The Hornberger Lab

Research

Defining the role of mTOR in the mechanical regulation of skeletal muscle mass

It is well recognized that mechanical stimuli play a major role in the regulation of skeletal muscle mass, and the maintenance of muscle mass contributes significantly to disease prevention and quality of life. Although the link between mechanical signals and the regulation of muscle mass has been recognized for decades, the molecular mechanisms that drive this process remain poorly defined. Intrigued by this gap in knowledge, we tried to address it by focusing on the mTOR signaling pathway. Our initial work indicated that signaling through mTOR is inhibited during periods of decreased mechanical loading (1) and activated during periods of increased mechanical loading (2). By using a pharmacological inhibitor of mTOR (rapamycin), we also obtained evidence which suggested that signaling through mTOR is necessary for the increase in myofiber cross-sectional area (i.e., radial growth) that occurs in response to chronic mechanical overload (2). However, like all pharmacological inhibitors, it remained possible that the effects of rapamycin were mediated through a non-specific / mTOR-independent mechanism. Several years later, we were able to address this by employing mice that possessed skeletal muscle-specific expression of a rapamycin-resistant mutant of mTOR (3). With these mice, we were able to confirm that mTOR is the rapamycin-sensitive element that confers the radial growth of myofibers and that the kinase activity of mTOR is necessary for this event.

Having established the above point, we next sought to determine whether the activation of mTOR is sufficient to induce the radial growth of myofibers. To accomplish this, we employed an in-vivo transient transfection method which enabled us to overexpress Rheb (a direct activator of mTOR signaling) in mouse skeletal muscles (Figure 1). Our results demonstrated that the overexpression of Rheb induced a robust radial growth response and that this effect was completely inhibited when mice were treated with rapamycin (4). Moreover, by using the transgenic mice that express the rapamycin-resistant mutants of mTOR, we were able to confirm that the radial growth-promoting effect of Rheb was mediated by mTOR and that the kinase activity of mTOR was necessary for this event (4). When combined, our research in this area has helped to establish two fundamentally important points: 1) signaling through mTOR is necessary for the radial growth of myofibers that occurs in response to chronic mechanical overload, and 2) the activation of mTOR is sufficient to induce the radial growth of myofibers.

Identifying the mechanisms through which mechanical stimuli activate mTOR
Over twenty years ago it became apparent that the activation of mTOR might play a key role in the pathway through which mechanical stimuli regulate muscle mass. Therefore, one of the major aims of our research has been focused on defining the mechanisms through which mechanical stimuli activate mTOR. When we initiated this research, the most widely accepted paradigm in the field suggested that mechanical stimuli induce the release of growth factors (e.g., IGF1) which, in turn, would promote the activation of mTOR via the canonical PI3K-dependent pathway. However, unexpectedly, our work revealed that mechanical stimuli activate mTOR through a unique PI3K-independent mechanism that is distinct from the core pathways employed by growth factors and nutrients (5, 6). Moreover, we obtained evidence which indicated that this PI3K-independent mechanism did not involve traditional signaling molecules such as iCa2+, ERK, PKA, PKC, or PLC (7). Instead, our work suggested that phosphatidic acid (PA) might be a critical component of the pathway (7). Indeed, our lab has provided further support for this conclusion by demonstrating that mechanical stimuli promote an increase in the levels of PA through a mechanism that requires the PA synthesizing enzyme called diacylglycerol kinase zeta (DGKζ) (8). Our work has also demonstrated that the magnitude of the mechanical activation of mTOR signaling is significantly reduced (⁓50%) in muscles that lack DGKζ (8, 9). This was an important observation because it not only indicated that DGKζ / PA plays an important role in the pathway through which mechanical stimuli activate mTOR, but it also suggested that additional regulatory mechanisms are involved. Inspired by this possibility, we sought to identify the location of mTOR in skeletal muscle, and our results indicated that mTOR is highly enriched on late endosomes / lysosomes (LEL) (10). This was an exciting observation because recent studies had suggested that the LEL is also enriched with the two direct regulators of mTOR (i.e., PA and Rheb). Importantly, the ability of Rheb to activate mTOR is inhibited by a protein called tuberin (TSC2), and we discovered that, under resting conditions, the LEL is enriched with TSC2, and that the association of TSC2 with the LEL is almost completely abolished in muscles that have been subjected to mechanical stimulation (10). Furthermore, through the generation of tamoxifen-inducible and skeletal muscle-specific knockout mice, we have shown that TSC2 and Rheb significantly contribute (⁓50%) to the pathway via which mechanical stimuli activate mTOR signaling (11). Thus, when combined, our work has helped to establish a new paradigm: mechanical stimuli activate mTOR signaling via a unique PI3K-independent mechanism that involves both a DGKζ-dependent increase in PA and a TSC2-dependent increase in the amount of active Rheb, at the LEL.

The development and use of innovative technologies
Our lab has always strived to develop and employ innovative techniques. For instance, it is well known that changes in skeletal muscle mass are regulated by alterations in the rate of protein synthesis and/or degradation. Hence, measurements of protein synthesis and degradation serve as key readouts for studies that are aimed at defining the mechanisms that regulate muscle mass. Traditionally, rates of protein synthesis have been measured with radioactively labeled amino acids. However, this approach is expensive, requires highly specialized equipment, is labor-intensive, and it can introduce numerous safety concerns. Thus, about a decade ago, we started to examine alternative (non-radioactive) approaches that could potentially be used to measure protein synthesis. During this period, we had immediate success with an immunological-based approach that measures the incorporation of puromycin into actively translated peptides (a.k.a., the SUnSET technique). Following numerous validation experiments, we demonstrated that our simple approach could be used to accurately visualize and quantify short-term rates of protein synthesis in vivo (12) (Figure 2). Furthermore, with the ability to visualize the rate of protein synthesis, our technique enabled us to perform various types of measurements that were not previously possible to obtain (20). Our technique was a particularly important advancement for the field because it essentially made cost-effective measurements of protein synthesis accessible to anyone that can run a western blot or perform immunohistochemistry.

Over the last few years, our lab has continued to push the technological boundaries of the field. For instance, we recently proposed that mechanically induced increases in muscle mass can be mediated by a combination of two distinct growth regulatory processes: i) the radial growth of myofibers which is driven by a mTORC1-dependent mechanism that we have coined as “the myofibril expansion cycle” (Figure 3), and ii) the longitudinal growth of myofibers which is driven by a mTORC1-independent mechanism that involves transverse Z-line splitting of sarcomeres at regions called sphenodes (Figure 4) (13).

In order to test our hypotheses we needed to be able to visualize where newly synthesized proteins (NSPs) accumulate at the sub-myofibril level. Initially, one might think that we could have used our SUnSET technique to accomplish this: however, the SUnSET technique labels truncated peptides, and such peptides would not be expected to possess the appropriate localization or half-life characteristics of their respective full-length proteins. Thus, to overcome this, we developed a technique that relies on the principles of bioorthogonal non-canonical amino acid tagging (BONCAT) with azidonorleucine (ANL) (14-16). Specifically, we crossed floxed STOP GFP-2A-MetRSL274G mice with mice that express CMV-Cre (15, 17). The presence of CMV-Cre resulted in mice with germline cells that no longer contained the STOP cassette in front of the GFP-2A-MetRSL274G and, in turn, enabled us to generate “MetRS” mice which ubiquitously co-express GFP and MetRSL274G in the absence of Cre (Figure 5A-B). Of note, the MetRSL274G allows ANL (a methionine analog) to be incorporated into NSPs and this is a critical point because ANL (i.e., the non-canonical amino acid) contains a reactive azide that, via a bioorthogonal azide-alkyne click reaction, can be covalently bound to effectively any tag of interest (15, 18). For instance, we have used alkyne-Alexa Fluor 594 to demonstrate that ANL-labeled NSPs can be detected with an exceptional signal-to-noise ratio in whole muscle cross-sections (Figure 5C). We have also subjected plantaris muscles to chronic mechanical overload and demonstrated that our approach could be used to visualize and quantify the MTE-induced accumulation of NSPs in whole muscle cross-sections (Figure 5D).

Having established the ability to visualize the accumulation of NSPs, we then needed to develop a rapid and cost-effective method for obtaining high-resolution sub-myofibril level images of skeletal muscle. To accomplish this, we took advantage of the rapid acquisition abilities of a wide-field fluorescence microscope and combined it with a computational approach called deconvolution. We refer to our collective methodology as FIM-ID (Fluorescence Imaging of Myofibrils with Image Deconvolution), and with the high-resolution images that it produced, we were able to demonstrate that the mechanically induced growth of skeletal muscle is mediated by both myofibril hypertrophy and the formation of new myofibrils (myofibrillogenesis).

The advent of FIM-ID and our BONCAT-based method of labelling NSPs have not only opened an entirely new area of research for our lab, but they have also enabled us to gain insight into the validity of the growth regulatory processes that we proposed. It’s important to emphasize that we are in the very early stages of these analyses and no definitive conclusions have been reached. However, as shown in Figure 6, we have already made some very provocative observations that are highly consistent with our hypotheses.

In addition to developing new methods, our lab has also consistently used state-of-the-art technologies to advance the field. For instance, in 2017 we published our first phosphoproteomics study in which we quantified nearly 6,000 different phosphorylation sites, and our analyses led to the identification of over 650 novel contraction-regulated phosphorylation events (20). After making refinements to our initial approach, we were able to quantify >10,000 phosphorylation sites per experiment, and we recently used our refined approach to: 1) identify the mechanically induced phosphorylation events that are regulated downstream vs. upstream / parallel to mTOR (21) and, 2) discover a signaling pathway that is activated specifically by resistance exercise and capable of inducing skeletal muscle growth (22). Notably, a technological breakthrough in our collaborator’s lab has now made it possible to quantify >35,000 phosphorylation sites per experiment and in the near future we will be using this approach to gain unprecedented insight into the phosphorylation events that are regulated by mechanical stimuli (23).

 

The development of animal models of resistance exercise
Many years ago, we needed to employ a rat model that could mimic a human progressive resistance exercise program. At that time, several candidate models had been described; but, in our opinion, all of them suffered from unacceptable limitations. Therefore, we developed a new model that allowed rats to engage in a physiologically relevant form of resistance exercise (ladder climbing), and with the model, the rate of progression through the training program could be tailored to each subject’s level of performance (24). Thus, with our new approach, it became possible to track each subject’s daily progression in strength as well as the concomitant increase in muscle mass that ultimately occurred. The development of this model was not only critical for the success of our own studies, but it is now being used by a variety of different labs (e.g., the model was used in nearly 200 different studies during the last 5 years).

The lessons learned while developing the rat model also helped us to develop a new mouse model of resistance exercise called weight pulling (25). Specifically, the weight pulling model utilizes a full-body/multi-joint exercise along with a training protocol that mimics a traditional human paradigm (three training sessions per week, ~8-12 repetitions per set, 2 min of rest between sets, approximately two maximal-intensity sets per session, last set taken to failure, and a progressive increase in loading that is based on the individual’s performance). Importantly, our work with the weight pulling model has revealed that, in numerous muscles throughout the body, it can induce the same type of long-term adaptations that occur in humans (e.g., radial growth of myofibers, myonuclear accretion, etc.), as well as the same type of acute responses that are thought to drive these long-term adaptations (e.g., activation of signaling through mTOR and the induction of protein synthesis) (22, 25) (click on the video below if you want to see a mouse do resistance exercise). Given how well the weight pulling model mimics human resistance exercise, it is our hope that this model will become widely adopted by the field and, in turn, increase the translational potential of the work that is being done in mouse-based studies.

 

  1. Hornberger, T. A., Hunter, R. B., Kandarian, S. C., and Esser, K. A. (2001) Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats. Am J Physiol Cell Physiol 281, C179-187
  2. Hornberger, T. A., McLoughlin, T. J., Leszczynski, J. K., Armstrong, D. D., Jameson, R. R., Bowen, P. E., Hwang, E. S., Hou, H., Moustafa, M. E., Carlson, B. A., Hatfield, D. L., Diamond, A. M., and Esser, K. A. (2003) Selenoprotein-deficient transgenic mice exhibit enhanced exercise-induced muscle growth. J Nutr 133, 3091-3097
  3. Goodman, C. A., Frey, J. W., Mabrey, D. M., Jacobs, B. L., Lincoln, H. C., You, J. S., and Hornberger, T. A. (2011) The role of skeletal muscle mTOR in the regulation of mechanical load-induced growth. J Physiol 589, 5485-5501
  4. Goodman, C. A., Miu, M. H., Frey, J. W., Mabrey, D. M., Lincoln, H. C., Ge, Y., Chen, J., and Hornberger, T. A. (2010) A phosphatidylinositol 3-kinase/protein kinase B-independent activation of mammalian target of rapamycin signaling is sufficient to induce skeletal muscle hypertrophy. Mol Biol Cell 21, 3258-3268
  5. Hornberger, T. A., Stuppard, R., Conley, K. E., Fedele, M. J., Fiorotto, M. L., Chin, E. R., and Esser, K. A. (2004) Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism. Biochem J 380, 795-804
  6. Hornberger, T. A., and Chien, S. (2006) Mechanical stimuli and nutrients regulate rapamycin-sensitive signaling through distinct mechanisms in skeletal muscle. J Cell Biochem 97, 1207-1216
  7. Hornberger, T. A., Chu, W. K., Mak, Y. W., Hsiung, J. W., Huang, S. A., and Chien, S. (2006) The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle. Proc Natl Acad Sci U S A 103, 4741-4746
  8. You, J. S., Lincoln, H. C., Kim, C. R., Frey, J. W., Goodman, C. A., Zhong, X. P., and Hornberger, T. A. (2014) The Role of Diacylglycerol Kinase zeta and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy. J Biol Chem 289, 1551-1563
  9. You, J. S., Dooley, M. S., Kim, C. R., Kim, E. J., Xu, W., Goodman, C. A., and Hornberger, T. A. (2018) A DGKzeta-FoxO-ubiquitin proteolytic axis controls fiber size during skeletal muscle remodeling. Science signaling 11
  10. Jacobs, B. L., You, J. S., Frey, J. W., Goodman, C. A., Gundermann, D. M., and Hornberger, T. A. (2013) Eccentric contractions increase the phosphorylation of tuberous sclerosis complex-2 (TSC2) and alter the targeting of TSC2 and the mechanistic target of rapamycin to the lysosome. J Physiol 591, 4611-4620
  11. Jacobs, B. L., McNally, R. M., Kim, K. J., Blanco, R., Privett, R. E., You, J. S., and Hornberger, T. A. (2017) Identification of mechanically regulated phosphorylation sites on tuberin (TSC2) that control mechanistic target of rapamycin (mTOR) signaling. J Biol Chem 292, 6987-6997
  12. Goodman, C. A., Mabrey, D. M., Frey, J. W., Miu, M. H., Schmidt, E. K., Pierre, P., and Hornberger, T. A. (2011) Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J 25, 1028-1039
  13. Jorgenson, K. W., Phillips, S. M., and Hornberger, T. A. (2020) Identifying the Structural Adaptations that Drive the Mechanical Load-Induced Growth of Skeletal Muscle: A Scoping Review. Cells 9
  14. Erdmann, I., Marter, K., Kobler, O., Niehues, S., Abele, J., Muller, A., Bussmann, J., Storkebaum, E., Ziv, T., Thomas, U., and Dieterich, D. C. (2015) Cell-selective labelling of proteomes in Drosophila melanogaster. Nature communications 6, 7521
  15. Alvarez-Castelao, B., Schanzenbacher, C. T., Hanus, C., Glock, C., Tom Dieck, S., Dorrbaum, A. R., Bartnik, I., Nassim-Assir, B., Ciirdaeva, E., Mueller, A., Dieterich, D. C., Tirrell, D. A., Langer, J. D., and Schuman, E. M. (2017) Cell-type-specific metabolic labeling of nascent proteomes in vivo. Nature biotechnology 35, 1196-1201
  16. Evans, H. T., Bodea, L. G., and Gotz, J. (2020) Cell-specific non-canonical amino acid labelling identifies changes in the de novo proteome during memory formation. Elife 9
  17. Schwenk, F., Baron, U., and Rajewsky, K. (1995) A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic acids research 23, 5080-5081
  18. Saleh, A. M., Wilding, K. M., Calve, S., Bundy, B. C., and Kinzer-Ursem, T. L. (2019) Non-canonical amino acid labeling in proteomics and biotechnology. J Biol Eng 13, 43
  19. Jorgenson, K. W., Hibbert, J. E., Sayed, R. K. A., Lange, A. N., Godwin, J. S., Mesquita, P. H. C., Ruple, B. A., McIntosh, M. C., Kavazis, A. N., Roberts, M. D., and Hornberger, T. A. (2024) A novel imaging method (FIM-ID) reveals that myofibrillogenesis plays a major role in the mechanically induced growth of skeletal muscle. Elife 12
  20. Potts, G. K., McNally, R. M., Blanco, R., You, J. S., Hebert, A. S., Westphall, M. S., Coon, J. J., and Hornberger, T. A. (2017) A map of the phosphoproteomic alterations that occur after a bout of maximal-intensity contractions. J Physiol 595, 5209-5226
  21. Steinert, N. D., Potts, G. K., Wilson, G. M., Klamen, A. M., Lin, K. H., Hermanson, J. B., McNally, R. M., Coon, J. J., and Hornberger, T. A. (2021) Mapping of the contraction-induced phosphoproteome identifies TRIM28 as a significant regulator of skeletal muscle size and function. Cell Rep 34, 108796
  22. Zhu, W. G., Thomas, A. C. Q., Wilson, G. M., McGlory, C., Hibbert, J. E., Flynn, C. G., Sayed, R. K. A., Paez, H. G., Meinhold, M., Jorgenson, K. W., You, J. S., Steinert, N. D., Lin, K. H., MacInnis, M. J., Coon, J. J., Phillips, S. M., and Hornberger, T. A. (2025) Identification of a resistance-exercise-specific signalling pathway that drives skeletal muscle growth. Nat Metab
  23. Lancaster, N. M., Sinitcyn, P., Forny, P., Peters-Clarke, T. M., Fecher, C., Smith, A. J., Shishkova, E., Arrey, T. N., Pashkova, A., Robinson, M. L., Arp, N., Fan, J., Hansen, J., Galmozzi, A., Serrano, L. R., Rojas, J., Gasch, A. P., Westphall, M. S., Stewart, H., Hock, C., Damoc, E., Pagliarini, D. J., Zabrouskov, V., and Coon, J. J. (2024) Fast and deep phosphoproteome analysis with the Orbitrap Astral mass spectrometer. Nature communications 15, 7016
  24. Hornberger, T. A., Jr., and Farrar, R. P. (2004) Physiological hypertrophy of the FHL muscle following 8 weeks of progressive resistance exercise in the rat. Canadian journal of applied physiology = Revue canadienne de physiologie appliquee 29, 16-31
  25. Zhu, W. G., Hibbert, J. E., Lin, K. H., Steinert, N. D., Lemens, J. L., Jorgenson, K. W., Newman, S. M., Lamming, D. W., and Hornberger, T. A. (2021) Weight Pulling: A Novel Mouse Model of Human Progressive Resistance Exercise. Cells 10