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. Our lab has been intrigued by this gap in knowledge and we have tried to address it by focusing on the mTOR signaling pathway. Our early 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 hypertrophy 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 concern by employing mice that possess myofiber-specific expression of a rapamycin-resistant mutant of mTOR (3). With these mice, we confirmed that mTOR is the rapamycin-sensitive element that confers the mechanically induced hypertrophic response (i.e., radial growth), and that the kinase activity of mTOR was necessary for this event.
Having established that signaling through mTOR is necessary for the radial growth of myofibers that occurs in response to chronic mechanical overload, we next sought to determine whether the activation of mTOR, in and of itself, is sufficient to induce this response. 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) (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. 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. When combined, our research in this area has helped to establish two fundamentally important points: i) signaling through mTOR is necessary for the radial growth of myofibers that occurs in response to chronic mechanical overload, and ii) the activation of mTOR is sufficient to induce the radial growth of myofibers.
Identifying the mechanisms through which mechanical stimuli activate mTOR
The aforementioned points indicate that the activation of mTOR might play a key role in the pathway through which mechanical stimuli regulate muscle mass. Therefore, a subset 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 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-independent 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 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 (10) (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 (10). 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.
Although the SUnSET technique is excellent for measuring short-term rates of protein synthesis, it does so by labeling truncated peptides and such peptides would not be expected to possess the appropriate localization or half-life characteristics of their respective full-length proteins. This is particularly problematic because one of our future goals is to identify where newly synthesized proteins (NSPs) accumulate during growth. Thus, to overcome the limitations of the SUnSET technique, we have been developing a new technique that utilizes the principles of bioorthogonal non-canonical amino acid tagging (BONCAT) with azidonorleucine (ANL) (11-13). Specifically, we have crossed floxed STOP GFP-2A-MetRSL274G mice with mice that express CMV-Cre (12, 14). 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 3A-B). Importantly, MetRSL274G allows ANL (a methionine analog) to be incorporated into NSPs (12, 15). This is very important 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). 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 3C). We have also subjected plantaris muscles to chronic mechanical overload and demonstrated that our approach could be used to visualize and quantify the overload-induced accumulation of NSPs in whole muscle cross-sections (Figure 3D).
Having worked out the basic conditions for our NSP imaging technique, we next set out to modify the protocol in a manner that would allow for high-resolution (≤10 nm) imaging of where the NSPs accumulate. Specifically, we placed MetRS mice on a control diet (i.e., no ANL) or our custom ANL diet, for 4 days. The plantaris muscles were then collected and subjected to a modified freeze-substitution and Lowicryl resin embedding procedure. Next, ultrathin longitudinal sections were subjected to a tailor-made post-embedding click reaction containing alkyne-biotin, and then the biotin was tagged with 1.4 nm FluoroNanogold-streptavidin. To help the reader see the gold particles, a prolonged gold enhancement procedure was used to amplify the nanogold to ~25 nm, and then the sections were imaged with transmission electron microscopy (Figure 4). The outcomes revealed a >30-fold signal-to-noise ratio and, based on the size of the tagging components, 100% of the signal (i.e., the center of the gold particles) should be located within 7 nm of the source (~½ the diameter of a myosin thick filament). It bears noting that we spent more than 4 years on the development/optimization of this technique and, to the best of our knowledge, it represents the only instance in which a post-embedding click reaction has been successfully combined with TEM imaging. Hence, with the use of our new technique, we will now be able to precisely define where NSPs accumulate during skeletal muscle growth (i.e., identify where and how growth occurs). Specifically, we will be able to test our hypothesis that a mechanically induced increase 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 5), 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 6). For additional information about these hypotheses please see our recent review (16).
In addition to our work in the field of protein synthesis, our group has also been working with cutting-edge technologies in phosphoproteomics. For instance, in 2017 we published our first phosphoproteomics study in which we successfully quantified close to 6,000 different phosphorylation sites, and our analyses led to the identification of over 650 novel contraction-regulated phosphorylation events (17). With refinements to our approach, we are now able to consistently analyze >10,000 phosphorylation sites per experiment, and we recently published two large-scale datasets which described: i) the phosphoproteomic alterations that occur following maximal-intensity contractions +/- rapamycin treatment (18), and ii) the time-dependent phosphoproteomic alterations that occur following the onset of immobilization (19). Of note, we have only scratched the surface of the results that are contained within these datasets, yet they have already led to exciting observations such as the identification of TRIM28 as a novel regulator of skeletal muscle size and function (18).
The development of animal models of resistance exercise
Our early work led to the development of a highly cited rat model for mimicking a human progressive resistance exercise program (ladder climbing) (20), and the lessons we learned while developing the rat model helped us develop a new mouse model of resistance exercise called weight pulling (21). 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 mTORC1 and the induction of protein synthesis). 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 nature of the work that is being done in mouse-based studies.
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