Sixty days of head-down tilt bed rest with or without artificial gravity do not affect the neuromuscular secretome.

Artificial gravity is a potential countermeasure to attenuate effects of weightlessness during long-term spaceflight, including losses of muscle mass and function, possibly to some extent attributable to disturbed neuromuscular interaction. The 60-day AGBRESA bed-rest study was conducted with 24 participants (16 men, 8 women; 33 ± 9 years; 175 ± 9 cm; 74 ± 10 kg; 8 control group, 8 continuous (cAG) and 8 intermittent (iAG) centrifugation) to assess the impact of bed rest with or without daily 30-min continuous/intermittent centrifugation with 1G at the centre of mass. Fasting blood samples were collected before and on day 6, 20, 40 and 57 during 6° head-down tilt bed rest. Concentrations of circulating markers of muscle wasting (GDF-8/myostatin; slow skeletal muscle troponin T; prostaglandin E2), neurotrophic factors (BDNF; GDNF) and C-terminal Agrin Fragment (CAF) were determined by ELISAs. Creatine kinase activity was assessed by colorimetric enzyme assay. Repeated-measures ANOVAs were conducted with TIME as within-subject, and INTERVENTION and SEX as between-subject factors. The analyses revealed no significant effect of bed rest or sex on any of the parameters. Continuous or intermittent artificial gravity is a safe intervention that does not have a negative impact of the neuromuscular secretome.

The State of the Art of Piezo1 Channels in Skeletal Muscle Regeneration.

Piezo1 channels are highly mechanically-activated cation channels that can sense and transduce the mechanical stimuli into physiological signals in different tissues including skeletal muscle. In this focused review, we summarize the emerging evidence of Piezo1 channel-mediated effects in the physiology of skeletal muscle, with a particular focus on the role of Piezo1 in controlling myogenic precursor activity and skeletal muscle regeneration and vascularization. The disclosed effects reported by pharmacological activation of Piezo1 channels with the selective agonist Yoda1 indicate a potential impact of Piezo1 channel activity in skeletal muscle regeneration, which is disrupted in various muscular pathological states. All findings reported so far agree with the idea that Piezo1 channels represent a novel, powerful molecular target to develop new therapeutic strategies for preventing or ameliorating skeletal muscle disorders characterized by an impairment of tissue regenerative potential.

High eEF1A1 Protein Levels Mark Aggressive Prostate Cancers and the In Vitro Targeting of eEF1A1 Reveals the eEF1A1-actin Complex as a New Potential Target for Therapy.

Although the eukaryotic elongation factor eEF1A1 plays a role in various tumours, there is little information on its prognosis/therapeutic value in prostate carcinoma. In high-grade and castration-resistant prostate carcinoma (CRPC), the identification of novel therapeutic markers/targets remains a priority. The expression of eEF1A1 protein was determined in formalin-fixed, paraffin-embedded prostate cancer and hyperplasia tissue by IHC. The role of eEF1A1 was investigated in a cellular model using a DNA aptamer (GT75) we previously developed. We used the aggressive CRPC cancer PC-3 and non-tumourigenic PZHPV-7 lines. Cytotoxicity was measured by the MTS assay and eEF1A1 protein levels by in-cell Western assays. The mRNA levels of eEF1A1 were measured by qPCR and ddPCR. Higher expression of eEF1A1 was found in Gleason 7-8 compared with 4-6 tissues (Gleason ≥ 7, 87% versus Gleason ≤ 6, 54%; p = 0.033). Patients with a high expression of eEF1A1 had a worse clinical outcome. In PC-3, but not in PZHPV-7, GT75 decreased cell viability and increased autophagy and cell detachment. In PC-3 cells, but not in PZHPV-7, GT75 mainly co-localised with the fraction of eEF1A1 bound to actin. Overexpression of the eEF1A1 protein can identify aggressive forms of prostate cancer. The targeting of eEF1A1 by GT75 impaired cell viability in PC-3 cancer cells but not in PZHPV-7 non-tumourigenic cells, indicating a specific role for the protein in cancer survival. The eEF1A1-actin complexes appear to be critical for the viability of PC-3 cancer cells, suggesting that eEF1A1 may be an attractive target for therapeutic strategies in advanced forms of prostate cancer.

A "noisy" electrical stimulation protocol favors muscle regeneration in vitro through release of endogenous ATP.

An in vitro system of electrical stimulation was used to explore whether an innovative "noisy" stimulation protocol derived from human electromyographic recordings (EMGstim) could promote muscle regeneration. EMGstim was delivered to cultured mouse myofibers isolated from Flexor Digitorum Brevis, preserving their satellite cells. In response to EMGstim, immunostaining for the myogenic regulatory factor myogenin, revealed an increased percentage of elongated myogenin-positive cells surrounding the myofibers. Conditioned medium collected from EMGstim-treated cell cultures, promoted satellite cells differentiation in unstimulated myofiber cell cultures, suggesting that extracellular soluble factors could mediate the process. Interestingly, the myogenic effect of EMGstim was mimicked by exogenously applied ATP (0.1 µM), reduced by the ATP diphosphohydrolase apyrase and prevented by blocking endogenous ATP release with carbenoxolone. In conclusion, our results show that "noisy" electrical stimulations favor muscle progenitor cell differentiation most likely via the release of endogenous ATP from contracting myofibres. Our data also suggest that "noisy" stimulation protocols could be potentially more efficient than regular stimulations to promote in vivo muscle regeneration after traumatic injury or in neuropathological diseases.

Head-down tilt bed rest with or without artificial gravity is not associated with motor unit remodeling.

PURPOSE: The objective of this study was to assess whether artificial gravity attenuates any long-duration head-down 60 bed rest (HDBR)-induced alterations in motor unit (MU) properties. METHODS: Twenty-four healthy participants (16 men; 8 women; 26-54 years) underwent 60-day HDBR with (n = 16) or without (n = 8) 30 min artificial gravity daily induced by whole-body centrifugation. Compound muscle action potential (CMAP), MU number (MUNIX) and MU size (MUSIX) were estimated using the method of Motor Unit Number Index in the Abductor digiti minimi and tibialis anterior muscles 5 days before (BDC-5), and during day 4 (HDT4) and 59 (HDT59) of HDBR. RESULTS: The CMAP, MUNIX, and MUSIX at baseline did not change significantly in either muscle, irrespective of the intervention (p > 0.05). Across groups, there were no significant differences in any variable during HDBR, compared to BDC-5. CONCLUSION: Sixty days of HDBR with or without artificial gravity does not induce alterations in motor unit number and size in the ADM or TA muscles in healthy individuals.

Effects of 21 days of bed rest and whey protein supplementation on plantar flexor muscle fatigue resistance during repeated shortening contractions.

PURPOSE: Space flight and bed rest (BR) lead to a rapid decline in exercise capacity. Whey protein plus potassium bicarbonate diet-supplementation (NUTR) could attenuate this effect by improving oxidative metabolism. We evaluated the impact of 21-day BR and NUTR on fatigue resistance of plantar flexor muscles (PF) during repeated shortening contractions, and whether any change was related to altered energy metabolism and muscle oxygenation. METHODS: Ten healthy men received a standardized isocaloric diet with (n = 5) or without (n = 5) NUTR. Eight bouts of 24 concentric plantar flexions (30 s each bout) with 20 s rest between bouts were employed. PF muscle size was assessed by means of peripheral quantitative computed tomography. PF muscle volume was assessed with magnetic resonance imaging. PF muscle force, contraction velocity, power and surface electromyogram signals were recorded during each contraction, as well as energy metabolism (31P nuclear magnetic resonance spectroscopy) and oxygenation (near-infrared spectroscopy). Cardiopulmonary parameters were measured during an incremental cycle exercise test. RESULTS: BR caused 10-15% loss of PF volume that was partly recovered 3 days after re-ambulation, as a consequence of fluid redistribution. Unexpectedly, PF fatigue resistance was not affected by BR or NUTR. BR induced a shift in muscle metabolism toward glycolysis and some signs of impaired muscle oxygen extraction. NUTR did not attenuate the BR-induced-shift in energy metabolism. CONCLUSIONS: Twenty-one days' BR did not impair PF fatigue resistance, but the shift to glycolytic metabolism and indications of impaired oxygen extraction may be early signs of developing reduced muscle fatigue resistance.

Local capillary supply in muscle is not determined by local oxidative capacity.

It is thought that the prime determinant of global muscle capillary density is the mean oxidative capacity. However, feedback control during maturational growth or adaptive remodelling of local muscle capillarisation is likely to be more complex than simply matching O2 supply and demand in response to integrated tissue function. We tested the hypothesis that the maximal oxygen consumption (MO2,max) supported by a capillary is relatively constant, and independent of the volume of tissue supplied (capillary domain). We demonstrate that local MO2,max assessed by succinate dehydrogenase histochemistry: (1) varied more than 100-fold between individual capillaries and (2) was positively correlated to capillary domain area in both human vastus lateralis (R=0.750, P<0.001) and soleus (R=0.697, P<0.001) muscles. This suggests that, in contrast to common assumptions, capillarisation is not primarily dictated by local oxidative capacity, but rather by factors such as fibre size, or consequences of differences in fibre size such as substrate delivery and metabolite removal.

The impact of bed rest on human skeletal muscle metabolism.

Insulin sensitivity and metabolic flexibility decrease in response to bed rest, but the temporal and causal adaptations in human skeletal muscle metabolism are not fully defined. Here, we use an integrative approach to assess human skeletal muscle metabolism during bed rest and provide a multi-system analysis of how skeletal muscle and the circulatory system adapt to short- and long-term bed rest (German Clinical Trials: DRKS00015677). We uncover that intracellular glycogen accumulation after short-term bed rest accompanies a rapid reduction in systemic insulin sensitivity and less GLUT4 localization at the muscle cell membrane, preventing further intracellular glycogen deposition after long-term bed rest. We provide evidence of a temporal link between the accumulation of intracellular triglycerides, lipotoxic ceramides, and sphingomyelins and an altered skeletal muscle mitochondrial structure and function after long-term bed rest. An intracellular nutrient overload therefore represents a crucial determinant for rapid skeletal muscle insulin insensitivity and mitochondrial alterations after prolonged bed rest.

Capillary rarefaction during bed rest is proportionally less than fibre atrophy and loss of oxidative capacity.

BACKGROUND: Muscle disuse from bed rest or spaceflight results in losses in muscle mass, strength and oxidative capacity. Capillary rarefaction may contribute to muscle atrophy and the reduction in oxidative capacity during bed rest. Artificial gravity may attenuate the negative effects of long-term space missions or bed rest. The aim of the present study was to assess (1) the effects of bed rest on muscle fibre size, fibre type composition, capillarization and oxidative capacity in the vastus lateralis and soleus muscles after 6 and 55 days of bed rest and (2) the effectiveness of artificial gravity in mitigating bed-rest-induced detriments to these parameters. METHODS: Nineteen participants were assigned to a control group (control, n = 6) or an intervention group undergoing 30 min of centrifugation (n = 13). All underwent 55 days of head-down tilt bed rest. Vastus lateralis and soleus biopsies were taken at baseline and after 6 and 55 days of bed rest. Fibre type composition, fibre cross-sectional area, capillarization indices and oxidative capacity were determined. RESULTS: After just 6 days of bed rest, fibre atrophy (-23.2 ± 12.4%, P < 0.001) and reductions in capillary-to-fibre ratio (C:F; 1.97 ± 0.57 vs. 1.56 ± 0.41, P < 0.001) were proportional in both muscles as reflected by a maintained capillary density. Fibre atrophy proceeded at a much slower rate between 6 and 55 days of bed rest (-11.6 ± 12.1% of 6 days, P = 0.032) and was accompanied by a 19.1% reduction in succinate dehydrogenase stain optical density (P < 0.001), without any further significant decrements in C:F (1.56 ± 0.41 vs. 1.49 ± 0.37, P = 0.459). Consequently, after 55 days of bed rest, the capillary supply-oxidative capacity ratio of a fibre had increased by 41.9% (P < 0.001), indicating a capillarization in relative excess of oxidative capacity. Even though the heterogeneity of capillary spacing (LogR SD) was increased after 55 days by 12.7% (P = 0.004), tissue oxygenation at maximal oxygen consumption of the fibres was improved after 55 days bed rest. Daily centrifugation failed to blunt the bed-rest-induced reductions in fibre size and oxidative capacity and capillary rarefaction. CONCLUSIONS: The relationship between fibre size and oxidative capacity with the capillary supply of a fibre is uncoupled during prolonged bed rest as reflected by a rapid loss of muscle mass and capillaries, followed at later stages by a more than proportional loss of mitochondria without further capillary loss. The resulting excessive capillary supply of the muscle after prolonged bed rest is advantageous for the delivery of substrates needed for subsequent muscle recovery.

Nerve-dependent distribution of subsynaptic type 1 inositol 1,4,5-trisphosphate receptor at the neuromuscular junction.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation-transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the lack of muscle electrical activity, as judged by partial reversal upon field electrical stimulation of in vitro denervated skeletal muscle fibers.

High plasma retinol binding protein 4 (RBP4) is associated with systemic inflammation independently of low RBP4 adipose expression and is normalized by transplantation in nonobese, nondiabetic patients with chronic kidney disease.

OBJECTIVE: Adipose-secreted retinol binding protein 4 (RBP4) circulates in free (active) and transthyretin (TTR)-bound forms and may be associated with obesity-related inflammation. Potential involvement of plasma and adipose RBP4 in systemic inflammation in the absence of obesity and diabetes is unknown. Inflammation reduces survival in chronic kidney disease (CKD) [particularly in maintenance haemodialysis (MHD)], and plasma RBP4 may increase with renal dysfunction. We investigated (i) potential associations between RBP4 and inflammation in CKD and (ii) the role of adipose tissue in this putative interaction. DESIGN: Cross-sectional. PATIENTS: Nonobese, nondiabetic patients with CKD undergoing conservative (CT: n = 10) or MHD treatment (n = 25) and healthy control subjects (C: n = 11). Renal transplant recipients (n = 5) were studied to further assess the impact of restored near-normal renal function. MEASUREMENTS: Plasma RBP4, TTR and C-reactive protein (CRP), adipose RBP4 expression. RESULTS: Plasma RBP4, TTR and CRP were highest in MHD (P < 0·05). Adipose RBP4 mRNA was, however, comparably low in CT and MHD (P < 0·05 vs C), and all parameters were normalized in transplant recipients (P < 0·05 vs MHD). In all subjects (n = 51), creatinine and TTR (P < 0·05) but not adipose RBP4 mRNA were associated with plasma RBP4. Plasma RBP4 but not its adipose expression was in turn associated positively (P < 0·05) with CRP independently of creatinine-TTR. CONCLUSIONS: High plasma RBP4 and inflammation are clustered in CKD in the absence of obesity and diabetes and are normalized by transplantation. Adipose RBP4 expression is not involved in plasma RBP4 elevation, which appears to be mainly because of passive accumulation, or in CKD-associated inflammation.

Hemodialysis induces p66(shc) gene expression in nondiabetic humans: correlations with oxidative stress and systemic inflammation.

OBJECTIVE: Oxidative stress and inflammation characterize hemodialysis (HD) and are associated with malnutrition, cardiovascular disease, and poor clinical outcome. p66(shc) stimulates oxidative stress and atherogenesis. The objective of the present study was to assess p66(shc) expression levels in HD and their associations with inflammatory and oxidative stress markers. DESIGN: p66(shc) messenger ribonucleic acid (mRNA) was compared with systemic oxidative stress and inflammation markers in control subjects and patients on HD before and after a single HD session in a cross-sectional analysis. SETTING: Outpatient hemodialysis unit. PATIENTS: The study included stable HD patients (n = 21, men/women: 18/3) who were on HD 3 times per week for a minimum of 8 weeks; age-matched control subjects (n = 22, men/women:17/5). MAIN OUTCOME MEASURE: mRNA levels of p66(shc), tumor necrosis factor α (TNF-α), and pentraxin 3 (PTX3), p66(shc) protein levels in white blood cells, lipid peroxidation (in the form of plasma thiobarbituric acid-reactive substance [TBARS]) and serum C-reactive protein. RESULTS: In patients on dialysis, of the p66(shc), TNF-α, and PTX3 mRNAs, p66(shc) protein levels were higher (P < .05) than in control subjects, as well as plasma TBARS and C-reactive protein (P < .05). p66(shc) mRNA directly correlated with TBARS (r = 0.69, P = .0005) and with TNF-α mRNA (r = 0.63, P = .003). These associations were confirmed in the whole study population (TBARS: r = 0.541, P = .0003; TNF-α: r = 0.581, P < .0001), whereas in the control group only the positive association between p66(shc) and TNF-α was detected. TNF-α was directly correlated with PTX3 both in HD patients (r = 0.72, P = .0005) and in the whole study group (r = 0.678, P < .0001). The dialysis session affected neither p66(shc) and TNF-α mRNA nor p66(shc) protein expression, whereas it further increased (P = .002) PTX3 mRNA. As compared with predialysis levels, TBARS were reduced (P < .05) after dialysis. In these conditions, p66(shc) remained directly correlated with TNF-α (r = 0.901, P < .0001). CONCLUSIONS: Increased p66(shc) gene expression correlates with TNF-α mRNA and with levels of markers of oxidative stress in HD. We suggest a novel link between HD-associated inflammation and p66(shc) gene expression contributing to systemic oxidative stress.

"Time window" effect of Yoda1-evoked Piezo1 channel activity during mouse skeletal muscle differentiation.

AIM: Mechanosensitive Piezo1 ion channels emerged recently as important contributors to various vital functions including modulation of the blood supply to skeletal muscles. The specific Piezo1 channel agonist Yoda1 was shown to regulate the tone of blood vessels similarly to physical exercise. However, the direct role of Piezo1 channels in muscle function has been little studied so far. We therefore investigated the action of Yoda1 on the functional state of skeletal muscle precursors (satellite cells and myotubes) and on adult muscle fibres. METHODS: Immunostaining, electrophysiological intracellular recordings and Ca2+ imaging experiments were performed to localize and assess the effect of the chemical activation of Piezo1 channels with Yoda1, on myogenic precursors, adult myofibres and at the adult neuromuscular junction. RESULTS: Piezo1 channels were detected by immunostaining in satellite cells (SCs) and myotubes as well as in adult myofibres. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of SCs. Moreover, in myotubes, Yoda1 induced significant [Ca2+ ]i transients, without detectable [Ca2+ ]i response in adult myofibres. Furthermore, although expression of Piezo1 channels was detected around the muscle endplate region, Yoda1 application did not alter either the nerve-evoked or spontaneous synaptic activity or muscle contractions in adult myofibres. CONCLUSION: Our data indicate that the chemical activation of Piezo1 channels specifically enhances the differentiation of skeletal muscle precursors, suggesting a possible new strategy to promote muscle regeneration.

Preliminary Observations on Skeletal Muscle Adaptation and Plasticity in Homer 2-/- Mice.

Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2-/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR (Reverse Transcription PCR), confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2-/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2-/- mice detected the exclusive presence of the adult ε-nAChR isoform excluding denervation. As for NMJ morphology, data were not conclusive, and further work is needed to ascertain whether the null Homer 2 phenotype induces any endplate remodelling. Within the context of adaptation and plasticity, the present data show that Homer 2 is a co-regulator of the normotrophic status in a muscle specific fashion.

Changes in contractile properties of skinned single rat soleus and diaphragm fibres after chronic hypoxia.

Hypoxia may be one of the factors underlying muscle dysfunction during ageing and chronic lung and heart failure. Here we tested the hypothesis that chronic hypoxia per se affects contractile properties of single fibres of the soleus and diaphragm muscle. To do this, the force-velocity relationship, rate of force redevelopment and calcium sensitivity of single skinned fibres from normoxic rats and rats exposed to 4 weeks of hypobaric hypoxia (410 mmHg) were investigated. The reduction in maximal force (P(0)) after hypoxia (p=0.031) was more pronounced in type IIa than type I fibres and was mainly attributable to a reduction in fibre cross-sectional area (p=0.044). In type IIa fibres this was aggravated by a reduction in specific tension (p=0.001). The maximal velocity of shortening (V (max)) and shape of the force velocity relation (a/P(0)), however, did not differ between normoxic and hypoxic muscle fibres and the reduction in maximal power of hypoxic fibres (p=0.012) was mainly due to a reduction in P(0). In conclusion, chronic hypoxia causes muscle fibre dysfunction which is not only due to a loss of muscle mass, but also to a diminished force generating capacity of the remaining contractile material. These effects are similar in the soleus and diaphragm muscle, but more pronounced in type IIa than I fibres.

Inhibitory effects of fenofibrate on apoptosis and cell proliferation in human endothelial cells in high glucose.

Fenofibrate has beneficial effects on the progression and clinical emergence of atherosclerosis in normoglycemic and in diabetic patients. Given the involvement of endothelium in these processes, we speculated that fenofibrate may influence endothelial cell apoptosis and proliferation, regulators of endothelium integrity. Fenofibrate effects on apoptosis and proliferation were studied in human umbilical vein endothelial cells under normal (5.5 mmol/l, NG) and high (22 mmol/l, HG) glucose with or without fenofibrate (50 micromol/l). Apoptosis was evaluated by annexin V, by poly(ADP-ribose) polymerase protein cleavage, and cyclooxygenase-2 (COX-2), Bax/Bcl-2, and p53 protein levels; proliferation was assessed by determining cell cycle phase distribution and the amounts of the cell cycle regulators E2F1, cyclin D1, E1, and A and the levels of the hyper-phosphorylated form of the retinoblastoma protein (ppRb). HG resulted in increased (p<0.05) apoptosis rate associated with COX-2 protein overexpression, without modification of Bax/Bcl2 ratio and p53 levels. Fenofibrate decreased apoptosis and normalized increased COX-2 expression in HG (p<0.05). Both in HG and NG, fenofibrate dramatically reduced cell proliferation (p<0.05) through a G1/G0 block mediated by the reduction in ppRb and the decrease in E2F1, cyclin E1, A, and D1 protein expression, with a mechanism that, for cyclin E1, occurred at the posttranscriptional level. In conclusion, our data show that fenofibrate reduces apoptosis caused by HG but severely interferes with endothelial cell proliferation both in NG and HG. The resulting effect may influence endothelium integrity in vivo and may impact the outcome of acute complications of atherosclerosis in diabetes.

A rapid and specific method to simultaneously quantify eukaryotic elongation factor 1A1 and A2 protein levels in cancer cells.

BACKGROUND: The two isoforms of the eukaryotic Elongation Factor 1A (eEF1A1 and eEF1A2), sustain the progression/aggressiveness of cancer cells. Thus, they are considered promising therapeutic targets and prognostic markers. It follows that their precise quantification is of utmost relevance in research and development. The simultaneous quantification of A1 and A2 proteins in the cells helps the comprehension of cancer biology mechanisms and response to drug treatments. However, the high homology at the amino-acidic level (92%) can cause antibodies cross-reaction. Moreover, the commonly employed western blotting just gives semi-quantitative data and does not allow the detection of both protein targets within the same cell. Thus, we developed an in cell western (ICW) technique to bypass the above limitations. METHODS: Firstly, relevant antibodies cross-reaction was excluded by immunohistochemistry on normal pancreatic tissue; then eEF1A1-A2 protein levels were quantitated by ICW in prostate and colorectal cancer cell lines in 96 well plates under different conditions, which include: 1) drug treatment, 2) siRNA silencing, 3) cell seeding density. RESULTS: We show that: 1) eEF1A1-A2 levels vary depending on the cell type following drug treatment, 2) ICW can accurately detect eEF1A1-A2 protein levels following siRNA silencing, 3) cell seeding density influences eEF1A1-A2 levels, depending on cell type. CONCLUSIONS: ICW is a valuable tool to specifically determine the intracellular level of eEF1A1-A2 proteins thus contributing to better define their role as potential therapeutic targets and prognostic markers in human tumors as well as for drug effects screening.

Reactive Jumps Preserve Skeletal Muscle Structure, Phenotype, and Myofiber Oxidative Capacity in Bed Rest.

Identification of countermeasures able to prevent disuse-induced muscle wasting is crucial to increase performance of crew members during space flight as well as ameliorate patient's clinical outcome after long immobilization periods. We report on the outcome of short but high-impact reactive jumps (JUMP) as countermeasure during 60 days of 6° head-down tilt (HDT) bed rest on myofiber size, type composition, capillarization, and oxidative capacity in tissue biopsies (pre/post/recovery) from the knee extensor vastus lateralis (VL) and deep calf soleus (SOL) muscle of 22 healthy male participants (Reactive jumps in a sledge, RSL-study 2015-2016, DLR:envihab, Cologne). Bed rest induced a slow-to-fast myofiber shift (type I ->II) with an increased prevalence of hybrid fibers in SOL after bed rest without jumps (control, CTRL, p = 0.016). In SOL, JUMP countermeasure in bed rest prevented both fast and slow myofiber cross-sectional area (CSA) decrements (p = 0.005) in CTRL group. In VL, bed rest only induced capillary rarefaction, as reflected by the decrease in local capillary-to-fiber ratio (LCFR) for both type II (pre vs. post/R + 10, p = 0.028/0.028) and type I myofibers (pre vs. R + 10, p = 0.012), which was not seen in the JUMP group. VO2 max Fiber (pL × mm-1 × min-1) calculated from succinate dehydrogenase (SDH)-stained cryosections (OD660 nm) showed no significant differences between groups. High-impact jump training in bed rest did not prevent disuse-induced myofiber atrophy in VL, mitigated phenotype transition (type I - >II) in SOL, and attenuated capillary rarefaction in the prime knee extensor VL however with little impact on oxidative capacity changes.

Calorie restriction modulates inactivity-induced changes in the inflammatory markers C-reactive protein and pentraxin-3.

CONTEXT: Energy balance and physical activity potentially influence systemic inflammation. OBJECTIVE: Our objective was to test the hypothesis that moderate energy restriction may prevent activation of inactivity-induced inflammatory response. DESIGN: Participants were studied four times at the end of 14-d periods of experimental bed rest or controlled ambulation, after receiving eucaloric or hypocaloric diets. SETTING: The study was conducted at the clinical research center of the German Space Agency. SUBJECTS: Nine healthy young volunteers participated. INTERVENTIONS: Energy intake was calibrated to physical activity and decreased by about 20% in hypocaloric conditions. MAIN OUTCOME MEASURES: Changes in body fat by dual-energy x-ray absorptiometry as well as plasma inflammatory markers and cytokine mRNA levels in blood cells were measured. RESULTS: Fat mass did not change significantly in eucaloric conditions and decreased in hypocaloric periods (-1.0 +/- 0.3 and -1.0 +/- 0.3 kg in ambulatory and bed rest, respectively). Bed rest in eucaloric conditions increased plasma C-reactive protein (CRP) (+143 +/- 53%) and both the ratios between plasma IL-6 and IL-10 (4+/-1 times) and white blood cell IL-6 and IL-10 mRNAs (5 +/- 1 times). Energy restriction prevented bed-rest-mediated increases in CRP and the IL-6 to IL-10 ratio. Bed rest increased (P = 0.03) long pentraxin-3 (PTX3) plasma concentration, without significant activity-by-diet interaction. In all conditions (n = 36), CRP and PTX3 were inversely correlated (r = -0.61; P < 0.001). Changes in fat mass, leptin, and IL-6 directly correlated with CRP and inversely correlated with PTX3. IL-10 inversely correlated with CRP and directly correlated with PTX3 (r = 0.52; P < 0.01). CONCLUSIONS: Calorie restriction prevents the inflammatory response induced by 14 d of bed rest. We suggest an inverse regulation of CRP and PTX3 in response to changes in energy balance.

Calorie restriction accelerates the catabolism of lean body mass during 2 wk of bed rest.

BACKGROUND: Muscle inactivity and low energy intake commonly occur in persons with acute or chronic disease, in astronauts during space flight, and during aging. OBJECTIVE: We used a crossover design to investigate the effects of the interactions of inactivity and calorie restriction on whole-body composition and protein kinetic regulation in 9 healthy volunteers. DESIGN: Lean body mass (LBM) was measured by using dual-energy X-ray absorptionmetry before and at the end of 14-d periods of bed rest (B) and controlled ambulation (A) in patients receiving eucaloric (E) or hypocaloric (H) (approximately 80% of total energy expenditure) diets. Whole-body leucine kinetics were determined at the end of the 4 study periods by using a standard stable-isotope technique in the postabsorptive state and during a 3-h infusion of a 0.13 g x kg LBM(-1) x h(-1) amino acid mixture. RESULTS: In the postabsorptive state, we found a significant (P = 0.04) bed rest x hypocaloric diet interaction for the rate of leucine oxidation, an index of net protein catabolism (A+E: 0.23 +/- 0.01; B+E: 25 +/- 0.01; A+H: 0.23 +/- 0.01; B+H: 0.28 +/- 0.01 micromol x min(-1) x kg LBM(-1)). Bed rest significantly (P < 0.01) decreased amino acid-mediated stimulation of nonoxidative leucine disappearance, an index of protein synthesis (A+E: 35 +/- 2%; B+E: 30 +/- 2%; A+H: 41 +/- 3%; B+H: 32 +/- 2%). B+H decreased LBM by 1.10 +/- 0.1 kg, which is significantly (P < 0.01) greater than the decrease seen with A+E, A+H, or B+E. CONCLUSION: Calorie restriction enhanced the catabolic response to inactivity by combining greater protein catabolism in the postabsorptive state with an impaired postprandial anabolic utilization of free amino acids.