Resistance Exercise Induced Changes Of Inflammatory Gene Expression Within Human Skeletal Muscle


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Resistance Exercise Induced Changes Of Inflammatory Gene Expression Within Human Skeletal Muscle

  1. 1. Eur J Appl Physiol (2009) 107:463–471 DOI 10.1007/s00421-009-1145-z O R I G I N A L A R T I CL E Resistance exercise-induced changes of inXammatory gene expression within human skeletal muscle Thomas W. Buford · Matthew B. Cooke · Darryn S. Willoughby Accepted: 24 July 2009 / Published online: 11 August 2009 © Springer-Verlag 2009 Abstract Aberrant local inXammatory signaling within obtained from the vastus lateralis of the dominant leg at skeletal muscle is now considered a contributing factor to baseline and 3 h following exercise. SigniWcant (p < 0.05) the development of sarcopenia. Recent evidence indicates up-regulation in mRNA content was observed for TNF , that chronic resistance training contributes to the control of IL1 , IL6, IL8, SOCS2, COX2, SAA1, SAA2, IKKB, cfos, locally derived inXammation via adaptations to repeated, and junB. Muscle mRNA content was not signiWcantly acute increases in pro-inXammatory mRNA within muscle. altered at the 0.05 level for IL2, IL5, IL10, or IL12 (p35). However, only a limited number of gene transcripts related Venous blood samples were also obtained at baseline as to the inXammatory process have been examined in the lit- well as at 3, 24, and 48 h post-exercise. Serum was ana- erature. The present study utilized an acute bout to examine lyzed for circulating TNF , IL1 , IL6, IL8, COX2, and the eVects of resistance exercise on several inXammatory- SAA with no signiWcant changes observed. These results related genes in 24 physically active, post-menopausal indicate that resistance exercise is capable of up-regulating women not currently undergoing hormone replacement transcription of numerous inXammatory mediators within therapy. Following a standard warm-up, participants com- skeletal muscle, and these appear to be worthy of future pleted a lower-body resistance exercise bout consisting of 3 examination in chronic studies. sets of 10 repetitions on machine squat, leg press, and leg extension exercises (80% intensity). Muscle biopsies were Keywords InXammation · Cytokines · Resistance training · mRNA · Sarcopenia · Menopause T. W. Buford Division of Medicine, Department of Aging and Geriatric Research, Institute on Aging, University of Florida, Introduction Gainesville, FL, USA M. B. Cooke · D. S. Willoughby Skeletal muscle mass and strength are important factors in Exercise and Biochemical Nutrition Laboratory, maintaining independence and quality of life in the elderly. Baylor University, Waco, TX, USA More than 20 years ago, Lexell et al. (1988) noted that there is a nearly 40% loss of muscle mass of people in their M. B. Cooke · D. S. Willoughby Exercise, Nutrition, and Resistance Training Research Unit, 1980s compared to when they were in their 1920s. As the Baylor University, Waco, TX, USA world population grows ever older, the loss of skeletal mus- cle function impacts ever-increasing numbers of people and D. S. Willoughby their ability to carry out daily tasks such as climbing stairs, Baylor Biomedical Science Institute, Baylor University, Waco, TX, USA rising from the toilet, or carrying groceries. Recent esti- mates indicate that approximately 45% of older Americans D. S. Willoughby (&) are sarcopenic (Melton et al. 2000) while approximately Exercise and Biochemical Nutrition Laboratory, 20% are functionally disabled (Manton and Gu. 2001). In Department of Health, Human Performance, and Recreation, Baylor University, Waco, TX 76798, USA addition to the individual loss of functional capabilities, the e-mail: economic impact of sarcopenia is also dramatic, as it has 123
  2. 2. 464 Eur J Appl Physiol (2009) 107:463–471 been estimated that the direct healthcare costs of sarcopenia related to the inXammatory process (several of which have in the US were approximately $18 billion at the turn of the yet to be investigated within skeletal muscle) in response to century (Janssen et al. 2004). Thus, interest in mechanisms acute resistance exercise. Thus, the purpose of this study that underlie and interventions that could alleviate age- was to test the hypothesis that a single bout of resistance related muscle dysfunction is signiWcant. exercise bout would signiWcantly alter the muscle mRNA In comparison to a number of other disease states, evi- content of a more complex set of inXammatory-related dence indicating the contribution of inXammation to sarco- genes than has previously been described. penia has steadily been accumulating over the last few years. In fact, a number of studies have now reported links between aberrant inXammatory/cytokine signaling and the Materials and methods development of sarcopenia (Haddad et al. 2005; Hamada et al. 2005; RoubenoV 2007; Visser et al. 2002). SpeciW- Participants cally, inXammatory dysfunction may contribute to sarcope- nia through multiple mechanisms including induction of the The present study employed 24 recreationally active caspases (Dirks and Leeuwenburgh 2006), activation of (consistent, structured exercise at least 3£/week) female nuclear factor kappa B (NFkB) and subsequent atrophy- subjects with an average (§SD) age of 54.54 (3.89) year, related gene expression (Mourkioti and Rosenthal 2005), or height of 159.67 (5.22) cm, and body mass of 72.43 via the impairment of skeletal muscle growth/repair mecha- (15.58) kg. Participants were required to attain written phy- nisms such as the growth hormone (GH)-insulin-like sician consent before being allowed to participate. Post- growth factor 1 (IGF1) axis (Frost and Lang 2005; Haddad menopausal status was veriWed by physician conWrmation et al. 2005). of amenorrhea concurrent with elevated serum levels of fol- In conjunction with the consumption of adequate pro- licle stimulating hormone (FSH) for at least the previous tein, resistance training provides a safe, cost-eVective 12 months (World Health Organization 1981; Rannevik method of preserving muscle mass and strength in older et al. 2008). Participants with contraindications to exercise individuals. Additionally, resistance training has been as outlined by the American College of Sports Medicine shown to provide anti-inXammatory beneWt to skeletal mus- were not allowed to participate. In addition, participants cle (Greiwe et al. 2001). It now appears possible, if not who were on estrogen replacement therapy were ineligible likely, that these adaptations are due to local changes in the to participate. All eligible participants signed university- muscle rather than systemic changes. Skeletal muscle has approved informed consent documents and approval was come to be viewed as an endocrine organ actively involved granted by the Institutional Review Board for Human in inXammation due to its ability to produce cytokines such Subjects. Additionally, all experimental procedures involved as interleukin 6 (IL6) and tumor necrosis factor alpha in the study conformed to the ethical consideration of the (TNF ) (Plomgaard et al. 2005; Steensberg et al. 2000). Helsinki Code. Investigators explained the purpose of the Further, more recent studies have indicated that exercise study, the protocol to be followed, and the experimental regimens including resistance training improve muscle procedures to be used prior to allowing prospective partici- strength (Bruunsgaard et al. 2004) and reduce inXammatory pants to enter the study. mRNA expression (Lambert et al. 2008) without altering plasma levels of IL6 or TNF . Yet the number and precise Experimental procedures role of endogenously produced inXammatory mediators within skeletal muscle has been incompletely delineated During the familiarization session, lower-body maximum and needs further investigation given the development of strength [one repetition maximum (1-RM)] was determined low-grade inXammation in aged skeletal muscle. for machine squat, leg press, and leg extension exercises. Although the information gained from acute exercise During the exercise testing session, participants underwent studies is typically less clinically relevant than that a resistance exercise bout consisting of 3 sets of 10 repeti- obtained from training studies; these investigations can pro- tions at 80% of 1RM on each of the three exercises. Exer- vide a wide range of valuable information concerning bio- cise order was consistent for all participants and between logical targets in training studies. In order to gain a more the familiarization to the exercise bout. Prior to beginning comprehensive understanding of how resistance training the bout, participants underwent a warm-up consisting of provides anti-inXammatory beneWt to the muscle, we con- 5 min cycling on a Monarch (Model 818E, Varberg, Sweden) ducted the present investigation to gain pilot data regarding cycle ergometer (50 rpm) and 2 sets of 10 repetitions (50% the transcriptional response of a number of inXammatory 1RM) on the leg press. mediators within skeletal muscle. We thus proposed to Prior to each testing session, total body mass (kg) was investigate the transcription of a number of other genes determined on a calibrated electronic scale with a precision 123
  3. 3. Eur J Appl Physiol (2009) 107:463–471 465 of §0.02 kg (Detecto, Webb City, MO, USA). In addition, the needle to collect a muscle piece. The biopsy needle was participants underwent assessment of heart rate and blood removed from the pilot hole and the muscle specimen was pressure upon arrival at each session as well as following removed from the biopsy needle using a sterile scalpel, sep- the exercise bout. Heart rate was determined by palpation arated from connective and/or adipose tissues, and immedi- of the radial artery using standard procedures, and blood ately frozen in liquid nitrogen and stored at ¡80°C for pressure was assessed by auscultation of the brachial artery further analyses. This whole biopsy procedure was repeated using a mercurial sphygmomanometer using standard clini- two to three times in order to obtain suYcient muscle tissue cal procedures. (»15 mg). This muscle collection approach was used as an Venous blood samples were obtained from the antecubital attempt to minimize scarring, invasiveness, and discomfort vein into a 10-mL collection tube using a standard Vacu- attributed to traditional needle muscle biopsy procedures. tainer apparatus at baseline as well as 3, 24, and 48 h post- Hayot et al. (2005) previously reported similar gene expres- exercise. Blood samples were then centrifuged at room sion via this technique as compared to the traditional Berg- temperature at 2,000 rpm for 15 min. The serum was strom technique while participants reported less pain and removed and frozen at ¡80°C for later analysis. Serum was discomfort. Additionally, the small sample size and the analyzed for serum amyloid A (SAA) using a DADE method of extraction limit the content of connective and/or Dimension RXL clinical chemistry analyzer (Dade-Dehring, adipose contained within the sample, and removal of these Inc, Newark, DE, USA). The analyzer was calibrated daily tissues is simpliWed compared to the Bergstrom technique. using liquid assay multiqual (Bio-Rad, Hercules, CA, Total cellular RNA was extracted from biopsy samples USA), and two levels of quality control with known con- with a monophasic solution of phenol and guanidine isothi- centrations were performed. In addition, serum tumor ocyanate contained within the TRI-reagent (Sigma Chemi- necrosis factor alpha TNF , IL1 , IL6, IL8, and cyclooxy- cal Co., St. Louis, MO, USA). Aliquots of total RNA were genase 2 (COX2) were assessed using commercially made then separated with agarose gel electrophoresis and moni- enzyme-linked immunoabsorbent assay (ELISA) kits tored under an ultraviolet light (Chemi-Doc XRS, Bio-Rad, (Invitrogen Corp., Carlsbad, CA, USA; EMD Chemicals Hercules, CA, USA) to verify RNA integrity and absence Inc., San Diego, CA, USA). Concentrations of each target of RNA degradation, indicated by prominent 28 and 18 s protein were determined using the colorimetric method at ribosomal RNA bands (data not shown), as well as an an optical density of 450 nm with a microplate reader OD260/OD280 ratio of approximately 2.0. The RNA samples (Wallac Victor 1420, Perkin Elmer, Boston, MA, USA). were stored at ¡80°C until later analysis (Willoughby Standard curves were generated using commercially avail- 2004). able microplate reader-compatible statistical software Two microgram of total skeletal muscle RNA were (MicroWin 2000, Microtek Laborsysteme GmbH, Overath, reverse-transcribed to synthesize cDNA using the iSscript Germany). cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Fine needle aspiration muscle biopsies were performed A reverse transcription reaction mixture [2 g of cellular at baseline and 3 h post-exercise. 3 h post-exercise was RNA, 5£ reverse transcription buVer (20 mM Tris–HCL, chosen as the post-exercise biopsy as most contraction- pH 8.3; 50 mM KCl; 2.5 mM MgCl2; 100 g of bovine induced mRNA increases occur between 3 and 12 h post- serum albumin/ml), a dNTP mixture containing 0.2 mM exercise (Bickel et al. 2005; Pilegaard et al. 2000; Yang each of dATP, dCTP, dGTP, and dTTP, 0.8 M MgCl2, et al. 2005) and due to the previously demonstrated spike in 0.5 g/ l of oligo(dT) 15 primer, and 25 / g of MMLV skeletal muscle mRNA expression of TNF , IL6, and IL8 RNAase H+ reverse transcriptase enzyme (Bio-Rad, Hercu- between 2 and 4 h post-resistance exercise (Louis et al. les, CA, USA)] was incubated at 25°C for 5 min, 42°C for 2007). Participants were instructed to refrain from exercise 30 min, heated to 85°C for 10 min, and then quick-chilled for at least 48 h prior to the initial biopsy. Muscle was on ice. The cDNA concentration was determined by using extracted from the lateral portion of the vastus lateralis an OD260 equivalent to 50 g/ l and the starting cDNA midway between the patella and iliac crest of the dominant template concentration was standardized by adjusting all leg using a TRU-CORE® 1 Automatic Reusable Biopsy samples to 200 ng prior to ampliWcation (Willoughby Instrument (Angiotech, Medical Device Technologies, 2004). INC., Gainsville, FL, USA). BrieXy, 1.0 ml of 1.0% Lido- The mRNA sequences of human skeletal muscle pub- caine HCl was injected subcutaneously prior to making a lished in the NCBI Entrez Nucleotide database (http:// small pilot hole with a 23 gauge sterile needle. The biopsy were used to construct oligonu- needle was placed into the biopsy device and the inserted cleotide PCR primers using Beacon Designer software into the previously made pilot hole (a depth of about (Bio-Rad, Hercules, CA, USA). Table 1 shows the sense 5–10 mm). A muscle sample was obtained by the activation sequence, anti-sequence, amplicon size, and NCBI acces- of a trigger button, which unloaded the spring and activated sion number for each gene assessed in the study. The sense 123
  4. 4. 466 Eur J Appl Physiol (2009) 107:463–471 Table 1 mRNA sequences of primers used in the real-time PCR procedure Primer name NCBI accession Sense sequence (5 ! 3 ) Anti-sense sequence (5 ! 3 ) Amplicon number size (bp) Beta-actin NM_001101 ATC GTG CGT GAC ATT AAG GTC ATC ACC ATT GGC AAT 102 IKKB NM_001556 AAC CAG CAT CCA GAT TGA C GCC ATC ATC CGT TCT ACC 156 Cox-2 M90100 TCC CTG AGC ATC TAC GGT TTG CAT CGC ATA CTC TGT TGT GTT CC 108 JunB NM_002229 TCT CTC TAC ACG ACT ACA AAC GAC AAT CAG GCG TTC CAG 197 TNF- NM_000594 CAG CAA GGA CAG CAG AGG AGT ATG TGA GAG GAA GAG AAC C 139 SAA1 M23698 CTC CTT GGT CCT GGG TGT C TTG TCT GAG CCG ATG TAA TTG G 126 SAA2 NM_030754 GGC AGG AGT GGC AGA GAC C TGA GAG CAG AGT GAA GAG GAA GC 83 cfos V01512 CGG CAG GAT GGA AGA GAC GCA GCA GTG AAG AGA AAC G 128 IL-1 NM_000576 TGA TGG CTT ATT ACA GTG GCA ATG GTA GTG GTG GTC GGA GAT TCG 140 IL-2 NM_000586 CAA GAA TCC CAA ACT CAC CAG CGT TGA TAT TGC TGA TTA AGT CC 179 IL-4 M13982 AGT TGA CCG TAA CAG ACA TC GAG CCG TTT CAG GAA TCG 182 IL-5 J03478 GGA TGT GGA ACC TGT AAC AAC TCA GTC TTT CTA ATG GG 192 IL-6 NM_000600 GGT CCA GTT GCC TTC TCC TGT CAA TTC GTT CTG AAG AGG 136 IL-8 NM_000584 AGA GAC AGC AGA GCA CAC GTT CTT TAG CAC TCC TTG GC 174 IL-10 NM_000572 GAA CCA AGA CCC AGA CAT C CAT TCT TCA CCT GCT CCA C 137 IL-12 AF101062 GCA GCC TCC TCC TTG TGG GGG AAC ATT CCT GGG TCT GG 94 and anti-sense primers were synthesized commercially (TAE) buVer to verify positive ampliWcation and the gel (Integrated DNA Technologies, Coralville, IA, USA). stained with ethidium bromide (present in the TAE buVer at -actin was used as an internal control standard for each 1 g/ml) and illuminated with UV transillumination reaction due to its consideration as a constitutively (Chemi-Doc XRS, Bio-Rad, Hercules, CA, USA). The rela- expressed “housekeeping gene,” and the fact that it has tive expression of mRNA was assessed by determining the been shown to be an appropriate external reference stan- ratio between the CT values of each target mRNA and the dard in real-time PCR in human skeletal muscle following CT values for -actin for each muscle sample obtained at acute exercise (Mahoney et al. 2004). each timepoint (Willoughby et al. 2007). Test–retest reli- Based on previous guidelines, 200 ng of cDNA were ability of performing this procedure of mRNA expression added to each of the 25 l PCR reactions using iQ SYBR on samples in this laboratory has demonstrated low mean Green Supermix (Bio-Rad, Hercules, CA, USA). SpeciW- coeYcients of variation and high reliability (1.6%, intra- cally, each PCR reaction contained the following mixtures: class r = 0.95). [10£ PCR buVer, 0.2 M dNTP mixture, 2.0 M of a cock- tail containing both the sense and anti-sense RNA oligonu- Statistical analyses cleotide primers, 2 mM MgCL2, 1.0 / l of hot-start iTaq DNA polymerase, SYBR Green I dye, and nuclease-free Data were analyzed initially for normality and homogeneity dH2O]. Each PCR reaction was ampliWed using real-time of variances. The ratios of the mRNA of target genes to the quantitative polymerase chaine reaction (PCR) (iCycler IQ mRNA of the housekeeping gene ( -actin) were then ana- Real-Time PCR Detection System, Bio-Rad, Hercules, CA, lyzed then analyzed using a single-factor multivariate analy- USA). The ampliWcation proWle was run for 40-cycles sis of variance (MANOVA) with repeated measures, with employing a denaturation step at 95°C for 30 s, primer univariate tests on each dependent variable conducted as fol- annealing at 58°C for 30 s, and extension at 72°C for 30 s. low-up tests to the MANOVA. Blood data were analyzed Fluorescence was measured after each cycle, resulting from using individual ANOVAs. SigniWcance was set at P < 0.05 the incorporation of SYBR green dye into the ampliWed (two-tailed) throughout. Statistical analyses were performed PCR product. The speciWcity of the PCR was demonstrated using the SPSS 16.0 for Windows software package. with absolute negative controls using separate PCR reac- tions containing no cDNA template or primers, and a single gene product was conWrmed using DNA melt curve analy- Results sis. Additionally, to assess positive ampliWcation of mRNA, aliquots (20 l) of the PCR reaction mixtures were electro- No signiWcant changes were observed for serum levels of phoresed in 1.5% agarose gels in 1£ Tris-Acetate-EDTA TNF , IL1 , IL6, IL8, COX2, or SAA for 48 h following 123
  5. 5. Eur J Appl Physiol (2009) 107:463–471 467 Table 2 Circulating serum levels of TNF- , IL-1 , IL-6, IL-8, COX2, and SAA at baseline as well as 3, 24, and 48 h post-exercise Protein Pre-exercise 3 h post-exercise 24 h post-exercise 48 h post-exercise p value TNF- (pg/mL) 10.77 § 6.98 9.24 § 8.49 12.20 § 8.16 12.90 § 8.44 0.408 IL-1 (pg/mL) 4.59 § 3.04 4.15 § 2.68 6.33 § 4.47 6.37 § 5.22 0.117 IL-6 (pg/mL) 3.93 § 4.39 2.60 § 3.19 3.28 § 3.32 3.83 § 3.82 0.585 IL-8 (pg/mL) 4.76 § 3.34 3.75 § 3.29 5.02 § 4.19 4.75 § 3.47 0.623 COX2 (ng/mL) 1.367 § 1.220 0.981 § 1.332 1.872 § 2.176 1.622 § 1.978 0.327 SAA (ng/mL) 412.25 § 283.64 421.08 § 247.58 588.46 § 290.63 532.00 § 292.74 0.084 Fig. 1 mRNA expression of TNF IL1 , IL6, and IL8 at baseline and Fig. 2 mRNA expression of IL2, IL5, IL10, and IL12 at baseline and 3 h post-exercise relative to -actin from muscle biopsies extracted 3 h post-exercise relative to -actin from muscle biopsies extracted from the vastus lateralis. *SigniWcantly diVerent from baseline mRNA from the vastus lateralis. No signiWcant changes were observed, expression at the p < 0.05 level. *9SigniWcantly diVerent from baseline although a weak trend for an increase in SAA was observed mRNA expression at the p < 0.005 level exercise; although a weak trend was observed for circulat- Discussion ing SAA (p = 0.084) (Table 2). Meanwhile, at 3 h post- exercise, elevations in skeletal muscle mRNA expression As the world’s population ages, the loss of skeletal muscle of 11 genes of interest were observed in comparison to mass and function in older individuals has become a signiW- -actin. Several of these were signiWcantly upregulated at cant societal problem. Resistance training provides a the p < 0.005 level, including IL1 (f = 27.47, p < 0.001), simple, cost-eVective, and feasible alternative method for IL6 (f = 32.39, p < 0.001), IL8 (f = 12.33, p = 0.001), preserving/improving skeletal muscle mass in older indi- COX2 (f = 9.57, p = 0.003), cfos (f = 33.63, p < 0.001) and viduals, and part of this beneWt appears to be via alterations junB (f = 28.50, p < 0.001) (Figs. 1, 3, and 4). Further, in skeletal muscle inXammatory signaling. Although sarco- mRNA of TNF (f = 7.90, p = 0.007), IKKB (f = 4.14, penia aVects both genders, women with sarcopenia have p = 0.048), suppressor of cytokine signaling 2 [(SOCS2) been reported to have lower functional capacities than com- (f = 6.48, p = 0.014)], SAA1 (f = 6.77, p = 0.012), and plimentary males (Bassey et al. 1992; Frontera et al. 1991). SAA2 (f = 4.94, p = 0.031) were signiWcantly elevated at While opposing evidence exists (Gower and Nyman 2000; the p < 0.05 level (Figs. 1, 3, and 4). No signiWcant changes TaaVe et al. 1995), it appears that the loss of endogenous were observed for expression of genes IL2 (f = 1.69, estrogens signiWcantly contributes to this functional decline p = 0.200), IL5 (f = 0.773, p = 0.384), IL10 (f = 0.067, (Douchi et al. 1998). One mechanism by which the loss of p = 0.796), or IL12 (f = 2.94, p = 0.093) (Fig. 2). estrogen has been reported to contribute to sarcopenia is 123
  6. 6. 468 Eur J Appl Physiol (2009) 107:463–471 through the increase in pro-inXammatory cytokines as estrogen withdrawal has been shown to increase these pro- teins (Greeves et al. 1999; Phillips et al. 1993). Primarily because of the eVects of menopause on bone mass, many women are prescribed hormone replacement therapy (HRT) in an attempt to minimize the detrimental eVects of estro- gen withdrawal. Yet some are now becoming worried about associated side eVects of HRT such as increased risk of breast cancer (Girasole et al. 1999; Kramer et al. 2004). For these women, resistance training provides a safe alternative that can provide beneWt to both skeletal muscle and bone. For these reasons, we chose post-menopausal women as our study population for examining the eVects of resistance exercise on inXammatory mRNA expression within muscle. As previously stated, previous investigations have sug- gested that skeletal muscle is capable of inducing changes in skeletal muscle mRNA expression independent of the circulation. Lambert and colleagues (2008) proposed that the changes in cytokine gene expression within muscle are derived from the muscle itself due to the unresponsiveness Fig. 3 mRNA expression of IKKB, cFos, and JunB at baseline and 3 h of circulating levels to exercise. Further, Akerstrom et al. post-exercise relative to -actin from muscle biopsies extracted from (2005) previously observed this response following acute the vastus lateralis. *SigniWcantly diVerent from baseline mRNA expression at the p < 0.05 level. *9SigniWcantly diVerent from baseline knee extensor exercise, as IL8 mRNA and protein expres- mRNA expression at the p < 0.005 level sion were increased within muscle while circulating levels remained unchanged. Our resistance training protocol was thus chosen in an attempt to produce mRNA alterations without inducing a systemic inXammatory response. Rippy and Marsden (2006). Choosing such a limited protocol left some risk for not observing signiWcance due to intensity insuYciency; however, only four genes (IL2, IL4, IL10, IL12) were not signiWcantly altered and none of these four have previously been reported to be altered by exercise. Meanwhile, we were successful in mirroring previous stud- ies by signiWcantly altering muscular mRNA expression without aVecting circulating protein levels of six represen- tative markers. The lack of a signiWcant change in circulat- ing levels of representative markers within the serum observed in the present study appears to indicate that the source of the mRNA increases is not due to peripheral blood mononuclear cells. While adipocytes and hepatocytes also produce a number of these mRNA, it appears highly unlikely that these cells contributed to the mRNA increases given the fact that the protocol utilized was focused on con- traction-induced changes and involved little metabolic per- turbation. These data taken together with previous evidence lead us to believe that muscle is the source of the additional mRNA and thus suggest several inXammatory mediators within skeletal muscle worthy of further research in con- junction with chronic exercise training. Additional evi- Fig. 4 mRNA expression of COX2, SOCS2, SAA1, and SAA2 at dence is necessary, however, to conWrm this hypothesis. baseline and 3 h post-exercise relative to -actin from muscle biopsies extracted from the vastus lateralis. *SigniWcantly diVerent from base- In response to acute resistance exercise, we observed line mRNA expression at the p < 0.05 level. *9SigniWcantly diVerent signiWcant (p < 0.05) changes in mRNA content of 11 from baseline mRNA expression at the p < 0.005 level genes pertinent to tissue inXammation. These data agree 123
  7. 7. Eur J Appl Physiol (2009) 107:463–471 469 with previous evidence showing transcriptional up-regula- indicate the same in response to resistance exercise, tion of several of these genes in response to resistance exer- although one study has reported otherwise (Puntschart et al. cise (Louis et al. 2007; Akerstrom et al. 2005; Hamada 1998). et al. 2005). Little or no evidence exists for the presence of As a whole, the eVects of exercise on these genes is several of these mRNA within skeletal muscle, nor for their interesting given the traditional paradigm that the direc- regulation by exercise. Most notable among these is the tional change of the mRNA for exercise-induced genes is expression of two isoforms of SAA. SAA is a generic term typically thought to occur in the same direction as the for a family of cytokine-induced acute-phase proteins pri- encoded protein with long-term adaptation being accom- marily produced by the liver. Its functions in inXammation plished by each short-term bout leading to a change in the include the capacity to induce chemotaxis, cell adhesion change in the steady state of that protein (Durham et al. and migration (Akerstrom et al. 2005) and the ability to act 2004). The inXammatory genes, however, appear to be as an extracellular matrix adhesion protein (Badolato et al. down-regulated in response to repeated, transient increases, 1995). While extra-hepatic expression has been reported similar to recent Wndings concerning ROS/antioxidant (Hershkoviz et al. 1997), no information exists concerning interactions with insulin signaling (CoVey and Hawley SAA within skeletal muscle of mammals. Here, we 2007). observed signiWcant increases in both SAA1 and SAA2 The exact mechanisms that underlie the seemingly mRNA expression following the exercise session. SAA “inverse” adaptations are yet to be delineated; however, production is known to be responsive to cytokines such as they appear to be necessary components of the muscle IL6 and TNF ; however, it appears that its upregulation repair process following mechanical stretch. While chronic may be contraction-responsive as circulating levels of these muscle inXammation induces catabolism, short-term cytokines were not altered and muscle translation of the inXammation is a necessary part of muscle repair. Recently, cytokines could not have occurred by 3 h post-exercise. Cheng and colleagues (2008) reported that damaged skele- Additionally, we observed signiWcant increases in an tal muscle expressed both mRNA and protein of interferon- isoform of suppressor of cytokine signaling (SOCS2), a (IFN ) and that IFN null mice showed signiWcantly signaling molecule for the NFkB cascade, and family mem- impaired muscle healing compared to normal mice. These bers of the activator protein 1 (AP1) transcription factor. authors concluded that endogenous IFN promotes muscle Some evidence exists for the eVects of exercise on these healing in part by stimulating the formation of new muscle mRNA; however, this evidence is far from conclusive Wbers. Additional evidence has also shown that COX2 is (Kovacevic et al. 2008). SOCS proteins are negative regu- necessary for proper muscle regeneration following muscle lators of cytokine signaling that are typically expressed at injury (Cheng et al. 2008). It seems plausible that this low levels in un-stimulated cells, but respond rapidly upon explanation applies to all of the examined inXammatory cytokine stimulation. They are also associated with growth mediators and that skeletal muscle plays an extremely hormone signaling in skeletal muscle. Given the known important role in control of inXammation and in its own stimulation of cytokines by exercise, it seems intuitive repair. While the mechanism of chronic down-regulation is that expression would increase. However, Haddad and unknown, it is possible that this adaptation is moderated by colleagues (2005) reported decreases in skeletal muscle the action of muscle-speciWc IGF1 (mIGF1), as a recent SOCS2 mRNA in response to exercise in young rats with transgenic model has shown that mIGF1 expression signiW- no changes in old rats. It is curious how the Haddad group cantly accelerates the regenerative process of injured observed no signiWcant increases in SOCS2 mRNA, while muscle and down-regulates pro-inXammatory cytokines the changes observed in this study were quite robust. It is (Bondesen et al. 2004, 2006). Further, mIGF1 enhanced likely that the either the exercise mode (isometric vs. iso- connective tissue remodeling and limited muscle Wbrosis, tonic) or relative intensity are behind this discrepancy; each positive eVects of chronic exercise. Given that mIGF1 although the possibility of a species-speciWc response does has been shown to be robustly increased by resistance train- exist. Meanwhile, NFkB and AP1 are redox-sensitive tran- ing (Pelosi et al. 2007), we propose here a model in which scription factors that regulate expression of numerous acute exercise resistance stimulates muscle inXammation inXammation-related genes. In addition, NFkB plays a early in the repair process, followed by a signal from well-documented role in muscle atrophy signaling mIGF1 to shut down transcription. (reviewed in (Haddad and Adams 2006). Acute aerobic We do acknowledge that several pertinent questions exercise has been shown to up-regulate signaling of both remain unanswered and need clariWcations within future NFkB (Kandarian and Jackman 2006) and AP1 (Ji et al. training studies. First, future investigations involving train- 2004) in skeletal muscle. Less is known concerning mRNA ing protocols should include a comparison group of expression, but our Wndings showing increases in transcrip- younger women as well as measurements of reproductive tion of inhibitor of kappa kinase , cfos, and junB appear to hormone status for comparison. Such a group was not 123
  8. 8. 470 Eur J Appl Physiol (2009) 107:463–471 included in the present study as the overall purpose was to Badolato R, Johnston JA, Wang JM et al (1995) Serum amyloid A identify molecular mediators worthy of investigation within induces calcium mobilization and chemotaxis of human mono- cytes by activating a pertussis toxin-sensitive signaling pathway. chronic studies, not to determine the overall eVectiveness of J Immunol 155:4004–4010 training on one particular group. Secondly, given the goal Bassey EJ, Fiatarone MA, O’Neill EF, Kelly M, Evans WJ, Lipsitz LA of the present study and thus the chosen protocol, we can- (1992) Leg extensor power and functional performance in very not conclude the maximal possible induction of mRNA old men and women. 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No sig- DNA binding activity of NF-kappaB in skeletal muscle nuclei. niWcant increases in serum proteins TNF , IL1 , IL6, IL8, J Appl Physiol 97:1740–1745 COX2, or SAA were observed, suggesting muscle to be the Frontera WR, Hughes VA, Lutz KJ, Evans WJ (1991) A cross- source of the mRNA for these cytokines. In addition, sectional study of muscle strength and mass in 45- to 78-yr-old men and women. J Appl Physiol 71:644–650 increases in several other inXammation-pertinent tran- Frost RA, Lang CH (2005) Skeletal muscle cytokines: regulation by scripts were also observed as a result of the resistance exer- pathogen-associated molecules and catabolic hormones. Curr cise. This is the Wrst demonstration of several of these Opin Clin Nutr Metab Care 8:255–263 parameters within skeletal muscle. 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