Nitrosative Stress in Astronaut Skeletal Muscle in Spaceflight.

Long-duration mission (LDM) astronauts from the International Space Station (ISS) (>180 ISS days) revealed a close-to-normal sarcolemmal nitric oxide synthase type-1 (NOS1) immunoexpression in myofibers together with biochemical and quantitative qPCR changes in deep calf soleus muscle. Nitro-DIGE analyses identified functional proteins (structural, metabolic, mitochondrial) that were over-nitrosylated post- vs. preflight. In a short-duration mission (SDM) astronaut (9 ISS days), s-nitrosylation of a nodal protein of the glycolytic flux, specific proteins in tricarboxylic acid (TCA) cycle, respiratory chain, and over-nitrosylation of creatine kinase M-types as signs of impaired ATP production and muscle contraction proteins were seen. S-nitrosylation of serotransferrin (TF) or carbonic anhydrase 3 (CA3b and 3c) represented signs of acute response microgravity muscle maladaptation. LDM nitrosoprofiles reflected recovery of mitochondrial activity, contraction proteins, and iron transporter TF as signs of muscle adaptation to microgravity. Nitrosated antioxidant proteins, alcohol dehydrogenase 5/S-nitrosoglutathione reductase (ADH5/GSNOR), and selenoprotein thioredoxin reductase 1 (TXNRD1) levels indicated signs of altered redox homeostasis and reduced protection from nitrosative stress in spaceflight. This work presents a novel spaceflight-generated dataset on s-nitrosylated muscle protein signatures from astronauts that helps both to better understand the structural and molecular networks associated to muscular nitrosative stress and to design countermeasures to dysfunction and impaired performance control in human spaceflight missions.

Muscle stiffness indicating mission crew health in space.

Muscle function is compromised by gravitational unloading in space affecting overall musculoskeletal health. Astronauts perform daily exercise programmes to mitigate these effects but knowing which muscles to target would optimise effectiveness. Accurate inflight assessment to inform exercise programmes is critical due to lack of technologies suitable for spaceflight. Changes in mechanical properties indicate muscle health status and can be measured rapidly and non-invasively using novel technology. A hand-held MyotonPRO device enabled monitoring of muscle health for the first time in spaceflight (> 180 days). Greater/maintained stiffness indicated countermeasures were effective. Tissue stiffness was preserved in the majority of muscles (neck, shoulder, back, thigh) but Tibialis Anterior (foot lever muscle) stiffness decreased inflight vs. preflight (p < 0.0001; mean difference 149 N/m) in all 12 crewmembers. The calf muscles showed opposing effects, Gastrocnemius increasing in stiffness Soleus decreasing. Selective stiffness decrements indicate lack of preservation despite daily inflight countermeasures. This calls for more targeted exercises for lower leg muscles with vital roles as ankle joint stabilizers and in gait. Muscle stiffness is a digital biomarker for risk monitoring during future planetary explorations (Moon, Mars), for healthcare management in challenging environments or clinical disorders in people on Earth, to enable effective tailored exercise programmes.

Space Omics and Tissue Response in Astronaut Skeletal Muscle after Short and Long Duration Missions.

The molecular mechanisms of skeletal muscle adaptation to spaceflight are as yet not fully investigated and well understood. The MUSCLE BIOPSY study analyzed pre and postflight deep calf muscle biopsies (m. soleus) obtained from five male International Space Station (ISS) astronauts. Moderate rates of myofiber atrophy were found in long-duration mission (LDM) astronauts (~180 days in space) performing routine inflight exercise as countermeasure (CM) compared to a short-duration mission (SDM) astronaut (11 days in space, little or no inflight CM) for reference control. Conventional H&E scout histology showed enlarged intramuscular connective tissue gaps between myofiber groups in LDM post vs. preflight. Immunoexpression signals of extracellular matrix (ECM) molecules, collagen 4 and 6, COL4 and 6, and perlecan were reduced while matrix-metalloproteinase, MMP2, biomarker remained unchanged in LDM post vs. preflight suggesting connective tissue remodeling. Large scale proteomics (space omics) identified two canonical protein pathways associated to muscle weakness (necroptosis, GP6 signaling/COL6) in SDM and four key pathways (Fatty acid ß-oxidation, integrin-linked kinase ILK, Rho A GTPase RHO, dilated cardiomyopathy signaling) explicitly in LDM. The levels of structural ECM organization proteins COL6A1/A3, fibrillin 1, FBN1, and lumican, LUM, increased in postflight SDM vs. LDM. Proteins from tricarboxylic acid, TCA cycle, mitochondrial respiratory chain, and lipid metabolism mostly recovered in LDM vs. SDM. High levels of calcium signaling proteins, ryanodine receptor 1, RyR1, calsequestrin 1/2, CASQ1/2, annexin A2, ANXA2, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) pump, ATP2A, were signatures of SDM, and decreased levels of oxidative stress peroxiredoxin 1, PRDX1, thioredoxin-dependent peroxide reductase, PRDX3, or superoxide dismutase [Mn] 2, SOD2, signatures of LDM postflight. Results help to better understand the spatiotemporal molecular adaptation of skeletal muscle and provide a large scale database of skeletal muscle from human spaceflight for the better design of effective CM protocols in future human deep space exploration.

Opposite Regulation of Homer Signal at the NMJ Postsynaptic Micro Domain between Slow- and Fast-Twitch Muscles in an Experimentally Induced Autoimmune Myasthenia Gravis (EAMG) Mouse Model.

Accelerated postsynaptic remodelling and disturbance of neuromuscular transmission are common features of autoimmune neurodegenerative diseases. Homer protein isoform expression, crosslinking activity and neuromuscular subcellular localisation are studied in mouse hind limb muscles of an experimentally induced autoimmune model of Myasthenia Gravis (EAMG) and correlated to motor end plate integrity. Soleus (SOL), extensor digitorum longus (EDL) and gastrocnemius (GAS) skeletal muscles are investigated. nAChR membrane clusters were studied to monitor neuromuscular junction (NMJ) integrity. Fibre-type cross-sectional area (CSA) analysis is carried out in order to determine the extent of muscle atrophy. Our findings clearly showed that crosslinking activity of Homer long forms (Homer 1b/c and Homer2a/b) are decreased in slow-twitch and increased in fast-twitch muscle of EAMG whereas the short form of Homer that disrupts Homer crosslinking (Homer1a) is upregulated in slow-twitch muscle only. Densitometry analysis showed a 125% increase in Homer protein expression in EDL, and a 45% decrease in SOL of EAMG mice. In contrast, nAChR fluorescence pixel intensity decreased in endplates of EAMG mice, more distinct in type-I dominant SOL muscle. Morphometric CSA of EAMG vs. control (CTR) revealed a significant reduction in EDL but not in GAS and SOL. Taken together, these results indicate that postsynaptic Homer signalling is impaired in slow-twitch SOL muscle from EAMG mice and provide compelling evidence suggesting a functional coupling between Homer and nAChR, underscoring the key role of Homer in skeletal muscle neurophysiology.

Nerve-dependent distribution of subsynaptic type 1 inositol 1,4,5-trisphosphate receptor at the neuromuscular junction.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation-transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the lack of muscle electrical activity, as judged by partial reversal upon field electrical stimulation of in vitro denervated skeletal muscle fibers.

Effects of Omega-3 and Antioxidant Cocktail Supplement on Prolonged Bed Rest: Results from Serum Proteome and Sphingolipids Analysis.

Physical inactivity or prolonged bed rest (BR) induces muscle deconditioning in old and young subjects and can increase the cardiovascular disease risk (CVD) with dysregulation of the lipemic profile. Nutritional interventions, combining molecules such as polyphenols, vitamins and essential fatty acids, can influence some metabolic features associated with physical inactivity and decrease the reactive oxidative and nitrosative stress (RONS). The aim of this study was to detect circulating molecules correlated with BR in serum of healthy male subjects enrolled in a 60-day BR protocol to evaluate a nutritional intervention with an antioxidant cocktail as a disuse countermeasure (Toulouse COCKTAIL study). The serum proteome, sphingolipidome and nitrosoproteome were analyzed adopting different mass spectrometry-based approaches. Results in placebo-treated BR subjects indicated a marked decrease of proteins associated with high-density lipoproteins (HDL) involved in lipemic homeostasis not found in the cocktail-treated BR group. Moreover, long-chain ceramides decreased while sphingomyelin increased in the BR cocktail-treated group. In placebo, the ratio of S-nitrosylated/total protein increased for apolipoprotein D and several proteins were over-nitrosylated. In cocktail-treated BR subjects, the majority of protein showed a pattern of under-nitrosylation, except for ceruloplasmin and hemopexin, which were over-nitrosylated. Collectively, data indicate a positive effect of the cocktail in preserving lipemic and RONS homeostasis in extended disuse conditions.

Preliminary Observations on Skeletal Muscle Adaptation and Plasticity in Homer 2-/- Mice.

Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2-/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR (Reverse Transcription PCR), confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2-/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2-/- mice detected the exclusive presence of the adult ε-nAChR isoform excluding denervation. As for NMJ morphology, data were not conclusive, and further work is needed to ascertain whether the null Homer 2 phenotype induces any endplate remodelling. Within the context of adaptation and plasticity, the present data show that Homer 2 is a co-regulator of the normotrophic status in a muscle specific fashion.

Nitrosative Redox Homeostasis and Antioxidant Response Defense in Disused Vastus lateralis Muscle in Long-Term Bedrest (Toulouse Cocktail Study).

Increased oxidative stress by reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major determinant of disuse-induced muscle atrophy. Muscle biopsies (thigh vastus lateralis, VL) obtained from healthy male subjects enrolled in the Toulouse Cocktail bedrest (BR) study were used to assess efficacy of an antioxidant cocktail (polyphenols, omega-3, vitamin E, and selenium) to counteract the increased redox homeostasis and enhance the antioxidant defense response by using label-free LC-MS/MS and NITRO-DIGE (nitrosated proteins), qPCR, and laser confocal microscopy. Label-free LC-MS/MS indicated that treatment prevented the redox homeostasis dysregulation and promoted structural remodeling (TPM3, MYH7, MYBPC, MYH1, MYL1, HRC, and LUM), increment of RyR1, myogenesis (CSRP3), and skeletal muscle development (MUSTN1, LMNA, AHNAK). These changes were absent in the Placebo group. Glycolysis, tricarboxylic acid cycle (TCA), oxidative phosphorylation, fatty acid beta-oxidation, and mitochondrial transmembrane transport were normalized in treated subjects. Proteins involved in protein folding were also normalized, whereas protein entailed in ion homeostasis decreased. NITRO-DIGE analysis showed significant protein nitrosylation changes for CAT, CA3, SDHA, and VDAC2 in Treatment vs. Placebo. Similarly, the nuclear factor erythroid 2-related factor 2 (Nrf-2) antioxidant response element (Nrf-2 ARE) signaling pathway showed an enhanced response in the Treatment group. Increased nitrosative redox homeostasis and decreased antioxidant defense response were found in post-BR control (Placebo, n = 10) vs. the antioxidant cocktail treated group (Treatment, n = 10). Taken together, increased nitrosative redox homeostasis and muscle deterioration during BR-driven physical inactivity were prevented, whereas decreased antioxidant nitrosative stress defense response was attenuated by Treatment suggesting positive effects of the nutritional intervention protocol in bedrest.

Reciprocal Homer1a and Homer2 Isoform Expression Is a Key Mechanism for Muscle Soleus Atrophy in Spaceflown Mice.

The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or microgravity are not yet fully understood. The transition of Homer isoforms may play a key role during neuromuscular junction (NMJ) imbalance/plasticity in space. Here, we investigated the expression pattern of Homer short and long isoforms by gene array, qPCR, biochemistry, and laser confocal microscopy in skeletal muscles from male C57Bl/N6 mice (n = 5) housed for 30 days in space (Bion-flight = BF) compared to muscles from Bion biosatellite on the ground-housed animals (Bion ground = BG) and from standard cage housed animals (Flight control = FC). A comparison study was carried out with muscles of rats subjected to hindlimb unloading (HU). Gene array and qPCR results showed an increase in Homer1a transcripts, the short dominant negative isoform, in soleus (SOL) muscle after 30 days in microgravity, whereas it was only transiently increased after four days of HU. Conversely, Homer2 long-form was downregulated in SOL muscle in both models. Homer immunofluorescence intensity analysis at the NMJ of BF and HU animals showed comparable outcomes in SOL but not in the extensor digitorum longus (EDL) muscle. Reduced Homer crosslinking at the NMJ consequent to increased Homer1a and/or reduced Homer2 may contribute to muscle-type specific atrophy resulting from microgravity and HU disuse suggesting mutual mechanisms.

Evaluation of an Antioxidant and Anti-inflammatory Cocktail Against Human Hypoactivity-Induced Skeletal Muscle Deconditioning.

Understanding the molecular pathways involved in the loss of skeletal muscle mass and function induced by muscle disuse is a crucial issue in the context of spaceflight as well as in the clinical field, and development of efficient countermeasures is needed. Recent studies have reported the importance of redox balance dysregulation as a major mechanism leading to muscle wasting. Our study aimed to evaluate the effects of an antioxidant/anti-inflammatory cocktail (741 mg of polyphenols, 138 mg of vitamin E, 80 µg of selenium, and 2.1 g of omega-3) in the prevention of muscle deconditioning induced by long-term inactivity. The study consisted of 60 days of hypoactivity using the head-down bed rest (HDBR) model. Twenty healthy men were recruited; half of them received a daily antioxidant/anti-inflammatory supplementation, whereas the other half received a placebo. Muscle biopsies were collected from the vastus lateralis muscles before and after bedrest and 10 days after remobilization. After 2 months of HDBR, all subjects presented muscle deconditioning characterized by a loss of muscle strength and an atrophy of muscle fibers, which was not prevented by cocktail supplementation. Our results regarding muscle oxidative damage, mitochondrial content, and protein balance actors refuted the potential protection of the cocktail during long-term inactivity and showed a disturbance of essential signaling pathways (protein balance and mitochondriogenesis) during the remobilization period. This study demonstrated the ineffectiveness of our cocktail supplementation and underlines the complexity of redox balance mechanisms. It raises interrogations regarding the appropriate nutritional intervention to fight against muscle deconditioning.

Reactive Jumps Preserve Skeletal Muscle Structure, Phenotype, and Myofiber Oxidative Capacity in Bed Rest.

Identification of countermeasures able to prevent disuse-induced muscle wasting is crucial to increase performance of crew members during space flight as well as ameliorate patient's clinical outcome after long immobilization periods. We report on the outcome of short but high-impact reactive jumps (JUMP) as countermeasure during 60 days of 6° head-down tilt (HDT) bed rest on myofiber size, type composition, capillarization, and oxidative capacity in tissue biopsies (pre/post/recovery) from the knee extensor vastus lateralis (VL) and deep calf soleus (SOL) muscle of 22 healthy male participants (Reactive jumps in a sledge, RSL-study 2015-2016, DLR:envihab, Cologne). Bed rest induced a slow-to-fast myofiber shift (type I ->II) with an increased prevalence of hybrid fibers in SOL after bed rest without jumps (control, CTRL, p = 0.016). In SOL, JUMP countermeasure in bed rest prevented both fast and slow myofiber cross-sectional area (CSA) decrements (p = 0.005) in CTRL group. In VL, bed rest only induced capillary rarefaction, as reflected by the decrease in local capillary-to-fiber ratio (LCFR) for both type II (pre vs. post/R + 10, p = 0.028/0.028) and type I myofibers (pre vs. R + 10, p = 0.012), which was not seen in the JUMP group. VO2 max Fiber (pL × mm-1 × min-1) calculated from succinate dehydrogenase (SDH)-stained cryosections (OD660 nm) showed no significant differences between groups. High-impact jump training in bed rest did not prevent disuse-induced myofiber atrophy in VL, mitigated phenotype transition (type I - >II) in SOL, and attenuated capillary rarefaction in the prime knee extensor VL however with little impact on oxidative capacity changes.

Bed Rest, Exercise Countermeasure and Reconditioning Effects on the Human Resting Muscle Tone System.

The human resting muscle tone (HRMT) system provides structural and functional support to skeletal muscle and associated myofascial structures (tendons, fascia) in normal life. Little information is available on changes to the HRMT in bed rest. A set of dynamic oscillation mechanosignals ([Hz], [N/m], log decrement, [ms]) collected and computed by a hand-held digital palpation device (MyotonPRO) were used to study changes in tone and in key biomechanical and viscoelastic properties in global and postural skeletal muscle tendons and fascia from a non-exercise control (CTR) and an exercise (JUMP) group performing reactive jumps on a customized sledge system during a 60 days head-down tilt bed rest (RSL Study 2015-2016). A set of baseline and differential natural oscillation signal patterns were identified as key determinants in resting muscle and myofascial structures from back, thigh, calf, patellar and Achilles tendon, and plantar fascia. The greatest changes were found in thigh and calf muscle and tendon, with little change in the shoulder muscles. Functional tests (one leg jumps, electromyography) showed only trends in relevant leg muscle groups. Increased anti-Collagen-I immunoreactivity found in CTR soleus biopsy cryosections was absent from JUMP. Results allow for a muscle health status definition after chronic disuse in bed rest without and with countermeasure, and following reconditioning. Findings improve our understanding of structural and functional responses of the HRMT to disuse and exercise, may help to guide treatment in various clinical settings (e.g., muscle tone disorders, neuro-rehabilitation), and promote monitoring of muscle health and training status in personalized sport and space medicine.

Effects of Plantar Vibration on Bone and Deep Fascia in a Rat Hindlimb Unloading Model of Disuse.

The deep fascia of the vertebrate body comprises a biomechanically unique connective cell and tissue layer with integrative functions to support global and regional strain, tension, and even muscle force during motion and performance control. However, limited information is available on deep fascia in relation to bone in disuse. We used rat hindlimb unloading as a model of disuse (21 days of hindlimb unloading) to study biomechanical property as well as cell and tissue changes to deep fascia and bone unloading. Rats were randomly divided into three groups (n = 8, each): hindlimb unloading (HU), HU + vibration (HUV), and cage-control (CON). The HUV group received local vibration applied to the plantar of both hind paws. Micro-computed tomography analyzed decreased bone mineral density (BMD) of vertebra, tibia, and femur in HU vs. CON. Biomechanical parameters (elastic modulus, max stress, yield stress) of spinal and crural fascia in HU were always increased vs. CON. Vibration in HUV only counteracted HU-induced tibia bone loss and crural fascia mechanical changes but failed to show comparable changes in the vertebra and spinal fascia on lumbar back. Tissue and cell morphometry (size and cell nuclear density), immunomarker intensity levels of anti-collagen-I and III, probed on fascia cryosections well correlated with biomechanical changes suggesting crural fascia a prime target for plantar vibration mechano-stimulation in the HU rat. We conclude that the regular biomechanical characteristics as well as tissue and cell properties in crural fascia and quality of tibia bone (BMD) were preserved by local plantar vibration in disuse suggesting common mechanisms in fascia and bone adaptation to local mechanovibration stimulation following hind limb unloading in the HUV rat.

Microgravity-Induced Transcriptome Adaptation in Mouse Paraspinal longissimus dorsi Muscle Highlights Insulin Resistance-Linked Genes.

Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least in part from paraspinal muscle deconditioning. At present no study investigated the complexity of the molecular changes in human or mouse paraspinal muscles exposed to microgravity. The aim of this study was to evaluate longissimus dorsi adaptation to microgravity at both morphological and global gene expression level. C57BL/N6 male mice were flown aboard the BION-M1 biosatellite for 30 days (BF) or housed in a replicate flight habitat on ground (BG). Myofiber cross sectional area and myosin heavy chain subtype patterns were respectively not or slightly altered in longissimus dorsi of BF mice. Global gene expression analysis identified 89 transcripts differentially regulated in longissimus dorsi of BF vs. BG mice. Microgravity-induced gene expression changes of lipocalin 2 (Lcn2), sestrin 1(Sesn1), phosphatidylinositol 3-kinase, regulatory subunit polypeptide 1 (p85 alpha) (Pik3r1), v-maf musculoaponeurotic fibrosarcoma oncogene family protein B (Mafb), protein kinase C delta (Prkcd), Muscle Atrophy F-box (MAFbx/Atrogin-1/Fbxo32), and Muscle RING Finger 1 (MuRF-1) were further validated by real time qPCR analysis. In conclusion, our study highlighted the regulation of transcripts mainly linked to insulin sensitivity and metabolism in longissimus dorsi following 30 days of microgravity exposure. The apparent absence of robust signs of back muscle atrophy in space-flown mice, despite the overexpression of Atrogin-1 and MuRF-1, opens new questions on the possible role of microgravity-sensitive genes in the regulation of peripheral insulin resistance following unloading and its consequences on paraspinal skeletal muscle physiology.

Bed rest and resistive vibration exercise unveil novel links between skeletal muscle mitochondrial function and insulin resistance.

AIMS/HYPOTHESIS: Physical inactivity has broad implications for human disease including insulin resistance, sarcopenia and obesity. The present study tested the hypothesis that (1) impaired mitochondrial respiration is linked with blunted insulin sensitivity and loss of muscle mass in healthy young men, and (2) resistive vibration exercise (RVE) would mitigate the negative metabolic effects of bed rest. METHODS: Participants (n = 9) were maintained in energy balance during 21 days of bed rest with RVE and without (CON) in a crossover study. Mitochondrial respiration was determined by high-resolution respirometry in permeabilised fibre bundles from biopsies of the vastus lateralis. A hyperinsulinaemic-euglycaemic clamp was used to determine insulin sensitivity, and body composition was assessed by dual-energy x-ray absorptiometry (DEXA). RESULTS: Body mass (-3.2 ± 0.5 kg vs -2.8 ± 0.4 kg for CON and RVE, respectively, p < 0.05), fat-free mass (-2.9 ± 0.5 kg vs -2.7 ± 0.5 kg, p < 0.05) and peak oxygen consumption ([Formula: see text]) (10-15%, p < 0.05) were all reduced following bed rest. Bed rest decreased insulin sensitivity in the CON group (0.04 ± 0.002 mg kgFFM-1 [pmol l-1] min-1 vs 0.03 ± 0.002 mg kgFFM-1 [pmol l-1] min-1 for baseline vs post-CON), while RVE mitigated this response (0.04 ± 0.003 mg kgFFM-1 [pmol l-1] min-1). Mitochondrial respiration (oxidative phosphorylation and electron transport system capacity) decreased in the CON group but not in the RVE group when expressed relative to tissue weight but not when normalised for citrate synthase activity. LEAK respiration, indicating a decrease in mitochondrial uncoupling, was the only component to remain significantly lower in the CON group after normalisation for citrate synthase. This was accompanied by a significant decrease in adenine nucleotide translocase protein content. CONCLUSIONS/INTERPRETATION: Reductions in muscle mitochondrial respiration occur concomitantly with insulin resistance and loss of muscle mass during bed rest and may play a role in the adaptations to physical inactivity. Significantly, we show that RVE is an effective strategy to partially prevent some of the deleterious metabolic effects of bed rest.

Gene Expression Profiling in Slow-Type Calf Soleus Muscle of 30 Days Space-Flown Mice.

Microgravity exposure as well as chronic disuse are two main causes of skeletal muscle atrophy in animals and humans. The antigravity calf soleus is a reference postural muscle to investigate the mechanism of disuse-induced maladaptation and plasticity of human and rodent (rats or mice) skeletal musculature. Here, we report microgravity-induced global gene expression changes in space-flown mouse skeletal muscle and the identification of yet unknown disuse susceptible transcripts found in soleus (a mainly slow phenotype) but not in extensor digitorum longus (a mainly fast phenotype dorsiflexor as functional counterpart to soleus). Adult C57Bl/N6 male mice (n = 5) flew aboard a biosatellite for 30 days on orbit (BION-M1 mission, 2013), a sex and age-matched cohort were housed in standard vivarium cages (n = 5), or in a replicate flight habitat as ground control (n = 5). Next to disuse atrophy signs (reduced size and myofiber phenotype I to II type shift) as much as 680 differentially expressed genes were found in the space-flown soleus, and only 72 in extensor digitorum longus (only 24 genes in common) compared to ground controls. Altered expression of gene transcripts matched key biological processes (contractile machinery, calcium homeostasis, muscle development, cell metabolism, inflammatory and oxidative stress response). Some transcripts (Fzd9, Casq2, Kcnma1, Ppara, Myf6) were further validated by quantitative real-time PCR (qRT-PCR). Besides previous reports on other leg muscle types we put forth for the first time a complete set of microgravity susceptible gene transcripts in soleus of mice as promising new biomarkers or targets for optimization of physical countermeasures and rehabilitation protocols to overcome disuse atrophy conditions in different clinical settings, rehabilitation and spaceflight.

Whey protein with potassium bicarbonate supplement attenuates the reduction in muscle oxidative capacity during 19 days of bed rest.

The effectiveness of whey protein plus potassium bicarbonate-enriched diet (WP+KHCO3) in mitigating disuse-induced changes in muscle fiber oxidative capacity and capillarization was investigated in a 21-day crossover design bed rest study. Ten healthy men (31 ± 6 yr) once received WP+KHCO3 and once received a standardized isocaloric diet. Muscle biopsies were taken 2 days before and during the 19th day of bed rest (BR) from the soleus (SOL) and vastus lateralis (VL) muscle. Whole-body aerobic power (V̇o2 max), muscle fatigue, and isometric strength of knee extensor and plantar flexor muscles were monitored. Muscle fiber types and capillaries were identified by immunohistochemistry. Fiber oxidative capacity was determined as the optical density (OD) at 660 nm of succinate dehydrogenase (SDH)-stained sections. The product of fiber cross-sectional area and SDH-OD (integrated SDH) indicated the maximal oxygen consumption of that fiber. The maximal oxygen consumption supported by a capillary was calculated as the integrated SDH in its supply area. BR reduced isometric strength of knee extensor muscles (P < 0.05), and the fiber oxidative capacity (P < 0.001) and V̇o2 max (P = 0.042), but had no significant impact on muscle capillarization or fatigue resistance of thigh muscles. The maximal oxygen consumption supported by a capillary was reduced by 24% in SOL and 16% in VL (P < 0.001). WP+KHCO3 attenuated the disuse-induced reduction in fiber oxidative capacity in both muscles (P < 0.01). In conclusion, following 19 days of bed rest, the decrement in fiber oxidative capacity is proportionally larger than the loss of capillaries. WP+KHCO3 appears to attenuate disuse-induced reductions in fiber oxidative capacity.

Vibration mechanosignals superimposed to resistive exercise result in baseline skeletal muscle transcriptome profiles following chronic disuse in bed rest.

Disuse-induced muscle atrophy is a major concern in aging, in neuromuscular diseases, post-traumatic injury and in microgravity life sciences affecting health and fitness also of crew members in spaceflight. By using a laboratory analogue to body unloading we perform for the first time global gene expression profiling joined to specific proteomic analysis to map molecular adaptations in disused (60 days of bed rest) human soleus muscle (CTR) and in response to a resistive exercise (RE) countermeasure protocol without and with superimposed vibration mechanosignals (RVE). Adopting Affymetrix GeneChip technology we identified 235 differently transcribed genes in the CTR group (end- vs. pre-bed rest). RE comprised 206 differentially expressed genes, whereas only 51 changed gene transcripts were found in RVE. Most gene transcription and proteomic changes were linked to various key metabolic pathways (glycolysis, oxidative phosphorylation, tricarboxylic acid (TCA) cycle, lipid metabolism) and to functional contractile structures. Gene expression profiling in bed rest identified a novel set of genes explicitly responsive to vibration mechanosignals in human soleus. This new finding highlights the efficacy of RVE protocol in reducing key signs of disuse maladaptation and atrophy, and to maintain a close-to-normal skeletal muscle quality outcome following chronic disuse in bed rest.

Disuse deterioration of human skeletal muscle challenged by resistive exercise superimposed with vibration: evidence from structural and proteomic analysis.

In the present bed rest (BR) study, 23 volunteers were randomized into 3 subgroups: 60 d BR control (Ctr); BR with resistive exercise (RE; lower-limb load); and resistive vibration exercise (RVE; RE with superimposed vibration). The aim was to analyze by confocal and electron microscopy the effects of vibration on myofibril and filament integrity in soleus (Sol) and vastus lateralis (VL) muscle; differential proteomics of contractile, cytoskeletal, and costameric proteins (TN-C, ROCK1, and FAK); and expression of PGC1α and atrophy-related master genes MuRF1 and MuRF2. RVE (but not RE) preserved myofiber size and phenotype in Sol and VL by overexpressing MYBPC1 (42%, P ≤ 0.01), WDR1 (39%, P ≤ 0.01), sarcosin (84%, P ≤ 0.01), and CKM (20%, P ≤ 0.01) and prevented myofibrillar ultrastructural damage as detectable by MuRF1 expression. In Sol, cytoskeletal and contractile proteins were normalized by RVE, and TN-C increased (59%, P ≤ 0.01); the latter also with RE (108%, P ≤ 0.01). In VL, the outcomes of both RVE (acting on sarcosin and desmin) and RE (by way of troponinT-slow and MYL2) were similar. RVE appears to be a highly efficient countermeasure protocol against muscle atrophy and ultrastructural and molecular dysregulation induced by chronic disuse.

Increased myofiber remodelling and NFATc1-myonuclear translocation in rat postural skeletal muscle after experimental vestibular deafferentation.

BACKGROUND: The vestibular system undergoes considerable modification during spaceflight [5]. This is paralleled by microgravity-induced muscle atrophy [6]. However, the possibility of vestibulo-autonomic regulatory mechanisms affecting skeletal muscle structure and function have not yet been addressed. OBJECTIVE: We hypothesise that the vestibular system affects anti-gravitational skeletal muscle phenotype composition, size and the transcriptional factor called nuclear factor of activated T cells (NFATc1). METHODS: In a laboratory study, we examined the morphological and histochemical properties including intramyocellular NFATc1 changes in slow-type soleus muscle of chemically labyrinthectomized rats (VLx; n=8) compared to a control group (Sham; n=6) after a period of one month. RESULTS AND CONCLUSION: Neurochemical vestibular deafferentation resulted in smaller myofibre sizes, altered myofibre phenotype composition, high yields of hybrid fibre formation, and reduced myonuclear NFATc1 accumulation as signs of slow-type myofibre atrophy, myofibre type remodelling, and altered nuclear transcriptional activity in the postural soleus muscle of rats. We propose that vestibulo-autonomic modification of skeletal muscles occurs during prolonged microgravity. Our findings are likely to have implications for vestibular rehabilitation in clinical settings.