Erythroid terminal differentiation (ETD) entails cell divisions coupled to decreasing cell size. The tight link between the number of cell divisions and red cell size is apparent in nutritional ...deficiencies or genetic variants in which fewer cycles result in larger red cells. Here we investigated novel EpoR functions, finding that EpoR signaling disrupts the relationship between cell cycle number and cell size, simultaneously promoting rapid cycling and the formation of larger red cells.
EpoR is essential for erythroblast survival, but it is unclear whether it has other non-redundant functions. To address this, we developed a genetic system in which we rescue mouse Epor -/- fetal liver progenitors from apoptosis by transduction with the anti-apoptotic protein Bcl-x L, and compare their ensuing differentiation with that of Epor -/- progenitors rescued with EpoR (Fig 1a). We found that the Bcl-x L survival signal, in the absence EpoR, supported formation of enucleated red cells. However, key ETD features were abnormal.
First, Bcl-x L-transduced Epor -/- erythroblasts underwent slower and fewer cell cycles (Figure 1b), differentiating prematurely into enucleated red cells. Premature induction of the cyclin-dependent-kinase inhibitor p27 KIP1 was in part responsible for the fewer cycles in the absence of EpoR signaling. We confirmed that EpoR also stimulates rapid cycling in wild-type erythroblasts in vivo, using a mouse transgenic for a live-cell reporter of cell cycle speed.
Second, using imaging flow cytometry, we found that Bcl-x L-transduced Epor -/- erythroblasts were smaller than EpoR-transduced Epor -/- cells (Fig 1c,d). By doubly transducing Epor -/- erythroblasts with both Bcl-x L and EpoR, we verified that EpoR absence, and not Bcl-x L overexpression, is responsible for the smaller size of Bcl-x L-transduced Epor -/- erythroblasts and reticulocytes.
Bcl-x L-transduced Epor -/- erythroblasts failed to upregulate the transferrin receptor, suggesting that iron deficiency may be responsible for their smaller size. However, neither iron supplementation, nor transduction with the transferrin receptor, rescued their smaller size. Iron regulates cell size through Heme-regulated eIF2α kinase (HRI). To definitively test the role of iron and HRI, we generated mice doubly deleted for both EpoR and HRI. We then rescued both Epor -/- and Epor -/-Hri -/- -fetal liver cells in parallel, by transduction with either Bcl-x L or EpoR. In agreement with the known role of HRI as a negative regulator of erythroblast size, both Bcl-x L- transduced and EpoR-transduced erythroblasts were larger on the Epor -/-Hri -/- genetic background. However, the difference in size between Bcl-x L and EpoR-rescued erythroblasts persisted in Epor -/-Hri -/- erythroblasts and reticulocytes (Fig 1c,d), conclusively showing that EpoR signaling regulates cell size independently of the HRI pathway.
EpoR promoted increased erythroblast and reticulocyte cell size in wild-type mice in vitro and in vivo, in response to Epo concentrations ranging from 10 to 10,000 mU/ml. We also evaluated the effect of Epo on red cell size in humans, in two independent studies, where healthy volunteers were administered Epo for either 3 weeks (20 IU /kg every 48 hours, 25 subjects, Study #1) or for 7 weeks (weekly Epo dosing that increased hemoglobin by 10 -15%; 24 subjects, Study #2). In a third intervention, 21 subjects participated in a randomized double-blind placebo-controlled crossover study in which 900 ml of whole blood was withdrawn from the treatment group by venipuncture. In all three studies, the increase in MCV in the treatment groups persisted long after Epo and reticulocyte levels returned to baseline (Figure 2). There was no correlation between MCV and the reticulocyte count, whose time courses were clearly divergent (r < 0.1, Pearson's product-moment correlation). Further, computational simulation suggests that the extent and duration of the increase in MCV is unlikely to be the result of skewing of the circulating red cell population in favor of younger, larger red cells.
Our work reveals a paradoxical EpoR-driven increase in erythroblast cycling simultaneously with increased erythroblast and red cell size. It suggests that EpoR alters the relationship between cell cycle and biomass in erythroblasts. It further suggests that hypoxia, anemia and other high-Epo syndromes are new diagnostic interpretations of increased MCV in the clinic.
Display omitted
No relevant conflicts of interest to declare.
Abstract only
Autologous blood transfusion (ABT) is illegal for competing athletes but misuse remains difficult to detect. Thus, the development of novel and sensitive methods is warranted. In this ...study, we explored whether ABT causes a shift of the urine metabolome. The hypothesis was that an untargeted metabolomics analysis of urine is able to identify novel biomarkers sensitive to ABT.
In a randomized, double‐blinded, placebo‐controlled cross over design (3 month wash‐out), twelve trained males donated 900 ml blood or were sham phlebotomized. Four weeks later, a transfusion of the stored red blood cells or a sham transfusion was completed. Urine was collected before phlebotomy and 2 h, 1, 2, 3, 5 and 10 days after transfusion and analyzed by UPLC‐QTOF‐MS.
Models of unique metabolites reflecting ABT was derived by partial least squares regression discriminant analysis. The strongest model appeared 2 h after transfusion (misclassification error: 6.3%, specificity: 98.8%). The remaining time points provided misclassification errors from ~20–50% with specificities of ~50–80%. Metabolite identification revealed secondary di‐2‐ethylhexyl phtalate metabolites as the strongest biomarkers for detection.
In conclusion, untargeted metabolomics of urine identified plasticizers as the strongest metabolites for ABT detection. Future research should validate the proposed models.
Support or Funding Information
The study was funded by Partnership for Clean Competition
PURPOSEAutologous blood transfusion is performance enhancing and prohibited in sport but remains difficult to detect. This study explored the hypothesis that an untargeted urine metabolomics analysis ...can reveal one or more novel metabolites with high sensitivity and specificity for detection of autologous blood transfusion.
METHODSIn a randomized, double-blinded, placebo-controlled, crossover design, exercise-trained men (n = 12) donated 900 mL blood or were sham phlebotomized. After 4 wk, red blood cells or saline were reinfused. Urine samples were collected before phlebotomy and 2 h and 1, 2, 3, 5, and 10 d after reinfusion and analyzed by ultraperformance liquid chromatography–quadrupole time-of-flight mass spectrometry. Models of unique metabolites reflecting autologous blood transfusion were attained by partial least-squares discriminant analysis.
RESULTSThe strongest model was obtained 2 h after reinfusion with a misclassification error of 6.3% and 98.8% specificity. However, combining only a few of the strongest metabolites selected by this model provided a sensitivity of 100% at days 1 and 2 and 66% at day 3 with 100% specificity. Metabolite identification revealed the presence of secondary di-2-ethylhexyl phtalate metabolites and putatively identified the presence of (iso)caproic acid glucuronide as the strongest candidate biomarker.
CONCLUSIONSUntargeted urine metabolomics revealed several plasticizers as the strongest metabolic pattern for detection of autologous blood transfusion for up to 3 d. Importantly, no other metabolites in urine seem of value for antidoping purposes.
EpoR signaling is essential for the survival of erythroid precursors during terminal differentiation, when these cells undergo 3-5 cell divisions that are coupled to a rapid loss in cell size. The ...absolute dependence of erythroblasts on EpoR signaling for survival makes it challenging to identify other essential functions of EpoR in these cells. Key open questions include a role for EpoR in cell cycle regulation; and a potential role for EpoR in the induction of erythroid genes.
To address these gaps, we developed a genetic system that allowed us to examine essential non-survival functions of EpoR signaling in erythroid differentiation. We rescued mouse Epor-/- fetal liver erythroid progenitors from apoptosis by transduction with the anti-apoptotic protein bcl-xL, comparing their ensuing differentiation with that of Epor-/- progenitors that were rescued by re-introduction of the EpoR. We found that the bcl-xL survival signal, in the absence of any EpoR signaling, was sufficient to allow erythroid terminal differentiation and formation of anucleated red cells. However, key features were abnormal. First, erythroblasts underwent slower and fewer cell cycles, generating fewer than half the number of red cells, showing an essential role for EpoR signaling in the regulation of cell cycle speed and number. Second, we found that EpoR signaling unexpectedly increased red cell size, in spite of the concurrent increase in the number of cell divisions. Using mice doubly deleted for both EpoR and HRI, we found that EpoR regulation of red cell size is independent of the well-described red cell size regulation by iron and heme. Healthy human volunteers that were administered Epo showed an increase in mean corpuscular volume (MCV) that persisted long after Epo and reticulocyte levels returned to baseline, supporting a role for the regulation of red cell size by Epo and suggesting new diagnostic interpretations for this common clinical test.
Together, our findings reveal novel functions for the EpoR in erythroid differentiation including an unexpected role in the regulation of red cell size.
Total hemoglobin mass (Hb
mass
) is routinely assessed in studies by the carbon monoxide (CO) rebreathing. Its clinical application is often hindered due to the consequent rise in carboxyhemoglobin ...(%HbCO) and the concern of CO toxicity. We tested the reproducibility of the CO rebreathing with a CO dose of 0.5 mL/kg body mass (CO
0.5
) compared to 1.5 mL/kg (CO
1.5
) and when shortening the CO rebreathing protocol. Therefore, CO rebreathing was performed 1×/day in eight healthy individuals on four consecutive days. On each day, either CO
0.5
(CO
0.5
-1 and CO
0.5
-2) or CO
1.5
(CO
1.5
-1 and CO
1.5
-2) was administered. Venous blood samples to determine %HbCO and quantify Hb
mass
were obtained prior to, and at 6 (T
6
), 8 (T
8
) and 10 min (T
10
) of CO rebreathing. This protocol was tested at sea level and at 2320 m to investigate the altitude-related measurement error. At sea level, the mean difference (95% limits of agreement) in Hb
mass
between CO
0.5
-1 and CO
0.5
-2 was 26 g (-26; 79 g) and between CO
1.5
-1 and CO
1.5
-2, it was 17 g (-18; 52 g)
.
The respective typical error (TE) corresponded to 2.4% (CO
0.5
) and 1.5% (CO
1.5
), while it was 6.5% and 3.0% at 2320 m. With CO
0.5
, shortening the CO rebreathing resulted in a TE for Hb
mass
of 4.4% (T
8
vs. T
10
) and 14.1% (T
6
vs T
10
) and with CO
1.5
, TE was 1.6% and 5.8%. In conclusion, the CO dose and rebreathing time for the CO rebreathing procedure can be decreased at the cost of a measurement error ranging from 1.5-14.1%.
Purpose
Angiotensin-converting enzyme (ACE) inhibitor treatment is widely applied, but the fact that plasma ACE activity is a potential determinant of training-induced local muscular adaptability is ...often neglected. Thus, we investigated the hypothesis that ACE inhibition modulates the response to systematic aerobic exercise training on leg and arm muscular adaptations.
Methods
Healthy, untrained, middle-aged participants (40 ± 7 yrs) completed a randomized, double-blinded, placebo-controlled trial. Participants were randomized to placebo (PLA: CaCO
3
) or ACE inhibitor (ACE
i
: enalapril) for 8 weeks and completed a supervised, high-intensity exercise training program. Muscular characteristics in the leg and arm were extensively evaluated pre and post-intervention.
Results
Forty-eight participants (
n
ACEi
= 23,
n
PLA
= 25) completed the trial. Exercise training compliance was above 99%. After training, citrate synthase, 3-hydroxyacyl-CoA dehydrogenase and phosphofructokinase maximal activity were increased in m. vastus lateralis in both groups (all
P
< 0.05) without statistical differences between them (all time × treatment
P
> 0.05). In m. deltoideus, citrate synthase maximal activity was upregulated to a greater extent (time × treatment
P
< 0.05) in PLA (51 33;69 %) than in ACE
i
(28 13;43 %), but the change in 3-hydroxyacyl-CoA dehydrogenase and phosphofructokinase maximal activity was similar between groups. Finally, the training-induced changes in the platelet endothelial cell adhesion molecule-1 protein abundance, a marker of capillary density, were similar in both groups in m. vastus lateralis and m. deltoideus.
Conclusion
Eight weeks of high-intensity whole-body exercise training improves markers of skeletal muscle mitochondrial oxidative capacity, glycolytic capacity and angiogenesis, with no overall effect of pharmacological ACE inhibition in healthy adults.
We examined whether a semi-automated carbon monoxide (CO) rebreathing method accurately detect changes in blood volume (BV) and total hemoglobin mass (tHb). Furthermore, we investigated whether a ...supine position with legs raised reduced systemic CO dilution time, potentially allowing a shorter rebreathing period. Nineteen young healthy males participated. BV and tHb was quantified by a 10-min CO-rebreathing period in a supine position with legs raised before and immediately after a 900 ml phlebotomy and before and after a 900 ml autologous blood reinfusion on the same day in 16 subjects. During the first CO-rebreathing, arterial and venous blood samples were drawn every 2 min during the procedure to determine systemic CO equilibrium in all subjects. Phlebotomy decreased (P < 0.001) tHb and BV by 166 ± 24 g and 931 ± 247 ml, respectively, while reinfusion increased (P < 0.001) tHb and BV by 143 ± 21 g and 862 ± 250 ml compared to before reinfusion. After reinfusion BV did not differ from baseline levels while tHb was decreased (P < 0.001) by 36 ± 21 g. Complete CO mixing was achieved within 6 min in venous and arterial blood, respectively, when compared to the 10-min sample. On an individual level, the relative accuracy after donation for tHb and BV was 102–169% and 55–165%, respectively. The applied CO-rebreathing procedure precisely detect acute BV changes with a clinically insignificant margin of error. The 10-min CO-procedure may be reduced to 6 min with no clinical effects on BV and tHb calculation. Notwithstanding, individual differences may be of concern and should be investigated further.
Blood volume (BV) is an important clinical parameter and is usually reported per kg of body mass (BM). When fat mass is elevated, this underestimates BV/BM. One aim was to study if differences in ...BV/BM related to sex, age, and fitness would decrease if normalized to lean body mass (LBM). The analysis included 263 women and 319 men (age: 10–93 years, body mass index: 14–41 kg/m2) and 107 athletes who underwent assessment of BV and hemoglobin mass (Hbmass), body composition, and cardiorespiratory fitness. BV/BM was 25% lower (70.3 ± 11.3 and 80.3 ± 10.8 mL/kgBM) in women than men, respectively, whereas BV/LBM was 6% higher in women (110.9 ± 12.5 and 105.3 ± 11.2 mL/kgLBM). Hbmass/BM was 34% lower (8.9 ± 1.4 and 11.5 ± 11.2 g/kgBM) in women than in men, respectively, but only 6% lower (14.0 ± 1.5 and 14.9 ± 1.5 g/kgLBM)/LBM. Age did not affect BV. Athlete's BV/BM was 17.2% higher than non‐athletes, but decreased to only 2.5% when normalized to LBM. Of the variables analyzed, LBM was the strongest predictor for BV (R2 = .72, p < .001) and Hbmass (R2 = .81, p < .001). These data may only be valid for BV/Hbmass when assessed by CO re‐breathing. Hbmass/LBM could be considered a valuable clinical matrix in medical care aiming to normalize blood homeostasis.
Optimized concurrent training regimes are warranted in physical training of military-, law enforcement- and rescue-personnel. This study investigated if four 15-min endurance training sessions weekly ...improve aerobic capacity and performance more than one 60-min endurance session weekly during the initial phase of a Basic Military Training program.
A randomized training intervention study with functional and physiological tests before and after the intervention.
Military conscripts (n=290) were randomly allocated to three groups completing 9 weeks training. Weekly training consisted of four endurance and four strength training sessions lasting 15min each (‘Micro-training’: MIC); one strength and one endurance session lasting 60min each (‘Classical-training’: CLA) or two 60min sessions of standard military training (‘Control-training’: CON).
Both 12-min (∼7−10%) and shuttle run performance (∼35−42%) improved (P≤0.001) similarly in all groups. Likewise, functional 2-min maximal repetition exercise capacity increased (P≤0.05) similarly in all groups (Lunges ∼17−24 %; PushUp ∼10−20%; AbdominalFlexions∼21−23%). Peak oxygen uptake changes depended on group (P≤0.05) with increases (P≤0.01) in MIC (7±7%, n=23) and CON (12±18%, n=17) and no changes in CLA. Maximal m. vastus lateralis citrate synthase activity decreased 14±26% (P≤0.001, n=18) in CLA. Likewise, maximal m. vastus lateralis 3-hydroxyacyl-CoA dehydrogenase activity decreased 8±17% in MIC (n=28) and 14±24% in CLA (n=18).
Four 15-min endurance training sessions weekly improves running performance and strength-endurance similarly to one 60min session. Peak oxygen uptake only increases with more than one endurance session weekly and leg muscle oxidative capacity appears reduced after basic military training.
PDF
