Hélène Gilson

Follistatin induces muscle hypertrophy through satellite cell proliferation and inhibition of both myostatin and activin

Follistatin (FS) inhibits several members of the TGF-beta superfamily, including myostatin (Mstn), a negative regulator of muscle growth. Mstn inhibition by FS represents a potential therapeutic approach of muscle atrophy. The aim of our study was to investigate the mechanisms of the FS-induced muscle hypertrophy. To test the role of satellite cells in the FS effect, we used irradiation to destroy their proliferative capacity. FS overexpression increased the muscle weight by about 37% in control animals, but the increase reached only 20% in irradiated muscle, supporting the role of cell proliferation in the FS-induced hypertrophy. Surprisingly, the muscle hypertrophy caused by FS reached the same magnitude in Mstn-KO as in WT mice, suggesting that Mstn might not be the only ligand of FS involved in the regulation of muscle mass. To assess the role of activin (Act), another FS ligand, in the FS-induced hypertrophy, we electroporated FSI-I, a FS mutant that does not bind Act with high affinity. Whereas FS electroporation increased muscle weight by 32%, the muscle weight gain induced by FSI-I reached only 14%. Furthermore, in Mstn-KO mice, FSI-I overexpression failed to induce hypertrophy, in contrast to FS. Therefore, these results suggest that Act inhibition may contribute to FS-induced hypertrophy. Finally, the role of Act as a regulator of muscle mass was supported by the observation that ActA overexpression induced muscle weight loss (-15%). In conclusion, our results show that satellite cell proliferation and both Mstn and Act inhibition are involved in the FS-induced muscle hypertrophy.

Mechanisms of muscle atrophy induced by glucocorticoids.

Background: Many pathological states characterized by muscle atrophy (e.g., sepsis, cachexia, starvation, metabolic acidosis and severe insulinopenia) are associated with an increase in circulating glucocorticoid (GC) levels, suggesting that GC could trigger the muscle atrophy observed in these conditions. GC-induced muscle atrophy results from decreased protein synthesis and increased protein degradation. The inhibitory effect of GCs on protein synthesis is thought to result mainly from the inhibition of the p70 ribosomal S6 protein kinase. The stimulatory effect of GCs on muscle proteolysis results from the activation of two major cellular proteolytic systems: ubiquitin proteasome and lysosomal systems. The decrease in muscle production of insulin-like growth factor I (IGF-I), a muscle anabolic growth factor, could contribute to GC-induced muscle atrophy. By activating the phosphatidylinositol-3-kinase/Akt pathway, IGF-I overrides GC action to stunt muscle atrophy. Evidence also indicates that increased production of myostatin, a catabolic growth factor, could play a critical role in GC-induced muscle atrophy. Conclusions: Recent progress in understanding the role of growth factors in GC-induced muscle atrophy allows investigation into new therapies to minimize this myopathy.

Higher activation of autophagy in skeletal muscle of mice during endurance exercise in the fasted state.

Activation of autophagy in skeletal muscle has been reported in response to endurance exercise and food deprivation independently. The purpose of this study was to evaluate whether autophagy was more activated when both stimuli were combined, namely when endurance exercise was performed in a fasted rather than a fed state. Mice performed a low-intensity running exercise (10 m/min for 90min) in both dietary states after which the gastrocnemius muscles were removed. LC3b-II, a marker of autophagosome presence, increased in both conditions, but the increase was higher in the fasted state. Other protein markers of autophagy, like Gabarapl1-II and Atg12 conjugated form as well as mRNA of Lc3b, Gabarapl1, and p62/Sqstm1 were increased only when exercise was performed in a fasted state. The larger activation of autophagy by exercise in a fasted state was associated with a larger decrease in plasma insulin and phosphorylation of Akt(Ser473), Akt(Thr308), FoxO3a(Thr32), and ULK1(Ser757). AMPKα(Thr172), ULK1(Ser317), and ULK1(Ser555) remained unchanged in both conditions, whereas p38(Thr180/Tyr182) increased during exercise to a similar extent in the fasted and fed conditions. The marker of mitochondrial fission DRP1(Ser616) was increased by exercise independently of the nutritional status. Changes in mitophagy markers BNIP3 and Parkin suggest that mitophagy was increased during exercise in the fasted state. In conclusion, our results highlight a major implication of the insulin-Akt-mTOR pathway and its downstream targets FoxO3a and ULK1 in the larger activation of autophagy observed when exercise is performed in a fasted state compared with a fed state.

The Type 1 Insulin-Like Growth Factor Receptor (IGF-IR) Pathway Is Mandatory for the Follistatin-Induced Skeletal Muscle Hypertrophy

Myostatin inhibition by follistatin (FS) offers a new approach for muscle mass enhancement. The aim of the present study was to characterize the mediators responsible for the FS hypertrophic action on skeletal muscle in male mice. Because IGF-I and IGF-II, two crucial skeletal muscle growth factors, are induced by myostatin inhibition, we assessed their role in FS action. First, we tested whether type 1 IGF receptor (IGF-IR) is required for FS-induced hypertrophy. By using mice expressing a dominant-negative IGF-IR in skeletal muscle, we showed that IGF-IR inhibition blunted by 63% fiber hypertrophy caused by FS. Second, we showed that FS caused the same degree of fiber hypertrophy in wild-type and IGF-II knockout mice. We then tested the role of the signaling molecules stimulated by IGF-IR, in particular the Akt/mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (S6K) pathway. We investigated whether Akt phosphorylation is required for the FS action. By cotransfecting a dominant-negative form of Akt together with FS, we showed that Akt inhibition reduced by 65% fiber hypertrophy caused by FS. Second, we evaluated the role of mTOR in FS action. Fiber hypertrophy induced by FS was reduced by 36% in rapamycin-treated mice. Finally, because the activity of S6K is increased by FS, we tested its role in FS action. FS caused the same degree of fiber hypertrophy in wild-type and S6K1/2 knockout mice. In conclusion, the IGF-IR/Akt/mTOR pathway plays a critical role in FS-induced muscle hypertrophy. In contrast, induction of IGF-II expression and S6K activity by FS are not required for the hypertrophic action of FS.

Functional assessment of skeletal muscle in intact mice lacking myostatin by concurrent NMR imaging and spectroscopy

Inhibiting myostatin (mstn) causes spectacular increase in muscle mass, spurring research for therapeutic approaches against neuromuscular disorders. Yet, possible functional deterioration and compromised force production have been reported in isolated muscle of null mstn(−/−) mice. We analyzed vascular and metabolic response to repeated electro-stimulated exercise in vivo in mstn−/− mice compared with FVB wild-type controls (WT), using interleaved multi-parametric functional nuclear magnetic resonance (NMR) imaging and spectroscopy. At steady-state exercise, specific force of plantar flexion, phosphocreatine consumption measured by phosphorus spectroscopy and maximum perfusion measured by arterial spin-labeled (ASL) NMR imaging were identical in both groups. After exercise, phosphorus spectroscopy revealed reduced oxidative mitochondrial capacity in mstn−/−, whereas early recovery perfusion was identical and oxygen extraction, estimated from the blood oxygen level-dependent (BOLD) contrast, was decreased when compared with WT. Hyperemia was prolonged, indicating specific regulation of the perfusional response in mstn−/− mice. Histology showed an increased proportion of type IIb fibers in hypertrophied muscles, but the distribution of capillary contacts per fiber between oxidative and glycolytic fibers was unaltered in mstn−/− compared with WT. These integrated results formed coherent evidence of a congruous, non-pathologic shift toward a more glycolytic metabolism in this model of mstn−/−.

Activation of ER stress by hydrogen peroxide in C2C12 myotubes.

The purpose of this study was to examine the link between oxidative stress and endoplasmic reticulum (ER) stress in myogenic cells. C2C12 myotubes were incubated with hydrogen peroxide (H2O2, 200 μM) and harvested 4h or 17 h after the induction of this oxidative stress. A massive upregulation of binding immunoglobulin protein (BiP) was found, indicating the presence of ER stress. Nevertheless, the three branches of the unfolded protein response (UPR) were not activated to the same extent. The double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK) branch was the most activated as shown by the increase of phospho-eukaryotic translation-initiation factor 2α (eIF2α, Ser51) and the mRNA levels of activating transcription factor 4 (ATF4), C/EBP homologous (CHOP) and tribbles homolog 3 (TRB3). The slight increase in the spliced form of X-box binding protein 1 (XBP1s) together with the decrease of the unspliced form (XBP1u) indicated a higher endoribonuclease activity of inositol-requiring 1α (IRE1α). The transcriptional activity of activating transcription factor 6 (ATF6) remained unchanged after incubation with H2O2. The mechanisms by which the three branches of UPR can be specifically regulated by oxidative stress are currently unresolved and need further investigations.

Multiparametric functional nuclear magnetic resonance imaging shows alterations associated with plasmid electrotransfer in mouse skeletal muscle.

In vivo gene electrotransfer is frequently used in preclinical gene therapy. Many studies have attempted to optimize protocols efficiency at the same time as reducing muscle damage. Most of them have reported histological evidence of muscle degeneration and completion of regeneration within 15 days. The functional consequences have rarely been addressed, which may reflect the lack of appropriate techniques. Yet, it is important to characterize the changes induced by the procedure itself because it may interfere with therapy. We used multiparametric functional (mpf)‐nuclear magnetic resonance (NMR) imaging to evaluate mice hindlimb muscle after electrotransfer of an empty plasmid.

SPONTANEOUS CP VIOLATION AND MINIMAL COUPLING TO SUPERGRAVITY

In the framework of minimal coupling to supergravity, it is found that the assumption of spontaneous /b CP/ violation allows one to generate naturally real couplings between the high-energy and the low-energy sectors of the theory, at the grand unification scale /b M//sub x/. These boundary conditions for the renormalization group equations imply sufficiently small imaginary parts of these couplings at the weak scale /b M//sub w/, to ensure that the experimental bound on the electric dipole moment of the neutron is respected.