New insights into the skeletal muscle phenotype of equine motor neuron disease: a quantitative approach.

Equine motor neuron disease (EMND) is a neurodegenerative disorder similar to the sporadic form of human amyotrophic lateral sclerosis. This study was conducted to quantify myofiber plasticity in response to EMND. Deep M. gluteus medius biopsy samples from eight horses with an ante mortem diagnosis of EMND, which in five cases was later confirmed by post mortem examination of spinal cord and peripheral nerves, were examined by combined methodologies of electrophoresis of myosin heavy chains (MyHC), muscle enzymes and substrate biochemistry, immunohistochemistry of MyHCs and sarcoendoplasmic Ca2+-ATPase (SERCA) isoforms, quantitative histochemistry of succinic dehydrogenase, glycerol-3-phosphate dehydrogenase, periodic acid-Schiff and capillaries, and photometric image analysis. The data were compared with muscle biopsies from healthy controls. Histopathological findings of EMND were observed in muscle biopsy specimens from all cases, but the severity and intra-biopsy extent varied from case to case. Compared with controls, muscle biopsy samples from EMND horses had a lower percentage of MyHC type I fibers, higher percentages of hybrid IIAX and pure IIX fibers, significant atrophy of all muscle fiber types, reduced oxidative capacity, increased glycolytic capacity, diminished intramuscular glycogen, lower capillary-to-fiber ratio, a higher ratio of myofibers expressing SERCA1a to SERCA2a isoforms, and a lower percentage of fibers expressing phospholamban. Objective discrimination of muscle biopsy specimens according to their healthy status (EMND vs controls) was possible on the basis of their muscular characteristics. A coordinated shift from slow to fast muscle characteristics in contractile and metabolic features of muscle fiber types, together with generalized myofiber atrophy, occurs in EMND and the extent of this change seems to be related to the duration of the disease.

Oral L-carnitine combined with training promotes changes in skeletal muscle.

The purpose of this study was to determine whether oral L-carnitine supplementation enhances the responses of skeletal muscle to training in seven 2-year-old Standardbreds. Four horses were supplemented with 10 g/day L-carnitine for 10 weeks and 3 horses served as controls. All horses were exercised regularly every second day on a treadmill for 5 weeks (training period) and housed in individual boxes for 5 additional weeks (detraining period). The training period consisted of 8 high- and 8 low-speed exercises carried out in alternating sequence. Gluteus medius muscle biopsies were taken at Weeks 0 (pretraining), 5 (post-training) and 10 (detraining). Muscular adaptations to training were observed mainly in the L-carnitine-supplemented horses and included an increase in the percentage of type IIA fibres (delta35%, P<0.05), atrophy of type I fibres (delta24%, P<0.01), a rise in the capillary-to-fibre ratio (delta40%, P<0.01) and an increase in the quantitative reaction of periodic acid Schiff stain (delta11%, P<0.05), used as an indicator of intrafibre glycogen content. After detraining, most of these adaptations reverted towards the pretraining situation. Therefore, exogenous carnitine has an additive effect on muscular responses to training and this should be favourable to improve athletic performance. Nevertheless, further studies are necessary to show whether muscle carnitine content is a limiting factor for fatty acid oxidation.

Effects of repeated biopsying on muscle tissue in horses.

The collection of muscle samples in horses to estimate their performance potential, adaptations to training and diagnosis of exertional myopathies is of increasing interest. This study was designed to examine whether repeated biopsying of the gluteus medius muscle at 7 week intervals alters morphology and muscle fibre variables in the sampling area. The gluteus had been biopsied 3 times previously, each biopsy separated by a 7 week interval. Seven weeks after the last biopsy, this study was initiated. Percutaneous needle biopsies were taken from left and right muscles at 2 depths (2 and 6 cm) and at 2 sites for each depth (22 and 25 cm from the tuber coxae) of 7 mature Thoroughbreds. The 22 cm site was located close (2 cm caudal) to an area of the muscle that had been biopsied previously. The 25 cm site was 5 cm apart from this area. A total of 52 samples were available for histology, electron microscopy and immunohistochemistry. The latter was used to study percentages, sizes and capillaries of muscle fibre types. Most muscle samples collected (75%) had normal morphology, but the remaining 25% showed signs of muscle repair. In some circumstances, they showed regenerative signs of complete muscle repair, while in others ineffective muscle repair (scar formation) was evident. Nevertheless, sufficient normal tissue was always available for measuring routine muscle variables. Samples collected 3 cm laterally apart showed large differences with regard to muscle fibre type variables examined. These results show that repeated muscle samples in intervals of 7 weeks do not have effects that would impair evaluation of muscle fibre variables for diagnosis of effectivity of conditioning programmes. Furthermore, they emphasise that the only way of keeping variations through sample collection technique small is to standardise all biopsying techniques accurately.

Polysaccharide storage myopathy in the M. longissimus lumborum of showjumpers and dressage horses with back pain.

This study was designed to investigate whether horses with clinical signs of back pain due to suspected soft tissue injuries were affected by polysaccharide storage myopathy (PSSM). Diagnosis of PSSM in muscle biopsies obtained from the M. longissimus lumborum of 5 showjumpers and 4 dressage horses with a history of back pain is reported. M. longissimus lumborum biopsies of these horses were characterised histopathologically and in 3/9 cases also by electron microscopy. Observations were compared with M. gluteus biopsies of the same horses, and with M. gluteus biopsies obtained from 6 Standardbreds with recurrent exertional rhabdomyolysis and from 6 healthy trotters. M. longissimus biopsies from horses with back pain showed pathognomonic signs of PSSM, i.e. high glycogen and/or abnormal complex amylase-resistant polysaccharide deposits. Similar features were found in M. gluteus biopsies of the same horses. Sections of horses with rhabdomyolysis had increased PAS stain when compared with healthy horses, but did not show amylase-resistant material. Qualitative observations were corroborated by quantitative histochemistry (optical densities) of sections stained with PAS and amylase PAS. This study demonstrated the presence of PSSM in the M. longissimus of showjumpers and dressage horses with back pain and indicates that epaxial muscle biopsy is an option in diagnosing back problems in horses when clinical examination and imaging techniques do not provide a precise diagnosis.

Co-ordinated expression of contractile and non-contractile features of control equine muscle fibre types characterised by immunostaining of myosin heavy chains.

Combined methodologies of immunohistochemistry, histochemistry and photometric image analysis were applied: (1) to characterise control equine skeletal muscle fibres according to their myosin heavy chain (MyHC) composition and (2) to determine on a fibre-to-fibre basis the correlation between contractile [i.e. MyHC(s), myofibrillar ATPase (mATPase) and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms], metabolic [i.e. succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities, glycogen and phospholamban (PLB) contents], and morphological [i.e. cross-sectional area (CSA), capillary and nuclear densities] features of individual myofibres. An accurate delineation of MyHC-based fibre types was obtained with the immunohistochemical method developed. This protocol showed a high sensitivity and objectivity to delineate hybrid fibres with overwhelming dominance of one MyHC isoform and, furthermore, it allowed a semiquantitative delineation of fast hybrid fibres according to the predominant MyHC isoform expressed. The phenotypic differences in contractile, metabolic and morphological properties seen between fibre types were related to MyHC content. Slow fibres had the lowest mATPase activity (related to shortening velocity), the highest SDH activity (oxidative capacity), the lowest GPD activity (glycolytic metabolism) and glycogen content, the smallest CSA, the greatest capillary and nuclear densities, and expressed slow SERCA isoform and PLB, but not the fast SERCA isoform. The reverse pattern was true for pure IID/X fibres, and type IIA fibres had intermediate properties. Hybrid IIAD/X fibres had mean values intermediate to those of their respective pure phenotypes. Discrimination of fibres according to their MyHC content was possible on the basis of their contractile and non-contractile profiles. These intrafibre interdependencies suggest that, even when controlled by different mechanisms, myofibres of control horses exhibit a high degree of co-ordination in their physiological, biochemical and anatomical features.

Coordinated changes of kinematics and muscle fibre properties with prolonged endurance training.

The objective of this study was to investigate the relationship between kinematic variables and muscle characteristics by determining (1) if heavy endurance training alters these variables and (2) if such modifications occur in a coordinated manner. Fifteen Andalusian stallions age 41-45 months were used. Five horses were used as controls and 10 horses underwent a training programme based on aerobic exercise for 8 months. Intensity of exercise was adjusted individually for each horse according to a standardised exercise test. Stride kinematic characteristics at the trot were analysed by videography and gluteus medius muscle biopsies were removed before and after the experiment. Muscle samples were analysed immunohistochemically for fibre types and fibre sizes. After training, stride frequency increased (6%, P < 0.05), whereas stride duration and stance time of the stride decreased (6 and 10% respectively, P < 0.05 in both). The percentage of type IIA fibres increased (25%, P < 0.01), whereas the percentage of type IIX fibres decreased (80%, P < 0.001). Training had no significant effect on the percentage of types IIAX and I fibres. After training, mean lesser fibre diameters of types I and IIA fibres increased (13 and 10%, respectively; P < 0.05). These results suggest that prolonged endurance training alters kinematic and muscle fibre properties in a coordinated manner, reflecting a greater stability and more efficient propulsion associated with an increase in muscle fatigue resistance and strength. This adaptive response has an impact on athletic performance.

Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level.

The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC beta-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies.

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.