Metabolomic responses to high-intensity interval exercise in equine skeletal muscle: effects of rest interval duration.

High-intensity interval training has attracted considerable attention as a time-efficient strategy for inducing physiological adaptations, but the underlying mechanisms have yet to be elucidated. By using metabolomics techniques, we investigated changes in the metabolic network responses in Thoroughbred horses to high-intensity interval exercise performed with two distinct (15 min or 2 min) rest intervals. The peak plasma lactate level was higher during high-intensity exercise with a 2 min rest duration than that with a 15 min rest duration (24.5±6.8 versus 13.3±2.7 mmol l-1). The arterial oxygen saturation was lower at the end of all exercise sessions with a 2 min rest duration than that with a 15 min rest duration. Metabolomic analysis of skeletal muscle revealed marked changes in metabolite concentrations in the first and third bouts of the 15 min rest interval conditions. In contrast, there were no metabolite concentrations or pathways that significantly changed during the third bout of exercise performed with a 2 min rest interval. Our findings suggest that the activity of each energy production system is not necessarily reflected by apparent changes in metabolite concentrations, potentially due in part to a better match between metabolite flux into and out of the pathway and cycle, as well as between metabolite production and disposal. This study provides evidence that changes in metabolite concentrations vary greatly depending on the number of repetitions and the length of rest periods between exercises, even if the exercises themselves are identical.

Field-training in young two-year-old thoroughbreds: investigating cardiorespiratory adaptations and the presence of exercise induced pulmonary hemorrhage.

BACKGROUND: Comparatively little is known regarding the initial cardiorespiratory response of young racehorses to training. The objectives were to compare physiological parameters before and after introductory training and determine whether young Thoroughbreds show endoscopic signs of exercise-induced pulmonary hemorrhage (EIPH). Ten Thoroughbreds (20-23 months) underwent 12-weeks of introductory training, including weekly speed sessions. Two 600 m high-speed exercise tests (HSET) were performed following weeks 4 and 12 while wearing a validated ergospirometry facemask. Peak oxygen consumption (V̇O2pk) and ventilatory parameters (tidal volume, VT; peak inspiratory and expiratory flow, PkV̇I, PkV̇E; respiratory frequency, Rf; minute ventilation, V̇E) were measured. The ventilatory equivalent of oxygen (V̇E/V̇O2) and the aerobic and anaerobic contributions to energy production were calculated. Maximal heart rate (HRmax) and HR at maximal speed (HRVmax) were determined. Post-exercise hematocrit, plasma ammonia and blood lactate were measured. Evidence of EIPH was investigated via tracheobronchoscopy post-exercise. Results were compared (paired t-test, P < 0.05). RESULTS: Horses were faster following training (P < 0.001) and V̇O2pk increased 28 ml/(kg total mass.min) (28 ± 16%; P < 0.001). Ventilatory (V̇E, P = 0.0015; Rf, P < 0.001; PkV̇I, P < 0.001; PkV̇E, P < 0.001) and cardiovascular parameters (HRmax, P = 0.03; HRVmax, P = 0.04) increased. The increase in V̇E was due to greater Rf, but not VT. V̇E/V̇O2 was lower (26 ± 3.6 vs 23 ± 3.7; P = 0.02), indicating improved ventilatory efficiency. Anaerobic contribution to total energy production increased from 15.6 ± 6.1% to 18.5 ± 6.3% (P = 0.02). Post-exercise hematocrit (P < 0.001), plasma ammonia (P = 0.03) and blood lactate (P = 0.001) increased following training. Horses showed no signs of EIPH. CONCLUSIONS: Young two-year-old Thoroughbreds responded well to introductory training without developing tracheobronchoscopic evidence of EIPH.

Cardiopulmonary function during supramaximal exercise in hypoxia, normoxia and hyperoxia in Thoroughbred horses.

Supramaximal exercise while inspiring different O2 gases may induce different responses in cardiopulmonary function at the same relative and/or absolute exercise intensity. The purpose of this study was to compare the effects of supramaximal exercise in hypoxia, normoxia and hyperoxia on cardiopulmonary function in Thoroughbred horses. Using a crossover design, five well-trained horses were made to run up a 6% grade on a treadmill at supramaximal speeds sustainable for approximately 110 sec (approximately 115% V̇O2max) while breathing normoxic gas (NO, 21% O2) or hypoxic gas (LO, 15.3% O2) in random order. Horses also ran at the same speed, incline and run time as in NO while breathing hyperoxic gas (HONO, 28.8% O2) and as in LO while breathing normoxic gas (NOLO). Runs were on different days, and cardiopulmonary variables were analyzed with repeated-measures ANOVA and the Holm-Sidák method for pairwise comparisons. Supramaximal speeds differed significantly between NO and LO (14.0 ± 0.5 [SD] m/sec vs. 12.6 ± 0.5 m/sec), but run times to exhaustion did not (112 ± 17 sec vs. 103 ± 14 sec). The V̇O2max in NO was higher than that in LO (165 ± 11 vs. 120 ± 15 ml (min× kg)), as was the arterial oxygen tension (66 ± 5 vs. 45 ± 2 Torr). Oxygen consumption was increased in HONO and NOLO compared with the values in NO and LO, respectively. Supramaximal exercise in hypoxia induces more severe hypoxemia and decreases V̇O2max compared with normoxia at the same relative intensity. Conversely, supramaximal exercise in hyperoxia alleviates hypoxemia and increases V̇O2 compared with normoxia at the same absolute intensity.

Hypoxic training increases maximal oxygen consumption in Thoroughbred horses well-trained in normoxia.

Hypoxic training is effective for improving athletic performance in humans. It increases maximal oxygen consumption (V̇O2max) more than normoxic training in untrained horses. However, the effects of hypoxic training on well-trained horses are unclear. We measured the effects of hypoxic training on V̇O2max of 5 well-trained horses in which V̇O2max had not increased over 3 consecutive weeks of supramaximal treadmill training in normoxia which was performed twice a week. The horses trained with hypoxia (15% inspired O2) twice a week. Cardiorespiratory valuables were analyzed with analysis of variance between before and after 3 weeks of hypoxic training. Mass-specific V̇O2max increased after 3 weeks of hypoxic training (178 ± 10 vs. 194 ± 12.3 ml O2 (STPD)/(kg × min), P<0.05) even though all-out training in normoxia had not increased V̇O2max. Absolute V̇O2max also increased after hypoxic training (86.6 ± 6.2 vs. 93.6 ± 6.6 l O2 (STPD)/min, P<0.05). Total running distance after hypoxic training increased 12% compared to that before hypoxic training; however, the difference was not significant. There were no significant differences between pre- and post-hypoxic training for end-run plasma lactate concentrations or packed cell volumes. Hypoxic training may increase V̇O2max even though it is not increased by normoxic training in well-trained horses, at least for the durations of time evaluated in this study. Training while breathing hypoxic gas may have the potential to enhance normoxic performance of Thoroughbred horses.

Cardiorespiratory and anesthetic effects of combined alfaxalone, butorphanol, and medetomidine in Thoroughbred horses.

This study evaluated induction of anesthesia and cardiorespiratory and anesthetic effects during maintained anesthesia with the combination of alfaxalone, medetomidine, and butorphanol. Alfaxalone (1.0 mg/kg) was administered to induce anesthesia after premedication with medetomidine (7.0 µg/kg), butorphanol (25 µg/kg), and midazolam (50 µg/kg) in six Thoroughbred horses. Intravenous general anesthesia was maintained with alfaxalone (2.0 mg/(kg∙hr)), medetomidine (5.0 µg/(kg∙hr)), and butorphanol (30 µg/(kg∙hr)) for 60 min. Electrical stimulation of the upper oral mucosa was used to assess anesthetic depth at 10 min intervals during anesthesia. Heart rate (HR), respiratory rate (RR), and mean arterial pressure (MAP) were measured. All horses became recumbent within 1 min after alfaxalone administration. Induction scores were 5 (best) in five horses and 4 in one horse. During the 60-min anesthesia, average HR, RR, and MAP were 35.8 ± 2.6 beat/min, 4.7 ± 0.6 breath/min, and 129 ± 3 mmHg, respectively. No horse moved with electrical stimulation; however, two horses experienced apnea (no respiration for 1 to 3 min). Recovery scores were 5 (best) in two horses and 3 in four horses. These results suggest that alfaxalone is effective for induction and maintenance of anesthesia and analgesia when combined with butorphanol and medetomidine for 60 min in Thoroughbreds. However, respiratory depression might require support.

Profiling of exercise-induced transcripts in the peripheral blood cells of Thoroughbred horses.

Transcriptome analyses based on DNA microarray technology have been used to investigate gene expression profiles in horses. In this study, we aimed to identify exercise-induced changes in the expression profiles of genes in the peripheral blood of Thoroughbred horses using DNA microarray technology (15,429 genes on 43,603 probes). Blood samples from the jugular vein were collected from six horses before and 1 min, 4 hr, and 24 hr after all-out running on a treadmill. After the normalization of microarray data, a total of 26,830 probes were clustered into four groups and 11 subgroups showing similar expression changes based on k-mean clustering. The expression level of inflammation-related genes, including interleukin-1 receptor type II (IL-1R2), matrix metallopeptidase 8 (MMP8), protein S100-A8 (S100-A8), and serum amyloid A (SAA), increased at 4 hr after exercise, whereas that of c-Fos (FOS) increased at 1 min after exercise. These results indicated that the inflammatory response increased in the peripheral blood cells after exercise. Our study also revealed the presence of genes that may not be affected by all-out exercise. In conclusion, transcriptome analysis of peripheral blood cells could be used to monitor physiological changes induced by various external stress factors, including exercise, in Thoroughbred racehorses.

In vivo measurements of flexor tendon and suspensory ligament forces during trotting using the thoroughbred forelimb model.

The purpose of this study was to create a lower forelimb model of the Thoroughbred horse for measuring the force in the superficial and deep digital flexor tendons (SDFT and DDFT), and the suspensory ligament (SL) during a trot. The mass, centers of gravity, and inertial moments in the metacarpus, pastern, and hoof segments were measured in 4 Thoroughbred horses. The moment arms of the SDFT, DDFT, and SL in the metacarpophalangeal (fetlock) and distal interphalangeal (coffin) joints were measured in 7 Thoroughbred horses. The relationship between the fetlock joint angle and the force in the SL was assessed in 3 limbs of 2 Thoroughbred horses. The forces in the SDFT, DDFT, and SL during a trot were also measured in 7 Thoroughbred horses. The mass of the 3 segments, and the moment arms of the SDFT and DDFT in the fetlock joint of the Thoroughbred horses were smaller than those of the Warmblood horses, whereas the other values were almost the same in the 2 types. The calculated force in the SDFT with this Thoroughbred model reached a peak (4,615 N) at 39.3% of the stance phase, whereas that in the DDFT reached a peak (5,076 N) at 51.2% of the stance phase. The force in the SL reached a peak (11,957 N) at 49.4% of the stance phase. This lower forelimb model of the Thoroughbred can be applied to studying the effects of different shoe types and change of hoof angle for the flexor tendon and SL forces.

The Effects of Inclination (Up and Down) of the Treadmill on the Electromyogram Activities of the Forelimb and Hind limb Muscles at a Walk and a Trot in Thoroughbred Horses.

It is important to know the effects of the inclination of a slope on the activity of each muscle, because training by running on a sloped track is commonly used for Thoroughbred racehorses. The effects of incline (from -6 to +6%) on the forelimbs and hind limbs during walking and trotting on a treadmill were evaluated by an integrated electromyogram (iEMG). The muscle activities in the forelimbs (5 horses) and hind limbs (4 horses) were measured separately. Two stainless steel wires were inserted into each of the brachiocephalicus (Bc), biceps brachii (BB), splenius (Sp), and pectoralis descendens (PD) in the forelimb experiment and into the longissimus dorsi (LD), vastus lateralis (VL), gluteus medius (GM), and biceps femoris (BF) in the hind limb experiment. The EMG recordings were taken at a sampling rate of 1,000 Hz. At a walk, the iEMG values for the forelimb were not significantly different under any of the inclinations. In the hind limb, the iEMG values for the GM and BF significantly decreased as the inclination decreased. At a trot, the iEMG values for the Bc in the forelimb significantly decreased as the inclination of the treadmill decreased. In the hind limb, the iEMG values for the LD, GM, and BF significantly decreased as the inclination decreased. Uphill exercise increased the iEMG values for the Bc, LD, GM, and BF, while downhill exercise resulted in little increase in the iEMG values. It was concluded that the effects of inclination on the muscle activities were larger for the uphill exercises, and for the hind limb muscles compared with the forelimb muscles.

Changes in muscle activation with graded surfaces during canter in Thoroughbred horses on a treadmill.

Understanding how muscle activity changes with different surface grades during canter is essential for developing training protocols in Thoroughbreds because canter is their primary gait in training and races. We measured the spatiotemporal parameters and the activation of 12 surface muscles in the leading limb side of 7 Thoroughbreds. Horses were equipped with hoof strain gauges and cantered at 10 m/s on a treadmill set to grades of -4%, 0%, 4%, and 8%, randomly, for 30 seconds each without a lead change. Integrated electromyography (iEMG) values during stance and swing phases were calculated and normalized to mean iEMG values during stride duration at 0% grade in each muscle. The iEMG values at each grade were compared using a generalized mixed model. Stride duration significantly decreased due to shorter swing duration on an 8% grade (P < 0.001) compared to all other grades, where no significant changes were observed. Compared to a 0% grade, the normalized iEMG values during the stance phase on an 8% grade in five muscles significantly increased (Musculus infraspinatus; +9%, M. longissimus dorsi (LD); +4%, M. gluteus medius (GM); +29%, M. biceps femoris; +47%, M. flexor digitorum lateralis; +16%). During the swing phase, the normalized iEMG values in six muscles significantly increased on an 8% grade compared to a 0% grade (M. splenius; +21%, M. triceps brachii; +54%, LD; +37%, GM; +24%, M. semitendinosus; +51%, M. extensor digitorum longus; +10%). No significant changes were observed in iEMG values on -4% and 4% grades compared to the 0% grade. Although +/- 4% grades had little effect on neuromuscular responses, 8% uphill canter reduced stride duration due to decreased swing duration and required increase of muscle activation during either stance and swing phase. Canter on an 8% grade might strengthen equine muscles to increase propulsive force and stride frequency.

Effects of pacing strategy on metabolic responses to 2-min intense exercise in Thoroughbred horses.

Evidence suggests that positive pacing strategy improves exercise performance and fatigue tolerance in athletic events lasting 1-5 min. This study investigated muscle metabolic responses to positive and negative pacing strategies in Thoroughbred horses. Eight Thoroughbred horses performed 2 min treadmill running using positive (1 min at 110% maximal O2 uptake [V̇O2max], followed by 1 min at 90% V̇O2max) and negative (1 min at 90% V̇O2max, followed by 1 min at 110% V̇O2max) pacing strategies. The arterial-mixed venous O2 difference did not significantly differ between the two strategies. Plasma lactate levels increased toward 2 min, with significantly higher concentrations during positive pacing than during negative pacing. Muscle glycogen level was significantly lower at 1 and 2 min of positive pacing than those of negative pacing. Metabolomic analysis showed that the sum of glycolytic intermediates increased during the first half of positive pacing and the second half of negative pacing. Regardless of pacing strategy, the sum of tricarboxylic acid cycle metabolites increased during the first half but remained unchanged thereafter. Our data suggest that positive pacing strategy is likely to activate glycolytic metabolism to a greater extent compared to negative pacing, even though the total workload is identical.

Heat acclimation improves exercise performance in hot conditions and increases heat shock protein 70 and 90 of skeletal muscles in Thoroughbred horses.

This study aimed to determine whether heat acclimation could induce adaptations in exercise performance, thermoregulation, and the expression of proteins associated with heat stress in the skeletal muscles of Thoroughbreds. Thirteen trained Thoroughbreds performed 3 weeks of training protocols, consisting of cantering at 90% maximal oxygen consumption (VO2max) for 2 min 2 days/week and cantering at 7 m/s for 3 min 1 day/week, followed by a 20-min walk in either a control group (CON; Wet Bulb Globe Temperature [WBGT] 12-13°C; n = 6) or a heat acclimation group (HA; WBGT 29-30°C; n = 7). Before and after heat acclimation, standardized exercise tests (SET) were conducted, cantering at 7 m/s for 90 s and at 115% VO2max until fatigue in hot conditions. Increases in run time (p = 0.0301), peak cardiac output (p = 0.0248), and peak stroke volume (p = 0.0113) were greater in HA than in CON. Pulmonary artery temperature at 7 m/s was lower in HA than in CON (p = 0.0332). The expression of heat shock protein 70 (p = 0.0201) and 90 (p = 0.0167) increased in HA, but not in CON. These results suggest that heat acclimation elicits improvements in exercise performance and thermoregulation under hot conditions, with a protective adaptation to heat stress in equine skeletal muscles.

AMP-activated protein kinase is required for exercise-induced peroxisome proliferator-activated receptor co-activator 1 translocation to subsarcolemmal mitochondria in skeletal muscle.

In skeletal muscle, mitochondria exist as two subcellular populations known as subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS mitochondria preferentially respond to exercise training, suggesting divergent transcriptional control of the mitochondrial genomes. The transcriptional co-activator peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and mitochondrial transcription factor A (Tfam) have been implicated in the direct regulation of the mitochondrial genome in mice, although SS and IMF differences may exist, and the potential signalling events regulating the mitochondrial content of these proteins have not been elucidated. Therefore, we examined the potential for PGC-1α and Tfam to translocate to SS and IMF mitochondria in human subjects, and performed experiments in rodents to identify signalling mechanisms regulating these translocation events. Acute exercise in humans and rats increased PGC-1α content in SS but not IMF mitochondria. Acute exposure to 5-aminoimidazole-4-carboxamide-1-ß-ribofuranoside in rats recapitulated the exercise effect of increased PGC-1α protein within SS mitochondria only, suggesting that AMP-activated protein kinase (AMPK) signalling is involved. In addition, rendering AMPK inactive (AMPK kinase dead mice) prevented exercise-induced PGC-1α translocation to SS mitochondria, further suggesting that AMPK plays an integral role in these translocation events. In contrast to the conserved PGC-1α translocation to SS mitochondria across species (humans, rats and mice), acute exercise only increased mitochondrial Tfam in rats. Nevertheless, in rat resting muscle PGC-1α and Tfam co-immunoprecipate with α-tubulin, suggesting a common cytosolic localization. These data suggest that exercise causes translocation of PGC-1α preferentially to SS mitochondria in an AMPK-dependent manner.

Effect of speed and leading or trailing limbs on surface muscle activities during canter in Thoroughbred horses.

Given that Thoroughbred horses' canter is an asymmetric gait, not only speed but also leading or trailing limbs could affect muscle activities. However, the muscle activity during a canter remains poorly understood. Hence, we aimed to investigate speed and lead-side (leading or trailing) effects on surface electromyography (sEMG) during a canter. The sEMG data were recorded from left Musculus brachiocephalicus (Br), M. infraspinatus (Inf), long head of M. triceps brachii (TB), M. gluteus medius (GM), M. semitendinosus (ST), and M. flexor digitorum longus of seven Thoroughbreds with hoof-strain gauges at the left hooves. Horses cantered on a flat treadmill at 7, 10, and 13 m/s for 25 s each without lead change. Subsequently, the horses trotted for 3 min and cantered at the same speed and duration in the opposite lead side ("leading" at the left lead and "trailing" at the right lead). The order of the lead side and speed was randomized. The mean of 10 consecutive stride durations, duty factors, integrated-EMG values (iEMG) for a stride, and muscle onset and offset timing were compared using a generalized mixed model (P < 0.05). Stride durations and duty factors significantly decreased with speed regardless of the lead side. In all muscles, iEMG at 13 m/s significantly increased compared with 7 m/s (ranging from +15% to +134%). The lead-side effect was noted in the iEMG of Br (leading > trailing, +47%), Inf (leading > trailing, +19%), GM (leading < trailing, +20%), and ST (leading < trailing, +19%). In TB, GM, and ST, muscle onset in trailing was earlier than the leading, while offset in the leading was earlier in Br. In conclusion, different muscles have different responses to speed and lead side; thus, both the lead side and running speed should be considered during training and/or rehabilitation including canter or gallop.

Risk Factors for Epistaxis in Thoroughbred Flat Races in Japan (2001-2020).

We investigated the risk factors for epistaxis in Japanese flat races over a 20-year period. The veterinary records of horses identified as having epistaxis by endoscopy on the race day, and the official racing records of all flat races from April to September between 2001 and 2020, were reviewed. The racecourses (n = 10), surface type, surface condition, race class, race distance, race year, sex, age, two training centers, ambient temperature, and body weight on race days were assessed using multivariable logistic regression (p < 0.05). Of 475,709 race starts, 616 (1.30 cases per 1000 starts; 95% confidence interval [CI], 1.20-1.40) included an epistaxis event. Nine variables were significantly associated with epistaxis. Seven of the variables have been reported in previous studies: lower ambient temperature, soft surface conditions, shorter racing distances (≤1400 m), increasing age, females and geldings compared to males, training center, and race year. However, two novel variables were identified as significantly associated with epistaxis, increasing body weight per 20 kg (p < 0.001, odds ratio [OR], 1.33; 95% CI, 1.25-1.41) and the racecourses that the horses were running at (p < 0.001, especially Sapporo [OR; 4.74, 95% CI, 3.07-7.31], Hakodate [OR, 4.66; 95% CI, 3.05-7.11], and Kokura [OR, 4.14; 95% CI, 2.65-6.48] compared to the reference racecourse [Kyoto]). These results can facilitate developing interventions to reduce epistaxis in flat racing.

Physiological and skeletal muscle responses to high-intensity interval exercise in Thoroughbred horses.

Introduction: The purpose of this study was to determine whether acute high-intensity interval exercise or sprint interval exercise induces greater physiological and skeletal muscle responses compared to moderate-intensity continuous exercise in horses. Methods: In a randomized crossover design, eight trained Thoroughbred horses performed three treadmill exercise protocols consisting of moderate-intensity continuous exercise (6 min at 70% VO2max; MICT), high-intensity interval exercise (6 × 30 s at 100% VO2max; HIIT), and sprint interval exercise (6 × 15 s at 120% VO2max; SIT). Arterial blood samples were collected to measure blood gas variables and plasma lactate concentration. Biopsy samples were obtained from the gluteus medius muscle before, immediately after, 4 h, and 24 h after exercise for biochemical analysis, western blotting and real-time RT-PCR. Effects of time and exercise protocol were analyzed using mixed models (p < 0.05). Results: Heart rate and plasma lactate concentration at the end of exercise were higher in HIIT and SIT than those in MICT (heart rate, HIIT vs. MICT, p = 0.0005; SIT vs. MICT, p = 0.0015; lactate, HIIT vs. MICT, p = 0.0014; SIT vs. MICT, p = 0.0003). Arterial O2 saturation and arterial pH in HIIT and SIT were lower compared with MICT (SaO2, HIIT vs. MICT, p = 0.0035; SIT vs. MICT, p = 0.0265; pH, HIIT vs. MICT, p = 0.0011; SIT vs. MICT, p = 0.0023). Muscle glycogen content decreased significantly in HIIT (p = 0.0004) and SIT (p = 0.0016) immediately after exercise, but not in MICT (p = 0.19). Phosphorylation of AMP-activated protein kinase (AMPK) in HIIT showed a significant increase immediately after exercise (p = 0.014), but the increase was not significant in MICT (p = 0.13) and SIT (p = 0.39). At 4 h after exercise, peroxisome proliferator-activated receptor γ co-activator-1α mRNA increased in HIIT (p = 0.0027) and SIT (p = 0.0019) and vascular endothelial growth factor mRNA increased in SIT (p = 0.0002). Discussion: Despite an equal run distance, HIIT and SIT cause more severe arterial hypoxemia and lactic acidosis compared with MICT. In addition, HIIT activates the AMPK signaling cascade, and HIIT and SIT elevate mitochondrial biogenesis and angiogenesis, whereas MICT did not induce any significant changes to these signaling pathways.

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).

Mitochondrial creatine kinase activity and phosphate shuttling are acutely regulated by exercise in human skeletal muscle.

Energy transfer between mitochondrial and cytosolic compartments is predominantly achieved by creatine-dependent phosphate shuttling (PCr/Cr) involving mitochondrial creatine kinase (miCK). However, ADP/ATP diffusion through adenine nucleotide translocase (ANT) and voltage-dependent anion carriers (VDACs) is also involved in this process. To determine if exercise alters the regulation of this system, ADP-stimulated mitochondrial respiratory kinetics were assessed in permeabilized muscle fibre bundles (PmFBs) taken from biopsies before and after 2 h of cycling exercise (60% ) in nine lean males. Concentrations of creatine (Cr) and phosphocreatine (PCr) as well as the contractile state of PmFBs were manipulated in situ. In the absence of contractile signals (relaxed PmFBs) and miCK activity (no Cr), post-exercise respiratory sensitivity to ADP was reduced in situ (up to 126% higher apparent K(m) to ADP) suggesting inhibition of ADP/ATP diffusion between matrix and cytosolic compartments (possibly ANT and VDACs). However this effect was masked in the presence of saturating Cr (no effect of exercise on ADP sensitivity). Given that the role of ANT is thought to be independent of Cr, these findings suggest ADP/ATP, but not PCr/Cr, cycling through the outer mitochondrial membrane (VDACs) may be attenuated in resting muscle after exercise. In contrast, in contracted PmFBs, post-exercise respiratory sensitivity to ADP increased with miCK activation (saturating Cr; 33% lower apparent K(m) to ADP), suggesting prior exercise increases miCK sensitivity in situ. These observations demonstrate that exercise increases miCK-dependent respiratory sensitivity to ADP, promoting mitochondrial-cytosolic energy exchange via PCr/Cr cycling, possibly through VDACs. This effect may mask an underlying inhibition of Cr-independent ADP/ATP diffusion. This enhanced regulation of miCK-dependent phosphate shuttling may improve energy homeostasis through more efficient coupling of oxidative phosphorylation to perturbations in cellular energy charge during subsequent bouts of contraction.

Effect of training and detraining on monocarboxylate transporter (MCT) 1 and MCT4 in Thoroughbred horses.

The aim of this study was to investigate the effects of training and detraining on the monocarboxylate transporter (MCT) 1 and MCT4 levels in the gluteus medius muscle of Thoroughbred horses. Twelve Thoroughbred horses were used for the analysis. For 18 weeks, all the horses underwent high-intensity training (HIT), with running at 90-110% maximal oxygen consumption (VO2 max ) for 3 min, 5 days week(-1). Thereafter, the horses either underwent detraining for 6 weeks by either 3 min of moderate-intensity training (MIT) at 70% VO2 max, 5 days week(-1) (HIT-MIT group) or stall rest (HIT-SR group). The horses underwent an incremental exercise test, VO2 max was measured and resting muscle samples were obtained from the middle gluteus muscle at 0, 18 and 24 weeks. The content of MCT1 and MCT4 proteins increased after 18 weeks of HIT. At the end of this period, an increase was noted in the citrate synthase activity, while phosphofructokinase activity remained unchanged. After 6 weeks of detraining, all these indexes returned to the pretraining levels in the HIT-SR group. However, in the HIT-MIT group, the increase in the MCT1 protein content and citrate synthase activity was maintained after 6 weeks of MIT, while the MCT4 protein content decreased to the pretraining value. These results suggest that the content of MCT1 and MCT4 proteins increases after HIT in Thoroughbred horses. In addition, the increase in the MCT1 protein content and oxidative capacity induced by HIT can be maintained by MIT of 70% VO2 max, but the increase in the MCT4 protein content cannot be maintained by MIT.

Effects of Fatigue on Stride Parameters in Thoroughbred Racehorses During Races.

Exercise intensity during races is considerably high. To understand how Thoroughbreds adapt to fatigue conditions, stride parameters for the first and second lap of the race (2400-m, turf) were compared. A high-speed video system was set in a right lateral position about 20 m before the finishing post, with a field view width of about 16 m. The stride frequency, the length between each limb (hind step, diagonal step, fore step, and airborne step), and stride length were measured and analyzed using a generalized linear mixed model. Compared with the first lap, the mean ± standard deviation values in the second lap for running speed (17.3 ± 1.3 to 16.0 ± 0.9 m/s, P < .01), stride frequency (2.34 ± 0.08 to 2.21 ± 0.09 strides/s, P < .01) and stride length (7.42 ± 0.52 to 7.25 ± 0.38 m, P = .04) significantly decreased. Furthermore, significant changes (P < .01) were observed in the diagonal step length (2.32 ± 0.34 to 1.88 ± 0.23 m), hind step (1.19 ± 0.09 to 1.26 ± 0.10 m) and airborne step length (2.43 ± 0.25 to 2.61 ± 0.18 m). When controlled for speed, stride frequency (P = .02) and diagonal step length (P < .01) decreased, while the length of the hind step (P < .01), fore step (P < .01), airborne step (P < .01), and stride (P = .02) increased with fatigue in the second lap. These results suggest that horses could not extend their body when fatigued.

Metabolomic analysis of skeletal muscle before and after strenuous exercise to fatigue.

Thoroughbreds have high maximal oxygen consumption and show hypoxemia and hypercapnia during intense exercise, suggesting that the peripheral environment in skeletal muscle may be severe. Changes in metabolites following extreme alterations in the muscle environment in horses after exercise may provide useful evidence. We compared the muscle metabolites before and after supramaximal exercise to fatigue in horses. Six well-trained horses ran until exhaustion in incremental exercise tests. Biopsy samples were obtained from the gluteus medius muscle before and immediately after exercise for capillary electrophoresis-mass spectrometry analysis. In the incremental exercise test, the total running time and speed of the last step were 10.4 ± 1.3 (mean ± standard deviation) min and 12.7 ± 0.5 m/s, respectively. Of 73 metabolites, 18 and 11 were significantly increased and decreased after exercise, respectively. The heat map of the hierarchical cluster analysis of muscle metabolites showed that changes in metabolites were clearly distinguishable before and after exercise. Strenuous exercise increased many metabolites in the glycolytic pathway and the tricarboxylic acid cycle in skeletal muscle. Targeted metabolomic analysis of skeletal muscle may clarify the intramuscular environment caused by exercise and explain the response of working muscles to strenuous exercise that induces hypoxemia and hypercapnia in Thoroughbred horses.