Tissue plasminogen and acute exercise
35
Journal of Exercise Physiologyonline
(JEPonline)
Volume 13 Number 6 December 2010
Managing Editor
Tommy Boone, PhD, MPH
Editor-in-Chief
Jon K. Linderman, PhD
Review Board
Todd Astorino, PhD
Julien Baker, PhD
Tommy Boone, PhD
Eric Goulet, PhD
Robert Gotshall, PhD
Alexander Hutchison, PhD
M. Knight-Maloney, PhD
Len Kravitz, PhD
James Laskin, PhD
Derek Marks, PhD
Cristine Mermier, PhD
Chantal Vella, PhD
Ben Zhou, PhD
Official
Research Journal of
the American Society of
Exercise Physiologists
(ASEP)
ISSN 1097-9751
Systems Physiology - Musculoskeletal
Tissue Plasminogen Activator and Plasminogen Activator Inhibitor1 Gene Expression in Muscle after Maximal Acute Aerobic Exercise
RICHARD L. CARPENTER1, JEFFREY T. LEMMER1, RYAN M.
FRANCIS1, JEREMY L. KNOUS1, MARK A. SARZYNSKI1,
CHRISTOPHER J. WOMACK1.
1Department
of Kinesiology, Michigan State University, East Lansing, MI
USA.
ABSTRACT
Carpenter RL, Lemmer JT, Francis RM, Knous JL, Sarzynski MA,
Womack CJ. Tissue Plasminogen Activator And Plasminogen Activator
Inhibitor-1 Gene Expression in Muscle After Maximal Acute Aerobic
Exercise. JEPonline 2010;13(6):35-44. Animal studies suggest
plasminogen activation in skeletal muscle is necessary for muscle repair.
However, plasminogen activators have been studied little in humans.
Therefore, the purposes of this study were to: 1) assess changes in
skeletal muscle gene expression of fibrinolytic and coagulation factors 2)
assess plasma activity of tPA and PAI-1 in response to an acute bout of
maximal aerobic exercise, 3) determine if there is any relationship
between muscle gene expression of tPA and PAI-1 with plasma activity
levels. Six healthy, college-aged males volunteered blood and muscle
samples prior to and immediately following a maximal treadmill exercise
test. Muscle tissue was homogenized and purified RNA underwent RTPCR using gene-specific primers for tPA and PAI-1 as well as
biotynylation for microarray. Blood was analyzed via biofunctional
immunosorbent assays for tPA and PAI-1 activity. A significant increase
in tPA mRNA was seen with exercise (p=0.038) while PAI-1 mRNA
showed no changes with the exercise. Muscle tPA activity showed no
changes with the exercise bout. Plasma tPA showed a significant
increase in activity (p<0.0001) while plasma PAI-1 activity had no
change with the exercise bout. In conclusion, tPA synthesis increases in
muscle following acute, high-intensity exercise and muscle production
does not significantly contribute to plasma tPA increases seen with
acute, high-intensity exercise.
Key Words: Microarray, Muscle Regeneration, Zymography,
Coagulation, Fibrinolysis
36
INTRODUCTION
Although exercise is an important part of cardiovascular disease prevention, it transiently increases
blood coagulation potential (28) and risk for cardiovascular events (21). Fibrinolysis is the dissolution
of fibrin networks associated with blood clots and counteracts coagulation. The fibrinolytic response
to exercise is crucial for maintaining low risk for cardiovascular events such as myocardial infarction
and stroke, which are typically caused by occlusive clots (8,17). Fibrinolysis is initiated by tissue
plasminogen activator (tPA) or urokinase plasminogen activator (uPA; primary activator in mice),
which converts plasminogen to plasmin, the active protease that degrades fibrin networks. tPA is
inhibited by plasminogen activator inhibitor-1 (PAI-1) providing fine regulation of fibrinolysis. Several
studies have observed increases in tPA and decreases in PAI-1 activities in plasma with maximal
exercise (3,14,24,26,28,29), suggesting fibrinolytic activity is increased with acute exercise possibly
providing protection from occlusive thrombi.
It has been shown in animal models that plasminogen activation occurs locally within skeletal muscle
following injury and this activation plays a role in muscle regeneration (23). More specifically,
plasminogen, tPA/uPA, and PAI-1 initially increase expression in skeletal muscle following muscle
injury in animals (22). Furthermore, inactivation of the plasminogen system in knockout mouse
models results in a severe muscle regeneration defect following chemically-induced muscle injury
(15,22). Additionally, PAI-1-deficient mice demonstrate accelerated recovery of muscle force, protein
levels, and morphology following injury (11). These results suggest local plasminogen activation in
skeletal muscle is important for recovery from muscle-damaging exercise or injury. A recent study
observed tPA and PAI-1 expression in skeletal muscle in men with metabolic syndrome after aerobic
training (9). Coagulation factors also may play a role as certain coagulation factors (Factor Xa and
Va) can accelerate tPA-induced plasminogen activation (19). However, responses of fibrinolytic and
coagulation factors in human skeletal muscle following muscle-damaging exercise has not been
assessed. Understanding the mechanisms of muscle repair and regeneration following muscle injury,
such as from intense exercise, could prove to be useful clinically for athletes or patients that are using
exercise for rehabilitation.
Expression of tPA has been found in the uterus, brain, and heart but plasma levels have traditionally
thought to be primarily from vascular endothelial cell production (10,25). As mentioned above, tPA
and PAI-1 expression has been observed in skeletal muscle of humans (9) and animals (15,22).
Increases in plasminogen activation in animal muscle following muscle injury combined with the
finding of local expression of plasminogen activators in skeletal muscle may suggest muscle
contributes to the large increase in tPA seen with exercise (3,14,24,26,28,29). This suggestion is
attractive as skeletal muscle provides a highly abundant tissue for release of tPA. However, the
contribution of muscle tPA and PAI-1 to plasma levels has not been studied.
Therefore, the purposes of this study were: 1) assess changes in skeletal muscle gene expression of
fibrinolytic and coagulation factors 2) assess plasma activity of tPA and PAI-1 in response to an acute
bout of maximal aerobic exercise, 3) determine if there is any relationship between muscle gene
expression of tPA and PAI-1 with plasma activity levels.
METHODS
Subjects
Six healthy college-aged males participated in this study. Subject characteristics are listed in Table 1.
Subjects were excluded if there was previous diabetes or cardiovascular disease including bleeding
disorders and hypertension. Subjects were not required to be trained and average VO 2max can be
37
seen in Table 1. Health and exercise history was determined by questionnaire. All subjects agreed to
participate in the study and signed informed consent. This study was approved by the Institutional
Review Board at Michigan State University.
Procedures
Exercise Testing
The maximal treadmill exercise test began at a speed of 2.5 miles per hour and increased at a rate of
0.5 mph per minute until reaching a maximum speed of 6.0 mph.
Table 1. Subject Characteristics.
Once this speed was reached, treadmill elevation increased
continuously at a rate of 3% per minute until subjects achieved
Variable
Value (n=6)
volitional exhaustion. All subjects had heart rate monitored via
Polar Heart Rate monitors (Polar Electro Inc., Lake Success, NY,
Age (yrs)
21.2  1.0
USA) and oxygen uptake measured continuously using indirect
Height (cm)
178.0  2.9
calorimetry (Sensor Medics 2900 Metabolic Cart, Yorba Linda,
CA, USA). The highest one-minute oxygen uptake achieved
Weight (kg)
72.1  4.8
during the test was defined as maximal oxygen uptake (VO 2max).
VO2max
To ensure a maximal effort, subjects achieved two out of the
60  2
(mL•kg-1 min-1)
following three established criteria (4): 1) achievement of 95% of
Values represented are mean ± SE
age-predicted maximal heart rate, 2) respiratory exchange ratio
and includes all subjects.
greater than or equal to 1.15, and 3) plateau of O 2 consumption
(ΔO2 ≤ 150 mL O2/min) (2).
Blood and Muscle Tissue Collection
All blood and muscle samples were obtained following a 12-hour fast. Samples were collected
between 7 and 10 a.m. to minimize the effects of diurnal variation in fibrinolysis (1). Prior to baseline
blood sampling, subjects assumed a semi-recumbent position for 30 minutes to eliminate postural
effects on fibrinolysis (27). Blood samples of 5 ml were collected from the antecubital vein into an
acidified citrate solution. Blood was spun to obtain platelet-poor plasma and stored at –80oC until
assayed.
The site for muscle biopsies was at the mid-line of the quadriceps in the vastus lateralis. Muscle
samples were obtained using a 5 mm Bergstrom biopsy needle and applied suction (5). The muscle
sample was immediately placed on an ice-chilled watch glass and dissected of all visible blood,
adipose, and connective tissue. A portion of the sample was flash frozen in liquid nitrogen and
another stored in RNAlater (Ambion, Austin, TX, USA). Both samples were stored at –80˚C until
analysis.
Following baseline collection, subjects completed the exercise test with Steri-strips, sterile gauze, and
an elastic pressure bandage over the biopsy incision. Immediately following the exercise test,
subjects were placed on the examination bed in a supine position where blood was drawn and
muscle tissue extracted from the same incision as the baseline biopsy. All blood and muscle samples
were collected within 4 minutes of cessation of the exercise test.
Muscle tPA and PAI-1 Expression Analysis
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
A portion of the muscle biopsy tissue was homogenized in Tri-Reagent (Molecular Research Center,
Inc., Cincinnati, OH, USA) using a polytron homogenizer. Total RNA was extracted and purified using
an RNeasy column per manufacturer’s instructions (RNeasy kit, Qiagen Inc., Chatsworth, CA, USA).
RNA was then subjected to DNase treatment (DNase Free, Ambion, Austin, TX, USA. RNA was
quantified spectrophotometrically at 260:280 nm to determine concentration and purity. Total RNA
38
(0.5 μg) was then reverse transcribed using the SuperScriptTM III First-Strand Synthesis System kit
(Invitrogen, Carlsbad, CA, USA) per manufacturer’s instructions. Equal amounts of cDNA were
amplified by PCR using gene-specific primers for tPA and PAI-1 using HotStarTaq Master Mix kit
(Qiagen Inc., Chatsworth, CA, USA). Primers used are as follows: tPA sense primer 5AGGAGCCAGATCTTACCAAGTGA-3 and anti-sense primer 5-CGCAGCCATGACTGATGTTG-3 for
a product size of 78 bp; PAI-1 (30) sense primer 5-GTATCTCAGGAAGTCCAGCC-3 and anti-sense
primer 5-TCTAAGGTAGTTGAATCCGAGC-3 for a product size of 396 bp. Touchdown PCR was
utilized with an intial annealing temperature of 76oC while denaturation occurred at 94oC for 45
seconds for both tPA and PAI-1 reactions. After the initial cycle of 76oC, the following four cycles
reduced the annealing temperature by 2oC until the final annealing temperature of 66 oC was obtained
for both tPA and PAI-1. Extension occurred at 72oC for 1 minute for tPA and PAI-1. The PCR was
optimized for PAI-1 to 40 cycles
and for tPA to 34 cycles. These
PCR conditions were optimized
with tPA and PAI-1 primers in our
lab and each sample was run in
duplicate. Amplified cDNA was
then loaded into a 2% agarose
gel and electrophoresed for 30
minutes at 120 V. A KODAK
2000R imaging station and
KODAK 1D image software
(version 4) were used to analyze
band intensity.
Microarray Gene Expression
A portion of the total RNA
described above was used for
microarray analysis. The preexercise samples for all subjects
were pooled as one sample such
that equal amounts of RNA from
each
subject
were
used
obtaining 5 μg of RNA for the Figure 1. Muscle tPA and PAI-1 mRNA Expression with Exercise.
pre-exercise sample (0.83 μg Muscle tissue was collected from subjects via percutaneous biopsy just
before and immediately following acute maximal aerobic exercise. Tissue
RNA/subject
with
6
total was homogenized and total RNA was collected and analyzed for mRNA
subjects) that was used on one content via semi-quantitative RT-PCR. A) Quantified tPA mRNA from muscle
microarray chip. The post- muscle tissue as seen with a representative gel from one subject. n=6. B)
exercise samples were also Quantified PAI-1 mRNA from muscle tissue as seen with a representative
pooled with equal amounts of gel from one subject . n=6. C) tPA mRNA from muscle tissue for each study
subject for pre- (white bars) and post-exercise (black bars). D) tPA mRNA
RNA from each subject totaling 5 from muscle tissue quantification with the exclusion of subject 2. n=5.
μg of RNA. These pooled RNA *Represents p<0.05.
samples
underwent
biotin
labeling using the BioArrayTM Single-Round RNA and Biotin Labeling System (Enzo, Life Sciences,
Farmingdale, NY, USA). The biotinylated RNA for pre- and post-exercise were then quantified to
determine concentration and purity. Biotinylated RNA quality was then analyzed using an Agilent
2100 Bioanalyzer and an Agilent RNA 6000 Pico chip (Agilent Technologies, Santa Clara, CA, USA)
per manufacturer’s instructions. Human genome expression of biotinylated RNA for pre- and postexercise pooled samples was then quantified using an Affymetrix (Santa Clara, CA, USA) U133 Plus
2.0 GeneChip kit per manufacturer’s instructions. Fragmentation and analysis were performed by the
39
Genomics Technology Support Facility at Michigan State University. Coagulation and fibrinolysis
proteins were selected from the NetAffx database on the Affymetrix website
(http://www.affymetrix.com/analysis/index.affx). Fibrinolysis pathway genes assessed were: tPA, uPA,
plasminogen, PAI-1, and fibrinogen. Coagulation pathway genes assessed were: coagulation factors
II, III, V, VII, VIII, IX, X, XI, XII, XIII, Von Willebrand factor, protein S, and protein C.
Muscle tPA Activity
Analysis:
Total protein was extracted
from the muscle tissue
homogenate
using
TriReagent™
and
was
resuspended in 0.1% SDS
following the manufacturer’s
instructions.
Protein
concentration was assessed
using the DC Protein Assay
Kit (Bio-RAD, Hercules, CA,
USA). Five micrograms of
protein was loaded into a 12%
polyacrylamide gel. A tPA
standard of 0.1 ng was used
as a positive control. Several
optimization studies were
performed with tPA standards
and tissue samples (data not
Figure 2. tPA and PAI-1 Protein Activity. A) Muscle tissue was collected
shown). The amount of protein
before and immediately following maximal acute aerobic exercise via
(5 µg) and gel conditions used
percutaneous biopsy. Tissue was homogenized and protein was isolated.
were
optimal
to
see
Equal amounts of protein were used in a zymography assay (see Materials
and Methods). Quantified muscle tPA activity is from gel as seen with a
differences between positive
representative banding result. B) Blood was collected before and immediately
controls. Gels then underwent
following acute aerobic exercise. tPA activity was measured via ELISA. C)
electrophoresis at 4oC for 5 h
Blood was analyzed for PAI-1 activity via ELISA.
at 180 V followed by two 30minute washes with 2.5% Triton-X to reactivate tPA. Between washes, gels were briefly rinsed with
distilled water. Gels were then incubated for 19 h at 37 oC with a collegenase soaking buffer (pH=7.5)
to allow any active tPA present to catalyze the breakdown of plasminogen copolymerized in the gel. A
0.2% stain stock solution of PhastGel Blue-R (Pharmacia, NY, NY, USA) was prepared according to
manufacturer’s protocol. A final 0.025% staining/destaining solution was prepared by mixing 26.5 mL
of stain stock with 184 mL destaining solution (1:3:6 glacial acetic acid:methanol:distilled water) (12).
Each gel was placed in 200 mL of the final staining/destaining solution and gently rocked for 4 h.
KODAK 2000R imaging station and KODAK 1D image software (version 4) was used to analyze band
net intensity to quantify plasminogen breakdown for each sample.
Plasma Analysis:
Plasma tPA and PAI-1 activities were quantified using bio-functional immunosorbent assays (Biopool
International, Ventura, CA, USA) per manufacturer’s instructions. Plasma concentrations of tPA
antigen were assessed using enzyme-linked immunosorbent assays (American Diagnostica,
Greenwich, CT, USA) per manufacturer’s instructions and were not corrected for plasma volume
changes.
40
Statistical Analyses
Changes in gene expression from pre- to post-testing by microarray were assessed by GeneChip
Operating Software (GCOS) version 3.1 and were performed by the Genomics Technology Support
Facility at Michigan State University. Changes in muscle and plasma dependent variables from preto post-testing were assessed using paired t-tests. Dependent variables included muscle mRNA (tPA
and PAI-1), muscle tPA activity, and plasma tPA and PAI-1 activities. Correlations between muscle
variables and plasma variables were assessed using Pearson correlation coefficient. These tests
were performed on SPSS version 11. Statistical significance was set at p < 0.05.
RESULTS
Muscle Tissue Analysis
RT-PCR Gene Expression
Mean values for all subjects for tPA and PAI-1 mRNA by RT-PCR are shown in Figure 1A and 1B.
There were no significant changes in tPA or PAI-1 expression from pre- to post-exercise. Figure 1C
illustrates gene expression for each individual subject illustrating the erroneously high resting value
and a differential response for tPA for subject 2 compared with Table 2. Gene expression by
other subjects. As such, when the analysis was performed microarray for fibrinolysis.
excluding subject #2, a significant increase (p<0.05) in muscle
Fold
P
tPA expression from pre- to post-exercise was observed (Figure Gene
Change
1D). Excluding subject #2 did not change results for PAI-1 mRNA.
tPA
0.812
0.500
uPA
0.758
0.888
Plasminogen (1)
0.812
0.468
Plasminogen (2)
1.00
0.747
PAI-1 (1)
1.00
0.854
Muscle tPA Activity
Muscle tPA activity, assessed by plasminogen gel zymography,
did not significantly change with exercise (Figure 2A). Exclusion
of subject 2 did not alter results for muscle tPA activity.
PAI-1 (2)
0.536
0.956
PAI-1 (3)
0.574
0.888
PAI-1 (4)
0.144
0.975
Plasma Analysis:
Plasma tPA activity showed a significant increase (Figure 2B)
with exercise while plasma PAI-1 activity showed no statistical
change (Figure 2C). There were no significant associations
between plasma variables and any muscle tissue variables
(p>0.05).
Fibrinogen β (1)
5.66
0.646
Fibrinogen β (2)
0.072
0.921
Fibrinogen γ
0.812
0.500
Fibrinogen α (1)
0.758
0.500
Fibrinogen α (2)
3.48
0.500
Fibrinogen α (3)
0.933
0.500
Microarray Gene Expression
While changes in gene expression were observed, there were no
changes in any fibrinolytic factors that were statistically significant
(Table 2) likely indicating expression changes were not large
enough to be measured by microarray analysis. Factor V was the
only coagulation factor seen to have a statistically significant
increase in gene expression in muscle tissue (Table 3).
DISCUSSION
Plasminogen activation clearly plays a role in recovery and
regeneration of muscle following injury. Inhibition of uPA results in Values are means for all subjects.
Numbers following each gene
reduced fusion of myoblasts and differentiation (6,18) and corresponds to the different probes
reduced proliferation (6,7). Additionally, chemical muscle damage used on the microarray chip.
increases gene expression of uPA and myogenic genes leading
to muscle repair and regeneration in mice (15). Further, uPA-deficient mice showed higher fibrin
deposition and impaired muscle repair following muscle damage (15). Eliminating fibrin deposits via
41
the snake venom ancrod following muscle damage resulted in increased muscle repair and
regeneration (22). A later study showed PAI-1 knockout mice have an advanced ability for muscle
repair and regeneration (11), indicating these plasminogen activators are critical in the face of muscle
damage. Studies regarding plasminogen activation in human
cells have only used uPA (6,7), however, results from our lab
Table 3. Gene expression by
suggest that tPA is the predominant plasminogen activator in
microarray for coagulation.
humans (13). Regardless, these plasminogen activators
Fold
Gene
P
appear to be critical for muscle repair following injury.
Change
Coag. Factor II
2.14
0.621
Coag. Factor III
0.354
0.500
Coag. Factor V (1)
0.574
0.805
Coag. Factor V (2)
6.96
0.213
Coag. Factor V (3)
2.14*
0.047
Coag. Factor VII (1)
1.52
0.169
Coag. Factor VII (2)
0.660
0.500
Coag. Factor VIII
0.933
0.500
Coag. Factor IX
1.41
0.500
Coag. Factor X
1.62
0.704
Coag. Factor XI (1)
1.00
0.186
Coag. Factor XI (2)
0.871
0.655
Coag. Factor XI (3)
3.48
0.500
Coag. Factor XI (4)
1.15
0.494
Coag. Factor XII (1)
0.250
0.532
Coag. Factor XII (2)
0.933
0.500
Coag. Factor XIII
0.812
0.500
Coag. Factor XIII β
0.812
0.805
Von Will Factor (1)
0.758
0.999
Von Will Factor (2)
0.871
0.500
Protein S
0.933
0.500
Protein C
0.707
0.787
Values are means for all subjects.
Numbers
following
each
gene
corresponds to the different probes used
on the microarray chip.
The present study may further the hypothesis that
plasminogen activation plays a role in muscle repair. An
increase in tPA has been shown in humans following a chronic
exercise program in men with metabolic disease (9). However,
the current study is the first to show an increase in tPA mRNA
in human skeletal muscle following acute, high-intensity
exercise (Figure 1D). Mice also show similar increases,
although with uPA, but mice also show an increase in PAI-1
mRNA (22). The present study found an increase in tPA but
not in PAI-1 suggesting a possible increase in fibrinolytic
potential. Despite these mRNA changes, we did not detect any
significant increase in tPA activity from whole muscle samples
(Figure 2A). An increase in Factor V gene expression was
seen following exercise even though the activated form of
Factor V is a known accelerant of tPA-induced plasminogen
activation (19). However, an increase in tPA activity may
require full protein synthesis as we only detected increases in
mRNA. It is possible the current exercise did not induce
enough muscle damage to increase tPA activity. However, a
recent study with a similar graded treadmill exercise test
showed significant increases immediately following the
exercise test in plasma levels of creatine kinase, lactate
dehydrogenase, and aspartate aminotransferase, all of which
are markers of muscle structural damage (20). Despite these
inferences, results may be more pronounced using protocols
that induce higher levels of muscle damage such as downhill
treadmill running.
Sample collection immediately following exercise completion
may contribute to the present findings. No significant changes
were detected via microarray for fibrinolytic genes despite
increases seen for tPA via PCR. Samples were collected from
subjects immediately following exercise, which likely limited
our detection of changes as up-regulation of genes is a timedependent process. This limitation accentuates the detected
increase in tPA gene expression via RT-PCR and further
increases would likely be seen if muscles were sampled
longer after the completion of the exercise bout. This time
point was chosen as plasma changes in tPA occur during this
time interval allowing us to compare muscle production and
plasma changes. There were significant increases in plasma
42
tPA immediately following exercise (Figure 2B). However, there were no significant associations
found between muscle tPA or PAI-1 mRNA and plasma changes suggesting muscle production of
these factors does not significantly contribute to plasma changes seen immediately following
exercise.
The present results have interesting implications but have limitations. Having only six subjects
decreases statistical power and is compounded by an outlier in a major outcome variable. This outlier
was decidedly excluded because his response (Figure 1C) was completely opposite to all other
subjects, opposite to well-known plasma tPA changes, and opposite to tPA mRNA responses from
animal studies. The differential response led us to conclude this subject was not classified correctly
from inclusion/exclusion criteria and therefore should be excluded in the results and conclusions.
Post-hoc power calculations showed 32% power to detect differences in tPA mRNA levels at a
significance of 0.05. Removing the subject decreased our power by 4%. Removing this subject
resulted in a statistically significant increase in tPA mRNA. In addition to this outlier, samples were
taken via biopsy of whole muscle, indicating possible contamination of analyzed muscle with protein
from microvessels and connective tissue. However, we found no change in muscle protein activity of
tPA, which was expected to be at the highest risk for contamination. Furthermore, we have observed
tPA expression in single muscle fibers in humans, suggesting these acute changes may be due to
inherent changes in the fiber, rather than adjacent tissue (13). The design of the current study also
required the post-exercise muscle biopsy to be collected at the same incision as the baseline biopsy
in order to collect tissue within the time required. A second collection in the same biopsy site may
induce gene expression changes primarily due to the tissue injury. However, previous data indicate
that up to three collections from the same site did not induce significant changes in gene expression
as detected by real-time PCR (16), suggesting the current data is valid.
CONCLUSIONS
Human skeletal muscle tissue showed an increase in expression of tPA following acute maximal
aerobic exercise similar to animal studies suggesting humans also increase plasminogen activation
following acute, high intensity exercise. Plasma tPA levels increased dramatically following exercise,
however, there was no association between muscle mRNA levels and plasma tPA activity suggesting
muscle does not contribute to plasma tPA activity seen with acute, high intensity exercise. Future
studies should include more subjects and use an exercise bout to induce higher muscle damage such
as downhill running.
ACKNOWLEDGMENTS
We would like to thank the Human Energy Research Laboratory (HERL) for equipment usage and
space. We would also like to thank the Genomics Technology Support Facility (GTSF) for their
assistance with microarray protocol and analysis. Portions of this study were supported by a Michigan
State University Intramural Research Grant Program. The granting agency had no input on the design
of the study, execution of the study, writing of the manuscript or submission of the manuscript.
Address for correspondence: Womack CJ, Ph.D., MSC 2303, James Madison University,
Harrisonburg, VA 22801. Phone (540)568-6515; FAX: (540)568-3338; Email: womackcx@jmu.edu.
43
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