Skeletal Muscle Unloading Research Articles

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.

Are Skeletal Muscle Changes during Prolonged Space Flights Similar to Those Experienced by Frail and Sarcopenic Older Adults?

Microgravity exposure causes several physiological and psychosocial alterations that challenge astronauts' health during space flight. Notably, many of these changes are mostly related to physical inactivity influencing different functional systems and organ biology, in particular the musculoskeletal system, dramatically resulting in aging-like phenotypes, such as those occurring in older persons on Earth. In this sense, sarcopenia, a syndrome characterized by the loss in muscle mass and strength due to skeletal muscle unloading, is undoubtedly one of the most critical aging-like adverse effects of microgravity and a prevalent problem in the geriatric population, still awaiting effective countermeasures. Therefore, there is an urgent demand to identify clinically relevant biological markers and to underline molecular mechanisms behind these effects that are still poorly understood. From this perspective, a lesson from Geroscience may help tailor interventions to counteract the adverse effects of microgravity. For instance, decades of studies in the field have demonstrated that in the older people, the clinical picture of sarcopenia remarkably overlaps (from a clinical and biological point of view) with that of frailty, primarily when referred to the physical function domain. Based on this premise, here we provide a deeper understanding of the biological mechanisms of sarcopenia and frailty, which in aging are often considered together, and how these converge with those observed in astronauts after space flight.

Metformin attenuates an increase of calcium-dependent and ubiquitin-proteasome markers in unloaded muscle.

Current study tested a hypothesis that during skeletal muscle unloading, calcium-dependent signaling pathways, markers of protein synthesis, and expression of E3 ubiquitin ligases can be regulated by metformin. Thirty-two male Wistar rats were randomly assigned into one of four groups: nontreated control (3C), control rats treated with metformin (3CM), 3 days of unloading/hindlimb suspension with placebo (3HS), and 3 days of unloading treated with metformin (3HSM). In soleus muscle of HS group level of phospho-AMP-activated protein kinase (p-AMPK) was decreased by 46% while ATP content was increased by 49% when compared with the control group. There was an increase of the level of phospho-CaMK II (483%) and an upregulation of Calcineurin (CaN), SERCA2a, and Calpain-1 mRNA expression (87%, 41%, and 62%, respectively, P < 0.05) in the HS group relative to the control. HS group also had increased mRNA expression of MuRF1, MAFbx, and ubiquitin (167%, 146%, and 191%, respectively, P < 0.05) when compared with the control soleus muscle. Metformin treatment impeded unloading-induced changes in soleus muscle. In conclusion, metformin treatment during 3 days of soleus muscle unloading: 1) prevented the decrease of p-AMPK and increase of ATP content; 2) affected regulation of calcium-dependent signaling pathways via level of CaMK II phosphorylation or CaMK II, CaN, SERCA2a, and Calpain-1 mRNA expression; 3) attenuated an increase in the expression of critical markers of ubiquitin-proteasome pathways MuRF1, MAFbx, and ubiquitin while not affecting the unloading-induced increase of ULK-1 marker of autophagic/lysosomal pathway.NEW & NOTEWORTHY Current study for the first time tested the hypothesis that during 3 days of soleus muscle unloading, calcium-dependent signaling pathways, markers of protein synthesis, and the expression of E3 ubiquitin ligases can be regulated by metformin. Treatment with metformin during unloading: prevented the decrease of p-AMPK and increase of ATP content, affected regulation of calcium-dependent signaling pathways, and attenuated an increase of critical markers of ubiquitin-proteasome pathways. Nevertheless, metformin treatment has not prevented soleus muscle atrophy.

The muscle proteome reflects changes in mitochondrial function, cellular stress and proteolysis after 14 days of unilateral lower limb immobilization in active young men.

Skeletal muscle unloading due to joint immobilization induces muscle atrophy, which has primarily been attributed to reductions in protein synthesis in humans. However, no study has evaluated the skeletal muscle proteome response to limb immobilization using SWATH proteomic methods. This study characterized the shifts in individual muscle protein abundance and corresponding gene sets after 3 and 14 d of unilateral lower limb immobilization in otherwise healthy young men. Eighteen male participants (25.4 ±5.5 y, 81.2 ±11.6 kg) underwent 14 d of unilateral knee-brace immobilization with dietary provision and following four-weeks of training to standardise acute training history. Participant phenotype was characterized before and after 14 days of immobilization, and muscle biopsies were obtained from the vastus lateralis at baseline (pre-immobilization) and at 3 and 14 d of immobilization for analysis by SWATH-MS and subsequent gene-set enrichment analysis (GSEA). Immobilization reduced vastus group cross sectional area (-9.6 ±4.6%, P <0.0001), immobilized leg lean mass (-3.3 ±3.9%, P = 0.002), unilateral 3-repetition maximum leg press (-15.6 ±9.2%, P <0.0001), and maximal oxygen uptake (-2.9 ±5.2%, P = 0.044). SWATH analyses consistently identified 2281 proteins. Compared to baseline, two and 99 proteins were differentially expressed (FDR <0.05) after 3 and 14 d of immobilization, respectively. After 14 d of immobilization, 322 biological processes were different to baseline (FDR <0.05, P <0.001). Most (77%) biological processes were positively enriched and characterized by cellular stress, targeted proteolysis, and protein-DNA complex modifications. In contrast, mitochondrial organization and energy metabolism were negatively enriched processes. This study is the first to use data independent proteomics and GSEA to show that unilateral lower limb immobilization evokes mitochondrial dysfunction, cellular stress, and proteolysis. Through GSEA and network mapping, we identify 27 hub proteins as potential protein/gene candidates for further exploration.

Effect of enhanced muscle tone on the expression of atrogenes and cytoskeletal proteins during postural muscle unloading

Skeletal muscle unloading leads to the decreased electrical activity and decline of muscle tone. Aims: Current study evaluated the effect of muscle tone preservation achieved by tetanus toxin (TeNT) treatment on signaling pathways regulating atrophic processes during unloading. Main methods: Four groups of rats were used: non-treated control (C), control rats with TeNT administration (CT), 7 days of unloading/hindlimb suspension with placebo (HS), and 7 days of unloading with TeNT administration (HST). Key findings: Absolute and relative force of tetanic contractions was decreased by 65% in soleus muscle of HS rats when compared with C. Treatment with TeNT significantly lessened force decline in soleus muscle of HST rats when compared with HS. TeNT administration increased myosin heavy chain I beta (MyHC Iβ) expression in CT rats and prevented MyHC Iβ loss in HST group when compared with C rats. Desmin content was lower by 31.4% (p < 0.05) in HS group when compared with HST. Calpain-1 expression was increased in HS group when compared with C, CT and HST. There was a decrease in p-p70S6K content (41%, p < 0,05) and an increase in p-eEF2 content (77%, p < 0,05) in HS group when compared with C, while there were no significant differences in the content of these proteins between HST, CT and C groups. Significance: Treatment with TeNT significantly diminished unloading-induced decline of soleus muscle mass and mechanical properties and affected the regulation of MyHC Iβ expression. These effects are mediated by signaling pathways regulating protein synthesis and degradation.

Role of Pannexin 1 ATP-Permeable Channels in the Regulation of Signaling Pathways during Skeletal Muscle Unloading.

Skeletal muscle unloading results in atrophy. We hypothesized that pannexin 1 ATP-permeable channel (PANX1) is involved in the response of muscle to unloading. We tested this hypothesis by blocking PANX1, which regulates efflux of ATP from the cytoplasm. Rats were divided into six groups (eight rats each): non-treated control for 1 and 3 days of the experiments (1C and 3C, respectively), 1 and 3 days of hindlimb suspension (HS) with placebo (1H and 3H, respectively), and 1 and 3 days of HS with PANX1 inhibitor probenecid (PRB; 1HP and 3HP, respectively). When compared with 3C group there was a significant increase in ATP in soleus muscle of 3H and 3HP groups (32 and 51%, respectively, p < 0.05). When compared with 3H group, 3HP group had: (1) lower mRNA expression of E3 ligases MuRF1 and MAFbx (by 50 and 38% respectively, p < 0.05) and MYOG (by 34%, p < 0.05); (2) higher phosphorylation of p70S6k and p90RSK (by 51 and 35% respectively, p < 0.05); (3) lower levels of phosphorylated eEF2 (by 157%, p < 0.05); (4) higher level of phosphorylated GSK3β (by 189%, p < 0.05). In conclusion, PANX1 ATP-permeable channels are involved in the regulation of muscle atrophic processes by modulating expression of E3 ligases, and protein translation and elongation processes during unloading.

The Role of Histone Deacetylases I and IIa (HDAC1, HDAC4/5) and the MAPK38 Signaling Pathway in the Regulation of Atrophic Processes under Skeletal Muscle Unloading

The Role of Histone Deacetylases I and IIa (HDAC1, HDAC4/5) and the MAPK38 Signaling Pathway in the Regulation of Atrophic Processes under Skeletal Muscle Unloading

Роль гистондеацетилаз I и IIa (HDAC1, HDAC4/5) и сигнального пути MAPK38 в регуляции атрофических процессов при функциональной разгрузке скелетных мышц

Various forms of muscle unloading can be found in patients with prolonged bed rest, with strokes and spinal lesions, during muscle immobilization in traumatology, in zero gravity, etc. During unloading, basically, postural muscles (for example, soleus) are affected. The rearrangement of skeletal muscles during unloading is based on their atrophy due to an increase in proteolysis and a decrease in the intensity of protein synthesis [1, 2]. The review is devoted to the study of the histone deacetylases I and IIa (HDAC1, HDAC 4/5) role, as well as the MAPK38 signaling pathway in the activation of transcription factors FOXO and myogenin, which are involved in the expression of E3 ubiquitin ligases genes atrogin-1 and MuRF-1 under skeletal muscle unloading.

Nox2 signaling and muscle fiber remodeling are attenuated by losartan administration during skeletal muscle unloading.

Reduced mechanical loading results in atrophy of skeletal muscle fibers. Increased reactive oxygen species (ROS) are causal in sarcolemmal dislocation of nNOS and FoxO3a activation. The Nox2 isoform of NADPH oxidase and mitochondria release ROS during disuse in skeletal muscle. Activation of the angiotensin II type 1 receptor (AT1R) can elicit Nox2 complex formation. The AT1R blocker losartan was used to test the hypothesis that AT1R activation drives Nox2 assembly, nNOS dislocation, FoxO3a activation, and thus alterations in morphology in the unloaded rat soleus. Male Fischer 344 rats were divided into four groups: ambulatory control (CON), ambulatory + losartan (40 mg kg−1 day−1) (CONL), 7 days of tail‐traction hindlimb unloading (HU), and HU + losartan (HUL). Losartan attenuated unloading‐induced loss of muscle fiber cross‐sectional area (CSA) and fiber‐type shift. Losartan mitigated unloading‐induced elevation of ROS levels and upregulation of Nox2. Furthermore, AT1R blockade abrogated nNOS dislocation away from the sarcolemma and elevation of nuclear FoxO3a. We conclude that AT1R blockade attenuates disuse remodeling by inhibiting Nox2, thereby lessening nNOS dislocation and activation of FoxO3a.

RNA sequencing (RNA-seq) analysis of gene expression provides new insights into hindlimb unloading-induced skeletal muscle atrophy.

BackgroundWeightlessness-induced skeletal muscle atrophy, accompanied by complex biochemical and physiological changes, has potentially damaged consequences. However, there is still an insufficient effective strategy to treat skeletal muscle atrophy. Therefore, exploring the molecular mechanisms regulating skeletal muscle atrophy and effective protection is necessary.MethodsRNA sequencing (RNA-seq) analysis was used to detect differentially expressed genes (DEGs) in the soleus muscle at 12, 24, 36 hours, three days, and seven days after hindlimb unloading in rats. Pearson correlation heatmaps and principal component analysis (PCA) were applied to analyze DEGs’ expression profiles. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for cluster analysis of DEGs. Ingenuity pathway analysis (IPA) was used to analyze specific biological processes further.ResultsAt different time points (12, 24, 36 hours, three days, seven days) after hindlimb unloading, the expression levels of 712, 1,109, 1,433, 1,162, and 1,182 genes in rat soleus muscle were upregulated, respectively, whereas the expression levels of 1,186, 1,324, 1,632, 1,446, and 1,596 genes were downregulated, respectively. PCA revealed that rat soleus muscle showed three different transcriptional phases within seven days after hindlimb unloading. KEGG and GO annotation indicated that the first transcriptional phase primarily involved the activation of stress responses, including oxidative stress, and the inhibition of cell proliferation and angiogenesis; the second transcriptional phase primarily involved the activation of proteolytic systems and, to a certain degree, inflammatory responses; and the third transcriptional phase primarily involved extensive activation of the proteolytic system, significant inhibition of energy metabolism, and activation of the aging process and slow-to-fast muscle conversion.ConclusionsDifferent physiological processes in rat skeletal muscles were activated sequentially after unloading. From these activated biological processes, the three transcriptional phases after skeletal muscle unloading can be successively defined as the stress response phase, the atrophic initiation phase, and the atrophic phase. Our study not only helps in the understanding of the molecular mechanisms underlying weightlessness-induced muscle atrophy but may also provide an important time window for the treatment and prevention of weightlessness-induced muscle atrophy.

Muscle unloading: A comparison between spaceflight and ground-based models.

Prolonged unloading of skeletal muscle, a common outcome of events such as spaceflight, bed rest and hindlimb unloading, can result in extensive metabolic, structural and functional changes in muscle fibres. With advancement in investigations of cellular and molecular mechanisms, understanding of disuse muscle atrophy has significantly increased. However, substantial gaps exist in our understanding of the processes dictating muscle plasticity during unloading, which prevent us from developing effective interventions to combat muscle loss. This review aims to update the status of knowledge and underlying mechanisms leading to cellular and molecular changes in skeletal muscle during unloading. We have also discussed advances in the understanding of contractile dysfunction during spaceflights and in ground-based models of muscle unloading. Additionally, we have elaborated on potential therapeutic interventions that show promising results in boosting muscle mass and strength during mechanical unloading. Finally, we have identified key gaps in our knowledge as well as possible research direction for the future.

Changes in Membrane Ceramide Pools in Rat Soleus Muscle in Response to Short-Term Disuse.

Lipid raft disruption is an early event during skeletal muscle unloading. Ceramide (Cer) serves as a signaling lipid that can contribute to lipid raft disturbance and muscle atrophy. Using biochemical and fluorescent approaches, the distribution of Cer and related molecules in the rat soleus muscle subjected to 12 h of hindlimb suspension (HS) was studied. HS led to upregulation of TNFα receptor 1 (TNFR1), Cer-producing enzymes, and acid and neutral sphingomyelinase (SMase) in detergent-resistant membranes (lipid rafts), which was accompanied by an increase in Cer and a decrease in sphingomyelin in this membrane fraction. Fluorescent labeling indicated increased Cer in the sarcoplasm as well as the junctional (synaptic) and extrajunctional compartments of the suspended muscles. Also, a loss of membrane asymmetry (a hallmark of membrane disturbance) was induced by HS. Pretreatment with clomipramine, a functional inhibitor of acid SMase, counteracted HS-mediated changes in the Cer/sphingomyelin ratio and acid SMase abundance as well as suppressed Cer accumulation in the intracellular membranes of junctional and extrajunctional regions. However, the elevation of plasma membrane Cer and disturbance of the membrane asymmetry were suppressed only in the junctional compartment. We suggest that acute HS leads to TNFR1 and SMase upregulation in the lipid raft fraction and deposition of Cer throughout the sarcolemma and intracellularly. Clomipramine-mediated downregulation of acid SMase can suppress Cer accumulation in all compartments, excluding the extrajunctional plasma membrane.

Massage Protects against Disuse Atrophy in Young Adult Rats

Maintenance of skeletal muscle (SkM) mass is essential for maintaining mobility and metabolic health. SkM unloading or disuse, such as extended bed rest or limb immobilization, leads to rapid SkM wasting. Interventions to prevent disuse atrophy have had limited success. We recently found that applying massage, in the form of cyclic compressive loading (CCL), during the reloading period after hindlimb unloading, results in a greater increase in SkM protein synthesis (MPS) and SkM fiber cross‐sectional area (CSA) compared to reloading alone in 10 month old male rats. The purpose of the current investigation was to determine if CCL has similar anabolic effects when applied during normal weight bearing (WB versus WBM) as an anabolic stimulus, or during hindlimb suspension (HS versus HSM) to prevent muscle loss. We hypothesized that CCL applied during WB or HS would enhance (WBM) or protect against SkM fiber CSA loss (HSM), respectively, by increasing ribosome biogenesis and MPS. Adult (10 month) male F344/Brown Norway rats underwent either HS (n=8) or WB (n=8) for 7 days. In the treatment groups, CCL was applied directly to the gastrocnemius muscle at a load of 4.5 N and 0.5 Hz for 30 min. every other day for a total of 4 bouts during HSM (n=9) or WBM (n=9). WBM had no significant differences (NSD) in any anabolic parameters assessed compared to WB. CSA was significantly lower in HS vs WB (2373.5±267.6 vs 3796.7±80.2 mm2), with HSM significantly minimizing the loss compared to HS (2737.4±64.0 vs 2373.5±267.6 mm2). We used D2O and modeling for non‐steady state conditions to measure myofibrillar protein synthesis (Ksyn) and breakdown (Kdeg), and ribosomal biogenesis (rRNA Ksyn) and breakdown (rRNA Kdeg). HS and HSM had significant decreases in myofibrillar Ksyn vs WB (13.5±0.63 and 22.8±2.1 vs 35.5±2.3 mg/day), with HSM being significantly greater than HS. Myofibrillar Kdeg was significantly elevated in HS and HSM vs WB (.030±.001 and .030±.001 vs .018±.001 1/t), with NSD between HS and HSM. Further, in HSM only, the contralateral non‐massaged gastrocnemius also had significantly greater myofibrillar Ksyn and CSA (22.5±1.8 mg/day and 2713.5±96.5 mm2) vs HS. There were NSD in rRNA Ksyn between any group, which was despite a significant decrease in total RNA in HS and HSM vs WB (520.8±25.3 and 545.2±35.9 vs 600.5±42.0 ng/mg muscle), with NSD between HS and HSM. However, rRNA Kdeg was significantly elevated in HS and HSM vs WB (.045±.000 and .040±.000 vs .006±.000 1/t), with HSM being significantly reduced vs HS. In summary, massage in weight bearing rats did not affect any anabolic parameter assessed. In contrast, there were strong and significant effects of massage during HS (HSM) by minimizing the loss of CSA through enhancing myofibrillar protein synthesis and protecting against ribosomal breakdown vs HS alone. These findings support the highly translatable potential of CCL to protect against SkM loss during disuse by increasing MPS in either the massaged or contralateral non‐massaged limb.Support or Funding InformationThis research was supported by NIH grant R01AT009268 and R21AG042699.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PO-064 A potential role of stretch-activated channels in anabolic mechanotransduction in the atrophied rat soleus muscle

Objective Prolonged immobilization or unloading of skeletal muscle causes muscle disuse atrophy, which is characterized by a reduction in muscle cross-sectional area and compromised contractile function. To date, the mechanisms of anabolic mechanotransduction in the atrophied mammalian skeletal muscle remain poorly understood. The aim of the present study was to assess a possible role of stretch-activated ion channels (SAC) in the propagation of a mechanical signal to anabolic signaling and protein synthesis (PS) in an isolated rat soleus muscle following mechanical unloading.&#x0D; Methods The mechanical unloading was performed via hindlimb suspension (HS). Twenty-eight male Wistar rats weighing were randomly assigned to the following 4 groups (n=7/group): 1) vivarium control (C), 2) control + SAC inhibitor (gadolinium) (C+Gd3+), 3) 7-day HS (HS), 4) 7-day HS + SAC inhibitor (HS+Gd3+). Following unloading, an isolated rat soleus was placed in an organ culture medium and subjected to a bout of eccentric contractions (EC). Upon completion of the EC, muscles were collected for Western blot analyses to determine the content of the key anabolic markers. The rate of PS was measured by SUnSET technique.&#x0D; Results EC-induced increase in PS was significantly less in the HS and HS+ Gd3+ groups vs. the C group. There was no statistically significant difference between the HS and HS+ Gd3+ groups in terms of EC-induced increase in muscle PS. A decrease in EC-induced phosphorylation of p70S6K, 4E-BP1, RPS6 and GSK-3beta in the 7-day unloaded soleus treated with SAC inhibitor did not differ from that of the 7-day unloaded soleus without SAC blockade. Thus, the inhibition of SAC with gadolinium did not lead to further decline in EC-induced phosphorylation of the key anabolic markers and muscle PS.&#x0D; Conclusions The results of the study suggest that attenuation of mTORC1-signaling and PS in response to EC in unloaded soleus muscle may be associated with inactivation of SAC. The study was supported by the RFBR grant # 16-34-60055.

Alterations in mitochondrial fission, fusion, and mitophagic protein expression in the gastrocnemius of mice after a sciatic nerve transection.

Paralysis and unloading of skeletal muscle leads to a rapid loss in muscle size, function and oxidative capacity. The reduction in metabolic capability after disuse leads to dysregulation and increased breakdown of mitochondria by mitophagy. Eight-week-old C57BL/6 male mice were given a sham surgery or sciatic nerve transection. Animals were euthanized at 7, 14, 21, or 35 days postsurgery. Whole gastrocnemius muscles were isolated from the animal, weighed and used for Western blotting. Markers of mitochondrial fusion were reduced while fission proteins were elevated following a sciatic nerve transection. There were elevations in phosphorylated unc-51-like kinase 1 (ULK1S555 ) and total expression of Beclin1, and of the mitophagy markers PINK1, p62, and microtubule-associated proteins 1A/1B light chain 3b (LC3-II). Paralysis of the gastrocnemius leads to a progressive elevation in expression of mitochondrial fission and mitophagic proteins. Rehabilitative or pharmaceutical interventions to limit excess mitophagy may be effective therapies to protect paralyzed muscle mass and function. Muscle Nerve 58: 592-599, 2018.

Partial or Complete Unloading of Skeletal Muscle Leads to Specific Alterations of Anabolic Signal Transduction

Consequences of disuse atrophy of skeletal muscle observed during spaceflight on astronaut health and performance are a focal point of space research. Decrements of both muscle mass and protein synthesis rates have been observed with exposure to varying muscle loading environments (1G > partial loading > 0G), and most of the reduced muscle mass can be attributed to diminished rates of synthesis. However, specific mechanisms behind unloading-dependent reductions of protein synthesis are not well defined. PURPOSE: To determine whether or not alterations of anabolic signal transduction was responsible for the changes previously observed in fractional synthesis rates with specific gravitational loading paradigms. METHODS: Female BALB/cByJ were normalized by bodyweight and assigned to normal cage ambulation (1G), partial weight bearing suspension titrated to approximately 33% bodyweight (G/3), partial weight bearing titrated to 16% bodyweight (G/6) and full unloading of hind limbs (0G) in specially designed cages. All mice were subjected to that loading environment for 21d prior to tissue harvest, and monitored daily. Immunoblotting of the gastrocnemius (n=23) was carried out to analyze alterations of anabolic signal transduction. Although numerous signaling intermediates were assessed, the focus of this abstract will be on ribosomal protein S6 kinase (p70-S6K). This important protein has served as a marker of protein synthesis signal transduction as well as the anabolic capacity in skeletal muscle. RESULTS: Regardless of loading paradigm, no differences were detected among groups for the activation of p70-S6K (as indicated by the phospho: total protein content). Total protein content, however, was ~27% lower than control in 0G and G/6 (P=0.008) with G/3 not being different from control (P>0.05). CONCLUSION: In combination with previous data (unpublished observations), ambulation at G/3 is sufficient to maintain anabolic signaling capacity when compared to G/6 or 0G, suggesting that a threshold level of stimulus is necessary to maintain anabolic capacity in muscle. These results may have important implications towards the development of strategies designed to counter the effects of partial/complete unloading on skeletal muscle based on how the anabolic capacity of muscle is affected.

Characterization of Novel lncRNAs Transcribed Near Contractile Protein‐Coding Genes During Skeletal Muscle Unloading

Unloading of skeletal muscle induces atrophy and a shift in slow fiber‐type muscles toward a fast‐fiber phenotype. This remodeling results in altered expression of contractile protein‐coding genes. It has become recognized that long noncoding RNAs (lncRNAs) are widely expressed throughout the genome and many have been shown to play a significant role in the transcriptional regulation of protein‐coding genes. We have identified four novel lncRNAs potentially involved in contractile phenotype shifts by examining the transcriptome response to seven days of hind‐limb unloading suspension (HS) in rat soleus (SOL) muscle using next generation sequencing data (RNA‐Seq) that was processed and analyzed with the Tuxedo Suite. A normal ambulatory group of rats served as controls (CON); (N=6). We hypothesized that these unannotated lncRNAs are differentially expressed during the phenotype shift and that they may regulate proximal protein‐coding genes. Transcription of the lncRNAs was examined with RT‐PCR and their abundance was assessed in both the sense (S) and antisense (AS) orientations, relative to the coding strand of the neighboring gene, by using gene‐specific primers during the RT and assessed at intervals of 3kb or less. The expression of these transcripts were characterized in CON and HS SOL through the use of both real‐time and endpoint PCR.We have identified lncRNAs near Myosin Heavy Chain 7/Slow Type 1 (Myh7), Myosin Regulatory Light Chain 2 (Myl2), Troponin I Type 1 (Tnni1), and Troponin C Type 1 (Tnnc1). There was a 38% decrease in the expression of Myh7 mRNA (p&lt;0.05) with HS. We discovered an AS transcript overlapping Myh7 that extends to ~−19kb upstream of the Myh7 transcription start site (TSS) and was either increased (p&lt;0.05) or unchanged in HS relative to CON SOL, depending on the target site. There is also an overlapping S lncRNA, but its expression averaged only 1/10th that of the AS lncRNA. In conjunction with an 84% HS‐induced decrease in Myl2 mRNA expression we discovered a S lncRNA that extends from −13kb relative to the Myl2 TSS and extends to the Myl2 coding region. It was decreased an average of 88% with HS at each interval measured. There was a single region 9kb upstream of Myl2 where transcription of AS RNA exceeded that of S RNA. At this site there was a 1.8‐fold increase in AS RNA in HS Sol (p&lt;0.05). In regards to TnnC1, we report that there is a S lncRNA that extends ~5kb downstream from the protein‐coding region that is an average of 2.5 fold higher in HS (measured at ~1kb intervals). The expression of this lncRNA is coupled with a 4‐fold increase in Tnnc1 pre‐mRNA (p&lt;0.05) and no change in mRNA levels. Upstream of Tnni1 we found varying levels of S and AS RNA. We found AS lncRNA that is transcribed from ~1.4 kb upstream of Tnni1 to ~8kb upstream. Its expression was 2.7‐fold higher in HS relative to CON. This was coupled with a 35% decrease in Tnni1 mRNA (not significant) and 52% decrease in pre‐mRNA (p=0.05) expression in HS Sol. In summary, we characterized four novel lncRNAs that are differentially expressed during muscle disuse. Further research is needed to determine if the reported increase in AS lncRNA regulates the observed decrease in protein‐coding mRNA expression.Support or Funding InformationMercer University School of Medicine and Navicent Health Foundation

Transcriptome Analysis Reveals Regulation of Novel lncRNAs Transcribed Near Contractile Protein‐Coding Genes During Skeletal Muscle Unloading

Unloading of skeletal muscle induces atrophy and a shift in slow fiber‐type muscles toward a fast‐fiber phenotype. This remodeling results in altered expression of contractile protein‐coding genes. It has become recognized that long noncoding RNAs (lncRNAs) are widely expressed throughout the genome and many have been shown to play a significant role in the transcriptional regulation of protein‐coding genes. The purpose of this study was to examine the transcriptome response to seven days of hindlimb unloading suspension (HS) in adult rat soleus (SOL) muscle using next generation sequencing (RNA‐Seq). A normal ambulatory group of rats served as the controls (CON); (N=6). We hypothesized that unannotated lncRNAs are differentially expressed during the phenotype shift and that they may regulate protein‐coding genes. Raw RNA‐Seq data was processed and analyzed using the Tuxedo suite. Analysis of the transcriptome data led to the selection of a subset of slow‐phenotype contractile protein‐coding genes that were also associated with the differential expression of novel lncRNAs transcribed within 10kb of the coding genes. Transcription of the lncRNAs was validated with RT‐PCR and their abundance was assessed in both the sense (S) and antisense (AS) orientations, relative to the neighboring S coding gene, by using gene‐specific primers during the RT.The following selected slow‐phenotype contractile protein‐coding genes were downregulated with HS as compared to CON SOL and were associated with upregulation of an lncRNA in the AS orientation: Troponin I Type 1 (TNNI1), Troponin C Type 1 (TNNC1), Myosin Heavy Chain 7/slow type 1 (MYH7), and Myosin Regulatory Light Chain 2 (MYL2). RT‐PCR was performed to target multiple locations in the lncRNA in CON and HS SOL. We report the following representative results of one specific region for each lncRNA. A decrease in TNNI1 mRNA in response to HS was associated with a 2.7‐fold (p&lt;0.05) increase in an AS lncRNA assessed ~2.2kb from the 5′‐end of TNNI1. Downregulation of TNNC1 mRNA with HS was associated with a 1.7‐fold (p&lt;0.05) increase in an AS lncRNA as measured ~2.7kb from the 3′‐end of TNNC1. A reduction in MYH7 mRNA with HS occurred with a 2.1‐fold increase (p&lt;0.05) in AS lncRNA, as measured 10kb from the 5′‐end of MYH7. A decline in MYL2 mRNA with HS was associated with a 12‐fold increase (p&lt;0.05) in AS lncRNA, as measured 9kb from the 5′‐end of MYL2. Further work will be required to delineate the function of these lncRNAs. In summary, we report on the discovery of four lncRNAs that are upregulated in response to muscle disuse unloading in conjunction with downregulation of adjacent contractile protein‐coding genes. The expression patterns are consistent with an inhibitory role of the AS lncRNAs on the protein coding genes. The divergent transcription of these newly identified lncRNAs may indicate their involvement in the muscle unloading‐induced coordinated shift in expression from slow to fast contractile proteins.Support or Funding InformationMedcen Community Health Foundation &amp; Mercer University School of Medicine

CrossTalk proposal: The dominant mechanism causing disuse muscle atrophy is decreased protein synthesis.

CrossTalk proposal: The dominant mechanism causing disuse muscle atrophy is decreased protein synthesis.

Mitochondrial ROS Amplify Nox2 Signaling and Atrophy in the Rat Soleus with 7 Days of Hindlimb Unloading (LB821)

Removal of mechanical loading during spaceflight, bedrest, or immobilization causes rapid and profound atrophy of skeletal muscle. Emerging data suggest that oxidative stress from mitochondrial sources may contribute to unloading‐induced skeletal muscle atrophy. We recently found that the Nox2 isoform of NADPH oxidase is also upregulated in the unloaded soleus muscle, associated with FoxO3a activation and fiber atrophy. We used a mitochondrial specific antioxidant peptide (SS‐31) to test the hypothesis that mitochondrial ROS are involved in feed‐forward or amplification signaling with Nox2 in response to mechanical unloading of the rat soleus muscle. F344 rats were divided (n=7/grp) into the following groups: ambulatory control (CON), ambulatory + SS‐31 (1.5 mg/kg/d) (AS), hindlimb unloaded for 7 days (HU), and HU + SS‐31 (HUS). SS‐31 significantly attenuated the reduction in soleus mass and soleus mass/body mass ratio elicited by HU. SS‐31 also abrogated an increase in Type II fibers during unloading. In addition, SS‐31 significantly blunted upregulation of Nox2 subunit p67phox in the unloaded soleus, using fluorescent signal intensity and p67phox membrane localization as markers. Our results are consistent with the postulate that mechanical unloading of skeletal muscle causes mitochondrial ROS to upregulate Nox2 and thus drive atrophy and fiber‐type shift from slow to fast. Supported by NASA (NNX13AE45G), NIH (AR054084), and the Huffines Institute.Grant Funding Source: NASA