Microgravity exposure as well as chronic muscle disuse are two of the main causes of physiological adaptive skeletal muscle atrophy in humans and murine animals in physiological condition. The aim of this study was to investigate at both morphological and global gene expression level skeletal muscle adaptation to microgravity in mouse soleus and extensor digitorum longus (EDL). Adult male mice C57BL/N6 were flown aboard the BION-M1 biosatellite for 30 days on orbit (BF) or housed in a replicate flight habitat on Earth (BG) as reference flight control. In this study we investigated for the first time gene expression adaptation to 30 days of microgravity exposure in mouse soleus and EDL highlighting potential new targets for improvement of countermeasures able to ameliorate or even prevent microgravity-induced atrophy in future spaceflights. Overall Design: C57BL/N6 mice were randomly divided in 3 groups: Bion Flown (BF) mice flown aboard the Bion M1 biosatellite in microgravity environment for 30 days; Bion Ground (BG) mice housed in the same habitat of flown animals but exposed to earth gravity; and Flight Control (FC) mice housed in a standard animal facility.

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 and tongue (as a new potential in-flight negative control) adaptation to microgravity at 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). Global gene expression analysis identified 89 transcripts differentially regulated in longissimus dorsi of BF vs. BG mice (False Discovery Rrate < 0,05 and fold change < -2 and > +2) while only a small number of genes were found differentially regulated in tongue muscle ( BF vs. BG = 27 genes). Overall Design: C57BL/N6 mice were randomly divided in 3 groups: Bion Flown (BF) mice flown aboard the Bion M1 biosatellite in microgravity environment for 30 days; Bion Ground (BG) mice housed in the same habitat of flown animals but exposed to earth gravity; and Flight Control (FC) mice housed in a standard animal facility.

Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts towards faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, microarray expression analysis was performed on gastrocnemius from mice flown on the STS-108 shuttle flight (11 days, 19 hours) versus mice maintained on earth for the same period. Additionally, to identify changes that were due to unloading and reloading, microarray analyses were conducted on calf muscle from ground-based mice subjected to hindlimb suspension (12 days) and mice subjected to hindlimb suspension plus a brief period of reloading (3.5 hours) to simulate the time between landing and sacrifice of the spaceflight mice.

It is well established that microgravity exposure causes significant muscle weakness and atrophy via muscle unloading. On Earth, muscle unloading leads to a disproportionate loss in muscle force and size with the loss in muscle force occurring at a faster rate. Although the exact mechanisms are unknown, a role for Ca2+ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump actively brings cytosolic Ca2+ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca2+ ([Ca2+]i). SERCA dysfunction contributes to elevations in [Ca2+]i, leading to cellular damage, and may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content, and reactive oxygen/nitrogen species (RONS) protein adduction in murine skeletal muscle after 35-37 days of spaceflight. In male and female soleus muscles, spaceflight led to drastic impairments in Ca2+ uptake despite significant increases in SERCA1a protein content. We attribute this impairment to an increase in RONS production and elevated total protein tyrosine (T) nitration and cysteine (S) nitrosylation. Contrarily, in the tibialis anterior (TA), we observed an enhancement in Ca2+ uptake, which we attribute to a shift towards a faster muscle fiber type (i.e., increased myosin heavy chain IIb and SERCA1a) without elevated total protein T-nitration and S-nitrosylation. Thus, spaceflight affects SERCA function differently between the soleus and TA. This dataset derives results from the calcium uptake (spectrofluorometry) and Western blot assays.

The spaceflight experiment was carried out using male C57BL/10J mice (8 weeks old at launch). Wild type mice (n=3) were launched by Space Shuttle Discovery and housed on the International Space Station (ISS) for 91 days. They returned to the Earth by Space Shuttle Atlantis. But only one mouse returned to the Earth alive. Whole brain was sampled from the mouse killed by inhalation of carbon dioxide at the Life Sciences Support Facility of Kennedy Space Center within 3-4 hours after landing. After the spaceflight experiment the on-ground experiment was also carried out at the Advanced Biotechnology Center in Genova Italy. A mouse with the same species sex and age was housed in mice drawer system (MDS) which was utilized for the spaceflight (SF) mice for 3 months as the ground control (GC). Another mouse was housed in normal vivarium cage as the laboratory control (LC). Amount of food and water supplementation and environmental conditions were simulated as the flight group. After 3 months brain was sampled from one mouse in group GC and LC respectively. Comprehensive analyses of gene expression were performed in the right brain. Total of 4,000 genes were analyzed. The expression levels of 60 genes significantly changed in response to SF compared with LC and/or GC. The 15 and 16 genes were up- (> 2 folds) and down-regulated (< 0.5 folds) respectively following SF vs. GC. The levels of 58 genes were significantly altered by housing in MDS in space and/or on the ground. Forty seven and 11 genes were significantly up- and down-regulated vs. LC. Twenty seven out of these genes responded to caging in MDS both in space and on the ground. Further 31 genes were influenced by housing in MDS on the Earth. Responses of the characteristics of brain to long-term gravitational unloading were investigated in mice.

Microphysiological systems provide the opportunity to model accelerated changes at the human tissue level in the extreme space environment. Spaceflight-induced muscle atrophy experienced by astronauts shares similar physiological changes to muscle wasting in older adults, known as sarcopenia. These shared attributes provide a rationale for investigating molecular changes in muscle cells exposed to spaceflight that may mimic the underlying pathophysiology of sarcopenia. We report the results from three-dimensional myobundles derived from muscle biopsies from young and older adults, integrated into an autonomous CubeLab™, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analyses comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation. The analyses also revealed downregulated differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were downregulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides an approach to studying the cell autonomous effects of spaceflight on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. We also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLab™ payloads on the ISS.

Spaceflight imposes the risk of skeletal muscle atrophy for astronauts. The understanding of muscle atrophy because of spaceflight is limited but continued efforts are essential for developing countermeasures of this effect. A distinct difference between spaceflight-induced muscle atrophy and other forms of atrophy is the additional effect of cosmic rays in outer space. To study spaceflight-induced muscle atrophy we performed two ground-based models of microgravity in a low dose radiation environment and studied transcriptional changes in rat soleus muscle using microarray technology.

Exposure to weightlessness in microgravity and elevated space radiation are associated with rapid bone loss in mammals, but questions remain about their mechanisms of action and relative importance. In this study, we tested the hypothesis that bone loss during spaceflight in Low Earth Orbit is primarily associated with site-specific microgravity unloading of weight-bearing sites in the skeleton. Microcomputed tomography and histological analyses of bones from mice space flown on ISS for 37 days in the NASA Rodent Research-1 experiment show significant site-specific cancellous and cortical bone loss occurring in the femur, but not in L2 vertebrae. The lack of bone degenerative effects in the spine in combination with same-animal paired losses in the femur suggests that space radiation levels in Low Earth Orbit or other systemic stresses are not likely to significantly contribute to the observed bone loss. Remarkably, spaceflight is also associated with accelerated progression of femoral head endochondral ossification. This suggests the microgravity environment promotes premature progression of secondary ossification during late stages of skeletal maturation at 21 weeks. Furthermore, mice housed in the NASA ISS Rodent Habitat during 1g ground controls maintained or gained bone relative to mice housed in standard vivarium cages that showed significant bone mass declines. These findings suggest that housing in the Rodent Habitat with greater topological enrichment from 3D wire-mesh surfaces may promote increased mechanical loading of weight-bearing bones and maintenance of bone mass. In summary, our results indicate that in female mice approaching skeletal maturity, mechanical unloading of weight-bearing sites is the major cause of bone loss in microgravity, while sites loaded predominantly by muscle activity, such as the spine, appear unaffected. Additionally, we identified early-onset of femoral head epiphyseal plate secondary ossification as a novel spaceflight skeletal unloading effect that may lead to premature long bone growth arrest in microgravity. This study derives results from femur and vertebrae using the micro computed tomography assay.

Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen in parallel the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground as well as plus and minus hyperosmolar sodium chloride providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state suggesting mechanisms by which spaceflight may negatively affect cell fitness.

Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen in parallel the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground as well as plus and minus hyperosmolar sodium chloride providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state suggesting mechanisms by which spaceflight may negatively affect cell fitness.