Hypergravity Exposure Research Articles

A concentric 3D-printed centrifuge rotor to study the effects of hypergravity on small organisms at three different but concurrent radial accelerations

For life to reach space, it must first escape Earth's gravity—hence exposure to hypergravity during launch, and interplanetary acceleration and deceleration. Long-term colonisation of space requires the establishment of sustainable, in situ, food production systems. Earthworms may be involved as a regolith/soil ameliorant, a source of protein, and for organic waste conversion. Earthworms, however, have hydrostatic skeletons that may be susceptible to hypergravity exposure. We investigated whether earthworms (Eisenia andrei) would survive prolonged centrifugal hypergravity using a table-top centrifuge. Our intention was to concurrently test three different hypergravity treatments (plus a 1 G control). Our concentric rotor concept, with appropriate design adaptations, allows concurrent testing of three different gravity treatments on small- and microorganisms, cell cultures, plants, microcosm, and chemical/biochemical reactions for extended periods of hours to months. Our concentric concept therefore is easily adaptable and may facilitate other terrestrial-based research on chemical, molecular, and organismal levels.•We designed a 3D-printed centrifuge rotor with three concentric rings, each ring with four separate 125 mL compartments.•This design was tested with an 8-day pilot run with four young worms per container at 1.7 G, 2.5 G, and 3.1 G, with a 1 G control.•All worms at all treatments gained body mass, the mean increases of which were almost indistinguishable between treatments, illustrating the utility of the design.

Is sleep affected after microgravity and hypergravity exposure? A pilot study.

Sleep is known to be affected in space travel and in residents of the international space station. But little is known about the direct effects of gravity changes on sleep, if other factors, such as sleep conditions, are kept constant. Here, as a first exploration, we investigated sleep before and after exposure to short bouts of microgravity and hypergravity during parabolic flights. Sleep was measured through actigraphy and self-report questionnaires in 20 healthy men and women before and after parabolic flight. Higher sleep fragmentation and more awakenings were found in the night after the flight as compared with the night before, which was discrepant from participants' reports showing better and longer sleep after the parabolic flight. Variable levels of experience with parabolic flights did not affect the results, nor did levels of scopolamine, a medication typically taken against motion sickness. Pre-existing sleep problems were related to sleep fragmentation and wake after sleep onset by a quadratic function such that participants with more sleep problems showed lower levels of sleep fragmentation and nighttime awakenings than those with few sleep problems. These novel findings, though preliminary, have important implications for future research, directed at prevention and treatment of sleep problems and their daytime consequences in situations of altered gravity, and possibly in the context of other daytime vestibular challenges as well.

An experimentally informed computational model of neurovestibular adaptation to altered gravity.

Transitions to altered gravity environments result in acute sensorimotor impairment for astronauts, leading to serious mission and safety risks in the crucial first moments in a new setting. Our understanding of the time course and severity of impairment in the early stages of adaptation remains limited and confounded by unmonitored head movements, which are likely to impact the rate of adaptation. Here, we aimed to address this gap by using a human centrifuge to simulate the first hour of hypergravity (1.5g) exposure and the subsequent 1g readaptation period, with precisely controlled head tilt activity. We quantified head tilt overestimation via subjective visual vertical and found ∼30% tilt overestimation that did not decrease over the course of 1h of exposure to the simulated gravity environment. These findings extended the floor of the vestibular adaptation window (with controlled vestibular cueing) to 1h of exposure to altered gravity. We then used the empirical data to inform a computational model of neurovestibular adaptation to changes in the magnitude of gravity, which can offer insight into the adaptation process and, with further tuning, can be used to predict the temporal dynamics of vestibular-mediated misperceptions in altered gravity.

Handheld Sonographic Cardiovascular Imaging Under Hypergravity Conditions.

INTRODUCTION: Real-time cardiovascular imaging during hypergravity exposure has been historically limited by technological and physical challenges. Previous efforts at sonographic hypergravity imaging have used fixed ultrasound probes; the use of hand-held ultrasound, particularly performed by minimally trained laypersons, has been less explored. Here we will discuss handheld sonography to self-visualize carotid vascular and cardiac changes during hypergravity.METHODS: Three subjects with variable ultrasound experience ranging from no familiarity to extensive clinical experience used handheld ultrasound at rest and under stepwise +Gz hypergravity exposures (maximum +3.5 Gz) to visualize carotid vascular changes. Subxiphoid cardiac ultrasound was obtained by the most experienced subject. Subjects had variable prior hypergravity experience; all were trained in anti-G straining techniques. Sonographically inexperienced subjects underwent a brief (< 5 min) familiarization with the ultrasound probe, user interface, and desirable viewing window immediately prior to centrifugation; real-time coaching was provided. Ultrasound images were correlated to self-reported symptoms and hemodynamic data.RESULTS: Handheld ultrasound performed as desired; all subjects were successful at obtaining ultrasound images with adequate capture of windows of interest. Subxiphoid imaging efforts were limited by probe overheating and associated with variable quality of imaging due to probe displacement from straining techniques; the subject noted transient, mild discomfort and ecchymosis after imaging in the subxiphoid region.DISCUSSION: Even individuals with minimal or no ultrasound experience successfully obtained usable images under centrifuge conditions. While there were some limitations, this technical demonstration provides initial validation of handheld sonography as an available tool for real-time cardiovascular imaging in a hypergravity environment.Blue RS, Ong KM. Handheld sonographic cardiovascular imaging under hypergravity conditions. Aerosp Med Hum Perform. 2024; 95(3):158-164.

Increased H3K9me3 and F-Actin Reorganization in the Rapid Adaptive Response to Hypergravity in Human T Lymphocytes.

Our study explored the impact of hypergravity on human T cells, which experience additional acceleration forces beyond Earth's gravity due to various factors, such as pulsatile blood flow, and technology, such as high-performance aircraft flights or spaceflights. We investigated the histone modifications Histone 3 lysine 4 and 9 trimethylation (H3K4me3 and H3K9me3, respectively), as well as the structural and cytoskeletal organization of Jurkat T cells in response to hypergravity. Histone modifications play a crucial role in gene regulation, chromatin organization and DNA repair. In response to hypergravity, we found only minimal changes of H3K4me3 and a rapid increase in H3K9me3, which was sustained for up to 15 min and then returned to control levels after 1 h. Furthermore, rapid changes in F-actin fluorescence were observed within seconds of hypergravity exposure, indicating filament depolymerization and cytoskeletal restructuring, which subsequently recovered after 1 h of hypergravity. Our study demonstrated the rapid, dynamic and adaptive cellular response to hypergravity, particularly in terms of histone modifications and cytoskeletal changes. These responses are likely necessary for maintaining genome stability and structural integrity under hypergravity conditions as they are constantly occurring in the human body during blood cell circulation.

The importance of gravity vector on adult mammalian organisms: Effects of hypergravity on mouse testis.

In the age of space exploration, the effect of hypergravity on human physiology is a relatively neglected topic. However, astronauts have several experiences of hypergravity during their missions. The main disturbance of altered gravity can be imputed to cell cytoskeleton alteration and physiologic homeostasis of the body. Testis has proved to be a particularly sensible organ, subject to environmental alteration and physiological disturbance. This makes testis an organ eligible for investigating the alteration following exposure to altered gravity. In our study, mice were exposed to hypergravity (3g for 14 days) in the Large Diameter Centrifuge machine (ESA, Netherland). We have observed a morphological alteration of the regular architecture of the seminiferous tubules of testis as well as an altered expression of factors involved in the junctional complexes of Sertoli cells, responsible for ensuring the morpho-functional integrity of the organ. The expression of key receptors in physiological performance, such as Androgen Receptors and Interstitial Cells Stimulating Hormone receptors, was found lower expressed. All these findings indicate the occurrence of altered physiological organ performance such as the reduction of the spermatozoa number and altered endocrine parameters following hypergravity exposure.

Peripheral skin cooling during hyper-gravity: hemodynamic reactions.

Introduction: Orthostatic dysregulation occurs during exposure to an increased gravitational vector and is especially common upon re-entering standard Earth gravity (1g) after an extended period in microgravity (0g). External peripheral skin cooling (PSC) has recently been described as a potent countermeasure against orthostatic dysregulation during heat stress and in lower body negative pressure (LBNP) studies. We therefore hypothesized that PSC may also be an effective countermeasure during hyper-gravity exposure (+Gz). Methods: To investigate this, we designed a randomized short-arm human centrifuge (SAHC) experiment ("Coolspin") to investigate whether PSC could act as a stabilizing factor in cardiovascular function during +Gz. Artificial gravity between +1g and +4g was generated by a SAHC. 18 healthy male volunteers completed two runs in the SAHC. PSC was applied during one of the two runs and the other run was conducted without cooling. Each run consisted of a 10-min baseline trial followed by a +Gz step protocol marked by increasing g-forces, with each step being 3min long. The following parameters were measured: blood pressure (BP), heart rate (HR), stroke volume (SV), total peripheral resistance (TPR), cardiac output (CO). Furthermore, a cumulative stress index for each subject was calculated. Results: +Gz led to significant changes in primary as well as in secondary outcome parameters such as HR, SV, TPR, CO, and BP. However, none of the primary outcome parameters (HR, cumulative stress-index, BP) nor secondary outcome parameters (SV, TPR, CO) showed any significant differences-whether the subject was cooled or not cooled. Systolic BP did, however, tend to be higher amongst the PSC group. Conclusion: In conclusion, PSC during +Gz did not confer any significant impact on hemodynamic activity or orthostatic stability during +Gz. This may be due to lower PSC responsiveness of the test subjects, or an insufficient level of body surface area used for cooling. Further investigations are warranted in order to comprehensively pinpoint the exact degree of PSC needed to serve as a useful countermeasure system during +Gz.

Hypergravity Increases Blood-Brain Barrier Permeability to Fluorescent Dextran and Antisense Oligonucleotide in Mice.

The earliest effect of spaceflight is an alteration in vestibular function due to microgravity. Hypergravity exposure induced by centrifugation is also able to provoke motion sickness. The blood-brain barrier (BBB) is the crucial interface between the vascular system and the brain to ensure efficient neuronal activity. We developed experimental protocols of hypergravity on C57Bl/6JRJ mice to induce motion sickness and reveal its effects on the BBB. Mice were centrifuged at 2× g for 24 h. Fluorescent dextrans with different sizes (40, 70 and 150 kDa) and fluorescent antisense oligonucleotides (AS) were injected into mice retro-orbitally. The presence of fluorescent molecules was revealed by epifluorescence and confocal microscopies in brain slices. Gene expression was evaluated by RT-qPCR from brain extracts. Only the 70 kDa dextran and AS were detected in the parenchyma of several brain regions, suggesting an alteration in the BBB. Moreover, Ctnnd1, Gja4 and Actn1 were upregulated, whereas Jup, Tjp2, Gja1, Actn2, Actn4, Cdh2 and Ocln genes were downregulated, specifically suggesting a dysregulation in the tight junctions of endothelial cells forming the BBB. Our results confirm the alteration in the BBB after a short period of hypergravity exposure.

Chronic Hypergravity Induces a Modification of Histone H3 Lysine 27 Trimethylation at TCRβ Locus in Murine Thymocytes.

Gravity changes are major stressors encountered during spaceflight that affect the immune system. We previously evidenced that hypergravity exposure during gestation affects the TCRβ repertoire of newborn pups. To identify the mechanisms underlying this observation, we studied post-translational histone modifications. We first showed that among the four studied post-translational histone H3 modifications, only lysine 27 trimethylation (H3K27me3) is downregulated in the thymus of mice exposed to 2× g for 21 days. We then asked whether the TCRβ locus chromatin structure is altered by hypergravity exposure. ChIP studies performed on four Vβ segments of the murine double-negative SCIET27 thymic cell line, which corresponds to the last maturation stage before V(D)J recombination, revealed increases in H3K27me3 after 2× g exposure. Finally, we evaluated the implication for the EZH2 methyltransferase in the regulation of the H3K27me3 level at these Vβ segments by treating SCIET27 cells with the GSK126-specific inhibitor. These experiments showed that the downregulation of H3K27me3 contributes to the regulation of the Vβ germline transcript expression that precedes V(D)J recombination. These data show that modifications of H3K27me3 at the TCRβ locus likely contribute to an explanation of why the TCR repertoire is affected by gravity changes and imply, for the first time, EZH2 in the regulation of the TCRβ locus chromatin structure.

Effects of short-term hypergravity on hematopoiesis and vasculogenesis in embryonic zebrafish

Microgravity and hypergravity-induced changes affect both molecular and organismal responses as demonstrated in various animal models. In addition to its inherent advantages, zebrafish have been shown to be incredibly resilient to altered gravity conditions. To understand the effects of altered gravity on animal physiology, especially the cardiovascular system, we used 2 h centrifugations to simulate short-term hypergravity and investigated its effects on zebrafish development. Morphological and in situ hybridization observations show a comparable overall development in both control and treated embryos. Spatiotemporal analysis revealed varied gene expression patterns across different developmental times. Genes driving primitive hematopoiesis (tal1, gata1) and vascular specificity (vegf, etv2) displayed an early onset of expression following hypergravity exposure. Upregulated expression of hematopoiesis-linked genes, such as runx1, cmyb, nos, and pdgf family demonstrate short-term hypergravity to be a factor inducing definitive hematopoiesis through a combinatorial mechanism. We speculate that these short-term hypergravity-induced physiological changes in the developing zebrafish embryos constitute a rescue mechanism to regain homeostasis.

Photoactive chlorpromazine and promazine drugs exposed to hypergravity conditions after interaction with UV laser radiation

Long-term space missions must be prepared to provide means to treat astronauts' illnesses that could occur at take-off, during flights or arriving on other planets. This could be achieved using multipurpose medicines. Phenothiazine derivatives, normally used to treat mental and emotional disorders, are photosensitive drugs and in solutions undergo molecular modifications by exposure to UV laser radiation, which leads to transformation of a parent-compound into photoproducts with increased antimicrobial activity when compared to unirradiated solutions. Since space missions involve, even if only for short durations, hypergravity environment transitions, their effects on unirradiated and laser irradiated chlorpromazine and promazine aqueous solutions were studied in this paper. The experiment was performed at the European Space Agency's Large Diameter Centrifuge, subjecting solutions at 20 times Earth's gravitational acceleration. Since, generally, little is known about medicines exposed to high-g levels, this research brings an insight into the impact of hypergravity on phenothiazines. To evaluate drug stability, samples were assessed pre- and post-hypergravity treatment by pH measurements, UV-Vis-NIR/FTIR spectroscopy and thin layer chromatography. No significant changes between uncentrifuged and centrifuged samples were evidenced subsequent to hypergravity exposure, emphasising the stability of unirradiated and laser irradiated phenothiazines, which may allow their use during future space missions.

Hypergravity Load Modulates Acetaminophen Nephrotoxicity via Endoplasmic Reticulum Stress in Association with Hepatic microRNA-122 Expression.

Hypergravity conditions may subject the kidney to intrinsic stress and lead to hemodynamic kidney dysfunction. However, the mechanisms underlying this phenomenon remain unclear. Accumulation of unfolded proteins in the endoplasmic reticulum (i.e., ER stress) is often observed in kidney diseases. Therefore, this study investigated whether hypergravity stress alters acetaminophen-induced renal toxicity in vivo, as well as the molecular mechanisms involved in this process. C57BL/6 mice were submitted to one or three loads of +9 Gx hypergravity for 1 h with or without acetaminophen (APAP) treatment. The protein levels of cell survival markers, including pAKT and pCREB, were decreased in the kidney after acetaminophen treatment with a single hypergravity load. Additionally, the combined treatment increased kidney injury markers, serum creatinine, and Bax, Bcl2, and Kim-1 transcript levels and enhanced ER stress-related markers were further. Moreover, multiple hypergravity loads enabled mice to overcome kidney injury, as indicated by decreases in serum creatinine content and ER stress marker levels, along with increased cell viability indices. Similarly, multiple hypergravity loads plus APAP elevated miR-122 levels in the kidney, which likely originated from the liver, as the levels of primary miR-122 increased only in the liver and not the kidney. Importantly, this phenomenon may contribute to overcoming hypergravity-induced kidney injury. Taken together, our results demonstrate that APAP-exposed mice submitted to a single load of hypergravity exhibited more pronounced kidney dysfunction due to increased ER stress, which may be overcome by repetitive hypergravity loads presumably due to increased production of miR-122 in the liver. Thus, our study provides novel insights into the mechanisms by which hypergravity stress plus APAP medication induce kidney injury, which may be overcome by repeated hypergravity exposure.

Hypergravity-induced changes in actin response of breast cancer cells to natural killer cells

Although immunotherapy holds promising cytotoxic activity against lymphoma or leukemia, the immunosuppressive mechanisms of solid tumors remain challenging. In this study, we developed and applied a hypergravity exposure system as a novel strategy to improve the responsiveness of breast cancer cells to natural killer (NK) cells for efficient immunotherapy. Following exposure to hypergravity, either in the presence or absence of NK cells, we investigated for changes in the cell cytoskeletal structure, which is related to the F-actin mediated immune evasion mechanism (referred to as “actin response”) of cancer cells. Breast cancer cell line MDA-MB-231 cells were exposed thrice to a 20 min hypergravitational condition (10 × g), with a 20 min rest period between each exposure. The applied hypergravity induces changes in the intracellular cytoskeleton structure without decreasing the cell viability but increasing the cytotoxicity of MDA-MB-231 from 4 to 18% (4.5-fold) at a 3:1 ratio (NK-to-target). Analyses related to F-actin further demonstrate that the applied hypergravity results in rearrangement of the cytoskeleton, leading to inhibition of the actin response of MDA-MB-231. Taken together, our results suggest that the mechanical load increases through application of hypergravity, which potentially improves efficiency of cell-based immunotherapies by sensitizing tumors to immune cell-mediated cytotoxicity.

Gravitational Experimental Platform for Animal Models, a New Platform at ESA's Terrestrial Facilities to Study the Effects of Micro- and Hypergravity on Aquatic and Rodent Animal Models.

Using rotors to expose animals to different levels of hypergravity is an efficient means of understanding how altered gravity affects physiological functions, interactions between physiological systems and animal development. Furthermore, rotors can be used to prepare space experiments, e.g., conducting hypergravity experiments to demonstrate the feasibility of a study before its implementation and to complement inflight experiments by comparing the effects of micro- and hypergravity. In this paper, we present a new platform called the Gravitational Experimental Platform for Animal Models (GEPAM), which has been part of European Space Agency (ESA)’s portfolio of ground-based facilities since 2020, to study the effects of altered gravity on aquatic animal models (amphibian embryos/tadpoles) and mice. This platform comprises rotors for hypergravity exposure (three aquatic rotors and one rodent rotor) and models to simulate microgravity (cages for mouse hindlimb unloading and a random positioning machine (RPM)). Four species of amphibians can be used at present. All murine strains can be used and are maintained in a specific pathogen-free area. This platform is surrounded by numerous facilities for sample preparation and analysis using state-of-the-art techniques. Finally, we illustrate how GEPAM can contribute to the understanding of molecular and cellular mechanisms and the identification of countermeasures.

Exposure to hypergravity during zebrafish development alters cartilage material properties and strain distribution.

Vertebrates have adapted to life on Earth and its constant gravitational field, which exerts load on the body and influences the structure and function of tissues. While the effects of microgravity on muscle and bone homeostasis are well described, with sarcopenia and osteoporosis observed in astronauts returning from space, the effects of shorter exposures to increased gravitational fields are less well characterized. We aimed to test how hypergravity affects early cartilage and skeletal development in a zebrafish model. We exposed zebrafish to 3 g and 6 g hypergravity from three to five days post-fertilization, when key events in jaw cartilage morphogenesis occur. Following this exposure, we performed immunostaining along with a range of histological stains and transmission electron microscopy (TEM) to examine cartilage morphology and structure, atomic force microscopy (AFM) and nanoindentation experiments to investigate the cartilage material properties, and finite element modelling to map the pattern of strain and stress in the skeletal rudiments. We did not observe changes to larval growth, or morphology of cartilage or muscle. However, we observed altered mechanical properties of jaw cartilages, and in these regions we saw changes to chondrocyte morphology and extracellular matrix (ECM) composition. These areas also correspond to places where strain and stress distribution are predicted to be most different following hypergravity exposure. Our results suggest that altered mechanical loading, through hypergravity exposure, affects chondrocyte maturation and ECM components, ultimately leading to changes to cartilage structure and function. Cite this article: Bone Joint Res2021;10(2):137-148.

Simulated Hypergravity Activates Hemostasis in Healthy Volunteers.

BackgroundHypergravity may promote human hemostasis thereby increasing thrombotic risk. Future touristic suborbital spaceflight will expose older individuals with chronic medical conditions, who are at much higher thromboembolic risk compared with professional astronauts, to hypergravity. Therefore, we tested the impact of hypergravity on hemostasis in healthy volunteers undergoing centrifugation.Methods and ResultsWe studied 20 healthy seated men before and after 15 minutes under 3 Gz hypergravity on a long‐arm centrifuge. We obtained blood samples for hemostasis testing before, immediately after, and 30 minutes after centrifugation. Tests included viscoelastic thromboelastometry, platelet impedance aggregometry, endothelial activation markers, blood rheology testing, microparticle analyses, and clotting factor analysis. Exposure to hypergravity reduced plasma volume by 12.5% (P=0.002) and increased the red blood cell aggregation index (P<0.05). With hypergravity, thrombelastographic clotting time of native blood shortened from 719±117 seconds to 628±89 seconds (P=0.038) and platetet reactivity increased (P=0.045). Hypergravity shortened partial thromboplastin time from 28 (26–29) seconds to 25 (24–28) seconds (P<0.001) and increased the activity of coagulation factors (eg, factor VIII 117 [93–134] versus 151 [133–175] %, P<0.001). Tissue factor concentration was 188±95 pg/mL before and 298±136 pg/mL after hypergravity exposure (P=0.023). Antithrombin (P=0.005), thrombin‐antithrombin complex (P<0.001), plasmin‐alpha2‐antiplasmin complex (0.002), tissue‐plasminogen activatior (P<0.001), and plasminogen activator inhibitor‐1 (P=0.002) increased with centrifugation. Statistical adjustment for plasma volume attenuated changes in coagulation.ConclusionsHypergravity triggers low‐level hemostasis activation through endothelial cell activation, increased viscoelasticity, and augmented platelet reactivity, albeit partly counteracted through endogenous coagulation inhibitors release. Hemoconcentration may contribute to the response.

Roles of Physiological Responses and Anthropometric Factors on the Gravitational Force Tolerance for Occupational Hypergravity Exposure.

Gravity in the head-to-toe direction, known as +Gz (G force), forces blood to pool in the lower body. Fighter pilots experience decreases in blood pressure when exposed to hypergravity in flight. Human centrifuge has been used to examine the G tolerance and anti-G straining maneuver (AGSM) techniques of military pilots. Some factors that may affect G tolerance have been reported but are still debated. The aim of this study was to investigate the physiological responses and anthropometric factors correlated with G tolerance. We retrospectively reviewed the training records of student pilots who underwent high G training. Variables were collected to examine their correlations with the outcome of 7.5G sustained for 15 s (7.5G profile). There were 873 trainees who underwent 7.5G profile training, 44 trainees (5.04%) could not sustain the test for 15 s. The group with a small heart rate (HR) increase (less than 10%) during the first 1–5 s of the 7.5G profile had a nearly ten-fold higher failing chance compared with the large HR increase group (adjusted odds ratio: 9.91; 95% confidence interval: 4.11–23.88). The chances of failure were inversely related to the HR increase percentage (p for trend <0.001). Factors, including body mass index, relaxed and straining G tolerance, and AGSM, were found to be negatively correlated with the outcome.

Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice

Investigating protein abundance profiles is important to understand the differences in the slow and fast skeletal muscle characteristics. The profiles in soleus (Sol) and extensor digitorum longus (EDL) muscles in mice exposed to 1 g or 3 g for 28 d were compared. The biological implications of the profiles revealed that hypergravity exposure activated a larger number of pathways involved in protein synthesis in Sol. In contrast, the inactivation of signalling pathways involved in oxidative phosphorylation were conspicuous in EDL. These results suggested that the reactivity of molecular pathways in Sol and EDL differed. Additionally, the levels of spermidine synthase and spermidine, an important polyamine for cell growth, increased in both muscles following hypergravity exposure, whereas the level of spermine oxidase (SMOX) increased in EDL alone. The SMOX level was negatively correlated with spermine content, which is involved in muscle atrophy, and was higher in EDL than Sol, even in the 1 g group. These results indicated that the contribution of SMOX to the regulation of spermidine and spermine contents in Sol and EDL differed. However, contrary to expectations, the difference in the SMOX level did not have a significant impact on the growth of these muscles following hypergravity exposure. SignificanceThe skeletal muscle-specific protein abundance profiles result in differences in the characteristics of slow and fast skeletal muscles. We investigated differences in the profiles in mouse slow-twitch Sol and fast-twitch EDL muscles following 28-d of 1 g and 3 g exposure by LC-MS/MS analysis and label-free quantitation. A two-step solubilisation of the skeletal muscle proteins increased the coverage of proteins identified by LC-MS/MS analysis. Additionally, this method reduced the complexity of samples more easily than protein or peptide fractionation by SDS-PAGE and offline HPLC while maintaining the high operability of samples and was reproducible. A larger number of hypergravity-responsive proteins as well as a prominent increase in the wet weights was observed in Sol than EDL muscles. The biological implications of the difference in the protein abundance profiles in 1 g and 3 g groups revealed that the reactivity of each molecular pathway in Sol and EDL muscles to hypergravity exposure differed significantly. In addition, we found that the biosynthetic and interconversion pathway of polyamines, essential factors for cell growth and survival in mammals, was responsive to hypergravity exposure; spermidine and spermine contents in Sol and EDL muscles were regulated by different mechanisms even in the 1 g group. However, our results indicated that the difference in the mechanism regulating polyamine contents is unlikely to have a significant effect on the differences in Sol and EDL muscle growth following hypergravity exposure.

Impact of g-Load Shift on Temporal Expression Pattern of Apoptosis-linked Proteins in the Rat Mammary Gland

Abstract Alteration in gravitational load impacts homeorhetic response in rat dams which affects neonatal pup survival. However, the effects of hypergravity (HG) exposure on the abundance of apoptosis-associated proteins in mammary epithelial cells (MECs) have not been characterized. Therefore, we examined whether chronic exposure to HG from midpregnancy alters the abundance of proapoptotic proteins in MECs during the late pregnancy and early lactation. A group of pregnant Sprague Dawley rats were exposed to either HG (2g) or normo-gravity (1g: stationary control [SC]) from days 11 to 20 of gestation (G20). Another set of animals were investigated from day 11 of pregnancy through days 1 and 3 (P1 and P3, respectively) postpartum. Quantitative (pixels [px]/lobule) immunohistochemistry at G20 of Cleaved Caspase-3 (CC-3), Tumor Protein p53 (P53), and vitamin D receptor (VDR) revealed that all the three proteins were increased (p&lt;0.01) in HG rats compared to SC animals. At P1, the HG group had twofold higher (p&lt;0.001) expression of CC-3 relative to the SC group. Approximately, 50% (p&lt;0.001) more VDR was detected in the HG cohorts than SC at P3. These results suggest that a shift in g-load upregulates the expression of key proapoptotic proteins during the pregnancy-to-lactation transition in the rat MECs.

Thermoregulation through the glutamatergic neurons in vestibular nucleus complex

The vestibular system is one of the sensory systems which contributes to the sense of balance and spatial orientation. This also participates in the autonomic nervous response, which stimulation of the peripheral vestibular organs induces sympathoexcitation. This response is observed in both rodents and human beings, and we have reported that vestibular system contributes to the arterial pressure response during postural change as one of the feedforward control system (Morita, Abe et al., Sci Rep, 2016). In the other autonomic nervous responses, stimulation of the otolith organs in the inner ear induces hypothermia; exposure to the hypergravity environment decreases body temperature by 8 degree Celsius in mice. This response was attenuated by vestibular lesion or genetic deletion of otolith. In order to elucidate the central mechanism of hypergravity‐induced hypothermia, we examined the role of Vglut2, Vgat and ChAT neurons in vestibular nucleus complex (VNC) on thermoregulation in mice. We used Vglut2‐cre, Vgat‐cre, and ChAT‐cre mice to manipulate each neuron in VNC. Based on the previous work (Abe et al., Nat Neurosci, 2017), AAV‐DIO type viral vector was injected in VNC to express photo sensors (channelrhodopsin or archaerhodopsin) for optogenetics or hM3D(Gq) for chemogenetics. Unilateral photostimulation of the Vglut2 neurons in VNC induced body tilt to the ipsilateral side, while photoinhibition induced body tilt to the contralateral side. In Vgat‐cre mice, opposite response was observed compared with Vglut2‐cre mouse. Photostimulation of ChAT neurons did not show any responses. Chemogenetics stimulation of Vglut2 neurons showed hypothermia with increasing in activity, while Vgat stimulation increased body temperature with decreasing in activity. Deletion of Vglut2 neurons in VNC using AAV2–DIO–taCasp3–TEVp attenuated hypothermia induced by hypergravity exposure. On the other hand, hypothermia was still observed by deletion of Vgat neurons in VNC. Interestingly, chemogenetics stimulation of Vglut2 neurons in VNC 2 days before hypergravity exposure, the hypothermia was attenuated. Taken together, hypothermia induced by hypergravity exposure is due to activation of Vglut2 neurons in VNC.Support or Funding InformationGrant‐in‐Aid for Scientific Research on Innovative Areas 15H05940 (HM) and 18H04974 (CA), Grant‐in‐Aid for Scientific Research (C) 18K06850 (HM), The Takeda Science Foundation (CA), The Uehara Memorial Foundation (CA), Kato Memorial Bioscience Foundation (CA), The Nakatomi Foundation (CA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.