Acute Loading and Aging Effects on Myostatin Pathway Biomarkers in Human Skeletal Muscle After Three...

Journal of Gerontology: BIOLOGICAL SCIENCES © The Author 2011. Published by Oxford University Press on behalf of The Gerontological Society of America.Cite journal as: J Gerontol A Biol Sci Med Sci. 2011 August;66A(8):855–865 All rights reserved. For permissions, please e-mail: [email protected]:10.1093/gerona/glr091 Advance Access published on June 10, 2011

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MYOSTATIN (MSTN) serves as a negative regulator of skeletal muscle mass by maintaining satellite cells in

a quiescent state (1–3) and is involved in skeletal muscle loss via glucocorticoid-induced skeletal muscle atrophy (4,5). Animal models have demonstrated the importance of MSTN in the regulation of skeletal muscle mass as male mice overexpressing MSTN have significantly less skeletal muscle mass compared with normal counterparts (6). Con-versely, MSTN (7,8) and follistatin (FST) (9) knockout mice and mice receiving an MSTN (10,11) or FST (12) blocking antibody have been found to be significantly stron-ger and to have significantly more skeletal muscle mass compared with normal counterparts. MSTN also serves to regulate skeletal muscle mass in an endocrine manner as serum MSTN has been found to be upregulated during states of skeletal muscle wasting in HIV-infected men (13).

Due to the importance of MSTN in the regulation of skel-etal muscle mass, the possibility exists that MSTN pathway signaling may be involved in the age-related loss of skeletal muscle mass, but few investigations have directly examined

the effects of age on the messenger RNA (mRNA), protein, or activity markers of the MSTN pathway. In 2002, Roth and colleagues (14) examined the effects of a 9-week unilat-eral heavy resistance training program on MSTN mRNA expression in younger (20–30 years; four men and four women) and older (65–75 years; three men and four women) adults. MSTN mRNA expression was found to be signifi-cantly downregulated following chronic resistance exercise with no significant age or gender differences. In 2006, Raue and colleagues (15) examined the effects of age and an acute bout of resistance exercise on MSTN mRNA expres-sion in younger (n = 8; 18–30 years) and older (n = 6; 80–89 years) women. Muscle biopsies were obtained prior to and 4 hours following resistance exercise. Baseline MSTN mRNA expression was significantly greater in older com-pared with younger women. Following training, there was a significant downregulation of MSTN mRNA in younger and older women with no significant difference in MSTN mRNA between groups. Finally, Jensky and colleagues (16) examined the effects of an acute bout of resistance exercise

Acute Loading and Aging Effects on Myostatin Pathway Biomarkers in Human Skeletal Muscle After Three

Sequential Bouts of Resistance Exercise

Vincent J. Dalbo,1 Michael D. Roberts,2 Kyle L. Sunderland,3 Chris N. Poole,3 Jeff R. Stout,3 Travis W. Beck,3 Mike Bemben,3 and Chad M. Kerksick3

1School of Medicine and Applied Sciences, Faculty of Sciences, Engineering, and Health, Institute for Health and Social Science Research, Central Queensland University, Rockhampton, Australia.

2Department of Biomedical Science, University of Missouri-Columbia. 3Department of Health and Exercise Science, University of Oklahoma, Norman.

Address correspondence to Chad M. Kerksick, PhD, Department of Health and Exercise Science, University of Oklahoma, Norman, OK 73019-6081. Email: [email protected]

To determine the influence of age and resistance exercise on myostatin pathway–related genes, younger (n = 10; 28 ± 5 years) and older (n = 10; 68 ± 6 years) men underwent four testing conditions (T1–T4). A baseline (T1) muscle sample was obtained, whereas the second and third biopsies were obtained 48 hours following the first and second training ses-sions (T2, T3), and a final biopsy was taken 24 hours following T3. The training sessions consisted of 3 sets of 10 repe-titions (80% of one repetition maximum) on leg press, hack squat, and leg extension exercises. Follistatin (FST) messenger RNA was greater in older compared with younger men at T1 and T2 (p < .05). Follistatin-like 3 (FSTL3) messenger RNA was greater in older compared with younger men at T1 and T4 (p < .05). In older men, there was a sig-nificant decrease in myostatin (MSTN) messenger RNA at T4 (p < .05). Older men contained less active (Ser-425 phos-phorylated) SMAD3 (p-SMAD3) protein than younger men at T3 and T4 (p < .05).Although it is well known that younger individuals possess a greater hypertrophic potential to resistance exercise, it appears that older individuals may paradoxically possess a more favorable resistance exercise response regarding myostatin pathway–related genes and a protein marker of pathway activity. Future research is warranted to examine the physiological significance of this age-dependent mechanism.

Key Words: Muscle—Molecular—Exercise—PCR—Resistance Training.

Received November 24, 2010; Accepted April 19, 2011

Decision Editor: Rafael de Cabo, PhD

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consisting of single leg eccentric knee extensions on select markers of MSTN pathway–related genes between younger (n = 10; 28 ± 5 years) and older (n = 10; 68 ± 6 years) men at baseline and 24 hours following exercise. At baseline, there was a trend for MSTN mRNA to be greater in older compared with younger men (p = .06) and older men had significantly greater levels of follistatin (FST), whereas no differences between groups were present for human small glutamine-rich tetratricopeptide repeat-containing protein (SGTA). Exercise had no impact on the expression of MSTN, FST, or SGTA 24 hours postexercise.

Factors influencing the expression of MSTN pathway–related genes are still poorly understood; however, the basic control points of the MSTN pathway have been outlined in detail (17,18). In short, MSTN is secreted in an inactive form as it is bound to a latency-associated peptide. An ac-tive form of MSTN is produced following proteolytic pro-cessing producing the mature form of MSTN (17). Mature MSTN is then able to bind to activin IIB receptors (ACVR2B) that creates a receptor heterodimer with activin receptor–like kinase (ALK) monomers and initiates a sig-naling cascade resulting in the phosphorylation (activation) of SMAD2/3 that aggregate with SMAD4. This active SMAD heterotrimer then translocates the nucleus (19) and interacts with SMAD binding partners in the DNA to regu-late gene transcription (20). Likewise, recent data support that SMAD complexes inhibit Akt-mTORC1 signaling and thus act to blunt muscle hypertrophy in mature myofibers by reducing muscle protein synthesis while concomitantly increasing the expression of atrogenes (21–24). Therefore, a paradigm exists illustrating the pleiotropic role of MSTN in promoting a catabolic milieu within skeletal muscle.

Interestingly, MSTN signaling is subjected to numerous regulatory steps. For instance, SGTA (25) and titin cap (TCAP) (26) prevent MSTN secretion, whereas follistatin-related gene (FSTL3) (27) and FST (28) bind to active MSTN and prevent binding to ACVR2B. The negative reg-ulators of MSTN are SMAD7 (19) and SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) (29). SMAD7 pre-vents the phosphorylation of SMAD2/3 (30,31), whereas SMURF1 is an E3 ubiquitin ligase that tags SMAD2/3 for degradation (17). Thus, not only is MSTN mRNA and pro-tein regulation of interest when examining the effects of exercise, but favorable alterations in these negative regula-tors of MSTN signaling may facilitate positive exercise ad-aptations as well.

Due to the importance of MSTN in the regulation of skel-etal muscle mass, it is important to understand the effects of age on the primary control mechanisms of MSTN pathway signaling. Therefore, the purpose of this investigation was twofold: (a) to examine if baseline differences in the expres-sion of MSTN pathway–related genes exist between youn-ger (18–35 years) and older (65–80 years) men and (b) to examine the effects of three repeated bouts of resistance ex-ercise on the expression of MSTN pathway–related genes

and a protein marker in MSTN pathway activity (Ser-425 phosphorylated SMAD3). Considering previously pub-lished work, it is hypothesized that the expression of MSTN pathway–related genes would be upregulated in older com-pared with younger men, but following acute resistance ex-ercise, the expression of MSTN pathway–related markers would be downregulated to a greater extent in older com-pared with younger men.

Methods

ParticipantsYounger (n = 10; 18–25 years) and older (n = 10; 60–75

years) men were recruited to participate in the investiga-tion. Eligibility inclusion criteria included the following: (a) Participants were apparently healthy and had not par-ticipated in a structured resistance exercise regimen consis-tently (ie, at least one time per week) 1 year prior to participation in this investigation. (b) Participants did not consume an ergogenic or nutritional supplement for at least 3 months prior to the start of the investigation. (c) Partici-pants did not have any absolute or relative contraindica-tions for exercise testing as outlined by the American College of Sports Medicine. This study was approved by the University of Oklahoma Health Sciences Center Institu-tional Review Board for Human Subjects and all partici-pants completed a written informed consent form, a prestudy health and exercise status questionnaire, received clearance from their personal physician to participate, and were screened for approval by a study physician.

Research DesignAn overview of the research design is presented in Figure 1.

All participants visited the laboratory on seven occasions over a 12-day period (T1 = Day 0/baseline, FAM1 = Day 2, FAM2 = Day 4, workout 1 = Day 7, T2 = Day 9, T3 = Day 11, T4 = Day 12). Prior to T1, participants were verbally screened for inclusion criteria before signing informed con-sent documents and receiving a physical examination by the study physician. During this visit (T1), participants reported to the laboratory between 6 am and 9 pm following an over-night fast, and a percutaneous muscle biopsy was obtained from the lateral aspect (ie, vastus lateralis) of their quadri-ceps region. Following the biopsy, participants warmed-up for 10 minutes on a cycle ergometer (ie, 60 rpm at a self-selected intensity) and performed a one repetition maxi-mum (RM) strength test for the bilateral leg press, hack squat, and leg extension using guidelines established by the National Strength and Conditioning Association (NSCA) (32). In accordance with NSCA standards, each one RM was determined within five lifts, and participants received 3-minute rest intervals between lifts. Two days following T1, participants returned to the laboratory for FAM1 and FAM2 whereby at each visit participants warmed-up for

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5 minutes on a cycle ergometer each time and performed two sets of 10 repetitions at a lifting intensity of 60% one RM for FAM1 and 70% one RM for FAM2 for each lower-body exercise while resting for 2–3 minutes between sets. Employment of familiarization sessions were performed: (a) To ensure that participants were gradually introduced to the training protocol employed during the training sessions, (b) to minimize the delayed onset of muscle soreness, (c) to help ensure each participant was entering the training proto-col with a similar baseline exposure to resistance exercise, and (d) to help establish proper lifting form with submaxia-mal loads prior to intiation of the training protocol.

Two days following FAM2, participants reported to the laboratory for the first of the three identical training ses-sions between 6 am and 9 pm following an overnight fast. For each session, participants warmed-up for 10 minutes on a cycle ergometer and performed three sets of 10 repetitions at a lifting intensity of 80% of their one RM for each lower body exercise with 2- to 3-min rest between sets. Consecu-tive biopsy sampling was completed starting 2 days (48 hours) following the first training session (prior to T2), whereas subsequent muscle samples were collected 48 hours after the second workout (prior to T3) and 24 hours after the third workout (T4).

Anthropometric DataParticipants changed into minimal clothing and were

barefoot for the measurement of body mass and height on a calibrated scale and standiometer (Detecto, Webb City, MO). For descriptive purposes, body composition was as-sessed utilizing a three site skinfold equation according to the recommendations of Jackson and Pollock (33).

Percutaneous Muscle BiopsiesUsing the schedule outlined in Figure 1, all biopsies were

collected halfway between the hip and patella at a depth between and 5 cm. To minimize repeated biopsy-induced muscle damage (34), biopsies were collected from alternating

legs during subsequent testing sessions in such a way that two muscle collections were obtained from each leg. To minimize the effect of muscle fiber distribution, the second biopsy on each leg was collected immediately distal to the initial biopsy insertion. The multiple muscle collection pro-cedures were standardized by using anatomical palpation, visual identification of the previous biopsy, and depth mark-ings on the needle. Immediately following the biopsy, the muscle tissue was removed from the Bergstrom needle, placed into a cryogenic storage tube, and frozen in liquid nitrogen (ie, flash frozen). Samples were then transferred into a −80ºC freezer for later analyses.

Muscle (Total RNA) DeterminationEach section of muscle was weighed (~30 mg) and ho-

mogenized using 500 mL of Tri reagent with a polypropyl-ene pestle and microcentrifuge tube using manufacturer’s instructions (Sigma Chemical, St Louis, MO). The resultant RNA pellet was dissolved in 50 mL of RNase-free water and stored at −80ºC until later analyses.

Total RNA concentrations for each sample were deter-mined using a high-sensitivity RNA analysis kit with the Experion Automated Electrophoresis platform (Bio-Rad Laboratories, Hercules, CA). This procedure has been pre-viously shown in our laboratory to yield undegraded RNA, free of DNA and proteins as indicated by prominent 28S and 18S ribosomal RNA bands (Figure 2). The preparation of reagents and the RNA ladder were performed according to the manufacturer’s instructions. Furthermore, all RNA samples and the RNA ladder were thawed on ice during the assay to preserve mRNA integrity, and all assays were per-formed in duplicate.

Real-Time Polymerase Chain Reaction to Detect Postexercise Expression of Genes of Interest

Following total RNA concentration determination, 200 ng of total RNA was reverse transcribed to synthesize cDNA. For each sample, a reverse transcription reaction

Figure 1. Study design overview: All workouts (dark gray and black bars) consisted of a 5-minute warm-up on a cycle ergometer followed by three sets of 10 repetitions for the bilateral leg press, hack squat, and leg extension exercises (intensities denoted) with 2–3 minutes of rest between sets and exercises.

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mixture (40 mL total) was prepared containing: (a) 200 ng of total cellular RNA diluted to 30 mL with RNase-free water, (b) 8 mL 5× reverse transcription buffer, a dNTP mixture containing dATP, dCTP, dGTP, and dTTP, MgCl2, RNase inhibitor, an oligo(dT)15 primer, and (c) 2 mL of MMLV reverse transcriptase enzyme (Bio-Rad Laboratories) were incubated at 42°C for 40 minutes, heated to 85°C for 5 minutes, and then quick-chilled on ice yielding the cDNA product. Then, 80 mL of RNase-free water was added to bring the cDNA solutions up to 120 mL, and cDNA solutions were subsequently frozen at −80ºC until semiquantitative real-time PCR was performed. Forward and reverse oligonucle-otide primer pairs were constructed using Beacon Designer software (Bio-Rad Laboratories) and commercially synthe-sized (Integrated DNA Technologies, Coralville, IA) (Table 1). Melt curves were periodically performed to ensure that a prominent polymerase chain reaction (PCR) product was being produced by each primer in the absence of primer dimmers (data not shown). Beta-2 microglobulin (B2M) and 28S were used as an internal reference for detecting the relative change in the quantity of target mRNA as B2M and 28S are constitutively expressed prior to and following re-

sistance exercise (35). Two microliters of cDNA were added to each of the separate PCR reactions for MSTN, ACVR2B, SGTA, TCAP, FST, FSTL3, SMURF1, B2M, and 28S. Each PCR reaction contained the following mixtures: 12.5 mL of SYBR Green Supermix (Bio-Rad, Hercules, CA), 1.5 mL of sense and antisense gene-specific primers, and 7.5 mL of nuclease-free dH2O. The PCR reactions were amplified with a thermal cycler (Bio-Rad Laboratories). The amplifi-cation sequence involved an initial 10-minute cycle at 95°C to activate the Taq polymerase followed by a 40-cycle pe-riod with a denaturation step at 95°C for 15 seconds and a primer annealing/extension step at 55°C for 45 seconds. It should be noted that all assays were performed in duplicate, and gene expression data was expressed using 2−∆CT method where ∆CT = (gene of interest − avg B2M and 28S). The coefficient of variation for values between duplicates were less than 5% for all genes of inter-est and housekeeping genes.

Immunoblotting ProceduresEach muscle section was weighed (~25 mg) and homog-

enized on ice with 300 mL of cell lysis homogenizing buffer

Figure 2. RNA automated electrophoresis electropherogram (N = 1). Past data from our laboratory demonstrating that our isolation techniques yields high-quality RNA as evidenced by the presence of 18S and 28S rRNA peaks and an RNA Quality Indicator (RQI) score of 7.0 as well as the lack of high molecular weight peaks past the 28S peak (indicative of DNA contamination).

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(Sigma). Prior to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), protein concentrations of cell lysis homogenates were determined spectrophotometri-cally using Bradford reagent (Bio-Rad Laboratories) against a standard curve using bovine serum albumin as the refer-ence protein. Following determination of protein concentra-tion for each sample, a total of 30 mg of protein was diluted in Laemmli sample loading buffer spiked with 5% 2-mer-captoethanol, heated at 95°C for 5 minutes, and loaded on handmade 12% polyacrylamide gels (Bio-Rad) for SDS-PAGE. Each run lasted approximately 1 hour at 150 V until the bromophenol blue tracer dye from the Laemmli buffer ran approximately 1 cm to the end of the gel. Following SDS-PAGE runs, proteins were eluted from the gels to ni-trocellulose membranes using a “wet transfer” in an elec-trotransfer blotting apparatus (Bio-Rad Laboratories) with Towbin electrotransfer buffer. Electrotransfers lasted 1 hour in duration at 100 V and 380–500 mA. Following electrotransfers, loading efficiencies were determined by staining nitrocellulose membranes with Ponceau S stain (Sigma). Membranes were destained in Tris-buffered saline spiked with 1% Tween-20 followed by immunoprobing ex-periments.

Immunoblotting was then performed using mouse mono-clonal antibodies against Ser-425 phosphorylated SMAD3 (p-SMAD3) (Cell Signaling, Danvers, MA). Nonspecific binding sites on the nitrocellulose membranes were blocked using Tris-buffered saline spiked with 1% Tween-20 (TBST) spiked with 5% nonfat blocking agent (NFBA) for 50 minutes at room temperature. Membranes were then in-cubated with the primary antibody solution (fold-diluted antibody in 5% NFBA in TBST) for 50 minutes at room temperature. The primary antibody solution was then de-canted, membranes were washed two times, 10 minutes per wash, with TBST, and membranes were incubated with a horseradish peroxidase–conjugated goat antimouse anti-body solution (10,000-fold dilution in TBST; Cell Signaling)

Table 1. Primer Sequences Used to Probe Genes of Interest Expressed in Skeletal Muscle Samples

Gene Primer Sequence (forward and reverse) GenBank Accession No.

MSTN 5′- GAC CAG GAG AAG ATG GGC TGA ATC CGT T-3′ NM_0052595′- CTC ATC ACA GTC AAG ACC AAA ATC CCT T-3′

ACVR2B 5′- GCC TTG CCA TCA GAT TGT G-3′ NM_0011065′- GCC ATC AGA ACC AGA TAT ACC -3′

SGTA 5′- TTG GGG TGA CGG TAG AAG AC -3′ NP_AJ2238285′- GTT GAG CTC GAT GGC TTT TC -3′

TCAP 5′- GAG ACT CCA TCG GTA CTA -3′ NM_0036735′- CCT TCC TAG ACT GTG ACA -3′

FST 5′-TGC CAC CTG AGA AAG GCT AC -3′ NM_0134095′- ACA GAC AGG CTC ATC CGA CT -3′

FSTL3 5′- TGC TCA GAA TCG CCT ACC-3′ NM_0058605′- CTC CGT GTT GTC CTC TCC -3′

SMURF1 5′- TGA AGG AAC GGT GTA TGA AG- 3′ NM_0204295′- CGG TGC TAT CTG TGT AAG G -3′

Notes: ACVR2B = activin IIB; MSTN = myostatin; FST = follistatin; FSTL3 = follistatin-like 3; SGTA = human small glutamine-rich tetratricopeptide repeat-containing protein; SMURF1 = SMAD-specific E3 ubiquitin protein ligase 1; TCAP = titin cap.

Table 2. Normality Distribution Tests for All Dependent Variables

Variable

p Value

T1 T2 T3 T4

mRNAMSTN* .136 .038 .039 .170ACVR2B* < .001 .024 < .001 .007SGTA* .150 .010 .039 .008FST* < .001 .003 < .001 < .001FSTL3* < .001 < .001 .363 .002SMURF1* .156 .111 .018 .029Proteinp-SMAD3* .032 .002 .016 .002

Notes: Data represent the Shapiro–Wilk statistic p values for each depen-dent variable. ACVR2B = activin IIB; MSTN = myostatin; FST = follistatin; FSTL3 = follistatin-like 3; SGTA = human small glutamine-rich tetratricopep-tide repeat-containing protein; SMURF1 = SMAD specific E3 ubiquitin protein ligase 1; p-SMAD3 = phosphorylated SMAD3.

*Data at one or multiple time points from T1 to T4 was not normally distrib-uted (p < .05).

for 45 minutes. Finally, the secondary antibody solution was decanted, membranes were washed two times, 10 min-utes per wash, with TBST, and membranes were incubated with an enhanced chemiluminescent reagent (Bio-Rad Lab-oratories) for 5 minutes. Following this step, membranes were placed in the gel documentation system (Bio-Rad Laboratories) and immunoprobed band densities were de-termined using densitometric software.

Statistical AnalysesIndependent samples t-tests were used to examine possi-

ble between group differences for normally distributed data (age, height, weight, and percent body fat). The Shapiro–Wilk statistic was performed for the mRNA and protein data at each time point to assess the data for normality (p < .05). Since all mRNA and protein data were nonnormally distributed (Table 2) nonparametric statistics were used for further analyses, which is consistent with previous literature

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[36,37]. The Mann–Whitney U statistic was used to deter-mine between group differences at each time point. The Friedman test was used to detect changes in data among all conditions over time and a Wilcoxin signed rank test with a Bonferroni correction was performed as a post hoc measure to determine which time points were significantly different within each age group. Statistical significance was deter-mined at p < .05 for all tests. As a result, when needed, sta-tistical significance was determined at p < .016 following the Bonferroni correction. All analyses were performed us-ing SPSS 17.0 (SPSS, Chicago, IL) and all data are pre-sented as means ± standard error.

Raue and colleagues (15) reported a significant differ-ence in the basal mRNA expression of MSTN between younger (n = 8; 23 ± 2 years; 1.90 ± 0.612 AU) and older women (n = 6; 85 ± 1 year; 2.70 ± 0.566 AU). As a result, the sample size calculations for a between-subjects study design yielded a minimum sample size of n = 8 for each group to attain a statistical power of 0.80. To minimize the probability of making a type II error, we recruited 10 sub-jects into both the younger and older age groups.

Results

Participant Demographics, Lifting, and Dietary Analyses

Participant demographics, strength, volume load and di-etary analyses are presented in Table 3. When compared with older men, younger men were significantly stronger (p < .05) for all exercises (leg press, hack squat, and leg exten-sion) and completed a greater volume load during the train-ing sessions (p < .05). Results from the 3-day food diaries indicated that relative caloric, protein, carbohydrate, and fat consumption were significantly greater (p < .05) in younger compared with older men.

Table 3. Participant Demographics, Strength, Training Volume, and Dietary Analyses

Variable Younger Men Older Men Significance

Demographics Age (y) 21.0 ± 0.5 66.4 ± 1.6 < .001* Height (cm) 178.4 ± 2.2 180.9 ± 2.2 .421 Weight (kg) 82.3 ± 4.2 94.2 ± 3.7 .046* Body fat (%) 15.4 ± 2.9 27.4 ± 1.8 < .001*Strength and training volume Leg press (kg) 271.1 ± 14.6 182.7 ± 14.9 < .001* Hack squat (kg) 170.0 ± 10.9 105.8 ± 10.4 .001* Leg extension (kg) 64.8 ± 4.0 46.7 ± 2.8 .002* Volume load (kg) 36,392 ± 1,894 23,724 ± 1,639 < .001*Nutrition Energy (kcal/kg/d) 33.4 ± 5.1 19.5 ± 1.9 .038* Protein (g/kg/d) 1.6 ± 0.2 0.8 ± 0.1 .030* Carbohydrate (g/kg/d) 4.1 ± 0.6 2.3 ± 0.3 .017* Fat (g/kg/d) 1.2 ± 0.2 0.6 ± 0.1 .006*

Note: *Significant between group difference, p < .05

Basal and Postexercise mRNA Expression of MSTN-Related Genes

MSTN mRNA expression values (means ± SE) are pre-sented in Figure 3A. No between-group differences existed for MSTN at any measured time point (T1: p = .89, T2: p = .38, T3: p = .94, T4: p = .26). There was a main effect for time (p < .001) and a significant downregulation of MSTN mRNA occurred in older men at T4 (p = .013) in compari-son with baseline expression. ACVR2B mRNA expression values (means ± SE) are presented in Figure 3B. No be-tween-group differences existed for ACVR2B mRNA at each time point (T1: p = .08, T2: p = .43, T3: p = .29, T4: p = .20) and no main effect for time was present (p = .32).

SGTA mRNA expression values (means ± SE) are pre-sented in Figure 4A. No between-group differences existed for SGTA mRNA at each time point (T1: p = .23, T2: p = .88, T3: p = .76, T4: p = .76). There was a main effect for time (p = .01); however, no significant within group changes occurred in SGTA mRNA expression values in younger or older men (p > .016) compared with the respective baseline values. FSTL3 mRNA expression values (means ± SE) are presented in Figure 4B. The mRNA expression of FSTL3 was significantly greater in older compared with younger men at T1 (p = .04) and T4 (p < .01). No main effect for time for FSTL3 mRNA expression was present (p = .42). FST mRNA expression values (means ± SE) are presented in Figure 4C. The expression of FST mRNA was signifi-cantly greater in older compared with younger men at T1 (p < .02) and T2 (p < .01). No time effect for FST mRNA was present (p = .44).

SMURF1 mRNA expression values (means ± SE) are presented in Figure 5. No between-group differences ex-isted for SMURF1 mRNA at each time point (T1: p = .08, T2: p = .82, T3: p = .50, T4: p = .68) and no time effect was present (p = .38).

TCAP mRNA was measured in both age groups at all time points. Unfortunately, we were unable to obtain a mea-surable result under the measurement conditions and avail-able supply of RNA. Consequently, we are unable to present any data for this target, likely due to appreciably low levels of intramuscular expression of this gene.

Basal and Postexercise Protein Levels of p-SMAD3Intramuscular p-SMAD3 protein levels expressed in arbi-

trary density units (ADUs) (means ± SE) are presented in Figure 6. The expression of p-SMAD3 was significantly lower in older compared with younger men at T3 (p < .01) and T4 (p < .01). No time effect was present (p = .55).

DiscussionThe purpose of the current investigation was twofold:

(a) to examine if baseline differences in the expression of MSTN pathway–related genes are present between younger and older men and (b) to examine the individual and

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summated effects of three sequential resistance exercise bouts on the expression of MSTN pathway–related genes in younger and older men. MSTN mRNA levels were de-

creased in older men at T4, 24 hours following repeated bouts of resistance exercise. These findings support the pre-vious findings of Kim and colleagues (38) who reported a

Figure 3. (A) Myostatin (MSTN) mRNA expression values expressed as means ± SE. † Significant within group difference from baseline, p < .016 (10 young and 10 old). (B) Activin IIB (ACVR2B) mRNA expression values are expressed as means ± SE. There were no significant between or within group differences, p > .05 (10 young and 10 old).

Figure 4. (A) Human small glutamine-rich tetratricopeptide repeat-containing protein (SGTA) mRNA expression values expressed as means ± SE. There were no significant between or within group differences, p > .05 (10 young and 10 old). (B) Follistatin-like 3 (FSTL3) mRNA expression values expressed as means ± SE. *Significant between group difference, p < .05 (10 young and 10 old). (C) Follistatin (FST) mRNA expression values expressed as means ± SE. *Significant between group difference, p < .05 (10 young and 10 old).

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significant load-mediated reduction in MSTN mRNA 24 hours following loading in both young and older adults. In-terestingly, baseline expression of FST and FSTL3 was greater in older men compared with younger men (Figure 4). Given the suggested inhibitory function associated with FST on MSTN activity, this outcome would suggest a more favorable disposition for older men regarding the negative control of MSTN on skeletal muscle hypertrophy (ie, ↑FST or FSTL3→↓ biologically active MSTN →↑ hypertrophy). Additional decreases in p-SMAD3 protein expression at T3 and T4 (p < .05) in older compared with younger men pro-vide additional evidence of an age-dependent pattern of ac-tivation within the MSTN signal transduction pathway secondary to external load. Whether this apparent favorable response in aged muscle occurs as part of a compensatory

Figure 5. SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) mRNA expression values expressed as means ± SE. There were no significant between or within group differences, p > .05 (10 young and 10 old).

Figure 6. Phosphorylated SMAD3 (p-SMAD3) protein levels expressed as means ± SE. *Significant between group difference, p < .05 (10 young and 10 old).

mechanism to preserve skeletal muscle mass with age re-mains to be fully determined.

Nutritional ConsumptionThere were significant differences in the relative energy

and protein consumption between younger and older men. This finding was not surprising as nationwide studies have suggested inadequate energy intake is common in healthy older adults (39). In this regard, protein intake has also been suggested to decrease with age (40). Work by Hulmi and colleagues in younger (25.7 ± 0.2 years) (41) and older (61.8 ± 0.5 years) (42) men has found protein consumption in the form of 15 g of whey protein isolate consumed im-mediately before and after a bout of resistance exercise to have no significant effect on the mRNA expression of MSTN (41,42), ACVR2B (41,42), and FSTL3 (41), 1 and 48 hours postexercise. Based on the findings from Hulmi and colleagues (41,42) and the finding that the older men in the current investigation were not protein deficient, it is un-likely that the difference in relative protein consumption influenced the results. However, to support this hypothesis, future investigations should examine rates of muscle pro-tein synthesis between younger and older adults under isoenergetic conditions in which macronutrient consump-tion is consistent across groups.

mRNA Expression of MSTN and ACVR2BIn concert with a number of previous investigations

(14,16,38,43), data from the present study revealed no sig-nificant age-related differences in MSTN mRNA expres-sion at baseline. Although Raue and colleagues (15) reported MSTN mRNA to be significantly greater in older compared with younger women, the advanced age of these partici-pants (83 ± 1 year) may likely have been a primary factor as several studies using similar-aged individuals as the current investigation reported no difference in baseline MSTN mRNA expression (14,16,38,43). Given that skeletal mus-cle degradation accelerates with aging (44), it is possible MSTN mRNA expression increased in these advanced age individuals.

In an acute manner and after a single resistance exercise bout, MSTN mRNA has been shown to be significantly downregulated after 1 (41,42,45), 2 (45), 4 (15,45), 12 (45), 24 (38,45,46), and 48 hours (15,42,46), whereas other in-vestigations have reported no change after 4 (47) and 48 hours (41,47) postexercise. Therefore, current literature suggests that MSTN mRNA expression experiences a de-creased regulation up to 24 hours with additional evidence suggesting this event may persist until 48 hours postexer-cise. In the current investigation (Figure 3A), older men ex-perienced a significant downregulation in MSTN 24 hours after the third or final exercise bout (T4; p = .013). These results support the finding that the regulation of MSTN mRNA decreases in response to an acute loading stimulus,

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but the temporal pattern between younger and older men may differ, especially as repeated exercise bouts are completed.

Currently, data are lacking which outlines the effects of age on ACVR2B mRNA expression. Findings from the present investigation indicate that expression of this gene may take on similar patterns between younger and older men (Figure 3B). Unlike MSTN, literature on ACVR2B re-veals little change in its mRNA expression patterns 15 min-utes (48), 1 hour (41,42) and 48 hours (42,47) following resistance exercise. However, one study did report a signifi-cant reduction in ACVR2B expression 1 hour following resistance exercise in older men (47). In the current investi-gation, ACVR2B mRNA expression was not different be-tween ages at any of the measured time points nor was the mRNA expression of ACVR2B influenced by repeated loading in younger or older men (Figure 3).

mRNA Expression of Active/Mature Myostatin-Binding Proteins FSTL3 and FST

The basal mRNA expression of FSTL3 (p = .038) and FST (p = .023) were significantly greater in older compared with younger men. Older men also had higher mRNA ex-pression of FSTL3 at T4 (p = .005) and had higher expres-sion of FST at T2 (p = .008) compared with younger men (Figures 4B and 4C). There was no exercise effect with re-gard to the mRNA expression of FSTL3 or FST in younger or older men. Given that no studies have examined the ef-fects of age alone on skeletal muscle FSTL3 mRNA expres-sion, the finding that older men express significantly more FSTL3 prior to exercise than younger men is novel and may suggest that compensatory MSTN inhibition pathways are increased with skeletal muscle aging. In concert with the present findings regarding changes in these genes of interest after loading exercise, Hulmi and colleagues (47] found FSTL3 mRNA expression to be unaffected 1 and 48 hours following an acute bout as well as following 21 weeks of resistance training in older men (63.2 ± 6.3 years). These findings confirmed earlier data from this research group il-lustrating the lack of change seen for FSTL3 mRNA 1 and 48 hours postexercise (47). Additionally, present data for FST mRNA expression is consistent with the work of Jensky and colleagues (16] who reported baseline differences in the mRNA expression of FST between younger (28 ± five years) and older (68 ± six years) men. Furthermore, the lack of any loading effect as seen in the present study for FST mRNA expression also confirms lack of changes seen in this gene target 24 hours following a single bout of resis-tance exercise (16).

mRNA Expression of SMURF1There were no significant between or within group differ-

ences for the mRNA expression of SMURF1 at any time point. This finding appears to be one of the first data re-ported for age and loading effects of SMURF1 mRNA

expression levels. In yet to be published data from our labo-ratory, we have found an up-regulation of SMURF1 mRNA expression up to 6 hours following a single resistance train-ing session. Nonetheless, SMURF1 mRNA expression seems to return to baseline levels 24–48 hours following resistance exercise. In this regard, future research should examine the physiological relevance of SMURF1 and its protein product with regard to blunting MSTN signaling in human skeletal muscle through resistance training.

Protein Levels of p-SMAD3Older men had significantly less protein for p-SMAD3

following T3 (p = .012) and T4 (p = .009) when compared with younger men; a finding yet to be reported in human exercise and aging literature (Figure 6). Decreased levels of p-SMAD3 at T3 and T4 in older compared with younger men may possibly be due to decreased levels of MSTN. Al-though immunoblotting for MSTN protein would have aided in this suggestion, the lack of adequate sample com-bined with technical issues related to the primary antibody we selected precluded this measurement. Regardless, de-creased MSTN mRNA (Figure 3A) supports these hypoth-eses at the transcriptional level. Decreased MSTN would subsequently reduce the available targets for SMAD2/3 co-alescing and eventual oligomerization with SMAD4 (18). Although more measurements are needed to confirm this suggestion, studies have reported that an increase in p-SMAD3 increases expression of genes that inhibit satellite cell activity (ie, proliferation and differentiation) (17), a re-sponse that could potentially influence hypertrophic poten-tial (49). As mentioned previously, an increase in MSTN signaling has also been linked to reducing mTORC1 signal-ing and increasing the mRNA expression of atrogenes (21–24). In this regard, future research should examine the importance of the current findings and determine if this ex-ercise-induced mechanism (ie, determining mTORC1 sig-naling and satellite cell activity in transgenic SMAD3-KO mice during synergist ablation) is integral for the pheno-typic adaptations experienced in older populations in re-sponse to resistance exercise.

It has previously been reported that SMADs serve a vital role in the regulation of MSTN. For instance, MSTN exerts its effects via a downstream signaling cascade in which SMAD2/3 aggregate with SMAD4, translocate the nucleus (19), and interact with SMAD binding partners on the DNA to regulate gene transcription (20). Conversely, SMAD7 has been found to reduce MSTN-induced transcription with the effects of SMAD7 being enhanced by the ccoexpression of SMURF1. SMAD7 is also influenced by MSTN by way of the interaction between SMAD2/3, SMAD4, and the SMAD binding element in the SMAD7 promoter, suggesting the MSTN signal transduction pathway is regulated by SMAD7 through a negative feedback mechanism (19). Thus, despite the limited evidence in our study suggesting that resistance

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DALBO ET AL.864

exercise favorably affects the expression of MSTN path-way–related genes, additional studies elucidating this mechanism are warranted to better understand the interac-tion between resistance exercise, aging, and myostatin sig-naling.

mRNA Expression of Early-Stage Myostatin-Binding Proteins SGTA and TCAP

In the current investigation, there were no significant between- or within-group differences for the mRNA expression of SGTA that is consistent with the results of Jensky and colleagues (16) who reported no age or exercise effect 24 hours following a bout of resistance exercise. The importance of TCAP in the MSTN pathway was demon-strated by Nicholas and colleagues who reported TCAP to have a high protein–protein interaction with MSTN using surface plasmon resonance kinetics (26). Furthermore, the overexpression of TCAP was found to decrease the secretion and downstream signaling of MSTN in C2C12 myoblasts by retaining it within the cells (26). Therefore, we sought to determine the mRNA expression of TCAP in response to aging and exercise but were unable to obtain a measurable result. Given the nonphysiological constraints of overex-pression cell culture models, it may be possible that the mRNA expression of TCAP does not occur to an appreciable level in human skeletal muscle.

ConclusionsAge-related baseline and resistance exercise responses of

the MSTN pathway occur within the skeletal muscle. In this regard, future studies should seek to qualify how aging or what physiological triggers exists which lead to this adap-tive response. Strengths of the present study lie in it being one of the more complete investigations to measure the ef-fects of aging and exercise on numerous biomarkers of MSTN signaling. In this regard, it appears MSTN mRNA expression is reduced in older muscle upon loading, a re-sponse that points to the favorable effects of resistance training. Baseline mRNA levels of FST and FSTL3 are higher in older muscle, a response suggestive of an age-specific adaptive response to minimize the influence of MSTN signaling and to sustain muscle mass. Additionally, age-specific adaptive responses lie with the significant re-duction seen for p-SMAD3, but only after repeated bouts of loading. Taken as a whole, one could surmise that as aging ensues the adaptive response by muscle works to reduce the influence of MSTN and other transforming growth factor-b as a compensatory mechanism to help preserve skeletal muscle mass.

Funding

The National Strength and Conditioning Foundation provided funds for this project through a Young Investigator Grant to the corresponding author.

Acknowledgments

We would like to thank the subjects that participated in this study as well as all laboratory assistants who assisted with data collection and analysis. We would also like to graciously thank the reviewers that took the time to critique this manuscript.

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