It is well recognized that mechanical stimuli play a major role in the regulation of skeletal muscle mass, and that the maintenance of muscle mass contributes significantly to disease prevention and the 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. Hence, the long-term goal of our research is to determine how skeletal muscles sense mechanical information and convert this stimulus into the molecular events that regulates changes in muscle mass (i.e., mechanotransduction).
Defining the role of mTOR in the mechanical regulation of skeletal muscle mass
A substantial amount of our work has focused on a single evolutionarily conserved kinase called the mammalian target of rapamycin (mTOR). For instance, we have shown 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 a mechanically-induced increase in muscle mass (2). However, like all pharmacological inhibitors, it remained possible that the effects of rapamycin were mediated through a non-specific / mTOR-independent mechanism. To address this concern we employed mice that possess myofiber specific expression of a rapamycin-resistant mutant of mTOR (3). With these mice we were able to confirm that mTOR was indeed the rapamycin-sensitive element that conferred the mechanically-induced increase in fiber size (i.e., hypertrophy). We also demonstrated that the kinase activity of mTOR was necessary for this event.
Having established that signaling through mTOR is necessary for mechanically-induced hypertrophy, we next sought to determine whether the activation of mTOR, in and of itself, was sufficient to induce hypertrophy. 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 (4). Our results demonstrated that the overexpression of Rheb induced a robust hypertrophic response, and this effect was completely inhibited when mice were treated with rapamycin. Moreover, by using the transgenic mice that express the rapamycin-resistant mutants of mTOR, we were able to confirm that the hypertrophic effects of Rheb were mediated by mTOR and that the kinase activity of mTOR was necessary for this event. When combined, our research in this area has helped to establish two fundamentally important points: i) signaling through mTOR is necessary for mechanically-induced hypertrophy, and ii) the activation of mTOR is sufficient to induce hypertrophy.
Identifying the mechanisms through which mechanical stimuli activate mTOR
The aforementioned outcomes indicated that the activation of mTOR might play a key role in the pathway through which mechanical stimuli regulate muscle mass. Therefore, we set out to definine 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 a canonical PI3K-dependent pathway. However, our work revealed that mechanical stimuli activate mTOR through a unique PI3K-independent mechanism (5). Moreover, we obtained evidence which indicated that this PI3K-indpendent mechanism did not involve traditional signaling molecules such as iCa2+, ERK, PKA, PKC, or PLC (6). Instead, our work suggested that phosphatidic acid (PA) might be a critical component of the pathway (6). Recently, we obtained further support for this conclusion by demonstrating that mechanical stimuli promote an increase in the levels of PA through a mechanism that requires a PA synthesizing enzyme called diacylglycerol kinase zeta (DGKζ). Our work has also demonstrated that the magnitude of the mechanical activation of mTOR signaling is significantly (≈50%) reduced in muscles that lack DGKζ (7, 8). 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) (9). 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 (9). Furthermore, through the generation of inducible and myofiber specific knockout mice, we recently demonstrated that both TSC2 and Rheb significantly contribute (≈50%) to the pathway via which mechanical stimuli activate mTOR signaling. Thus, when combined, our work in this area 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 use and development of innovative models / technologies
Our lab has always strived to develop better model systems and analytical techniques. For instance, one of our studies required us to employ an animal model that could mimic a human progressive resistance-exercise program. Several candidate models had previously been described in the literature, however, all of them had unacceptable limitations. Therefore, we developed a new model that allowed animals to engage in a physiologically relevant form of resistance-exercise, and with our model, the rate of progression through the training program could be tailored to each individuals level of performance (10). Thus, with our new approach, it became possible to track each individual’s daily progression in strength, and the concomitant hypertrophic response that ultimately developed. The development of this model was critical for the success of our own studies, and it is now being employed by numerous other labs (e.g., the model has been cited in over 200 publications in the last 5 years).
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 labelled amino acids. However, this approach is expensive, requires highly specialized equipment, is labor intensive, and it can introduce numerous safety concerns. Therefore, a few years 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 (SUnSET). Following numerous validation experiments, we demonstrated that our simple approach could be used to accurately quantify rates of protein synthesis in vivo (11). 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 (11, 12). 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.
In addition to developing new analytical techniques, our lab has also consistently pushed the technological boundaries of our field. For instance, we recently used mass spectrometry to identify six mechanically regulated phosphorylation sites on TSC2. We then set out to define the functional significance of these sites by employing an in vivo rescue-based approach in which we first inducibly knocked endogenous TSC2 out of myofibers, and then used electroporation to re-express various forms of TSC2 (e.g., wild-type vs. phosphodefective mutants). The approach enabled us to demonstrate that the phosphorylation sites we identified were necessary for the role that TSC2 plays in the mechanical activation of mTOR signaling (13). To the best of our knowledge, we are the only group that has ever utilized this type of in vivo rescue-based approach.
Finally, during the last few years, our lab has done an extensive amount of work with cutting-edge technologies in phosphoproteomics. For instance, we recently published our first phosphoproteomics study in which we successfully quantified close to 6000 different phosphorylation sites, and our analyses led to the identification of over 650 novel contraction regulated phosphorylation events (14). With refinements to our approach, we are now able to consistently analyze >10000 phosphorylation sites per experiment, and we are in the process of completing several large-scale datasets. These datasets include: i) the phosphoproteomic alterations that occur following contractions +/- rapamycin treatment, ii) a time course of the phosphoproteomic alterations that occur during immobilization (i.e., muscle disuse), and iii) the phosphoproteoimc alterations that occur following endurance versus resistance exercise. The results from these discovery-based projects have already led to several exciting observations and we expect that additional outcomes from these projects will substantially expand the breadth of our research program, and ultimately move us closer to our goal of defining how mechanical signals are converted into the molecular events that regulate muscle mass.
- 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.
- 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.
- 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.
- 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.
- 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. https://www.ncbi.nlm.nih.gov/pubmed/15030312
- 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.
- 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.
- 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.
- 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.
- 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 29, 16-31.
- 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.
- Goodman, C. A., Kotecki, J. A., Jacobs, B. L., and Hornberger, T. A. (2012) Muscle fiber type-dependent differences in the regulation of protein synthesis. PloS one 7, e37890.
- 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.
- 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.