mTORC1 mediates fiber type-specific regulation of protein synthesis and muscle size during denervation

Abstract

Skeletal muscle denervation occurs in diverse conditions and causes severe muscle atrophy. Signaling by mammalian target of rapamycin complex 1 (mTORC1) plays a central role in the maintenance of skeletal muscle mass by regulating net protein balance; yet, its role in denervation-induced atrophy is unclear. In this study, by using skeletal muscle-specific and inducible raptor knockout mice, we demonstrate that signaling through mTORC1 is activated during denervation and plays an essential role in mitigating the atrophy of non-type IIB muscle fibers. Measurements of protein synthesis rates of individual fibers suggest that denervation increases protein synthesis specifically in non-type IIB muscle fibers and that mTORC1 is required for this event. Furthermore, denervation induced a more pronounced increase in the level of phosphorylated ribosomal S6 protein in non-type IIB muscle fibers than in type IIB muscle fibers. Collectively, our results unveil a novel role for mTORC1 in mediating a fiber type-specific regulation of muscle size and protein synthesis during denervation.

Fig. 1. Signaling through mTORC1 contributes to the regulation of ubiquitin-mediated proteolytic pathway during denervation.

A Skeletal muscle-specific inducible raptor knockout (iRAmKO) mice and control littermates (WT) were subjected to denervation (right leg, D or DNV) and sham (contralateral left leg, C or CTL) surgeries. Tibialis anterior (TA) and gastrocnemius (GAST) muscles were collected 7 days after surgery and analyzed by western blotting for the indicated proteins. N.S., non-specific band. B–D The levels of raptor and phosphorylated (P)/total (T) p70 ratio (B), P/T Akt ratio, MAFbx, and MuRF1 (C), and global ubiquitination (Ubi), K48-linked ubiquitination (K48-Ubi), and actin (D), were quantified and expressed relative to WT/CTL. n = 4–6 mice/group. Data are presented as mean ± s.e.m. Surgery effects (Sur, *p < 0.05, **p < 0.01, ***p < 0.001), genotype effects (Gen, ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001), and interaction between Sur and Gen (Int), by two-way mixed ANOVA.

Fig. 2. Signaling through mTORC1 alleviates atrophy of non-type IIB muscle fibers, but not of type IIB fibers, during denervation.

A Skeletal muscle-specific inducible raptor knockout (iRAmKO) mice and control littermates (WT) were subjected to denervation (right leg, DNV) and sham (contralateral left leg, CTL) surgeries. Tibialis anterior (TA), extensor digitorum longus (EDL), and gastrocnemius (GAST) muscles were collected 14 days after surgery and analyzed by immunohistochemistry for cross-sectional area (CSA) of type IIB and non-type IIB muscle fibers. Type IIB muscle fibers were identified by type IIB myosin heavy chain (MHCIIB). B, C Representative cross-sectional images (B) and fiber CSA distribution (C) of the denervated and CTL muscles (EDL) from WT and iRAmKO mice. Scale bar: 70 µm. n = 4–9 mice/group. Data are presented as mean ± s.e.m. Surgery effects (^‡p < 0.05, ^‡‡p < 0.01, ^‡‡‡p < 0.001) by paired t test. Genotype effects (*p < 0.05, **p < 0.01, ***p < 0.001), fiber type effects (^††p < 0.01, ^†††p < 0.001), and interaction between genotype and fiber type effects (Int), by two-way mixed ANOVA. ^§P ≤ 0.05 vs. iRAmKO by unpaired t test.

Fig. 3. Signaling through mTORC1 enhances protein synthesis and the level of phosphorylated S6 preferably in non-type IIB muscle fibers.

A, B Skeletal muscle-specific inducible raptor knockout (iRAmKO) mice and control littermates (WT) were subjected to denervation (right leg, DNV) and sham (contralateral left leg, CTL) surgeries. At 14 days post surgery, the mice were injected with puromycin, and extensor digitorum longus (EDL) muscles were collected 30 min after the injection. The samples were analyzed by immunohistochemistry for the total intensity of puromycin-labeled peptides (Puro) per type IIB and non-type IIB muscle fiber cross-section (CS) (A) or for the total intensity of phosphorylated (P) S6 protein per type IIB and non-type IIB muscle fiber CS (B). Type IIB muscle fibers were identified by type IIB myosin heavy chain (MHCIIB). Note that EDL muscles from the right and left legs were frozen together in OCT for immunohistochemistry on the same slide. Scale bar: 50 µm (A) or 30 µm (B). n = 4–5 mice/group. Data are presented as mean ± s.e.m. Surgery effects (*p < 0.05, **p < 0.01), fiber type effect (^††p < 0.01), and interaction (Int) among surgery (S), fiber type (F), and genotype (G) effects, by three- and two-way mixed ANOVA.

Fig. 4. Signaling through mTORC1 regulates muscle fiber type proportion during denervation.

Skeletal muscle-specific inducible raptor knockout (iRAmKO) mice and control littermates (WT) were subjected to denervation (right leg, DNV) and sham (contralateral left leg, CTL) surgeries. Extensor digitorum longus (EDL) and tibialis anterior (TA) muscles were collected 14 days after surgery and analyzed by immunohistochemistry for the percentage of type IIB and non-type IIB muscle fibers. Type IIB muscle fibers were identified by type IIB myosin heavy chain (MHCIIB). n = 5–7 mice/group. Data are presented as mean ± s.e.m. Surgery effects (*p < 0.05, **p < 0.01) and interaction (Int) among surgery (S), fiber type (F), and genotype (G) effects, by three- and two-way mixed ANOVA.