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.     

A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients

Somatic mosaicism of the expanded CTG repeat in myotonic dystrophy type 1 is age-dependent, tissue-specific and expansion-biased, contributing toward the tissue-specificity and progressive nature of the symptoms. Previously, using regression modelling of repeat instability we showed that variation in the rate of somatic expansion in blood DNA contributes toward variation in age of onset, directly implicating somatic expansion in the disease pathway. Here, we confirm these results using a larger more genetically homogenous Costa Rican DM1 cohort (p < 0.001). Interestingly, we also provide evidence that supports subtle sex-dependent differences in repeat length-dependent age at onset and somatic mutational dynamics. Previously, we demonstrated that variation in the rate of somatic expansion was a heritable quantitative trait. Given the important role that DNA mismatch repair genes play in mediating expansions in mouse models, we tested for modifier gene effects with 13 DNA mismatch gene polymorphisms (one each in MSH2, PMS2, MSH6 and MLH1; and nine in MSH3). After correcting for allele length and age effects, we identified three polymorphisms in MSH3 that were associated with variation in somatic instability: Rs26279 (p = 0.003); Rs1677658 (p = 0.009); and Rs10168 (p = 0.031). However, only the association with Rs26279 remained significant after multiple testing correction. Although we revealed a statistically significant association between Rs26279 and somatic instability, we did not detect an association with the age at onset. Individuals with the A/A genotype for Rs26279 tended to show a greater propensity to expand the CTG repeat than other genotypes. Interestingly, this SNP results in an amino acid change in the critical ATPase domain of MSH3 and is potentially functionally dimorphic. These data suggest that MSH3 is a key player in generating somatic variation in DM1 patients and further highlight MSH3 as a potential therapeutic target.     

Genealogical study of myotonic dystrophy in Istria (Croatia)

High prevalence of myotonic dystrophy (DM) of 18.1 per 100,000 has been found in Croatian region Istria, a region where a great mixture of nations occurred over the last three centuries. The objective of this study was to test the hypothesis of common ancestry in Istrian DM families. Pedigrees were constructed on the basis of extensive family history obtained from the patients in all Istrian DM families. Church records were consulted in order to improve genealogical reconstruction. Additionally, we performed haplotype analyses with two intragenic and three extragenic DNA polymorphic markers. A common ancestor couple for three of nine nucleus families was found eight generations backward, which was supported by haplotype analysis. In spite of finding an evidence of common ancestry in Croatian Istria we argue that the phenomenon of founder effect is not sufficient to explain the high DM prevalence in Istria.     

Oxidative stress in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults. The genetic basis of DM1 consists of a mutational expansion of a repetitive trinucleotide sequence (CTG). The number of triplets expansion divides patients in four categories related to the molecular changes (E1, E2, E3, E4). The pathogenic mechanisms of multi-systemic involvement of DM1 are still unclear. DM1 has been suspected to be due to premature aging, that is known to be sustained by increased free radicals levels and/or decreased antioxidants activities in neurodegenerative disorders. Recently, the gain-of-function at RNA level hypothesis has gained great attention, but oxidative stress might act in the disease progression. We have investigated 36 DM1 patients belonging to 22 unrelated families, 10 patients with other myotonic disorders (OMD) and 22 age-matched healthy controls from the clinical, biochemical and molecular point of view. Biochemical analysis detected blood levels of superoxide dismutase (SOD), malonilaldehyde (MDA), vitamin E (Vit E), hydroxyl radicals (OH) and total antioxidant system (TAS). Results revealed that DM1 patients showed significantly higher levels of SOD (+40%; MAL (+57%; RAD 2 (+106%; and TAS (+20%; than normal controls. Our data support the hypothesis of a pathogenic role of oxidative stress in DM1 and therefore confirm the detrimental role played by free radicals in this pathology and suggest the opportunity to undertake clinical trials with antioxidants in this disorder.     

Anatomy of a founder effect: myotonic dystrophy in Northeastern Quebec

Founder effects are largely responsible for changes in frequency profiles of genetic variants in local populations or isolates. They are often recognized by elevated incidence of certain hereditary disorders as observed in regions of Charlevoix and Saguenay-Lac-Saint-Jean (SLSJ) in Northeastern Quebec. Dominantly transmitted myotonic dystrophy (DM1) is highly prevalent in SLSJ where its carrier rate reaches 1/550, compared with 1/5,000 to 1/50,000 elsewhere. To shed light on the origin of DM1 in this region, we have screened 50 nuclear DM1 families from SLSJ and studied the genetic variation in a 2.05 Mb (2.9 cM) segment spanning the site of the expansion mutation. The markers analyzed included 22 biallelic SNPs and two microsatellites. Among 50 independent DM1 chromosomes, we distinguished ten DM1-associated haplotypes and grouped them into three haplotype families, A, B and C, based on the relevant extent of allele sharing between them. To test whether the data were consistent with a single entry of the mutation into SLSJ, we evaluated the age of the founder effect from the proportion of recombinant haplotypes. Taking the prevalent haplotype A1_21 (58%) as ancestral to all the disease-associated haplotypes in this study, the estimated age of the founder effect was 19 generations, long predating the colonization of Nouvelle-France. In contrast, considering A1_21 as ancestral to the haplotype family A only, yielded the estimated founder age of nine generations, consistent with the settlement of Charlevoix at the turn of 17th century and subsequent colonization of SLSJ. We conclude that it was the carrier of haplotype A (present day carrier rate of 1/730) that was a driver of the founder effect, while minor haplotypes B and C, with corresponding carrier rates of 1/3,000 and 1/10,000, respectively, contribute DM1 to the incidence level known in other populations. Other studies confirm that this might be a general scenario in which a major driver mutation/haplotype issued from a founder effect is found accompanied by distinct minor mutations/haplotypes occurring at background population frequencies.Electronic Supplementary Material Supplementary material is available for this article at     

High conservation of the trinucleotide [CTG]n repeat at the myotonic dystrophy locus in nonhuman primates

Myotonic dystrophy is due to instability of a [CTG] repeat in the myotonin-protein kinase gene. We have sequenced the complete 3′ untranslated region of this gene which contains the repeat, in seven nonhuman primates. We found that the genomic organisation was conserved, suggesting that this region has important regulatory functions. These data also argue that the human state is derived from a primate ancestor in which the mutational event did not involve the loss of cryptic sequences interrupting or surrounding the repeat, but likely affected only the original length of the repeat.     

The hallmarks of myotonic dystrophy type 1 muscle dysfunction

Myotonic dystrophy type 1 (DM1) is the most prevalent form of muscular dystrophy in adults and yet there are currently no treatment options. Although this disease causes multisystemic symptoms, it is mainly characterised by myopathy or diseased muscles, which includes muscle weakness, atrophy, and myotonia, severely affecting the lives of patients worldwide. On a molecular level, DM1 is caused by an expansion of CTG repeats in the 3′ untranslated region (3′UTR) of the DM1 Protein Kinase (DMPK) gene which become pathogenic when transcribed into RNA forming ribonuclear foci comprised of auto complementary CUG hairpin structures that can bind proteins. This leads to the sequestration of the muscleblind-like (MBNL) family of proteins, depleting them, and the abnormal stabilisation of CUGBP Elav-like family member 1 (CELF1), enhancing it. Traditionally, DM1 research has focused on this RNA toxicity and how it alters MBNL and CELF1 functions as key splicing regulators. However, other proteins are affected by the toxic DMPK RNA and there is strong evidence that supports various signalling cascades playing an important role in DM1 pathogenesis. Specifically, the impairment of protein kinase B (AKT) signalling in DM1 increases autophagy, apoptosis, and ubiquitin–proteasome activity, which may also be affected in DM1 by AMP-activated protein kinase (AMPK) downregulation. AKT also regulates CELF1 directly, by affecting its subcellular localisation, and indirectly as it inhibits glycogen synthase kinase 3 beta (GSK3β), which stabilises the repressive form of CELF1 in DM1. Another kinase that contributes to CELF1 mis-regulation, in this case by hyperphosphorylation, is protein kinase C (PKC). Additionally, it has been demonstrated that fibroblast growth factor-inducible 14 (Fn14) is induced in DM1 and is associated with downstream signalling through the nuclear factor κB (NFκB) pathways, associating inflammation with this disease. Furthermore, MBNL1 and CELF1 play a role in cytoplasmic processes involved in DM1 myopathy, altering proteostasis and sarcomere structure. Finally, there are many other elements that could contribute to the muscular phenotype in DM1 such as alterations to satellite cells, non-coding RNA metabolism, calcium dysregulation, and repeat-associated non-ATG (RAN) translation. This review aims to organise the currently dispersed knowledge on the different pathways affected in DM1 and discusses the unexplored connections that could potentially help in providing new therapeutic targets in DM1 research.     

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.     

High-fat diet induced adiposity and insulin resistance in mice lacking the myotonic dystrophy protein kinase

Myotonic dystrophy 1 (MD1) is caused by a CTG expansion in the 3′-unstranslated region of the myotonic dystrophy protein kinase (DMPK) gene. MD1 patients frequently present insulin resistance and increased visceral adiposity. We examined whether DMPK deficiency is a genetic risk factor for high-fat diet-induced adiposity and insulin resistance using the DMPK knockout mouse model. We found that high-fat fed DMPK knockout mice had significantly increased body weights, hypertrophic adipocytes and whole-body insulin resistance compared with wild-type mice. This nutrient-genome interaction should be considered by physicians given the cardiometabolic risks and sedentary lifestyle associated with MD1 patients.     

Myotonic dystrophy type 1 (DM1): A triplet repeat expansion disorder

Myotonic dystrophy is a progressive multisystem genetic disorder affecting about 1 in 8000 people worldwide. The unstable repeat expansions of (CTG)n or (CCTG)n in the DMPK and ZNF9 genes cause the two known subtypes of myotonic dystrophy: (i) myotonic dystrophy type 1 (DM1) and (ii) myotonic dystrophy type 2 (DM2) respectively. There is currently no cure but supportive management helps equally to reduce the morbidity and mortality and patients need close follow up to pay attention to their clinical problems. This review will focus on the clinical features, molecular view and genetics, diagnosis and management of DM1.     

Direct effects of the pathogenic mutation on satellite cell function in muscular dystrophy

Skeletal muscle is maintained and repaired by resident stem cells called muscle satellite cells, but there is a gradual failure of this process during the progressive skeletal muscle weakness and wasting that characterises muscular dystrophies. The pathogenic mutation causes muscle wasting, but in conditions including Duchenne muscular dystrophy, the mutant gene is not expressed in satellite cells, and so muscle maintenance/repair is not directly affected. The chronic muscle wasting, however, produces an increasingly hostile micro-environment in dystrophic muscle. This probably combines with excessive satellite cell use to eventually culminate in an indirect failure of satellite cell-mediated myofibre repair. By contrast, in disorders such as Emery-Dreifuss muscular dystrophy, the pathogenic mutation not only instigates muscle wasting, but could also directly compromise satellite cell function, leading to less effective muscle homeostasis. This may again combine with excessive use and a hostile environment to further compromise satellite cell performance. Whichever the mechanism, the ultimate consequence of perturbed satellite cell activity is a chronic failure of myofibre maintenance in dystrophic muscle. Here, we review whether the pathogenic mutation can directly contribute to satellite cell dysfunction in a number of muscular dystrophies.     

中国羌族人群强直性肌营养不良症基因CTG重复序列多态性研究

强直性肌营养不良症是由于MT-PK基因3'非编码区CTG三核苷酸重复序列的过度扩展所致。正常人群中CTG的拷贝数为5-30,而患者在50以上,且具民族差异。目前尚无我国羌族人群的有关资料。为了解中国羌族人群该基因3'UT R CTG三核苷重复序列的分布情况,作者采用PCR、聚丙烯酰胺凝胶电泳、银染和测序等技术,对60例正常羌族人的CTG重复序列进行了分析。共发现8种等位基因,其中CTG拷贝数为5的等位基因最为常见,占30 .83%,其余依次为13拷贝(22.5%)、12(19.17%)、11(15.83%)、14(5.83%)和15(4.17%);拷贝数大于15的等位基因极少,仅检测到一例,为27拷贝;CT G拷贝数在6-10之间的等位基因也很少,仅发现一例为9拷贝,而该等位基因在其它人群尚无报道。60名个体中共发现纯合子18例,其中9例为5/5,2例为11/11,2例为12/12,4例13/13和1例为15/15,杂合率为70%。本系统的多态信息量(PIC)为0.77。羌族和汉族人群该位点的多态性无显著差异。 Abstract:Myotonic dystrophy is associated with an increased number of CTG repeats in the 3’UTR of the myotonic protein kinase gene(MT-PK) located on chromosome 19q13.3.The triplet repeats region of the gene of 60 healthy Qiang subjects from Sichuan province was analyzed by polymerase chain reaction and polyacrylamide gel electrophoresis.A total of 8 alleles were found ranging in size from 5 to 27copies with the most common allele of 5 copies(30.83%).The other major alleles were 11,12 and 13 copies with frequency of 15.83%,19.17% and 22.5%,respectively.An allele of 9 copies was found in a Qiang individual which has never been reported before in other populations.Only 5.83% of alleles were longer than 14 copies and one longer than 15 copies.Heterozygote frequency in this population was 70%.The CTG repeats is highly informative with a PIC value of 0.77.There is no significant difference between Qiang and Han population in the distribution of the CTG allele frequencies.     

Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture

Satellite cells represent a cellular source of regeneration in adult skeletal muscle. It remains unclear why a large pool of stem myoblasts in denervated muscle does not compensate for the loss of muscle mass during post-denervation atrophy. In this study, we present evidence that satellite cells in long-term denervated rat muscle are able to activate synthesis of contractile proteins after single fusions in situ. This process of early differentiation leads to formation of abnormally diminutive myotubes. The localization of such dwarf myotubes beneath the intact basal lamina on the surface of differentiated muscle fibers shows that they form by fusion of neighboring satellites or by the progeny of a single satellite cell following one or two mitotic divisions. We demonstrated single fusions of myoblasts using electron microscopy, immunocytochemical labeling and high resolution confocal digital imaging. Sequestration of nascent myotubes by the rapidly forming basal laminae creates a barrier that limits further fusions. The recruitment of satellite cells in the formation of new muscle fibers results in a progressive decrease in their local densities, spatial separation and ultimate exhaustion of the myogenic cell pool. To determine whether the accumulation of aberrant dwarf myotubes is explained by the intrinsic decline of myogenic properties of satellite cells, or depends on their spatial separation and the environment in the tissue, we studied the fusion of myoblasts isolated from normal and denervated muscle in cell culture. The experiments with a culture system demonstrated that the capacity of myoblasts to synthesize contractile proteins without serial fusions depended on cell density and the availability of partners for fusion. Satellite cells isolated from denervated muscle and plated at fusion-permissive densities progressed through the myogenic program and actively formed myotubes, which shows that their myogenic potential is not considerably impaired. The results of this study suggest that under conditions of denervation, progressive spatial separation and confinement of many satellite cells within the endomysial tubes of atrophic muscle fibers and progressive interstitial fibrosis are the important factors that prevent their normal differentiation. Our findings also provide an explanation of why denervated muscle partially and temporarily is able to restore its functional capacity following injury and regeneration: the release of satellite cells from their sublaminal location provides the necessary space for a more active regenerative process.     

Slipped (CTG).(CAG) repeats of the myotonic dystrophy locus: surface probing with anti-DNA antibodies

At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x相似文献   

A novel role for non-muscle gamma-actin in skeletal muscle sarcomere assembly

Existing models describing sarcomere assembly have arisen primarily from studies using cardiac muscle. In contrast to cardiac muscle, skeletal muscle differentiation is characterised by dramatic changes in protein expression, from non-muscle to muscle-specific isoforms before organisation of the sarcomeres. Consequently, little is understood of the potential influence of non-muscle cytoskeletal proteins on skeletal sarcomere assembly. To address this issue, transfectant (gamma33-B1) and control mouse C2 myoblasts were differentiated to form myotubes, and various stages of skeletal sarcomere assembly were studied. Organisation of non-muscle gamma-actin and co-localisation with sarcomeric alpha-actinin, an early marker of sarcomere assembly and a major component of Z lines, was noted. gamma-Actin was also identified in young myotubes with developing sarcomeric myofibrils in regenerating adult mouse muscle. Localisation of gamma-actin in a different area of the myotube to the muscle-specific sarcomeric alpha-actin also indicated a distinct role for gamma-actin. The effects of aberrant gamma-actin expression in other myoblast lines, further suggested a sequestering role for gamma-actin. These observations make the novel suggestion that non-muscle gamma-actin plays a role in skeletal sarcomere assembly both in vitro and in vivo. Consequently, a modified model is proposed which describes the role of gamma-actin in skeletal sarcomere assembly.