Overexpression of CUGBP1 in Skeletal Muscle from Adult Classic Myotonic Dystrophy Type 1 but Not from Myotonic Dystrophy Type 2

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are progressive multisystemic disorders caused by similar mutations at two different genetic loci. The common key feature of DM pathogenesis is nuclear accumulation of mutant RNA which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of two RNA binding proteins, MBNL1 and CUGBP1. However, DM1 and DM2 show disease-specific features that make them clearly separate diseases suggesting that other cellular and molecular pathways may be involved. In this study we have analysed the histopathological, and biomolecular features of skeletal muscle biopsies from DM1 and DM2 patients in relation to presenting phenotypes to better define the molecular pathogenesis. Particularly, the expression of CUGBP1 protein has been examined to clarify if this factor may act as modifier of disease-specific manifestations in DM. The results indicate that the splicing and muscle pathological alterations observed are related to the clinical phenotype both in DM1 and in DM2 and that CUGBP1 seems to play a role in classic DM1 but not in DM2. In conclusion, our results indicate that multisystemic disease spectrum of DM pathologies may not be explained only by spliceopathy thus confirming that the molecular pathomechanism of DM is more complex than that actually suggested.     

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.     

Myotonic dystrophy protein kinase is critical for nuclear envelope integrity

Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.     

Myotonic dystrophy: RNA pathogenesis comes into focus

Myotonic dystrophy (DM)--the most common form of muscular dystrophy in adults, affecting 1/8000 individuals--is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1, on chromosome 19, and DM2, on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes myotonic dystrophy type 1(DM1) has been controversial. The recent discovery that myotonic dystrophy type 2 (DM2) is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain-of-function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. We discuss the pathogenic mechanisms that have been proposed for the myotonic dystrophies, the clinical and molecular features of DM1 and DM2, and the characterization of murine and cell-culture models that have been generated to better understand these diseases.     

Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.     

Tibialis Anterior Muscle Needle Biopsy and Sensitive Biomolecular Methods: A Useful Tool in Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG repeat expansion in 3’UTR of DMPK gene. This mutation causes accumulation of toxic RNA in nuclear foci leading to splicing misregulation of specific genes. In view of future clinical trials with antisense oligonucleotides in DM1 patients, it is important to set up sensitive and minimally-invasive tools to monitor the efficacy of treatments on skeletal muscle. A tibialis anterior (TA) muscle sample of about 60 mg was obtained from 5 DM1 patients and 5 healthy subjects through a needle biopsy. A fragment of about 40 mg was used for histological examination and a fragment of about 20 mg was used for biomolecular analysis. The TA fragments obtained with the minimally-invasive needle biopsy technique is enough to perform all the histopathological and biomolecular evaluations useful to monitor a clinical trial on DM1 patients.Key words: Myotonic dystrophy type 1, tibialis anterior; needle biopsy, alternative splicing, clinical trial     

ZNF9 Activation of IRES-Mediated Translation of the Human ODC mRNA Is Decreased in Myotonic Dystrophy Type 2

Myotonic dystrophy types 1 and 2 (DM1 and DM2) are forms of muscular dystrophy that share similar clinical and molecular manifestations, such as myotonia, muscle weakness, cardiac anomalies, cataracts, and the presence of defined RNA-containing foci in muscle nuclei. DM2 is caused by an expansion of the tetranucleotide CCTG repeat within the first intron of ZNF9, although the mechanism by which the expanded nucleotide repeat causes the debilitating symptoms of DM2 is unclear. Conflicting studies have led to two models for the mechanisms leading to the problems associated with DM2. First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function. Instead repeat-containing RNAs are thought to sequester proteins in the nucleus, causing misregulation of normal cellular processes. In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation. Here we examine the normal in vivo function of ZNF9. We report that ZNF9 associates with actively translating ribosomes and functions as an activator of cap-independent translation of the human ODC mRNA. This activity is mediated by direct binding of ZNF9 to the internal ribosome entry site sequence (IRES) within the 5′UTR of ODC mRNA. ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient. These data identify ZNF9 as a regulator of cap-independent translation and indicate that ZNF9 activity may contribute mechanistically to the myotonic dystrophy type 2 phenotype.     

TWEAK Regulates Muscle Functions in a Mouse Model of RNA Toxicity

Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, is caused by toxic RNAs produced from the mutant DM protein kinase (DMPK) gene. DM1 is characterized by progressive muscle wasting and weakness. Therapeutic strategies have mainly focused on targeting the toxic RNA. Previously, we found that fibroblast growth factor-inducible 14 (Fn14), the receptor for TWEAK, is induced in skeletal muscles and hearts of mouse models of RNA toxicity and that blocking TWEAK/Fn14 signaling improves muscle function and histology. Here, we studied the effect of Tweak deficiency in a RNA toxicity mouse model. The genetic deletion of Tweak in these mice significantly reduced muscle damage and improved muscle function. In contrast, administration of TWEAK in the RNA toxicity mice impaired functional outcomes and worsened muscle histopathology. These studies show that signaling via TWEAK is deleterious to muscle in RNA toxicity and support the demonstrated utility of anti-TWEAK therapeutics.     

Entactin promotes adhesion and long-term maintenance of cultured regenerated skeletal myotubes.

The basal lamina protein, laminin, has been shown to promote migration and proliferation of cultured skeletal myoblasts, resulting in increased myotube formation. However, skeletal myotubes adhere poorly to a laminin substrate, and long-term cultures of skeletal myotubes on laminin have not been achieved. We have found that cultured satellite cells from bupivacaine-damaged rat skeletal muscle actively proliferate and differentiate on a diluted Matrigel substrate composed of laminin, type IV collagen, heparan sulfate proteoglycan, and entactin. Myotubes cultured on diluted Matrigel are contractile and have never been observed to detach from the culture dish; rather, myotubes generally atrophy after 2-3 weeks in culture. Antibodies directed against the various protein components of Matrigel were used to determine the role of each component in enhancing muscle differentiation. Anti-laminin impaired satellite cell adhesion, whereas antibodies against either type IV collagen or heparan sulfate proteoglycan had no effect. Anti-entactin did not inhibit attachment, proliferation, or fusion of cultured satellite cells; however, myotubes exposed to anti-entactin failed to adhere to the culture dish after spontaneous myotube contractions began. We conclude that entactin is responsible for long-term maintenance and maturation of contractile skeletal myotubes on a diluted Matrigel substrate. This is the first study to assign a biological function for entactin in myogenesis.     

Altered signal transduction pathways and induction of autophagy in human myotonic dystrophy type 1 myoblasts

Congenital myotonic dystrophy type 1 (CDM1) affects patients from birth and is associated with mental retardation and impaired muscle development. CDM1 patients carry 1000–3000 CTG repeats in the DMPK gene and display defective skeletal muscles differentiation, resulting in reduced size of myotubes and decreased number of satellite cells. In this study, human myoblasts in culture deriving from control and DM1 embryos (3200 CTG repeats) were analyzed using both a biochemical and electron microscopic approach, in order to provide new insights into the molecular mechanisms underlying such alteration. Interestingly, electron microscopy analysis showed not only ultrastructural features of abnormal differentiation but also revealed the presence of autophagic vacuoles in DM1 myoblasts not undergoing differentiation. In accordance with the electron microscopic findings, the autophagic markers LC3 and ATG5, but not apoptotic markers, were significantly up regulated in DM1 myoblasts after differentiating medium addition. The induction of autophagic processes in DM1 myoblasts was concomitant to p53 over-expression and inhibition of the mTOR–S6K1 pathway, causatively involved in autophagy. Moreover biochemical alterations of the two main signal transduction pathways involved in differentiation were observed in DM1 myoblasts, in particular decreased activation of p38MAPK and persistent activation of the MEK–ERK pathway. This work, while demonstrating that major signaling pathways regulating myoblasts differentiation are profoundly deranged in DM1 myoblasts, for the first time provides evidence of autophagy induction, possibly mediated by p53 activation in response to metabolic stress which might contribute to the dystrophic alterations observed in the muscles of congenital DM1 patients.     

Myotonic dystrophy and myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) was designated as a gene responsible for myotonic dystrophy (DM) on chromosome 19, because the gene product has extensive homology to protein kinase catalytic domains. DM is the most common disease with multisystem disorders among muscular dystrophies. The genetic basis of DM is now known to include mutational expansion of a repetitive trinucleotide sequence (CTG)n in the 3'-untranslated region (UTR) of DMPK. Full-length DMPK was detected and various isoforms of DMPK have been reported in skeletal and cardiac muscles, central nervous tissues, etc. DMPK is localized predominantly in type I muscle fibers, muscle spindles, neuromuscular junctions and myotendinous tissues in skeletal muscle. In cardiac muscle it is localized in intercalated dises and Purkinje fibers. Electron microscopically it is detected in the terminal cisternae of SR in skeletal muscle and the junctional and corbular SR in cardia muscle. In central nervous system, it is located in many neurons, especially in the cytoplasm of cerebellar Purkinje cells, hippocampal interneurons and spinal motoneurons. Electron microscopically it is detected in rough endoplasmic reticulum. The functional role of DMPK is not fully understood, however, it may play an important role in Ca2+ homeostasis and signal transduction system. Diseased amount of DMPK may play an important role in the degeneration of skeletal muscle in adult type DM. However, other molecular pathogenetical mechanisms such as dysfunction of surrounding genes by structural change of the chromosome by long trinucleotide repeats, and the trans-gain of function of CUG-binding proteins might be responsible to induce multisystemic disorders of DM such as myotonia, endocrine dysfunction, etc.     

Isolation,Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.     

Expression and localization of myotonic dystrophy protein kinase in human skeletal muscle cells determined with a novel antibody: possible role of the protein in cytoskeleton rearrangements during differentiation

Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.     

Ectopic expression of cyclin D3 corrects differentiation of DM1 myoblasts through activation of RNA CUG-binding protein, CUGBP1

Differentiation of myocytes is impaired in patients with myotonic dystrophy type 1, DM1. CUG repeat binding protein, CUGBP1, is a key regulator of translation of proteins that are involved in muscle development and differentiation. In this paper, we present evidence that RNA-binding activity of CUGBP1 and its interactions with initiation translation complex eIF2 are differentially regulated during myogenesis by specific phosphorylation and that this regulation is altered in DM1. In normal myoblasts, Akt kinase phosphorylates CUGBP1 at Ser28 and increases interactions of CUGBP1 with cyclin D1 mRNA. During differentiation, CUGBP1 is phosphorylated by cyclinD3-cdk4/6 at Ser302, which increases CUGBP1 binding with p21 and C/EBPbeta mRNAs. While cyclin D3 and cdk4 are elevated in normal myotubes; DM1 differentiating cells do not increase these proteins. In normal myotubes, CUGBP1 interacts with cyclin D3/cdk4/6 and eIF2; however, interactions of CUGBP1 with eIF2 are reduced in DM1 differentiating cells and correlate with impaired muscle differentiation in DM1. Ectopic expression of cyclin D3 in DM1 cells increases the CUGBP1-eIF2 complex, corrects expression of differentiation markers, myogenin and desmin, and enhances fusion of DM1 myoblasts. Thus, normalization of cyclin D3 might be a therapeutic approach to correct differentiation of skeletal muscle in DM1 patients.     

Comparison of the proliferation and differentiation of myogenic satellite cells and embryonic myoblasts derived from the turkey

• 1.1. Embryonic and posthatch turkey skeletal muscle development was compared in in vitro studies using clonal-derived embryonic myoblasts and satellite cells.
• 2.2. Although population doubling times were similar between the two lines (25.4 hr for satellite cells and 26.4 hr for embryonic myoblasts), embryonic myoblasts consistently began log phase growth 24 hr earlier than satellite cells.
• 3.3. Differentiation (fusion) of embryonic myoblasts was maximized by 36 hr in Dulbecco's Modified Eagle's Medium containing 1% horse serum compared with 72 hr for satellite cells.
• 4.4. When administered a serum-free medium which supports proliferation of turkey satellite cells, embryonic myoblasts differentiated to form myotubes.

     

Alternative splicing of PDLIM3/ALP, for α-actinin-associated LIM protein 3, is aberrant in persons with myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder of muscular dystrophy characterized by muscle weakness and wasting. DM1 is caused by expansion of CTG repeats in the 3′-untranslated region (3′-UTR) of DM protein kinase (DMPK) gene. Since CUG-repeat RNA transcribed from the expansion of CTG repeats traps RNA-binding proteins that regulate alternative splicing, several abnormalities of alternative splicing are detected in DM1, and the abnormal splicing of important genes results in the appearance of symptoms. In this study, we identify two abnormal splicing events for actinin-associated LIM protein 3 (PDLIM3/ALP) and fibronectin 1 (FN1) in the skeletal muscles of DM1 patients. From the analysis of the abnormal PDLIM3 splicing, we propose that ZASP-like motif-deficient PDLIM3 causes the muscular symptoms in DM. PDLIM3 binds α-actinin 2 in the Z-discs of muscle, and the ZASP-like motif is needed for this interaction. Moreover, in adult humans, PDLIM3 expression is highest in skeletal muscles, and PDLIM3 splicing in skeletal muscles is regulated during human development.     

Elevated Muscle-Specific miRNAs in Serum of Myotonic Dystrophy Patients Relate to Muscle Disease Progress

The discovery of reliable and sensitive blood biomarkers is useful for the diagnosis, monitoring and potential future therapy of diseases. Recently, microRNAs (miRNAs) have been identified in blood circulation and might have the potential to be used as biomarkers for several diseases and clinical conditions. Myotonic Dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy primarily characterized by muscle myotonia, weakness and atrophy. Previous studies have shown an association between miRNAs and DM1 in muscle tissue and, recently, in plasma. The aim of this study was to detect and assess muscle-specific miRNAs as potential biomarkers of DM1 muscle wasting, an important parameter in the disease’s natural history. Disease stable or progressive DM1 patients with muscle weakness and wasting were recruited and enrolled in the study. RNA isolated from participants’ serum was used to assess miRNA levels. Results suggest that the levels of muscle-specific miRNAs are correlated with the progression of muscle wasting and weakness observed in the DM1 patients. Specifically, miR-1, miR-133a, miR133b and miR-206 serum levels were found elevated in DM1 patients with progressive muscle wasting compared to disease stable DM1 patients. Based on these results, we propose that muscle-specific miRNAs might be useful molecular biomarkers for monitoring the progress of muscle atrophy in DM1 patients.