Myostatin precursor is present in several tissues in teleost fish: a comparative immunolocalization study

In this study, the distribution of myostatin was investigated during larval and postlarval developmental stages of Sparus aurata(sea bream), Solea solea(sole) and Brachydanio rerio(zebrafish) by immunohistochemistry using antisera raised against a synthetic peptide located within the precursor region of sea bream myostatin. All the three species examined showed the strongest immunoreactivity in red skeletal muscle in juveniles and adults. During larval development of sea bream, strong staining was detected in skin and brain. Immunoreactivity was also found in muscle, pharynx, gills, pancreas and liver. From metamorphosis, immunoreactivity was identifiable in the oesophagus, in the apical portion of the stomach epithelium, in the intestinal epithelium and in renal tubules. In larval zebrafish at hatching, the most intense myostatin immunoreactivity was evident in the skin epithelium. Immunoreactivity was also found in the retina and brain. In the adult, an intense immunostaining occurred in the gastrointestinal tract as well as in the ovary. In sole larvae, immunoreactivity was found in liver and intestine. Our results support the hypothesis suggested earlier that myostatins in fish have retained a different partition (compared with mammals) of the expression patterns and functions which characterized the ancestral gene before the duplication event that gave rise to growth differentiation factor-11 (GDF-11) and GDF-8 (myostatin).     

No classical type IIB fibres in dog skeletal muscle

Summary To analyse the fibre type composition of adult dog skeletal muscle, enzyme histochemistry, immunohistochemistry for type I, IIA and IIB myosins, and peptide mapping of myosin heavy chains isolated from typed single fibres were combined. Subdivision of type II fibres into two main classes according to the activity of the m-ATPase after acidic and alkaline preincubation proved to be rather difficult and was only consistently achieved after a very careful adjustment of the systems used. One of these sub-classes of type II fibres stained more strongly for m-ATPase activity after acidic and alkaline preincubation, was oxidative-glycolytic and showed a strong reaction with an anti-type IIA myosin. The other one, however, although unreactive with anti-IIA myosin, was also oxidative-glycolytic, and only showed a faint reaction with an anti-type IIB myosin. Peptide mapping of the myosin heavy chains of typed single fibres revealed two populations of heavy chains among the type II fibre group. Thus, in dog muscle, we are confronted with the presence of two main classes of type II fibres, both oxidative-glycolytic, but differing in the structure of their myosin heavy chains. In contrast to some reports in the literature, no classical type IIB fibres could be detected.     

Properties of the vastus lateralis muscle in relation to age and physiological function in master cyclists aged 55–79 years

In this study, results are reported from the analyses of vastus lateralis muscle biopsy samples obtained from a subset (n = 90) of 125 previously phenotyped, highly active male and female cyclists aged 55–79 years in regard to age. We then subsequently attempted to uncover associations between the findings in muscle and in vivo physiological functions. Muscle fibre type and composition (ATPase histochemistry), size (morphometry), capillary density (immunohistochemistry) and mitochondrial protein content (Western blot) in relation to age were determined in the biopsy specimens. Aside from an age‐related change in capillary density in males (r = ?.299; p = .02), no other parameter measured in the muscle samples showed an association with age. However, in males type I fibres and capillarity (p < .05) were significantly associated with training volume, maximal oxygen uptake, oxygen uptake kinetics and ventilatory threshold. In females, the only association observed was between capillarity and training volume (p < .05). In males, both type II fibre proportion and area (p < .05) were associated with peak power during sprint cycling and with maximal rate of torque development during a maximal voluntary isometric contraction. Mitochondrial protein content was not associated with any cardiorespiratory parameter in either males or females (p > .05). We conclude in this highly active cohort, selected to mitigate most of the effects of inactivity, that there is little evidence of age‐related changes in the properties of VL muscle across the age range studied. By contrast, some of these muscle characteristics were correlated with in vivo physiological indices.     

Histochemical and immunohistochemical properties of skeletal muscle fibres fromRana andXenopus

Summary Modern histochemical and immunohistochemical techniques have been used to type skeletal muscle fibres from threeRana species andXenopus laevis.Differing myosin properties and metabolic capacities (representing various contractile properties) define a minimum of four fibre types inRana and five inXenopus. TheRana andXenopus types are sufficiently similar so that a single nomencclature can be applied to them. This nomenclature uses an initial letter indicating the probable contractile performance (F=fast-twitch, S=slow-twitch and T=tonic), and a number indicating rank order of presumed shortening velocity.The largest, fastest fibres-F1-have low oxidative and, at best, moderate glycolytic capacities. Commonly adjacent to them are smaller, F2 fibres with variable but at least moderate metabolic capacities. F3 fibres are rarer and have on average the highest oxidative capacity, and at least moderate glycolytic capacity. They usually occur in the reddest parts of the muscle and, inRana, only in the vicinity of tonic fibres.Metabolically weak, classical amphibian tonic fibres (T5) occur in bothXenopus andRana, but onlyXenopus also has an S4 fibre type. This has moderate metabolic capacity and myosin properties suggesting it is probably capable of slow shortening as well as tonic hold. Immunohistochemically, S4 fibres are most similar to avian slow-twitch fibres.     

Preparation of type-specific antimyosin antibodies and determination of their specificity by biochemical and immunohistochemical methods

This paper reports the preparation of specific anti-slow myosin antibodies (anti-I) and anti-fast myosin antibodies (anti-IIA) raised against myosins from sheep and guinea pig masseter muscles. The specificity of the antibodies has been studied by immunodiffusion in agar and by the GEDELISA test using slow-twitch (type I), fast-twitch red (type IIA) and fast-twitch white (type IIB) myofibrils isolated from guinea pig muscles. The principal specificity of the anti-I and anti-IIA antibodies was for the heavy chains of type I and IIA myosins, respectively. A smaller reaction with the corresponding light chains was also detected. Immunohistochemical staining of muscle sections using these antibodies confirmed their fibre type specificity.     

Cellular localisation of insulin-like growth factor binding protein-2 (IGFBP-2) during development of the marine fish, Sparus aurata

The spatial localisation of insulin-like growth-factor-binding protein-2 (IGFBP-2) and its mRNA was investigated during larval and post-larval developmental stages of the gilthead seabream (Sparus aurata) by immunohistochemistry and in situ hybridisation with specific antisera and riboprobes. During larval development, immunoreactivity was found in skin, muscle, gills, pharynx, intestine, liver and olfactory epithelium. After metamorphosis, immunoreactivity was found in the oesophageal epithelium (the strongest reaction) and in red skeletal muscle, heart muscle, the thymus and the epithelium of renal tubules. In the adult, immunostaining with IGFBP-2 antibody was also found in the saccus vasculosus, ovary and testis. IGFBP-2 mRNA was detected by in situ hybridisation mainly in the intestine, skeletal musculature and ovary. These results show that IGFBP-2 protein and mRNA are expressed in a variety of seabream tissues, suggesting that IGFBP-2 regulates the actions of IGFs on these tissues during development and growth.This work was supported by grants from the University of Padua (Progetto di Ateneo, 2001) and by the US–Israel Binational Agricultural Research and Development Fund (BARD, Project IS-2769-96R)     

Developmental programming of skeletal muscle phenotype/metabolism

Skeletal muscle is a highly dynamic and malleable tissue that is able to adapt to different stimuli placed upon it, both during gestation and after birth, ultimately resulting in anatomical changes to muscle fibre composition. Variation in nutrient supply throughout gestation is common, whether in livestock or in the human. The specific effects of maternal nutrition on foetal development are at the forefront of scientific research. However, results describing how different maternal feeding strategies affect skeletal muscle fibre development in the offspring are not fully consistent, even where the same time windows during gestation have been examined. The aim of this study is to determine the effects of increased maternal nutrition (above the recommended levels) on the Musculus semitendinosus phenotype of progeny. In all, 24 pregnant sows were assigned to one of four feeding regimes during gestation; T1 (control group): 30 MJ digestible energy per day (MJ DE/day) throughout gestation, T2: same as that for T1 but increased to 60 MJ DE/day from 25 to 50 days of gestation (dg), T3: same as that for T1 but increased to 60 MJ DE/day from 50 to 80 dg, T4: same as that for T1 but increased nutrition to 60 MJ DE/day from 25 to 80 dg. Light- and heavy-weight littermate pairs of the same sex were selected at birth and individually fed to slaughter (c. 158 days). Histochemical and immunohistochemical staining were used to identify the predominantly oxidative (deep) and less oxidative (superficial) regions of the M. semitendinosus, and to determine total fibre number and proportions of fibre types. The results demonstrate that increased maternal nutrition alters skeletal muscle phenotype in the offspring by changing fibre-type proportions, leading to an increased oxidative capacity due to an increase in Type IIA fibres. No change in total muscle area, total muscle fibre number, or fibre cross-sectional area is observed. The precise molecular mechanism(s) by which these findings occur is being investigated.     

Muscle development in cultured blackspot seabream Pagellus bogaraveo: preliminary histochemical and immunohistochemical data on the fibre types

We studied muscle ontogeny and fibre type characteristics in the blackspot seabream, a new species for commercial aquaculture. Myosin ATPase and SDH histochemistry and immunohistochemistry were tested at different ontogenetic stages, using a panel of antibodies to myosin isoforms and parvalbumin. In general, deep white muscle was parvalbumin‐positive, and superficial 'red' muscle was parvalbumin‐negative at all ages examined. At 6 days of age (transition from endogenous to exogenous feeding) three layers of muscle fibres were observed with different antimyosin reactivities: superficial monolayer, presumptive slow red (present only as a small group of fibres adjacent to the lateral line nerve), and presumptive fast‐white (forming the bulk of the muscle). The superficial monolayer and presumptive slow fibres were positive for SDH. At 60 days of age (transition from live to artificial feeding) an additional fibre type was identified: a typical 'pink' or intermediate layer. In juveniles, the axial muscle consisted mainly of fast white fibres covered by a slow‐red layer and between them a pink layer. Surprisingly, the red layer could be resolved into two distinct types by myosin immunostaining. Red fibres were also present along the horizontal septum, near the notochord. Both red and white muscle layers showed a mosaic appearance, which was confirmed by ATPase reaction. The work was financed by British Council, CRUP, and FCT (PhD Grant SFRH‐BD‐14068–2003).