Fucose expression in skeletal muscle: a lectin histochemical study

Summary Binding sites for three fucose specific lectins, Aleuria aurantia agglutinin (AAA), Lotus tetragonolobus agglutinin (LTA) and Ulex europeus I agglutinin (UEA I), were investigated in sections from normal human and rat muscles, in muscle from patients with Duchenne muscular dystrophy (DMD) and in denervated and devascularized rat muscle. In normal human and rat muscle AAA detected fucosylated glycocompounds in the sarcoplasm, sarcolemma, interfibre connective tissue and vascular structures. In normal human muscle addition of fucose to the AAA incubation medium or treatment of the sections with formaldehyde followed by periodic oxidation before lectin incubation strongly inhibited the staining at all sites other than endothelial cells. In normal rat muscle the same staining procedures strongly inhibited the AAA binding at all sites other than the sarcolemma. Incubation with LTA resulted in a diffuse reaction around the vascular structures in rat muscle, while in human muscle a moderate, homogeneous staining was present in all muscle fibres. Treatment of the sections with formaldehyde and periodic acid before incubation with LTA resulted in strongly labelled muscle capillaries in both human and rat muscle. The only elements in the muscle tissues that were stained with UEA I were human endothelial cells. In denervated and devascularized rat muscle incubation with AAA revealed a novel fucose expression that appeared intracellularly in some necrotic fibres. The AAA-positive fucose residues in the sarcolemma of normal muscle fibres that were resistant to periodic acid oxidation could not be shown by AAA in denervated muscle. In DMD muscle a cryptic sarcolemmal fucose expression could be shown with AAA. It is suggested that both the sarcoplasm and sarcolemma of diseased muscle fibres show altered fucose expression.     

Tropomyosin 4 defines novel filaments in skeletal muscle associated with muscle remodelling/regeneration in normal and diseased muscle

The organisation of structural proteins in muscle into highly ordered sarcomeres occurs during development, regeneration and focal repair of skeletal muscle fibers. The involvement of cytoskeletal proteins in this process has been documented, with nonmuscle gamma-actin found to play a role in sarcomere assembly during muscle differentiation and also shown to be up-regulated in dystrophic muscles which undergo regeneration and repair [Lloyd et al.,2004; Hanft et al.,2006]. Here, we show that a cytoskeletal tropomyosin (Tm), Tm4, defines actin filaments in two novel compartments in muscle fibers: a Z-line associated cytoskeleton (Z-LAC), similar to a structure we have reported previously [Kee et al.,2004], and longitudinal filaments that are orientated parallel to the sarcomeric apparatus, present during myofiber growth and repair/regeneration. Tm4 is upregulated in paradigms of muscle repair including induced regeneration and focal repair and in muscle diseases with repair/regeneration features, muscular dystrophy and nemaline myopathy. Longitudinal Tm4-defined filaments also are present in diseased muscle. Transition of the Tm4-defined filaments from a longitudinal to a Z-LAC orientation is observed during the course of muscle regeneration. This Tm4-defined cytoskeleton is a marker of growth and repair/regeneration in response to injury, disease state and stress in skeletal muscle.     

The slow skeletal muscle troponin T gene is expressed in developing and diseased human heart

Cardiac muscle development is characterised by the activation of contractile protein genes and subsequent modulation of expression resulting, ultimately, in the formation of a mature four-chambered organ. Myocardial gene expression is also altered in the adult in response to pathological stimuli and this is thought to contribute to the altered contractile characteristics of the diseased heart. We have examined the expression of the slow skeletal troponin T (TnT) gene in the human heart during development and in disease using whole mount in situ hybridisation and real-time quantitative (TaqMan) polymerase chain reaction (PCR). Slow skeletal TnT mRNA shows transitory and regional expression in the early foetal heart, which occurs at different times in atria and ventricles. In ventricular myocardium, expression is seen in the outer epicardial layer at a time when the coronary circulation is being established. Expression was detected at low levels in the adult human heart and was significantly increased in end-stage heart failure. Similarly, expression was readily detectable during early rat heart development and was up-regulated in pressure overload hypertrophy in adult. Together these data show for the first time that slow skeletal TnT mRNA is readily detectable during early human heart development. They further suggest that slow skeletal TnT may be responsive to myocardial stress and that elevated levels may contribute to myocardial dysfunction in adult disease. (Mol Cell Biochem 263: 91–97, 2004)     

Proximal skeletal muscle alterations in streptozotocin-diabetic rats: a histochemical and morphometric analysis.

The response of rat quadriceps muscle fibers to chronic streptozotocin (STZ) diabetes was studied. Transverse sections of rectus femoris muscle from diabetic and weight-matched control rats were assayed for myofibrilar adenosine triphosphatase (ATPase) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR). A quantitative analysis was carried out by an automatic interactive analysis system focused on the fiber type size and distribution. STZ-induced diabetes caused important effects in this muscle, with changes in the distribution of oxidative enzyme reactions, type I fiber hypertrophy, and type II fiber atrophy, which was greater in type IIB than in type IIA. It is concluded that hypoinsulinism produces morphological alterations in proximal skeletal muscle fibers that are similar to those of neurogenic myopathy. Thus the pathological changes in these mammalian muscle fibers could explain the clinical syndrome seen in diabetic patients called "diabetic symmetrical proximal motor neuropathy," perhaps the least understood of the major neuropathic complications of diabetes.     

Sarcoglycan and integrin localization in normal human skeletal muscle: a confocal laser scanning microscope study

Many studies have been performed on the sarcoglycan sub-complex and a7B and b1D integrins, but their distribution and localization patterns along the non-junctional sarcolemma are still not clear. We have carried out an indirect immunofluorescence study on surgical biopsies of normal human skeletal muscle, performing double localization reactions with antibodies to sarcoglycans, integrins and sarcomeric actin. Our results indicate that the tested proteins colocalize with each other. In a few cases, a-sarcoglycan does not colocalize with the other sarcoglycans and integrins. We also demonstrated, by employing antibodies to all the tested proteins, that these proteins can be localized to regions of the sarcolemma corresponding either to the I-band or A-band. Our results seem to confirm the hypothesis of a correlation between the region of the sarcolemma occupied by costameric proteins and the metabolic type (fast or slow) of muscle fibers. On this basis, we suggest that slow fibers are characterized by localization of costameric proteins to I-bands, while fast fibers are characterized by localization of costameric proteins to A-bands. The results open a new line of research in understanding interactions between the components of the DGC and vinculin-talin-integrin complexes in the context of different fiber types. Moreover, the same results may be extended to skeletal muscle fibers affected by neuromuscular diseases to detect possible structural alterations.     

Calpain inhibitor in rabbit skeletal muscle: an immunochemical and histochemical study

Summary Calpastatin, the endogenous inhibitor of calcium-activated neutral proteases (calpains; EC 3.4.22.17), was studied in the rabbit vastus lateralis muscle by means of immunochemical and immunohistochemical techniques. Immunoaffinity chromatography using an antibody raised against the 34-kDa monomer of the 68-kDa dimeric inhibitor allowed us to isolate three main proteins (130-, 100- and 80-kDa). These proteins strongly inhibited calpain activity in muscle homogenate (I₅₀ at about 50 g/ml). Immunohistochemical experiments showed that calpastatin-related immunoreactivity was present in all fibre types (oxidative, glycolytic, oxidative-glycolytic) at both surface and cytoplasmic level. However, a few (20%) of the slow-twitch, oxidative fibres (5% of the total fibres), did not contain the cytoplasmic inhibitor. Specific immunoreactivity for calpastatin was also associated with the interstitial connective tissue. These results suggest that (i) calpastatin in skeletal muscle, as in other tissues, is present as a mixture of proteins of various molecular weights and (ii) the inhibitor may act not only in the cytoplasm but also at the surface or extracellular level.     

Calcium currents in normal and dystrophic human skeletal muscle cells in culture

Human muscle cells obtained from biopsy specimens were grown in a primary culture system and electrophysiologically studied. Whole cell patch-clamp recordings revealed the presence of two types of calcium currents: (i) a low-threshold (-60 mV) one (ICa, T) with fast activation and inactivation kinetics (time-to-peak: 39 ms at -30 mV); and (ii) a high-threshold (-10 mV) one (ICa,L) with slower kinetics (time-to-peak: 550 ms at 20 mV). These two types of calcium currents could be also distinguished by their pharmacological characteristics since ICa,L was sensitive to the antagonist and agonist dihydropyridine derivatives contrary to ICa,T which was completely resistant to these compounds. These functional calcium channels existed both in normal and Duchenne dystrophic (DMD) human skeletal muscle cells in culture. We discuss a possible role of these two types of calcium channels in the myoplasmic calcium accumulation observed in the Duchenne muscular dystrophy.     

Trypanosoma cruzi: histochemical characterization of parasitized skeletal muscle fibers

C57B1/6 mice were infected with Brasil strain Trypanosoma cruzi trypomastigotes. The leg muscles of the mice were serial-sectioned with a cryostat, and individual fibers were classified histochemically as type I or type II on the basis of succinic dehydrogenase or adenosine triphosphatase activity. Although markedly more type II fibers were present in the leg muscles, the percentage of infected type I fibers was nearly five-fold higher than type II. This is the first demonstration of a preferential in vivo distribution of T. cruzi in muscle fibers based upon muscle type.     

Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle

Fluorescent antibody staining experiments with both isolated myofibrils and muscle fibers grown in culture show that AMP deaminase is bound to the myofibril in the A band. The strongest staining occurs at each end of the A band. The approximate width of the fluorescent stripes and their relation to the A band remains constant as a function of sarcomere length. Removal of enzyme from the myofibrils leads to loss of staining, and readdition of purified enzyme restores the original staining pattern. A histoenzymatic method for the detection of AMP deaminase activity in cultured fibers gives comparable localization. The results are consistent with the previous observation (Ashby, B. and C. Frieden. 1977.J. Biol. Chem. 252:1869--1872) that AMP deaminase forms a tight complex in solution with subfragment-2 (S-2) of myosin or with heavy meromyosin (HMM).     

The value of enzyme histochemical techniques in classifying fibre types of human skeletal muscle

Summary Fibre-type classification of human skeletal muscle into type I and type II fibres is mostly based on their slight or strong staining with the myosin adenosine triphosphatase reaction. In order to evaluate the reliability of this screening technique a combined histochemical and biochemical study was performed on normal and diseased skeletal muscle of human subjects. In the present investigation activities of enzymes which play a role in the aerobic and anaerobic pathways and which can characterize fibre type, were examined in muscle specimens, with no apparent disease of the neuromuscular system. Special attention is given to the maximal activities of phosphofructokinase and fructose-1,6-diphosphatase, the rate limiting enzymes for the regulation of the glycolysis and glyconeogenesis, respectively. A most important feature of the biochemical findings is the constancy of the activity ratios of the examined enzymes. From these results and from the histochemical results it can be concluded that in apparently normal adult human skeletal muscle the ATP-ase technique for type I and type II typing is reliable. For fibres with an intermediate intensity of staining with the myosin ATPase technique of typing it is also necessary to apply other enzyme histochemical techniques.     

Calpain inhibitor in rabbit skeletal muscle: an immunochemical and histochemical study.

Calpastatin, the endogenous inhibitor of calcium-activated neutral proteases (calpains; EC 3.4.22.17), was studied in the rabbit vastus lateralis muscle by means of immunochemical and immunohistochemical techniques. Immunoaffinity chromatography using an antibody raised against the 34-kDa monomer of the 68-kDa dimeric inhibitor allowed us to isolate three main proteins (130-, 100- and 80-kDa). These proteins strongly inhibited calpain activity in muscle homogenate (I50 at about 50 micrograms/ml). Immunohistochemical experiments showed that calpastatin-related immunoreactivity was present in all fibre types (oxidative, glycolytic, oxidative-glycolytic) at both surface and cytoplasmic level. However, a few (20%) of the slow-twitch, oxidative fibres (5% of the total fibres), did not contain the cytoplasmic inhibitor. Specific immunoreactivity for calpastatin was also associated with the interstitial connective tissue. These results suggest that (i) calpastatin in skeletal muscle, as in other tissues, is present as a mixture of proteins of various molecular weights and (ii) the inhibitor may act not only in the cytoplasm but also at the surface or extracellular level.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. V. A differential diagnostic method for the histochemical classification of glycoproteins

Summary A differential diagnostic scheme is described for the division of colonic epithelial glycoproteins into eleven histochemically distinct classes. The scheme depends upon the use of seven histochemical techniques which, collectively, permit the differential staining ofO-sulphate ester, sialic acid and its side chainO-acyl variants and vicinal diols located on carbohydrate residues other than sialic acids. Elements of the scheme also provide a general approach to the classification of epithelial glycoproteins in anatomic sites other than the colon.Application of the scheme permitted the classification of the epithelial glycoproteins in the mucosa 0.5–5.0 cm from human colonic tumours and provided direct confirmation of previous observations that changes from normal in the relative proportions of either side chainO-acylated sialic acids or sialic acids andO-sulphate esters can occur independently of one another.     

Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57+/-22% of whole homogenate 5'-nucleotidase activity and was not significantly different from the other groups. A weak inverse correlation was found between AMP deaminase and cN-I activities (r2=0.18, p<0.01). There were no significant differences between different groups in the activities of cN-I, whole homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities of whole homogenate nucleotidase, total cN and cN-I. The results indicate no changes in the relative expression or catalytic behaviour of cN-I in AMP deaminase-deficient human skeletal muscle, but suggest that increased turnover of AMP by cN-I in working skeletal muscle is due to higher substrate availability of AMP.