The role of raptor in the mechanical load-induced regulation of mTOR signaling, protein synthesis, and skeletal muscle hypertrophy

Abstract

It is well known that an increase in mechanical loading can induce skeletal muscle hypertrophy, and a long standing model in the field indicates that mechanical loads induce hypertrophy via a mechanism that requires signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Specifically, it has been widely proposed that mechanical loads activate signaling through mTORC1 and that this, in turn, promotes an increase in the rate of protein synthesis and the subsequent hypertrophic response. However, this model is based on a number of important assumptions that have not been rigorously tested. In this study, we created skeletal muscle specific and inducible raptor knockout mice to eliminate signaling by mTORC1, and with these mice we were able to directly demonstrate that mechanical stimuli can activate signaling by mTORC1, and that mTORC1 is necessary for mechanical load-induced hypertrophy. Surprisingly, however, we also obtained multiple lines of evidence that indicate that mTORC1 is not required for a mechanical load-induced increase in the rate of protein synthesis. This observation highlights an important shortcoming in our understanding of how mechanical loads induce hypertrophy and illustrates that additional mTORC1-independent mechanisms play a critical role in this process.-You, J.-S., McNally, R. M., Jacobs, B. L., Privett, R. E., Gundermann, D. M., Lin, K.-H., Steinert, N. D., Goodman, C. A., Hornberger, T. A. The role of raptor in the mechanical load-induced regulation of mTOR signaling, protein synthesis, and skeletal muscle hypertrophy.

Keywords: exercise; growth; mTORC1; mechanotransduction; rapamycin.

Figure 1

Characterization of skeletal muscle specific and inducible raptor knockout mice. A) Skeletal muscle specific and inducible raptor knockout mice (iRAmKO⁺), along with control littermates (iRAmKO⁻), were treated with 2 mg/d of tamoxifen for 5 d (Tam). At 7, 14, or 21 d post-Tam, TA muscles were collected and subjected to Western blot analysis. Values are expressed relative to the time-matched iRAmKO⁻ samples. B) Western blot analysis of TA muscles from 21 d post-Tam mice. Values represent the ratios of Cox IV and Cytochrome C (Cyt C) to tubulin (Tub) and are expressed relative to the iRAmKO⁻ samples. C) Muscle weight to bodyweight ratio of TA muscles at 21 d post-Tam. D) Cross-sectional area (CSA) of each fiber type (i.e., type IIa, IIx, and IIb) in the TA muscles at 21 d post-Tam was determined, and then the average of these values was used to calculate the “type II fiber CSA” (individual fiber type data are shown in Supplemental Fig. S2). E) Representative images of the cross sections that were stained for laminin (white) as well as type IIa and type IIb fibers. Scale bars, 100 μm. F) Average voluntary distance run during a 5-d period in mice that were 17–21 d post-Tam. G) Photograph of 21 d post-Tam mice. All values represent the group means + sem; n = 4–8/group as indicated in the graphs and Supplemental Fig. S11 which contains additional quantitative information. *P ≤ 0.001, significantly different from the time-matched iRAmKO⁻.

Figure 2

Raptor/mTORC1 is necessary for, at least a subset of, the RSmTOR-dependent signaling events that are activated by maximal-intensity contractions. A, B) Wild-type FVB (A) and FVB (B) mice with skeletal muscle specific expression of a rapamycin-resistant mutant of mTOR (RR-mTOR) were treated with rapamycin (RAP⁺) or the solvent vehicle (RAP⁻), and then the tibialis anterior (TA) muscles were stimulated with a bout of maximal-intensity contractions (MIC⁺) or the control condition (MIC⁻). At 1 h after stimulation, the TA muscles were collected and subjected to Western blot analysis. Values represent the phosphorylated to total ratio (P:T) expressed relative to the genotype-matched control group (i.e., RAP⁻ and MIC⁻). C) At 21 d after being treated with 2 mg/d of tamoxifen for 5 d (Tam), TA muscles from skeletal muscle specific and inducible raptor knockout mice (iRAmKO⁺), and the control littermates (iRAmKO⁻), were subjected to a bout of MIC, or the control condition, and then analyzed as previously described. Values represent the group means for the P:T ratios, or the percentage of 4EBP1 in the γ form (γ:T), expressed relative to the control group (i.e., iRAmKO⁻ and MIC⁻); n = 4–6/group. Horizontal bar above the values indicates a main effect for RAP (A) or iRAmKO (C). *P < 0.05, significant effect of MIC within the given level of RAP or iRAmKO; ^†P < 0.05, significant interaction between RAP and MIC (A), or iRAmKO and MIC (C). Additional quantitative information can be found in Supplemental Fig. S11.

Figure 3

Maximal-intensity contractions enhance the targeting of mTOR to the LEL system via a raptor-dependent mechanism. At 21 d after being treated with tamoxifen (2 mg/d for 5 d), the tibialis anterior muscles from skeletal muscle specific and inducible raptor knockout mice (iRAmKO⁺), as well as control littermates (iRAmKO⁻), were stimulated with a bout of maximal-intensity contractions (MIC⁺) or the control condition (MIC⁻). At 1 h after stimulation, the muscles were collected and subjected to immunohistochemistry for mTOR and LAMP2 as a marker of the LEL system. A, B) Representative grayscale and merged images of the signals for mTOR and LAMP2 in muscles from iRAmKO⁻ (A) and iRAmKO⁺ (B) mice. Scale bars, 10 μm. C, D) Images from iRAmKO⁻ (C), and iRAmKO⁺ (D) mice were analyzed for the number of pixels that were intensely positive for both mTOR and LAMP2 (i.e., colocalized), and the results were expressed as a percentage of the mean value obtained in the control samples. All values represent the group means + sem; n = 27–31 images/group as indicated in graphs. *P ≤ 0.005, significant effect of MIC.

Figure 4

The synergist ablation model of mechanical overload induces reexpression of raptor in the muscle fibers of iRAmKO⁺ mice. At 21 d after being treated with tamoxifen (2 mg/d for 5 d), the plantaris muscles from skeletal muscle specific and inducible raptor knockout mice (iRAmKO⁺), as well as control littermates (iRAmKO⁻), were subjected to synergist ablation (SA⁺) or a sham (SA⁻) surgery. During a 7-d recovery period, the mice were treated with daily injections of tamoxifen as detailed in the Materials and Methods, and then the muscles were collected. A) Western blot analysis of mTOR and raptor. Values are expressed relative to the control group (i.e., iRAmKO⁻ and 7-d SA⁻). B–D) Cross sections of the muscles were subjected to immunohistochemistry for eMHC, mTOR, and LAMP2. B) Representative images of the muscles from iRAmKO⁻ and iRAmKO⁺ mice that had been subjected to SA. Scale bars, 10 μm. C) Frequency scatterplots in I–IV were generated by comparing the intensity of signal for mTOR vs. LAMP2 in every pixel of the analyzed images. Frequency scatterplots in V, VI were generated by only analyzing the pixels within, or outside, of the eMHC positive fibers, respectively. D) Pearson’s correlation coefficients (CC) between the signals for mTOR and LAMP2. Values represent the group means + sem in the graph; n = 7-9/group as indicated in the graph and Supplemental Fig. S11 which contains additional quantitative information. Horizontal bar indicates a main effect for iRAmKO. *P ≤ 0.001, significant effect of SA within the given level of iRAmKO.

Figure 5

Myotenectomy is an effective model for mechanical overload-induced hypertrophy, and it does not induce the re-expression of raptor in skeletal muscle fibers of iRAmKO⁺ mice. A) The plantaris (PLT) muscles of wild-type C57BL6 mice were subjected to synergist ablation (SA⁺), myotenectomy (MTE⁺), or their respective sham surgeries as a control condition. After 14 d of recovery, cross sections of the PLT muscles were subjected to immunohistochemistry for laminin and eMHC, and then the total number of eMHC-positive fibers in the entire cross section was determined. Scale bars, 100 μm. B) The PLT muscles of wild-type C57BL6 mice were subjected to MTE or a sham surgery as the control condition, and then the mice were treated with daily injections of 1.5 mg/kg rapamycin (RAP⁺) or the solvent vehicle (RAP⁻). After 14 d of recovery, the PLT muscles were collected and subjected to immunohistochemistry for fiber type identification. The mean CSA of each fiber type (i.e., type IIa, IIx, and IIb) was determined, and then the average of these values was used to calculate the “type II fiber CSA” (individual fiber type data is shown in Supplemental Fig. S5). C–F) At 21 d after being treated with tamoxifen (2 mg/d for 5 d), skeletal muscle specific and inducible raptor knockout mice (iRAmKO⁺), along with control littermates (iRAmKO⁻), were subjected to MTE or a sham surgery, treated with daily injections of tamoxifen as described in Materials and Methods, and then the PLT muscles were collected after a 3-, 7-, or 14-d recovery period. Nonsurgically treated mice were used for a 0 d MTE⁻ condition. C) Western blot analysis of mTOR and raptor. D) The amount of raptor at each time point was expressed as a percentage of the control condition (i.e., iRAmKO⁻ and 0 d MTE⁻). E, F) Muscles from the 7-d time point were subjected to immunohistochemistry for dystrophin, mTOR, and LAMP2. E) Representative images for each group with the images on the right revealing a magnified region of the images on the left. Scale bars, 10 μm. Arrows indicate mTOR positive cells that resided outside of the dystrophin boundary. F) Pearson’s CC between the signal for mTOR and LAMP2. All values represent the group means + sem in the graph; n = 5-10/group as indicated in the graphs and Supplemental Fig. S11 which contains additional quantitative information. Horizontal bar above the values indicates a main effect for iRAmKO. *P ≤ 0.005, significantly different from the control condition; ^•P ≤ 0.005, significantly different from the time-matched iRAmKO⁻ group.

Figure 6

Raptor is necessary for myotenectomy-induced hypertrophy. At 21 d after being treated with tamoxifen (2 mg/d for 5 d), skeletal muscle specific and inducible raptor knockout mice (iRAmKO⁺), and control littermates (iRAmKO⁻), were subjected to myotenectomy (MTE) or a sham surgery, treated with daily injections of tamoxifen as described in Materials and Methods, and then the plantaris muscles were collected after a 7- or 14-d recovery period. Cross sections were subjected to immunohistochemistry for laminin and fiber type identification. A) The mean CSA of each fiber type (i.e., type IIa, IIx, and IIb) was determined, and then the average of these values was used to calculate the “type II fiber CSA.” The resulting values were expressed relative to the mean value obtained in the genotype-matched sham group (individual fiber type data is shown in Supplemental Fig. S6). B) Representative images of cross sections that were stained for type IIa and type IIb fibers. Scale bars, 100 μm. Values represent the group means + sem; n = 5–20 muscles/group as indicated in the graph. Horizontal bar indicates a main effect for iRAmKO. *P ≤ 0.001, significant effect of MTE within the given level of iRAmKO; ^▪P ≤ 0.001, significantly different from the genotype-matched 7-d MTE group.

Figure 7

Raptor/mTORC1 is not necessary for a mechanical overload-induced increase in protein synthesis. At 21 d after being treated with tamoxifen (2 mg/d for 5 d), skeletal muscle specific and inducible raptor knockout mice (iRAmKO⁺), and control littermates (iRAmKO⁻), were subjected to MTE or a sham surgery, and then treated with daily injections of tamoxifen during a 7-d recovery period. At 30 min before collection, the mice were injected with puromycin, and then the plantaris muscles were subjected to Western blot analysis to quantify the amount of puromycin-labeled peptides (i.e., the rate of protein synthesis). The membranes were also stained with Coomassie to verify equal loading of protein in all lanes. Values in the graph are expressed relative to the control group (i.e., iRAmKO⁻ and MTE⁻) and represent the group means ± sem; n = 9–12/group as indicated in the graph. Horizontal bar above the values indicates a main effect for iRAmKO. *P ≤ 0.01, significant effect of MTE within the given level of iRAmKO.