The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice

Abstract

The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate that the activation of mTOR signaling is a viable target for therapies that are aimed at preventing muscle atrophy during periods of mechanical unloading.

Keywords: Atrophy; Contraction; Disuse; Rapamycin; Rheb; Skeletal muscle.

Fig. 1.

Immobilization decreases the global rates of protein synthesis and muscle mass, but activates mTOR signaling and cap-dependent translation. (A) Representative image of the caudal end of a mouse that was subjected to unilateral hindlimb immobilization (IM). (B-D) Mice were subjected to IM for 3 or 7 days, or a non-immobilized control condition (IM 0), and injected with puromycin at 30 min prior to muscle collection for the measurement of protein synthesis by SUnSET. Various lower hindlimb muscles (EDL, extensor digitorum longus; TA, tibialis anterior; PLT, plantaris; GAST, gastrocnemius; SOL, soleus) were weighed to obtain (B) the muscle weight to body weight ratio, and then subjected to western blot analysis for (C) puromycin-labeled peptides, (D) phosphorylated (P) (T389) and total (T) p70, and P (T308)- and T-PKB. The total amount of puromycin-labeled peptides (i.e. protein synthesis), T-p70, P-PKB, T-PKB and P:T ratios of p70 and PKB were expressed relative to the values obtained in the muscle-matched IM 0 control groups. (E) Mouse TA muscles were co-transfected with GFP, and a dual-luciferase bicistronic reporter of cap-dependent translation, and immediately subjected to IM or the non-immobilized control condition (CNT). At 3 days post-transfection, the muscles were collected and luciferase activities produced by cap-dependent translation of Renilla luciferase (REN) and cap-independent translation of firefly luciferase (FF) were measured to obtain the REN:FF ratio. All values are presented as the mean (+s.e.m. in graphs, n=3-8 muscles per group). * versus the muscle-matched control groups, P≤0.05.

Fig. 2.

Rapamycin exacerbates immobilization-induced decreases in protein synthesis and muscle mass. Mice were subjected to unilateral hindlimb immobilization for 3 or 7 days (IM+), or a non-immobilized control condition (IM− or CNT), and received an acute (day 3) or chronic (day 7) administration of rapamycin (RAP+) or the vehicle (RAP− or VEH) as described in the Materials and Methods. At 30 min prior to the collection of the EDL muscles, mice were injected with puromycin. The muscles were (A) subjected to western blot analysis for phosphorylated (P) (T389) and total (T) p70 and P (S240/244)- and T-S6, (B) analyzed for the muscle weight (MW) to body weight (BW) ratio, (C,D) subjected to immunohistochemistry for laminin to obtain the cross-sectional area (CSA) (≥120 fibers per muscle), or (E,F) subjected to western blot analysis for puromycin-labeled peptides (i.e. protein synthesis) and ubiquitylated proteins, respectively. The values in A, E and F were expressed relative to the values obtained in the time-matched IM−/RAP− (A) or CNT/VEH groups (E,F). All values are presented as the mean (+s.e.m. in graphs, n=3-12 muscles per group). * versus the time- and drug-matched IM− or CNT groups, ^# versus the time- and mobility-matched RAP− or VEH groups, P≤0.05.

Fig. 3.

In immobilized muscles, isometric contractions enhance mTOR signaling and rescue the decrease in protein synthesis via a rapamycin-sensitive mechanism. (A-C) Mice were subjected to unilateral hindlimb immobilization for 3 days (IM+), or a non-immobilized control condition (IM−), and then injected with rapamycin (RAP+) or the vehicle (RAP−) and puromycin (puro) at the indicated time points. Following the RAP−/+ injections, mice were subjected to a bout of isometric contractions (IC+) or the sham condition (IC−). Upon collection, EDL muscles were subjected to western blot analysis for (B) phosphorylated (P) and total (T) p70, T-S6 and (C) puromycin-labeled peptides (i.e. protein synthesis). (D,E) Mice were treated as in A except for the injections, and the EDL muscles were analyzed for (D) total RNA to muscle weight (MW) ratio and (E) 28S+18S rRNA content. The amount of P-p70, T-p70, puromycin-labeled peptides and 28S+18S rRNA was expressed relative to the values obtained in the IC−/IM−/RAP− groups. All values are presented as the mean (+s.e.m. in graphs, n=3-6 muscles per group). * versus the drug- and mobility-matched IC− groups, ^# versus the contraction-matched IM−/RAP− groups, ^† versus the contraction-matched IM+/RAP− groups, P≤0.05.

Fig. 4.

In immobilized muscles, Rheb overexpression enhances mTOR signaling, cap-dependent translation and protein synthesis, and rescues the decrease in fiber size. (A) Mouse TA muscles were co-transfected with GST-p70 and GFP or GFP-Rheb, and immediately subjected to unilateral hindlimb immobilization (IM). After 3 days, the muscles were collected and subjected to western blot analysis for phosphorylated (P) and total (T) GST-p70, GFP and GFP-Rheb. The P:T ratio for GST-p70 was expressed relative to the values obtained in GFP (control) group. (B) Mouse TA muscles were co-transfected with a dual-luciferase bicistronic reporter of cap-dependent translation and GFP or GFP-Rheb, and immediately subjected to IM. At 3 days post-transfection, the muscles were collected, and luciferase activities produced by cap-dependent translation of Renilla luciferase (REN) and cap-independent translation of firefly luciferase (FF) were measured to obtain the REN:FF ratio. (C) Mouse TA muscles were transfected with GFP or GFP-Rheb, and immediately subjected to IM. After 3 days, the mice were injected with puromycin as in Fig. 1 and the muscles were subjected to immunohistochemistry for GFP and puromycin-labeled peptides. The puromycin staining intensity in transfected (positive) fibers was expressed relative to the value obtained in non-transfected fibers from the same section and plotted on histograms (≥160 fibers per muscle). (D) Mouse TA muscles were transfected with GFP or GFP-Rheb, and immediately subjected to IM or a non-immobilized control condition (CNT). After 7 days, the muscles were collected and subjected to immunohistochemistry for GFP and laminin to obtain the cross-sectional area (CSA) of the transfected (positive) and non-transfected (negative) fibers within each muscle (≥100 fibers per muscle). All values are presented as the mean (+s.e.m. in graphs, n=3-4 muscles per group). ^† versus GFP groups, * versus the plasmid- and transfection-matched CNT groups, ^# versus the mobility-matched GFP-Rheb negative groups as well as the mobility-matched GFP positive groups, P≤0.05.

Fig. 5.

eIF4F formation is enhanced by immobilization in a rapamycin-sensitive manner, but not further enhanced by isometric contractions. Mice were treated as in Fig. 3 and pre-cleared homogenates from EDL muscles were subjected to immunoprecipitation (IP) of eIF4E followed by western blot analysis for 4E-BP1, eIF4G and eIF4E to obtain the ratio of (A) 4E-BP1:eIF4E and (B) eIF4G:eIF4E. (C) Whole homogenates (WH) were subjected to western blot analysis for the total (T) and phosphorylated (P) forms of various proteins. All values were expressed relative to the values obtained in the IC−/IM−/RAP− group and presented as the mean (+s.e.m. in graphs, n=3-6 muscles per group). * versus the drug- and mobility-matched IC− groups, ^# versus the contraction-matched IM−/RAP− groups, ^† versus the contraction-matched IM+/RAP− groups, P≤0.05. IC, isometric contractions; IM, immobilization; RAP, rapamycin.

Fig. 6.

Immobilization induces fiber-type-dependent decreases in protein synthesis and fiber size that are not associated with the level of mTOR activity. Mice were subjected to unilateral hindlimb immobilization (IM) for 3 days, or a non-immobilized control condition (CNT), and injected with puromycin as in Fig. 1. (A) EDL muscles obtained from CNT and IM mice were frozen adjacent to one another and subjected to immunohistochemistry for different isoforms of myosin heavy chain (MHC; 1, yellow; 2A, blue; 2X, red; 2B, green) and puromycin-labeled peptides, phosphorylated (P)-S6 (S240/244), or total (T)-S6. (B-D) Fiber-type-specific measurements of (B) the fiber cross-sectional area (CSA) normalized to body weight (BW), (C) the puromycin staining intensity (i.e. protein synthesis), and (D) the ratio of P-S6 normalized to T-S6. All values were expressed relative to the values obtained in the fiber-type-matched CNT muscles and presented as the mean+s.e.m. (n=48-360 fibers per group from four independent pairs of muscles). * versus the fiber-type-matched CNT groups, a-d versus one another, P≤0.05.