Effects of a Maximal Exercise Followed by a Submaximal Exercise Performed in Normobaric Hypoxia (2500 m), on Blood Rheology, Red Blood Cell Senescence, and Coagulation in Well-Trained Cyclists

exercise impacts the rheological properties of red blood cells (RBC) and their senescence state; however, there is a lack of data on the effects of exercise performed in hypoxia on RBC properties. This crossover study compared the effects of acute hypoxia vs. normoxia on blood rheology, RBC senescence, and coagulation during exercise.

69002 Bron, France *Correspondence: elie.nader@univ-lyon1.fr Abstract: Acute normoxic exercise impacts the rheological properties of red blood cells (RBC) and their senescence state; however, there is a lack of data on the effects of exercise performed in hypoxia on RBC properties. This crossover study compared the effects of acute hypoxia vs. normoxia on blood rheology, RBC senescence, and coagulation during exercise. Nine trained male cyclists completed both a session in normoxia (FiO 2= 21%) and hypoxia (FiO 2= 15.3% 2500 m). The two sessions were randomly performed, separated by one week, and consisted of an incremental and maximal exercise followed by a 20 min exercise at the rst ventilatory threshold (VT1) on a home-trainer. Blood samples were taken before and after exercise to analyze hematological parameters, blood rheology (hematocrit, blood viscosity, RBC deformability and aggregation), RBC senescence markers (phosphatidylserine (PS) and CD47 exposure, intraerythrocyte reactive oxygen species (ROS), and calcium content), and blood clot viscoelastic properties. Hemoglobin oxygen saturation (SpO 2) and blood lactate were also measured. In both conditions, exercise induced an increase in blood viscosity, hematocrit, intraerythrocyte calcium and ROS content, and blood lactate concentration. We also observed an increase in blood clot amplitude, and a signi cant drop in SpO 2during exercise in the two conditions. RBC aggregation and CD47 exposure were not modi ed. Exercise in hypoxia induced a slight decrease in RBC deformability which could be related to the slight increase in mean corpuscular hemoglobin concentration (MCHC). However, the values of RBC deformability and MCHC after the exercise performed in hypoxia remained in the normal range of values. In conclusion, acute hypoxia does not amplify the RBC and coagulation changes induced by an exercise bout. Keywords:hemorheology; endurance; hemostasis; altitude; eryptosis; cycling 1. Introduction Through their effects on the regulation of blood ow and tissue perfusion, the physical and dynamic properties of blood and its constituents (i.e., blood rheology) may impact on endurance performance [1,2]. Red blood cells (RBCs) need to be highly deformable to easily ow through the smallest capillaries (whose diameters are sometimes less than that of the RBC itself) and transport oxygen to the

on the regulation of blood ow and tissue perfusion, the physical and dynamic properties of blood and its constituents (i.e., blood rheology) may impact on endurance performance [1,2]. Red blood cells (RBCs) need to be highly deformable to easily ow through the smallest capillaries (whose diameters are sometimes less than that of the RBC itself) and transport oxygen to the tissues [3,4]. In addition, any loss of Metabolites2023,13, 179.

Metabolites2023,13, 179 2 of 14 RBC deformability or any increase in RBC aggregation may lead to a rise in blood viscosity which would be accompanied by a rise of vascular resistance [5]. Due to the important role played by blood rheology on tissue perfusion and possibly on endurance performance [1,2], several studies have investigated the effect of an acute cycling maximal and/or submaximal exercise on blood rheology. Most of the studies have reported an increase in blood viscosity at the end of the exercise, mainly in relation with the rise in hematocrit subsequent to water loss [6,7], a uid shift from the vascular to the extravascular compartment, or even the release of stored RBCs into the spleen following adrenergic- related stress [8,9]. The changes in RBC deformability and RBC aggregation properties during submaximal and maximal cycling exercises may also participate in the changes in blood viscosity [8,10–12]. It has been observed that a decrease in RBC deformability during a short and intense cycling exercise resulted in a rise of blood viscosity [10]. In vitro studies have reported that several physiological factors modulated by exercise can affect the RBC rheological properties as well as their senescence state. The increase in blood lactate concentration and the decrease in pH can lead to activation of nonselective cation channels located on the membrane of RBCs, leading to RBC dehydration, and, there- fore, reducing their deformability [13,14]. Similarly, through lipid and protein oxidation, accumulation of reactive oxygen species (ROS) can damage RBCs, leading to a decrease in RBC deformability and an increase in the strength of RBC aggregates [15,16]. Oxidative stress is also involved in triggering senescence [17]. RBC senescence is characterized, among other things, by a calcium (Ca 2+ ) entry into RBCs, leading to RBC shrinkage and breakdown of the cell membrane asymmetry with translocation of phosphatidylserine (PS) from the inner lea et of the cell membrane to the RBC surface [18]. However, studies on the effects of exercise on RBC senescence markers are rather scarce and have been conducted mainly during running exercises [19,20]. The externalization of PS may also promote the

to RBC shrinkage and breakdown of the cell membrane asymmetry with translocation of phosphatidylserine (PS) from the inner lea et of the cell membrane to the RBC surface [18]. However, studies on the effects of exercise on RBC senescence markers are rather scarce and have been conducted mainly during running exercises [19,20]. The externalization of PS may also promote the activation of coagulation through the formation of a tenase and prothrombinase complex [21,22] and could explain the ndings reported in some studies showing a shortening of blood clot formation time during exercise using rotational thromboelastography [23]. The altitude environment is becoming increasingly common among endurance athletes to optimize their performance following the stimulation of erythropoiesis. Hypoxic condi- tions (simulated or real) are characterized by a drop in inspiratory oxygen pressure, leading to a decrease in O2binding to hemoglobin and thus a drop in arterial O2saturation [24]. Acute hypoxia has been found to promote systemic oxidative stress in humans [25,26]. During exercise in hypoxia, the reduction in arterial O2desaturation was reported to posi- tively correlate with plasma markers of oxidative stress [27]. In nonacclimatized to altitude athletes, performing a maximal and/or a submaximal exercise in hypoxia results in a greater increase in blood lactate concentration compared to the same exercise performed at sea level [28,29].In vitrohypoxic studies on RBC reported a decrease in RBC deformability (FiO2= 0%, time of exposure: 60 min) [30] and a rise in PS externalization (FiO2= 5%, time of exposure: 24 h) [31,32] after a severe hypoxic exposure. One could, thus, expect greater changes in RBC rheology and senescence markers in athletes exercising in hypoxia compared to normoxic conditions. Moreover, an increase in prothrombin expression after exposure to hypobaric hypoxia (30 min at 2400 m) has been observed [33]. Similarly, one could suspect additional effects of hypoxia and exercise on the formation of clot. The aim of this study was to compare the effects of an exercise conducted in normobaric hypoxia (FiO2= 15.3% 2500 m) vs. normoxia on blood rheology, RBC senescence and blood coagulation in endurance-trained cyclists. We hypothesized that the exercise performed in hypoxia would

observed [33]. Similarly, one could suspect additional effects of hypoxia and exercise on the formation of clot. The aim of this study was to compare the effects of an exercise conducted in normobaric hypoxia (FiO2= 15.3% 2500 m) vs. normoxia on blood rheology, RBC senescence and blood coagulation in endurance-trained cyclists. We hypothesized that the exercise performed in hypoxia would result in greater changes on those parameters compared to the same exercise conducted in normoxia. 2. Materials and Methods 2.1. Subjects Nine endurance-trained male cyclists (30.0 8.5 years; 66.2 7.5 kg; 177.0 2.8 cm) voluntarily took part in this study. After being informed of the experimental procedure

Metabolites2023,13, 179 3 of 14 and the possible risks associated with the experiment, all participants signed a consent form. The experimental protocol was approved by the local ethics committee (Lyon, France, L16-47), and all the procedures performed during this study were in agreement with the Declaration of Helsinki. Participants practiced cycling for at least 5 years and trained at least three times a week. Subjects were nonsmokers, presented no known cardiovascular, metabolic, or pulmonary pathology, and had not been exposed to an altitude environment (real or simulated) in the three months preceding the intervention. 2.2. Protocol This crossover study was conducted in a laboratory at sea level (Respiratory Functional Exploration Service, Hospices Civils de Lyon, Croix Rousse Hospital, Lyon, France). Each participant randomly performed a session in normoxia (FiO2= 21%) and a session in normobaric hypoxia (FiO2= 15.3% 2500 m). Normobaric hypoxia was generated by adding nitrogen (N2) to the inspired air, thus decreasing the fraction of oxygen in the ambient air (Alti-Trainer200, Sport and Medical Technology). The participants performed two sessions separated by one week. Tests were executed at the same time of day to avoid the effects of circadian rhythm. During each session, participants performed a maximal incremental test followed by a 20 min submaximal exercise at the rst ventilatory threshold (VT1). The tests were carried out on a home-trainer (Saris H3 direct drive), in a laboratory where the temperature was kept constant between 20 and 25 C. Athletes were asked to refrain from any physical activity the day before the tests. Subjects were weighted before and after exercise. Water intake was not allowed during the tests. Venous blood samples were drawn in a sitting position from the antecubital vein, at rest before the maximal incremental test (Pre) and 3 min after the end of the submaximal exercise at VT1 (Post) in EDTA or citrate tubes (Vacutainer, Becton Dickinson, Rutherford, NJ, USA), for blood rheological, RBC senescence, hematological, and coagulation parameters. 2.3. Maximal Incremental Test In order to determine the maximal oxygen consumption (VO2max), the maximal aerobic power (MAP), and the rst ventilatory threshold (VT1) during each of

and 3 min after the end of the submaximal exercise at VT1 (Post) in EDTA or citrate tubes (Vacutainer, Becton Dickinson, Rutherford, NJ, USA), for blood rheological, RBC senescence, hematological, and coagulation parameters. 2.3. Maximal Incremental Test In order to determine the maximal oxygen consumption (VO2max), the maximal aerobic power (MAP), and the rst ventilatory threshold (VT1) during each of the two sessions, the athletes rst performed a maximal incremental test on the home-trainer. This test started with a 3 min warm up at 90 watts. The pedaling frequency had to be maintained between 70 and 90 RPM. The power was increased by 30 watts per minute, until exhaustion. Gas exchanges and ventilation were measured using a breath-by-breath automated exercise metabolic system (COSMED, Rome, Italy), and heart rate monitoring was performed by electrocardiography. VO2maxwas considered when the usual criteria were met (i.e., VO2 plateau despite an increase in the intensity of the effort, heart rate close to the theoretical maximum heart rate (220-age +/ 10%), and respiratory exchange ratio higher than 1.1) [34]. The rst ventilatory threshold (VT1) was calculated using the methods of Wasserman et al. [35] and Beaver et al. (V-slope method [36]). A 6 min active recovery period at 90 watts was then respected for all athletes. 2.4. Submaximal Exercise at VT1 After the recovery period, the athletes performed a 20 min submaximal exercise at VT1. The pedaling frequency had to be maintained between 70 and 90 RPM. Gas exchanges, ventilation, and heart rate were measured in the same way as during the incremental test. 2.5. Hemoglobin Saturation During both sessions, the noninvasive pulse ear oximetry (Medisoft, Sorinnes, Bel- gium) method was used to assess hemoglobin saturation (SpO2) during the maximal incremental test and the submaximal exercise at VT1. This method has been proven to be valid and reliable for measurement of signi cant falls in SpO2during exercise [37]. A signi cant desaturation was considered when the fall in SpO2was 4% compared to resting condition [38].

at VT1. This method has been proven to be valid and reliable for measurement of signi cant falls in SpO2during exercise [37]. A signi cant desaturation was considered when the fall in SpO2was 4% compared to resting condition [38].

Metabolites2023,13, 179 4 of 14 2.6. Hematological Parameters, Fibrinogen, Lactate, and Glucose Hematological parameters were measured using an hematological analyzer (Excell 2280, Drew Scienti c, Miami Lakes, FL, USA). Analyses were performed within one hour after blood sampling to avoid RBC deterioration. Fibrinogen was measured using a stan- dard coagulation method (ACLTOP750, Werfen). Blood lactate and glucose concentrations were analyzed (Pre and Post) with a drop of blood collected at the nger level with a lactate meter (Nova Biomedical, Cheshire, UK) and a glucometer (Abbot Diabetes Care, Oxon, UK), respectively. 2.7. Blood Rheological Parameters 2.7.1. Blood Viscosity and Hematocrit Blood viscosity was measured after complete blood oxygenation, at native hemat- ocrit (Hct), 25 C and several shear rates (11.5; 22.5; 45; 90; 225 s 1 ) using a cone/plate viscometer (Brook eld DVII+ with CPE40 spindle, Brook eld Engineering Labs, Natick, MA, USA), as recommended [39]. Hematocrit was determined by the micromethod after blood microcentrifugation at 1500 g for ve minutes at 20 C (Pico 17, Thermo Scienti c, Illkirch, France). 2.7.2. Red Blood Cell Deformability Red blood cell deformability under isotonic condition was assessed at 37 C and at several shear stresses (from 0.3 to 30 Pa) by laser diffraction analysis (ektacytometry), using the laser-assisted optical rotational cell analyzer (LORRCA MaxSis, RR Mechatronics, Hoorn, The Netherlands). The system has been described elsewhere in detail [39]. Brie y, 5 L of blood were mixed with 1 mL polyvinylpyrrolidone (PVP; viscosity = 27.1 cP). The blood suspension was placed into a Couette system and increasing shear stresses were applied on the blood suspension. A laser beam was projected from the stationary cylinder through the RBC suspension, and the resulting diffraction pattern was captured by a video camera and analyzed by a computer in order to calculate an elongation index (EI), which re ects RBC deformability. The maximum elongation index (EImax) and the shear stress required to deform RBCs by half (SS1/2) were determined by a Lineviewer Burk model [40]. 2.7.3. Red Blood Cell Aggregation Red blood cell aggregation properties were determined at 37 C and at a standardized Hct (i.e., 40%)

computer in order to calculate an elongation index (EI), which re ects RBC deformability. The maximum elongation index (EImax) and the shear stress required to deform RBCs by half (SS1/2) were determined by a Lineviewer Burk model [40]. 2.7.3. Red Blood Cell Aggregation Red blood cell aggregation properties were determined at 37 C and at a standardized Hct (i.e., 40%) and after complete oxygenation of the blood, by syllectometry (changes in backscattered light intensity over time) using the laser-assisted optical rotational cell analyzer (LORRCA MaxSis, RR Mechatronics, Hoorn, The Netherlands). This method has been described in detail by Baskurt et al. [39]. The minimum shear rate required to break the RBC aggregates formed (min; RBC aggregates strength) was determined by an iteration procedure [39]. 2.8. Red blood Cell Senescence Assessment 2.8.1. RBCs Preparation Blood collected in citrate tubes was centrifuged (1000 g, 10 min at 20 C) and plasma and buffy coat were discarded. RBCs were washed in PBS, and then resuspended at 0.4% Hct in PBS buffer containing 2.5 mM Ca 2+ . 2.8.2. Phosphatidylserine (PS) Exposure PS exposure on the outer membrane lea et of RBCs was evaluated by Annexin V-FITC binding to this phospholipid. RBCs suspensions were incubated, protected from light, 30 min at 37 C with Annexin V-FITC (1:200 dilution, Beckman Coulter, Pasadena, CA, USA). Immediately after incubation, samples were diluted and analyzed by FACS (BD Accuri C6, Franklin Lakes, NJ, USA). PS exposure was measured in the FITC channel (with an excitation wavelength of 488 nm and an emission wavelength of 530 nm) according to the manufacturer's instructions. For each sample, 50,000 events gated for the appropriate

Metabolites2023,13, 179 5 of 14 FSC were counted. PS externalization was assessed by the percentage of Annexin V FITC- positive RBCs. 2.8.3. Intracellular Reactive Oxygen Species (ROS) Intracellular RBC ROS was determined using 2 0 ,7 0 –dichloro uorescin diacetate (DCFDA, Sigma-Aldrich, Saint-Quentin-Fallavier, France). RBC suspensions at 0.4% Hct were incu- bated 30 min at 37 C in the dark with 20 M of DCFDA (Sigma-Aldrich, Saint-Quentin- Fallavier, France). Immediately after incubation, samples were diluted and analyzed by FACS (BD Accuri C6, Franklin Lakes, NJ, USA) according to the manufacturer's instruc- tions. Median uorescence intensity (MFI) of the 50,000 gated events was recorded to quantify ROS levels. 2.8.4. Intracellular Calcium (Ca 2+ ) RBC Ca 2+ content was measured with Fluo3/AM (Biotium, Fremont, CA, USA) probe. RBC suspensions were incubated 30 min at 37 C with 5 M of Fluo3/AM. Immediately after incubation, samples were diluted and analyzed by FACS (BD Accuri C6, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. MFI of the 50,000 gated events was recorded to quantify Ca 2+ levels. 2.8.5. CD47 Exposure CD47 (anti-erythrophagocytosis protein) membrane exposure was assessed by incubat- ing RBC suspensions for 30 min at 37 C with anti-CD47 antibody (1:34 dilution, Miltenyi, Cologne, Germany). Immediately after incubation, samples were diluted and analyzed by FACS (BD Accuri C6, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. MFI of the 50,000 gated events was recorded to quantify CD47 exposure levels. 2.9. Rotational Thromboelastometry Coagulation activation and blood clot polymerization parameters were determined with rotational thromboelastometry (ROTEM ® delta, Werfen, TEM International, Kampala, Germany) in NATEM and EXTEM mode at 37 C for 45 min. For both NATEM and EXTEM mode, samples were recalci ed with 0.2 M CaCl2 solution. Then, only in the EXTEM mode, tissue factor was added to explore the extrinsic pathway of coagulation. Several parameters were analyzed: (1) coagulation time (CT), which corresponds to the time required to reach an amplitude of 2 mm after the beginning of the test; (2) clot formation time (CFT), which corresponds to the time required to reach an amplitude of 20 mm

This study compares the effects of acute hypoxia vs. normoxia on blood rheology and RBC senescence during exercise.