To investigate temporal changes in capillarization and increases
in mitochondrial enzyme activity, rabbit tibialis anterior muscles
underwent chronic low-frequency stimulation for up to 50 days.
Capillary density (CD), capillary-to-fiber ratio (C/F), intercapillary
distance (ICD), and mean capillary area (MCA), as well as several other
parameters of capillarization, were examined. In addition, tissue
levels of mRNA specific to vascular endothelial growth factor (VEGF)
were assessed by reverse transcriptase-polymerase chain reaction.
Citrate synthase (CS) activity, a marker of aerobic-oxidative metabolic
potential, was measured in the same muscles. Significant increases in
CD and C/F, respectively, and decreases in ICD and MCA were observed
after 2 days. These changes reached stable maxima by 14 days. The
increases in capillarization occurred in a fiber-type-specific manner,
affecting type IId fibers before types IIda and IIa. VEGF mRNA levels increased in a bimodal time pattern with a first elevation (2.5-fold) after 1 day and a second (9-fold) after 6-8 days.
Increases in CS were first noted after 8 days. Obviously, increases in
capillarization as induced by enhanced contractile activity precede
increases in the aerobic-oxidative potential of energy metabolism.
electrical stimulation; mitochondrial enzyme activity; morphometry; vascular endothelial growth factor
 |
INTRODUCTION |
NUMEROUS STUDIES HAVE shown that chronic low-frequency
stimulation (CLFS) transforms fast-twitch, fast-fatigable muscles into slow-twitch, fatigue-resistant muscles (for review, see Ref. 22). In
rabbit extensor digitorum longus (EDL) and tibialis anterior (TA)
muscles, this process encompasses sequential fiber-type transitions in
the order of type IId/x
type IIa
type I. These
fiber-type transitions correspond to changes in myosin heavy chain
(MHC) isoform composition in the order of MHC IId/x
MHC IIa
MHC I
(18). In addition, CLFS elicits profound alterations
in the metabolic profile of the muscle fibers, such as severalfold
increases in mitochondrial volume and enzyme activities of terminal
substrate oxidation, concomitant with reduced glycogenolytic and
glycolytic enzyme activities (22). CLFS has also been shown to have
strong effects on capillarization, leading to pronounced increases in capillary density (CD) and enhanced perfusion and oxygen supply (3, 4,
9, 14-17, 20). Together, these changes may contribute to the enhanced aerobic-oxidative capacity and fatigue resistance of
chronically stimulated muscles.
EDL and TA muscles of the rabbit are fast-twitch muscles with a mixed
fiber population. As judged from MHC analyses, the two muscles
predominantly contain type IId/x and a smaller fraction of type IIa
fibers (1). These two fiber types differ with regard to their
myofibrillar protein isoform patterns and their metabolic profiles
(21). In view of these differences, the question arises whether CLFS
equally affects both fiber populations and, generally, whether changes
in capillarization and mitochondrial enzyme activity follow similar or
different time courses. It is not easy to answer this question on the
basis of the existing literature because, due to different experimental
conditions, studies from different laboratories are difficult to
compare. With the exception of an investigation from our laboratory,
previous morphometric studies on capillarization and quantitative
measurements of mitochondrial enzyme activities have not been conducted
on the same samples in the same experimental series. The results of our
study indicated that stimulation-induced increases in capillarization
may precede increases in mitochondrial enzyme activities (24).
The purpose of the present study was to investigate in more detail
temporal changes in capillarization and oxidative enzyme activity in TA
muscles of rabbit exposed to CLFS for different durations. The animals
were stimulated between 1 and 50 days. To relate changes in
capillarization to specific fiber types, analyses were performed on
fibers histochemically classified according to their myofibrillar
actomyosin ATPase (mATPase) activity. Because muscles stimulated for
longer than 8 days exhibit pronounced histochemical signs of fiber-type
transitions, the analysis of changes in capillarization with regard to
fiber types was restricted to the early phase of CLFS. With the use of
a newly developed software for image analysis, changes in
capillarization were assessed on the basis of several parameters. In
addition to the commonly investigated parameters such as CD,
capillary-to-fiber ratio (C/F), and number of capillaries around fibers
(CAF), we examined intercapillary distance (ICD) and diffusion distance
(DD), the capillary arrangement around fibers (CA), and a newly defined
capillary supplying factor (SUPF). The study of these parameters
appeared to be meaningful because muscle fibers are not a uniform
population with regard to size and shape, as well as with regard to the
CA. To correlate morphometrically assessed alterations in
capillarization with the expression of the vascular endothelial growth
factor (VEGF), thought to control adaptive changes of the capillary
bed, we studied changes in tissue levels of VEGF mRNA by reverse
transcription-polymerase chain reaction (RT-PCR). Finally, citrate
synthase (CS) activity, a mitochondrial marker of terminal substrate
oxidation and aerobic-oxidative potential, was biochemically determined
in the same samples used for morphometry. Because quantitative
microphotometric measurements of succinate dehydrogenase (SDH) activity
in single muscle fibers had previously been performed on the same
muscles investigated in the present study, these results were included,
since they provided additional information on the time-dependent
changes in aerobic-oxidative metabolic potential.
 |
METHODS |
Animals and muscles.
Adult male White New Zealand rabbits (initial body wt 3,170 ± 290 g
and 4,540 ± 130 g after 50 days of CLFS) were used. CLFS (10 Hz, 12 h/day, 1 h on-1 h off) of the left TA muscle was performed via
electrodes implanted laterally to the peroneal nerve (26). After
different periods of stimulation (0, 2, 4, 6, 8, 10, 14, 21, and 50 days), the animals (3 for each time point) were killed and TA muscles
from both the stimulated and contralateral hindlimb were excised and
weighed and thin longitudinal fiber bundles were dissected from the
superficial layer of the midbelly region. The fiber bundles were
divided transversely, with half for biochemical studies and the other
half for morphometric analysis. The bundles were quickly frozen in a
slightly stretched position in melting isopentane (
159°C).
Cross sections (9 µm thick) of composite blocks from stimulated and
the corresponding contralateral muscles were cut on a microtome in a
cryostat at
25°C. For the study of changes in VEGF
expression, animals stimulated for up to 14 days were used. To assess
the early changes, additional rabbits stimulated for 1 and 3 days (3 for each time point) were included in the study.
Histochemical analysis.
Serial cross sections of TA muscles were mounted on glass coverslips,
air dried, and subsequently stained for mATPase as previously described
(8). Capillaries were identified by two methods. Method 1 was the same as used for
mATPase, but the preincubation step was performed at pH 3.80. Method 2 was based on a modification of the immunohistochemical staining for fibronectin, originally described by Erzen and Maravic (5). A mouse anti-human monoclonal antibody (Boehringer Mannheim) was used in the primary immunoreaction, and a peroxidase-coupled anti-mouse immunoglobulin Fab fragment from
sheep (Boehringer Mannheim) was used for visualization. Cryosections were air dried and fixed for 10 min in acetone at
20°C. In
accordance with manufacturer's instructions, sections were then
incubated for 20 min in 5% fetal calf serum in phosphate-buffered
saline (PBS) for inhibition of nonspecific reaction. Subsequently, the sections were covered with the primary antibody (1:4 diluted in PBS,
containing 1% bovine serum albumin) and incubated for 60 min in a
humid chamber at room temperature. After sections were washed in PBS,
they were incubated for 60 min at room temperature with the secondary
antibody (1:60 in PBS, containing 1% bovine serum albumin). After an
extensive washing in PBS, bound peroxidase was visualized by incubation
in a solution containing 0.05% 3,3'-diaminobenzidine (Sigma), 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.3), 0.08%
H2O2,
and 0.06% NiCl2. After a washing
in PBS, the sections were dehydrated and embedded in Entellan (Merck).
Control sections were treated in the same way, except the incubation
with the primary antibody was omitted.
Morphometry.
Measurements were taken with ×25 magnifying objective. Evaluation
of morphometric data was performed by image analysis using a Leitz
Orthoplan (Wetzlar, Germany) microscope with a scanning stage and an
attached video camera (Bosch model TYK 91D, Vidicon tube; linearity
95%). A personal computer (Intel 586, 100 MHz, 1 GB hard disk, 16 MB
RAM, 250 MB streamer) with a frame grabber (Vision EZ, Data
Translation) was used for analysis. The resolution was 768 × 512 pixels with 8 bits/pixel, corresponding to 256 gray levels. Digitized
pictures were displayed in pseudocolors on the computer monitor, and
single fiber areas were marked by surrounding their circumference with
a computer mouse. Up to six different fiber types could be marked on
the computer screen by selecting different color codes. Similarly, the
computer mouse was used for marking the capillaries. Capillaries in the
periphery of the field of vision, i.e., marginal to peripheral fibers,
were delineated and marked in a specific color. Video pictures were
independently displayed on a monochrome monitor to compare original and
digitized pictures on the computer monitor. Digitized pictures,
additional information, and computed results (fiber masks, fiber-type
labels, x and
y coordinates, point of gravity,
capillary marks, comments, and so forth) were stored on the hard disk
of the computer.
Morphometric characteristics of muscle fibers.
In addition to fiber number and percentage of individual
mATPase-defined fiber types, we evaluated the following
fiber geometries (Fig. 1):
1) cross-sectional area (CSA),
2) maximum and minimum diameters
(DIAmax and
DIAmin, respectively), and
3) fiber shape factor. The computer
program was designed to automatically evaluate these characteristics
from the marked capillaries and fiber areas.

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Fig. 1.
Schematic representation of morphometric parameters related to fiber
cross-sectional area (CSA) and capillarization in cross-sectioned
skeletal muscle. CA, capillary arrangement; CAF, capillary around
fiber; DIAmax,
DIAmin, maximum and minimum
diameters, respectively; DD, diffusion distance; ICD, intercapillary
distance; PG, point of gravity; SUPF, supplying factor.
A-H
represent areas of fiber subdivided into 4 segments of equal length,
which partition the total fiber area into 8 sectors.
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The CSA was defined as
The
marked areas were evaluated as pixels and transformed into micrometers
squared.
For DIAmin and
DIAmax, the coordinates of the
point of gravity and the area of the fiber cross section were used to
calculate an equivalent ellipse.
DIAmax was calculated from the
longest distance between the point of gravity and the polygon. With the use of DIAmax and area of the
ellipse, DIAmin was calculated
using the equation (30)
The shape factor was defined as the ratio
DIAmax/DIAmin
according to Ref. 28.
Morphometric evaluation of capillarization was based on the following
characteristics. 1) CD,
which was defined as the number of capillaries per millimeter squared.
The number of capillaries was counted for all fibers in the field of
vision. For fibers in the periphery of the field cut by the frame of
the vision field, an area corresponding to one-half of the area of the
marginal fiber cross section was added. In contrast to restricting
capillary counting to the field of vision ("closed field
evaluation"), this "open field" evaluation reduces errors due
to the so-called edge effect. Other characteristics used for
morphometric evaluation of capillarization were
2) C/F, defined as the number of capillaries per individual fiber, and 3) CAF
type, defined as the number of capillaries in contact with a specific
fiber type. To assign capillaries to individual fibers, the computer
program constructs frames (10 pixels large) around every fiber, and
capillaries falling into this frame are automatically assigned to the
corresponding fiber. Other characteristics included
4) ICD, defined as the distance in
micrometers between next neighbor capillaries (2);
5) DD, defined as the mean value of
the distances (in µm) between point of gravity of a given fiber and
all capillaries in contact with it;
6) mean capillary area (MCA),
defined as the total CSA of all muscle fibers in a vision field divided
by the number of capillaries in the same field (the value is given in
µm2); and
7) SUPF, which relates to the areas
supplied by a given capillary that is in contact with several fibers of
different types and/or size. The domain area of the capillary
is calculated from the sum of areas supplied in the adjacent fibers.
These areas are limited by the point of gravity and the half distance
between a specific capillary and its next neighbor.
8) CA, describing the topography of
capillaries around muscle fibers, was also used for morphometric
evaluation of capillarization. For this type of evaluation, the fiber
area is transformed into an ellipse and DIAmax is subdivided into four
segments of equal length that partition the total fiber area into eight
sectors, with four (A,
D, E,
H) in the two end regions and four
(B,
C, F,
G) in the middle region as referred
to DIAmax. The program assigns a
given capillary according to its coordinates to a single sector and
also computes the ratio between capillaries in end and middle regions
of the fiber.
CS activity.
Frozen muscle tissue was pulverized under liquid
N2 in a small steel mortar and
homogenized (1:20, wt/vol) in a cold 100 mM sodium-potassium phosphate
buffer (pH 7.2) containing 2 mM EDTA. The homogenate was sonicated
three times for 30 s under intense cooling, stirred for 15 min on ice,
and centrifuged for 10 min in a refrigerated Eppendorf centrifuge. The
supernatant fraction was decanted and used for determining CS activity
in a coupled optical test (24).
Preparation of total RNA.
Frozen muscle was pulverized under liquid
N2 and homogenized (1:20, wt/vol)
in cold TriStar reagent (AGS, Heidelberg, Germany). Total RNA was
isolated according to the producer's instructions using three
modifications. 1) Proteins and
unsoluble material were removed after homogenization by 10-min
centrifugation at 12,000 g (+4°C).
2) Phase separation was performed
using 1-bromo-3-chloropropane (Fluka, Buchs, Switzerland).
3) For RNA precipitation, 0.25 ml isopropanol and 0.25 ml of a solution containing 1.2 M sodium citrate
and 0.8 M NaCl were used. Pellets were resuspended in 40 µl of
diethyl pyrocarbonate (DEPC)-treated
H2O, and RNA concentration was
assessed spectrophotometrically. Each preparation was diluted to 1 µg
RNA/µl in DEPC-treated H2O.
Oligonucleotide primers.
Oligonucleotide primers specific to a sequence common to VEGF1 (GenBank
accession no. S38083), VEGF2 (GenBank accession no. S38100), and VEGF3
(GenBank accession no. S37052) from mouse (positions 184-409) were
derived and compared for sequence similarity with other species. Sense
primer and antisense primers were derived from sequences identical in
mouse, rat, guinea pig, pig, bovine, macaque, and human. The sense
primer was GTG GAC ATC TTC CAG GAG TA; the antisense primer was TCT TTG
GTC TGC ATT CAC A. For
-skeletal actin (GenBank accession no.
J00692), the sense primer was (23) CGC GAC ATC AAA GAG AAG CT; the
antisense primer was GGG CGA TGA TCT TGA TCT TC.
cDNA synthesis, PCR, and product detection.
cDNA synthesis was performed with 2 µl of RNA stock solution in 10 µl volume using the following assay mixture: 10 units avian myeloblastosis virus reverse transcriptase (Pharmacia, Uppsala, Sweden), 12.5 units RNAguard (Pharmacia), 0.625 mM each deoxynucleoside triphosphate, 1 µM specific antisense primer, 50 mM
Tris · HCl (pH 8.3), 8 mM
MgCl2, and 30 mM KCl. Incubation
lasted for 60 min at 50°C. PCR was performed on several dilutions
of the RT assay. One microliter of template was transferred into 24 µl of the PCR incubation mixture for VEGF consisting of 10 mM
Tris · HCl (pH 9.0), 1.5 mM
MgCl2, 50 mM KCl, 0.2 µM
antisense and sense primers, 0.25 mM dNTPs, and 0.75 units
Taq polymerase (Pharmacia). The assay
mixture for
-skeletal actin PCR differed in the amount of
MgCl2 (2 mM) and a lower pH of
8.3. The annealing temperature was 57°C for VEGF and 59°C for
-skeletal actin. The number of cycles was 28 for VEGF and 22 for
actin. Product detection [226 nucleotides for VEGF, 367 for
-skeletal actin (23)] was performed after
electrophoresis on a 6% polyacrylamide gel by ethidium bromide staining. The signals were photographically documented and evaluated by
densitometry. At least two measurements were performed on each sample
(animal and time point). Changes in mRNA amounts were evaluated by
comparison with the amounts of products amplified from cDNA dilutions
of control muscles. Dilutions of 1:10, 1:20, 1:40, and 1:100 were used
throughout. To correct for changes in total RNA, the results were
normalized to actin mRNA levels in the same preparations.
The specificity of the amplified PCR product for VEGF was verified by
sequence analysis (Fig. 2). Comparison with known
sequences from other mammals yielded the following sequence identities: 92% for cynomolgus monkey (VEGF165, GenBank accession no. S82167) and
92% for human (VEGF gene Homo
sapiens, GenBank accession no. X62568; human heparin-binding VEGF
mRNA, GenBank accession no. M32977; human vascular permeability factor
mRNA, GenBank accession no. M27281). Sequence identity amounted to 87%
for the three VEGFs from mouse.

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Fig. 2.
cDNA sequence of the amplified reverse transcription-polymerase chain
reaction (RT-PCR) product specific to rabbit vascular endothelial
growth factor (VEGF) mRNA (GenBank accession no. AF022179). Sequences
from which sense and antisense primers were derived are printed in
bold.
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Statistical analyses.
A paired t-test was used to determine
if differences existed between methods
1 and 2 for capillary
staining in serial sections. An unpaired
t-test was applied to evaluate
differences in fiber types, morphometric data, and VEGF mRNA levels
between stimulated and control muscles for animals stimulated for
different time periods. Time-dependent changes in capillary
characteristics and CS activity were analyzed using a general linear
model. When a significant
F ratio was obtained, the Duncan's
multiple range test (SAS Statistical Software Package, SAS
Institute) was used to determine differences between time
points. The acceptable level of significance was set at
P < 0.05.
 |
RESULTS |
Changes in fiber-type distribution.
As judged from mATPase histochemistry, low-frequency stimulation
induced changes in fiber-type distribution as early as 4 days after
stimulation onset. In 4-day-stimulated muscle, the fraction of type IId
fibers was reduced by ~12%, but this decrease was compensated for by
a corresponding increase in type IIda fibers (Table
1). Whereas no
further changes were observed in the distribution of type IId and type
IIda fibers at 6 days, a major shift from type IId to type IIda fibers
occurred between 6 and 8 days. Type IId fibers could no longer be
delineated in 8-day-stimulated muscles. By this time, the fiber
composition was characterized by a shift from type IIda to type IIa
(Table 1).
Changes in fiber CSA and shape.
Early changes in fiber-type distribution were accompanied by
alterations in CSA. In agreement with previous results on rabbit adductor magnus and EDL muscles (8), type IId fibers in normal TA
muscle were found to be larger than type IIa fibers (Table 2). This pattern was markedly altered in
4-day-stimulated muscles in which type IIda and type IIa fibers
displayed a significantly enlarged CSA
(P < 0.01). On the basis of previous
observations (10, 19), this increase most likely resulted from a
transient edema. Compared with the 4-day values, the CSA of type IIda
and IIa fibers were smaller again in 6-day-stimulated muscles. Compared with 2-day-stimulated muscles, the mean CSA of type IId fibers was
unaltered at 4 days, although it was elevated relative to the
contralateral muscles. In 6-day-stimulated muscles, the CSA of type IId
fibers was also markedly reduced (P < 0.001), and this reduction continued with the growing populations
of type IIda and type IIa fibers. Changes in
DIAmin displayed similar trends
but were less pronounced than the changes in CSA (Table 2).
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Table 2.
Cross-sectional area and minimum diameter of fiber types in
contralateral and low-frequency-stimulated TA muscles
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When no distinction was made between the different fiber types, changes
in mean values of CSA were characterized by an initial transient
increase followed by a significant decrease (Fig. 3). Similar changes occurred in whole muscle weight, namely a transient increase during the first 4 days followed by a pronounced decrease. Thereafter, the weight of the stimulated muscle was stable. However, compared with the contralateral muscle, which, due to the animals' growth, increased in weight, it became evident that longer stimulation periods led to pronounced decreases in muscle weight (Fig. 3). On
average, 50-day-stimulated TA muscles weighed 40% less than the
corresponding contralateral muscles.

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Fig. 3.
Time-dependent changes in muscle weight ( ) and in CSA ( ) of
chronically stimulated rabbit tibialis anterior (TA) muscle.
Inset: weight changes of stimulated
muscles were referred to the weight of contralateral muscles.
* Significant differences between time points
(P < 0.05).
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As reflected by alterations in the fiber shape factor, CLFS led to a
10% reduction in the ratio between fiber
DIAmax and
DIAmin (Table
3). As indicated by a decrease in CA, the
distribution of the capillaries around muscle fibers also changed such
that the number of capillaries adjacent to the middle region of fibers increased in 8-day-stimulated muscle. Therefore, the ratio between capillaries in the end and middle regions of the fiber declined. However, longer stimulation periods did not lead to further decreases.
Histochemical identification of capillaries.
Two approaches were chosen for histochemically identifying capillaries
in muscle cross sections, mATPase staining after acid preincubation and
immunohistochemical staining of fibronectin. To compare the reliability
of the two methods, mATPase and fibronectin stainings were evaluated on
serial sections from all contralateral and stimulated muscles. As
documented by the results from 12,696 fibers and 34,251 capillaries,
both methods yielded identical results in the muscles under study
(Table 4).
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Table 4.
Comparison between histochemically and immunohistochemically
identified capillaries in serial cross sections of
contralateral and low-frequency-stimulated TA muscles
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Changes in CD, C/F, ICD, and MCA.
CLFS induced pronounced increases in capillarization. This was
reflected by steep elevations in CD and C/F, as well as by decreases in
ICD and MCA (Figs. 4 and 5). An almost
immediate effect of CLFS was observed in 2-day-stimulated muscles.
Although increases in CD reached significance only by 6 days (Fig. 4), the decreases in ICD and MCA were significant 2 days after the onset of
stimulation (Figs. 4 and 5). These changes continued, reaching their
maxima in muscles stimulated for 2 wk. Longer stimulation periods did
not lead to further changes.

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Fig. 4.
Time course of changes in intercapillary distance ( ) and capillary
density ( ) in low-frequency-stimulated rabbit TA muscle.
* Significant differences between time points
(P < 0.05).
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Fig. 5.
Time course of changes in mean capillary area ( ) and
capillary-to-fiber ratio ( ) in low-frequency-stimulated rabbit TA
muscle. * Significant differences between time points
(P < 0.05).
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The stimulation-induced changes in C/F displayed a bimodal time course.
Increases were first observed in 2-day-stimulated muscles. No further
change was noted until day 8. However,
a second increase occurred between 8 and 10 days (Fig. 5). This second rise in C/F was higher than the first increase after 2 days. Most likely, the transient increase in CSA due to an edema (Fig. 3) partially masked an initial increase in the C/F, which could be expected from the rise in CD (Fig. 4).
This interpretation is in agreement with the changes in ICD (Fig. 4).
Two-day-stimulated muscles exhibited significantly reduced ICD values,
but no further decrease was seen after 4 days. However, ICD was
progressively reduced in muscles stimulated for more than 4 days.
Minimum values were reached after 14 days when the ICD had decreased by
~45% compared with 0-day control muscles. No further decreases
occurred with CLFS up to 50 days (Fig. 4).
The changes in MCA (Fig. 5) resembled those of ICD. With the exception
of a transient break at 4 days, there was a progressive decrease in
MCA, reaching its lowest value after 14 days. CLFS for longer time
periods did not lead to further reductions.
Fiber-type-specific changes in capillarization.
To investigate fiber-type-specific responses to CLFS, changes in
capillarization were evaluated with regard to specific fiber types.
Fiber-type-specific patterns of capillarization were obvious in
unstimulated muscles. Thus, as judged from Table
5, type IId fibers had fewer
capillaries and longer DDs than IIda and IIa fibers. The difference
between fiber types became smaller in stimulated muscles. First,
increases in CAF and SUPF, as well as decreases in DD, were noted in
type IId fibers after 2 days. These changes progressed in 6-day- and
8-day-stimulated muscles, although type IId fibers could no longer be
delineated from type IIda fibers at 8 days. As seen in Table 5, type
IIda fibers exhibited first changes in these three parameters after 6 days. These early changes seemed to be less pronounced in type IIa
fibers, with significant increases in SUPF only after 8 days.
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Table 5.
Number of capillaries around fiber, supplying factor, and diffusion
distance in different fiber types of contralateral and
low-frequency-stimulated TA muscles
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Stimulation-induced changes in VEGF mRNA content.
Stimulation led to pronounced increases in the tissue level of mRNA
specific to VEGF (Fig. 6). Its increase followed a
bimodal time course with a first elevation (2.5-fold) in
1-day-stimulated muscles and a second rise (9-fold) by
days 6-8. Thereafter, VEGF mRNA
started to decay. Compared with its initial level, it was still
2.9-fold elevated in 14-day-stimulated muscles.

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Fig. 6.
Time course of changes in VEGF mRNA. Tissue levels of VEGF mRNA and
-skeletal actin mRNA were determined in total RNA preparations by
RT-PCR. To correct for stimulation-induced changes in total RNA
content, VEGF mRNA data were referred to -skeletal actin mRNA levels
and set equal to one for unstimulated muscles at zero time point. ,
Contralateral; , stimulated. * Significant differences between
stimulated and control muscles (P < 0.05).
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Stimulation-induced increases in CS activity.
To correlate changes in capillarization with stimulation-induced
increases in the aerobic-oxidative potential of energy metabolism, CS
activity was measured in homogenates of the same muscles used for
histochemistry and morphometric analysis. Low-frequency stimulation for
up to 50 days induced a fourfold increase in CS activity. First,
significant increases were observed in muscles exposed to low-frequency
stimulation for periods longer than 8 days (Fig. 7).
A steep rise in activity occurred between 8 and 14 days. Thereafter, it
continued to increase at a lower rate. However, as indicated from the
slope of the curve, the maximum value was not yet attained by 50 days.
Obviously, the rise in total CS activity occurred much later than the
increases in capillarization. This observation is confirmed by the
increase in SDH activity previously investigated by quantitative
microphotometry in large numbers of single fibers from the same muscles
used in the present study (29). The increase in SDH activity,
therefore, has been included in Fig. 7. Except for an initial decrease
in CS, the time-dependent increases of the two enzyme activities were
nearly identical. Most likely, the initial decrease in CS activity
resulted from a transitory edema of the stimulated muscle. This
decrease was not observed by microphotometric determination of SDH
activity because measurements were performed on single fibers and
results were computed per unit fiber area.

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Fig. 7.
Time course of changes in citrate synthase activity ( ) and succinate
dehydrogenase (SDH) activity ( ) of low-frequency-stimulated rabbit
TA muscle. Values for SDH are from a previous study (29) in which
enzyme activity was assessed microphotometrically on the same muscles
analyzed in the present study. * Significant differences between
time points (P < 0.05).
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DISCUSSION |
The present study focuses on the temporal patterns of adaptive changes
in fiber morphology, capillarization, and aerobic-oxidative potential
of energy metabolism in response to sustained contractile activity by
CLFS. As shown in previous studies, CLFS leads to marked decreases in
muscle fiber CSA, as well as to enhanced capillarization and elevations
in mitochondrial enzyme activities of terminal substrate oxidation (for
review, see Ref. 22). These changes are thought to contribute to an
enhanced resistance to fatigue, a conspicuous property of chronically
stimulated muscle. Our interest to elucidate in more detail the time
course of these changes originates from a previous study in which we
showed that stimulation-induced elevations in CS activity and
improvements in resistance to fatigue did not occur in a strictly
coordinate manner, especially during the first days when improvements
in resistance to fatigue preceded the rise in CS activity (27). Maximum
increase in resistance to fatigue was observed after 14 days, whereas
CS continued to rise with longer stimulation periods. This discrepancy
suggested that additional factors, such as early increases in perfusion and capillarization, might contribute to enhanced fatigue resistance. Increases in capillarization had previously been observed in 2-day- (24) and 4-day-stimulated rabbit TA muscles (3).
The present results confirm and extend these findings. However,
increases in capillarization and mitochondrial enzyme activities seem
to follow different time courses. This discrepancy is most conspicuous
during the first days of CLFS but is evident also with prolonged
stimulation. Whereas significant changes in ICD, C/F, and MCA first
occur as soon as after 2 days and progress with ongoing stimulation,
increases in enzyme activities of terminal substrate oxidation become
evident only several days later. Significant elevations in CS and SDH
activities are recorded after stimulation periods exceeding 8 days.
However, as previously shown by studies on key metabolites of energy
metabolism, 8-day-stimulated TA muscle exhibits properties suggesting
that its energy supply is no longer "glycolytic" but is
increasingly based on oxidative phosphorylation (7). Because this
change occurs before substantial increases in mitochondrial enzyme
activities, we suggest that an elevated capacity for oxygen supply by
increased capillarization is a major factor contributing to this
qualitative switch of energy metabolism.
Our findings on the effects of sustained contractile activity suggest
that elevations in the capacity of mitochondrial end oxidation follow,
but do not precede, enhanced capillarization. In other words, the
differences between the temporal patterns of improved capacities for
local oxygen supply and oxygen consumption clearly point to sequential
inductions, i.e., improved oxygen supply first and mitochondrial
induction second.
C/F has been reported to be tightly coupled to muscle mitochondrial
volume density in 28-day-stimulated fast-twitch muscle of rat (20).
However, as shown in the present study, increases in capillarization
and mitochondrial volume density must not necessarily occur in parallel
to attain a stable ratio in long-term-stimulated muscle. Interestingly,
CS and cytochrome oxidase activities reach maximum elevations in rat TA
muscle only by 30 days of CLFS (11, 27). In rabbit muscle, the
possibility must also be considered that damage and regeneration of
some fibers, passing through the myotube stage (19), may transiently
perturb the otherwise tight relationship between the aerobic-oxidative
potential of muscle fibers and the size of their capillary bed.
The continuing rise of mitochondrial enzyme activities after
capillarization has attained a maximum cannot be explained by the
present data but possibly relates to a qualitative switch of energy
supply. For example, due to continuing elevations in enzyme activities
involved in activation and transport and oxidation of fatty acids (24,
25), the latter may be utilized as major fuel in long-term-stimulated
muscles. It is noteworthy that, also, the activity levels of CS
(present study) and SDH (previous study, Ref. 29) have not reached
their maximum by 14 days but continue to rise with stimulation up to 50 days (Fig. 6). In view of these increases, the plateau of the CD, MCA,
and ICD values by 14 days supports the notion that maximum
capillarization has been attained by this time. This interpretation is
in agreement with data on blood flow in low-frequency-stimulated rabbit
muscles. Thus blood flow in contracting ankle flexors reaches maximum
values by day 14 (13).
The improvement in local oxygen supply relates to two complementary
processes, angiogenesis and decrease in fiber CSA. In the present
study, both seem to be more or less complete after 2-3 wk. As
indicated by the changes in absolute and relative weight (Fig. 3) and
transitory increases in CSA (Table 2), the muscle develops an edema
during the first days of stimulation, which seems to obscure the
initial increase in capillarization, as reflected by decreases in ICD
and MCA (Figs. 4 and 5).
Local increases in blood pressure and endothelial injury have been
suggested to be important factors initiating the formation of new
capillaries (12). In low-frequency-stimulated TA muscles of rat,
capillary growth has been related to an increased expression of an
endothelial cell-stimulating angiogenic factor (4). An independent
study on low-frequency-stimulated EDL and TA muscles of rat
demonstrated by Northern blot analyses that the mRNA specific to VEGF
was elevated during 3 wk. VEGF mRNA peaked in 4-day-stimulated muscles
(6-fold elevation) and declined thereafter (9). An even higher increase
(9-fold) is shown in the present study for rabbit muscle.
Interestingly, the increase in VEGF mRNA follows a bimodal time course.
Although our results refer to mRNA data, VEGF protein(s) might also
rise in a bimodal manner, following the changes in mRNA levels. This
suggestion is deduced from the bimodal time course of changes in the
C/F ratio with increases after 2 and 10 days (Fig. 5).
Three different splice variants have been suggested for VEGF in mouse
and four in the human (6). Therefore, the possibility exists that the
bimodal increase in VEGF mRNA in rabbit muscle relates to differential
expression of splice variants. The latter variants are not
distinguished by the sequence under study because this sequence seems
to code for a region common to all splice variants. A bimodal temporal
pattern could also be explained by assuming that an initial increase of
VEGF results from mRNA stabilization, whereas the second and major rise
results from enhanced transcription.
In summary, we show that CLFS leads to increases in capillarization as
soon as 2 days after the onset of stimulation. Changes in the CAF,
SUPF, and DD occur in a fiber-type-specific manner, affecting type IId
fibers before types IIda and IIa. Increases in VEGF mRNA precede the
changes of the capillary bed. As mirrored by the changes in CD, C/F,
ICD, and MCA, enhanced capillarization plateaus by 2-3 wk of CLFS,
whereas mitochondrial CS and SDH activities continue to increase in the
same muscles. These different time courses indicate that improvements
in oxygen supply precede the adaptive increases in aerobic-oxidative
potential of energy metabolism.
We thank Dr. Ted Putman and Michael Schuler for discussions and Elmi
Leisner for excellent technical assistance in the stimulation experiments.
This study was supported by the Deutsche Forschungsgemeinschaft,
Sonderforschungsbereich 156.
Address for reprint requests: D. Pette, Faculty of Biology, Univ. of
Konstanz, D-78457 Constance, Germany.