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Identification of a resistance-exercise-specific signalling pathway that drives skeletal muscle growth

Nature Metabolism volume 7, pages 1404–1423 (2025)Cite this article

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Abstract

Endurance and resistance exercise lead to distinct functional adaptations: the former increases aerobic capacity and the latter increases muscle mass. However, the signalling pathways that drive these adaptations are not well understood. Here we identify phosphorylation events that are differentially regulated by endurance and resistance exercise. Using a model of unilateral exercise in male participants and deep phosphoproteomic analyses, we find that a prolonged activation of a signalling pathway involving MKK3b/6, p38, MK2 and mTORC1 occurs specifically in response to resistance exercise. Follow-up studies in both male and female participants reveal that the resistance-exercise-induced activation of MKK3b is highly correlated with the induction of protein synthesis (R = 0.87). Additionally, we show that in mice, genetic activation of MKK3b is sufficient to induce signalling through p38, MK2 and mTORC1, along with an increase in protein synthesis and muscle fibre size. Overall, we identify core components of a signalling pathway that drives the growth-promoting effects of resistance exercise.

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Fig. 1: Overview of the phosphoproteomic alterations that occur after a bout of endurance versus resistance exercise in humans.
Fig. 2: Cluster-based identification of phosphorylation events that are specific to endurance and resistance exercise in humans.
Fig. 3: Identification of kinases that are reproducibly inferred as being regulated by endurance and/or resistance exercise in humans.
Fig. 4: Prediction and validation of a signalling pathway that is activated specifically by resistance exercise in humans.
Fig. 5: Changes in MKK3b(S218) phosphorylation are highly correlated with the resistance-exercise-induced increase in myofibrillar protein synthesis.
Fig. 6: Mouse models of endurance and resistance exercise lead to distinct adaptations.
Fig. 7: Mouse models affirm that prolonged activation of signalling through MKK3/4/6, p38, MK2 and mTORC1 occurs specifically in response to resistance exercise.
Fig. 8: Genetic activation of MKK3b and MKK6 is sufficient to induce resistance-exercise-specific signalling events, protein synthesis and growth.

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Data availability

All processed data are available in the article and supplementary materials. The RAW files for the proteomics and phosphoproteomics data are available on MassIVE (https://massive.ucsd.edu/ProteoSAFe/index.jsp) under identifier MSV000093793. All of the supplementary software can be found on GitHub https://github.com/wenyuanzhuuw/PhosphoMS.git. Source data are provided with this paper.

Code availability

The supplementary software can be found on GitHub https://github.com/wenyuanzhuuw/PhosphoMS.git. Supplementary Software 1 comprises the modified PhosR programming code in R language that was used to remove batch effects and generate the Post-PhosR dataset in Supplementary Table 3. Supplementary Software 2 comprises the modified PhosR programming code in R language that was used to remove batch effects and generate the Post-PhosR dataset in Supplementary Table 6. Supplementary Software 3 comprises the modified PhosR programming code in R language that was used to generate the substrate kinase scores shown in Supplementary Fig. 2. Supplementary Software 4 comprises the CellProfiler pipeline that was used to determine the capillary density in whole muscle cross-sections.

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Acknowledgements

The research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH) under Awards AR074932 and AR082816 to T.A.H., and the National Institute of General Medical Sciences of NIH under award P41GM108538 to J.J.C. Support to T.A.H. was also provided by the Novo Nordisk Bio Innovation Hub Green House programme, and we offer special thanks for scientific advice to the members of the Bio Innovation team, including S. B. Jørgensen, J. B. Roland, B. F. Hansen and M. K. Jensen. The work was also supported in part by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2020-06346) to S.M.P. Additionally, S.M.P. received support from the Canada Research Chairs programme (CRC-2021-00495) during this work. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Department of Comparative Biosciences, University of Wisconsin–Madison, Madison, WI, USA

    Wenyuan G. Zhu, Jamie E. Hibbert, Corey GK. Flynn, Ramy K. A. Sayed, Hector G. Paez, Marius Meinhold, Kent W. Jorgenson, Jae-Sung You, Nathaniel D. Steinert, Kuan-Hung Lin & Troy A. Hornberger

  2. School of Veterinary Medicine, University of Wisconsin–Madison, Madison, WI, USA

    Wenyuan G. Zhu, Jamie E. Hibbert, Corey GK. Flynn, Ramy K. A. Sayed, Hector G. Paez, Marius Meinhold, Kent W. Jorgenson, Jae-Sung You, Nathaniel D. Steinert, Kuan-Hung Lin & Troy A. Hornberger

  3. Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada

    Aaron C. Q. Thomas, Chris McGlory, Martin J. MacInnis & Stuart M. Phillips

  4. Department of Chemistry, University of Wisconsin–Madison, Madison, WI, USA

    Gary M. Wilson & Joshua J. Coon

  5. Department of Biomolecular Chemistry, University of Wisconsin–Madison, Madison, WI, USA

    Gary M. Wilson & Joshua J. Coon

  6. National Center for Quantitative Biology of Complex Systems, University of Wisconsin–Madison, Madison, WI, USA

    Gary M. Wilson & Joshua J. Coon

  7. Morgridge Institute for Research, University of Wisconsin–Madison, Madison, WI, USA

    Gary M. Wilson & Joshua J. Coon

  8. Department of Medicine, Queen’s University, Kingston, Ontario, Canada

    Chris McGlory

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Contributions

W.G.Z., A.C.Q.T., G.M.W., C.M., K.W.J., S.M.P. and T.A.H. conceived and designed the experiments. W.G.Z., A.C.Q.T., G.M.W., J.E.H., C.M., K.W.J., N.D.S., K.-H.L., M.J.M., R.K.A.S., J.-S.Y. and T.A.H. performed the experiments. W.G.Z., A.C.Q.T., C.M., H.G.P., M.M., J.S.Y. and T.A.H. analysed the data. W.G.Z., A.C.Q.T., G.M.W., C.G.F., K.W.J., J.J.C., S.M.P. and T.A.H. contributed materials and/or analysis tools. W.G.Z., A.CQ.T., G.M.W. and T.A.H. wrote the paper.

Corresponding author

Correspondence to Troy A. Hornberger.

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Competing interests

T.A.H. received a research grant from Novo Nordisk. This could be perceived as a potential conflict of interest; however, Novo Nordisk and T.A.H. do not have any agreements that could lead to a financial gain or loss from this publication.

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Nature Metabolism thanks Sue Bodine, Mark Febbraio and Benjamin Parker for their contribution to the peer review of this article. Primary Handling Editors: Jean Nakhle and Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 GO term enrichment in phosphopeptide clusters 1 and 2.

1D enrichment analysis of gene ontology (GO) terms when using the membership score of the phosphopeptides for ‘cluster 1’ (a), or ‘cluster 2’ (b) as defined in Fig. 2. All GO terms were assigned a rank-based score between -1 and 1, with a negative score indicating under-representation of the term and a positive score indicating over-representation. Redundant GO terms were then removed with REVIGO. In the graphs, the score for each term was plotted against the –Log10 of its respective q-value. The displayed dots indicate GO terms with a q-value of < 0.05. GO terms of interest are highlighted in each graph, and a full list of the outcomes is provided in the Supplementary Table 4.

Extended Data Fig. 2 Reproducibly of the endurance and resistance exercise-induced changes in the phosphorylation of MAPKAPK substrates.

Heatmaps of the mean exercise-induced change in the phosphorylation of the known and/or predicted substrates of the MAPKAPK’s that were identified in both the current study and the phosphopeptide dataset of Blazev et al.10. Also shown is the number of participants (n) that the mean values were obtained from in each dataset.

Extended Data Fig. 3 Quantitative results from the western blots in Fig. 4.

Biopsies from the experimental interventions described in Fig. 4c were subjected to western blot analysis as shown in Fig. 4d. a-k, For each participant, the phospho to total protein ratio (P/T) for the indicated signaling event in each biopsy was determined and then expressed relative to the mean value observed in the pre-exercise biopsies. Values in the graphs are presented as the group mean ± SEM, the number of samples per group is indicated at the bottom of the bars in the graphs. The data was analyzed with one-way mixed ANOVA. The q-value for each statistically significant pairwise comparison is annotated in the graphs with a * being used when P < 0.0001.

Source data

Extended Data Fig. 4 Long-term adaptations in the mouse model of endurance exercise.

Mice were subjected to 13 weeks of training with treadmill running (TR) or a mock (control) paradigm. The average weekly (a) body weight, and (b) workload per training session, as well as (c) the number of times the rear of the animal was touched during each training session. Individual data points are displayed with hollow symbols and the weekly means for each group are displayed with solid symbols. n = 10 per group for a-c. d-r, After 13 weeks of training, the mice were subjected to measurements of (d) grip strength, and (e) tibia length (TL). The mass of the (f) individual epididymal (Epi.) fat pads, (g) interscapular brown adipose tissue (iBAT), (h) adrenal glands, and (i) heart were measured and normalized to TL. j, The mass of individual muscles (MM) including the gastrocnemius (GAST), plantaris (PLT), soleus (SOL), flexor digitorum longus (FDL), pectoralis major (PEC), triceps brachii lateral head (Tri-Lat), triceps brachii long head (Tri-Long), and the forearm flexor complex (FF) were all normalized to TL and expressed relative to the mean value observed in the control group. k, Mid-belly cross-sections of the FDL muscles were subjected to immunohistochemistry (IHC) for laminin and fiber type identification (that is, Type I, IIA, IIX, or IIB), scale bars = 500 µm. The entire cross-section was used to determine (l), the average cross-sectional area (CSA) of the different fiber types, and (m) the proportion of the fibers that were represented by each fiber type. n, Mid-belly cross-sections of the FDL muscles were subjected to IHC for laminin and CD31 to identify capillaries, scale bars = 25 µm. o, The entire cross-section was used to determine the average number of capillaries per fiber. p-r, FDL muscles were subjected to western blot analysis for (q) members of the five OXPHOS complexes (that is, CI–CV), and (r) other mitochondrial (mito.) proteins. For each sample, the individual protein content was normalized to the total amount of protein loaded on the gel and then expressed relative to the mean of the control group. Values in the graphs are presented as the group mean ± SEM, for d-r the number of samples per group is indicated at the bottom of the bars in the graphs. The data were analyzed with two-way repeated measures (RM) ANOVA (a), one-way RM ANOVA (b,c), paired t-tests (d-i, and o), or two-way ANOVA (j, l, m, q, r). ■ Significantly different from week 1, P < 0.05. The specific P-values for all other statistically significant pairwise comparisons are annotated in the graphs.

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Extended Data Fig. 5 Long-term adaptations in the mouse model of resistance exercise.

Flexor digitorum longus (FDL) muscles were collected from mice that had completed 13 weeks of training with weighted pulling (WP) or an unweighted (control) paradigm as previously reported by Zhu et al.34. a, Mid-belly cross-sections of the FDL muscles were subjected to immunohistochemistry for laminin and CD31 to identify capillaries, scale bars = 25 µm. b, The entire cross-section was used to determine the average number of capillaries per fiber. c-e, FDL muscles were subjected to western blot analysis for (d) members of the five OXPHOS complexes (that is, CI–CV), and (e) other mitochondrial (mito.) proteins. For each sample, the individual protein content was normalized to the total amount of protein loaded on the gel and then expressed relative to the mean of the control group. Values in the graphs are presented as the group mean ± SEM, the number of samples per group is indicated at the bottom of the bars in the graphs. The data were analyzed with two-sided paired t-tests (b), or two-way ANOVA (d, e).

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Extended Data Fig. 6 A rapid and robust activation of signaling through MKK3/4/6, p38, and MK2 occurs specifically in response to resistance exercise in mice.

a, Schematic of how C57BL6 mice were subjected to endurance exercise with treadmill running (TR), resistance exercise with weight pulling (WP), or their respective mock-trained (control) conditions. b, FDL muscles from the mice were collected immediately after the last training bout and subjected to western blot analysis for the phospho (P) and total (T) levels of the indicated proteins. Long isoform of MK2 (L), short isoform of MK2 (S). c, For each sample, the phospho to total protein ratio (P/T) for each signaling event was determined and expressed relative to the mean value observed in the treadmill control group. Values in the graphs are presented as the group mean ± SEM, the number of samples per group is indicated at the bottom of the bars in the graphs. The data were analyzed with two-way ANOVA or a Student’s t-test. ■ Significant difference between the TR control and TR trained group when the planned comparison was analyzed with a Student’s t-test, P < 0.05. The P-value for each statistically significant pairwise comparison is annotated in the graphs with a * being used when P < 0.0001.

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Extended Data Fig. 7 Quantitative results from the western blots in Fig. 7.

FDL muscles from the experimental conditions described in Fig. 7a were subjected to western blot analysis as shown in Fig. 7e. a-k, For each sample, the phospho to total protein ratio (P/T) for each signaling event was determined and then expressed relative to the mean value observed in the treadmill (TR) control group. Values in the graphs are presented as the group mean ± SEM, the number of samples per group is indicated at the bottom of the bars in the graphs. The data were analyzed with two-way ANOVA. The P-value for each statistically significant pairwise comparison is annotated in the graphs with a * being used when P < 0.0001.

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Extended Data Fig. 8 Quantitative results from the western blots in Fig. 8.

TA muscles were subjected to western blot analysis as described in Fig. 8b. a-i, For each sample, the total (T) protein level, or the phospho to total protein ratio (P/T) for the indicated signaling event, was determined and then expressed relative to the mean value observed in the LacZ control group. Values in the graphs are presented as the group mean ± SEM, n = 4 per group unless otherwise indicated at the bottom of the bars in the graphs. The data were analyzed with one-way ANOVA. The P-value for each statistically significant pairwise comparison is annotated in the graphs with a * being used when P < 0.0001.

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Extended Data Fig. 9 Genetic activation of MKK3b induces hypertrophy through a mechanism that is only partially dependent on mTORC1.

a, Schematic describing how electroporation was used to transfect mouse tibialis anterior (TA) muscles. Specifically, the TA muscles of male and female C57BL6 mice were co-transfected with plasmid DNA encoding tdTomato and LacZ as a control condition, Rheb as a direct activator of mTORC1, or with constitutively active (c.a.) mutant of MKK3b. Following electroporation, the mice were given daily intraperitoneal (IP) injections of the drug rapamycin (1.5 mg/kg) to inhibit signaling through mTORC1 or the solvent vehicle as a control. b, At 7 days post-transfection, the muscles were collected and subjected to immunohistochemistry for laminin to identify the periphery of the transfected (tdTomato positive) vs. non-transfected (tdTomato negative) fibers, scale bars = 50 µm. c, The cross-sectional area (CSA) of randomly selected fibers were measured, and then the values in the transfected fibers were expressed relative to the mean of the values observed in the non-transfected (control) fibers within each sample (n = 60–155 transfected and non-transfected fibers per sample). Values in the graphs are presented as the group mean ± SEM, the number of samples per group is indicated at the bottom of the bars in the graphs (605–889 fibers per group). The data were analyzed with two-way ANOVA. The P-value for each statistically significant pairwise comparison is annotated in the graphs with a * being used when P < 0.0001. Panel a created with BioRender.com.

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Extended Data Fig. 10 The genetic activation of MKK3b prevents immobilization-induced atrophy.

a, Illustration of how electroporation was used to co-transfect the left and right tibialis anterior (TA) muscles of male and female C57BL6 mice with plasmid DNA encoding tdTomato and constitutively active (c.a.) MKK3b or LacZ as a control. Immediately following electroporation, the right hindlimb was subjected to immobilization while the left hindlimb was untouched and used for the control condition. b, At 7 days post electroporation, the TA muscles were collected and cross-sections were subjected to immunohistochemistry for laminin to identify the periphery of the transfected (tdTomato positive) and non-transfected (tdTomato negative) fibers, scale bars = 100 µm. The mean cross-sectional area (CSA) of the transfected and non-transfected fibers in each sample was determined from n = 58–130 transfected and non-transfected fibers per sample, and the resulting values for each sample were expressed relative to the mean value obtained in the sex-matched control group (that is, the tdTomato negative fibers from vehicle-treated muscles that were co-transfected with LacZ). Values in the graphs are presented as the group mean ± SEM, the number of samples per group is indicated at the bottom of the bars in the graphs (669–1269 fibers per group). The data were analyzed with two-way RM ANOVA. The P-value for each statistically significant pairwise comparison is annotated in the graphs with a * being used when P < 0.0001. Panel a created with BioRender.com.

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Supplementary information

Supplementary Information

Final approved protocol for the human trials, Supplementary Figs. 1–4 and related uncropped western blot images

Reporting Summary

Supplementary Tables 1–11

Supplementary Tables 1–11

Supplementary Software 1

The modified PhosR programming code in R language that was used to remove batch effects and generate the Supplementary Table 3 Post-PhosR dataset.

Supplementary Software 2

The modified PhosR programming code in R language that was used to remove batch effects and generate the Supplementary Table 6 Post-PhosR dataset.

Supplementary Software 3

The modified PhosR programming code in R language that was used to generate the substrate kinase scores shown in Extended Figure 2.

Supplementary Software 4

The CellProfiler pipeline that was used to determine the capillary density in whole muscle cross-sections.

Supplementary Data 1

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Supplementary Data 2

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Supplementary Data 3

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Zhu, W.G., Thomas, A.C.Q., Wilson, G.M. et al. Identification of a resistance-exercise-specific signalling pathway that drives skeletal muscle growth. Nat Metab 7, 1404–1423 (2025). https://doi.org/10.1038/s42255-025-01298-7

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