Deregulated microRNAs in myotonic dystrophy type 2

Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms.     

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.     

Decreased DMPK transcript levels in myotonic dystrophy 1 type IIA muscle fibers

Concentrations of free polyamines were investigated in Trypanosoma granulosum cultured in a semidefined medium containing traces of polyamines. Spermidine content peaked in early logarithmic growth while putrescine was not detectable. Unlike African trypanosomes and Leishmania, spermine was measured at equivalent amounts to spermidine in mid to late logarithmic stage cells. Addition of d,l-alpha-difluoromethylornithine to cultures did not decrease polyamine content nor was ornithine decarboxylase activity detected. In contrast, incubation of parasites with tritiated putrescine showed rapid uptake and subsequent conversion to spermidine and spermine. At late logarithmic growth, parasites contained glutathione (77% of total sulphydryl groups) and ovothiol A as major low molecular mass thiols with glutathionylpolyamine conjugates undetectable. However, the addition of exogenous putrescine elevated trypanothione and glutathionylspermidine content to 48% of total sulphydryl groups. Correspondingly, the addition of exogenous cadaverine increased homotrypanothione content. This first report of polyamines and low molecular mass thiols in Trypanosoma granulosum indicates intriguing similarities with the metabolism of the human pathogen Trypanosoma cruzi.     

Alternative splicing of human insulin receptor gene (INSR) in type I and type II skeletal muscle fibers of patients with myotonic dystrophy type 1 and type 2

INSR, one of those genes aberrantly expressed in myotonic dystrophy type 1 (DM1) and type 2 (DM2) due to a toxic RNA effect, encodes for the insulin receptor (IR). Its expression is regulated by alternative splicing generating two isoforms: IR-A, which predominates in embryonic tissue, and IR-B, which is highly expressed in adult, insulin-responsive tissues (skeletal muscle, liver, and adipose tissue). The aberrant INSR expression detected in DM1 and DM2 muscles tissues, characterized by a relative increase of IR-A versus IR-B, was pathogenically related to the insulin resistance occurring in DM patients. To assess if differences in the aberrant splicing of INSR could underlie the distinct fiber type involvement observed in DM1 and DM2 muscle tissues, we have used laser capture microdissection (LCM) and RT-PCR, comparing the alternative splicing of INSR in type I and type II muscle fibers isolated from muscle biopsies of DM1, DM2 patients and controls. In the controls, the relative amounts of IR-A and IR-B showed no obvious differences between type I and type II fibers, as in the whole muscle tissue. In DM1 and DM2 patients, both fiber types showed a similar, relative increase of IR-A versus IR-B, as also evident in the whole muscle tissue. Our data suggest that the distinct fiber type involvement in DM1 and DM2 muscle tissues would not be related to qualitative differences in the expression of INSR. LCM can represent a powerful tool to give a better understanding of the pathogenesis of myotonic dystrophies, as well as other myopathies.     

Non-B DNA conformations formed by long repeating tracts of myotonic dystrophy type 1, myotonic dystrophy type 2, and Friedreich's ataxia genes, not the sequences per se, promote mutagenesis in flanking regions

The expansions of long repeating tracts of CTG.CAG, CCTG.CAGG, and GAA.TTC are integral to the etiology of myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), and Friedreich's ataxia (FRDA). Essentially all studies on the molecular mechanisms of this expansion process invoke an important role for non-B DNA conformations which may be adopted by these repeat sequences. We have directly evaluated the role(s) of the repeating sequences per se, or of the non-B DNA conformations formed by these sequences, in the mutagenic process. Studies in Escherichia coli and three types of mammalian (COS-7, CV-1, and HEK-293) fibroblast-like cells revealed that conditions which promoted the formation of the non-B DNA structures enhanced the genetic instabilities, both within the repeat sequences and in the flanking sequences of up to approximately 4 kbp. The three strategies utilized included: the in vivo modulation of global negative supercoil density using topA and gyrB mutant E. coli strains; the in vivo cleavage of hairpin loops, which are an obligate consequence of slipped-strand structures, cruciforms, and intramolecular triplexes, by inactivation of the SbcC protein; and by genetic instability studies with plasmids containing long repeating sequence inserts that do, and do not, adopt non-B DNA structures in vitro. Hence, non-B DNA conformations are critical for these mutagenesis mechanisms.     

Leptin in type 2 diabetic or myotonic dystrophic women.

BACKGROUND: Insulin resistance is an important determinant of circulating leptin concentrations in humans, but its independent contribution on plasma leptin levels are controversial. In the present study, we characterized plasma leptin levels and their regulation in women with 2 different insulin resistance states: type 2 diabetes and myotonic dystrophy disease, and in controls. MATERIAL AND METHODS: We studied 3 groups of women: 21 type 2 diabetic patients, 20 myotonic dystrophic patients and a control group of 20 normoglycemic subjects, matched in age and body mass index. Body composition, fasting glucose and insulin, IGF-I, IGF-binding protein-3 and leptin were studied. Body composition was measured using a bioelectrical impedance analyser. Insulin sensitivity (in percentage) was modeled according to a computer-based homeostasis model assessment model. Data are expressed in mean +/- SEM. RESULTS: In both groups of patients, glucose concentrations were higher in type 2 diabetic patients than in myotonic dystrophic patients, and insulin concentrations and insulin sensitivity were similar in the 2 groups of patients (82.4 +/- 18.6% in type 2 diabetic patients vs. 69.7 +/- 9.7% in myotonic dystrophic patients, p = 0.2) and lower than in controls. Serum leptin and leptin/fat mass ratio were higher in myotonic dystrophic patients than in type 2 diabetic patients (30 +/- 4.9 ng/ml vs. 17.7 +/- 2.6 ng/ml, p = 0.03 and 2.32 +/- 0.69 ng/ml/kg vs. 1.07 +/- 0.2 ng/ml/kg, p = 0.02, respectively) or those found in controls. In type 2 diabetic patients, leptin concentrations were correlated with body mass index and body fat, and in myotonic dystrophic patients leptin concentrations were correlated with age, body mass index, fasting insulin and lower insulin sensitivity, whereas leptin concentrations were not correlated with body fat. CONCLUSIONS: These findings suggest that leptin concentrations and regulation in myotonic dystrophic patients are different from type 2 diabetes.     

Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy

In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.     

Redox imbalance in peripheral blood of type 1 myotonic dystrophy patients

Objectives: The aim of our study was to determine if redox imbalance caused by the activities of antioxidant enzymes existed in erythrocytes of type 1 myotonic dystrophy (DM1) patients.

Methods: The activities of erythrocyte superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were measured in 30 DM1 patients and 15 healthy controls (HCs). The obtained values were correlated with the Muscular Impairment Rating Scale (MIRS) score and creatine kinase (CK).

Results: Superoxide dismutase and catalase activities were lower in DM1 patients compared to HCs. A positive correlation was found between disease duration and MIRS score as well as with glutathione reductase activity. In DM1 patients, there were positive correlations between catalase, glutathione peroxidase, and glutathione reductase activities. After sub-dividing DM1 patients according to CK levels, superoxide dismutase activity was still statistically different from HCs. However, catalase activity was significantly lower only in DM1 patients with increased CK.

Discussion: Undesirable alterations in antioxidant enzyme activities during DM1 disease progression may result in conditions favoring oxidative stress and changes in metabolism which together could contribute to muscle wasting.     

Routinely frozen biopsies of human skeletal muscle are suitable for morphological and immunocytochemical analyses at transmission electron microscopy

The aim of the present investigation was to evaluate whether routinely frozen biopsies of human skeletal muscle may be suitable for morphological and immunocytochemical analyses at transmission electron microscopy. The fixation/embedding protocols we successfully used for decades to process fresh mammalian tissues have been applied to frozen muscle biopsies stored for one to four years in liquid nitrogen. After 2.5% glutaraldehyde -2% paraformaldehyde - 1% OsO₄ fixation and embedding in epoxy resin, the ultrastructural morphology of myofibres and satellite cells as well as of their organelles and inclusions proved to be well preserved. As expected, after 4% paraformaldehyde - 0.5% glutaraldehyde fixation and embedding in LR White resin, the morphology of membrane-bounded organelles was relatively poor, although myofibrillar and sarcomeric organization was still recognizable. On the contrary, the myonuclei were excellently preserved and, after conventional staining with uranyl acetate, showed an EDTA-like effect, i.e. the bleaching of condensed chromatin, which allows the visualization of RNP-containing structures. These samples proved to be suitable for immunocytochemical analyses of both cytoskeletal and nuclear components, whereas the poor mitochondrial preservation makes unreliable any in situ investigation on these organelles.Keeping in mind the limitations found, these results open promising perspectives in the study of frozen skeletal muscle samples stored in the tissue banks; this would be especially interesting for rare muscle diseases, where the limited number of biopsies suitable for ultrastructural investigation has so far represented a great restriction in elucidating the cellular mechanisms responsible for the pathological phenotype.Key words: frozen biopsy, electron microscopy, fixation, immunocytochemistry, skeletal muscle.     

Cyclooxygenase-2 expression in skeletal muscle of knockout mice suffering Duchenne muscular dystrophy

The purpose of the present study was to investigate the role of cyclooxygenase-2 (COX-2) expression in fibrotic lesion in mdx mice. A total of six male C57BL/10 mice and six C57BL/10-DMD/mdx were distributed into two groups: control and animals with Duchenne muscular dystrophy (DMD). The medial part of gastrocnemius muscle was evaluated being the specimens stained with hematoxylin and eosin (H&E) and Sirius Red under normal and polarized light to differentiate type I (red and yellow) and III (green) collagen. COX-2 expression was assessed by immunohistochemistry. The results revealed histopathological changes in C57BL/10-DMD/mdx as depicted by regenerating fibers. Sirius Red stain showed a substantial increase in the amount of type I collagen of mdx mice. DMD induced a strong COX-2 immunoexpression in intercellular space. Taken together, our results are consistent with the notion that necrotic and fibrotic lesions are able to increase COX-2 expression in DMD.     

Localization of calpain 3 in human skeletal muscle and its alteration in limb-girdle muscular dystrophy 2A muscle

Calpain 3/p94, the skeletal muscle-specific isoform of the calpain large subunit family, is a protein product of the gene responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Through yeast two-hybrid experiments, calpain 3 has been shown to bind to titin in myofibrils [Sorimachi et al. (1995) J. Biol. Chem. 270, 31158-31162]. However, because of extensive autolysis activity, calpain 3 localization in skeletal muscle has been undefined. In this study, we generated a polyclonal antibody against an N-terminal 98-amino-acid calpain 3 fragment, which is not homologous to the corresponding regions of other conventional calpains. This antibody stained myofibrils with a unique repeated doublet-pattern. Confocal microscopic observation with marker antibodies confirmed that calpain 3 is localized in the N2 region of myofibrils. Furthermore, using this antibody, we examined the localization of calpain 3 in LGMD2A muscles.     

Mitochondrial dysfunction in human skeletal muscle biopsies of lipid storage disorder

Mitochondria regulate the balance between lipid metabolism and storage in the skeletal muscle. Altered lipid transport, metabolism and storage influence the bioenergetics, redox status and insulin signalling, contributing to cardiac and neurological diseases. Lipid storage disorders (LSD s) are neurological disorders which entail intramuscular lipid accumulation and impaired mitochondrial bioenergetics in the skeletal muscle causing progressive myopathy with muscle weakness. However, the mitochondrial changes including molecular events associated with impaired lipid storage have not been completely understood in the human skeletal muscle. We carried out morphological and biochemical analysis of mitochondrial function in muscle biopsies of human subjects with LSD s (n  = 7), compared to controls (n  = 10). Routine histology, enzyme histochemistry and ultrastructural analysis indicated altered muscle cell morphology and mitochondrial structure. Protein profiling of the muscle mitochondria from LSD samples (n  = 5) (vs. control, n  = 5) by high‐throughput mass spectrometric analysis revealed that impaired metabolic processes could contribute to mitochondrial dysfunction and ensuing myopathy in LSD s. We propose that impaired fatty acid and respiratory metabolism along with increased membrane permeability, elevated lipolysis and altered cristae entail mitochondrial dysfunction in LSD s. Some of these mechanisms were unique to LSD apart from others that were common to dystrophic and inflammatory muscle pathologies. Many differentially regulated mitochondrial proteins in LSD are linked with other human diseases, indicating that mitochondrial protection via targeted drugs could be a treatment modality in LSD and related metabolic diseases.

Cover Image for this Issue: doi: 10.1111/jnc.14177 .