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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

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.
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DMM019414F4: 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.

Mentions: The results from Fig. 3 suggest that, in immobilized muscles, the activation of mTOR signaling can induce an increase in the rate of protein synthesis, which might help to prevent disuse atrophy. To more directly investigate this possibility, we used overexpression of Rheb as a means for inducing the activation of mTOR signaling in immobilized muscles. As shown in Fig. 4A, we confirmed that the overexpression of Rheb in immobilized muscles was sufficient to induce the activation of mTOR signaling, as revealed by an increase in the T389 phosphorylation of co-transfected GST-p70. Furthermore, we determined that the overexpression of Rheb was sufficient to induce an increase in both cap-dependent translation and the global rate of protein synthesis (Fig. 4B,C). Finally, and most significantly, we demonstrated that the overexpression of Rheb robustly increased the size of fibers and prevented the atrophy that occurred during immobilization (Fig. 4D). Combined, these results indicate that the forced activation of mTOR signaling during immobilization can prevent disuse atrophy, at least in part, by promoting an increase in protein synthesis via enhanced cap-dependent translation.Fig. 4.


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)

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.
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Related In: Results  -  Collection

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DMM019414F4: 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.
Mentions: The results from Fig. 3 suggest that, in immobilized muscles, the activation of mTOR signaling can induce an increase in the rate of protein synthesis, which might help to prevent disuse atrophy. To more directly investigate this possibility, we used overexpression of Rheb as a means for inducing the activation of mTOR signaling in immobilized muscles. As shown in Fig. 4A, we confirmed that the overexpression of Rheb in immobilized muscles was sufficient to induce the activation of mTOR signaling, as revealed by an increase in the T389 phosphorylation of co-transfected GST-p70. Furthermore, we determined that the overexpression of Rheb was sufficient to induce an increase in both cap-dependent translation and the global rate of protein synthesis (Fig. 4B,C). Finally, and most significantly, we demonstrated that the overexpression of Rheb robustly increased the size of fibers and prevented the atrophy that occurred during immobilization (Fig. 4D). Combined, these results indicate that the forced activation of mTOR signaling during immobilization can prevent disuse atrophy, at least in part, by promoting an increase in protein synthesis via enhanced cap-dependent translation.Fig. 4.

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