Signalling pathways regulating muscle mass in ageing skeletal muscle: the role of the IGF1-Akt-mTOR-FoxO pathway.

During ageing skeletal muscles undergo a process of structural and functional remodelling that leads to sarcopenia, a syndrome characterized by loss of muscle mass and force and a major cause of physical frailty. To determine the causes of sarcopenia and identify potential targets for interventions aimed at mitigating ageing-dependent muscle wasting, we focussed on the main signalling pathway known to control protein turnover in skeletal muscle, consisting of the insulin-like growth factor 1 (IGF1), the kinase Akt and its downstream effectors, the mammalian target of rapamycin (mTOR) and the transcription factor FoxO. Expression analyses at the transcript and protein level, carried out on well-characterized cohorts of young, old sedentary and old active individuals and on mice aged 200, 500 and 800 days, revealed only modest age-related differences in this pathway. Our findings suggest that during ageing there is no downregulation of IGF1/Akt pathway and that sarcopenia is not due to FoxO activation and upregulation of the proteolytic systems. A potentially interesting result was the increased phosphorylation of the ribosomal protein S6, indicative of increased activation of mTOR complex1 (mTORC1), in aged mice. This result may provide the rationale why rapamycin treatment and caloric restriction promote longevity, since both interventions blunt activation of mTORC1; however, this change was not statistically significant in humans. Finally, genetic perturbation of these pathways in old mice aimed at promoting muscle hypertrophy via Akt overexpression or preventing muscle loss through inactivation of the ubiquitin ligase atrogin1 were found to paradoxically cause muscle pathology and reduce lifespan, suggesting that drastic activation of the IGF1-Akt pathway may be counterproductive, and that sarcopenia is accelerated, not delayed, when protein degradation pathways are impaired.

Meningoencefalite por Herpesvirus bovino 5 em Minas Gerais: relato de caso clínico / Meningoencephalitis by Bovine herpesvirus 5 in Minas Gerais state: clinical case report

Relata-se um caso de meningoencefalite causada por Herpesvirus bovino 5 (BoHV-5) heritabilityem uma vaca com cinco anos de idade. O animal manifestou quadro clínico inicial de síndrome medular baixa, caracterizada por incoordenação dos membros pélvicos, sinais estes ainda não descritos para a enfermidade. Dentro de pouco tempo a doença evoluiu para síndrome cerebral, e o óbito ocorreu seis dias após o inicio dos sintomas. Na histopatologia, evidenciou-se meningoencefalite difusa, não supurada, e a confirmação do diagnóstico foi feita por reação em cadeia de polimerase e sequenciamento do segmento parcial da glicoproteína G do vírus. O trabalho confirma a presença do BoHV-5 em Minas Gerais, descreve características clínicas novas para a enfermidade e ressalta sua importância no diagnóstico diferencial das neuropatias bovinas.

A clinical case of meningoencephalitis by Bovine herpesvirus 5 (BoHV-5) in a five-year-old cow was reported. The disease began with low spinal cord signs, characterized by incoordination, and these symptoms had never been related to this illness before. Signs of a brain syndrome were observed and the cow died in six days. At the histopathology, a spread non-supurative meningoencephalitis was diagnosed, and the virus identification was made by PCR and partial sequence of the glycoprotein G. This study confirm the BoHV-5 presence in the State of Minas Gerais, Brazil, describes new clinic characteristics, and show the importance of the disease in the differentiate diagnosis with others bovine central nervous system affections.

Avaliação clínica, ultra-sonográfica e teloscópica das papilas mamárias de búfalas / Clinical, ultrasonographic, and theloscopic evaluations of the water buffaloes mammary papillae

As papilas mamárias de 23 búfalas foram avaliadas por meio de exame clínico e ultra-sonográfico. Em oito animais do mesmo grupo, as papilas mamárias foram submetidas ao exame teloscópico. Os resultados mostraram que o exame ultra-sonográfico e a teloscopia podem ser utilizados para a avaliação das papilas mamárias na espécie bubalina. As restrições ao uso desses exames, com a metodologia e os equipamentos empregados, foram: a identificação ultra-sonográfica dos ductos papilares foi precária para as papilas mais curtas, e em matrizes jovens, a resistência do ducto papilar limita o uso de telescopia axial.

All the mammart papillae from 23 water buffaloes were clinically and ultrasonographically examined, and eight animals from the same herd were submitted to theloscopic examination. The results showed that ultrasonography and theloscopy are satisfactory methods to evaluate the mammary papillae of the water buffalo. However, the ultrasonographic identification of the streak canal of the shorter papillae was poor and, it was not possible to proceed with axial theloscopy in some animals, specially the primiparous.

Desempenho de bezerros alimentados com dietas líquidas à base de leite integral ou soro de leite / Performance of calves fed on liquid diets based on whole milk and whey

Quinze bezerros da raça Holandesa, desaleitados aos 30 dias de idade, foram utilizados para avaliar os efeitos da inclusão de fontes protéicas alternativas, em substituição às proteínas do leite integral, na formulação de sucedâneos do leite. Os animais foram divididos em três grupos: o grupo controle (C) - recebeu leite em pó integral reconstituído; o grupo sucedâneo SL (soro/leite) recebeu 41,6 por cento da proteína bruta (PB) vinda do concentrado protéico de soro (CPS), 23,1 por cento do soro de leite e 35,3 por cento do leite em pó integral; e o grupo sucedâneo S (soro) recebeu 68 por cento da PB vinda do CPS e 32 por cento do soro de leite. O consumo de concentrado, feno, sal mineral e água foi mensurado diariamente, enquanto o ganho de peso foi avaliado semanalmente. Não houve diferença (P>0,05) para o consumo de concentrado, sal mineral e água entre os grupos, nos períodos avaliados. O consumo de feno e o ganho de peso foram inferiores nos animais do grupo S (P<0,05). A substituição de 100 por cento do leite integral por CPS e soro de leite influenciou negativamente o desempenho.

Effects of transcutaneous short-term electrical stimulation on M. vastus lateralis characteristics of healthy young men.

Fifteen healthy, untrained male subjects (mean age +/- SD, 22 +/- 5 years) were used to examine the plasticity of myosin heavy chain phenotype, size, oxidative capacity and capillarization of skeletal muscle fibre types with short-term electrical stimulation (ES). Ten subjects were electro-stimulated on both quadriceps muscles with a frequency of 45-60 Hz, with 12 s of stimulation followed by 8 s of recovery for a total of 30 min per day, 3 days per week for 6 weeks. The remaining five subjects served as controls. Two vastus lateralis muscle biopsy samples were removed from each subject before (week 0) and after (week 6) ES training. A standardized exercise test on a cycle ergometer was performed by each subject before and after the experimental period and several indicators of whole-body aerobic capacity were estimated. The so-called electromyographic threshold was also determined during the tests. Muscle biopsy samples were analysed by electrophoresis, immunohistochemistry and quantitative histochemistry. Myosin heavy chain (MHC) composition, muscle fibre type distribution, fibre areas, oxidative capacity and capillaries of each fibre type were estimated. Muscular changes with ES revealed an increase of fibres expressing MHC-IIA, and a decrease of fibres expressing MHC-IIX and MHC-I, as well as an increase of the oxidative capacity and mean number of capillaries of fast-twitch (type II) fibres with minimal muscle fibre hypertrophy. These adaptations seem related to a bi-directional transformation from both MHC isoforms I and IIX towards the MHC-IIA isoform. The aerobic performance and electromyographic variables at the whole-body level were not altered by ES. These results indicate that the particular short-term ES training protocol tested in the present study induces significant adaptations in histochemical and metabolic machineries of human skeletal muscle. The results also offer new perspectives for realistic applications of ES in various clinical situations and sport training.

Early and long-term changes of equine skeletal muscle in response to endurance training and detraining.

Twenty-four 4-year-old Andalusian (Spanish breed) stallions were used to examine the plasticity of myosin heavy chain (MHC) phenotype and the metabolic profile in horse skeletal muscle with long-term endurance-exercise training and detraining. Sixteen horses underwent a training programme based on aerobic exercises for 8 months. Afterwards, they were kept in paddocks for 3 months. The remaining eight horses were used as controls. Three gluteus medius muscle biopsy samples were removed at depths of 20, 40 and 60 mm from each horse before (month 0), during (month 3) and after (month 8) training, and again after 3 months of detraining (month 11). MHC composition was analysed by electrophoresis and immunohistochemistry with anti-MHC monoclonal antibodies. Fibre areas, oxidative capacity and capillaries were studied histochemically. The activities of key muscle enzymes of aerobic (citrate synthase and 3-hydroxy-acyl-CoA-dehydrogenase) and anaerobic (phosphofructokinase and lactic dehydrogenase) metabolism and the intramuscular glycogen and triglyceride contents were also biochemically analysed. Early changes with training (3 months) included hypertrophy of type IIA fibres, a reduction of MHC-IIX with a concomitant increase of MHC-IIA, a rise in the number of high-oxidative fibres and in the activities of aerobic muscle enzymes and glycogen content. Long-term changes with training (8 months) were a further decline in the expression of MHC-IIX, an increase of slow MHC-I, additional increases of high-oxidative fibres, capillary density, activities of aerobic enzymes and endogenous glycogen; intramuscular lipid deposits also increased after 8 months of training whereas the activities of anaerobic enzymes declined. Most of exercise-induced alterations reverted after 3 months of detraining. These results indicate that endurance-exercise training induces a reversible transition of MHC composition in equine muscle in the order IIX-->IIA-->I, which is coordinated with changes in the metabolic properties of the muscle. Furthermore, a dose-response relationship was evident between the duration (in total) of training and the magnitude of muscle adaptations.

Distribution of fast myosin heavy chain-based muscle fibres in the gluteus medius of untrained horses: mismatch between antigenic and ATPase determinants.

The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in muscle biopsies from the gluteus medius of adult untrained horses by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies and standard myofibrillar ATPase (mATPase) histochemistry. Percutaneous needle biopsies were taken at 3 depths (20, 40 and 60 mm) from 4 4-y-old Andalusian stallions. The percentage of 'pure' I MHC fibres increased whereas that for pure IIX MHC fibres decreased from the most superficial to the deepest sampling site. Within the fast fibres, types IIA and IIAX MHC-classified fibres were proportionately more abundant in the deepest sampling site than in the superficial region of the muscle. The immunohistochemical and histochemical characterisation of a large number of single fibres (n = 1375) was compared and correlated on a fibre-to-fibre basis. The results showed that 40% of the fibres analysed were pure type I (expressing only MHC-I); they showed correct matching between their antigenic and mATPase determinants. In contrast, within the fast fibres, a considerable proportion of fibres were found showing a mismatch between their immunohistochemical and mATPase profiles. The most common mismatched fibre phenotypes comprised fibres displaying coexpression of both fast MHCs when analysed by immunocytochemistry, but showing an mATPase profile similar to typical IIX fibres (moderate mATPase reaction after preincubation at pH 4.4). Considered altogether, the total mismatched fibres represented only 4.2% of the whole fast fibre population in the superficial region of the muscle, but their proportion increased to 15.6% and 38.4% in the middle and deep regions, respectively, of gluteus medius. It is concluded that a considerable number of hybrid fast MHC IIAX fibres are present in the gluteus medius of untrained horses, suggesting that equine type II fibres have probably been misclassified in numerous previous publications based on the use of histochemistry alone. This has important implications in attempts to study the physiological properties of fast fibre types adequately in horses.

Neural regulation of myosin gene expression in regenerating skeletal muscle.

Excitation-transcription coupling, namely the process whereby plasma membrane depolarization leads to gene activation or inactivation, is still a black box for most muscle genes. Muscle regeneration is a useful model system to ask basic questions concerning the triggering signals and the transduction pathways involved in activity-dependent gene regulation. We report ongoing research in our laboratory concerning (1) myosin heavy chain changes in regenerating muscle in the presence and absence of the nerve, as well as changes induced by electrical stimulation, (2) identification of activity response elements in the promoter of a slow myosin light chain gene, and (3) potential approaches to define the transduction pathways induced by neural or electrical activity and implicated in muscle gene regulation.

Myosin isoforms and muscle fiber characteristics in equine gluteus medius muscle.

BACKGROUND: To date, four different myosin heavy chain (MyHC) isoforms have been identified in adult skeletal muscle of a number of species: types I, IIa, IIx or IId, and IIb. The aim of this study was to investigate the distribution of various MyHC isoforms in the equine gluteus medius and gluteus profundus muscles in relation with several morphometric variables of muscle fibers. METHODS: Samples from different depths of the gluteus medius muscle (2, 4, 6, and 8 cm) and gluteus profundus muscle of five sedentary horses were examined by MyHC gel electrophoresis, monoclonal antibodies staining against fast, slow and neonatal MyHC isoforms, myosin adenosine triphosphatase (m-ATPase) activity, nicotinamide adenine dinucleotide tetrazolium reductase, alpha-glycerophosphate dehydrogenase, and alpha-amylase-PAS. Data about relative frequencies, sizes, and capillaries of the various histochemical fiber types were collected by morphometry. RESULTS: Three MyHC isoforms were present in the gluteus medius muscle. Two of them comigrated with type I and IIa MyHC isoforms of rat diaphragm (used as a control). The third isoform showed an electrophoretic mobility closer to type IIx than to the IIb MyHC isoform of rat diaphragm. Only two MyHC isoforms (type I and IIa) were detected in the gluteus profundus muscle. In both muscles, type I fibers (high m-ATPase activity at pH 4.5) only reacted with the anti slow-MyHC antibody and both type IIA and IIB fibers (low and moderate m-ATPase activity at pH 4.5, respectively) only reacted with the anti fast-MyHC antibody. No cross-reactivity of fibers positive for both antibodies was found except for the scarce type IIC fibers. Fiber types and capillaries were heterogeneously distributed across the gluteus medius muscle. The deeper regions of this muscle were found to contain a higher percentage of type I fibers, a large number of capillaries and a lower proportion of type IIB fibers compared to the superficial regions of the muscle. The gluteus profundus muscle had more abundant and larger type I fibers than the deepest sampling site of the gluteus medius muscle. CONCLUSIONS: These results show the existence of three different MyHC isoforms in the equine gluteus medius muscle and that fiber types and MyHC isoforms are heterogeneously distributed within this muscle. The distribution of slow-twitch and fast-twitch MyHCs among the fibers determined by immunohistochemistry was in agreement with histochemically identified type I and type II fibers, respectively.

Activities of selected aerobic and anaerobic enzymes in the gluteus medius muscle of endurance horses with different performance records.

Biopsies of the gluteus medius muscle were taken at three different depths from 36 endurance horses aged 8.42 +/- 2.85 years and of both sexes. Twenty of the horses were considered to be excellent performers on the basis of the mean speed of their three fastest records in endurance events over the previous two or three years, whereas 16 were moderate performers. The biopsy samples were analysed for the activities of the enzymes citrate synthase (an indicator of citric acid cycle activity), 3-OH-acyl-CoA-dehydrogenase (an indicator of lipid oxidation) and lactate dehydrogenase (an indicator of anaerobic metabolism). The 20 excellent performers had higher activities of citrate synthase (P < 0.001) and 3-OH-acyl-CoA-dehydrogenase (P < 0.02) than the 16 moderate performers. The activities of citrate synthase and 3-OH-acyl-CoA-dehydrogenase increased by 65 per cent and 75 per cent, respectively, and the activity of lactate dehydrogenase decreased by 23 per cent in the samples taken at successively greater depths. There was a strong linear relationship between the ratios of the activities of lactate dehydrogenase/citrate synthase and lactate dehydrogenase/3-OH-acyl-CoA-dehydrogenase and the depth from which the samples were taken for both performance groups (P < 0.001). The intercepts of the regression lines were higher in the moderate than in the excellent performers (P < 0.001 and P < 0.01 for the two ratios), showing that the endurance horses with the better performance record had a greater aerobic capacity and a relatively lower anaerobic capacity in the gluteus medius muscle than the horses with a poorer record.

Muscle fiber type composition and fiber size in successfully and unsuccessfully endurance-raced horses.

Triplicate biopsies from three different depths of the gluteus medius muscle were obtained in 36 endurance-raced horses, aged 8.42 +/- 2.85 yr. Twenty of the horses were considered excellent endurance performers according to the mean speed of their three fastest records in endurance events for the past 2 or 3 years, whereas 16 were moderate performers, with a mean racing speed < 12.5 km/h (in 120- to 180-km endurance rides), < 14 km/h (in 80- to 120-km endurance rides), or < 13.5 km/h (in 40- to 60-km endurance rides). Significant differences in muscle fiber type composition and fiber size were recorded; excellent performers had a higher percentage and a larger size of type I and type IIa fibers (high and low myosin adenosinetriphosphatase activity at pH 4.5, respectively) and a lower percentage of type IIb fibers (moderate myosin adenosinetriphosphatase activity at pH 4.5), including both type IIb oxidative (moderate to high NADH-tetrazolium reductase activity) and IIb nonoxidative (low NADH-tetrazolium reductase activity). The differences in distribution of myofiber types and in fiber sizes were more marked in the deeper parts compared with the superficial regions of muscle. Our results also imply a greater homogeneity among the fiber type sizes across the muscle in horses with a superior endurance performance than in horses that had been poorly or moderately endurance raced. Thus the results show that fiber type proportions and fiber size in equine skeletal muscle are directly related to the athletic ability of the horse for endurance events.

Changes in cross-sectional area and capillary supply of the muscle fiber population in equine gluteus medius muscle as a function of sampling depth.

The right and left gluteus medius muscles of 4 mature Andalusian stallions were examined by repeated needle biopsy over a specific area and depth, as well as at different depths, to determine whether the cross-sectional area and capillary supply of the various fiber types are homogeneous throughout the muscle. The muscle biopsy specimens were histochemically analyzed for fiber types (myofibrillar adenosine triphosphatase) and capillaries (amylase-periodic acid-Schiff method). Differences between contralateral sites were not identifiable for any of the analyzed variables. Differences between sampling depths were larger than those between sample sites. The percentage of type-1 fibers increased, and that of type-2B fibers decreased as a function of increased sampling depth, but the relative frequency of type-2A fibers did not change with depth. The mean cross-sectional area of type-1 and type-2A fibers increased significantly, and that of type-2B fibers decreased between the most superficial and the deepest sampling sites. The capillary density and the mean number of capillaries per fiber increased as a function of sampling depth, but significant changes were not recorded between sampling depths for overall mean fiber area. The mean number of capillaries in contact with fibers of each histochemical type increased significantly with increasing sampling depth only for type-1 fibers. However, the number of capillaries in contact with fibers changed significantly, relative to the fiber area, as a function of sampling depth; for type-2B fibers this variable increased, whereas for type-2A and type-1 fibers, it decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Variability of muscle fibre composition and fibre size in the horse gluteus medius: an enzyme-histochemical and morphometric study.

To determine the variability in fibre types and fibre sizes in the equine gluteus medius muscle, biopsy specimens were removed from 5 sites, at 4 different depths, within the right and left muscles of 3 Andalusian stallions. The percentage, lesser fibre diameter and cross-sectional area of the various fibre types were measured systematically in myosin ATPase and NADH-tetrazolium reductase-stained, serial cryostat sections of these multiple samples. Significant differences in muscle fibre type composition were recorded, with a lower percentage of type I fibres (high myosin ATPase activity at pH 4.5) being observed towards superficial regions of the muscle and a greater percentage towards the deep areas. Type II B fibres (moderate myosin ATPase activity at pH 4.5), including both II B nonoxidative (low NADH-TR activity) and II B oxidative (moderate NADH-TR activity), displayed the opposite tendency, and the percentage of type II A fibres (low myosin ATPase activity at pH 4.5) did not change with depth. Types I and II A fibres in the deep regions were larger than superficially, whereas the II B fibres in the deep regions were smaller than in the superficial parts of the muscle. The results also imply that type I fibres tend to be larger than type II fibres in the deep regions. The size of type I fibres is more homogeneous in the deep parts than in the superficial regions of the muscle, while II B fibres vary more in size in the peripheral portions than in deep regions. A single biopsy taken from the gluteus medius muscle of the horse is therefore a poor representative of the whole muscle and care should be exercised in sampling and interpreting data obtained from limited biopsy of this muscle. The pattern of variation in fibre types and fibre sizes between the different depths of the muscle probably reflect different functional demands on the gluteus medius muscle.