Voluntary running induces fiber type-specific
angiogenesis in mouse skeletal muscle
Richard E. Waters, Svein Rotevatn, Ping Li, Brian H. Annex and Zhen Yan
Am J Physiol Cell Physiol 287:1342-1348, 2004. First published Jul 14, 2004;
doi:10.1152/ajpcell.00247.2004
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Am J Physiol Cell Physiol 287: C1342–C1348, 2004.
First published July 14, 2004; doi:10.1152/ajpcell.00247.2004.
Voluntary running induces fiber type-specific angiogenesis
in mouse skeletal muscle
Richard E. Waters,1 Svein Rotevatn,2 Ping Li,1 Brian H. Annex,1,3 and Zhen Yan1
1
Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham 27710; and 3Division of
Cardiology, Department of Medicine, Durham Department of Veterans Affairs Medical Center, Durham, North Carolina 27705;
and 2Department of Heart Disease, Haukeland University Hospital, N-5021 Bergen, Norway
Submitted 17 May 2004; accepted in final form 8 July 2004
adaptation; capillary density; endothelial cells; fiber type transformation; vascular endothelial growth factor
ADULT MAMMALIAN SKELETAL MUSCLE is composed of multiple
fiber types with diverse functional capabilities. Tonically active muscles have mainly slow-twitch fibers with an oxidative
phenotype, whereas phasically active muscles have a predominance of fast-twitch fibers with a glycolytic phenotype. Slowtwitch oxidative skeletal myofibers have greater capillary density than fast-twitch glycolytic myofibers (5, 15, 28, 38). One
of the unique features of mammalian skeletal muscle is its
remarkable ability to adapt to altered functional demands by
changing these phenotypic profiles (8, 46). Various stimuli
including innervation/neuromuscular activity, mechanical
loading/unloading, physical activity, or even hormonal levels
and aging can trigger alterations in various functional components, including contractile protein elements, proteins of oxidative phosphorylation, as well as the vasculature (22, 26, 27,
Address for reprint requests and other correspondence: Z. Yan, Div. of
Cardiology, Dept. of Medicine, Duke Univ. Medical Center, 4321 Medical
Park Dr., Suite 200, Duke Univ. Independence Park Facility, Durham, NC
27704 (E-mail: zhen.yan@duke.edu).
C1342
29, 41, 45). More recently, genetic engineering-mediated manipulation of expression of functional proteins has also been
shown to induce phenotypic adaptation in skeletal muscle (23),
providing mechanistic clues to the tightly regulated physiological structure in and around myofibers as an integrative unit for
function.
The functional adaptability of muscle fiber type and capillarity has been most thoroughly explored in animal models of
chronic motor nerve stimulation, in which a dramatic increase
in capillary formation and an impressive fiber type transition
have been reported (11, 17, 29, 37). After 4 wk of continuous
electrical stimulation of the rabbit tibialis anterior muscle via
the motor nerve, the number of capillaries per unit of crosssectional area doubles (11, 17), and this predominantly fasttwitch muscle (only ⬃6% slow-twitch type I fibers before
stimulation) is transformed into a muscle containing almost
entirely slow-twitch oxidative fibers (37). As a result, the
previously fatigue-susceptible muscle becomes highly fatigueresistant. The adaptive process begins early, with a significant
increase in capillary density after only 2– 4 days of stimulation
(11, 16, 29). A reduction in fast-twitch glycolytic fibers (type
IIb) and an accumulation of fast-twitch oxidative fibers (type
IIa) appear to occur simultaneously, or shortly thereafter (between 2 and 12 days), whereas slow-twitch oxidative fibers do
not become a major component of the tibialis anterior muscle
until day 21 of stimulation (12, 29).
In humans and various animal models, endurance exercise, a
more physiological model of increased muscle activity, also
induces fast-to-slow fiber type switching and enhanced angiogenesis (25, 30 –32, 39, 40, 44). In contrast to chronic motor
nerve stimulation, less is known about the time course of
muscular adaptations that occur during endurance exercise.
Several studies have reported increased capillarity in skeletal
muscle after long-term exercise training (3, 25, 30, 31, 33, 40).
Other studies have described fiber type transformation after
exercise (primarily type IIb to IIa), although the fiber type
switching is less robust than the changes seen with chronic
motor nerve stimulation (4, 14, 18, 47). It is possible that the
relationship between the adaptations induced by endurance
exercise is considerably different from that induced by chronic
motor nerve stimulation. Understanding the temporal relationship between exercise-induced angiogenesis and other adaptive
processes may provide valuable information regarding the
functional dependence of one process on the other. If exerciseinduced angiogenesis always precedes adaptation in the myoThe costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Waters, Richard E., Svein Rotevatn, Ping Li, Brian H. Annex,
and Zhen Yan. Voluntary running induces fiber type-specific angiogenesis in mouse skeletal muscle. Am J Physiol Cell Physiol 287:
C1342–C1348, 2004. First published July 14, 2004; doi:10.1152/
ajpcell.00247.2004.—Adult skeletal muscle undergoes adaptation in
response to endurance exercise, including fast-to-slow fiber type
transformation and enhanced angiogenesis. The purpose of this study
was to determine the temporal and spatial changes in fiber type
composition and capillary density in a mouse model of endurance
training. Long-term voluntary running (4 wk) in C57BL/6 mice
resulted in an approximately twofold increase in capillary density and
capillary-to-fiber ratio in plantaris muscle as measured by indirect
immunofluorescence with an antibody against the endothelial cell
marker CD31 (466 ⫾ 16 capillaries/mm2 and 0.95 ⫾ 0.04 capillaries/
fiber in sedentary control mice vs. 909 ⫾ 55 capillaries/mm2 and
1.70 ⫾ 0.04 capillaries/fiber in trained mice, respectively; P ⬍ 0.001).
A significant increase in capillary-to-fiber ratio was present at day 7
with increased concentration of vascular endothelial growth factor
(VEGF) in the muscle, before a significant increase in percentage of
type IIa myofibers, suggesting that exercise-induced angiogenesis
occurs first, followed by fiber type transformation. Further analysis
with simultaneous staining of endothelial cells and isoforms of myosin heavy chains (MHCs) showed that the increase in capillary contact
manifested transiently in type IIb ⫹ IId/x fibers at the time (day 7) of
significant increase in total capillary density. These findings suggest
that endurance training induces angiogenesis in a subpopulation of
type IIb ⫹ IId/x fibers before switching to type IIa fibers.
EXERCISE-INDUCED ANGIOGENESIS IN SKELETAL MUSCLE
MATERIALS AND METHODS
Animals. Male C57BL/6J mice (8 wk of age) were obtained
commercially (The Jackson Laboratory), housed in temperature-controlled quarters (21°C) with a 12:12-h light-dark cycle, and provided
with water and chow (Purina) ad libitum. For endurance training, the
mice were housed individually in cages (13 ⫻ 13 ⫻ 30 cm) equipped
with running wheels (11 cm in diameter) and were allowed to run
voluntarily. The voluntary running cages were modified from a
previous model (1) by replacing the mechanical switch on the running
wheel with a magnetic reed switch (Radio Shack) connected to the
Dataquest Acquisition and Analysis System (Data Sciences International). The mice ran for 1, 3, 7, 14, or 28 days (n ⫽ 6 for each time
point). A group of mice housed individually in running cages with
locked running wheels comprised a sedentary group (n ⫽ 6). Separate
groups of mice that either were sedentary or voluntarily ran for 3 or
7 days (n ⫽ 5 for each time point) were used for quantification of
VEGF protein. All mice were killed, and the plantaris muscles were
harvested for tissue sectioning 24 h after the last bout of running. All
procedures involving these animals conformed to the guidelines for
use of laboratory animals published by the U.S. Department of Health
AJP-Cell Physiol • VOL
and Human Services and were approved by the Duke University
Animal Use Committee.
Indirect immunofluorescence. Immunohistochemistry techniques
were used for fiber type determination and capillary density analysis.
Briefly, plantaris muscles were harvested and saturated in 30% sucrose-phosphate-buffered saline solution (PBS) for ⬃2 h, placed in
optimal cutting temperature (OCT) tissue-freezing medium (Miles
Pharmaceuticals, West Haven, CT), and frozen in liquid nitrogencooled isopentane. Frozen sections (6 ␮m) were cut in a cryostat on
microscope slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA).
Slides were allowed to come to room temperature, fixed in 4%
paraformaldehyde-PBS for 10 min at 4°C, and permeabilized with
0.3% Triton X-100-PBS for 10 min at 4°C. Blocking solution [5%
normal goat serum (NGS)-PBS] was applied for 30 min at room
temperature, followed by incubation with MHC I antibody (BF-F8)
diluted 1:100 in 5% NGS-PBS at 4°C overnight. Three consecutive
washes with PBS for 5 min each were followed by sequential
incubation with cyanine Cy5-conjugated goat anti-mouse IgG secondary antibody (1:50) at room temperature for 30 min. The muscle
sections were then washed three times with PBS, fixed in 4% paraformaldehyde for 2 min at 4°C, and blocked with 5% NGS-PBS for 30
min. Sequential staining, as described above, was then performed with
MHC IIa antibody (SC-71, 1:100) followed by rhodamine red-Xconjugated goat anti-mouse IgG and antibody against endothelial cells
(rat anti-mouse monoclonal CD-31, 1:25; Serotec) followed by fluorescein-conjugated goat anti-rat IgG. Images were captured under the
confocal microscope (Olympus).
After immunohistochemical staining, the percentage of type I fibers
(cyanine Cy5 stained), type IIa fibers (rhodamine red-X stained), and
type IIb ⫹ IId/x (unstained) fibers was determined. The entire plantaris muscle cross section was analyzed at ⫻20 magnification, with
care taken to ensure comparable cross section locations. Similarly, the
capillary density was determined by counting the total number of
capillaries (fluorescein stained) on the entire muscle cross section at
⫻20 magnification, and results are expressed as capillaries per unit
area of muscle tissue (a hemocytometer was used to standardize area
measurements). To further ensure that the analysis of capillary density
was not subject to error from muscle atrophy or interstitial edema,
capillary density was also determined by dividing the number of
capillaries by the number of muscle fibers to yield the capillary-tofiber ratio (4). To determine capillary contacting, the number of
capillaries in contact with a muscle fiber, capillaries around each
muscle fiber type (I, IIa, IIb ⫹ IId/x) were counted (total of ⬎600
fibers were counted for each type to calculate the means). This was
done on sedentary and 3-day, 7-day, and 28-day running samples.
Type IIa fiber cross-sectional areas were measured by using Scion
Image software (n ⬎ 20 for each section). The staining, photography,
and image analysis were performed by a single operator with no
knowledge of the coding system.
Endothelial alkaline phosphatase staining. Capillary density was
also assessed by endogenous endothelial alkaline phosphatase staining
on frozen sections as previously described (42). Briefly, slides were
fixed in 4% paraformaldehyde and incubated with nitro blue tetrazolium (NBT)-5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt
(BCIP) (GIBCO-BRL; Grand Island, NY) for 1 h, followed by eosin
staining. Capillaries appear dark blue against a red background.
Capillary density was measured by counting six random high-power
(⫻40 magnification) fields or a minimum of 200 fibers on an inverted
light microscope. Photographs were taken with an Optometrics analog
camera and Adobe Premier Version 5.1, and these images were
analyzed with an NIH Image analysis system. For the reasons described above, the capillary density was again expressed both as
capillaries per unit area and capillaries per muscle fiber. As was done
for the immunofluorescence, the staining, photography, and image
analysis were performed by a single operator with no knowledge of
the coding system.
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fiber, i.e., mitochondrial biogenesis and switching of myosin
heavy chain (MHC) isoform expression, it may suggest that
angiogenesis plays a permissive role for, or a functional role in,
promoting the later events.
A feature of exercise-induced angiogenesis that has recently
been appreciated is the heterogeneity of the response among
muscles of different fiber types to physiological stimuli (13, 15,
25, 40, 48). The existing evidence is consistent with the notion
that fast-twitch skeletal muscle displays phenotypic changes
more readily than slow-twitch muscle (48). The apparent
determining factor is the muscle phenotypic profile, but other
factors such as type, intensity, and duration of the exercise
regime may also contribute to the differential responsiveness
(20). Because most of the previous comparisons were between
different groups of muscles or different regions of the same
muscle, it is impossible to know whether the differences are
due to a difference in neuromuscular recruitment or a difference in fiber type composition. Thus it is desirable to define the
kinetics and the interrelationship of the myofiber adaptation
within individual muscles between different subtypes of myofibers.
Angiogenic growth factors such as vascular endothelial
growth factor (VEGF) may play important roles in skeletal
muscle adaptation after increased contractile activity. Fiber
type-specific differential expression of angiogenic growth factor expression has been investigated. For example, mRNA
expression of multiple angiogenic growth factors and related
receptors demonstrate fiber type specificity (30). Of particular
interest is the finding that treadmill running induces significant
increases in VEGF mRNA and protein, with the increased
VEGF mRNA being specific to type IIb myofibers (7). However, it is not known whether the fiber type-specific changes in
the mRNAs of angiogenic factors lead to a fiber type-specific
increase in capillary density.
The purpose of this study was to advance our understanding
of the temporal and spatial features of endurance exerciseinduced angiogenesis and fiber type switching in skeletal
muscle. We used a mouse model of endurance training because
recent developments in mouse genetics and molecular biology
provide useful tools to elucidate cellular and molecular mechanisms of skeletal muscle adaptation.
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EXERCISE-INDUCED ANGIOGENESIS IN SKELETAL MUSCLE
All techniques and materials used in these experiments were in
accordance with the provided protocols.
Statistics. All data are presented as means ⫾ SE. Comparisons
were performed between the sedentary and endurance-trained mice by
unpaired Student’s t-test and among different time points during
voluntary running by ANOVA followed by the Tukey test. P ⬍ 0.05
was accepted as statistically significant.
Downloaded from ajpcell.physiology.org on May 19, 2009
Fig. 1. Long-term voluntary running induces skeletal muscle adaptation in
mice. A: immunofluorescence staining of endothelial cells with anti-CD31
antibody and fluorescein-conjugated secondary antibody (green) in plantaris
muscle (⫻20 magnification) of sedentary and 28-day trained mice. Bars ⫽ 100
␮m. B: immunofluorescence staining of myosin heavy chain (MHC) type IIa
with anti-MHC IIa antibody and rhodamine red-X-conjugated secondary
antibody (red) and with anti-MHC I antibody and cyanine Cy5-conjugated
secondary antibody (blue). Unstained fibers are MHC type IIb ⫹ IId/x fibers.
Bars ⫽ 100 ␮m.
Quantification of VEGF protein. Plantaris muscles were harvested
from each mouse. For protein extraction, muscle samples were
weighed (14 –16 mg) and then homogenized with the use of a Pro 200
model polytron (Monroe, CT) in 400 ␮l of homogenization buffer
containing 10 mM Tris (pH 7.4) and 100 mM NaCl. The suspension
was then centrifuged twice at 8,000 g at 4°C for 15 min. The protein
content of the supernatant was determined by Bradford assay (9).
Total VEGF concentration in the plantaris muscle supernatant was
determined with a solid-state enzyme-linked immunosorbent assay
(ELISA) system with a mouse Quantikine VEGF ELISA Kit (R & D
Systems, Minneapolis, MN), as previously described (21). Assay
sensitivity is ⬍3.0 pg/ml. Results are expressed in picograms per
milligram of muscle based on standard recombinant protein curves.
Table 1. Capillary density and fiber type composition of
plantaris muscles in sedentary and 28-day trained mice
Capillary density
Capillary/mm2
Capillary-to-fiber ratio
Fiber type composition
Type I, %
Type IIa, %
Type IIb ⫹ IId/x, %
Sedentary
Trained
466⫾16
0.95⫾0.04
909⫾55*
1.70⫾0.04*
1.7⫾0.2
15.3⫾1.1
83.0⫾1.0
1.8⫾0.2
31.7⫾1.9*
66.5⫾1.8*
Values are means ⫾ SE; n ⫽ 6 mice/group. *P ⬍ 0.001 vs. sedentary.
AJP-Cell Physiol • VOL
Fig. 2. Exercise-induced angiogenesis precedes fast-to-slow fiber type switching in mouse skeletal muscle. A: average daily running distance during a time
course of 28 days of voluntary running; n ⫽ 35, 28, 21, 14, and 7 for day 1,
between days 2 and 3, between days 4 and 7, between days 8 and 14, and
between days 15 and 28, respectively. B: changes in capillary-to-fiber ratio in
plantaris muscle at different running time points (n ⫽ 6 –7 per time point).
Values are means ⫾ SE. **P ⬍ 0.01. Sed, sedentary. C: changes in %type IIa
fibers in plantaris muscle at different running time points (n ⫽ 6 –7 per time
point). Values are means ⫾ SE. ***P ⬍ 0.001.
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EXERCISE-INDUCED ANGIOGENESIS IN SKELETAL MUSCLE
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standing of the temporal changes of capillary density and fiber
type composition. The average running distance increased
steadily and reached a plateau around day 8 (Fig. 2A). The
increase in capillary-to-fiber ratio became statistically significant at day 7, where the number of capillaries per fiber
increased from 1.01 ⫾ 0.04 at day 3 to 1.44 ⫾ 0.08 at day 7
(P ⬍ 0.001), suggesting that active angiogenesis occurred
between day 3 and day 7 (Fig. 2B). On the other hand, a
significant change in fiber type occurred after 14 days of
running, where the percentage of type IIa fibers increased from
21.2 ⫾ 1.2% at day 7 to 32.7 ⫾ 1.0% at day 14 (P ⬍ 0.001),
Downloaded from ajpcell.physiology.org on May 19, 2009
Fig. 3. Voluntary running results in increased vascular endothelial growth
factor (VEGF) protein concentration in plantaris muscle. VEGF protein concentration was measured in plantaris muscles from sedentary mice and mice
having run for 3 or 7 days (n ⫽ 5 for each group). Values are means ⫾ SE.
*P ⬍ 0.05.
RESULTS
Long-term voluntary running induces significant increase in
capillary density in plantaris muscle. We first sought to determine whether endurance exercise results in enhanced angiogenesis in active skeletal muscle. The average daily running
distance for the training group was 10.2 ⫾ 0.7 km. This
long-term voluntary running resulted in significant increases in
indexes of capillary density in plantaris muscle as determined
by indirect immunofluorescence with an antibody specific for
the endothelial marker CD31 (Fig. 1A and Table 1). The
capillary density increased from 466 ⫾ 16 capillaries/mm2 in
sedentary mice to 909 ⫾ 55 capillaries/mm2 in trained mice
(P ⬍ 0.001). The capillary-to-fiber ratio increased from 0.95 ⫾
0.04 in sedentary mice to 1.70 ⫾ 0.04 in trained mice (P ⬍
0.001). Similar results (1.16 ⫾ 0.08 capillaries/fiber in sedentary mice vs. 1.79 ⫾ 0.08 capillaries/fiber in trained mice; P ⬍
0.001) were obtained when the endogenous alkaline phosphatase staining assay was used (not shown).
Voluntary running induces significant increase in percentage of type IIa myofibers in plantaris muscle. To assess
changes in fiber type composition in plantaris muscle, we
performed indirect immunofluorescence using antibodies specific for two MHC isoforms (I and IIa). The percentage of type
IIa myofibers in plantaris muscle increased significantly from
15.3 ⫾ 1.1% in the sedentary mice to 31.7 ⫾ 1.9% in the
trained mice (P ⬍ 0.001; Fig. 1B and Table 1). Conversely, the
percentage of type IIb ⫹ IId/x myofibers decreased significantly from 83.0 ⫾ 1.0% in the sedentary mice to 68.5 ⫾ 1.8%
in the trained mice (P ⬍ 0.001). The percentage of type I
myofibers was low in plantaris muscle and remained unchanged (1.7 ⫾ 0.2% in sedentary mice vs. 1.8 ⫾ 0.2% in
trained mice, P ⬎ 0.05; Table 1). There were also no significant differences in the average cross-sectional area of type IIa
fibers between sedentary control and trained mice (1,059.7 ⫾
116.4 and 1,053.1 ⫾ 122.6 ␮m2, respectively; P ⬎ 0.05).
Increased capillary density occurs before fiber type switching. We performed analyses for capillary density and fiber type
composition in plantaris muscle during a time course (1, 3, 7,
14, and 28 days) of voluntary running to gain a better underAJP-Cell Physiol • VOL
Fig. 4. Exercise-induced angiogenesis is spatially regulated. A: immunofluorescence staining of endothelial cells (green) and type IIa fibers (red) (unstained fibers are type IIb ⫹ IId/x fibers) in plantaris muscle of sedentary mice
and mice having run for 7 days. Increased capillary contacts per fiber can be
appreciated in 2 type IIb ⫹ IId/x fibers adjacent to the type IIa fibers in day 7
running section. B: quantification of the capillary contacts per fiber for type
IIb ⫹ IId/x fibers (n ⫽ 6 –7 for each time point). Values are means ⫾ SE.
**P ⬍ 0.01. C: quantification of the capillary contacts per fiber for type IIa
fibers (n ⫽ 6 –7 for each time point). Values are means ⫾ SE. *P ⬍ 0.05.
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C1346
EXERCISE-INDUCED ANGIOGENESIS IN SKELETAL MUSCLE
DISCUSSION
Mammalian skeletal muscle has remarkable plasticity to
respond to both physiological and pathological stimuli. This
adaptability is achieved primarily through changes in contractile protein composition, oxidative and glycolytic metabolism,
and vascularity, which in turn directly impact the exercise
capacity of the muscle (22, 26, 27, 29, 41, 45). Exercise
training is perhaps the most widely employed physiological
stimulus to skeletal muscle, but the mechanisms that underlie
skeletal muscle adaptation are not completely understood. The
major finding of our study is that exercise-induced changes in
skeletal muscle vascularity occur before a significant alteration
in fiber type composition and that changes in VEGF protein
occur before changes in capillary density. Moreover, we demonstrate fiber type specificity to the angiogenic response at the
cellular level in skeletal muscle, with capillary density increasing specifically around type IIb ⫹ IId/x fibers before switching
to type IIa fibers, a finding that has not been previously
described.
Chronic motor nerve stimulation is an “extreme” model of
increased contractile activity and perhaps more accurately
represents a model of fiber type transformation. Studies using
chronic motor nerve stimulation have clearly delineated the
changes in capillary density and fiber type composition (11, 17,
29, 37). It is not known whether the temporal relationship
between enhanced angiogenesis and fiber type switching is an
inherently programmed event for skeletal muscle in response to
increased contractile activity. In this study, we used a voluntary
running model to examine the temporal and spatial changes in
peripheral skeletal muscle induced by endurance exercise in
mice. Consistent with a previous report in other species (30),
capillary density in active skeletal muscle in mice showed a
sigmoid increase during long-term endurance exercise (Fig.
2B). As has been reported for chronic motor nerve stimulation
(29), this enhanced angiogenesis occurs before changes in
contractile protein composition. Therefore, the sequential
changes in vascularity and fiber type composition are recapitulated in the mouse model of endurance exercise.
The apparent temporal order of the changes in capillary
density and fiber type composition in plantaris muscle during
exercise training may be due to the differences in the inherent
kinetics of the proteins and the cellular components involved.
Contractile proteins, such as MHC, have a relatively long
half-life (34) and, with the techniques used in this study, it may
take a significantly longer time to show detectable changes in
MHC than in endothelial cells. Functionally, it is possible that
increased capillary density plays a permissive role for the
ultimate phenotypic changes in the myofibers. However, these
findings and the proposed scenario do not dispute the notion
that an orchestrated signaling cascade through hard-wired circuitry built in the skeletal muscle at the onset of exercise
training is directly involved in the highly coordinated adaptive
processes of angiogenesis and fiber type switching.
This study is the first to demonstrate that VEGF protein in
active skeletal muscle is increased in response to endurance
exercise in mice. We show that VEGF protein remains elevated
for at least 24 h after cessation of running when mice are
allowed to run voluntarily for 3 or 7 days (Fig. 2D). This
temporal pattern of VEGF protein expression is consistent with
a long-standing hypothesis that induced VEGF mediates vasculature remodeling in response to endurance exercise. VEGF,
among many angiogenic factors that are upregulated in contracting skeletal muscles and have potential functional roles in
Fig. 5. Schematic diagram of the sequential cellular
adaptation in skeletal muscle during endurance training.
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suggesting that the period between these time points is when
the majority of fiber type switching occurred (Fig. 2C).
Increased VEGF protein expression precedes increased capillary density. Because the increase in capillary density became
significant after day 7, we sought to determine whether the
enhanced angiogenesis was associated with an increase of
VEGF protein in the muscle. VEGF protein concentration in
plantaris muscle was 6.3 ⫾ 1.3, 11.5 ⫾ 2.0, and 10.3 ⫾ 1.4
pg/mg soluble muscle protein in the sedentary and 3-day and
7-day trained mice, respectively (P ⬍ 0.05 for both day 3 and
day 7 mice vs. sedentary mice; Fig. 3). Because capillary
density did not change significantly until day 7, the increase in
VEGF protein concentration in plantaris muscle preceded the
increase in capillary density.
Transient increase in capillary contacting around type IIb ⫹
IId/x myofibers precedes fiber type switching. To define the
spatial feature of the enhanced angiogenesis, we performed
triple-color indirect immunofluorescence for MHC I, MHC IIa,
and CD31. The unstained muscle fibers were considered type
IIb ⫹ IId/x. Fiber type-specific capillary contacting was determined by counting the capillaries around individual fibers of
different types (Fig. 4A). Capillary contacts per fiber for type
IIb ⫹ IId/x fibers increased from 1.59 ⫾ 0.09 in sedentary mice
to 2.06 ⫾ 0.11 (P ⬍ 0.01) at day 7 of running, a time point
before the majority of fiber type conversion, and decreased
toward the sedentary control level (1.82 ⫾ 0.08 capillaries/
fiber; P ⬎ 0.05 vs. sedentary mice) at 28 days of running, a
time point following the majority of fiber type conversion (Fig.
4B). In contrast, capillary contacts per fiber for type IIa fibers
remained unchanged at day 7 (3.92 ⫾ 0.12 capillaries/fiber in
sedentary mice vs. 4.03 ⫾ 0.10 capillaries/fiber at day 7; P ⬎
0.05) and increased at day 28 (4.35 ⫾ 0.10 capillaries/fiber;
P ⬍ 0.05 vs. sedentary) (Fig. 4C). Thus increased capillary
density in plantaris muscle during long-term voluntary running
could be attributed to an early (around day 7) increase in
capillary density in type IIb ⫹ IId/x fibers followed by a later
(after 7 days) combinatory increase of capillary density in type
IIa fibers and percentage of type IIa fibers.
EXERCISE-INDUCED ANGIOGENESIS IN SKELETAL MUSCLE
AJP-Cell Physiol • VOL
muscle (25) is consistent with the notion that capillary density
in slow-twitch oxidative and fast-twitch oxidative fibers may
increase further after long-term endurance training.
Because modulating exercise capacity has the potential to
impact an enormous number of clinical conditions, therapeutic
manipulations that are intended to alter the response to exercise
would benefit from an advanced understanding of the temporal
and spatial changes that occur in muscle in response to exercise. Although the mechanisms responsible for the fiber type
specificity of the angiogenic responses are currently unknown
and warrant further investigation, there are important implications of these findings. First, the differential adaptive response
within a muscle among different fiber types has improved our
understanding of the cellular mechanisms of heterogeneity in
adaptability between glycolytic muscle and oxidative muscle.
Second, our data suggest that angiogenesis is one of the early
steps in skeletal muscle adaptation. Data from our investigation
will also be critical for the refinement of studies that can be
performed to inhibit the angiogenic response to exercise to
directly test the mechanistic link between angiogenesis and
skeletal muscle plasticity.
ACKNOWLEDGMENTS
We are appreciative of the excellent technical support from C. Z. Ireland,
A. M. Pippen, and M. Zhang. We are thankful to Dr. H. Tibbals at the
University of Texas Southwestern Medical Center for assisting in the modification of the mouse running cage.
GRANTS
This work was supported by American Heart Association (AHA) Scientist
Development Grant 0130261N (to Z. Yan) and in part by AHA Established
Investigator Grant EI0140126N and a Department of Veterans Affairs Merit
Review Grant (to B. H. Annex).
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