Dear Editor,
Several recent clinical studies have indicated that dietary supplementation with branched-chain amino acids (BCAA), particularly with leucine, is an effective anti-atrophic therapy (
Bauer et al., 2015;
Tsien et al., 2015;
English et al., 2016). In animal models, BCAA can prevent denervation (
Ribeiro et al., 2015), hindlimb suspension (
Maki et al., 2012;
Jang et al., 2015) or dexamethasone-induced (
Yamamoto et al., 2010) muscle atrophy. General control nonderepressible 2 kinase (GCN2) is a well-known amino-acid sensor. Under conditions of amino-acid deprivation, the increased level of uncharged transfer RNA (tRNA) activates GCN2 through binding to the histadyl-tRNA synthetase-like domain (
Wek et al., 1995). Upon activation, GCN2 phosphorylates eukaryotic initiation factor 2 alpha at Ser51, which leads to translational arrest and restoration of amino acid homeostasis (
Wek et al., 1995;
Sood et al., 2000). As amino acids are potent modulators of protein turnover in skeletal muscle, we proposed that GCN2 may affect denervation-induced muscle atrophy, but the detail mechanism remains unclear.
To investigate the impact of GCN2 on the development of muscle atrophy, we performed sciatic denervation procedure on hindlimb muscles in wild-type (WT) and Gcn2−/− mice. On day 7 after denervation, WT tibial anterior (TA) muscle mass decreased to 70.9% ± 1.8% of the contralateral level, while GCN2-deficient TA muscle mass remained at 83.1% ± 1.6% of its contralateral level (Fig. 1A). GCN2 deficiency also significantly attenuated the muscle mass loss in atrophied gastrocnemius (GAS) and extensor digitorum longus (EDL) muscles. Similar results were observed on day 14 after denervation (Fig. S1). Wheat germ agglutinin (WGA) staining of muscle cryosections demonstrated that GCN2-deficient TA muscles had a better preservation of myofiber size in response to denervation (Fig. 1B). After denervation, myofiber size distribution calculated from WT TA muscles showed a leftward shift from its contralateral conditions. However, such shift was delayed in atrophied GCN2-deficient TA muscles (Fig. 1C). To further verify whether activation of GCN2 contributes to muscle atrophy, we over-expressed GCN2 in flexor digitorum brevis (FDB) muscles using in vivo electroporation. The transfection efficiency was confirmed by Western blot (Fig. S2). After denervation for ten days, the diameter of WT FDB myofiber decreased from 37.2 ± 0.9 μm to 24.2 ± 0.7 μm, while the diameter of Gcn2−/− FDB myofiber was maintained at 32.3 ± 0.7 μm. Overexpression of GCN2 resulted in a further reduction in the diameter of FDB myofibers in both WT and Gcn2−/− mice. However, the reduction was more dramatically in Gcn2−/− FDB muscle and the significant difference in the diameter of atrophied FDB myofiber between WTand Gcn2−/− mice was diminished after transfected with the GCN2 plasmid (Fig. 1D).
Emerging evidence suggests that the protein degradation in muscle atrophy is mediated by FoxO3a, an important regulator of
Atrogin-
1,
MuRF-
1 and
LC3 (
Sandri et al., 2004;
Zhao et al., 2007;
Guo et al., 2016). Upon atrophy stimuli, FoxO3a shuffled into the myofiber nucleus, which leads to transcriptional activation (
Sandri et al., 2004). Through Western blotting and immunostaining, we observed that the phosphorylation level of FoxO3a at Ser207 were increased in TA muscles of WT mice after denervation, and FoxO3 was mainly located in the nucleus. However, the denervation-induced phosphorylation and nuclear accumulation of FoxO3a were significantly less in GCN2-deficient muscles compared with that in WT muscles (Fig. 2A–B). We also observed significant increases in protein expression of MuRF-1 and Atrogin-1, as well as the ratio of conversion of LC3 into the activated forms (LC3-II/I) in WT atrophic TA muscles. However, increases in E3 ubiquitin ligases expression and activation of autophagy were statistically less remarkable in atrophic
Gcn2−/− muscles than in WT (Fig. S3).
To investigate whether GCN2 activation directly causes FoxO3a nuclear translocation in muscle atrophy, we generated a stable C2C12 cell line (mGCN2-C2C12) with doxycycline (Dox)-controlled expression of flag-tagged mouse GCN2 (Fig. 2C), and found that GCN2 overexpression induced by Dox (Dox+) significantly increased the ratio of FoxO3a in the nucleus (Fig. 2D). Furthermore, co-immunoprecipitation experiments using lysates from FoxO3a plasmid-transfected mGCN2-C2C12 cells with Dox demonstrated that GCN2-Flag and FoxO3a-EGFP were specifically co-precipitated with anti-GFP and anti-Flag antibodies, respectively (Fig. 2E), demonstrating that GCN2 and FoxO3a can physically interact with each other in cells. Using the differentiated mGCN2-C2C12 cells, we also found that overexpression of GCN2 exacerbated dexamethasone-induced upregulation of Atrogin-1 and LC3-II in differentiated C2C12 cells (Fig. S4).
It has been demonstrated dietary deprivation of essential amino acids, which activates GCN2 via increasing uncharged tRNA levels (
Wek et al., 1995), caused diffuse atrophy in the rectus femoris muscles (
Kamata et al., 2014). In contrast, leucine or other BCAA supplementation, which attenuates GCN2 activity (
Wek et al., 1995), has been regarded as a potential pharmaconutrient for the treatment of numerous muscle wasting conditions (
Bauer et al., 2015;
Tsien et al., 2015;
English et al., 2016). In agreement with those findings, we demonstrated that GCN2 deletion attenuates, whereas GCN2 overexpression exacerbates denervation-induced muscle atrophy. Furthermore, the detrimental effect of GCN2 in denervation-induced atrophy was related to FoxO3a activation, which upregulates genes involved in both the ubiquitin-proteasome pathway and autophagy in muscle atrophy (
Sandri et al., 2004;
Bertaggia et al., 2012;
Wei et al., 2013;
Guo et al., 2016). Thus, reducing GCN2 activity may be a potential therapeutic approach for the clinical treatment of muscle atrophy.
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