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 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

Natação e aspectos morfológicos do músculo esquelético em processo de reparo após criolesão Swimming and morphology of skeletal muscle repair process after cryoinjury

O objetivo do estudo foi investigar a influência da natação sobre as alterações morfológicas do músculo esquelético em processo de reparo após criolesão. Foram usados 45 ratos divididos em cinco grupos: controle (n=5); sham (n=5), adaptação (n=5), criolesionados e tratados com natação sacrificados após 7, 14 e 21 dias (n=15); criolesionados e sem tratamento aquático sacrificados após 7, 14 e 21 dias (n=15). As sessões de natação foram realizadas 6 vezes por semana com 90 min de duração cada. Ao término do protocolo os animais foram sacrificados e a análise morfológica da área da lesão foi realizada. A análise morfológica semiquantitativa demonstrou que os músculos do grupo controle apresentaram aspecto histológico normal. O grupo sham apresentou edema, mionecrose e infiltrado inflamatório em grau 1. Nos grupos 7, 14 e 21 dias, não existiram diferenças estatisticamente significativas nas 4 etapas de remodelamento tecidual avaliadas (infiltrado inflamatório, edema, necrose e fibras musculares imaturas) entre os grupos lesionados quando comparados aos grupos com lesão e tratamento aquático. Em conclusão, foi possível verificar que a natação não causou alterações morfológicas durante o reparo do músculo esquelético após criolesão.<br>The aim of study was investigate the influence of swimming on the morphological changes in skeletal muscle repair process following cryoinjury. There were used 45 rats divided into 5 groups: control (n=5), sham (n=5), adaptation (n=5), cryolesioned treated with swimming and sacrificed after 7, 14 and 21 days (n=15), untreated and cryolesioned sacrificed after 7, 14, and 21 days (n=15). Animals swan for 90 min/ each session and 6 times a week. At the end of the protocol, the animals were sacrificed and morphological analysis of the lesion area was performed. The semi-quantitative morphological analysis showed that the muscles in the control group exhibited normal histological aspects while the sham group exhibited edema, myonecrosis and inflammatory infiltrate grade 1. In groups 7, 14, and 21 days, the results indicated that there were no statistically significant differences in four stages of tissue remodeling evaluated (inflammatory infiltration, edema, necrosis, and immature muscle fibers) between the injured groups compared to groups with lesion and treated with swimming. In conclusion, it was verified that swimming did not alter morphological aspects of skeletal muscle during the repair process following cryoinjury

Subcellular Fractionation of Brain Tissue from Small Tissue Explants

For several decades, neurobiologists have used subcellular fractionation methods to analyze the molecular structure and some functional features of the cells in the central nervous system. Indeed, the brain tissue is built through the networking of neuronal, glial, and vascular cells in an intermingled meshwork of micrometer-sized structures. Subcellular fractionation protocols allow for the separation of specific compartments such as synapses (called “synaptosomes”), synaptic plasma membranes, and synaptic vesicles for their analysis at the molecular level. Most current protocols were established to process large amounts of tissue as required in previous experimental paradigms. Here, we provide a protocol to prepare synaptosomes from as little as 10 mg of tissue or a full fractionation to enrich crude synaptic vesicles and synaptic plasma membranes from 20 mg of tissue. This protocol will be useful to anyone aiming at addressing specific questions regarding local microcircuits in combination with connectomics and proteomics approaches

Phosphorylation of nucleoporins: Signal transduction-mediated regulation of their interaction with nuclear transport receptors

The nuclear pore complex (NPC) is composed of ∼30 unique proteins, collectively referred to as nucleoporins or Nups. While metazoan Nups are known to be phosphorylated during mitosis to cause disassembly of the NPC, what is less clear is whether Nups are phosphorylated and regulated by extracellular stimuli in interphase cells. Our multi-step phosphoproteomic approach revealed a number of physiologically relevant extracellular signal-regulated kinase (ERK) targets, including Nups containing FG repeats (FG Nups) that provide binding sites for nuclear transport receptors (NTRs) during the NPC passage. The phosphorylation of FG Nups by ERK does not affect the overall architecture of the NPC but directly inhibits their interactions with NTRs and regulates the permeability barrier properties of the NPC. Such regulation at the levels of transport machinery is expected to have a broad impact on cellular physiology through the spatiotemporal control of signaling events. Until recently, many studies have focused on cellular signaling-mediated phosphorylation of individual cargo proteins, such as transcription factors. An understanding of the effects of signaling pathways on nucleocytoplasmic transport machinery is only beginning to emerge