Lack of muscle mTOR kinase activity causes early onset myopathy and compromises whole-body homeostasis

Abstract

Background: The protein kinase mechanistic target of rapamycin (mTOR) controls cellular growth and metabolism. Although balanced mTOR signalling is required for proper muscle homeostasis, partial mTOR inhibition by rapamycin has beneficial effects on various muscle disorders and age-related pathologies. Besides, more potent mTOR inhibitors targeting mTOR catalytic activity have been developed and are in clinical trials. However, the physiological impact of loss of mTOR catalytic activity in skeletal muscle is currently unknown.

Methods: We have generated the mTORmKOKI mouse model in which conditional loss of mTOR is concomitant with expression of kinase inactive mTOR in skeletal muscle. We performed a comparative phenotypic and biochemical analysis of mTORmKOKI mutant animals with muscle-specific mTOR knockout (mTORmKO) littermates.

Results: In striking contrast with mTORmKO littermates, mTORmKOKI mice developed an early onset rapidly progressive myopathy causing juvenile lethality. More than 50% mTORmKOKI mice died before 8 weeks of age, and none survived more than 12 weeks, while mTORmKO mice died around 7 months of age. The growth rate of mTORmKOKI mice declined beyond 1 week of age, and the animals showed profound alterations in body composition at 4 weeks of age. At this age, their body weight was 64% that of mTORmKO mice (P < 0.001) due to significant reduction in lean and fat mass. The mass of isolated muscles from mTORmKOKI mice was remarkably decreased by 38-56% (P < 0.001) as compared with that from mTORmKO mice. Histopathological analysis further revealed exacerbated dystrophic features and metabolic alterations in both slow/oxidative and fast/glycolytic muscles from mTORmKOKI mice. We show that the severity of the mTORmKOKI as compared with the mild mTORmKO phenotype is due to more robust suppression of muscle mTORC1 signalling leading to stronger alterations in protein synthesis, oxidative metabolism, and autophagy. This was accompanied with stronger feedback activation of PKB/Akt and dramatic down-regulation of glycogen phosphorylase expression (0.16-fold in tibialis anterior muscle, P < 0.01), thus causing features of glycogen storage disease type V.

Conclusions: Our study demonstrates a critical role for muscle mTOR catalytic activity in the regulation of whole-body growth and homeostasis. We suggest that skeletal muscle targeting with mTOR catalytic inhibitors may have detrimental effects. The mTORmKOKI mutant mouse provides an animal model for the pathophysiological understanding of muscle mTOR activity inhibition as well as for mechanistic investigation of the influence of skeletal muscle perturbations on whole-body homeostasis.

Keywords: Body composition; Glycogen; Mitochondria; Myopathy; mTOR kinase activity.

Figure 1

Characterization of mTOR mutant mice. (A) Strategy to generate the mTOR mutant mouse models. (B) Growth curve of mTORmKI (n = 11), mTORmKO (n = 11), mTORmKOKI (n = 10), and Control (n = 11) male mice (n ≥ 10 per genotype) between week 1 and 6. (C) Morphology of mTORmKI, Control, mTORmKO, and mTORmKOKI mice at 4 weeks of age. (D) Survival curve of mTOR mutant and control mice (n = 23). (E) Growth curve of mTORmWT (n = 11), mTORmKO (n = 11), mTORmKOWT (n = 10), and Control (n = 11) male mice between week 4 and 19. (F) Morphology of mTORmKO, mTORmKOWT, and Control mice at 23 weeks of age. Data indicate mean ± SD. ^*/# P < 0.05; ^**/## P < 0.01; ^***/### P < 0.001, ^* is mTOR mutant vs. Control, ^# is mTORmKOKI vs. mTORmKO.

Figure 2

mTORmKOKI mice exhibit exacerbated dystrophic features. (A) Body weight as well as SOL and TA mass from 6‐w mTORmKO (n = 7) and mTORmKOKI (n = 8) mice relative to Controls (n = 7). (B) SOL and TA muscle mean fibre cross‐sectional area (CSA) in 6‐w Control, mTORmKO, and mTORmKOKI male mice. Fibre CSA was determined on TRITC‐labelled WGA‐stained sections as described in the methods section. This analysis includes a minimum of 400 myofibres per SOL muscle and 1200 myofibres per TA muscle from three mice per genotype. (C) Representative Hematoxylin & Eosin & Saffron (upper panel), Gomori trichrome (middle panel), and Sudan black (lower panel) staining of soleus muscle sections from 6‐w Control, mTORmKO, and mTORmKOKI mice. Black thick arrows indicate regenerated muscle fibres with centrally placed nuclei. Thin arrow indicates fibrosis. Images are representative of five sections from three mice per genotype. Bar, 50 μm. (D) Percentage of centrally nucleated fibres (CNF) in SOL and TA muscles from 6‐w Control, mTORmKO, and mTORmKOKI mice. A minimum of 500 myofibres per SOL muscle and 2800 per TA muscle from three mice per genotype was analysed. (E) Relative mRNA levels of myogenin, IGFII, and MyH8 in SOL muscles from 6‐w mTOR mutant mice. Controls (n = 6); mTORmKO (n = 6); mTORmKOKI (n = 12). Data indicate mean ± SEM. ^*/# P < 0.05; ^**/## P < 0.01; ^***/### P < 0.001, ^* is mTOR mutant vs. Control, ^# is mTORmKOKI vs. mTORmKO.

Figure 3

Muscle mTOR kinase activity is required for muscle dystrophin expression and mitochondria function. (A, B) Relative mRNA levels of dystrophin (A) and PGC‐1α (B) in SOL muscles from 4‐w Control (n = 7–13), mTORmKOKI (n = 8–12), and mTORmKO (n = 7–11) mice, and in SOL muscles from 6‐w Control (n = 6), mTORmKOKI (n = 6–12), and mTORmKO (n = 6) mice.Data indicate mean ± SEM. ^*/# P < 0.05; ^**/## P < 0.01; ^***/### P < 0.001, ^* is mTOR mutant vs. Control, ^# is mTORmKOKI vs. mTORmKO. (C) Western blot analysis showing myoglobin and complex IV protein levels in SOL muscle from 4‐w and 6‐w Control, mTORmKOKI, and mTORmKO mice (n = 3 mice per age and genotype). α‐Tubulin and GAPDH were used as loading control in muscles from 4‐w and 6‐w mice, respectively. (D) Succinate dehydrogenase (upper panel) and cytochrome oxidase (lower panel) histochemical staining demonstrating defects in the mitochondrial respiratory chain in muscles from 4‐w mTORmKOKI mice, specifically. Images are representative of five sections from three mice per genotype. Bar, 300 μm. (E) Relative mRNA levels of PPARα (peroxisome proliferator‐activated receptor‐α); PPARδ (peroxisome proliferator‐activated receptor‐δ); FABP3 (Fatty‐acid‐binding protein 3); CPT2 (Carnitine palmitoyltransferase II); MCAD (medium‐chain acyl‐CoA dehydrogenase); LCAD (long‐chain acyl‐CoA dehydrogenase); HADH (Hydroxyacyl‐Coenzyme A dehydrogenase) in SOL muscles from 6‐w control (n = 5), mTORmKO (n = 5), and mTORmKOKI (n = 10) mice. Data indicate mean ± SEM. ^*/# P < 0.05; ^**/## P < 0.01; ^***/### P < 0.001, ^* is mTOR mutant vs. Control, ^# is mTORmKOKI vs. mTORmKO.

Figure 4

Biochemical characterization of mTOR mutant muscles. (A, B) Hindlimb, TA, and GC muscle extracts from the specified mice at various ages were immunoblotted with the indicated antibodies to examine mTOR signalling (n = 3 mice per age and genotype). α‐Tubulin was used as loading control in muscles from P2, 1‐w, 2‐w, and 4‐w mice, and GAPDH was used as loading control in muscles from 6‐w mice. (C) Representative polysome profiles of GC muscles from 4‐w and 6‐w Control, mTORmKOKI, and mTORmKO mice fractionated by sucrose density ultracentrifugation as described in the methods section. The concentration of ribosomes was continuously monitored at 254 nm from top to bottom. The monosome peak is marked as 80S. (D) Analysis of the phosphorylation of mTOR at S2448 and AS160 at S588 in mTOR mutant muscles (n = 3 mice per age and genotype). α‐Tubulin was used as loading control.

Figure 5

mTORmKOKI mice display massive glycogen accumulation but mild alterations of whole‐body glucose homeostasis. (A) Quantification of muscle glycogen content in TA and GC muscles from the indicated mice at 6 weeks of age (n = 3 mice per genotype). Results are expressed in μg of glycogen per mg of tissue. (B) Representative electron micrographs of longitudinal section of TA muscles from the indicated mice at 6 weeks of age (n = 3 per genotype). Electron micrographs were taken at higher magnifications in mTORmKOKI muscles to visualize the accumulation of glycogen granules. (C) Western blot analysis showing GPh protein levels in TA and GC muscles from 6‐w Control and mTOR mutant mice (n = 3 mice per genotype). (D) Fed blood glucose levels (n = 12–17 mice per genotype) and (E) fed serum insulin levels (n = 8–10 mice per genotype) in Control and mTOR mutant mice from 4 weeks of age. (F) Glucose tolerance (n = 9–11 mice per genotype) and (G) insulin tolerance (n = 6–12 mice per genotype) tests in 4‐w mTOR mutant mice and Controls after 5 h fasting. (F) and (G) Insets show calculated glucose areas under the curve (AUC _{0–120 min}). Data indicate mean ± SEM. ^*/# P < 0.05; ^**/## P < 0.01; ^***/### P < 0.001, ^* is Control vs. mTOR mutant, ^# is mTORmKOKI vs. mTORmKO.