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.

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.

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.

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.

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.

Homer protein family regulation in skeletal muscle and neuromuscular adaptation.

Scaffolding adaptor proteins of the Homer family have recently been implicated in regulation of a large number of physiological processes owing to their remarkable ability to coordinate a complex network of different molecular players within the same signaling pathway. However, because of their unique molecular properties that also allow functional modulation of a plethora of different interacting protein partners, Homers seem to play additional and important roles in the integration of several molecular players belonging to different signaling pathways and thus allowing crosstalk. The role of the Homer protein family has been previously extensively investigated in neuronal tissue where it was first discovered as a new protein family being upregulated in response to brain seizures (Brakeman P.R., et al., Nature 1997, 386, 284-288.). Recently, the role of Homers was also proposed in skeletal muscle physiology. For instance, it has been shown that Homers regulate both the myogenic differentiation program and the open probability (Po) of several ion channels. Furthermore, by knocking out Homer1, one of the three Homer genes, mice carrying such deletion displayed a pronounced skeletal muscle myopathy associated with altered transient receptor potential activity and calcium homeostasis. Homer expression has now been further characterized at the neuromuscular junction in skeletal muscle. Apart from their known role at central synapses, Homers are important physiological determinants in differentiation, development, and adaptation in skeletal muscle and the neuromuscular system and thus integrating motor neuron control, for example, with downstream calcium signaling pathways in muscle fibers.

Expression and regulation of Homer in human skeletal muscle during neuromuscular junction adaptation to disuse and exercise.

Protein calcium sensors of the Homer family have been proposed to modulate the activity of various ion channels and nuclear factor of activated T cells (NFAT), the transcription factor modulating skeletal muscle differentiation. We monitored Homer expression and subcellular localization in human skeletal muscle biopsies following 60 d of bedrest [Second Berlin Bedrest Study (BBR2-2)]. Soleus (SOL) and vastus lateralis (VL) biopsies were taken at start (pre) and at end (end) of bedrest from healthy male volunteers of a control group without exercise (CTR; n=9), a resistive-only exercise group (RE; n=7), and a combined resistive/vibration exercise group (RVE; n=7). Confocal analysis showed Homer immunoreactivity at the postsynaptic microdomain of the neuromuscular junction (NMJ) at bedrest start. After bedrest, Homer immunoreactivity decreased (CTR), remained unchanged (RE), or increased (RVE) at the NMJ. Homer2 mRNA and protein were differently regulated in a muscle-specific way. Activated NFATc1 translocates from cytoplasm to nucleus; increased amounts of NFATc1-immunopositive slow-type myonuclei were found in RVE myofibers of both muscles. Pulldown assays identified NFATc1 and Homer as molecular partners in skeletal muscle. A direct motor nerve control of Homer2 was confirmed in rat NMJs by in vivo denervation. Homer2 is localized at the NMJ and is part of the calcineurin-NFATc1 signaling pathway. RVE has additional benefit over RE as countermeasure preventing disuse-induced neuromuscular maladaptation during bedrest.

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.

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.

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.

Long term bed rest with and without vibration exercise countermeasures: effects on human muscle protein dysregulation.

The present investigation, the first in the field, was aimed at analyzing differentially, on individual samples, the effects of 55 days of horizontal bed rest, a model for microgravity, on myosin heavy and myosin light chain isoforms distribution (by SDS) and on the proteome (by 2-D DIGE and MS) in the vastus lateralis (VL), a mixed type II/I (∼50:50%) head of the quadriceps and in the calf soleus (SOL), a predominantly slow (∼35:65%) twitch muscle. Two separate studies were performed on six subjects without (BR) and six with resistive vibration exercise (RVE) countermeasures, respectively. Both VL and SOL underwent in BR decrements of ∼15% in cross-sectional area and of ∼22% in maximal torque that were prevented by RVE. Myosin heavy chain distribution showed increased type I and decreased type IIA in BR both in VL and in SOL, the opposite with RVE. A substantial downregulation of proteins involved in aerobic metabolism characterized both in SOL and VL in BR. RVE reversed the pattern more in VL than in SOL, whereas proteins involved in anaerobic glycolysis were upregulated. Proteins from the Z-disk region and from costamers were differently dysregulated during bed rest (both BR and RVE), particularly in VL.

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.

Atypical fast SERCA1a protein expression in slow myofibers and differential S-nitrosylation prevented by exercise during long term bed rest.

We monitored changes in SERCA isoform specific expression and S-nitrosylation in myofibers of lower limb soleus (SOL) and vastus lateralis (VL) muscle biopsies before and after 60 days of voluntary long term bed rest (BR) without (BR-CTRL group, n = 8) and with exercise countermeasure (BR-EX group, n = 8). Before BR, a typical myofiber type-specific distribution of fast and slow SERCA1/2a isoforms was seen. After BR, a subpopulation (approx. 15%) of slow myofibers in BR-CTRL additionally expressed the fast SERCA1a isoform which was not seen in BR-EX. After BR, SERCA1a S-nitrosylation patterns analyzed by the biotin-switch assay decreased in disused SOL only but increased in both muscles following exercise. Differential SERCA1a S-nitrosylation and SERCA1a/2a co-expression in subsets of slow myofibers should be considered as signs of an altered cytosolic Ca(2+) homeostasis following chronic muscle disuse. Exercise preserved myofiber type-specific SERCA1a expression and S-nitrosylation in VL and SOL in a different way, suggesting muscle-specific responses to the countermeasure protocol applied during bed rest.

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.

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.

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.

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.