Combinatorial therapeutic activation with heparin and AICAR stimulates additive effects on utrophin A expression in dystrophic muscles.

Upregulation of utrophin A is an attractive therapeutic strategy for treating Duchenne muscular dystrophy (DMD). Over the years, several studies revealed that utrophin A is regulated by multiple transcriptional and post-transcriptional mechanisms, and that pharmacological modulation of these pathways stimulates utrophin A expression in dystrophic muscle. In particular, we recently showed that activation of p38 signaling causes an increase in the levels of utrophin A mRNAs and protein by decreasing the functional availability of the destabilizing RNA-binding protein called K-homology splicing regulatory protein, thereby resulting in increases in the stability of existing mRNAs. Here, we treated 6-week-old mdx mice for 4 weeks with the clinically used anticoagulant drug heparin known to activate p38 mitogen-activated protein kinase, and determined the impact of this pharmacological intervention on the dystrophic phenotype. Our results show that heparin treatment of mdx mice caused a significant ∼1.5- to 3-fold increase in utrophin A expression in diaphragm, extensor digitorum longus and tibialis anterior (TA) muscles. In agreement with these findings, heparin-treated diaphragm and TA muscle fibers showed an accumulation of utrophin A and ß-dystroglycan along their sarcolemma and displayed improved morphology and structural integrity. Moreover, combinatorial drug treatment using both heparin and 5-amino-4-imidazolecarboxamide riboside (AICAR), the latter targeting 5' adenosine monophosphate-activated protein kinase and the transcriptional activation of utrophin A, caused an additive effect on utrophin A expression in dystrophic muscle. These findings establish that heparin is a relevant therapeutic agent for treating DMD, and illustrate that combinatorial treatment of heparin with AICAR may serve as an effective strategy to further increase utrophin A expression in dystrophic muscle via activation of distinct signaling pathways.

Muscle-specific microRNA-206 targets multiple components in dystrophic skeletal muscle representing beneficial adaptations.

Over the last several years, converging lines of evidence have indicated that miR-206 plays a pivotal role in promoting muscle differentiation and regeneration, thereby potentially impacting positively on the progression of neuromuscular disorders, including Duchenne muscular dystrophy (DMD). Despite several studies showing the regulatory function of miR-206 on target mRNAs in skeletal muscle cells, the effects of overexpression of miR-206 in dystrophic muscles remain to be established. Here, we found that miR-206 overexpression in mdx mouse muscles simultaneously targets multiple mRNAs and proteins implicated in satellite cell differentiation, muscle regeneration, and at the neuromuscular junction. Overexpression of miR-206 also increased the levels of several muscle-specific mRNAs/proteins, while enhancing utrophin A expression at the sarcolemma. Finally, we also observed that the increased expression of miR-206 in dystrophin-deficient mouse muscle decreased the production of proinflammatory cytokines and infiltration of macrophages. Taken together, our results show that miR-206 acts as a pleiotropic regulator that targets multiple key mRNAs and proteins expected to provide beneficial adaptations in dystrophic muscle, thus highlighting its therapeutic potential for DMD.

Converging pathways involving microRNA-206 and the RNA-binding protein KSRP control post-transcriptionally utrophin A expression in skeletal muscle.

Several reports have previously highlighted the potential role of miR-206 in the post-transcriptional downregulation of utrophin A in cultured cells. Along those lines, we recently identified K-homology splicing regulator protein (KSRP) as an important negative regulator in the post-transcriptional control of utrophin A in skeletal muscle. We sought to determine whether these two pathways act together to downregulate utrophin A expression in skeletal muscle. Surprisingly, we discovered that miR-206 overexpression in cultured cells and dystrophic muscle fibers causes upregulation of endogenous utrophin A levels. We further show that this upregulation of utrophin A results from the binding of miR-206 to conserved sites located in the 3'-UTR (untranslated region) of KSRP, thus causing the subsequent inhibition of KSRP expression. This miR-206-mediated decrease in KSRP levels leads, in turn, to an increase in the expression of utrophin A due to a reduction in the activity of this destabilizing RNA-binding protein. Our work shows that miR-206 can oscillate between direct repression of utrophin A expression via its 3'-UTR and activation of its expression through decreased availability of KSRP and interactions with AU-rich elements located within the 3'-UTR of utrophin A. Our study thus reveals that two apparent negative post-transcriptional pathways can act distinctively as molecular switches causing repression or activation of utrophin A expression.

Activation of p38 signaling increases utrophin A expression in skeletal muscle via the RNA-binding protein KSRP and inhibition of AU-rich element-mediated mRNA decay: implications for novel DMD therapeutics.

Several therapeutic approaches are currently being developed for Duchenne muscular dystrophy (DMD) including upregulating the levels of endogenous utrophin A in dystrophic fibers. Here, we examined the role of post-transcriptional mechanisms in controlling utrophin A expression in skeletal muscle. We show that activation of p38 leads to an increase in utrophin A independently of a transcriptional induction. Rather, p38 controls the levels of utrophin A mRNA by extending the half-life of transcripts via AU-rich elements (AREs). This mechanism critically depends on a decrease in the functional availability of KSRP, an RNA-binding protein known to promote decay of ARE-containing transcripts. In vitro and in vivo binding studies revealed that KSRP interacts with specific AREs located within the utrophin A 3' UTR. Electroporation experiments to knockdown KSRP led to an increase in utrophin A in wild-type and mdx mouse muscles. In pre-clinical studies, treatment of mdx mice with heparin, an activator of p38, causes a pronounced increase in utrophin A in diaphragm muscle fibers. Together, these studies identify a pathway that culminates in the post-transcriptional regulation of utrophin A through increases in mRNA stability. Furthermore, our results constitute proof-of-principle showing that pharmacological activation of p38 may prove beneficial as a novel therapeutic approach for DMD.

Brain-derived neurotrophic factor expression is repressed during myogenic differentiation by miR-206.

Brain-derived neurotrophic factor (BDNF) is required for efficient skeletal-muscle regeneration and perturbing its expression causes abnormalities in the proliferation and differentiation of skeletal muscle cells. In this study, we investigated the mechanism of BDNF suppression that occurs during myogenic differentiation. BDNF is expressed at the mRNA level as two isoforms that differ in the length of their 3'UTRs as a result of alternative cleavage and polyadenylation. Sequence analysis revealed the presence of three miR-206 target sites in the long BDNF 3'UTR (BDNF-L), whereas only one site was found in the short mRNA BDNF 3'UTR (BDNF-S). miR-206 is known to regulate the differentiation of C2C12 myoblasts and its expression is induced during the transition from myoblasts to myotubes. We thus examined whether miR-206-mediated suppression is responsible for the expression pattern of BDNF during myogenic differentiation. BDNF-L was suppressed to a greater extent than BDNF-S during differentiation of C2C12 myoblasts. Transfection of a miR-206 precursor decreased activity of reporters representative of the BDNF-L 3'UTR, but not BDNF-S 3'UTR, and repressed endogenous BDNF mRNA levels. This suppression was found to be dependent on the presence of multiple miR-206 target sites in the BDNF-L 3'UTR. Conversely, suppression of miR-206 levels resulted in de-repression of BDNF 3'UTR reporter activity and increased endogenous BDNF-L mRNA levels. A receptor for BDNF, p75(NTR) , was also suppressed during differentiation and in response to miR-206, but this appeared to not be entirely mediated via a miR-206 target site its 3'UTR. Based on these observations, BDNF represents a novel target through which miR-206 controls the initiation and maintenance of the differentiated state of muscle cells. These results further suggest that miR-206 might play a role in regulating retrograde signaling of BDNF at the neuromuscular junction.

Down-regulation of Akt/mammalian target of rapamycin signaling pathway in response to myostatin overexpression in skeletal muscle.

Myostatin, a member of the TGF-beta family, has been identified as a master regulator of embryonic myogenesis and early postnatal skeletal muscle growth. However, cumulative evidence also suggests that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression and that myostatin may contribute to muscle mass loss in adulthood. Two major branches of the Akt pathway are relevant for the regulation of skeletal muscle mass, the Akt/mammalian target of rapamycin (mTOR) pathway, which controls protein synthesis, and the Akt/forkhead box O (FOXO) pathway, which controls protein degradation. Here, we provide further insights into the mechanisms by which myostatin regulates skeletal muscle mass by showing that myostatin negatively regulates Akt/mTOR signaling pathway. Electrotransfer of a myostatin expression vector into the tibialis anterior muscle of Sprague Dawley male rats increased myostatin protein level and decreased skeletal muscle mass 7 d after gene electrotransfer. Using RT-PCR and immunoblot analyses, we showed that myostatin overexpression was ineffective to alter the ubiquitin-proteasome pathway. By contrast, myostatin acted as a negative regulator of Akt/mTOR pathway. This was supported by data showing that the phosphorylation of Akt on Thr308, tuberous sclerosis complex 2 on Thr1462, ribosomal protein S6 on Ser235/236, and 4E-BP1 on Thr37/46 was attenuated 7 d after myostatin gene electrotransfer. The data support the conclusion that Akt/mTOR signaling is a key target that accounts for myostatin function during muscle atrophy, uncovering a novel role for myostatin in protein metabolism and more specifically in the regulation of translation in skeletal muscle.

Ectopic expression of myostatin induces atrophy of adult skeletal muscle by decreasing muscle gene expression.

Myostatin is a master regulator of myogenesis and early postnatal skeletal muscle growth. However, myostatin has been also involved in several forms of muscle wasting in adulthood, suggesting a functional role for myostatin in the regulation of skeletal muscle mass in adult. In the present study, localized ectopic expression of myostatin was achieved by gene electrotransfer of a myostatin expression vector into the tibialis anterior muscle of adult Sprague Dawley male rats. The corresponding empty vector was electrotransfected in contralateral muscle. Ectopic myostatin mRNA was abundantly present in muscles electrotransfected with myostatin expression vector, whereas it was undetectable in contralateral muscles. Overexpression of myostatin elicited a significant decrease in muscle mass (10 and 20% reduction 7 and 14 d after gene electrotransfer, respectively), muscle fiber cross-sectional area (15 and 30% reduction 7 and 14 d after gene electrotransfer, respectively), and muscle protein content (20% reduction). No decrease in fiber number was observed. Overexpression of myostatin markedly decreased the expression of muscle structural genes (myosin heavy chain IIb, troponin I, and desmin) and the expression of myogenic transcription factors (MyoD and myogenin). Incidentally, mRNA level of caveolin-3 and peroxisome proliferator activated receptor gamma coactivator-1alpha was also significantly decreased 14 d after myostatin gene electrotransfer. To conclude, our study demonstrates that myostatin-induced muscle atrophy elicits the down-regulation of muscle-specific gene expression. Our observations support an important role for myostatin in muscle atrophy in physiological and physiopathological situations where myostatin expression is induced.

Regulation of Akt-mTOR, ubiquitin-proteasome and autophagy-lysosome pathways in response to formoterol administration in rat skeletal muscle.

Administration of ß2-agonists triggers skeletal muscle anabolism and hypertrophy. We investigated the time course of the molecular events responsible for rat skeletal muscle hypertrophy in response to 1, 3 and 10 days of formoterol administration (i.p. 2000µg/kg/day). A marked hypertrophy of rat tibialis anterior muscle culminated at day 10. Phosphorylation of Akt, ribosomal protein S6, 4E-BP1 and ERK1/2 was increased at day 3, but returned to control level at day 10. This could lead to a transient increase in protein translation and could explain previous studies that reported increase in protein synthesis following ß2-agonist administration. Formoterol administration was also associated with a significant reduction in MAFbx/atrogin-1 mRNA level (day 3), suggesting that formoterol can also affect protein degradation of MAFbx/atrogin1 targeted substrates, including MyoD and eukaryotic initiation factor-3f (eIF3-f). Surprisingly, mRNA level of autophagy-related genes, light chain 3 beta (LC3b) and gamma-aminobutyric acid receptor-associated protein-like 1 (Gabarapl1), as well as lysosomal hydrolases, cathepsin B and cathepsin L, was significantly and transiently increased after 1 and/or 3 days, suggesting that autophagosome formation would be increased in response to formoterol administration. However, this has to be relativized since the mRNA level of Unc-51-like kinase1 (Ulk1), BCL2/adenovirus E1B interacting protein3 (Bnip3), and transcription factor EB (TFEB), as well as the protein content of Ulk1, Atg13, Atg5-Atg12 complex and p62/Sqstm1 remained unchanged or was even decreased in response to formoterol administration. These results demonstrate that the effects of formoterol are mediated, in part, through the activation of Akt-mTOR pathway and that other signaling pathways become more important in the regulation of skeletal muscle mass with chronic administration of ß2-agonists.

High-resolution pQCT analysis at the distal radius and tibia discriminates patients with recent wrist and femoral neck fractures.

We depict a fragility bone state in two primitive osteoporosis populations using 3D high-resolution peripheral in vivo QCT (HR-pQCT). Postmenopausal women (C, controls, n = 54; WF, wrist, n = 50; HF, hip, n = 62 recent fractured patients) were analyzed for lumbar and hip DXA areal BMD (aBMD), cancellous and cortical volumetric BMD (vBMD), and microstructural and geometric parameters on tibia and radius by HR-pQCT. Principal component analysis (PCA) allowed extracting factors that best represent bone variables. Comparison between groups was made by analysis of covariance (ANCOVA). Two factors (>80% of the entire variability) are extracted by PCA: at the radius, the first is a combination of trabecular parameters and the second of cortical parameters. At the tibia, we found the reverse. Femoral neck aBMD is decreased in WF (8.6%) and in HF (18%) groups (no lumbar difference). WF showed a approximately 20% reduction in radius trabecular vBMD and number. Radius cortical vBMD and thickness decrease by 6% and 14%, respectively. At the tibia, only the cortical compartment is affected, with approximately 20% reduction in bone area, thickness, and section modulus and 6% reduction in vBMD. HF showed same radius trabecular alterations than WF, but radius cortical parameters are more severely affected than WF with reduced bone area (25%), thickness (28.5%), and vBMD (11%). At the tibia, trabecular vBMD and number decrease by 26% and 17.5%, respectively. Tibia cortical bone area, thickness, and section modulus showed a >30% decrease, whereas vBMD reduction reached 13%. Geometry parameters at the tibia displayed the greatest differences between healthy and fractured patients and between wrist and hip fractures.