KU LEUVEN GROEP BIOMEDISCHE WETENSCHAPPEN FACULTEIT BEWEGINGS- EN REVALIDATIEWETENSCHAPPEN

Autophagy as the main proteolytic pathway in human skeletal muscle in response to acute hypoxia

door Stijn Van Driessche masterproef aangeboden tot het behalen van de graad van Master of Science in de lichamelijke opvoeding en de bewegingswetenschappen

o.l.v. Prof. Dr. M. Thomis, promotor E. Masschelein, copromotor

LEUVEN, 2013

KU LEUVEN GROEP BIOMEDISCHE WETENSCHAPPEN FACULTEIT BEWEGINGS- EN REVALIDATIEWETENSCHAPPEN

Autophagy as the main proteolytic pathway in human skeletal muscle in response to acute hypoxia

door Stijn Van Driessche masterproef aangeboden tot het behalen van de graad van Master of Science in de lichamelijke opvoeding en de bewegingswetenschappen

o.l.v. Prof. Dr. M. Thomis, promotor E. Masschelein, copromotor

LEUVEN, 2013 Opgesteld volgens de richtlijnen van Medicine and Science in Sports and Exercise

WOORD VOORAF

Vanaf het begin van mijn studies aan de faculteit FABER was ik reeds geïnteresseerd in het wetenschappelijke. Ik ging op zoek naar alle mogelijke opties en afstudeerrichtingen in de sportwereld en vond al snel de mogelijkheid om de optie ‘Research in Biomedical Kinesiology’ te volgen. Een kans die ik niet wilde laten schieten en waarvoor de motivatie alleen maar groeit. Via de onderzoeksstage ‘Twins @ Everest Base Camp studie : Genetische determinanten van fysiologische adaptaties op extreme hoogte’ zag ik een unieke kans om meer te weten te komen over hoe wetenschappelijk onderzoek in de praktijk in zijn werk gaat. Op die manier kreeg ik meer inzicht in de fysiologie van het menselijk lichaam en meer bepaald de aanpassingen op hoogte. Het onderwerp van lichaamsadaptaties aan bepaalde omgevingsomstandigheden maakt het voor mij als outdoor(hiking, duiken…) en duursporter (afstandslopen en wielrennen) zeer boeiend en leerrijk. Dit stimuleert mij om hier meer over te weten te komen. Ook deze masterproef zie ik als een unieke kans en misschien ook als een begin van verder onderzoek. De theoretische achtergrond van deze masterproef was niet altijd eenvoudig, maar heeft mijn interesse kunnen opwekken om mij verder te verdiepen binnen het domein van de inspanningsfysiologie.

Een welgemeend woord van dank gaat uit naar mijn copromotor Evi Masschelein en promotor Prof. M. Thomis. Hun mening, nuchtere instelling en precieze raadgevingen hebben me geholpen bij het realiseren van deze masterproef. Graag wil ik mijn copromotor nog expliciet bedanken om mij in te leiden in het wetenschappelijk onderzoek binnen deze onderzoeksgroep en om mij steeds met lachend gezicht bij te staan gedurende dit academiejaar. Daarnaast wil ik nog de proefpersonen voor deze studie bedanken, alsook Prof. L. Deldicque voor haar inbreng in deze studie. Tot slot wil ik ook mijn familie, vrienden en vriendin bedanken. Zonder hun steun was dit alles uiteraard niet mogelijk geweest. Dankjewel!

Leuven, mei 2013

S. V.D.

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SITUERING De masterproef is gekaderd binnen het onderzoek van de ‘Exercise Physiology Research Group’. Hun onderzoek focust zich op de studie van het spiermetabolisme en spiercontractiliteit als gevolg van inspanning en training. Dit houdt mede het onderzoek in naar de effecten van inspanning op de cellulaire mechanismen en signaling pathways in skeletspiercellen. De onderzoeksgroep streeft er naar de metabole en functionele respons binnen de context van fitheids- en gezondheidsverbetering bij verschillende populaties te onderzoeken. Een specifiek onderzoeksthema binnen de groep omvat het onderzoek naar de regulerende rol van omgevingsfactoren, zoals hypoxie, op de fysiologische responsen tijdens rust en inspanning.

Hypoxie, pathologisch of als gevolg van de omgeving, doet zich voor wanneer de zuurstofdruk in het bloed te laag is om het hemoglobine te satureren, met als gevolg een zuurstoftekort op weefselniveau. Het onderzoeken van gezonde mensen in hypoxie kan ons helpen om de moleculaire mechanismen en aanpassingen van het lichaam als gevolg van het lage zuurstofgehalte in de omgeving beter te verstaan. Dit kan ook interessant zijn om pathologische situaties geassocieerd met hypoxie beter te leren begrijpen. Voorbeelden hiervan zijn anemie, chronisch obstructief longlijden, alsook chronisch hartfalen (Lundby et al. 2009).

Een fenomeen dat vaak wordt opgemerkt op grote hoogte is een daling in dwarsdoorsnede van het spierweefsel (Hoppeler et al. 1990, Mizuno et al. 2008), dit als gevolg van een disbalans in het proteïnemetabolisme. Het evenwicht tussen proteïne synthese en afbraak in de spieren is een belangrijke determinant voor het behoud van skeletspiermassa. De exacte mechanismen die dit verlies in spiermassa reguleren tijdens blootstelling aan hypoxie zijn echter nog onduidelijk.

Daarom onderzoeken wij het effect van acute omgevingshypoxie en inspanning op het proteïnemetabolisme in de skeletspier van de mens. D’Hulst et al. onderzochten eerder al het effect van 4u acute blootstelling aan normobare hypoxie (11% O₂) op het proteïne metabolisme in de skeletspier bij de mens in rust (D’Hulst et al. 2013). De hoofdbevinding van deze studie was dat acute omgevingshypoxie leidde tot een hogere respons voor PKB en S6K1, merkers van proteïne synthese. De hogere respons kon echter een gevolg zijn van een verhoogde plasma insuline concentratie als reactie op een maaltijd.

Omdat het nog verre van duidelijk is hoe de moleculaire mechanismen van het verlies in skeletspiermassa werken, onderzoeken we verder het effect van hypoxie op het spiermetabolisme. iii

Om het effect van hypoxie verder op te helderen, ongeacht het effect van een maaltijd, vergelijken we het effect van acute omgevingshypoxie op het spiermetabolisme in rust met dat na inspanning en bekijken we nog steeds wat er gebeurt in vergelijking met condities in normoxie. Vergeleken met de studie van D’Hulst et al., focust de huidige studie zich meer op merkers van proteïne afbraak als respons op 6u30 acute blootstelling aan normobare hypoxie (11% O₂) en in combinatie met een bijkomende submaximale inspanning

(20 minuten fietsen aan 1.2 W/kg lichaamsgewicht). De

experimentele testen werden uitgevoerd in de normobare hoogtekamer te Leuven.

De huidige studie kan een nieuw licht brengen op de moleculaire mechanismen die achter het verlies van skeletspiermassa liggen als gevolg van acute blootstelling aan omgevingshypoxie en in combinatie met een submaximale inspanning. De bevindingen van deze studie kunnen ons helpen om de regulatie van het proteïnemetabolisme in de spieren beter te begrijpen en kunnen belangrijk zijn voor bergbeklimmers op hoogte, alsook voor patiënten die lijden aan een hypoxie-gerelateerde ziekte, want beiden ondergaan spieratrofie.

Referenties: D'Hulst G, Jamart C, Van Thienen R, Hespel P, Francaux M, Deldicque L. Effect of acute environmental hypoxia on protein metabolism in human skeletal muscle. Acta Physiol. 2013, doi: 10.1111/apha.12086. Hoppeler H, Kleinert E, Schlegel C, Claassen H, Howald H, Kayar SR, Cerretelli P. Morphological adaptations of human skeletal muscle to chronic hypoxia. Int J Sports Med 1991, 11:S3–S9. Lundby C, Calbet J A L, Robach P The response of human skeletal muscle tissue to hypoxia. Cell Mol Life Sci 2009, 66: 3615-3623. Mizuno M, Savard GK, Areskog N, Lundby C and Saltin B. Skeletal Muscle Adaptations to Prolonged Exposure to Extreme Altitude: A Role of Physical Activity? High Alt. Med. & Biol. 2008, 9(4):311-317.

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Autophagy as the main proteolytic pathway in human skeletal muscle in response to acute hypoxia

Abstract

Background: A phenomenon often observed at high altitude is muscle atrophy as a result of a disbalance between muscle protein synthesis and breakdown. However the exact mechanisms underlying this loss of muscle mass are still unclear. Purpose: We hypothesized that hypoxia would decrease markers of muscle protein synthesis both at rest and during exercise, in combination with an activation of markers of protein breakdown. Methods: Twenty-three subjects (age: 24.9 ± 0.9 years, VO₂peak: 54.7 ± 1.7 ml.kg-1.min-1) participated in an experimental trial in normoxic (20.9 %O2, NOR) and hypoxic (11.0 %O2, HYP) conditions with a 2-wk wash-out period. A biopsy was taken from m. vastus lateralis before (Pre-ex) and immediately after (Post-ex) a 20-min submaximal cycling bout at 1.2 W/kg body weight. Western blots were used to determine anabolic signaling phosphorylation (PKB, S6K1, 4E-BP1) and expression of key signaling molecules in both the ubiquitin-proteasome (MuRF-1, MAFbx) and the autophagylysosome pathway (LC3b, p62). Results: Phosphorylation status of PKB, S6K1 and 4E-BP1 were similar between NOR and HYP, but decreased Post-ex (P < 0.05). No effect of HYP and exercise was found in expression of MuRF-1 and MAFbx. However, in HYP LC3b protein expression increased Pre-ex (P < 0.05) and returned to basal levels Post-ex (P < 0.05). Expression of p62 was decreased in HYP compared with NOR Pre-ex (P < 0.05), this decrement was further enhanced Post-ex (P < 0.05). Conclusion: The phosphorylation of PKB, S6K1 and 4E-BP1 is not modified by hypoxia, indicating that anabolic signaling is not altered by hypoxia. On the contrary, the autophagy-lysosome pathway is activated by hypoxia at rest and after exercise. Our results suggest that the up regulation of catabolic pathways could favor muscle atrophy during chronic hypoxia.

Keywords: altitude, exercise, anabolic signaling, catabolic signaling, protein metabolism

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Introduction

Hypoxia, pathological or environmental, occurs when the oxygen pressure in the blood is too low to saturate the hemoglobin causing an oxygen deficit at the tissue level. Investigating healthy humans at high altitude, which is a state of hypoxia, can help us understand the molecular mechanisms and adaptations of the body as a consequence of the low oxygen environment. This can also be interesting to help us understand pathological situations associated with hypoxia including anemia, chronic obstructive pulmonary disease (COPD) as well as chronic heart failure (24). Whatever the origin of hypoxia may be, environmental or pathological, all nucleated cells are oxygen sensitive and able to adapt to a lowered oxygen tension (pO₂) (24).

A phenomenon often observed at high altitude is a decrease in muscle fiber cross sectional area (16, 27). It has been speculated that this loss of muscle mass can be due to a hypoxia-induced down regulation of muscle protein synthesis. For instance, in COPD patients, there has been found a reduced protein synthesis, which could be a direct effect of hypoxia (20). The balance between muscle protein synthesis and degradation is an important determinant of the maintenance of skeletal muscle mass. However the exact mechanisms regulating this loss of muscle mass during exposure to hypoxia are unclear.

Protein translation and cell growth in skeletal muscle are mainly regulated by the kinase mTOR that controls the key regulators of protein synthesis, p70 ribosomal S6 protein kinase (S6K) and 4E binding protein 1 (4E-BP1) (6, 15). mTOR exists of two distinct complexes mTORC1 and mTORC2. Mechanical stimulation of muscles, like during exercise, is sufficient to activate mTORC1 (34), but the exact mechanism by which mechanical stimulation activates mTOR remains inconclusive. mTOR can be activated via several factors like nutrients, exercise and growth factors, such as insulin and IGFs (9). These factors can cause a signal transduction cascade with endpoint Rheb, which in its GTP-bound state is an essential activator of mTORC1. Akt/protein kinase B (PKB), an upstream regulator of mTOR, can phosphorylate tuberous sclerosis complex 2 (TSC2) which inhibits the GAP activity for Rheb. RhebGTP is then able to promote mTORC1 activity. The initiation of translation to start protein synthesis starts via the recruitment of a small 40S ribosome to the m⁷G cap structure of the 5’ end of the mRNA. This translational process is promoted by the eIF4F complex. Phosphorylation of 4E-BP1, one of the downstream substrates of mTOR, leads to inhibition of this negative regulator and as a consequence will positively regulate the translational process by increasing formation of the eIF4F complex (34). Another downstream substrate of mTOR, S6K1, functions as a positive regulator of protein translation initiation. It can be speculated that a catabolic condition, like hypoxia, could lead 3

to a decrease in protein synthesis via inhibition of mTORC1. It is already known that hypoxia can activate AMP-activated protein kinase (AMPK), a sensor for the energetic status of the cell. AMPK can phosphorylate TSC2 at different sites than Akt, stimulating the GAP activity of Rheb (23). Another way hypoxia can inhibit mTORC1 is via expression of ‘regulated in development and DNA damage responses 1’ (REDD1) (4). The latter promotes the assembly of TSC1/2 complex and its GAP-activity (34).

Until now, research has mainly focused on muscle protein synthesis and less is known about muscle protein breakdown in hypoxia. In skeletal muscle, there are two major proteolytic pathways: the ubiquitin-proteasome pathway (UPP) and the autophagy-lysosome pathway (ALP). In the UPP, proteins destined for degradation are labeled with a covalent bond of multiple ubiquitin monomers and degraded by the 26S proteasome (32). The ubiquitination of proteins involves action of three enzymes, E1 as an ubiquitin-activating enzyme, E2 as an ubiquitin-conjugating enzyme, and E3 as an ubiquitin-ligating enzyme that provides specificity to the proteins that will be degraded (14). Two muscle-specific ligases are Muscle Ring Finger 1 (MuRF-1) and Muscle atrophy F-box (Mafbx/MAFbx) which are up regulated in conditions of muscle atrophy (32). Degradation of cell proteins can also be regulated by ALP. In autophagy, double membrane vesicles are generated that engulf portion of the cytoplasm, organelles, glycogen and protein aggregates (31). The formed autophagosomes bind to lysosomes, forming autolysosomes for degradation of their contents by lysosomal hydrolases. Autophagosome formation is under control of some autophagy-related genes (ATG) like ATG5, ATG12, and ATG8 also known as microtubule-associated protein 1 light chain 3 (LC3). LC3 is essential for membrane biogenesis and/or closure of the membrane (32). Very recently, it has been shown that p62 plays a key role in the formation of autophagosomes (18). Ubiquitinated proteins are delivered to autophagosomes via p62 that binds the polyubiquitin chains and LC3 (31). LC3 and p62 can thus be indicators of autophagic activity and interesting to investigate.

Forkhead box O (FoxO) has been identified as a critical transcription factor controlling UPP and ALP. FoxO itself is regulated by PKB. Under catabolic conditions, like oxidative stress, PKB is dephosphorylated, which leads to FoxO dephosphorylation and translocation to the nucleus for transcription (14). So both the UPP and ALP contribute to muscle loss and are regulated by FoxO. During oxidative stress FoxO3-mediated transcription of the atrophy-related ubiquitin ligases, MAFbx and MuRF-1, could be enhanced, causing muscle loss (31, 22). To date, only limited data is available about protein degradation in response to hypoxia. To clarify and summarize the different pathways visually, a simplified map is shown in figure 1.

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Effector activated Effector inhibited

Fig. 1. Simplified map of the signaling pathways of protein synthesis and breakdown in human skeletal muscle cells as a result of hypoxia, insulin, exercise or growth factors. Markers of protein synthesis are S6 kinase 1 (S6K1) and 4E binding protein 1 (4E-BP1) and are regulated via mammalian target of rapamycin (mTOR). mTOR can be inhibited via hypoxia through activation of AMP dependent proteinkinase (AMPK) or expression of regulated in development and DNA damage responses 1 (REDD1), which is promoted by hypoxia inducible factor (HIF). mTOR can be activated via proteinkinase B (PKB), which can inhibit Forkhead box O (FoxO). The ubiquitin proteasome pathway (UPP) and autophagy lysosome pathway (ALP) are regulated by FoxO. Markers for the UPP are Muscle Ring Finger 1 (MuRF-1) and Muscle Atrophy F-box (MAFbx). Markers for the ALP are microtubule-associated protein 1 light chain 3 (LC3) and p62.

Against this background, we investigated the effect of acute hypoxia on markers of muscle protein synthesis and protein breakdown. Moreover, we examined this not only under resting conditions but also under conditions of a catabolic challenge (a 20min. cycling bout at 1.2 W/kg body weight). We hypothesized that hypoxia would decrease markers of muscle protein synthesis both in rest and during exercise, in combination with an increase in protein breakdown. In a hypoxic facility, high altitude (5000m – 11% O₂) was simulated in a standardized manner.

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Materials and methods

Subjects. Ten monozygotic (MZ) twin pairs and one MZ triplet participated in the study (Table 1), which was approved by the KU Leuven Biomedical Ethics Committee and was in accordance with The Declaration of Helsinki. Subjects gave their written, informed consent after they were fully informed of all experimental procedures and risks associated with the experiments. The subjects were medically screened and were all in good health. None of them were smokers or had a BMI > 30. The training status in each twin pair was similar. None of the subjects were exposed for more than 7 days to an altitude higher than 1500 m during a period of 6 months preceding the study. No one had any kind of pathology that is a contra-indication for hypoxia and/or exercise or took any kind of medication that is a contra-indication for hypoxia and/or exercise. All subjects were asked to continue their normal sport activities throughout the study. They were instructed to restrain from heavy exercise and alcohol for two days preceding the experimental sessions. The food on the day of testing was standardized and provided by the research team to minimize the influence of nutritional variability ((breakfast: 600-700 kcal (70% carbohydrates, 20% fat, 10% proteins) and lunch: 800-1000 kcal (60% carbohydrates, 25% fat, 15% proteins)).

Table 1. Subject characteristics Age

24.9 ± 0.9 years

Weight

77.3 ± 1.6 kg

VO₂max (in normoxia)

54.7 ± 1.7 ml.kg-1.min-1

Values are means ± SE (n = 23) for age, weight and VO₂max.

Study design. This study was performed in the hypoxic facility (Sporting Edge, U.K.) within the Research Center for Exercise and Health at FABER, K.U.Leuven. Oxygen level, temperature and relative humidity were automatically controlled. The climate chamber creates a hypoxic environment via the principle of normobaric hypoxia: the percentage of oxygen in the ambient air is reduced by molecular oxygen filtration causing the percentage of ambient nitrogen to increase while atmospheric pressure at sea level (760mmHg) is maintained. Prior to the experimental sessions of the study, subjects underwent a medical screening including a medical questionnaire, resting electrocardiogram, and measurement of blood pressure and weight. All subjects were required to attend the hypoxic facility on two separate occasions with a wash-out period of two weeks. The first experimental day was performed in normoxia (FiO2 = 20.93 %, NOR). During the second experimental day they were exposed to severe hypoxia (FiO2 = 11 %, HYP), which corresponds to 5000m altitude. They first rested for 5 hours in a comfortable chair. During this period FiO2 was maintained at 20.93% 6

in NOR. In HYP, FiO2 was gradually decreased from 20.93% to 11% over a period of 5 hours. After the 5-h period, oxygen level was held constant for another 1.5 hours at 20.93% and 11% respectively, where after they were enrolled in an experimental protocol (Fig. 2).

Fig. 2. Experimental protocol of the normoxic and hypoxic condition over time. Conditions are expressed as 0 and 5000 meters above sea level respectively in normoxia and hypoxia. The time is expressed in hours and minutes. Muscle biopsies were taken Pre- and Post-exercise. (EX=20min submaximal cycling at 1.2 W/kg body weight)

A percutaneous needle biopsy (50-200mg) was taken Pre-exercise (Pre-ex) from m. vastus lateralis through a 5-mm incision in the skin under local anesthetic (1–2 ml Lidocaine). This was followed by a 20-min submaximal exercise bout on a cycle ergometer (Avantronic Cyclus II, Leipzig, Germany) at a constant workload of 1.2 W∙kg-1 body weight. Immediately post exercise (Post-ex) another muscle biopsy was taken through the same incision as the Pre-ex biopsy. The muscle biopsy samples were immediately frozen in liquid nitrogen and stored at -80°C until biochemical and histological analyses at a later date. During the experimental sessions oxygen hemoglobin saturation (SpO₂) and muscle tissue oxygenation evaluated via the tissue oxygenation index (TOI) were continuously measured. SpO2 was measured by non-invasive registration using a pulse oximetry sensor placed on the forehead of the subject, approximately 2cm above the left eyebrow (Nellcor N-600-x, Oxismart, Mallinckrodt, St. Louis, MO). Muscle tissue oxygenation was monitored by near infrared spectroscopy (NIRS), a non-invasive and non-ionizing method. For local muscle oxygenation, the optodes were placed on the vastus lateralis muscle of the right leg.

Western Blot. The frozen biopsy samples were homogenized 3 x 5s with a Polytron mixer in ice-cold buffer (1:10, w/v) [50 mM Tris-HCl pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM glycerophosphate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 1

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mM DTT, 0.1 % Triton-X 100 and a complete protease inhibitor tablet (Roche Applied Science, Vilvoorde, Belgium)]. Homogenates were then centrifuged at 10000 g for 10 min at 4°C. The supernatant was collected and immediately stored at -80°C. The protein concentration was measured using the DC protein assay kid (Bio-Rad laboratories, Nazareth, Belgium). Proteins (30-80 µg) were diffused during SDS-PAGE (8-15% gels) and transferred to PVDF membranes via Electrotransfer. Subsequently, the membranes were then blocked for 1 hour in TBST with 5% non-fat milk powder and afterwards incubated overnight (4°C) with the following antibodies (1:1000): phospho-PKB Ser473, total PKB, phospho-S6K1 Thr389, total S6K1, phospho-4E-BP1 Thr37/46, total 4EBP1, phospho-FoxO1/3a Thr24/32, total FoxO1/3a, total eEF2, LC3b, HIF-1α (Cell Signaling), MuRF-1 (Santa Cruz), MAFbx (Emelca Bioscience), p62 (Progen), REDD1 (Bioconnect). Horseradish peroxidaseconjugated, anti-rabbit (1:5000) or anti-guinea pig (1:5000) secondary antibodies (Sigma-Aldrich, Bornem, Belgium) were used for chemiluminescent detection of proteins. Membranes were scanned and quantified with Genetools and Genesnap softwares (Syngene, Cambridge, UK), respectively. Then, membranes were stripped and reprobed with the antibody for the total form of the respective protein to ascertain the relative amount of the phosphorylated protein compared with the total form throughout the whole experiment. The results are presented as the ratio protein of interest/eEF2 or as the ratio phosphorylated/total forms of the proteins when the phosphorylation status of the protein was measured. To analyze the data, each value was expressed relative to the mean value of the basal sample (NOR Pre-ex) for each protein.

Statistical analysis. A two-way repeated measures ANOVA was used to determine statistical significance of time (effects of exercise), condition (normoxia versus hypoxia) and time x condition interaction effects. A Bonferroni multiple comparison test was used as post hoc test. A P-value of 0.05 was set as threshold for significance. Results are expressed as means ± SE. All statistical procedures were done via the statistical program SigmaStat.

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Results Oxygen hemoglobin saturation (SpO₂) and tissue oxygenation index (TOI) During the whole experiment SpO₂ and TOI were lower in HYP than in NOR (Table 2). SpO₂ was similar Pre- and Post-ex in the normoxic condition. Compared with NOR, in HYP SpO₂ was significantly decreased by 22% (P < 0.05) Pre-ex and even lower Post-ex (-28%, P < 0.05). TOI followed the same pattern as SpO₂. TOI was significantly decreased in HYP compared with NOR by 3.5% (P < 0.05) Pre-ex and by 6% (P < 0.05) Post-ex. In contrast with SpO₂, TOI was significantly different in Pre- and Post-ex within NOR. Table 2. Arterial oxygen hemoglobin saturation and tissue oxygenation index values in response to acute hypoxia and submaximal exercise NOR HYP SpO2 (%) Pre-ex 99.5 ± 0.1 77.6 ± 0.8* Post-ex 98.4 ± 0.2 70.7 ± 0.6*# TOI (%) Pre-ex 68.3 ± 0.2 64.8 ± 0.2* Post-ex 64 ± 0.2# 58 ± 0.4*# Mean arterial oxygen hemoglobin saturation (SpO₂) and tissue oxygenation index (TOI) values during normoxia # (NOR) and hypoxia (HYP) at rest (Pre-ex) and after exercise (Post-ex). Values are means ± SEM (n=23). p