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Folia Neuropathologica
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Original paper

Dysfunctional lamins as mediators of oxidative stress in Emery-Dreifuss muscular dystrophy

Irena Niebroj-Dobosz, Beata Sokołowska, Agnieszka Madej-Pilarczyk, Michał Marchel, Irena Hausmanowa-Petrusewicz

Folia Neuropathol 2017; 55 (3): 193-198
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Introduction

A deficit of lamins A/C or emerin in skeletal muscles and the heart causes a rare, genetically transmitted disease known as Emery-Dreifuss muscular dystrophy (EDMD). The cell defect is generalized, but skeletal muscles, heart and joints are all selectively affected, with the ultimate effects being skeletal muscle atrophy, joint contractures and dilated cardiomyopathy, which is at the onset either clinically silent or preceded by conduction block and arrhythmias [13,14]. Sudden death both in patients and in carriers is not a rare event.
Lamins serve as mediators of oxidative stress [20]. There is a relationship between lamin mutations and altered metabolism of radical oxygen species (ROS). Laminopathies are correlated with oxidative stress at the cellular level. No data have hitherto been available on antioxidant/oxidant status in EDMD.
The aim of the study described here was therefore to assess serum for total oxidant status (TOS), which represents the oxidative/antioxidative balance. The antioxidant capacity (TAC) measures the total antioxidant capacity of biomolecules. The relevant data obtained were compared to the clinical status of the EDMD patients.

Material and methods

A total of 29 patients with EDMD were examined. Twelve of these had autosomal-dominant EDMD associated with laminopathy (AD-EDMD), while 17 had X-linked EDMD (X-EDMD) associated with emeri­nopathy. The diagnoses of EDMD were confirmed clinically, by indirect immunofluorescence analysis of muscle nuclei, immunochemically by Western blotting examination of a muscle biopsy specimen, as previously described [5,15], and by molecular testing of LMNA and EMD genes, respectively (Tables I and II). Dilated cardiomyopathy was present in all the cases. However, no subjective cardiac symptoms, even when there was evident bradycardia, were present. In some patients cardiac involvement was already detected at the time of the first neurological examination. In AD-EDMD patients the left ventricle was affected. There were also disturbances of contractility or conductivity (Table I). Dilated cardiomyo­pathy was a feature common to all studied cases detected already at the first examination. Cardiac symptoms appeared earlier in the AD-EDMD than in the X-EDMD group. In the latter cardiac involvement was moderate/mild overall, to severe/very severe in some cases (Table II). With disease onset, conductance and atrial involvement predominate, necessitating implantation of a pacemaker in the majority of patients. In the follow-up clinical examination dilated cardiomyopathy was found to develop later in X-EDMD than AD-EDMD sufferers.
The control group consisted of 20 healthy age-matched subjects with no history of muscle or cardiac disease.
Blood was selected for routine biochemical analyses and centrifuged at 3000 rpm for 10 minutes. Serum was separated and frozen at –30°C until used, usually up to 30 days both in patients and controls. Total oxidant status was determined using a Real Assay Diagnostics kit 4. The total antioxidant capacity was determined using an OxiSelect kit (Cell Biolabs, Inc.) [8]. Routine hematoxylin-eosin staining, anti-lamin and anti-emerin staining and Western blotting muscle biopsy with anti-lamin and anti-emerin antibodies [16] were also performed.

Statistical analysis

Analyses were performed using the commercial statistical package Statistica ver. 9.0. In line with the non-normal data distribution the medians and interquartile ranges are presented. The Kruskal-Wallis analysis of variance was applied, followed by the Mann-Whitney U test for a two-group comparison. The relationships among variables were analyzed by Spearman’s correlation coefficients. Analysis of the receiver-operating characteristic (ROC) curves was also performed. The probability value of p < 0.05 was regarded as statistically significant.

Results

Levels for serum TOS and total TAC reflect the redox balance between antioxidation and oxidation in EDMD. Total antioxidant capacity values in serum were significantly below those of the control in the cases of both EDMD groups, being at the level of 83.3% of patients in the case of AD-EDMD and 82.4% of patients in the case of X-EDMD (Table III). Slightly decreased values were present in both groups in 16.7 and 17.6% of the patients, respectively (appearing especially in mildly progressing cases). The values of TOS were also reduced in the EDMD patients (Table III). The ROC analysis demonstrated that the area under the curve (AUC) value of serum TAC equals 0.890, while the AUC value for TOS reached 1.000 (Fig. 1). No correlation was observed between TAC, TOS, and the cardiological or neurological parameters. A relationship between TOS level and disease progression was observed (Table IV, p = 0.032).

Discussion

Free radicals are responsible for chemical modifications of and molecular changes in proteins, lipids, carbohydrates and nucleotides, as well as for modulation of gene expression. In biological membranes they induce changes in enzymes, and other proteins, as well as in the oxidation of –SH groups, the peroxidation of lipids, and the oxidation of polyunsaturated fatty acids. They also affect membrane transport systems. Accumulation of ROS can lead to DNA damage and the production of oxidized proteins [16,18]. Free radicals are also produced in different physiological processes under normal conditions. ROS action is typically limited by physiologically active antioxidant defense mechanisms [22]. Where the production of oxidative damaged molecules is enhanced, this is followed by an impairment of defense that leads to the accumulation of toxic products. Several chemical compounds are able to act as antioxidants; the impact is greater when the antioxidants are acting in a set [8,21,22] and when glutathione, the most essential key agent in cell defense, is included [1,24].
Lamina provides mechanical support for the nucleus [9], modulates gene expression [2,11,17], modulates the oxidative stress response [12], acts in intracellular redox homeostasis [20 ], and participates in transcription and chromatin organization [2] and DNA repair [4,6,7]. Laminopathies share features including defects in the DNA damage response [4]. In emerinopathies, apart from the deficit of emerin, a decrease of lamins is also noted [16].
A relationship between LMNA mutation and altered ROS metabolism has already been proposed [19]. ROS have a direct damaging effect, affecting the lamin structure [16], and giving rise to persistent DNA damage [7] and telomere shortening [6]. In laminopathic cells during prelamin A accumulation ROS levels are elevated [3,10,15]. Persistent prelamin accumulation and lamin A/C depletion are likewise associated with elevated ROS levels [20].
Details of redox biology in EDMD are not known, yet. When established it is likely to be helpful as regards both the introduction of early stage pharmacological treatment and decision-making in respect of cardiac device implantations and consideration even of heart transplantation. Thus far, the only TAC member assessed in EDMD has been blood glutathione, which is present in reduced amounts in EDMD with LMNA mutation, and is associated with early cardiac involvement [4]. More details of redox biology in EDMD may serve as a basis for prognoses regarding progression of the disease, as well as in monitoring for the effectiveness of an antioxidant therapy. Such therapy would be useful in restoring the balance between the production of free radicals and the antioxidant capacity, with resultant reduced severity of the disease.
The results presented here for TOS and TAC levels support the opinion that oxidative stress is involved in the pathogenesis of EDMD. This finding could lead to new directions of future therapeutic intervention.

Acknowledgments

Analyses were carried out using the computational infrastructure of the Biocentrum-Ochota project (POIG.02.03.00-00-0030/09).

Disclosure

Authors report no conflict of interest.

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Copyright: © 2017 Mossakowski Medical Research Centre Polish Academy of Sciences and the Polish Association of Neuropathologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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