Generation of allogenic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected mouse DMD-induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD-iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Remarkably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation, and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

MME+ fibro-adipogenic progenitors are the dominant adipogenic population during fatty infiltration in human skeletal muscle.

Fatty infiltration, the ectopic deposition of adipose tissue within skeletal muscle, is mediated via the adipogenic differentiation of fibro-adipogenic progenitors (FAPs). We used single-nuclei and single-cell RNA sequencing to characterize FAP heterogeneity in patients with fatty infiltration. We identified an MME+ FAP subpopulation which, based on ex vivo characterization as well as transplantation experiments, exhibits high adipogenic potential. MME+ FAPs are characterized by low activity of WNT, known to control adipogenic commitment, and are refractory to the inhibitory role of WNT activators. Using preclinical models for muscle damage versus fatty infiltration, we show that many MME+ FAPs undergo apoptosis during muscle regeneration and differentiate into adipocytes under pathological conditions, leading to a reduction in their abundance. Finally, we utilized the varying fat infiltration levels in human hip muscles and found less MME+ FAPs in fatty infiltrated human muscle. Altogether, we have identified the dominant adipogenic FAP subpopulation in skeletal muscle.

Resistance exercise enhances long-term mTORC1 sensitivity to leucine.

OBJECTIVE: Exercise enhances the sensitivity of mammalian target of rapamycin complex 1 (mTORC1) to amino acids, in particular leucine. How long this enhanced sensitivity lasts, and which mechanisms control enhanced leucine-mediated mTORC1 activation following exercise is currently unknown. METHODS: C57BL/6J mice were exercised for one night in a resistance-braked running wheel after a 12-day acclimatization period. Mice were gavaged with a submaximal dose of l-leucine or saline acutely or 48 h after exercise cessation, following 3 h food withdrawal. Muscles were excised 30 min after leucine administration. To study the contribution of mTORC1, we repeated those experiments but blocked mTORC1 activation using rapamycin immediately before the overnight running bout and one hour before the first dose of leucine. mTORC1 signaling, muscle protein synthesis and amino acid sensing machinery were assessed using immunoblot and qPCR. Leucine uptake was measured using L-[14C(U)]-leucine tracer labeling. RESULTS: When compared to sedentary conditions, leucine supplementation more potently activated mTORC1 and protein synthesis in acutely exercised muscle. This effect was observed in m. soleus but not in m. tibialis anterior nor m. plantaris. The synergistic effect in m. soleus was long-lasting as key downstream markers of mTORC1 as well as protein synthesis remained higher when leucine was administered 48 h after exercise. We found that exercise enhanced the expression of amino acid transporters and promoted uptake of leucine into the muscle, leading to higher free intramuscular leucine levels. This coincided with increased expression of activating transcription factor 4 (ATF4), a main transcriptional regulator of amino acid uptake and metabolism, and downstream activation of amino acid genes as well as leucyl-tRNA synthetase (LARS), a putative leucine sensor. Finally, blocking mTORC1 using rapamycin did not reduce expression and activation of ATF4, suggesting that the latter does not act downstream of mTORC1. Rather, we found a robust increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, suggesting that the integrated stress response pathway, rather than exercise-induced mTORC1 activation, drives long-term ATF4 expression in skeletal muscle after exercise. CONCLUSIONS: The enhanced sensitivity of mTORC1 to leucine is maintained at least 48 h after exercise. This shows that the anabolic window of opportunity for protein ingestion is not restricted to the first hours immediately following exercise. Increased mTORC1 sensitivity to leucine coincided with enhanced leucine influx into muscle and higher expression of genes involved in leucine sensing and amino acid metabolism. Also, exercise induced an increase in ATF4 protein expression. Altogether, these data suggest that muscular contractions switch on a coordinated program to enhance amino acid uptake as well as intramuscular sensing of key amino acids involved in mTORC1 activation and the stimulation of muscle protein synthesis.

Patient-reported outcomes evaluation and assessment of facilitators and barriers to physical activity in the Transplantoux aerobic exercise intervention.

BACKGROUND: Transplantoux's MVT exercise intervention prepares organ transplant recipients to cycle or hike up France's Mont Ventoux. We aimed to assess (i) MVT's effects on patient-reported outcomes (PROs) and (ii) perceived barriers and facilitators to physical activity. METHODS: Using a hybrid design, a convenience sample of transplant recipients participating in MVT (n = 47 cycling (TxCYC); n = 18 hiking (TxHIK)), matched control transplant recipients (TxCON, n = 213), and healthy MVT participants (HCON, n = 91) completed surveys to assess physical activity (IPAQ), health-related quality of life (HRQOL; SF-36 and EuroQol VAS), mental health (GHQ-12), and depressive symptomatology, anxiety, and stress (DASS-21) at baseline, then after 3, 6 (Mont Ventoux climb), 9, and 12 months. TxCYC and TxHIK participated in a 6-month intervention of individualized home-based cycling/hiking exercise and a series of supervised group training sessions. Barriers and facilitators to physical activity (Barriers and Motivators Questionnaire) were measured at 12 months. RESULTS: Regarding PROs, except for reducing TxHIK stress levels, MVT induced no substantial intervention effects. For both TxCYC and TxHIK, between-group comparisons at baseline showed that physical activity, HRQOL, mental health, depressive symptomatology and stress were similar to those of HCON. In contrast, compared to TxCYC, TxHIK, and HCON, physical activity, HRQOL and mental health were lower in TxCON. TxCON also reported greater barriers, lower facilitators, and different priority rankings concerning physical activity barriers and facilitators. CONCLUSION: Barely any of the PROs assessed in the present study responded to Transplantoux's MVT exercise intervention. TxCON reported distinct and unfavorable profiles regarding PROs and barriers and facilitators to physical activity. These findings can assist tailored physical activity intervention development. TRIAL REGISTRATION: Clinical trial notation: The study was approved by the University Hospitals Leuven's Institutional Review Board (B322201523602).

Exercise-induced angiogenesis is dependent on metabolically primed ATF3/4+ endothelial cells.

Exercise is a powerful driver of physiological angiogenesis during adulthood, but the mechanisms of exercise-induced vascular expansion are poorly understood. We explored endothelial heterogeneity in skeletal muscle and identified two capillary muscle endothelial cell (mEC) populations that are characterized by differential expression of ATF3/4. Spatial mapping showed that ATF3/4+ mECs are enriched in red oxidative muscle areas while ATF3/4low ECs lie adjacent to white glycolytic fibers. In vitro and in vivo experiments revealed that red ATF3/4+ mECs are more angiogenic when compared with white ATF3/4low mECs. Mechanistically, ATF3/4 in mECs control genes involved in amino acid uptake and metabolism and metabolically prime red (ATF3/4+) mECs for angiogenesis. As a consequence, supplementation of non-essential amino acids and overexpression of ATF4 increased proliferation of white mECs. Finally, deleting Atf4 in ECs impaired exercise-induced angiogenesis. Our findings illustrate that spatial metabolic angiodiversity determines the angiogenic potential of muscle ECs.

High-Intensity Training for 6 Months Safely, but Only Temporarily, Improves Exercise Capacity in Selected Solid Organ Transplant Recipients.

BACKGROUND: Organ transplantation is a life-saving intervention that improves quality of life of patients with irreversible organ failure. Although exercise training immediately after transplantation has been suggested to be beneficial, such interventions remain rare in stable transplant recipients, whereas effects of high-intensity training (HIT) are even less frequently investigated. Moreover, sustainability of such interventions has not yet been reported. We investigated the effects of a 6-month, cycling-based HIT program on physical performance in long-term stable solid organ transplant (SOT) recipients, with follow-up evaluation after 6 months. METHODS: Forty-two adult, stable, and selected SOT recipients participated in a 6-month individualized home- and group-based HIT program. Exercise capacity (VO2max), maximal power (Wmax), and body mass index were measured before, at the end, and 6 months after completion of the intervention. RESULTS: The study comprised 12 heart, 7 lung, 8 liver, and 15 kidney recipients (mean age, 41.4 ± 11.1 years; median time posttransplant, 3.4 [1.7-8.0] years). For 6 months, VO2max increased in the heart, lung, and kidney groups, Wmax increased in the heart group, and body mass index decreased in the liver group. Six months after the HIT program, the achieved gain in exercise capacity had disappeared in all groups. CONCLUSION: Despite voluntary participation selection bias, our observations indicate that HIT is safe and may result in a beneficial effect on physical performance in selected, stable SOT recipients. However, there was no sustained beneficial effect once training stopped. Larger scale and longer term studies are still required to investigate longevity of improvement and overall beneficial effects on clinical outcomes.

Dampened Muscle mTORC1 Response Following Ingestion of High-Quality Plant-Based Protein and Insect Protein Compared to Whey.

Increased amino acid availability acutely stimulates protein synthesis partially via activation of mechanistic target of rapamycin complex 1 (mTORC1). Plant-and insect-based protein sources matched for total protein and/or leucine to animal proteins induce a lower postprandial rise in amino acids, but their effects on mTOR activation in muscle are unknown. C57BL/6J mice were gavaged with different protein solutions: whey, a pea-rice protein mix matched for total protein or leucine content to whey, worm protein matched for total protein, or saline. Blood was drawn 30, 60, 105 and 150 min after gavage and muscle samples were harvested 60 min and 150 min after gavage to measure key components of the mTORC1 pathway. Ingestion of plant-based proteins induced a lower rise in blood leucine compared to whey, which coincided with a dampened mTORC1 activation, both acutely and 150 min after administration. Matching total leucine content to whey did not rescue the reduced rise in plasma amino acids, nor the lower increase in mTORC1 compared to whey. Insect protein elicits a similar activation of downstream mTORC1 kinases as plant-based proteins, despite lower postprandial aminoacidemia. The mTORC1 response following ingestion of high-quality plant-based and insect proteins is dampened compared to whey in mouse skeletal muscle.

Exercise promotes satellite cell contribution to myofibers in a load-dependent manner.

BACKGROUND: Satellite cells (SCs) are required for muscle repair following injury and are involved in muscle remodeling upon muscular contractions. Exercise stimulates SC accumulation and myonuclear accretion. To what extent exercise training at different mechanical loads drive SC contribution to myonuclei however is unknown. RESULTS: By performing SC fate tracing experiments, we show that 8 weeks of voluntary wheel running increased SC contribution to myofibers in mouse plantar flexor muscles in a load-dependent, but fiber type-independent manner. Increased SC fusion however was not exclusively linked to muscle hypertrophy as wheel running without external load substantially increased SC fusion in the absence of fiber hypertrophy. Due to nuclear propagation, nuclear fluorescent fate tracing mouse models were inadequate to quantify SC contribution to myonuclei. Ultimately, by performing fate tracing at the DNA level, we show that SC contribution mirrors myonuclear accretion during exercise. CONCLUSIONS: Collectively, mechanical load during exercise independently promotes SC contribution to existing myofibers. Also, due to propagation of nuclear fluorescent reporter proteins, our data warrant caution for the use of existing reporter mouse models for the quantitative evaluation of satellite cell contribution to myonuclei.

Endothelial Lactate Controls Muscle Regeneration from Ischemia by Inducing M2-like Macrophage Polarization.

Endothelial cell (EC)-derived signals contribute to organ regeneration, but angiocrine metabolic communication is not described. We found that EC-specific loss of the glycolytic regulator pfkfb3 reduced ischemic hindlimb revascularization and impaired muscle regeneration. This was caused by the reduced ability of macrophages to adopt a proangiogenic and proregenerative M2-like phenotype. Mechanistically, loss of pfkfb3 reduced lactate secretion by ECs and lowered lactate levels in the ischemic muscle. Addition of lactate to pfkfb3-deficient ECs restored M2-like polarization in an MCT1-dependent fashion. Lactate shuttling by ECs enabled macrophages to promote proliferation and fusion of muscle progenitors. Moreover, VEGF production by lactate-polarized macrophages was increased, resulting in a positive feedback loop that further stimulated angiogenesis. Finally, increasing lactate levels during ischemia rescued macrophage polarization and improved muscle reperfusion and regeneration, whereas macrophage-specific mct1 deletion prevented M2-like polarization. In summary, ECs exploit glycolysis for angiocrine lactate shuttling to steer muscle regeneration from ischemia.

Hallmarks of frailty and osteosarcopenia in prematurely aged PolgA(D257A/D257A) mice.

BACKGROUND: Frailty is a geriatric syndrome characterized by increased susceptibility to adverse health outcomes. One major determinant thereof is the gradual weakening of the musculoskeletal system and the associated osteosarcopenia. To improve our understanding of the underlying pathophysiology and, more importantly, to test potential interventions aimed at counteracting frailty, suitable animal models are needed. METHODS: To evaluate the relevance of prematurely aged PolgA(D257A/D257A) mice as a model for frailty and osteosarcopenia, we quantified the clinical mouse frailty index in PolgA(D257A/D257A) and wild-type littermates (PolgA(+/+) , WT) with age and concertedly assessed the quantity and quality of bone and muscle tissue. Lastly, the anabolic responsiveness of skeletal muscle, muscle progenitors, and bone was assessed. RESULTS: PolgA(D257A/D257A) accumulated health deficits at a higher rate compared with WT, resulting in a higher frailty index at 40 and 46 weeks of age (+166%, +278%, P < 0.0001), respectively, with no differences between genotypes at 34 weeks. Concomitantly, PolgA(D257A/D257A) displayed progressive musculoskeletal deterioration such as reduced bone and muscle mass as well as impaired functionality thereof. In addition to lower muscle weights (-14%, P < 0.05, -23%, P < 0.0001) and fibre area (-20%, P < 0.05, -22%, P < 0.0001) at 40 and 46 weeks, respectively, PolgA(D257A/D257A) showed impairments in grip strength and concentric muscle forces (P < 0.05). PolgA(D257A/D257A) mutation altered the acute response to various anabolic stimuli in skeletal muscle and muscle progenitors. While PolgA(D257A/D257A) muscles were hypersensitive to eccentric contractions as well as leucine administration, shown by larger downstream signalling response of the mechanistic target of rapamycin complex 1, myogenic progenitors cultured in vitro showed severe anabolic resistance to leucine and robust impairments in cell proliferation. Longitudinal micro-computed tomography analysis of the sixth caudal vertebrae showed that PolgA(D257A/D257A) had lower bone morphometric parameters (e.g. bone volume fraction, trabecular, and cortical thickness, P < 0.05) as well as reduced remodelling activities (e.g. bone formation and resorption rate, P < 0.05) compared with WT. When subjected to 4 weeks of cyclic loading, young but not aged PolgA(D257A/D257A) caudal vertebrae showed load-induced bone adaptation, suggesting reduced mechanosensitivity with age. CONCLUSIONS: PolgA(D257A/D257A) mutation leads to hallmarks of age-related frailty and osteosarcopenia and provides a powerful model to better understand the relationship between frailty and the aging musculoskeletal system.

PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase.

mTORC1 is an important regulator of muscle mass but how it is modulated by oxygen and nutrients is not completely understood. We show that loss of the prolyl hydroxylase domain isoform 1 oxygen sensor in mice (PHD1KO) reduces muscle mass. PHD1KO muscles show impaired mTORC1 activation in response to leucine whereas mTORC1 activation by growth factors or eccentric contractions was preserved. The ability of PHD1 to promote mTORC1 activity is independent of its hydroxylation activity but is caused by decreased protein content of the leucyl tRNA synthetase (LRS) leucine sensor. Mechanistically, PHD1 interacts with and stabilizes LRS. This interaction is promoted during oxygen and amino acid depletion and protects LRS from degradation. Finally, elderly subjects have lower PHD1 levels and LRS activity in muscle from aged versus young human subjects. In conclusion, PHD1 ensures an optimal mTORC1 response to leucine after episodes of metabolic scarcity.

Voluntary Resistance Running as a Model to Induce mTOR Activation in Mouse Skeletal Muscle.

Long-term voluntary resistance running has been shown to be a valid model to induce muscle growth in rodents. Moreover, the mammalian target of rapamycin complex 1 (mTORC1) is a key signaling complex regulating exercise/nutrient-induced alterations in muscle protein synthesis. How acute resistance running affects mTORC1 signaling in muscle and if resistance applied to the wheel can modulate mTORC1 activation has not yet been fully elucidated. Here, we show that both acute resistance running and acute free running activated mTORC1 signaling in the m. gastrocnemius, m. soleus, and m. plantaris, but not in m. tibialis anterior of mice when compared to sedentary controls. Furthermore, only the low threshold oxidative part in the m. gastrocnemius showed increased mTORC1 signaling upon running and acute heavy-load resistance running evoked higher downstream mTORC1 signaling in both m. soleus and m. plantaris than free running without resistance, pointing toward mechanical load as an important independent regulator of mTORC1. Collectively, in this study, we show that voluntary resistance running is an easy-to-use, time-efficient and low stress model to study acute alterations in mTORC1 signaling upon high-load muscular contractions in mice.

Twin Resemblance in Muscle HIF-1α Responses to Hypoxia and Exercise.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of myocellular adaptation to exercise and hypoxia. However, the role of genetic factors in regulation of HIF-1 responses to exercise and hypoxia is unknown. We hypothesized that hypoxia at rest and during exercise stimulates the HIF-1 pathway and its downstream targets in energy metabolism regulation in a genotype-dependent manner. Eleven monozygotic twin (MZ) pairs performed an experimental trial in both normoxia and hypoxia (FiO2 10.7%). Biopsies were taken from m. vastus lateralis before and after a 20-min submaximal cycling bout @~30% of sea-level VO2max. Key-markers of the HIF-1 pathway and glycolytic and oxidative metabolism were analyzed using real-time PCR and Western Blot. Hypoxia increased HIF-1α protein expression by ~120% at rest vs. +150% during exercise (p < 0.05). Furthermore, hypoxia but not exercise increased muscle mRNA content of HIF-1α (+50%), PHD2 (+45%), pVHL (+45%; p < 0.05), PDK4 (+1200%), as well as PFK-M (+20%) and PPAR-γ1 (+60%; p < 0.05). Neither hypoxia nor exercise altered PHD1, LDH-A, PDH-A1, COX-4, and CS mRNA expressions. The hypoxic, but not normoxic exercise-induced increment of muscle HIF-1α mRNA content was about 10-fold more similar within MZ twins than between the twins (p < 0.05). Furthermore, in resting muscle the hypoxia-induced increments of muscle HIF-1α protein content, and HIF-1α and PDK4 mRNA content were about 3-4-fold more homogeneous within than between the twins pairs (p < 0.05). The present observations in monozygotic twins for the first time clearly indicate that the HIF-1α protein as well as mRNA responses to submaximal exercise in acute hypoxia are at least partly regulated by genetic factors.

A genetic predisposition score associates with reduced aerobic capacity in response to acute normobaric hypoxia in lowlanders.

Given the high inter-individual variability in the sensitivity to high altitude, we hypothesize the presence of underlying genetic factors. The aim of this study was to construct a genetic predisposition score based on previously identified high-altitude gene variants to explain the inter-individual variation in the reduced maximal O2 uptake (ΔVo2max) in response to acute hypoxia. Ninety-six healthy young male Belgian lowlanders were included. In both normobaric normoxia (Fio2=20.9%) and acute normobaric hypoxia (Fio2=10.7%-12.5%) Vo2max was measured. Forty-one SNPs in 21 genes were genotyped. A stepwise regression analysis was applied to detect a subset of SNPs to be associated with ΔVo2max. This subset of SNPs was included in the genetic predisposition score. A general linear model and regression analysis with age, weight, height, hypoxic protocol group, and Vo2max in normoxia as covariates were used to test the explained variance of the genetic predisposition score. A ROC analysis was performed to discriminate between the low- and high ΔVo2max subgroups. A stepwise regression analysis revealed a subset of SNPs [rs833070 (VEGFA), rs4253778 (PPARA), rs6735530 (EPAS1), rs4341 (ACE), rs1042713 (ADRB2), and rs1042714 (ADRB2)] to be associated with ΔVo2max. The genetic predisposition score was found to be an independent predictive variable with a partial explained variance of 23% (p<0.0001). A ROC analysis showed significant discriminating accuracy (AUC=0.78, 95% confidence interval=0.64-0.91) between the low- and high ΔVo2max subgroups. This six-SNP based genetic predisposition score showed a significantly predictive value for ΔVo2max.

High twin resemblance for sensitivity to hypoxia.

PURPOSE: Physiological responses to hypoxia vary between individuals, and genetic factors are conceivably involved. Using a monozygotic twin design, we investigated the role of genetic factors in physiological responses to acute hypoxia. METHODS: Thirteen pairs of monozygotic twin brothers participated in two experimental sessions in a normobaric hypoxic facility with a 2-wk interval. In one session, fraction of inspired O2 (FiO2) was gradually reduced to 10.7% (approximately 5300 m altitude) over 5 h. During the next 3 h at 10.7%, FiO2 subjects performed a 20-min submaximal exercise bout (EXSUB, 1.2 W·kg) and a maximal incremental exercise test (EXMAX). An identical control experiment was done in normoxia. Cardiorespiratory measurements were continuously performed, and 8-h urine output was collected. RESULTS: Compared with normoxia, hypoxia decreased (P < 0.05) arterial O2 saturation (%SpO2) at rest (-22%) and during exercise (-28%). Furthermore, V˙O2max (-39%), HRmax (HR, -8%), maximal pulmonary ventilation (V˙Emax, -11%), and urinary norepinephrine excretion (-31%) were reduced (P < 0.05) whereas HR at rest (25%) and during EXSUB (16%) and V˙E at rest (38%) and during EXSUB (70%) were increased (P < 0.05). However, hypoxia-induced changes (Δ) were not randomly distributed between subjects. Between-pair variance was substantially larger than within-pair variance (P < 0.05) for Δ%SpO2 at rest (approximately threefold) and during exercise (approximately fourfold), ΔV˙O2max (approximately fourfold), ΔHR during exercise (approximately seven- to eightfold), hypoxic ventilatory response (approximately sixfold), and Δ urinary norepinephrine output (approximately threefold). Incidence of acute mountain sickness (AMS) also yielded significant twin similarity (P < 0.05). AMS subjects showed approximately 50% greater drop in urinary norepinephrine and lower hypoxic ventilator response than AMS individuals. CONCLUSIONS: Our data suggest that genetic factors regulate cardiorespiratory responses, exercise tolerance, and pathogenesis of AMS symptoms in acute severe hypoxia. Hypoxia-induced sympathetic downregulation was associated with AMS.

Acute environmental hypoxia induces LC3 lipidation in a genotype-dependent manner.

Hypoxia-induced muscle wasting is a phenomenon often described with prolonged stays at high altitude, which has been attributed to altered protein metabolism. We hypothesized that acute normobaric hypoxia would induce a negative net protein balance by repressing anabolic and activating proteolytic signaling pathways at rest and postexercise and that those changes could be partially genetically determined. Eleven monozygotic twins participated in an experimental trial in normoxia and hypoxia (10.7% O2). Muscle biopsy samples were obtained before and after a 20-min moderate cycling exercise. In hypoxia at rest, autophagic flux was increased, as indicated by an increased microtubule-associated protein 1 light chain 3 type II/I (LC3-II/I) ratio (+25%) and LC3-II expression (+60%) and decreased p62/SQSTM1 expression (-25%; P<0.05), whereas exercise reversed those changes to a level similar to that with normoxia except for p62/SQSTM1, which was further decreased (P<0.05). Hypoxia also increased Bnip3 (+34%) and MAFbx (+18%) mRNA levels as well as REDD1 expression (+439%) and AMP-activated protein kinase phosphorylation (+22%; P<0.05). Among the molecular responses to hypoxia and/or exercise, high monozygotic similarity was found for REDD1, LC3-II, and LC3-II/I (P<0.05). Our results indicate that environmental hypoxia modulates protein metabolism at rest and after moderate exercise by primarily increasing markers of protein breakdown and, more specifically, markers of the autophagy-lysosomal system, with a modest genetic contribution.

Dietary nitrate improves muscle but not cerebral oxygenation status during exercise in hypoxia.

Exercise tolerance is impaired in hypoxia, and it has recently been shown that dietary nitrate supplementation can reduce the oxygen (O(2)) cost of muscle contractions. Therefore, we investigated the effect of dietary nitrate supplementation on arterial, muscle, and cerebral oxygenation status, symptoms of acute mountain sickness (AMS), and exercise tolerance at simulated 5,000 m altitude. Fifteen young, healthy volunteers participated in three experimental sessions according to a crossover study design. From 6 days prior to each session, subjects received either beetroot (BR) juice delivering 0.07 mmol nitrate/kg body wt/day or a control drink (CON). One session was in normoxia with CON (NOR(CON)); the two other sessions were in hypoxia (11% O(2)), with either CON (HYP(CON)) or BR (HYP(BR)). Subjects first cycled for 20 min at 45% of peak O(2) consumption (VO(2)peak; EX(45%)) and thereafter, performed a maximal incremental exercise test (EX(max)). Whole-body VO(2), arterial O(2) saturation (%SpO(2)) via pulsoximetry, and tissue oxygenation index of both muscle (TOI(M)) and cerebral (TOI(C)) tissue by near-infrared spectroscopy were measured. Hypoxia per se substantially reduced VO(2)peak, %SpO(2), TOI(M), and TOI(C) (NOR(CON) vs. HYP(CON), P < 0.05). Compared with HYP(CON), VO(2) at rest and during EX(45%) was lower in HYP(BR) (P < 0.05), whereas %SpO(2) was higher (P < 0.05). TOI(M) was ~4-5% higher in HYP(BR) than in HYP(CON) both at rest and during EX(45%) and EX(max) (P < 0.05). TOI(C) as well as the incidence of AMS symptoms were similar between HYP(CON) and HYP(BR) at any time. Hypoxia reduced time to exhaustion in EX(max) by 36% (P < 0.05), but this ergolytic effect was partly negated by BR (+5%, P < 0.05). Short-term dietary nitrate supplementation improves arterial and muscle oxygenation status but not cerebral oxygenation status during exercise in severe hypoxia. This is associated with improved exercise tolerance against the background of a similar incidence of AMS.

Simultaneous determination of nitrite and nitrate in human plasma by on-capillary preconcentration with field-amplified sample stacking.

A simple method for the determination of nitrite and nitrate in human plasma has been developed using CZE with minimal sample preparation. Field-amplified sample stacking (FASS) was used to achieve submicromolar detection by dilution of the plasma sample with deionized water. In CZE, the separation of nitrite and nitrate was achieved within 10 min without adding EOF modifier. The optimal condition was achieved with 50 mM phosphate buffer at pH 9.3. The ninefold diluted plasma samples were injected hydrodynamically for 40 s into a 60 cm×75 µm id uncoated fused-silica capillary. The separation voltage was 20 kV (negative potential) and UV detection was performed at 214 nm. The linearity curves for nitrite and nitrate were obtained by the standard addition method. The estimated LODs for nitrite and nitrate in ninefold diluted plasma sample were 0.05 and 0.07 µM, respectively. The LODs for nitrite and nitrate in original plasma samples were 0.45 and 0.63 µM. The intra- and inter-day precisions for both analytes were <2.6% and the recovery ranged between 92.3 and 113.3%. It was found that nitrite was more stable than nitrate in the plasma after the sample preparation. This proposed method was applied to a number of human plasma samples and the measured nitrite and nitrate concentrations in human plasma were consistent with the literature ranges.