Potential regulation of human muscle plasticity by MLC2 post-translational modifications during bed rest and countermeasures Laurence Stevens ⇑,1 , Bruno Bastide 1 , Julie Hedou, Caroline Cieniewski-Bernard, Valérie Montel, Laetitia Cochon, Erwan Dupont, Yvonne Mounier Université Lille Nord de France, Université Lille 1 – Sciences et Technologies, Laboratoire Activité Physique, Muscle et Santé, EA 4488, IFR 114, Institut de Recherches Pluridisciplinaires de Biologie et de Biotechnologies, 59655 Villeneuve d’Ascq cedex, France article info Article history: Received 18 July 2013 and in revised form 8 October 2013 Available online 31 October 2013 Keywords: Human soleus Inactivity Myosin Light Chain 2 Phosphorylation O-glycosylation Countermeasures abstract This study investigated the effects of a 60-day bed rest with or without countermeasures on muscular phenotype and post-translational modifications of the regulatory Myosin Light Chain 2 (MLC2) protein. Soleus biopsies were obtained from female subjects before and after bed rest. Control subjects were assigned only to bed rest (BR), BR + Ex subjects were submitted to combined aerobic and resistive exer- cises, and BR + Nut to nutritional leucine and valine diet. We determined Myosin Heavy Chains (MHC) and MLC2 composition of muscles using 1D SDS–PAGE. MLC2 phosphorylation was measured on 2D gels and O-N-Acetyl Glucosaminylation (O-GlcNAc) level of MLC2 was determined. Our results showed a slow-to-fast shift of MHC and MLC2 isoforms in BR and BR + Nut while BR + Ex combinations prevented these phenotype changes. After BR, the MLC2 phosphorylation state was increased while the global MLC2 glycosylation level was decreased. Exercises prevented the variations of phosphorylation and glycosyla- tion observed after BR whereas nutrition had no effects. These results suggested an interplay between phosphorylation and glycosylation of MLC2, which might be involved in the development of muscle atro- phy and associated changes. These findings of differential responses to exercises and nutrition protocols were discussed with implications for future prescription models to preserve muscle against long-term unloading. Ó 2013 Elsevier Inc. All rights reserved. Introduction Proteomics research enabled to understand the complex and dynamic protein expression patterns in the various mammalian cell types. Among many other mechanisms like gene up- or down- regulation, post-translational modifications play a crucial role in striated muscle protein structure, function, signalling and regula- tion. Post-translational events are known to control protein func- tion [1,2], and to regulate protein–protein interactions. In striated muscles, post-translational modifications could be in- volved in regulating actin–myosin interaction and contractile func- tion [3–5]. In skeletal muscle, phosphorylation represents one of the most frequent peptide post-translational modifications [6]. The only sites available for phosphorylation in sarcomeric myosins are located on the Myosin Light Chains (MLC) 2 2 or regulatory light chains (in Ser 14 for the slow MLC2 isoform and in Ser 15 for the fast one; [7]) MLC2 phosphorylation is not essential for skeletal muscle contraction but is an important regulatory mechanism since it can produce changes in thick filament structure and enhance cross- bridge attachment [8]. For instance, in rabbit skinned muscle fibres, MLC2 phosphorylation increased Ca 2+ -sensitivity by inducing a shift to submaximal intracellular Ca 2+ concentration in the force-pCa rela- tionship [8–11]. We also demonstrated in rat skeletal muscles that changes in MLC2 phosphorylation were in relation to skeletal muscle plasticity since an increase of MLC2 phosphorylation was associated with slow-to-fast transition regardless of whether hypertrophy or atrophy develops [12,13]. It has been suggested that striated muscle physiology might be influenced, for instance, by glycosylation [5], nitrosylation [14] or deacetylation [15]. Among glycosylation, O-Linked N-acetyl-D- glucosaminylation (termed O-Glc-NAcylation or O-GlcNAc) is a dynamic, cytosolic and nuclear glycosylation that consists in the transfer of a unique monosaccharide, O-N-acetylglucosamine, on the Ser/Thr hydroxyl group of a protein. It is dependent both on glucose flow through the hexosamine biosynthesis pathway and on phosphorylation, because of the existence of a balance between phosphorylation and O-GlcNAc [16]. Our group has previously reported that, in rat skeletal muscle, proteins of the glycolytic 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.10.016 ⇑ Corresponding author. E-mail address: Laurence.Stevens@univ-lille1.fr (L. Stevens). 1 L. Stevens and B. Bastide have contributed equally to this work. 2 Abbreviations used: MLC, myosin light chains; HDT, head-down tilt; SS, supine squat; CP, calf press. Archives of Biochemistry and Biophysics 540 (2013) 125–132 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi