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Human physiological and heat shock protein 72 adaptations during the initial phase of humid-heat acclimation
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Journal of Thermal Biology 32 (2007) 341–348 www.elsevier.com/locate/jtherbio
Human physiological and heat shock protein 72 adaptations during the initial phase of humid-heat acclimation Helen C. Marshalla, Samantha A. Campbellb, Craig W. Robertsb, Myra A. Nimmoa, a
Strathclyde Institute of Pharmacy and Biomedical Sciences, Faculty of Science, University of Strathclyde, 199 Cathedral Street, Glasgow G4 0QU, UK Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, 27 Taylor St, Glasgow G4 0NR, UK
b
Received 12 December 2006; accepted 17 April 2007
Abstract 1. The timescale and integration of human whole body and cellular Hsp72 adaptations during the initial phase of human-heat acclimation were determined. 2. Two exercise humid-heat exposures on consecutive days lowered exercise rectal temperature (Po0.05) and heart rate (Po0.01). 3. Sweat rate was increased (Po0.001) during exercise through an improved maintenance of sweating, and sweat Na+ reabsorption was enhanced (Po0.05). 4. These adaptations were accompanied by a reduced Hsp72 mRNA response with no change in protein level. 5. Two prolonged, low-intensity exercise-heat bouts on consecutive days are sufficient to initiate physiological and Hsp72 mRNA adaptations, although Hsp72 protein adaptation may require a greater exercise intensity or longer acclimation period. r 2007 Elsevier Ltd. All rights reserved. Keywords: Acclimation; Heat shock protein 72; Humid-heat stress
1. Introduction The process of acclimation to a hot environment is commonly judged on the response of heart rate (HR), core and skin temperatures, and sweat rate (SR). Acclimation, however, is a complex process involving adaptations not only at whole body but also cellular levels. The most inducible and thermosensitive marker of cellular stress is heat shock protein (Hsp) 72 (Mizzen and Welch, 1988). Human peripheral blood mononuclear cells (PBMCs) are commonly used to monitor Hsp72 changes with exercise (Fehrenbach et al., 2001, 2003; Shin et al., 2004) and heat stress (Hunter-Lavin et al., 2004) as these cells respond rapidly compared with skeletal muscle which has been reported to take 48 h to respond (Morton et al., 2006a). The majority of studies report physiological changes after 6–10 consecutive days of exercise-heat stress (Kirby and Convertino, 1986; Nielsen et al., 1997; Patterson et al., 2004), with no analysis of changes over the first few days. Corresponding author. Tel.: +44 141 548 5791; fax:+44 141 548 3129.
E-mail address: [email protected] (M.A. Nimmo). 0306-4565/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2007.04.003
For many, an acclimation period of 6–10 consecutive days is impractical, yet the minimum number of exposures required to gain a thermoregulatory benefit remains unclear. Graphical representations suggest heart rate, core temperature and SR adaptations may start to occur in the early phase of acclimation (Lind and Bass, 1963; Strydom et al., 1966; Convertino et al., 1980; Nielsen et al., 1997), with quantitative data indicating a reduction in exercise heart rate by the third exercise-heat exposure (Wyndham et al., 1976; Greenleaf et al., 1981; Yamazaki and Hamasaki, 2003) and enhanced SR on day five (Greenleaf et al., 1981). Recent preliminary work in our laboratory, however, found an enhanced SR following a single exercise-heat bout, although no changes in heart rate or rectal temperature were observed (Milne & Nimmo, 2004). Whether these reductions in cardiovascular and thermoregulatory strain are accompanied by Hsp72 adaptations is unclear. Existing human literature suggests the response of intracellular Hsp72 reflects earlier stresses (Ryan et al., 1991; Fehrenbach et al., 2001) and could hence be a potential marker for assessing adaptation to repeated exercise-heat exposures. An initial indication of this is
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found in the study of Fehrenbach et al. (2001), which reports both core temperature and leukocyte Hsp72 adaptations following two identical exercise-heat exposures, separated by six days rest. This finding is somewhat surprising, however, considering exercise bouts in the heat, separated by six days, have previously been found to have no acclimation effect on physiological variables (Barnett and Maughan, 1993). Although human studies are lacking, research in rats has reported myocardial Hsp72 adaptations to the initial stage of passive heat acclimation (continuous exposure to 34 1C for two days) (Maloyan et al., 1999), and exercise bouts on three consecutive days in a temperate environment (Taylor et al., 1999). Comparison of these studies to humans, however, is not robust as the Hsp72 response appears to be both species (earlier skeletal muscle Hsp72 response in rats (Skidmore et al., 1995) versus humans (Morton et al., 2006a)) and tissue specific (Flanagan et al., 1995; Skidmore et al., 1995). The present human study, therefore, aims to determine both physiological and molecular (PBMC Hsp72 expression at the mRNA and protein levels) adaptations following two exercise humid-heat exposures on consecutive days, in order to determine whether this exposure is sufficient to gain thermoregulatory benefits and whether these are evident at the cellular level. 2. Materials and methods 2.1. Subjects Seven males (mean (SD); age, height, body mass and _ 2peak ): 30 (4) yr, 1.80 (0.08) m, 73.8 peak oxygen uptake (VO (8.5) kg and 3.88 (0.43) L min1, respectively) volunteered to participate in the present study. Subjects had undertaken no formal acclimation or spent any time in a hot environment over the preceding 2 months. The study was conducted in accordance with the Declaration of Helsinki, approved by the Local Ethics Committee and subjects provided written informed consent. Subjects attended the laboratory at a consistent time in the morning on five separate occasions. 2.2. Experimental design Subjects completed a 2 h cycle in a hot-humid environment (mean (SD); 38.0 (0.1) 1C, 60.0 (0.1)% relative humidity (RH), wind 0.6 (0.1) m s1) on three consecutive _ 2peak , was days. Workload, corresponding to 38% VO determined from preliminary tests involving submaximal exercise of varying intensity followed by a continuous, incremental cycling test to volitional exhaustion on an electronically braked cycle ergometer (Lode Excalibur, Groningen, The Netherlands). Expired gases were measured continuously during the preliminary tests using an automated gas analysis system (Jaeger Oxycon Pro 4.6, Hoechberg, Germany). The load and pedal cadence (70 rpm) were kept constant between the three main trials.
On days 1 and 3, a baseline arterialised-venous blood sample was collected after 30 min seated rest, and an exercise blood sample was collected following 2 h upright cycling. Posture was controlled to allow for the calculation of plasma volume (PV) changes. Sweat was collected from a sweat patch every 30 min. Heart rate (HR), rectal temperature (Tre) and skin temperatures (Tsk) were regularly observed during all three exercise-heat exposures and recorded at rest and on completion of exercise on days 1 and 3. Time of testing was consistent within a subject. Subjects were all nonsmokers, abstained from caffeine and alcohol ingestion and refrained from strenuous physical activity for 48 h prior to testing. They recorded their dietary intake and physical activity for 24 h prior to the first main trial which was replicated for the subsequent trials. Two hours prior to exercise the subjects consumed a meal replacement drink (Complan, H. J. Heinz Co Ltd., Hayes, UK) which was reconstituted by adding 2.86 mL kg1 body mass of semiskimmed milk to 0.81 g kg1 body mass of powder. To ensure euhydration, subjects consumed 10 mL kg1 body mass of water the evening before testing and consumed 10 mL kg1 body mass of water minus the volume of milk in the meal replacement drink 2 h prior to testing. On arrival to the laboratory subjects voided and euhydration status was confirmed by the measurement of urine specific gravity (Armstrong et al., 1994; Hoffman et al., 1994); Multistix SG; Bayer Diagnostics, Newburg, UK). Nude body mass was recorded (72 g; Sartorius, Go¨ttingen, Germany), a rectal thermistor was inserted, and a heart rate monitor was strapped to the chest. Subjects entered a thermoneutral environment (mean (SD); 27.0 (0.4) 1C; 27.5 (3.7)% RH) wearing shorts, socks and trainers where an antecubital vein was cannulated while subjects were supine. Subjects then moved to a seated position for 30 min, during which skin thermistors were attached and the cannulated arm placed in a heated box (50 1C) during the final 10 min. A pilot study had identified the temperature and duration of forearm heating as being sufficient to attain arterialisation, which was found to occur naturally during exercise in the heat. Resting HR, Tre and Tsk were recorded in the seated position before heating of the forearm. The left forearm was cleaned with deionised water and a sweat patch attached as previously described (Brisson et al., 1991; Hayden et al., 2004). Subjects then entered the heat chamber where exercise commenced immediately. Subjects cycled at an average workload _ 2peak over the 2 h corresponding to 43.7 (3.9)% VO _ 2 drift. Sweat loss, as determined exposures, due to VO during a dehydrated familiarisation trial, was replaced with a 20 mmol L1 sodium-chloride solution (Gisolfi et al., 1990). Fluid was provided every 15 min and was weighted towards the first hour of exercise to promote gastric emptying (Costill and Saltin, 1974); 20% (0 min), 20% (15 min), 15% (30 min), 15% (45 min), 15% (60 min), 5% (75 min), 5% (90 min) and 5% (105 min). On completion of exercise, the subjects towel dried and nude body mass
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was re-measured, allowing an estimation of whole-body sweat loss. 2.3. Experimental procedures Tre was measured using a rectal thermistor (Edale Instruments Ltd., Roedoure, UK) inserted 10 cm beyond the anal sphincter. Skin thermistors (Edale Instruments Ltd.) attached to the forearm, chest, thigh and calf on the right side of the body were used to measure Tsk. Temperatures were recorded via a 1000 series 8-bit Squirrel data logger (Type 1002, Grants, Cambridge, UK). Weighted mean skin temperature ðT¯ sk Þ was calculated according to Ramanathan (1964). HR was monitored and recorded with the use of a heart rate monitor (Polar Vantage, Kempele, Finland). Whole body sweat loss was determined by a change in body mass, corrected for fluid intake and urinary losses. Regional sweat was aspirated from a single forearm (middorsal) site on the left side of the body and regional SR determined gravimetrically by the change in mass of the aspirating syringe (70.01 g; A & D Instruments, Abingdon, UK) (method CV ¼ 6:3%; Hayden et al., 2004). The four sweat samples (0–30, 30–60, 60–90 and 90–120 min) were combined and a 2 h sweat sample was then frozen (70 1C) for later analysis. Blood was collected via dry syringes from the indwelling catheter (Insyte 20 G, Becton Dickinson, Madrid, Spain). Whole blood (10 mL) was transferred to a lithium heparin tube and used immediately for the isolation and counting of the PBMCs. PBMC pellets were frozen at 70 1C in aliquots of 1 106 cells. Haematocrit was determined immediately from whole blood. Plasma and serum were obtained by centrifugation and aliquots of plasma, serum and whole blood were frozen (70 1C) for later analysis. Sweat and serum: Sweat and serum electrolytes were determined with the use of indirect Ion Selective Electrodes (Pentra 400; Horiba ABX, Montpellier, France) and osmolality was determined using freezing-point depression (Osmomat 030; Gonotec, Berlin, Germany). Blood analysis: The analysis of plasma aldosterone was performed using a commercially available kit (Euro DPC, Gwynedd, UK) for radioimmunoassay. Plasma catecholamines, epinephrine and norepinephrine, were extracted by absorption on alumina, separated by reverse-phase liquid chromatography and measured with electrochemical detection (Waters UK Ltd., Herts, UK). Haematocrit was measured in triplicate by the microcapillary technique and
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corrected for trapped plasma (Chaplin and Mollison, 1952). Haemoglobin was determined in whole blood using the cyanomethemoglobin method (Randox, Dublin, Ireland) on a spectrophotometer (Cecil Aquarius; Thistle Scientific, Uddingston, UK). Isolation of cells from whole blood: PBMCs were isolated from whole blood by Ficoll-Histopaques (1.077 g mL1) (Sigma, Poole, UK) density centrifugation. The cells were washed in Hanks’ balanced salt solution (Sigma, Poole, UK) then re-suspended in RPMI 1640 with 2.1 mM L-Glutamine (Cambrex, Wokingham, UK). A cell count was carried out on a haemocytometer (Neubauer; Fisher, Loughborough, UK) after which cell pellets (1 106 cells) were frozen (70 1C) prior to analysis for Hsp72 mRNA and Hsp72 protein. Hsp72 gene expression in PBMCs: Total RNA was isolated from the PBMCs using TrizolTM reagent (InvitrogenTM, Paisley, UK) based on the acid-guanidium thiocyanate–phenol–chloroform method of Chomczynski and Sacchi (1987). Complimentary DNA (cDNA) was synthesised from the total RNA using SuperScriptTM III RNase H Reverse Transcriptase (InvitrogenTM, Paisley, UK) and random hexamer primers (Promega, Southampton, UK). All cDNA was stored at 20 1C until further analysis. Transcripts for Hsp72 mRNA were quantified by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Primer sequences for Hsp72 and RibS29 (internal control) were designed with the aid of MacVectorTM 7.0 (Oxford Molecular) and are presented, along with the GenBank accession numbers for the sequences used for primer design, in Table 1. Hsp72 is encoded by both Hsp70-1 and Hsp70-2 genes (Tavaria et al., 1996) hence, the Hsp72 primers were designed to detect both genes. Real-time RT-PCR (Mx3000P; Stratagene, La Jolla, CA), using SYBRs-Green (Sigma, Poole, UK) as the detection method, was used for the duplicate amplification of the cDNA samples. The thermal profile consisted of an initial 3 min denaturation at 95 1C, 40 PCR cycles (95 1C, 30 s; 64 1C, 30 s; 72 1C, 30 s), and a final incubation at 95 1C for 1 min. A melting curve analysis was performed at the end of each real-time run to verify the purity of the reaction products. The standard curve method was used for calculating the number of transcripts for both Hsp72 and RibS29 in the cDNA samples. Hsp72 mRNA expression is presented as a ratio to the internal control (Hsp72/RibS29). RibS29 was selected as the internal control as it has been shown in human PBMCs to remain unchanged upon
Table 1 GenBank Accession Numbers and primer sequences for Hsp72 and RibS29 Gene
GenBank
50 Sense 30
50 Antisense 30
Hsp70-1 Hsp70-2 RibS29
M11717 M59830 B035313
TAC AAG GGG GAG ACC AAG GCA TTC
TGC GAG TCG TTG AAG TAG GC
TCT CGC TCT TGT CGT GTC TGT TC
GGT TTT TCA TTG AGT AGA TGC CCC
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presentation of, and during recovery from, exertional heat injury (Sonna et al., 2004). Hsp72 protein expression in PBMCs: Hsp72 protein was semi-quantified by SDS-PAGE and Western blotting. A discontinuous gel system was used for SDS-PAGE involving a 5% stacking gel and a 12% separating gel using the method of Laemmli (1970). PBMC proteins from 375,000 cells were separated electrophoretically (InvitrogenTM Electrophoresis System) at 200 V for around 1 h. Proteins were transferred electrophoretically to nitrocellulose membrane (HybondTM ECLTM; Amersham Biosciences, Bucks, UK) at 100 V for 2 h. Membranes were stained with Ponceau S (Sigma, Poole, UK) to visually control protein transfer and to mark the position of marker proteins. They were then blocked with 5% skimmed milk in phosphate buffered saline and Tween-20 (0.1%) (PBS-T) and probed with a 1:3000 dilution of a mouse monoclonal antibody specific for Hsp72 (SPA-810; StressGen, Victoria, BC, Canada) overnight at 4 1C. Following washing in PBS-T, the membrane was incubated with an anti-mouse IgG HRP-linked antibody (Cell Signalling Technology, Beverly, MA) at a 1:2000 dilution, for 1 h at room temperature. The membrane was incubated for approximately 30 s with chemiluminescence substrate (SuperSignal West Pico; Perbio Science UK Ltd., Northumberland, UK) and exposed to photographic film (Amersham, Bucks, UK). Fold change in Hsp72 protein was calculated with the use of a densitometer (GS-800; Bio-Rad, Herts, UK) and image analysis software (Quantity One; Bio-Rad, Herts, UK). 2.4. Data analyses and statistics All blood and sweat measures were performed in duplicate with the exception of haematocrit, which was performed in triplicate, and Hsp72 protein expression which was determined singularly. Changes in PV with exercise and between days were estimated using the method outlined by Dill and Costill (1974). A commercial statistics package (SPSS version 12.0) was used for determining statistical significance. Data are presented as means (SD) in the text and tables, and as means (SEM) in the figures. A one-way analysis of variance (ANOVA) was carried out on resting nude body mass and urine specific gravity on all three days. A two-factor (trial by time) repeated measures ANOVA was performed to ascertain any treatment or exercise effects on Tre, HR, T¯ sk , blood variables, PBMC counts and Hsp72 mRNA levels, and 30 min forearm SRs, between the data of days 1 and 3. A Tukey’s HSD post-hoc test was used to locate differences when the ANOVA revealed a significant difference. Hsp72 protein data were not normally distributed, as determined by the Levene test, hence this data set was analysed by a Friedman’s two-way ANOVA. A paired sample Student’s t-test was used to analyse delta T¯ sk , sweat variables and percentage change in PV. The significance level was set at Po0.05 and n ¼ 7.
3. Results Subjects were euhydrated with no difference in urine specific gravity (day 1, 1.006 (0.002); day 2, 1.008 (0.004); day 3, 1.006 (0.002)) or nude body mass (day 1, 73.84 (8.47) kg; day 2, 73.87 (8.73) kg; day 3, 73.67 (8.38) kg) upon arrival at the laboratory on all three days. Exercise increased Tre, T¯ sk and HR on both days 1 and 3 (Po0.01) (Table 2). Resting Tre and HR were the same on both days while exercise levels were lower on day 3 (Po0.05) (Table 2). T¯ sk was unaffected by two exerciseheat exposures at rest or during exercise (Table 2), however, the exercise-induced increase was lower on day 3 compared with day 1 (Po0.05). A similar percentage change in PV resulted from the exercise-heat exposure on days 1 and 3 (day 1: 10.2 (3.1)%; day 3: 6.5 (3.7)%). Resting PV was estimated to expand by 6.0 (6.1)% following two days acclimation. Concentrations of plasma aldosterone and catecholamines, and PBMC counts were therefore corrected for PV expansion on day 3 (concentration 1.06) and for PV decline during exercise on both days (day 1: concentration 0.898; day 3: concentration 0.935). Whole body SR was greater (12 (5)%) on day 3 (1.29 (0.17) L h1) compared with day 1 (1.15 (0.15) L h1) (Po0.001). Similarly, average forearm SR over the 2 h exercise-heat bout increased (31 (32)%) from day 1 (9.16 (4.29) mL cm2 min1) to day 3 (11.71 (5.45) mL cm2 min1) (Po0.05). Forearm SR on day 1 did not significantly change during the 30 min periods, however, on day 3 the 60–90 min sample was greater than that collected during the initial 30 min. The elevation of sweating was maintained into the last 30 min producing a significant increase compared to the similar period on day 1 (Po0.05) (Fig. 1). Sweat [Na+], [Cl] (Fig. 2) and osmolality (day 1: 153 (36) mOsmol kg1; day 3: 140 (31) mOsmol kg1) were lower on day 3 compared with day 1 (Po0.05). No differences in serum [Na+], [Cl] and osmolality were found between days (Table 3). Plasma aldosterone levels were enhanced following exercise-heat stress on both days (Po0.001) (Table 4) with a greater concentration at rest and following exercise on day 3 than on day 1 (Po0.001 ) (Table 4). Table 2 Resting and 2 h exercise rectal temperature (Tre), mean skin temperature ðT¯ sk Þ, and heart rate (HR) during days 1 and 3
Tre (1C)—rest Tre (1C)—exercise T¯ sk (1C)—rest T¯ sk (1C)—exercise HR (beats min1)—rest HR (beats min1)—exercise
Day 1
Day 3
36.8 (0.1) 38.9 (0.4)* 33.0 (0.2) 37.4 (0.5)* 58 (4) 143 (20)*
36.7 (0.1) 38.7 (0.2)*,** 33.1 (0.2) 37.1 (0.5)* 55 (7) 134 (16)*,**
Values are means (SD). * Significant difference from rest (Po0.001), significant difference from day 1 (Po0.05).
**
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Table 4 Resting and 2 h exercise plasma epinephrine (Ep) and norepinephrine (NE) concentration on days 1 and 3
1
Aldosterone (ng dL )—rest Aldosterone (ng dL1)—exercise Ep (nmol L1)—rest Ep (nmol L1)—exercise NE (nmol L1)—rest NE (nmol L1)—exercise
Day 1
Day 3
8.0 (4.8) 37.5 (17.0)* 0.02 (0.04) 0.13 (0.14) 1.0 (0.2) 5.8 (0.5)*
14.5 (5.5)** 54.9 (19.6)*,** 0.08 (0.06) 0.14 (0.09) 1.5 (0.3) 5.6 (0.9)*
Values are means (SD). * Significant difference from rest (Po0.001), significant difference from day 1 (Po0.05).
**
Fig. 1. Forearm (f/arm) sweat rate (SR) during each 30 min stage of the 2 h exercise-heat exposure on day 1 (solid line) and day 3 (dashed line). *, Significant difference from 0 to 30 min SR (Po0.05); y, significant difference from day 1 (Po0.05). Values are means (SEM).
Fig. 2. Sweat sodium and chloride concentrations during day 1 (solid bar) and day 3 (striped bar). *, Significant difference from day 1 (Po0.05). Values are means (SEM).
Table 3 Resting and 2 h exercise serum electrolyte concentrations and osmolarity on days 1 and 3
[Na+] (mmol L1)—rest [Na+] (mmol L1)—exercise [Cl] (mmol L1)—rest [Cl] (mmol L1)—exercise Osmolality (mOsmol kg1)—rest Osmolality (mOsmol kg1)—exercise
Day 1
Day 3
135.99 (1.10) 137.36 (2.11) 99.38 (2.75) 99.24 (4.20) 284 (6) 283 (5)
135.63 (1.16) 136.28 (1.00) 99.04 (1.66) 97.74 (1.70) 283 (3) 281 (3)
Values are means (SD).
Plasma norepinephrine concentration increased with exercise (Po0.001) on both days 1 and 3 with no difference between days at rest or during exercise (Table 4). Plasma epinephrine showed no change with exercise or between days (Table 4). PBMC counts were increased with exercise on both days (day 1: rest 6.8 (2.5) cells 108 L1, exercise 11.4 (3.0) cells 108 L1; day 3: rest 6.8 (2.6) cells 108 L1, exercise 11.8 (3.6) cells 108 L1) (Po0.01). No difference in PBMC counts were detected between days, in either resting or exercise samples.
Fig. 3. Hsp72 mRNA (normalised to RibS29) (a) and protein levels (b) at rest (solid bar) and following exercise (striped bar) on days 1 and 3. *, Significant difference from rest (Po0.05). Values are means (SEM).
No significant difference in PBMC Hsp72 mRNA was revealed between days 1 and 3, however, a significant difference was found with time. Post-hoc analysis detected an increase in mRNA levels on day 1 (Po0.05) with no change found on day 3 (Fig. 3a). Nonparametric analysis found no difference in Hsp72 protein levels with exercise or between days (Fig. 3b). 4. Discussion Exercise-heat stress on two consecutive days resulted in reduced thermal and cardiovascular strain during exercise. These adaptations were paralleled by an attenuated
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increase in PBMC Hsp72 mRNA levels during exercise on day 3, with no significant adaptation at the protein level. A reduction in exercise HR and Tre, and an enhanced PV, were found after two exercise-heat exposures. This decrease in exercise HR was potentially due to the expanded PV (Senay et al., 1976). The fall in exercise Tre was not due to a lowered resting level, as previously indicated (Buono et al., 1998), suggesting an improved mechanism of heat loss occurred during exercise in the current study. This study is the first to definitively identify an increase in SR after only two exercise-heat sessions, supportive of our preliminary data showing SR to be enhanced after a single exposure (Milne and Nimmo, 2004). The mechanism responsible for the enhanced SR involves the maintenance of sweating toward the end of exercise (Fig. 1). This was not a response to differences in catecholamine concentration and, as a uniform increase in SR was not observed, enhanced neurotransmitter sensitivity of the sweat gland or increased sweat gland size (Sato et al., 1990) are unlikely mechanisms. Nor is it likely that previously inactive sweat glands were recruited (Collins and Weiner, 1964; Sargent II et al., 1965). It is therefore proposed that a delay in the onset of hidromeiosis, resulting from the adaptive ability of the sweat glands (Fox et al., 1967; Ogawa et al., 1982; Candas et al., 1983), improved the maintenance of sweating following two consecutive exercise-heat exposures. A fall in sweat [Na+] and [Cl] occurred despite the increased SR. This was not merely a reflection of altered serum composition, indicating an enhanced reabsorption following two days exercise-heat stress. Aldosterone, involved in the reabsorption of Na+ at the duct of the sweat gland (Conn and Arbor, 1963; Sato and Dobson, 1970), was likely responsible for the improved sweat Na+ retention as its level was enhanced on day 3, both at rest and following exercise. The genomic action of the elevated aldosterone with exercise on day 1 is thought to have stimulated the synthesis of sweat duct Na+ channels and pumps, with the elevated exercise and resting levels on day 3 possibly upregulating the activity of these Na+ transporters (reviewed in Booth et al., 2002). In response to exercise, Hsp72 mRNA increased significantly on day 1, however, by day 3 there was no significant increase, indicative of a reduced Hsp72 mRNA response. This is possibly due to a reduced level of stress, as suggested by the improved thermoregulatory and cardiovascular function, and is consistent with a trend noted by Fehrenbach et al. (2001). The data of the present study suggest the Hsp72 mRNA response to exercise is a potential marker of acclimation, however, resting levels will not provide sufficient discrimination. Hsp72 protein at rest, or in response to exercise, was unaffected by two consecutive exercise-heat exposures. The acute exercise-heat bout on day 1 of the present study enhanced Hsp72 mRNA and norepinephrine levels, the latter a suggested mediator of Hsp72 protein induction in rats (Meng et al., 1996; Heneka et al., 2003), but failed to
enhance Hsp72 protein levels. In contrast, previous studies have demonstrated an enhanced monocyte Hsp72 protein level immediately following exercise-heat stress, with peak levels found 24 h after exercise (Fehrenbach et al., 2001, 2003). The contrasting findings may be related to cell type analysed as the PBMCs in the current study included monocytes and lymphocytes. Lymphocyte Hsp72 level is minimally affected by exercise stress (Fehrenbach et al., 2000), hence the chance of detecting elevated PBMC Hsp72 is lower than when analysing monocyte Hsp72. In addition, this bias would be exaggerated during exercise due to the exercise-heat enhancement of PBMCs being dominated by an increase in lymphocytes (Fehrenbach et al., 2003). A further possibility relates to the intensity of exercise. Although the present combination of exercise and heat stress resulted in the attainment of core temperatures previously found to elevate monocyte Hsp72 protein (Fehrenbach et al., 2001, 2003), the intensity of exercise is _ 2peak versus 70–75% lower than in these studies (44% VO _ VO2peak ). This suggests that hyperthermia, as reflected by core temperature, is not the major factor involved in the regulation of monocyte Hsp72, supported by the finding that increases in systemic and local temperature per se are not involved in the regulation of the human skeletal muscle Hsp72 response (Morton et al., 2006b). This is the first study to examine the human physiological and Hsp72 adaptations that occur during the initial phase (two days) of human-heat acclimation. The study has identified that, in these particular exercise and environmental conditions, exercise-heat stress must be conducted on at least two consecutive days for the attainment of reduced thermal and cardiovascular strain and that the enhanced SR is most likely due to a decrease in hidromeiosis. It has also shown that the physiological adaptations are matched with a reduction in cellular stress as evidenced by a reduction in the response of Hsp72 mRNA to exercise, although these adaptations were not reflected in PBMC Hsp72 protein levels. Acknowledgements The authors would like to thank Moira Watson for performing the PBMC separations and cell counts. We would also like to thank the participants for volunteering and completing this demanding study. The authors are grateful to Glasgow City Council, Glasgow School of Sport and the West of Scotland Institute of Sport for financial support for this study. References Armstrong, L.E., Maresh, C.M., Castellani, J.W., et al., 1994. Urinary indices of hydration status. Int. J. Sport. Nutr. 4, 265–279. Barnett, M.A., Maughan, R.J., 1993. Response of unacclimatized males to repeated weekly bouts of exercise in the heat. Br. J. Sports Med. 27, 39–44. Booth, R.E., Johnson, J.P., Stockand, J.D., 2002. Aldosterone. Adv. Physiol. Educ. 26, 8–20.
ARTICLE IN PRESS H.C. Marshall et al. / Journal of Thermal Biology 32 (2007) 341–348 Brisson, G.R., Boisvert, P., Peronnet, F., Perrault, H., Boisvert, D., Lafond, J.S., 1991. A simple and disposable sweat collector. Eur. J. Appl. Physiol. 63, 269–272. Buono, M.J., Heaney, J.H., Canine, K.M., 1998. Acclimation to humid heat lowers resting core temperature. Am. J. Physiol. 274, R1295–R1299. Candas, V., Libert, J.P., Vogt, J.J., 1983. Sweating and sweat decline of resting men in hot humid environments. Eur. J. Appl. Physiol. 50, 223–234. Chaplin, H., Mollison, P.L., 1952. Correction for plasma trapped in the red cell column of the hematocrit. Blood 7, 1227–1238. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. Collins, K.J., Weiner, J.S., 1964. The effect of heat acclimatisation on the activity and numbers of sweat-glands: a study on Indians and Europeans. J. Physiol. 177, 16P–17P. Conn, J.W., Arbor, A., 1963. Aldosteronism in man. J. Am. Med. Assoc. 183, 775–781. Convertino, V.A., Greenleaf, J.E., Bernauer, E.M., 1980. Role of thermal and exercise factors in the mechanism of hypervolemia. J. Appl. Physiol. 48, 657–664. Costill, D.L., Saltin, B., 1974. Factors limiting gastric emptying during rest and exercise. J. Appl. Physiol. 37, 679–683. Dill, D.B., Costill, D.L., 1974. Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration. J. Appl. Physiol. 37, 247–248. Fehrenbach, E., Passek, F., Niess, A.M., et al., 2000. HSP expression in human leukocytes is modulated by endurance exercise. Med. Sci. Sports Exercise 32, 592–600. Fehrenbach, E., Niess, A.M., Veith, R., Dickhuth, H.-H., Northoff, H., 2001. Changes of HSP72-expression in leukocytes are associated with adaptation to exercise under conditions of high environmental temperature. J. Leukocyte Biol. 69, 747–754. Fehrenbach, E., Veith, R., Schmid, M., Dickhuth, H.-H., Northoff, H., Niess, A.M., 2003. Inverse response of leukocyte heat shock proteins and DNA damage to exercise and heat. Free Radical Res. 37, 975–982. Flanagan, S.W., Ryan, A.J., Gisolfi, C.V., Moseley, P.L., 1995. Tissuespecific HSP70 response in animals undergoing heat stress. Am. J. Physiol. 268, R28–R32. Fox, R.H., Goldsmith, R., Hampton, I.F.G., Hunt, T.J., 1967. Heat acclimatization by controlled hyperthermia in hot-dry and hot-wet climates. J. Appl. Physiol. 22, 39–46. Gisolfi, C.V., Summers, R.W., Schedl, H.P., 1990. Intestinal absorption of fluids during rest and exercise. In: Gisolfi, C.V., Lamb, D.R. (Eds.), Fluid homeostasis during exercise, Perspectives in Exercise Science and Sports Medicine. WBC Brown & Benchmark, Dubuque, IA, pp. 129–180. Greenleaf, J.E., Sciaraffa, D., Shvartz, E., Keil, L.C., Brock, P.J., 1981. Exercise training hypotension: implications for plasma volume, renin, and vasopressin. J. Appl. Physiol. 51, 298–305. Hayden, G., Milne, H.C., Patterson, M.J., Nimmo, M.A., 2004. The reproducibility of closed-pouch sweat collection and thermoregulatory responses to exercise-heat stress. Eur. J. Appl. Physiol. 91, 748–751. Heneka, M.T., Gavrilyuk, V., Landreth, G.E., O’Banion, M.K., Weinberg, G., Feinstein, D.L., 2003. Noradrenergic depletion increases inflammatory responses in brain: effects on IkappaB and HSP70 expression. J. Neurochem. 85, 387–398. Hoffman, J.R., Maresh, C.M., Armstrong, L.E., et al., 1994. Effects of hydration state on plasma testosterone, cortisol and catecholamine concentrations before and during mild exercise at elevated temperature. Eur. J. Appl. Physiol. 69, 294–300. Hunter-Lavin, C., Davies, E.L., Bacelar, M.M.F.V.G., Marshall, M.J., Andrew, S.M., Williams, J.H.H., 2004. Hsp70 release from peripheral blood mononuclear cells. Biochem. Biophys. Res. Commun. 324, 511–517.
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Kirby, C.R., Convertino, V.A., 1986. Plasma aldosterone and sweat sodium concentrations after exercise and heat acclimation. J. Appl. Physiol. 61, 967–970. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lind, A.R., Bass, D.E., 1963. Optimal exposure time for development of acclimatization to heat. Fed. Proc. 22, 704–708. Maloyan, A., Palmon, A., Horowitz, M., 1999. Heat acclimation increases the basal HSP72 level and alters its production dynamics during heat stress. Am. J. Physiol. 276, R1506–R1515. Meng, X., Brown, J.M., Ao, L., Banerjee, A., Harken, A.H., 1996. Norepinephrine induces cardiac heat shock protein 70 and delayed cardioprotection in the rat through alpha1 adrenoreceptors. Cardiovasc. Res. 32, 374–383. Milne, H.C., Nimmo, M.A., 2004. The effect of an acute exercise-heat stress on subsequent human thermoregulatory and sweat variables. J. Physiol. 557P, C65. Mizzen, L.A., Welch, W.J., 1988. Characterization of thermotolerant cell. I. Effects of protein synthesis activity and the regulation of heat-shock protein 70 expression. J. Cell. Biol. 106, 1105–1116. Morton, J.P., MacLaren, D.P., Cable, N.T., et al., 2006a. Time course and differential responses of the major heat shock protein families in human skeletal muscle following acute nondamaging treadmill exercise. J. Appl. Physiol. 101, 176–182. Morton, J., MacLaren, D., Cable, T., McArdle, A., Drust, B., 2006b. Time-course and differential expression of heat shock proteins in human skeletal muscle following non-damaging treadmill exercise: is heat a mechanism of activation? ECSS conference abstract (Abstract ID: 761). Nielsen, B., Strange, S., Christensen, N.J., Warberg, J., Saltin, B., 1997. Acute and adaptive responses in humans to exercise in a warm, humid environment. Pflugers Arch. 434, 49–56. Ogawa, T., Asayama, M., Miyagawa, T., 1982. Effects of sweat gland training by repeated local heating. Jpn. J. Physiol. 32, 971–981. Patterson, M.J., Stocks, J.M., Taylor, N.A.S., 2004. Sustained and generalised extracellular fluid expansion following heat acclimation. J. Physiol. 559, 327–334. Ramanathan, N.L., 1964. A new weighting system for mean surface temperature of the human body. J. Appl. Physiol. 19, 531–533. Ryan, A.J., Gisolfi, C.V., Moseley, P.L., 1991. Synthesis of 70K stress protein by human leukocytes: effect of exercise in the heat. J. Appl. Physiol. 70, 466–471. Sargent II, F., Smith, C.R., Batterton, D.L., 1965. Eccrine sweat gland activity in heat acclimation. Int. J. Biometeorol. 9, 229–231. Sato, K., Dobson, R.L., 1970. The effect of intracutaneous d-aldosterone and hydrocortisone on human eccrine sweat gland function. J. Invest. Dermatol. 54, 450–462. Sato, F., Owen, M., Matthes, R., Sato, K., Gisolfi, C.V., 1990. Functional and morphological changes in the eccrine sweat gland with heat acclimation. J. Appl. Physiol. 69, 232–236. Senay, L.C.J., Mitchell, D., Wyndham, C.H., 1976. Acclimatisation in a hot, humid environment: body fluid adjustments. J. Appl. Physiol. 40, 786–796. Shin, Y.-O., Oh, J.-K., Sohn, H.-S., et al., 2004. Expression of exerciseinduced Hsp70 in long-distance runner’s leukocytes. J. Thermal. Biol. 29, 769–774. Skidmore, R., Gutierrez, J.A., Guerriero Jr., V., Kregel, K.C., 1995. HSP70 induction during exercise and heat stress in rats: role of internal temperature. Am. J. Physiol. 268, R92–R97. Sonna, L.A., Wenger, C.B., Flinn, S., Sheldon, H.K., Sawka, M.N., Lilly, C.M., 2004. Exertional heat injury and gene expression changes: a DNA microarray analysis study. J. Appl. Physiol. 96, 1943–1953. Strydom, N.B., Wyndham, C.H., Williams, C.G., et al., 1966. Acclimatisation to humid heat and the role of physical conditioning. J. Appl. Physiol. 21, 636–642.
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Wyndham, C.H., Rogers, G.G., Senay, L.C., Mitchell, D., 1976. Acclimatisation in a hot, humid environment: cardiovascular adjustments. J. Appl. Physiol. 40, 779–785. Yamazaki, F., Hamasaki, K., 2003. Heat acclimation increases skin vasodilation and sweating but not cardiac baroreflex responses in heatstressed humans. J. Appl. Physiol. 95, 1567–1574.
Journal of Thermal Biology 32 (2007) 341–348 www.elsevier.com/locate/jtherbio
Human physiological and heat shock protein 72 adaptations during the initial phase of humid-heat acclimation Helen C. Marshalla, Samantha A. Campbellb, Craig W. Robertsb, Myra A. Nimmoa, a
Strathclyde Institute of Pharmacy and Biomedical Sciences, Faculty of Science, University of Strathclyde, 199 Cathedral Street, Glasgow G4 0QU, UK Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, 27 Taylor St, Glasgow G4 0NR, UK
b
Received 12 December 2006; accepted 17 April 2007
Abstract 1. The timescale and integration of human whole body and cellular Hsp72 adaptations during the initial phase of human-heat acclimation were determined. 2. Two exercise humid-heat exposures on consecutive days lowered exercise rectal temperature (Po0.05) and heart rate (Po0.01). 3. Sweat rate was increased (Po0.001) during exercise through an improved maintenance of sweating, and sweat Na+ reabsorption was enhanced (Po0.05). 4. These adaptations were accompanied by a reduced Hsp72 mRNA response with no change in protein level. 5. Two prolonged, low-intensity exercise-heat bouts on consecutive days are sufficient to initiate physiological and Hsp72 mRNA adaptations, although Hsp72 protein adaptation may require a greater exercise intensity or longer acclimation period. r 2007 Elsevier Ltd. All rights reserved. Keywords: Acclimation; Heat shock protein 72; Humid-heat stress
1. Introduction The process of acclimation to a hot environment is commonly judged on the response of heart rate (HR), core and skin temperatures, and sweat rate (SR). Acclimation, however, is a complex process involving adaptations not only at whole body but also cellular levels. The most inducible and thermosensitive marker of cellular stress is heat shock protein (Hsp) 72 (Mizzen and Welch, 1988). Human peripheral blood mononuclear cells (PBMCs) are commonly used to monitor Hsp72 changes with exercise (Fehrenbach et al., 2001, 2003; Shin et al., 2004) and heat stress (Hunter-Lavin et al., 2004) as these cells respond rapidly compared with skeletal muscle which has been reported to take 48 h to respond (Morton et al., 2006a). The majority of studies report physiological changes after 6–10 consecutive days of exercise-heat stress (Kirby and Convertino, 1986; Nielsen et al., 1997; Patterson et al., 2004), with no analysis of changes over the first few days. Corresponding author. Tel.: +44 141 548 5791; fax:+44 141 548 3129.
E-mail address: [email protected] (M.A. Nimmo). 0306-4565/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2007.04.003
For many, an acclimation period of 6–10 consecutive days is impractical, yet the minimum number of exposures required to gain a thermoregulatory benefit remains unclear. Graphical representations suggest heart rate, core temperature and SR adaptations may start to occur in the early phase of acclimation (Lind and Bass, 1963; Strydom et al., 1966; Convertino et al., 1980; Nielsen et al., 1997), with quantitative data indicating a reduction in exercise heart rate by the third exercise-heat exposure (Wyndham et al., 1976; Greenleaf et al., 1981; Yamazaki and Hamasaki, 2003) and enhanced SR on day five (Greenleaf et al., 1981). Recent preliminary work in our laboratory, however, found an enhanced SR following a single exercise-heat bout, although no changes in heart rate or rectal temperature were observed (Milne & Nimmo, 2004). Whether these reductions in cardiovascular and thermoregulatory strain are accompanied by Hsp72 adaptations is unclear. Existing human literature suggests the response of intracellular Hsp72 reflects earlier stresses (Ryan et al., 1991; Fehrenbach et al., 2001) and could hence be a potential marker for assessing adaptation to repeated exercise-heat exposures. An initial indication of this is
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found in the study of Fehrenbach et al. (2001), which reports both core temperature and leukocyte Hsp72 adaptations following two identical exercise-heat exposures, separated by six days rest. This finding is somewhat surprising, however, considering exercise bouts in the heat, separated by six days, have previously been found to have no acclimation effect on physiological variables (Barnett and Maughan, 1993). Although human studies are lacking, research in rats has reported myocardial Hsp72 adaptations to the initial stage of passive heat acclimation (continuous exposure to 34 1C for two days) (Maloyan et al., 1999), and exercise bouts on three consecutive days in a temperate environment (Taylor et al., 1999). Comparison of these studies to humans, however, is not robust as the Hsp72 response appears to be both species (earlier skeletal muscle Hsp72 response in rats (Skidmore et al., 1995) versus humans (Morton et al., 2006a)) and tissue specific (Flanagan et al., 1995; Skidmore et al., 1995). The present human study, therefore, aims to determine both physiological and molecular (PBMC Hsp72 expression at the mRNA and protein levels) adaptations following two exercise humid-heat exposures on consecutive days, in order to determine whether this exposure is sufficient to gain thermoregulatory benefits and whether these are evident at the cellular level. 2. Materials and methods 2.1. Subjects Seven males (mean (SD); age, height, body mass and _ 2peak ): 30 (4) yr, 1.80 (0.08) m, 73.8 peak oxygen uptake (VO (8.5) kg and 3.88 (0.43) L min1, respectively) volunteered to participate in the present study. Subjects had undertaken no formal acclimation or spent any time in a hot environment over the preceding 2 months. The study was conducted in accordance with the Declaration of Helsinki, approved by the Local Ethics Committee and subjects provided written informed consent. Subjects attended the laboratory at a consistent time in the morning on five separate occasions. 2.2. Experimental design Subjects completed a 2 h cycle in a hot-humid environment (mean (SD); 38.0 (0.1) 1C, 60.0 (0.1)% relative humidity (RH), wind 0.6 (0.1) m s1) on three consecutive _ 2peak , was days. Workload, corresponding to 38% VO determined from preliminary tests involving submaximal exercise of varying intensity followed by a continuous, incremental cycling test to volitional exhaustion on an electronically braked cycle ergometer (Lode Excalibur, Groningen, The Netherlands). Expired gases were measured continuously during the preliminary tests using an automated gas analysis system (Jaeger Oxycon Pro 4.6, Hoechberg, Germany). The load and pedal cadence (70 rpm) were kept constant between the three main trials.
On days 1 and 3, a baseline arterialised-venous blood sample was collected after 30 min seated rest, and an exercise blood sample was collected following 2 h upright cycling. Posture was controlled to allow for the calculation of plasma volume (PV) changes. Sweat was collected from a sweat patch every 30 min. Heart rate (HR), rectal temperature (Tre) and skin temperatures (Tsk) were regularly observed during all three exercise-heat exposures and recorded at rest and on completion of exercise on days 1 and 3. Time of testing was consistent within a subject. Subjects were all nonsmokers, abstained from caffeine and alcohol ingestion and refrained from strenuous physical activity for 48 h prior to testing. They recorded their dietary intake and physical activity for 24 h prior to the first main trial which was replicated for the subsequent trials. Two hours prior to exercise the subjects consumed a meal replacement drink (Complan, H. J. Heinz Co Ltd., Hayes, UK) which was reconstituted by adding 2.86 mL kg1 body mass of semiskimmed milk to 0.81 g kg1 body mass of powder. To ensure euhydration, subjects consumed 10 mL kg1 body mass of water the evening before testing and consumed 10 mL kg1 body mass of water minus the volume of milk in the meal replacement drink 2 h prior to testing. On arrival to the laboratory subjects voided and euhydration status was confirmed by the measurement of urine specific gravity (Armstrong et al., 1994; Hoffman et al., 1994); Multistix SG; Bayer Diagnostics, Newburg, UK). Nude body mass was recorded (72 g; Sartorius, Go¨ttingen, Germany), a rectal thermistor was inserted, and a heart rate monitor was strapped to the chest. Subjects entered a thermoneutral environment (mean (SD); 27.0 (0.4) 1C; 27.5 (3.7)% RH) wearing shorts, socks and trainers where an antecubital vein was cannulated while subjects were supine. Subjects then moved to a seated position for 30 min, during which skin thermistors were attached and the cannulated arm placed in a heated box (50 1C) during the final 10 min. A pilot study had identified the temperature and duration of forearm heating as being sufficient to attain arterialisation, which was found to occur naturally during exercise in the heat. Resting HR, Tre and Tsk were recorded in the seated position before heating of the forearm. The left forearm was cleaned with deionised water and a sweat patch attached as previously described (Brisson et al., 1991; Hayden et al., 2004). Subjects then entered the heat chamber where exercise commenced immediately. Subjects cycled at an average workload _ 2peak over the 2 h corresponding to 43.7 (3.9)% VO _ 2 drift. Sweat loss, as determined exposures, due to VO during a dehydrated familiarisation trial, was replaced with a 20 mmol L1 sodium-chloride solution (Gisolfi et al., 1990). Fluid was provided every 15 min and was weighted towards the first hour of exercise to promote gastric emptying (Costill and Saltin, 1974); 20% (0 min), 20% (15 min), 15% (30 min), 15% (45 min), 15% (60 min), 5% (75 min), 5% (90 min) and 5% (105 min). On completion of exercise, the subjects towel dried and nude body mass
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was re-measured, allowing an estimation of whole-body sweat loss. 2.3. Experimental procedures Tre was measured using a rectal thermistor (Edale Instruments Ltd., Roedoure, UK) inserted 10 cm beyond the anal sphincter. Skin thermistors (Edale Instruments Ltd.) attached to the forearm, chest, thigh and calf on the right side of the body were used to measure Tsk. Temperatures were recorded via a 1000 series 8-bit Squirrel data logger (Type 1002, Grants, Cambridge, UK). Weighted mean skin temperature ðT¯ sk Þ was calculated according to Ramanathan (1964). HR was monitored and recorded with the use of a heart rate monitor (Polar Vantage, Kempele, Finland). Whole body sweat loss was determined by a change in body mass, corrected for fluid intake and urinary losses. Regional sweat was aspirated from a single forearm (middorsal) site on the left side of the body and regional SR determined gravimetrically by the change in mass of the aspirating syringe (70.01 g; A & D Instruments, Abingdon, UK) (method CV ¼ 6:3%; Hayden et al., 2004). The four sweat samples (0–30, 30–60, 60–90 and 90–120 min) were combined and a 2 h sweat sample was then frozen (70 1C) for later analysis. Blood was collected via dry syringes from the indwelling catheter (Insyte 20 G, Becton Dickinson, Madrid, Spain). Whole blood (10 mL) was transferred to a lithium heparin tube and used immediately for the isolation and counting of the PBMCs. PBMC pellets were frozen at 70 1C in aliquots of 1 106 cells. Haematocrit was determined immediately from whole blood. Plasma and serum were obtained by centrifugation and aliquots of plasma, serum and whole blood were frozen (70 1C) for later analysis. Sweat and serum: Sweat and serum electrolytes were determined with the use of indirect Ion Selective Electrodes (Pentra 400; Horiba ABX, Montpellier, France) and osmolality was determined using freezing-point depression (Osmomat 030; Gonotec, Berlin, Germany). Blood analysis: The analysis of plasma aldosterone was performed using a commercially available kit (Euro DPC, Gwynedd, UK) for radioimmunoassay. Plasma catecholamines, epinephrine and norepinephrine, were extracted by absorption on alumina, separated by reverse-phase liquid chromatography and measured with electrochemical detection (Waters UK Ltd., Herts, UK). Haematocrit was measured in triplicate by the microcapillary technique and
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corrected for trapped plasma (Chaplin and Mollison, 1952). Haemoglobin was determined in whole blood using the cyanomethemoglobin method (Randox, Dublin, Ireland) on a spectrophotometer (Cecil Aquarius; Thistle Scientific, Uddingston, UK). Isolation of cells from whole blood: PBMCs were isolated from whole blood by Ficoll-Histopaques (1.077 g mL1) (Sigma, Poole, UK) density centrifugation. The cells were washed in Hanks’ balanced salt solution (Sigma, Poole, UK) then re-suspended in RPMI 1640 with 2.1 mM L-Glutamine (Cambrex, Wokingham, UK). A cell count was carried out on a haemocytometer (Neubauer; Fisher, Loughborough, UK) after which cell pellets (1 106 cells) were frozen (70 1C) prior to analysis for Hsp72 mRNA and Hsp72 protein. Hsp72 gene expression in PBMCs: Total RNA was isolated from the PBMCs using TrizolTM reagent (InvitrogenTM, Paisley, UK) based on the acid-guanidium thiocyanate–phenol–chloroform method of Chomczynski and Sacchi (1987). Complimentary DNA (cDNA) was synthesised from the total RNA using SuperScriptTM III RNase H Reverse Transcriptase (InvitrogenTM, Paisley, UK) and random hexamer primers (Promega, Southampton, UK). All cDNA was stored at 20 1C until further analysis. Transcripts for Hsp72 mRNA were quantified by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Primer sequences for Hsp72 and RibS29 (internal control) were designed with the aid of MacVectorTM 7.0 (Oxford Molecular) and are presented, along with the GenBank accession numbers for the sequences used for primer design, in Table 1. Hsp72 is encoded by both Hsp70-1 and Hsp70-2 genes (Tavaria et al., 1996) hence, the Hsp72 primers were designed to detect both genes. Real-time RT-PCR (Mx3000P; Stratagene, La Jolla, CA), using SYBRs-Green (Sigma, Poole, UK) as the detection method, was used for the duplicate amplification of the cDNA samples. The thermal profile consisted of an initial 3 min denaturation at 95 1C, 40 PCR cycles (95 1C, 30 s; 64 1C, 30 s; 72 1C, 30 s), and a final incubation at 95 1C for 1 min. A melting curve analysis was performed at the end of each real-time run to verify the purity of the reaction products. The standard curve method was used for calculating the number of transcripts for both Hsp72 and RibS29 in the cDNA samples. Hsp72 mRNA expression is presented as a ratio to the internal control (Hsp72/RibS29). RibS29 was selected as the internal control as it has been shown in human PBMCs to remain unchanged upon
Table 1 GenBank Accession Numbers and primer sequences for Hsp72 and RibS29 Gene
GenBank
50 Sense 30
50 Antisense 30
Hsp70-1 Hsp70-2 RibS29
M11717 M59830 B035313
TAC AAG GGG GAG ACC AAG GCA TTC
TGC GAG TCG TTG AAG TAG GC
TCT CGC TCT TGT CGT GTC TGT TC
GGT TTT TCA TTG AGT AGA TGC CCC
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presentation of, and during recovery from, exertional heat injury (Sonna et al., 2004). Hsp72 protein expression in PBMCs: Hsp72 protein was semi-quantified by SDS-PAGE and Western blotting. A discontinuous gel system was used for SDS-PAGE involving a 5% stacking gel and a 12% separating gel using the method of Laemmli (1970). PBMC proteins from 375,000 cells were separated electrophoretically (InvitrogenTM Electrophoresis System) at 200 V for around 1 h. Proteins were transferred electrophoretically to nitrocellulose membrane (HybondTM ECLTM; Amersham Biosciences, Bucks, UK) at 100 V for 2 h. Membranes were stained with Ponceau S (Sigma, Poole, UK) to visually control protein transfer and to mark the position of marker proteins. They were then blocked with 5% skimmed milk in phosphate buffered saline and Tween-20 (0.1%) (PBS-T) and probed with a 1:3000 dilution of a mouse monoclonal antibody specific for Hsp72 (SPA-810; StressGen, Victoria, BC, Canada) overnight at 4 1C. Following washing in PBS-T, the membrane was incubated with an anti-mouse IgG HRP-linked antibody (Cell Signalling Technology, Beverly, MA) at a 1:2000 dilution, for 1 h at room temperature. The membrane was incubated for approximately 30 s with chemiluminescence substrate (SuperSignal West Pico; Perbio Science UK Ltd., Northumberland, UK) and exposed to photographic film (Amersham, Bucks, UK). Fold change in Hsp72 protein was calculated with the use of a densitometer (GS-800; Bio-Rad, Herts, UK) and image analysis software (Quantity One; Bio-Rad, Herts, UK). 2.4. Data analyses and statistics All blood and sweat measures were performed in duplicate with the exception of haematocrit, which was performed in triplicate, and Hsp72 protein expression which was determined singularly. Changes in PV with exercise and between days were estimated using the method outlined by Dill and Costill (1974). A commercial statistics package (SPSS version 12.0) was used for determining statistical significance. Data are presented as means (SD) in the text and tables, and as means (SEM) in the figures. A one-way analysis of variance (ANOVA) was carried out on resting nude body mass and urine specific gravity on all three days. A two-factor (trial by time) repeated measures ANOVA was performed to ascertain any treatment or exercise effects on Tre, HR, T¯ sk , blood variables, PBMC counts and Hsp72 mRNA levels, and 30 min forearm SRs, between the data of days 1 and 3. A Tukey’s HSD post-hoc test was used to locate differences when the ANOVA revealed a significant difference. Hsp72 protein data were not normally distributed, as determined by the Levene test, hence this data set was analysed by a Friedman’s two-way ANOVA. A paired sample Student’s t-test was used to analyse delta T¯ sk , sweat variables and percentage change in PV. The significance level was set at Po0.05 and n ¼ 7.
3. Results Subjects were euhydrated with no difference in urine specific gravity (day 1, 1.006 (0.002); day 2, 1.008 (0.004); day 3, 1.006 (0.002)) or nude body mass (day 1, 73.84 (8.47) kg; day 2, 73.87 (8.73) kg; day 3, 73.67 (8.38) kg) upon arrival at the laboratory on all three days. Exercise increased Tre, T¯ sk and HR on both days 1 and 3 (Po0.01) (Table 2). Resting Tre and HR were the same on both days while exercise levels were lower on day 3 (Po0.05) (Table 2). T¯ sk was unaffected by two exerciseheat exposures at rest or during exercise (Table 2), however, the exercise-induced increase was lower on day 3 compared with day 1 (Po0.05). A similar percentage change in PV resulted from the exercise-heat exposure on days 1 and 3 (day 1: 10.2 (3.1)%; day 3: 6.5 (3.7)%). Resting PV was estimated to expand by 6.0 (6.1)% following two days acclimation. Concentrations of plasma aldosterone and catecholamines, and PBMC counts were therefore corrected for PV expansion on day 3 (concentration 1.06) and for PV decline during exercise on both days (day 1: concentration 0.898; day 3: concentration 0.935). Whole body SR was greater (12 (5)%) on day 3 (1.29 (0.17) L h1) compared with day 1 (1.15 (0.15) L h1) (Po0.001). Similarly, average forearm SR over the 2 h exercise-heat bout increased (31 (32)%) from day 1 (9.16 (4.29) mL cm2 min1) to day 3 (11.71 (5.45) mL cm2 min1) (Po0.05). Forearm SR on day 1 did not significantly change during the 30 min periods, however, on day 3 the 60–90 min sample was greater than that collected during the initial 30 min. The elevation of sweating was maintained into the last 30 min producing a significant increase compared to the similar period on day 1 (Po0.05) (Fig. 1). Sweat [Na+], [Cl] (Fig. 2) and osmolality (day 1: 153 (36) mOsmol kg1; day 3: 140 (31) mOsmol kg1) were lower on day 3 compared with day 1 (Po0.05). No differences in serum [Na+], [Cl] and osmolality were found between days (Table 3). Plasma aldosterone levels were enhanced following exercise-heat stress on both days (Po0.001) (Table 4) with a greater concentration at rest and following exercise on day 3 than on day 1 (Po0.001 ) (Table 4). Table 2 Resting and 2 h exercise rectal temperature (Tre), mean skin temperature ðT¯ sk Þ, and heart rate (HR) during days 1 and 3
Tre (1C)—rest Tre (1C)—exercise T¯ sk (1C)—rest T¯ sk (1C)—exercise HR (beats min1)—rest HR (beats min1)—exercise
Day 1
Day 3
36.8 (0.1) 38.9 (0.4)* 33.0 (0.2) 37.4 (0.5)* 58 (4) 143 (20)*
36.7 (0.1) 38.7 (0.2)*,** 33.1 (0.2) 37.1 (0.5)* 55 (7) 134 (16)*,**
Values are means (SD). * Significant difference from rest (Po0.001), significant difference from day 1 (Po0.05).
**
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Table 4 Resting and 2 h exercise plasma epinephrine (Ep) and norepinephrine (NE) concentration on days 1 and 3
1
Aldosterone (ng dL )—rest Aldosterone (ng dL1)—exercise Ep (nmol L1)—rest Ep (nmol L1)—exercise NE (nmol L1)—rest NE (nmol L1)—exercise
Day 1
Day 3
8.0 (4.8) 37.5 (17.0)* 0.02 (0.04) 0.13 (0.14) 1.0 (0.2) 5.8 (0.5)*
14.5 (5.5)** 54.9 (19.6)*,** 0.08 (0.06) 0.14 (0.09) 1.5 (0.3) 5.6 (0.9)*
Values are means (SD). * Significant difference from rest (Po0.001), significant difference from day 1 (Po0.05).
**
Fig. 1. Forearm (f/arm) sweat rate (SR) during each 30 min stage of the 2 h exercise-heat exposure on day 1 (solid line) and day 3 (dashed line). *, Significant difference from 0 to 30 min SR (Po0.05); y, significant difference from day 1 (Po0.05). Values are means (SEM).
Fig. 2. Sweat sodium and chloride concentrations during day 1 (solid bar) and day 3 (striped bar). *, Significant difference from day 1 (Po0.05). Values are means (SEM).
Table 3 Resting and 2 h exercise serum electrolyte concentrations and osmolarity on days 1 and 3
[Na+] (mmol L1)—rest [Na+] (mmol L1)—exercise [Cl] (mmol L1)—rest [Cl] (mmol L1)—exercise Osmolality (mOsmol kg1)—rest Osmolality (mOsmol kg1)—exercise
Day 1
Day 3
135.99 (1.10) 137.36 (2.11) 99.38 (2.75) 99.24 (4.20) 284 (6) 283 (5)
135.63 (1.16) 136.28 (1.00) 99.04 (1.66) 97.74 (1.70) 283 (3) 281 (3)
Values are means (SD).
Plasma norepinephrine concentration increased with exercise (Po0.001) on both days 1 and 3 with no difference between days at rest or during exercise (Table 4). Plasma epinephrine showed no change with exercise or between days (Table 4). PBMC counts were increased with exercise on both days (day 1: rest 6.8 (2.5) cells 108 L1, exercise 11.4 (3.0) cells 108 L1; day 3: rest 6.8 (2.6) cells 108 L1, exercise 11.8 (3.6) cells 108 L1) (Po0.01). No difference in PBMC counts were detected between days, in either resting or exercise samples.
Fig. 3. Hsp72 mRNA (normalised to RibS29) (a) and protein levels (b) at rest (solid bar) and following exercise (striped bar) on days 1 and 3. *, Significant difference from rest (Po0.05). Values are means (SEM).
No significant difference in PBMC Hsp72 mRNA was revealed between days 1 and 3, however, a significant difference was found with time. Post-hoc analysis detected an increase in mRNA levels on day 1 (Po0.05) with no change found on day 3 (Fig. 3a). Nonparametric analysis found no difference in Hsp72 protein levels with exercise or between days (Fig. 3b). 4. Discussion Exercise-heat stress on two consecutive days resulted in reduced thermal and cardiovascular strain during exercise. These adaptations were paralleled by an attenuated
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increase in PBMC Hsp72 mRNA levels during exercise on day 3, with no significant adaptation at the protein level. A reduction in exercise HR and Tre, and an enhanced PV, were found after two exercise-heat exposures. This decrease in exercise HR was potentially due to the expanded PV (Senay et al., 1976). The fall in exercise Tre was not due to a lowered resting level, as previously indicated (Buono et al., 1998), suggesting an improved mechanism of heat loss occurred during exercise in the current study. This study is the first to definitively identify an increase in SR after only two exercise-heat sessions, supportive of our preliminary data showing SR to be enhanced after a single exposure (Milne and Nimmo, 2004). The mechanism responsible for the enhanced SR involves the maintenance of sweating toward the end of exercise (Fig. 1). This was not a response to differences in catecholamine concentration and, as a uniform increase in SR was not observed, enhanced neurotransmitter sensitivity of the sweat gland or increased sweat gland size (Sato et al., 1990) are unlikely mechanisms. Nor is it likely that previously inactive sweat glands were recruited (Collins and Weiner, 1964; Sargent II et al., 1965). It is therefore proposed that a delay in the onset of hidromeiosis, resulting from the adaptive ability of the sweat glands (Fox et al., 1967; Ogawa et al., 1982; Candas et al., 1983), improved the maintenance of sweating following two consecutive exercise-heat exposures. A fall in sweat [Na+] and [Cl] occurred despite the increased SR. This was not merely a reflection of altered serum composition, indicating an enhanced reabsorption following two days exercise-heat stress. Aldosterone, involved in the reabsorption of Na+ at the duct of the sweat gland (Conn and Arbor, 1963; Sato and Dobson, 1970), was likely responsible for the improved sweat Na+ retention as its level was enhanced on day 3, both at rest and following exercise. The genomic action of the elevated aldosterone with exercise on day 1 is thought to have stimulated the synthesis of sweat duct Na+ channels and pumps, with the elevated exercise and resting levels on day 3 possibly upregulating the activity of these Na+ transporters (reviewed in Booth et al., 2002). In response to exercise, Hsp72 mRNA increased significantly on day 1, however, by day 3 there was no significant increase, indicative of a reduced Hsp72 mRNA response. This is possibly due to a reduced level of stress, as suggested by the improved thermoregulatory and cardiovascular function, and is consistent with a trend noted by Fehrenbach et al. (2001). The data of the present study suggest the Hsp72 mRNA response to exercise is a potential marker of acclimation, however, resting levels will not provide sufficient discrimination. Hsp72 protein at rest, or in response to exercise, was unaffected by two consecutive exercise-heat exposures. The acute exercise-heat bout on day 1 of the present study enhanced Hsp72 mRNA and norepinephrine levels, the latter a suggested mediator of Hsp72 protein induction in rats (Meng et al., 1996; Heneka et al., 2003), but failed to
enhance Hsp72 protein levels. In contrast, previous studies have demonstrated an enhanced monocyte Hsp72 protein level immediately following exercise-heat stress, with peak levels found 24 h after exercise (Fehrenbach et al., 2001, 2003). The contrasting findings may be related to cell type analysed as the PBMCs in the current study included monocytes and lymphocytes. Lymphocyte Hsp72 level is minimally affected by exercise stress (Fehrenbach et al., 2000), hence the chance of detecting elevated PBMC Hsp72 is lower than when analysing monocyte Hsp72. In addition, this bias would be exaggerated during exercise due to the exercise-heat enhancement of PBMCs being dominated by an increase in lymphocytes (Fehrenbach et al., 2003). A further possibility relates to the intensity of exercise. Although the present combination of exercise and heat stress resulted in the attainment of core temperatures previously found to elevate monocyte Hsp72 protein (Fehrenbach et al., 2001, 2003), the intensity of exercise is _ 2peak versus 70–75% lower than in these studies (44% VO _ VO2peak ). This suggests that hyperthermia, as reflected by core temperature, is not the major factor involved in the regulation of monocyte Hsp72, supported by the finding that increases in systemic and local temperature per se are not involved in the regulation of the human skeletal muscle Hsp72 response (Morton et al., 2006b). This is the first study to examine the human physiological and Hsp72 adaptations that occur during the initial phase (two days) of human-heat acclimation. The study has identified that, in these particular exercise and environmental conditions, exercise-heat stress must be conducted on at least two consecutive days for the attainment of reduced thermal and cardiovascular strain and that the enhanced SR is most likely due to a decrease in hidromeiosis. It has also shown that the physiological adaptations are matched with a reduction in cellular stress as evidenced by a reduction in the response of Hsp72 mRNA to exercise, although these adaptations were not reflected in PBMC Hsp72 protein levels. Acknowledgements The authors would like to thank Moira Watson for performing the PBMC separations and cell counts. We would also like to thank the participants for volunteering and completing this demanding study. The authors are grateful to Glasgow City Council, Glasgow School of Sport and the West of Scotland Institute of Sport for financial support for this study. References Armstrong, L.E., Maresh, C.M., Castellani, J.W., et al., 1994. Urinary indices of hydration status. Int. J. Sport. Nutr. 4, 265–279. Barnett, M.A., Maughan, R.J., 1993. Response of unacclimatized males to repeated weekly bouts of exercise in the heat. Br. J. Sports Med. 27, 39–44. Booth, R.E., Johnson, J.P., Stockand, J.D., 2002. Aldosterone. Adv. Physiol. Educ. 26, 8–20.
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