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Aerobic Exercise Augments Muscle

Transcriptome Profile of Resistance Exercise
Article in AJP Regulatory Integrative and Comparative Physiology April 2016
Impact Factor: 3.11 DOI: 10.1152/ajpregu.00035.2016

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Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 13, 2016). doi:10.1152/ajpregu.00035.2016

Aerobic Exercise Augments Muscle Transcriptome Profile of

Resistance Exercise

Tommy R. Lundberg1, Rodrigo Fernandez-Gonzalo2, Per A. Tesch2, Eric Rullman1, 3, Thomas

Gustafsson1.

University Hospital, Stockholm, Sweden,

Stockholm, Sweden, 3 Department of Cardiology, Karolinska University Hospital, Stockholm, Sweden.

Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet and Karolinska
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Department of Physiology & Pharmacology, Karolinska Institutet,

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Running Head: Gene Signature of Concurrent Exercise

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Address for Correspondance:

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Tommy Lundberg, PhD

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Karolinska Institutet, Department of Laboratory Medicine

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Division of Clinical Physiology

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Karolinska University Hospital, Huddinge

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14186 Stockholm, Sweden

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E-mail: tommy.lundberg@ki.se

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Phone: +46(0)8-585 867 71

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Copyright 2016 by the American Physiological Society.

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Abstract

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Recent reports suggest that aerobic exercise may boost the hypertrophic response to short-

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term resistance training. This study explored the effects of an acute aerobic exercise bout on

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the transcriptional response to subsequent resistance exercise. Ten moderately trained men

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performed ~45 min cycling on one leg followed by 4x7 maximal knee extensions for each leg,

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15 min later. Thus, one limb performed aerobic and resistance exercise (AE+RE), while the

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opposing leg did resistance exercise only (RE). Biopsies were obtained from m. vastus

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lateralis of each leg 3-h after the resistance exercise bout. Using DNA microarray, we

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analyzed differences (1.5-fold, FDR 10%) in gene expression profiles for the two modes of

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exercise. There were 176 genes up- (127) or down-regulated (49) by AE+RE compared with

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RE. Among the most significant differentially expressed genes were established markers for

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muscle growth and oxidative capacity, novel cytokines, transcription factors and microRNAs.

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The most enriched functional categories were those linked to carbohydrate metabolism and

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transcriptional regulation. Upstream analysis revealed that VEGF, CREB, TET2 and mTOR

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were regulators highly activated by AE+RE, whereas JnK, Nf, MAPK and several

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miRNAs were inhibited. Thus, aerobic exercise alters the skeletal muscle transcriptional

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signature of resistance exercise to initiate important gene programs promoting both myofiber

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growth and improved oxidative capacity. These results provide novel insight into human

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muscle adaptations to diverse exercise modes and offer the very first genomic basis

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explaining how aerobic exercise may augment, rather than compromise muscle growth

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induced by resistance exercise.

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Key words: Affymetrix, endurance, gene expression, microarray, strength

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Introduction

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It is often put forth that aerobic endurance-type exercise (e.g. running and cycling) interferes

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with muscle adaptations to strength/resistance exercise executed as part of the same training

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program (11, 12). Conversely, we have reported that concurrent aerobic exercise may in fact

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boost skeletal muscle hypertrophic response to resistance exercise, accompanied by molecular

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signaling responses that could favor increased net protein turnover (25, 27). At the same time,

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the idea that aerobic exercise alone, particularly cycle training, may produce hypertrophy, has

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been given more attention (17, 32). These reports are supported by studies showing that low-

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load exercise may produce muscle growth and increased strength (17, 31). However, the

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mechanisms explaining how aerobic exercise could act synergistically with resistance

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exercise facilitating muscle growth, remain largely unexplored.

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Any change in muscle mass arises from an altered balance between protein synthesis and

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breakdown, favoring either muscle hypertrophy or atrophy (19). However, the precise

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molecular networks underpinning altered protein balance and changes in muscle mass in

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response to diverse exercise modes such as aerobic and resistance exercise, are not completely

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understood. Based on acute measurements, we (24) and others (7) reported that concurrent

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exercise may produce a more anabolic molecular and protein synthetic response compared

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with resistance exercise alone. Indeed, muscle hypertrophy is typically accompanied by

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mTOR-activation and increased muscle protein synthesis (29), yet these markers appear to

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have minute predictive value to explain large individual variations in exercise-induced

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hypertrophy (8, 30). Thus, the reductionistic single marker approach from acute studies

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remains somewhat unsatisfactory to explain mechanisms governing muscle adaptations to

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chronic exercise.

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As newly introduced high-throughput techniques allow for in-depth analysis of the mRNA

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responses to exercise, the transcriptional regulation of muscle hypertrophy has received

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increased attention lately. In particular, microarray analysis makes it possible to

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simultaneously assess a large number of muscle transcripts involved in muscle regenerative

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processes in response to acute and chronic exercise (1, 35, 38). Studies have shown that

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young individuals and/or high-responders to resistance exercise may show a different

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signature with regard to myogenic growth transcripts, microRNAs (miRNA) and key

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transcription factors than old individuals and/or low responders (1, 16). Moreover, while it

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appears that several hundred genes co-vary with gains in lean mass, there are apparent

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distinctions between acute and chronic exercise, as well as between aerobic- and resistance-

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type exercise (33). To date, no study has examined muscle gene expression profile to gain

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insight about the molecular regulation responsible for adaptations to combined aerobic and

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resistance exercise.

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In an attempt to reveal the molecular basis of why concurrent aerobic and resistance exercise

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may produce different phenotypic adaptations (e.g. greater hypertrophy) than resistance

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exercise alone, we examined subjects who had undertaken 5 weeks of resistance exercise with

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(AE+RE) or without (RE) concurrent aerobic exercise. The specific aim was to analyze the

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acute transcriptome response to a novel exercise bout of either AE+RE or RE. Gene

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expression profiles were assessed by means of microarray analysis. As 5 weeks training using

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the current AE+RE regimen produced substantially greater muscle hypertrophy than RE (27),

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we hypothesized that an acute AE+RE bout would elicit a gene signature indicative of greater

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muscle mass accretion, compared with RE.

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Methods

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Experimental design

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After familiarization with the protocols and exercise equipment, ten participants performed an

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acute exercise bout of knee extensor AE+RE for one leg, and RE only for the contralateral

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limb (Fig. 1). The leg assigned to AE+RE was randomized in a counter-balanced manner. A

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DNA microarray analysis was performed in biopsies obtained from m. vastus lateralis of both

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legs 3 h after the resistance exercise bout. Subsequently, and as we have reported previously

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(27), the subjects completed a 5-week training program using these identical exercise modes

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(AE+RE vs RE).

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Subjects

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Ten men (26 5 yrs, 183 7 cm and 77 9 kg) volunteered for the study. Subjects were

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moderately trained college students performing recreational exercise 2-3 dwk-1, (e.g. running

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and team sports). At the time of the study, they had not experienced any lower limb injury for

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the last 6 months or performed structured/intense resistance training in the past year. After

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being informed about study procedures, risks and discomforts, subjects gave their written

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informed consent to participate. The study experiments were approved by the Regional

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Ethical Review Board in Stockholm.

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Exercise experiments

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Exercise equipment, protocols, and their efficacy in stimulating robust muscle adaptations to

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5 weeks training, have been described in detail elsewhere (25, 27). After a standardized

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warm-up, the aerobic exercise bout was performed in the morning of the experimental day

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(~8.00 AM). It consisted of a 40 min isolated and dynamic knee extensions on a modified

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one-legged ergometer (model 828E, Monark Exercise AB, Varberg, Sweden) at ~70% of

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maximal workload (previously determined by a maximal incremental test) with a cadence of

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60 rpm. Rate of perceived exertion was assessed regularly to confirm strenuous effort. Upon

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completion of the 40 min, the workload was increased (~20 W) until subject failed to

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maintain target cadence (~2-4 min). After 15 min recovery, resistance exercise for both legs

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(one leg at a time) was carried out. Four sets of 7 maximal repetitions were performed on a

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knee extension iso-inertial flywheel ergometer (YoYo Technology Inc., Stockholm, Sweden

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(37)), with 2 min rest between sets. This exercise device uses the inertia of a spinning

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flywheel (inertia, 0.11 kg/m2) to offer unlimited resistance during coupled concentric and

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eccentric muscle actions. The range of motion was from 90 knee flexion to almost full

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extension (i.e. 180). Power was measured in all repetitions. During all tests, subjects received

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strong verbal encouragement from research personnel.

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Muscle biopsies and diet control

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Three hours after completion of the resistance exercise bout, biopsies were obtained from m.

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vastus lateralis of both legs. Under local anesthesia, a 5-mm Bergstrm needle with suction

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was employed to obtain muscle tissue samples (2). After excess blood, connective tissue

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and/or fat had been removed; the tissue was frozen in liquid nitrogen and stored at -80C until

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further analysis. Subjects received a standardized dinner the night before the experiments

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(pasta, tomato sauce, and juice; 2.21 g carbohydrates/kg body weight (bw), 0.22 g protein/kg

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bw, and 0.04 g fat/kg bw). In addition, standardized breakfast 2 hr prior to the aerobic

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exercise bout was provided to participants (1.01 g carbohydrates/kg bw, 0.31 g protein/kg bw,

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and 0.24 g fat/kg bw; Ensure Plus, Abbott Laboratories, Maidenhead, UK). Subjects then

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remained fasted during the 3 h resting time prior to biopsy collection.

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RNA extraction and microarray analysis

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Total RNA was extracted from ~20-mg wet muscle samples using a bead beater device

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(BioSpec Products, Inc., Bartlesville, OK) and TRIzol (Invitrogen Life Technologies,

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Carlsbad, CA). Total RNA was then purified using PureYield RNA Midiprep System
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(Promega Corporation, Madison, WI). Each of the 20 samples were subjected to analysis on

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the Affymetrix HuGene-2.1-st platform. Hybridization, washing, staining and scanning of the

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arrays were performed according to the manufacturers instructions (Affymetrix). To control

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the quality, in addition to the standard quality assessments including scaling factors and chip

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housekeeper 5/3-ratios, all individual arrays were examined using hierarchical clustering

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and Normalized Unscaled Standard Error (NUSE, a variance based metric to identify outliers

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prior to statistical analysis). All chips passed these QC-steps. The probe-set level intensities

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for the arrays were normalized using the Robust Multi-array Analysis method (RMA)

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implemented within the R statistical software environment using the Oligo package

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(Bioconductor project). Raw data and RMA normalized expression values are publicly

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available through Gene Expression Omnibus (GEO) accession number GSE74194. Prior to

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differential expression analysis probe-sets without available annotation and probe-sets

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displaying low-variance across samples (IQR <0.5) were excluded from further analysis.

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Annotation was carried out in the R environment using hugene21sttranscriptcluster.db, and

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the annotation and genefilter packages from Bioconductor. Differential expression was

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estimated through pairwise-statistical analysis of microarrays (SAM) using 1000 permutations

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on the R-platform. Significantly up- and down-regulated probe-sets were analyzed for gene-

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ontology enrichment using the web-based Database for Annotation, Visualization and

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Integrated Discovery (DAVID) version 6.7 (13, 14). Probe-sets with a FDR of 10% and a

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fold change of 1.5-fold were analyzed for enrichment of biological functions. To correct

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for possible selection bias of genes, only probe-sets used in the SAM analysis were used as

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background. Enriched ontologies with a FDR of 10% were categorized as significant. For

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further pathway analysis, we used the web-based bioinformatics tool Ingenuity Pathway

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Analysis (IPA, http://www.ingenuity.com, Qiagen). IPA is a knowledge database generated

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from peer-reviewed scientific publications that enables discovery of biological networks in

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gene expression data and determines the biological functions most significant to those

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networks. Affymetrix transcript identifiers were uploaded onto IPA and were re-annotated

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and queried against the verified IPA knowledge database, and probe-sets analyzed in the

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SAM-analysis were used as a reference set. Both up- and down-regulated probe-sets were

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considered, and interactions annoted as predicted with high confidence or experimentially

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validated were used in the IPA analysis. Enrichment of the focus genes in networks in IPA is

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assessed via Fishers exact test. Furthermore, the software identifies top functions associated

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with each network via enrichment scores (z-score), highlighting the predicted biological

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significance of the results.

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Validation of microarray

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Real-time PCR for five selected mRNA targets was carried out on the same RNA extract as

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the array, using TaqMan probes and primers as previously reported (27). Target gene

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expression was reported as a ratio to reference genes (GAPDH and 18S) using the 2-CT

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formula. Real-time PCR data were then scaled and plotted (Fig. 2) together with the

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corresponding normalized probe-set expression levels of the array (Probe-set ID: 16974830,

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17009093, 16906471, 16683771 & 17080788).

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Results

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Exercise responses and functional measures

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During the acute resistance exercise bout, the leg that had completed aerobic exercise 15 min

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earlier, produced 10% lower peak power than the rested RE-leg. The end-point results of the

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5-wk training program have been reported previously (27). In brief, after 5 weeks training,

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gains in peak power (~20%) were similar between AE+RE and RE. Likewise, the increase in

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peak torque did not differ between AE+RE (10%) and RE (12%). With regard to muscle size,

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AE+RE increased (p < 0.05) quadriceps muscle volume by 6%, compared with 3% for RE.

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AE+RE, but not RE, increased endurance performance (22%) and citrate synthase activity

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(18%).

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Gene expression analysis

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After annotation and filtering 11,869 probe-sets were analyzed using pairwise SAM analysis.

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Of these probe-sets, 49 were down- and 127 up-regulated (1.5-fold) by AE+RE as compared

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with RE, having a Q-value (corresponding to FDR) of 10% (See Supplemental Digital

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Content 1, Differential Expression Analysis). Analysis of biological functions significantly

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enriched amongst the up-regulated genes using DAVID identified seven ontologies, all of

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which can be defined as variations of carbohydrate metabolism (Table 1). In contrast, 11

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biological functions were identified as enriched among the down-regulated genes. Most

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notably were variations of response to hormone stimulus, MAPKKK cascade, regulation

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of skeletal muscle tissue development and angiogenesis (Table 1). Some key down-

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regulated genes found in several of these clusters were transcriptional and epigenetic

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regulators such as HDAC and TXNIP. The 25 most significantly differentially expressed

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genes (up- and down-regulated by AE+RE compared with RE) are displayed in Table 2.

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Several of these genes are well-known markers involved in muscle adaptations to exercise

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(e.g. AMPK, PGC-1, MuRF-1, myostatin). Some cytokines (IL18 and IL-311RA) and
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several miRNAs (mir-133, mir-1, mir-623) appeared among the down-regulated genes. As

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indicated earlier, we compared five exercise-relevant differentially expressed genes (PGC-1,

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VEGF, myostatin, MuRF-1, atrogin-1) from the array with q-PCR data previously published

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from the same RNA (27). In both the microarray and the q-PCR, the five genes were found to

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be significantly differentially expressed in the corresponding manner (Fig. 2).

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Upstream analysis

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Ingenuitys Upstream Regulator Analysis in IPA is a tool that predicts upstream regulators

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from gene expression data based on the literature and compiled in the Ingenuity Knowledge

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Base. A Fishers Exact Test p-value was calculated to assess the significance of enrichment of

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the gene expression data for the genes downstream of an upstream regulator. In addition,

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directionality and magnitude of the differentially expressed genes was calculated to a z-score

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of directional consistency. A high positive z-score indicates activation for that pathway

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whereas a negative z-score is associated with an inhibition by AE+RE as compared with RE.

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The top-ranking regulatory molecules based solely on overlap (i.e. p-value of Fishers Exact

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test) were MYC and TNF- (-log10 p-value >10), followed by HGF, IL1 and FOXO3.

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However, z-score of all of these pathways were in the range of -0.6 0.2 illustrating that

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while many genes in these pathways are differentially expressed, the directionality of the

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altered gene expression is inconsistent with an activation or inhibition of the pathway.

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When considering the regulatory molecules with a gene expression pattern consistent with

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what is expected (i.e. z-score >2), the #1 pathway predicted to be activated was VEGF (p-

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value 3e-7, z-score 2.7) and miR-181a which was predicted to be inhibited (p-value 1.7e-7, z-

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score -2.6), see Figure 3 and 4, and Table 3. The pathways predicted to be activated second to

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VEGF were CREB (p-value 5.6e-4, z-score 2.2), TET2 (p-value 6.9e-4, z-score 2.0) and

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mTOR (p-value 5.3e-3, z-score 2.4). The second and third most inhibited regulators were JnK

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(p-value 4.5e-5, z-score -2.4) and Nf, followed by MAP2K4 and MAP3K8 (p-value 7.8e-5,
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z-score -2.5). See Figure 3 and 4, Table 3, and Supplemental Digital Content 2 (Upstream

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Analysis).

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Discussion

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This study provides the first comprehensive gene expression analysis comparing concurrent

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aerobic and resistance exercise (AE+RE) with resistance exercise alone (RE). The main

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finding was that an exhaustive aerobic exercise insult, performed prior to resistance exercise,

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augmented important gene signatures that regulate contrasting functions such as oxidative

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metabolism on one hand, and myofiber growth and tissue regeneration on the other. Thus, we

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have characterized a molecular signature of concurrent exercise that could aid in explaining

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how aerobic exercise may boost, rather than compromise, muscle hypertrophy to chronic

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resistance training.

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While we and others have reported on single molecular markers in response to concurrent

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aerobic and resistance exercise (7, 24, 27), neither study provided an in-depth transcriptional

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map of this exercise mode. We hypothesized that there must be substantial differences in the

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transcriptome response of an exercise regime (AE+RE) producing twice as robust

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hypertrophy as RE alone (27). Indeed, our results showed that 176 genes were differently

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expressed 1.5-fold when aerobic exercise preceded a bout of resistance exercise. This would

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suggest that these genes, and their associated targets and regulators, play important roles in

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eliciting phenotype differentiation depending on exercise stimulus imposed over 5 weeks.

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In an effort to explore the rather substantial difference in expression across exercise modes,

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we performed analysis for enrichment of gene ontologies related to biological functions.

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Perhaps not surprising, the most activated biological functions induced by AE+RE were

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processes involved in carbohydrate metabolism. Indeed, it has been shown that aerobic

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exercise promotes gene programs involved in glucose homeostasis and oxidative metabolism

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(28). Similarly, the gene showing the most significant difference in expression across exercise

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modes was AMPK, a well-known regulator of carbohydrate metabolism. Furthermore, PGC12

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1 was highly up-regulated by AE+RE, confirming that this gene and its exercise-responsive

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splice variants are up-regulated by both aerobic and resistance exercise, and more so by

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combined exercise (26, 42). The fact that AMPK and PGC-1 were prominent genes in this

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unbiased expression analysis further underlines their role in promoting an oxidative

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phenotype (20). It also fits with the impressive increase in endurance performance and CS

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activity reported by us following AE+RE training (27).

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Among functions being down-regulated by AE+RE compared with RE, it is worth noting we

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observed genes involved in regulation of skeletal muscle tissue development. Genes

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clustered in this group have a negative effect on muscle hypertrophy, and one of the most

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down-regulated gene was myostatin, an established negative regulator of hypertrophy (36). In

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addition, we found key genes involved in muscle protein breakdown to be up-regulated, e.g.

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MuRF-1 and atrogin-1. This adds further credibility to the recent notion that these ubiquitin

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ligase proteins may be critical in the healthy skeletal muscle remodeling process, in fact

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facilitating contraction-induced muscle growth (15). Overall, the response of key genes and

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associated biological functions activated by AE+RE concords with the greater muscle mass

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accretion following the concurrent exercise regime.

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To identify transcriptional regulators potentially explaining the observed gene expression

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differences between AE+RE and RE, we used IPA upstream regulator analysis. The analysis

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revealed that factors centralized around the key regulators VEGF, CREB, TET2 and mTOR

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were highly activated by AE+RE, whereas JnK, Nf, MAPK and several miRNAs were

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inhibited regulators. While mTOR is a growth-inducer, its role in regulating muscle mass is

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not entirely unequivocal. Thus, recent research suggest high responders to resistance exercise

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display an inhibited mTOR-activation signature (33), and that the muscle anabolic response to

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exercise is not solely dependent on mTOR activation (34). Nevertheless, mTOR aligns with

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muscle hypertrophy (29) and is deemed crucial for the hypertrophic response and for
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protection against atrophy (3). As our first hand physiological data would be hard to challenge

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(27), the demonstration of greater activation of mTOR following AE+RE than RE would be

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indicative of a greater protein synthetic response. Ultimately, this aids in explaining the more

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robust hypertrophic response to concurrent execise.

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MAPK- and Nf-associated signaling can be both adaptive and maladaptive in skeletal

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muscle depending on the conditions (18). However, given that exercise seems potent to

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increase MAPK and Nf in the non-diseased state (18, 41), it strikes that MAPK/JnK and

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Nf were identified as inhibited regulators in the current study. In support of this notion,

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among the top genes being down-regulated by AE+RE, we found genes involved in myokine

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(e.g. IL-18, IL-31RA) and MAPK/ERK signaling (ATF3). Thus, while activation of these

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genes in response to acute exercise appears to benefit muscle adaptations, chronic activation

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could very well exacerbate protein breakdown and hence muscle wasting (18). In light of this,

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the AE+RE-induced down-regulation of these genes was perhaps due to a volume-related

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attenuation of MAPK-signaling (22).

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A less studied factor in human skeletal muscle remodeling is the transcriptional regulator

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CREB, which was significantly activated as an upstream regulator in the current analysis. It

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has been reported that high-intensity exercise activates CREB in mice, and the activation of

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this transcriptional complex induces anabolic changes driving muscle hypertrophy during

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subsequent recovery (4). Furthermore, over-expressing cells with CREB-regulated co-

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regulators induced hypertrophy in transgenic mouse models and cultured myotubes (4). Our

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findings spur future studies to examine weather CREB and its associated targets play a key

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role in exercise-induced adaptations of human skeletal muscle.

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miRNAs are accepted post-transcriptional regulators of protein translation which appear to

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block translation of transcribed genes in human skeletal muscle (9). Several miRNA routes

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346

were inhibited by AE+RE in the upstream regulator analysis. In further support of this notion,

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some of the most established myomirs (miR-133 and miR-206/miR1) were significantly

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less expressed after AE+RE compared with RE. Hence, it is tempting to speculate that the

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blunted miRNA expression in AE+RE could reverse inhibitory effects on target gene

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transcription. This in turn may allow for more efficient protein translation from available

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mRNA transcripts, resulting in more robust muscle protein accretion following the combined

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exercise paradigm. Interestingly, miRNAs also seem to regulate TET2 signaling (6), which

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was heavily activated by AE+RE in the current study. To the authors knowledge, we are the

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first to depict the TET2 pathway as a potential regulator of adaptations to combined training.

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TET2 is a master epigenetic regulator of smooth muscle plasticity (21), yet its role in human

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skeletal muscle is relatively unknown. The TET2 transcript was fairly abundant and

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detectable in all samples, yet it was discarded from further analysis due to low variability

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across samples (SD ~0.2). Post-hoc analysis of TET2 did not indicate it being differentially

359

expressed. Therefore, a potential role for TET2 in the context of skeletal muscle response to

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exercise does not seem to involve transcriptional regulation of TET2 itself. However, given

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that TET2 may be under heavy miRNA regulation (6), recalling we discovered several

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suppressed miRNAs in our gene list, future studies should explore the potential interaction

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between miRNA, TET2 and muscle adaptive responses to exercise in more detail.

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The ontology analysis surprisingly revealed that AE+RE, while improving endurance

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capacity, down-regulated the biological function angiogenesis. This was due to the fact that

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several known angiogenic factors were down-regulated by combined exercise. In contrast, the

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pro-angiogenic growth factor VEGF (10) showed substantially greater expression after

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AE+RE than RE, and this was confirmed through q-PCR. This highlights the risk of drawing

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conclusions about complex biological processes based on measurements of single factors. In

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further support of a suppressed angiogenic response, we have reported that 5 weeks AE+RE
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371

(25) did not induce capillarization (23). Thus, albeit the training period may have been too

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short for vascular growth to occur, it cannot be excluded that our finding indicates an

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interference effect in vascular remodeling induced by adding resistance exercise to aerobic

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exercise.

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It is obvious that in order to assess the effects of adding aerobic exercise prior to resistance

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exercise, the resistance exercise protocol needs to be matched and hence total work performed

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is substantially greater in the AE+RE leg. Although this is an essential part of the design for

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this and other comparable concurrent exercise studies, it does not rule out the possibility that

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differences in workloads account for the different gene expression profiles across legs. As the

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primary goal of this study was to comprehensively explore gene expression profiles of the two

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exercise modes, we did not assess protein-related changes of any specific target, nor did we

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specifically profile transcripts in the polysome pool (5). However, we appreciate that further

383

validation work could have revealed even more significant information. The specific one-

384

legged model was selected because it allows for a unique inter-individual design with

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repeated-measure analysis of samples originating from the same individual and hence the

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same genetic variance, training history and nutrient status. We acknowledge that the acute

387

nature of the imposed exercise challenges and subsequent biopsy sampling time-point could

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produce large noise and non-specific transcriptional responses (39). This was reflected in our

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data set where members of several key regulatory pathways were differentially expressed in a

390

discordant manner i.e. both up- and down-regulation. While this highlights the complexity in

391

interpreting acute exercise data, it also emphasizes a major strength of the current study, i.e.

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first hand data of muscle adaptations to the exercise modes under study (40). In the absence of

393

such knowledge, it becomes virtually impossible to interpret the acute molecular response to

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exercise.

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Perspectives and Significance

397

We provide novel data revealing that the skeletal muscle, subjected to concurrent exercise,

398

shows striking transcriptional responses when initiating gene programs generally thought to

399

be antagonistic with each other. In addition to providing unbiased support for factors with

400

established roles in regulating muscle growth and an oxidative phenotype in response to

401

concurrent exercise (e.g. mTOR, myostatin, AMPK, PGC-1), our in-depth transcriptional

402

map also identified several new candidate genes and putative networks that markedly differed

403

in expression between AE+RE and RE. Thus, central regulators such as CREB, TET2,

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MAPK, Nf, and several miRNAs appeared as novel factors that could be part in regulating

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vast differences in types and magnitudes of adaptations across combined exercise paradigms.

406

The current findings also highlight the difficulty in assessing single markers as a proxy of

407

physiological adaptations if no end-point measures of chronic adaptations are at hand.

408

Collectively, our findings should stimulate further exhaustive investigations of the

409

transcriptional and molecular basis of exercise-induced muscle adaptations using different

410

exercise combinations in various populations.

411
412

Acknowledgements

413

The authors would like to thank BEA - the core facility for Bioinformatics and Expression

414

Analysis service facility at Novum, Karolinska Institutet in Huddinge, for their helpful

415

contribution. This study was supported by grants from the Swedish National Centre for

416

Research in Sports (PAT), the European Space Agency (PAT, TG), the Marianne & Marcus

417

Wallenberg Foundation (TG), the Swedish Medical Research Council (TG), and the Swedish

418

Society for Medical Research (SSFM, ER).

419
420
17

421

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truncated splice variants, NT-PGC-1alpha and PGC-1alpha4, increase with both endurance

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Supplemental Digital Content

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Supplemental Digital Content 1.xls: Differential Expression analysis

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Supplemental Digital Content 2.xls: Upstream Analysis

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Figure legends

565
566

Figure 1. Schematics of the acute exercise experiment. Muscle biopsies from each leg (B)

567

were obtained 3 h after an acute bout of resistance exercise with (AE+RE) or without (RE)

568

preceding aerobic exercise.

569
570

Figure 2. Validation of the microarray and previously reported real-time PCR data. Lines

571

represent individual real-time PCR observations whereas the microarray data are depicted as

572

boxplots. These are plotted as median, first and fourth quartile, and range.

573
574

Figure 3. Upstream regulator analysis by Ingenuity pathway analysis (IPA). The figures

575

illustrate the top-ranking pathways predicted as activated in Upstream regulator analysis.

576

Genes in red were greater expressed in AE+RE compared with RE whereas genes in green

577

were lower expressed. An orange line indicates predicted up-regulation (by AE+RE) whereas

578

a blue line indicates predicted down-regulation. A yellow line indicates expression being

579

contradictory to the prediction.

580
581

Figure 4. Upstream regulator analysis by Ingenuity pathway analysis (IPA). The figures

582

illustrate the top-ranking pathways predicted as inhibited in Upstream regulator analysis.

583

Genes in red were greater expressed in AE+RE compared with RE whereas genes in green

584

were lower expressed. An orange line indicates predicted up-regulation (by AE+RE) whereas

585

a blue line indicates predicted down-regulation. A yellow line indicates expression being

586

contradictory to the prediction.

24

Myostatin

MuRF-1

Atrogin-1

VEGF

PGC-1

Table 1. Gene ontology (GO) categories related to biological processes induced by AE+RE
compared with RE as generated by DAVID.
GO term
Up-regulated processes
GO:0010906~regulation of glucose metabolic process
GO:0010675~regulation of cellular carbohydrate metabolic process
GO:0006109~regulation of carbohydrate metabolic process
GO:0019318~hexose metabolic process
GO:0015980~energy derivation by oxidation of organic compounds
GO:0006006~glucose metabolic process
GO:0005996~monosaccharide metabolic process
Down-regulated processes
GO:0007167~enzyme linked receptor protein signaling pathway
GO:0010033~response to organic substance
GO:0009725~response to hormone stimulus
GO:0042127~regulation of cell proliferation
GO:0009719~response to endogenous stimulus
GO:0007169~transmembrane receptor protein tyrosine kinase signaling pathway
GO:0001525~angiogenesis
GO:0009266~response to temperature stimulus
GO:0051789~response to protein stimulus
GO:0000165~MAPKKK cascade
GO:0048641~regulation of skeletal muscle tissue development

Fold
Enrichment

FDR (%)

15.71
14.89
14.89
4.67
7.69
5.30
4.12

2.81
3.30
3.30
5.27
5.59
7.58
9.47

6.66
4.08
5.43
3.62
4.94
6.77
7.61
12.18
11.63
6.48
23.10

0.06
0.32
0.76
0.85
1.32
2.40
5.43
5.72
6.48
9.44
9.21

Table 2. The 25 top up- and down-regulated genes by AE+RE compared with RE out of 176
differentially expressed genes (FDR 10%).
Top genes up-regulated by AE+RE compared with RE
Fold change Gene symbol
Entrez Gene Name
10.85
PRKAG2
protein kinase, AMP-activated, gamma 2 non-catalytic subunit
2.96
TRIM63
tripartite motif containing 63, E3 ubiquitin protein ligase
2.87
KCNQ4
potassium channel, voltage gated KQT-like subfamily Q, member 4
2.85
RPS6KA2-IT1 RPS6KA2 intronic transcript 1
2.75
PPARGC1A
peroxisome proliferator-activated receptor gamma, coactivator 1 alpha
2.61
CHST15
carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferase 15
2.61
FAM102A
family with sequence similarity 102, member A
2.60
SNAI3
snail family zinc finger 3
2.48
MAFF
v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F
2.39
ESRRG
estrogen-related receptor gamma
2.37
CES3
carboxylesterase 3
2.37
SLC20A1
solute carrier family 20 (phosphate transporter), member 1
2.36
NR4A3
nuclear receptor subfamily 4, group A, member 3
2.36
PFKFB2
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2
2.32
GPR157
G protein-coupled receptor 157
2.32
SLC25A33
solute carrier family 25 (pyrimidine nucleotide carrier), member 33
2.26
SLC38A1
solute carrier family 38, member 1
2.26
SYNJ2
synaptojanin 2
2.25
SLC16A6
solute carrier family 16, member 6
2.18
PXDC1
PX domain containing 1
2.16
LOC101928327 uncharacterized LOC101928327
2.14
CIART
circadian associated repressor of transcription
2.11
ANGPTL2
angiopoietin-like 2
2.08
MIR3147
microRNA 3147
2.05
BHLHE40
basic helix-loop-helix family, member e40
Top genes down-regulated by AE+RE compared with RE
Fold change Gene symbol
Entrez Gene Name
0.36
OTUD1
OTU deubiquitinase 1
0.41
ATF3
activating transcription factor 3
0.42
SERPINE1
serpin peptidase inhibitor, clade E, member 1
0.44
IL18
interleukin 18
0.45
IL31RA
interleukin 31 receptor A
0.47
KCNQ5-IT1
KCNQ5 intronic transcript 1
0.47
GADD45A
growth arrest and DNA-damage-inducible, alpha
0.47
ARRDC4
arrestin domain containing 4
0.48
NEXN-AS1
NEXN antisense RNA 1
0.48
CYR61
cysteine-rich, angiogenic inducer, 61
0.51
BTG2
BTG family, member 2
0.51
IER5
immediate early response 5
0.52
RUNX1-IT1
RUNX1 intronic transcript 1
0.53
mir-133
microRNA 133b
0.54
CTGF
connective tissue growth factor
0.54
ENAH
enabled homolog (Drosophila)
0.54
CA4
carbonic anhydrase IV
0.55
FOS
FBJ murine osteosarcoma viral oncogene homolog
0.56
PLAU
plasminogen activator, urokinase
0.56
SPNS2
spinster homolog 2 (Drosophila)
0.57
PFKFB3
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3
0.57
SLC8A3
solute carrier family 8 (sodium/calcium exchanger), member 3
0.57
TIAM2
T-cell lymphoma invasion and metastasis 2
0.57
ARID5A
AT rich interactive domain 5A (MRF1-like)
0.58
MSTN
myostatin

Table 3. The top ranked activated/inhibited central regulators in the IPA analysis.
Predicted
activation
state

Zscore

P-value of
overlap

miR-181a-5p (and other miRNAs w/seed ACAUUCA)

Inhibited

-2.57

0.000000167

Vegf

Activated

2.67

0.000000303

Jnk

Inhibited

-2.35

0.0000447

NFkB (complex)

Inhibited

-2.52

0.0000775

MAP2K4

Inhibited

-2.19

0.000101

MAP3K8

Inhibited

-2.00

0.000107

miR-141-3p (and other miRNAs w/seed AACACUG)

Inhibited

-2.81

0.000341

miR-291a-3p (and other miRNAs w/seed AAGUGCU)

Inhibited

-3.25

0.000544

Creb

Activated

2.18

0.000561

miR-17-5p (and other miRNAs w/seed AAAGUGC)

Inhibited

-2.82

0.000633

TET2

Activated

2.00

0.000685

MTOR

Activated

2.41

0.00525

Upstream regulator