Here we describe the phenotype of mice in which mTORC1 is constitutively active in skeletal muscle (TSCmKO) and compare it to mice with inactivated mTORC1 signaling (RAmKO). While the oxidative changes in TSCmKO mice were largely the opposite of those observed in RAmKO mice and affected all examined muscles, the effect of mTORC1 activation on muscle size was unexpected as all muscles except soleus muscles were slightly but significantly smaller. Thus, our work highlights the existence of several feed-forward or auto-inhibitory loops that allow fine-tuning of the signaling networks involved in the control of muscle mass (Figure 7E).
Based on the current concepts, mTORC1 activation should result in an increase in muscle mass and muscle fiber size. This view is based on the findings that activation of the mTORC1 upstream components PKB/Akt or IGF-1 receptor causes an increase in muscle mass [5, 10, 11, 52, 53] and that this increase is rapamycin-sensitive [11, 53]. Moreover, overexpression of Rheb in single muscle fibers by electroporation leads to hypertrophy of the transfected fibers  and whole body knockout of the mTORC1 target S6K1 results in smaller muscle fibers . Consistent with these experiments, acute knockdown of TSC1/2 by shRNA resulted in slightly bigger muscle fibers in soleus or TA muscles, confirming that transient activation of the mTORC1 pathway is sufficient to induce muscle fiber growth. However, under conditions of prolonged activation of mTORC1 in TSCmKO mice, all muscles examined, with the exception of soleus, were smaller than in control mice. As mTORC1 targets are activated and protein synthesis in EDL muscle of TSCmKO mice is increased, the atrophy induced by chronic mTORC1 activation is likely related to the feedback inhibition of activated S6K onto IRS1, which in turn, decreases activation of PKB/Akt. This tight feedback control of S6K on IRS1-PKB/Akt was also observed in mice deficient for raptor or mTOR in some tissues including skeletal and heart muscle [16, 54–56] but not in others . Similarly, deletion of TSC1 strongly decreases activation of PKB/Akt in cultured mouse embryonic fibroblasts , whereas it does not at all affect PKB/Akt phosphorylation in some tissues [58, 59]. These data indicate that the feedback control of S6K depends on the cellular context and our data now show that this feedback is particularly strong in skeletal muscle.
Consistent with decreased inhibition of FoxO transcription factors by PKB/Akt, TA muscle from TSCmKO mice express high levels of MuRF1 and atrogin-1/MAFbx, involved in protein degradation through the proteasome [7, 8]. Hence, the atrophy observed in muscles of the TSCmKO mice is likely caused by the prevalence of the FoxO pathway over mTORC1 activation. This differs from the muscle hypertrophy observed using the transient, partial activation of mTORC1 with shRNA electroporation. Thus, the atrophy response caused by the sustained, saturated mTORC1 activation by genetic Tsc1 deletion may unveil a long-term adaptation of the FoxO pathway. Consistently, transient overexpression of Rheb does not seem to affect PKB/Akt phosphorylation , further supporting the idea that muscle atrophy in TSCmKO mice is related to the indirect PKB/Akt-dependent activation of FoxO pathways.
Importantly, contrasting with the atrophic phenotype of most muscles, sustained activation of mTORC1 leads to increased mass of soleus muscle in TSCmKO mice. Although PKB/Akt was similarly inhibited in soleus and TA muscles, expression of MuRF1 and atrogin-1/MAFbx was not increased in soleus muscle, indicating that an additional regulatory mechanism suppresses their expression, thereby overruling the regulation by PKB/Akt. This differential regulation of MuRF1 and atrogin-1/MAFbx expression did not seem to be mediated by PGC1α, previously identified as a negative regulator of FoxO , because there was no significant difference in PGC1α/β expression between soleus and TA muscles from TSCmKO mice.
With different atrophy and hypertrophy paradigms, we also demonstrate that mTORC1 plays a critical and complex role in muscle plasticity. Using shRNA electroporation, we show that transient activation of mTORC1 is sufficient to limit denervation-induced atrophy and to enhance fiber hypertrophy upon re-innervation. Similarly, TSCmKO mice display atrophy resistance to denervation in soleus muscle, which shows only moderate expression of the E3 ubiquitin ligases MuRF1 and atrogin-1/MAFbx. By contrast, long-term activation of mTORC1 did not protect TA muscle from atrophy and did not exacerbate the hypertrophy response to overloading of plantaris muscle. These results indicate that the increased protein synthesis by mTORC1 hyperactivation is not sufficient to maintain muscle mass in cases where the FoxO-MuRF1-atrogin-1/MAFbx axis is active due to the absence of PKB/Akt signaling. Importantly, both transient and long-term inactivation of mTORC1 increased denervation-induced atrophy and prevented muscle growth associated with re-innervation or overloading, indicating that increased protein synthesis is required even when the catabolic proteasomal activity is reduced. Thus, our results provide genetic evidence that muscle growth requires mTORC1.
In our previous work, we demonstrated that raptor-deficient skeletal muscles show a strongly decreased oxidative capacity due to changes in mitochondrial function . This loss of oxidative capacity correlated with a substantial decrease in the transcript levels of Pgc1α, consistent with the direct regulation of Pgc1α expression by mTOR , and could be restored by transgenic expression of PGC1α . Contrary to the expectations and the effect of mTORC1 activation in embryonic fibroblasts , all examined muscles of TSCmKO mice showed a decreased expression of Pgc1α but increased levels of Pgc1β. Thus, the increase in the oxidative capacity in TSCmKO mice may be mediated by PGC1β. Indeed, PGC1β has also been shown to be sufficient to increase oxidative capacity in skeletal muscle despite the concomitant reduction in PGC1α expression . Moreover, depletion of both PGC1α and PGC1β results in much more severe loss of oxidative capacity than depletion of either protein alone . The reason for the unexpected down-regulation of Pgc1α transcripts in TSCmKO mice might be the counter-regulation of PGC1α and PGC1β. We show here that overexpression of PGC1β in C2C12 myotubes results in a strong suppression of the endogenous Pgc1α expression and, conversely, Pgc1β knockdown leads to increased expression of Pgc1α transcripts. These data indicate that the total amount of both PGC1 co-activators is tightly controlled in skeletal muscle.