Calpain-dependent degradation of cytoskeletal proteins as a key mechanism for a reduction in intrinsic passive stiffness of unloaded rat postural muscle.

In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14 days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14 days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical analysis and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle was significantly reduced after 7- and 14-day HS compared to the control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and -3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content, and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle.

Regulation of Protein Synthesis in Inactivated Skeletal Muscle: Signal Inputs, Protein Kinase Cascades, and Ribosome Biogenesis.

Disuse atrophy of skeletal muscles is characterized by a significant decrease in the mass and size of muscle fibers. Disuse atrophy develops as a result of prolonged reduction in the muscle functional activity caused by bed rest, limb immobilization, and real or simulated microgravity. Disuse atrophy is associated with the downregulation of protein biosynthesis and simultaneous activation of protein degradation. This review is focused on the key molecular mechanisms regulating the rate of protein synthesis in mammalian skeletal muscles during functional unloading.

Early Deсline in Rat Soleus Passive Tension with Hindlimb Unloading: Inactivation of Cross-bridges or Activation of Calpains?

The study was aimed at testing the hypotheses about the role of cross-bridges and calpains in reduction of rat soleus passive tension under conditions of hindlimb unloading. For this purpose, we used an inhibitor of µ-calpain PD 150606 as well as a blocker of actomyosin interaction (blebbistatin). It was found for the first time that a decrease in passive tension of rat soleus after 3-day hindlimb unloading is associated with the activity of µ-calpain and does not depend on the processes of cross-bridges formation.

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.

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.

[EFFECT OF SHORT-TERM DRY IMMERSION ON PROTEOLYTIC SIGNALING IN HUMAN SOLEUS MUSCLE].

The signaling processes initiating proteolytic events in m. soleus of humans during short-term exposure in the non-weight bearing conditions were analyzed. Dry immersion (DI) was used to induce weight deprivation over 3 days. Western blotting was used to define the IRS-1 content, total and phosphorylated neuronal NO-synthase (nNOS), AMP-activated protein kinase (AMPK) that control the anabolic and catabolic pathways, and concentrations of cytoskeletal protein desmin and Ca²âº-activated protease calpin. Already on day-3 of DI calpain-dependent proteolysis manifests itself by reductions in both the total content and level of nNOS phosphorilation. Moreover, AMPK phosphorilation was decreased drastically.

Human muscle signaling responses to 3-day head-out dry immersion.

To date little is known about catabolic NO-dependent signaling systems in human skeletal muscle during early stages of gravitational unloading. The goal of the study was to analyze signaling pathways that determine the initial development of proteolytic events in human soleus muscle during short-term gravitational unloading (simulated microgravity). Gravitational unloading was simulated by 3-day head-out dry immersion. Before and after the immersion the samples of soleus muscle were taken under local anesthesia, using biopsy technique. The content of desmin, IRS-1, phospho-AMPK, total and phospho-nNOS in soleus of 6 healthy men was determined using Western-blotting before and after the dry-immersion. Three days of the dry immersion resulted in a significant decrease in desmin, phospho-nNOS and phospho-AMPK as compared to the pre-immersion values. The results of the study suggest that proteolytic processes in human soleus at the early stage of gravitational unloading are associated with inactivation of nNOS. Reduction in AMPK phosphorylation could serve as a trigger event for the development of primary atrophic changes in skeletal muscle.

[Effect of 30-day space flight and subsequent readaptation on the signaling processes in m. longissimus dorsi of mice].

Some steps of anabolic and catabolic signaling pathways were investigated in postural/tonic m. longissimus dorsi of mice following the 30-day orbital flight of biosatellite "Bion-M1" and 8-day recovery. Western blotting was used for determining insulin receptor substrate 1 (IRS-1) and AMR-activated protein kinase (AMPK) involved in reciprocal regulation of anabolic and catabolic pathways, as well as E3-ligase MURF-1, and elongation factor eEF2. Functioning of the IGF-1-dependent IRS-1 signaling pathway was activated in the recovery period only. Though the content of ubiquitinligase MURF-1 showed an increase after flight, on completion of the recovery period it did not exceed the pre-flight level unambiguously.

Structure and functional characteristics of rat's left ventricle cardiomyocytes under antiorthostatic suspension of various duration and subsequent reloading.

The goal of the research was to identify the structural and functional characteristics of the rat's left ventricle under antiorthostatic suspension within 1, 3, 7 and 14 days, and subsequent 3 and 7-day reloading after a 14-day suspension. The transversal stiffness of the cardiomyocyte has been determined by the atomic force microscopy, cell respiration--by polarography and proteins content--by Western blotting. Stiffness of the cortical cytoskeleton increases as soon as one day after the suspension and increases up to the 14th day, and starts decreasing during reloading, reaching the control level after 7 days. The stiffness of the contractile apparatus and the intensity of cell respiration also increases. The content of non-muscle isoforms of actin in the cytoplasmic fraction of proteins does not change during the whole experiment, as does not the beta-actin content in the membrane fraction. The content of gamma-actin in the membrane fraction correlates with the change in the transversal stiffness of the cortical cytoskeleton. Increased content of alpha-actinin-1 and alpha-actinin-4 in the membrane fraction of proteins during the suspension is consistent with increased gamma-actin content there. The opposite direction of change of alpha-actinin-1 and alpha-actinin-4 content suggests their involvement into the signal pathways.

[Parameters of fibers cell respiration and desmin content in rat soleus muscle at early stages of gravitational unloading].

The aim of the work was to study the parameters of fibers cell respiration and desmin content in Wistar rat soleus muscle after 1, 3, 7 and 14 days of gravitational unloading. Gravitational unloading was simulated by antiorthostatic hindlimb suspension. The parameters of cell respiration were determined using the polarography, and desmin content was assessed by means of Western blotting. The results showed that the intensity of cell respiration is reduced after three days of gravitational unloading, reaches a minimum level after seven days and slightly increases by the fourteenth day of hindlimb unloading, as well as the content of desmin, which, however, to the fourteenth day returns to the control level. Taking into account that mitochondrial function depends on the state of cytoskeleton the data allow us to assume that early reduction of the intensity of cell respiration under unloading could be caused by degradation of the protein desmin that determines intracellular localization of mitochondria.

[Desmin content and cell breathing in rats' m. soleus fibers after 3- and 7-day recovery from 14-day suspension].

Cell breathing and relative desmin content were investigated in m. soleus fibers of rats after 3 and 7 days of recovery from 14-day suspension according to the Morey - Holton modification of the Ilyin - Novikov procedure. The cell breathing parameters were determined with the help of polarography; desmin content was measured using the western-blot technique. The results evidence that cell breathing intensity subsided during 14 days of gravitational unloading, reached minimum after 3 days of recovery and regained baseline values after 7 days of recovery. Post suspension desmin content did not differ from control values, made a significant drop in 3 days and returned to baseline values in 7 days of recovery. These data suggest an interdependence between cell breathing and desmin content in m. soleus fibers of rats as during gravitation unloading, so in the period of recovery.

[SIGNALING PATHWAYS REGULATING PROTEIN SYNTHESIS IN RAT SOLEUS MUSCLE DURING EARLY STAGES OF HINDLIMB UNLOADING].

The impact of mechanical unloading (hindlimb suspension for 1-, 3- and 7 days) on the protein synthesis intensity and as well as intracellular signaling pathways controlling mRNA translation was investigated. The content of the key signaling molecules of the various anabolic signaling pathways was determined by Western-blotting. The rate of protein synthesis was assessed using in vivo SUnSET technique. Hindlimb suspension (HS) within 24 hours resulted in a significant (p < 0.05) decrease in p-4E-BP 1 content and increase in p-p70s6k content in soleus muscle. Follo- wing three days of HS the content of p-AKT and p-90RSK1 was decreased (p <0.05). After 7 days of HS phosphorylation level of AKT and GSK-3beta was significantly reduced (p < 0.05) compared to the control group. We also observed a significant decrease in the amount of 28S rRNA in soleus following 3 and 7 days of HS. The rate of protein synthesis after 3 and 7 days of HS was decreased (p < 0.05) in comparison with the basal level. Our data suggest that the decrease in the protein synthesis rate in rat soleus during early stages of simulated microgravity could be associated with the impaired ribosome biogenesis as well as reduced activity of the mTORC1-independent signaling pathways.