Disuse-Induced Muscle Fatigue: Facts and Assumptions.

Skeletal muscle unloading occurs during a wide range of conditions, from space flight to bed rest. The unloaded muscle undergoes negative functional changes, which include increased fatigue. The mechanisms of unloading-induced fatigue are far from complete understanding and cannot be explained by muscle atrophy only. In this review, we summarize the data concerning unloading-induced fatigue in different muscles and different unloading models and provide several potential mechanisms of unloading-induced fatigue based on recent experimental data. The unloading-induced changes leading to increased fatigue include both neurobiological and intramuscular processes. The development of intramuscular fatigue seems to be mainly contributed by the transformation of soleus muscle fibers from a fatigue-resistant, "oxidative" "slow" phenotype to a "fast" "glycolytic" one. This process includes slow-to-fast fiber-type shift and mitochondrial density decline, as well as the disruption of activating signaling interconnections between slow-type myosin expression and mitochondrial biogenesis. A vast pool of relevant literature suggests that these events are triggered by the inactivation of muscle fibers in the early stages of muscle unloading, leading to the accumulation of high-energy phosphates and calcium ions in the myoplasm, as well as NO decrease. Disturbance of these secondary messengers leads to structural changes in muscles that, in turn, cause increased fatigue.

Six-day dry immersion leads to downregulation of slow-fiber type and mitochondria-related genes expression.

The soleus muscle in humans is responsible for maintaining an upright posture and participating in walking and running. Under muscle disuse, it undergoes molecular signaling changes that result in altered force and work capacity. The triggering mechanisms and pathways of these changes are not yet fully understood. In this article, we aimed to detect the molecular pathways that are involved in the unloading-induced alterations in the human soleus muscle under 6-days of dry immersion. A 6-day dry immersion led to the downregulation of mitochondrial biogenesis and dynamics markers, upregulation of calcium-dependent CaMK II phosphorylation, enhanced PGC1α promoter region methylation, and altered muscle micro-RNA expression, without affecting p-AMPK content or fiber-type transformation.NEW & NOTEWORTHY Dry immersion dysregulates mitochondrial genes expression, affects mi-RNA expression and PGC1 promoter methylation.

The Effect of SERCA Activation on Functional Characteristics and Signaling of Rat Soleus Muscle upon 7 Days of Unloading.

Skeletal muscle abnormalities and atrophy during unloading are accompanied by the accumulation of excess calcium in the sarcoplasm. We hypothesized that calcium accumulation may occur, among other mechanisms, due to the inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity. Consequently, the use of the SERCA activator will reduce the level of calcium in the sarcoplasm and prevent the negative consequences of muscle unloading. Wistar rats were randomly assigned into one of three groups (eight rats per group): control rats with placebo (C), 7 days of unloading/hindlimb suspension with placebo (7HS), and 7 days of unloading treated with SERCA activator CDN1163 (7HSC). After seven days of unloading the soleus muscle, the 7HS group displayed increased fatigue in the ex vivo test, a significant increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein, slow-type myosin mRNA, and the percentage of slow-type muscle fibers. All of these changes were prevented in the 7HSC group. Moreover, treatment with CDN1163 blocked a decrease in the phosphorylation of p70S6k, an increase in eEF2 phosphorylation, and an increase in MuRF-1 mRNA expression. Nevertheless, there were no differences in the degree of fast and slow muscle fiber atrophy between the 7HS and 7HSC groups. Conclusion: SERCA activation during 7 days of unloading prevented an increase in soleus fatigue, the decrease of slow-type myosin, mitochondrial markers, and markers of calcium homeostasis but had no effect on muscle atrophy.

A Prochlorperazine-Induced Decrease in Autonomous Muscle Activity during Hindlimb Unloading Is Accompanied by Preserved Slow Myosin mRNA Expression.

Skeletal muscle disuse leads to pathological muscle activity as well as to slow-to-fast fiber-type transformation. Fast-type fibers are more fatigable than slow-type, so this transformation leads to a decline in muscle function. Prochlorperazine injections previously were shown to attenuate autonomous rat soleus muscle electrical activity under unloading conditions. In this study, we found that prochlorperazine blocks slow-to-fast fiber-type transformation in disused skeletal muscles of rats, possibly through affecting calcium and ROS-related signaling.

Inhibition of mTORC1 differentially affects ribosome biogenesis in rat soleus muscle at the early and later stages of hindlimb unloading.

Prolonged inactivity of skeletal muscles due to limb immobilization, bedrest, and exposure to microgravity results in a significant muscle atrophy. Inactivity-induced muscle atrophy is caused by a downregulation of protein synthesis (PS) and increased proteolysis. Mechanistic target of rapamycin complex 1 (mTORC1) is considered to be one of the main regulators of translational capacity (quantity of ribosomes), a key determinant of PS. Using a specific mTORC1 inhibitor (rapamycin) we aimed to determine if mTORC1 activity would influence ribosome biogenesis in rat soleus muscle at both early and later stages of mechanical unloading. Wistar rats were subjected to 1- and 7-day hindlimb suspension (HS) with and without rapamycin injections (1.5 mg/kg) and compared to weight-bearing control animals. The key markers of ribosome biogenesis were assessed by RT-PCR or agarose gel electrophoresis. The rate of PS was measured by SUnSET method. Both 1-day and 7-day HS resulted in a significant downregulation of ribosome biogenesis markers (c-Myc, 47S pre-rRNA, 18S + 28S rRNAs) and the rate of PS. Rapamycin administration during 1-day HS fully prevented a decrease in 47S pre-rRNA expression and amount of 18S + 28S rRNAs (without affecting c-Myc mRNA expression) and partially attenuated a decline in PS. Rapamycin treatment during 7-day HS significantly decreased p70S6K phosphorylation but failed to rescue a reduction in both the markers of ribosome biogenesis and the rate of PS. All together, our results suggest that mTORC1 inhibition at the initial (1 day), but not later (7 days) stage of HS can be beneficial for the maintenance of translational capacity (ribosome biogenesis) and the rate of PS in rat soleus muscle.

Roles of ATP and SERCA in the Regulation of Calcium Turnover in Unloaded Skeletal Muscles: Current View and Future Directions.

A decrease in skeletal muscle contractile activity or its complete cessation (muscle unloading or disuse) leads to muscle fibers' atrophy and to alterations in muscle performance. These changes negatively affect the quality of life of people who, for one reason or another, are forced to face a limitation of physical activity. One of the key regulatory events leading to the muscle disuse-induced changes is an impairment of calcium homeostasis, which leads to the excessive accumulation of calcium ions in the sarcoplasm. This review aimed to analyze the triggering mechanisms of calcium homeostasis impairment (including those associated with the accumulation of high-energy phosphates) under various types of muscle unloading. Here we proposed a hypothesis about the regulatory mechanisms of SERCA and IP3 receptors activity during muscle unloading, and about the contribution of these mechanisms to the excessive calcium ion myoplasmic accumulation and gene transcription regulation via excitation-transcription coupling.

The Role of Glycogen Synthase Kinase-3 in the Regulation of Ribosome Biogenesis in Rat Soleus Muscle under Disuse Conditions.

It is well-established that prolonged exposure to real or simulated microgravity/disuse conditions results in a significant reduction in the rate of muscle protein synthesis (PS) and loss of muscle mass. Muscle protein synthesis is largely dependent upon translational capacity (ribosome content), the regulation of which is poorly explored under conditions of mechanical unloading. Glycogen synthase kinase-3 (GSK-3) (a negative regulator of PS) is known to be activated in rat soleus muscle under unloading conditions. We hypothesized that inhibition of GSK-3 activity under disuse conditions (hindlimb suspension, HS) would reduce disuse-induced downregulation of ribosome biogenesis in rat soleus muscle. Wistar rats were randomly divided into four groups: (1) vivarium control (C), (2) vivarium control + daily injections (4 mg/kg) of AR-A014418 (GSK-3 inhibitor) for 7 days, (3) 7-day HS, (4) 7-day HS + daily injections (4 mg/kg) of AR-A014418. GSK-3beta and glycogen synthase 1 (GS-1) phosphorylation levels were measured by Western-blotting. The key markers of ribosome biogenesis were assessed via agarose gel-electrophoresis and RT-PCR. The rate of muscle PS was assessed by puromycin-based SUnSET method. As expected, 7-day HS resulted in a significant decrease in the inhibitory Ser9 GSK-3beta phosphorylation and an increase in GS-1 (Ser641) phosphorylation compared to the C group. Treatment of rats with GSK-3 inhibitor prevented HS-induced increase in GS1 (Ser641) phosphorylation, which was indicative of GSK-3 inhibition. Administration of GSK-3 inhibitor partly attenuated disuse-induced downregulation of c-Myc expression as well as decreases in the levels of 45S pre-rRNA and 18S + 28S rRNAs. These AR-A014418-induced alterations in the markers of ribosome biogenesis were paralleled with partial prevention of a decrease in the rate of muscle PS. Thus, inhibition of GSK-3 during 7-day HS is able to partially attenuate the reductions in translational capacity and the rate of PS in rat soleus muscle.

Metformin Attenuates Slow-to-Fast Fiber Shift and Proteolysis Markers Increase in Rat Soleus after 7 Days of Rat Hindlimb Unloading.

Muscle unloading leads to signaling alterations that cause muscle atrophy and weakness. The cellular energy sensor AMPK can regulate myofiber-type shift, calcium-dependent signaling and ubiquitin-proteasome system markers. We hypothesized that the prevention of p-AMPK downregulation during the first week of muscle unloading would impede atrophy development and the slow-to-fast shift of soleus muscle fibers, and the aim of the study was to test this hypothesis. Thirty-two male Wistar rats were randomly assigned to four groups: placebo control (C), control rats treated with metformin (C + M), 7 days of hindlimb suspension (HS) + placebo (7HS), and 7 days of HS + metformin administration (7HS + M). In the soleus of the 7HS rats, we detected a slow-to-fast fiber-type shift as well as a significant downregulation of MEF-2D and p300 in the nuclei. In the 7HS group, we also found decreases in p-ACC (AMPK target) protein level and in the expression of E3 ubiquitin ligases and p-CaMK II protein level vs. the C group. The 7-day metformin treatment for soleus muscle unloading (1) prevented slow-to-fast fiber-type shift; (2) counteracted changes in the p-ACC protein level; (3) hindered changes in the nuclear protein level of the slow myosin expression activators MEF-2D and p300, but did not affect NFATc1 signaling; and (4) attenuated the unloading-induced upregulation of MuRF-1, atrogin-1, ubiquitin and myostatin mRNA expression, but did not prevent soleus muscle atrophy. Thus, metformin treatment during muscle disuse could be useful to prevent the decrease in the percentage of slow-type fatigue-resistant muscle fibers.

NOS-dependent effects of plantar mechanical stimulation on mechanical characteristics and cytoskeletal proteins in rat soleus muscle during hindlimb suspension.

The study was aimed at investigating the mechanisms and structures which determine mechanical properties of skeletal muscles under gravitational unloading and plantar mechanical stimulation (PMS). We hypothesized that PMS would increase NO production and prevent an unloading-induced reduction in skeletal muscle passive stiffness. Wistar rats were hindlimb suspended and subjected to a daily PMS and one group of stimulated animals was also treated with nitric oxide synthase (NOS) inhibitor (L-NAME). Animals received mechanical stimulation of the feet for 4 h a day throughout 7-day hindlimb suspension (HS) according to a scheme that mimics the normal walking of the animal. Seven-day HS led to a significant reduction in soleus muscle weight by 25%. However, PMS did not prevent the atrophic effect induced by HS. Gravitational unloading led to a significant decrease in maximum isometric force and passive stiffness by 38% and 31%, respectively. The use of PMS prevented a decrease in the maximum isometric strength of the soleus muscle. At the same time, the passive stiffness of the soleus in the PMS group significantly exceeded the control values by 40%. L-NAME (NOS inhibitor) administration attenuated the effect of PMS on passive stiffness and maximum force of the soleus muscle. The content of the studied cytoskeletal proteins (α-actinin-2, α-actinin-3, desmin, titin, nebulin) decreased after 7-day HS, but this decrease was successfully prevented by PMS in a NOS-dependent manner. We also observed significant decreases in mRNA expression levels of α-actinin-2, desmin, and titin after HS, which was prevented by PMS. The study also revealed a significant NOS-dependent effect of PMS on the content of collagen-1a, but not collagen-3a. Thus, PMS during mechanical unloading is able to maintain soleus muscle passive tension and force as well as mRNA transcription and protein contents of cytoskeletal proteins in a NOS-dependent manner.

The Role of GSK-3ß in the Regulation of Protein Turnover, Myosin Phenotype, and Oxidative Capacity in Skeletal Muscle under Disuse Conditions.

Skeletal muscles, being one of the most abundant tissues in the body, are involved in many vital processes, such as locomotion, posture maintenance, respiration, glucose homeostasis, etc. Hence, the maintenance of skeletal muscle mass is crucial for overall health, prevention of various diseases, and contributes to an individual's quality of life. Prolonged muscle inactivity/disuse (due to limb immobilization, mechanical ventilation, bedrest, spaceflight) represents one of the typical causes, leading to the loss of muscle mass and function. This disuse-induced muscle loss primarily results from repressed protein synthesis and increased proteolysis. Further, prolonged disuse results in slow-to-fast fiber-type transition, mitochondrial dysfunction and reduced oxidative capacity. Glycogen synthase kinase 3ß (GSK-3ß) is a key enzyme standing at the crossroads of various signaling pathways regulating a wide range of cellular processes. This review discusses various important roles of GSK-3ß in the regulation of protein turnover, myosin phenotype, and oxidative capacity in skeletal muscles under disuse/unloading conditions and subsequent recovery. According to its vital functions, GSK-3ß may represent a perspective therapeutic target in the treatment of muscle wasting induced by chronic disuse, aging, and a number of diseases.

Plantar Mechanical Stimulation Maintains Slow Myosin Expression in Disused Rat Soleus Muscle via NO-Dependent Signaling.

It was observed that gravitational unloading during space missions and simulated microgravity in ground-based studies leads to both transformation of slow-twitch muscle fibers into fast-twitch fibers and to the elimination of support afferentation, leading to the "switching-off" of postural muscle motor units electrical activity. In recent years, plantar mechanical stimulation (PMS) has been found to maintain the neuromuscular activity of the hindlimb muscles. Nitric oxide (NO) was shown to be one of the mediators of muscle fiber activity, which can also promote slow-type myosin expression. We hypothesized that applying PMS during rat hindlimb unloading would lead to NO production upregulation and prevention of the unloading-induced slow-to-fast fiber-type shift in rat soleus muscles. To test this hypothesis, Wistar rats were hindlimb suspended and subjected to daily PMS, and one group of PMS-subjected animals was also treated with nitric oxide synthase inhibitor (L-NAME). We discovered that PMS led to sustained NO level in soleus muscles of the suspended animals, and NOS inhibitor administration blocked this effect, as well as the positive effects of PMS on myosin I and IIa mRNA transcription and slow-to-fast fiber-type ratio during rat hindlimb unloading. The results of the study indicate that NOS activity is necessary for the PMS-mediated prevention of slow-to-fast fiber-type shift and myosin I and IIa mRNA transcription decreases during rat hindlimb unloading.

NO-Dependent Mechanisms of Myosin Heavy Chain Transcription Regulation in Rat Soleus Muscle After 7-Days Hindlimb Unloading.

It is known that nitric oxide (NO) may affect myosin heavy chain (MyHC) isoform mRNA transcription in skeletal muscles. The content of NO in soleus muscles decreases during rat hindlimb unloading as well as slow MyHC mRNA transcription. We aimed to detect which signaling pathways are involved in NO-dependent prevention of hindlimb-suspension (HS)-induced changes in MyHCs' expression pattern. Male Wistar rats were divided into four groups: cage control group (C), hindlimb suspended for 7 days (7HS), hindlimb suspended for 7 days with L-arginine administration (7HS+A) (500 mg/kg body mass), and hindlimb suspended for 7 days with both L-arginine (500 mg/kg) and NO-synthase inhibitor L-NAME administration (50 mg/kg) (7HS+A+N). L-arginine treatment during 7 days of rat HS prevented HS-induced NO content decrease and slow MyHC mRNA transcription decrease and attenuated fast MyHC IIb mRNA transcription increase; it also prevented NFATc1 nuclear content decrease, calsarcin-2 expression increase, and GSK-3ß Ser 9 phosphorylation decrease. Moreover, L-arginine administration prevented the HS-induced myh7b and PGC1α mRNAs content decreases and slow-type genes repressor SOX6 mRNA transcription increase. All these slow fiber-type protective effects of L-arginine were blocked in HS+A+N group, indicating that these effects were NO-dependent. Thus, NO decrease prevention during HS restores calcineurin/NFATc1 and myh7b/SOX6 signaling.

Effects of Plantar Mechanical Stimulation on Anabolic and Catabolic Signaling in Rat Postural Muscle Under Short-Term Simulated Gravitational Unloading.

It is known that plantar mechanical stimulation (PMS) is able to attenuate unloading-induced skeletal muscle atrophy and impaired muscle function. However, molecular mechanisms underlying the effect of PMS on skeletal muscle during unloading remain undefined. The aim of the study was to evaluate the effects of PMS on anabolic and catabolic signaling pathways in rat soleus at the early stages of mechanical unloading. Wistar rats were randomly assigned to ambulatory control, hindlimb suspension (HS) for 1 or 3 days, and HS for 1 or 3 days with PMS. The key anabolic and catabolic markers were assessed by western blotting and RT-PCR. Protein synthesis (PS) rate was estimated using SUnSET technique. PMS attenuated a 1-day HS-induced decrease in 4E-BP1, GSK-3ß, and AMPK phosphorylation. PMS also partially prevented a decrease in PS, phosphorylation of GSK-3ß, nNOS, and an increase in eEF2 phosphorylation after 3-day HS. PMS during 1- and 3-day HS prevented MuRF-1, but not MAFbx, upregulation but did not affect markers of ribosome biogenesis (18S + 28S rRNA, c-myc) as well as AKT phosphorylation. Thus, PMS during 3-day HS partially prevented a decrease in the global rate of PS in rat soleus muscle, which was accompanied by attenuation of MuRF-1 mRNA expression as well as changes in GSK-3ß, nNOS, and eEF2 phosphorylation.