International Journal of Molecular Sciences Article Acute Cycling Exercise Induces Changes in Red Blood Cell Deformability and Membrane Lipid Remodeling Travis Nemkov 1,†, Sarah C. Skinner 2,3,4,†, Elie Nader 3,4 , Davide Stefanoni 1 ,Mélanie Robert 3,4,5, Francesca Cendali 1 , Emeric Stauffer 3,4,6, Agnes Cibiel 5, Camille Boisson 3,4 , Philippe Connes 3,4,7,* and Angelo D’Alessandro 1,* 1 Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; [email protected] (T.N.); [email protected] (D.S.); [email protected] (F.C.) 2 UNC Blood Center, University of North Carolina, Chapel Hill, NC 27599, USA; [email protected] 3 Inter-University Laboratory of Biology of Motor Function EA7424, Vascular Biology and the Red Blood Cell Team, Claude Bernard University Lyon 1, University de Lyon 1, 69100 Villeurbanne, France; [email protected] (E.N.); [email protected] (M.R.); [email protected] (E.S.); [email protected] (C.B.) 4 Laboratory of Excellence (Labex GR-Ex), PRES Sorbonne, 75015 Paris, France 5 Erytech Pharma, 69008 Lyon, France; [email protected] 6 Department of Functional Respiratory Testing, Croix-Rousse Hospital, Hospices Civils of Lyon, 69002 Lyon, France 7 Institute of Universities of France (IUF), 75015 Paris, France * Correspondence: [email protected] (P.C.); [email protected] (A.D.) † These authors contributed equally to this work. Abstract: Here we describe the effects of a controlled, 30 min, high-intensity cycling test on blood rhe- ology and the metabolic profiles of red blood cells (RBCs) and plasma from well-trained males. RBCs Citation: Nemkov, T.; Skinner, S.C.; demonstrated decreased deformability and trended toward increased generation of microparticles Nader, E.; Stefanoni, D.; Robert, M.; after the test. Meanwhile, metabolomics and lipidomics highlighted oxidative stress and activation Cendali, F.; Stauffer, E.; Cibiel, A.; of membrane lipid remodeling mechanisms in order to cope with altered properties of circulation Boisson, C.; Connes, P.; et al. Acute resulting from physical exertion during the cycling test. Of note, intermediates from coenzyme Cycling Exercise Induces Changes in A (CoA) synthesis for conjugation to fatty acyl chains, in parallel with reversible conversion of Red Blood Cell Deformability and Membrane Lipid Remodeling. Int. J. carnitine and acylcarnitines, emerged as metabolites that significantly correlate with RBC deforma- Mol. Sci. 2021, 22, 896. https:// bility and the generation of microparticles during exercise. Taken together, we propose that RBC doi.org/10.3390/ijms22020896 membrane remodeling and repair plays an active role in the physiologic response to exercise by altering RBC properties. Received: 8 December 2020 Accepted: 11 January 2021 Keywords: red blood cell; deformability; cycling; exercise; metabolomics; lipidomics Published: 18 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- Maintaining adequate blood flow and tissue perfusion is crucial during exercise iations. because macronutrients, oxygen, carbon dioxide, and metabolic waste products are all transported in the blood. Owing to increased muscle oxygen demand during exercise, exercise-induced physiological changes (e.g., increased heart rate) may increase the me- chanical and shear stresses experienced by red blood cells (RBCs) in the bloodstream; such Copyright: © 2021 by the authors. stress may modulate blood rheology and RBC properties, which could in turn impact on Licensee MDPI, Basel, Switzerland. blood flow and tissue perfusion. For example, RBCs must be highly deformable to pass This article is an open access article through the smallest capillaries and deliver oxygen to the tissues [1]. Moreover, decreased distributed under the terms and RBC deformability and increased RBC aggregation can cause blood viscosity to increase, conditions of the Creative Commons thereby altering blood flow and impacting exercise performance [2,3]. Previous studies Attribution (CC BY) license (https:// have shown that acute cycling exercise decreases RBC deformability [4–11]. One cause creativecommons.org/licenses/by/ of this change could be directly or indirectly related to lactate accumulation. Evidence 4.0/). Int. J. Mol. Sci. 2021, 22, 896. https://doi.org/10.3390/ijms22020896 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 896 2 of 21 suggests that increased lactate ion concentration and decreased pH could lead to RBC de- hydration and activate cationic channels in the RBC membrane, resulting in RBC shrinkage, thereby decreasing RBC deformability [12–15]. In addition, findings from several studies suggest that oxidative stress could also contribute to reductions in RBC deformability via membrane lipid and protein oxidation [2,16]. Oxidative stress is also a recognized trigger of eryptosis or suicidal erythrocyte death [17]. Eryptosis is characterized by increased intracellular Ca2+, cell membrane blebbing, cell shrinkage, and phosphatidylserine (PS) exposure on the RBC membrane surface. These effects have a significant dependence upon metabolic alterations, which are uniquely elicited by different stressors including nutrient depletion, Ca2+ overload, and hy- perthermia [18]. The onset of eryptosis is associated with the release of microparticles (MPs), membrane-bound vesicles 0.1–1 µm in diameter, which can also be influenced by increased shear stress [19–21]. Although shear stress and oxidative stress are both elevated during exercise, previous studies have indicated that short maximal exercise tests do not cause RBC-MPs or markers of eryptosis to increase [22,23]. Similarly, a recent study of endurance- trained athletes showed that a 10 km running trial did not result in elevated levels of RBC-MPs or markers of eryptosis [24]. However, the effect of a prolonged, high-intensity cycling exercise on erythrocyte PS exposure or RBC-MP concentration remains unknown. As RBCs are devoid of organelles, they are uniquely dependent upon metabolic- associated pathways to cope with oxidative stress. This relationship has been well char- acterized in RBCs stored in blood banks for transfusion medicine, where accumulating effects of oxidative stress result in cellular damage referred to as the “storage lesion” [25]. In this setting, accumulation of oxidative damage results in decreased deformability and generation of RBC-MPs. A recent study also demonstrated the key role of oxidative stress in RBC deformability and the release of RBC-MPs in the context of sickle cell anemia [26]. The application of omics technologies, including metabolomics, lipidomics, and proteomics, has contributed significantly to the understanding of RBC responses to oxidative stress [27]. Recently, these techniques were also employed to study the complex physiological re- sponses to exercise [28]. These methods have been used to evaluate changes in metabolites related to lipid and carbohydrate metabolism at variable exercise intensities and durations in body fluids including blood, plasma/serum, saliva, and urine [29]. Studies have shown that metabolomics and lipidomics can reveal alterations in oxidative stress [30,31], changes in fuel use during exercise [32–34], and distinct metabolic phenotypes corresponding to physiological parameters such as VO2max [35] and lactate clearance capacity [36]. While these studies focused predominantly on metabolic alterations present in plasma, serum, or whole blood, no studies have focused on metabolic alterations in red blood cells (RBCs) dur- ing acute exercise specifically or how these cells interact with the circulatory environment of the plasma. In order to develop a better understanding of the metabolic milieu associated with changes in RBCs during exercise, this study aimed to examine the effects of high-intensity, prolonged exercise on concentrations of metabolites and lipid markers in the plasma and RBC compartments of blood in association with changes in RBC physiology. 2. Results 2.1. Exercise Testing The maximal oxygen uptake (VO2max) and maximal aerobic power (MAP) values obtained from the graded exercise test indicated that all eight subjects had a high level of aerobic fitness (Table1). During the 30 min submaximal exercise, subjects pedaled at an average power that corresponded to 71% of MAP. The exercise significantly reduced body weight (−0.88 ± 0.44 kg) and decreased blood oxygen saturation (SpO2)(−6.0% ± 3.1%). Int. J. Mol. Sci. 2021, 22, 896 3 of 21 Table 1. Subject characteristics. n 8 Age (years) 35 ± 8 Weight (kg) 65.8 ± 6.4 Height (cm) 176.1 ±2.2 VO2max (mL/min/kg) 70.5 ± 5.8 HRmax (BPM) 188 ± 11 MAP (W) 373 ± 39 Percent HRmax at VT1 (%) 85.95 ± 3.4 Percent MAP at VT1 (%) 69.88 ± 5.0 Exercise duration (min) 20.1 ± 1.8 VO2max, maximal oxygen uptake; HRmax, heart rate max; MAP, maximal aerobic power; VT1, ventilatory threshold. 2.2. Distinct Metabolite Profiles in RBC and Plasma Fractions Blood from eight well-trained male athletes was sampled before and 3 min after a 30 min submaximal cycling test (Figure1A). As expected, whole-blood lactate levels increased in a subject-specific manner during the test (Figure1B). Lactate is the obliga- tory byproduct of glycolysis and is produced in part to regenerate nicotinamide adenine dinucleotide (NAD+) for the maintenance of glycolytic flux. While lactate measurements during sport performance studies are typically performed using whole blood,