EV-mediated promotion of myogenic differentiation is dependent on dose, collection medium, and isolation method.

Extracellular vesicles (EVs) have been implicated in the regulation of myogenic differentiation. C2C12 murine myoblast differentiation was reduced following treatment with GW4869 or heparin (to inhibit exosome biogenesis and EV uptake, respectively). Conversely, treatment with C2C12 myotube-conditioned medium enhanced myogenic differentiation. Ultrafiltration-size exclusion liquid chromatography (UF-SEC) was used to isolate EVs and non-EV extracellular protein in parallel from C2C12 myoblast- and myotube-conditioned medium. UF-SEC-purified EVs promoted myogenic differentiation at low doses (≤2 × 108 particles/mL) and were inhibitory at the highest dose tested (2 × 1011 particles/mL). Conversely, extracellular protein fractions had no effect on myogenic differentiation. While the transfer of muscle-enriched miRNAs (myomiRs) has been proposed to mediate the pro-myogenic effects of EVs, we observed that they are scarce in EVs (e.g., 1 copy of miR-133a-3p per 195 EVs). Furthermore, we observed pro-myogenic effects with undifferentiated myoblast-derived EVs, in which myomiR concentrations are even lower, suggestive of a myomiR-independent mechanism underlying the observed pro-myogenic effects. During these investigations we identified technical factors with profound confounding effects on myogenic differentiation. Specifically, co-purification of insulin (a component of Opti-MEM) in non-EV LC fractions and polymer precipitated EV preparations. These findings provide further evidence that polymer-based precipitation techniques should be avoided in EV research.

Investigating Eukaryotic Elongation Factor 2 Kinase/Eukaryotic Translation Elongation Factor 2 Pathway Regulation and Its Role in Protein Synthesis Impairment during Disuse-Induced Skeletal Muscle Atrophy.

The principal mechanism underlying the reduced rate of protein synthesis in atrophied skeletal muscle is largely unknown. Eukaryotic elongation factor 2 kinase (eEF2k) impairs the ability of eukaryotic translation elongation factor 2 (eEF2) to bind to the ribosome via T56 phosphorylation. Perturbations in the eEF2k/eEF2 pathway during various stages of disuse muscle atrophy have been investigated utilizing a rat hind limb suspension (HS) model. Two distinct components of eEF2k/eEF2 pathway misregulation were demonstrated, observing a significant (P < 0.01) increase in eEF2k mRNA expression as early as 1-day HS and in eEF2k protein level after 3-day HS. We set out to determine whether eEF2k activation is a Ca2+-dependent process with involvement of Cav1.1. The ratio of T56-phosphorylated/total eEF2 was robustly elevated after 3-day HS, which was completely reversed by 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and decreased by 1.7-fold (P < 0.05) by nifedipine. Transfection of C2C12 with cytomegalovirus promoter (pCMV)-eEF2k and administration with small molecules were used to modulate eEF2k and eEF2 activity. More importantly, pharmacologic enhancement of eEF2 phosphorylation induced phosphorylated ribosomal protein S6 kinase (T389) up-regulation and restoration of global protein synthesis in the HS rats. Taken together, the eEF2k/eEF2 pathway was up-regulated during disuse muscle atrophy involving calcium-dependent activation of eEF2k partly via Cav1.1. The study provides evidence, in vitro and in vivo, of the eEF2k/eEF2 pathway impact on ribosomal protein S6 kinase activity as well as protein expression of key atrophy biomarkers, muscle atrophy F-box/atrogin-1 and muscle RING finger-1.

Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties.

Therapies that restore dystrophin expression are presumed to correct Duchenne muscular dystrophy (DMD), with antisense-mediated exon skipping being the leading approach. Here we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. Dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed 2 weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed, with dystrophin uniformly distributed at the sarcolemma of corrected myofibers at all doses. Serum microRNA biomarkers (i.e., miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase levels were restored toward wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e., Young's modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression and that both serum microRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in DMD.

Gaining insight into the role of FoxO1 in the progression of disuse-induced skeletal muscle atrophy.

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.

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy.

The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although 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 MS (HiRIEF-LC-MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 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 that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated 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.

Uniform sarcolemmal dystrophin expression is required to prevent extracellular microRNA release and improve dystrophic pathology.

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-XistΔhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-XistΔhs 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-XistΔhs 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-XistΔhs mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-XistΔhs 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.

Key Markers of mTORC1-Dependent and mTORC1-Independent Signaling Pathways Regulating Protein Synthesis in Rat Soleus Muscle During Early Stages of Hindlimb Unloading.

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-3ß, 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.

Reduced expression of MyHC slow isoform in rat soleus during unloading is accompanied by alterations of endogenous inhibitors of calcineurin/NFAT signaling pathway.

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 GSK3ß 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 GSK3ß 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.

No-dependent signaling pathways in unloaded skeletal muscle.

The main focus of the current review is the nitric oxide (NO)-mediated signaling mechanism in unloaded skeletal. Review of the published data describing muscles during physical activity and inactivity demonstrates that NO is an essential trigger of signaling processes, which leads to structural and metabolic changes of the muscle fibers. The experiments with modulation of NO-synthase (NOS) activity during muscle unloading demonstrate the ability of an activated enzyme to stabilize degradation processes and prevent development of muscle atrophy. Various forms of muscle mechanical activity, i.e., plantar afferent stimulation, resistive exercise and passive chronic stretch increase the content of neural NOS (nNOS) and thus may facilitate an increase in NO production. Recent studies demonstrate that NO-synthase participates in the regulation of protein and energy metabolism in skeletal muscle by fine-tuning and stabilizing complex signaling systems which regulate protein synthesis and degradation in the fibers of inactive muscle.

Calpain-dependent regulation of the skeletal muscle atrophy following unloading.

Unloading causes rapid skeletal muscle atrophy due to increased protein degradation via activation of calpains and decreased protein synthesis. Our study elucidated role of calpain-1 in the regulation of ubiquitin proteasome pathway (UPP) and anabolic processes mediated by Akt-mTOR-p70S6K and MAPK-Erk (p90RSK) signaling. We hypothesized that blocking calpain will inhibit activation of UPP and decrease protein degradation resulting in reduction of unloading-induced skeletal muscle atrophy. Rats were divided into three groups: non-treated control (C), three day hindlimb suspension with (HSPD) or without (HS) treatment with calpain inhibitor PD150606. When compared with control PD150606 treatment during unloading: 1) attenuated loss of muscle mass, 2) prevented accumulation of calpain-1 (1.8-fold in HS vs 1.3-fold in HSPD) and ubiquitin (2.3-fold in HS vs 0.7-fold in HSPD) mRNA and ubiquitinated proteins (1.6-fold in HS vs 0.8-fold in HSPD), 3) prevented decrease in the pAkt (0.4-fold in HS vs 1-fold in HSPD) and pFOXO3 (0.2-fold in HS vs 1.2-fold in HSPD) levels, 4) prevented increase in MAFbx (3.8-fold in HS vs 1.3-fold in HSPD) and eEF2k (1.8-fold in HS vs 0.6-fold in HSPD) mRNA. Our study indicates that blocking of calpain during unloading decreases skeletal muscle atrophy by inhibiting UPP activation and preserving anabolic signaling.

L-arginine supplementation protects exercise performance and structural integrity of muscle fibers after a single bout of eccentric exercise in rats.

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.

Attenuation of unloading-induced rat soleus atrophy with the heat-shock protein inducer 17-(allylamino)-17-demethoxygeldanamycin.

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.