UC San Diego
UC San Diego Previously Published Works
Title
Skeletal myofiber VEGF is essential for the exercise training response in adult mice.
Permalink
https://escholarship.org/uc/item/4kk7r8sc
Journal
American journal of physiology. Regulatory, integrative and comparative physiology,
306(8)
ISSN
0363-6119
Authors
Delavar, Hamid
Nogueira, Leonardo
Wagner, Peter D
et al.
Publication Date
2014-04-01
DOI
10.1152/ajpregu.00522.2013
Peer reviewed
eScholarship.org Powered by the California Digital Library
University of California
Skeletal myofiber VEGF is essential for the exercise training response
in adult mice
Hamid Delavar,
1
Leonardo Nogueira,
1
Peter D. Wagner,
1
Michael C. Hogan,
2
Daniel Metzger,
2
and Ellen C. Breen
1
1
Department of Medicine, University of California, San Diego, La Jolla, California;
2
Institut de Génétique et de Biologie
Moléculaire et Cellulaire (IGBMC), INSERM, CNRS, Université de Strasbourg, Collège de France, Illkirch, France
Submitted 25 November 2013; accepted in final form 10 February 2014
Delavar H, Nogueira L, Wagner PD, Hogan MC, Metzger D,
Breen EC. Skeletal myofiber VEGF is essential for the exercise
training response in adult mice. Am J Physiol Regul Integr Comp
Physiol 306: R586 –R595, 2014. First published February 12, 2014;
doi:10.1152/ajpregu.00522.2013.—Vascular endothelial growth fac-
tor (VEGF) is exercise responsive, pro-angiogenic, and expressed in
several muscle cell types. We hypothesized that in adult mice, VEGF
generated within skeletal myofibers (and not other cells within mus-
cle) is necessary for the angiogenic response to exercise training. This
was tested in adult conditional, skeletal myofiber-specific VEGF
gene-deleted mice (skmVEGF⫺/⫺), with VEGF levels reduced by
⬎80%. After 8 wk of daily treadmill training, speed and endurance
were unaltered in skmVEGF⫺/⫺ mice, but increased by 18% and
99% (P ⬍ 0.01), respectively, in controls trained at identical absolute
speed, incline, and duration. In vitro, isolated soleus and extensor
digitorum longus contractile function was not impaired in
skmVEGF⫺/⫺ mice. However, training-induced angiogenesis was inhib-
ited in plantaris (wild type, 38%, skmVEGF⫺/⫺ 18%, P ⬍ 0.01), and
gastrocnemius (wild type, 43%, P ⬍ 0.01; skmVEGF⫺/⫺, 7%, not
significant). Capillarity was maintained (different from VEGF gene
deletion targeted to multiple cell types) in untrained skmVEGF⫺/⫺
mice. Arteriogenesis (smooth muscle actin⫹, artery number, and
diameter) and remodeling [vimentin⫹,5=-bromodeoxycytidine
(BrdU)⫹, and F4/80⫹ cells] occurred in skmVEGF⫺/⫺ mice, even
in the absence of training. skmVEGF⫺/⫺ mice also displayed a
limited oxidative enzyme [citrate synthase and -hydroxyacyl CoA
dehydrogenase (-HAD)] training response; -HAD activity levels
were elevated in the untrained state. These data suggest that myofiber
expressed VEGF is necessary for training responses in capillarity and
oxidative capacity and for improved running speed and endurance.
angiogenesis; exercise; metabolism; peripheral vascular disease
EXERCISE INTOLERANCE is exhibited by most patients with pe-
ripheral arterial disease, chronic obstructive pulmonary dis-
ease, and diabetes and may stem from poor peripheral circu-
lation. In these chronic conditions, reduced oxygen and nutri-
ent availability to skeletal myofiber may limit the ability to
exercise. This is supported by findings in chronic heart failure
(CHF) and chronic obstructive pulmonary disease (COPD)
patients who display lower peak oxygen consumption, reduced
skeletal muscle capillary density, and capillary-to-fiber ratio, as
well as lower skeletal muscle oxygen tension (16, 26). In
addition, inhibited vascular endothelial growth factor (VEGF)
signaling has been reported in locomotor skeletal muscle of
such patients (4, 8, 23). VEGF is important in both muscle
capillary maintenance and in the angiogenic response to exer-
cise training (36, 43). This makes VEGF and other angiogenic
factors, linked with improving oxygen metabolism, attractive
targets for the development of therapeutics to treat peripheral
vascular dysfunction. However, VEGF is expressed by several
cell types within muscle, including the myofiber itself, endo-
thelial cells, satellite cells, and white blood cells.
The formation of new capillaries within the muscle depends
on dynamic cell-cell interactions (2, 12, 15). An increase in the
number of satellite cells parallels the formation of new capil-
laries in response to exercise training (13). These myogenic
precursor cells are in turn supported by factors secreted by
macrophages in contact with the capillary endothelial cells (12)
and, a shift in the balance of pro-arteriogenic (M2) versus
inflammatory (M1) macrophages has been suggested to play a
role in the formation of collateral arteries in ischemic muscle
(41). In endothelial cells, VEGF is regulated by an autocrine
mechanism and functions to preserve vascular integrity (pre-
vent endothelial cell apoptosis) (27, 32). In several nonskeletal
muscle organs analyzed to date, endothelium-expressed VEGF
does not appear to be essential for maintaining capillary num-
ber (27). It is thus still unclear which VEGF-producing cell
types in muscle are essential for regulating vascular structure.
This is despite the fact that myofiber-expressed VEGF repre-
sents 80 –90% of the VEGF content in hindlimb skeletal
muscle. Knowing the cellular origin of VEGF, essential to
maintaining capillarity, is important for targeting therapeutic
strategies to the responsible cell type.
Previous studies from our laboratory suggest that VEGF has
an important role in maintaining the number of skeletal muscle
capillaries under sedentary (cage-confined) conditions (43).
The injection of an AAV/Cre virus into a localized region of
VEGFloxP mouse gastrocnemius resulted in a loss of two-
thirds of the capillaries in the transfected region (43), but this
approach deletes VEGF from all cell types (including muscle
fibers and capillary endothelial cells) in that region. In a
subsequent study, using a Cre/LoxP cross-breeding strategy to
delete VEGF just within the mouse myofiber, its lifelong
deletion in both cardiac and skeletal myofibers resulted in
substantial reduction in the number of capillaries measured in
the heart and gastrocnemius. These mice performed very
poorly when exercise tested on a treadmill (35) and were
unable to form new skeletal muscle capillaries or improve
overall exercise capacity with aerobic training (36). However,
in these studies, it remains unclear whether the poor outcomes
in adulthood are due to improper vascular system development
during late embryogenesis and/or early postnatal life (35). This
is important clinically, because in many human diseases of
relevance, such as COPD, the vascular system likely develops
normally, with disease appearing later in adulthood.
Address for reprint requests and other correspondence: E. C. Breen, Dept. of
Medicine, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA
92093 (e-mail: ebreen@ucsd.edu).
Am J Physiol Regul Integr Comp Physiol 306: R586–R595, 2014.
First published February 12, 2014; doi:10.1152/ajpregu.00522.2013.
0363-6119/14 Copyright
©
2014 the American Physiological Society http://www.ajpregu.orgR586
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (137.110.076.185) on July 3, 2018.
Copyright © 2014 American Physiological Society. All rights reserved.
In the present study we hypothesized that VEGF expressed
by skeletal myofibers, which have developed a normal vascular
system, is essential for the angiogenic response to exercise
training. Furthermore, it is predicted that an inability to in-
crease the number of capillaries surrounding myofibers would
limit O
2
availability to mitochondria and prevent improvement
of oxidative metabolism and exercise capacity that normally
occurs in response to training. To test this hypothesis, the
VEGF gene was conditionally deleted in skeletal myofibers of
adult VEGFLoxP mice via a tamoxifen-inducible HSA-Cre-
ER
T2
system (40). Wild-type (WT) and skeletal myofiber
VEGF-deficient mice (skmVEGF⫺/⫺) were subjected to a
progressive treadmill exercise training schedule for 8 wk.
Vascular structure (capillarity and arteriogenesis), oxidative
and glycolytic enzyme capacity, fiber composition, and muscle
contractile function were evaluated and related to the exercise
capacity of WT and skmVEGF⫺/⫺ mice.
MATERIALS AND METHODS
Experimental animals. Mice homozygous for the VEGFLoxP trans-
gene (19) and heterozygous for HSA-Cre-ER
T2
(40) were maintained
for this study. Mice were housed in standard mouse cages in a
pathogen-free vivarium, with a 12:12-h light-dark cycle, and fed a
standard mouse diet (Harlan Tekland 8604, Madison, WI) and water
ad libitum. This study was approved by the University of California,
San Diego, Animal Care and Use Committee and conducted in
accordance to guidelines outlined by the Guide for the Care and Use
of Laboratory Animals (National Insitutes of Health).
Genotyping. The presence or absence of the HSA-Cre-ER
T2
trans
-
gene in each mouse was determined by PCR analysis using tail DNA
and forward 5=-CTAGAGCCTGTTTTGCACGTTC-3= and reverse
primers 5=-TGCAAGTTGAATAACCGGAAA-3= under the follow-
ing conditions: one 2-min polymerase activation incubation at
95°C, 35 cycles of 30 s denaturation at 94°C, 30 s annealing at
52.1°C, 60 s elongation at 72°C, followed by one 8-min elongation
period at 72°C.
Experimental design. HSA-Cre-ER
T2
-positive (pre-skmVEGF⫺/⫺)
and control littermates (VEGF⫹/⫹) were randomly assigned to ex-
ercise-trained (EX) or cage-confined untrained (UN) groups for a total
of four experimental groups (n ⫽ 9 –10 mice per group). Adult (4 mo
old) pre-skmVEGF⫺/⫺ and VEGF ⫹/⫹ mice were weighed and
treadmill-exercise tested for maximal speed and endurance on 2
consecutive days. All mice then received tamoxifen from day 0–4 (1
mg/day for 5 days ip). On day 21, post-tamoxifen mice were retested
for speed and endurance. skmVEGF⫺/⫺ EX and VEGF⫹/⫹ EX
mice were exercise trained or remained cage confined for 8 wk.
Maximal speed and endurance were retested upon completion of this
8-wk period. Mice were again weighed. Soleus, plantaris, gastrocne-
mius, and extensor digitorum longus (EDL) muscles from one leg
were isolated, weighed, frozen in liquid nitrogen, and stored at ⫺80°C
for analysis or mounted for preparation of cryosections.
Exercise testing and training. Twenty-four to seventy-two hours
before exercise testing, mice were familiarized on the treadmill
(model CL-4, Omnitech, Columbus, OH) by running for 10 min at
10 –15 cm/s on a 10° incline. Maximal speed was measured by
running mice at 33 cm/s for 1 min and increasing the speed by 3– 4
cm/s each minute until exhaustion. Endurance capacity was deter-
mined by running mice at 33 cm/s (50% of skmVEGF⫺/⫺ average
maximal speed) until exhaustion. Mice were deemed exhausted at the
point they could not remain on the treadmill and sat on shock grid
(ⱕ0.3 milliamps) for 10 s. Exercise training consisted of1hof
treadmill running 5 days per week, increasing speed from 28 to 34
cm/s over 8 wk. All mice groups received the same absolute speed and
duration throughout 8 wk of exercise training.
Ex vivo muscle contractile function and fatigue resistance. Con-
tractile properties of the EDL and soleus were measured ex vivo as
previously described (33, 44). EDL and soleus mounted in experi-
mental chambers (800MS, Danish Myo Technology, Aarhus, Den-
mark) were perfused with Tyrode solution (in mM: 121 NaCl, 5 KCl,
0.4 NaH
2
PO
4
, 1.8 CaCl
2
, 0.5 MgCl
2
, 24 NaHCO
3
, 5.5 glucose, and
0.1 EGTA) continuously bubbled with 95% O
2
-5% CO
2
(pH 7.4,
22°C). Muscles were electrically stimulated (S88X stimulator, Grass
Technologies) using square-wave pulses (16 V; EDL: 300 ms train
duration, 0.5 ms pulse duration; soleus: 500 ms train duration, 0.5 ms
pulse duration) with single twitches to set the length for maximal
twitch tension (L
0
). After 15 min, contractile function was evaluated
by stimulating the muscles at different frequencies (EDL muscle:
1–150 Hz; soleus: 1–120 Hz) with 100-s intervals between contrac-
tions. After the force-frequency protocol, muscles rested for 10 min.
Fatigue resistance was determined by a series of repeated tetanic
contractions at a frequency that evoked near-maximal tetanic tension
development (70 Hz for EDL and 50 Hz for soleus) with increasing
train frequency (1 contraction each 8, 4, 3, 2, and 1 s) each minute (for
EDL) or every 2 min (for soleus) until the initial force decreased to
50% (fatigue point). Tension development (in mN) was normalized
with respect to the muscle cross-sectional area (mN/mm
2
). Cross-
sectional area was calculated dividing muscle mass (mg) by the
product of L
0
(mm) times muscle density (1.06 mg/mm
3
).
VEGF protein levels. VEGF protein levels were measured by
ELISA (VEGF Mouse ELISA, R&D Systems, La Jolla, CA) and
normalized to total protein levels (Bio-Rad DC protein assay).
Skeletal muscle morphology and immunohistochemistry. Capillar-
ies and fibers in 10-m cryosections were detected using the Capillary
Lead-ATPase method (39). Images were captured using a Hamamatsu
Nanozoomer Slide Scanning System. Total capillary number, total
fiber number, fiber areas, and type were calculated using ImageJ
software. 5=-Bromodeoxycytidine (BrdU) was injected (50 mg/kg ip)
daily for 6 consecutive days before animals were euthanized. For
immunohistochemistry 10-ìm cryosections were fixed with 4% para-
formaldehyde for 10 min at room temperature, rinsed with PBS, and
incubated overnight at 4°C with the following primary antibody
concentrations to detect proliferating cells, fibroblasts, and smooth
muscle cells:BrdU mouse mAB (0.2 mg/ml, B35128, Life Technol-
gies), vimentin rabbit mAB (1:200, no. 5741, Cell Signaling),
␣-smooth muscle antibody (1:200, A2547, Sigma), and F4/F80⫹
(1:100, no. 14-5989, eBiosceince, San Diego, CA). BrDU sections
were treated with 2 M HCl at 56°C for 30 min and washed with PBS
before primary antibody incubation. Signals were detected with anti-
mouse and anti-rat AlexaFlour 488 or anti-rabbit AlexaFluor 546
(Invitrogen, Molecular Probes, Carlsbad, CA) and mounted with
ProLong Gold antifade reagent with 4=,6-diamidino-2-phenylindole
(DAPI, P-36931; Invitrogen, Carlsbad, CA). Images were collected
with an Olympus FV100 confocal microscope.
Metabolic enzyme analysis. Metabolic enzyme activities [expressed
in units (U) of catalyzing mmol substrate·mg tissue
⫺1
·min
⫺1
in
muscle homogenates] were measured at room temperature as previ-
ously described (44).
Statistical analysis. A two-way ANOVA was used to detect dif-
ference between exercise condition and genotype. A Tukey post hoc
test was used to analyze specific differences between the four exper-
imental groups. P ⬍ 0.05 is considered significant. All data are
presented as means ⫾ SE.
RESULTS
Conditional ablation of the VEGF gene in adult skeletal
myofibers. VEGF protein levels, measured at the end of the
training period, were decreased in the soleus, plantaris, gas-
trocnemius, and EDL by 80, 86, 85, and 76% (P ⬍ 0.01),
respectively, in untrained skmVEGF⫺/⫺ mice compared with
WT, untrained VEGF⫹/⫹ mice. After 8 wk of progressive
R587VEGF-DEPENDENT RESPONSES TO EXERCISE TRAINING
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00522.2013 • www.ajpregu.org
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (137.110.076.185) on July 3, 2018.
Copyright © 2014 American Physiological Society. All rights reserved.
treadmill training was completed, VEGF protein levels were
higher in the soleus, plantaris, gastrocnemius, and EDL by 23,
21, 19, and 25% (P ⬍ 0.01), respectively, in WT exercise-
trained mice (VEGF⫹/⫹ EX mice) compared with untrained,
WT mice. No differences in VEGF protein levels were ob-
served in the soleus, plantaris, gastrocnemius, or EDL in
skmVEGF⫺/⫺ mice after 8 wk of training (Fig. 1) compared
with untrained skmVEGF⫺/⫺ mice.
Exercise capacity in response to treadmill exercise training.
Maximal running speed was unchanged from pre-tamoxifen
level in VEGF⫹/⫹ and skmVEGF⫺/⫺ mice 21 days after
administration of tamoxifen (tested before exercise training).
Maximal speed increased by 18% in VEGF⫹/⫹ mice after 8
wk of training (VEGF⫹/⫹ UN, 60.11 ⫾ 1.5 cm/s; VEGF⫹/⫹
EX, 69.8 ⫾ 1.1 cm/s; P ⬍ 0.01). No difference in maximal
speed of skmVEGF⫺/⫺ mice was observed after training
(skmVEGF⫺/⫺ UN, 57.4 ⫾ 1.1 cm/s; skmVEGF⫺/⫺ EX,
56.2 ⫾ 4.4 cm/s) (Fig. 2A). On day 22 (but not at the later
11-wk time point) post-tamoxifen, skmVEGF⫺/⫺ mice exhib-
ited a 30% reduction in the time to exhaustion compared
with untrained, VEGF⫹/⫹ mice (skmVEGF⫺/⫺, 60.2 ⫾ 6.1
min; VEGF⫹/⫹, 86.1 ⫾ 6.6 min; P ⬍ 0.01). However, the
skmVEGF
⫺/⫺ EX group did not improve endurance capacity
with exercise training. WT VEGF⫹/⫹ mice increased the time
to reach exhaustion by 99% (time to exhaustion: VEGF⫹/⫹
UN, 84.1 ⫾ 7.8 min, VEGF ⫹/⫹ EX, 167.3 ⫾ 5.1 min, P ⬍
0.01) (Fig. 2B).
Body weights and skeletal muscle mass. Body mass
decreased by 11.2% in VEGF⫹/⫹ mice in response to 8 wk of
exercise training (VEGF ⫹/⫹ UN, 24.43 ⫾ 0.70 g;
VEGF ⫹/⫹ EX, 21.68 ⫾ 0.27 g; P ⬍ 0.01) but did not change
in skmVEGF⫺/⫺ mice. No change in muscle mass-to-body
weight ratio (mg/g) was observed in the soleus, plantaris,
gastrocnemius, or EDL between groups (Table 1).
Ex vivo muscle contractile function. Maximal tetanic force
(Fig. 3, A and C) and the force-frequency relationship (Fig. 3,
B and D) were not affected by exercise training in the soleus
and EDL. Maximal tetanic force was reduced in the soleus
muscle of skmVEGF⫺/⫺ EX mice, yet this did not change the
force-frequency relationship (Fig. 3, A and B). EDL isolated
from exercise-trained skmVEGF⫺/⫺ revealed an increase in
the time to fatigue, or improved fatigue resistance, compared
with the other three groups (Fig. 3E). Exercise training or
VEGF gene deletion did not alter isolated soleus fatigue
resistance (Fig. 3E).
Angiogenic response to exercise training. After training,
VEGF⫹/⫹ EX mice increased capillary-to-fiber ratio in the
plantaris (40%) and gastrocnemius (43%). Capillary density in
VEGF⫹/⫹
mice also increased in the soleus (27%), plantaris
(38%), and gastrocnemius (49%). Fiber area was unchanged
between untrained and exercise-trained VEGF⫹/⫹ mice. In
the skmVEGF⫺/⫺ mice, the only muscle to demonstrate an
angiogenic response to training was the plantaris. Capillary-to-
fiber ratio and capillary density were increased by 18% and
25%, respectively, in the plantaris of skmVEGF⫺/⫺ mice.
skmVEGF⫺/⫺ gastrocnemius mean fiber area was reduced by
18% and accompanied by a 6% increase in capillary density
(Table 2).
Arteriogenesis and skeletal muscle remodeling. The number
of ␣-smooth muscle actin (␣SMA⫹) arteries per gastrocne-
mius complex cross-sectional area increased with both exercise
training and VEGF gene ablation. There was no interaction
between these two conditions (Fig. 4, B and D). Analysis of
arterial size revealed more arteries with diameters in the 30- to
60-m range in skmVEGF⫺/⫺ mice, both with and without
exercise training, compared with the VEGF⫹/⫹ groups.(Fig.
4, B and E). BrdU-positive cells were present in areas of
increased vimentin⫹ fibroblasts (Fig. 4A). Quantification of
Fig. 1. VEGF levels in hindlimb muscles from wild-type and skeletal myofiber VEGF gene-ablated mice. VEGF protein in the soleus (A), gastrocnemius (B),
plantaris (C), and extensor digitorum longus (EDL) from VEGF⫹/⫹ and skeletal myofiber-specific VEGF gene-deleted (skmVEGF⫺/⫺) mice (D) that were
either exercise trained (TRAINED) or remained cage-confined (UNTRAINED). Values are means ⫾ SE, P ⬍ 0.01. *P indicates a significant difference between
genotype within the same UNTRAINED or TRAINED group, #P indicates a significant difference between exercise condition within the same genotype.
R588 VEGF-DEPENDENT RESPONSES TO EXERCISE TRAINING
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00522.2013 • www.ajpregu.org
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (137.110.076.185) on July 3, 2018.
Copyright © 2014 American Physiological Society. All rights reserved.
BrDU⫹ cell per myofiber revealed increases in the gastrocne-
mius of UN and EX skmVEGF⫺/⫺ mice compared with
UN and EX VEGF⫹/⫹ groups, respectively (skmVEGF⫺/⫺
UN, 41 ⫾ 2%; VEGF⫹/⫹ UN, 11 ⫾ 2%; P ⬍ 0.01.
skmVEGF⫺/⫺ EX, 50 ⫾ 3%; VEGF⫹/⫹ EX, 29 ⫾ 2%; P ⬍
0.01). Exercise training, alone, also increased BrdU incorpo-
ration in VEGF⫹/⫹ muscles (Table 3). Vimentin-positive
cells around the myofibers and in vessel walls also increased
with VEGF gene deletion and were augmented with exercise
training. Vimentin⫹ cells per myofiber increased in both
UN and EX skmVEGF⫺/⫺ mice compared with VEGF⫹/⫹
UN and VEGF⫹/⫹ EX groups in the gastrocnemius
(skmVEGF⫺/⫺ UN, 51 ⫾ 7%; VEFG⫹/⫹ UN, 26 ⫾ 4%, P ⬍
0.01. skmVEGF⫺/⫺ EX, 87 ⫾ 1%; VEGF⫹/⫹ EX, 35 ⫾ 4%;
P ⬍ 0.01). Gastrocnemius sections from exercise-trained sk-
mVEGF⫺/⫺ mice displayed clusters of numerous vimentin
⫹/
BrdU⫹ cells around vessels (Table 3). In addition, numerous
inflammatory macrophages (F4/80⫹ cells) were present in
untrained skmVEGF⫺/⫺ (Fig. 4C).
Metabolic response to exercise training. Citrate synthase
(CS) activity increased 49%, 44%, 54%, and 13% in the soleus,
plantaris, gastrocnemius, and EDL, respectively, of VEGF⫹/⫹
mice after training. skmVEGF⫺/⫺ mice increased CS activity
by only 21%, 22%, and 28% in the soleus, plantaris, and
gastrocnemius, respectively. -Hydroxyacyl CoA dehydroge-
nase (-HAD) activity, a marker for fatty acid -oxidation,
increased in the soleus (36%), plantaris (29%), gastrocnemius
(28%), and EDL (14%) (P ⬍ 0.01) in VEGF⫹/⫹ mice with
training. -HAD activity was not increased in response to
training in skmVEGF⫺/⫺ mice but was higher in the plantaris
and gastrocnemius in the untrained condition compared with
WT mice. Activity of the glycolytic enzyme phosphofructoki-
nase (PFK) increased in both VEGF⫹/⫹ and skmVEGF⫺/⫺
mice after training in the soleus (VEGF⫹/⫹, 17%; skmVEGF⫺/⫺,
11%), plantaris (VEGF⫹/⫹, 21%; skmVEGF⫺/⫺, 21%), gas-
trocnemius (VEGF⫹/⫹, 21%; skmVEGF⫺/⫺, 19%), and
EDL (VEGF⫹/⫹, 20%; skmVEGF⫺/⫺, 22%) (Fig. 5). Fiber-
type composition was unchanged in the soleus, plantaris, or
gastrocnemius. However, the EDL showed an increase (P ⬍
0.01) in type IIa fibers and reduction in IIb fibers in UN
skmVEGF⫺/⫺ mice compared with control UN VEGF⫹/⫹
mice (UN skmVEGF⫺/⫺, 11.6 ⫾ 1.2%; UN VEGF⫹/⫹,
6.4 ⫾ 0.6%). Eight weeks of exercise training increased (P ⬍
0.01) type IIa fibers in both skmVEGF⫺/⫺ and VEGF⫹/⫹
mice (skmVEGF⫺/⫺ EX, 13.1 ⫾ 1.4%; VEGF⫹/⫹ EX,
14.1 ⫾ 2.1%) (data not shown).
DISCUSSION
This study suggests that VEGF expressed by skeletal myo-
fibers is essential in adult mice to improve exercise capacity in
response to an 8-wk treadmill running program. The angio-
genic adaptation to treadmill running was completely or par-
tially inhibited depending on muscle type. Furthermore,
the training-induced increase in oxidative metabolic capa-
city, namely CS and -HAD activity, was attenuated in
skmVEGF⫺/⫺ mice. In contrast, the glycolytic response to exer-
cise training (PFK enzyme activity) was not altered, and
Fig. 2. Treadmill exercise capacity. Maximal speed (A) and endurance (B) were
tested in VEGF⫹/⫹ and skmVEGF⫺/⫺ mice at the following time points:
pre-tamoxifen (TAM) (day 0), 21 days post-tamoxifen to conditionally delete
the VEGF gene, and 8 wk after cage confinement or exercise training (day 78).
Values are means ⫾ SE, P ⬍ 0.01. *P indicates a significant difference
between genotype within the same untrained or exercissed group, #P indicates
a significant difference after exercise training within the same genotype (n ⫽
9 –10).
Table 1. Body weights and skeletal muscle mass
VEGF⫹/⫹ skmVEGF⫺/⫺
Untrained (n ⫽ 9) Exercise Trained (n ⫽ 10) Untrained (n ⫽ 9) Exercise Trained (n ⫽ 10)
Body mass, g 24.4 ⫾ 0.7 21.7 ⫾ 0.2* 23.0 ⫾ 0.7 22.2 ⫾ 0.4
Soleus, mg/g 0.31 ⫾ 0.01 0.31 ⫾ 0.01 0.30 ⫾ 0.01 0.29 ⫾ 0.01
Plantaris, mg/g 0.74 ⫾ 0.08 0.77 ⫾ 0.06 0.68 ⫾ 0.05 0.68 ⫾ 0.03
Gastrocnemius, mg/g 4.08 ⫾ 0.19 4.57 ⫾ 0.23 3.73 ⫾ 0.23 4.07 ⫾ 0.16
EDL, mg/g 0.32 ⫾ 0.01 0.36 ⫾ 0.02 0.33 ⫾ 0.01 0.30 ⫾ 0.03
Values are means ⫾ SE; n is number of mice. skmVEGF⫺/⫺, skeletal myofiber-specific vascular endothelial growth factor (VEGF) gene-deleted mice; EDL,
Extensor digitorum longus. *P indicates a significant difference after exercise training within the same genotype (n ⫽ 9 –10).
R589VEGF-DEPENDENT RESPONSES TO EXERCISE TRAINING
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00522.2013 • www.ajpregu.org
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (137.110.076.185) on July 3, 2018.
Copyright © 2014 American Physiological Society. All rights reserved.
