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

Abstract

BackgroundChronic 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.MethodsTo 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.ResultsA 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.Conclusions2D-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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-017-0525-x) contains supplementary material, which is available to authorized users.

<p><em>CYP1B1</em>, <em>VEGFA</em>, <em>BCL2</em>, and <em>CDKN1A</em> Affect the Development of Chronic Obstructive Pulmonary Disease</p>

“…*P<0.05. a biomarker of cell cycle arrest and premature ageing, is upregulated in COPD patients, which is related to skeletal muscle wasting. 31,32 Skeletal muscle dysfunction is common in COPD patients. A previous study identified 18 genes, including CDKN1A, that were upregulated in the quadriceps of COPD patients relative to that in healthy controls.…”

Physical Activity and Underlying Muscle Biology in Chronic Obstructive Pulmonary Disease (COPD)

“…2). Beside muscle underuse 44 , several factors have been described as putative mechanisms leading to muscle dysfunction/atrophy, namely systemic inflammation 45 , oxidative stress 46 , cell hypoxia 47 , hypercapnia, low levels of anabolic hormones (testosterone) and growth factors (growth hormone [GH], insulin-like growth factor I [IGF-I]), use of glucocorticoids, vitamin D deficiency, impaired energy balance 5 , accelerated ageing and cellular senescence [48][49][50][51][52] , and nutritional depletion 53 .…”

Protein analysis: key to the future

“…In a study published in Respiratory Research in 2017, Lakhdar and colleagues utilized 2D-DIGE combined with mass spectrometry to identify proteins that could be involved in the process of muscle wasting in chronic obstructive pulmonary disease. By using this technique, they elucidated differences in the expression of various proteins in the wasted vastus lateralis muscle that are relevant to the disease process [14]. …”