Previous investigations on molecular responses and adaptations in skeletal muscle to concurrent training have focused almost exclusively on markers of post-exercise translational efficiency (i.e., mTORC1 signalling and rates of MPS)9,10,11,12,13,14,15,16,17,18. For the first time, we present data on the regulation of translational capacity (i.e., ribosome biogenesis) in skeletal muscle with concurrent training compared with resistance training performed alone. The major findings were that training-induced changes in markers of ribosome biogenesis, including total RNA content and expression of some mature rRNA species (i.e., 5.8S and 28S, but not 18S) were more favourable following concurrent training compared with resistance training alone, and irrespective of the endurance training intensity employed. These responses occurred despite a single bout of resistance exercise, when performed post-training, further inducing both mTORC1- and ribosome biogenesis-related signalling (i.e., TIF-1 and UBF phosphorylation) compared with concurrent exercise. These observations also contrasted with our findings regarding changes in muscle fibre-type specific hypertrophy, which was greater in type I muscle fibres for the resistance training group, suggesting a disconnect between training-induced changes in markers of ribosome biogenesis and muscle fibre hypertrophy.

To investigate the effects of concurrent versus single-mode resistance training on markers of skeletal muscle ribosome biogenesis, we measured training-induced changes in total RNA content and basal expression of mature ribosome species 5.8S, 18S, and 28S, as well as early post-exercise changes in mature rRNA expression. Contrary to our hypothesis, resistance training alone induced small decreases in the levels of both the 5.8S and 28S rRNAs in the basal state post-training, while the training-induced change in both of these mature rRNA species was greater with concurrent exercise compared with resistance training alone. Neither training protocol induced any changes in 18S rRNA expression. Previous work in humans has observed basal increases in 5.8S, 18S, and 28S rRNA expression in human skeletal muscle after 8 weeks of resistance training, all of which were reduced 1 h following a single session of resistance exercise performed post-training34. The present data contrast with these findings by suggesting that resistance training performed alone was an insufficient stimulus to increase mature rRNA content, whereas concurrent exercise was sufficient to increase mature 5.8S and 28S expression after a single post-training exercise bout.

Consistent with the training-induced changes in both 5.8S and 28S rRNA expression with resistance training performed alone, a small reduction in basal total RNA content in skeletal muscle was observed within this cohort. Despite this paradoxical finding, it is interesting to note total RNA content was higher at pre-training for the RT group compared with both the HIT+RT and MICT groups (1.6- and 1.3-fold, respectively). The reason for this between-group discrepancy at baseline is not immediately clear, given we previously showed no differences in baseline lean mass measured via DXA or lower-body 1-RM strength in these participants44, suggesting other factors may have influenced the between-group differences in baseline skeletal muscle RNA content. It is also possible that the training program provided an insufficient stimulus to at least maintain this elevated basal RNA content for the RT group. Studies demonstrating robust increases in total RNA content concomitantly with rodent skeletal muscle hypertrophy typically employ supraphysiological methods for inducing muscle hypertrophy, such as synergist ablation36,39,46,47, a stimulus that is clearly not replicated by resistance training in human models. Participant training status may also impact upon training-induced changes in ribosome biogenesis in humans. The participants in the present study were actively engaging in resistance and/or endurance training for at least 1 year prior to commencing the study, suggesting a higher training status compared with those of Figueiredo et al.34 who were likely untrained (although this was not made explicitly clear) and asked to refrain from resistance training for 3 weeks prior to the study34. It is also possible that between-group differences in training volume, which was clearly higher for the concurrent training groups compared with the RT group, may have impacted upon the training-induced changes in total skeletal muscle RNA content.

Despite the observed changes in skeletal muscle RNA content, resistance training alone was sufficient to increase type I, but not type II, muscle fibre CSA. The lack of any substantial type II fibre hypertrophy is likely due, at least in part, to the specific nature of the resistance training program employed, which was perhaps better-oriented for enhancing maximal strength rather than lean mass44. Indeed, previously-published data indicates that the resistance training protocol employed in the present study was effective in improving maximal strength and measures of lean mass44, although these changes did not transfer to detectable type II fibre hypertrophy. Nevertheless, in agreement with previous research2,4, the training-induced increase in type I muscle fibre CSA was attenuated with concurrent exercise, albeit only when incorporating HIT, compared with resistance training performed alone. Despite these between-group differences in fibre-type specific hypertrophy, we could find no evidence that the training-induced changes in lean mass or muscle fibre CSA were correlated with changes in total RNA content of skeletal muscle (data not shown). The apparent disconnect between training-induced changes in total RNA content and markers of muscle hypertrophy, both at the whole-body and muscle-fibre levels, suggests further investigation is required into relationship between changes in translational capacity and resistance training-induced hypertrophy in human skeletal muscle, particularly in the context of concurrent training.

To circumvent the potentially confounding influence of training status on the mode-specificity of post-exercise molecular responses in skeletal muscle21,22, we investigated potential interference to mTORC1 signalling following exercise protocols that participants were accustomed to via eight weeks of prior training. In contrast to previous studies in untrained or relatively training-unaccustomed participants14,16,17,18, we observed enhanced mTORC1 signalling after resistance training compared with concurrent exercise, including greater mTOR and p70S6K1 phosphorylation at 1 h post-exercise, and rps6 phosphorylation at 3 h post-exercise. These observations contrast with previous data, including our own20, showing no differences in mTORC1 signalling responses to single bouts of resistance exercise, performed alone or after a bout of continuous endurance exercise13. It has been suggested that any tendency for mTORC1 signalling responses (e.g., p70S6KThr389 phosphorylation) to be further enhanced by concurrent exercise (relative to resistance exercise alone) before training, as shown in a previous study14, were lessened when exercise was performed in a training-accustomed state13. Taken together, these data lend support to the notion the molecular signals initiated by exercise in skeletal muscle become more mode-specific with repeated training, and increases in post-exercise mTORC1 signalling with concurrent exercise may be attenuated when performed in a training-accustomed state.

While the observed mTORC1 signalling responses were consistent with the paradigm of enhanced mode-specificity of molecular responses with repeated training, the finding of greater AMPK phosphorylation following resistance exercise compared with concurrent exercise was unexpected, given the energy-sensing nature of AMPK signalling and its role in promoting an oxidative skeletal muscle phenotype48. This observation may suggest an adaptive response whereby endurance training rendered subjects in the concurrent training groups less susceptible to exercise-induced metabolic perturbation in skeletal muscle, manifesting in an attenuated post-exercise AMPK phosphorylation response. A similar phenomenon has been observed in human skeletal muscle after only 10 days of endurance training, whereby post-exercise increases in AMPK activity following a single pre-training exercise bout are attenuated compared with the same exercise bout performed before training49. It should also be acknowledged that while AMPK Thr172 phosphorylation alone does not necessarily reflect changes in AMPK activity per se, ACC Ser79 phosphorylation is generally accepted as a marker for AMPK activity50,51. Since we observed greater increases in ACC Ser79 phosphorylation with concurrent exercise versus resistance exercise alone during the post-training exercise trial, this may instead reflect further increases in AMPK activity in response to concurrent exercise. Nevertheless, the present data suggest further work is required to define the mode-specificity of AMPK signalling in skeletal muscle and the effect of repeated training on these responses.

In addition to mediating transient changes in translational efficiency, accumulating evidence suggests mTORC1 also plays a key role in regulating ribosome biogenesis (and therefore translational capacity) in skeletal muscle by regulating all three classes of RNA polymerases (RNA Pol-I to -III)25. In agreement with mTORC1 signalling responses, the phosphorylation of upstream regulators of RNA Pol-I-mediated rDNA transcription, including UBF and TIF-1A, was increased more by resistance exercise alone than when combined with endurance exercise in the form of either HIT or MICT. Previous work has demonstrated single sessions of resistance exercise to induce robust increases in TIF-1A Ser649 phosphorylation and UBF protein content in human skeletal muscle at 1 h post-exercise, both in untrained and trained states34. Moreover, whereas a single session of resistance exercise did not influence UBF Ser388 phosphorylation, this response was elevated in the basal state post-training34. The present data add to the growing body of evidence that resistance exercise is a potent stimulus for increasing the phosphorylation of regulators of Pol-I-mediated rDNA transcription, and suggest these early signalling responses may be similarly attenuated when resistance exercise is combined with endurance exercise. These responses also indicate an apparent disconnect between the upstream signalling responses in the post-training exercise trial related to 45S pre-rRNA transcription (i.e., TIF-1A and UBF phosphorylation), and the basal training-induced changes in markers of ribosomal content (i.e., total RNA and expression of mature rRNA species). While these responses appear paradoxical, they may suggest that although short-term concurrent training may optimise ribosome biogenesis adaptation versus resistance training performed alone, ribosome biogenesis may instead be further enhanced by longer-term resistance training performed alone. This notion aligns with recent discussion regarding the progression of adaptation with concurrent versus single-mode training, suggesting early adaptation to combined resistance and endurance training may initially be complimentary, whereas longer-term training exacerbates interference to hallmark resistance training adaptations52. Clearly, longer-term training studies are likely required to fully elucidate the effect of concurrent training versus resistance training alone on ribosome biogenesis adaptation in skeletal muscle.

Despite the present findings regarding signalling responses upstream of 45S pre-rRNA transcription, the expression of 45S pre-RNA, but not mature ribosome species, was increased only after concurrent exercise during the post-training exercise trial. Previous work in humans has reported basal increases in 45S pre-rRNA after 8 weeks of resistance training34, and 4 h after a single session of resistance exercise performed in both untrained and trained states33. Notably, post-exercise expression of 45S pre-rRNA was less pronounced in the trained compared with untrained state33. While no substantial basal changes in 45S pre-rRNA expression were observed in the present study, the change in 45S pre-rRNA levels between pre- and post-training was greater for both concurrent training groups compared with RT performed alone. Concurrent exercise also increased 45S pre-rRNA levels at 3 h post-exercise, with little effect of single-mode resistance exercise. These observations may be explained by the muscle sampling time points employed in the present study. Increased post-exercise 45S pre-rRNA levels have been previously shown 4 h after resistance exercise33, whereas a reduction in 45S rRNA levels has been demonstrated 1 h post- resistance exercise in trained, but not untrained, states34. The possibility therefore exists that resistance exercise may increase 45S rRNA expression at a later timepoint post-exercise, and the sampling time points employed herein were not extensive enough to measure any exercise-induced increases in 45S pre-rRNA expression.

The regulation of several Pol-I associated proteins was also measured at the transcriptional level, including TIF-1A, POLR1B, UBF, and cyclin D1. Concurrent exercise, irrespective of endurance training intensity, was sufficient to increase POLR1B mRNA expression at 3 h post-exercise, but only MICT+RT and RT alone increased TIF-IA mRNA content at this timepoint. Previous work in human skeletal muscle has demonstrated no effect of a single session of resistance exercise performed in either untrained or trained states on the mRNA expression of either TIF-1A or POLR1B at either 1 h34 or 4 h33 post-exercise. Eight weeks of resistance training has previously been shown to increase basal UBF mRNA expression, which was reduced 1 h following a single session of resistance exercise performed post-training34. Although we observed no basal training-induced increases in UBF mRNA expression for any training group, a similar reduction in UBF mRNA content was noted 3 h post-exercise for the RT group. Increased cyclin D1 mRNA was also seen at rest post-training for the HIT+RT group, which was maintained at 3 h post-exercise. Figueiredo et al.34 have shown eight weeks of resistance training decreased post-training levels of cyclin D1 mRNA compared with pre-training, with a small increase induced at 1 h post-exercise by a single session of post-training resistance exercise. It therefore appears HIT is a more potent stimulus for increasing levels of cyclin D1 mRNA compared with resistance exercise alone or MICT, although an acute reduction in cyclin D1 protein levels was also seen 1 h following a single bout of HIT+RT. Previous work has shown increases in cyclin D1 mRNA during long-term (3 months) resistance training53, which may suggest an increase in satellite cell activation and proliferation during the training intervention53,54, although direct measures of these markers were not made in the present study.

The rRNA primers used in the present study were specifically designed to differentiate between mature rRNA expression and the expression of these sequences when still bound to the polycistrionic 45S rRNA precursor (i.e., 5.8S, 18S and 28S [span] rRNA)34. Previous work using these primer sequences has shown basal training-induced increases in mature rRNA expression did not occur concomitantly with increased expression of rRNA transcripts still bound to the 45S precursor (i.e., 5.8S, 18S and 28S [span]), suggesting a training-induced increase in mature rRNA content, rather than increased 45S precursor expression34. In contrast, we observed simultaneous post-exercise increases in the expression of both mature rRNA transcripts and those still bound to the 45S precursor (i.e., ‘span’ rRNA transcripts). It is therefore possible our observed changes in these markers may be reflective solely of changes in 45S pre-rRNA content, and not the mature forms of these rRNAs. However, it is also possible this may relate to the post-exercise time course examined in the present study. In support of this notion, it was shown that a single session of resistance exercise was sufficient to increase only the expression of rRNA transcripts still bound to the 45S pre-rRNA, and not mature rRNA species, even after 48 h of post-exercise recovery55. It is therefore plausible that the post-exercise time courses examined in the present study were not extensive enough to measure early post-exercise changes in mature rRNA expression. Clearly, further work is required to investigate the time course of rRNA regulation with training in human skeletal muscle.

Although we have investigated various upstream regulators of 45S pre-rRNA transcription, it is possible other factors may have been differentially regulated by concurrent versus single-mode resistance training and may have contributed to the observed changes in ribosome biogenesis markers. For example, CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase) is directly phosphorylated by p70S6K1 and controls the first three steps in de novo pyrimidine synthesis56, a necessary process for accommodating the increased demand for RNA and DNA synthesis to support cellular growth. To our knowledge, the regulation of CAD has, however, not been investigated in the context of training-induced skeletal muscle hypertrophy in humans. Future studies should also consider the potential role of CAD in the regulation of skeletal muscle growth in response to resistance and/or concurrent training.