Hindlimb Unweighting Research Articles

Estrogen receptor α regulates phenotypic switching and proliferation of vascular smooth muscle cells through the NRF1-OMI-mitophagy signaling pathway under simulated microgravity.

Vascular remodeling during microgravity exposure results in postflight cardiovascular deconditioning and orthostatic intolerance in astronauts. To clarify the underlying mechanism, we investigated whether estrogen receptor α (ERα)-NRF1-OMI-mitophagy signaling was involved in the dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) under simulated microgravity. Phenotypic markers, mtDNA copy number and mitochondrial biogenesis, mitochondrial dynamics and mitophagy in rat thoracic artery smooth muscle cells were examined. Four-week hindlimb unweighting (HU) was used to simulate microgravity in rats and 10% serum was used to induce VSMCs dedifferentiation in vitro. The effects of ERα-NRF1-OMI signaling on mitophagy, phenotypic switching and proliferation of VSMCs, and cerebrovascular remodeling in HU rats were studied by genetic manipulation and chronic drug intervention. We found that ERα is positively associated with contractile phenotype switching but inversely correlated with synthetic phenotype switching and proliferation of VSMCs both in vivo and in vitro. During the dedifferentiation process of VSMCs, reduced mtDNA copy number, disturbed mitochondrial biogenesis and respiration, and perturbed fission-fusion-mitophagy signaling were detected, which were reversed by ERα overexpression. Mechanistically, the ERα downstream protein OMI preserved the mitochondrial Parkin level by increasing its protein stability, thereby protecting mitophagy. In line with this, we found that activating ERα signaling by propyl pyrazole triol (PPT) could alleviate the synthetic phenotype switching and proliferation of HU rat cerebral VSMCs by reestablishing fission-fusion-mitophagy hemostasis. The current study clarified a novel mechanism by which inhibited ERα-NRF1-OMI-mitophagy signaling resulted in synthetic phenotype switching and proliferation of VSMCs and cerebrovascular remodeling under simulated microgravity.

The OPG/RANKL/RANK system modulates calcification of common carotid artery in simulated microgravity rats by regulating NF-κB pathway.

Functional and structural adaptation of common carotid artery could be one of the important causes of postflight orthostatic intolerance after microgravity exposure, the mechanisms of which remain unclear. Recent evidence indicates that long-term spaceflight increases carotid artery stiffness, which might present a high risk to astronaut health and postflight working ability. Studies have suggested that vascular calcification is a common pathological change in cardiovascular diseases that is mainly manifested as an increase in vascular stiffness. Therefore, this study investigated whether simulated microgravity induces calcification of common carotid artery and to elucidate the underlying mechanisms. Four-week-old hindlimb-unweighted (HU) rats were used to simulate the deconditioning effects of microgravity on cardiovascular system. We found that simulated microgravity induced vascular smooth muscle cell (VSMC) osteogenic differentiation and medial calcification, increased receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) and RANK expression, and enhanced NF-κB activation in rat common carotid artery. In vitro activation of the RANK pathway with exogenous RANKL, a RANK ligand, increased RANK and osteoprotegerin (OPG) expression in HU rats. Moreover, the expression of osteogenic markers and activation of NF-κB in HU rats were further enhanced by exogenous RANKL but suppressed by the RANK inhibitor osteoprotegerin fusion protein (OPG-Fc). These results indicated that the OPG/RANKL/RANK system modulates VSMC osteogenic differentiation and medial calcification of common carotid artery in simulated microgravity rats by regulating the NF-kB pathway.

FGGY carbohydrate kinase domain containing is expressed and alternatively spliced in skeletal muscle and attenuates MAP kinase and Akt signaling.

Skeletal muscle atrophy can result from a range of physiological conditions, including denervation, immobilization, hindlimb unweighting, and aging. To better characterize the molecular genetic events of atrophy, a microarray analysis revealed that FGGY carbohydrate kinase domain containing (Fggy) is expressed in skeletal muscle and is induced in response to denervation. Bioinformatic analysis of the Fggy gene locus revealed two validated isoforms with alternative transcription initiation sites that we have designated Fggy-L-552 and Fggy-S-387. Additionally, we cloned two novel alternative splice variants, designated Fggy-L-482 and Fggy-S-344, from cultured muscle cells suggesting that at least four Fggy splice variants are expressed in skeletal muscle. Quantitative RT-PCR was performed using RNA isolated from muscle cells and primers designed to distinguish the four alternative Fggy transcripts and found that the Fggy-L transcripts are more highly expressed during myoblast differentiation, while the Fggy-S transcripts show relatively stable expression in proliferating myoblasts and differentiated myotubes. Confocal fluorescent microscopy revealed that the Fggy-L variants appear to localize evenly throughout the cytoplasm, while the Fggy-S variants produce a more punctuate cytoplasmic localization pattern in proliferating muscle cells. Finally, ectopic expression of Fggy-L-552 and Fggy-S-387 resulted in inhibition of muscle cell differentiation and attenuation of the MAP kinase and Akt signaling pathways. The identification and characterization of novel genes such as Fggy helps to improve our understanding of the molecular and cellular events that lead to atrophy and may eventually result in the identification of new therapeutic targets for the treatment of muscle wasting.

MERG1A Protein Abundance Increases in the Atrophied Skeletal Muscle of Denervated Mice, But Does Not Affect NFκB Activity.

Skeletal muscle atrophy may occur with disease, injury, decreased muscle use, starvation, and normal aging. No reliably effective treatments for atrophy are available, thus research into the mechanisms contributing to muscle loss is essential. The ERG1A K+ channel contributes to muscle loss by increasing ubiquitin proteasome proteolysis (UPP) in the skeletal muscle of both unweighted and cachectic mice. Because the mechanisms which produce atrophy vary based upon the initiating factor, here we investigate atrophy produced by denervation. Using immunohistochemistry and immunoblots, we demonstrate that ERG1A protein abundance increases significantly in the Gastrocnemius muscle of rodents 7 days after both sciatic nerve transection and hind limb unweighting. Further, we reveal that ectopic expression of a Merg1a encoded plasmid in normal mouse Gastrocnemius muscle has no effect on activity of the NFκB transcription factor family, a group of proteins which contribute to muscle atrophy by modulation of the UPP. Further, although NFκB activity increases significantly after denervation, we show that expression of a plasmid encoding a dominant negative Merg1a mutant in Gastrocnemius muscle prior to denervation, has no effect on NFκB activity. Thus, although the ERG1A K+ channel increases UPP, it does not do so through modulation of NFκB transcription factors.

Mitochondrial Oxidative Stress Enhances Vasoconstriction by Altering Calcium Homeostasis in Cerebrovascular Smooth Muscle Cells under Simulated Microgravity

ObjectiveExposure to microgravity results in postflight cardiovascular deconditioning in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been reported during this process. To elucidate the mechanism for this condition, we investigated whether mitochondrial oxidative stress regulates calcium homeostasis and vasoconstriction in hindlimb unweighted (HU) rat cerebral arteries. MethodsThree-week HU was used to simulate microgravity in rats. The contractile responses to vasoconstrictors, mitochondrial fission/fusion, Ca2+ distribution, inositol 1,4,5-trisphosphate receptor (IP3R) abundance, and the activities of voltage-gated K+ channels (KV) and Ca2+-activated K+ channels (BKCa) were examined in rat cerebral vascular smooth muscle cells (VSMCs). ResultsAn increase of cytoplasmic Ca2+ and a decrease of mitochondrial/sarcoplasmic reticulum (SR) Ca2+ were observed in HU rat cerebral VSMCs. The abundance of fusion proteins (mitofusin 1/2 [MFN1/2]) and fission proteins (dynamin-related protein 1 [DRP1] and fission-mitochondrial 1 [FIS1]) was significantly downregulated and upregulated, respectively in HU rat cerebral VSMCs. The cerebrovascular contractile responses to vasoconstrictors were enhanced in HU rats compared to control rats, and IP3 R protein/mRNA levels were significantly upregulated. The current densities and open probabilities of KV and BKCa decreased and increased, respectively. Treatment with the mitochondrial-targeted antioxidant mitoTEMPO attenuated mitochondrial fission by upregulating MFN1/2 and downregulating DRP1/FIS1. It also decreased IP3R expression levels and restored the activities of the KV and BKCa channels. MitoTEMPO restored the Ca2+ distribution in VSMCs and attenuated the enhanced vasoconstriction in HU rat cerebral arteries. ConclusionThe present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity.

Regulation of the cerebrovascular smooth muscle cell phenotype by mitochondrial oxidative injury and endoplasmic reticulum stress in simulated microgravity rats via the PERK-eIF2α-ATF4-CHOP pathway

Microgravity exposure results in vascular remodeling and cardiovascular dysfunction. Here, the effects of mitochondrial oxidative stress on vascular smooth muscle cells (VSMCs) in rat cerebral arteries under microgravity simulated by hindlimb unweighting (HU) was studied. Endoplasmic reticulum (ER)-resident transmembrane sensor proteins and phenotypic markers of rat cerebral VSMCs were examined. In HU rats, CHOP expression was increased gradually, and the upregulation of the PERK-eIF2α-ATF4 pathway was the most pronounced in cerebral arteries. Furthermore, PERK/p-PERK signaling, CHOP, GRP78 and reactive oxygen species were augmented by PERK overexpression but attenuated by the mitochondria-targeting antioxidant MitoTEMPO. Meanwhile, p-PI3K, p-Akt and p-mTOR protein levels in VSMCs were increased in HU rat cerebral arteries. Compared with the control, HU rats exhibited lower α-SMA, calponin, SM-MHC and caldesmon protein levels but higher OPN and elastin levels in cerebral VSMCs. The cerebral VSMC phenotype transition from a contractile to synthetic phenotype in HU rats was augmented by PERK overexpression and 740Y-P but reversed by MitoTEMPO and the ER stress inhibitors tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). In summary, mitochondrial oxidative stress and ER stress induced by simulated microgravity contribute to phenotype transition of cerebral VSMCs through the PERK-eIF2a-ATF4-CHOP pathway in a rat model.

BKCa compensates impaired coronary vasoreactivity through RhoA/ROCK pathway in hind-limb unweighted rats.

Previous studies have demonstrated cardiac and vascular remodeling induced by microgravity exposure. Yet, as the most important branch of vasculatures circulating the heart, the coronary artery has been seldomly studied about its adaptations under microgravity conditions. Large-conductance Ca2+-activated potassium channel (BKCa) and the Ras homolog family member A (RhoA)/Rho kinase (ROCK) pathway play key roles in control of vascular tone and mediation of microgravity-induced vascular adjustments. Therefore, we investigated the adaptation of coronary vasoreactivity to simulated microgravity and the role of BKCa and the RhoA/ROCK pathway in it. Four-week-old hind-limb unweighted (HU) rats were adopted to simulate effects of microgravity. Right coronary artery (RCA) constriction was measured by isometric force recording. The activity and expression of BKCa and the RhoA/ROCK pathway were examined by Western blot, patch-clamp recordings, and immunoprecipitation. We found HU significantly decreased RCA vasoconstriction to KCl, serotonin, and U-46619, but increased protein expression and current densities of BKCa, inhibition of which by iberiotoxin (IBTX) further decreased RCA vasoconstriction (P < 0.05). Expression of RhoA and ROCK as well as active RhoA and phosphorylation of myosin light chain (MLC) at Ser19 and MLC phosphatase target-1 at Thr696 were significantly increased by HU, and ROCK inhibitor Y-27632 exerted greater suppressing effect on HU RCA vasoconstriction than that of control (P < 0.05). BKCa opener NS1619 increased HU RCA vasoconstriction, which was blocked by both RhoA and ROCK inhibitor, similar to the effect of IBTX. These results indicate that HU impairs coronary vasoconstriction but enhances BKCa activity acting as a protective mechanism avoiding excessive decrease of coronary vasoreactivity through activation of the RhoA/ROCK pathway.-Wu, Y., Yue, Z., Wang, Q., Lv, Q., Liu, H., Bai, Y., Li, S., Xie, M., Bao, J., Ma, J., Zhu, X., Wang, Z. BKCa compensates impaired coronary vasoreactivity through RhoA/ROCK pathway in hind-limb unweighted rats.

Effect of a combination of astaxanthin supplementation, heat stress, and intermittent reloading on satellite cells during disuse muscle atrophy.

We examined the effect of a combination of astaxanthin (AX) supplementation, repeated heat stress, and intermittent reloading (IR) on satellite cells in unloaded rat soleus muscles. Forty-nine male Wistar rats (8-week-old) were divided into control, hind-limb unweighting (HU), IR during HU, IR with AX supplementation, IR with repeated heat stress (41.0-41.5 °C for 30 min), and IR with AX supplementation and repeated heat stress groups. After the experimental period, the antigravitational soleus muscle was analyzed using an immunohistochemical technique. Our results revealed that the combination of dietary AX supplementation and heat stress resulted in protection against disuse muscle atrophy in the soleus muscle. This protective effect may be partially due to a higher satellite cell number in the atrophied soleus muscle in the IR/AX/heat stress group compared with the numbers found in the other groups. We concluded that the combination treatment with dietary AX supplementation and repeated heat stress attenuates soleus muscle atrophy, in part by increasing the number of satellite cells.

Oxidative Stress Contributes to Fracture/Cast-Induced Inflammation and Pain in a Rat Model of Complex Regional Pain Syndrome

Clinical evidence suggests that vitamin C (Vit C) may protect against the development of complex regional pain syndrome (CRPS) after fracture or surgery. Tibia fracture followed by 4 weeks of cast immobilization (fracture/cast) in rats results in nociceptive, vascular, and bone changes resembling clinical CRPS. In this study, fracture/cast rats were treated with the oxidative stress inhibitors Vit C, N-acetyl cysteine, or 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl to examine their effects on CRPS-related nociceptive and vascular changes. Administration of these agents significantly reduced fracture/cast-induced cutaneous allodynia by 64 to 78%, muscle hyperalgesia by 34 to 40%, and hind limb unweighting by 48 to 89%. Treatments with Vit C and N-acetyl cysteine reduced the oxidative stress markers malondialdehyde in the skin, muscle, and sciatic nerve, and lactate in the gastrocnemius muscle of the fracture/cast limb. Furthermore, Vit C treatment inhibited the post-fracture upregulation of substance P and calcitonin gene-related peptide in the sciatic nerve and the increased expression of the pain-related inflammatory mediators, including interleukin (IL)-6, and nerve growth factor in the skin and IL-1β, and IL-6 in the muscle of the post-fracture/cast limb. These data suggest that oxidative stress may contribute to the nociceptive features of the rat CRPS model.Perspective:Vit C reduced the CRPS-like signs, oxidative stress, and the upregulation of neuropeptide production and inflammatory mediators observed after tibia fracture and casting in rats. Limiting oxidative stress by use of Vit C or alternative strategies could reduce the risk of developing CRPS after surgery or other forms of trauma.

Focal adhesions are involved in simulated-microgravity-induced basilar and femoral arterial remodelling in rats.

Recent studies have suggested that microgravity-induced arterial remodelling contributes to post-flight orthostatic intolerance and that multiple mechanisms are involved in arterial remodelling. However, the initial mechanism by which haemodynamic changes induce arterial remodelling is unknown. Focal adhesions (FAs) are dynamic protein complexes that have mechanotransduction properties. This study aimed to investigate the role of FAs in simulated-microgravity-induced basilar and femoral arterial remodelling. A 4-week hindlimb-unweighted (HU) rat model was used to simulate the effects of microgravity, and daily 1-hour intermittent artificial gravity (IAG) was used to prevent arterial remodelling. After 4-week HU, wall thickness, volume of smooth muscle cells (SMCs) and collagen content were increased in basilar artery but decreased in femoral artery (P < 0.05). Additionally, the expression of p-FAK Y397 and p-Src Y418 was increased and reduced in SMCs of basilar and femoral arteries, respectively, by HU (P < 0.05). The number of FAs was increased in basilar artery and reduced in femoral artery by HU (P < 0.05). Furthermore, daily 1-hour IAG prevented HU-induced differential structural adaptations and changes in FAs of basilar and femoral arteries. These results suggest that FAs may act as mechanosensors in arterial remodelling by initiating intracellular signal transduction in response to altered mechanical stress induced by microgravity.

ALTERATIONS OF ECCENTRICALLY EVOKED PROTEIN SYNTHESIS IN ISOLATED RAT SOLEUS FOLLOWING GRAVITATIONAL UNLOADING: ROLE OF FAK

ALTERATIONS OF ECCENTRICALLY EVOKED PROTEIN SYNTHESIS IN ISOLATED RAT SOLEUS FOLLOWING GRAVITATIONAL UNLOADING: ROLE OF FAK

Disuse skeletal muscle atrophy in humans. Proteomic and molecular adaptations

Disuse skeletal muscle atrophy in humans. Proteomic and molecular adaptations

Estrogen inhibits the overgrowth of Escherichiacoli in the rat intestine under simulated microgravity.

Microgravity can affect many aspects of intestinal homeostasis, leading to an increased risk of colitis. Estrogen, the most frequently affected hormone when under simulated microgravity, regulates the permeability of the colonic mucosa barrier. The associations between alterations in intestinal microbiota and increased susceptibility under microgravity have not been thoroughly elucidated. The aim of the present study was to evaluate the changes in intestinal microbiota under simulated microgravity and to investigate the protective effect of estrogen against those changes. The hindlimb unweighting (HU) model was used to simulate microgravity in rats. Estrogen was administered via intramuscular injection. Amplicons of the V3 variable regions of bacterial 16S rDNA were analyzed using denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Several specific bacterial groups were assayed using quantitative-polymerase chain reaction. Bacterial translocation was evaluated by detecting serum lipopolysaccharide (LPS) and LPS binding protein (LBP) levels. DGGE profiles generated by universal primers revealed minor, though specific, changes in bacterial communities under simulated microgravity, particularly the band matching the sequence of Escherichia coli (E. coli). The quantification of 16S RNA revealed increased numbers of Bacteroides fragilis, E. coli and Fusobacterium nucleatum; however, Bifidobacteria longum significantly decreased under microgravity. Estrogen inhibited the overgrowth of E. coli, and decreased the levels of LBS and LBP under simulated microgravity. These results demonstrated that simulated microgravity alters the intestinal microflora and may contribute to bacterial translocation in the gut mucosa. The data also suggested that further investigations evaluating the administration of estrogen to protect against microgravity-associated diseases may be required.

The ERG1 K+ Channel Protein Is More Abundant in the Denervated Skeletal Muscle of Rodents

Skeletal muscle atrophy is the loss of muscle mass and strength that occurs with normal aging, neural and muscle injuries, starvation and diseases such as diabetes, neuropathies, muscular dystrophy, cancer and sepsis cachexia, etc. The atrophy of skeletal muscle can be incapacitating and is related to increased disability, morbidity and mortality in people. We have shown that the ERG1a alternative splice variant of the ERG1 K+ channel is up‐regulated in the skeletal muscle of mice experiencing atrophy as a consequence of cancer cachexia and disuse (i.e., hind limb unweighting, a condition in which muscle action potentials are reduced). In fact, the ERG1 protein is up‐regulated after 4 days of hind limb unweighting, which is prior to a significant loss in muscle cross sectional area, strongly suggesting ERG1 is involved in the onset of muscle loss. Further, we have shown that the ERG1a channel contributes to protein breakdown in muscle by increasing both ubiquitin proteasome proteolysis (UPP) and intracellular calcium concentration with a subsequent increase in calpain activity. Because skeletal muscle atrophy occurs rapidly in denervated skeletal muscle, we hypothesized that ERG1 would also increase in denervated muscles. To test this hypothesis, we performed sciatic nerve transection on mice and rats and collected gastrocnemius and soleus muscles at 0, 4, 7 and 14 days after surgery. Decreased muscle weight and myofiber cross sectional area demonstrate that the muscles were successfully denervated and underwent atrophy. Immunoblot with anti‐ERG1 antibodies demonstrates that indeed the ERG1 protein increases post denervation in mice. Further, immunohistochemistry demonstrates that ERG1 protein also increases in denervated soleus of rats. Interestingly, confocal microscopy analysis of these tissues shows that the ERG1 protein is localized in the sarcolemma and t‐tubules of denervated rat skeletal muscle as it is in heart. The t‐tubule location places it in the vicinity of the calcium release apparatus where it can affect calcium release. We conclude that ERG1 protein is upregulated in the t‐tubules and sarcolemma of denervated muscle and, because it is up‐regulated in other atrophy models, ERG1 is a consistent marker of skeletal muscle atrophy.Support or Funding InformationThis research was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number NIH NIAMS 1R03AR053706‐01A2 to ALP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Effects of Hindlimb Unweighting on MBP and GDNF Expression and Morphology in Rat Dorsal Root Ganglia Neurons.

With the development of technology and space exploration, studies on long-duration space flights have shown that microgravity induces damage to multiple organs, including the dorsal root ganglia (DRG). However, very little is known about the effects of long-term microgravity on DRG neurons. This study investigated the effects of microgravity on lumbar 5 (L5) DRG neurons in rats using the hindlimb unweighting (HU) model. Male (M) and female (F) Sprague-Dawley rats were randomly divided into M- and F-control (CON) groups and M- and F-HU groups, respectively (n=10). At the end of HU treatment for 4 weeks, morphological changes were detected. Myelin basic protein (MBP) and degenerated myelin basic protein (dgen-MBP) expressions were analyzed by immunofluorescence and western blot assays. Glial cell line-derived neurotrophic factor (GDNF) protein and mRNA expressions were also analyzed by immunohistochemistry, western blot, and RT-PCR analysis, respectively. Compared with the corresponding CON groups, the HU groups exhibited slightly loose junctions between DRG neurons, some separated ganglion cells and satellite cells, and lightly stained Nissl bodies that were of smaller size and had a scattered distribution. High levels of dgen-MBP and low MBP expressions were appeared and GDNF expressions were significantly decreased in both HU groups. Changes were more pronounced in the F-HU group than in the M-HU group. In conclusion, HU treatment induced damage of L5 DRG neurons, which was correlated with decreased total MBP protein expression, increased dgen-MBP expression, and reduced GDNF protein and mRNA expression. Importantly, these changes were more severe in F-HU rats compared with M-HU rats.

NADPH Oxidase Accounts for Changes in Cerebrovascular Redox Status in Hindlimb Unweighting Rats.

The roles of cerebrovascular oxidative stress in vascular functional remodeling have been described in hindlimb-unweighting (HU) rats. However, the underlying mechanism remains to be established. We investigated the generation of vascular reactive oxygen species (ROS), Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and manganese superoxide dismutase (MnSOD) and glutathione peroxidase-1 (GPx-1) mRNA levels in cerebral and mesenteric smooth muscle cells (VSMCs) of HU rats. ROS production increased in cerebral but not in mesenteric VSMCs of HU rats compared with those in control rats. Nox2 and Nox4 protein and mRNA levels were increased significantly but MnSOD/GPx-1 mRNA levels decreased in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly more in cerebral but not in mesenteric arteries of HU rats. NADPH oxidase inhibition with apocynin attenuated cerebrovascular ROS production and partially restored Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and MnSOD/GPx-1 mRNA levels in cerebral VSMCs of HU rats. These results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravit.

NADPH Oxidase Accounts for Changes in Cerebrovascular Redox Status in Hindlimb Unweighting Rats

ObjectiveThe roles of cerebrovascular oxidative stress in vascular functional remodeling have been described in hindlimb-unweighting (HU) rats. However, the underlying mechanism remains to be established. MethodsWe investigated the generation of vascular reactive oxygen species (ROS), Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and manganese superoxide dismutase (MnSOD) and glutathione peroxidase-1 (GPx-1) mRNA levels in cerebral and mesenteric smooth muscle cells (VSMCs) of HU rats. ResultsROS production increased in cerebral but not in mesenteric VSMCs of HU rats compared with those in control rats. Nox2 and Nox4 protein and mRNA levels were increased significantly but MnSOD/GPx-1 mRNA levels decreased in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly more in cerebral but not in mesenteric arteries of HU rats. NADPH oxidase inhibition with apocynin attenuated cerebrovascular ROS production and partially restored Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and MnSOD/GPx-1 mRNA levels in cerebral VSMCs of HU rats. ConclusionThese results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravity.

Myostatin blockade with a fully human monoclonal antibody induces muscle hypertrophy and reverses muscle atrophy in young and aged mice.

BackgroundLoss of skeletal muscle mass and function in humans is associated with significant morbidity and mortality. The role of myostatin as a key negative regulator of skeletal muscle mass and function has supported the concept that inactivation of myostatin could be a useful approach for treating muscle wasting diseases.MethodsWe generated a myostatin monoclonal blocking antibody (REGN1033) and characterized its effects in vitro using surface plasmon resonance biacore and cell-based Smad2/3 signaling assays. REGN1033 was tested in mice for the ability to induce skeletal muscle hypertrophy and prevent atrophy induced by immobilization, hindlimb suspension, or dexamethasone. The effect of REGN1033 on exercise training was tested in aged mice. Messenger RNA sequencing, immunohistochemistry, and ex vivo force measurements were performed on skeletal muscle samples from REGN1033-treated mice.ResultsThe human monoclonal antibody REGN1033 is a specific and potent myostatin antagonist. Chronic treatment of mice with REGN1033 increased muscle fiber size, muscle mass, and force production. REGN1033 prevented the loss of muscle mass induced by immobilization, glucocorticoid treatment, or hindlimb unweighting and increased the gain of muscle mass during recovery from pre-existing atrophy. In aged mice, REGN1033 increased muscle mass and strength and improved physical performance during treadmill exercise.ConclusionsWe show that specific myostatin antagonism with the human antibody REGN1033 enhanced muscle mass and function in young and aged mice and had beneficial effects in models of skeletal muscle atrophy.

The response of apoptotic and proteolytic systems to repeated heat stress in atrophied rat skeletal muscle.

We examined the effect of repeated heat stress on muscle atrophy, and apoptotic and proteolytic regulation in unloaded rat slow- and fast-type skeletal muscles. Forty male Wistar rats (11 week-old) were divided into control (CT), hindlimb unweighting (HU), intermittent weight-bearing during HU (HU + IWB), and intermittent weight-bearing with heat stress during HU (41–41.5°C for 30 min; HU + IWB + HS) groups. The HU + IWB + HS and HU + IWB groups were released from unloading for 1 h every second day, during which the HU + IWB + HS group underwent the heating. Our results revealed that repeated bouts of heat stress resulted in protection against disuse muscle atrophy in both soleus and plantaris muscles. This heat stress–induced protection against disuse-induced muscular atrophy may be partially due to reduced apoptotic activation in both muscles, and decreased ubiquitination in only the soleus muscle. We concluded that repeated heat stress attenuated skeletal muscle atrophy via suppressing apoptosis but the response to proteolytic systems depend on the muscle phenotype.

Caveolae regulate vasoconstriction of conduit arteries to angiotensin II in hindlimb unweighted rats.

Weightlessness induces the functional remodelling of arteries, but the changes to angiotensin II (Ang II)-elicited vasoconstriction and the underlying mechanism have never been reported. Caveolae are invaginations of the cell membrane crucial for the contraction of vascular smooth muscle cells, so we investigated the adaptation of Ang II-elicited vasoconstriction to simulated weightlessness and the role of caveolae in it. The 4 week hindlimb unweighted (HU) rat was used to simulate the effects of weightlessness. Ang II-elicited vasoconstriction was measured by isometric force recording. The morphology of caveolae was examined by transmission electron microscope. The binding of the angiotensin II type 1 receptor (AT1 ) and caveolin-1 (cav-1) was examined by coimmunoprecipitation and Western blot. We found that the maximal developing force (E(max)) of Ang II-elicited vasoconstriction was decreased in abdominal aorta by 30.6%, unchanged in thoracic aorta and increased in carotid artery by 17.9% after HU, while EC50 of the response was increased in all three arteries (P < 0.05). AT1 desensitization upon activation was significantly reduced by HU in all three arteries, as was the number of caveolae (P < 0.05). Furthermore, Ang II promoted the binding of AT1 and cav-1 significantly in control but not HU arteries. Both the number of caveolae and the binding of AT1 and cav-1 in HU arteries were restored by cholesterol pretreatment which also reinstated the change in EC50 as well as the level of AT1 desensitization. These results indicate that modified caveolae in vascular smooth muscle cells could interfere with the binding of AT1 and cav-1 mediating the adaptation of Ang II-elicited vasoconstriction to HU.