Vorinostat Improves Myotonic Dystrophy Type 1 Splicing Abnormalities in DM1 Muscle Cell Lines and Skeletal Muscle from a DM1 Mouse Model.

Myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy, is caused by an abnormal expansion of CTG repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The expanded repeats of the DMPK mRNA form hairpin structures in vitro, which cause misregulation and/or sequestration of proteins including the splicing regulator muscleblind-like 1 (MBNL1). In turn, misregulation and sequestration of such proteins result in the aberrant alternative splicing of diverse mRNAs and underlie, at least in part, DM1 pathogenesis. It has been previously shown that disaggregating RNA foci repletes free MBNL1, rescues DM1 spliceopathy, and alleviates associated symptoms such as myotonia. Using an FDA-approved drug library, we have screened for a reduction of CUG foci in patient muscle cells and identified the HDAC inhibitor, vorinostat, as an inhibitor of foci formation; SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) spliceopathy was also improved by vorinostat treatment. Vorinostat treatment in a mouse model of DM1 (human skeletal actin-long repeat; HSALR) improved several spliceopathies, reduced muscle central nucleation, and restored chloride channel levels at the sarcolemma. Our in vitro and in vivo evidence showing amelioration of several DM1 disease markers marks vorinostat as a promising novel DM1 therapy.

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.

HuR Mediates Changes in the Stability of AChR ß-Subunit mRNAs after Skeletal Muscle Denervation.

Acetylcholine receptors (AChRs) are heteromeric membrane proteins essential for neurotransmission at the neuromuscular junction. Previous work showed that muscle denervation increases expression of AChR mRNAs due to transcriptional activation of AChR subunit genes. However, it remains possible that post-transcriptional mechanisms are also involved in controlling the levels of AChR mRNAs following denervation. We examined whether post-transcriptional events indeed regulate AChR ß-subunit mRNAs in response to denervation. First, in vitro stability assays revealed that the half-life of AChR ß-subunit mRNAs was increased in the presence of denervated muscle protein extracts. A bioinformatics analysis revealed the existence of a conserved AU-rich element (ARE) in the 3'-untranslated region (UTR) of AChR ß-subunit mRNA. Furthermore, denervation of mouse muscle injected with a luciferase reporter construct containing the AChR ß-subunit 3'UTR, caused an increase in luciferase activity. By contrast, mutation of this ARE prevented this increase. We also observed that denervation increased expression of the RNA-binding protein human antigen R (HuR) and induced its translocation to the cytoplasm. Importantly, HuR binds to endogenous AChR ß-subunit transcripts in cultured myotubes and in vivo, and this binding is increased in denervated versus innervated muscles. Finally, p38 MAPK, a pathway known to activate HuR, was induced following denervation as a result of MKK3/6 activation and a decrease in MKP-1 expression, thereby leading to an increase in the stability of AChR ß-subunit transcripts. Together, these results demonstrate the important contribution of post-transcriptional events in regulating AChR ß-subunit mRNAs and point toward a central role for HuR in mediating synaptic gene expression. SIGNIFICANCE STATEMENT: Muscle denervation is a convenient model to examine expression of genes encoding proteins of the neuromuscular junction, especially acetylcholine receptors (AChRs). Despite the accepted model of AChR regulation, which implicates transcriptional mechanisms, it remains plausible that such events cannot fully account for changes in AChR expression following denervation. We show that denervation increases expression of the RNA-binding protein HuR, which in turn, causes an increase in the stability of AChR ß-subunit mRNAs in denervated muscle. Our findings demonstrate for the first time the contribution of post-transcriptional events in controlling AChR expression in skeletal muscle, and points toward a central role for HuR in mediating synaptic development while also paving the way for developing RNA-based therapeutics for neuromuscular diseases.

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.

Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1α axis.

Slower, more oxidative muscle fibers are more resistant to the dystrophic pathology in Duchenne muscular dystrophy (DMD) patients as well as in the preclinical mdx mouse model of DMD. Therefore, one therapeutic strategy for DMD focuses on promoting expression of the slow, oxidative myogenic program. In the current study, we explored the therapeutic potential of stimulating the slow, oxidative phenotype in mdx mice by feeding 6-wk-old animals with the natural phenol resveratrol (RSV; ~100 mg·kg(-1)·day(-1)) for 6 wk. Sirtuin 1 (SIRT1) activity and protein levels increased significantly, as well as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) activity, in the absence of alterations in AMPK signaling. These adaptations occurred concomitant with evidence of a fast, glycolytic, to slower, more oxidative fiber type conversion, including mitochondrial biogenesis and increased expression of slower myosin heavy chain isoforms. These positive findings raised the question of whether increased exposure to RSV would result in greater therapeutic benefits. We discovered that an elevated RSV dose of ~500 mg·kg(-1)·day(-1) across a duration of 12 wk was clearly less effective at muscle remodeling in mdx mice. This treatment protocol failed to influence SIRT1 or AMPK signaling and did not result in a shift towards a slower, more oxidative phenotype. Taken together, this study demonstrates that RSV can stimulate SIRT1 and PGC-1α activation, which in turn may promote expression of the slow, oxidative myogenic program in mdx mouse muscle. The data also highlight the importance of selecting an appropriate dosage regimen of RSV to maximize its potential therapeutic effectiveness for future application in DMD patients.

Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle.

A therapeutic approach for Duchenne muscular dystrophy (DMD) is to up-regulate utrophin in skeletal muscle in an effort to compensate for the lack of dystrophin. We previously hypothesized that promotion of the slow, oxidative myogenic program, which triggers utrophin up-regulation, can attenuate the dystrophic pathology in mdx animals. Since treatment of healthy mice with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) enhances oxidative capacity and elicits a fast-to-slow fiber-type transition, we evaluated the effects of chronic AMPK stimulation on skeletal muscle phenotype and utrophin expression in mdx mice. Daily AICAR administration (500 mg/kg/day, 30 days) of 5-7-week-old mdx animals induced an elevation in mitochondrial cytochrome c oxidase enzyme activity, an increase in myosin heavy-chain type IIa-positive fibers and slower twitch contraction kinetics in the fast, glycolytic extensor digitorum longus muscle. Utrophin expression was significantly enhanced in response to AICAR, which occurred coincident with an elevated ß-dystroglycan expression along the sarcolemma. These adaptations were associated with an increase in sarcolemmal structural integrity under basal conditions, as well as during damaging eccentric contractions ex vivo. Notably, peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) and silent information regulator two ortholog 1 protein contents were significantly higher in muscle from mdx mice compared with wild-type littermates and AICAR further increased PGC-1α expression. Our data show that AICAR-evoked muscle plasticity results in beneficial phenotypic adaptations in mdx mice and suggest that the contextually novel application of this compound for muscular dystrophy warrants further study.

Ets-2 repressor factor silences extrasynaptic utrophin by N-box mediated repression in skeletal muscle.

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPalpha/beta to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest "repressing repressors" as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.

Multiple regulatory events controlling the expression and localization of utrophin in skeletal muscle fibers: insights into a therapeutic strategy for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most prevalent inherited muscle disease and results from mutations/deletions in the X-linked dystrophin gene. Although several approaches have been envisaged to counteract the effects of this progressive disease, there is currently no cure available. One strategy consists in utilizing a protein normally expressed in DMD muscle which, once expressed at appropriate levels and at the correct subcellular location, could compensate for the lack of dystrophin. A candidate for such a role is the dystrophin-related protein now referred to as utrophin. In contrast to dystrophin, which is expressed along the length of healthy muscle fibers, utrophin accumulates at the neuromuscular junction in both normal and DMD fibers. Several years ago, we began a series of experiments to determine the mechanisms responsible for the expression of utrophin at the neuromuscular synapse. Initially, we showed that utrophin transcripts accumulate preferentially within the postsynaptic sarcoplasm. To determine whether selective transcription of the utrophin gene accounts for this synaptic accumulation of utrophin mRNAs, we injected several utrophin promoter-reporter constructs directly into mouse muscle and demonstrated the preferential synaptic expression of the reporter gene. These results suggested that local transcriptional activation of the utrophin gene is responsible for the accumulation of utrophin mRNAs at the neuromuscular junction. In these studies, we also demonstrated that an N-box motif contained within the utrophin promoter plays a critical role in directing the synapse-specific expression of the utrophin gene. Additionally, our studies have shown that the ets-factors GABP alpha and beta are part of a protein complex that can bind to the N-box motif to transactivate the gene in muscle cells in culture and in vivo. In these experiments, we also noted that the nerve-derived trophic factors agrin and ARIA/heregulin regulate expression of utrophin via the activation of GABP alpha and beta which in turn, transactivate the utrophin gene via the N-box motif. Although these studies demonstrate that transcriptional activation can regulate utrophin mRNA levels, it is possible that additional mechanisms are also involved. In particular, the association of mRNAs with cytoskeletal elements and RNA-binding proteins may contribute to the accumulation of utrophin transcripts within the postsynaptic sarcoplasm. In recent studies, we have begun to examine this and we have now identified specific regions within the 3' untranslated region that are necessary for targeting and stabilizing utrophin mRNAs in skeletal muscle cells. A series of in vivo studies have also led us to conclude that post-transcriptional mechanisms are indeed important in regulating the abundance of utrophin transcripts in muscle. Together, these studies should lead to the identification of cis- and trans-acting elements regulating transcription of the utrophin gene as well as the stability and targeting of its mRNA in muscle cells. The results should therefore, identify specific targets that may become important in designing specific pharmacological interventions directed at increasing the expression of utrophin into extrasynaptic regions of DMD muscle fibers. In addition, these findings will contribute to our basic understanding of the cellular and molecular events involved in the formation, maintenance and plasticity of the neuromuscular synapse.

A reduction in the human adenovirus virion size through use of a shortened fibre protein does not enhance muscle transduction following systemic or localised delivery in mice.

We have investigated whether reducing the overall size of adenovirus (Ad), through use of a vector containing a shortened fibre, leads to enhanced distribution and dissemination of the vector. Intravenous or intraperitoneal injection of Ad5SlacZ (12 nm fibre versus the normal Ad5 37 nm fibre) or Ad5SpKlacZ (shortened fibre with polylysine motif in the H-I loop of fibre knob domain) led to similar levels of lacZ expression compared to Ad5LlacZ (native Ad5 fibre) in the liver of treated animals, but did not enhance extravasation into the tibialis anterior muscle. Direct injection of the short-fibre vectors into the tibialis anterior muscle did not result in enhanced spread of the vector through muscle tissue, and led to only sporadic transgene expression in the spinal cord, suggesting that modifying the fibre length or redirecting viral infection to a more common cell surface receptor does not enhance motor neuron uptake or retrograde transport.

The RNA-binding protein HuR binds to acetylcholinesterase transcripts and regulates their expression in differentiating skeletal muscle cells.

During myogenic differentiation, acetylcholinesterase (AChE) transcript levels are known to increase dramatically. Although this increase can be attributed in part to increased transcriptional activity, posttranscriptional mechanisms have also been implicated in the high levels of AChE mRNA in myotubes. In this study, we observed that transfection of a luciferase reporter construct containing the full-length AChE 3'-untranslated region (UTR) resulted in significantly higher (5-fold) luciferase activity in differentiated myotubes versus myoblasts. RNA-electrophoretic mobility shift assays (REMSAs) performed with a full-length AChE 3'-UTR probe and the AU-rich element revealed that the intensity of RNA-binding protein complexes increased as myogenic differentiation proceeded. Using several complementary approaches including supershift REMSA, mRNA-binding protein pull-down assays, and immunoprecipitation followed by reverse transcription-PCR, we found that the mRNA-stabilizing protein HuR interacts directly with AChE transcripts. Stable overexpression of HuR in C2C12 cells increased the expression of endogenous AChE transcripts as well as that of the luciferase reporter construct containing the AChE 3'-UTR. In vitro stability assays performed with protein extracts from these cells versus controls resulted in a slower rate of AChE mRNA decay. The down-regulation of HuR expression mediated through small interfering RNA further confirmed the role of HuR in the regulation of AChE mRNA levels. Taken together, these studies demonstrate that HuR interacts with the AChE 3'-UTR to regulate posttranscriptionally the expression of AChE mRNA during myogenic differentiation.