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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.
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Aclimatación , Proteínas HSP70 de Choque Térmico , Músculo Esquelético , Condicionamiento Físico Animal , Animales , Caballos/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Condicionamiento Físico Animal/métodos , Condicionamiento Físico Animal/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Aclimatación/fisiología , Masculino , Calor , Regulación de la Temperatura Corporal/fisiología , Consumo de Oxígeno/fisiología , Respuesta al Choque Térmico/fisiologíaRESUMEN
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.
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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).
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In this study, the pattern of myosin heavy chain (MHC) isoforms expression in skeletal muscles of the trunk, forelimb and hindlimb in Polar Bear (PB) Ursus maritimus; American Black Bear (AmBB), Ursus americanus and Asian Black Bear (AsBB), Ursus thibetanus was analysed by immunohistochemistry and SDS-PAGE. Results showed that slow (MHC-I) and fast (MHC-II) isoforms exist in muscles of bears. Type II fibres were classified further into Type IIa and IIx in PB but not in AsBB and AmBB. The distribution of Type I and Type II fibres in the trunk, forelimb and hindlimb varied based on muscle type and animal species. The proportions of Type I fibres formed approximately one-third of muscle composition in PB (trunk, 32.0%; forelimb, 34.7%; hindlimb, 34.5%) and a half in both AsBB and AmBB whereas Type IIa and IIx formed approximately two-third in PB (trunk, 68.0%; forelimb, 65.3%; hindlimb, 65.5%) and a half of Type II in both AmBB and AsBB. PB is a good swimmer, lives in Arctic Ocean on slippery ice catching aquatic mammals such as seals and is larger in size compared to the medium sized AmBB (living in forest) and AsBB (arboreal). The results suggest that in bears, there is greater diversity in MHC isoforms II, being expressed in selected fast contracting skeletal muscles in response to variety of environments, weight bearing and locomotion.
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Cadenas Pesadas de Miosina , Ursidae , Animales , Cadenas Pesadas de Miosina/análisis , Cadenas Pesadas de Miosina/metabolismo , Ursidae/metabolismo , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismoRESUMEN
This study aimed to verify the effects of platelet lysate (PL) administration on the repair of injured horse tissue. Skeletal muscle injuries were induced in 26 Thoroughbreds by bupivacaine administration. PL or saline was administered 1 day (1D) after injury. Muscle samples from 22 horses injected with PL or saline were obtained by needle biopsy at 2, 3, 4, or 7 days (2D, 3D, 4D, or 7D, respectively) after injury, and growth-factor concentrations and muscle regeneration-associated gene expression levels were determined. Intact samples were similarly collected before injury, and samples of injured muscle not treated with PL or saline (sham samples) were also obtained at 1D, 2D, 3D, 4D, and 7D as references for comparison. Samples from the remaining 4 horses were obtained by surgical incision following euthanasia at 5 days (5D) and 7D after injury, followed by histological analysis. Although increased growth factor levels caused by PL administration were observed for up to 1-day post-administration (2D), gene expressions were enhanced for up to 6 days post-administration (7D). Moreover, the number of embryonic myosin heavy chain (MHC-e)-positive myofibrils at 5D was higher in the PL-treated group than in the saline-treated group, whereas no significant between-group difference in the number of myofibrils was recorded at 7D. Thus, PL administration in muscle injury upregulated the expression of various genes associated with muscle regeneration and promoted morphological regeneration within 6 days of treatment, although growth factor levels from PL decreased at the injected site by approximately 2 days post-administration.
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Enfermedades de los Caballos , Enfermedades Musculares , Animales , Bupivacaína/efectos adversos , Eutanasia Animal , Enfermedades de los Caballos/metabolismo , Caballos , Músculo Esquelético/metabolismo , Enfermedades Musculares/metabolismo , Enfermedades Musculares/veterinaria , RegeneraciónRESUMEN
This study characterised muscle fibres in trunk, forelimb and hindlimb muscles of three bat species: little Japanese horseshoe (Rhinolophus cornutus), greater horseshoe (Rhinolophus ferrumequinum) and Egyptian fruit (Rousettus aegyptiacus). Twenty-seven muscles from trunk, forelimb and hindlimb were dissected, weighed and analysed by immunohistochemistry and sodium didecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and determined their cross-sectional areas (CSA). Results showed that Type IIa and Type IIa/x made the highest proportion of total muscle mass. Moderate proportion was formed by Type IIb. Type I and IIx appeared at very low levels in all bats. Type IIb was the only fibre type detected in patagial muscles in wing membrane of greater horseshoe while other fibre types were not observed. Type I muscle fibres were very few and appeared infrequently in fifteen muscles of Egyptian fruit and in only one muscle in each, greater horseshoe and little Japanese horseshoe. Type IIx was also detected in three muscles in greater horseshoe and only one muscle in Egyptian fruit but none in little Japanese horseshoe. The highest average CSA µm2 was detected in Type IIb and values were 734.2µm2 for LHB; 1537.9µm2 for GHB and 1,720.9µm2 for EFB. Lowest and undetermined values were observed for Type I and IIx. These data demonstrate that Type IIa, IIa/x and IIb form significant proportion of adult bat muscle mass and Type IIb is the largest fibre type. The distribution pattern is suggestive of specialised functions of the fibres in relation to orientation and speed of bats during flight.
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Quirópteros , Fibras Musculares Esqueléticas , Animales , Miembro Anterior , Miembro Posterior , Músculo EsqueléticoRESUMEN
Moles are a strictly fossorial Soricomorpha species and possess a suite of specialized adaptations to subterranean life. However, the contractile function of skeletal muscles in moles remains unclear. We compared muscle fiber-type distribution in two mole species (the large Japanese mole and lesser Japanese mole) with that in four other Soricomorpha species that are semi-fossorial, terrestrial, or semi-aquatic (the Japanese shrew-mole, house shrew, Japanese white-toothed shrew, and Japanese water shrew). For a single species, the fiber-type distribution in up to 38 muscles was assessed using immunohistochemical staining and/or gel electrophoresis. We found that slow and fatigue-resistant Type I fibers were absent in almost all muscles of all species studied. Although, the two methods of determining the fiber type did not give identical results, they both revealed that fast Type IIb fibers were absent in mole muscles. The fiber-type distribution was similar among different anatomical regions in the moles. This study demonstrated that the skeletal muscles of moles have a homogenous fiber-type distribution compared with that in Soricomorpha species that are not strictly fossorial. Mole muscles are composed of Type IIa fibers alone or a combination of Type IIa and relatively fast Type IIx fibers. The homogenous fiber-type distribution in mole muscles may be an adaptation to structurally simple subterranean environments, where there is no need to support body weight with the limbs, or to move at high speeds to pursue prey or to escape from predators. Anat Rec, 302:1010-1023, 2019. © 2018 Wiley Periodicals, Inc.
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Adaptación Fisiológica , Topos/fisiología , Contracción Muscular/fisiología , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Animales , Topos/anatomía & histología , Musarañas/anatomía & histología , Musarañas/fisiologíaRESUMEN
Inexperienced vigorous exercise, including eccentric contraction (ECC), causes muscle pain and damage. Similar prior light exercise suppresses the development of muscle pain (repeated-bout effect), but the molecular mechanisms behind this are not sufficiently understood. In this study, the influence of a nondamaging preconditioning ECC load (Precon) on muscle pain-related molecules and satellite cell-activating factors was investigated at the mRNA expression level. Nine-week-old male Wistar rats (n=36) were divided into 2 groups: a group receiving only a damaging ECC (100 contractions) load (non-Precon) and a group receiving a nondamaging ECC (10 contractions) load 2 days before receiving the damaging ECC load (Precon). ECC was loaded on the left leg, and the right leg was regarded as the intact control (CTL). The medial head of the gastrocnemius muscle from all rats was excised 2 or 4 days after the damaging ECC loading, and the relative mRNA expression levels of muscle pain- and satellite cell-related molecules were quantitated using real-time RT PCR. Precon suppressed increases in MHC-embryonic and MHC-neonatal mRNA expressions. Enhancement of HGF, Pax7, MyoD, and myogenin mRNA expression was also suppressed, suggesting that Precon decreased the degree of muscle damage and no muscle regeneration or satellite cell activation occurred. Similarly, increases in mRNA expression of muscle pain-related molecules (BKB2 receptor, COX-2, and mPGEC-1) were also suppressed. This study clearly demonstrated that at the mRNA level, prior light ECC suppressed muscle damage induced by later damaging ECC and promoted recovery from muscle pain.
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Regulación de la Expresión Génica/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Contracción Muscular/fisiología , Mialgia/rehabilitación , Condicionamiento Físico Animal/métodos , Animales , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Masculino , Músculo Esquelético/patología , Músculo Esquelético/fisiología , Mialgia/etiología , Proteína MioD/genética , Proteína MioD/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Prostaglandina-E Sintasas/genética , Prostaglandina-E Sintasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor de Bradiquinina B2/genética , Receptor de Bradiquinina B2/metabolismo , Factores de TiempoRESUMEN
We clarified in our previous study that hypoxic training promotes angiogenesis in skeletal muscle, but the mechanism of angiogenesis in skeletal muscle remains unknown. In this study, we investigated the influence of differences in hypoxia exposure on angiogenesis in skeletal muscles at differing ages and metabolic characteristics at which the production of reactive oxygen species and nitric oxide may differ. Ten-week-old (young) and 20-month-old (old) mice were separated into control (N), continuous hypoxia (H), and intermittent hypoxia (IH) groups. The H group was exposed to 16% O2 hypoxia for 5 days and the IH group was exposed to 16% O2 hypoxia at one-hour intervals during the light period for 5 days. After completion of hypoxia exposure, the soleus and gastrocnemius muscles were immediately excised, and mRNA expression of angiogenesis- and satellite cell-related genes was investigated using real-time RT-PCR. In addition, muscle fiber type composition, muscle fiber area, number of satellite cells, and capillary density were measured immunohistochemically. In the young soleus muscle, the muscle fiber area was decreased in the H group, and mRNA expression of satellite cell activation-related MyoD, MHCe, and BDNF was significantly increased. On the other hand, in the old soleus muscle, nNOS and VEGF-A mRNA expression, and the capillary density were significantly increased in the H group. In the superficial portion of the gastrocnemius, mRNA expression of FGF2, an angiogenic factor secreted by satellite cells, was significantly increased in the young IH group. In addition, a positive correlation between VEGF-A mRNA expression and nNOS mRNA expression in the soleus muscle and eNOS mRNA expression in the superficial portion of the gastrocnemius was noted. These data demonstrated that age, hypoxia exposure method and muscle metabolic characteristics are related, which results in significant differences in angiogenesis.
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Músculo Esquelético/citología , Músculo Esquelético/fisiología , Neovascularización Fisiológica , Células Satélite del Músculo Esquelético/citología , Animales , Peso Corporal , Hipoxia de la Célula , Regulación de la Expresión Génica , Ratones , Tamaño de los Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
This study investigated the effects of AdipoRon, which is an agonist for adiponectin receptor 1 (AdipoR1) and AdipoR2, on the protein content, myotube diameter, and number of nuclei per myotube of C2C12 cells and skeletal muscle mass in C57BL/6J mice. AdipoRon suppressed the protein content, myotube diameter, and number of nuclei per myotube of C2C12 cells of C2C12 myotubes in a dose-dependent manner. Adiponectin-associated decline of protein content, diameter, and number of nuclei per myotube in C2C12 myotubes was partially rescued by knockdown of AdipoR1 and/or AdipoR2. Phosphorylation level of AMPK showed a trend to be increased by AdipoRon. A significant increase in phosphorylation level of AMPK was observed at 20 µM AdipoRon. Knockdown of AdipoR1 and/or AdipoR2 rescued AdipoRon-associated decrease in protein content of C2C12 myotubes. AdipoRon-associated increase in phosphorylation level of AMPK in C2C12 myotubes was suppressed by knockdown of AdipoR1 and/or AdipoR2. Successive intravenous injections of AdipoRon into mice caused a decrease in the wet weight of plantaris muscle (PLA), but not in soleus muscle (SOL). Mean fiber cross-sectional area of PLA, but not of SOL, was significantly decreased by AdipoRon administration. On the one hand, the expression level of phosphorylated AMPK and ubiquitinated protein in SOL and PLA muscles was upregulated by AdipoRon administration. On the other hand, AdipoRon administration induced no changes in the expression level of puromycin-labeled proteins in both SOL and PLA muscles. Expression level of adiponectin in extensor digitorum longus (EDL) muscle was increased by aging, but not in SOL muscle. Aging had no effect on the expression level of AdipoR1 and AdipoR2 in both muscles. Phosphorylation level of AMPK in EDL was increased by aging, but not SOL muscle. Results from this study suggest that high level of circulating adiponectin may induce skeletal muscle atrophy, especially fast-type muscle.
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Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Receptores de Adiponectina/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Western Blotting , Línea Celular , Relación Dosis-Respuesta a Droga , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/ultraestructura , Piperidinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Adiponectina/agonistasRESUMEN
Variants of the Myostatin gene have been shown to have an influence on muscle hypertrophy phenotypes in a wide range of mammalian species. Recently, a Thoroughbred horse with a C-Allele at the g.66493737C/T single-nucleotide polymorphism (SNP) has been reported to be suited to short-distance racing. In this study, we examined the effect of the Myostatin SNP on muscle fiber properties in young Thoroughbred horses during a training period. To investigate the effect of the Myostatin SNP on muscle fiber before training, several mRNA expressions were relatively quantified in biopsy samples from the middle gluteal muscle of 27 untrained male Thoroughbred horses (1.5 years old) using real-time RT-PCR analysis. The remaining muscle samples were used for immunohistochemical analysis to determine the population and area of each fiber type. All measurements were revaluated in biopsy samples of the same horses after a 5-month period of conventional training. Although the expressions of Myostatin mRNA decreased in all SNP genotypes, a significant decrease was found in only the C/C genotype after training. While, expression of VEGFa, PGC1α, and SDHa mRNAs, which relate to the biogenesis of mitochondria and capillaries, was significantly higher (54-82%) in the T/T than the C/C genotypes after training. It is suggested that hypertrophy of muscle fiber is directly associated with a decrease in Myostatin mRNA expression in the C/C genotype, and that increased expressions of VEGFa, PGC1α, and SDHa in the T/T genotype might be indirectly caused by the Myostatin SNP.
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Regulación de la Expresión Génica/fisiología , Caballos/fisiología , Fibras Musculares Esqueléticas/fisiología , Miostatina/genética , Condicionamiento Físico Animal , Polimorfismo de Nucleótido Simple , Animales , Genotipo , Caballos/genética , Masculino , Miostatina/fisiologíaRESUMEN
The transfer of sweat to the skin surface without leakage is important for the homeostatic regulation of skin and is impaired in atopic dermatitis. Although the precise composition of the leakage barrier remains obscure, there is a large contribution from claudins, the major components of tight junctions. In humans, claudin-1, -3, and -15 are expressed on sweat ducts, and claudin-3 and -10 are expressed on secretory coils. Although only two claudins are expressed in murine sweat glands, we found that the expression of claudin-3 is conserved. Atopic dermatitis lesional skin had decreased claudin-3 expression in sweat glands, which was accompanied by sweat leakage. This critical role in water barrier function was confirmed in Cldn3-/- and Cldn3+/- mice and those with experimentally decreased claudin-3. Our results show the crucial role of claudin-3 in preventing sweat gland leakage and suggest that the pathogenesis of dermatoses accompanied by hypohidrosis involves abnormally decreased claudin-3.
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Claudina-3/metabolismo , Dermatitis Atópica/patología , Glándulas Sudoríparas/patología , Uniones Estrechas/patología , Acetilcolina/farmacología , Adulto , Animales , Claudina-3/genética , Femenino , Voluntarios Sanos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sudor/metabolismo , Glándulas Sudoríparas/citología , Glándulas Sudoríparas/efectos de los fármacos , Sudoración/efectos de los fármacos , Sudoración/fisiología , Uniones Estrechas/metabolismo , Pérdida Insensible de Agua/fisiología , Adulto JovenRESUMEN
Platelet-rich plasma (PRP) therapy is promising for treating skeletal muscle injuries in human athletes by promoting muscle regeneration. It might also be useful for treating muscle injuries in equine athletes. In the present study, muscle regeneration induced by injection of PRP into intact muscle of Thoroughbred was investigated. Autologous PRP and saline were injected twice into intact left and right gluteus medius muscles of seven clinically healthy Thoroughbreds. Muscle samples were collected from the injection sites by needle biopsy at 2 and 7 days after PRP injection. Immunohistochemical staining to identify the types of myosin heavy chains (MHCs) and satellite cells was performed to compare morphological changes among intact (pre-injection), saline-, and PRP-injected muscles. The expression of marker genes related to muscle regeneration (MHC-I, MHC-II, and embryonic MHC [MHC-e]), satellite cell activity (CK, Pax7, MyoD, and myogenin), and proinflammatory and promyogenic cytokines (IL-6, IGF-1, and HGF) was analyzed and compared between saline- and PRP-injected muscles. There were no obvious morphological differences among the three treatments. There were no significant differences in gene expression associated with satellite cell activity between saline and PRP injection at 7 days after injection. MHC genes showed significantly higher expression levels with PRP than with saline, including MHC-e at 2 days and MHC-I at 7 days after injection. It is suggested that injection of PRP into intact skeletal muscle does not induce specific morphological changes, but upregulate the expression of genes related to muscle regeneration.
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BACKGROUND: We examined the effect of transient hypoxic exposure during high-intensity exercise on satellite cell activation and angiogenesis in Thoroughbred skeletal muscle. METHODS: Six Thoroughbreds horses (6.3±0.8 years old) ran on a treadmill in normoxia (N; FIO2=21%) and hypoxia (H; FIO2=16%) at the same speed for the same duration, and oxygen consumption and plasma lactate and hemoglobin concentrations were measured. In addition, muscle biopsy samples were obtained from gluteus medius muscle before exercise and immediately, 4 hours, 24 hours, 3 days and 7 days after exercise, and immunohistochemical analysis and relative quantitative analysis of mRNA expression using real-time RT-PCR were performed. RESULTS: Oxygen consumption during exercise in H was 29% lower than that in N, and plasma lactate concentration in H was 47% higher than that in N. The mRNA expressions of satellite cell activation-related factors slightly increased, but the mRNA expressions of factors related to angiogenesis and mitochondrial biogenesis slightly decreased. Fluorescence-stained basal lamina evaluation in stacked images 7 days after exercise showed no difference in capillary density between the two groups. CONCLUSIONS: These results suggest that transient hypoxic exposure during exercise increases the contribution of the glycolytic energy supply and promotes satellite cell activation, but does not facilitate angiogenesis and mitochondrial biogenesis.
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Hipoxia/etiología , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal , Esfuerzo Físico/fisiología , Animales , Prueba de Esfuerzo , Hemoglobinas/análisis , Caballos , Lactatos/sangre , Masculino , Consumo de Oxígeno/fisiologíaRESUMEN
Hypoxic training is believed to increase endurance capacity in association with hypoxia inducible factor-1α (HIF-1α), a modulator of vascular endothelial growth factor-A (VEGF-A), and to influence activation of satellite cells (SCs). However, the effect of hypoxic training on SC activation and its relation to angiogenesis has not been thoroughly investigated. Eight Thoroughbred horses were subjected to normoxic (FIO2 = 21%) or hypoxic (FIO2 = 15%) training for 3 days/week (100% [Formula: see text]) for 4 weeks. Incremental exercise tests (IET) were conducted on a treadmill under normoxia and the maximal oxygen consumption ([Formula: see text]) and running distance were measured before and after each training session. Muscle biopsy samples were obtained from the gluteus medius muscle at 6 scheduled times before, during, and one week after IET for immunohistochemical analysis and real-time RT-PCR analysis. Running distance and [Formula: see text], measured during IET, increased significantly after hypoxic training compared with normoxic training. Capillary density and mRNA expression related to SC activation (e.g., myogenin and hepatocyte growth factor) and angiogenesis (VEGF-A) increased only after hypoxic training. These results suggest that increases in mRNA expression after training enhance and prolong SC activation and angiogenesis and that nitric oxide plays an important role in these hypoxia-induced training effects.
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Músculo Esquelético/metabolismo , Animales , Peso Corporal , Estudios Cruzados , Femenino , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Caballos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunohistoquímica , Masculino , Músculo Esquelético/patología , Miogenina/genética , Miogenina/metabolismo , Consumo de Oxígeno , Resistencia Física , ARN Mensajero/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cu/Zn superoxide dismutase (SOD1) knockout (KO) mice are known as an aging model in some aspects, but the damage and regeneration process of each fiber type have not been sufficiently studied. In this study, we investigated the damage and satellite cell state of the gastrocnemius muscle in SOD1 KO mice (6 months old) using immunohistochemical staining and real-time RT-PCR. The proportion of central nuclei-containing Type IIx/b fibers in the deep and superficial portions of the gastrocnemius muscle was significantly higher in SOD1 KO than control mice. The number of satellite cells per muscle fiber decreased in all muscle fiber types in the deep portion of the gastrocnemius muscle in SOD1 KO mice. In addition, the mRNA expression levels of Pax7 and myogenin, which are expressed in satellite cells in the activation, proliferation, and differentiation states, significantly increased in the gastrocnemius muscle of SOD1 KO mice. Furthermore, mRNA of myosin heavy chain-embryonic, which is expressed in the early phase of muscle regeneration, significantly increased in SOD1 KO mice. It was suggested that muscle is damaged by reactive oxygen species produced in the mitochondrial intermembrane space in Type IIxb fibers, accelerating the proliferation and differentiation of satellite cells through growth factors in SOD1 KO mice.
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Fibras Musculares Esqueléticas/metabolismo , Superóxido Dismutasa-1/deficiencia , Animales , Peso Corporal , Recuento de Células , Inmunohistoquímica , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Satélite del Músculo Esquelético/metabolismo , Superóxido Dismutasa-1/metabolismoRESUMEN
We compared the time-course of satellite cell (SC) activation between eccentric and concentric contractions in the vastus lateralis (VL) muscle after step exercise. Young adults participated in a 30-min step up/down exercise which mainly involved concentric contractions with the right VL muscle and eccentric contractions with the left VL muscle. The concentric and eccentric contraction phases of the VL muscles were identified by changes in the electromyogram (EMG) and knee joint angle. Biopsy samples were taken from both VL muscles at three time periods: before the exercise and 2 and 5 days after the exercise. We found that the numbers of SCs were significantly increased in the type IIa fibers of the left VL at 2 and 5 days after the exercise. The expression of both hepatocyte growth factor (HGF) and myogenic differentiation 1 (MyoD) mRNA had significantly increased in the left VL at 2 and 5 days after the exercise and in the right VL at 5 days after the exercise. The expression of transient receptor potential canonical (TRPC) 1 mRNA also increased in the left VL at 2 days after exercise. These results indicate that eccentric contraction can effectively activate SC proliferation for up to 5 days after exercise. Similar changes in HGF, MyoD and TRPC1 mRNA expression suggest that HGF/c-Met signal activation through cation influx has a major impact on skeletal muscle SC activation in response to eccentric exercise.
Asunto(s)
Contracción Muscular , Músculo Cuádriceps/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Fenómenos Biomecánicos , Biopsia , Proliferación Celular , Electromiografía , Femenino , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Articulación de la Rodilla/fisiología , Masculino , Proteína MioD/genética , Proteína MioD/metabolismo , Músculo Cuádriceps/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rango del Movimiento Articular , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Factores de Tiempo , Regulación hacia Arriba , Adulto JovenRESUMEN
Despite the growing numbers of successful applications in regenerative medicine, biotechnologies for evaluating the quality of cells remain limited. To evaluate the cultured cells non-invasively, image-based cellular assessment method holds great promise. However, although there are various image-processing algorithms, very few studies have focused to prove the effectiveness of phase contrast images with risk assessment example that reflects actual difficulties in regenerative medicine products. In this study, we developed a simple image-processing method to recognize the number of dividing cells in time-course phase-contrast microscopic images, and applied this method to assess the irregular proliferation behavior in normal cells. Practically, as a model, rapid proliferating human fibrosarcoma cells were mixed in normal human fibroblasts in the same culture dish, and their sarcoma existence was evaluated. As a result, the existence of sarcoma population in normal cell sample could be feasibly detected within earliest period of cell culture by their irregular rise of accumulated counts of dividing cells. Our image-processing technique also illustrates the technical effectiveness of combining intra-frame and inter-frame image processing to accurately count only the dividing cells. Our concept of focused counting of dividing cells shows a successful example of image-based analysis to quickly and non-invasively monitor the regular state of regenerative medicine products.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Microscopía de Contraste de Fase , Medicina Regenerativa , Algoritmos , Recuento de Células , División Celular , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Fibroblastos/citología , Fibrosarcoma/patología , HumanosRESUMEN
The ultraviolet B (UVB) component of sunlight can cause severe damage to skin cells and even induce skin cancer. Growing evidence indicates that the UVB-induced signaling network is complex and involves diverse cellular processes. In this study, we investigated the role of c-Jun NH2 -terminal kinase-associated leucine zipper protein (JLP), a scaffold protein for mitogen-activated protein kinase (MAPK) signaling cascades, in UVB-induced apoptosis. We found that UVB-induced skin epidermal apoptosis was prevented in Jlp knockout (KO) as well as in keratinocyte-specific Jlp KO mice. Analysis of the repair of UVB-induced DNA damage over time showed no evidence for the involvement of JLP in this process. In contrast, UVB-stimulated p38 MAPK activation in the skin was impaired in both Jlp KO and keratinocyte-specific Jlp KO mice. Moreover, topical treatment of UVB-irradiated mouse skin with a p38 inhibitor significantly suppressed the epidermal apoptosis in wild-type mice, but not in Jlp KO mice. Our findings suggest that JLP in skin basal keratinocytes plays an important role in UVB-induced apoptosis by modulating p38 MAPK signaling pathways. This is the first study to show a critical role for JLP in an in vivo response to environmental stimulation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis/efectos de los fármacos , Fragmentación del ADN , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Epidermis/patología , Epidermis/efectos de la radiación , Imidazoles/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/patología , Queratinocitos/efectos de la radiación , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos C57BL , Ratones Noqueados , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
To investigate the age-related changes in satellite cell (SC) proliferation in vivo, we used a compensatory activation (CAC) model of the hemi-diaphragm muscle. Young (2-month), adult (14-month) and old (24-month) rats were randomly divided into control and CAC groups. In the CAC group, denervation surgery in the left hemi-diaphragm was performed to induce CAC of the right hemi-diaphragm. Six days after the surgery, the CAC diaphragm muscle was removed and separated into two blocks for immunohistochemical staining and real time RT-PCR procedures. The number of SCs in type I and IIa fibers were not affected significantly by the CAC in any age groups, but that in type IIx/b fibers was significantly increased in the young and adult groups. As compared to the age-matched control group, the Pax7 mRNA expression level was significantly higher in the young and adult CAC groups, but not in the old CAC group. These results may suggest that the mechanism of SC proliferation in type IIx/b fibers is impaired in aged diaphragm muscles.