Acute exercise in a hot environment increases heat shock protein 70 and peroxisome proliferator-activated receptor γ coactivator 1α mRNA in Thoroughbred horse skeletal muscle

Heat acclimatization or acclimation training in horses is practiced to reduce physiological strain and improve exercise performance in the heat, which can involve metabolic improvement in skeletal muscle. However, there is limited information concerning the acute signaling responses of equine skeletal muscle after exercise in a hot environment. The purpose of this study was to investigate the hypothesis that exercise in hot conditions induces greater changes in heat shock proteins and mitochondrial-related signaling in equine skeletal muscle compared with exercise in cool conditions. Fifteen trained Thoroughbred horses [4.6 ± 0.4 (mean ± SE) years old; 503 ± 14 kg] were assigned to perform a treadmill exercise test in cool conditions [COOL; Wet Bulb Globe Temperature (WBGT), 12.5°C; n = 8] or hot conditions (HOT; WBGT, 29.5°C; n = 7) consisting of walking at 1.7 m/s for 1 min, trotting at 4 m/s for 5 min, and cantering at 7 m/s for 2 min and at 90% of VO2max for 2 min, followed by walking at 1.7 m/s for 20 min. Heart rate during exercise and plasma lactate concentration immediately after exercise were measured. Biopsy samples were obtained from the middle gluteal muscle before and at 4 h after exercise, and relative quantitative analysis of mRNA expression using real-time RT-PCR was performed. Data were analyzed with using mixed models. There were no significant differences between the two groups in peak heart rate (COOL, 213 ± 3 bpm; HOT, 214 ± 4 bpm; p = 0.782) and plasma lactate concentration (COOL, 13.1 ± 1.4 mmoL/L; HOT, 17.5 ± 1.7 mmoL/L; p = 0.060), while HSP-70 (COOL, 1.9-fold, p = 0.207; HOT, 2.4-fold, p = 0.045), PGC-1α (COOL, 3.8-fold, p = 0.424; HOT, 8.4-fold, p = 0.010), HIF-1α (COOL, 1.6-fold, p = 0.315; HOT, 2.2-fold, p = 0.018) and PDK4 (COOL, 7.6-fold, p = 0.412; HOT, 14.1-fold, p = 0.047) mRNA increased significantly only in HOT at 4 h after exercise. These data indicate that acute exercise in a hot environment facilitates protective response to heat stress (HSP-70), mitochondrial biogenesis (PGC-1α and HIF-1α) and fatty acid oxidation (PDK4).


Introduction
Heat acclimatization or acclimation training has generally been practiced in humans to reduce physiological strain, decrease aerobic metabolic rate, and improve exercise performance in the heat (1)(2)(3)(4)(5).Studies in humans and rodents have shown that these adaptations involve changes at the cellular level, including improved cytoprotection against heat stress, stimulated mitochondrial biogenesis, enhanced fatty acid oxidation, and angiogenesis promotion (6)(7)(8)(9).
Heat shock proteins (HSPs) are classified as molecular chaperones that play a central role in the cellular stress response to a hot environment, and increased expression of HSPs has a protective function against cell injury associated with adverse stresses (10,11).In addition, Henstridege et al. demonstrated that genetic overexpression of HSP-70 upregulates oxidative metabolism in rodent skeletal muscle and that HSP-70 may play an important role in the regulation of mitochondrial biogenesis (12).HSP-90, a member of the HSPs, also has specific characteristics and stimulates the activation of signaling transduction pathways distinct from those stimulated by HSP-70.Katschinski et al. demonstrated that the chaperone activity of HSP-90 is involved in hypoxic-inducible factor-1α (HIF-1α) stabilization in vitro (13).HIF-1α regulates vascular endothelial growth factor (VEGF), which is implicated as important modulator of angiogenesis and facilitates migration.Both HSP-70 and HSP-90 augment proportionally increased cellular heat stress (14), in particular, HSP-70 gene expression increases synergistically when combined with exercise and hot conditions in rats (15).In addition, Morton et al. indicated that there is a sex specific HSP adaptation caused by estrogen in human skeletal muscle (16), and Gillum et al. have demonstrated that male increased HSP-72 more than female in peripheral blood mononuclear cells in response to running at 65% VO 2peak for 60 min in the heat (ambient temperature, 42°C; relative humidity, 20%) (17).In contrast, Mee et al. have demonstrated that there is no sex difference in leucocyte HSP72 mRNA after cycling at 65% VO 2peak for 90 min under a hot environment (ambient temperature, 40°C; relative humidity, 40%) in humans (18).However, there is limited information concerning the sex differences on HSPs in Thoroughbred horses.
Several studies have demonstrated that chronic heat stress can upregulate mitochondrial-related factors in vitro and in vivo, including humans and rodents.Repeated exposure to high temperature (40°C) can induce mitochondrial biogenesis and mitochondrial oxidative efficiency in C2C12 myotubes (19, 20).In addition, Maunder et al. demonstrated that endurance training under environmental heat stress (ambient temperature, 33°C; relative humidity, 80%) improves endurance performance and induces mitochondrial adaptation in humans (6).The increased energy demands associated with physiological strain activate the expression of the muscle peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, gene expression, which can induce pyruvate dehydrogenase kinase 4 (PDK4) gene expression via estrogen-related receptor α (21).PDK4 facilitates the inactivation of pyruvate dehydrogenase complex, promoting an increase in mitochondrial fatty acid oxidation and a concurrent decrease in glucose oxidation (22).In terms of acute exercise, Tamura et al. demonstrated that post-exercise in the chamber where ambient temperature regulated 40°C upregulates PGC-1α expression in mouse skeletal muscle (7).In contrast, Heesch et al. demonstrated that acute exercise in a hot environment (ambient temperature, 33°C; relative humidity, 60%) blunts mitochondrial biogenesis related gene expression both immediately and at 3 h postexercise in humans (23).Therefore, the effect of acute exercise in a hot environment on mitochondrial adaptations is still controversial and needs further investigation.
Thoroughbreds have a high capacity for energy production and produce heat that elevates core temperature by approximately 1°C per minute during strenuous exercise (24).Furthermore, when these horses exercise in hot and humid environments (ambient temperature, 32-24°C; relative humidity, 80-85%), including racing or competition during daytime in summer, where heat dissipation is challenging, their core and muscle temperatures can rise to over 42°C during high-intensity exercise (25).Previously, we reported that exercise in an environment with an ambient temperature that exceeds body temperature causes a significant elevation of core body temperature and leads to decreased running economy (26).In addition, several other studies have demonstrated that exercise in a hot environment increases physiological strain and impairs exercise performance (25, 27).As a countermeasure to reduce these deleterious effects of heat stress, it is recommended that horses train in hot conditions similar to human athletes (28), and several studies have demonstrated that repeated exposure to heat stress can improve aerobic exercise performance and reduces metabolic rate in horses (29,30).Therefore, we hypothesized that heat acclimatization or acclimation training would induce not only physiological adaptations but also cellular adaptations in skeletal muscle in horses.Several studies reported that the mRNA expression of heat shock proteins and mitochondria-associated proteins is increased after acute exercise in temperate conditions (31)(32)(33) and that there are no sex differences in the enzyme activities related to oxidative capacity in equine skeletal muscle (34).However, there is limited information concerning the differences in the acute signaling responses of equine skeletal muscle after exercise in a hot environment compared to exercise in a cool environment.Understanding these skeletal muscle responses can help construct a training strategy for races and competitions in a hot environment.
The purpose of this study was to investigate the hypothesis that exercise in hot conditions induces greater changes in heat shock proteins and mitochondrial-related signaling in equine skeletal muscle compared to exercise in cool conditions.

Oxygen consumption
The procedure for measuring oxygen consumption has been described previously (35)(36)(37).Horses wore a 25 cm diameter openflow mask on the treadmill, with a rheostat-controlled blower drawing air.Air flowed through 25 cm diameter tubing and across a pneumotachograph (LF-150B, Vise Medical, Chiba, Japan) connected to a differential pressure transducer (TF-5, Vise Medical, Chiba, Japan); this ensured that the bias flows during measurements were identical to those used during calibrations.Oxygen and CO 2 concentrations were measured by an O 2 and CO 2 analyzer (MG-360, Vise Medical, Chiba, Japan), and calibrations were performed to calculate rates of O 2 consumption and CO 2 production with electronic mass flow meters (CR-300, Kofloc, Kyoto, Japan) using the N 2dilution/CO 2 -addition mass-balance technique (38).Gas analyzer and mass flow meter outputs were recorded on personal computer and analyzed with commercial hardware and software (DI-720 and Windaq Pro+, DATAQ, Akron, OH).VO 2 was calculated for the final 30 s of each step at the incremental exercise test.

Heart rate and plasma lactate concentration
A heart rate monitor (S810, Polar, Kempele, Finland) was attached around the thorax and mean heart rate was calculated for the final 30 s of each step of the exercise test.Venous blood was collected from the jugular vein via an 18-gauge needle into a heparin tube immediately after exercise.Blood samples were centrifuged (AX-511, Tomy industrial, Tokyo, Japan) at 1740 ×g for 10 min to measure plasma lactate concentration with a lactate analyzer (Biosen S-Line, EKF-diagnostic GmbH, Barleben, Germany).

Muscle biopsy
Before exercise (pre) and 4 h after exercise, muscle samples (~50 mg wet weight) were obtained from the same area (the two sampling points were approximately 2 cm apart) at the mid-section of the gluteus medius muscle and from the same depth (5 cm below the skin surface) by needle biopsy under local anesthesia (Lidocaine, Fujisawa pharmaceutical, Osaka, Japan).All muscle samples were immediately frozen by liquid nitrogen and stored at −80°C until analyzed.

RNA isolation and real time RT-PCR
The procedure for RT-PCR has been described previously (39).Total RNA was extracted from each muscle sample with a TRIZOL regimen (Molecular Probes, Breda, Netherlands).The purity and quantity of total RNA were determined by measuring the absorbance of aliquots at 260 and 280 nm.Total RNA was then treated for 30 min at 37°C with TURBO DNase (Ambion, Austin, TX, United States) to remove genomic DNA from samples.DNase-treated RNA (0.5 μg) was used to synthesize first-stand cDNA with an Exscript™ RT reagent Kit (Takara, Tokyo, Japan).Thereafter, the cDNA products were analyzed by real-time PCR using the SYBR Green PCR Master Mix protocol in a StepOne™ Real Time PCR System (Applied Biosystems Japan, Tokyo, Japan).
The amplification program included an initial denaturation step at 95°C for 10 min, 40 cycles of denaturation at 95°C for 30 s, and annealing/extension at 58°C for 1 min.The amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was estimated as an internal control (40).Each mRNA was normalized to GAPDH by subtracting the cycle threshold (Ct) value of GAPDH from the Ct value of the gene target [ΔCt (target)].The relative expression of the target gene was calculated as the relative quantification value for the pre value.Following the relative expression, dissociation curve analysis detected no nonspecific amplification in cDNA samples.The sequences of the specific primers used in this study are presented in Table 2.Each PCR primer was designed by Primer Express ® software (Applied Biosystems Japan, Tokyo, Japan), and oligonucleotides were purchased from FASMAC (FASMAC, Kanagawa, Japan).

Statistical procedures
All data are presented as mean ± standard error (SE).Differences in heart rate and plasma lactate concentration between COOL and HOT during exercise were analyzed using Student's t-test.We analyzed mRNA expressions using mixed model with time, group and sex as fixed effects and individual horse as a random effect.When a significant effect or interaction was observed, Tukey's tests were used as post hoc tests.For all analyses, significance was set as p ≤ 0.05.Statistical software (JMP 16.2.0,SAS Institute Inc., Cary, NC) was used for all data analyses.

Heart rate and plasma lactate concentration
There were no differences between groups in peak heart rate during exercise (COOL, 213 ± 3 bpm; HOT, 214 ± 4 bpm; p = 0.782; Figure 1A).Plasma lactate concentration immediately after exercise in HOT tended to be higher than that in COOL, but the difference was not significant (COOL, 13.1 ± 1.4 mmoL/L; HOT, 17.5 ± 1.7 mmoL/L; p = 0.060; Figure 1B).

Discussion
Our main finding in this study is that exercise in a hot environment upregulates the expression of HSP-70 mRNA and PGC-1α mRNA in Thoroughbred horses.These results indicate that the combination of heat exposure and exercise facilitate the activation of heat stress responses and mitochondrial adaptations in Thoroughbred horses.
HSP-70 is classified as molecular chaperone that plays a central role in the cellular stress response to hot environments and has a protective function against cell injury associated with adverse stresses (41).Previous studies have shown that acute thermal stress with an increase in body temperature enhances HSP-70 mRNA expression in various species, including humans and rodents (7,15,42,43).Heat shock factor 1, which is a direct transcriptional activator that induces heat shock gene transcription following heat stress, directly activates the expression of PGC-1α, a master regulator in mitochondrial biogenesis (44, 45).Although we did not measure body core temperature during exercise in this study, previous studies have demonstrated that pulmonary artery temperature during exercise under hyperthermic conditions (WBGT 28°C) is 1-2°C higher than that under cool conditions (WBGT 15°C) in horses (25-27).Therefore, it is reasonable to assume that core and muscle temperature in HOT was also higher than that in COOL, inducing sufficient thermal stress to upregulate the expression of these genes.Furthermore, the expression of PGC-1α mRNA is enhanced in an intensity-dependent manner (46)(47)(48).In horses, several studies have demonstrated that VO 2 during submaximal exercise in hot conditions is higher compared to that at the same speed in cool conditions (26, 27), and increased relative workload may also contribute the upregulation of PGC-1α mRNA.Therefore, additional increases in core and muscle temperatures with the combination of heat exposure and exercise enhance HSP-70 and PGC-1α mRNA responses, which may be beneficial for cytoprotection and mitochondrial biogenesis.
While the expression of HSP-70 mRNA increased following exercise, no significant change was observed in HSP-90 mRNA.HSP-90, similar to HSP-70, plays a crucial role in cytoprotection and acts as biochemical sensors for various stressors, including heat shock and exercise (41).The extent of HSPs induction following a single bout of exercise depends on several factors, including exercise modality, duration, intensity, and training status of Heart rate (A) at the end of exercise and plasma lactate concentration immediately after exercise (B) in COOL (blue) and HOT (red).In the present study, we detected increased PDK4 mRNA coincident with increased PGC-1α mRNA in Thoroughbred horses training in hot conditions (21,22).Several studies have demonstrated that muscle glycogen utilization is reduced with heat acclimation (8,50).Thus, the improved muscle energy metabolism with heat acclimation may involve a shift in energy fuel utilization from glucose oxidation to fatty acid oxidation due to the upregulation of PDK4.
In a hot environment, cutaneous blood flow increases through peripheral vasodilation for heat dissipation in horses and humans (51, 52).During prolonged low-to-moderate intensity exercise, muscle  , also demonstrated a correlation between the increase in VEGF mRNA and plasma lactate concentration after exercise (56).In humans, exercise in a hot environment often enhances glycolysis in skeletal muscle, which may be probably due to reduced blood flow and oxygen delivery to working muscle (57).In this study, we observed no differences in the plasma lactate concentration between HOT and COOL.Therefore, the reduction in muscle blood flow and HIF-1α induction may be insufficient to enhance angiogenesis in this study.We observed no changes in PFK, MCT1, and MCT4 mRNA expressions in this study.PFK is a kinase enzyme that phosphorylates fructose 6-phosphate and is the main rate-controlling enzyme of glycolysis.MCT1 and 4 are key factors in mediating lactate transport.There were no differences in the plasma lactate concentration between HOT and COOL, which is reasonable in that gene expressions related to the glycolysis pathway and lactate transport were not enhanced in HOT.Previous studies in humans and horses have showed an augmented intramuscular carbohydrate utilization and an increased blood lactate concentration when comparing prolonged submaximal exercise in a hot environment with that in a cool environment (1,27,58).In contrast, Maxwell et al. demonstrated no differences in the rate of muscle glycogenolysis and blood lactate accumulation during intermittent high-intensity exercise between hot and cool environments (59,60).These results, including our results, indicate that the increased glycolysis and blood lactate accumulation may depend on several factors, including the duration and intensity of exercise, training status of subjects, environmental temperature, and the timing of sample collection.
We acknowledge there are limitations in this study.First, we obtained muscle samples only at 4 h after exercise and did not observe time-course changes of gene expressions over time.Because the time-course changes of gene expressions vary with target markers (61), we could detect more mRNA changes if we collected muscle samples at several time points.Second, we only observed changes in mRNA expression following acute exercise.In humans, 10 days or longer heat acclimation are recommended to induce optimize adaptations (62).Some reports in horses have demonstrated that heat acclimation have beneficial effects on physiological responses similar to humans (29,63).However, these studies did not evaluate cellular adaptations in skeletal muscle.Therefore, further studies are necessary to fully investigate the long-term effects of heat acclimatization or acclimation on skeletal muscle adaptations.
In summary, we have demonstrated that HSP-70, PGC-1α, HIF-1α, and PDK4 mRNA significantly increased after high-intensity exercise in hot conditions.These increases in gene expressions may facilitate a protective response to heat stress, mitochondrial biogenesis, and fatty acid oxidation.Therefore, our results suggest that heat acclimatization or acclimation for competitions and races in hot conditions is effective in improving aerobic energy metabolism, as well as reducing heat stress.Repeated exposure to heat stress, such as training in natural hot environment or in artificial heated environment, may be effective strategy for competitions and races held in hot environments.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

FIGURE 2 Fold
FIGURE 2Fold changes in the mRNA expression of HSP-70 (A) and HSP-90 (B) at 4 h after exercise compared to before exercise (pre) in COOL (blue) and HOT (red).*Significant differences from pre (p < 0.05).

FIGURE 3 Fold
FIGURE 3Fold changes in the mRNA expression of PGC-1α (A), PDK4 (B), and SDHa (C) at 4 h after exercise compared to before exercise (pre) in COOL (blue) and HOT (red).*Significant differences from pre (p < 0.05).

FIGURE 4
FIGURE 4    Changes in the mRNA expression of HIF-1α (A), VEGF (B) and ANGPT1 (C), and CD31 (D) at 4 h after exercise compared to before exercise (pre) in COOL (blue) and HOT (red).*Significant differences from pre (p < 0.05).

FIGURE 5
FIGURE 5Changes in the mRNA expression of PFK (A), MCT1 (B), and MCT4 (C) at 4 h after exercise compared to before exercise (pre) in COOL (blue) and HOT (red).
Temperature Humidity Index (THI), 62.8] 2 days/ week for 4 weeks and walked for 1 h/day in a walker for 5 days/week and were kept in a 400 m 2 paddock for 6 h/day prior to the experiment.After training, the horses performed preliminary incremental exercise tests in cool conditions to determine their VO 2max .Following a warm up at 4 m/s for 3 min, the horses began exercising up a 6% incline for 2 min each at 1.7, 4, 6, 8, 10, 12 and 13 m/s until the houses could not maintain their position at the front of the treadmill with human encouragement.

TABLE 1
Characteristics of fifteen horses used in the present study.