ Our equations calculate synthesis and breakdown in two ways. The first uses change in mass and models synthesis (Ksyn) as a zero‐order equation with units of mass/day, whereas breakdown (Kdeg) is a first‐order equation dependent on mass with units of inverse time (1/t). ²⁵ Our second approach calculates FSR and fractional breakdown rate (FBR) (FSR adjusted and FBR adjusted, respectively) in units of %/day. The adjusted value refers to an adjustment for the change in protein pool size determined from the change in muscle mass. Because these calculations are made at discreet time points, we increased the N (N = 7) at Days 15 and 60. However, when analysing the myofibrillar data, we noted that somewhere between Days 30 and 60, a plateau of fraction new was reached, and therefore, values calculated at Day 60 would underestimate synthesis rates. Therefore, we only calculated Ksyn and Kdeg for proteins at Day 15.

We used a discovery‐based proteomics approach as previously described ²⁷ on total GA muscle and insoluble/soluble GA muscle fractions. Prepared samples were run on a Q Exactive™ Plus Hybrid Quadrupole‐Orbitrap™ Mass Spectrometry System (Thermo Fisher Scientific). After identification of individual proteins with Mascot Daemon software, we analysed data with d2ome software ²⁸ and used rates of tracer incorporation into peptides to calculate protein synthesis and individual protein synthesis rates (k, 1/day) as performed previously in our lab. ²⁴ We used Gene Ontology (GO) for bioinformatics analysis.

Proteasomal activity

We determined ATP‐dependent chymotrypsin‐like activity (26S) proteasomal activity using 25 mg of frozen powdered GA muscle as previously described. ⁸

Western blotting

For western blotting, we used the same portions (∼30–50 mg) of GA and SOL muscles that were used for protein synthesis analysis by saving the cytosolic protein fraction from differential centrifugation. We prepared primary antibody dilutions as follows: lysyl oxidase (LOX) (Novus Biologicals, NBP2‐24877, 1:1000), advanced glycation end products (AGEs) (Abcam, ab23722, 1:500), total ribosomal protein S6 (rpS6) (Cell Signaling Technology [CST] 2217, 1:1000) and phospho‐rpS6 (CST 4858, 1:1000), were used with a goat anti‐rabbit, horseradish peroxidase (HRP)‐linked secondary antibody (CST 7074) at 1:5000. K48‐Ubiquitin D9D5 (CST 8081, 1:1000), Poly Ubiquitin P4D1 (CST 3936, 1:1000), SQSTM1/p62 D6M5X (CST 23214, 1:1000), primary antibodies were used with LI‐COR secondary antibodies IRDye 800CW Goat anti‐Rabbit IgG 926‐32211 and IRDye 800CW Goat anti‐Mouse IgG 926‐32210.

Statistical analysis

When comparing adult to old, we used an unpaired two‐tailed t test. For other analyses, we used a two‐way (age by time) analysis of variance (ANOVA). When there were significant main effects, we used either Dunnett's post hoc test to compare changes from the WB control condition (as in Figure 1 ) or the Bonferroni adjustment for all other comparisons. We prepared all statistical analyses and figures using GraphPad Prism 9 (San Diego, CA). We reported all values as mean ± SEM with individual data points shown for each rat. We assumed significance at P < 0.05.

Results

There was a main effect of time and age on the body mass of adult and old rats. When compared to WB, the body mass of old rats was lower after HU and at Day 15 of RE, whereas adult rats were not lighter after HU (Figure 1 B ). There was a main effect of time and age on both SOL and GA muscle mass of adult and old rats. The mass of the SOL (predominantly Type I fibres) and GA (mixed fibre‐type) muscles was lower than WB after HU in both adult and old muscles. Following 15 days of reloading, the mass of the GA muscle of adult rats was not different from WB indicating a complete recovery of mass. In contrast, the GA muscle mass of old rats was still lower at 15 days of reloading but returned to baseline by 60 days (Figure 1 C ). We do not have data for TA muscle during WB; however, muscle mass for TA muscle during HU and RE is shown in (Figure  S1A ). There was a main effect of time and age on both SOL and GA muscle myofibre size in both adult and old rats. Mean fibre CSA of both the SOL and GA muscles was smaller after HU. Following 15 days of RE, SOL muscle CSA of both adult and old was not different from WB. In the GA muscle, however, fibre CSA was similar to WB by 15 days of RE in adult but not in old rats. In old rats, GA muscle CSA was still lower than WB at 60 days of RE (Figure 1 D,E ). Plots of the distribution of myofibre size revealed a leftward shift following HU in all muscles and highlight the delayed recovery of myofibre size in old GA muscles (Figure 1 F ). Together, these data showed that old muscle, particularly old GA muscle, had limited mass and fibre size recovery during RE.

To determine indicators of translation, we measured total RNA concentration, synthesis and breakdown during RE in adult and old muscles. The SOL muscle had a main effect of age where RNA concentration was lower in old than adult (Figure 2 A ). We did not have SOL muscle tissue at the end of HU to be able to calculate the concentration change at Days 15 and 60 that is necessary for calculating Ksyn and Kdeg. In the TA muscle, there was a main effect of age such that RNA concentration was greater in old muscle than adult muscle. This effect was largely driven by a difference at Day 60 (Figure 2 A ). Importantly, in both the GA and TA muscles, there was a main effect of time with a general decrease during RE except Day 60 RE in old TA muscle where there was no difference from HU (Figure 2 A ). The RNA concentration of old SOL, GA and TA muscles were equal or greater than adult SOL, GA and TA muscles during RE. The synthesis of RNA (k) over 60 days of RE was higher in old than adult in all three muscles (Figure 2 B ). This finding was supported by calculations of Ksyn in GA and TA muscles, which expresses the data in absolute quantities (ng/mg/day) (Figure 2 C,D ). The RNA synthesis data demonstrate that ribosomal biogenesis was not impaired in old muscle and was even greater than adult muscle.

Figure 2.

Translational potential/capacity is similar or higher in old muscle than adult muscle during reloading (RE). (A) Total RNA concentration per mg of muscle at the end of hindlimb unloading (HU) and during 15 and 60 days of RE in soleus (SOL), gastrocnemius (GA) and tibialis anterior (TA) muscles (n = 2–6). (B) Fraction new and synthesis rate of RNA measured throughout 60‐day labelling period in SOL, GA and TA muscles (n = 15–20). Calculated rates of RNA (C) synthesis (ribosomal biogenesis) (Ksyn) and (D) degradation (Kdeg) at 15 and 60 days of RE in GA and TA muscles (n = 4–7). Data are means ± SEM. *P < 0.05, ^** P < 0.01 and ^**** P < 0.0001.

By any measurement of synthesis and breakdown, there were no differences between adult and old SOL muscles (Figure 3 A–C ). In the GA and TA muscles, the myofibrillar protein synthesis rate was higher in old muscle than adult muscle during the RE period (Figure 3 A–C ). When calculating Ksyn and Kdeg, which uses the change in protein mass of myofibrillar protein that was synthesized during the labelling period, there was no difference (GA muscle) or a decrease (TA muscle) in Ksyn between adult and old and greater Kdeg in old GA muscle (Figure 3 B ). When expressing the synthesis rate as a fraction of the proteins synthesized (FSR), and accounting for the changes in protein pool size (Adj), both the FSR and the FBR were higher in the old GA muscle than the adult GA muscle (Figure 3 C ). In the TA muscle, there was no difference between adult and old in FSR and a greater FBR in old muscle (Figure 3 C ). Last, mTOR complex 1 (mTORC1) target rpS6 had elevated phosphorylation at site Ser235/236 during the reloading period in the old GA muscle compared to adult GA muscle, with no differences observed in SOL muscle (Figure 3 D ). These data suggest that limitations in old muscle to recover after disuse are not due to an inability of old muscle to synthesize myofibrillar proteins.

Figure 3.

Old muscle has higher protein synthesis and degradation rates and higher mTOR signalling than adult muscle during reloading (RE). Soleus (SOL), gastrocnemius (GA) and tibialis anterior (TA) muscles were fractionated, and the myofibrillar fractions of each muscle were assessed in (A). (A) Fraction new and synthesis rate of myofibrillar proteins measured throughout 60‐day labelling period (n = 17–21). (B) Ksyn and Kdeg (which accounts for the mass of protein that was made) during RE (n = 3–6). (C) Adjusted fractional synthesis rate (FSR) and fractional breakdown rate (FBR) of myofibrillar proteins during RE. Adjusted FSR and FBR account for protein pool size (muscle mass) and are expressed as a fraction of the proteins synthesized (n = 3–6). (D) Representative immunoblot and quantification of phospho‐ribosomal protein S6 (rpS6) and total rpS6, a target of S6K1, in SOL and GA muscles during RE (n = 9–11). Immunoblot quantification is relative to adult RE 15 days. Data are means ± SEM. *P < 0.05, ^** P < 0.01, ^*** P < 0.001 and ^**** P < 0.0001.

Collagen synthesis rates were lower in old SOL muscles than adult SOL muscles during RE, while no differences in collagen synthesis were observed in adult and old GA and TA muscles (Figure 4 A ). The fraction of the collagen protein pool that remained dynamic across the different muscles was 11–29%, which means 71–89% was resistant to turnover (Figure 4 A ). This finding was corroborated by OHP concentration where the soluble fraction approximated 10% of the insoluble. In most collagen fractions, there was a main effect of age where old had lower soluble collagen and higher insoluble collagen than adult (Figure 4 B,C ). In SOL muscle, old muscle also had greater total collagen than adult muscle (Figure 4 D ). Finally, the abundance of the cross‐linking enzyme LOX was not higher in old muscle than adult muscle, although AGEs, a post‐translational modification that increases cross‐linking, were more abundant in the old GA muscle than adult GA muscle (Figure 4 E ).

Figure 4.

Collagen proteostatic maintenance is impaired in extracellular matrix (ECM) of old muscle after disuse. (A) Collagen protein fraction new/fraction resistant to turnover and synthesis rate for soleus (SOL), gastrocnemius (GA) and tibialis anterior (TA) muscles throughout 60‐day reloading (RE) period (n = 14–20). Hydroxyproline (OHP) assay measuring content of (B) non‐cross‐linked and immature cross‐linked (pepsin soluble), (C) cross‐linked (pepsin insoluble) and (D) total collagen in SOL, GA and TA muscles at the end of the hindlimb unloading (HU) period and at 15 and 60 days of RE (n = 2–7). (E) Representative immunoblot and quantification of relative abundance of advanced glycation end products (AGEs) and lysyl oxidase (LOX) in SOL and GA muscles during RE period (n = 9–12). All immunoblot quantification is relative to adult RE 15 days. Data are means ± SEM. *P < 0.05, ^** P < 0.01 and ^*** P < 0.001.

Because we only saw limited recovery in GA muscle, not SOL muscle, and due to limited amount of SOL muscle tissue, the subsequent data in Figures 5 and 6 were collected on GA muscle only. We determined if detergent‐insoluble proteins, which are enriched in protein aggregates, accumulated in old muscle. We showed that there was a greater ratio of insoluble over soluble proteins in old GA muscle than adult GA muscle (Figure 5 A ). We assessed different protein degradative mechanisms by measuring markers for autophagy and proteasome. As a marker of autophagy, there was a main effect of time and age in the insoluble fraction where p62 (sequestosome 1 [SQSTM1]) was greater in old than adult. Interestingly, this higher level of p62 in the insoluble fraction of old muscle compared to adult muscle was apparent during HU and remained higher at 15 days of RE. There was also a main effect of time and age in both the soluble and insoluble fractions for ubiquitinated proteins. Compared to adult, ubiquitinated proteins accumulated in old muscle during HU (Figure 5 B ). Proteins that are tagged with lysine‐48 (K48)‐linked polyubiquitin are identified by the proteasome for degradation. There was a main effect of time, where soluble appeared to be lower during RE. There was a main effect of time and age in the insoluble fraction for K48‐ubiquitinated proteins, with old being greater than adult. We saw an accumulation of K48‐ubiquitinated proteins in the insoluble fraction of old muscle compared to adult muscle during WB, after HU and at RE Day 15 (Figure 5 C ). We next measured β₅ 26S (ATP‐dependent) proteasomal activity and showed a main effect of time in absolute activity, and a main effect of time and age, with old greater than adult, in % change from WB (Figure 5 D,E ).

Figure 5.

Old gastrocnemius (GA) muscle has more detergent‐insoluble proteins and an accumulation of insoluble ubiquitinated proteins after disuse. All gels and immunoblots are from adult and old GA muscles. (A) Representative Coomassie Blue stain and total protein quantification of soluble and insoluble fractions during reloading (RE) Days 15 and 60 combined (n = 11). (B) Representative immunoblots and quantification of autophagy‐related proteins (p62 and ubiquitin) in the soluble and insoluble fractions during weight bearing (WB), hindlimb unloading (HU) and 15 and 60 days of RE (n = 3–6). (C) Representative immunoblot and quantification of K48‐Ub in the soluble and insoluble fractions during WB, HU and 15 and 60 days of RE. Immunoblot quantification is represented relative to (A) or fold change from (B, C) adult WB (n = 2–6). (D, E) Quantification of 26S (ATP‐dependent) proteolytic activity in adult and old GA muscles during WB, HU and RE periods (n = 3–5). (D) Activity normalized to adult WB. (E) Activity represented as % change from WB within respective age. Data are means ± SEM. *P < 0.05, ^** P < 0.01 and ^*** P < 0.001.

Figure 6.

Different content and synthesis rates of proteins in old compared to adult gastrocnemius (GA) muscle aggregates. All data were obtained from adult and old GA muscles. (A) Relative synthesis rates (old − adult) for individual proteins identified in whole GA muscle during reloading (RE). Proteins to the right of mid‐line (above zero) have higher synthesis rates in old muscle than adult muscle, while proteins to the left of mid‐line have slower synthesis rates. Relative synthesis rates calculated as old − adult. (B) Top 20 proteins with slowest (blue) and fastest (red) synthesis rates in old muscle relative to adult. Table showing the enriched muscle‐ and proteostasis‐related Gene Ontology (GO) biological processes identified based on proteins with higher synthesis rates in old muscle. (C) Number of detergent‐insoluble proteins identified in GA muscle during weight bearing (WB), hindlimb unloading (HU) and at 15 and 60 days of RE. Venn diagrams depicting number of unique and shared proteins identified in detergent‐insoluble fractions of GA muscle during WB, HU and RE (combined Days 15 + 60). (D) Main biological signatures of insoluble proteins identified in old or adult muscles during WB, HU and RE (combined Days 15 + 60). The table indicates the number of times the identified terms appear in the top 50 enriched GO pathways for that given group. (E) Relative synthesis rates (old − adult) of individual proteins identified in detergent‐insoluble fraction during RE. (F) List of unique insoluble proteins and their biological function. These proteins are found only in old muscle during RE and are not present in adult during RE.

Using D₂O labelling, we showed that ~‐78% of proteins identified by discovery proteomics had higher rates of synthesis in old GA muscle than adult GA muscle during RE (Figure 6 A and Table S1 ). We identified the top 20 proteins that had the greatest change in synthesis (increase or decrease) in old muscle compared to adult muscle during the RE period (Figure 6 B ). We identified the GO terms for all identified proteins that had changes in their relative synthesis rates in old compared to adult (old − adult) that were ≥0.015 k (1/day) (Table  S2 ). Many of the top enriched GO terms were related to metabolic processes, which may indicate that increased metabolism supported the higher rate of protein synthesis in old muscle. Two of the top 6 enriched processes for proteins with higher synthesis rates were related to protein folding/refolding while three of the top 13 were related to skeletal muscle organization (Figure 6 B ). To understand which proteins were accumulating in aggregates, we performed proteomic analysis of detergent‐insoluble proteins in adult and old GA muscles. Adult with HU had a 17% and old a 28% greater number of insoluble proteins identified than WB (Figure 6 C ). We further analysed the insoluble proteins and identified the unique and shared proteins between adult and old during WB, HU and RE conditions (Figure 6 C ). From these unique proteins, we identified the top 50 GO biological processes with the highest fold enrichment values under each condition (WB, HU and RE) (Table  S3 ) and compared between adult and old. There were three major differentially enriched signatures in the following biological categories: (1) neuron, pre‐synapse and post‐synapse, and synaptic vesicle; (2) catabolic processes, autophagy and endoplasmic reticulum (ER) unfolded protein response; and (3) exocytosis (Figure 6 D ). We identified 47 proteins that were D₂O labelled during RE and in the insoluble protein fraction (Table  S4 ). We showed that 33 (85%) of these proteins had higher synthesis rates in old muscle than adult muscle (Figure 6 E ). Four of the 47 proteins identified were only found in the old muscle; all other proteins were either shared between adult and old or unique to adult. Each of those four proteins only found in the old insoluble fraction has functional roles important to skeletal muscle maintenance or growth, contractile function and degradation: eukaryotic translation elongation factor 1 alpha 2 (EF1A2), four and a half LIM domains 1 (FHL1), myosin light chain 2 (MLRV) and voltage‐dependent anion channel 1 (VDAC1) (Figure 6 F ).

Discussion

We hypothesized that an inability to synthesize myofibrillar proteins is not the primary cause of reduced recovery in old muscle after disuse but is instead due to an imbalance or impairment in other proteostatic mechanisms such as protein folding or protein degradation. We found that old GA muscle has limited recovery of muscle mass during RE despite having higher translational capacity and myofibrillar protein synthesis rates than adult GA muscle. We also showed that collagen was accumulating in aged SOL muscle during RE from a slowing of breakdown, and this finding was supported by old muscle having less soluble and more insoluble collagen than adult muscle. Our data showed that limited recovery of muscle mass during RE is in part due to higher protein degradation, as measured with long‐term isotope labelling, and/or compromised proteostasis as evidenced by the accumulation of ubiquitinated insoluble/aggregate proteins. Last, we showed that the synthesis of individual proteins related to protein folding/refolding, protein degradation and neural‐related biological processes was higher in old muscle during RE than adult muscle. Therefore, proteostatic mechanisms other than myofibrillar protein synthesis are likely underlying the poor recovery of muscle after disuse in the old.

Maintenance of proteostasis is complex as it involves multiple proteostatic mechanisms (i.e., protein synthesis, folding and degradation) working in concert. One hallmark of impaired proteostasis is the accumulation of protein aggregates. ¹⁷ Protein aggregates can accumulate due to aberrant protein synthesis leading to improperly translated proteins, ¹⁶ , ¹⁷ impaired protein folding, ¹² , ¹³ or compromised protein degradation. ¹⁴ , ¹⁵ Cellular cargo tagged for degradation, such as ubiquitinated aggregate proteins, can accumulate due to improper autophagic clearance or due to a lack of recognition of specific cellular debris even without any obvious impairment in autophagic flux or proteasomal function. ²¹ In old muscle, protein breakdown rates were higher in GA and TA muscles than adult muscle during RE. Yet we showed an increase in detergent‐insoluble/aggregate proteins in old muscle compared to adult muscle. These data demonstrated that although protein breakdown rates were higher in old muscle, certain insoluble proteins are still accumulating during HU and RE. We also showed an accumulation of the autophagy adaptor p62 in the insoluble fraction of old muscle in addition to an accumulation of ubiquitinated proteins in old muscle compared to adult muscle. Although we did not measure autophagic flux, we suggest that certain proteins were not cleared sufficiently through autophagy leading to the accumulation of insoluble protein aggregates in old muscle during HU and RE. Lysosomal exocytosis promotes cellular clearance, ²⁹ which may function as a way of clearing misfolded or aggregate proteins to help alleviate the burden for autophagy. Interestingly, in our GO analysis of insoluble proteins, four exocytosis‐related biological pathways were enriched in adult, but not in old. This suggests that exocytosis may be supporting clearance of insoluble and aggregate proteins in the adult during RE but may be inadequate in the old. It is important to note that there were many unique outcomes between the SOL, GA and TA muscles regardless of age (e.g., recovery of mass, myofibrillar protein synthesis and degradation, mTORC1 signalling and collagen dynamics) that likely arose due to differing muscle fibre‐type composition and/or functional use between these muscles, which is supported by previous studies. ⁵ , ⁸ SOL muscle recovers the best and is the most actively recruited out of the three muscles measured, while TA and GA muscles have more Type IIb fibres, which are the least active/recruited fibres. Future studies should investigate how these different muscle types/functions contribute to recovery.

The proteasome serves essential functions in proteostasis by degrading proteins and peptides within a cell. ³⁰ Although β₅ 26S proteolytic activity was lower in old muscle than adult muscle during WB, it was higher in old GA muscle than adult GA muscle after HU. A previous study did not identify the same increase in proteasomal activity, but the response to RE was like ours. ⁵ This difference in proteasomal response to disuse may arise from the difference in muscles tested between studies. In our study, we assessed GA muscle for proteasomal assays, while Baehr et al. used SOL and TA muscles. Each of these muscles differs in fibre‐type composition and functional use during ambulation. Despite the increase in proteasomal activity with HU and RE, K48‐ubiquitinated proteins, which are recognized by the proteasome for degradation, ³¹ accumulated during HU and RE in old muscle. These data suggest that the proteasome may not be clearing K48‐ubiquitinated cargo effectively, resulting in the accumulation of proteasomal targets. Together, our data suggest that limited recovery of GA muscle mass during RE is in part due to increased myofibrillar protein breakdown, but that accumulation of ubiquitinated insoluble/aggregate proteins still occurs, in impaired proteostasis.

Extracellular matrix (ECM) largely consists of collagen proteins and has important structural and signalling roles in skeletal muscle. ³² However, excessive ECM accumulation leads to fibrosis and impaired muscle function. ³³ Collagen proteins have a long half‐life and are therefore especially susceptible to the accumulation of AGEs, which increase cross‐linking, making collagen resistant to degradation and increasing tissue stiffness. ³⁴ We showed that old GA muscle has an accumulation of AGEs compared to adult GA muscle during RE, indicating more collagen cross‐linking in old muscle. We also showed that collagen concentration is increased in old SOL muscle compared to adult SOL muscle and that old SOL muscle also has less soluble and more insoluble collagen. It was surprising that we did not find these same changes in collagen concentration in old GA muscle. As we previously demonstrated, ¹¹ a surprisingly small amount of collagen (11–29%) continues to turn over in old skeletal muscle. It is likely that the less dynamic nature of the collagen has a large impact on muscle ageing because there is a lower adaptive capacity. Our data indicate that decreases in collagen breakdown over time are likely causing the accumulation of collagen in old muscle. Further, these results indicate that there is impaired proteostatic maintenance in the collagen of muscle ECM with age, which may contribute to a fibrosis‐like state potentially limiting regrowth of muscle mass. ³⁵

Old muscle did not recover mass as well as adult muscle and even failed to completely recover in the GA muscle over 60 days, despite having similar or higher anabolic potential and higher myofibrillar protein synthesis than adult muscle. We found that old GA, SOL and TA muscles had higher rates of RNA synthesis than adult GA, SOL and TA muscles and that RNA concentration was similar (GA and SOL muscles) or higher (TA muscle) in old muscles during RE. Together, these results demonstrate that ribosomal biogenesis was not impaired in old muscle and was even greater than adult muscle in certain muscles during RE despite a lack of recovery of muscle mass. To combat loss of myofibrillar protein mass with disuse, mTORC1 stimulation is commonly targeted (e.g., amino acid feeding) in old muscle to stimulate protein synthesis. ⁹ , ¹⁰ The rationale to stimulate mTOR activity to improve recovery of muscle mass in the elderly after periods of disuse is based on studies performed in adult and young muscle, when mTOR activity is not chronically elevated. ³⁶ Our data showed that mTOR signalling, translational capacity and protein synthesis were the same or higher in old muscle than adult muscle during RE. Models with chronically elevated mTOR, such as the TSC1 skeletal muscle knockout mouse, resemble old muscle with muscle fibre atrophy and impairments in muscle strength. ³⁷ When mTOR is inhibited in these TSC1mKO mice with rapamycin, the mice have restored myofibre size and strength. ³⁷ To our knowledge, there are no studies that demonstrate that there is a loss of accuracy of protein synthesis (i.e., translational fidelity) with ageing. There is, however, evidence that shows that rodents with longer lifespan have greater translational fidelity, ³⁸ that increases in translational fidelity can extend lifespan, ³⁹ and that impairments in translational fidelity can lead to premature ageing. ⁴⁰ We therefore speculate that the higher rates of protein synthesis in the old muscle contribute to greater misfolding and protein aggregation. Therefore, our data support that efforts to further activate mTOR activity in old muscle could actually worsen proteostatic stress leading to poor recovery.

Because muscle function is determined by more than myofibrillar proteins, we examined the synthesis rates of individual proteins to further understand differences between adult and old muscles. We showed that during RE, nearly 80% of identified proteins in the GA muscle had higher rates of synthesis in old muscle than adult muscle, supporting our fractional data. Of the proteins with the highest rates of synthesis and greatest fold enrichment, two of the top 6 GO terms were grouped into ‘protein refolding’ and ‘de novo protein folding’. These changes indicate a compensatory response in muscles from old rats to the large number of improperly translated and misfolded proteins. After HU, adult muscle had a 17% higher abundance of insoluble proteins than WB, while old muscle had 28% more. With D₂O labelling, we found that some proteins that were synthesized during RE accumulated in the aggregates during the RE period. This observation indicates that the RE phase is a period of proteostatic dysregulation and a potential period for intervention. However, at this point, we do not know if the dysregulation was also present before HU, which could contribute to the proteostatic dysregulation and subsequent recovery during RE. We also found that there were differences in the insoluble proteins present in adult and old muscles during RE. Interestingly, the insoluble proteins that were only found in old muscle have functional roles important to skeletal muscle maintenance or growth (EF1A2 and FHL1), contractile function (MLRV) and autophagy/apoptosis (VDAC1). Taken together, we showed that older muscle is synthesizing proteins at a higher rate but that some of these proteins are ending up in aggregates indicating proteostatic dysregulation.

This study is not without limitations, which impair our ability to draw some conclusions. We were only able to measure RNA concentration during the RE period and not with HU in SOL muscle because of a shortage of muscle tissue. Similarly, we were unable to measure RNA Ksyn and Kdeg in SOL muscle. Further, we only measured autophagic markers and not autophagic flux because that requires administration of a drug such as colchicine in a live animal. It would also be beneficial for future studies to investigate the impact that fibre type/muscle activity and recruitment has on recovery. Finally, coverage of the skeletal muscle proteome remains a technical challenge, thus limiting the full determination of which proteins are subject to proteostatic stress. The primary reason for this challenge is the high abundance of myofibrillar proteins that can hinder proteomic identification of other proteins. Although our questions for this study focused on the different responses to reloading between adult and old muscles, it is important to note that there were many unique outcomes between the SOL, GA and TA muscles regardless of age (e.g., recovery of mass, myofibrillar protein synthesis and degradation, mTORC1 signalling and collagen dynamics) that likely arose due to differing fibre type and functional use between these muscles.

In conclusion, we demonstrated that limitations in recovery of muscle after disuse atrophy in old muscle were not due to an inability to synthesize proteins but instead from other compromised proteostatic mechanisms. We showed that there is a greater accumulation of insoluble ECM in old muscle than adult muscle during RE and that this accumulation is likely caused by increased cross‐linking and decreased breakdown rates. We also showed that during RE, there is a greater accumulation of insoluble protein aggregates in old muscle than adult muscle. These aggregates are at least partially caused by high rates of protein synthesis and that the accumulation continues with proteins synthesized during RE. We further showed that although protein breakdown rates were higher in old muscle than adult muscle, the degradative processes of old muscle might be compromised as evident by accumulation of adaptor proteins in the aggregates. Because mTOR activity was higher in old muscle than adult muscle, we suspect that this overactivity contributes to the proteostatic stress. We suggest that future studies on therapeutic approaches for the impaired recovery of old muscle after disuse should shift from a sole focus on increasing protein synthesis to improving other mechanisms of proteostasis.

Conflict of interest statement

The authors declare no conflicts of interest.

Supporting information

Figure S1. TA muscle mass during HU and RE. A) Wet muscle mass during HU and RE days 15 and 60 (n = 3–7). There is no data for WB. WB, weight bearing; HU, hindlimb unloading; RE, reloading. Data are means ± SEM. **p < 0.01.

Methods S1. Supplemental Methods

Table S1. Synthesis Rates of Individual Proteins Identified in Total GA Muscle During RE

Table S2. GO Terms for Proteins with Higher Synthesis Rates in Old Muscle Compared to Adult (O‐A)

Table S3. List of GO terms for insoluble proteins identified in Adult WB

Table S4. Synthesis Rates of Individual Proteins Identified in Insoluble Fraction of GA Muscle During RE

Fuqua J. D., Lawrence M. M., Hettinger Z. R., Borowik A. K., Brecheen P. L., Szczygiel M. M., et al (2023) Impaired proteostatic mechanisms other than decreased protein synthesis limit old skeletal muscle recovery after disuse atrophy, Journal of Cachexia, Sarcopenia and Muscle, 14, 2076–2089, 10.1002/jcsm.13285