I. B. Kozlovskaya

New titin isoforms in skeletal muscles of mammals.

The existence of a third type of filaments was assumed back in the 1950s. However, it was not until the late 1970s to early 1980s that two research groups found two bands in the patterns of macroporous (2 − 3%) sodium dodecylsulfate (SDS) gel electrophoresis of striated muscles corresponding to proteins with molecular weights more than 1000 kDa [3, 4]. These proteins were called titin 1 ( α -connectin) and titin 2 ( β -connectin, a proteolytic fragment of titin 1). This was the beginning of intense studies on the structure and properties of titin. To date, the sarcomere location of titin filaments has been determined. Titin is tightly bound to myosin-containing (thick) filaments in the Adisk. Titin molecules run freely through the I-disk region and form an elastic joint between the ends of thick filaments and the Z-membrane. Amino acid sequence analysis demonstrated that more than 90% of a titin molecule is accounted for by fibronectin-like (FN) and immunoglobulin-like (Ig) domains forming a β -folded structure. Unique sequences with no homologs known thus far account for the remaining 10% [5].

Polymorphism of skeletal muscle titin under the extreme conditions of hibernation and microgravity: the diagnostic value of titin isoforms for choosing approaches to the correction of "hypogravity muscle syndrome".

88 Studies on the systemic and cellular mechanisms of the organization of the tonic function and adaptive transformations in the motor control systems in response to changing environment [1] have yielded evidence for the independence of the tonic muscle system of mammals, which is closed and has its own structures and mechanisms at all levels of motor control from receptors (generating afferent impulses from zones normally in contact with the support) to effectors (tonic muscle fibers). This conclusion was confirmed by the results of morphological and functional studies on the muscle apparatus and the patterns of the nervous control of muscle plasticity. The studies demonstrated marked atrophic changes in the tonic muscles of the skeletal musculus soleus in humans and rats under the conditions of support unload: the shift of the myosin phenotype towards an increased expression of the fast isoforms of heavy chains and a decrease in the amounts of titin and nebulin, sarcomere cytoskeletal proteins [2–4].

The contents of desmin and α-actinin-1 in the human soleus muscle after seven-day “dry” immersion

Measurement of the transversal stiffness in various regions of the muscle fiber allows the state of local structures to be assessed at the level of both the con tractile apparatus and the sarcolemma [6]. Earlier studies of this type on the effect of seven day “dry” immersion on the state of the human soleus muscle fibers [7] have demonstrated that the changes in the contractile apparatus can be prevented by a plantar mechanical stimulator compensating for gravitational unloading, developed by Kozlovskaya et al. [10]. Elec tromyostimulation is another yet least efficient method.

Isoform composition of proteins of myosin filaments in cardiac muscle of Mongolian gerbils (Meriones unguiculatus) after space flight.

14 The results obtained earlier within the Basic Research Program of the Division of Biological Sci ences of the Russian Academy of Sciences “Physio logical Mechanisms of Regulation of the Internal Environment and Organization of Behavior of Living Systems” revealed the key role of support afferent pulses in the functional organization of the muscle tone in mammals [1, 2]. The absence of a support in real or simulated microgravity results in the develop ment of the “muscle hypogravity syndrome” in skele tal postural muscles, which manifests itself in the development of muscle atrophy accompanied by a reduction in the muscle fiber volume and destruction of the myofibrillar apparatus as well as a decrease in the tone, endurance level, and general working capac ity of muscles [1]. The studies performed within the above mentioned program showed that a decrease in the content of proteins of the sarcomeric cytoskeleton (N2A form of titin, nebulin, and X protein), shift of the myosin phenotype towards an increase in the amount of fast isoforms of myosin heavy chains, and a decrease in the number of isoforms of myosin light chains 2 in human and rat m. soleus in microgravity may contribute to the development of the “muscle hypogravity syndrome” [2–7]. The development of this syndrome in human and rat m. soleus is also accompanied by the degradation of the high molecu lar weight form of titin, which, according to our elec trophoretic and immunological data [7], may be the intact N2A isoform of this protein. Such studies have not yet been performed in car diac muscle proteins. The goal of this work was to study the quantitative changes occurring in the intact titin isoforms, N2B and N2BA titin forms, α and β myosin heavy chains, myosin light chains 1 and 2, and in the myosin binding protein C (MyBP C) in the cardiac muscle of Mongolian gerbils (Meriones unguiculatus) under conditions of 12 day space flight.

Immunohistochemical confirmation of localization of the ribosomal protein L26 in the terminal buttons of rat motor axon.

313 Normal functioning of motoneurons is impossible without a reliable system of synthesis and delivery of various protein molecules (channels, pumps, receptors, enzymes, etc.) to target sites. In axon terminals (motor nerve endings), a special role is fulfilled by exocytotic proteins, which ensure the release of mediator quanta [2]. It is believed that axonal proteins are synthesized in the motoneuron perikaryon and then are distributed over the axon by the mechanism of anterograde axonal transport. It was shown that many proteins are transferred by the mechanism of slow axonal transport, the rate of which is on the order of several millimeters per day. The distance to which relevant compounds are transported varies significantly, and the transport of molecules by the mechanism of slow axonal transport may take several days and even weeks, with allowance for the fact that the length of human neural outgrowths may by more than 1 m [1]. The results of morphological studies demonstrating that ribosomes, cell structures responsible for protein synthesis, are located in the perikaryon are usually adduced as convincing evidence for protein synthesis in the neuron body [5]. However, in terms of this hypothesis, it is difficult to explain how short-lived protein molecules can reach the motor nerve ending and fulfill their functions. Data confirming the existence of intraaxonal protein synthesis (the presence of mRNA, ribosomes, and de novo synthesized protein molecules) have been published recently [3, 4]. However, data on protein synthesis in the terminal buttons of motoneuron axons are missing. In this study, performed at the electron-microscopic level using antibodies against ribosomal proteins, we verified the hypothesis on the presence of the protein synthesis machinery in rat motoneuron terminal. Experiments were performed with mature male Wistar rats weighing 260–280 g. A fragment of the diaphragm of anesthetized animals was excised at the phrenic nerve input under a microsection microscope. To perform immunohistochemical reaction, the material was fixed as described earlier [6] with some modifications. The fixative contained 2.0% paraformaldehyde and 0.5% glutaraldehyde prepared in 0.1 M phosphate buffer (pH 7.3). Then, the samples were postfixed in 0.5% OsO 4 and then dehydrated in a series of alcohols and acetone. To maintain antigenic properties, the tissue was impregnated in the LR White resin (Medium Grade Acrylic Resin, TED PELLA Inc.). To study the nerve ending ultrastructure, the tissue was impregnated with the epoxy resin Epon by the conventional procedure. Immunohistochemical reaction was performed in ultrathin sections mounted on nickel grids. Ribosomes were localized using rabbit polyclonal antibodies against the ribosomal protein L26 (Abcam, Great Britain) in a dilution of 1 : 200. The secondary antibodies bound to 5-nm colloidal gold particles (TED PELLA) were used in a dilution of 1 : 50. The control sections were treated only with the secondary antibodies. The samples were examined under a JEOL 1200EX transmission electron microscope at a working voltage of 80 kV. Motor axon terminal buttons with a characteristic structure—aggregations of a large number of mitochondria and synaptic vesicles in the vicinity of active zones as well as postsynaptic folds of muscle fiber plasma membrane—can be seen on the electron-microscopic images of tissue fixed by the conventional procedure, which is used for studying the animal cell ultrastructure (Fig. 1a). PHYSIOLOGY

Changes in the titin isoform composition in the cardiac muscle of spontaneously hypertensive rats and its restoration after a course of low-intensity red-orange irradiation.

320 Titin is a giant elastic sarcomeric protein of the skeletal and heart muscles in vertebrates. Titin molecules with a length of over 1 μ m and molecular weight of 3000–3700 kDa span half sarcomere from the M-line to Z-disc, forming the third filament system in myofibrils. The N-termini of titin molecules from the adjacent sarcomeres overlap at the Z-disc, while the C-termini overlap at the M-line. At the A-band of sarcomere, titin is bound to myosin filaments. Titin spans freely the I-band, connecting the ends of myosin filaments with the Z-disc [1]. There are several isoforms of titin with isovariants differing in the length of the elastic part of the molecule in the I-disc of sarcomere [2]. In the heart muscle, titin is expressed in two isoforms, namely, short N2B isoform with a molecular weight of ~3000 kDa and long N2BA isoform with molecular weights of its variants amounting to 3200–3350 kDa. Titin is a multifunctional protein. It forms the backbone for assembly of myosin filaments and the sarcomere during myofibrillogenesis, regulates the length of myosin filaments, and is responsible for their arrangement in the center of the sarcomere [1, 3]. Titin is involved in the regulation of muscle contraction, changing the enzymatic properties of myosin and its Ca 2+ sensitivity [4, 5]. The data on titin localization and its binding to other proteins suggest that it is involved in the intracellular signaling. It is demonstrated that the N-terminal domains of titin interact in the sarcomere Z-disc with the protein telethonin, which is bound to minK β -subunit of the stretch-activated potassium channel. It is assumed that the passive tension developed by titin during sarcomere stretch can influence the activity of ion channels [1]. Presumably, the titin kinase domain, located in the vicinity of its C-end, is involved in myofibrillogenesis, since it phosphorylates the protein telethonin, localized to the Z-disc, at the early stages of myocyte differentiation. In mature muscles, titin kinase activity can modulate the muscle contraction via phosphorylation of myosin light chains. It was assumed that the titin kinase domain can be involved in the regulation of the muscle gene expression and protein turnover in the sarcomere [1]. In addition, elastic titin molecules can function as the sensors of sarcomere extension and transmit the information about changes in its length to myosin and actin filaments, thereby regulating their interaction [7].

Eukaryotic elongation factor 2 kinase activation in M. soleus under 14-day hindlimb unloading of rats

Functional unloading of m. soleus of male Wistar rats was found to cause a reduction in protein synthesis. The level of phosphorylation of the translation elongation factor 2 (eEF2) and the eEF2 kinase (eEF2k) activity in m. soleus after 14 days of unloading were assessed. Rats were divided into the control group (C) and the group with hindlimb unloading for 14 days (HU14). The level of eEF2 phosphorylation in group HU14 was 80%, whereas in the control is was 40%. The indices of eEF2k expression and protein content in group HU14 increased compared to group C.

Genomic analysis of mouse lumbar spinal cord after 30-day space flight on biosatellite Bion-M1.

1 One of the adverse factors affecting human in space is microgravity. This is not surprising, because the evo lution of all living systems proceeded under conditions of the Earth’s gravity. The negative effect of micro gravity develops in space flight but is clinically expressed after the return of astronauts to the Earth, under normal gravity conditions. All body systems adapted to the flight conditions, where the weight is virtually absent, are unable to function normally under conditions of the Earth’s gravity for a long time.

Effect of hindlimb unloading on myelinated fibers in the mouse lumbar spinal cord

266 The evolution of living systems occurred under the conditions of earth gravity. Prolonged absence of grav ity disturbs the function of almost all organs and sys tems of living organisms at the molecular, cellular and tissue levels. Skeletal musculature is most sensitive to the state of weightlessness. For example, as soon as the first day under the conditions of microgravity, the dis turbances that underlie the hypogravitational motor syndrome are developed in the skeletal muscles main taining posture [1–4]. In consideration of the fact that motor neurons are directly involved in the control of functional and morphological characteristics of skele tal muscles [5], their key role in the pathogenesis of hypogravitational motor syndrome seems to be quite likely. However, the effects of microgravity on the spinal cord as a whole and the structural and functional char acteristics of motor neurons, glial cells, and elements of microvasculature remain insufficiently explored.

Phosphorylation of elongation factor and its kinase expression in Rat m. soleus under early stage of hindlimb unloading.

283 In microgravity, tonic postural muscles of animals and humans undergo atrophy. Grounddbased simulaa tions of the effects of microgravity [1] revealed not only a significant increase in the expression of gene of various proteolytic systems [2], but also a decrease in the activity of complexes regulating protein synthesis, such as mTOR [3] and p70S6Kl [4], in the antigravity soleus muscle (m. soleus). The intensity of protein synthesis is determined by a group of factors regulating the processes of initiation and elongation of the polypeptide chain on the ribosome. In this study, we considered the mechanisms of signaling factors reguu lating protein synthesis elongation in rat m. soleus in hypogravity unloading during a shorttterm (33day) antiorthostatic suspension. Protein synthesis elongaa tion depends directly on the activity of the elongation factor eEF2. It was shown that a 25% decrease in the eEF2 phosphorylation level in skeletal muscles leads to an increase in protein synthesis by 40% [5]. This elongation factor is blocked by its specific kinase eEF2K [6]. The activity of Ca 2+ /calmodulinndepenn dent eEF2 kinase is affected by excess Ca 2+ accumuu lated during muscle contraction [7]. In addition, cAMPPdependent kinase can lead to eEF2K activaa tion [8]. The activity of eEF2K is blocked by kinases p90 (RSKl) and p70S6K, which, in turn, are regulated by ERK1/2 kinases and mTORCl complex, respecc tively [9]. After a 33day hindlimb suspension, the activity of p70S6K remained almost unchanged, whereas the activity of p90 (RSK1) significantly decreased [10]. Experiments with shorttterm gravitaa tional unload make it possible to detect early changes caused by hypogravity in the mechanisms of intracell lular signaling and can help identify the triggering mechanisms of muscle atrophy. Male 33monthhold Wistar rats were divided into two groups: control (group C, n = 10) and hindlimbb unloaded for 3 days according to the Novikov–Ilyin technique with Morey–Holton modification (3HS group, n = 8). After the experiment, the animals were sacrificed under avertin anesthesia, and m. soleus was isolated from both hindlimbs. Both muscles were weighed on an analytical balance. Then, m. soleus of one leg was frozen immediately in liquid nitrogen and stored at –85°C, and the muscle of the contralateral hindlimb was used to determine the dry weight. Western blot. Samples were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Santa Cruz, United States). The homogenates were centrifuged at 12 000 g for 10 min and …