Insulin-like growth factor immunoreactivity
in muscle after acute eccentric contractions
increases
ZHEN YAN, RAY B. BIGGS, AND FRANK
W. BOOTH
Department
of Physiology and Cell Biology, The University of Texas Medical School, Houston, Texas 77225
YAN, ZHEN,
RAY B. BIGGS,
AND FRANK
W. BOOTH.
Insulinlike growth factor immunoreactivity
increases in muscle after
acute eccentric contractions.
J. Appl. Physiol. 74(l): 410-414,
1993.-The
purpose of the study was to note whether insulinlike growth factor (IGF) immunoreactivity
increased after eccentric contractions.
IGF immunoreactivity
in the rat tibialis
anterior muscle was measured on 5 successive days (4-5 rats/
group, n = 28) after an acute bout of 192 eccentric contractions
elicited by electrical stimulation.
The muscle tissue sections
were immunocytochemically
processed with rabbit anti-human
IGF-I serum. Immunoreactivity
was analyzed with videomicroscopy and computer-aided
image processing. Four days after eccentric contractions,
IGF immunoreactivity
was significantly
higher than control [0.081 t 0.073 (SD) absorbance at 480 nm
vs. 0.026 t 0.018; P < 0.051. The increases in IGF-I immunoreactivity were mostly within the muscle fibers. These results
suggest that an acute bout of eccentric exercise increases IGF-I
immunoreactivity
in rat type II muscle 4 days postexercise.
exertion;
resistance
exercise;
skeletal
muscle
RESISTANCE
TRAINING
results
in an enhanced muscle strength in humans (4,6,10,13) and muscle hypertrophy in animals (19, 21). Eccentric contraction is defined as a forcible stretching or lengthening of
the active muscle during cross-bridge cycling (1, 12). A
single acute bout of 192 eccentric contractions increases
the synthesis rates (41-65%) of total mixed protein and
of myofibril protein 12-17 and 36-41 h after ending exercise (21). However, the mechanisms initiating and maintaining the increase in muscle protein synthesis rate
after eccentric exercise are not known. In one study, a
150% increase of insulin-like growth factor- (IGF) I
mRNA was found in the soleus and plantaris muscles on
the 2nd day of compensatory overload-induced hypertrophy in the rat (5). There are no reports on IGF immunoreactivity after eccentric exercise. On the other hand,
it is clear that IGF-I peptide can increase muscle protein
synthesis rate. In one study, a 40% increase in protein
synthesis was shown in L6 myoblasts during a 4-h exposure to IGF-I in tissue culture (2). In a second study, the
inclusion of IGF-I peptide in the maintenance medium of
cultured primary myofibers for 4-5 days produced fibers
of larger diameter and with more myonuclei per fiber
length than those produced in maintenance medium
without IGF-I (17). These fibers had more myosin accumulation, higher myosin synthesis rates, and lower myosin degradation rates. In a third report, protein synthesis
rate was increased more if muscle fibers were rhythmically stretched than if they underwent no mechanical ac-
tivity at a given concentration of IGF-I in cultured myofibers (18). In summary, muscle protein synthesis rates
are increased by eccentric contractions and by IGF-I. On
the basis of these. observations, it is possible that an increase in IGF-I concentration could be in the signal pathway from eccentric contraction to the observed increase
in muscle protein synthesis. The purpose of the present
study was to test whether IGF immunoreactivity
increased after eccentric exercise.
MATERIALS
AND
METHODS
Animal Care
Adult female Sprague-Dawley rats (Harlan, Indianapolis, IN) were housed in animal quarters maintained at
21°C with a 12:12-h light-dark cycle. Water and rodent
laboratory chow (Purina) were provided ad libitum. Experimental protocols had been approved by the Institutional Animal Care and Use Committee of The University of Texas Health Science Center at Houston.
ECCENTRIC
410
0161-7567/93
$2.00 Copyright
Experimental Design
Rats were assigned randomly to either one of five postexercise groups or a control nonexercised group, for
which five subsets existed. Each subset consisted of one
animal from the control group and one animal from each
of the five postexercise groups. The five postexercise
groups consisted of rats that were killed 1, 2, 3, 4, and 5
days after a single bout of 192 eccentric contractions.
Within each subset, rats were killed on the same morning
and their muscles were processed at the same time for
immunocytochemistry.
Eccentric Contraction
Rats were anesthetized with pentobarbital sodium (48
mg/kg body wt). Animals were exercised with a model of
nonvoluntary
electrically contracted skeletal muscle
(19-21). Subcutaneous platinum electrodes were positioned bilaterally along the lower leg muscles of the right
hindlimb. The foot was secured to a pulley-bar apparatus
with the rat supported on a platform above the foot lever.
The muscle contraction of the anesthetized rat was induced with a Grass S48 electrical stimulator with l-ms
pulses at 100 Hz and 30 V with a 2.5-s train duration
every 15th s for six trains. After each set of six trains
(contractions), there was a rest period of 1 min (first 16
sets) or 30 s (last 16 sets). A longer rest duration was
provided after every 4th set (3.5 min in the first 16 sets
0 1993 the American
Physiological
Society
GROWTH
FACTOR
IN
1. IGF-I immunocytoreactivity in the tibialis
anterior muscle at various times after 192
eccentric contractions
TABLE
Days
Control
IGF-I
n
26-tl8
5
Values are means
no. of animals.
IGF,
1
25217
4
2
43&28
5
Postexercise
3
42t33
4
+ SD expressed
in optical
insulin-like
growth
factor.
4
81+73*
5
5
61+58
5
density units X 10m3. n,
* P < 0.05 vs. control.
and 3 min in the last 16 sets). This experimental regimen
was identical to the muscle stimulation protocol described elsewhere in which an increase in protein synthesis rate was reported after eccentric contraction (21).
Muscle stimulation causes both the anterior and posterior muscle compartments of the lower leg to contract,
with a net plantar flexion resulting in a lengthening of
the contracting tibialis anterior muscle (eccentric contraction).
Muscle Preparation
The tibialis anterior muscle was fixed in situ as follows. On the day rats were killed, they were anesthetized
with pentobarbital sodium (48 mg/kg) and a laparotomy
was performed. A 150 mM phosphate buffer solution
(150 mM NaCl, 14 mM potassium phosphate, pH of 7.4)
was infused through a catheter into the dorsal artery at a
rate of 0.5 ml/min. After 2 min of infusion, the 130 mM
phosphate buffer was discontinued and the fixative solution [130 mM phosphate buffer (130 mM NaCl, 14 mM
potassium phosphate), 4% paraformaldehyde, 0.5% glutaraldehyde, 100 PM MgCl,, and 100 ,uM CaCl,, pH of
7.41 was infused for 15 min while the right ankle was
taped at a 90’ angle. At the end of infusion, the right
tibialis anterior muscle was removed from the rat and
placed for 45 min in a vial containing the aforementioned
fixative solution without glutaraldehyde. The fixative solution was replaced with phosphate-glycine solution (130
mM phosphate buffer solution with 50 mM glycine, pH
7.4) for 30 min, and the sample was washed successively
with 5,10, and 20% sucrose in 130 mM phosphate buffer
solutions, pH 7.4, with the time of each sucrose wash
varying from 15 min to 3 h. Muscle blocks were then
mounted with OCT compound embedding medium
(Miles, Elkhart, IN) and stored at -2OOC. In a cryostat,
-g-pm-thick
sections were cut and placed onto DL-polylysine-coated glass slides. For each rat, five sections were
placed onto a slide (3 to receive primary and secondary
antibodies; 1 to receive secondary antibody only, the reagent control; and 1 to receive Goldner’s modification of
Masson’s trichrome stain) (9). Slides were stored overnight in phosphate buffer at 4°C.
Immunocytochemistry
Slides from the same subset were processed at the
same time. Muscle sections on slides underwent the following procedures at room temperature. Sections were
blocked with 3% normal goat serum (NGS). The primary
antibody was contained in anti-IGF-I/somatomedin
C
MUSCLE
HYPERTROPHY
411
rabbit antiserum (UB-189) (8) and was a gift of the Hormone Distribution Program of the National Hormone
and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; Baltimore, MD). The anti-human-IGF-I
rabbit antiserum
used in the present study has a 0.5% cross-reactivity with
IGF-II and it cross-reacts minimally with 10v6 M insulin
(Hormone Distribution Program, NIDDK). The terminology “IGF immunoreactivity”
is used in the present
paper to describe the reaction product obtained with this
antiserum, because a small portion of the 200% increase
in IGF immunoreactivity on the 4th day after 192 eccentric contractions could be due to cross-reactivity with
IGF-II. The antiserum was diluted l:l,OOO in 3% NGS
and was placed on the muscle sections for 2 h. After unbound IGF-I antibody was washed away, the Vectastain
ABC immunoperoxidase kit (Vector Laboratories, Burlington, CA) was used to localize IGF-I antibody bound to
antigen and as a reagent control on the other half of the
muscle sections. The kit’s biotinylated goat anti-rabbit
immunoglobulin G (diluted 1200 in 3% NGS) was put on
all muscle sections. After a wash, sections were exposed
to 3% H,O, in methanol. After the sections were washed
again, the kit’s avidin-biotinylated
horseradish peroxidase macromolecular complex was added. After the next
wash, a peroxidase substrate [3-amino-9-ethylcarbazole
(AEC) working solution] was put onto the muscle section. The resultant red insoluble reaction product was
quantified by image processing.
Image Processing
Setup and calibration. A videomicroscope, which consists of a BH-2 microscope (Olympus, Lake Success, NY)
and a color video camera ‘(Microimage Video Systems,
World Video, Boyertown, PA), was used for image processing. Once the image was loaded, it was analyzed using
Jandel Video Analysis Software (JAVA 1.3) on a personal computer. The instruments were warmed up
for r10 min to ensure a steady light intensity. An ~4 lens
was used when loading the image with the cube selection
knob at the position that allowed 80% of the light to enter
the photo tube. The lens aperture was adjusted to 0.10
and the light source aperture to maximum. No filter was
selected, and the position of the lens was adjusted by
focusing an actual slide. Before the analysis of a slide, the
videomicroscope was calibrated internally. To accomplish this, the light intensity was adjusted so that the
absolute gray level detected at the center of the image in
the absence of a slide was 150 absolute units.
Analysis of the image. For each cross section of the
whole muscle, the average gray level for the whole muscle
cross section was determined by measuring random sites,
the proportion of which represented the whole muscle
cross section, i.e., if 20% of the muscle cross section were
very lightly stained, then one random site was selected
from the very lightly stained area; if the remaining 80%
of the muscle cross section were moderately stained, then
four random sites were selected in these areas (approach
1). (A random site consisted of -50 muscle fibers.) If the
entire muscle cross section was evenly stained, four to
eight random sites within the whole muscle cross section
GROWTH
FACTOR
IN
were selected (approach 2). The mean of the random sites
within each whole muscle cross section was calculated.
The mean gray level for each rat was obtained from the
mean gray levels of two to three whole muscle cross-section gray levels on the same slide. The mean gray level of
each rat was converted to an optical density at 480 nm
with use of a standard curve, which was generated as
described below. Each rat’s mean optical density was
corrected by the optical density of the reagent control.
After analysis of all rats, the mean for each time point
was obtained. In some of the slides in approaches 1 and 2,
either extracellular space increased because of the eccentric exercise or muscle fibers shrank during either the
fixing procedure or the histochemical
treatment procedure, resulting in an increase of extracellular space and a
possible increase of the image intensity within the cells.
To eliminate the effect of shrinkage in these slides, we
measured the average gray level of muscle bundles, including the extracellular
space, instead of the average
gray level in the muscle fiber.
Standard curue establishment. The standard solution
was prepared by the same reaction as in the immunocytochemical treatment of the sections. Briefly, a reaction
cocktail was made by adding 200 ~1 of 30% H,O, and’100
,LJ of 14% AEC (in N,N-dimethylformamide)
to 5 ml of
Na-acetate buffer (pH 5.2). The reaction was started by
adding 30 ~1 of goat anti-rabbit immunoglobulin
G conjugated with horseradish peroxidase (protein concn 13.9
mg/ml). The solution was incubated at room temperature for 10 min and spun in a clinical centrifuge at 4,000
rpm for 3 min. The pellet was redissolved in 5 ml of 95%
ethanol by vigorous vortex. Then the solution was filtered through a 0.2-pm filter to exclude any undissolved
precipitate. A serial dilution was made with 95% ethanol
and was used for establishing a standard curve. The gray
levels of the standards were determined under the videomicroscope by pipetting -200 ~1 of each standard dilution to fill the well (0.177 cm deep) of a hanging drop
slide, which was then covered by a glass cover slip. Immediately after the measurement
under the videomicroscope, the optical densities of each standard dilution
were measured at 480 nm with a Beckman DU-50 spectrophotometer.
(The spectrum of this solution was made
using the spectrophotometer,
and a peak absorbance at
480 nm was found.) A standard curve and mathematical
equation were derived for converting the gray level to
optical density at 480 nm. The standard curve generated
by this approach was linear (r = 0.998) for the absorbance range of the samples in the present study.
Statistics
The error mean square value from an analysis of variance was used to calculate a least significant differences
test for groups with unequal replication
(16). P < 0.05
was designated as significant.
MUSCLE
HYPERTROPHY
413
RESULTS
The purpose of the semiquantification
of IGF-I immunoreactivity
was to objectively distinguish
any differences among experimental groups rather than to indicate
subjectively that one group stained more intensely than
another. This assay does not give absolute quantities of
IGF-I peptide. The sensitivity of the assay enables detection of concentration
differences of AEC of the magnitude of 0.002%. An external standard of known gray level
permitted
calibration
of the image-processing
system
each day. Thus, variations due to the image processing
per se were minimal.
The IGF-I immunoreactivity
in the tibialis anterior
muscle was -200%
greater (P < 0.05) 4 days after an
acute bout of eccentric contractions than the control values (Table 1,. Fig. 1). Although IGF-I immunoreactivity
started to increase 2 days postexercise, the value only
reached the significant level 4 days postexercise. It is
unlikely that variations in the repeatability
of gray level
measurements
contributed to the large standard deviations (Table l), because a correlation
of 0.998 existed
between AEC concentration
and gray level. The large
standard deviations for individual group means (Table 1)
could be due to any or all of the following reasons: 1)
variations in the thickness of muscle sections (the microtome did not cut sections of consistent thickness), 2) variations among days for the immunocytochemistry,
because each subset (a subset consists of 1 animal from
each experimental
group) underwent immunocytochemistry on different days [the rate of color development for
a positive control (sections of marcaine-damaged
muscle) varied for each subset], and 3) the response to the
eccentric contraction varied among animals within a day
(e.g., 2 of the 5 rats within day 4 had IGF immunoreactivity levels similar to the control values, while the other 3
rats had optical densities 2,3, and 20 times their control
values). Others have observed nonresponders to eccentric exercise (R. Armstrong and G. Dudley, personal communication).
Nevertheless the variation in IGF immunoreactivity did not prevent the detection of a significant
increase in IGF-I immunoreactivity
in type II muscle 4
days after an acute bout of 192 eccentric contractions.
The eccentric contraction-mediated
increase of IGF
immunoreactivity
was within and sometimes outside the
muscle fibers (Fig. 1). However, the increase in IGF immunoreactivity
is not always consistent within the muscle. Because of the nature of this study, it is not possible
to elucidate the origin of the increased availability
of
IGF-I peptide.
Some of the postexercise sections showed focal accumulation of inflammatory
cells, which others have interpreted to mean that the eccentric contractions caused
focal damage to muscle fibers (7, 15). At this point, we
cannot exclude the possibility
that events associated
with accumulation of inflammatory
cells could play a role
414
in the increase
postexercise.
GROWTH
of IGF-I
immunoreactivity
FACTOR
IN
in the muscle
DISCUSSION
The new information
provided in the present study is a
200% increase in IGF immunoreactivity
(4 days) after
the 192 eccentric contractions
and the large number of
fibers showing the increase in IGF immunoreactivity
in
those muscles having an increased IGF response. A previous report examined IGF-I mRNA (5), but not the immunoreactivity
of the IGF-I peptide, after continuous
overload, rather than an acute eccentric exercise.
The experimental
model of compensatory
overload-induced hypertrophy,
which was used to obtain the increase in IGF-I mRNA, differs from a single acute bout of
eccentric exercise both in the duration of the exercise
stimulus and the amount of postexercise
recovery (3).
Whereas compensatory
overload-induced
hypertrophy
is
essentially
a continuous
exercise stimulus (2,880 min of
application in 2 days) with no long period of recovery, the
192 eccentric contractions,
which were employed in the
present study, consist of a total of 6.4 min of contraction
followed by days of recovery. Because of this difference,
it is not certain that IGF immunoreactivity
would be increased after 192 eccentric contractions.
Furthermore
the increased IGF-I mRNA quantities observed in compensatory
overload-induced
muscle do not definitively
prove an increase in IGF immunoreactivity,
because protein level is a dual function of synthesis and degradation.
Thus a direct measurement
of an increase in IGF immunoreactivity
had not been noted postexercise
in any contractile model in vivo.
Increases of 41-56% in mixed and myofibrillar
protein
synthesis rates were found as early as 12-17 h and maintained to at least 36-41 h after 192 eccentric contractions
by the tibialis anterior muscle of the rat (21). IGF immunoreactivity
was not increased at a time point (24 h after
192 eccentric contractions;
present study) that is between the previously reported times (12-17 h and 36-41 h
after 192 eccentric contractions)
(21) in which protein
synthesis rates were found to be increased. However, an
alternative
mode by which IGF-I could play a role in the
increased protein synthesis
rate by 12 h, or up to 41 h
after 192 eccentric contractions,
is by increasing the skeletal muscle sensitivity
and/or responsiveness
to IGFs.
This is supported by two recent reports of increases in
both sensitivity
and responsiveness
to IGF-I for glucose
and amino acid transport
in the epitrochearis
muscles of
rats 3.5 h after a 2-h swim (11, 14).
We thank Mildred
Lai and Jason Lai for assistance
in establishing
immunocytochemistry
in our laboratory,
Dr. Adrian
Sheldon for assistance in image processing,
and Drs. Chris Kirby
and Craig Stump for
assistance
in critique
of the manuscript.
The hormone
distribution
program
of the National
Institute
of Diabetes and Digestive
and Kidney
Diseases provided
the funding
to permit the gift of the anti-IGF-I
antiserum.
Research
funding
was provided by National
Institute
of Arthritis
and Musculoskeletal
and Skin
Diseases Grant AR-19393.
MUSCLE
HYPERTROPHY
Address for reprint
requests:
F. W. Booth, Dept. of Physiology
and
Cell Biology,
The University
of Texas Medical
School, PO Box 20708,
Houston,
TX 77225.
Received
15 January
1992; accepted
in final
form
10 August
1992.
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