Physiol Genomics 40: 141–149, 2010.
First published November 10, 2009; doi:10.1152/physiolgenomics.00151.2009.
Differential genomic responses in old vs. young humans despite similar levels
of modest muscle damage after resistance loading
Anna E. Thalacker-Mercer,1,2 Louis J. Dell’Italia,1,3 Xiangqin Cui,4 James M. Cross,5
and Marcas M. Bamman1,2,3,6
Departments of 1Physiology and Biophysics, 2Nutrition Sciences, 3Medicine, 4Biostatistics,5Surgery, University
of Alabama at Birmingham; and 6Geriatric Research, Education, and Clinical Center, Birmingham Department of Veterans
Affairs Medical Center, Birmingham, Alabama
Submitted 2 September 2009; accepted in final form 9 November 2009
resistance exercise; aging; microarray; inflammation
WHILE SKELETAL MUSCLE IS RECOGNIZED as an exceptionally plastic tissue, aging skeletal muscle demonstrates an impaired
capacity for regeneration when exposed to the same injury
stimulus as young muscle, as shown definitively in rodent
models (13, 14, 39). This creates a concern for older adults as
they transition toward frailty and/or attempt to recover from
surgery, because sarcopenia (i.e., age-related skeletal muscle
atrophy) is exacerbated in these patients and they are unable to
restore affected muscle mass to presurgery levels despite intensive physical intervention (48).
Address for reprint requests and other correspondence: M. M. Bamman,
UAB Dept. of Physiology and Biophysics, 1530 3rd Ave S., Birmingham, AL
35294-0005 (e-mail: [email protected]).
Postnatal skeletal muscle repair and regeneration following
acute injury (e.g., high-intensity exercise bout, trauma, surgery,
drug-induced necrosis) involves a complex array of coordinated activities that is incompletely understood. On the other
hand, it is widely accepted that muscle progenitor cell [satellite
cell (SC)] recruitment is a key component of the process,
indicating that at least some aspects of developmental (secondary) myogenesis are recapitulated during postnatal muscle
regeneration (35, 54). The nominally quiescent SCs reside just
outside of the myofiber sarcolemma beneath the basal lamina
and are thus architecturally positioned to respond rapidly to
cues from the circulation, the extracellular matrix, and secreted
factors exiting nearby myofibers (28). The appropriate regeneration response involves SC proliferation; progression along a
myogenic lineage to differentiated, fusion-competent myoblasts; and fusion to injured, repairing myofibers as nuclear
donors or, with overt damage (e.g., trauma, surgery), myoblasts
fuse with one another to form multinucleated myotubes that
then develop into replacement myofibers. These SC functions
and their molecular regulation are described in a number of
excellent reviews (10, 35, 51, 54). Although the precise mechanisms are not fully understood, SC function is clearly impaired with advancing age as shown in both human (8) and rat
(4) primary SCs. Furthermore, in vivo aging rodent studies
have demonstrated that SC recruitment is impaired during
atrophy countermeasures (23) and SC-mediated regeneration
following damage is less effective (13, 14), which is at least
partially caused by the aging SC niche (14). Concurrent to the
initiation of regenerative mechanisms after skeletal muscle
injury is an induction of inflammatory and stress responses (19,
27, 55) including an infiltration of macrophages (2, 12). These
responses may “jump-start” regeneration; however, prolonged
or hyperelevated proinflammatory cytokine signaling impedes
the process (42). Furthermore, inhibiting the conversion of
invading macrophages to the anti-inflammatory M2 phenotype
impairs muscle regeneration (52). A better understanding of
the principal mechanisms driving skeletal muscle regeneration
is essential to fully appreciate key alterations in the aging
muscle that cause disruption.
Microarray analysis allows for comprehensive and simultaneous assessment of all skeletal muscle transcripts and provides a unique opportunity to identify age-dependent changes
in the skeletal muscle transcript profile after a stressor or injury
stimulus [e.g., unaccustomed resistance loading (RL)] that
might have otherwise been unidentified. While a few transcript
profiling studies of resistance exercise-mediated changes have
been published (31, 38, 41, 50, 60), none has tested differential
age responses to contraction-mediated damage across the entire
141
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Thalacker-Mercer AE, Dell’Italia LJ, Cui X, Cross JM, Bamman
MM. Differential genomic responses in old vs. young humans despite
similar levels of modest muscle damage after resistance loading. Physiol
Genomics 40: 141–149, 2010. First published November 10, 2009;
doi:10.1152/physiolgenomics.00151.2009.—Across numerous model
systems, aging skeletal muscle demonstrates an impaired regenerative
response when exposed to the same stimulus as young muscle. To better
understand the impact of aging in a human model, we compared changes
to the skeletal muscle transcriptome induced by unaccustomed highintensity resistance loading (RL) sufficient to cause moderate muscle
damage in young (37 yr) vs. older (73 yr) adults. Serum creatine kinase
was elevated 46% 24 h after RL in all subjects with no age differences,
indicating similar degrees of myofiber membrane wounding by age.
Despite this similarity, from genomic microarrays 318 unique transcripts
were differentially expressed after RL in old vs. only 87 in young
subjects. Follow-up pathways analysis and functional annotation revealed
among old subjects upregulation of transcripts related to stress and
cellular compromise, inflammation and immune responses, necrosis, and
protein degradation and changes in expression (up- and downregulation)
of transcripts related to skeletal and muscular development, cell growth
and proliferation, protein synthesis, fibrosis and connective tissue function, myoblast-myotube fusion and cell-cell adhesion, and structural
integrity. Overall the transcript-level changes indicative of undue inflammatory and stress responses in these older adults were not mirrored in
young subjects. Follow-up immunoblotting revealed higher protein expression among old subjects for NF-␬B, heat shock protein (HSP)70, and
IL-6 signaling [total and phosphorylated signal transducer and activator
of transcription (STAT)3 at Tyr705]. Together, these novel findings
suggest that young and old adults are equally susceptible to RL-mediated
damage, yet the muscles of older adults are much more sensitive to this
modest degree of damage—launching a robust transcriptome-level response that may begin to reveal key differences in the regenerative
capacity of skeletal muscle with advancing age.
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AGE DIFFERENCES IN GENOMIC RESPONSES TO MUSCLE DAMAGE
transcriptome, because these studies were conducted in younger
men only (38, 60), were long-term training studies (41, 50), or did
not assess changes in the whole transcriptome (31).
The purpose of this study was therefore to determine, by
genomewide microarray analysis including functional gene
networks and pathways analysis, whether young and old adults
respond differentially to a standardized bout of unaccustomed
RL sufficient to induce moderate muscle damage. Our overarching hypothesis was that any age differences in the transcriptome-level response may reveal key factors responsible for the
muscle regeneration impairment among the old.
METHODS
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Forty-six young and older men and women were studied (subject
characteristics in Table 1). Health history and physical activity questionnaires were completed by all participants during screening. Participants in the older group also passed a comprehensive physical
exam and a diagnostic, graded exercise stress test with 12-lead ECG.
Subjects were excluded for any musculoskeletal or other disorders
that might have affected their ability to complete the RL bout and
testing for the study; obesity [body mass index (BMI) ⬎ 30.0 kg/m2];
knee extensor resistance training within the past 5 yr; and treatment
with exogenous testosterone or other pharmacological interventions
known to influence muscle mass or muscle recovery. The study was
approved by the Institutional Review Boards of both the University of
Alabama at Birmingham (UAB) and the Birmingham Department of
Veterans Affairs (VA) Medical Center. All subjects provided written
informed consent before participation.
Unaccustomed resistance loading bout. Mechanical overload of the
knee extensor muscle group was accomplished by a standardized RL
bout designed specifically to induce moderate myofiber wounding,
because subjects were untrained and unaccustomed to the RL. The RL
bout consisted of 9 sets of ⬃10 repetitions of bilateral, concentriceccentric knee extension contractions against a constant external load
on a conventional weight-stack knee extension machine. Subjects
were instructed and encouraged throughout to perform the concentric
phase of each repetition as rapidly as possible, followed by their best
attempt to control the eccentric lowering phase. The external resistance set for each subject was equivalent to 40% of bilateral maximum
voluntary isometric contraction (MVC) force determined by a load
cell and regression procedures as we have previously described (46).
As demonstrated by ⬎1,000 tests in our laboratory of combined
bilateral MVC and dynamic one-repetition maximum (1RM) knee
extension strength testing, the external load equivalent to 40% MVC
approximates 60% 1RM (data not shown). The “intensity” of each
contraction based solely on external load would therefore be considered moderate. However, key features of this protocol that increased
overall intensity included the explosive concentric phase of each
repetition and brief rest periods (⬍90 s) between sets. We have
previously shown (46) by electrogoniometry that 10 successive repetitions of this protocol induce velocity-dependent muscle fatigue,
particularly among old subjects.
Muscle biopsy and tissue preparation procedures. Percutaneous
needle biopsy of the vastus lateralis was taken with a 5-mm Bergstrom
biopsy needle under local anesthetic (1% lidocaine) by previously
described methods (11, 21). Muscle biopsies were performed in the
morning after an overnight fast at baseline (pre-RL) and 24 h after RL
(post-RL) in the resting state. The post-RL muscle biopsy was taken
from the contralateral leg to avoid any residual effects of the pre-RL
biopsy. Each biopsy was quickly blotted with gauze and dissected to
remove any visible connective and/or adipose tissue. Muscle samples
were divided into ⬃30-mg portions, snap frozen in liquid nitrogen,
and stored at ⫺80°C. Blood draws (10 ml from an antecubital vein)
were performed in the fasting state at the same two time points, and
serum was isolated and stored at ⫺80°C.
RNA isolation and microarray analysis. Genomic microarrays were
performed on 16 subjects (8 young, 8 old) before and after RL; groups
were balanced by sex (4 men, 4 women per age group). Total RNA
was isolated and further purified from the frozen muscle samples with
TRI Reagent (Molecular Research Center, Cincinnati, OH) and
RNeasy Mini Kits (Qiagen, Valencia, CA), respectively, according to
the manufacturers‘ instructions. RNA concentration was determined
with a spectrophotometer (NanoDrop ND-1000). An aliquot from
each sample of total RNA was shipped to either the Agilent Center for
Excellence (Santa Clara, CA) or Cogenics (Morrisville, NC) for
microarray analysis. Before sample processing, RNA quantity and
quality of each sample were determined by spectrophotometry and an
Agilent Bioanalyzer, respectively. The transcript profile of each purified RNA sample was determined with Agilent human 4X44K
single-color arrays (Agilent Technologies, Santa Clara, CA) containing ⬎41,000 gene transcripts and the standard single-color array
protocol at Agilent’s Center for Excellence and Cogenics. For standardization, pre- and post-RL samples within each subject were
analyzed in the same batch and each batch included samples from
both young and old subjects.
Immunoblotting. Standard immunoblotting was performed with established methods in our laboratory (3, 33) on samples from 32 subjects
(16 young, 16 old); groups were balanced by sex (8 men, 8 women per
age group). Muscle protein lysate was extracted from frozen muscle
samples (average 30 –35 mg) as detailed previously (3, 32, 33). Protein
concentrations were determined by the bicinchoninic acid (BCA) technique with bovine serum albumin as a standard. Samples were run on
4 –12% Bis-Tris (Invitrogen) SDS-PAGE gel matrices with 30 ␮g of total
protein loaded into each well, which was determined as ideal by preliminary experiments. Samples within subjects across time were loaded in
adjacent lanes. Proteins were transferred to polyvinylidene difluoride
(PVDF) membranes at 100 mA for 12 h. Primary antibodies of selected
proteins of interest based on microarray results and gene network analyses included total signal transducer and activator of transcription
(STAT)3, phospho-STAT3(Ser727), phospho-STAT3(Tyr705), total
NF-␬B p50, total I␬B␣, phospho-I␬B␣(Ser32/36), phospho-I␬␬␣/
␤(Ser176/180), heat shock protein (HSP)27, and HSP70 (Cell Signaling
Technologies, Beverly, MA). Appropriate primary antibody concentrations were determined in preliminary experiments and were 1:1,000
(vol/vol). Horseradish peroxidase-conjugated secondary antibody was
used at 1:50,000 (wt/vol), followed by chemiluminescent detection in a
Bio-Rad ChemiDoc imaging system with band densitometry performed
with Bio-Rad Quantity One (software package 4.5.1, Bio-Rad Laboratories, Hercules, CA). Parameters for image development were consistent
across all membranes by predefined saturation criteria as described
previously (3).
Serum cytokines and creatine kinase. Circulating concentrations of
proinflammatory cytokines IL-6, IL-1␤, IL-8, and TNF-␣ were determined by ELISA on 39 subjects [20 young (10 men, 10 women); 19
old (9 men, 10 women)] with MS2400 Human Pro-Inflammatory
4-Plex II Ultra-Sensitive Kits (Meso Scale Discovery, Gaithersburg,
MD) and standard procedures. Samples were measured in triplicate.
Serum creatine kinase (CK) activity was determined in 39 subjects [19
young (10 men, 9 women); 20 old (10 men, 10 women)] by a standard
enzymatic rate method with the SYNCHRON DxC 800 system in the
UAB Hospital Chemistry Laboratory. The baseline muscle biopsy was
taken a minimum of 1 wk before the 24-h post-RL biopsy to prevent
any residual effects of the baseline biopsy on circulating CK or
cytokine levels.
Data analysis. Baseline differences between the older and younger
adults in subject characteristics and protein expression were determined by independent t-tests. A 2 ⫻ 2 (age ⫻ RL) repeated-measures
ANOVA was used to test age, time, and age ⫻ time interaction effects
for serum cytokines, CK levels, and muscle protein expression/
phosphorylation with age and in response to RL (pre-RL vs. post-RL).
Tukey’s honestly significant difference (HSD) tests were performed
post hoc as appropriate. Significance was accepted at P ⬍ 0.05.
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AGE DIFFERENCES IN GENOMIC RESPONSES TO MUSCLE DAMAGE
RESULTS
There was a 46% increase in serum CK 24 h after RL in both
the older and younger adults (P ⬍ 0.001, Table 1), indicating
that the RL bout achieved the goal of modest damage to the
quadriceps. Importantly, the degree of myofiber membrane
wounding as indexed by serum CK was nearly identical in the
two age groups. Additionally, post-RL there was an increase in
circulating IL-6 in both the older and younger adults (P ⬍
0.005, Table 1). There were no changes in circulating TNF-␣
or IL-8 levels (P ⬎ 0.05, Table 1). The multiplex plate that was
used to determine changes in serum cytokine levels was not
sensitive enough to sufficiently detect IL-1␤ in all samples.
Transcript profile results and functional annotation. Average fold changes of the genes found to be differentially
expressed (P ⬍ 0.001) within each age group after RL are
summarized in Supplemental Table S1.1 After unaccustomed
RL, 351 genes were differentially expressed (128 downregulated and 223 upregulated) among the older adults. Of these,
318 were unique transcripts (for internal validation, the Agilent
chip contains more than one probe set targeting the same gene
in some cases—thus we discarded redundant results). By
contrast, only 91 transcripts (87 unique) were differentially
1
The online version of this article contains supplemental material.
expressed after RL in the younger group (36 downregulated
and 55 upregulated). Furthermore, of the 318 in old and 87 in
young subjects that were altered by RL, only 2 transcripts were
commonly differentially expressed after RL in both young and
old subjects: profilin 1 (PFN1; ⫹1.2-fold in old, ⫹1.1-fold in
young) and tubulin ␣-8 (TUBA8; ⫹1.6-fold in old, ⫹1.2-fold
in young). Using DAVID and GO databases, we functionally
annotated the 318 and 87 unique differentially expressed transcripts for the older and younger adults, respectively, and these
results are displayed in Supplemental Table S1.
Among old subjects, 25 of the differentially expressed genes
were found to be associated with inflammation and stress. Of note,
only four genes fell into this functional classification among
young subjects. Stress responses in the older group included
downregulation (range ⫺1.4-fold to ⫺2.1-fold) for several members of the antioxidant metallothionein family (MT1X, MT1G,
MT1H, MT1B, MT1A), while four HSP family members were
upregulated 1.3- to 1.6-fold (HSPA9, HSPE1, HSPB1, HSPH1).
Among older adults, 11 proteolysis-related genes were differentially expressed after RL (7 upregulated ⫹1.2 to ⫹1.5-fold, 4
downregulated ⫺1.2 to ⫺1.5-fold) compared with only 3 among
young adults. In older adults these included four components of the
ubiquitin-proteasome—an E2 conjugating enzyme (UBE2G1), ubiquitin-specific peptidases 54 and 28 (USP54, USP28), and
ubiquitin family domain containing 1 (UBFD1)—as well as
two autophagy-related genes (ATG3, ATG4A). Among genes
involved in transcriptional processes, altered expression was
found for 17 genes in old and 5 in young adults. In the older
adults, 10 of the 11 differentially expressed genes involved in
translation were upregulated including the ␦-subunit of eIF2B
(⫹1.3-fold), eIF4E (⫹1.4-fold), and ribosomal protein S6
kinase-like 1 (RPS6KL1, ⫹1.5-fold), while no changes in this
functional group were seen among young adults. Nine of the
differentially expressed genes among old adults were functionally grouped under structural integrity, including myosin binding protein H (MYBPH), which showed the most robust
change across all transcripts (⫹4.1-fold). Under the broad
grouping of genes related to cell cycle/cell proliferation, 13
genes were differentially expressed in old and 8 in young
adults, yet none were common in both age groups.
Network analysis. IPA was used to further characterize the
age-specific responses to RL by clustering and scoring functional
networks of differentially expressed transcripts. The maximum
number of nodes allowed in an IPA network is 35. Among the
older adults, we focused on the network with the highest score of
Table 1. Subject characteristics and serum levels of creatine kinase and inflammatory cytokines before and 24 h after
resistance loading
Old
Young
Pre-RL
Subject characteristics
Age, yr
Weight, kg
LBM, kg
Creatine kinase, U/l
IL-6, pg/ml
TNF-␣, pg/ml
IL-8, pg/ml
73 ⫾ 1*
72.4 ⫾ 2.1
43.97 ⫾ 1.90*
117.3 ⫾ 23.0
1.44 ⫾ 0.21
4.11 ⫾ 0.63
10.88 ⫾ 1.48
Post-RL
Pre-RL
Post-RL
171.1 ⫾ 31.5†
1.78 ⫾ 0.25†
4.21 ⫾ 0.68
10.90 ⫾ 1.33
37 ⫾ 1
76.6 ⫾ 2.6
50.35 ⫾ 2.30
128.4 ⫾ 14.8
1.12 ⫾ 0.14
3.68 ⫾ 0.39
10.58 ⫾ 2.68
187.7 ⫾ 26.6†
1.40 ⫾ 0.21†
3.75 ⫾ 0.40
11.21 ⫾ 2.41
Values are means ⫾ SE for 22 and 24 (subject characteristics), 20 and 19 (creatine kinase levels) and 19 and 20 (IL-6, TNF-␣, IL-8 levels) old and young
subjects, respectively. RL, resistance loading; LBM, lean body mass. *Different from young, P ⬍ 0.05. †Different from baseline, P ⬍ 0.05.
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Microarray data analysis was performed as follows. Normalization
of raw data and comparative analysis between expression profiles
were carried out with Genespring GX 10.0 (Agilent Technologies).
The raw data were normalized with quantile normalization and filtering of the transcripts by “present” and “marginal” flags. A “present,”
“absent,” or “marginal” was designated to a transcript based on signal
intensity and background noise. A transcript was kept if it had a
“present” or “marginal” call for 100% of the arrays in at least one of
the two treatment groups (pre-RL or post-RL). This reduced the
number of transcripts from 41,078 to 25,597 in the older adults and to
25,337 in the younger adults. A paired t-test was used to determine
RL-mediated differences in gene expression within each age group
(old and young) at a stringent P value of ⬍0.001 to minimize type I
errors. To provide biologically meaningful information to the transcript profile changes, all differentially expressed transcripts were
functionally annotated with the Database for Annotation, Visualization, and Integrative Discovery (DAVID, david.abcc.ncifcrf.gov) and
Gene Ontology (GO, www.geneontology.org), and their interacting
roles in networks, cellular functions, and canonical pathways were
further analyzed with Ingenuity Pathways Analysis (IPA) 5.0 (Ingenuity Systems, Redwood City, CA).
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AGE DIFFERENCES IN GENOMIC RESPONSES TO MUSCLE DAMAGE
Protein-level analysis. Proteins were selected for follow-up
immunoblotting based on microarray results that suggested
stress- and cytokine-mediated inflammation among old subjects after RL. While no RL-mediated changes in protein
content or phosphorylation state were found at the 24 h time
point, some remarkable age differences in the resting muscle were
identified and are described below. These age differences in
protein expression at baseline suggest that the muscles of old
adults are “primed” for a stress response and therefore may have
a heightened sensitivity to cellular stress and inflammation.
IL-6 signaling is transduced largely via phosphorylation and
activation of the transcription factor STAT3. While circulating
IL-6 increased similarly (⬃25%) in old and young subjects
after RL, STAT3 mRNA increased (1.4-fold) among old subjects only. We therefore quantified total and phosphorylated
STAT3 protein in muscle lysate and found remarkably higher
baseline levels of both total (⫹114%, P ⬍ 0.001) (Fig. 2A) and
phosphorylated (Tyr705 ⫹96%, P ⬍ 0.005) (Fig. 2B) STAT3
protein in old versus young subjects. Phosphorylation at
Tyr705 induces STAT3 dimerization and nuclear localization.
Fig. 1. Ingenuity Pathways Analysis (IPA) Network 1 (score ⫽ 49) clustered 29 (of 35 in the network) of the resistance loading (RL)-mediated differentially
expressed genes in older adults (n ⫽ 8) that are directly and indirectly interacting and relevant to stress and cellular compromise (a); inflammation and immune
responses (b); necrosis (c); protein synthesis (d); protein degradation (e); skeletal and muscular development (f); cell growth and proliferation (g);
myoblast-myotube fusion and cell-cell adhesion (h); fibrosis and connective tissue function (i); and structural integrity (j). Red, upregulated; green,
downregulated; white, not differentially expressed but related to this network. *Transcript represented by multiple probe sets. EG, Entrez Gene No. or GeneID.
Two heat shock proteins (HSPs) inserted into the network by IPA, HSP27 and HSP70, are synonymous with the differentially expressed HSPB1 and HSPA9,
respectively. Such redundant entries occur by default in IPA’s automated networking function if a gene within, or synonymous with, a larger “group” enters the
network (HSPA9 is synonymous with, or within, the HSP70 group, which contains 11 members; HSPB1 is synonymous with, or within, the HSP27 group, which
contains 4 members).
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49 (P ⫽ 10⫺49) in which 29 of the 35 possible transcripts were
differentially expressed after RL (Fig. 1 and Table 2). In agreement with the largest list of transcripts grouped via functional
annotation, the two “hubs” of this network (NF-␬B and p38
MAPK) gave a strong indication that inflammation and stress lay
at the center of the primary skeletal muscle responses to unaccustomed RL among older adults. The translated protein products of
transcripts within this network play key roles in stress and cellular
compromise (HSPH1, HSPA9, HSP70, HSP27, HSPB1,
SMARCA4, PLXNA1, NRP1); inflammation and immune responses (ZFP36, TRAF7, MAP2K3, MAP3K5, NRP1); necrosis
(MAP3K5); protein synthesis (CTNNA1, JUP); protein degradation (TRAF7, ubiquitin, MAP2K3, MAP3K5); skeletal and muscular development (DYRK1B, MAP2K3, MYBPH, NRP1,
SMARCA4, JUP, PLXNA1, NPTX1); cell growth and proliferation (TCP1, CCT5, FADS3, COL4A3); myoblast-myotube fusion and cell-cell adhesion (DYRK1B, CTNNA1, JUP); fibrosis
and connective tissue function (PLXNA1, NRP1, COL4A3,
MAP2K3); and structural integrity (MYBPH, SMARCA4,
COL4A3, CTNNA1, JUP).
AGE DIFFERENCES IN GENOMIC RESPONSES TO MUSCLE DAMAGE
Table 2. Summary of resistance loading-mediated
differentially expressed genes (P ⬍ 0.001) in older adults
(n ⫽ 8) identified in Network 1
Name
C8ORF32
CCT5
COL4A3
CTNNA1
DYRK1B
SMC5
STX4
TCP1
TRAF7
ULK1
ZFAND5
ZFP36
Caspase*
IFN Beta*
NF-␬B*
P38 MAPK*
Rb*
Ubiquitin*
Chromosome 8 open reading frame 32
Chaperonin containing TCP1, subunit 5 (ε)
Collagen, type IV, ␣3 (Goodpasture antigen)
Catenin (cadherin-associated protein), ␣1, 102 kDa
Dual-specificity tyrosine-(Y)-phosphorylation
regulated kinase 1B
Fatty acid desaturase 3
Heat shock 70-kDa protein 9 (mortalin)
Heat shock 27-kDa protein 1
Heat shock 105-kDa/110-kDa protein 1
Inhibitor of growth family, member 1
Junction plakoglobin
Mitogen-activated protein kinase kinase 3
Mitogen-activated protein kinase kinase kinase 5
Myosin binding protein H
Neuronal pentraxin I
Neuropilin 1
Nuclear mitotic apparatus protein 1
Plexin A1
S100 calcium binding protein A2
SWI/SNF-related, matrix-associated, actindependent regulator of chromatin, subfamily a,
member 4
Structural maintenance of chromosomes 5
Syntaxin 4
T-complex 1
TNF receptor-associated factor 7
Unc-51-like kinase 1
Zinc finger, AN1-type domain 5
Zinc finger protein 36, C3H type, homolog
Caspase
Interferon ␤
Nuclear factor-␬B
p38-Mitogen-activated protein kinase
Retinoblastoma
Ubiquitin
1.2
1.4
⫺1.7
1.2
⫺1.4
DISCUSSION
1.2
1.3
1.5
1.6
⫺1.2
⫺1.5
1.7
1.4
4.1
⫺2.4
1.5
⫺1.3
1.3
1.8
1.2
1.3
1.2
1.3
1.3
⫺1.3
⫺1.4
⫺1.5
NA
NA
NA
NA
NA
NA
*Nondifferentially expressed transcript identified by Ingenuity Pathways
Analysis as either a hub or a connecting node that connects other nodes within
the network. NA, not applicable.
Ser727 phosphorylation, also required for transcriptional activation, was not different by age (not shown). STAT3 total
protein increased 35% in young subjects after RL (P ⬍ 0.05)
but remained substantially lower than the total STAT3 levels
seen among old subjects.
Muscle transcript-level changes suggested the potential for
increased RL-mediated muscle TNF activity including a 1.3fold upregulation of TNF receptor-associated factor 7 (TRAF7)
in older adults and a 1.2-fold upregulation of TRAF and TNF
receptor-associated protein (TTRAP) in young adults. Furthermore, NF-␬B, a transcription factor largely responsible for
mediating the stress-activated catabolic effects of TNF-␣
downstream of the TNF receptor, emerged as a central hub in
Network 1 for the older adults. We therefore assessed protein
levels of NF-␬B (p50) and found 26% higher protein content in
old versus young adults (P ⬍ 0.05) at baseline (Fig. 2C) but no
age differences in the phosphorylation states of I␬B␣ and
I␬␬␣/␤ (not shown).
The transcriptome response of older adults revealed robust
upregulation of several HSP family members, suggesting a
reaction to cellular stress. Consequently we assayed muscle
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protein lysate for two of the HSPs appearing in Network
1—70-kDa (HSPA9 or HSP70) and 27-kDa (HSPB1 or
HSP27) HSPs—after finding transcript-level increases of ⫹1.3
and ⫹1.5-fold, respectively, post-RL among older adults. Similar to the transcription factors mediating inflammation
(STAT3, NF-␬B), HSP70 protein content was greater (41%)
among old versus young adults at baseline (P ⬍ 0.01, Fig. 2D).
There were no age differences for HSP27 (not shown). Representative immunoblots are shown in Fig. 2E.
40 •
Differentially expressed genes in older and younger adults,
increased serum CK, and increased circulating IL-6 suggest
that the unaccustomed high-intensity RL bout was sufficient to
induce moderate damage in the skeletal muscle and initiate
changes in the skeletal muscle transcriptome, particularly
among older adults. We find it remarkable that, after the same
mechanical stress, 318 genes were differentially expressed
among old versus only 87 genes among young adults, with the
two age groups showing similar changes in only two genes.
These age differences in response to RL occurred despite
similar degrees of myofiber membrane wounding as indexed
by nearly identical percent increases in serum CK. The value of
this finding should not be overlooked, as it indicates that the
myofibers of older adult muscle were not more susceptible to
mechanical membrane damage, yet they responded to the insult
with a remarkably different gene expression profile that may
help us begin to understand why regenerative function is
impaired in the old as shown in rodent models (13, 14, 39).
Follow-up analyses centered on the a priori assumption that
mechanical load-induced transcriptome changes unique to the
old may underlie causes of age-related regeneration impairment, thus identifying attractive candidates for targeted studies.
We therefore focused on the IPA network with the highest
score (i.e., significance) among the old only. The two hubs of
this network, NF-␬B and p38 MAPK, suggest that inflammation and stress lay at the center of the primary skeletal muscle
responses among the old.
Repair and regeneration. Skeletal muscle regeneration following injury appears dependent on the recruitment of resident
SCs and/or other stem cell populations capable of differentiation along the myogenic lineage to fusion-competent myoblasts
(15). These myoblasts then fuse as nuclear donors to damaged
myofibers or, at sites of necrosis or severe damage, fuse to one
another to form myotubes that differentiate into replacement
fibers (10). We identified a few differentially expressed transcripts in the older adults 24 h after RL that may negatively
impact SC-mediated processes during the subsequent recovery
period (e.g., 48 –96 h). For example, neuronal pentraxin 1
(NPTX1) and DYRK1B/Mirk (minibrain-related kinase) were
downregulated ⫺2.4 and ⫺1.4 fold, respectively. NPTX1 is
transcriptionally activated by the myogenic transcription factor
MyoD in proliferating myoblasts (59), while DYRK1B is a
Rho-induced kinase highly expressed in skeletal muscle that
promotes myoblast fusion (18), and its expression normally
increases coordinate with myogenin during myogenesis. Cellcell adhesion is modulated by catenin ␣1 (CTNNA1) and one
of its binding partners, junction plakoglobin (JUP). Although
CTNNA1 increased slightly (1.2-fold), JUP was downregulated (⫺1.5-fold). Furthermore, calpastatin, a calpain inhibitor,
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FADS3
HSPA9
HSPB1
HSPH1
ING1
JUP
MAP2K3
MAP3K5
MYBPH
NPTX1
NRP1
NUMA1
PLXNA1
S100A2
SMARCA4
Fold
Change
Description
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AGE DIFFERENCES IN GENOMIC RESPONSES TO MUSCLE DAMAGE
inhibits fusion and is normally downregulated during differentiation (6, 7) but was increased (1.3-fold). Collectively the
differential expression of these genes at 24 h—among only the
older adults—may negatively impact myoblast differentiation
and fusion later during recovery when these processes are
expected to be more prominent (e.g., 48 –96 h).
Inflammatory and stress responses. We interpret several transcript-level alterations found only in the older adults to be suggestive of undue stress and inflammatory signaling within the
muscle, which may be partially responsible for muscle regeneration impairment. Pavlath (43) showed quite clearly in mice that
overt inflammation impairs regeneration, as IFN-␥-mediated muscle inflammation induced expression of class I major histocompatibility complex (MHC) in regenerating myofibers and attenuated regeneration. Analyzing a small number of target transcripts,
two human studies indicate that aging alters the expression of proand anti-inflammatory genes in response to unaccustomed RL (20,
27). In the present study, the two main hubs within Network 1,
NF-␬B and p38 MAPK (Fig. 1), are intracellular intermediates of
cytokine/stress signaling that are linked to numerous differentially
expressed transcripts in Network 1. TNF-␣ and IL-1, two wellrecognized proinflammatory cytokines with both systemic and
local tissue effects, share common signaling pathways (NF-␬B,
SAPK/JNK, p38 MAPK). The upregulation after RL of TRAF7,
a TNF receptor signal transducer and component of the ubiquitin
ligase complex mediating protein degradation, along with higher
Physiol Genomics • VOL
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levels of NF-␬B protein in old versus young muscle despite no
age differences in circulating TNF-␣ or IL-1, suggests heightened
sensitivity in the older muscles to cytokine levels and/or cellular
stresses. This concept is supported by recent evidence in rat
primary SCs from old (vs. young) animals showing increased
susceptibility to TNF-␣ induced proapoptotic signaling (36). Furthermore, in mice aging has been associated with greater stressinduced muscle expression of TNF-␣, IL-1, and IL-6 (40). Muscle
expression of TNF-␣ or IL-1 is also elevated in aging humans
(25), and in aging rats increased TNF-␣ signaling (via NF-␬B)
induces apoptotic signaling, primarily in type II myofibers (47).
Both TNF-␣ and IL-1 suppress protein synthesis in myoblasts and
do so via a common second messenger, ceramide (53). Additional
rodent data provide evidence that TNF-␣ promotes muscle protein
degradation by increasing expression of the E3 ubiquitin ligase
MuRF1 (1) and concomitantly impairs muscle protein synthesis
by inhibiting mammalian target of rapamycin (mTOR)-mediated
translation initiation (34). In the present study, PANX1, which
regulates the release of IL-1 isoforms ␣ and ␤ (44), was robustly
upregulated among old (2.9-fold) adults. Like TNF, IL-1 has been
shown to induce muscle atrophy and inhibit protein synthesis, at
least in part by blunting the expression of the ε-subunit of eIF2B
(eIF2Bε) (16). Finally, MAP kinases in Network 1 that were
upregulated include MAPKK3 (MAP2K3; 1.7-fold) and MAPKKK5 (MAP3K5; 1.4-fold); both are intermediates in the transduction of several cytokine-mediated inflammatory and cell stress
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Fig. 2. Effects of age and RL on protein content or phosphorylation [optical density, arbitrary units (AU)] of the selected target proteins including: signal
transducer and activator of transcription (STAT3) (A); phospho-STAT3 (B); NF-␬B (C); and 70-kDa heat shock protein (HSP70) (D). E: representative
immunoblots. Immunoblotting was performed with samples from 16 young and 16 older adults. *Different from young at baseline, P ⬍ 0.05; †age ⫻ RL
interaction, P ⬍ 0.05.
AGE DIFFERENCES IN GENOMIC RESPONSES TO MUSCLE DAMAGE
Physiol Genomics • VOL
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underwent a single bout of high-intensity eccentric RL, only
one MT was differentially expressed and was downregulated
(38). The oxidative nature of endurance exercise compared
with RL suggests that the MTs may perhaps play different roles
in stress management during recovery. Why MT expression
was downregulated 24 h after RL among old subjects only is as
yet unclear; however, this may be yet one more indication that
the RL stimulus was much more of a stress in the old since the
present literature suggests that a substantially higher degree of
mechanical stress is required to elicit MT downregulation in
the young (38).
Protein metabolism. Among older adults we found significantly increased mRNA expression of factors involved in
translation, including eIF2B␦ (1.3-fold), eIF4E (1.4-fold), and
ribosomal protein S6 kinase-like 1 (RPS6KL1; 1.5-fold); on
the other hand, expression of the upstream negative regulator—protein phosphatase 2A (PP2A)—was concurrently increased (1.4-fold). PP2A inhibits protein synthesis by dephosphorylating the mTOR targets 4EBP1 and p70S6K1, and, in
fact, there is some evidence that mTOR’s kinase action on
these two targets may not be direct but rather results from
restraining PP2A via mTOR phosphorylation (inhibition) of
PP2A (45). Additionally, PP2A may impair protein synthesis
by directing ubiquitination of p70S6K1 (58). AMP-activated
protein kinase (AMPK) signaling is also known to counteract
Akt/mTOR signaling, and the ␥-2 subunit of AMPK
(PRKAG2) increased 1.5-fold among the old. The chaperone
HSP27, also upregulated among the old, inhibits protein synthesis by binding eIF4G and thus preventing assembly of the
eIF4F cap-binding initiation complex (17).
Among older subjects, several components of the Ub-proteasome system were differentially expressed after RL, including the
peptidases USP28 (⫹1.5-fold) and USP54 (⫺1.3-fold), an E2
conjugating enzyme (UBE2G1; ⫺1.4-fold), and UBFD1 (⫺1.2fold). At this point the nature of these transcript-level changes in
the translational machinery and Ub-proteasome system among
only the older subjects is unclear, yet these data provide further
evidence that the RL bout was interpreted by the muscles of old
subjects as a much more potent stimulus of transcriptional activity.
Structural integrity of skeletal muscle. Some noteworthy
genes that were differentially expressed only in older subjects
after RL support the concept that the muscles of older subjects
may have experienced a degree of stress far exceeding that in
young subjects despite being exposed to the exact same stressor.
For example, gene expression of MyBPH was robustly elevated
(4.1-fold) in the old only, as was myosin head domain containing
1 (MYOHD1; 1.4-fold). MyBPH is an integral myosin binding
partner in the A band of myofibrils that interacts with the myosin
rods and titin to provide structural integrity to the contractile
apparatus. Reduced MyBPH expression is associated with muscle
weakness in age-related disorders (30). Interestingly, localization
of MyBPH to the contractile apparatus is directed by its C
terminal domain consisting of two fibronectin type III motifs (24),
and our microarray analysis also revealed a 1.6-fold increase
among the old in the expression of fibronectin type III domain
containing 3B (FNDC3B). As shown in mice, MyBPH is upregulated in the young after more intense eccentric loading (5), again
suggesting age differences in the degree of mechanical stress
required to activate many of these transcriptional responses (with
young muscles requiring greater stress than old). MyBPH expreswww.physiolgenomics.org
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responses including SAPK/JNK signaling, necrosis, and caspasemediated apoptosis. The upregulation of MAP3K5 in muscle after
unaccustomed RL persists for at least 48 h (38).
Increased expression of IL-6 is a common finding following
mechanically induced muscle damage (55). There is some
debate as to whether IL-6 signaling is beneficial or inhibitory
during the acute response to mechanical stress. In mice, IL-6
has recently been shown to increase the expression of HSP72
and HSP25 in response to stress (29), suggesting an acute
protective effect. On the other hand, chronic exposure to
elevated muscle IL-6 induces atrophy (26), blunts growth (9),
and is commonly considered a potential contributor to agerelated sarcopenia. Congruent with the TNF/NF-␬B results, our
muscle protein-level analyses indicate much greater IL-6-associated signaling within muscle among older adults, despite
no age differences in circulating IL-6. STAT3, the key transcription factor in IL-6 mediated JAK/STAT3 signaling as well
as glucocorticoid receptor signaling, was upregulated 1.4-fold.
Furthermore, a higher overall STAT3 protein level and state of
STAT3 phosphorylation (Tyr705) among older adults suggests
greater inflammatory signaling in the old even at rest. These
findings again suggest that the older muscle may be more
sensitive to any given amount of IL-6. Antimyogenic effects of
IL-6 may be mediated at least partially by induced expression
of the muscle E3 ubiquitin ligase atrogin-1/MAFbx and TNF-␣
(9). Recently in humans it has also been noted that acute
mechanical stress robustly activates skeletal muscle STAT3
signaling (56), and, relevant to the present study, the investigators found that STAT3 phosphorylation was fivefold greater
in old versus young while protein expression of the STAT3
antagonist suppressor of cytokine signaling (SOCS)-3 was
repressed in the old (57).
Numerous members of the HSP family were upregulated
among older adults as highlighted in Network 1. HSPs may
play a role in degrading damaged proteins and maintaining
cellular integrity after mechanical load-induced muscle injury.
That we found higher HSP70 protein in the resting muscles of
older adults and a greater HSP response to RL at the mRNA
level suggests that the muscles of the old may be more actively
remodeling to maintain integrity at rest, and may sense a
greater need for HSP-mediated protection in response to the
mechanical perturbation imposed by RL. Among young adults,
the single HSP transcript that was upregulated (heat shock
70-kDa protein 1-like, HSPA1L) in the present study has been
reported to be robustly induced in young men 3 h after a bout
of 300 maximal eccentric contractions (38). Why this particular HSP transcript was unchanged in old subjects and appears
to be consistently upregulated in young subjects after RL is
unclear at this point. Overall, however, a far greater HSP
response at the mRNA level was found among the old.
Five metallothionein (MT) isoforms (1A, 1B, 1G, 1H, and
1X) were downregulated (1.4- to 2.1-fold) in the older adults
after RL. MTs play a protective role against oxidative stress,
apparently as free radical scavengers, as heavy metal “buffers,”
and/or by upregulating the expression of antioxidant enzymes
(37). Five additional transcripts that function in metal ion
binding were downregulated only among the old, including
phosphodiesterase 11A (PDE11A), which was down 2.5-fold
(see Supplemental Table S1). In prior studies of young men,
seven MTs were upregulated after endurance exercise, presumably from the oxidative stress (37); however, when young men
147
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AGE DIFFERENCES IN GENOMIC RESPONSES TO MUSCLE DAMAGE
Physiol Genomics • VOL
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groupings including inflammation and stress, repair, structural
integrity, protein metabolism, apoptosis, and proliferation among
others. These novel data provide an important basis for future
investigations aimed to target age differences in specific cellular
processes that may help us to better understand the impact of
aging on muscle regenerative function.
ACKNOWLEDGMENTS
We thank the participants for their efforts, and we thank S. C. Tuggle for
administering the resistance loading stimulus.
GRANTS
This research was supported by a VA Merit Review Award (M. M.
Bamman) and National Institutes of Health Grants 5R01-AG-017896 (M. M.
Bamman), 5P50-HL-077100 (L. J. Dell’Italia), 2T32-DK-062710 (A. E. Thalacker-Mercer), and GCRC M01-RR-00032.
DISCLOSURES
No conflicts of interest are declared by the author(s).
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Limitations. While we feel that the results revealed from this
study reveal valuable information on postexercise inflammatory
and regenerative responses in older adults, there are limitations.
We recognize that changes in the skeletal muscle transcript profile
in response to exercise are transient (38) and that the single time
point of 24 h after RL limits our ability to detect early, more
transient changes. Differential responses between older and
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RNA isolated from tissues of mixed cellularity such as skeletal
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isolated from muscle tissue is derived from myonuclei, age differences in the abundance of other contributing cell types could be
an important factor that was not controlled. This possible confounder, however, should have been minimized since our analysis
focused on within-subject changes in response to RL (rather than
cross-sectional age group comparisons). Microarray technology
itself is not without inherent limitations, including background
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great care to control for these issues by employing quantile
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signal intensity and background noise. Finally, the number of
samples/subjects analyzed via microarray did not allow us to
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certainly constrained the number of significant findings. It is clear,
however, that the RL-mediated changes in the transcript profile
between the older and younger adults were different.
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capacity of skeletal muscle with advancing age. With nearly
fourfold more genes differentially expressed in old versus young
adults in response to RL, we found the muscle transcriptome of
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