Low baseline ribosome‐related gene expression and resistance training‐induced declines in ribosome‐related gene expression are associated with skeletal muscle hypertrophy in young men and women

Ribosomes are essential cellular machinery for protein synthesis. It is hypothesised that ribosome content supports muscle growth and that individuals with more ribosomes have greater increases in muscle size following resistance training (RT). Aerobic conditioning (AC) also elicits distinct physiological adaptations; however, no measures of ribosome content following AC have been conducted. We used ribosome‐related gene expression as a proxy measure for ribosome content and hypothesised that AC and RT would increase ribosome‐related gene expression. Fourteen young men and women performed 6 weeks of single‐legged AC followed by 10 weeks of double‐legged RT. Muscle biopsies were taken following AC and following RT in the aerobically conditioned (AC+RT) and unconditioned (RT) legs. No differences in regulatory genes (Ubf, Cyclin D1, Tif‐1a and Polr‐1b) involved in ribosomal biogenesis or ribosomal RNA (45S, 5.8S, 18S and 28S rRNAs) expression were observed following AC and RT, except for c‐Myc (RT > AC+RT) and 5S rRNA (RT < AC+RT at pre‐RT) with 18S external transcribed spacer and 5.8S internal transcribed spacer expression decreasing from pre‐RT to post‐RT in the RT leg only. When divided for change in leg‐lean soft tissue mass (ΔLLSTM) following RT, legs with the greatest ΔLLSTM had lower expression in 11/13 measured ribosome‐related genes before RT and decreased expression in 9/13 genes following RT. These results indicate that AC and RT did not increase ribosome‐related gene expression. Contrary to previous research, the greatest increase in muscle mass was associated with lower changes in ribosome‐related gene expression over the course of the 10‐week training programme. This may point to the importance of translational efficiency rather than translational capacity (i.e. ribosome content) in mediating long‐term exercise‐induced adaptations in skeletal muscle.


| INTRODUCTION
Resistance exercise training increases human muscle mass and strength (Joanisse et al., 2020).Repeated transient spikes in muscle protein synthesis following resistance exercise lead to an accumulation of sarcoplasmic and myofibrillar proteins in skeletal muscle that results in hypertrophy over time (Phillips, 2000).Ribosomes are cellular machinery that are essential for protein translation and regulate protein synthesis through changes in content (translational capacity) and activity (translational efficiency) (Chaillou et al., 2014;Wen et al., 2016).
Ribosomal biogenesis is a complex process that requires the coordination of upstream transcriptional regulators, which act downstream to coordinate the formation of the pre-initiation complex on the ribosomal DNA (rDNA) (Brook et al., 2019;Chaillou et al., 2014;Figueiredo & McCarthy, 2019;Mayer & Grummt, 2006).
Current literature suggests that translational capacity is an important determinant of adaptation to resistance training.Ribosome content increases alongside increases in muscle mass (i.e.hypertrophy) to support the adaptive response to load-induced stimuli; however, the strongest evidence to support this is in rodents (Kirby et al., 2015;Mobley, Holland, et al., 2018;Nakada et al., 2016;West et al., 2016West et al., , 2019)), where some is in humans but is less compelling (Figueiredo et al., 2015;Hammarström et al., 2020;Stec et al., 2016).
Recent evidence highlights the high variation (Figueiredo et al., 2021;Gibbons et al., 2014) in ribosomal adaptation to resistance training and some have even suggested that there may be no change or a decrease in ribosome and RNA content (Fyfe et al., 2018;Mobley, Haun, et al., 2018;Smith et al., 2023)  Although typically investigated in the context of resistance training, ribosome content may contribute to adaptations to aerobic exercise conditioning.Aerobic conditioning leads to a fibre-type shift towards more oxidative type 1 fibres, mitochondrial biogenesis and microvascular perfusion (Hoier & Hellsten, 2014;Irrcher et al., 2003;Jornayvaz & Shulman, 2010).Remodelling processes characteristic of aerobic conditioning involve the turnover and synthesis of new proteins, for example, myosin heavy chain proteins, formation of oxidative enzymes and protein complexes during mitochondrial biogenesis, and proliferation and sprouting of endothelial cells through the extracellular matrix to form new capillary beds (Hoier & Hellsten, 2014;Irrcher et al., 2003;Jornayvaz & Shulman, 2010).
Although ribosome content does not change following an acute bout of aerobic exercise (Figueiredo et al., 2021), 12 weeks of highintensity interval training augmented the abundance of both ribosomal and mitochondrial proteins (Robinson et al., 2017).Therefore, skeletal muscle remodelling resulting from aerobic conditioning may require increased protein translation supported by elevated ribosome content (i.e.translational capacity).
No studies have directly investigated ribosomal changes following aerobic conditioning.Therefore, the purpose of this study was to determine the impact of both aerobic conditioning and resistance training on ribosomal biogenesis by determining ribosome-related gene expression as an indirect measure for ribosome content.A secondary aim was to investigate the impact of aerobic 'pre-conditioning' on ribosomal adaptations following resistance training.We hypothesised that ribosome-related gene expression would increase following both aerobic conditioning and resistance training to support the adaptive responses to the respective training modes.We also aimed to explore how changes to ribosome-related gene expression are related to changes in muscle mass with resistance training.

| Participants
Participant characteristics have been described previously (Brown et al., 2022;Thomas et al., 2022), where one female participant was excluded due to low RNA concentration yields (n = 13; five females and eight males).Participants were 21 ± 2 years old, had a body mass index of 25.3 ± 4.8 kg/m 2 , a double-legged VO 2 peak of 39.7 ± 7.2 mL/ min/kg, an average single-legged VO 2 peak of 31.4 ± 6.4 mL/min/kg and an average leg-lean soft tissue mass (LLSTM) of 9233 ± 2534 g.
Participants had not participated in formal aerobic or resistance training programmes at least 6 months before the study commencement, were non-smokers and had no history of diabetes, use of non-steroidal antiinflammatory drugs or statins, respiratory disease and/or major orthopaedic disability.Participant's baseline characteristics

| Study design
This project was part of a larger study described previously (Brown et al., 2022;Thomas et al., 2022).Briefly, participants completed 6 weeks of single-legged cycling where legs were randomised to be aerobically conditioned (AC+RT) or act as a non-conditioned control leg (RT) (Figure 1).The aerobic conditioning period was sufficient in inducing several aerobic adaptations (Brown et al., 2022;Thomas et al., 2022).Following aerobic conditioning, participants underwent double-legged, lower-body resistance training for 10 weeks.
Baseline fitness was assessed with a VO 2 peak test on a cycleergometer (Excalibur Sport, version 2.0; Lode).Before aerobic conditioning, participants underwent single-legged VO 2 peak tests as described previously (Abbiss et al., 2011;Burns et al., 2014;MacInnis et al., 2017a).The mean average of the single-legged VO 2 peak tests (one from each leg) was used to determine the initial workload (50%) for aerobic training (continuous, single-legged cycling three times/week for 6 weeks with workload increasing 2%-4% every four sessions).
Following 6 weeks of aerobic conditioning (pre-RT), participants were familiarised with double-legged resistance-training exercises (squat, leg press, leg extension, hamstring curl and calf raises).One-repetition maximum (1RM) tests were performed for squat, leg press and leg extension and the initial workload was set at 70%-80% 1RM.
Participants' resistance trained three times/week for 10 weeks, performing 3 × 8-12 repetitions of each exercise (with the last set performed until failure).Weight was increased between sessions when the participant exceeded 12 repetitions in the final set.1RM was tested again following 10 weeks of resistance training (post-RT).

| Muscle biopsies
Skeletal muscle biopsies were obtained pre-RT and post-RT (in both RT and AC+RT legs) from the vastus lateralis following a 10-12 h overnight fast and at least 48 h following the last training session (Tarnopolsky et al., 2011).Approximately 150 mg of muscle was obtained from each biopsy and oriented in cross-sections, mounted in optimal cutting temperature compound (Tissue-Tek; Sakura Finetek) and frozen in isopentane cooled with liquid nitrogen.Embedded samples were then stored at -80°C for subsequent immunohistochemical analysis.The remaining tissue was freed of blood and connective tissue, immediately frozen in liquid nitrogen and stored at -80°C for subsequent analyses.

| Body composition
Dual-energy X-ray absorptiometry (DXA-GE Lunar iDXA; Aymes Medical) scans were taken pre-RT and post-RT after a 10-12 h overnight fast to assess whole-body and LLSTM.

| Muscle homogenisation, RNA isolation
Approximately 20 mg flash-frozen skeletal muscle was homogenised using 1 mL TRIzol ® reagent in Lysing Matrix D tubes (MP Biomedicals), with FastPrep-24™ Tissue and Cell Homogenizer (MP Biomedicals) for 40 s at 6 m/s.RNA was isolated using the TRIzol ® manufacturer's protocol.RNA yields and 260/280 ratios were determined using a NanoDrop 2000™ Spectophotometer (Thermo Scientific).random hexamers and oligo-dT primers in 10 µL reaction volumes as per the manufacturer's instructions.Revere transcription was carried out using a SimpliAmp™ Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific).cDNA was then diluted to a final concentration of 10 ng/µL and stored at −20°C until subsequent analysis.
Primers (Table 1) were resuspended in 1X TE Buffer (10 mM Tris-HCl and 0.11 mM EDTA) and stored alongside the commercially available gene expression assays (Table 1) at −20°C before use.The primers for ribosome markers and the 45S pre-rRNA assay have been used previously (Figueiredo et al., 2015(Figueiredo et al., , 2021)).RT-qPCR reactions for individual primers and the 45S pre-rRNA assay (Qiagen) were prepared in triplicates of 12.5 µL reaction volumes containing RT 2 Sybr Green qPCR Master Mix (cat.#330500; Qiagen), forward and reverse primers (or 45S pre-rRNA assay) and 10 ng cDNA.Moreover, 384-well PCR plates were prepared using the epMotion 5075 Eppendorf automated pipetting system (Eppendorf).Taqman gene expression assays were prepared in triplicates of 10.0 µL reaction volumes containing Taqman™ Fast Advanced Master Mix (Thermo-Fisher; cat.#4444556), Taqman™ gene expression assays (Thermo-Fisher) and 10 ng cDNA.Taqman™ gene expression assays were also conjugated to NFQ-MGB quencher and ROX as a passive reference.
Samples were normalised to ß2M (ΔC t ; either respective SYBR or Taqman™ ß2M) which was not statistically different at any timepoint and to the pre-RT (RT leg) timepoint (ΔΔC t ).Statistical analysis was performed on the 2 −ΔΔC t value (Livak & Schmittgen, 2001).Fold change was calculated for graphical purposes by relating all values to the pre-RT (RT leg) average.

| Statistical analyses
Statistical analysis was performed using Jamovi 1.6.23 analysis software, and graphs were prepared using GraphPad Prism (Version 9.0) and BioRender.com.Individual participants for specific measures were deemed outliers when two or more values were two standard deviations (SDs) from the mean and removed from analyses.Deterministic imputation methods were used to impute missing/removed data for participants with only one missing sample.Results are reported as mean ± SD, the n for each analysis is reported in the corresponding figure legend.Two-way repeated-measures analysis of variance, where factors of time (pre-RT and post-RT) and condition (RT and AC+RT) were used to determine differences in gene expression, and Tukey's honest significant difference test was used to account for multiple post hoc comparisons.Independent samples t tests were used to compare HIGH and LOW responder groups.Correlations were run using Pearson's correlation coefficient (binomial) between changes in LLSTM and markers of ribosomal biogenesis.
T A B L E 1 Primer sequences and commercially available gene expression assays for quantitative real-time polymerase chain reaction.To ascertain the reliability of the ribosome-related gene expression data, we ran correlations between ribosome-related markers in each individual and at every time point using a correlation matrix, as the expression of these markers is highly related within individuals (Gibbons et al., 2014(Gibbons et al., , 2015)).Of the 21 possible correlations between the rRNAs transcribed on the 45S pre-rRNA, 20 were correlated to each other (TableS1), suggesting that the trends observed in the data are valid.The difference in c-Myc expression where no correlations were observed with the other markers may be due to its role in other cellular processes in addition to ribosomal biogenesis, including metabolism, angiogenesis, DNA repair, cell growth, proliferation and apoptosis, amongst other processes (van Riggelen et al., 2010).
Differences in 5S rRNA expression may be due to being transcribed independently from 45S pre-rRNA (Mayer & Grummt, 2006;van Riggelen et al., 2010;von Walden et al., 2016;Wen et al., 2016) and its contribution to the mitochondrial ribosome could explain why its expression tended to be greater in the conditioned leg following aerobic conditioning (Smirnov et al., 2008(Smirnov et al., , 2010(Smirnov et al., , 2011)).

| [RNA]
No significant effects of time (pre-RT and post-RT; p = 0.279) or condition (RT and AC+RT; p = 0.573) were observed for RNA concentration (Figure 2b) from pre-RT to post-RT or between the RT and AC+RT legs.

| Responder analyses
Due to the high variability between individuals in ribosome-related gene expression (Figueiredo et al., 2021;Gibbons et al., 2014), we created further separation in the participant cohort by dividing legs into higher (HIGH) or lower (LOW) responders to resistance training (Phillips et al., 2013;Roberts et al., 2018;Stec et al., 2015).
We analysed legs independently as no 'cross-over' effects have been observed with unilateral training, making the legs physiologically distinct following aerobic conditioning (MacInnis et al., 2017b).
We then ranked the legs from highest to lowest change in (Δ) LLSTM from pre-RT to post-RT, taking the top 10 (HIGH) and bottom 10 (LOW) values to rank higher and lower responders, with the middle 6 removed to ensure separation between groups.
Therefore, in some cases, one participant could be represented by two data points (one from each leg).In the HIGH group, 6/10 were from the AC+RT leg and 4/10 from the RT leg, whereas in the LOW group, 4/10 were from the AC+RT leg and 6/10 from the RT leg.
HIGH (1314 ± 196 g) had a significantly greater ΔLLSTM from pre-RT to post-RT compared to LOW (128 ± 204 g) (Figure 3a; At pre-RT, LOW had significantly greater expression of all ribosomal markers (all p < 0.05) except for c-Myc compared to HIGH and had a trend for greater 18 S rRNA (p = 0.0920) expression (Figure 3b).

| DISCUSSION
To our knowledge, this study is the first to investigate the impact of

| Ribosomal biogenesis with aerobic conditioning
Aerobic training increases oxidative capacity through shifts in muscle fibre-type, mitochondrial biogenesis and microvascular perfusion F I G U R E 2 Markers of ribosomal biogenesis and ribosome content.(a) Overview of upstream ribosomal biogenesis signalling and rRNA contributions to mature ribosomal subunits.C-Myc promotes the transcription of ribosomal biogenesis regulators UBF and TIF-1A and binds RNA polymerase 1B (POLR-1B) at the rDNA promoter region.UBF becomes activated downstream of mechanistic target of rapamycin 1, through cyclin-D1-dependent activation of CDK4, and can then facilitate the interaction between POLR-1B (tethered by TIF-1A) and the rDNA promoter region.POLR-1B then transcribes the 45S pre-rRNA, which is subsequently processed into its mature rRNAs and contributes (alongside ribosomal proteins) to their respective ribosomal subunits.5S rRNA is transcribed independently from the 45S pre-rRNA via RNA polymerase III and contributes (alongside the 5.8S and 28S rRNAs) to the large ribosomal subunit (60S).Following subunit assembly and upon translation initiation, the small (40S) and large ribosomal subunits form the mature 80S ribosome in the cytosol.( Hoier & Hellsten, 2014;Irrcher et al., 2003;Jornayvaz & Shulman, 2010).Our protocol induced aerobic adaptations to the muscle exclusive to the conditioned leg (Brown et al., 2022;Thomas et al., 2022) and no change in ribosome-related gene expression with aerobic conditioning suggest that the basal ribosome pool is sufficient to support aerobic adaptations.Only one other study has examined ribosome content following aerobic conditioning, where 12 weeks of high-intensity interval training increased the phosphorylation of proteins involved in translational pathways and mitochondrial-related protein abundance (Robinson et al., 2017).The increased expression of proteins involved in protein translation suggests an increase in translational capacity following aerobic conditioning; however, this may have occurred to support the increase in muscle mass that was observed (Figueiredo et al., 2015;Hammarström et al., 2020;Nader et al., 2005;Nakada et al., 2016;Robinson et al., 2017;von Walden et al., 2012;von Walden et al., 2016).Ribosomal and mitochondrial biogenesis may have competing signalling mechanisms, which implies that adaptation to one may blunt adaptation to the other (Mesquita et al., 2021).In accordance, aerobic exercise does not result in acute increases in ribosome content (Figueiredo et al., 2021), this could help to explain why ribosome-related gene expression did not increase following chronic aerobic conditioning.
While we did not observe any changes in ribosome content following aerobic conditioning, we have previously reported a greater increase in ribosome-related gene expression in the aerobically conditioned leg following an acute bout of eccentric contractions (Brown et al., 2022).Although bouts of aerobic exercise have been shown not to increase ribosome content (Figueiredo et al., 2021) aerobic conditioning can prime the muscle to augment ribosomal biogenesis following a resistance-like exercise stimulus (Brown et al., 2022).Therefore, while ribosome content is not altered with aerobic exercise or conditioning, it appears beneficial for supporting acute resistance exercise-like adaptations.Unfortunately, we could not measure any other acute time points and henceforth focused on the chronic adaptations that occurred.

| Ribosome content with resistance training
Resistance training increased LLSTM and fibre cross-sectional area as previously reported (Thomas et al., 2022), but this was not accompanied by a change in ribosome-related gene expression, contrary to our hypotheses.It is generally accepted that ribosome content increases alongside muscle hypertrophy to support the increase in protein synthetic demands.While it is possible that ribosome content changes to support other cell types within skeletal muscle, it is more likely that the majority of changes reported regarding ribosome content are due to what is observed within muscle cells specifically as they are by far the most abundant cell type (Figueiredo et al., 2015;Hammarström et al., 2020;Nader et al., 2005;Nakada et al., 2016;von Walden et al., 2016).However, it is also suggested that acute ribosomal biogenesis increases within the first 4-6 resistance training sessions and levels off after that (Hammarström et al., 2022).Some studies in humans with baseline measurements and longer post-training time points have reported no changes or decreased ribosome content (Fyfe et al., 2018;Mobley et al., 2018;Smith et al., 2023).Another study performed either high or low volumes of resistance exercise on each limb, and while the limb  et al., 2020).Indeed, when RNA content per muscle weight is used as a measure of ribosome content, there is an increase relative to baseline after 3 weeks of resistance training, but this difference is washed out after 6 weeks (Brook et al., 2016).However, when RNA is made relative to DNA this increase persists.The differences in how RNA content is expressed are subtle and indicate that two things can be true.While there may be greater ribosome content relative to DNA content to support translation during hypertrophy, there appear to be fewer ribosomes that govern more volume in hypertrophied muscle.
The rDNA transcribed spacer regions decreased with resistance training in the aerobically non-conditioned leg only.Little emphasis has been placed on rRNA transcriber spacer regions, with measures either not being included (Figueiredo et al., 2020(Figueiredo et al., , 2021;;Hammarström et al., 2020;Mobley, Haun, et al., 2018), showing no change (Figueiredo et al., 2015) or decreasing (Fyfe et al., 2018) expression following resistance training.The spacer regions are transcribed alongside 18S, 5.8S and 28 S rRNA as 45S pre-rRNA and are cleaved by nucleolin to form mature rRNAs (Figueiredo & McCarthy, 2019;Kusnadi et al., 2015;Wen et al., 2016) and are likely degraded once excised from the rRNA.We suggest that although no changes in rRNA were observed following resistance training, a decreased expression of the spacer regions suggests higher ribosomal turnover in the nonconditioned leg, which may be a result of the aerobically conditioned leg being more adapted to exercise stimuli and may have been similar if the training of the conditioned leg was extended.adaptations (Burd et al., 2010(Burd et al., , 2012;;Figueiredo et al., 2020;West et al., 2010;Wilkinson et al., 2008).In addition, DXA does not directly measure muscle mass; however, most LLSTM in the lower extremities is composed of skeletal muscle and is highly correlated to the gold standard measure of muscle mass using magnetic resonance imaging (Haun et al., 2019).

| HIGH versus LOW responder analyses
Higher  (Kirby et al., 2015;Nakada et al., 2016) and humans (Figueiredo et al., 2015;Hammarström et al., 2020;Mobley, Haun, et al., 2018;Stec et al., 2016).Ribosomal biogenesis occurs following an acute bout of resistance exercise and in response to a novel stimulus (Figueiredo et al., 2016(Figueiredo et al., , 2021;;Nakada et al., 2016;von Walden et al., 2012) but then subsequently decreases.While or to conserve cellular energy (Warner, 1999).This 'refinement' mechanism has also been referred to as 'ribosome specialisation' (Chaillou, 2019) and has been shown to control myogenesis in vitro (Warner, 1999).We propose that increasing translational efficiency is favoured for optimal adaptive responses and that increases in following resistance training.The discordance in findings regarding ribosome content and adaptations to resistance training may result from several factors, such as the training status of individuals and/or the limited selection of sampling times that require further investigation.
Isolated RNA samples were reversed transcribed into complementary DNA (cDNA) using an RT 2 First Strand Kit (Qiagen), containing F I G U R E 1 Overview of study design.Participants (n = 14) underwent 6 weeks of single-legged AC followed by 10 weeks of double-legged resistance training.Testing and measures taken following AC/preresistance training (pre-RT) and postresistance training (post-RT).AC, aerobic conditioning.
chronic aerobic conditioning and aerobic conditioning before resistance training on ribosome-related gene expression.Contrary to our hypotheses and previous literature, no change in ribosome-related gene expression was detected following aerobic conditioning or resistance training.We observed an association between ribosome-related gene expression and accretion of lean mass (ΔLLSTM) following resistance training.Legs with the greatest increase in muscle mass following resistance training (HIGH) had lower ribosome-related gene expression content before training and a smaller change in ribosome content following resistance training compared to legs with the smallest increase in muscle mass (LOW).These findings suggest that greater translational capacity (i.e.ribosome content), contrary to previous reports, is not associated with increases in muscle mass following long-term (10 weeks) resistance training.

F
I G U R E 4 Correlations between change in ribosome content and muscle hypertrophy from pre-RT to post-RT.Pearson's correlations between (a) ΔCyclin D1 (n = 18) and (b) ΔPolr-1b (n = 18) messenger RNA, (c) Δ45S pre-rRNA (n = 19), (d) Δ18S ETS (n = 20), (e) Δ5.8S ITS (n = 19), (f) Δ28S ITS (n = 19), (g) Δ18S rRNA (n = 20) and (h) Δ5.8S rRNA (n = 20) expression in RT (circle) and AC+RT (square) legs.AC, aerobic conditioning; ETS, external transcribed spacer; ITS, internal transcribed spacer; rRNA, ribosomal RNA; RT, resistance training; ΔLLSTM, change in leg-lean soft tissue mass.completing high-volume training had higher ribosomal RNA expression early in the training programme, the extended training period either eliminated the observed differences or in some instances resulted in the limb completing low volume training having higher ribosomal RNA expression than the limb completing high volume training (Hammarström et al., 2020).Therefore, our measurements of ribosome-related gene expression at baseline and a longer training protocol of 10 weeks likely do not capture the initial changes to ribosome content in the training programme, which are likely important in supporting the initial increase in size.Although our sampling points cannot capture these changes, it is important to recognise that long-term adaptations to resistance training result in either no change from initial or potentially a decrease in ribosome content.It is also important to note that 'decrease' in ribosome content is relative to muscle mass, whereby as muscle mass increases, there is a dilution of the ribosome pool (Joanisse Legs with greater changes in LLSTM following resistance training had lower ribosome-related gene expression (11/13 markers) before resistance training and a smaller change (9/13 markers) throughout training.In addition, the change in LLSTM with resistance training was negatively associated with increases in ribosome-related gene expression.It is important to note that legs were treated independently, and therefore individual participant and training status were not considered.While these analyses are not ideal due to the utilisation of multiple samples from the same participant, unilateral training induces distinct physiological adaptations specific to the trained leg (MacInnis et al., 2017b; Thomas et al., 2022) and the unilateral model has been used numerous times to measure training

Figueiredo
Figueiredo et al. (2015) demonstrated an increase in resting ribosome-related gene expression alongside muscle hypertrophy following 8 weeks of resistance training, similar studies show either no change or a decrease in markers of ribosome-related gene expression following 8 and 12 weeks of resistance training(Fyfe et al., 2018;Mobley, Haun, et al., 2018) likely due to higher training volumes and/or training periods(Hammarström et al., 2020).Therefore, because our resistance training intervention lasted 10 weeks we were not able to capture the earlier increase in ribosome content, which may have occurred, to support muscle hypertrophy.Delayed increases in translational machinery support the notion that as muscle hypertrophy occurs, ribosome content increases initially, but over time, the translational machinery becomes more refined and allows for a decrease in ribosome content while likely increasing translational efficiency.Indeed, 20 weeks of resistance training revealed downregulated ribosomal gene abundance in those with the greatest increase in lean mass and greater expression in those with the least change in lean mass(Phillips et al., 2013).It is therefore plausible that extending the training programme for those who increased ribosome content (e.g.lower responders) would eventually decrease ribosome content.Our findings are consistent with the hypotheses presented byMcGlory et al. (2017) andJoanisse et al. (2020) and likely also offer a refinement mechanism translational capacity are acute and transient in response to a novel stimulus.No changes in ribosome-related gene expression were observed with aerobic conditioning or resistance training, which may be a testament to the timeframe and lack of acute sampling time points.Lower baseline ribosome-related gene expression and a smaller change in ribosome content are associated with the greatest increase in LLSTM following resistance training.Although it was not directly measured in the current study, we propose, in agreement with previous hypotheses(Joanisse et al., 2020;McGlory et al., 2017) that translational capacity increases acutely and temporarily in response to a novel stimulus and then decreases following exercise training as protein translation more closely parallels a refined transcriptional response.Therefore, this may suggest that translational efficiency rather than chronic increases in ribosomal content to expand capacity may be most important in dictating cellular adaptations to exercise training, and future work in this area is warranted.5.1 | Future directionsRecent work has identified rDNA gene copy number as a potential mechanism explaining inter-individual variability in ribosome-related gene expression(Figueiredo et al., 2021); however, future research should continue to measure how rDNA gene copy number influences ribosomal adaptation to acute and chronic exercise stimuli.Research in humans should also focus on measuring translational efficiency in addition to capacity and using better, more accurate measures of translational capacity (e.g.RNA synthesis) and efficiency (e.g.acute phosphorylation of ribosome-related protein abundance).More time points should be added in future studies measuring translational capacity to capture the initial increase in ribosome content(Hammarström et al., 2022) and subsequent decrease following the initial onset of exercise training.Finally, more research should explore how ribosome content is impacted by aerobic conditioning.AUTHOR CONTRIBUTIONS Alex Brown, Gianni Parise and Sophie Joanisse contributed to the conceptualisation and design of the study.Aaron C. Q. Thomas, Chris McGlory, Sophie Joanisse, Stuart M. Phillips and Dinesh Kumbhare collected tissue.Alex Brown and Sophie Joanisse performed experiments and analysed data.Alex Brown, Gianni Parise, Sean Y. Ng and Sophie Joanisse interpreted results.Alex Brown, Gianni Parise, Aaron C. Q. Thomas, Sean Y. Ng, Chris McGlory, Stuart M. Phillips, Dinesh Kumbhare and Sophie Joanisse revised the manuscript and approved the final, submitted version.