Global Mrna Sequencing of Human Skeletal Muscle
Total Page：16
File Type：pdf, Size：1020Kb
MOLMET433_proof ■ 3 February 2017 ■ 1/14 Original article 61 62 1 63 2 64 3 Global mRNA sequencing of human skeletal 65 4 66 5 muscle: Search for novel exercise-regulated 67 6 68 7 Q10 myokines 69 8 70 9 71 10 72 11 1 1 1 1 1 2 3 Q9 S. Pourteymour , K. Eckardt , T. Holen , T. Langleite , Sindre Lee , J. Jensen , K.I. Birkeland , 73 12 1 1,4,* Q1 C.A. Drevon , M. Hjorth 74 13 75 14 ABSTRACT 76 15 77 16 Objective: Skeletal muscle is an important secretory organ, producing and releasing numerous myokines, which may be involved in mediating 78 17 beneficial health effects of physical activity. More than 100 myokines have been identified by different proteomics approaches, but these 79 18 techniques may not detect all myokines. We used mRNA sequencing as an untargeted approach to study gene expression of secreted proteins in 80 19 skeletal muscle upon acute as well as long-term exercise. 81 20 Methods: Twenty-six middle-aged, sedentary men underwent combined endurance and strength training for 12 weeks. Skeletal muscle bi- 82 21 opsies from m. vastus lateralis and blood samples were taken before and after an acute bicycle test, performed at baseline as well as after 12 83 22 weeks of training intervention. We identified transcripts encoding secretory proteins that were changed more than 1.5-fold in muscle after 84 23 exercise. Secretory proteins were defined based on either curated UniProt annotations or predictions made by multiple bioinformatics methods. 85 24 Results: This approach led to the identification of 161 candidate secretory transcripts that were up-regulated after acute exercise and 99 that 86 25 where increased after 12 weeks exercise training. Furthermore, 92 secretory transcripts were decreased after acute or long-term physical 87 26 activity. From these responsive transcripts, we selected 17 candidate myokines sensitive to short- and/or long-term exercise that have not been 88 27 described as myokines before. The expression of these transcripts was confirmed in primary human skeletal muscle cells during in vitro dif- 89 28 ferentiation and electrical pulse stimulation (EPS). One of the candidates we identified was colony-stimulating factor 1 (CSF1), which influences 90 29 macrophage homeostasis. CSF1 mRNA increased in skeletal muscle after acute exercise, which was accompanied by a rise in circulating CSF1 91 30 protein. In cultured muscle cells, EPS promoted a significant increase the expression and secretion of CSF1. 92 31 Conclusion: We identified 17 new, exercise-responsive transcripts encoding secretory proteins. We further identified CSF1 as a novel myokine, 93 32 which is secreted from cultured muscle cells and up-regulated in muscle and plasma after acute exercise. 94 33 Ó 2017 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 95 34 96 35 Keywords Exercise; Myokine; Colony stimulating factor 1; RNA sequencing; Skeletal muscle secretome 97 36 98 37 1. INTRODUCTION promoting increased hepatic glucose output and enhanced glucose 99 38 uptake in muscle cells [6]. 100 39 Skeletal muscle was recognized as a secretory organ about 15 years More than 100 myokines have been identified [7], and the skeletal 101 40 ago [1]. Proteins and peptides produced by and released from skeletal muscle secretome is predicted to include more than 300 proteins [8]. 102 41 muscles are termed myokines, and several myokines play important In several proteomics studies, scientists have identified peptides in 103 42 roles in muscle physiology as well as in tissue cross talk [2e4]. Thus, medium conditioned by cultured human [9e12] or murine myocytes 104 43 interest in the secretory function of the skeletal muscle has increased [13e16]. To explain some of the positive effects of physical activity, 105 44 markedly during the last decade. Physical activity alters secretion of several studies focused on identifying myokines that are elevated in 106 45 many myokines, several of which may play a role in mediating response to exercise or muscle contraction [11,17e20]. 107 46 beneficial health effects of physical activity. The aim of this study was to identify novel myokines regulated by acute 108 47 Interleukin 6 (IL6) is the most extensively studied myokine, and is or long-term exercise. Many myokines, such as IL6 and other cyto- 109 48 secreted from skeletal muscle during acute physical activity [5]. IL6 kines, have a low abundance in basal conditions, and are therefore 110 49 may act as an energy sensor in skeletal muscle during exercise, hard to detect with untargeted proteomics. We used global mRNA 111 50 sequencing to detect all genes expressed in human skeletal muscle 112 51 113 52 114 53 1Department of Nutrition, Institute for Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway 2Department of Physical Performance, Norwegian School 3 115 54 of Sport Sciences, Oslo, Norway Department of Endocrinology, Morbid Obesity and Preventive Medicine, Oslo University Hospital and Faculty of Medicine, University of Oslo, Oslo, Norway 4Diabetes and Metabolism Division, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia 116 55 Q2 117 56 *Corresponding author. Diabetes and Metabolism Division, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia. E-mail: [email protected] 118 57 (M. Hjorth). 119 58 Received December 8, 2016 Revision received January 16, 2017 Accepted January 19, 2017 Available online xxx 120 59 121 60 http://dx.doi.org/10.1016/j.molmet.2017.01.007 122 MOLECULAR METABOLISM - (2017) 1e14 Ó 2017 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 www.molecularmetabolism.com MOLMET433_proof ■ 3 February 2017 ■ 2/14 Original article 1 biopsies. In addition, we used cultures of primary human skeletal training was assessed as the mRNA expression at B1 vs. A1 63 2 muscle cells to monitor the expression of novel myokine candidates (Figure 1A,F). 64 3 during differentiation and after electrical pulse stimulation (EPS). To identify transcripts encoding secreted proteins, we used the Met- 65 4 azSecKB knowledgebase [30]. MetazSecKB identifies secretory pro- 66 5 2. METHODS teins based on either curated evidence of secretion (annotated and 67 6 reviewed in the UniProtKB/Swiss-Prot dataset) or being “highly likely” 68 7 2.1. Human trial to be secreted based on computationally predicted secretory protein 69 8 A human exercise intervention trial (NCT01803568) was performed as sequences, without containing transmembrane domains or endo- 70 9 described before [21]. The National Committee for Research Ethics plasmic reticulum (ER) retention signals, by several tools (SignalP4, 71 10 North (Tromsø, Oslo, Norway) approved the trial. This study adhered to Phobius, TargetP and WoLF PSORT). 72 11 the standards set by the Declaration of Helsinki. 73 12 Briefly, 26 sedentary (<1 bout of exercise/week during the previous 2.4. Cell culture 74 13 year) men aged 40e65 y with BMI 26 Æ 4.0 kg/m2, were included in Biopsies from either m. obliquus internus abdominis or m. vastus 75 14 the trial. They were initially recruited to a control group (n ¼ 13) with lateralis from three male donors (age 33e62 y) were used to isolate 76 15 normal glucose metabolism and a dysglycemic group (n ¼ 13) with primary human satellite cells [31]. Myoblasts were proliferated to 77 16 fasting serum glucose 5.6 mmol/L and/or 2 h glucose 7.8 mmol/L passage 4e5 and differentiated [27]. EPS (1 Hz, 2 ms, 11.5 V) was 78 17 based on an oral glucose tolerance test. Two subjects had normal applied to the cells after 5 days of differentiation for 24 h or after 6 days 79 18 glucose levels at screening, but both had a glucose infusion rate of of differentiation for 1e6 h. EPS should mimic muscle contraction 80 À À 19 4.4 mg min 1 kg 1 during the euglycemic hyperinsulinemic clamp similar to in vivo exercise [32]. Supernatants were collected and RNA 81 20 and were included in the dysglycemic group. Here we did not inves- isolated for further analysis. 82 21 tigate group differences, and all subjects were therefore included 83 22 (n ¼ 26). 2.5. Protein analyses 84 23 The participants underwent 12 weeks of supervised exercise training We measured CSF1 concentration in the supernatant of cultured 85 24 with two interval sessions (bicycle) and two whole-body strength- myotubes and plasma samples using a human CSF1 ELISA Kit 86 25 training sessions per week [21]. Bicycle tests (45 min at 70% of (DMC00B; R&D Systems Minneapolis; Minnesota, United States). Total 87 26 88 VO2max) were conducted before and after the long-term training protein concentration of plasma was measured by BC Assay (Protein 27 intervention [21]. Blood samples and biopsies from m. vastus lateralis assay kit, Uptima, Montlucon, France). 89 28 were collected before, immediately after, and 2 h after the acute bi- 90 29 cycle tests (Figure 1A). 2.6. RNA isolation, cDNA synthesis and gene expression analyses 91 30 Total RNA was isolated from cultured cells using the RNeasy Mini kit 92 31 2.2. High throughput mRNA sequencing (Qiagen, Hilden, Germany). RNA was reversely transcribed into cDNA 93 32 RNA was isolated from muscle biopsies and reverse-transcribed into using the High Capacity cDNA Revers Transferase kit (ThermoFisher, 94 33 cDNA. RNA integrity was determined using Agilent RNA 6000 Nano Foster City, California, United States). Quantitative real-time PCR was 95 34 Chips and a Bioanalyzer 2100. Deep sequencing was performed with performed using the CFX96Ô Real-Time System (Bio-Rad, Hercules, 96 35 the Illumina HiSeq 2000 system with multiplexed design [22].