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

You JS, Anderson GB, Dooley MS, Hornberger TA - Dis Model Mech (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin - Madison, 2015 Linden Drive, Madison, WI 53706, USA Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin - Madison, 2015 Linden Drive, Madison, WI 53706, USA.

No MeSH data available.


Related in: MedlinePlus

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.
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DMM019414F1: 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.

Mentions: In this study, we employed a new mouse model of unilateral hindlimb immobilization that externally immobilizes both the knee and ankle joints in a highly convenient, stable and safe manner (Fig. 1A and supplementary material Movie 1). With this method, we found that the mass of the five major muscles involved in ankle joint movement were significantly reduced after 7 days of immobilization (Fig. 1B) (note: the animal body weight decreases slightly during the first 2 days of immobilization, supplementary material Fig. S1). Furthermore, with the surface sensing of translation (SUnSET) technique, we found that all of the muscles displayed a significant decrease in the amount of puromycin-labeled peptides during the course of immobilization, which demonstrates that immobilization induced a decrease in the global rates of protein synthesis (Fig. 1C). Combined, these results validate the effectiveness of our immobilization model and indicate that the decreases in muscle mass were due, at least in part, to a decrease in the rate of protein synthesis.Fig. 1.


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

You JS, Anderson GB, Dooley MS, Hornberger TA - Dis Model Mech (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582099&req=5

DMM019414F1: 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.
Mentions: In this study, we employed a new mouse model of unilateral hindlimb immobilization that externally immobilizes both the knee and ankle joints in a highly convenient, stable and safe manner (Fig. 1A and supplementary material Movie 1). With this method, we found that the mass of the five major muscles involved in ankle joint movement were significantly reduced after 7 days of immobilization (Fig. 1B) (note: the animal body weight decreases slightly during the first 2 days of immobilization, supplementary material Fig. S1). Furthermore, with the surface sensing of translation (SUnSET) technique, we found that all of the muscles displayed a significant decrease in the amount of puromycin-labeled peptides during the course of immobilization, which demonstrates that immobilization induced a decrease in the global rates of protein synthesis (Fig. 1C). Combined, these results validate the effectiveness of our immobilization model and indicate that the decreases in muscle mass were due, at least in part, to a decrease in the rate of protein synthesis.Fig. 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin - Madison, 2015 Linden Drive, Madison, WI 53706, USA Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin - Madison, 2015 Linden Drive, Madison, WI 53706, USA.

No MeSH data available.


Related in: MedlinePlus