[Translocation of MuRF isoforms to nuclei in postural muscle during gravitational unloading].

Ubiquitin-proteasomal proteolytic pathway is one of the key signaling pathways determining protein degradation in muscle fibers. Among the E3 ubiquitin ligases, rate limiting enzymes of the ubiquitin-proteasomal pathway, the most interesting ones are the MuRF isoforms: MuRF-1 and MuRF-2. There are some pieces of evidence that these enzymes are also involved in the regulation of gene expression in skeletal muscle under some specific conditions (i. e. muscle disuse). We supposed that it was disuse that brought about to altered localization of MuRFs in postural muscle fibers and their translocation to nuclei. In the study using the conventional simulation model of the gravitational unloading (rat hindlimb suspension according to Ilyin and Novikov modified by Morey-Holton) we found that from the 3rd day till 14th day of unloading the content of MuRF-1 and MuRF-2 in the nuclear fraction 4-5 fold increased in unloaded soleus as compared to the control values. These data obtained by means of electrophoresis and western blot of the nuclear fraction of rat soleus were confirmed in the immunohistochemical study of co-localization of MuRF-1 and MuRF-2 antibodies and DAPI nuclear stain on transverse frozen sections of soleus muscle. Thus in the present study we observed the phenomenon of MuRF isoforms accumulation in nuclei of soleus muscle fibers during simulated gravitational unloading.

[Effect of branched-chain amino acids mixture on repair of atrophied rats' skeletal muscles].

Loss of muscle mass (atrophy) is a regular consequence of gravitational unloading. In hypogravity, muscle atrophy ensues from depression of protein synthesis and increase of its degradation. Both of these processes underlie also atrophy caused by muscle immobilization, cancer cachexia, cardiomyopathy and age-related sarcopenia. At the same time, atrophy of skeletal muscles due to chronic alcohol intoxication is the outcome of protein synthesis depression primarily. The study was part of a program aimed to develop a technique for atrophied muscle recovery by activation of anabolic signal pathways. A similar approach is applied to correct hypogravity-induced atrophy; however, analysis of effectiveness of this approach to the treatment of alcoholic muscle atrophy will be more "pure", as proteolytic processes in intoxicated animals are on the level typical of the intact ones. The issue in hand was whether injection of branched-chain amino acids (BCAA) would facilitate repair of m. gastrocnemius medialis in rats in the period of 30 days after 16-week alcoholic intoxication. BCAA was shown to enhance regain size by fast muscular fibers, full recovery of phosphorylated p90RSK concentrations without consequential effects on general proliferative processes and myonuclear number in rat's m. gastrocnemius medialis.

[Effects of recombinant mechano-dependent growth factor against the background of chronic alcoholisation in rats].

In alcoholised rats, proliferation of satellite cells consistently decreased as well as the number of myonuclei, while phosphorylation of p90RSK became reduced. The mechano-growth factor abministration increased the proliferate activity of the myogenic precursors and restored the myonuclei pool. Phosphorylation of p90RSK increased too.

[Insulin-like growth factor-I splice-variants expression and myosatellite cells in the rat m. soleus under unloading and passive stretch].

Hindlimb unloading produces atrophy and suppresses proliferative processes in postural muscles. Muscle stretch applied simultaneously with hindlimb suspension is known to prevent soleus muscle atrophy. In the rat experiments, we assessed the number of M-cadherin (satellite cell marker molecule)-labeled cells per one myofiber cross-section. After 2-week hindlimb suspension the number of labeled cells decreases by 33% as compared to the control group. The amount of labeled cells was 2.5-fold greater in passive stretch group in comparison with the hindlimb suspended animals and 1.7-fold greater as compared to the control group. We suppose that that proliferation of satellite cells with subsequent incorporation of their nuclei in myofiber is sufficient for increasing the protein synthesis. The insulin-like growth factor (IGF-I) is involved in regulation of protein turnover and exerts potent mitogenic and differentiating effects. We investigated the level of IGF-I expression in soleus muscle tissue after 14 day hindlimb suspension with stretch and observed no changes as compared to the control or 14 day hindlimb suspended group. We conclude that the muscle IGF-I and satellite cells incorporation do not make essential contribution to passive stretch preventive action under the conditions of simulated weightlessness.

[Insulin-like growth factor 1 and the key markers of proteolysis during the acute period of readaptation of the muscle atrophied as a result of unloading].

It has been shown that, after prolonged disuse, the accumulation of muscle mass and the recovery of soleus fibers volume are caused by water accumulation rather than protein synthesis intensification. At the same time, expression rate of the main markers of the activity of ubiquitin-proteasome system remained increased on the 3rd day of reloading and decreased to the control by the 7th day. Both the quantity of the insulin-like growth factor 1 and the number of satellite cells fused with muscle fibers and of myonuclei began to increase only on the 7th day of reloading. The data obtained evidenced a significant inertness of the postural muscle during its adaptation to the load (normal gravity) after prolonged disuse.

Skeletal muscle activity and the fate of myonuclei.

Adult skeletal muscle fiber is a symplast multinuclear structure developed in ontogenesis by the fusion of the myoblasts (muscle progenitor cells). The nuclei of a muscle fiber (myonuclei) are those located at the periphery of fiber in the space between myofibrils and sarcolemma. In theory, a mass change in skeletal muscle during exercise or unloading may be associated with the altered myonuclear number, ratio of the transcription, and translation and proteolysis rates. Here we review the literature data related to the phenomenology and hypothetical mechanisms of the myonuclear number alterations during enhanced or reduced muscle contractile activity. In many cases (during severe muscle and systemic diseases and gravitational unloading), muscle atrophy is accompanied by a reduction in the amount of myonuclei. Such reduction is usually explained by the development of myonuclear apoptosis. A myonuclear number increase may be provided only by the satellite cell nuclei incorporation via cell fusion with the adjacent myofiber. It is believed that it is these cells which supply fiber with additional nuclei, providing postnatal growth, work hypertrophy, and repair processes. Here we discuss the possible mechanisms controlling satellite cell proliferation during exercise, functional unloading, and passive stretch.

[The cellular effects of functional unloading and passive stretch on m. soleus of dystrophin-deficient mdx mice].

Dystrophin, subsarcolemmal protein communicating muscle fiber cytoskeleton to extracellular matrix, is believed to participate in mechanical signal transduction. Recent works testify possible signaling role of this protein to prevent development ofproteolytic processes accompanying muscle fiber atrophy and to stimulate the passive stretch anabolic effects. The experiment was carried out to assess the role of dystrophin in these processes. The study was performed on two months old C57 black and mdx (dystrophin-deficient) mice. Passive stretch resulted in attenuating atrophy development in two fiber types of both C57 black and mdx mice, at the same time fiber type slow-to-fast transformation did not occur in mdx soleus. We established ablatitious effect of chronic hindlimb unloading on SC proliferative activity in soleus muscle and drastic increase of proliferation under effect of passive stretch. We observed no relationship between altered dystrophin synthesis and satellite cell proliferation activity in soleus muscle under conditions of simulated microgravity and concurrent passive stretch. It is concluded that altered dystrophin synthesis partly retarded slow myofibers atrophy and had virtually no effect on passive stretch preventive action. Thus, the hypothesis about dystrophin key role in downregulation of atrophy signaling mechanisms has not found its confirmation concerning gravitational unloading atrophy.

[The contribution of muscle progenitor cells to maintaining morphological characteristics of unweighted rat soleus muscle during passive stretch].

Skeletal muscle work hypertrophy is usually connected with muscle progenitor SC (satellite cells) activation with subsequent incorporation their nuclei into myofibers. Passive stretch of unloaded muscle was earlier established to prevent atrophic processes and be accompanied by enhanced protein synthesis. We hypothesized that elimination of SC proliferation capacity by gamma-irradiation would partly preavent stretched muscle fiber capability to maintain their size under condition of gravitational unloading. To assess the role of muscle progenitor (satellite) cells in development of passive stretch preventive effect SC proliferation was suppressed by local exposure to ionizing radiation (2500 Rad) and then subsequent hindlimb suspension or hindlimb suspension with concomitant passive stretch were carried out. Reduction of myofiber cross-sectional area and decrease in myo-nuclei number accompanying unloaded muscle atrophy were completely abolished by passive stretch both in irradiated and sham-treated animals. We concluded that satellite cells did not make essential contribution to passive stretch preventive action under condition of simulated weightlessness.

[Effects of creatine phosphokinase competitive inhibitor on system and tissue energy metabolism in rats in the norm and during unloading].

In an experiment with rats system and intracellular energy metabolism was assessed by cell reactions to chronic injection of beta-guanidine propionic acid (beta-GPA) stimulating AMP-dependent protein kinase (AMPK). Suspension was shown to inhibit the succinate dehydrogenase (SDH) activity, reduce glycogen in both types of muscle fibers, and stimulate the activity of alpha-glycerophosphate dehydrogenase (alpha-GPDH) in fast fibers. Supplementing the rat chow with beta-GPA did not modify these parameters during suspension; however, the blood urea level increased considerably in the suspended and control rats. In the controls, beta-GPA as well as suspension, stimulates growth of the aspartate aminotranspherase activity (AST) in blood. Yet, the suspension and beta-GPA injection had no additive effect. Moreover, their effects were opposite in rats subjected to suspension + beta-GPA. Glucose concentration was observed to become lowered in blood of resting rats treated with beta-GPA. This effect can be associated with a more intensive insulin-dependent glucose transport to muscles. The additional glucose, because of increased demand by fibers, underwent to oxidation and did not replenish the intracellular carbohydrate deposits These data suggest energy metabolism shifting toward activation of the processes of disintegration of substrates for energy production due to a sharp growth of energy demand.

Muscle progenitor cell proliferation during passive stretch of unweighted soleus in dystrophin deficient mice.

Dystrophin, subsarcolemmal protein communicating muscle fiber cytoskeleton to extracellular matrix, is believed to be involved in mechanical signal transduction. The experiment was carried out to assess the role of dystrophin in passive stretch-induced preventing unloaded muscle fiber atrophy and possible linkage between this protein and muscle progenitor (satellite cells) proliferation activity. The study was performed on two months old C57 black and mdx (dystrophin-deficient) mice. Passive stretch resulted in attenuating atrophy development in two fiber types of both C57 black and mdx mice. Altered dystrophin synthesis in mdx mice had virtually no effect on passive stretch preventive action. Thus the hypothesis about dystrophin key role in mediating stretch-induced hypertrophy effects didn't find its confirmation concerning gravitational unloading atrophy. Chronic hindlimb unloading downregulated SC proliferative activity in soleus muscle, passive stretch drastically increased proliferation both in C57 and mdx mice. Thus we observed no relationship between altered dystrophin synthesis and satellite cell proliferation activity in soleus muscle under conditions of simulated microgravity and concurrent passive stretch.