Acclimatization to 4100 m does not change capillary density or mRNA expression of potential angiogenesis regulatory factors in human skeletal muscle
1 The Copenhagen Muscle Research Centre at Rigshospitalet section 7652,
Blegdamsvej 9, 2100 Copenhagen Ø, Denmark
2 The August Krogh Institute, University of Copenhagen, Denmark
3 Rigshospitalet Section 9312, University of Las Palmas de Gran Canaria,
Spain
4 Department of Physical Education, University of Las Palmas de Gran
Canaria, Spain
* Author for correspondence (e-mail: carsten{at}cmrc.dk)
Accepted 3 August 2004
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Summary |
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Key words: transcription, skeletal muscle, capillary density, hypoxia, HIF-1, VEGF
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Introduction |
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The signal transduction pathways underlying capillarization are undoubtedly
complex. The transcription factor hypoxia-inducible factor-1
(HIF-1
) is increased with hypoxic exposure in many tissues, including
hypoxic rat muscle (Stroka et al.,
2001
). HIF-1
regulates many of the genes that are augmented
in hypoxia, and it is thought that the overall purpose of these genes and
their products is to increase O2 delivery to the cells.
HIF-1
has been recognized to cause an increased expression of at least
one important angiogenic factor, vascular endothelial growth factor (VEGF;
Jin et al., 2000
). VEGF mRNA
levels have been reported to increase with hypoxia in mouse brain
(Chávez et al., 2000
;
Kuo et al., 1999
) but also to
decrease in rat muscle (Olfert et al.,
2001b
). In human muscle, VEGF is increased with acute exercise
(Gustafsson et al., 2002
;
Richardson et al., 1999
).
However, it has not previously been studied whether VEGF expression is
increased in human muscle in response to hypoxia, and simultaneous
determination of these signalling factors and morphological characterization
of capillary density in human skeletal muscle has not previously been
performed in lowlanders acclimatizing to altitude or in high-altitude
natives.
The purpose of the present study was twofold. First, to test whether
skeletal muscle HIF-1 and VEGF mRNA content and morphologically
determined capillarization increase in lowlanders during the course of
acclimatization to high altitude compared with sea level. Second, to test
whether expression of HIF-1
and VEGF mRNA and capillarization are
higher in high-altitude natives compared with the levels observed in
lowlanders at both sea level and after prolonged acclimatization. Human
skeletal muscle is an interesting model because (1) it may be studied
repeatedly in the same subject by intravital biopsies, (2) skeletal muscle
microvasculature is characterized by homogenous structure, which facilitates
evaluation of changes, and (3) oxygen extraction from capillary blood is
higher in exercising skeletal muscle than in any other organ. To address
specifically whether any changes in capillarization were caused by hypoxia
per se, care was taken to avoid cold exposure, malnutrition,
gastroenteritis and detraining.
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Materials and methods |
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After being given both written and oral information on the experimental protocol and procedures, the subjects gave their informed, written consent to participate. The study conformed with guidelines laid down in the Declaration of Helsinki and was approved by the Copenhagen and Frederiksberg Ethics Committees (KF11-050/01) and by El Tribunal de Honor del Colegio Médico Departamental de La Paz and the Ministerio de Previsión Social y Salud Pública, La Paz, Bolivia.
Acclimatization and conditions in high altitude
The subjects were flown from Copenhagen (sea level) to La Paz in Bolivia.
Here, acclimatization started with two nights in a hotel at 3800 m, from where
the subjects moved to a well-equipped heated apartment in El Alto at 4100 m,
where they resided for the next 8 weeks. Subjects had ample access to food and
followed a varied diet rather similar to their usual diet before the
expedition. Subjects remained active throughout the stay with activities such
as cycling, soccer, basketball, hiking and rock climbing, to match their
reported activity level at sea level. The Bolivian Aymara high-altitude
natives were all born and raised around El Alto and La Paz and had lived their
whole lives at altitudes between 3700 and 4100 m. They were all employed or
active students and were physically active. The oxygen uptake per body mass at
high altitude was similar in the lowlanders and high-altitude natives.
Experimental procedure
All sampling was performed in hospital settings, either at sea level at the
Copenhagen Muscle Research Centre or at high altitude in a clinic at 4100 m
situated a few kilometres from the apartment. After local anaesthesia
(lidocaine), a resting muscle biopsy was obtained from the middle portion of
the vastus lateralis muscle. The muscle specimen was immediately frozen in
liquid nitrogen, transported to Copenhagen on dry ice and stored at
80°C for later analysis. The lowlanders had biopsies taken at sea
level (SLR) and after 2- (CH2) and 8-weeks (CH8) exposure to 4100 m of
altitude. The Aymara natives had one resting biopsy taken at high altitude.
The subjects were not physically active 24 h prior to biopsies.
Arterial blood sampling
Blood samples were obtained at rest from the femoral artery at sea level
and after breathing 12.6% O2 balanced in N2 for 15 min
at sea level (acute hypoxia) and after 2 and 8 weeks of acclimatization to
4100 m. Blood sampling from the high-altitude natives was done at 4100 m. For
further details see Lundby et al.
(2004).
RNA isolation, reverse transcription and PCR
To determine the mRNA content of HIF-1 and VEGF, total RNA
isolation, reverse transcription (RT) and PCR were carried out as follows.
Total RNA was isolated from
25 mg of muscle tissue by a modified
guanidinium thiocyanatephenolchloroform extraction method
adapted from Chomczynski and Sacchi
(1987
), as described previously
(Pilegaard et al., 2000
). RNA
was resuspended overnight (4°C) in 2 µl mg1 original
tissue mass in diethyl pyrocarbonate (DEPC)-treated H2O containing
0.1 mmol l1 EDTA. RT of 11 µl of total RNA sample was
performed using the Superscript II RNase H system (Invitrogen, Carlsbad, CA,
USA) as previously described (Pilegaard et
al., 2000
). RT products were diluted in nuclease-free
H2O to a total volume of 150 µl.
The mRNA content of the selected genes was determined by fluorescence-based
real-time PCR (ABI PRISM 7700 Sequence Detection System; Applied Biosystems,
Foster City, CA, USA). Forward (FP) and reverse (RP) primers and TaqMan probes
were designed from human specific sequence data (Entrez-NIH and Ensembl,
Sanger Institute) using computer software (Primer Express; Applied
Biosystems). The following sequences were used to amplify a fragment of
HIF-1 FP: 5'-GCCCCAGATTCAGGATCAGA-3'; RP:
5'-TGGGACTATTAGGCTCAGGTGAAC-3'; probe:
5'-ACCTAGTCCTTCCGATGGAAGCACTAGACAA-3'. The following sequences
were used to amplify a fragment of VEGF FP:
5'-CTTGCTGCTCTACCTCCACCAT-3'; RP:
5'-AGGAACAGATAAAAGAGAAAAGGCATT-3'; probe:
5'-CCAAGGTGTGCGACTGCTGCGAC-3'. The probes were 5'
6-carboxyfluorescein (FAM) and 3'
6-carboxy-N,N,N',N'-tetramethylrhodamine (TAMRA)
labelled. Prior optimization was conducted for each set of self-designed
oligos determining optimal primer concentrations, probe concentration and
verifying the efficiency of the amplification. For each of the target genes,
the expected size of the PCR product was confirmed on a DNA 2.5% agarose gel.
ß-actin was also amplified for use as an endogenous control using a
pre-developed assay reaction (Applied Biosystems). PCR amplification was
performed (in triplicate) in a total reaction volume of 25 µl. The reaction
mixture consisted of 2.5 µl diluted template, forward and reverse primers
and probe as determined from the prior optimization, 2x TaqMan Universal
MasterMix optimized for TaqMan reactions (Applied Biosystems; containing
AmpliTaq Gold DNA polymerase, AmpErase uracil N-glycosylase, dNTPs
with dUTP, ROX as passive reference and buffer components) and nuclease-free
water. The following cycle profile was used for all genes: 50°C for 2 min
+ 95°C for 10 min + 40 cycles of 95°C for 15 s + 60°C for 1 min.
Serial dilutions were made from a representative sample, and these samples
were amplified together with the unknown samples and used to construct a
standard curve. The mRNA contents were normalized to ß-actin mRNA levels,
and samples expressed relative to the sea level samples, which were set to 1.
ß-actin mRNA has previously been shown not to change in bronchial
epithelium and leukocytes in healthy subjects exposed to hypoxia
(Mairbaurl et al., 2003
).
Capillarization and fibre distribution
Serial sections (10 µm) of the muscle biopsy samples were cut in a
cryostat (Zeiss, HM 560) at 20°C, and routine ATPase histochemistry
analysis was performed after pre-incubation at pH 4.37, 4.60 and 10.30
(Brooke and Kaiser, 1970). Five
different fibre types were defined: types 1, 1/2a, 2a, 2ax and 2x
(Andersen and Aagaard, 2000
).
Capillary density was determined using the double staining method combining
Ulex europaeus agglutinin I lectin (UEA-I) and a collagen IV antibody
as previously described (Qu et al.,
1997
). The serial sections of the various ATPase and capillary
stainings were visualized and analysed for fibre type percentage, fibre size
and capillary density, expressed as capillaries fibre1 and
capillaries mm2, using a TEMA image analyzing system (TEMA,
Hadsund, Denmark) as earlier described in detail
(Andersen and Aagaard, 2000
;
Qu et al., 1997
). An average
of 67±7 fibres was included in the analysis of fibre type and size. An
average of 197±9 capillaries around 59±3 fibres was included in
the separate analysis of capillary density.
Statistics
Values reported are mean ± S.D. One-way analysis of
variance (ANOVA) for repeated measurements was used to evaluate the effect of
duration at altitude. Student's t-test was used for comparisons
between the lowlanders and the Aymara high-altitude natives. To conform to
normal distribution criteria, mRNA ratios were log transformed before
statistical analysis. The significance level was P<0.05, using the
Bonferroni correction where relevant.
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Results |
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Body mass, fibre size and fibre type
Body mass was 78.4±9.1 kg at sea level and 74.6±9.6 and
77.4±10.2 kg after 2 and 8 weeks acclimatization, respectively. At sea
level, the mean fibre area was 6492±1448 µm2 in the
lowlanders and was not significantly changed after 8 weeks of acclimatization
to 4100 m (6060±1765 µm2)
(Table 2). The mean fibre area
for the high-altitude natives was 4474±497 µm2
(P<0.05 vs lowlanders), a 31% lower mean fibre area
compared with lowlanders before altitude exposure. The muscle fibre type
distribution was not affected by altitude exposure in the lowlanders; while
high-altitude natives tended to have a decreased fraction of type 1 fibres,
this did not reach statistical significance
(Table 3).
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|
Capillarization
At sea level, lowlanders had 4.0±0.6 capillaries
fibre1, and this ratio was not significantly changed with
acclimatization (Table 2). By
comparison, the high-altitude natives had 2.4±0.3 capillaries
fibre1 (P<0.05 compared with lowlanders).
Despite a smaller mean fibre area in the high-altitude natives, the capillary
density tended to be smaller in high-altitude natives than in lowlanders, and
the difference reached significance when comparing with the 8-week value for
the lowlanders (P<0.05).
HIF-1 and VEGF mRNA content
No significant changes were found in HIF-1 and VEGF mRNA content
after 2 and 8 weeks hypoxic exposure in the lowlanders, and HIF-1
and
VEGF mRNA levels were similar in the high-altitude natives
(Fig. 1).
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Discussion |
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Body mass and mean muscle fibre area
Previous high-altitude mountaineering expeditions have reported 510%
reductions in body mass (Hoppeler et al.,
1990), probably caused by reductions in food intake
(Guilland and Klepping, 1985
;
Kayser, 1992
) and
gastroenteritis (Kayser,
1994
). To prevent undernutrition-induced weight loss, Butterfield
et al. (1992
) imposed
hypercaloric diets on subjects sojourning on the top of Pike's Peak for 3
weeks (4300 m). Despite the hypercaloric diet, these subjects lost a
significant 3% of body mass during the 3 weeks, most likely an effect of
inactivity at the spatially restricted high-altitude laboratory on Pike's
Peak. Two previous human biopsy studies have reported a decrease of
2026% in mean fibre area during an 8-week expedition to the Himalayas
(Hoppeler et al., 1990
) and in
Operation Everest II (OEII), a chamber study lasting 7 weeks
(MacDougall et al., 1991
). It
is likely that the catabolic state associated with gastroenteritis contributed
to the muscle atrophy reported with Himalayan mountaineering, and the reported
muscle fibre atrophy in OEII could have been at least partly caused by
decreased physical activity while the subjects were confined to the hypobaric
chamber. It has been hypothesized that hypoxia causes overall downregulation
of protein synthesis in skeletal muscle with the purpose of decreasing fibre
size to facilitate oxygen diffusion
(Hochachka et al., 1996
).
However, decreased fibre size is not a universal finding. Five months at 3800
m caused no change in the cross-sectional area of pigeon muscle fibres
compared with those at sea level (Mathieu-Costello et al., 1997), and rat
skeletal muscle fibre cross-sectional area was preserved after 79 weeks
of exposure to 1213% O2
(Abdelmalki et al., 1996
;
Olfert et al., 2001b
). In
humans, it was recently reported that a 21-day expedition to Mt Denali, USA
(reaching the summit at 6194 m after 18 days climbing effort from 2160 m) did
not induce any significant change in mean fibre area
(Green et al., 2000
).
By design, our subjects remained physically active and controlled their own diet. In contrast to previous studies, this regimen was accompanied by a non-significant 1% reduction in whole body mass and a non-significant 4% reduction in mean muscle fibre area during 8 weeks of acclimatization to 4100 m. An unchanged fibre area was an important prerequisite for interpretation of the capillary density in human skeletal muscle before and after hypoxic exposure. If fibre size had been reduced by hypoxia per se or alternative mechanisms, this would improve oxygen diffusion and diminish any potential hypoxia-induced stimulation of growth factor expression and neoformation of capillaries.
Compared with the lowlanders, the mean fibre area in the high-altitude
natives was significantly lower (by 30%). This is in agreement with previous
published data from a similar experimental population
(Desplanches et al., 1996).
Whether this is related to high-altitude residence or perhaps an evolutionary
adaptation remains unknown but, interestingly, similar small muscle fibre
areas have been observed in high altitude Sherpas from the Himalayas
(Kayser et al., 1991
).
Fibre type distribution
Typically, untrained subjects have an approximately equal distribution of
the major fibre types in the vastus lateralis muscle
(Saltin and Gollnick, 1983).
The Danish lowlanders studied in the present investigation were physical
education college students active in a variety of sporting activities and this
may, to some extent, explain the rather high distribution of type 1 fibres
(Table 3). However, although
physically active, their
O2max
(approximately 54 ml min1 kg1) indicates
that they were not competing athletes. It has been speculated that it would be
favourable to modify the fibre composition of the muscle to more oxidative
fibres in hypoxic conditions. However, in agreement with previous studies
(Green et al., 1989
), we did
not find any changes in muscle fibre type histochemistry with acclimatization
in the lowlanders. The Aymara high-altitude natives tended to have a smaller
fraction of type I fibres compared with the lowlanders, which may in part, but
not entirely, correspond to their smaller mean fibre area.
HIF-1 and VEGF
The biological activity of HIF-1 is determined by the expression and
activity of the HIF-1 subunit. Hypoxia has been demonstrated to
increase HIF-1
mRNA expression
(Wang et al., 1995
;
Wiener et al., 1996
;
Yu et al., 1998
). Perhaps even
more importantly, hypoxia disrupts the usual cytoplasmic HIF-1
ubiquination (inactivation), and instead HIF-1 is stabilized and translocates
to the nucleus (Semenza,
2000
). Analysis of rat brain exposed to hypoxia revealed an
increase in HIF-1
protein after 6 h exposure that persisted for 14 days
(Chávez et al., 2000
).
It has also been shown that the response of HIF-1
to hypoxia is very
tissue specific and that brain and spleen are much more sensitive than liver
and kidney (Stroka et al.,
2001
). Recently, human muscle HIF-1
mRNA was found to
increase following intermittent exposure to normobaric hypoxia, equivalent to
an altitude of 3850 m (Vogt et al.,
2001
). In the present investigation, we observed no increases in
HIF-1
mRNA after either 2 or 8 weeks of hypoxic exposure. While most
animal studies finding increased HIF-1
mRNA levels have been conducted
under severe hypoxia, with FIO2s (fractional
O2 concentrations in incurrent gas) of 0.00 or 0.01
(Wang et al., 1995
;
Yu et al., 1998
), the human
study by Vogt et al. (2001
)
was conducted with a somewhat milder hypoxic exposure than used in the present
study. The most obvious differences between the present study and that of Vogt
et al. were the intermittent nature of hypoxia used by Vogt and co-workers,
and the fact that Vogt et al. combined the hypoxic exposure with exercise
whereas our subjects abstained from exercise for 24 h prior to the biopsy
procedure. Exercise in a hypoxic environment may cause severe arterial and
tissue deoxygenation (Calbet et al.,
2003
), and this could explain the increases in HIF-1
mRNA
found by Vogt et al. (2001
).
It would have been an advantage if we had been able to quantify the
HIF-1
protein in our biopsies, but western blotting analysis of this
particular protein proved difficult in pilot experiments and was most likely
related to the limited amount of muscle tissue. The VEGF gene is well
accepted to be a target gene for HIF-1
and the fact that VEGF mRNA did
not change significantly is an important indirect indication that HIF-1
activity was not increased in skeletal muscle in our study.
VEGF mRNA levels have been found to increase in human muscle following an
exercise bout (Gustafsson et al.,
2002) and in humans exercising in hypoxia
(Vogt et al., 2001
); however,
it was unknown whether hypoxia per se would lead to an increased VEGF
mRNA content in resting human skeletal muscle. With prolonged hypoxic
exposure, an increase in VEGF mRNA was found in rat brain after 6 h of 10%
oxygen exposure (Chávez et al.,
2000
) and remained elevated for 14 days but had returned to
normoxic levels after 21 days. In rat skeletal muscle, a surprising
attenuation of resting VEGF mRNA was reported after 8-weeks exposure to 12%
O2 (Olfert et al.,
2001a
,b
).
In the present study, there was a trend towards an increase in VEGF mRNA
levels after 2 weeks. However, this change did not reach statistical
significance, and the VEGF mRNA levels at 8 weeks and in high-altitude natives
were very similar to the levels obtained at sea level in the lowlanders. It is
noteworthy that HIF-1
mRNA and VEGF mRNA were easily detectable in all
biopsies.
Capillarization
Even after prolonged exposure to high altitude, we observed no indication
of neoformation of capillaries in human skeletal muscle. Similar findings have
been reported in animal skeletal muscle after hypoxic exposure
(Abdelmalki et al., 1996;
Olfert et al.,
2001a
,b
;
Sillau and Banchero, 1977
,
1979
;
Sillau et al., 1980
;
Snyder et al., 1985
). Two
animal studies report increased muscle fibre capillarization after exposure to
hypoxia (Cassin et al., 1971
;
Smith and Marshall, 1999
).
However, at least part of the results can be attributed to body mass
differences in the animals. In humans exposed to altitudes between 5000 and
8500 m for 38 weeks, no net capillary neoformation has been reported
(Green et al., 2000
;
Hoppeler et al., 1990
;
MacDougall et al., 1991
). It
is unknown whether neoformation of capillaries in human skeletal muscle would
occur during acclimatization of lowlanders beyond 8 weeks. Interestingly, an
increased capillary to fibre ratio has been found in animals living
permanently at high altitude (Hepple et
al., 1998
; Leon-Velarde et
al., 1993
; Mathieu-Costello
and Agey, 1997
; Sillau and
Banchero, 1979
). However, in the present study, the capillary to
fibre ratio in the Aymara natives was lower than that found in the lowlanders,
which is in agreement with previous reported data from a similar population
(Desplanches et al., 1996
). It
could be speculated that if the natives had a similar mean muscle fibre area
to the lowlanders, this would require an increased capillary number (in order
to achieve a similar capillarization/mm2). Maybe the
capillarization per mm2 of muscle tissue is of greater importance
than capillarization/muscle fibre and could explain why the natives have a
lower capillarization per muscle fibre, i.e. due to the smaller mean area, the
tissue is sufficiently supplied as it is.
Conclusion
In summary, HIF-1 and VEGF mRNA levels in lowlanders are not
increased after 2- and 8-weeks exposure to 4100 m, and similar levels are
found in high-altitude natives living permanently at this altitude. Eight
weeks of acclimatization did not induce any detectable angiogenesis in human
skeletal muscle. Skeletal muscle fibre type distribution and mean muscle fibre
area are not altered by altitude acclimatisation. Altogether, these findings
indicate that, at 4100 m, the acclimatization-dependent increase in
haemoglobin concentration is sufficient to maintain adequate levels of
muscular oxygen supply and that angiogenesis is not necessary in order to
preserve oxygen delivery to skeletal muscle.
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