The role of skeletal muscle mTOR in the regulation of mechanical load-induced growth

Abstract

Chronic mechanical loading (CML) of skeletal muscle induces compensatory growth and the drug rapamycin has been reported to block this effect. Since rapamycin is considered to be a highly specific inhibitor of the mammalian target of rapamycin (mTOR), many have concluded that mTOR plays a key role in CML-induced growth regulatory events. However, rapamycin can exert mTOR-independent actions and systemic administration of rapamycin will inhibit mTOR signalling in all cells throughout the body. Thus, it is not clear if the growth inhibitory effects of rapamycin are actually due to the inhibition of mTOR signalling, and more specifically, the inhibition of mTOR signalling in skeletal muscle cells. To address this issue, transgenic mice with muscle specific expression of various rapamycin-resistant mutants of mTOR were employed. These mice enabled us to demonstrate that mTOR, within skeletal muscle cells, is the rapamycin-sensitive element that confers CML-induced hypertrophy, and mTOR kinase activity is necessary for this event. Surprisingly, CML also induced hyperplasia, but this occurred through a rapamycin-insensitive mechanism. Furthermore, CML was found to induce an increase in FoxO1 expression and PKB phosphorylation through a mechanism that was at least partially regulated by an mTOR kinase-dependent mechanism. Finally, CML stimulated ribosomal RNA accumulation and rapamycin partially inhibited this effect; however, the effect of rapamycin was exerted through a mechanism that was independent of mTOR in skeletal muscle cells. Overall, these results demonstrate that CML activates several growth regulatory events, but only a few (e.g. hypertrophy) are fully dependent on mTOR signalling within the skeletal muscle cells.

Figure 1. Dose-dependent effect of rapamycin on mechanical load-induced hypertrophy

Plantaris muscles from wild-type mice were subjected to sham or synergist ablation (SA) surgeries. Following surgery, the mice were administered a daily regime of vehicle (RAP⁻) or 0.3–3.0 mg kg⁻¹ rapamycin (RAP⁺) injections. A–D, after 14 days, plantaris muscles were collected and mid-belly cross-sections were subjected to immunohistochemistry for laminin. E, the average fibre cross-sectional area (CSA) for each muscle was determined from 150 randomly selected fibres. Values represent the mean + SEM, n = 3–6 muscles per group. *Significantly different from the vehicle sham condition, P≤ 0.05.

Figure 2. Confirmation of the phenotypes in RR-mTOR and RRKD-mTOR transgenic mice

A, lysates from muscles of wild-type, RR-mTOR and RRKD-mTOR mice were subjected to Western blot analysis or immunoprecipiation (i.p.) for the FLAG-tag followed by Western blot analysis with the indicated antibodies. B, quantification of total mTOR with the data expressed as a percentage of the values obtained in wild-type samples. C and D, mice were subjected to sham or synergist ablation (SA) surgeries. Following surgery, the mice were administered a daily regime of either vehicle (RAP⁻) or 0.6 mg kg⁻¹ rapamycin (RAP⁺) injections. At 7 days post-surgery, the plantaris muscles were collected and subjected to Western blot analysis. Representative images and quantification of the Western blot analysis for phosphorylation of the p70^S6k on the threonine 389 residue (P-p70(389)) (C) and total p70^S6k (D). The values at the top of each blot represent the amount of each protein when expressed relative to the values obtained in the vehicle treated sham muscles of the respective genotype. Values are expressed as the mean (+SEM in graphs), n = 3–11 per group. †Significantly different from wild-type, *Significant effect of SA within a given drug treatment, ‡significant effect of RAP within the SA groups, P≤ 0.05.

Figure 3. The role of mTOR in mechanical load-induced hypertrophy

Plantaris muscles from wild-type, RR-mTOR, and RRKD-mTOR mice were subjected to sham or synergist ablation (SA) surgeries. Following the surgery, the mice were administered a daily regime of either vehicle (RAP⁻) or 0.6 mg kg⁻¹ rapamycin (RAP⁺) injections. A–L, at 14 days post surgery, the plantaris muscles were collected and cross-sections from the mid-belly were subjected to immunohistochemistry for laminin. M, the average fibre cross-sectional area (CSA) for each muscle was determined from 150 randomly selected fibres. All values are expressed as the mean +SEM, n = 4–7 muscles/group. *Significant effect of SA within a given drug treatment, #significant effect of RAP within the sham groups, ‡significant effect of RAP within the SA groups, P≤ 0.05. Scale bars in the images represent a length of 100 μm.

Figure 4. The role of mTOR in mechanical load-induced hyperplasia

Plantaris muscles from wild-type, RR-mTOR, and RRKD-mTOR mice were subjected to sham or synergist ablation (SA) surgeries. Following the surgery, the mice were administered a daily regime of either vehicle (RAP⁻) or 0.6 mg kg⁻¹ rapamycin (RAP⁺) injections. A–L, at 14 days post-surgery, the plantaris muscles were collected and cross-sections from the mid-belly were subjected to immunohistochemistry for laminin (red) and embryonic myosin heavy chain (MHC-emb+, green). M and N, the total number of fibres per muscle cross-section (M) and the total number of MHC-emb+ fibres per muscle cross-section (N) were manually counted. All values are expressed as the mean + SEM, n = 4–6 muscles per group. *Significant effect of SA within a given drug treatment, P≤ 0.05. Scale bars in the images represent a length of 100 μm.

Figure 5. The role of mTOR in mechanical load-induced signalling events

Plantaris muscles from wild-type, RR-mTOR and RRKD-mTOR mice were subjected to sham or synergist ablation (SA) surgeries. Following surgery, the mice were administered a daily regime of either vehicle (RAP⁻) or 0.6 mg kg⁻¹ rapamycin (RAP⁺) injections. At 7 days post-surgery, the plantaris muscles were collected and subjected to Western blot analysis with the indicated antibodies. The values at the top of each blot represent the mean amount of each protein when expressed relative to the values obtained in the vehicle treated sham muscles of the respective genotype, n = 3–6 per group. *Significant effect of SA within a given drug treatment, #significant effect of RAP within the sham groups, ‡significant effect of RAP within the SA groups, P≤ 0.05.

Figure 6. The role of mTOR in mechanical load-induced events associated with ribosome biogenesis

Plantaris muscles from wild-type, RR-mTOR, and RRKD-mTOR mice were subjected to sham or synergist ablation (SA) surgeries. Following surgery, the mice were administered a daily regime of either vehicle (RAP⁻) or 0.6 mg kg⁻¹ rapamycin (RAP⁺) injections. At 7 days post-surgery, the plantaris muscles were collected and subjected to RNA isolation or Western blot analysis. A, total RNA concentration was determined by spectrophotometric analysis. B and C, representative images and quantification of the 28S + 18S rRNA bands (B) and Western blot analysis for expression of UBF (C). The values at the top of the blots represent the amount of rRNA or UBF when expressed relative to the values obtained in the vehicle treated sham muscles of the respective genotype. D, representative image and quantification of the Western blot analysis for Rb protein. The values at the top of the blot represent the ratio of hyperphosphorylated Rb (ppRb) to total Rb (ppRB + pRb) and were expressed relative to the values obtained in the vehicle treated sham muscles of the respective genotype. All values are expressed as the mean (+SEM in graphs), n = 4–6 per group. *Significant effect of SA within a given drug treatment, #significant effect of RAP within the sham groups, ‡significant effect of RAP within the SA groups, P≤ 0.05.

Figure 7. Summary of the effect of rapamycin on various mechanical load-induced growth regulatory events

Growth regulatory events activated by mechanical loading in wild-type animals were categorized as rapamycin sensitive, partially rapamycin sensitive or rapamycin insensitive. The events that showed sensitivity to rapamycin were further separated by their dependence of signalling by mTOR, within the skeletal muscle cells, as determined by the outcomes observed with RR-mTOR and RRKD-mTOR mice. Note, all mTOR-dependent events that were identified in this study required mTOR kinase activity and are therefore referred to as mTOR kinase-dependent events.