Background/Aims: The purpose of the study was to assess the amount of rRNA and phosphorylation status of the key markers of mTORC1-dependent (70s6k, 4E-BP1) and mTORC1-independent (GSK-3β, AMPK) signaling pathways controlling protein synthesis in rat soleus during early stages of mechanical unloading (hindlimb suspension (HS) for 1-, 3- and 7 days). Methods: The content of the key signaling molecules of various anabolic signaling pathways was determined by Western-blotting. The amount of 28S rRNA was evaluated by RT-PCR. The rate of protein synthesis was assessed using in-vivo SUnSET technique. Results: HS for 3 and 7 days induced a significant (p<0.05) decrease in the rate of global protein synthesis in soleus muscle in comparison with control. HS within 24 hours resulted in a significant (p<0.05) decrease in p-4E-BP1 content, p-AMPK content and increase in p-p70s6k content in rat soleus muscle. Following three days of HS the content of p-AKT was decreased (p<0.05). After 7 days of HS the phosphorylation level of AKT and GSK-3beta was significantly reduced (p<0.05) compared to control. We also observed a significant decrease in the amount of 28S rRNA in rat soleus following 1, 3 and 7 days of HS. Conclusion: Taken together, the results of our study suggest that a decline in the global rate of protein synthesis in rat soleus during early stages of simulated microgravity is associated with impaired ribosome biogenesis as well as reduced activity of mTORC1-independent signaling pathways.

Background: Duchenne muscular dystrophy (DMD) is a fatal muscle‐wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex‐miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex‐miRNAs (e.g. miR‐1, miR‐133a, miR‐206, and miR‐483) are highly up‐regulated in the serum of DMD patients and dystrophic animal models and are restored to wild‐type levels following exon skipping‐mediated dystrophin rescue in mdx mice. As such, ex‐miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex‐miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle.Methods: Candidate ex‐miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide‐PMO (PPMO) exon skipping conjugates and in mdx‐Xist^Δhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X‐chromosome inactivation. miRNA profiling was performed in mdx‐Xist^Δhs mice using the FirePlex methodology and key results validated by small RNA TaqMan RT‐qPCR. The muscles from each animal model were further characterized by dystrophin western blot and immunofluorescence staining.Results: The restoration of ex‐myomiR abundance observed following PPMO treatment was not recapitulated in the high dystrophin‐expressing mdx‐Xist^Δhs group, despite these animals expressing similar amounts of total dystrophin protein (~37% of wild‐type levels). Instead, ex‐miRNAs were present at high levels in mdx‐Xist^Δhs mice regardless of dystrophin expression. PPMO‐treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx‐Xist^Δhs muscles showed non‐homogeneous dystrophin staining and sporadic regenerating foci.Conclusions: Uniform dystrophin expression is required to prevent ex‐miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle.

Expression of FoxO transcription factors increases during certain forms of atrophy. In a dephosphorylated state, FoxOs participate in ubiquitin-mediated proteasomal degradation through the transcriptional activation of E3-ubiquitin ligases such as MAFbx/atrogin-1 and MuRF1. There is exhaustive research demonstrating that FoxO3a is sufficient to induce MAFbx/atrogin-1 and MuRF-1 expressions. In contrast, the data are conflicting on the requirement of FoxO1 signaling in the activation of the E3-ubiquitin ligases. Moreover, no reports currently exist on the particular role of FoxO1 in the molecular mechanisms involved in the progression of physiological muscle wasting. Here, we have applied the most extensively used rodent model of microgravity/functional unloading to stimulate disuse-induced skeletal muscle atrophy such as rat hindlimb suspension (HS). We showed that inhibition of FoxO1 activity by a selective inhibitor AS1842856 completely reversed an increase in expression of MuRF-1, but not MAFbx/atrogin-1, observed upon HS. Furthermore, we demonstrated that FoxO1 induced upregulation of another E3-ubiquitin-ligase of a MuRF protein family MuRF-2 in skeletal muscle subjected to disuse. Prevention of the MuRF increase upon HS impeded upregulation of transcript expression of a negative regulator of NFATc1 pathway calsarcin-2, which was associated with a partial reversion of MyHC-IId/x and MyHC-IIb mRNA expressions. Importantly, FoxO1 inhibition induced a marked increase in p70S6k phosphorylation, an important stage in the initiation of protein translation, concomitant with the restoration of global protein synthesis in the skeletal muscle of the HS rats. Examination of eIF3f expression and the eEF2k/eEF2 pathway, other factors controlling translation initiation and elongation respectively, did not reveal any impact of FoxO1 on their activity. Lastly, we observed a decrease in transcript levels of Sesn3, but not Sesn1 and Sesn2, upon disuse, which was completely reversed by FoxO1 inhibition. These data demonstrate that FoxO1 signaling contributes to the development of disuse-induced skeletal muscle atrophy, including slow to fast MyHC isoform shift, mostly through upregulation of MuRF-1 and MuRF-2 expression. Furthermore, FoxO1 inhibition is required to recover Sesn3 mRNA expression in atrophic conditions, which likely contributes to the enhanced p70S6k activity and restoration of the protein synthesis rate.

We hypothesized that pharmacological induction of HSP70 would attenuate soleus atrophy development under 3 d of rat hindlimb unloading. Male Wistar rats were divided into control (C; n =7), 3‐d hindlimb unloading (HUL; n =7), HUL with HSP90 inducer administration, 17‐allylamino‐17‐emethoxygel‐danamycin (17‐AAG; 60 mg/kg, HUL+17‐AAG, n =8). The relative weight of soleus muscle to body weight [soleus wt (mg)/body wt (g)] in the HUL group was less than that of the C and HUL + 17‐AAG groups ( P <0.05). We revealed HSP90, HSP70 mRNA decrease in the HUL group (but not the HUL+ 17‐AAG group) vs. C ( P <0.05). The unloading resulted in significant increases of μ‐calpain and conjugated ubiquitin (Ub) levels (proteins as well as mRNAs) vs. the C group, whereas 17‐AAG administration prevented these alterations (studied by SDS‐PAGE and RT‐PCR). pFOXO3 protein was decreased in the HUL group vs. C, but not in HUL + 17‐AAG. Content of E3‐lygase (MuRF‐1, MAFbx) mRNA was increased in both suspended groups. In summary, 17‐AAG administration attenuates soleus muscle atrophy, μ‐calpain, and Ub increases under hindlimb unloading as well as decrease of pFOXO3.—Lomonosova, Y. N., Shenkman, B S., Nemirovskaya, T. L. Attenuation of unloading‐induced rat soleus atrophy with the heat‐shock protein inducer 17‐(allylamino)‐17‐demethoxygeldanamycin. FASEB J. 26, 4295–4301 (2012). www.fasebj.org