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Respiratory Research
Published in: Respiratory Research 1/2017

Open Access 01-12-2017 | Research

2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls

Authors: Ramzi Lakhdar, Ellen M. Drost, William MacNee, Ricardo Bastos, Roberto A. Rabinovich

Published in: Respiratory Research | Issue 1/2017

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Abstract

Background

Chronic obstructive pulmonary disease (COPD) is associated with several extra-pulmonary effects of which skeletal muscle wasting is one of the most common and contributes to reduced quality of life, increased morbidity and mortality. The molecular mechanisms leading to muscle wasting are not fully understood. Proteomic analysis of human skeletal muscle is a useful approach for gaining insight into the molecular basis for normal and pathophysiological conditions.

Methods

To identify proteins involved in the process of muscle wasting in COPD, we searched differentially expressed proteins in the vastus lateralis of COPD patients with low fat free mass index (FFMI), as a surrogate of muscle mass (COPDL, n = 10) (FEV1 33 ± 4.3% predicted, FFMI 15 ± 0.2 Kg.m−2), in comparison to patients with COPD and normal FFMI (COPDN, n = 8) and a group of age, smoking history, and sex matched healthy controls (C, n = 9) using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) technology, combined with mass spectrometry (MS). The effect of silencing DOT1L protein expression on markers of cell arrest was analyzed in skeletal muscle satellite cells (HSkMSCs) in vitro and assessed by qPCR and Western blotting.

Results

A subset of 7 proteins was differentially expressed in COPDL compared to both COPDN and C. We found an increased expression of proteins associated with muscle homeostasis and protection against oxidative stress, and a decreased expression of structural muscle proteins and proteins involved in myofibrillogenesis, cell proliferation, cell cycle arrest and energy production. Among these was a decreased expression of the histone methyltransferase DOT1L. In addition, silencing of the DOT1L gene in human skeletal muscle satellite cells in vitro was significantly related to up regulation of p21 WAF1/Cip1/CDKN1A, a marker of cell arrest and ageing.

Conclusions

2D-DIGE coupled with MS identified differences in the expression of several proteins in the wasted vastus lateralis that are relevant to the disease process. Down regulation of DOT1L in the vastus lateralis of COPDL patients may mediate the muscle wasting process through up regulation of markers of cell arrest and senescence.
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Metadata
Title
2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls
Authors
Ramzi Lakhdar
Ellen M. Drost
William MacNee
Ricardo Bastos
Roberto A. Rabinovich
Publication date
01-12-2017
Publisher
BioMed Central
Published in
Respiratory Research / Issue 1/2017
Electronic ISSN: 1465-993X
DOI
https://doi.org/10.1186/s12931-017-0525-x

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