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.

Cytoskeletal and contractile proteins degenerate during functional unloading of muscle. The ratio of myosin heavy chain (MHC) expression changes simultaneously. We have supposed that NO can be a signal molecule related to the regulation of protein metabolism upon muscle unloading. To test this hypothesis, Wistar rats underwent functional unloading for 14 days without and with peroral administration of L-arginine (500 mg/kg) as NO precursor. Significant decreases in m. soleus mass, NO, nNOS, dystrophin, Hsp90, p-S6K, and type I MHC mRNA contents were found in the group of animals with unloading without preparation compared to those in control and in the group with unloading and administration of L-arginine; at the same time, increased contents of atrogin-1/MAFbx and MuRF-1 (p < 0.05) were found. No difference in the IGF-1 mRNA content between all three groups was found. Atrophy was significantly less pronounced in the group with unloading and L-arginine administration compared to that without the amino acid, and no destruction of cytoskeletal proteins was observed. We conclude that administration of L-arginine upon functional unloading decreases the extent of m. soleus atrophy, prevents the decrease in it of type I MHC mRNA, and blocks destructive changes in some cytoskeletal proteins. Such effect can be due to the absence of increase in this group of the content of some ubiquitin ligases and decreased intensity of the p70S6 kinase synthesis marker.

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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.

Under muscle disuse conditions decrease of expression of MyHC of slow type, and sometimes of type IIa, as well as upregulation of expression of IIb and IId/x isoforms were observed. Through dephosphorylation and entry of NFAT molecules to the nucleus calcineurin/NFATc1 signaling pathway promotes upregulation of the slow MyHC expression. We supposed that downregulation of calcineurin pathway took place during unloading. The study was aimed to analyze the states of the myonuclear NFAT inhibitors calsarcin I (CSI) and calsarcin II (CSII) (also referred to as myozenin II and I) and GSK3β in rat soleus during hindlimb suspension (HS). Male Wistar rats were subjected to 3, 7 and 14 day of HS. We found that after 3 days of HS the content of CSII mRNA twofold increased in soleus as compared to the controls. This level was increased by more than fivefold (as compared to control) after 2 weeks of HS. The increase of CSII mRNA expression may be explained as the mechanism of stabilization of fast phenotype. We found that from the 3 day till 14 day of HS the content of MuRF-1 and MuRF-2 in the nuclear fraction fourfold to fivefold increased in HS soleus. We supposed that nuclear import of the MuRFs allows to promote CSII expression during unloading. We also observed the decline of the phosphorylated GSK3β content in the nuclear extract of the soleus tissue. Thus decline of slow MyHC expression characteristic for the unloading conditions is accompanied with the increased expression and activation of the factors known to prevent NFAT accumulation in the myonuclei.

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-emethoxygeldanamycin (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.

Eccentric exercise is known to disrupt sarcolemmal integrity and induce damage of skeletal muscle fibers. We hypothesized that L-arginine (L-Arg; nitric oxide synthase (NOS) substrate) supplementation prior to a single bout of eccentric exercise would diminish exercise-induced damage. In addition, we used N-nitro-L-arginine methyl ester hydrochloride (L-NAME; NOS inhibitor) to clarify the role of native NOS activity in the development of exercise-induced muscle damage. Rats were divided into four groups: non-treated control (C), downhill running with (RA) or without (R) L-Arg supplementation and downhill running with L-NAME supplementation (RN). Twenty four hours following eccentric exercise seven rats in each group were sacrificed and soleus muscles were dissected and frozen for further analysis. The remaining seven rats in each group were subjected to the exercise performance test. Our experiments showed that L-Arg supplementation prior to a single bout of eccentric exercise improved subsequent exercise performance capacity tests in RA rats when compared with R, RN and C rats by 37%, 27% and 13%, respectively. This outcome is mediated by L-Arg protection against post-exercise damage of sarcolemma (2.26- and 0.87-fold less than R and RN groups, respectively), reduced numbers of damaged muscle fibers indicated by the reduced loss of desmin content in the muscle (15% and 25% less than R and RN groups, respectively), and diminished µ-calpain mRNA up-regulation (42% and 30% less than R and RN groups, respectively). In conclusion, our study indicates that L-Arg supplementation prior to a single bout of eccentric exercise alleviates muscle fiber damage and preserves exercise performance capacity.

The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. While a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n=3. High-resolution isoelectric focusing liquid chromatography-tandem mass spectrometry (HiRIEF-LC-MS/MS) was used to quantify the expression of 4,974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to wild-type (C57BL/6) controls at each age. Furthermore, 1,795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function which have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators which together are indicative of crosstalk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Up-regulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was up-regulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging.