Gene Expression and Structural Skeletal Responses to Long-Duration Simulated Microgravity in Rats

Spaceflight has deleterious effects on skeletal structure and function, specifically causingprofound loss in bone mass, density, and strength, as well as changes in expression levels of genes related to oxidative stress [Hyeon et al., Smith et al.]. It is known that bone resorption remains elevated after spaceflight and that bone density and strength fail to recover completely even years following spaceflight [Smith et al., Carpenter et al.]. However, our current understanding of the signaling pathways and molecular mechanisms that control bone loss and that link oxidative stress, bone resorption, and mechanical unloading of skeletal tissue is incomplete. Here, we aim to examine skeletal responses to simulated long-duration spaceflight on bone loss using the ground-based hindlimb unloading (HU) model in adult (9 months old) male rats. We hypothesized that simulated microgravity leads to the temporal regulation of oxidative-defense genes and pro-osteoclastogenic factors, showing progression and eventual plateau during long-term unloading, and that transient changes at early timepoints in these pathways precede skeletal adaptations to long-duration unloading. We will identify oxidativestress and bone resorption-related changes using global gene expression analysis (Affymetrix arrays) for both acute (within 14 days) and long-term timepoints (90 days). We will also use quantitative PCR to examine changes in expression of genes related to oxidative metabolism (e.g. Nrf2, SOD-1), bone turnover (resorption and formation markers, e.g. TRAP, osteocalcin respectively, SOST), and osteoclastogenesis (e.g. RANKL, OPG) at both early and late timepoints. We will then use detailed microarchitectural and structural analysis through microcomputed tomography to relate gene expression changes with structural changes in bone, expecting that plateaus in gene expression correlate with long-term changes in bone microarchitecture

Effects of Multiple Bouts of Long-duration Hindlimb Unloading and Recovery on Rat Plantaris Muscle

Exposure to microgravity results in a rapid reduction of muscle mass. However, few studies exist designed to examine the effects of multiple long-term exposures to microgravity with alternating recovery periods on skeletal muscle. To determine what happens to the recovery of skeletal muscle when faced with subsequent unloading and recovery periods. Male Sprague-Dawley (6 mo) were assigned to the following groups as shown in figure 1 below: 28d hindlimb unloading (1HU), 28d HU session followed by a 56d recovery bout of normal cage ambulation at 1g (1HU+REC), 2 cycles of 28d HU with a 56d recovery period between unloadings (2HU), 2 cycles of 28d HU as in the 2HU group, but followed by an additional 56d recovery at 1g (2HU+REC), and an age- and housing-matched control group (CON). On the final day of the experimental period, plantaris muscles were excised and weighed. The 1HU+REC (0.548 ± 0.012), 2HU+REC (0.562 ± 0.015), and CON (0.550 ± 0.013) showed no statistical difference (p\u3e0.05) between each other. The 1 HU (0.442 ± 0.020) and 2 HU (0.431 ± 0.011) groups were significantly less (p\u3c0.001) than recovery and aged control animals but were not significantly different from each other. The results show that the plantaris muscle presented reduction of muscle mass with initial and subsequent exposures to microgravity. However, with the recovery period, animals were able to regain lost muscle mass, similar to age-matched controls. These findings would be relevant for astronauts participating in multiple long-duration missions throughout their career

Effects of Voluntary Resistance Exercise Training During Recovery From Hindlimb Unloading on Rat Gastrocnemius Muscle

As research continues to examine the deleterious impact of long-duration spaceflight on human muscle mass and function, there remain gaps in our knowledge of muscle physiology, especially in examining how muscle’s ability to recover or rehabilitate from unloading may alter the results of multiple exposures to microgravity followed by 1g recovery. The purpose of this study was to analyze the effects of resistance exercise training of gastrocnemius muscle mass and anabolism during the initial recovery period immediately following a bout of unloading, as well as to examine the role that exercise may have on a subsequent period of weightlessness. This was achieved in rodent models of simulated spaceflight (0g), recovery (1g), and resistance training (\u3e1g) using male Sprague-Dawley (6 mo) rats randomly assigned to the following groups: 28d hindlimb unloading (HU), 28d HU followed by a 56d recovery period of normal cage ambulation at 1g (1HU+REC), 2 cycles of 28d HU with a 56d recovery period between unloading (2HU), 2HU followed by an additional 56d recovery at 1g (2HU+REC), or an age- and housing-matched control group (CON). In addition, following the initial 28d HU period, two groups of animals were given 7d recovery at 1g followed by a 7wk (3 sessions/wk) moderate-intensity, moderate-volume voluntary resistance exercise program (EX) in which the animals were trained to perform a squat-like motion with full extension of the lower limb and resistance was applied incrementally by weighted pouches over the scapula to ~65% bodyweight. At the conclusion of the experiments, gastrocnemius muscles were carefully excised, weighed, and evaluated for cumulative (24h) rates of protein synthesis (FSR). Values of both muscle mass and FSR were lower than control during periods of unloading (p\u3c0.05), but with recovery, control values were reached for mass and surpassed for FSR. Interestingly, there was no significant difference between the mass of 2HU and 2HU+EX (p\u3e0.05), and both were diminished in comparison to control animals, suggesting that benefits of exercise during periods of ambulatory reloading after disuse/microgravity may not be additive. In conclusion, our data suggest that given adequate recovery, microgravity-induced losses of muscle mass can be fully restored to control values, and this adaptational response persists even with multiple exposures. These findings may have important implications not only for career astronauts, but also for individuals who have been subjected to casting of a limb or a period of bed rest following severe injury or illness

Effects of Hindlimb Unloading and Ionizing Radiation on Murine Gene Expression in Skin and Bone

Long duration spaceflight causes a negative calcium balance and reduces bone density in astronauts. The underlying mechanisms of spaceflight-induced bone loss and the possible influences of both microgravity and radiation are not fully understood although emerging evidence suggests that these two factors may interact to result in increased bone loss. Previously, gene expression analysis of hair follicles from astronauts, as well as skin from space-flown mice, revealed changes in the expression of genes related to DNA damage and oxidative stress responses. These results resemble the responses of bone to spaceflight-like radiation and simulated weightlessness by hindlimb unloading (HU). Hence in this study, we initiated studies to determine whether skin can be used to predict the responses of bone to simulated microgravity and radiation. We examined oxidative stress and growth arrest pathways in mouse skin and long bones by measuring gene expression levels via quantitative polymerase chain reaction (qPCR). To investigate the effects of irradiation andor HU on gene expression, we used skin and femora (cortical shaft) from the following treatment groups: control (normally loaded, sham-irradiated) (CT), hindlimb unloading (HU), 56Fe radiation (IR) and both HU+IR. Animals were euthanized 11 days post-IR, and results were analyzed by 1-way ANOVA. In skin samples, Cdkn1a was decreased to the same extent in HU and HU+IR (47 of CT). In addition, HU reduced FoxO3 expression (46 of CT) and IR increased Gadd45g expression 135 compared CT in skin. But in bone, HU increased FoxO3 expression 31 compared the level of CT. These results suggest that radiation and simulated weightlessness regulated simliar oxidative stress and cell cycle arrest genes in both skin and bone, although the time course and direction of changes may differ. This research may lead to the development of a relatively simple diagnostic tool for bone loss with the advantage that hair follicles and skin are relatively easy to acquire from subjects

Acute Effects of Simulated Space Radiation and Micro-Gravity on Cancellous Bone Loss in Mice Tibiae

Space radiation and micro-gravity are the two major obstacles impeding human exploration of Mars and beyond. Long-duration space flights expose astronauts to high doses of high linear energy transfer (LET) radiation as well as prolonged periods of skeletal disuse due to weightlessness. One important consequence of both radiation exposure and micro-gravity is acute bone loss. However, biological responses to different radiation types and combined radiation and micro-gravity environments remain unknown. Thus, the purpose of this study is to compare the acute effects of different radiation species and simulated weightlessness on bone degeneration for the purpose of developing accurate risk assessments of prolonged space flight. Mouse models were used to simulate space flight-relevant doses of different radiation types as well as weightlessness via hind-limb unloading. Three groups of mice (n 9) were irradiated with 1 Gy (Gray) H+, 1 Gy 56Fe, and 1 Gy combined H+ and 56Fe (dual ion) respectively and compared to sham irradiated (n 9) and 2 Gy 56Fe irradiated positive controls (n 6). Two groups of mice (n 9) were hind-limb unloaded for three days and then either sham irradiated or dual ion irradiated respectively, followed by subsequent hind-limb unloading for 11 days. Cancellous tissue from tibiae metaphyses were harvested 11 days post-irradiation for ex vivo micro-computed tomography analysis. Microarchitecture parameters including bone volume to total volume ratio (BVTV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.S), and connectivity density (Conn.D) will be quantified using a novel automated segmentation procedure developed in our lab. The anticipated results will be instrumental in developing counter-measures against micro-gravity and radiation-induced bone loss. Moreover, possible synergistic effects may provide insight into underlying mechanisms mediating biological response

Decrements of Muscle Protein Synthesis with Unloading are Not Due to Insufficient Concentrations of Intramuscular Leucine

Skeletal muscle mass and strength play critical roles in quality of life, and significant muscle atrophy contributes to reduced function and can exacerbate some disease states. It is well-known that persistent reductions of mechanical loading in skeletal muscle result in degeneration. Generally, reductions of muscle protein synthesis are, at least in part, a major culprit with muscle loss under these conditions, and numerous countermeasures such as exercise and nutritional supplements, known to stimulate protein synthesis have been designed to maintain muscle mass under those conditions. Amino acid supplementation, particularly with branched-chain amino acids (BCAAs), has been suggested as a countermeasure to deter muscle loss during spaceflight and bed rest, suggesting that these important protein precursors are not sufficiently available to support muscle protein synthesis during mechanical unloading. The purpose of this study was to examine the effect of muscle loading/unloading on the free amino acid pool of skeletal muscle in order to determine if concomitant alterations of the amino acid availability impact known changes in muscle protein synthesis under these conditions. We hypothesized reduced protein synthesis during periods of chronic unloading would be due to rate-limiting concentrations of one or more amino acids in the cytosolic free pool. Specific amino acid concentrations of 29 amino acids commonly found in the skeletal muscle cytosolic free-pool were assessed with high-performance liquid chromatography (HPLC) in gastrocnemius muscles taken from male Sprague Dawley rats that were assigned to various hindlimb unloading groups or ambulatory controls, with and without exercise countermeasures. Of the 29 amino acids tested, only one amino acid (nonessential aspartic acid) displayed an instance of concentrations significantly below control values (p ≤ 0.05). Surprisingly, each of the BCAAs, known agonists of muscle protein synthesis, displayed significant elevations in free-pool concentrations in unloaded muscle, even though muscle protein synthesis, and ultimately muscle mass were diminished. Leucine, a potent stimulant of muscle protein synthesis was over two times higher than the leucine concentrations of control muscles, suggesting that leucine was not sufficient to stimulate protein synthesis under conditions of microgravity. It also indicates that amino acid supplementation as a countermeasure may be ineffective, as circulating levels of available BCAAs are already elevated. These results suggest that additional efforts are required to find a suitable defense against muscle atrophy due to mechanical unloading

Gene Expression and Structural Skeletal Responses to Long-Duration Simulated Microgravity in Rats

In this study, we aim to examine skeletal responses to simulated long-duration spaceflight (90 days) and weight-bearing recovery on bone loss using the ground-based hindlimb unloading (HU) model in adolescent (3-month old) male rats. We hypothesized that simulated microgravity leads to the temporal regulation of oxidative defense genes and pro-bone resorption factors, where there is a progression and eventual plateau; furthermore, early transient changes in these pathways precede skeletal adaptations

Mitigating HZE Radiation-Induced Deficits in Marrow-Derived Mesenchymal Progenitor Cells and Skeletal Structure

Future long-duration space exploration beyond the earths magnetosphere will increase human exposure to space radiation and associated risks to skeletal health. We hypothesize that oxidative stress resulting from radiation exposure causes progressive bone loss and dysfunction in associated tissue. In animal studies, increased free radical formation is associated with pathological changes in bone structure, enhanced bone resorption, reduced bone formation and decreased bone mineral density, which can lead to skeletal fragility

Ionizing Radiation Affects Gene Expression in Mouse Skin and Bone

Future long-duration space exploration beyond low earth orbit will increase human exposure to space radiation and microgravity conditions as well as associated risks to skeletal health. In animal studies, radiation exposure (greater than 1 Gy) is associated with pathological changes in bone structure, enhanced bone resorption, reduced bone formation and decreased bone mineral density, which can lead to skeletal fragility. Definitive measurements and detection of bone loss typically require large and specialized equipment which can make their application to long duration space missions logistically challenging. Towards the goal of developing non-invasive and less complicated monitoring methods to predict astronauts' health during spaceflight, we examined whether radiation induced gene expression changes in skin may be predictive of the responses of skeletal tissue to radiation exposure. We examined oxidative stress and growth arrest pathways in mouse skin and long bones by measuring gene expression levels via quantitative polymerase chain reaction (qPCR) after exposure to total body irradiation (IR). To investigate the effects of irradiation on gene expression, we used skin and femora (cortical shaft) from the following treatment groups: control (normally loaded, sham-irradiated), and IR (0.5 Gy 56Fe 600 MeV/n and 0.5 Gy 1H 150 MeV/n), euthanized at one and 11 days post-irradiation (IR). To determine the extent of bone loss, tibiae were harvested and cancellous microarchitecture in the proximal tibia quantified ex vivo using microcomputed tomography (microCT). Statistical analysis was performed using Student's t-test. At one day post-IR, expression of FGF18 in skin was significantly greater (3.8X) than sham-irradiated controls, but did not differ at 11 days post IR. Expression levels of other genes associated with antioxidant response (Nfe2l2, FoxO3 and Sod1) and the cell cycle (Trp53, Cdkn1a, Gadd45g) did not significantly differ between the control and IR groups at either time point. Radiation exposure resulted in a 27.0% increase in FGF18-positive hair follicles at one day post-IR and returned to basal levels at 11 days post-IR. A similar trend was observed from FGF18 gene expression analysis of skin. In bone (femora), there was an increase in the expression of the pro-osteoclastogenic cytokine, MCP-1, one day after IR compared to non-irradiated controls. FGF18 expression in skin and MCP- 1 expression in bone were found to be positively correlated (P less than 0.002, r=0.8779). Further, microcomputed tomography analysis of tibia from these animals showed reduced cancellous bone volume (-9.9%) at 11 days post- IR. These results suggest that measurements of early radiation induced changes in FGF18 gene expression in skin may have value for predicting subsequent loss of cancellous bone mass. Further research may lead to the development of a relatively simple diagnostic tool for bone loss, with the advantage that hair follicles and skin are relatively easy to acquire from human subjects

DEPTOR Expression Correlates with Muscle Protein Synthesis

Mammalian target of rapamycin (mTOR) has long been declared a focal point of muscle protein synthesis. mTORC1 (an mTOR complex consisting of mTOR, raptor, PRAS40, and mLST8) has been associated with regulation of protein translation in muscle, altering expression and activity levels of key downstream targets S6K1 and eIF-4E-BP1. mTORC1 has been shown to be affected by various stimuli, including nutritional status, growth factors, and mechanical loading. But in past incidents we have found disconnects in muscle protein synthesis and mTOR signaling, stimulating discussions that mTOR content and activation alone may not be able to fully account for muscle protein synthesis. Gaining popularity as a target for anti-cancer therapies, we became interested in DEPTOR, an endogenous inhibitor of mTORC1. Pharmacological inhibition of DEPTOR in cell culture and mouse studies has displayed increases of anabolic signaling in response to atrophic circumstances. We present two unique catabolic conditions in which we explore DEPTOR expression and muscle protein synthesis and demonstrate the first known data proposing that DEPTOR expression is not only influenced by physiological stimuli, including mechanical loading and insulin sensitivity, but that DEPTOR expression strongly correlates with 24-hr cumulative muscle protein synthesis rates. In one study, male Sprague Dawley rats were subjected to various conditions of musculoskeletal unloading, reloading, and overload, in which hindlimb unloading (HU) was utilized to mimic chronic disuse atrophy (28-d), followed by ambulatory reloading (56-d post HU) with and without the addition of resistance exercise prescribed to assist in recovery (3 sessions/wk for 7-wks; progressive increases in added resistance up to ~60% BW). DEPTOR expression was assessed via Immunoblotting. 24-hr cumulative muscle protein synthesis (FSR) was measured via stable isotope labeling and quantified by gas chromatogram/mass spectrometry. DEPTOR demonstrated a strong negative correlation with FSR in the gastrocnemius (r = - 0.93261; p \u3c0.01). In our second study, male obese Zucker rats were divided into their lean and obese phenotypes, as well as placed into sedentary and resistance exercised groups. DEPTOR and FSR were assessed as described above following operant conditioning and four progressive exercise sessions over 9-d. Gastrocnemius DEPTOR/FSR was again significant (r = - 0.75723; p\u3c0.01). Collectively, these results are the first to associate physiologic changes in DEPTOR expression with alterations of FSR, which may have important implications towards the design of therapeutic targets for the control of muscle mass or in evaluating muscle anabolism