Although it is well known that administration of the selective β(2)-adrenergic agonist clenbuterol (CB) protects muscle following denervation (DEN), the underlying molecular mechanism remains unclear. We report that in vivo treatment with CB (3 mg/kg sc) for 3 days induces antiproteolytic effects in normal and denervated rat soleus muscle via distinct mechanisms. In normal soleus muscle, CB treatment stimulates protein synthesis, inhibits Ca(2+)-dependent proteolysis, and increases the levels of calpastatin protein. On the other hand, the administration of CB to DEN rats ameliorates the loss of muscle mass, enhances the rate of protein synthesis, attenuates hyperactivation of proteasomal and lysosomal proteolysis, and suppresses the transcription of the lysosomal protease cathepsin L and of atrogin-1/MAFbx and MuRF1, two ubiquitin (Ub) ligases involved in muscle atrophy. These effects were not associated with alterations in either IGF-I content or Akt phosphorylation levels. In isolated muscles, CB (10(-6) M) treatment significantly attenuated DEN-induced overall proteolysis and upregulation in the mRNA levels of the Ub ligases. Similar responses were observed in denervated muscles exposed to 6-BNZ-cAMP (500 μM), a PKA activator. The in vitro addition of triciribine (10 μM), a selective Akt inhibitor, did not block the inhibitory effects of CB on proteolysis and Ub ligase mRNA levels. These data indicate that short-term treatment with CB mitigates DEN-induced atrophy of the soleus muscle through the stimulation of protein synthesis, downregulation of cathepsin L and Ub ligases, and consequent inhibition of lysosomal and proteasomal activities and that these effects are independent of Akt and possibly mediated by the cAMP/PKA signaling pathway.

AimTo identify microRNAs (miRs) involved in the regulation of skeletal muscle mass. For that purpose, we have initially utilized an in silico analysis, resulting in the identification of miR‐29c as a positive regulator of muscle mass.MethodsmiR‐29c was electrotransferred to the tibialis anterior to address its morphometric and functional properties and to determine the level of satellite cell proliferation and differentiation. qPCR was used to investigate the effect of miR‐29c overexpression on trophicity‐related genes. C2C12 cells were used to determine the impact of miR‐29c on myogenesis and a luciferase reporter assay was used to evaluate the ability of miR‐29c to bind to the MuRF1 3′UTR.ResultsThe overexpression of miR‐29c in the tibialis anterior increased muscle mass by 40%, with a corresponding increase in fibre cross‐sectional area and force and a 30% increase in length. In addition, satellite cell proliferation and differentiation were increased. In C2C12 cells, miR‐29c oligonucleotides caused increased levels of differentiation, as evidenced by an increase in eMHC immunostaining and the myotube fusion index. Accordingly, the mRNA levels of myogenic markers were also increased. Mechanistically, the overexpression of miR‐29c inhibited the expression of the muscle atrophic factors MuRF1, Atrogin‐1 and HDAC4. For the key atrogene MuRF1, we found that miR‐29c can bind to its 3′UTR to mediate repression.ConclusionsThe results herein suggest that miR‐29c can improve skeletal muscle size and function by stimulating satellite cell proliferation and repressing atrophy‐related genes. Taken together, our results indicate that miR‐29c might be useful as a future therapeutic device in diseases involving decreased skeletal muscle mass.

Although we have recently demonstrated that plasma catecholamines induce antiproteolytic effects on skeletal muscle (Graça FA, Gonçalves DAP, Silveira WA, Lira EC, Chaves VE, Zanon NM, Garófalo MAR, Kettelhut IC, Navegantes LCC. Am J Physiol Endocrinol Metab. 305: E1483-E1494, 2013), the role of the muscle sympathetic innervation and, more specifically, norepinephrine (NE) in regulating the ubiquitin (Ub)-proteasome system (UPS) remains unknown. Based on previous findings that chemical sympathectomy acutely reduces UPS activity, we hypothesized that muscle NE depletion induces adrenergic supersensitivity in rat skeletal muscles. We report that surgical sympathetic denervation (SDEN), a condition in which only muscle NE from both hindlimbs is depleted, transiently reduced the overall proteolysis and the UPS activity (∼25%) in both soleus and extensor digitorum longus muscles. This antiproteolytic response was accompanied by increased activity of adenylyl cyclase (112%), levels of cyclic adenosine monophosphate (cAMP; 191%), and the serine phosphorylation of cAMP response element-binding protein (32%). In extensor digitorum longus from normal rats, NE (10(-4) M) in vitro increased the levels of cAMP (115%) and the serine phosphorylation of both cAMP response element-binding protein (2.7-fold) and forkhead box class O1 transcription factor. Similar effects were observed in C2C12 cells incubated with forskolin (10 μM). In parallel, NE significantly reduced the basal UPS (21%) activity and the mRNA levels of atrophy-related Ub-ligases. Similar responses were observed in isolated muscles exposed to 6-BNZ-cAMP (500 μM), a specific PKA activator. The phosphorylation levels of Akt were not altered by SDEN, NE, forskolin or 6-BNZ-cAMP. Our results demonstrate that SDEN induces muscle adrenergic supersensitivity for cAMP leading to the suppression of UPS, and that the suppressive effects of NE on UPS activity and expression of Ub-ligases can be mediated by the activation of cAMP/PKA signaling, with the inhibition of forkhead box class O1 transcription factor.

Background Stimulation of β 2 ‐adrenoceptors can promote muscle hypertrophy and fibre type shift, and it can counteract atrophy and weakness. The underlying mechanisms remain elusive. Methods Fed wild type (WT), 2‐day fasted WT, muscle‐specific insulin (INS) receptor (IR) knockout (M‐IR −/− ), and MKR mice were studied with regard to acute effects of the β 2 ‐agonist formoterol (FOR) on protein metabolism and signalling events. MKR mice express a dominant negative IGF1 receptor, which blocks both INS/IGF1 signalling. All received one injection of FOR (300 μg kg −1 subcutaneously) or saline. Skeletal muscles and serum samples were analysed from 30 to 240 min. For the study of chronic effects of FOR on muscle plasticity and function as well as intracellular signalling pathways, fed WT and MKR mice were treated with formoterol (300 μg kg −1 day −1 ) for 30 days. Results In fed and fasted mice, one injection of FOR inhibited autophagosome formation (LC3‐II content, 65%, P ≤ 0.05) that was paralleled by an increase in serum INS levels (4‐fold to 25‐fold, P ≤ 0.05) and the phosphorylation of Akt (4.4‐fold to 6.5‐fold, P ≤ 0.05) and ERK1/2 (50% to two‐fold, P ≤ 0.05). This led to the suppression (40–70%, P ≤ 0.05) of the master regulators of atrophy, FoxOs, and the mRNA levels of their target genes. FOR enhanced (41%, P ≤ 0.05) protein synthesis only in fed condition and stimulated (4.4‐fold to 35‐fold, P ≤ 0.05) the prosynthetic Akt/mTOR/p70S6K pathway in both fed and fasted states. FOR effects on Akt signalling during fasting were blunted in both M‐IR −/− and MKR mice. Inhibition of proteolysis markers by FOR was prevented only in MKR mice. Blockade of PI3K/Akt axis and mTORC1, but not ERK1/2, in fasted mice also suppressed the acute FOR effects on proteolysis and autophagy. Chronic stimulation of β 2 ‐adrenoceptors in fed WT mice increased body (11%, P ≤ 0.05) and muscle (15%, P ≤ 0.05) growth and downregulated atrophy‐related genes (30–40%, P ≤ 0.05), but these effects were abolished in MKR mice. Increases in muscle force caused by FOR (WT, 24%, P ≤ 0.05) were only partially impaired in MKR mice (12%, P ≤ 0.05), and FOR‐induced slow‐to‐fast fibre type shift was not blocked at all in these animals. In MKR mice, FOR also restored the lower levels of muscle SDH activity to basal WT values and caused a marked reduction (57%, P ≤ 0.05) in the number of centrally nucleat...

Decline in skeletal muscle cell size (myofiber atrophy) is a key feature of cancer-induced wasting (cachexia). In particular, atrophy of the diaphragm, the major muscle responsible for breathing, is an important determinant of cancer-associated mortality. However, therapeutic options are limited. Here, we have used Drosophila transgenic screening to identify muscle-secreted factors (myokines) that act as paracrine regulators of myofiber growth. Subsequent testing in mouse myotubes revealed that mouse Fibcd1 is an evolutionary-conserved myokine that preserves myofiber size via ERK signaling. Local administration of recombinant Fibcd1 (rFibcd1) ameliorates cachexia-induced myofiber atrophy in the diaphragm of mice bearing patient-derived melanoma xenografts and LLC carcinomas. Moreover, rFibcd1 impedes cachexia-associated transcriptional changes in the diaphragm. Fibcd1-induced signaling appears to be muscle selective because rFibcd1 increases ERK activity in myotubes but not in several cancer cell lines tested. We propose that rFibcd1 may help reinstate myofiber size in the diaphragm of patients with cancer cachexia.