[Change in localization of cellular vesicular apparatus during differentiation of myoblasts into myotubules in cell culture].

The study of changes in the intracellular processes during differentiation of myoblasts into myotubules is of great importance for understanding several fundamental problems of cell biology. At first, this concerns the spatial organization of vacuolar apparatus that reflects the alterations in the properties of cell membranes, cytoskeleton elements and dynamics of vesicular transport in the course of differentiation. The distribution of acidic membrane organelles (lysosomes, late endosomes, Golgi cisternae) during the myotubule formation was revealed. It was shown that perinuclear localization of acidic organelles in myoblasts was replaced by diffuse distribution of these structures in the whole volume of myotubules. Using lipophilic fluorescent dyes, RH 414 and di-8-ANEPPS, the process of formation and dynamics of endocytic vesicles in myoblasts and myotubules was investigated. In the present work, semiconductive nanocrystals, quantum dots (QDs), conjugated with TAT-peptide, which belongs to cell-penetrating peptides, were used to characterize nonspecific endocytosis. It was shown that QDs--TAT complexes penetrate myoblasts but do not penetrate myotubules even after 24 h incubation, which might be connected with plasma membrane changes during the process of skeletal muscle differentiation.

[Untargeted quantum dots in confocal microscopy of living cells].

The problem of non-specific binding of quantum dots (QDs) with cells is very important but not fully understood taking into account the possible application of QDs in medical and fundamental studies. The interactions of untargeted CdSe/ZnS QDs with isolated frog muscle fibres, HeLa cells and J774 cells were investigated. The observations were performed on living cells using laser confocal microscopy (Leica TCS SL). The QDs covered with polyethylenglycol without any functional reactive groups with emission maximum at 565 nm were used in the study. This type of QDs is suggested to prevent an interaction of QDs with biological molecules. It has been shown that QDs do not enter HeLa cells, T-system and sarcoplasm of skeletal muscle fibres. However, during long-term incubation J774 cells can uptake QDs. The data obtained has demonstrated the diversity of interactions of untargeted QDs with different cell types and are important for understanding of the problems of non-selective uptake and cytotoxicity of QDs.

[AO distribution and fluorescence spectra in myoblasts and single muscle fibres].

Using spectral scanning regime of Leica TCS SL confocal microscope, acridine orange (AO) fluorescence spectra in nuclei and cytoplasms of living myoblasts L6J1 and frog single muscle fibres have been studied. AO fluorescence spectra in salt solutions dependent on free AO concentrations and in AO complexes with DNA have also been obtained for comparison. Myoblasts nuclei fluoresced in green spectral region with maximum at approximately 530 nm (corresponding AO monomers fluorescence), nucleoli fluoresced most brightly. Nuclear chromatin fluoresced not uniformly in these cells. We saw similar to myoblasts AO emission in nucleoli and nuclei of frog single muscle fibres. The uniformed weak green fluorescence was observed for myoblast cytoplasm. As to the muscle fibres sarcoplasm, we saw also AO green fluorescence in A-discs. In myoblasts and muscle fibre cytoplasm we saw the fluorescent red, yellow and green granules which were acidic organelles. The comparison of AO fluorescence spectra in living cells with fluorescence spectra of different AO concentrations and complexes of AO with DNA in buffer solutions allows estimation of AO concentration in acidic granules which is of interest in the investigation of cellular organelles functions in the processes of intracellular transport, adaptation, apoptosis and a number of pathological conditions.

[Confocal-microscopic study of skeletal muscle fibre membrane organelles during Zenker's (spreading) necrosis].

The changes of T-system and cellular acidic organelles during spreading (Zenker's) necrosis of frog skeletal muscle fibres have been investigated using laser confocal microscopy and several vital fluorescent dyes acridine orange, RH 414, DiOC6(3), rhodamine 123, fluorescein dextran. The formation of numerous vacuoles as a result of local T-system swelling is most characteristic for initial steps of Zenker's necrosis. Vacuoles can attain tens microns in length. They are located both near nuclear poles and between myofibres. Vacuoles maintain connections with the extracellular space up to the moment of contraction knot rejection, and under definite conditions (glycerol influx to fibre) vacuoles are reversible. They deform nuclei and sarcoplasmic reticulum cisternae. Cellular acidic organelles, accumulating acridine orange (lysosomes, late endosomes, Golgi apparatus cisternae) are situated in direct vicinity with normal and vacuolated T-system. The increase in acidic organelles number and size occur during the pathological process development, and tendency to vacuoles clusterization may be seen. Vacuolation of T-system during necrosis is not followed by vacuole content acidification. The role of cellular acidic organelles and of T-system vacuolation in the development of different muscle pathological changes is discussed.

Reversible vacuolation of T-tubules in skeletal muscle: mechanisms and implications for cell biology.

The majority of investigations of the transverse tubules (T-system) of skeletal muscle have been devoted to their role in excitation-contraction coupling, with particular reference to contact with the sarcoplasmic reticulum and the mechanism of Ca2- release. By contrast, this review is concerned with structural and functional aspects of the vacuolation of T-tubules. It covers experimental procedures used in reversible vacuolation induced by the efflux-influx of glycerol and other small nonelectrolytes, sugars, and ions. The characteristics of the phenomenon, associated alterations in muscle function, and the swelling of analogous structures in nonmuscle cells are considered. Possible functions of reversible vacuolation in water balance, transport, membrane repair, muscle pathology, and fatigue are considered, and the potential application of reversible vacuolation in the transfection of skeletal muscle is discussed. In relation to the possible mechanisms involved in reversible vacuolation, particular attention is given to the dynamic and structural aspects of the opening and closing of T-tubules, the origin of vacuolar membranes, and the localized character of tubular swelling.

The effects of glycerol and urea on the ultrastructure and contractility of fast and slow rat skeletal muscles.

The influence of the influx and efflux of glycerol and urea (400 mmol/l) on the amplitude of isometric twitches and the ultrastructure of isolated fast (EDL) and slow (SOL) muscles of young rats was studied. The influx of non-electrolytes was accompanied by a temporary decrease in the twitch tension. The removal of non-electrolytes resulted in a stable reduction of twitches. Both effects were less pronounced in glycerol experiments on slow muscles. The inhibition of twitches after the removal of non-electrolytes was associated with selective alterations of the T-system: swelling, vacuolation, and lysis of T-tubules. Quantitative analysis of the T-system showed that the extent of these changes may vary for different fibres, and the intensity of morphological alteration of the T-system generally correlated with the degree of twitch inhibition. Reloading of muscles with non-electrolytes tended to improve the T-system structure in some fibres and led to a partial restoration of the amplitude of twitches.

[Unusual transformation of myosin filaments in the frog skeletal muscle fiber]. / Neobychnaia transformatsiia miozinovykh nitei v skeletnom myshechnom volokne liagushki.

An ususual alteration of A-band of the frog fast skeletal muscle fiber in the course of the speading (Zenker's) necrosis in calcium-free medium is described, Myosin filaments in distal parts of A-band stick together to from ribbons approximately 0.5 mkm long. The ribbons separate from the central portion of A-band and displace into I-bands up to Z-line. On transverse section, structure of I-bands looks similar to that of vertebrate smooth muscle as reported by Small and Squire (1972).

[Stereologic analysis of vacuolization of the T-system of frog muscle fibers, detected using confocal fluorescence microscopy]. / Stereologicheskii analiz vakuolizatsii T-sistemy myshechnykh volokon liagushki, vyiavleiaemoi s pomoshch'iu konfokal'noi fluorestsentnoi mikroskopii.

The confocal fluorescence microscopy has been used for quantitative evaluation of the T-system reversible vacuoles produced by efflux of 80-120 mM glycerol from frog skeletal muscle fibers. The fibers were stained by membrane probe RH414 and by water-soluble dye fluorescein dextran that marks the vacuolar lumen. Using morphometrical and stereological methods the volume and surface densities of vacuoles were measured on single optical sections and Z-series during a 30 min glycerol efflux. Various methods of measurements (three-dimensional reconstruction of vacuoles, computer morphometry, point counting method) applied to the same Z-series provide similar results. The vacuolar membranes stained by RH414 look like bright rings 0.3-0.4 micron in width. It is concluded that the real position of vacuolar membrane corresponds to the middle of the vacuolar envelope. The measurements of the external dimensions of the envelope overestimate the stereological parameters up to 50%. The volume density of vacuoles reaches 10% within 20-30 min of glycerol efflux. It means that the volume of the T-system may increase by 25-30 times compared to the control value (0.3-0.4%). The surface density of vacuoles during reversible vacuolation is equal to 0.20-0.35 micron-1 and does not exceed the surface density of normal T-system. The sufficiency of membrane material for the T-system reversible vacuolation is discussed in addition to the role of geometrical factor in this phenomenon.

[Effect of glycerin and urea on rat skeletal muscle twitches]. / Vliianie glitserina i mocheviny na odinochnye sokrashcheniia skeletnykh myshts krysy.

The dynamics of changes in the amplitude of isometric twitches of isolated EDL and SOL muscles of young rats influenced by low molecular non-electrolytes has been studied. Incubation of EDL in hypertonic glycerol or urea solution (400 mM) leads to a 80 and 60% decrease of twitches, respectively, within 15 minutes. During the following 1--2 hours the twitches restore the initial level or exceed it by 60% (urea). Removal of glycerol or urea causes a stable reduce of contraction up to 5--10% within 1 hour. Reincubation in these non-electrolytes increases twitches 3--4 fold during 1.5--2 hours. Alterations of SOL twitches in urea are the same as of EDL, while glycerol even in concentration of 600 mM produces only small changes. This appears to be determined by a higher permeability of slow muscle fibers to glycerol as compared to EDL. It is suggested that the decrease of twitches and their restore in non-electrolyte solution are determined by the hypertonic effect of the latter while reduction of twitches during non-electrolyte removal is caused by alteration of T-system.

[Myofibril breakdown during spreading damage. III. Isolated rat muscles]. / Razrushenie miofibrill vo vremia rasprostraniaiushchegosia povrezhdeniia. III. Izolirovannye myshtsy krysy.

Ultrastructural changes have been studied in fast (EDL) and slow (SOL) isolated muscles of rats during Zenker's degeneration (ZD). The general pattern of ZD is similar in both the muscles and proceeds in the same way as in the fast skeletal muscles of amphibians: vacuolation of the T-system and of the sarcoplasmic reticulum occur prior to necrosis, and then contraction knots are formed. The latter detach from the rest of the fiber, and myofibrils degrade into clots of electron dense material. Differences in the development of ZD in this muscle are as follows: vacuolation is more strongly pronounced in EDL, contraction and supercontraction of sarcomeres being more characteristic of SOL. In this ZD region of both the muscles, additional membranes are found in some intracrystal spaces of mitochondria, this phenomenon occuring more frequently in SOL. Selective lysis of the Z-line during ZD has been detected only in single EDL Fibres.

[The effect of membrane-transport inhibitors on the vacuolization of skeletal muscle fibers induced by glycerin removal from them]. / Vliianie ingibitorov membrannogo transporta na vakuolizatsiiu skeletnykh myshechnykh volokon, vyzvannuiu vykhodom iz nikh glitserina.

Effects of inhibitors of volume regulation on reversible vacuolation of skeletal muscle transverse sarcotubules have been studied. The drugs include inhibitors of sodium pump (ouabain), electrodiffusion Cl- and K+ channels (SITS, DIDS, niflumic acid, Ba2+), anion-cation cotransport and antiport (bumetanide, furosimide, amiloride), calcium channels (verapamil, nifedipine), stretch-activated ion channels (Gd3+) and water channels (Hg2+). The drugs were used at concentrations usually inhibiting the regulatory volume response in nonmuscle cells. The vacuolation-devacuolation cycles of frog muscle fibers were observed under the light microscope during efflux and entry of glycerol (90 mM). Neither of used inhibitors produced any noticeable effect on development and disappearance of vacuolation. A conclusion is made that development of vacuoles from the T-system during glycerol removal is not associated with activation of ion transport pathways that leads to regulatory volume decrease or increase in other cell types. The mechanisms of T-tubule vacuolation are discussed.

[Myofibril breakdown during spreading damage. II. A calcium-free medium]. / Razrushenie miofibrill vo vremia rasprostraniaiushchegosia povrezhdeniia. II. Beskal'tsievaia sreda.

Peculiarities of Zenker's degeneration (ZD) have been investigated in fast muscle fibres of the frog incubated in a Ringer solution free of Ca++ (R--Ca) with a normal or increased (by 100 mM) concentration of KCl. ZD in these solutions is distinguished by a 10--90 minutes delay of the appearance of the primary contraction knot and cessation of ZD development in the majority of fibres after formation of several (1--5) contraction knots. In the presence of 0.5 mM EDTA in R--Ca, after a few typical contraction knots are formed, fibres commonly fall into large fragments that retain cross-striation. Contracted or super-contracted state of sarcomeres in detached contraction knots and at the necrosis boundary, as well as an increasing lysis of contactile material and proliferation of fibre membrane structures in the region of ZD arrested boundary, are characteristic of ultrastructural changes during ZD in calcium-free solutions.

[Disruption of myofibrils during spreading degeneration. I. Increased Ca2+ concentration]. / Razrushenie miofibrill vo vremia rasprostraniaiushchegosia povrezhdeniia. I. Povyshennaia kontseatratsiia Ca2+

The breakdown of sarcomeres of frog's twitch skeletal muscles during Zenker's (spreading) degeneration has been studied. The speed of propagation of the destruction process was accelerated by increasing CaCl2 concentration in Ringer's solution up to 8mM. An hour after local injury, the fibres were fixed just before separation of the next retraction clot or at the stage of granular destruction (Fig. 1). The dominating features of the ultrastructure of a fibre at the necrotic boundary are the coagulation of small bundles of supercontracted myofibrils and breakdown of uncontracted sarcomeres into separate A- and I-bands and then into small bundles of A- and I-protofibrils (Fig. 2,3). The same breakdown of sarcomers is observed in several small regions at a distance of about 100 micron from the necrotic boundary (Fig. 5). Besides this, fusion of a few myofibrils followed by the disappearence of M- and Z-bands occurs in the same region of the fibre (Fig. 4, 6). The diameter of the majority of myofibrils decreases towards the necrotic boundary due to longitudinal splitting and loss of peripheral protofibrils, presumably, as a result of lysis (Fig. 7).

[Functional role of vacuolization of the T-system of skeletal muscle fibers]. / Funktsional'naia rol' vakuolizatsii T-sistemy skeletnykh myshechnykh volokon.

T-tubules of skeletal muscle fibres easily transform into large vacuoles under the influence of various factors. These include osmotic shock produced by the efflux of small molecular weight molecules (e.g. glycerol), hypertonic shock, muscle fatigue and muscle damage. In most cases, vacuolation is reversible but the molecular mechanisms involved are not clear. Also, the functional role of reversible vacuolation has not been established. However, three possibilities may be considered. (1) Redistribution of ions and water between the cytoplasm and the extracellular space comprised by the T-system. Thus, the formation of large vacuoles may be a mechanisms for rapid osmoregulation that corresponds to regulated volume decrease in other types of cell. However, in our hands, inhibitors of various pathways that participate in volume regulation had no effect on reversible vacuolation. (2) Resealing of mechanical damage of the plasma membrane. This is usually accompanied by the development near the damaged membrane of numerous vacuoles which we have observed by confocal microscopy and use of a hydrophobic dye (RH414), to arise in part from T-tubules. (3) By confocal microscopy, it has also been shown that extracellular fluorescein dextran (Mr = 10,000), and both plasmid DNA (pUC18) and sonicated high molecular weight DNA stained with YOYO, enter vacuoles derived from T-tubules. This finding may indicate that reversible vacuolation, in the absence of membrane damage, could provide a pathway from the extracellular environment to the cytoplasm that is additional or complimentary to endocytosis; it may also be particularly relevant to the ability of muscle to be transfected by the direct injection of DNA. These several observations strongly indicate that the function of the T-system in skeletal muscle fibers is not restricted to excitation-contraction coupling.

[Localization of acid organelles in frog skeletal muscle fibers]. / Lokalizatsiia kislykh organoidov v skeletnykh myshechnykh voloknakh liagushki.

By means of fluorescent and phase-contrast microscopy the distribution of acid membrane organelles in normal and vacuolated frog skeletal muscle fibers has been studied. The vacuolation of the T-system was produced by loading and subsequent removal of glycerol (80-110 mM), or it appeared as a result of Zenker's necrosis. Acridine orange (AO) was used as a marker for acid intracellular compartments. AO accumulated in granules localized near the nuclear poles (more seldom around the nucleus)' and in the intermyofibrillar spaces. Typically the AO granules make up short longitudinal chains or regular pairs, where the distances between neighboring granules are short-dated to sarcomere lengths. Almost all granules emit in red, but about one third of them simultaneously emit in green, which is characteristic of AO monomers. In the vicinity of necrotic boundary or under the influence of brefeldin A, a green component of fluorescence appears in most granules. Treatment with monensin leads to granule disappearance. Vacuoles accompanying the glycerol treatment or developing of necrosis do not accumulate AO and exert no effect on the localization of AO-granules. The nature of cellular organelles accumulating AO in skeletal muscle fibers is discussed.

[State of the contractile apparatus during the development of a pathological process in the muscles. II. The effect of Ca2+ on the morphology of spreading necrosis caused by UV light]. / Izuchenie sostoianiia sokratitel'nogo apparata pri razvitii patologicheskogo protsessa v myshtsakh. II. Vliianie Ca2+ na morfologiiu rasprostraniaiushchegosia nekroza, vyzvannogo UF svetom.

Using phase-contrast technique and electron microscopy, a study was made of morphological changes of contractile system of striated muscle fibre during the spreading necrosis caused by ultraviolet light damage. It has been shown that the degree of manifestation of destructive changes in the contractile system depends upon Ca2+-ion concentration. The ultrastructural study of the damage region, under condition of muscle fibre stretching, made it possible to reveal the initial stages of formation of this pathological process. A possible contribution of intracellular membranous structures in spreading the destructive process along the muscle fibre is discussed.

[Sarcomere supercontraction during Zenker's necrosis of skeletal muscle fibers]. / Sverkhsokrashchenie sarkomerov vo vremia tsenkerovskogo nekroza skeletnykh myshechnykh volokon.

The ultrastructure and localization of supercontracted sarcomeres during the spreading (Zenker's) necrosis of twitch and tonic frog fibres and the fast- and slow-twitch rat fibres are described. The formation of supercontracted sarcomeres proceeds mainly by the sliding mechanism. Contracted bands are characterized by homogenization of myosin and actin filaments and by the disappearance of Z-band structures. In the calcium-free medium some myosin filaments pass through the whole depth of the contracted band or are seen bending and deviating backwards upon approaching the contracted band. In calcium-free solution supercontracted sarcomeres often form narrow strips (2-5 sarcomere long), crossing the fibres. The transverse elignment of sarcomeres is typical of supercontracted portion of fibres.