Ultrastructural localization of concanavalin A-binding sites in satellite cells of human skeletal muscle

Summary Electron-microscopic cytochemical studies on satellite cells of normal human skeletal muscle were carried out using the concanavalin Aperoxidase (Con A-HRP) coupling method. Con A-binding sites, which probably correspond to glycoproteins, were found to be associated with the cell surface, smooth surfaced vesicles, nuclear envelope and endoplasmic reticulum of the satellite cells and were also identified at the cell surface of the adjacent muscle fiber. The possible relationships of these observations to the functions of satellite cells are discussed.     

Ultrastructural localization of anionic phospholipids in skeletal muscle plasma membrane

Summary A cytochemical study of acid phosphatase (AcPase) in the lateral prostate of the rat was performed to investigate whether AcP-ase in the secretory apparatus can be distinguished from AcP-ase in lysosomes and their related structures. Two types of AcP-ase were observed in the rat lateral prostate. One was found in the secretory apparatus (Golgi saccules and some Golgi vesicles, condensing and secretory vacuoles), and reacted well with naphthol AS-BI phosphate (N AS-BI P) as substrate; the other was found in the lysosomes and Golgi-associated endoplasmic-reticulum-lysosome system (GERL)-like structure, and reacted well with -glycerophosphate (GP) as substrate. Although the AcP-ase which reacted well with N AS-BI P was also observed in certain portions of pleomorphic lysosomes, it was concluded that it was the same as the AcP-ase found in the condensing and secretory vacuoles, since a lysosome engulfing a condensing vacuole was often observed. Therefore, it is concluded that the AcP-ase in the secretory apparatus in the rat lateral prostate is different from the AcP-ase in lysosomes. Condensing vacuoles appear to originate from particular portions of Golgi saccules, but not from the GERL or GERL-like structure, at least in the rat lateral prostate.     

Ultrastructural localization of anionic phospholipids in skeletal muscle plasma membrane

Polymyxin B was used as a probe to label anionic phospholipids in skeletal muscle plasma membrane. This antibiotic produces muscle surface membrane lesions that can be identified in both thin sections and freeze-fracture replicas. The membrane perturbations assumed a patchy distribution with a preferential localization at the level of the I band and A-I bands junction. Intramembraneous particles were also observed within the lesions. We consider the possibility that microdomains of anionic phospholipids in muscle plasma membrane may function in the binding of Ca++.     

Maintenance of myoglobin concentration in human skeletal muscle after heavy resistance training

The aim of this study was to determine the effects of 8 weeks of resistance training (RT) on the myoglobin concentration ([Mb]) in human skeletal muscle, and to compare the change in the [Mb] in two different RT protocols. The two types of protocol used were interval RT (IRT) of moderate to low intensity with a high number of repetitions and a short recovery time, and repetition RT (RRT) of high intensity with a low number of repetitions and a long recovery time. A group of 11 healthy male adults voluntarily participated in this study and were divided into IRT (n = 6) and RRT (n = 5) groups. Both training protocols were carried out twice a week for 8 weeks. At the completion of the training period, the one-repetition maximal force values and isometric force were increased significantly in all the subjects, by about 38.8% and 26.0%, respectively (P < 0.01). The muscle fibre composition was unchanged by the 8 weeks of training. The muscle fibre cross-sectional areas were increased significantly by both types of training in all fibre types (I, IIa and IIb, mean + 16.1 %, P < 0.05). The [Mb] showed no significant changes at the completion of the training [IRT from 4.63 (SD 0.63) to 4.48 (SD 0.72), RRT from 4.47 (SD 0.75) to 4.24 (SD 0.80) mg x g(-1) wet tissue] despite a significant decrease in citrate synthase activity [IRT from 5.27 (SD 1.45) to 4.49 (SD 1.48), RRT from 5.33 (SD 2.09) to 4.85 (SD 1.87) micromol x min(-1) x g(-1) wet tissue; P < 0.05] observed after both protocols. These results suggested that myoglobin and mitochondria enzymes were regulated by different mechanisms in response to either type of RT. Moreover, the maintained [Mb] in hypertrophied muscle should preserve oxygen transport from capillaries to mitochondria even when diffusion distance is increased.     

Ultrastructural localization of dystrophin in human muscle by using gold immunolabelling

Immunolabelling with a 5 nm gold probe was used to localize dystrophin at the ultrastructural level in human muscle. The primary antibody was monoclonal, raised against a segment (amino acids 1181-1388) from the rod domain of dystrophin. The antibody (Dy4/6D3) is specific for dystrophin and shows no immunoreactivity with any protein from mdx mouse muscle or from patients with a gene deletion spanning part of the molecule recognized by the antibody (Nicholson et al. 1989 a; England et al. 1990). Using this antibody, labelling was almost entirely confined to a narrow 75 nm rim at the periphery of the muscle fibres. Histograms of the distance from the gold probe to the cytoplasmic face of the plasma membrane and of the distance between gold probes (nearest neighbour in a plane parallel with the plasma membrane) displayed modes at approximately 15 nm and 120 nm, respectively. The distribution of the probe was the same in longitudinal and transverse sections of the muscle. These observations suggest that the rod portion of the dystrophin molecule is normally arranged close to the cytoplasmic face of the plasma membrane and that the molecules form an interconnecting network. Labelling was not associated with the transverse tubular system.     

Ultrastructural localization of calsequestrin in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections

The ultrastructural localization of calsequestrin in rat skeletal muscle (gracilis) was determined by indirect immunoferritin labeling of ultrathin frozen sections. Calsequestrin was found in the lumen of transversely and longitudinally oriented terminal cisternae but was absent from most of the longitudinal sarcotubules and the fenestrated sarcoplasmic reticulum. Calsequestrin was occasionally observed in vesicular structures found in the central region of the I band. Since calsequestrin is believed to provide the major site of Ca2+ sequestration in the sarcoplasmic reticulum, the present results support the view that Ca2+, transported to the lumen of the sarcoplasmic reticulum, is preferentially sequestered in the terminal cisternae, but they also suggest that additional Ca2+ sequestration may occur near the center of the I band.     

Immunohistochemical localization of xanthine oxidase in human cardiac and skeletal muscle

The generation of a monoclonal antibody specific to xanthine oxidase and its use in the distribution of the enzyme in human tissue is described. Xanthine oxidase was purified from human and bovine milk by a rapid method, allowing for minimal proteolytic degradation, and the purified enzyme preparations were used for the immunization of BALB/c mice as well as for the subsequent selection of hybridomas. The hybridoma clone X1–7, IgG (2a, -light chain) was selected for further analysis and demonstrated to precipitate xanthine oxidase from human liver and skeletal muscle extracts. As determined by SDS-polyacrylamide gel electrophoresis of eluates from affinity chromatography, the X1–7 antibody bound to a main protein of 155 kDa, from human milk and skeletal muscle, and to proteins of 155, 143 and 95 kDa from human liver. Immunohistochemical studies, using two of the monoclonal antibodies with differing epitope specificity, revealed xanthine oxidase to be localized mainly in the vascular smooth muscle cells but also in a proportion of endothelial cells of capillaries and smaller vessels in both human cardiac and skeletal muscle. Immunoreactivity was additionally observed in human macrophages and mast cells. The results of the present study confirm previous reports of the presence of xanthine oxidase in capillary endothelial cells, but also demonstrates additional localization of the enzyme in vascular smooth muscle cells, macrophages and mast cells. The current findings verify that the distribution of xanthine oxidase in human tissue includes cardiac and skeletal muscle.     

Isolation and properties of myoglobin from murine skeletal muscle

Myoglobin (Mb) was isolated from skeletal muscle of JCL-ICR mice by heat denaturation-gel filtration combined with ion exchange chromatography or chromatofocusing by which isoelectric point of the main component was estimated as 7.63 +/- 0.09 (20 degrees C). The Mb was homogeneous by gel electrophoretic and ultracentrifugal analysis. The molecular weight by sedimentation equilibrium was 1.80 X 10(4) and essentially identical with the values by the iron analysis (1.82 X 10(4)) and amino acid composition (1.78 X 10(4)) in which one residue of cysteine was found per molecule. The spectroscopic properties of deoxy-, oxy-, carboxy- and ferri-derivatives of the protein were determined in ultraviolet, Soret and visible regions. The pK' of acid-alkaline transition of the ferri-form was estimated as 8.57 +/- 0.30 (30 degrees C) from the pH-dependent spectral changes. The oxygen equilibrium studies revealed complete absence of such allosteric properties as heme-heme interaction, anion effect and Bohr effect. Oxygen tension for the half-oxygenation (P50) was 0.69 +/- 0.06 Torr (20 degrees C) and its temperature-dependent change gave the delta H degrees of -14.1 kcal/mole.     

Histochemical localization of lactate dehydrogenase isoenzymes in human skeletal muscle fibers.

Lactate dehydrogenase (LDH) activity was histochemically localized in fibers of the vastus lateralis muscle of men and for comparative purpose in the soleus and plantaris muscleo of rats. Human muscle fibers were identified as fast twitch (FT) or slow twitch (ST) from the histochemical stain for myofibrillar adenosine triphosphatase activity. Rat skeletal muscle fibers were classified as fast-twitch-oxidative-glycolytic (FOG), fast-twitch-glycolytic (FG), or slow-twitch-oxidative (SO) on the basis of NADH-diaphorase and myofibrillar adenosine triphosphatase activities. Heart-type (H) LDH was identified by inhibition of the muscle-type (M) isozyme with 4 M urea. Total LDH as estimated histochemically was highest in the human FT and rat FG fibers. This was predominantly the M-LDH isozyme. ST fibers of human and SO fibers of rat skeletal muscle had the least total LDH but the most H-LDH activity. The FOG fibers of rat muscle contained a total LDH activity intermediate to that of the FG and SO fibers and a combination of H- and M-LDH. There were no fibers in the human muscle samples studied that had LDH activities similar to the FOG fibers of rat muscle.     

Ultrastructural localization of dystrophin in chicken smooth muscle.

We investigated the presence of dystrophin in gizzard smooth muscle by immunofluorescence assay, immunoblot detection and an immunogold electron microscopy technique. Western blot analyses, using antibodies raised against sequences 1173-1728 and 3357-3660 of the dystrophin molecule, revealed the presence of a major intact 400 kDa protein band and an immunofluorescence localization restricted to the periphery of the smooth muscle cells. We were able to precisely determine the dystrophin distribution along the plasmalemma whereas caldesmon molecules were present in the cytoplasm. The most commonly observed distance between two neighbouring dystrophin molecules suggested a self-associating arrangement. We discuss these findings in relation to the function of dystrophin in the smooth muscle cell structure.     

Ultrastructural localization of pyruvate dehydrogenase in rat heart muscle

Summary The present study attempts to localize pyruvate dehydrogenase activity in rat heart muscle by electron microscopy. The pyruvate and Mg²⁺ dependent reduction of ferricyanide was used as an indicator for enzyme activity. The reaction product, copper ferrocyanide, was found in the inner membrane, the intracristal and intermembrane spaces of mitochondria.This work was supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany.Supported by a Fellowship from the Alexander von Humboldt-Stiftung, Bad Godesberg, Germany.     

A new role for myoglobin in cardiac and skeletal muscle function

For many years, myoglobin was considered as an intracellular globin involved in oxygen transport and storage in cardiac and skeletal muscles. Following the discovery of its ability to convert nitrite into nitric oxide during hypoxia, myoglobin was shown to play a new role in the hypoxic signaling pathway that regulates mitochondrial functions of the electron-transport chain. This review presents experimental evidence that supports this concept and discusses the significance of this newly reported ability for cardiac and skeletal muscle functions.     

Role of myoglobin in the oxygen supply to red skeletal muscle.

The contribution of nyoglobin to the oxygen uptake of red skeletal muscle was estimated from the difference in oxygen uptake with and without functional myoglobin. The oxygen uptake of bundles (25 mm long, 0.5 mm mean diameter) of muscle fibers teased from pigeon breast muscle was measured in families of steady states of oxygen pressure from 0 to 250 mm Hg. The oxygen-binding function of myoglobin, in situ in muscle fiber bundles, was abolished by treatment with nitrite of hydroxylamine, which convert oxymyoglobin in situ to high spin ferric myoglobin, or with phenylhydrazine, which converts oxymyoglobin to denatured products, or with 2-hydroxyethylhydrazine, which appears to remove myoglobin from the muslce. The oxygen uptake was again measured. At higher oxygen pressure, where oxygen availability does not limit the respiration of the fiber bundles, oxygen uptake is not affected by any of the four reagents, which is evidence that mitochondrial oxygen uptake is not impaired. At lower oxygen pressure, where oxygen uptake is one-half maximal, the steady state oxygen consumption is roughly halved by abolishing functional myoglobin. Under the steady state conditions studied, the storage function of myoglobin, being static, vanishes and the transport function stands revealed. We conclude from these experiments that myoglobin may transport a significant fraction of the oxygen consumed by muscle mitochondria.