* Department of Transfusion Medicine, and
Medical Clinic and Polyclinic, Department of Sports Medicine, University of Tuebingen, Tuebingen, Germany
Correspondence: Elvira Fehrenbach, Ph.D., Abteilung Transfusionsmedizin, Eberhard-Karls-Universitaet Tuebingen, Otfried-Mueller-Str. 4/1, D-72076 Tuebingen, Germany. E-mail: elvira.fehrenbach{at}med.uni-tuebingen.de
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Key Words: heat shock protein • hyperthermia • acclimatization • thermotolerance • in vitro
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Elevated, external temperature exerts a synergistic impact on the stress response during intensive exercise. Furthermore, external heat results in detrimental effects on performance during endurance exercise [8 ]. One major factor limiting performance seems to be hyperthermia, which develops during exercise under conditions of increased external temperature and may exceed 40–41°C following strenuous exercise [9 ].
Conversely, repeated exercise bouts at high ambient temperature improve tolerance and performance, referred to as acclimatization. Maintenance of a high level of sweating, lowered heart rate, and internal body temperature constitutes the classic description of the heat-acclimated individual during exercise at high ambient temperature [10 ]. Exercising under conditions of elevated, external temperature appears to be the most effective method for developing heat acclimatization. Nevertheless, it is not clear which exact cellular and molecular mechanisms mediate adaptation to exercise in the heat.
The induction of HSP72 may be a protective mechanism of leukocytes against heat and exercise stress. Protective functions of HSP72 include chaperoning protein assembly and degradation, translocation, and stabilization to protect cellular homeostasis [1 , 6 , 11 , 12 ]. Furthermore, the HSP72-expression level may have important implications in adaptation processes as a result of exercise- and additional temperature-related stress. In addition, it may provide information about earlier stress situations of a cell [12 13 14 15 ]. The phenomenon, acquired thermotolerance, has been associated with the accumulation of HSP induced by a short exposure to a nonlethal heat treatment mediating a transient resistance to the cytotoxic effects of a subsequent, otherwise lethal shock [16 , 17 ]. Therefore, assessing adaptation to training or to heat conditions may be monitored by the cell’s content of HSP72, which may function as a cellular thermometer and a marker of recent thermal stress [7 , 15 , 17 , 18 ]. Because of this, we investigated the expression of HSP72 in peripheral leukocytes of nonheat-acclimated endurance runners under high ambient temperature. We examined the influence of environmental heat and two consecutive, intensive endurance exercise runs (CR1, CR2) on the expression of the inducible HSP72 in leukocytes at mRNA and protein levels by comparing two groups exercising with the same intensity at different ambient temperatures. Group HH performed both runs at 28°C, whereas group NH completed CR1 at 18°C and CR2 at 28°C room temperature. We wanted to know if thermotolerance or adaptation was induced by one bout of exercise under high ambient temperature conditions. Furthermore, we investigated the effect of an additional heat shock (HS) in vitro on HSP72-expression in leukocytes, which had already been stressed by intensive exercise in vivo, to obtain more insight into the mechanisms of stress response after prolonged, heavy exercise.
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Table 1. Physical Data of the Subjects
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Maximal oxygen consumption (VO2max) was assessed using an
additional ramp test until exhaustion on the treadmill. Subjects
started running at 8 km·h-1 for 30 s, and
thereafter, running speed was increased by 1 km·h-1
every 30 s. Ventilation (VE), VO2, and
respiratory exchange ratio (R) were assessed using breath-by-breath
analysis (Oxycon alpha, Jaeger, Germany) with a mouth-piece where the
flowmeter was attached. Criteria for assessment of VO2max
were a R 
1.1 and a plateau in VO2 (increase
150 ml
per 30 s) paralleled by an increase in workload
[20
].
Continuous run
The main investigation consisted of two continuous runs (CR1,
CR2) on the treadmill, lasting 60 min and performed 6 days apart
(Fig. 1
, design of study). Running velocity during the run was adjusted to
90% of the IAT as assessed before. The exercise procedure was
performed in a climatized room, where dry-bulb temperature and relative
humidity were kept constant at 28°C/50% or 18°C/50%. Group NH
(n=6) completed the first run (CR1) at 18°C and 1 week
later, the second trial (CR2) at 28°C room temperature. Group HH
(n=6) performed both exercise tests at 28°C environmental
temperature. All subjects refrained from competitions or vigorous
training at least 7 days before the test and in the week of rest
between the runs. A dietary protocol revealed a comparable fluid intake
24 h before the test. The subjects were allowed to drink water
during the run.
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Figure 1. Schematic illustration of the design of the study. Two groups of
athletes (HH, NH) performed two continuous runs (CR1, CR2) on a
treadmill at the indicated room temperatures (RT), exercise intensity
(VIAT=running velocity at the IAT), relative humidity (rH),
and duration. Oxygen consumption, core temperature, and heart rate were
assessed continuously during the runs, lactate at beginning and end.
Blood samples were drawn pre-exercise and 0-h, 24-h, and 48-h
post-exercise.
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Analytical methods
Flow cytometry
The leukocytes were analyzed by intracellular, indirect
immunofluorescence using the HSP72-specific monoclonal antibody (mAb)
SPA-810 [specific for the inducible form of human HSP70;
immunoglobulin (Ig)G1, clone C92F3A-5; StressGen Biotechnologies Corp.,
Victoria, BC, Canada].
In a 1 ml sample of ethylenediaminetetraacetate (EDTA)-treated blood, erythrocytes were lysed by incubating with 10 ml 1 x fluorescein-activated cell sorter (FACS) Lysing Solution (Becton Dickinson, San Jose, CA) for 15 min at room temperature. Next, cells were fixed at room temperature in a solution containing formaldehyde (reagent A) following the manufacturer’s instructions (Fix & Perm kit, An der Grub, Vienna) and washed twice. Then the cells were permeabilized with reagent B and at the same time, incubated with the primary HSP-specific mAb or isotype-matched, nonbinding antibody (negative control), and incubated for 15 min at room temperature. Previously, antibody concentrations were tested to give maximum counts of positive cells (1 µg/test). After washing the cells twice and incubating in the presence of the secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse F(ab')2 IgG (Dianova, Hamburg, Germany), the cells were analyzed using the flow cytometer EPICS-XL-MLC (Coulter, Krefeld, Germany). Dead cells were excluded by electronic-gating and fluorescence histograms, area-corrected to 10,000 cells. The lymphocyte, monocyte, and granulocyte populations were differentiated according to granularity and size in the forward- versus side-scattergram and then gated. Data were presented as percent-positive cells (%) and mean fluorescent channel (MFC) for each of the three special gates. They were corrected for background fluorescence by the negative controls using isotype-matched, nonbinding, primary antibody.
Isolation of RNA and reverse transcriptase-polymerase chain
reaction (RT-PCR)
Low-level, constitutive synthesis of HSP72 in human peripheral
blood mononuclear cells suggests that a semi-quantitative assay will be
necessary to detect enhanced synthesis after heat stress
[21
].
Cytoplasmic RNA for RT-PCR analyses was isolated from whole blood with the RNeasy-blood kit (Qiagen, Hilden, Germany). RNA (200 ng) was reverse-transcribed (10' 20°C, 15' 42°C, 5' 99°C, 5' 5°C) and amplified (3' 95°C, 1' 95°C, 1' 55°C, 1' 72°C) in the thermal cycler (PTC200, M.J. Research, Watertown, MA) using specific primers for HSP70B (StressGen Biotechnologies Corp.) [22 ] and ß-actin [23 ]. RT and subsequent amplification by the PCR were performed using a GeneAmp RNA PCR kit (Perkin Elmer, Foster City, CA). The RT master mix contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl2, 1 mM each deoxyribonucleoside triphosphates (dNTPs), 1 U/µL RNase inhibitor, 2.5 U/µL RT, and 2.5 µL oligo d(T). The final RT-reaction volume was 20 µL. The PCR master mix contained 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.25 mM MgCl2, 200 µM each dNTPs, 2.5 U/100 µL AmpliTaq DNA polymerase, and 0.15 µM of each primer. A final 25 µL PCR reaction solution contained 5 µL RT product (cDNA) and 20 µL PCR master mix. Control experiments were performed for each primer pair to determine the range of cycles in which a given amount of cDNA would be amplified in a linear fashion: HSP72, 30 cycles; actin, 27 cycles. Furthermore, a dilution assay was performed to determine the proper input-RNA concentration. The resulting amplified products for HSP70B (234 bp) were confirmed by sequence analysis (SEQLAB, Goettingen, Germany). Photographs of ethidium bromide-stained DNA gels (2%) were scanned by the Lumi-Imager-System (Boehringer Mannheim, Mannheim, Germany), which allowed semi-quantitative analyses of the specific HSP72-mRNA expression. The data generated were normalized to transcript levels for the constitutively expressed ß-actin gene.
In vitro stimulation with HS
Immediately after exercise, EDTA blood of the athletes was
incubated for 2 h in a waterbath heated to 42°C (HS). Control
blood samples of the same individuals were incubated at 37°C for
2 h instead of the HS exposure. The samples were prepared for
further analyses as described above.
Statistical methods
All statistical analyses and descriptional methods were computed
by the statistical software package JMP (JMP3.1-software, SAS Institute
Inc., Cary, NC) for PC. Data in Tables 1
and 2
are expressed as means and SD. Comparisons of repeated
measurements were tested for significance by the Wilcoxon signed ranks
test. The nonparametric test of Mann-Whitney was used to evaluate
significant differences between the groups HH and NH. A p
value of <0.05 was regarded as significant.
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Table 2. Lactate Values, Core Temperature, Relative Oxygen Consumption, and
Heart Rate Immediately after Two Continuous Runs (CR1, CR2) of Two
Athlete Groups HH (28°C/28°C) and NH (18°C/28°C)
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Body-core temperature showed significant differences between the groups and CR1/CR2 (Table 2) . Core temperature was diminished in group HH after the second run in the heat compared with CR1. In contrast, we found an increased body-core temperature in group NH after CR2. Core temperature was lower after the first exercise (CR1) at 18°C (NH) than at 28°C (HH) but lower in HH compared with NH after CR2.
Mean relative VO2max (%VOi2max) revealed no significant changes (Table 2) .
The maximal rise in heart rate at the end of exercise, presented here as deviation of maximal heart rate from heart rate at IAT, was significantly higher in HH than NH after CR1 and vice versa after CR2 (Table 2) . Deviation of heart rate in group HH at the end of CR2 was lower than after CR1, although not significant.
HSP72-expression
Flow cytometry revealed a significant increase in
expression of HSP72 protein after both exercise sessions (CR1, CR2) in
group HH. This was apparent in percent-positive cells (%) and mean
fluorescence channel (mfc; p<0.05; Fig. 2
). Monocytes and granulocytes showed comparable increases with a
maximum at 24 h after CR1 and only slightly decreased after
48 h. The 48-h values were significantly higher than the
pre-exercise expression levels (p<0.05). The second round
of exercise (CR2) induced a further increase in HSP72 protein, mainly
causing an increase in antigen density (mfc; Fig. 2
). The maximal
expression after 24 h was followed by a steep decrease resulting
in nearly basal expression levels 48 h after the second trial.
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Figure 2. Cytoplasmic expression of HSP72 in monocytes and granulocytes
of group HH determined by flow cytometry. The values before, and 0 h, 24 h, and 48 h, after the two exercise sessions (CR1, CR2)
at 28°C (group HH) are presented as mean fluorescence intensity
(mfc), a marker of antigen density and percent-positive cells. Data are
displayed as means ± SD. *, Significant changes
during exercise.  , Significant differences between CR1 and CR2.
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Figure 3. Changes of the HSP72-protein expression (differences of mfc to
pre-exercise values) after two continuous runs (CR1, CR2) at indicated
ambient temperatures in monocytes comparing the two groups of athletes
(HH and NH). Data are displayed as means ± SD. *,
Significant changes during exercise. #, Significant differences between
groups.
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Figure 4. HSP72-mRNA expression pre-exercise, 0 h, and 24 h after two
exercise trials (CR1, CR2) under high external temperature (28°C,
group HH) analyzed by semi-quantitative RT-PCR. In the upper panel, the
specific mRNA values are described in relative units normalized to
transcript levels of ß-actin. The single points of the individual
athletes were presented and connected with lines. In the lower panel,
original ethidium bromide-stained PCR-gels of two athletes are shown as
representative examples. HSP72 and actin were amplified under
conditions to allow relative comparisons for a given mRNA. M =
size marker; bp = base pairs.
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Figure 5. Relative expression of HSP72 mRNA comparing the two athlete groups HH
and NH before, 0 h, and 24 h after two continuous runs (CR1,
CR2) at the indicated ambient temperatures detected by RT-PCR. HSP72
and actin were amplified under conditions to allow relative comparisons
for a given mRNA. Data are displayed as means ± SD.
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Figure 6. Influence of HS in vitro on HSP72-expression in already
exercise-stressed monocytes. Analysis of antigen density (mfc) by flow
cytometry. Blood samples, drawn immediately after exercise, were
exposed to additional, experimental HS (2 h, 42°C). Data are
presented as a HS-induced increase compared with the native,
exercise-stressed values. Results are presented as means ±
SD.
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The more pronounced down-regulation of HSP72 protein and the minor increase at mRNA level after the second trial (CR2) in group HH may indicate parameters of adaptive mechanisms in leukocytes induced by intensive, prolonged exercise under conditions of increased temperature. Group NH, which performed the first run under cooler conditions, revealed no such effects after the second run. The content of HSP72 might provide information about earlier stress situations of a cell, including heat and/or exercise [12 , 14 , 15 , 30 ]. Therefore, HSP72-expression could be a useful marker for assessing adaptation to training or to heat conditions [26 , 31 , 32 ].
Parallel to the effects on HSP level, one bout of endurance exercise in the heat reduced the rise in body-core temperature and cardiac drift during a subsequent, identical trial of endurance exercise (HH, CR2) in nonheat-acclimated runners. This reflects the simultaneous adaptation of the thermoregulatory and cardiocirculatory capacity [33 ]. In contrast, these adaptive effects were not induced by a previous exercise session at a lower ambient temperature (group NH after CR2). Core temperature and heart rate were even higher after CR2 in NH compared with HH. The changes of HSP72 expression in group HH as a result of the second exercise trial may result in part from the more favorable thermo- and cardiocirculatory capacity, leading to less stress in general. Comparable %VO2max, lactate values, and leukocyte counts revealed that the intensity of exercise was equal in both heat sessions. It is suggested that the high ambient temperature is one factor responsible for thermotolerance and adaptive effects on HSP72 level in leukocytes of these nonheat-acclimated athletes. Additional, secondary mechanisms, such as reactive oxygen intermediates (ROI) or cytokines, may be involved. Cytokines, ROI, and nitric oxide have been shown to increase HSP synthesis [2 3 4 5 6 , 34 ]. Thus, the simultaneous regulation of cardiocirculatory/thermoregulatory and HS responses may be interpreted as partially decompensated hemodynamics, which reduce heat dissipation, stimulate thermal stress, and up-regulate HSP synthesis. However, core temperature seems not to be the only factor responsible for HSP induction when looking at the significantly higher core temperature in group NH after CR2 compared with HH. It cannot be excluded completely that the differences in expression between the two heat sessions were related to the exercise rather than to the heat input. This, however, seems less probable because such regularly and well-trained athletes are used to similar exercise protocols. It has already been documented that physically fit men acclimated to heat more rapidly than less-fit men [10 ]. Heat-clamping experiments revealed that exercise and a rise of core temperature contribute to changes in white-cell counts immediately after exercise, potentially mediated by cortisol and growth-hormone changes [35 ]. A relationship between heat exposure and its impact on hormonal and immune responses to exercise has been suggested [36 , 37 ]. In our study, we also found reduced increases in granulocyte and monocyte counts and growth hormone concentrations in the second trial [38 ].
In this context, it could be discussed that thermotolerance on HSP72 level may contribute to acclimatization of athletes to heat on a cellular level and be related to adaptation processes, such as increases in plasma volume and sweating rate, efficient nutrient metabolism, lower core temperature, and heart rate [33 ].
Conversely, adaptation to heat after a single exposure seems questionable because acclimatization processes are described as resulting from several daily, repeated exercise protocols at respective elevated, external temperatures [39 ]. However, these studies also revealed the beginning of heat acclimatization on the first day of exposure and that 4–5 days are sufficient to reach about 75% of the maximal, individual adaptive capacity. Furthermore, even endurance-exercise training in temperate conditions is shown to contribute, in part, to adaptation processes to high ambient temperature, and to offer some protection [40 ]. Moreover, the relatively high intensity of stress input (60-min exercise at 90% IAT plus heat of 28°C) performed during our exercise protocol has to be considered, resulting in maximal core temperatures of up to 40.5°C. The classic physiological adjustments during exercise-heat acclimatization, heart rate, and internal temperatures were reduced as a result of the second exercise trial at high environmental temperature (HH). This was not the case if the run was preceded by a run at lower ambient temperature (NH).
Influence of in vitro HS
Additional HS in vitro applied to blood samples
directly after exercise resulted in a further increase in
HSP72-expression at protein and mRNA levels when compared with the
exercise-induced HSP level. This enhanced HS response may be
interpreted as residual, protective resources, which immune cells have
after exercise to protect against denaturing heat and additional,
extraordinary stress. The experimental HS temperature of 42°C was
2°C above the mean-core temperature achieved by the exercise
protocol. Such a high temperature in vivo will be reached by
exercise solely in the skeletal musculature [28
].
Exposure of cells to high temperatures causes their membranes to
undergo a rapid decrease in molecular order, which is an important
factor in determining HS gene expression [41
].
Accumulation of specific HSP at or in the membrane during the transient
HS response causes a rigidification of the heat-fluidized membrane.
Thus, the membrane returns to its pre-exercise state
[42
]. It has been shown in epithelial cells that
sufficient heat applied to induce HSP70 synthesis attenuated the
heat-induced alterations. This suggests that HSP may be involved in
stabilizing cytoskeleton and/or tight-junction proteins
[43
, 44
].
The reduced heat responses, which occur immediately after the second exercise sessions compared with the responses after the first trials in both groups, independent of ambient temperatures, are noteworthy. Ryan et al. [15 ] found that leukocytes obtained after exercise and incubated at 41°C demonstrated a decreased HSP72 synthesis compared with pre-exercise incubations. They suggested that the thermal history of a cell might be evaluated by the cell’s content of HSP72. We questioned if HSP72-expression in leukocytes might be used as an indicator of earlier sets of exercise. The exercise-induced attenuation of the HS response in vitro in both groups after CR2 compared with CR1 may be interpreted as short-time and early adaptation in terms of tolerance to repeated exercise. The differences of exercise-related core temperatures and preceding environmental heat conditions between the groups seem to be of minor importance for this in vitro response. The response of HSP72-expression to additional HS in vitro might be used as an indicator of earlier, severe stress conditions.
In summary, we conclude that even one bout of intensive, endurance exercise on a treadmill under elevated, ambient temperature results in adaptation of cardiocirculatory/ thermoregulatory capacity and of HSP72-expression in leukocytes of nonheat-acclimatized, endurance runners. Furthermore, HSP72 thermotolerance is induced and may afford some protection against subsequent stress. The content of HSP72 in leukocytes may represent an indicator of earlier exercise and temperature-related stress conditions. This marker possibly allows conclusions about adaptation to exercise under high ambient temperature.
Received March 30, 2000; revised October 11, 2000; accepted January 5, 2001.
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