Am J Physiol Cell Physiol 312: C638–C650, 2017. First published March 8, 2017; doi:10.1152/ajpcell.00106.2016.

RESEARCH ARTICLE

␣7␤1 Integrin regulation of gene transcription in skeletal muscle following an acute bout of eccentric exercise Ziad S. Mahmassani,1 Kook Son,2 Yair Pincu,1 Michael Munroe,1 Jenny Drnevich,3 Jie Chen,2 and Marni D. Boppart1 1

Submitted 19 April 2016; accepted in final form 28 February 2017

Mahmassani ZS, Son K, Pincu Y, Munroe M, Drnevich J, Chen J, Boppart MD. ␣7␤1 Integrin regulation of gene transcription in skeletal muscle following an acute bout of eccentric exercise. Am J Physiol Cell Physiol 312: C638 –C650, 2017. First published March 8, 2017; doi:10.1152/ajpcell.00106.2016.—The ␣7␤1 integrin is concentrated at the costameres of skeletal muscle and provides a critical link between the actin cytoskeleton and laminin in the basement membrane. We previously demonstrated that expression of the ␣7BX2 integrin subunit (MCK:␣7BX2) preserves muscle integrity and enhances myofiber cross-sectional area following eccentric exercise. The purpose of this study was to utilize gene expression profiling to reveal potential mechanisms by which the ␣7BX2-integrin contributes to improvements in muscle growth after exercise. A microarray analysis was performed using RNA extracted from skeletal muscle of wild-type or transgenic mice under sedentary conditions and 3 h following an acute bout of downhill running. Genes with false discovery rate probability values below the cutoff of P ⬍ 0.05 (n ⫽ 73) were found to be regulated by either exercise or transgene expression. KEGG pathway analysis detected upregulation of genes involved in endoplasmic reticulum protein processing with integrin overexpression. Targeted analyses verified increased transcription of Rpl13a, Nosip, Ang, Scl7a5, Gys1, Ndrg2, Hspa5, and Hsp40 as a result of integrin overexpression alone or in combination with exercise (P ⬍ 0.05). A significant increase in HSPA5 protein and a decrease in CAAT-enhancer-binding protein homologous protein (CHOP) were detected in transgenic muscle (P ⬍ 0.05). In vitro knockdown experiments verified integrin-mediated regulation of Scl7a5. The results from this study suggest that the ␣7␤1 integrin initiates transcription of genes that allow for protection from stress, including activation of a beneficial unfolded protein response and modulation of protein synthesis, both which may contribute to positive adaptations in skeletal muscle as a result of engagement in eccentric exercise. integrin; eccentric exercise; microarray; heat shock protein; stress

protein heterodimers consisting of ␣ and ␤ subunits. Integrins link the cytoskeleton inside the cell with the extracellular matrix outside of the cell, and are thus appropriately positioned to facilitate cellular adhesion and migration of a variety of mononuclear cells (49). Integrins can also preserve cellular integrity and transmit information in a

INTEGRINS ARE TRANSMEMBRANE

Address for reprint requests and other correspondence: M. D. Boppart, Beckman Institute for Advanced Science and Technology, 405 N. Mathews Ave., MC-251, Urbana, IL 61801 (e-mail: [email protected]). C638

bidirectional manner across the membrane in multinuclear cells that lack capacity for migration, including skeletal muscle. The ␣7␤1 integrin is highly expressed in the sarcolemma of skeletal muscle, specifically at the Z bands that provide sarcomere stabilization and myofiber integrity during contraction (2, 6). Loss of the ␣7␤1 integrin results in progressive myopathy that is exacerbated as a result of lengthening contractions (6, 10, 11) and provides the basis for a congenital myopathy in humans (24). Conversely, overexpression of the ␣7 integrin subunit (MCK:␣7BX2) alone or as a dimer with ␤1, protects against exercise-induced skeletal muscle damage and associated inflammation in both wild-type (WT) mice and mouse models of muscular dystrophy (5, 6, 10, 11, 32, 33, 34). We have previously reported that an acute bout of eccentric exercise confers an increase in ␣7 integrin gene expression, including all of the primary isoforms (A/B, X1/X2), at 3 h and a subsequent increase in ␣7 and/or ␤1D integrin protein in both mouse (WT) and human skeletal muscle 24 h after exercise (6, 14). All studies conducted to date would support the conclusion that upregulation of the ␣7 integrin serves to provide reinforcement and protection against mechanical strain associated with contraction. However, the simplistic view of the integrin as a structural protein is challenged by prior observations of enhanced satellite cell content and myofiber growth in ␣7 integrin transgenic mice after eccentric exercise (34, 53). Thus, our current work focuses on elucidating novel signaling pathways and patterns of gene expression that may provide a better understanding of mechanisms by which the integrin can initiate muscle growth and adaptation after exercise. As a first step in addressing a role for the ␣7 integrin in the regulation of myofiber protection and growth after exercise, global gene profiling was completed using RNA extracted from sex- and age-matched WT and ␣7 integrin transgenic mice (MCK:␣7BX2; ␣7Tg) at 3 h following an acute bout of eccentric exercise. Results from this study suggest a role for the ␣7 integrin in the upregulation of genes related to stress resistance and protein homeostasis. MATERIALS AND METHODS

Animals and downhill running exercise. All mice were housed in the animal facility at the University of Illinois in a temperaturecontrolled specific pathogen-free animal room maintained on a 12: 12-h light-dark cycle. The animals were fed standard laboratory chow and water ad libitum. Protocols for animal use were approved by the

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Department of Kinesiology and Community Health and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois; 2Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois; and 3Roy J. Carver Biotechnology Center, High Performance Biological Computing, University of Illinois at Urbana-Champaign, Urbana, Illinois

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Genomes (KEGG) pathways were pulled directly from the KEGG on October 19, 2016, using the KEGGREST package [v1.14.0 (47a)]. The 50-mer probes on the array were designed many years ago based on the mouse annotation known at the time. Based on current annotation, some probes no longer interrogate any known gene, one probe can interrogate two or more (usually related) genes, and more than one probe on the array can interrogate the same gene. Additionally, up to 60% of the genes in the genome are not expressed in a particular tissue, which can be assessed by the detection P value. Therefore, the 45,281 probes on the array were collapsed down to 12,971 unique known genes in the following sequence: 1) removing probes that did not have at least 3/24 samples with detection value of P ⬍ 0.05; 2) removing probes that did not map to any Entrez gene identification; 3) using the lowest Entrez gene ID for probes that mapped to related genes; and 4) selecting a single probe with the lowest overall ANOVA P value for probes that mapped to the same gene. Correction for multiple hypothesis testing using the false discovery rate (FDR) method (3) was carried out on the raw P values for the 12,971 unique genes. Overrepresentation testing for selected gene sets was performed using the GOstats package (18) (v2.40.0) using conditional testing, which removes effects of significant child terms before testing parental terms to reduce redundancy. Quantitative RT-PCR. Both female and male samples (total n ⫽ 10 –12/group) were included in follow-up quantitative PCR (qPCR) and Western blotting experiments. Unused RNA (500 ng) from the same extraction procedure mentioned above were reverse-transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). PCR for specific target genes was performed using the 7900HT Fast Real-Time PCR System with TaqMan Universal PCR Master Mix (Applied Biosystems). Hypoxanthine phosphoribosyltransferase (Hprt) was used as the housekeeping gene, and data are expressed relative to this expression using the ⌬⌬Ct method. Female WT SED mice were used as the reference group. TaqMan primers are reported in Table 1 (Applied Biosystems). In vitro analysis. Five small hairpin RNAs (shRNAs) in the pLKO.1-puro vector were purchased from Sigma-Aldrich (MISSION TRC) for mouse Itga7 and tested to determine efficacy. Clone IDs are as follows: shITGA7 no. 1, TRCN0000066192; shITGA7 no. 2, TRCN0000066191; shITGA7 no. 3, TRCN0000442150; shITGA7 no. 4, TRCN0000066189; and shITGA7 no. 5, TRCN0000066188). A shRNA clone with scrambled hairpin sequence (shScramble), a negative control, and lentivirus packaging were previously described (20). shITGA7 no. 4 resulted in a 50% knockdown of ␣7 integrin protein compared with scrambled control and was chosen for further

Table 1. List of TaqMan gene expression assays Gene Name/Symbol

Assay ID

Activating transcription factor 6 (Atf6) Angiogenin, ribonuclease, RNase A family, 5 (Ang) DnaJ (Hsp40) homolog, subfamily B, member 9 (Dnajb) Glycogen synthase 1, muscle (Gys1) Heat shock protein (HSP72) 1A (Hspa1a) Heat shock protein (Grp78/BiP) 5 (Hspa5) Heat shock protein 90, ␤ (Grp94), member 1 (Hsp90b1) Hypoxanthine guanine phosphoribosyl transferase (Hprt) Integrin ␣7 (Itga7) Mouse Integrin ␣7 (Itga7) Rat Metallothionein 1 (Mt1) Metallothionein 1 (Mt2) Muscle, skeletal, receptor tyrosine kinase (Musk) Nitric oxide synthase interacting protein (Nosip) N-myc downstream regulated gene 2 (Ndrg2) Peroxisome proliferative activated receptor, ␥, coactivator 1 ␣ (Ppargc1a) Ribosomal protein L13A (Rpl13a) Solute carrier family 7 (cationic amino acid transporter, y⫹ system), member 5 (Slc7a5)

Mm01295319_m1 Mm00833184_s1 Mm01622956_s1 Mm01962575_s1 Mm01159846_s1 Mm00517691_m1 Mm00441926_m1 Mm01545399_m1 Mm00434400_m1 Rn01529354_m1 Mm00496660_g1 Mm00809556_s1 Mm01346929_m1 Mm00481528_m1 Mm00443481_g1

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Mm01208835_m1 Mm01612986_gH Mm00441516_m1

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Institutional Animal Care and Use Committee of the University of Illinois at Urbana-Champaign. Mice overexpressing the rat ␣7 integrin subunit under the control of a muscle-specific promoter (MCK: ␣7BX2; ␣7Tg or TG) and littermate controls were bred and maintained as previously described (5). Five-week-old WT and ␣7Tg (TG) mice were randomly assigned to a sedentary (SED) or an acute exercise group. Females (n ⫽ 6/group) and males (n ⫽ 5– 6/group) were equally represented in each of the four groups. Acute exercise consisted of a single bout of downhill running (⫺20°, 17 m/min, 40 min) on a treadmill (Exer-6M; Columbus Instruments, Columbus, OH). Speed was gradually increased from 10 to 17 m/min over the course of the first 2 min as a warm-up, and mice were encouraged to run without the use of electrical shock. Mice were euthanized via carbon dioxide asphyxiation 3 h after exercise. The gastrocnemius-soleus complexes were either rapidly dissected and frozen in liquid nitrogen for isolation of RNA, or in precooled isopentane for evaluation of tissue sections by microscopy. Extraction of RNA. Frozen gastrocnemius-soleus complexes from both male and female mice were manually ground in liquid nitrogen using a mortar and pestle. Total RNA was extracted from the frozen muscle powder in TRIzol (TriPure QIAzol; Qiagen, Valencia, CA). Muscle powder was homogenized in TRIzol and run through a QiaShredder spin column. Chloroform was added at room temperature for 2 min before a 12,000 g spin at 4°C for 15 min. The aqueous phase was removed and used to extract RNA using a Qiagen RNeasy Mini kit according to the manufacturer’s protocol. Once extracted, RNA from only the female mice groups were delivered to the Roy J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign in two aliquots containing 500 ng RNA. One aliquot was used to check the quality of the RNA using the 2100 Bioanalyzer (Agilent, Santa Clara, CA) and the other aliquot was used for microarray analysis. Microarray and data analysis. Total RNA (250 ng) from each female sample was labeled using the TotalPrep RNA Amplification Kit (Ambion, Austin, TX) for cDNA synthesis followed by in vitro transcription to synthesize biotin-labeled cRNA. Biotin-labeled cRNA (750 ng) was hybridized overnight to MouseWG-6 v2.0 BeadChips (Illumina, San Diego, CA). Hybridized biotinylated cRNA was detected with streptavidin-Cy3 (Amersham Biosciences, Little Chalfont, UK) and scanned with an iScan reader (Illumina). The Illumina MouseWG-6 v2.0 BeadChip contains 45,281 unique 50-mer probes, which interrogate ~30,800 genes. Each unique probe is represented by an average of 43 beads on the array. Illumina’s GenomeStudio (V2011.1) Gene Expression Module (V1.9.0) was used to average the individual bead-level fluorescence values per probe without background correction or normalization and to compute a detection P value, which tests whether the bead-level values for each probe were larger than the negative control beads. The probe-level expression values and detection P value were then imported into R software (40) (v3.3.1) for further quality control assessment, normalization, and statistical analysis using the limma (44) package (v3.30.0). The arrays were processed with the neqc (43) algorithm, which performs a normexp model-based background correction and quantile normalization, then transforms the data to the log2 scale. Microarray files were uploaded to the Gene Expression Omnibus database (accession number GSE79907). Differential gene expression was evaluated by first fitting a mixedeffect 2 ⫻ 2 factorial ANOVA model (45) on all 45,281 probes on the array, which adjusted for the correlation of an observed batch effect in the data (46). The overall ANOVA F-test, main effects of transgene and exercise, the interaction effect and post hoc pairwise comparison between the different experimental groups were pulled from the model. Updated mappings from probes to Entrez Gene identification (ID) numbers, symbols, gene names and gene ontology terms were taken from Bioconductor’s (28) illuminaMousev2.db_1.26.0 annotation package. Updated mappings to Kyoto Encyclopedia of Genes and

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RESULTS

Verification of transgene expression and clustering analysis. The mouse-specific 50-mer probe for ␣7␤1 integrin on the microarray has enough sequence divergence to prevent hybridization of rat ␣7␤1 integrin RNA. Thus, both mouse and rat ␣7␤1 integrin RNA and protein were evaluated using qPCR and Western blotting methods. Total ␣7␤1 protein was increased approximately fourfold in both male and female ␣7Tg mouse muscle compared with WT controls (transgene main effect, P ⬍ 0.05) (male and female data collapsed, data not

shown). Rat ␣7 integrin gene expression was higher in male transgenic mice compared with female (sex ⫻ transgene interaction, P ⬍ 0.05) (Fig. 1A). Endogenous mouse ␣7 integrin gene expression was elevated in both male and female transgenic mice compared with WT mice (twofold, transgene main effect, P ⬍ 0.05), and the total amount was higher in males than females independent of transgene expression (sex main effect, P ⬍ 0.05) (Fig. 1B). Exercise did not increase endogenous mouse integrin expression at this early time point. The overall ANOVA F-test detected only 73 genes with FDR values below the traditional cutoff of P ⬍ 0.05 (Table 2). However, when gene expression changes are not widespread it can be difficult to find any with very low FDR P values because of the sheer number of genes tested. To gain a better understanding of the overall patterns of expression changes due to transgene and exercise effects, we created a heat map of 274 genes that had overall ANOVA raw values of P ⬍ 0.005, which was equivalent to an FDR rate of 0.24 (Fig. 1C). Four main clusters of genes were evident, splitting into groups of those generally affected by the transgene only (black and purple clusters, 57 and 92 genes, respectively) and those generally affected by exercise only (green and yellow clusters, 72 and 53 genes, respectively) although the effect appears stronger in ␣7Tg mice compared with WT mice. Overrepresentation testing for KEGG pathways was completed separately for each of the four gene clusters. The purple cluster, which had higher expression due to the transgene, had overrepresentation of genes in the protein processing in the endoplasmic reticulum (ER) pathway (Sec61g, Xbp1, Map2k7, Herpud1, Ssr3, and Syvn1). The green cluster, which had higher expression due to exercise, had overrepresentation of genes in the estrogen signaling pathway (Adcy4, Fkbp5, Hspa8, Hsp90a1, Hspa1a, and Hsp90b1), mineral absorption pathway (Atp1a1, Mt1, and Mt2), insulin resistance pathway (Socs3, Ppargc1a, Stat3, and Ppp1r3c), and thyroid hormone synthesis pathway (Adcy4, Atp1a1, and Hsp90b1). Neither the yellow or black clusters had any pathways overrepresented at raw P ⬍ 0.005. We further assessed the pairwise responses for the 274 genes by using the same raw value cutoff of P ⬍ 0.005 to compare the response to exercise in WT vs. ␣7Tg mice (Fig. 1D) and the effect of the transgene in sedentary vs. exercised mice (Fig. 1E). Consistent with the visual interpretation of the heat map, transcription was altered to a greater extent in response to exercise in ␣7Tg muscle (87 genes) compared with that in WT mice (24 genes), although of these, only 12 and 6 genes, respectively, reached the raw P ⬍ 0.005 threshold for the interaction effect (interpreted here to be the difference in response to exercise). Only eight genes showed a similar response to exercise at this threshold. The transgene effect was more similar under sedentary and exercised conditions, with 52 genes shared between them. Of the 63 genes with a raw value of P ⬍ 0.005 in the sedentary condition, only 11 reached the same threshold for the interaction effect (interpreted here to be the difference in transgene effect) and likewise, of the 52 genes in the exercised condition, only 5 reached the threshold for the interaction effect. Targeted verification by qPCR was completed on genes previously documented to regulate muscle growth or protection from damage, the majority listed in Table 2 (most responsive to exercise or transgene expression).

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analysis. C2C12 myoblasts were virally transduced and selected in 3 ug/ml of puromycin for 3 days. After selection, the cells were induced to differentiate into myotubes in the presence of either gelatin (collagen) or Matrigel (laminin). RNA (using RLT buffer) and protein (using SDS lysis buffer) were collected from subsets of myotubes formed from a scrambled control transduction, as well as shITGA7 no. 3 and no. 4 constructs. RNA extraction was performed using a Qiagen RNeasy Micro kit and converted to cDNA. The same method of reverse transcription was used as described above. Preamplification steps were performed on genes identified to have transgene main effects from the in vivo study. Gene expression was performed using RT-PCR, and protein expression was determined via Western blot. Evaluation of protein expression. Powdered tissue was homogenized in ice-cold RIPA buffer containing 50 mM Tris·HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF (in 100% EtOH), Roche Complete Mini and Roche PhosSTOP. The homogenates were rotated at 4°C for 1 h and centrifuged at 14,000 g for 15 min at 4°C. The detergent soluble fraction in the supernatant was removed and the concentration of protein was determined with the Bradford protein assay using BSA for the standard curve. Equal amounts of protein (30 ␮g) were reduced, separated by SDS-PAGE using 8% acrylamide gels, and transferred to nitrocellulose membranes. Equal protein loading was verified by Ponceau S staining after transfer, and a single prominent band was used to normalize target protein. Membranes for detection of ␣7 were blocked overnight in Tris-buffered saline (pH 7.8) with Tween 20 (TBST) containing 5% nonfat dry milk (NFDM), followed by a primary incubation in purified ␣7 CDB 347 polyclonal rabbit anti-rat antibody (1:1,000) for 1 h. Membranes for p-IRE1␣ (Ser724), CAAT-enhancer-binding protein homologous protein (CHOP), and immunoglobulin binding protein (BiP) were blocked for 1 h in 5% NFDM in TBST, whereas p-eIF2␣ (Ser51) was blocked in 5% BSA. Primary antibody (1:1,000) was applied in 5% NFDM overnight. The following antibodies were used: ␣7 integrin CDB polyclonal rabbit anti-rat antibody, CHOP (L63F7) (2895S; Cell Signaling), GRP78/HSPA5/BiP (ADISPA-826-D; Enzo Life Sciences), IRE1␣ (Ser724) (NB100 –2323; Novus Biologicals), eIF2␣ (Ser51) (119A11) (3597S; Cell Signaling), and SLC7A5 (bs-10125R; Bioss Antibodies). Horseradish peroxidase-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was applied for 1 h at 1:20,000 for ␣7 integrin, and 1:3,000 for the other four proteins. Bands were detected using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL) and a Bio-Rad ChemiDoc XRS system (Bio-Rad, Hercules, CA). Quantification was completed using Quantity One software (Bio-Rad). Statistical analysis. Analysis of the array data was completed as described above. For all other experiments, averaged data are presented as means ⫾ SE. Two-way ANOVA was completed to determine whether an interaction or main effects were present as a result of transgene expression, sex, and exercise (SPSS, version 16). t-Tests were completed to determine whether gene expression was significantly different between scramble and knockdown. Data was considered significant at P ⬍ 0.05.

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A

C 1400

Itga7 (Rat) 2^(-ΔΔCT)

1200

WT Female WT Male TG Female TG Male

1000

*†

D

*†

*

*

800 600 400 200 Sedentary

Exercise

B *†

3 Itga7 (Mouse) 2^(-ΔΔCT)

2.5 2

†

*

1.5

E

*†

†

*

1 0.5 0 Sedentary

Exercise

Fig. 1. Verification of transgene expression and impact of the ␣7 integrin on gene clustering after acute downhill running. A and B: sex differences were evident in rat (A) and mouse (B) ␣7␤1 integrin gene expression. C: heat map of 274 genes with the most evidence for change within female groups. Red represents upregulation and blue represents downregulation. The four clusters are identified by color, including upregulation by the transgene (purple), downregulation by the transgene (black), upregulation by exercise (green), and downregulation by exercise (yellow). D and E: Venn diagrams representing differential response to exercise (D) and overexpression of the ␣7 integrin subunit (E) in female WT and ␣7Tg skeletal muscle. *Transgene main effect (P ⱕ 0.05). †Sex main effect (P ⱕ 0.05). All values are means ⫾ SE, and all genes were normalized to hypoxanthine phosphoribosyltransferase (Hprt). Data are expressed relative to WT-SED females.

Effect of acute eccentric exercise on skeletal muscle gene expression. Several genes were responsive to the acute bout of eccentric exercise in both WT and ␣7Tg muscle and were verified by qPCR, including metallothionein 1 (Mt1) and Mt2 (3.5- to 4-fold, exercise main effect, P ⬍ 0.05) (Fig. 2, A and B), peroxisome proliferator-activated receptor-␥ coactivator (PGC)-1␣ (Ppargc1a) (3-fold, exercise main effect, P ⬍ 0.05) (Fig. 2C), and muscle-specific kinase (Musk) (2-fold, exercise main effect, P ⬍ 0.05) (Fig. 2D). Although not included in our list of the top 73 genes, heat shock protein 90b1 (Hsp90b1) (1.5- to 2-fold, exercise main effect, P ⬍ 0.05) (Fig. 2E) and Hspa1a (a member of the Hsp70 family) (3- to 4-fold, exercise main effect, P ⬍ 0.05) (Fig. 2F) were also detected in the array and verified. Effect of ␣7 integrin overexpression on skeletal muscle gene expression. Several genes regulated by transgene expression were selected for verification by qPCR, including ribosomal protein L13a (Rpl13a) (20%, transgene main effect, P ⬍ 0.05) and nitric oxide synthase interacting protein (Nosip) (2-fold, transgene main effect, P ⬍ 0.05) (Fig. 3, A and B). Gene expression of angiogenin (Ang) and solute-linked carrier 7A5 (Slc7a5), both regulators of protein translation, were increased as a result of integrin overexpression, and this effect was augmented by exercise (1.5- to 2.5-fold, transgene and

exercise main effects, P ⬍ 0.05) (Fig. 3, C and D). Conversely, glycogen synthase (Gys1) and n-myc downstreamregulated gene 2 (Ndrg2) gene expression were increased in ␣7Tg mice, yet this effect was attenuated as a result of exercise (transgene ⫻ exercise interaction, P ⬍ 0.05) (Fig. 3, E and F). The microarray results indicated that branchedchain amino acid transaminase 2 (Bcat2) was elevated in both basal and exercise transgenic groups over WT mice (Table 2), but qPCR did not validate this finding. Due to the effect of the integrin on genes related to protein translation, we evaluated 18s and 28s rRNA (normalized to total RNA using data provided by the Agilent Bioanalyzer). No significant differences in rRNA content were detected between WT and ␣7Tg mice either in the absence or the presence of acute exercise. Hsp40 (Dnajb9) was increased in ␣7Tg mice and preferentially expressed in males (transgene and sex main effects, P ⬍ 0.05) (Fig. 4, A and B). Because the results from KEGG analysis and the fact that chaperone protein-related gene expression has been reported to be elevated in ␣7Tg mice in other microarrays (32), we also evaluated expression of heat shock 70-kDa protein 5 (Hspa5), also known as immunoglobulin binding protein (BiP) and the 78-kDa glucoseregulated protein (Grp78), and activating transcription fac-

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Table 2. The top 73 responsive genes (ANOVA FDR P ⬍ 0.05) Entrez ID

Symbol

ANOVA FDR P Value

Main Effect

FDR P Value

Direction

ILMN_1251254 ILMN_1219510 ILMN_2783557 ILMN_2890743 ILMN_2794608 ILMN_2740006 ILMN_1230327 ILMN_2596522 ILMN_2619156 ILMN_2815889 ILMN_2501719 ILMN_2971314 ILMN_2603834 ILMN_2667101 ILMN_1217519 ILMN_3102085 ILMN_1215969 ILMN_3135697 ILMN_2875251 ILMN_1221081 ILMN_2485340 ILMN_3160146 ILMN_1219154 ILMN_2912111 ILMN_1236577 ILMN_2598576 ILMN_2768053 ILMN_2918402 ILMN_2667463 ILMN_1249498 ILMN_2710139 ILMN_2711948 ILMN_1255422 ILMN_2662648 ILMN_3002505 ILMN_1239578 ILMN_1224768 ILMN_2846255 ILMN_2550291 ILMN_1252338 ILMN_1217966 ILMN_2813487 ILMN_2709864 ILMN_1254516 ILMN_1245437 ILMN_2773395 ILMN_2668886 ILMN_2900827 ILMN_2829636 ILMN_3102277 ILMN_1259294 ILMN_2597332 ILMN_2595732 ILMN_2697552 ILMN_2972585 ILMN_2631903 ILMN_2903511 ILMN_2771991 ILMN_2769734 ILMN_1249654 ILMN_1217308 ILMN_2667091 ILMN_2516383 ILMN_2696270 ILMN_1259368 ILMN_2476881 ILMN_1232067 ILMN_1245263 ILMN_2907972 ILMN_2495289 ILMN_2741070 ILMN_1242427 ILMN_2811918

14936 22121 233189 66394 12036 246694 14325 17748 16495 101612 72088 71755 53415 67023 71960 69217 217864 66869 11727 211135 269181 626359 17750 234358 72368 11546 77945 102247 77090 56193 19017 20539 12457 225115 64296 17158 98878 66522 66923 545123 384775 18626 226043 18203 68188 12845 218989 76900 27362 18198 68472 58248 18030 18578 52118 69301 50783 29811 59045 53609 16618 53412 107513 59047 80287 72899 234388 77106 72297 110172 259026 104099 243963

Gys1 Rpl13a Ctu1 Nosip Bcat2 Hps5 Ftl1 Mt1 Kcna7 Grwd1 Ush1c Dhdh Htatip2 Use1 Myh14 Plekha4 Rcor1 Zfp869 Ang D130040H23Rik Mgat4a Wdr93 Mt2 Zfp930 Borcs8 Parp2 Rpgrip1 Gpat4 Ocel1 Plek Ppargc1a Slc7a5 Noct Svil Abhd8 Man2a1 Ehd4 Pgpep1 Pbrm1 Cyp2d11 1700003H04Rik Per1 Cbwd1 Ntan1 Sympk Comp Tmem260 Ssbp4 Dnajb9 Musk Tmem126b 1700123O20Rik Nfil3 Pde4b Pvr Tescl Lsm4 Ndrg2 Stard3 Clasrp Klk1b26 Ppp1r3c Ssr1 Pnkp Apobec3 Macrod2 Ccdc124 Tmem181a B3gnt3 Slc35b1 Olfr378 Itga9 Zfp473

3.48E-14 2.73E-13 2.84E-06 1.11E-05 2.82E-05 4.29E-05 4.61E-05 0.000125 0.000125 0.000196 0.000249 0.000249 0.000324 0.000355 0.000355 0.000365 0.000701 0.00075 0.00125 0.002 0.00421 0.00558 0.00558 0.006 0.006 0.00618 0.00709 0.00722 0.00979 0.0104 0.0109 0.0117 0.0131 0.0139 0.0139 0.0139 0.0139 0.0139 0.0141 0.0146 0.0205 0.0209 0.0215 0.0262 0.0273 0.0275 0.0294 0.0315 0.0331 0.0339 0.0339 0.0339 0.0339 0.0353 0.0381 0.0382 0.0382 0.0382 0.0387 0.0411 0.044 0.044 0.0441 0.0456 0.0456 0.046 0.0469 0.0471 0.0471 0.0471 0.0471 0.0481 0.0481

Transgene Transgene Transgene Transgene Transgene Transgene Transgene Exercise Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Exercise Transgene Transgene Transgene Transgene Transgene* Transgene Transgene Exercise Exercise Exercise Exercise* Transgene Exercise Exercise* Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene Exercise Exercise* Transgene Exercise* Exercise* Transgene Transgene Transgene Transgene Transgene Transgene Transgene Transgene* Transgene Transgene Exercise* Transgene Transgene Transgene Transgene Transgene Transgene Exercise* Transgene

1.62E-15 1.21E-14 1.63E-07 6.51E-07 1.99E-06 2.36E-06 2.55E-06 6.74E-05 9.86E-06 1.18E-05 1.52E-05 1.52E-05 1.95E-05 2.78E-05 3.90E-05 3.48E-05 7.78E-05 5.98E-05 0.000254 0.000421 0.000447 0.0129 0.00399 0.000453 0.000487 0.000613 0.00119 0.0863 0.00121 0.00155 0.014 0.014 0.014 0.158 0.00155 0.0168 0.0509 0.0015 0.00412 0.0015 0.00412 0.0339 0.012 0.00591 0.00412 0.00305 0.0108 0.00418 0.00711 0.0291 0.104 0.00498 0.0766 0.126 0.00711 0.00711 0.0108 0.00711 0.0263 0.00711 0.0073 0.11 0.00711 0.00711 0.0942 0.00711 0.00655 0.00711 0.00891 0.00711 0.0124 0.0509 0.00875

Up Up Down Up Up Down Up Up Down Up Up Down Down Up Down Down Down Up Up Up Down Up Up Up Down Up Up Up Down Up Up Up Up Up Down Up Up Up Down Up Up Up Up Down Up Down Up Down Up Up Up Up Up Up Up Down Down Up Up Up Down Up Down Down Up Down Up Up Up Up Up Up Up

FDR, false discovery rate. *Main effect FDR P ⬎ 0.05. AJP-Cell Physiol • doi:10.1152/ajpcell.00106.2016 • www.ajpcell.org

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Probe ID

C643

INTEGRIN AND GENE EXPRESSION

‡

4.5

‡

B 5

4

4.5

3.5

4

3

3.5

2.5 2 1.5

TG

3 2.5 2 1.5

1

1

0.5

0.5

0 Sedentary

C

0

Exercise

Sedentary

D

‡

4

3

3.5

Exercise

‡

2.5

3 2.5

Musk 2^(-ΔΔCT)

Ppargc1a 2^(-ΔΔCT)

WT

2 1.5

2 1.5 1

1 0.5

0.5 0

0 Sedentary

E

2.5

Sedentary

Exercise

‡

F

6

‡

5 Hspa1a/Hsp70 2^(-ΔΔCT)

2 Hsp90b1 2^(-ΔΔCT)

Exercise

1.5 1 0.5

4 3 2 1

0 Sedentary

Exercise

0 Sedentary

Exercise

Fig. 2. Acute eccentric exercise increases gene expression in skeletal muscle independent of the transgene. Mt1, Mt2, Ppargc1a, Musk, Hsp90b1, and Hspa1a gene expression was examined in skeletal muscle (A–F). ‡Exercise main effect (P ⱕ 0.05). All values are means ⫾ SE, and all genes were normalized to Hprt and are expressed relative to WT-SED.

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Mt2 2^(-ΔΔCT)

Mt1 2^(-ΔΔCT)

A

C644

INTEGRIN AND GENE EXPRESSION

A

2.5

1 Nosip 2^(-ΔΔCT)

Rpl13a 2^(-ΔΔCT)

*

*

1.2

0.8 0.6 0.4

Sedentary

D

Exercise

‡

2.5

*

2

*

Slc7a5 2^(-ΔΔCT)

Ang 2^(-ΔΔCT)

1

*

2.5

1.5 1

1.5

*

1 0.5

0.5 0

0 Sedentary

Exercise

Transgene x Exercise Interaction

Sedentary

F

2

1.8

1.8

1.6

1.6

1.4

1.4 Ndrg2 2^(-ΔΔCT)

Gys1 2^(-ΔΔCT)

1.5

Exercise

‡

3

2

*

2

0 Sedentary

E

WT TG

1.2 1 0.8

Exercise

Transgene x Exercise Interaction

1.2 1 0.8

0.6

0.6

0.4

0.4

0.2

0.2 0

0 Sedentary

Exercise

Sedentary

Exercise

Fig. 3. The ␣7 integrin regulates skeletal muscle gene expression. Rpl13a, Nosip, Ang, Slc7a5, Gys1, and Ndrg2 gene expression was examined in skeletal muscle (A–F). *Transgene main effect (P ⱕ 0.05). ‡Exercise main effect (P ⱕ 0.05). All values are means ⫾ SE, and all genes were normalized to Hprt and are expressed relative to WT-SED.

tor 6 (Atf6). Hspa5 was significantly increased in ␣7Tg mice and preferentially expressed in males and in response to exercise (transgene, exercise and sex main effects, P ⬍ 0.05) (Fig. 4, C and D). Atf6 was significantly increased in

male transgenic mice and in response to exercise (transgene, exercise and sex main effects, P ⬍ 0.05) (Fig. 4F), but it was not altered in females, resulting in no apparent effect when male and female groups were combined (Fig. 4E).

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0

2

*

0.5

0.2

C

3

B

1.4

C645

INTEGRIN AND GENE EXPRESSION

A

1.6 1.4

WT

*

B

*

2.5

TG

2

1.2 Hsp40 2^(-ΔΔCT)

1 0.8 0.6 0.4

1.5

*

†

†

†

†

1 0.5

0.2 0

0 Sedentary

C

3

3 Hspa5/BiP 2^(-ΔΔCT)

Hspa5/BiP 2^(-ΔΔCT)

1

2 1.5

0.5

0.5

0

0

F 5

1.6

4.5

1.4

4

1.2

3.5

0.8 0.6

†

Exercise

Transgene x Sex Interaction

1.8

1

†

†

Sedentary

Exercise

2

†

*

‡

2.5

1

Atf6 2^(-ΔΔCT)

Atf6 2^(-ΔΔCT)

*

3.5

*

1.5

Exercise

Transgene x Sex Interaction

*

Sedentary

E

D

‡

2.5 2

Sedentary

Exercise

3

*

‡

* †

† †

†

2.5 2 1.5

0.4

1

0.2

0.5 0

0 Sedentary

Exercise

Sedentary

Exercise

Fig. 4. The ␣7 integrin augments expression of genes necessary for protection from stress. Gene expression of Hsp40 (Dnajb9), Hspa5, and Atf6 combined (A, C, and E, respectively) and by sex (B, D, and F, respectively) was examined in skeletal muscle. *Transgene main effect (P ⱕ 0.05). ‡Exercise main effect (P ⱕ 0.05). †Sex main effect (P ⱕ 0.05). All values are means ⫾ SE and all genes were normalized to Hprt. Combined data are expressed relative to WT-SED and sex differences are expressed relative to WT-SED Female.

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Hsp40 2^(-ΔΔCT)

*

WT Female WT Male TG Female TG Male

C646

INTEGRIN AND GENE EXPRESSION

DISCUSSION

We have previously established that ␣7␤1 integrin mRNA and protein are significantly enhanced in mouse and human skeletal muscle in response to an acute bout of eccentric exercise (6). The integrin protein is localized to the sarcolemma, particularly at the costamere and myotendinous junction, suggesting that this structure provides stabilization and protection from damage in response to strain associated with eccentric lengthening. Indeed, transgenic mice created to overexpress the ␣7BX2 integrin subunit in skeletal muscle display protection from damage and inflammation after exercise. Coincident with enhanced integrity is a phenotype of increased satellite cell quantity, new fiber synthesis, and increased myofiber cross-sectional area following single or repeated bouts of eccentric exercise (34, 53). The purpose of this study was to investigate global gene transcription of skeletal muscle in this unique animal model to reveal potential mechanisms by which the ␣7␤1 integrin can facilitate the positive benefits of eccentric exercise. The results suggest an important role for the integrin in upregulation of genes that confer resistance to stress and modulation of protein translation. The downhill running model used for this experiment resulted in increased expression of several genes including Mt1,

Mt2, Ppargc1a, Musk, Hsp90b1, and Hspa1a (Hsp70), which when analyzed together, suggest that this exercise paradigm was sufficiently stressful to elicit an early adaptive response in both WT and ␣7Tg mice. The precise function of the metallothioneins are not fully understood, but they are believed to possess physiological metal (zinc and copper) binding capacity and serve as antioxidants (1, 22, 41, 42). In concert with PGC-1␣, an essential regulator of mitochondrial biogenesis, both are likely enhanced in skeletal muscle to provide protection against oxidative stress associated with increased metabolism during exercise. In addition to metabolic stress, lengthening during eccentric exercise can damage the sarcolemma and muscle ultrastructure. The upregulation of muscle-specific kinase (Musk), a necessary component of the neuromuscular junction (9, 21), as well as important heat shock proteins, may be necessary to stabilize the myofiber and ensure proper innervation and protein folding immediately following exercise. The fact that Mt1, Mt2, and Ppargc1a gene expressions are equally enhanced in both WT and ␣7Tg muscle suggest that the integrin may not be an essential regulator of protection from oxidative stress after exercise. Overall, relatively few transcripts were altered with overexpression of the ␣7␤1 integrin in skeletal muscle in the present array (73 genes), a finding similar to that of a previous report (32). Despite the lack of global change in gene expression, the ␣7␤1 integrin appears to increase expression of key genes related to stress resistance, protein translation, and amino acid transport in a manner that is either unaltered by exercise (Rpl13a, Nosip, Hsp40), further enhanced by exercise (Ang, Slc7a5), or suppressed by exercise (Gys1, Ndrg2). Under conditions of nutrient stress and hypoxia, RPL13a can increase translation of mRNAs with an internal ribosome entry site (IRES), and this cap-independent mechanism is used to translate proteins such as molecular chaperones, growth factors, and anti-inflammatory cytokines that are necessary for cellular survival (26, 30, 31, 35, 47). Microarray results for Rpl13a demonstrate an elevation of more than 1,000-fold in an ␣7Tg muscle as compared with WT regardless of exercise. The increase was much less dramatic upon verification with qPCR, yet it was still significant. Angiogenin can similarly increase cap-independent translation during times of stress and serve as a transcription factor for rRNA during periods of growth after stress (17, 29, 38, 51). Interestingly, Ang was substantially increased as a result of both integrin overexpression and exercise in ␣7Tg muscle. Finally, transcription of the LAT1 amino acid transport protein, Slc7a5, also was increased in response to integrin overexpression and exercise, an event that may contribute to increased muscle protein synthesis postexercise (15). Minimal information exists regarding NOSIP and NDRG2 in skeletal muscle, yet studies suggest localization at the cell membrane and regulation of nitric oxide and mTORC1 signaling, respectively (8, 13, 16, 19). Taken together, the results from this study suggest a predominant role for the ␣7␤1 integrin in the modulation of protein translation in a manner that may provide protection from stress and increased growth following eccentric exercise. HSP40 (DnaJ) stimulates ATPase activity of HSP70, and thus regulates the function of this important chaperone protein (36, 39). Hsp40 gene expression was significantly increased in ␣7Tg muscle before and after exercise. Results from KEGG analysis and previous array data (32) suggested regulation of

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Due to the complex role of BiP in the regulation of protein folding and coordination of the unfolded protein response (UPR), BiP protein expression and UPR-associated protein activation were assessed in both male and female mice (representative blots are shown in Fig. 5A). BiP protein was significantly increased in ␣7Tg mice, independent of exercise and sex (60%, transgene main effect, P ⬍ 0.05) (Fig. 5B). Significant alterations in UPR-mediated signaling, including inositol-requiring enzyme 1 ␣ (IRE-1␣) and eukaryotic initiation factor 2 ␣ (eIF2␣) phosphorylation, were not detected at 3 h after eccentric exercise in WT or ␣7Tg mice (Fig. 5, C and D), but downstream mediator of stress induced apoptosis CAAT-enhancer-binding protein homologous protein (CHOP) was significantly reduced in in ␣7Tg mice (20%, transgene main effect, P ⬍ 0.05) (Fig. 5E) (52). No differences in UPR-mediated signaling were detected between males and females. Knockdown of the ␣ 7 integrin regulates Slc7a5 transcription. We validated two constructs that could effectively knockdown ␣7 integrin protein expression in C2C12 myotubes (shITGA7 no. 3 and no. 4) compared with scramble control, and construct no. 4 was used for further analysis (Fig. 6A). Whereas transcription of Slc7a5 and Ndrg2 was significantly decreased with ␣7 integrin knockdown, several genes were not consistently altered, including Rpl13a, Nosip, Gys1, Hsp40, Hspa5/BiP, and Atf6 (Fig. 6C). Ang gene expression increased with ␣7 integrin knockdown. The experiment was repeated using Matrigel (laminin) as a substrate to ensure proper integrin linkage in the scramble control (Fig. 6D). Slc7a5 expression remained decreased, but Ang was no longer significant, and Ndrg2 was increased in response to knockdown. Given the consistent change in Slc7a5 transcription, we evaluated SLC7A5 protein expression in the muscle of WT and ␣7Tg mice. We found that protein expression was increased in ␣7Tg mice compared with WT mice (transgene and exercise main effects, P ⱕ 0.05) (Fig. 6B).

C647

INTEGRIN AND GENE EXPRESSION

α7TG

WT

A MW

SED F M

F

EX M

SED F M

78 kDa

EX F

M BiP Ponceau S

110 kDa

p-IRE-1α

38 kDa

P-eIF2α Ponceau S

27 kDa

CHOP Ponceau S

B

2

*

*

1.6 1.4 1.2 1 0.8 0.6 0.4 0

0.8 0.6 0.4

Exercise

Transgene Main Effect (P=0.091)

Sedentary

E

Exercise

1.4 1.2

CHOP Protein Fold change from WT-R

1.2 IRE-1α Phosphorylation Fold change from WT-R

1

0 Sedentary

1.4

Transgene x Exercise Interaction (p=0.071)

0.2

0.2

C

1.4 1.2

eIF2α Phosphorylation Fold change from WT-R

BiP (HSPA5) Protein Fold change from WT-R

1.8

D

1 0.8 0.6 0.4 0.2

1 0.8

*

*

0.6 0.4 0.2

0

0 Sedentary

Exercise

Sedentary

Exercise

Fig. 5. The ␣7 integrin stimulates a beneficial UPR in skeletal muscle. A: representative immunoblots of unfolded protein response (UPR)-associated proteins. B–E: summary of data for total BiP (HSPA5) (B), phospho-IRE-1␣ (C), phospho-eIF2␣ (D), and total CHOP (E). *Transgene main effect (P ⱕ 0.05). All values are means ⫾ SE and all proteins were normalized to Ponceau S staining and are expressed relative to WT-SED. M, male; F, female.

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Ponceau S

C648

INTEGRIN AND GENE EXPRESSION

A

B Scramble shITGA7#3 Scramble #3

shITGA7#4

Ponceau S

1

**

0.8

**

0.6 0.4

* *

1.2 1 0.8 0.6 0.4 0.2

0.2 0

0

Scramble shITGA7 #3

C

1.6

shITGA7 #4

Sedentary

Exercise

shITGA7 #4 Gene Expression (Gelatin)

**

1.4

2^(-ΔΔCT)

Fig. 6. In vitro evaluation of ␣7 integrinmediated gene transcription. A: validation of two constructs that allow for ␣7 integrin knockdown in myotubes. B: SLC7A5 protein expression was examined in skeletal muscle. C and D: evaluation of target gene transcription with ␣7 integrin knockdown. *Ttransgene main effect (P ⱕ 0.05). ‡Exercise main effect (P ⱕ 0.05). **Different from scramble control (P ⱕ 0.05). All values are means ⫾ SE, and all genes are normalized to Hprt and are expressed relative to scramble control.

1.4

1.2 1

**

0.8

**

0.6 0.4 0.2

**

0

D

shITGA7 #4 Gene Expression (Matrigel)

**

1.4

2^(-ΔΔCT)

1.2

**

1 0.8 0.6 0.4

**

0.2 0

protein processing in the ER. Thus, we examined two other transcripts related to the UPR, including Hspa5 (BiP) and Atf6. BiP is located in the lumen of the ER and serves as an important molecular chaperone that can fold and assemble newly synthesized protein. Accumulation of unfolded proteins in the ER stimulates the release of BiP from transmembrane receptor proteins and initiates several events that serve to ensure cellular integrity during short periods of growth or stress (adaptive UPR) or serve to commit the cell to apoptosis during prolonged stress (maladaptive UPR). BiP expression was increased in ␣7Tg muscle in the rested state and after exercise, a result that was more evident in males. Atf6 expres-

sion was also significantly increased in both WT and ␣7Tg males compared with females, and further enhancement was detected with integrin overexpression and exercise. Results reported by Wu et al. (50) demonstrate that PGC-1␣ serves as a coactivator with ATF6 to enhance BiP expression in vitro. Our data suggest this mechanism may allow for increased BiP mRNA and protein in male mice after exercise. However, the lack of PGC-1␣ mRNA in the rested state (Fig. 2C) suggests that an alternative coactivator may be necessary to account for the enhancement in BiP during rest conditions. Intracellular signaling pathways in the ER provide the mechanistic basis for UPR following the initiation of stress, includ-

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α7 Integrin Protein

1.2

‡

WT TG

1.6 SLC7A5 Protein Fold change from WT-R

α7 Integrin (129kDa)

INTEGRIN AND GENE EXPRESSION

paradigms that can help preserve muscle mass and function across the lifespan. ACKNOWLEDGMENTS We thank Dr. Mark Band and Tanya Akraiko in the Functional Genomics Unit of the Roy J. Carver Biotechnology Center, University of Illinois, for hybridizing the Illumina BeadArrays. GRANTS This work was partially supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants R21 AR-065578 to M. D. Boppart and R01 AR-048914 to J. Chen. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Z.S.M., Y.P., and M.D.B. conceived and designed the research; Z.S.M., K.S., Y.P., and M.M. performed experiments; Z.S.M., K.S., J.D., and M.D.B. analyzed data; Z.S.M., K.S., J.C., and M.D.B. interpreted results of experiments; Z.S.M. and M.D.B. prepared figures; Z.S.M., J.D., and M.D.B. drafted manuscript; Z.S.M., K.S., Y.P., M.M., J.D., J.C., and M.D.B. edited and revised manuscript; Z.S.M., K.S., Y.P., M.M., J.D., J.C., and M.D.B. approved final version of manuscript. REFERENCES 1. Aschner M, Cherian MG, Klaassen CD, Palmiter RD, Erickson JC, Bush AI. Metallothioneins in brain—the role in physiology and pathology. Toxicol Appl Pharmacol 142: 229 –242, 1997. doi:10.1006/taap. 1996.8054. 2. Bao ZZ, Lakonishok M, Kaufman S, Horwitz AF. Alpha 7 beta 1 integrin is a component of the myotendinous junction on skeletal muscle. J Cell Sci 106: 579 –589, 1993. 3. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 57: 289 –300, 1995. 5. Boppart MD, Burkin DJ, Kaufman SJ. ␣7␤1 Integrin regulates mechanotransduction and prevents skeletal muscle injury. Am J Physiol Cell Physiol 290: C1660 –C1665, 2006. doi:10.1152/ajpcell.00317.2005. 6. Boppart MD, Volker SE, Alexander N, Burkin DJ, Kaufman SJ. Exercise promotes ␣7 integrin gene transcription and protection of skeletal muscle. Am J Physiol Regul Integr Comp Physiol 295: R1623–R1630, 2008. doi:10.1152/ajpregu.00089.2008. 7. Boppart MD, Burkin DJ, Kaufman SJ. Activation of AKT signaling promotes cell growth and survival in ␣7␤1 integrin-mediated alleviation of muscular dystrophy. Biochim Biophys Acta 1812: 439 –446, 2011. doi:10.1016/j.bbadis.2011.01.002. 8. Burchfield JG, Lennard AJ, Narasimhan S, Hughes WE, Wasinger VC, Corthals GL, Okuda T, Kondoh H, Biden TJ, Schmitz-Peiffer C. Akt mediates insulin-stimulated phosphorylation of Ndrg2: evidence for cross-talk with protein kinase C ␪. J Biol Chem 279: 18623–18632, 2004. doi:10.1074/jbc.M401504200. 9. Burden SJ. SnapShot: neuromuscular junction. Cell 144: 826 –826.e1, 2011. doi:10.1016/j.cell.2011.02.037. 10. Burkin DJ, Kaufman SJ. The ␣7␤1 integrin in muscle development and disease. Cell Tissue Res 296: 183–190, 1999. doi:10.1007/s004410051279. 11. Burkin DJ, Wallace GQ, Nicol KJ, Kaufman DJ, Kaufman SJ. Enhanced expression of the ␣7␤1 integrin reduces muscular dystrophy and restores viability in dystrophic mice. J Cell Biol 152: 1207–1218, 2001. doi:10.1083/jcb.152.6.1207. 13. Dedio J, König P, Wohlfart P, Schroeder C, Kummer W, MüllerEsterl W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J 15: 79 –89, 2001. doi:10.1096/fj.00-0078com. 14. De Lisio M, Farup J, Sukiennik RA, Clevenger N, Nallabelli J, Nelson B, Ryan K, Rahbek SK, de Paoli F, Vissing K, Boppart MD. The acute response of pericytes to muscle-damaging eccentric contraction and protein supplementation in human skeletal muscle. J Appl Physiol (1985) 119: 900 –907, 2015. doi:10.1152/japplphysiol.01112.2014. 15. Dickinson JM, Drummond MJ, Coben JR, Volpi E, Rasmussen BB. Aging differentially affects human skeletal muscle amino acid transporter

AJP-Cell Physiol • doi:10.1152/ajpcell.00106.2016 • www.ajpcell.org

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ing 1) protein kinase RNA-like ER kinase (PERK) autophosphorylation and subsequent phosphorylation of the ␣ subunit of eIF2 (resulting in attenuation of protein translation), 2) inositol-requiring enzyme-1␣ (IRE-1␣) phosphorylation and subsequent cleavage and translocation of the transcription factor XBP1 (resulting in upregulation of chaperone proteins and elements necessary for protein degradation), and 3) ATF6 cleavage and translocation to the nucleus (resulting in upregulation of chaperone proteins) (25, 27). In the current study, trends for an increase in IRE-1␣ phosphorylation and a decrease in eIF2␣ phosphorylation were detected in ␣7Tg muscle at 3 h postexercise, suggesting some ability for the integrin to regulate UPR in the context of exercise. These changes may account for the UPR observed following acute resistance exercise in humans (37). Although UPR signaling was not highly regulated with transgene expression or exercise, CHOP protein expression was significantly attenuated in ␣7Tg muscle independent of exercise. This observation is in disagreement with results reported by Liu et al. (32) that report an increase in Chop mRNA in ␣7Tg muscle. However, the results are in agreement with studies that report the ability of the ␣7 integrin to protect against apoptosis (32) both in myoblasts in culture and in the muscle fiber of mice with muscular dystrophy (7). The fact that a maladaptive UPR is present during rapid growth associated with chronic loading suggests that UPR may not provide the mechanistic basis for ␣7 integrin-mediated growth following eccentric exercise (23). However, further studies are necessary to address this important question. The extent to which the integrin can accelerate or further enhance growth observed in response to chronic loading has not been evaluated and is the focus of our current work. We evaluated the ability for the ␣7 integrin to directly regulate transcription using an in vitro knockdown approach. We incorporated two different substrates, including gelatin (collagen) and Matrigel (laminin), to account for the influence of integrin activation on gene transcription in the scrambled controls. Slc7a5 gene transcription was significantly decreased with ␣7 integrin knockdown, regardless of substrate. However, this was the only gene out of nine tested that was consistently altered in vitro. We have previously documented that myogenic and perivascular stem cells are enhanced in ␣7 integrin transgenic muscle under sedentary conditions and after exercise (34, 48). Thus, we speculate that the gene signature reported in this study may be influenced by an alteration in mononuclear cell composition. In vitro assessment will be necessary to directly link sarcolemmal integrin with transcriptional control in the absence and/or presence of mechanical cues. Regardless, it is clear from this study that the ␣7 integrin can directly and rapidly control Slc7a5 transcription, and enhancement of this vital amino acid transporter in muscle may significantly contribute to initiation of growth after exercise. The results from this study provide seminal information regarding the molecular basis by which the ␣7␤1 integrin may facilitate protection from damage as well as promote myofiber growth following eccentric exercise. Future work will strive to elucidate a role for an adaptive UPR and Slc7a5 transcription in the promotion of growth postexercise and investigate the effect of aging on integrin protein expression, activation, and signaling. This information will be essential for improving our current understanding of anabolic resistance and creating new

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17.

18. 19.

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25. 26. 27. 28.

29. 30.

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