2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls
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 was...
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doaj-46bb7c5dce5742c7828512fe95fab7052020-11-25T00:39:56ZengBMCRespiratory Research1465-993X2017-05-0118111010.1186/s12931-017-0525-x2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controlsRamzi Lakhdar0Ellen M. Drost1William MacNee2Ricardo Bastos3Roberto A. Rabinovich4ELEGI Colt Laboratory, Centre for Inflammation Research, The Queen’s Medical Research Institute, University of EdinburghELEGI Colt Laboratory, Centre for Inflammation Research, The Queen’s Medical Research Institute, University of EdinburghELEGI Colt Laboratory, Centre for Inflammation Research, The Queen’s Medical Research Institute, University of EdinburghInstitut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of BarcelonaELEGI Colt Laboratory, Centre for Inflammation Research, The Queen’s Medical Research Institute, University of EdinburghAbstract 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.http://link.springer.com/article/10.1186/s12931-017-0525-xCOPDSkeletal muscle dysfunction/wastingProteomic analysis2D-DIGEDOT1LAgeing |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Ramzi Lakhdar Ellen M. Drost William MacNee Ricardo Bastos Roberto A. Rabinovich |
spellingShingle |
Ramzi Lakhdar Ellen M. Drost William MacNee Ricardo Bastos Roberto A. Rabinovich 2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls Respiratory Research COPD Skeletal muscle dysfunction/wasting Proteomic analysis 2D-DIGE DOT1L Ageing |
author_facet |
Ramzi Lakhdar Ellen M. Drost William MacNee Ricardo Bastos Roberto A. Rabinovich |
author_sort |
Ramzi Lakhdar |
title |
2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls |
title_short |
2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls |
title_full |
2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls |
title_fullStr |
2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls |
title_full_unstemmed |
2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls |
title_sort |
2d-dige proteomic analysis of vastus lateralis from copd patients with low and normal fat free mass index and healthy controls |
publisher |
BMC |
series |
Respiratory Research |
issn |
1465-993X |
publishDate |
2017-05-01 |
description |
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. |
topic |
COPD Skeletal muscle dysfunction/wasting Proteomic analysis 2D-DIGE DOT1L Ageing |
url |
http://link.springer.com/article/10.1186/s12931-017-0525-x |
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