CH2-units on (poly-)ethylene glycol radially dehydrate cytoplasm of resting skinned skeletal muscle

Observing the optical cross-section and electron micrographs of mechanically skinned fibres of frog skeletal muscle, we found that ethylene glycols (EGs) of small (mono-, di-, tri- and tetra-EGs; M(r) 62-194) and medium (poly-EGs; M(r) 900 and 3350) molecular weights efficiently dehydrate the fibres to shrink them radially without microscopic inhomogeneity. The medium-sized poly-EGs at 30% weight/weight concentration absorbed almost all the evaporable water from the fibre. Passive tension measurement at near slack sarcomere spacing indicated that this dehydration by EGs did not accompany longitudinal fibre shrinkage. Chemically relevant fully hydric alcohols (glycerol, threitol, ribitol and mannitol; M(r) 92-182) showed no appreciable dehydrating ability on fibres. An intimate correlation was found between fibre dehydration and CH(2)-concentration of the solutions. Viscosity measurements indicated that the hydrodynamic radii of the alcohols were comparable to those of the small EGs. Therefore, hydrodynamic radii are not a primary determinant of the dehydrating ability. Additionally, CH(2)-concentration of EGs but not alcohols was found to correlate intimately with the measured viscosity of the bulk solution of EGs. These results suggested that the interaction between water molecules and CH(2)-units in crowded cytoplasm of skeletal muscle affects cytoplasm as a whole to realize anisotropic fibre shrinkage.     

The properties of the extraocular muscles of the frog. I. Mechanical properties of the isolated superior oblique and superior rectus muscles.

The mechanical properties of two extraocular muscles (superior oblique and superior rectus muscles) of the frog were studied and compared with those of a frog's skeletal muscle (iliofibularis muscle) which contains the same types of muscle fibres as the oculorotatory muscles. The extraocular muscles are very fast twitching muscles. They exhibit a smaller contraction time, a smaller half-relaxation time, a higher fusion frequency, and a lower twitch-tetanus ratio than the skeletal muscles. The maximum isometric tetanic tension produced per unit cross-sectional area is lower in the extraocular muscles than in skeletal muscles. However, the extraocular muscles show a higher fatigue resistance than the skeletal muscles. With respect to the dynamic properties there are some differences between the various oculorotatory muscles of the frog. The superior rectus muscle exhibits a faster time-course of the contraction, a higher fusion frequency, and a higher fatigability than the superior oblique muscle. An increase of the extracellular K+-concentration evokes sustained contractures not only in the extraocular muscles but also in the iliofibularis muscle; between these muscles there are no striking differences in the mechanical threshold of the whole muscle preparation. The mechanical threshold depends on the Ca++-concentration of the bathing solution and it is found in a range between 12.5 and 17.5 mM K+ in a normal Ringer solution containing 1.8 mM Ca++. The static-mechanical properties of the extraocular muscles of the frog and the dependence of the active developed tension on the muscle extension are very similar to those which are known to exist in the extraocular muscles of other vertebrates. In tetanic activated frog's oculorotatory muscles a linear relationship exists between length and tension. A variation of the stimulation frequency does not change the slope of this curve but causes parallel shifts of the curve. The peculiar properties of the extraocular muscles of the frog are discussed with respect to the muscle fibre types in these muscles and to the diameter of the muscle fibres.     

Comparative aspects of muscle fibre size and succinic dehydrogenase distribution in the longissimus dorsi muscle of several species of East African mammals

The distribution of succinic dehydrogenase enzyme activity was investigated in frozen sections of longissimus dorsi muscle taken from several species of East African game animal (giraffe, hartebeest, wildebeest, oryx, gerenuk and dik-dik) as well as local zebu cattle. Muscle fibres were classified as red (high succinic dehydrogenase activity), white (low activity) or intermediate. The mean diameter and percentage distribution of each fibre type were noted as well as the overall mean muscle fibre diameter (MFD) for each species. The diameters of red muscle fibres were found to be between 54 and 62% of the diameters of the white muscle fibres for all species with MFD differences between species being up to over 100%. The variation in MFD was found to be significantly and positively correlated with live weight, when zebu values were omitted. It was found that the variation in MFD was significantly related to the diameter of muscle fibre types and not to the percent distribution of these fibre types.     

Localization of lactate dehydrogenase isozymes in human muscle tissues by the mixed aggregation immunocytochemical technique.

Lactate dehydrogenase (LDH) isozyme composition and localization was determined in sections of skeletal, heart and smooth muscle by the mixed aggregation immunocytochemical method using first antibody directed against purified human LDH-A4 (M4) or LDH-B4 (H4) followed by the enzymes LDH-A4 and LDH-B4, respectively. An even distribution of the two monomers in all fibres was seen with heart muscle and smooth muscle. Heart muscle had a low concentration of A-monomers and a high concentration of B-monomers, whereas the smooth muscle had equal concentrations of the two monomers. In contrast, skeletal muscle from m. quadriceps femoris was found to be composed of two muscle fibre types, one containing mainly A-, the other mainly B-monomers. On the basis of succinate dehydrogenase activity it was shown that the red (type 1) fibres contain mainly B-monomers and the white (type 2) fibres mainly A-monomers of LDH.     

Adaptation of skeletal muscle fibers to endurance training, confinement or selection for body weight and physical endurance of laboratory mice

By the help of histological methods the changes in skeletal muscle of laboratory mice, which had different levels of exercise during postweaning period (training, confinement) or had been selected for body weight and endurance fitness (Du-6+LB), were investigated. The animal groups with the better endurance fitness (Du-6+LB, trained) had a higher total number of muscle fibres. An increased aerobic capacity of metabolism for the trained and selected animals, a decreased one for the confined animals resulted, indicated by the composition of muscle fibre types of M. rectus femoris. The appearance of stress-induced pathological changes of muscle fibres was observed, with the highest extent in the confined group with decreased endurance fitness. Similar correlations between structure of muscle and fitness in domestic animals are to be expected.     

Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy

The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5–7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20–25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34–40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration.     

Skeletal muscle abnormalities associated with occupational exposure to mercury vapours

There is scarce information on the possible effects of chronic exposure to mercury on skeletal muscle. Dental personnel are frequently exposed to inhalation of metallic mercury vapours. The skeletal muscle of five technicians and one dentist (females, age 36-55) was studied. All of them presented symptoms of chronic mercury poisoning. Needle biopsy was taken from the quadriceps femoris muscle and samples were prepared for light microscope histochemistry and for transmission electron microscopy. Selective atrophy of type IIB muscle fibres was found in patients, and in one of them there was fibre grouping. Most of the muscles showed increased fibre area per capillary. Atrophy was confirmed by the ultrastructural study, demonstrating increase of intermyofibrillar spaces, loss of myofibrils or complete disappearance in some fibres, and sarcolemmal foldings. Splitting of the fibres was also found. Some capillaries were altered, showing endothelial infoldings into the lumen, thickened basement membrane and partial or total occlusion. The alterations found in muscle may be secondary to nerve damage, to ischemia caused by capillary lesion and/or to a direct effect of mercury on muscle fibre proteins.     

Scanning electron microscopy of heart muscle freeze-dried from dimethylsulfoxide for simultaneous demonstration of cell morphology and microvascular function

Scanning electron microscopy was used to examine cryofracture surfaces of ventricular myocardium from glutaraldehyde fixed rat and rabbit hearts subjected to intravascular injection of polymerizing acrylic resin. This allowed simultaneous observation of morphological features of cardiac muscle cells and the functional state of their associated small blood vessels. Because the resin injected to identify capillaries accessible to flow might be soluble in commonly used tissue dehydrating agents, alternative preparation methods using the cryoprotectants dimethylsulfoxide (DMSO) and glycerol were investigated. Provided a high performance backscattered electron detector and simple environmental cell were used to abolish specimen charging and circumvent potential instrument contamination, immersion in 2.82 M DMSO for 12 hr prior to cryofracture and freeze-drying gave the best results. The SEM appearance of specimens dehydrated in this way differed little from that of specimens prepared by ethanol dehydration and freeze-drying or by acetone dehydration and critical-point drying. Tissue shrinkage was 26.5 +/- 9.4%, comparable to that found after standard methods using solvent dehydration and critical-point drying.     

Scanning Electron Microscopy of Heart Muscle Freeze-Dried from Dimethylsulfoxide for Simultaneous Demonstration of Cell Morphology and Microvascular Function

Scanning electron microscopy was used to examine cryofracture surfaces of ventricular myocardium from glutaraldehyde fixed rat and rabbit hearts subjected to intravascular injection of polymerizing acrylic resin. This allowed simultaneous observation of morphological features of cardiac muscle cells and the functional state of their associated small blood vessels. Because the resin injected to identify capillaries accessible to flow might be soluble in commonly used tissue dehydrating agents, alternative preparation methods using the cryoprotectants dimethylsulfoxide (DMSO) and glycerol were investigated. Provided a high performance backscattered electron detector and simple environmental cell were used to abolish specimen charging and circumvent potential instrument contamination, immersion in 2.82 M DMSO for 12 hr prior to cryofracture and freeze-drying gave the best results. The SEM appearance of specimens dehydrated in this way differed little from that of specimens prepared by ethanol dehydration and freeze-drying or by acetone dehydration and critical-point drying. Tissue shrinkage was 26.5 ± 9.4%, comparable to that found after standard methods using solvent dehydration and critical-point drying.     

The number of satellite cells in slow and fast fibres from human vastus lateralis muscle

The aim of this investigation was to study the distribution of satellite cells in slow (type I fibres) and fast (type II fibres) fibres from human vastus lateralis muscle. This muscle is characterised by a mixed fibre type composition and is considered as the site of choice for biopsies in research work and for clinical diagnosis. Biopsy samples were obtained from five healthy young volunteers and a total of 1,747 type I fibres and 1,760 type II fibres were assessed. Satellite cells and fibre type composition were studied on serial muscle cross-sections stained with specific monoclonal antibodies. From a total of 218 satellite cells, 116 satellite cells were found in contact with type I fibres (53.6±8% of the satellite cells associated to type I fibres) and 102 satellite cells in contact with type II fibres (46.4±8% of the satellite cells associated to type II fibres). There was no significant difference (P=0.4) between the percentages of satellite cells in contact with type I and with type II fibres. Additionally, there was no relationship between the mean number of satellite cells per fibre and the mean cross-sectional area of muscle fibres. In conclusion, our results show that there is no fibre type-specific distribution of satellite cells in a human skeletal muscle with mixed fibre type composition.     

Myosin isoenzymes in single muscle fibres of Xenopus laevis: analysis of five different functional types

An analysis has been performed of the native myosin isoenzyme composition of isolated skeletal muscle fibres from Xenopus laevis with well-defined isotonic contraction properties. Fast twitch 'white' (type 1) fibres contained three isomyosins; fast twitch 'red' (type 2) fibres showed two major myosin bands with migration velocities very similar to those of the two slower bands in type 1. Slow twitch (type 3) fibres yielded a single, slowly migrating band as did slow tonic (type 5) fibres, whereas the myosin from type 4 (very slow twitch, 'intermediate') fibres migrated with a somewhat higher mobility. The results suggest that amphibian skeletal muscle may possess the principal fibre types found in mammals and birds.     

Metabolic and physiologic characteristics of skeletal muscle determine its response to clenbuterol treatment

beta-Adrenoceptor agonists are reported to induce skeletal muscle hypertrophy and hence serve as valuable adjunct to the treatment of wasting disorders. In the present study, we attempted to find out whether metabolic and physiologic characteristics of fibres are important in determining skeletal muscle response to clenbuterol (an adrenergic receptor agonist) therapy, as proposed in the treatment of wasting disorders. The treatment of mice with clenbuterol (2 mg/kg body wt for 30 days) resulted in skeletal muscle hypertrophy, more common amongst fast-twitch glycolytic fibres/muscle, with increase in body mass and a parallel rise in muscle mass to body mass ratio. Measurement of fibre diameters in soleus (rich in slow-twitch oxidative fibres), ALD or anterior latissimus dorsi (with a predominance of fast-twitch glycolytic fibres) and gastrocnemius (a mixed-type of muscle) from clenbuterol-treated mice for 30 days revealed noticeable increase in the per cent population of narrow slow-twitch fibre and a corresponding decline in white-type or fast-twitch glycolytic fibres in gastrocnemius and ALD. As revealed by counting of muscle cells in soleus, narrow red fibres declined with corresponding increase in white-type glycolytic fibres population. A significant decline in the succinic dehydrogenase activity was observed, thereby suggesting abnormality in oxidative activity of skeletal muscles in response to clenbuterol therapy.     

Effects of aging and gender on the spatial organization of nuclei in single human skeletal muscle cells

The skeletal muscle fibre is a syncitium where each myonucleus regulates the gene products in a finite volume of the cytoplasm, i.e., the myonuclear domain (MND). We analysed aging‐ and gender‐related effects on myonuclei organization and the MND size in single muscle fibres from six young (21–31 years) and nine old men (72–96 years), and from six young (24–32 years) and nine old women (65–96 years), using a novel image analysis algorithm applied to confocal images. Muscle fibres were classified according to myosin heavy chain (MyHC) isoform expression. Our image analysis algorithm was effective in determining the spatial organization of myonuclei and the distribution of individual MNDs along the single fibre segments. Significant linear relations were observed between MND size and fibre size, irrespective age, gender and MyHC isoform expression. The spatial organization of individual myonuclei, calculated as the distribution of nearest neighbour distances in 3D, and MND size were affected in old age, but changes were dependent on MyHC isoform expression. In type I muscle fibres, average NN‐values were lower and showed an increased variability in old age, reflecting an aggregation of myonuclei in old age. Average MND size did not change in old age, but there was an increased MND size variability. In type IIa fibres, average NN‐values and MND sizes were lower in old age, reflecting the smaller size of these muscle fibres in old age. It is suggested that these changes have a significant impact on protein synthesis and degradation during the aging process.     

The effect of tetraethylammonium and the decrease in the extracellular chloride concentration on membrane depolarization and contraction of skeletal muscle fiber induced by a hyperpotassium solution

1.--The tetraethylammonium (TEA) effects on K+ contracture and membrane depolarization are compared in both crab and frog skeletal muscle fibres. 2.--The mechanical tension of the contracture is reduced by the TEA in frog skeletal muscle fibre; it is increased in crab skeletal fibre. 3.--When no mechanical phenomenon is observed in frog skeletal muscle, the amplitude and the velocity of membrane depolarization induced by an increase of outward K+ concentration is reduced by the TEA. These effects are in opposition in crab muscle fibre. 4.--In crab muscle fibre, the results obtained tend to show that the C1-ions are not distributed on each side of the membrane according to Donnan equilibrium.     

Biochemical verification of quantitative histochemical analysis of succinate dehydrogenase activity in skeletal muscle fibres

Summary The purpose of this investigation was to examine critically the validity of a computerized quantitative microphotometric histochemical technique for the determination of succinate dehydrogenase (SDH) activity in skeletal muscle fibres. Sections from the anterior costal diaphragm were removed from Fischer-344 rats (n = 12) and assayed histochemically to determine SDH activity. The SDH activity in individual muscle fibres was computed using a computerized microphotometric histochemical technique which involves measurement of the optical density of deposited diformazan derived from nitroblue tetrazolium within the fibres. To validate the histochemical technique, whole muscle SDH activities were calculated from the histochemical procedure and were compared to SDH activities determined from whole muscle homogenates via a standard quantitative biochemical assay. The mean within-day variability of the computerized microphotometric histochemical technique of determining SDH activity was 6% (range = 0.5–10.9%) for an area containing ~50 fibres and 6.1% (range = 1.05–14.9%) for an individual muscle fibre. Similarly, the mean between-day variability of the microphotometric histochemical technique of determining SDH activity was 5.9% (range = 2.6–13.9%) for an area containing ~50 fibres and 6.6% (range = 2.2–13.9%) for an individual muscle fibre. The inter-class correlation coefficient between biochemically determined SDH activity and histochemically determined SDH activity was r = 0.83 (p < 0.05). Collectively, these data demonstrate that the quantitative histochemical technique of Blanco et al. (1988) is both valid and reliable in the determination of SDH activity in skeletal muscle fibres.     

Skeletal muscle characteristics of reindeer (Rangifer tarandus L.)

Fibre type composition, fibre areas, capillaries, enzyme activities and intramuscular substrates were analysed on skeletal muscle samples from reindeer. The muscles contained 10-20% Type I fibres and a higher percentage of Type IIB (40-60%) than Type IIA fibres (20-40%). All fibre types revealed medium or dark staining intensity for oxidative capacity. Glycolytic capacity was greatest in Type IIB fibres. All fibres stained for glycogen, while Type I and IIA fibres stained for lipids. The mean number of capillaries in contact with fibres of each type, relative to fibre type area was high in all muscle types. The metabolic profile of reindeer muscle indicates that energy, to a great extent, is produced through oxidative pathways.     

Fibre type specificity of haem oxygenase-1 induction in rat skeletal muscle.

The expression of the inducible haem oxygenase (HO-1) gene was examined in different skeletal muscles. Rats were treated with haemin and a time course of HO-1 mRNA expression was determined in soleus and extensor digitorum longus (EDL) muscles. Fibre type composition and tissue myoglobin content were also measured. We found that HO-1 mRNA expression markedly increased in soleus (type I fibres) muscle but was only slightly affected in EDL (type II fibres). HO-1 expression directly correlated with both percentage of red fibres and tissue myoglobin. These data demonstrate that HO-1 gene expression follows a fibre type-specific pattern which might indicate an important role for this protein in the maintenance of skeletal muscle function.     

Localization of hyaluronan in various muscular tissues

Summary The histochemical distribution of hyaluronan (hyaluronic acid, HYA) was analysed in various types of muscles in the rat by use of a hyaluronan-binding protein (HABP) and the avidin-biotin/peroxidase complex staining procedure. Microwave-aided fixation was used to retain the extracellular location of the glycosaminoglycan. In skeletal muscles, HYA was detected in the connective tissue sheath surrounding the muscles (epimysium), in the septa subdividing the muscle fibre bundles (perimysium) and in the connective tissue surrounding each muscle fibre (endomysium). HYA was heterogeneously distributed in all striated muscles. In skeletal muscles with small fibre dimensions (e.g., the lateral rectus muscle of the eye and the middle ear muscles), HYA was predominantly accumulated around the individual muscle fibres. Perivascular and perineural connective tissue formations were distinctly HYA-positive. In cardiac muscles, HYA was randomly distributed around the branching and interconnecting muscle fibres. In comparison, smooth muscle tissue was devoid of HYA.     

The histochemical profiles of fibre types in porcine skeletal muscle

Using a variety of histochemical methods -mATPase staining after alkaline and acid preincubations, NADH-TR and alpha-MGPDH- we have investigated the fibre types in porcine skeletal muscle. The results reveal that four major fibre types -I, IIA, IIB and II*- can be separated histochemically in Longissimus lumborum muscle of Landrace pigs. The histochemical properties of the muscle fibre type II* are very similar to that of type IIX described in other mammals. The existence of IIX fibres in pig muscle has been recently demonstrated by molecular biology techniques and our results validate the use of histochemistry (mATPase) as an easy methodology to differentiate the three fast myosins (type II fibres) in pig muscle.     

Fine structure of myomyous junctions in the mouse skeletal muscles

The reaction product of acetylcholinesterase (AChE) activity is known to be specifically localized at a neuromuscular junction and a muscle-tendon junction of the striated skeletal muscles. In addition to the two junctions, we recently found some linear precipitates due to AChE activity running transversely across a fibre of the semitendinosus, rectus abdominis, gastrocnemius, tibialis anterior and diaphragm muscles in mice. Under an electron microscope, the linear precipitates were seen at the extracellular side of the muscle fibre endings. Most of the endings contacted each other to form a junction, which has been called the 'myomyous junction (M-Mj)'. The patterns of the M-Mj were grouped into three types: (1) a junction in which all contacts were firm, without any connective tissue, and invaginated deeply; (2) the ones in which numerous collagen fibres were visible in the space between the two separate opposing muscle fibres; (3) an intermediate type between (1) and (2), i.e. a junction with partial contacts. The muscle fibre ending forming M-Mj was constructed of finger-like processes like that of a muscle-tendon junction. However, the processes of a M-Mj adhered so closely to each other that no collagen fibrils could penetrate into their folds.