DM1 Skeletal Muscle Research Articles

Small Molecule Screening Identifies HSP90 as a Modifier of RNA Foci in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG triplet repeat expansion within the 3' untranslated region of the DMPK gene. Expression of the expanded allele generates RNA containing long tracts of CUG repeats (CUGexp RNA) that form hairpin structures and accumulate in nuclear RNA foci; however, the factors that control DMPK expression and the formation of CUGexp RNA foci remain largely unknown. We performed an unbiased small molecule screen in an immortalized human DM1 skeletal muscle myoblast cell line and identified HSP90 as a modifier of endogenous RNA foci. Small molecule inhibition of HSP90 leads to enhancement of RNA foci and upregulation of DMPK mRNA levels. Knockdown and overexpression of HSP90 in undifferentiated DM1 myoblasts validated the impact of HSP90 with upregulation and downregulation of DMPK mRNA, respectively. Furthermore, we identified p-STAT3 as a downstream mediator of HSP90 impacting levels of DMPK mRNA and RNA foci. Interestingly, differentiated cells exhibited an opposite effect of HSP90 inhibition displaying downregulation of DMPK mRNA through a mechanism independent of p-STAT3 involvement. This study has revealed a novel mediator for DMPK mRNA and foci regulation in DM1 cells with the potential to identify targets for future therapeutic intervention.

Combinatorial effects of ion channel mis-splicing as a cause of myopathy in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder caused by an unstable expanded CTG repeat located in the 3'-UTR of the DM1 protein kinase (DMPK) gene. The pathogenic mechanism results in misregulated alternative splicing of hundreds of genes, creating the dilemma of establishing which genes contribute to the mechanism of DM1 skeletal muscle pathology. In this issue of the JCI, Cisco and colleagues systematically tested the combinatorial effects of DM1-relevant mis-splicing patterns in vivo and identified the synergistic effects of mis-spliced calcium and chloride channels as a major contributor to DM1 skeletal muscle impairment. The authors further demonstrated the therapeutic potential for calcium channel modulation to block the synergistic effects and rescue myopathy.

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.

A single dose of exercise stimulates skeletal muscle mitochondrial plasticity in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the second most common muscular dystrophy after Duchenne and is the most prevalent muscular dystrophy in adults. DM1 patients that participate in aerobic exercise training experience several physiological benefits concomitant with improved muscle mitochondrial function without alterations in typical DM1-specific disease mechanisms, which suggests that correcting organelle health is key to ameliorate the DM1 pathology. However, our understanding of the molecular mechanisms of mitochondrial turnover and dynamics in DM1 skeletal muscle is lacking. Skeletal muscle tissue was sampled from healthy and DM1 mice under sedentary conditions and at several recovery time points following an exhaustive treadmill run. We demonstrate that DM1 patients exhibit an imbalance in the transcriptional apparatus for mitochondrial turnover and dynamics in skeletal muscle. Additionally, DM1 mice displayed elevated expression of autophagy and mitophagy regulators. A single dose of exercise successfully enhanced canonical exercise molecular pathways and skeletal muscle mitochondrial biogenesis despite failing to alter the cellular pathology in DM1 mice. However, treadmill running stimulated coordinated organelle fusion and fission signaling, as well as improved alternative splicing of Optic atrophy 1. Exercise also evoked autophagy and mitophagy pathways in DM1 skeletal muscle resulting in the normalized expression of autophagy- and lysosome-related machinery responsible for the clearance of dysfunctional organelles. Collectively, our data indicate that mitochondrial dynamics and turnover processes in DM1 skeletal muscle are initiated with a single dose of exercise, which may underlie the adaptive benefits previously documented in DM1 mice and patients.

Peptide-Conjugated PMOs for the Treatment of Myotonic Dystrophy.

Antisense oligonucleotides (ASOs) have shown great therapeutic potential in the treatment of many neuromuscular diseases including myotonic dystrophy 1 (DM1). However, systemically delivered ASOs display poor biodistribution and display limited penetration into skeletal muscle. The conjugation of cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligonucleotides (PMOs), a class of ASOs with a modified backbone, can be used to enhance ASO skeletal muscle penetration. Peptide-PMOs (P-PMOs) have been shown to be highly effective in correcting the DM1 skeletal muscle phenotype in both murine and cellular models of DM1 and at a molecular and functional level. Here we describe the synthesis and conjugation of P-PMOs and methods for analyzing their biodistribution and toxicity in the HSA-LR DM1 mouse model and their efficacy both in vitro and in vivo using FISH and RT-PCR splicing analysis.

Combinatorial treatment with exercise and AICAR potentiates the rescue of myotonic dystrophy type 1 mouse muscles in a sex-specific manner.

Targeting AMP-activated protein kinase (AMPK) is emerging as a promising strategy for treating myotonic dystrophy type 1 (DM1), the most prevalent form of adult-onset muscular dystrophy. We previously demonstrated that 5-aminomidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and exercise, two potent AMPK activators, improve disease features in DM1 mouse skeletal muscles. Here, we employed a combinatorial approach with these AMPK activators and examined their joint impact on disease severity in male and female DM1 mice. Our data reveal that swimming exercise additively enhances the effect of AICAR in mitigating the nuclear accumulation of toxic CUGexp RNA foci. In addition, our findings show a trend towards an enhanced reversal of MBNL1 sequestration and correction in pathogenic alternative splicing events. Our results further demonstrate that the combinatorial impact of exercise and AICAR promotes muscle fiber hypertrophy in DM1 skeletal muscle. Importantly, these improvements occur in a sex-specific manner with greater benefits observed in female DM1 mice. Our findings demonstrate that combining AMPK-activating interventions may prove optimal for rescuing the DM1 muscle phenotype and uncover important sex differences in the response to AMPK-based therapeutic strategies in DM1 mice.

Acute exercise stimulates AMPK and corrects some disease characteristics in the skeletal muscle of myotonic dystrophy type 1 mice

Myotonic Dystrophy type I (DM1) is the most prevalent adult form of muscular dystrophy and is characterized by myotonia, skeletal muscle weakness and wasting. The disease pathogenesis is characterized by a CTG microsatellite repeat expansion, leading to the dysregulation of pre-messenger RNA splicing of numerous muscle genes. There is currently no cure for this disorder, but recent evidence indicate that exercise is a safe and modestly effective therapy. AMP-activated protein kinase (AMPK) is critical to the acute responses and chronic adaptations to physical activity at the cellular and molecular level in the healthy condition, as well as in various disease states. However, the AMPK-signaling response to exercise in DM1 remains largely unknown. Therefore, the purpose of this study is to examine whether a single bout of exercise 1) activates AMPK and its downstream signalling network in DM1 skeletal muscle, and 2) modulates the DM1 molecular signature. Wild-type (WT) and HSA-LR (DM1) mice ran on a motor-driven treadmill until the inability to continue exercise was objectively determined, and the molecular response to physical activity at various timepoints post-exercise was examined using Western blotting, immunofluorescence microscopy, and qPCR techniques. WT mice ran for 937.6 ± 193.8 meters, which was significantly greater than DM1 mice at 573.3 ± 181.4meters. In the skeletal muscle of WT mice, AMPK activation status was significantly augmented immediately after exercise. This coincided with an increase (p < 0.05) in peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) transcript levels, as well as significant elevations in unc-51-like kinase 1 (ULK1) and calcium/calmodulin-dependent protein kinase type 2β (CAMKIIβ) activation. In DM1 mice, acute exercise also significantly stimulated AMPK, however, PGC-1α, ULK1 and CAMKIIβ were unaffected. In resting muscle, several markers related to autophagy downstream of AMPK, for example ULK1 activation status, p62 protein level, and the lipidated form of microtubule-associated protein 1-light chain 3, were significantly elevated in DM1 mice compared to their WT counterparts. These normalized after exercise. Acute physical activity did not impact either the prevalence of toxic myonuclear foci formed by the CTG microsatellite repeat or the proportion of mis-spliced muscle-specific chloride channel in DM1 mice. Collectively, these results indicate that while exercise-induced AMPK phosphorylation was preserved in the skeletal muscle of DM1 mice, markers of upstream and downstream AMPK signalling were attenuated in response to acute physical activity. Nonetheless, our data also suggest that exercise-evoked AMPK activation normalizes perturbations in the autophagy pathway observed in DM1 muscle. Thus, skeletal muscle AMPK stimulation after a single bout of exercise is very likely necessary but insufficient to elicit beneficial structural and functional adaptations in DM1.

Bioengineered in vitro 3D model of myotonic dystrophy type 1 human skeletal muscle

Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models and in vitro two-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first in vitro 3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy.

Overexpression of Staufen1 in DM1 mouse skeletal muscle exacerbates dystrophic and atrophic features

In myotonic dystrophy type 1 (DM1), the CUG expansion (CUGexp) in the 3' untranslated region of the dystrophia myotonica protein kinase messenger ribonucleic acid affects the homeostasis of ribonucleic acid-binding proteins, causing the multiple symptoms of DM1. We have previously reported that Staufen1 is increased in skeletal muscles from DM1 mice and patients and that sustained Staufen1 expression in mature mouse muscle causes a progressive myopathy. Here, we hypothesized that the elevated levels of Staufen1 contributes to the myopathic features of the disease. Interestingly, the classic DM1 mouse model human skeletal actin long repeat (HSALR) lacks overt atrophy while expressing CUGexp transcripts and elevated levels of endogenous Staufen1, suggesting a lower sensitivity to atrophic signaling in this model. We report that further overexpression of Staufen1 in the DM1 mouse model HSALR causes a myopathy via inhibition of protein kinase B signaling through an increase in phosphatase tensin homolog, leading to the expression of atrogenes. Interestingly, we also show that Staufen1 regulates the expression of muscleblind-like splicing regulator 1 and CUG-binding protein elav-like family member 1 in wild-type and DM1 skeletal muscle. Together, data obtained from these new DM1 mouse models provide evidence for the role of Staufen1 as an atrophy-associated gene that impacts progressive muscle wasting in DM1. Accordingly, our findings highlight the potential of Staufen1 as a therapeutic target and biomarker.

HNRNPA1-induced spliceopathy in a transgenic mouse model of myotonic dystrophy

Studies on myotonic dystrophy type 1 (DM1) have led to the RNA-mediated disease model for hereditary disorders caused by noncoding microsatellite expansions. This model proposes that DM1 disease manifestations are caused by a reversion to fetal RNA processing patterns in adult tissues due to the expression of toxic CUG RNA expansions (CUGexp) leading to decreased muscleblind-like, but increased CUGBP1/ETR3-like factor 1 (CELF1), alternative splicing activities. Here, we test this model in vivo, using the mouse HSALR poly(CUG) model for DM1 and recombinant adeno-associated virus (rAAV)-mediated transduction of specific splicing factors. Surprisingly, systemic overexpression of HNRNPA1, not previously linked to DM1, also shifted DM1-relevant splicing targets to fetal isoforms, resulting in more severe muscle weakness/myopathy as early as 4 to 6 wk posttransduction, whereas rAAV controls were unaffected. Overexpression of HNRNPA1 promotes fetal exon inclusion of representative DM1-relevant splicing targets in differentiated myoblasts, and HITS-CLIP of rAAV-mycHnrnpa1-injected muscle revealed direct interactions of HNRNPA1 with these targets in vivo. Similar to CELF1, HNRNPA1 protein levels decrease during postnatal development, but are elevated in both regenerating mouse muscle and DM1 skeletal muscle. Our studies suggest that CUGexp RNA triggers abnormal expression of multiple nuclear RNA binding proteins, including CELF1 and HNRNPA1, that antagonize MBNL activity to promote fetal splicing patterns.

Pharmacological and physiological activation of AMPK improves the spliceopathy in DM1 mouse muscles.

Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder caused by a triplet repeat expansion in the 3' untranslated region of dystrophia myotonica protein kinase mRNAs. Mutant mRNAs accumulate in the nucleus of affected cells and misregulate RNA-binding proteins, thereby promoting characteristic missplicing events. However, little is known about the signaling pathways that may be affected in DM1. Here, we investigated the status of activated protein kinase (AMPK) signaling in DM1 skeletal muscle and found that the AMPK pathway is markedly repressed in a DM1 mouse model (human skeletal actin-long repeat, HSALR) and patient-derived DM1 myoblasts. Chronic pharmacological activation of AMPK signaling in DM1 mice with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) has multiple beneficial effects on the DM1 phenotype. Indeed, a 6-week AICAR treatment of DM1 mice promoted expression of a slower, more oxidative phenotype, improved muscle histology and corrected several events associated with RNA toxicity. Importantly, AICAR also had a dose-dependent positive effect on the spliceopathy in patient-derived DM1 myoblasts. In separate experiments, we also show that chronic treatment of DM1 mice with resveratrol as well as voluntary wheel running also rescued missplicing events in muscle. Collectively, our findings demonstrate the therapeutic potential of chronic AMPK stimulation both physiologically and pharmacologically for DM1 patients.

Mechanisms of skeletal muscle wasting in a mouse model for myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease resulting in severe muscle weakening and wasting. DM1 is caused by expansion of CTG repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. We have developed an inducible, skeletal muscle-specific mouse model of DM1 (CUG960) that expresses 960 CUG repeat-expressing animals (CUG960) in the context of human DMPK exons 11-15. CUG960 RNA-expressing mice induced at postnatal day 1, as well as adult-onset animals, show clear, measurable muscle wasting accompanied by severe histological defects including central myonuclei, reduced fiber cross-sectional area, increased percentage of oxidative myofibers, the presence of nuclear RNA foci that colocalize with Mbnl1 protein, and increased Celf1 protein in severely affected muscles. Importantly, muscle loss, histological abnormalities and RNA foci are reversible, demonstrating recovery upon removal of toxic RNA. RNA-seq and protein array analysis indicate that the balance between anabolic and catabolic pathways that normally regulate muscle mass may be disrupted by deregulation of platelet derived growth factor receptor β signaling and the PI3K/AKT pathways, along with prolonged activation of AMP-activated protein kinase α signaling. Similar changes were detected in DM1 skeletal muscle compared with unaffected controls. The mouse model presented in this paper shows progressive skeletal muscle wasting and has been used to identify potential molecular mechanisms underlying skeletal muscle loss. The reversibility of the phenotype establishes a baseline response for testing therapeutic approaches.

Macroscopic and microscopic diversity of missplicing in the central nervous system of patients with myotonic dystrophy type 1.

Myotonic dystrophy type I (DM1) is a multiorgan disease caused by CTG-repeat expansion in the DMPK gene. Sequestration of the splicing factor MBNL1 results in aberrant splicing in many genes in DM1 skeletal muscle, whereas MBNL2 plays a leading role in missplicing in the central nervous system (CNS) of patients with DM1. Splicing misregulation of most MBNL2-regulated genes occurs in the temporal cortex but not in the cerebellum of autopsied patients with DM1. To understand the diversity at macroscopic and microscopic levels in CNS of patients with DM1. Using autopsied brain tissues, we examined alternative splicing ratios of MBNL2-regulated genes and expression levels of potential splicing factors. We found differences in splicing abnormalities among tested regions of the CNS from patients with DM1. In the frontal and temporal cortices and the hippocampus, many genes were aberrantly spliced, but severity differed among the brain regions. By contrast, there were no significant differences in the ratio of splicing variants for most of the genes in the cerebellar cortex and spinal cord between DM1 and control samples. We failed to find any change in the amount of potential factors (MBNL and CUGBP proteins and DMPK mRNA) which explain the modest missplicing in the cerebellum. LASER capture microdissection demonstrated splicing misregulation in the molecular layer of the cerebellum but not in the granular layer. This is the first study to reveal missplicing in a functional cell layer of DM1 and to compare splicing misregulation in a wide region of the CNS using statistical analysis.

LDB3 splicing abnormalities are specific to skeletal muscles of patients with myotonic dystrophy type 1 and alter its PKC binding affinity

Myotonic dystrophy type 1 (DM1) is caused by transcription of CUG repeat RNA, which causes sequestration of muscleblind-like 1 (MBNL1) and upregulation of CUG triplet repeat RNA-binding protein (CUG-BP1). In DM1, dysregulation of these proteins contributes to many aberrant splicing events, causing various symptoms of the disorder. Here, we demonstrate the occurrence of aberrant splicing of LIM domain binding 3 (LDB3) exon 11 in DM1 skeletal muscle. Exon array surveys, RT-PCR, and western blotting studies demonstrated that exon 11 inclusion was DM1 specific and could be reproduced by transfection of a minigene containing the CTG repeat expansion. Moreover, we found that the LDB3 exon 11-positive isoform had reduced affinity for PKC compared to the exon 11-negative isoform. Since PKC exhibits hyperactivation in DM1 and stabilizes CUG-BP1 by phosphorylation, aberrant splicing of LDB3 may contribute to CUG-BP1 upregulation through changes in its affinity for PKC.

Reexpression of pyruvate kinase M2 in type 1 myofibers correlates with altered glucose metabolism in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is caused by expansion of CTG repeats in the 3' UTR of the DMPK gene. Expression of CUG expansion (CUG(exp)) RNA produces a toxic gain of function by disrupting the functions of RNA splicing factors, such as MBNL1 and CELF1, leading to splicing changes associated with clinical abnormalities. Progressive skeletal muscle weakness and wasting is one of the most prominent clinical features in DM1; however, the underlying mechanisms remain unclear. Here we report that the embryonic M2 isoform of pyruvate kinase (PKM2), a key enzyme contributing to the Warburg effect in cancer, is significantly induced in DM1 tissue and mouse models owing to aberrant splicing. Expression of PKM2 in DM1 skeletal muscle is restricted to the type 1 fibers, which are particularly susceptible to wasting in DM1. Using antisense oligonucleotides to shift PKM splicing toward increased PKM2 expression, we observed increased glucose consumption with reduced oxidative metabolism in cell culture and increased respiratory exchange ratio in mice, suggesting defects in energy metabolism conferred by PKM2 expression. We propose that PKM2 expression induces changes in type 1 fibers associated with muscle atrophy and muscle weakness in DM1.

The RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing

In myotonic dystrophy type 1 (DM1), dystrophia myotonica protein kinase messenger ribonucleic acids (RNAs; mRNAs) with expanded CUG repeats (CUG(exp)) aggregate in the nucleus and become toxic to cells by sequestering and/or misregulating RNA-binding proteins, resulting in aberrant alternative splicing. In this paper, we find that the RNA-binding protein Staufen1 is markedly and specifically increased in skeletal muscle from DM1 mouse models and patients. We show that Staufen1 interacts with mutant CUG(exp) mRNAs and promotes their nuclear export and translation. This effect is critically dependent on the third double-stranded RNA-binding domain of Staufen1 and shuttling of Staufen1 into the nucleus via its nuclear localization signal. Moreover, we uncover a new role of Staufen1 in splicing regulation. Overexpression of Staufen1 rescues alternative splicing of two key pre-mRNAs known to be aberrantly spliced in DM1, suggesting its increased expression represents an adaptive response to the pathology. Altogether, our results unravel a novel function for Staufen1 in splicing regulation and indicate that it may positively modulate the complex DM1 phenotype, thereby revealing its potential as a therapeutic target.

CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1

The neuromuscular disease myotonic dystrophy type I (DM1) affects multiple organ systems with the major symptoms being severe muscle weakness, progressive muscle wasting and myotonia. The causative mutation in DM1 is a CTG repeat expansion in the 3'-untranslated region of the DM protein kinase (DMPK) gene. RNA transcribed from the expanded allele contains the expanded CUG repeats and leads to the nuclear depletion of Muscleblind-like 1 (MBNL1) and to the increased steady-state levels of CUG-binding protein 1 (CUGBP1). The pathogenic effects of MBNL1 depletion have previously been tested by the generation of MBNL1 knockout mice, but the consequence of CUGBP1 overexpression in adult muscle is not known. In a DM1 mouse model expressing RNA containing 960 CUG repeats in skeletal muscle, CUGBP1 up-regulation is temporally correlated with severe muscle wasting. In this study, we generated transgenic mice with doxycycline-inducible and skeletal muscle-specific expression of CUGBP1. Adult mouse skeletal muscle overexpressing CUGBP1 reproduces molecular and physiological defects of DM1 tissue. The results from this study strongly suggest that CUGBP1 has a major role in DM1 skeletal muscle pathogenesis.

Expanded CTG repeats within the DMPK 3′ UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy

Severe skeletal muscle wasting is the most debilitating symptom experienced by individuals with myotonic dystrophy type 1 (DM1). We present a DM1 mouse model with inducible and skeletal muscle-specific expression of large tracts of CTG repeats in the context of DMPK exon 15. These mice recapitulate many findings associated with DM1 skeletal muscle, such as CUG RNA foci with Muscleblind-like 1 (MBNL1) protein colocalization, misregulation of developmentally regulated alternative splicing events, myotonia, characteristic histological abnormalities, and increased CUGBP1 protein levels. Importantly, this DM1 mouse model recapitulates severe muscle wasting, which has not been reported in models in which depletion of MBNL1 is the main feature. Using these mice, we discovered previously undescribed alternative splicing events that are responsive to CUGBP1 and not MBNL, and these events were found to be misregulated in individuals with DM1. Our results indicate that increased CUGBP1 protein levels are associated with DMPK-CUG RNA expression, suggesting a role for CUGBP1-specific splicing or cytoplasmic functions in muscle wasting.

Increased Steady-State Levels of CUGBP1 in Myotonic Dystrophy 1 Are Due to PKC-Mediated Hyperphosphorylation

The genetic basis of myotonic dystrophy type 1 (DM1) is a CTG expansion in the 3' untranslated region (UTR) of DMPK. The pathogenic mechanism involves an RNA gain of function in which the repeat-containing transcripts accumulate in nuclei and alter the functions of RNA-binding proteins such as CUG-binding protein 1 (CUGBP1). CUGBP1 levels are increased in DM1 myoblasts, heart, and skeletal muscle tissues and in some DM1 mouse models. However, the molecular mechanisms for increased CUGBP1 in DM1 are unclear. Here, we demonstrate that expression of DMPK-CUG-repeat RNA results in hyperphosphorylation and stabilization of CUGBP1. CUGBP1 is hyperphosphorylated in DM1 tissues, cells, and a DM1 mouse model. Activation of PKC is required for CUGBP1 hyperphosphorylation in DM1 cells, and PKCalpha and betaII directly phosphorylate CUGBP1 in vitro. These results indicate that inappropriate activation of the PKC pathway contributes to the pathogenic effects of a noncoding RNA.

Regulation of chloride ion conductance during skeletal muscle development and in disease. Focus on “Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1”

productive investigations of disease mechanisms that also reveal new information about normal regulation elicit a particularly satisfying sense of a two-for-one deal. In the case of Lueck et al. (Ref. [13][1]; see page 1291 of this issue), a detailed study of the molecular basis for the myotonia (