The role of MAP-kinase p38 in the m. soleus slow myosin mRNA transcription regulation during short-term functional unloading.

The unloading of postural muscles leads to the changes in myosins heavy chains isoforms (MyHCs) mRNAs transcription pattern, that cause severe alterations of muscle functioning. Several transcription factors such as NFATc1 and TEAD1 upregulate slow MyHC mRNA transcription, and p38 MAP kinase can phosphorylate NFAT and TEAD1, causing their inactivation. However, the role p38 MAP kinase plays in MyHCs mRNAs transcription regulation in postural soleus muscle during unloading remains unclear. We aimed to investigate whether pharmacological inhibition of p38 MAPK during rat soleus unloading would prevent the unloading-induced slow-type MyHC mRNA transcription decrease by affecting calcineurin/NFATc1 or TEAD1 signaling. Male Wistar rats were randomly assigned to three groups: cage control (C), 3-day hindlimb suspended group (3HS) and 3-day hindlimb suspended group with the daily oral supplementation of 10 mg/kg p38 MAPK inhibitor VX-745 (3HS + VX-745). 3 days of hindlimb suspension caused the significant decreases of slow MyHC and slow-tonic myh7b mRNAs transcription as well as the decrease of NFATc1-dependent MCIP1.4 mRNA transcription in rat soleus muscles compared to the cage control. P38 MAP-kinase inhibition during hindlimb suspension completely prevented slow MyHC mRNA content decrease and partially prevented slow-tonic myh7b and MCIP1.4 mRNAs transcription decreases compared to the 3HS group. We also observed NFATc1 and TEAD1 myonuclear contents increases in the 3HS + VX-745 group compared to both 3HS and C groups (p < 0.05). Therefore, we found that p38 inhibition counteracts the unloading-induced slow MyHC mRNA transcription downregulation and leads to the activation of calcineurin/NFAT signaling cascade in unloaded rat soleus muscles.

Inhibition of Histone Deacetylase 1 Prevents the Decrease in Titin (Connectin) Content and Development of Atrophy in Rat m. soleus after 3-Day Hindlimb Unloading.

We studied the effect of histone deacetylase 1 (HDAC1) inhibition on titin content and expression of TTN gene in rat m. soleus after 3-day gravitational unloading. Male Wistar rats weighing 210±10 g were randomly divided into 3 groups: control, 3-day hindlimb suspension, and 3-day hindlimb suspension and injection of HDAC1 inhibitor CI-994 (1 mg/kg/day). In hindlimb-suspended rats, the muscle weight/animal body weight ratio was reduced by 13.8% (p<0.05) in comparison with the control, which attested to the development of atrophic changes in the soleus muscle. This was associated with a decrease in the content of NT-isoform of intact titin-1 by 28.6% (p˂0.05) and an increase in TTN gene expression by 1.81 times (p˂0.05) in the soleus muscle. Inhibition of HDAC1 by CI-994 during 3-day hindlimb suspension prevented the decrease in titin content and development of atrophy in rat soleus muscle. No significant differences in the TTN gene expression from the control were found. These results can be used when finding the ways of preventing or reducing the negative changes in the muscle caused by gravitational unloading.

Inhibition of Histone Deacetylases 4 and 5 Reduces Titin Proteolysis and Prevents Reduction of TTN Gene Expression in Atrophied Rat Soleus Muscle after Seven-Day Hindlimb Unloading.

The effect of HDACs 4 and 5 on the level of atrophy, calpain-1 and titin content, and TTN gene expression in rat soleus after 7-day gravitational unloading (hindlimb suspension model) was studied. The development of atrophic changes induced by gravitational unloading in rat soleus was accompanied by an increase in the calpain-1 content, an increase in titin proteolysis, and a decrease in the mRNA content of the protein. Inhibition of HDACs 4 and 5 did not eliminate the development of unloading-induced atrophy but significantly prevented proteolysis of titin and the decrease in the TTN gene expression.

Signaling targets of alcoholic intoxication in human skeletal muscle.

Intracellular signaling pathways were investigated in skeletal muscle cells at the early stages of alcohol addiction manifestations. No muscle fiber atrophy was observed in m. vastus lateralis of male patients. No significant changes in the signaling mechanisms that control protein degradation were detected as well. However, the concentration of the insulin-like growth factor (IGF-1) in blood plasma as well as the content of markers of intracellular signaling pathways regulating protein synthesis were significantly reduced compared to the control group.

[Physiological basis of alcohol-induced skeletal muscle injury].

Alcohol-induced muscle damage (AIMD) - an umbrella term that includes all forms of alcoholic myopathy developing in acute or chronic alcohol intoxication. The most common form of destruction of skeletal muscle in alcoholism is a chronic alcoholic myopathy, which develops independently of other manifestations of alcoholism, such as polyneuropathy, malabsorption syndrome, liver damage, but can be combined with them. The basis of the destruction of skeletal muscle in chronic AIPM is atrophy of muscle fibers. Mainly affects muscle fiber type II with less destruction of type ! fibers. Currently, the pathogenesis of chronic alcoholic myopathy is studied. The imbalance of protein synthesis and proteolysisand increased apoptosis rate are discussed.

[The Development of Clinical and Morphological Manifestations of Chronic Alcoholic Myopathy in Men with Prolonged Alcohol Intoxication].

Chronic alcoholic myopathy occurs in 40-60% of patients who abuse alcohol, and is accompanied by decreased performance, proximal paresis and atrophy of skeletal muscles. However, it is unknown what is important in the development of the disease: duration of alcohol abuse, or the dose of ethanol consumed. Unknown dynamics of the pathological process in skeletal muscle. We examined male patients identified with alcoholic myopathy and without it, evaluated the duration of alcohol abuse, intake of ethanol, morphological characteristics m.quadriceps vastus lateralis and the content of IGF-1 in plasma. It has been shown that chronic alcoholic myopathy develops after 10 years of alcohol abuse; proximal paresis is observed only in patients with atrophy of muscle fibers, thus there is a transformation of myosin phenotype from slow to fast. The decrease IGF-1 in plasma detected at the early stages of the Church, including in patients without clinical manifestations of proximal paresis and morphological signs of atrophy of muscle fibers.

Role of NO-synthase in regulation of protein metabolism of stretched rat m. soleus muscle during functional unloading.

Gravitational unloading causes atrophy of muscle fibers and can lead to destruction of cytoskeletal and contractile proteins. Along with the atrophic changes, unloaded muscle frequently demonstrates significant shifts in the ratio of muscle fibers expressing fast and slow myosin heavy chain isoforms. Stretching of the m. soleus during hindlimb suspension prevents its atrophy. We supposed that neuronal NO-synthase (NOS) (which is attached to membrane dystrophin-sarcoglycan complex) can contribute to maintenance of protein metabolism in the muscle and prevent its atrophy when m. soleus is stretched. To test this hypothesis, we used Wistar rats (56 animals) in experiments with hindlimb suspension during 14 days. The group of hindlimb suspended rats with stretched m. soleus was injected with L-NAME to block NOS activity. We found that m. soleus mass and its protein content in hindlimb-suspended rats with stretched m. soleus were preserved due to prevention of protein degradation. NOS is involved in maintenance of expression of some muscle proteins. Proliferation of satellite cells in stretched m. soleus may be due to nNOS activity, but maintenance of muscle mass upon stretching is regulated not by NOS alone.

Protective effect of L-Arginine administration on proteins of unloaded m. soleus.

Cytoskeletal and contractile proteins degenerate during functional unloading of muscle. The ratio of myosin heavy chain (MHC) expression changes simultaneously. We have supposed that NO can be a signal molecule related to the regulation of protein metabolism upon muscle unloading. To test this hypothesis, Wistar rats underwent functional unloading for 14 days without and with peroral administration of L-arginine (500 mg/kg) as NO precursor. Significant decreases in m. soleus mass, NO, nNOS, dystrophin, Hsp90, p-S6K, and type I MHC mRNA contents were found in the group of animals with unloading without preparation compared to those in control and in the group with unloading and administration of L-arginine; at the same time, increased contents of atrogin-1/MAFbx and MuRF-1 (p < 0.05) were found. No difference in the IGF-1 mRNA content between all three groups was found. Atrophy was significantly less pronounced in the group with unloading and L-arginine administration compared to that without the amino acid, and no destruction of cytoskeletal proteins was observed. We conclude that administration of L-arginine upon functional unloading decreases the extent of m. soleus atrophy, prevents the decrease in it of type I MHC mRNA, and blocks destructive changes in some cytoskeletal proteins. Such effect can be due to the absence of increase in this group of the content of some ubiquitin ligases and decreased intensity of the p70S6 kinase synthesis marker.

Calcium-dependent signaling mechanisms and soleus fiber remodeling under gravitational unloading.

The decrease in postural muscle fiber size, diminishing of their contractile properties, slow-to-fast shift in myosin heavy chain expression pattern are known to be the main consequences of gravitational unloading. The Ca(2+) role in these processes has been studied for about 20 years. Ingalls et al. [J Appl Physiol 87(1):382-390, 1999] found the resting Ca(2+) level increase in soleus fibers of hindlimb unloaded mice. Results obtained in our laboratory showed that systemic or local application of nifedipine (L-type Ca(2+) channels' blocker) prevents Ca(2+) accumulation in fibers. Thus, activation of dihydropyridine calcium channels can be supposed to promote resting Ca(2+) loading under disuse. So, calcium-dependent signaling pathways may play an important role in the development of some key events observed under unloading. Since 90th the increased activities of Ca(2+)-dependent proteases (calpains) were considered as the crucial effect of hypogravity-induced muscle atrophy, which was proved later. We observed maintenance of titin and nebulin relative content in soleus muscle under unloading combined with Ca(2+) chelators administration. Nifedipine administration was shown to considerably restrict the slow-to-fast transition of myosin heavy chains (MHC) under unloading (at the RNA level and at the protein level as well). To clarify the role of calcineurin/NFAT signaling system in MHC pattern transition under unloading, we blocked this pathway by cyclosporine A application. Hereby, we demonstrated that calcineurin/NFAT pathway possesses a stabilizing function counteracting the myosin phenotype transformation under gravitational unloading.

The role of L-type calcium channels in the accumulation of Ca2+ in soleus muscle fibers in the rat and changes in the ratio of myosin and serca isoforms in conditions of gravitational unloading.

Gravitational unloading is known to produce changes in the expression of a number of contractile and regulatory proteins in the soleus muscle. This applies particularly to isoforms of myosin heavy chains (MHC) and SERCA sarcoplasmic reticulum calcium pumps. Unloading increases the resting levels of extracellular calcium in soleus muscle fibers. The present study addresses verification of the hypothesis that changes in the expression of MHC and SERCA isoforms in gravitational unloading are linked with the accumulation of calcium ions in the myoplasm of muscle fibers. It is suggested that specific blockade of L-type calcium channels using nifedipine decreases the myoplasmic calcium ion concentration, thus preventing the development of changes in the expression of MHC and SERCA isoforms. A total of 36 male Wistar rats were divided into three groups: a control group, an unloading group using the Morley-Holton soleus muscle functional unloading model, and an unloading + nifedipine group, where animals received daily nifedipine (7 mg/kg/day) with their drinking water on the background of suspension. The results showed that blockade of L-type calcium channels on the background of gravitational unloading significantly decreased the extent of calcium ion accumulation in the myoplasm of soleus muscle fibers, which partly prevented the transformation of muscle fibers (in relation to the fast and slow isoforms of MHC and SERCA) to the rapid type. There was no nuclear translocation of the greater part of transcription factor NFATc1, as seen on unloading.

Effects of weightlessness and movement restriction on the structure and metabolism of the soleus muscle in monkeys after space flight.

After humans and animals have been in conditions of real and modeled weightlessness, the most marked changes are seen in the "slow" tonic muscles, particularly soleus. Studies of the effects of weightlessness and movement restriction on the soleus muscle in monkeys demonstrated significant reductions in the sizes of slow and rapid fibers due mainly to the actions of real weightlessness (rather than movement restriction in the space capsule). Protein loss in soleus muscle fibers in monkeys following space flight was more marked than loss of other components, including water. The level of atrophy of soleus muscle fibers in these conditions was greater than the decrease in the number of capillaries. Succinate dehydrogenase activity in soleus muscle fibers decreased proportionally to the reduction in fiber size.

Characteristic of changes in the structure and metabolism of the vastus lateralis muscles in monkeys after space flight.

Monkeys subjected to space flight were found to have significant decreases in the sizes of slow and rapid fibers in the vastus lateralis muscle, due not only to weightlessness but also, to some extent, to restriction of movement activity within the capsule. The quantity of total protein in muscle fibers did not decrease. The respiratory peak in the pool of vastus lateralis muscle fibers decreased after space flight, as did the activity of oxidative enzymes (particularly in rapid fibers of the vastus lateralis muscle).

Effects of deafferentation on the size and myosin phenotype of muscle fibers on stretching of the rat soleus muscle in conditions of gravitational unloading.

The aim of the present work was to assess the contributions of the reflex and local components to preventing decreases in the size and changes in the ratio of fibers containing the slow and fast isoforms of myosin heavy chains during chronic stretching of a postural muscle in rats in conditions of gravitational unloading. A unilateral surgical deafferentation method was used. The results demonstrated that deafferentation of the hindlimb had no effect on preventing reductions in muscle fiber size in conditions of chronic muscle stretching in conditions of gravitational unloading. The results obtained from these experiments did not support the hypothesis that the predominant contribution to preventing the development of atrophic changes comes from activation of muscle afferents in chronic stretching of the unloaded muscle. Deafferentation of both suspended animals and those with normal motor activity led to increases in the proportion of soleus muscle fibers containing the slow isoforms of myosin heavy chain.

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.

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.

Influence of single hindlimb support on fiber characteristics of unloaded skeletal muscle.

The unweighting and/or immobilization in the shortened position leads to muscle atrophy, slow-to-fast transformation and biochemical alterations in muscle fibers. This process could be prevented by the stretching or loaded contraction. On the other hand Kozlovskaya et al. demonstrated triggering of postural synergies due to using artificial support simulator in spaceflight. The study was aimed to test if the plantar support stimulation might prevent muscle atrophy, slow-to-fast transformation of muscle fibers and oxidative potential alteration during gravitational unloading.