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Doctoral Thesis

Erythropoietin's impact on endurance performance undernormoxic condition and upon acclimatization to moderatealtitude

Author(s): Schuler, Beat

Publication Date: 2009

Permanent Link: https://doi.org/10.3929/ethz-a-005772724

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DISS. ETH NO. 18183

Erythropoietin’s impact on endurance performance under normoxic condi-tion and upon acclimatization to moderate altitude

A dissertation submitted to

ETH ZURICH

for the degree of

Doctor of Natural Sciences

presented by

Beat Schuler

Dipl. Natw. ETH

Date of birth 16 June 1972

citizen of Schübelbach (SZ)

accepted on the recommendation of

Prof. Dr. Urs Boutellier Prof. Dr. Max Gassmann

Prof. Dr. Jean-Claude Perriard

2009

Table of contents

1. Summary............................................................................................................................ 1

2. Zusammenfassung............................................................................................................. 3

3. General introduction......................................................................................................... 5

3.1. Hypoxia...................................................................................................................... 5

3.2. Adaptational mechanism to hypoxic environment ..................................................... 6

3.3. Erythropoietin............................................................................................................ 8 3.3.1. Structure of erythropoietin ............................................................................... 8 3.3.2. Structure of erythropoietin receptor ................................................................. 8 3.3.3. Function............................................................................................................ 9 3.3.4. Exercise ……………………………………………………………………...10

3.3.4.1. Impact of recombinant human erythropoietin.................................. 12 3.3.4.2. Altitude acclimatization ................................................................... 14

3.4. Aim of the project..................................................................................................... 16

4. Study 1: Optimizing transmitter implantation and postoperative care to improve telemetric signal in exercising mice ............................................................................... 17

4.1. Abstract.................................................................................................................... 17

4.2. Introduction ............................................................................................................. 17

4.3. Methods.................................................................................................................... 19

4.4. Results...................................................................................................................... 22

4.5. Discussion................................................................................................................ 26

5. Study 2: Optimal hematocrit for maximal exercise performance in erythropoietin-treated mice...................................................................................................................... 29

5.1. Abstract.................................................................................................................... 29

5.2. Introduction ............................................................................................................. 29

5.3. Materials and Methods ............................................................................................ 31

5.4. Results...................................................................................................................... 35

5.5. Discussion................................................................................................................ 43

6. Study 3: Timing the arrival at 2340 m altitude for aerobic performance ................. 49

6.1. Abstract.................................................................................................................... 49

6.2. Introduction ............................................................................................................. 49

6.3. Methods.................................................................................................................... 51

6.4. Results...................................................................................................................... 54

6.5. Discussion................................................................................................................ 57

6.6. Perspectives ............................................................................................................. 59

7. General discussion and conclusions............................................................................... 61

8. References ........................................................................................................................ 64

9. List of abbreviations ....................................................................................................... 78

10. Curriculum Vitae ............................................................................................................ 79

11. Publications...................................................................................................................... 80

12. Acknowledgements.......................................................................................................... 82

Beat Schuler 2009 1

1. Summary Hypoxia is the main stimulus of erythropoietin (Epo) expression. The oxygen (O2)-sensing

protein termed hypoxia-inducible factor (Hif) has been identified as a key regulator of Epo.

At normoxia, Hif-α, a subunit of Hif, is degraded but stabilized under hypoxic condition, in

which it enhances Epo gene expression resulting in increased erythrocyte production and in

an increased O2 transport capacity of blood. Within the normal physiological range, maxi-

mal O2 uptake (V& O2max) increases in parallel with increasing blood O2 transport capacity at

normoxia. Therefore, some endurance athletes live at moderate altitude and train at low

altitude to improve sea level performance. Others do misuse recombinant human Epo

(rhEpo), originally developed for the treatment of anemia. An unwanted side effect of an

Epo-induced increase in the blood’s O2 transport capacity is an increased blood viscosity

due to the elevated hematocrit (Htc) levels. The higher blood viscosity strains the cardio-

vascular system and may limit endurance performance. On the other hand, our transgenic

mice line (tg6) overexpressed Epo constitutively and therefore reached Htc levels between

0.8 and 0.9 and interestingly, there were no signs of pathological alterations in three month

old mice. To study the impact of various Htc levels on exercise performance and the car-

diovascular system, telemetry and indirect calorimetry in exercising mice were combined

and improved in a first study. Telemetry offers the possibility to monitor arterial blood

pressure from conscious, freely moving laboratory mice; however, its use has been limited

because of high morbidity and mortality particularly in genetically modified animals. Of-

ten, either a weak telemetric signal is observed during exercise or even none at all is ob-

served. Here, we show an optimized transmitter implantation technique to improve the

telemetric signal in exercising mice. Moreover, a new postoperative intensive care regime

and analgesia were used to reduce morbidity and mortality. The new tool is useful for in-

vestigators who plan to measure cardiovascular function in mice using telemetry, but the

surgical procedure remains challenging.

The aim of the second study was to investigate the effect of varying Htc levels on exercise

performance and the vascular system. To this end, wild type mice (wt) and tg6 mice were

injected with the novel erythropoiesis stimulating protein (NESP; wtNESP), or the hemo-

lysis inducing compound phenylhydrazine (PHZ; tg6PHZ), respectively. Highest V& O2max

and best time to exhaustion were reached at Htc values of 0.58 and 0.57 for wtNESP mice,

and 0.68 and 0.66 for tg6PHZ, respectively. Maximal stroke volume was observed at simi-

lar Htc levels. Interestingly, V& O2max of wtNESP was most closely related to whole body

hemoglobin in an Htc range from 0.4 to 0.55. V& O2max was correlated with blood viscosity.

2 Beat Schuler 2009

We conclude that (1.) tgPHZ adapt better to varying Htc levels than wtNESP do, (2.) en-

durance performance is primarily limited by O2 delivery and (3) the general observation

that V& O2max is strongly correlated with whole body hemoglobin is only valid within the

physiological Htc range and when Htc levels are increased.

Acute hypoxia induces a reduction in V& O2max. During altitude acclimatization above 4100

m, arterial O2 content increases due to the increasing hemoglobin concentration without

effecting V& O2max. The data on moderate altitude are controversial. Thus, a third study in-

vestigated the hypothesis that V& O2max and performance increase upon altitude acclimatiza-

tion to 2340 m. Therefore, eight elite cyclists trained during a period of 21 days according

to the “live high-train low” approach. Performance parameters were mainly improved

within the first fourteen days, whereas in the following week, only a slight improvement

was observed. These results suggest that athletes who plan to compete around this altitude

have to arrive at least fourteen days before the beginning of the competition in order to be

prepared optimally for the competition day.

Beat Schuler 2009 3

2. Zusammenfassung Hypoxie ist der Hauptstimulus der Erythropoietin (Epo)-Expression. Der Sauerstoff (O2)-

sensitive hypoxie-induzierbare Faktor (Hif) wurde als wichtigster Regulator von Epo iden-

tifiziert. Unter normoxischen Bedingungen wird Hif-α, eine Untereinheit von Hif, schnell

abgebaut, bleibt aber bei Hypoxie stabil. Als Folge steigt die Epo-Expression und somit die

Erythrozytenproduktion und O2-Transportkapazität des Blutes an. Unter Normoxie nimmt

die maximale O2-Aufnahme (V& O2max) bei normalen physiologischen Bedingungen parallel

zur O2-Transportfähigkeit des Blutes zu. Darum halten sich manche Ausdauerathleten auf

moderater Höhe auf und trainieren alternierend in tieferen Lagen („live high-train low“),

um ihre Leistung auf Meereshöhe zu verbessern. Gewisse skrupellose Athleten missbrau-

chen rekombinantes humanes Epo (rhEpo), welches in erster Linie für die Behandlung von

anämischen Krankheiten entwickelt wurde, zur Steigerung ihrer Leistungsfähigkeit. Eine

unerwünschte Nebenerscheinung ist die Zunahme der Blutviskosität, welche durch die

Zunahme des Hämatokrit (Htc)-Spiegels hervorgerufen wird. Die erhöhte Blutviskosität

belastet das kardiovaskuläre System ernorm und könnte die Ausdauerleistung limitieren.

Interessanterweise, wurden aber keine pathologischen Veränderungen in 3 Monaten alten

transgenen Mäusen (tg6) gefunden. Diese Mausline überexprimiert humanes Epo konstitu-

tiv und erreicht deshalb Htc-Werte zwischen 0.8 und 0.9. Um den Einfluss verschiedener

Htc-Werte im Bezug auf Ausdauerleistung und kardiovaskuären System zu untersuchen,

wurden in einer ersten Studie vorgängig Telemetrie und indirekte Kaliometrie während

körperlicher Belastung kombiniert bzw. verbessert, um sportmedizinisch relevante Parame-

ter bei Mäusen messen zu können. Die Telemetrie ermöglicht die Messung des Blutdrucks

in wachen, sich frei bewegenden Labortiermäusen. Allerdings ist dessen Anwendbarkeit

durch eine hohe Mobilität und Mortabilität der Tiere, und mit keinem oder nur schlechten

telemetrischen Signal während körperlicher Betätigung limitiert. Darum zeigen wir hier

eine optimierte Implantationstechnik um das telemetrische Signal während körperlicher

Betätigung zu verbessern. Zudem wurde ein neues postoperatives Behandlungsregime und

verbesserte Analgesie etabliert, um die Morbidität und Mortalität zu reduzieren. Diese

neue Implantations- und Behandlungsmethode ist für Forscher wichtig, die die kardio-

vaskulären Funktionen in Mäusen mit der Telemetrie-Technik untersuchen möchten. Den-

noch bleibt die Tansmitterimplantation anspruchsvoll.

Das Ziel der zweiten Studie war es den Einfluss verschiedener Htc-Werte auf die sportli-

che Leistungsfähigkeit sowie kardiovaskuläre System zu untersuchen. Für diesen Zweck

wurden Wildtyp (wt)- und tg6-Mäuse mit dem Erythropoiese stimulierenden Protein

4 Beat Schuler 2009

(NESP; wtNESP) respektive, dem Hämolyse induzierenden Phenylhydrazin (PHZ;

tg6PHZ) behandelt. Höchster V& O2max und beste Zeit bis zur Erschöpfung wurden bei Htc-

Werten von 0.58 und 0.57 für wtNESP, bzw. 0.68 und 0.66 für tg6PHZ gemessen. Maxi-

males Herzschlagvolumen wurde bei ähnlichen Htc-Werten beobachtet. Interessanterweise

korrelierte V& O2max nur bei wtNESP-Mäusen in einem Htc-Bereich von 0.4 bis 0.55 am

stärksten mit der gesamten Hämoglobinmenge. Zudem wurde eine Abhängigkeit zwischen V& O2max und Blutviskostät beobachtet. Wir schlossen daraus, dass (1.) sich tg6PHZ-Mäuse

besser an die veränderten Htc-Werte anpassen konnten als die wtNESP-Mäuse, (2.) die

Ausdauerleistung hauptsächlich von der O2-Verfügbarkeit limitiert ist und (3) V& O2max stark

mit der gesamten Hämoglobinkonzentration korreliert. Allerdings gilt letzteres nur inner-

halb des physiologischen Htc-Bereichs und sofern die Htc-Werte erhöht wurden.

Akute Hypoxie verursacht eine Reduktion von V& O2max. Während der Höhenakklimatisie-

rung oberhalb von 4100 m, steigt der arterielle O2-Gehalt im Blut aufgrund der Zunahme

der Hämoglobinkonzentration auf oder über Meereshöheniveau an, wobei V& O2max davon

unbeeinflusst ist. Doch sind die Daten auf mittlerer Höhe kontrovers. Während in einigen

Studien V& O2max nicht anstieg, berichten andere von einer minimalen Zunahme. Darum

wurde in der dritten Studie die Hypothese untersucht, ob V& O2max und Leistung während

der Höhenakklimaisation an 2340 m ansteigen. 8 Eliteradfahrer trainierten während 21

Tagen gemäss dem „live-high-train low“ Prinzip. Die leistungsbezogenen Parameter ver-

besserten sich hauptsächlich in den ersten 14 Tagen während in den darauf folgenden 7

Tagen nur noch ein leichter Anstieg beobachtet wurde. Diese Resultate legen nahe, dass

Sportler, die auf etwa dieser Höhe einen Wettkampf betreiben möchten, sich mindestens 14

Tage vor Wettkampfbeginn auf diese Höhe begeben müssen, um am Wettkampftag best-

möglichst vorbereitet zu sein.

Beat Schuler 2009 5

3. General introduction

3.1. Hypoxia Adequate oxygen (O2) supply is essential to the aerobic metabolism of most eukaryotic

organisms. O2 participates in the cellular metabolism to ensure energy production in the

cell as substrate for an optimal oxidation. Even a slight reduction in O2 availability (hy-

poxia) seriously impairs this energy methabolism in humans. O2 may become limited by

anemia or cardiovascular, pulmonary or haematological diseases, but also by exercise and

exposure to high altitude.

While the percentage of O2 in the atmosphere below 10,000 m altitude is constant at 20-

21%, O2 partial pressure (pO2) falls exponentially with increasing altitude. With decreasing

pO2, inspiratory and alveolar partial pressure (pO2) is also reduced. For instance, at sea

level the mean alveolar pO2 is about 100 mmHg, whereas it drops to about 46 mmHg at an

altitude of 5000 m. As a response to hypoxic environment, several hypoxic sensors are

activated to maintain homeostasis. Tab. 3.1 shows altitude related to Patm as well as pO2.

Altitude [m] Patm [mmHg] pO2 [mmHg]

0 760 100

2000 596 82

3000 526 67

4000 462 50

5000 405 46

6000 354 40

7000 308 35

8000 267 32

9000 231 30

Tab. 3.1. Altitude related to the atmosphere pressure [Patm] as well as the alveolar oxy-

gen partial pressure [pO2] (modified from West, 1995)

6 Beat Schuler 2009

3.2. Adaptational mechanism to hypoxic environment The physiological adjustments to hypoxia occur at systemic and cellular levels. When pO2

falls, homeostatic mechanisms of the respiratory and cardiovascular systems, such as an

increase in ventilation and heart rate, are immediately activated to deliver adequate O2 to

the organism. When impaired O2 supply is prolonged, the response includes changes in

gene expression in the cell. Altitudes between 3500 and 5000 m induce the required en-

zyme synthesis for increased glycolysis, Krebs cycle, respiratory chain and several glucose

membrane transporters (Rynafarje et al., 1962; Ou and Tenney, 1970; West and Mangan,

1970). Above 5000 m, enzyme activity is reduced and the organism undergoes a loss of

muscle mass and body weight (Hoppeler et al., 1990; Kayser et al., 1993; Steinacker et al.,

1996).

A key molecular global regulator of hypoxia is hypoxia-inducible factor-1 (Hif-1). Hif-1 is

a transcription factor which regulates many genes influencing angiogenesis, erythropoiesis,

glycolysis, iron metabolism, cell survival and growth (Semenza, 2001). More than 100

genes are known to be directly or indirectly regulated by Hif-1 (Kotch et al., 1999; Wang

et al., 1995). Table 3.2 shows the most abundant of the identified Hif-1 target genes. It has

been shown that Hif-1 induces broad hypoxia at systemic and cellular level in order to

compensate for the energy deficit in the cell. Moreover, the Hif-1 pathway is critical in

development, physiology and disease (Jelkmann, 2007; Krishnan et al., 2008; Soliz et al.,

2005; Tovari et al., 2008).

Hif-1 is a heterodimeric transcription factor composed of Hif-1α and Hif-1β subunits

(Hopfl et al., 2004) and is expressed in all tissue of many species such as drosophila, fish,

C. elegans and mammals (Abbrecht and Littell, 1972; Epstein et al., 2001; Soitamo et al.,

2001).

Whereas the β subunit is a non-responsive nuclear protein and thus, stable, the accumula-

tion of α subunit is controlled by cellular O2 concentration (Hopfl et al., 2004; Wang et al.,

1995). At normoxia, Hif-1α is immediately degraded, but this process is inhibited under

hypoxic condition and therefore, it causes a response to hypoxia. As a consequence of hy-

poxia induced stabilization, Hif-1α translocates to the nucleus and heterodimerizes with

Hif-1β to form a function at Hif-1 complex, which interacts with the hypoxia response ele-

ment located within the promoter and enhancer of the O2-dependent target gene expression

(Hopfl et al., 2004). These genes are particularly relevant for homeostasis at the cellular

and systemic levels. The most of the identified Hif-1 target genes are listed in Tab. 3.2.

One of the best studied Hif target genes is erythropoietin (Epo).

Beat Schuler 2009 7

- Adenylate kinase 3 (AK-33)

- Adrenomedullin (ADM)

- Aldolase A (ALDA)

- Aldolase C (ALDC)

- ANF/GPI

- Autocrine mobility factor/ (AMF/GPI),

- α1B-adrenergic receptor (α1B-AR)

- Carbonic anhydrase 9 (CA-9)

- Cathepsin D (CATHD)

- Ceruloplasmin

- c-MET

- Collagen type V (α1)

- Cyclin G2

- Differentiated embryo–chondrocyte expressed gene 1,2 (DEC1,2)

- Ecto-5’-nucleotidase

- Endocrine-gland-derived VEGF (EG-VEGF)

- Endoglin (ENG)

- Endothelin-1 (ET-1)

- Enolase 1 (ENO1)

- Erythropoietin (EPO)

- ETS-1

- Ferrochelatase (FECH)

- Fibronectin 1 (FN1)

- Glucose transporter 1,3 (GLUT1,3)

- Glyceraldehyde-3-P-dehydrogenase (GAPDH)

- Haem oxygenase-1 (HO-1)

- Hexokinase 1, 2 (HK1, 2)

- Inhibitor of differentiation/DNA binding 2 (ID2)

- Insulin-like growth-factor (IGF2)

- Insulin-like growth-factor-binding-protein 1, 2, 3 (IGF-BP1, 2, 3)

- Intestinal trefoil factor

- Kreatin 14, 18, 19 (KRT14, 18, 19)

- Lactate dehydrogenase A (LDHA)

- LDL-receptor-related protein (LRP1)Leptin (LEP)

- LDL-receptor-related protein 1 (LRP1)

- Metalloproteinase (MMP2)

- MIC2

- Multidrugresistance (MDR1)

- NIP3

- Nitric oxide synthase 2 (NOS2)

- NIX

- NUR77

- Phosphofructokinase L (PFKL)

- Phosphoglyceratekinase 1 (PGK1)

- 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKBF3)

- Plasminogen-activator inhibitor 1 (PAI1)

- Prolyl-4-hydroxylase α

- Pyruvate kinase (PKM)

- p35srj

- RTP801

- Transferin

- Transferin receptor

- Transforming growth factor- α (TGF-α)

- Transforming growth factor- β3 (TGF- β3)

- Transglutaminase 2

- Triosehposphate isomerase (TPI)

- Urokinase plasminogen activator receptor (UPAR)

- Vascular growth factor (VEGF)

- Vimentin (VIM)

- WAF-1

Tab. 3.2. Some HIF-1 target genes, adapted from Kotch et al. (1999), Wang et al. (1995)

and own update

8 Beat Schuler 2009

More recently, further Hif-α isoforms have been identified, namely Hif-2α and Hif-3α

(Luo et al., 1997). These subunits are also O2-labile and can dimerise with Hif-1β under

hypoxic condition but are different with respect to their tissue-specific mRNA expression

pattern (Wiesener et al., 2002). Recent studies show that Hif-2α controlls Epo gene expres-

sion (Warnecke et al., 2004).

3.3. Erythropoietin

3.3.1. Structure of erythropoietin The secreted human Epo protein is a glycoprotein hormone 34 kDa in weight and consists

of 165 amino acids (60% of the total molecule) and 4 carbohydrate chains (40% of the total

molecule) (Egrie and Browne, 2001; Jelkmann, 2004; Wen et al., 1993). The carbohydrate

chains are essential to the biological stability and affinity of the circulating protein (Egrie

et al., 2003). However, several investigators have studied its tertiary structures (Bazan,

1990; Cheetham et al., 1998; Syed et al., 1998). All these models are based on two antipar-

allel pairs of α-helical bundles with interconnecting variably sized loops. Human and

mouse Epo are 80% identical in amino acid sequences (McDonald et al., 1986; Shoemaker

and Mitsock, 1986, Wen et al., 1993). The Epo gene in human beings is located on chro-

mosome 7 between 7q21 and 7q22 as a single copy and contains 5 exons (Koury and Bon-

durant, 1992; Powell et al. 1986; Wang et al., 1995). Epo is expressed in most tissue. This

gene is activated by a variety of stressors, including hypoxia (Jelkmann, 2004). When O2

supply drops, Epo expression induces an exponential increase in Epo plasma levels (Jelk-

mann, 2003), but this inducibility is tissue specific with the strongest effect in kidney and

brain (Koury and Bondurant, 1992; Siren et al., 2001).

3.3.2. Structure of erythropoietin receptor The Epo protein binds to specific receptors present in the membrane of the target cells

(Jelkmann et al., 2008). In general, the number of Epo receptors ranges from approxi-

mately 1000-3000/cell (Broudy et al., 1988; D’Andrea and Zon, 1990; Koury and Bondu-

rant; 1992). The human gene is located on chromosome 7 and has 8 exons (Budarf et al.,

1990; Maouche et al., 1991). Exons 1-5 encode the extracellular domain. Exon 6 encodes

the transmembrane and exons 7, 8 encode the cytoplasmatic domain. Interestingly, the

gene is transcribed continuously (Wickrema et al., 1992), but hypoxia may have an impact

on its up-regulation. The resulting protein has a molecular mass of 66 kDa and is a 484

amino-acids glycoprotein of the cytokine receptor superfamily which is characterized by

Beat Schuler 2009 9

ligand-inducible dimerization (Bailey et al., 1993; D’Andreas et al., 1989; Jelkmann, 2005;

2008). Two of the membrane-spanning Epo receptor molecules form a dimer to which the

Epo molecule binds (Jelkmann, 2004).

3.3.3. Function Epo is a cytokine whose main function is producing erythrocytes by stimulating the prolif-

eration, differentiation and maturation of the progenitors in the bone marrow, and prevent-

ing the apoptosis of these erythroid progenitors, by binding to and activating the Epo re-

ceptor on the surface of the cell (Jelkmann et al., 2008). It is rapidly up-regulated when

pO2 is reduced in the venous blood and tissue. Hypoxia leads to reduced pO2, which results

in Hif-1 induced up-regulation of the Epo gene expression, mainly occurring in the kidney.

Accordingly, the Epo plasma level is elevated and finally, the number of circulating eryth-

rocytes in the bloodstream is increased.

Apart from the kidney, Epo is produced in (fetal) liver, brain, lung, spleen, bone marrow,

male and female reproductive organs and also in numerous cancer cells (Fandrey and

Bunn, 1993; Hermine et al., 1991; Jelkmann et al., 2008; Maxwell et al., 1993, 1997; Tan

et al., 1992; Yasuda et al., 1998). Additionally, Epo receptor expression has been observed

in the brain, retina, and heart and in fractions of skeletal muscle fibers (Grimm et al., 2002;

Junk et al., 2002; Lundby et al., 2008a; Wu et al., 1999). The non-hematopoietic functions

of Epo are associated with these tissues. Thus, Epo is involved in the modulation of re-

sponses to injuries such as cerebral ischemia, cardiac infarction and retina degeneration

(Gassmann et al., 2003; Marzo et al., 2008; Wiessner et al., 2001, Wright et al., 2004).

Nevertheless, the physiological impact of Epo remains largely unclear. A recently pub-

lished paper showed that Epo-levels in plasma and brain are involved in the ventilatory

response to acute and chronic hypoxia (Soliz et al., 2005) and recombinant human Epo

(rhEpo) treated humans feel improvements in mood (Miskowiak et al., 2008) as well as

perceived physical conditioning (Ninot et al., 2006). Furthermore, it was reported that Epo

promotes the enhancement of the vascular endothelial growth factor (VEGF) expression in

tissue (Alvarez et al., 1998), resulting in increased skeletal muscle capillary growth. In a

recent published study performed on rodents, it was reported that muscle fiber types

change toward a more oxidative phenotype when Epo treatment occurs during training

(Cayla et al., 2008). Interestingly, no alterations in skeletal muscle angiogenesis or VEGF

mRNA levels were found after long time Epo treatment, nor was a shift in muscle fiber

type found (Lundby et al., 2008a).

10 Beat Schuler 2009

3.3.4. Exercise Endurance performance, normally characterized as maximal O2 uptake (V& O2max), is af-

fected by a series of steps in the transport of O2 from the atmosphere to the mitochondria

of the muscle. Inhibition of each step in the O2 transport system potentially leads to an im-

pediment of the O2 flux. These steps include central factors such as O2 diffusion from the

lung into the blood (pulmonary system), cardiac output and O2 carrying capacity of the

blood, and peripheral factors such as skeletal muscle characteristics such as muscle capil-

lary density, O2 diffusion gradient from the surface of red blood cells to sarcolemma and

mitochondrial enzyme activity (Fig. 3.1). The issue of which factors limit V& O2max remains

a subject to intensive debate.

Fig. 3.1. Potential factors which limit maximal oxygen uptake (V& O2max)

The difficulty to identify limiting factors is the fact that some variables can easily be influ-

enced by endurance training while others can not be modified. Thus, there is no single

variable limiting endurance performance, but there are a number of components reflecting

a cascade mechanism.

Pulmonary system Cardiac output

Peripheral factors

Blood O2 carrying capacity

Beat Schuler 2009 11

However, the blood’s O2 transport capacity is of special interest in this thesis. According to

Fick’s law, V& O2max depends on cardiac output and the arteriovenous O2 difference. This

implies that all factors influencing these physiological quantities may exert endurance lim-

iting effects. The most important related to blood supply are the blood volume and whole

body hemoglobin. Several studies have shown that there is a strong correlation between V& O2max and blood volume as well as whole body hemoglobin, but not hemoglobin concen-

tration [Hb] (Åstrand, 1952, 1977; Ekblom and Hermansen, 1968; Kanstrup and Ekblom,

1984; Heinicke et al., 2001). Note that most human studies are conducted with subjects

reaching hematocrit (Htc) levels up to 0.5. Thus, it is not known whether the relationships

exist at Htc values higher than 0.5, too.

If V& O2 is maintained during endurance exercise, it is equal to the product of the athlete’s V& O2max and the percent of V& O2max that can be maintained during performance (Coyle et al.,

1995). The percentage of V& O2max is related to the V& O2 at the lactate. Artificially raised

plasma volume, which increases whole blood volume but keeps red cell numbers constant

and lower [Hb], has no effect on V& O2max in trained subjects (Kanstrup and Ekblom, 1982),

while submaximal exercise duration may be prolonged (Coyle et al., 1990; Luetkemeier

and Thomas, 1994). Note hypervolemic plasma expansion in untrained subjects induces

significantly an increment in V& O2max due to increased stroke volume (Warburton et al.,

2004). Otherwise, an increment of whole body hemoglobin mainly affects V& O2max. Endur-

ance training at sea level improves cardiac output and muscular vascularisation (Gaudard

et al., 2003), but its impact on the stimulation of erythropoiesis is controversial. Some stud-

ies performed in untrained and trained athletes from different disciplines showed that sub-

maximal and maximal exercise have no impact on circulating plasma levels (Berglund et

al., 1988; Gareau et al., 1991, Ricci et al., 1990; Schmidt et al., 1991). In contrast, other

studies found a slight increase in the Epo plasma level after several hours of exercise

(Ricci et al., 1988; Schobersberger et al., 2000; Schwandt et al., 1991). One explanation for

the increasing Epo plasma level might be that these changes are due to changes in plasma

volume and another explanation might be that hormones in the blood show circadian

rhythm with a nadir in the morning. Thus, measurements have to be done at the same time

of day (Jelkmann, 2003; Schmidt et al., 1991, 1993). However, the number of circulating

reticulocytes is immediately increased after strenuous exercise (Schmidt et al., 1988).

Jelkman (2003) suggested that the reduced renal blood flow is only a minor factor in Epo

production, but stress hormones such as cortisol and catecholamines may be more impor-

tant in the regulation of erythrocyte production.


To improve their sea level endurance performance, some endurance athletes live at moder-

ate altitude (2000-3000 m above sea level) and train at low altitude to increase [Hb] due to

hypoxia induced stimulation of erythropoiesis (Levine and Stray-Gundersen, 1997; Wilber

et al., 2007). Unscrupulous athletes, coaches, and practitioners artificially increase the O2

transport capacity of the blood and thus exercise performance by using rhEpo and blood

transfusions of red blood cells.

3.3.4.1. Impact of recombinant human erythropoietin

In 1987, rhEpo became commercially available. It was developed for the treatment of ane-

mia occurring as a consequence of ailments such as chronic renal failure, HIV-infection

and cancer. Unfortunately, rhEpo is also well known as a doping agent in endurance sports,

and even worse, might be a target for gene doping. Since its launch onto the market, sev-

eral studies have investigated its effects on [Hb] and V& O2max In the first published study,

Ekblom and Berglund (1991) reported that injecting 20-40 IU/kg body mass rhEpo over a

period of 6 weeks, three times per week, induces an increment of 11.2% in [Hb], resulting

in an 8.9% improvement in V& O2max. The effect of injected rhEpo is even more pronounced

at submaximal exercise intensities than at maximal exercise intensities (Thomsen et al.,

2007). Similar results are observed when [Hb] is elevated by blood infusion (Brien and

Simon, 1987; Buick et al., 1980; Ekblom et al., 1972; 1976; Robinson et al., 1966; Turner

et al., 1993; Williams et al., 1981). For instance, Ekblom and co-workers (1972) demon-

strated that autologous blood re-infusion of 800-1200 ml results in an increase in [Hb] of

13%, whereas V& O2max and endurance time were increased by 9% and 23% respectively.

Thus, it may seem obvious that the ergogenic effect of rhEpo administration and blood

infusion on the exercise capacity is mediated through the increasing [Hb].

An increment in [Hb] is associated with elevated Htc levels and thus with blood viscosity

(Crowell and Smith, 1967; Vogel et al., 2003). Elevated blood viscosity may unfavorably

affect the microcirculatory blood flow and O2 delivery to the tissue (Crowell and Smith,

1967). Therefore, the peripheral resistance to blood flow within the vascular system is

regulated not only by the calibre of the vessels, but also by the viscous characteristics of

the blood (El-Sayed et al., 2005). Blood viscosity is linearly correlated over an Htc range

of 0.2 to 0.6, beyond which the increment in whole blood viscosity becomes disproportion-

ately higher with increasing Htc levels (Chien et al., 1966). Note that blood viscosity is

globally measured, but it may vary locally within the different parts of the vascular system

when blood flow is forced (Dorandy, 1979). Disruption of the normal rheological proper-

ties of the blood is considered an independent risk factor for the cardiovascular system and


might also reduce exercise performance and may be life-threatening (Jelkmann, 2003).

Therefore, our transgenic mouse line termed tg6 overexpressing human Epo cDNA

reached Htc values of up to 0.9 (Ruschitzka et al., 2000) and showed a dramatically re-

duced exercise performance (Heinicke et al., 2006; Wagner et al., 2001). This implies that

there is an optimal Htc value at which the O2 carrying capacity of blood is maximized

without compromising cardiac output due to the elevated blood viscosity associated with

higher Htc levels (Crowell et al., 1959, Guyton and Richardson, 1961; Richardson and

Guyton, 1959; Villafuerte et al., 2004). It was proposed that V& O2max is limited by 70% of

blood O2 transport capacity within the circulatory system; all other systems being respon-

sible for the remaining 30% (DiPrampero and Ferretti; 1990). Thus, there may potentially

exist an optimal Htc for maximal endurance performance. But no data are available for

higher vertebrates.

If Htc levels sharply rise, cardiac output falls (Richardson and Guyton, 1959). Interest-

ingly, the tg6 mice showed no changes in resting cardiac output (Vogel et al., 2003). Ani-

mals grow up normal and showed no symptoms of thromboembolism, but had a lifespan

that was a third shorter than that of their wild type (wt; Wagner et al., 2001) and developed

organ failures such as degenerative processes in the liver, kidney, hepatic system, and

nerve and skeletal muscle fibers (Heinicke et al., 2006). However, these findings indicate

that adaptive mechanisms enacted in response to excessive erythrocytosis exist. Adaptive

processes in response to excessive erythrocytosis include increased plasma nitric oxide

levels, elevated erythrocyte deformability and reduction of erythrocyte’s lifetime (Bogda-

nova et al., 2007; Ruschitzka et al., 2000; Vogel et al., 2003). Interestingly, there are also

humans who cope with excessive erythrocytosis. In one case, due to an autosomal domi-

nant erythrocytosis, a Finnish cross-country skier reached Htc levels of up to 0.68 (Ju-

vonen et al., 1991), won several Olympic gold medals and showed no organ failures. In

another case, some miners in the South American Andes - living and working at an altitude

of 5960 m above sea level and exposed to cobalt - were reported to have an acute Htc level

of between 0.75 and 0.91 (Jefferson et al., 2002).

To study the impact of varying Htc levels on exercise performance and the cardiovascular

system, the technology to measure blood pressure as well as heart rate during submaximal

and maximal exercise in mice was improved by using telemetry in the first study. In gen-

eral, the transmitter implantation is associated with a high failure rate. Thus, a further aim

was to improve the surgery to reduce that number. The second study investigated whether

there is an Htc value which allows maximal endurance performance for whole body exer-


cise. To this end, Htc levels of wt mice were increased by graduated application of novel

erythropoiesis stimulating protein (NESP). On the other hand, the Htc of our tg6 mice was

decreased in steps to different levels by the application of phenylhydrazine, a compound

inducing hemolysis (Lim et al., 1998). We postulate that the optimal Htc levels may not

identical for wt and tg6 mice. Thus, it was hypothesized that tg6 can adapt better to various

Htc levels than wt mice can.

3.3.4.2. Altitude acclimatization

When humans are exposed to hypoxia, besides the physiological changes that occur in the

muscular, respiratory, cerebral, cardiovascular and hormonal systems, and the changes in

fluid and electrolyte balances, the O2 transport capacity is particularly affected. This is

perhaps the reason why one of the most frequently studied adaptations to high altitude is

the increase in red blood cells.

Within two hours of acute onset of hypobaric hypoxia, circulating Epo plasma levels begin

to increase significantly (Eckardt et al., 1989). The peak is reached after between 24 and 28

hours of a stay at moderate (1500-3000 m) and high (> 3000 m) altitude (Abbrecht and

Littell, 1972; Gunga et al., 1994; Heinicke et al., 2003; Milledge and Cotes, 1985). After

this time, plasma Epo levels decline to close to pre-exposure levels, but may remain

slightly elevated when subjects keep staying at higher than normal altitudes (Heinicke et

al., 2003; Mairbaurl et al., 1990; Milledge and Cotes, 1985). Mountain climbers usually

undergo a gradual increase in the degree of hypoxia until they reach a high altitude destina-

tion. Therefore, increasing Epo levels are detected not only after an ascent from sea level

to altitude, but also when ascending from moderate altitude to high altitude. The effect of

acute exposure to hypoxia on Epo levels is maintained after 3 weeks of acclimatization,

and even after life long intermittent residency at high altitude (Heinicke et al., 2003).

However, despite the reduced Epo level in serum with prolonged altitude, erythropoiesis

remains stimulated (Milledge and Cotes, 1985). This is indicated by elevated reticulocyte

counts and decreased serum iron and ferritin (Mairbaurl et al., 1990). Note that [Hb] in-

creases during the first 24-48 hours of exposure to altitude due to a reduction in plasma

volume and that this increase is not due to the enhanced erythropoiesis. The latter is caused

by an enhancement of diuresis and displacement of water from the vascular to the ex-

travascular and intracellular spaces.

When humans are acutely exposed to high altitude, V& O2max is reduced as a function of the

reduction of arterial O2 saturation (SaO2; Fulco et al., 1998; Wehrlin and Hallen, 2006).


During altitude acclimatization [Hb] continues to increase in order to make up for the loss

in arterial O2 content (CaO2) due to decreased arterial SaO2 (Calbet et al., 2003a; Mair-

baurl, 1994). Accordingly, CaO2 is normalized or is even higher than at sea level during

acclimatization above 4100 m altitude (Calbet et al., 2003b; Ceretelli, 1976; Lundby et al.,

2004), whereas V& O2max remained depressed. Interestingly, V& O2max still does not increase

when CaO2 is elevated by NESP injection after acute hypoxia at 12.6% O2 (= 4100 m

above sea level; Lundby and Damsgaard, 2006a). NESP has been developed as an Epo-

analogue with prolonged survival in the circulation (MacDougall, 2000) and thus, it has a

longer half-life than rhEPO (NESP: 24-26h, rhEpo: 4-8h; Jelkmann, 2002).

However, there is a discrepancy in the ergogenic effect of increasing [Hb] observed at sea

level compared to that observed at high altitude and this suggests that an altitude threshold

exists over which increasing [Hb] has no advantageous effect on endurance performance,

but below this threshold increasing [Hb] improves endurance performance. However, Cal-

bet and co-workers (2002) reported that during maximal exercise on a cycle ergometer test

after 9 weeks at 5260 m, systemic O2 delivery was 10% lower than during maximal exer-

cise at sea level, due to a reduction in peak cardiac output during exercise in hypoxia. Al-

though the CaO2 was almost normalized by the increasing [Hb] to pre-altitude values, V& O2max was only partly improved and remained 30% below the values observed at nor-

moxia. This implies that part of the systemic O2 delivery gained with acclimatization is not

made available to the exercising muscle (Calbet et al., 2003b).

While it seems clear that V& O2max does not improve during altitude acclimatization above

4100 m, the data below are controversial. On one hand, five studies reported no change or

a slight increment in endurance after 14 and 20 days training and living at 2300 m respec-

tively (Adams et al., 1975; Daniels and Oldridge, 1970; Faulkner et al., 1967; 1968; Pugh,

1967). On the other hand, V& O2max and endurance performance increase after 19 days of

acclimatization to 2300 m and 21 days to 1822 m respectively (Jensen et al., 1993; Saltin

1967). The difficulties of these studies can mainly be explained by different training re-

gimes, different physiological organic systems in the human body reacting to training at

altitude at different times, the different degree of reaction of various systems and the vary-

ing overall training status of the studied athlete. Levine and Stray-Gundersen (2005) stated

the success of altitude training depends on two factors: 1.) live high enough to initiate and

maintain erythropoiesis to improve blood O2 transport capacity and thus V& O2max, and 2.)

train low enough to avoid intensified training stimulus, which results in an opposite effect

– such as reduced speeds and reduced power output. Thus, athletes should live in hypoxia,


but perform their training at sea level conditions in order to perform well at sea level. It

was reported that there are two groups (Levine and Stray-Gundersen, 2005). One group,

termed responders, displayed a larger increase in plasma Epo concentrations at altitude

compared to the concentrations in the second group of “non-responders”. The higher Epo

concentrations found in responders led to increased total red blood cell volume and V& O2max

values at sea level in contrast to the data from the non responders. The variation of increase

in Epo concentrations could be explained by the genetic differences of individuals (Chap-

man et al., 1998).

The third study of this thesis investigates the impact of acclimatization to moderate altitude

on exercise performance. To exclude detraining, elite athletes had to train according to the

“live high-train low” procedure (Levine and Stray-Gundersen, 2005). The outcome of this

study is particularly interesting for athletes who plan to participate in a competition at

around this altitude in order to be optimally prepared for competition day.

3.4. Aim of the project The aims of the present work were:

In mice - to establish and improve the telemetry technique during exercise as well as the transmit-

ter implantation to reduce the failure quota of the animals dramatically.

- to investigate the Htc level at which mice reach maximal exercise performance by titrat-

ing Htc levels of wt and tg6 mice.

- to examine the hypothesis that mice with chronically elevated Htc adapt better to the

excessive erythrocytosis compared to acutely NESP-injected animals.

In humans - to test the hypothesis that V& O2max and endurance performance increase after acclimati-

zation to an altitude of 2360 m.


4. Study 1: Optimizing transmitter implantation and postoperative care to improve telemetric signal in exer-cising mice

4.1. Abstract Genetically modified laboratory mice are increasingly used to study cardiovascular physi-

ology and diseases. The measurement of blood pressure in the free roaming, unanesthe-

tized and unstressed mouse is most reliably and accurately performed with telemetry.

However, implantation of telemetric transmitters can cause serious postoperative complica-

tions and death, in particular if animals with genetically induced abnormalities undergo

such major surgery. Moreover, data recording can be hampered if the measurements have

to be carried out in an exercising rodent.

Here we show an optimized telemetric transmitter implantation technique (fixation of the

transmitter body on the back of the animal with stainless steel wires) for measuring arterial

blood pressure during maximal exercise on a treadmill. This technique is used on trans-

genic mice that constitutively overexpress erythropoietin (Epo) resulting in hematocrit up

to 0.9 and on the corresponding wildtype mice. In addition, we have established a regime

for postoperative intensive care and analgesia: warmth, subcutaneous fluid therapy (600 μl)

and analgesics (flunixine 5 mg/kg bodyweight) twice per day, and offering high energy

liquid in a drinking bottle. The postoperative care was performed for 14 days and led to

substantially improved morbidity and mortality. The refined postoperative care and surgi-

cal technique were particularly successful in our genetically modified mice with severely

compromised physiological capacities.

4.2. Introduction The mouse has become the most commonly used animal model to study aspects of cardio-

vascular physiology important for drug development, safety, pharmacology and basic re-

search goals (Kramer and Kinter, 2003). Thus, the ability to record cardiovascular parame-

ters in mice has become an important tool for understanding the response of the cardiovas-

cular system in various experimental approaches (Kurtz et al., 2005). Under certain cir-

cumstances, it is necessary to investigate cardiovascular performance under different chal-

lenging body conditions. Treadmill exercise tests are a commonly used clinical approach in

human beings to induce cardiovascular stress in order to detect cardiovascular abnormali-

ties which may not be observed at rest (Sullivan and Hawthorne, 1995). Simultaneously,


metabolic parameters usually determined by indirect calorimetry are often measured during

exercise (Ba et al., 2008). In contrast to the situation in humans, cardiovascular function

and metabolic parameters have been very rarely measured directly in rodents during sub-

maximal and maximal exercise.

Radiotelemetry is the unique approach to measure cardiovascular parameters in unanesthe-

tized, freely moving small rodents using this method, physiological parameters are effi-

ciently recorded and the results are reliable and objective compared to the results obtained

using previous measuring techniques described in the literature (Butz and Davisson, 2001;

Clement et al., 1989; Feng et al., 2008; Kramer and Kinter, 2003; Kubota et al., 2006;

Whitesall et al., 2004). However, the disadvantages are the high costs of the equipment and

the need for experience in the microsurgical technique for implantation of the transmitters.

Usually, the pressure-sensing catheter tip is implanted in the thoracic aorta via the left ca-

rotid artery and the transmitter body is subcutaneously placed along the right flank (Butz

and Davisson, 2001). Thus, animals have to carry the transmitter’s weight unilaterally.

This is particularly uncomfortable during exercise, resulting in the abandonment of the

exercise test. Moreover, only weak telemetric signals or even no signals at all are obtained

during treadmill exercise due to the long distance between the transmitter (located at the

flank of the body of the mouse) and the receiver plate (placed over the treadmill). How-

ever, in spite of the advancement of the surgical technique over the last years, there is still

a high morbidity and mortality if telemetric transmitters are implanted in mice. From their

specific phenotypical characteristics, they often react by showing a severe impairment of

their general condition and symptoms of suffering as a result of the trauma of implantation

(Brown et al., 2006; Chen et al., 2005). Thus, even if the surgical procedure appears to be

successful, a quick recovery from anesthesia and intensive postoperative medical support

are necessary to prevent severe physiological aberrations and possible high death rates af-

ter transmitter implantation in genetically modified mice.

Here we show an alternative approach that places the transmitter in the midline of the

mouse’s back. The aim is to minimize the negative impact of the transmitter’s weight on

the running performance while obtaining telemetric signals in a constant and reliable man-

ner during maximum exercise. Furthermore, the positive effects of the postoperative anal-

gesia and intensive care regimen on the survival rate was demonstrated in our transgenic

mouse line termed tg6 that constitutively overexpresses human erythropoietin (Epo) cDNA

resulting in hematocrit levels of up to 0.9, and in the corresponding wild type (wt) mice.


4.3. Methods

Mouse model

The tg6 mice were generated by pronuclear microinjection of the full-length human Epo

cDNA driven by the human platelet-derived growth factor (PDGF) B-chain promoter as

described previously (Rutschitzka et al., 2000). The resulting tg6 mouse line B6D2-

TgN(PDGFBEPO)321Zbz showed increased Epo levels in plasma and brain (Soliz et al,

2007; Wiessner et al., 2001). Breeding was performed by mating hemizygous males to wt

C57BL/6 females. As expected, one half of the offspring was hemizygous for the transgene

while the other half was wt and served as controls.

Animals and housing conditions

Male animals were used at the age of 30.9 ± 5.3 days. Mice were kept in standard rodent

cages with food and water supplied ad libitum in 12:12 hour light-dark cycle. The experi-

mental protocols were approved by the Kantonales Veterinäramt Zurich and were per-

formed in accordance with the Swiss animal protection laws and institutional guidelines.

Side effects of carotid artery occlusion

To rule out that the occlusion of the carotid artery would induce ischemia in the brain or

even stroke, seven age matched (wt: n = 5, tg6: n = 2) mice underwent a preliminary ex-

periment. Anesthesia and surgical conditions as aseptic precautions, handling animals un-

der laminar flow and use of sterile instruments were set according to the procedures for

transmitter implantation (details see below). After removal of hairs and disinfection of the

anterior neck region, the skin was incised, connective tissues and muscles were prepared

and the left common carotid artery was exposed. The artery was ligated with two silk su-

tures (PERMA-Handseide 6-0, Ethicon, Norderstedt, Germany), a distance of 5mm apart

from each other (similar to the ligations applied for the catheter fixation). Connective tis-

sues and muscle layers were closed with resorbable sutures (VICRYL 6-0, Ethicon, Nor-

derstedt, Germany) and animals were allowed to recover on a warmed mat (38°C). Ani-

mals were screened daily for symptoms of cerebral ischemia and stroke by use of a pub-

lished neurological deficit score system (Huang et al., 1994).

Mice were sacrificed at different time points after occlusion of the left common carotid

artery: three mice (wt: n = 2, tg6: n = 1) at 36 hours, 2 mice (wt: n = 1, tg6: n = 1) at 72

hours and 2 mice (wt: n = 2) at 7 days. Brains were isolated, fixed in 4% neutral buffered

formalin and processed routinely for neurohistological examination (brains were cut sys-


tematically in layers to take sections at certain distances). Tissue sections (2 μm) were

stained with hematoxylin and eosin and evaluated by neuropathologists, who were blinded

to the treatment protocol.

Surgical procedures

The implantations were carried out under aseptic conditions in a laminar flow hood using

sterile equipment. Inhalation anesthesia was initiated with 7-8% and maintained with 3.5-

4% sevoflurane (Sevorane®, Abbot, Cham, Switzerland) in pure oxygen (O2). After re-

moval of hairs and disinfection of the anterior neck region, a longitudinal skin incision of 1

cm was performed, the connective tissues and muscles were prepared and the left common

carotid artery (Arteria carotis communis sinistra) was exposed. The artery was ligated with

a silk suture (PERMA-Handseide 6-0, Ethicon, Norderstedt, Germany) caudal to the bifur-

cation in the internal and external branches. A second suture was placed around the artery

at a distance of 4-6 mm below the bifurcation and the blood flow was stopped by retracting

the suture. A third suture was loosely placed around the artery between the other two.

Then, a hole was cut in the artery using fine bladed scissors and the catheter of the

TA11PA-C10 transmitter (DataSciences International, St. Paul, MN, USA) was inserted

into the vessel, while the second suture was opened to allow the tip of the catheter to be

introduced into the thoracic aorta. The catheter was then fixed with the sutures in the ar-

tery. The muscle layers and connective tissues were restored by resorbable sutures

(VICRYL 6-0, Ethicon, Norderstedt, Germany). By blunt dissection with an atraumatic

scissor a pocket underneath the skin was prepared, which reached from the right side of the

skin incision to the area between the shoulders and the midline of the thorax on the back of

the animal. The transmitter body was put between the skin and the subdermal connective

tissue at the right edge of the skin incision and was advanced in a dorsal direction until it

was located on the back of the mouse. The transmitter body was fixed there by 3 to 4 loops

of surgical stainless steel sutures (3-0 2xTS+FS; Ethicon, Norderstedt, Germany), which

were laid from one side to the other through the skin in the subdermal connective tissues

underneath the transmitter body. One to two loops of stainless steel wire were placed be-

hind the transmitter body to prevent its dislocation to the lower back. Finally, the skin inci-

sion in the neck was closed with resorbable sutures and the animals were allowed to re-

cover for 1-2 hours on a heated, water bath surface of the operating table (38°C).


Analgesia and postoperative care regimens

As analgesics either buprenorphine (Temgesic®, Essex Chemie AG, Lucerne, Switzerland)

was used in a dosage of 0.1 mg/kg body weight or flunixine (Biokema Flunixine®, Bi-

okema SA, Crissier-Lausanne, Switzerland) was applied at 5 mg/kg body weight. Analge-

sics were administered subcutaneously twice per day (i.e. every 12 hours) for 7 days; the

first injection of analgesics was performed during anesthesia for transmitter implantation.

To support fluid homeostasis during surgery, 1 ml of saline at a temperature of 36°C was

injected intraperitoneally after inducing anesthesia.

Three different postoperative care regimens were compared

In the first group, mice were treated with buprenorphine (wt: n = 2, tg6: n = 2); in the sec-

ond group flunixine was used as analgesic (wt: n = 1, tg6: n = 4).

A third group received flunixine as an analgesic for 7 days and an additional fluid therapy

of 300 μl glucose (5%) and 300 μl saline (0.9%), injected subcutaneously twice per day for

14 days (wt: n = 47, tg6: n = 48). Before injection, the mice were weighed. All analgesic

agents including glucose liquid and saline were heated up to body temperature before in-

jection. In the third group, all animals’ cages were kept on a heating mat during the whole

postoperative period of 2 weeks. In addition, only the third group had free access to glu-

cose (15%), offered in a second water bottle, and to high-energy, wet food (Solid Drink®-

Energy, Triple A Trading, Tiel, Netherlands). The high-energy food and glucose-

containing water bottles were given in the animals’ cages from 2 to 3 days before surgery

until 2 weeks afterwards. O2 was introduced via a tube into the cages (Fig. 4.1). After this

period, the implanted animals were transferred to a non-implanted ovarectomized Crl:CD

(ICR) female.

Fig. 4.1. Cages kept on the heat-ing pad and perfused with poor oxygen via red as well as white tub-ings.


Telemetric signal verification during maximal exercise

The telemetric signal was measured during maximal exercise using a Simplex II metabolic

rodent treadmill, fitted with Oxymax gas analyzer (Columbus Instruments, Columbus, OH,

USA). For the maximal incremental exercise test, mice were placed in the exercise cham-

ber and allowed to equilibrate for 30 min. Treadmill activity was initiated at 2.5 m/min and

0° inclination for 10 min and then, increased by 2.5 m/min and 2.5° every 3 min thereafter

until exhaustion. Mice were gently encouraged to run for as long as possible by the use of a

mild electric grid at the end of the treadmill (0.2 mA, pulse 200 ms, 1 Hz). Exhaustion was

defined as the inability to continue regular treadmill running despite a repeated electric

stimulus to the mice.

Statistics

All data were analyzed using StatView software (Version 4.57, Abacus Concepts, Berke-

ley, California, USA). Results are expressed as means ± standard deviation (SD).

4.4. Results All animals scored 0 (i.e. showed no behavioral signs of neurological deficits) after liga-

tion of the left common carotid artery. Systematic histological examination of brains con-

firmed no abnormalities that would hint at ischemia or even infarction of the brain. A total

number of 50 wt and 54 tg6 mice were used with a body weight between 18g and 27g

(mean: wt = 21.6 ± 1.6g; tg6 = 22.2 ± 2.1g) on implantation day (Fig. 4.2). Mice were

treated either with buprenorphine (first group) or flunixine (second group). In addition to

flunixine, third group received a warmth fluid therapy and had free access to high energy

liquid. No animal from the first (wt: n = 2; tg6: n = 2) or second (wt: n = 1; tg6: n = 4)

groups reached the final time point between 7 and 13 weeks, as the mice were euthanized

due to complications because they exhibited hypothermia, apathy, exsiccosis or an obvi-

ously moribund state (Fig. 4.3 a, b, c). In contrast to animals of the first and second group,

no animal of the thrid group (wt: n = 47 wt; tg6: n= 48) showed such complications. But,

three wt mice were euthanized between 23 and 34 days after implantation as these animals

were fighting with their female companions. Four tg6 mice showed hematoma in the right

neck and shoulder area at 1-2 hours after surgery. Three of these animals could be rescued

by re-opening the wound and removing the blood clots, while additional saline (1 ml) was

injected intraperitoneally. Despite removing the haematoma, one of these animals died 6

days after the implantation from repeated bleeding from the implantation site. Two tg6

mice were euthanized 23 and 41 days after the implantation because the telemetric signals


hinted at signs of blood clot formation at the tip of the catheter. This was confirmed during

necropsy. The thrombus formation was supposed to be a consequence of the repeated use

of the transmitters. Despite these limitations, the methodology was rather successful. Fig.

4.4 shows the relationship between establishing and refining the postoperative care regi-

mens and the survival time. The increasing experience in postoperative care was associated

with survival times of up to 100 days. After that time, survival time remained unaltered.

body

wei

ght o

f im

plan

tatio

n[g

]

survival time [day(s)]

15

20

25

30

0 10 20 30 40 50 60

tg6 with buprenorphin (n = 2)wt with buprenorphin (n = 2)tg6 with flunixin (n = 4)wt with flunixin (n = 1)tg6 with flunixin and glucose/saline (n = 48)wt with flunixin and glucose/saline (n = 47)

body

wei

ght o

f im

plan

tatio

n[g

]


15

20

25

30

0 10 20 30 40 50 60


Fig. 4.2. Correlation between weight at implantation and survival time. Each value rep-

resents a single animal.

In all animals, the transmitter body was fixed in the midline of the mouse’s back. In two

animals from the third group, the transmitter body had to be re-fixed one day after the im-

plantation because it had turned and moved to the upper neck region, where it could ham-

per the movement of the mouse’s head. In general, the mice showed no signs of reduced

physical activity or restricted head movement. In contrast to the commonly used fixation

technique of the transmitter body, the modified fixation method improved the telemetric

signal, as the distance between the telemetric receiver plate and the transmitter body was

shortened (Fig 4.5).


final

bod

yw

eigh

t[g]

final

bod

yw

eigh

t[g]

all animalsfin

al b

ody

wei

ght[

g]


15

20

25

30

0 10 20 30 40 50 60


15

20

25

30

0 10 20 30 40 50 60

wt animals

wt with flunixine and glucose/saline in experiment (n = 44)wt with flunixin and glucose/saline early sacrificed (n = 3)wt with flunixin early sacrificed (n = 1)wt with buprenorphin early sacrificed (n = 2)

15

20

25

30

0 10 20 30 40 50 60

tg6 animals

tg6 with flunixine and glucose/saline in experiment (n = 45) tg6 with flunixin and glucose/saline early sacrificed (n = 3)tg6 with flunixin early sacrificed (n = 4)tg6 with buprenorphin early sacrificed (n = 2)

(a)

(b)

(c)

final

bod

yw

eigh

t[g]

final

bod

yw

eigh

t[g]

all animalsfin

al b

ody

wei

ght[

g]


15

20

25

30

0 10 20 30 40 50 60


15

20

25

30

0 10 20 30 40 50 60

wt animals



15

20

25

30

0 10 20 30 40 50 60

tg6 animals



(a)

(b)

(c)

Fig. 4.3. Correlation between final body weight and survival time of (a) all animals, (b) wt mice, and (c) tg6 mice. Each value represents a single animal.


surv

ival

time

[day

(s)]

Progression in the refinement of the postoperative care regime [day(s)]


0

10

20

30

40

50

60

0 50 100 150 200 250 300

surv

ival

time

[day

(s)]

Progression in the refinement of the postoperative care regime [day(s)]


0

10

20

30

40

50

60

0 50 100 150 200 250 300

Fig. 4.4. Correlation between the survival time and the progression in the refinement of

the postoperative care regime. Each value represents a single animal.

Fig 4.5. Enclosed treadmill and telemetry receiver plate


4.5. Discussion The approach of inducing a genetically modified mouse model of compromised phenotype

to perform maximum exercise in order to obtain real-time blood pressure data was realized

by elaborating refined methods for telemetric transmitter implantation and postoperative

intensive care.

When telemetric transmitters for measuring blood pressure in mice first became available,

a technique of implantation was developed, in which the catheter was implanted in the ab-

dominal aorta and the transmitter body was located in the abdominal cavity (Kramer et al.,

2000; Mills et al., 2000; Van Vliet et al., 2000). This implantation method was of limited

success, because it induced thrombosis and embolia at high rates leading to the death of

more than half of the mice implanted within 2 after days of surgery. Therefore, implanta-

tion of the catheter in the thoracic aorta arch via the common carotid artery was soon pre-

ferred. Because of the limited length of the catheter, the transmitter body must be placed

under the skin of the back, from where it moved in most cases to the right body wall of the

mouse (Butz and Davisson, 2001; Carlson and Wyss, 2000). Thus, fixation of the transmit-

ter body with sutures on the muscles of the animals’ backs was proposed, but induced dif-

ficulties and additional injury, because a second skin incision at the shoulder region was

necessary in addition to the one in the neck (that was used for placement of the catheter).

Thus, it was generally preferred that the transmitter body was introduced via the wound to

the right flank, without any further fixation. This technique, in which the transmitter body

hung at the lateral body wall of the mouse, seemed feasible for recordings at rest and dur-

ing short-time exercise, e.g. for 15 minutes of treadmill running (Davis et al., 2003). How-

ever, we found in a previous pilot experiment (not shown), that transmitter signals were

weak, undetectable or disturbed by artifacts during incremental exercise to exhaustion on

the treadmill. Moreover, the lateral placement of the transmitter body, which lay in front of

the right hind leg, seemed to hamper movement and consequently the exercise perform-

ance of the animal. Thus, we put the transmitter body through the wound in the neck under

the skin of the back approximately between the shoulders. The stainless wire loops be-

tween the transmitter and the connective tissue fixed it in the midline over the axis. One or

two loops behind the transmitter body prevented it from moving to the lower back and

meant that the catheter tip could be withdrawn from the arotic arch. The drawbacks of this

method were seen only in the genetically modified mice, in which the tissue damage from

suturing with the wires or preparing the pocket for the transmitter injured subcutaneous

vessels, which led to prolonged bleeding due to the impaired blood clotting properties of


these transgenic mice. As a consequence, prominent haematoma were exhibited in the area

of the right shoulder and the neck 1 to 2 hours after surgery had been completed. Remov-

ing the haematoma by re-opening the wound in the neck and using an intraperitoneal injec-

tion of additional saline could rescue three of these mice, while one died at six days fol-

lowing implantation from repeated bleeding and hypovolumenia. Apart from this technical

aspect, three wt mice were euthanized before reaching the final time point because of seri-

ous injuries after fighting with their female cage mates. In these cases, there was no direct

relationship with the new transmitter body implantation technique. Taken together, the

fixation of the transmitter in the back of the mouse allowed us to measure reliable signals

at any time point during maximum exercise.

The use of genetically modified mice with phenotypes that hint at deficiencies in physio-

logical and bodily adaption capacities is increasing. Such specific disabilities in the tg6

mice were particularly impairing the outcome of our surgical efforts at the beginning of the

study. This observation is supported by the finding that the final body weights of wt mice

from the first and second groups were not different from those from the third group. In

contrast, the genetically modified mice showed clear symptoms of exsiccosis, hypother-

mia, and energy deficits, which led to severely depressed general condition and overall

appearance, in particular at 2-9 days following surgery. From this, we took supportive

postoperative means to improve the condition of animals, which led to a high survival rate.

We could not find a relationship between body weight and survival rate, which was sug-

gested by others (Johnston et al., 2007). Also, we suppose that administering of warmth for

a prolonged period of 7 days after implantation was a beneficial intervention, because this

would save energy for the animal, as has been proposed by others (Van Vliet et al., 2006).

In addition, we consider that the injection of warmed fluid at 12 hour intervals for seven

days was a key intervention supporting survival. Indeed, this intervention implies more

stress for the animals but the positive effect of this therapy appears to outbalance. Finally,

we suggest to provide energy in the form of glucose 15% as the animals consumed this

liquid in high amounts postoperatively. With these methods, we could overcome the prob-

lems which the phenotype of tg6 mice presented and which are most probably the reason

for the poor survival rate at the beginning of our study. The postoperative intensive care

and the analgesic regimens described in this study may be useful for others who are con-

fronted with similar difficulties when implanting probes in genetically modified mice.


Chronic side effects of telemetric transmitter implantation

The effect of the occlusion of one carotid artery (caused by inserting the catheter of a tele-

metric transmitter) on arterial blood pressure in general was described in a study of Carlson

and Wyss in 2000. With simultaneous measurements in the femoral artery, the authors

found a slight elevation of the arterial blood pressure, which returned to baseline within 30

seconds and thus can be estimated as an acute reaction that has no influence on the long-

term, chronic measurements after the recovery phase from transmitter implantation (Carl-

son and Wyss, 2000). However, it is known that mice show different blood flow and vessel

conformation depending on the strain (Van Vliet et al., 2006). To rule out that ligation of

the left common carotid artery induced any ischemia or even infarction in the brain, in a

preliminary experiment this vessel was occluded in age matched wt and tg6 mice. In these

mice, no symptoms of neurological deficits were detected. Systematic histological exami-

nation by experts in neuropathology showed no aberrations in the brain at various time

points due to left common carotid artery occlusion, confirming no detectable influence of

this manipulation on the experimental outcome in wt C57BL/6 and tg6 mice.

The present findings indicate that the placement of the transmitter body between the shoul-

ders of the mice is an important feature to improve the detection and recording of the tele-

metric signals, allowing monitor the cardiovascular function in the exercising rodent. How-

ever, the difficulties of surgical procedure remain challenging. The small size and delicate

nature of the arteries of the mouse require excellent hand-eye coordination and steady

hands in order to catheterize the vessel successfully. Intensive postoperative care and anal-

gesia improves the survival and body condition after implantation of probes, which is par-

ticularly important in genetically modified mice with impaired bodily capacities.


5. Study 2: Optimal hematocrit for maximal exercise per-formance in erythropoietin-treated mice

5.1. Abstract This study was performed to investigate the impact of varying hematocrit (Htc) levels on

endurance performance and the cardiovascular system using the telemetry technique.

Therefore, two strategies were combined. Htc levels of wild type (wt) mice were acutely

elevated by applying novel erythropoiesis stimulating protein (NESP; wtNESP). On the

other hand, Htc levels of our transgenic mice line (tg6) that reached Htc levels of up to 0.9

due to constitutive overexpression of erythropoietin (Epo) were reduced by the hemolysis-

inducing compound phenylhydrazine (PHZ; tg6PHZ). Highest maximal oxygen (O2) up-

take (V& O2max) and best time to exhaustion were reached at Htc values of 0.58 and 0.57 for

wtNESP, and 0.68 and 0.66 for tg6PHZ, respectively. Interestingly, the closest correlation

between V& O2max and whole body hemoglobin was only found in an Htc range from 0.4 to

0.55 in wtNESP. Maximal stroke volume was reached at Htc values of 0.58 for wtNESP

and 0.68 for tgPHZ, whereas maximal heart rate was unaffected at varying Htc levels.

Blood viscosity correlated with V& O2max. In conclusion, tg6PHZ adapted better to varying

Htc values than wtNESP did. Furthermore, the close relationship between V& O2max and

whole blood hemoglobin is only valid in the physiologically occuring Htc range and when

Htc levels were increased by NESP-injection.

5.2. Introduction The rate of maximal oxygen (O2) uptake (V& O2max) is mainly limited by the O2 delivery to

the skeletal muscle (Di Prampero and Ferreti, 1990; Turner et al., 1993). Accordingly,

when hemoglobin concentration [Hb] is increased by blood reinfusion or recombinant hu-

man erythropoietin (rhEpo) administration, V& O2max increases (Ekblom and Berglund,

1991; Robertson et al. 1988). In line with this, V& O2max is impaired, when [Hb] is acutely

reduced isovolemically (Lundby et al., 2008b, Woodson et al., 1978).

Although the role of [Hb] as an O2 transport carrier in circulating blood systems seems to

be clear, its impact on the endurance performance over a wide hematocrit (Htc) range is

still poorly understood. Increasing [Hb] results in elevated Htc levels that lead to an expo-

nential rise in blood viscosity and are associated with higher peripheral vascular resistance

in the tissue. Acutely increasing Htc levels are inversely related to the cardiac output (Cro-

well and Smith, 1967; Stone et al., 1968; Richardson and Guyton, 1959), which may theo-


retically reduce V& O2max (Connes et al., 2006). Surprisingly, cardiac output remains un-

changed when Htc levels are chronically elevated (Vogel, 2003; Wagner et al., 2001). This

was observed in our transgenic mouse line termed tg6 that due to constitutively overex-

pression of human erythropoietin (Epo) cDNA reached Htc values of 0.8 to 0.9, neverthe-

less had a reduced exercise performance (Heinicke et al., 2006; Wagner et al., 2001). En-

hanced erythropoiesis was also found in endurance athletes that due to a mutation in Epo

receptor reached Htc levels of up to 0.68 (Juvonen et al., 1991). This endurance athlete

won several Olympic gold medals and showed no signs of obvious organ failures. These

findings indicate that some adaptive mechanisms to chronic excessive erythrocytosis exist.

However, some authors suggest that there is an optimal Htc value for maximal blood O2

transport capacity to the tissue due to counteracting the effect of increased blood O2 bind-

ing activity and viscosity (Crowell and Smith, 1968; Gaehtgens et al., 1979; Guyton and

Richardson; 1961; Villafuerte et al., 2003).

In higher vertebrates, Gaehtgens and co-workers (1979) investigated the effect of varying

Htc levels in isolated canine gastrocnemius muscle during isotonic rhythmic exercise. A

plateau of maximal V& O2 and contractile power in this ex-vivo set-up was found at Htc lev-

els of between 0.4 and 0.7 with a slight tendency between 0.5 and 0.7. Below 0.4 and

above 0.7, both parameters decreased. The problem of this investigation is that optimal Htc

values may vary under different circumstances due to non-newtonian behavior of the

blood. Factors affecting this variation include the species, the organs involved, and

whether the organism is resting or exercising (Connes et al., 2004, Gaehtgens et al., 1979;

Kusunoki et al., 1981; Lee et al., 1994; Tu et al., 1997; Villafuerte et al., 2003). Thus, these

results do not necessarily reflect the situation in exercising mammals and humans.

The present study tested the hypothesis if there is an optimal Htc value that allows

maximal systemic endurance performance. These levels might not be identical in subjects

having an acutely or chronically increased Htc. Thus, we hypothesize that subjects suffer-

ing from excessive erythrocytosis can adapt better to varying Htc levels than subjects hav-

ing acutely increased Htc. For this purpose, wild typ (wt) mice were injected or not with

the novel erythropoiesis stimulating protein (NESP). On the other hand, tg6 mice were

treated or not with the hemolysis-inducing compound phenylhydrazine (PHZ). At the end

of a period of 4 and 3 weeks, respectively, metabolic and cardiovascular measurements

were performed at rest and during endurance performance, while whole blood analysis

including rheology was carried out at rest.


5.3. Materials and Methods

Mouse models

The tg6 mice line was generated as described previously (Rutschizka et al., 2000). Com-

pared to wt control, the tg6 mouse line had a 10 to 12-fold increase in plasma Epo-levels,

resulting in Htc levels of up to 0.9 (Bogdanova et al. 2007; Rutschizka et al., 2000; Vogel

et al., 2003). About half of the offspring was hemizygous for the transgene and was used

for the hemolysis-inducing experiments, while the other half was used as wt for the hemo-

concentration experiments. Mice were 12 weeks old during the first exercise test (Tab.

5.1). In total, 41 wt- and 40 tg6-mice were investigated. No weight loss occurred during

the study period. Mice were kept in standard rodent cages (T3) with food and water sup-

plied ad libitum in 12:12-hour light-dark cycle. The experimental protocols were approved

by the Kantonales Veterinäramt Zürich and were performed in accordance with the Swiss

animal protection laws and institutional guidelines.

Age (weeks) 1 2 3 4 6 7 8 9 10 11 125

tg6/tg6PHZ

wt/wtNESP

IS R EP

AVE

TD

PPPE

AA

RINV

TD

AAA

NNNNC C C C

Age (weeks) 1 2 3 4 6 7 8 9 10 11 125

tg6/tg6PHZ

wt/wtNESP

IS R EP

AVE

TD

PPPE

AA

RINV

TD

AAA

NNNNC C C C

Fig. 5.1. Timeline of animal age for the experimental set-up illustrated. S: Splenectomy;

I: Implantation of telemetric blood pressure transmitter; R: Postoperative re-covery; N: NESP injection and blood sampling; C: NESP injection; P: Phenyl-hydrazine injection and blood sampling; E: Phenylhydrazine injection; A: Treadmill adaptation; V: Incremental exercise test; T: Constant workload test; D: Terminal measurements.

Experimental design

Fig. 5.1 shows the experimental design. At an age of 3 weeks, only tg6 mice were splenec-

tomised to keep Htc levels low, since extramedullary erythropoiesis mainly occurs in the

spleen (Vogel et al., 2003). One week later, telemetric blood pressure transmitters were

implanted in 20 wt and 19 tg6 mice, which were 4 weeks old. In the remaining animals,

dummy transmitters were implanted (wt: n = 21; tg6: n = 21). Adjustments of the Htc lev-


els were started in 8 and 9 week old animals respectively. At an age of 12 weeks, the main

experiments were conducted including incremental as well as constant workload exercise

tests (see below) followed by measurements of the arterial O2 saturation (SaO2), [Hb],

Htc, blood viscosity, plasma and blood volume. To exclude the impact of circadian rhythm

all measurements were performed at the same time of day.

Htc adjustments wt + wtNESP

NESP (Aranesp, Darbepoietin Alpha, Amgen Europe B.V., Breda, Netherlands) of 3.125

μg/kg to 12.5 μg/kg (wtNESP) or saline 0.9% was subcutaneously (s.c.) injected twice a

week to increase and maintain the Htc levels. To this end, the animals were anaesthetized

with 7-8% sevoflurane (SecoraneTM, Abbot, Cham, Switzerland) in pure O2 to avoid un-

controlled moving of the blood pressure catheter (Arras et al., 2001). After the injections

blood sample (10 μl) was taken from the tail vein.

tg6 + tg6PHZ

Htc was adjusted to a range between 0.3 and 0.9 by s.c. administration of freshly prepared

PHZ that causes chemical hemolysis (Lim et al., 1998; Vannucchi et al., 2001). To this

end, PHZ hydrochloride (Sigma, P6926, Switzerland) was prepared as previously de-

scribed (Lim et al., 1998). Animals received two PHZ injections (0.125 to 1.2 mg/10g

body weight; tgPHZ) or saline 0.9% (tg6) spaced 2 days after the first injection to decrease

the Htc. To maintain Htc levels PHZ was injected at a concentration between 0.065 and 0.5

mg/10g body weight every third day. Mice were anaesthetized and blood samples were

taken after as described above.

Surgical procedures: Splenectomy (tg6 mice only) and implantation of telemetric transmitter.

Inhalation anesthesia was induced as described above. Anesthesia was maintained with

3.5-4% sevoflurane (Arras et al., 2001). Preliminary experiments showed that decreased

Htc levels in our tg6 mice recovered within days after the PHZ injection. Compared to wt,

tg6 mice showed that massive extramedullary erythropoiesis occurred in the spleen (Gass-

mann et al. 2008; Vogel et al, 2003). To maintain constant Htc levels, the spleens of 3

week old tg6 males were removed by left side-abdominal laparatomy as described previ-

ously (Vogel et al, 2003). One week later, blood pressure sensors were implanted in tg6

and wt mice of the same age. After shaving and disinfecting the neck, the left common

carotid artery was isolated. The transmitter’s catheter was inserted into the artery and


pushed forward until the tip was just inside the thoracic aorta. The transmitter body was

fixed under the skin. Splenectomy and blood pressure transmitter implantation were carried

out under aseptic conditions. Mice were allowed to recover for two weeks. Measurements

were performed using a TA11PA-C10 transmitter (DataSciences International, St. Paul,

MN, USA). Data were generated by the Dataquest A.R.T 3.0 software (DataSciences In-

ternational, St. Paul, MN, USA).

Measurements Exercise tests

The exercise tests were performed on an Instrument Simplex II metabolic rodent treadmill

(Columbus Instruments, Columbus, OH, USA) connected to an Oxymax gas analyzer (Co-

lumbus Instruments, Columbus, OH, USA). This system enables the measurement of V& O2

and carbon dioxide production (V& CO2), thus indirect calorimetry. Respiratory exchange

ratio (RER) was calculated as V& CO2/ V& O2. Before performing each exercise test, the gas

analyzer was calibrated with a high precision gas mixture. Mice were gently encouraged to

run on the belt until exhaustion with the use of a mild electric shock from the shock grid at

the end of the treadmill (0.2 mA, pulse 200 ms, 1 Hz).

To determine V& O2max, systolic blood pressure, mean arterial blood pressure and heart rate

were telemetrically monitored at rest and during exercise. Cardiac output depends on the

stroke volume and heart rate. Unfortunately, there is no device available to measure the

cardiac output telemetrically. The advantage of an implantable transmitter is the ability to

monitor cardiovascular parameters in conscious freely moving animals directly. But stroke

volume has to be assessed indirectly using O2 pulse, which correlates closely with the

stroke volume during effort, at least in healthy humans (Bhambhani, 1995; Crisafulli et al.,

2007). O2 pulse was calculated from division of V& O2max by heart rate. As an indicator of

myocardial V& O2 rate pressure product was calculated from the multiplication of heart rate

and systolic blood pressure.

10 μl blood samples were taken three hours before initiation of the exercise test from the

tail vein for the Htc determination. Mice were placed on individual treadmill lines. After

acclimatization for at least one hour, basal heart rate, blood pressure, V& O2 and V& CO2 were

monitored over the last five minutes of this period. Mice began running at 2.5 m/min and

0° inclination for 10 min. The intensity was then increased by 2.5 m/min and 2.5° every 3

min thereafter until exhaustion. Exhaustion was defined as the inability to continue regular

treadmill running despite the repeated stimulus to the mice. V& O2max was achieved when


V& O2 did not increase in spite of an increase in work load. For reported values at maximal

exercise, 1-min averages of blood pressure and heart rate were taken during last minute of

exercise, while for V& O2max the highest 1-min interval was considered. From the maximal

value rate, pressure product and O2 pulse were calculated. After 24 hours at rest, all mice

performed a constant workload exercise test to exhaustion. The workload was set to 80%

of the maximal attained workload of the incremental exercise set. Before performing the

time to exhaustion test, blood samples were taken at rest and the animal warmed up for 10

min at 20% followed by an additional 10 min at 40% of the maximal attained power output

of the V& O2max -test.

Terminal measurements

The day after performing the constant work load test, mice were anesthetized with a s.c.

injection of a mixture of 100 mg/kg ketamine (Ketasol-100TM, Dr. Graub, Bern, Switzer-

land), 20 mg/kg xylazine (RompunTM, Bayer, Leverkusen, Germany) and 3 mg/kg acepro-

mazine (SedalinTM, Chassot, Belp, Bern, Switzerland). Catheters were introduced into the

left femoral artery and vein. Arterial blood was collected in a heparinised capillary 35 min

after the injection of the anesthesia and the arterial acid-base status and SaO2 was immedi-

ately measured. Plasma volume and whole blood volume were measured by the injection

of Evans blue directly after blood sampling for the arterial acid-base status and SaO2 de-

termination. Twenty minutes later, animals were bled to death to measure blood viscosity,

blood volume, plasma volume, Htc, whole body haemoglobin and [Hb].

Blood analysis

Htc was measured in duplicate of heparinized blood using micro centrifuge (Autokrit II,

Pharmap, Geneva, Switzerland). [Hb] were determined with the automatic blood analyzer

Abbott Cell Dyn 3500 (Abbott Diagnostic Division CA, USA). Whole body hemoglobin

was calculated from the [Hb] and blood volume. SaO2 was evaluated by a gas analyzer

(AHVL Compact 3, AHL List, Graz, Austria).

Quantification of whole blood volume has been previously described (Vogel at al., 2003).

Briefly, 10 μl Evans blue solution (1% in saline) was injected into a femoral vein catheter.

Twenty minutes after the injection, 10 μl samples of blood were drawn into heparinised

capillaries. Absorbance of the dye in the plasma volume was read at 620 nm with a Nano-

Drop spectrometer (NanoDrop products, Wilmington, USA). Evans blue concentrations

were derived from a calibration curve and used to calculate plasma volume. Blood volume

was calculated from plasma volume and Htc.


Blood viscosity was measured in heparinised blood samples with a rotation viscoimeter

DV-II+PRO (BROOKFIELD, Brookfield Engineering Laboratories, Middleboro, MA,

USA) using Rheocalc software (BROOKFLIELD, Brookfield Engineering Laboratories,

Middleboro, MA, USA) as previously described (Vogel et al., 2003). But, due to the non-

newton fluid characteristics of blood, only blood temperature at 37º C and shear rates of

450 s-1 were compared.

Statistics


ley, California, USA). The relationship between the two parameters was analyzed with

linear or polynomial regression. Significances were performed by a one-way analysis of

variance (ANOVA). Results are expressed as mean ± standard deviation (SD). Statistical

difference was set at P < 0.05.

5.4. Results Male wt and tg6 mice were about 8 and 9 weeks old, respectively, at the beginning of the

corresponding injections and showed differences in Htc levels (Tab. 5.1). While wt males

had an Htc of 0.46 ± 0.03, the Epo overexpressing transgenic tg6 males developed exces-

sive erythrocytosis, and showed Htc values of 0.78 ± 0.06. No differences in resting mean

arterial blood pressure, heart rate, V& O2 or RER were observed between wt and tg6 at the

beginning of the incremental exercise test.

Type Weight Age Htc V& O2 RER Mean arte-rial blood pressure

Heart rate

[g] [Days] [ml kg-1min-1] [mmHg] [beats·min-1]

wt/wtNESP 25.0 ± 1.5 84.1 ± 5.2 0.46 ± 0.03 48.1 ± 2.7 0.81 ± 0.01 108.3 ± 6.7 513.8 ± 38.8

tg6/tg6PHZ 24.9 ± 1.6 86.9 ± 4.0 0.78 ± 0.06 50.1 ± 3.5 0.82 ± 0.01 103.4 ± 14.7 525.6 ± 55.0

Tab 5.1. Weight, age, and cardiac and metabolic parameters at baseline before perform-ing an incremental exercise test. Note that the body weight was measured prior to hematocrit (Htc) manipulation. Values represent means ± SD. V& O2: O2 up-take; RER: Respiratory exchange ratio.


The impact of Htc manipulation

The results of wt/wtNESP and tg6/tg6PHZ after about 4 and 3 weeks of treatment, respec-

tively, are depicted in Fig. 5.2. It shows [Hb], SaO2, plasma and blood volume in relation

to the Htc value for each individual mouse used in this study. Note that the blood samples

analyzed were taken from anaesthetized animals prior to euthanizing them.

0.9

Htc

0.9

Htc

y = 30.208x + 0.266 R2 = 0.9256; P < 0.0001

y = 26.283x + 2.8308R2 = 0.9071; P < 0.0001

5

(a) [Hb]

10

15

20

25

30

[Hb]

[g·d

l-1]

0.3 0.80.70.60.50.4

□□

□ □ □□□ □

□□□

△

△

△ △△ △△

60

(b) SaO2

70

80

90

100

Sao 2

[%]

0.3 0.80.70.60.50.4

△

□□

□

□

□□

□

□

□

□□

△

△△

△△

△

■ wtNESP▲ tg6PHZ

□ wt△ tg6

Normal physiological

range

0.9

Htc

0.9

Htc

y = 30.208x + 0.266 R2 = 0.9256; P < 0.0001

y = 26.283x + 2.8308R2 = 0.9071; P < 0.0001

5

(a) [Hb]

10

15

20

25

30

[Hb]

[g·d

l-1]

0.3 0.80.70.60.50.4

□□

□ □ □□□ □

□□□

△

△

△ △△ △△

60

(b) SaO2

70

80

90

100

Sao 2

[%]

0.3 0.80.70.60.50.4

△

□□

□

□

□□

□

□

□

□□

△

△△

△△

△


□ wt△ tg6■ wtNESP▲ tg6PHZ

□ wt△ tg6


range

Fig. 5.2.1. Relationship between hematocrit (Htc) and (a) hemoglobin concentration

([Hb]) as well as (b) arterial oxygen saturation (SaO2) during terminal determi-nation in wtNESP and tg6PHZ mice. Values represent individual values. Re-gression plot of wt/wtNESP: ⋅⋅⋅⋅⋅ and tg6/tg6PHZ: ⎯.


0.9

Htc

0.9

Htc

□□

y = 1002.4x2 - 799.69x + 257.15 R2 = 0.87; P < 0.0001

y = 436.25x2 + 200.17x + 105.43R2 = 8529; P < 0.0001

0

(b) Blood volume

100

200

300

400

500

Blo

odvo

lum

e[m

l·kg-1

]

0.3 0.80.70.60.50.4

0

(a) Plasma volume

20

40

60

80

100

Plas

ma

volu

me

[ml·k

g-1]

0.3 0.80.70.60.50.4

□

△□

□

□ □□

□□

□□□ △△ △

△ △ △

△ △△

△

△△

△

□□

□□

□□□□ □


range


□ wt△ tg6

0.9

Htc

0.9

Htc

□□

y = 1002.4x2 - 799.69x + 257.15 R2 = 0.87; P < 0.0001

y = 436.25x2 + 200.17x + 105.43R2 = 8529; P < 0.0001

y = 1002.4x2 - 799.69x + 257.15 R2 = 0.87; P < 0.0001

y = 436.25x2 + 200.17x + 105.43R2 = 8529; P < 0.0001

0

(b) Blood volume

100

200

300

400

500

Blo

odvo

lum

e[m

l·kg-1

]

0.3 0.80.70.60.50.4

0

(a) Plasma volume

20

40

60

80

100

Plas

ma

volu

me

[ml·k

g-1]

0.3 0.80.70.60.50.4

□

△□

□

□ □□

□□

□□□ △△ △

△ △ △

△ △△

△

△△

△

□□

□□

□□□□ □


range



□ wt△ tg6

Fig. 5.2.2. Relationship between hematocrit (Htc) and (a) plasma volume as well as (b)

whole blood volume during terminal determination in wtNESP and tg6PHZ mice. Values represent individual values. Regression plot of wt/wtNESP: ⋅⋅⋅⋅⋅ and tg6/tg6PHZ: ⎯.

As shown in Fig. 5.2.1a, there was a linear increase in [Hb] as Htc values increased in all

wt and tg6 mice used. While SaO2 (Fig. 5.2.1b) and plasma volume (Fig. 5.2.2a) did not

significantly change in either treated or untreated wt or tg6 mice, elevated Htc levels were

paralleled by dramatically increased blood volumes (Fig. 5.2.2b). As reflected by a calcu-

lated (degree two) polynomial equation, alterations at lower Htc values had a lower impact


on blood volume changes than was the case at higher Htc levels. Indeed, the increment of

blood volume at Htc levels from 0.4 to 0.5 was about 19 ml/kg for wtNESP and 10 ml/kg

for tgPHZ, while the increment between 0.6 and 0.7 was about 37 and 50 ml/kg, respec-

tively.

Optimal Htc for maximal endurance performance

Endurance performance consists of the product of the subject’s V& O2max and exercise dura-

tion at a certain percentage of V& O2max that the subject can undertake until exhaustion (Bas-

sett and Howley, 2000). Thus, to investigate the impact of varying Htc levels on endurance

performance, individual data of V& O2max (Fig. 5.3.1) and time to exhaustion (Fig. 5.3.2)

were plotted against Htc.

VO2max·

y = -252.78x2 + 292.75x + 59.812R2 = 0.7319; P < 0.0001

y = -337.58x2 + 461.8x - 14.515R2 = 0.7771; P < 0.0001

△△△△

□

60

80

100

120

140

160

60

80

100

120

140

160

0.3 0.90.80.70.60.50.4Htc

VO

2max

[ml·k

g-1·m

in-1

]

·

Max

. wtN

ESP

Max

. tg6

PHZ

□ □□□□

□□

□□

□ △△ △

△


□ wt△ tg6

0.3 0.90.80.70.60.50.4

Htc

Max

. wtN

ESP

Max

. tg6

PHZ

VO

2max

[ml·k

g-1·m

in-1

]

·

VO2max·

y = -252.78x2 + 292.75x + 59.812R2 = 0.7319; P < 0.0001

y = -337.58x2 + 461.8x - 14.515R2 = 0.7771; P < 0.0001

△△△△

□

60

80

100

120

140

160

60

80

100

120

140

160

0.3 0.90.80.70.60.50.4Htc

0.3 0.90.80.70.60.50.4Htc

VO

2max

[ml·k

g-1·m

in-1

]

·

Max

. wtN

ESP

Max

. tg6

PHZ

□ □□□□

□□

□□

□ △△ △

△



□ wt△ tg6

0.3 0.90.80.70.60.50.4

Htc

Max

. wtN

ESP

Max

. tg6

PHZ

VO

2max

[ml·k

g-1·m

in-1

]

·

Fig. 5.3.1. Relationship between hematocrit (Htc) and maximal oxygen uptake (V& O2max)

in wtNESP and tg6PHZ mice. Values represent individual values. Regression plot of wt/wtNESP: ⋅⋅⋅⋅⋅ and tg6/tg6PHZ: ⎯.


y = -575.87x2 + 764.75x - 170.32R2 = 0.7719; P < 0.0001

y = -1225.5x2 + 1389x - 298.72R2 = 0.7691; P < 0.0001

Time to exhaustion

0

30

60

90

120

□

△

△

△

△ △△△

△

□

□ □

□

□

□

□

□

□

□

Tim

e to

exh

aust

ion

[min

]

0

30

60

90

120

0.3 0.90.80.70.60.50.4

Htc

Max

. wtN

ESP

Max

. tg6

PHZ


□ wt△ tg6

0.3 0.90.80.70.60.50.4

Htc

Tim

e to

exh

aust

ion

[min

]

Max

. wtN

ESP

Max

. tg6

PHZ

y = -575.87x2 + 764.75x - 170.32R2 = 0.7719; P < 0.0001

y = -1225.5x2 + 1389x - 298.72R2 = 0.7691; P < 0.0001

Time to exhaustion

0

30

60

90

120

□

△

△

△

△ △△△

△

□

□ □

□

□

□

□

□

□

□

Tim

e to

exh

aust

ion

[min

]

0

30

60

90

120

0.3 0.90.80.70.60.50.4

Htc

Max

. wtN

ESP

Max

. tg6

PHZ



□ wt△ tg6

0.3 0.90.80.70.60.50.4

Htc

Tim

e to

exh

aust

ion

[min

]

Max

. wtN

ESP

Max

. tg6

PHZ

Fig. 5.3.2. Relationship between hematocrit (Htc) and time to exhaustion in wtNESP and

tg6PHZ mice. Values represent individual values. Regression plot of wt/wtNESP: ⋅⋅⋅⋅⋅ and tg6/tg6PHZ: ⎯.

V& O2max and time to exhaustion in both mouse lines showed a well shaped function and

were more affected by Htc alterations at lower and higher Htc levels. Furthermore, time to

exhaustion was more sensitive to Htc alterations than V& O2max. Mathematical calculations

showed that best V& O2max and time to exhaustion were reached at Htc values of 0.58 and

0.57 for wtNESP, respectively, and 0.68 and 0.66 for tg6PHZ-- mice, respectively.

Effect of varying blood volume, whole body hemoglobin and viscosity on endur-ance performance

Besides Htc, blood volume and whole body hemoglobin play an important role in cardio-

vascular performance (Åstrand, 1952, 1977; Ekblom and Hermansen, 1968; Kanstrup and

Ekblom, 1984). To determine the impact of the blood volume and whole body hemoglobin

which is required to reach maximal endurance performance, both parameters were corre-


lated with V& O2max. As shown in Fig. 5.4a, b, V& O2max of wtNESP and tg6PHZ expressed as

a function of blood volume or whole body hemoglobin behaved as a polynomial second

degree equation. The graphs illustrate that about twice as much blood volume or whole

body hemoglobin was necessary to reach maximal V& O2max in tgPHZ compared to the lev-

els required in wtNESP. When comparing all blood parameters to V& O2max, the best correla-

tion was found with various Htc levels. This was not the case, however, with an Htc range

of 0.4 to 0.55, in which whole haemoglobin correlated most closely with V& O2max in

wtNESP mice (R2 = 0.513; P < 0.001).

□□□□ □□□

□

△

□□

□□

(a)

VO

2max

[ml·k

g-1·m

in-1

]

Blood volume

y = -0.001x2 + 0.5474x + 76.706R2 = 0.4472; P < 0.0001

y = -0.0022x2 + 0.6703x + 93.356R2 = 0.6891; P < 0.0001

60

80

100

120

140

160

50 100 150 200 250 300 350 400 450Blood volume [ml·kg-1]

(b)

VO

2max

[ml·k

g-1·m

in-1

]

Whole body hemoglobin

y = -14.648x2 + 48.38x + 108.38R2 = 0.4006; P < 0.0001

y = -40.341x2 + 61.123x + 122.03R2 = 0.635; P < 0.0001

60

80

100

120

140

160

30 0.5 1 1.5 2 2.5

Whole body hemoglobin [g]

·

□△

△

△△△

△ △ △□□

□□

□

□

△△

△△ △□

□□


□ wt△ tg6

·


□ wt△ tg6

□□□□ □□□

□

△

□□

□□

(a)

VO

2max

[ml·k

g-1·m

in-1

]

Blood volume

y = -0.001x2 + 0.5474x + 76.706R2 = 0.4472; P < 0.0001

y = -0.0022x2 + 0.6703x + 93.356R2 = 0.6891; P < 0.0001

60

80

100

120

140

160

50 100 150 200 250 300 350 400 450Blood volume [ml·kg-1]

(b)

VO

2max

[ml·k

g-1·m

in-1

]

Whole body hemoglobin

y = -14.648x2 + 48.38x + 108.38R2 = 0.4006; P < 0.0001

y = -40.341x2 + 61.123x + 122.03R2 = 0.635; P < 0.0001

60

80

100

120

140

160

30 0.5 1 1.5 2 2.5

Whole body hemoglobin [g]

·

□△

△

△△△

△ △ △□□

□□

□

□

△△

△△ △□

□□



□ wt△ tg6

·



□ wt△ tg6

Fig. 5.4. Relationship between maximal oxygen uptake (V& O2max) and (a) whole blood volume, and (b) whole body hemoglobin in wtNESP and tg6PHZ mice, respec-tively. Values represent individual values. Regression plot of wt/wtNESP: ⋅⋅⋅⋅⋅ and tg6/tg6PHZ: ⎯.


Polyzythemic condition impairs the blood flow due to Htc-dependent increasing blood

viscosity and thus, less O2 is transported to the tissue (Crowell et al., 1959) which may

result in reduced exercise performance. We observed a correlation between blood viscosity

and V& O2max (Fig. 5.5). Compared with wtNESP, Epo-overexpressing tg6 mice reached

maximal V& O2max at a higher blood viscosity. Furthermore, blood viscosity versus Htc of wt

and tg6 mice was in line with previously published data (Vogel et al., 2003).

□□□□□□

□

△

VO

2max

[ml·k

g-1·m

in-1

]

Viscosity

y = -1.7469x2 + 24.414x + 64.448R2 = 0.5419; P < 0.0001

y = -1.2907x2 + 12.664x + 113.88R2 = 0.4339; P < 0.0001

60

80

100

120

140

160

2 4 6 8 10 12Viscosity [mPas]

Max

. wtN

ESP

Max

. tg6

PHZ

△△

△ △△△□

□□

□



□ wt△ tg6

·

Fig. 5.5. Relationship between maximal oxygen uptake (V& O2max) and blood viscosity in

wtNESP and tg6PHZ mice. Values represent individual values. Regression plot of wt/wtNESP: ⋅⋅⋅⋅⋅ and tg6/tg6PHZ: ⎯.

Increasing mean arterial blood pressure, constant heart rate and altered stroke volume, with rising Htc levels at V&O 2max.

As the heart is the main generator of systemic blood circulation, mean arterial blood pres-

sure, heart rate and stroke volume were investigated. Mean arterial blood pressure rose

with increasing Htc levels in wtNESP and tg6PHZ (Fig. 5.6a). Overall, wtNESP reached

higher mean arterial blood pressure values compared to those in tg6PHZ. Heart rate did not

alter with increasing Htc levels and also did not alter from group to group (Fig. 5.6b).

Previous studies found a correlation between the stroke volume and O2 pulse during exer-

cise in healthy humans (Bhambhani, 1995; Crisafulli et al., 2007). Fig. 5.6c shows O2 pulse

as a function of Htc at V& O2max. O2 pulse of wtNESP and tg6PHZ rises slightly with in-

creasing Htc levels and reaches a calculated maximum at Htc values of 0.58 for wt and


50

(a)

100

150

200M

ean

arte

rialb

lood

pres

sure

[mm

Hg]

Mean arterial blood pressure

0.3 0.90.80.70.60.50.4Htc

y = 66.831x2 + 35.568x + 81.608 R2 = 0.6901; P < 0.0001

y = 132.89x2 - 21.554x + 106.07R2 = 0.8132; P < 0.0001

□

△△△

△

△

△

□□

□

□□

△

50

(b)

100

150

200

Hea

rt ra

te [b

eats

·min

-1]

Heart rate

0.3 0.80.70.60.50.4Htc

0.9

△

□

□□

□

□

□

△△△△

△

60

(c)

70

80

90

100

O2

pulse

[ml·k

g-1·b

eats

-1]

O2 pulse

0.3 0.90.80.70.60.50.4

Htc

y = -0.3596x2 + 0.4871x + 0.0177 R2 = 0.5757; P < 0.01

y = -0.306x2 + 0.3456x + 0.084R2 = 0.3662; P < 0.05

Max

. wtN

ESP

Max

. tg6

PHZ

△

□△△

△△

△

□

□

□□

□


□ wt△ tg6

50

(a)

100

150

200M

ean

arte

rialb

lood

pres

sure

[mm

Hg]

Mean arterial blood pressure

0.3 0.90.80.70.60.50.4Htc

y = 66.831x2 + 35.568x + 81.608 R2 = 0.6901; P < 0.0001

y = 132.89x2 - 21.554x + 106.07R2 = 0.8132; P < 0.0001

□

△△△

△

△

△

□□

□

□□

△

50

(b)

100

150

200

Hea

rt ra

te [b

eats

·min

-1]

Heart rate

0.3 0.80.70.60.50.4Htc

0.9

△

□

□□

□

□

□

△△△△

△

60

(c)

70

80

90

100

O2

pulse

[ml·k

g-1·b

eats

-1]

O2 pulse

0.3 0.90.80.70.60.50.4

Htc

y = -0.3596x2 + 0.4871x + 0.0177 R2 = 0.5757; P < 0.01

y = -0.306x2 + 0.3456x + 0.084R2 = 0.3662; P < 0.05

Max

. wtN

ESP

Max

. tg6

PHZ

△

□△△

△△

△

□

□

□□

□



□ wt△ tg6

Fig. 5.6. Relationship between hematocrit (Htc) and (a) mean arterial blood pressure, (b)

heart rate and (c) oxygen (O2) pulse at maximal oxygen uptake (V& O2max) in wtNESP and tg6PHZ mice. Values represent individual values. Regression plot of wt/wtNESP: ⋅⋅⋅⋅⋅ and tg6/tg6PHZ: ⎯.


0.68 for tg6, and decreases with higher values. Fitted curves display polynomial second

degree characteristics. In comparison to wtNESP, tg6PHZ showed a greater impact of

varying Htc levels on O2 pulse.

Evidence of increasing myocardial V&O2 with rising Htc levels at V&O2max

To study the impact of the myocardial V& O2 on V& O2max, rate pressure product was corre-

lated with Htc levels. Myocardial V& O2 increased with increasing Htc values in both groups

(Fig. 5.7). Both graphs showed a similar slope, but wtNESP mice had higher rate pressure

product values at corresponding Htc levels than tg6PHZ did, indicating that the heart of

wtNESP had a higher myocardial O2 supply requirement.

Rate pressure product

50

90

130

170

Rat

e pr

essu

repr

oduc

t10

00·[m

mH

g·be

ats-1

·min

-1]

0.3 0.80.70.60.50.4

Htc0.9

y = 41876x2 + 46022x + 66746 R2 = 0.7488; P < 0.0001

y = 53811x2 + 39477x + 78548R2 = 0.8038; P < 0.0001

□

△△ △

△

△

△

□□

□□

□


□ wt△ tg6

Rate pressure product

50

90

130

170

Rat

e pr

essu

repr

oduc

t10

00·[m

mH

g·be

ats-1

·min

-1]

0.3 0.80.70.60.50.4

Htc0.9

y = 41876x2 + 46022x + 66746 R2 = 0.7488; P < 0.0001

y = 53811x2 + 39477x + 78548R2 = 0.8038; P < 0.0001

□

△△ △

△

△

△

□□

□□

□



□ wt△ tg6

Fig. 5.7. Relationship between hematocrit (Htc) and rate pressure product in wtNESP

and tg6PHZ mice. Values represent individual values. Regression plot of wt/wtNESP: ⋅⋅⋅⋅⋅ and tg6/tg6PHZ: ⎯.

5.5. Discussion This is the first resport demonstrating optimal Htc values for maximal systemic endurance

performance in mice. Interestingly, acutely increasing Htc levels resulted in lower optimal

Htc values than those measured when Htc values were chronically increased. In addition,

the data showed that (1) the optimal Htc levels for maximal stroke volume and maximal

systemic exercise performance were similar but mouse line dependent; (2) mean arterial

blood pressure and rate pressure product increased with increasing Htc levels, whereas


heart rate remained unaffected; and (3) blood volume was dramatically elevated at higher

Htc levels.

Some terrestrial vertebrate species, such as dogs and horses respond to strenuous exercise

by splenic contraction due to their very high O2 demand for behaviour and survival (Dane

et al., 2006; Fedde and Wood, 1993). Splenic contraction releases extra erythrocytes into

the circulation to increase the blood O2-carrying capacity. As a consequence, Htc is ele-

vated from approximately 0.4 at rest to approximately 0.6 at exercise (Wagner et al., 1996;

Wu et al., 1996), resulting in improved exercise performance. These parameters return

quickly to resting values when the animals stop exercising to avoid constant overload of

the cardiovascular system (Wagner et al., 1995; Wu et al., 1996). Considering our findings,

it is tempting to speculate that these species elevate temporary their Htc levels close to the

optimal value Htc for maximal systemic endurance performance to enhance their exercise

performance.

In the present study, V& O2max and time to exhaustion initially rose with increasing Htc and

reached a maximum value. After this point, both parameters began to decrease. Maximal V& O2max and time to exhaustion values were found at Htc levels of 0.58 and 0.57 for

wtNESP, and 0.68 and 0.66 for tg6PHZ, respectively. These findings are in agreement with

the optimal Htc hypothesis (Crowell et al., 1959; Gaehtgens et al., 1979; Guyton and

Richardson; 1961; Villafuerte et al., 2003). Buick et al. speculated (1980) that optimal sys-

temic Htc is in excess of the commonly observed 0.45. These calculations were based on

the condition existing in the circulatory system at rest, since exercise induces changes in

vessel diameters, blood flow, internal temperature and blood distribution. However, there

was no experimental proof. Gaethgens and co-workers (1979) showed in isolated dog mus-

cle that during rhythmic isotonic exercise, various Htc levels lead to a plateau of maximal V& O2 and contractile power, but with a slight tendency toward maximal values in an Htc

range between 0.5 and 0.7. Indeed, the result may not be transferable to systemic exercise,

since each organ may have its own individual optimal Htc (Gaehtgens et al., 1979; Lee et

al., 1994; Tu et al., 1997). Therefore, the systemic Htc value has to be interpreted as an

average value of all organ specific optimal Htc values to provide the whole organism with

adequate O2. During exercise, O2 supply is disturbed because the impact of the skeletal

muscle is increasing and thus, a shift from the optimal physiological to the optimal Htc

value for best endurance performance may be observed. Endurance athletes make use of

this knowledge by the combination of living at moderate altitude and training at low alti-

tude (termed “live high-train low”) to improve their performance at sea level and moderate


altitude due to increased [Hb] (Levine and Stray-Gundersen, 1997; Schuler et al., 2007).

Particularly unscrupulous athletes artificially increase their circulating red blood cell num-

ber by misusing Epo, resulting in improved endurance performance by doping abuse

(Eichner, 2007; Warburton et al., 2000). This is not only unfair and criminal, but also life-

threatening.

At first glance, the observed relationship between V& O2max and Htc in our study is in con-

tradiction to the one observed in human studies. Several investigators have shown that

there is a strong correlation between V& O2max and whole body hemoglobin and blood vol-

ume (Åstrand, 1952, 1977; Ekblom and Hermansen, 1968; Kanstrup and Ekblom, 1984;

Heinicke et al., 2001; Warburton et al., 2000) but not Htc. It has to be mentioned that most

human studies are carried out with Htc levels of up to 0.5. Interestingly, if only the animals

in the present study in an Htc range of 0.4 and 0.55 were considered, we also found the

closest relationship between V& O2max and whole body haemoglobin in wtNESP animals.

Thus, the potential ergogenic effect of altered total body hemoglobin to improve endurance

performance seems only to be valid in an Htc range of 0.4 to 0.55. At higher Htc levels,

other factors may be more important.

The changes in the blood volume at higher Htc values are caused by the dramatically in-

creased number of erythrocytes, since plasma volume remained unchanged. Elevated blood

volume enhances end-diastolic volume (preload) which results in increased stroke volume,

leading to enhancement of V& O2max as long as heart rate is not altered. This finding is in line

with a previous study (Kanstrup and Ekblom, 1982). But, after reaching maximum at 0.57

for wtNESP and 0.68 for tg6PHZ, the blood viscosity may play a major role. Winslow and

Monge (1958) showed that blood viscosity increases exponentially with Htc levels of

above 0.55. High viscosity increases arterial blood pressure and diminishes venous return

due to the increased peripheral resistance (Richardson and Guyton 1959; Villafuerte et al.,

2004). Thus, the changes in stroke volume are not only a function of whole blood volume,

but also simultaneously of blood viscosity and peripheral resistance.

The cardiac output is calculated by the multiplication of stroke volume and heart rate.

Since the maximal heart rate in all our mice remained unaffected by the varying Htc level,

cardiac output alterations were induced by the stroke volume. Other studies have already

reported that stroke volume is a main regulator of the cardiac output in healthy human be-

ings during maximal exercise (Ekblom and Hermansen, 1968; Grimby et al., 1966;

Mitchell et al., 1958; Saltin and Stenberg, 1964).


When Htc is acutely raised, cardiac output begins to fall (Crowell and Smith, 1967;

Richardson and Guyton, 1959; Robertson et al., 1988; Stone et al., 1968). Otherwise, car-

diac output increases, when Htc acutely decreases (Kanstrup and Ekblom, 1982; Lundby et

al., 2008b; Richardson and Guyton, 1959). Interestingly, we did not observe this phenome-

non in our mice. The discrepancy in our study may be explained by the different species

used and experiments performed. Most likely, the wtNESP and tgPHZ animals could com-

pensate for the increasing blood viscosity since Htc levels stayed constant at a certain level

for two weeks. This may have provided sufficient time for the cardiovascular system of

both mouse lines to adapt. Since maximal stroke volume of tg6PHZ was reached at higher

Htc levels compared to those for wtNESP, we conclude that tg6PHZ can adapt better to

varying Htc levels. Physiological adaptations to excessive erythrocytosis are also observed

in humans. In the case of sports medicine there was the case of a Finnish cross-country

skier with autosomal dominant erythrocytosis resulting in Htc levels of up to 0.68. He won

several Olympic gold medals (Juvonen et al., 1991). Based on results of our study, we can

conclude that Htc of this athlete may be equal to the optimal Htc for maximal endurance

performance of tg6PHZ.

Adaptational mechanisms to excessive erythroytosis include peripheral vasodilatation and

regulation of blood viscosity (Bogdanova et al., 2007; Ruschitzka et al., 2000; Vogel et al.,

2003). Vasodilatation is induced by the enhancement of endothelial nitric oxide synthase

activity, which results in peripheral vasodilatation despite concomitant increased endothe-

lial-1 levels (Quaschning et al., 2003), whereas blood viscosity is regulated by the flexibil-

ity of the erythrocytes (Vogel et al., 2003). Of note, the regulation of blood viscosity ap-

pears to be at least as important as vasodilatation (Bogdanova et al., 2007). The simplest

way to regulate blood viscosity is to increase the level of juvenile erythrocytes in the circu-

latory blood system, since reticulocytes have a higher deformability than erythrocytes

(Shiga et al., 1990). Deformability is of crucial importance for microcirculation, in which

cells have to deform to pass through capillaries but also for the enhancement of O2-release

(Stuart and Nash, 1990). In response to PHZ treatment (Lim et al., 1998), the number of

erythrocytes decreases, and Epo plasma and the population of reticulocytes dramatically

increase (Cherukuri et al., 2004; Criswell et al., 2000; Rothman et al., 1970). Thus, tg6PHZ

may have a higher population of juvenile erythrocytes than wtNESP have. This would in-

duce a shift to a higher optimal Htc value.

The close correlation between V& O2max and blood viscosity in the present study confirms

the classical explanation of the existence of critical Htc to reduce cardiac output due to the


increased viscosity and hence limit endurance performance. These findings support the

notion that V& O2max is primarily limited by O2 delivery to the exercising muscle (Calbet et

al., 2005; Ekblom et al., 1976; Krip et al., 1997).

Maximal heart rate at exhaustion was unaffected by the Htc level. This implies that sympa-

thetic nervous activity remained unchanged at maximal exercise and thus, other factors

such as heart contractility were not influcenced by the NESP and PHZ treatment. More-

over, the fact that rate pressure product and blood pressures were increasing with incre-

mental elevation of Htc levels shows that the heart did not reach its maximal work capacity

at optimal Htc. This is against the “Central governor theory” as the limiting factor of V& O2max (Noakes, 1997, 1998). The theory proposes that the central nervous system regu-

lates the extent of skeletal muscle recruitment avoiding myocardial ischemia and therefore,

maximal cardiac output is never reached. As consequence, V& O2max is limited by the maxi-

mal rate of work which the heart allowed. In the present study, maximal cardiac output was

decreased after reaching a maximum, whereas rate pressure product was constitutively

increased with increasing Htc levels. This suggests that maximal cardiac output is estab-

lished by a regulatory mechanism and not by the heart that is working at its maximum. Our

result is in line with a recent published study that heart working capacity does not limit

exercise performance (Brink-Elfegoun et al., 2007). It was demonstrated that two maximal

combined arm and leg exercise tests performed at identical V& O2max and cardiac output re-

sult in different rate pressure products.

Apart from the cardiovascular limitation, we found no organ degeneration of the wtNESP

and tg6PHZ mice, despite the fact that previous studies showed that excessive erythrocyto-

sis leads to multiple organ failures (Heinicke et al., 2006). In contrast to that study, our

study used younger mice.

It has been reported that Epo promotes angiogenesis by enhancing vascular endothelial

growth factor (VEGF) in the issue (Alvarez Arroyo et al., 1998; Bellomo et al., 2006). Its

importance in the skeletal muscle is the capillary growth. Interestingly, no difference in

skeletal muscle capillary density was found between wt and tg6 (Gassmann et al., 2008)

and thus, it may have no impact on the endurance performance of wtNESP and tg6PHZ.

In summary, the results of the present study confirm the optimal Htc hypothesis during

systemic exercise in mice. The reason for this is that blood viscosity increases with in-

creasing Htc levels. Furthermore, the animals with chronic excessive erythrocytosis

adapted better to different Htc levels than acutely NESP-injected animals did. At nor-


moxia, the heart can tolerate higher rate pressure product at higher Htc levels. Thus, the

optimal Htc values for maximal endurance performance were independent of the working

capacity of the heart. V& O2max is mainly limited by O2 delivery.

Acknowledgments

This study was supported by funds from the Forschungskredit, University of Zurich, Zu-

rich, Switzerland, and the Swiss National Science Foundation.


6. Study 3: Timing the arrival at 2340 m altitude for aero-bic performance

This study has been published:

Schuler B, Thomsen JJ, Gassmann M, and Lundby C. Timing the arrival at 2340 m

altitude for aerobic performance. Scand J Med Sci Sports 17(5): 588-594, 2007.

6.1. Abstract This study tested the hypothesis that V& O2max and performance increase upon altitude ac-

climatization at moderate altitude. Eight elite cyclists were studied at sea level, and after 1

(Day 1), 7 (Day 7), 14 (Day 14) and 21 (Day 21) days of exposure to 2340 m. Capillary

blood samples were taken on these days before performing two consecutive maximal exer-

cise trials. Acclimatization increased hemoglobin concentration ([Hb]) and arterial oxygen

content (CaO2). On Day 1, V& O2max and time to exhaustion (at 80% of sea level maximal

power output) decreased by 12.8% (P < 0.05) and 25.8% (P < 0.05), respectively, com-

pared to the corresponding sea level values. Subsequently, these parameters increased by

3.2% (P < 0.05) and 6.0% (P < 0.05) from Day 1 to Day 7, by 4.8% (P < 0.05) and 5.7% (P

< 0.05) from Day 7 to Day 14, and by 0.7% (P > 0.05) and 1.4% (P > 0.05) from Day 14 to

Day 21, respectively. These data suggest that endurance athletes competing at altitudes

around 2340 m should expose themselves to this altitude at least 14 days prior to competi-

tion.

6.2. Introduction Most sporting competitions are held near sea level, where the majority of athletes also re-

side. A few exceptions include the 1968 Olympic games held in Mexico City at around

2300 m altitude, the 1995 road cycling championships in Bogotá at 2640 m, and the 2000

winter Olympics at 1250-2003 m near Salt Lake City. Also, in 2006, the Olympic ski races

of the Turin games were held near Sestriere at altitudes between 1509 and 2800 m. Thus,

although the majority of competitions are generally held near sea level, some events take

place at higher altitudes. The present investigation focuses on the importance of timing the

arrival at altitude in order to optimize competitive performance.

In humans V& O2max is limited by approximately 70% by oxygen (O2) delivery, and all other

systems are responsible for the remaining 30% (Di Prampero & Ferretti, 1990). Moreover,

experimental data show that V& O2max is limited by maximal O2 delivery, which in turn de-


pends on maximal cardiac output and maximal O2 extraction (Calbet et al., 2004a, 2005).

Accordingly, when arterial O2 content (CaO2) is increased by hyperoxia (Amann et al.,

2006; Knight et al, 1993; Nielsen et al., 1998), erythropoietin (Epo) administration

(Ekblom and Berglund, 1991) or autologous blood infusions (Thomson et al., 1982), V& O2max increases. Conversely, when CaO2 is acutely reduced due to moderate hypoxia, V& O2max decreases (Lundby et al., 2004). Thus, there is good evidence that a close relation-

ship between CaO2 and V& O2max within a given individual exists. With acclimatization to

altitudes at or above 4100 m, however, CaO2 is resumed, or values even higher than sea

level values are observed without a concomitant normalization in V& O2max (Calbet et al.,

2003b; Lundby et al., 2004). More recently, it was shown that V& O2max, upon acute hypoxic

exposure to 4100 m, does not increase despite increases in CaO2 that were achieved by

enhanced erythropoiesis upon application of the novel erythropoiesis stimulating protein

(NESP; Lundby and Damsgaard, 2006a). On the other hand, exercise performance can be

increased by erythrocyte infusion at 2255 m and 3566 m (Robertson et al., 1982, 1988).

Thus, it is tempting to speculate that there is an altitude threshold over which increased

hemoglobin concentration ([Hb]) has no beneficial effect on V& O2max. Below this threshold

it is very likely that an increased [Hb] results in an improvement in V& O2max compared to

what occurs at sea level. The fact that V& O2max does not fully recover after acclimatization

at high altitudes may be a consequence of a reduced peak leg blood flow (Calbet et al.,

2003b; Lundby et al., 2006b).

Most altitude sporting events are conducted at considerably lower altitudes, but unfortu-

nately data on acclimatization, V& O2max and performance at these altitudes are sparse. Prior

to the Olympic games in 1968, Faulkner and co-workers (1967) found no increase in

swimming performance after 14 days exposure to 2300 m and also found similar results in

runners (Faulkner et al., 1968). Accordingly, Adams and colleagues (1975) found only

minimal increases in 2-mile run times (statistics not reported) after 20 days of living and

training at 2300 m, and similar data for V& O2max have been presented after about 4-5 weeks

at a similar altitude (Daniels & Oldridge, 1970; Pugh, 1967). On the other hand, Saltin

(1967) reported that if altitude related sicknesses are avoided, V& O2max and performance are

increased after 19 days of exposure to 2300 m (but are still lower than sea level values),

and similar results have been presented after 21 days at 1822 m (Jensen et al., 1993). Dif-

ferences in the outcomes of the above mentioned studies could be related to a deficit in

iron-stores prior to altitude exposure (Stray-Gundersen et al., 1992). Another explanation

for V& O2max and/or performance not to increase with acclimatization could also be related to


decrement in training intensities at altitude. This potentially offsets the effects of acclima-

tization on O2 transport. Accordingly, Levine and Stray-Gundersen (1997) suggest that

“live high-train low” is the optimal approach. Indeed, in a recent review it was suggested

that altitude acclimatization lasting 10 days or longer can lead to improvements in endur-

ance performance at altitude (Fulco et al., 2000). However, in order to test this hypothesis

we took the opportunity to quantify V& O2max and performance in eight iron supplemented

elite cyclists at sea level, and after 1, 7, 14 and 21 days of exposure to 2340 m. To maintain

training intensity during the altitude exposure period, all training was performed below

1100m of altitude as suggested by the “live high-train low” approach.

6.3. Methods

Subjects

Eight elite bike racers participated in the study, and their anthropometric data are presented

in Tab. 6.1. They were all sea level residents with no prior exposure to high altitude for

the last 6 months. During the study the subjects did not participate in competitions, and

their training regimes two weeks prior, and during, the experiments are reported in Tab.

6.2. Two weeks before altitude exposure, iron (100 mg/day, Ferro Duretta, Astra Zeneca)

supplementation in all athletes was initiated and was continued during the whole study

period. After being given both written and oral information on the experimental protocol

and procedures, the subjects gave their informed, written consent to participate. The study

conformed to the guidelines laid down in the Declaration of Helsinki.

Protocol

All subjects were studied on five occasions: In Malaga, Spain, at sea level, and after 1 day

(Day 1) of exposure to 2340 m altitude at Centro de Alto Rendimiento in Sierra Nevada,

Spain, and again at the same location after 7 (Day 7), 14 (Day 14), and 21 (Day 21) days of

altitude exposure. Thus, the experiments were conducted within 23 days. The subjects

spent 16-24 hours at altitude before being tested on Day 1. All training was performed be-

low 1100 m altitude. On training days all subjects were transported to low altitude by car

(approximately 30 min) and also transported back by car (approximately 45 min) after the

training session. Therefore, on a daily basis, the subjects spent approximately 19 hours at

2340 m, and the remaining time below approximately 1100 m. At the altitude facilities the

subjects had the choice of a large variety of food and beverages, and they were free to con-

sume what they preferred.


Tab. 6.1. Individual anthropometric data of the subjects at sea level. Additionally body mass on Day 1, Day 7, Day 14, Day 21. Values are means ± sd.

Tab. 6.2. Average training hours per week of all subjects expressed as % of maximal heart rate (%HR) one and two weeks prior to the experiments at sea level (Sea level 1 and Sea level 2), and during week one, two, and three at altitude (Alti-tude 1, 2, and 3, respectively). Values are means ± sd.

Each subject was investigated at the same time of the day on all occasions, but some were

studied in the morning; others were monitored throughout the afternoon/evening. After

blood sampling, all subjects performed a maximal cycle ergometer (Ergomedic 839E,

Monark, Varberg, Sweden) test. After 15 minutes of warm-up at 200 W, the workload was

increased by 30 W every minute until exhaustion. After at least 6 hours of rest the subjects

performed a constant workload exercise test to exhaustion. The workload (339.7 ± 15.8 W)


was the same in all conditions and calculated from 80% of the maximal attained workload

at sea level, and corresponded to 92.7 ± 1, 89.8 ± 1.3, 85.1 ± 0.8, and 84.3 ± 1.6% of

maximal power output on Day 1, Day 7, Day 14, and Day 21, respectively. Prior to the

time to exhaustion test, subjects warmed-up for 15 minutes at 200 W.

Measurement

Blood samples were taken before the initiation of the first exercise test, after 30 min of

supine resting. Hemoglobin concentration ([Hb]) and hematocrit (Htc) were measured in

duplicate using a HemoCue Hemoglobin Photometer (HemoCue AB, Ängelholm, Sweden)

and Mikro 12-24 Centrifuge (Hettich Zentrifugen, Tuttlingen, Germany). They were meas-

ured from venous blood obtained from an antecubital vein. Arterial oxygenation (SaO2)

was determined by a Nellcor pulse oxymeter (Nellcor, Hyward, CA, U.S.A.), and the CaO2

was calculated as CaO2 = [Hb] x 1.34 x SaO2. Blood samples for Epo quantification were

also obtained from the antecutital vein. These samples were centrifuged for 15 minutes at

3500 rpm and -4°C. The plasma was stored at -20°C for later analysis via ELISA (R&D

Systems, Minneapolis, MN, USA).

Pulmonary V& O2 and CO2 production (V& CO2) were continuously measured (Quark b2, Cos-

med Srl., Rome, Italy). Before each test, ambient conditions were measured, and then the

gas analyzer and the flowmeter were calibrated with high precision gases. During sub-

maximal and maximal exercise the V& O2 were recorded as averages of 15 second intervals.

Gross mechanical efficiency (%) was determined from the ratio of power output (kJ·min-1)

to energy expended (kJ·min-1), as calculated from V& O2 and the respiratory exchange ratio

(RER).

Statistics


ley, CA, USA). Statistical comparisons were performed by one-way ANOVA for repeated

measurements. The Fisher’s PLSD post hoc test was used to assess differences between

values at sea level and those from the different days at altitude, and from the previous

measuring day during acclimatization (e.g. Day 7 to Day 1, Day 14 to Day 7 and Day 21 to

Day 14). Correlations between the parameters were computed using linear regression. Sta-

tistical differences in ANOVA, post hoc test and linear regression were considered signifi-

cant when P < 0.05. Results are presented as means ± standard deviation (sd).


6.4. Results Individual anthropometric data of the subjects are given in Tab 6.1. There were no statisti-

cally significant differences in body weights observed at sea level, and on Day 1, Day 7,

Day 14, and Day 21.

Hematological parameters

Plasma Epo levels peaked on Day 1 at altitude, and remained slightly, but not statistically

significantly, elevated during the acclimatization period (Fig. 6.1 a). [Hb] and Htc in-

creased during the period from Day 1 until the end of the altitude exposure period by

15.1% and 13.4%, respectively (Fig. 6.1b, c). Compared to its value at sea level, the

amount of CaO2 decreased by 8.7% on Day 1, but increased by 15.6% after 21 days of alti-

tude exposure (Fig. 6.1d).

Fig. 6.1. Individual and mean resting values for (a) erythropoietin (Epo), (b) hemoglo-

bin concentration ([Hb]), (c) hematocrit (Htc), and (d) arterial O2 content (CaO2) at sea level (SL) and on Day 1 through 21 (Day 1, Day, 7, Day 14, Day 21, respectively) of acclimatization. a P < 0.05 compared to SL; b P < 0.05 compared to, e.g. Day 7 to Day 1, Day 14 to Day 7 and Day 21 to Day 14.


Exercise performance parameters

Acute hypoxic exposure decreased maximal power output by 13.8% resulting in an accord-

ingly lower V& O2max of 12.8% (Fig. 6.2a, b). With acute hypoxic exposure, time to exhaus-

tion declined by 25.8% (Fig. 6.2c).

Fig. 6.2. Individual and mean values for (a) maximal power output, (b) maximal oxygen uptake (V& O2max), and (c) time to exhaustion at sea level (SL) and on Day 1 through 21 (Day 1, Day, 7, Day 14, Day 21) of acclimatization. a P < 0.05 com-pared to SL; b P < 0.05 compared to, e.g. Day 7 to Day 1, Day 14 to Day 7 and Day 21 to Day 14.

Acclimatization produced a marked improvement in all exercise performance parameters.

At the end of the 3 week altitude period, maximal power output, V& O2max and time to ex-

haustion had increased by 10.0%, 8.9% and 13.6% when compared to the levels on Day 1.

The highest increase was observed between Day 7 and Day 14, and the lowest between

Day 14 and Day 21 (non-significant change). There was a correlation betweenV& O2max and

time to exhaustion (Fig. 6.3). Moreover, the changes in [Hb] were correlated with the

changes in V& O2max and time to exhaustion (Fig. 6.4a, b).


Fig. 6.3. Correlation between maximal oxygen uptake (V& O2max) and time to exhaustion.

Values are means. SL: sea level.

Fig. 6.4. Correlation between the percentage of changes in (a) hemoglobin concentration

[Hb] and maximal oxygen uptake (V& O2max), and (b) between [Hb] and time to exhaustion of, e.g., sea level (SL) to Day 1, Days 1-7, Days 7-14, Days 14-21. Values are means.


Gross mechanical efficiency (%) remained constant at all time points for a given workload,

and was on average 25.3 ± 0.9 at sea level, and 25.2 ± 1.0 (Day 1), 25.3 ± 1.2 (Day 7), 24.8

± 1 (Day 14), and 25.1 ± 0.7 (Day 21) at altitude.

6.5. Discussion The main finding of the present study is that with acclimatization to 2340 m (i) V& O2max and

time to exhaustion improve over time and (ii) this improvement occurs within the first 14

days of altitude exposure. Our data provides convincing evidence that athletes who plan to

compete in endurance events at this altitude should expose themselves to the altitude of

competition for at least 14 days prior to the competition.

In the present study an improvement in all measured exercise performance parameters was

found during acclimatization to moderate altitude. In all athletes, the main improvement in

performance was observed within the first 14 days of altitude exposure. In the following

week, however, five of eight athletes improved V& O2max even further, whereas eight contin-

ued to improve time to exhaustion slightly. The observation that V& O2max increases with

prolonged altitude acclimatization, is in agreement with data obtained by Jensen and co

(1993). That study reported that three weeks of acclimatization to 1822 m is accompanied

by an increase in V& O2max and 6-min work capacity during rowing. Our results and those of

Jensen et al. (1993), however, are not in agreement with the results of other studies deter-

mining acclimatization and performance at 2300 m (Adams et al. 1975; Daniels &

Oldridge, 1970; Faulkner et al., 1967, 1968; Pugh, 1967). The data from the latter studies

can be divided into two groups: Either V& O2max and/or performance remained constant dur-

ing acclimatization, or were slightly increased during the stay at altitude. For instance,

Faulkner and co-workers (1968) found no change in V& O2max and time trial performance

after three weeks of acclimatization to 2300 m altitude. The main difference between the

present study and those performed in the past is that our athletes performed all training at

altitudes lower than 1100 m. As suggested by the “live high-train low” concept, this ap-

proach minimizes the loss of training intensity which would normally be associated with

traditional high altitude training regimes (Levine and Stray-Gundersen, 1997). Thus,

whereas subjects in some of the previous studies may have experienced reduced training

intensities during the altitude exposures, this was not the case in the present study. The

study by Jensen et al. (1993) was conducted at an altitude where the decrement in V& O2max,

and therefore also training intensities, was less than in the remaining studies. Therefore, it

is tempting to speculate that these subjects were able to keep training intensities closer to


their normal sea level values, and therefore also minimized the loss of training stimulus,

while still achieving the effects of hypoxia on, for example, hemoglobin mass. Another

factor that may explain the differences in results is that we supplemented all subjects with

100 mg of daily iron in order to facilitate hemoglobin production. None of the previous

studies states that iron was supplemented. Yet another explanation for the different results

may be the presence of altitude associated diseases. From studies performed in preparation

for the 1968 Olympic Games, Saltin (1968) reported that 19 days of acclimatization to ap-

proximately 2300 m led to increments in V& O2max in the athletes who were not affected by

illness, whereas the infected athletes did not improve performance. In the case of Mexico,

the illness was most likely associated with infections obtained from polluted drinking wa-

ter and not with acute mountain sickness.

The altitude associated increase (%) in [Hb] correlated well with the increment (%) in

V& O2max (R2 = 0.948; P < 0.05) and time to exhaustion (R2 = 0.971; P < 0.05), and thus,

seems to be a reasonable candidate for the increase in performance (Fig. 6.4a, b). This is in

agreement with Robertson et al. (1982, 1988), who showed that erythrocyte infusion re-

sults in an improvement in exercise performance at altitudes of 2255 m and 3566 m. In the

present study the increase in [Hb] was somewhat greater than in previous studies con-

ducted with athletes at similar altitudes. This could be related to the relatively low [Hb]

observed at sea level compared to that observed in other studies (Kime et al. 2003; Veic-

steinas et al., 1984). The difference may be explained by different initial conditions and

different training regimes. The relatively low [Hb] could make the subjects in the present

study more sensitive to iron supplementation. Unfortunately, we can not access the relative

contribution of iron supplementation or acclimatization to the total improvements in [Hb].

It has to be mentioned, however, that only athletes with iron deficiency anemia may have a

beneficial effect on performance from iron supplementation (Zoller and Vogel, 2004).

Note that in this study the subjects were supplemented daily with iron (100 mg) starting

two weeks prior to, and during, the study period in order to minimize the possible influ-

ence of iron deficiency on V& O2max, performance and [Hb].

Although Epo increased rapidly, it is unlikely that the early increase in CaO2 was due to

increases in haemoglobin synthesis, since it is well known that plasma volume decreases

almost immediately with exposure to high altitude (Gunga et al., 1994), and that it remains

depressed for several weeks (Alexander et al., 1967; Pugh, 1964; Reynafarje et al., 1959).

Thus, the increase in V& O2max and performance could also reflect a time dependent recovery

in blood volume. However, plasma volume expansion does not affect V& O2max after 9 weeks


of acclimatization to high altitude (Calbet et al., 2004b). Unfortunately, we did not have

the opportunity to measure blood volume in the present study.

In previous ”sleep/live high-train low studies”, performed to investigate subsequent per-

formance at sea level, an increase in muscle buffer capacity and ergometer cycling econ-

omy (Gore et al., 2001) and running economy (Saunders et al., 2004) have been reported.

Others, however, have not observed changes in running economy (Levine and Stray-

Gundersen, 1997, 2005). Thus, this issue remains a topic of great debate (Levine and

Stray-Gundersen, 2005). In a recent study including over 100 subjects from different stud-

ies and altitudes ranging from 2500 to 5260 m, however, it was concluded that low to high

altitudes do not influence exercise economy (Lundby et al., 2007). Whether the differences

in results stem from methodological issues such as cycling vs. running and net vs. gross

economy still remains to be clarified.

In the present study we did not quantify muscle buffer capacity, but found no differences in

ergometer cycling economy. Thus, at least in the present investigation, performance incre-

ments were unrelated to changes in economy. Of note is that previous studies evaluated

economy at sea level after termination of the altitude training regimes, whereas we com-

pleted the trials at altitude.

It should be noted that all time to exhaustion experiments were conducted at the same ab-

solute exercise intensity, and that this elicited different relative exercise intensities, i.e.

80% at sea level, and values ranging from 92.2% on Day 1 at altitude to 84.3% on Day 21.

Therefore, the improvements in time to exhaustion could be related to decreases in relative

exercise intensities alone, but unfortunately, we did not have the opportunity to conduct a

second time to exhaustion test in all conditions in which the workload was matched to

elicit a relative intensity of 80% as at sea level.

6.6. Perspectives The results of this study show that the “live high-train low” approach can be recommended

to improve V& O2max and performance at moderate altitude. Accordingly, athletes who plan

to compete at 2340 m altitude are advised to expose themselves to this altitude for at least

14 days prior to the competition. Although elite cyclists were investigated in this study, it

also seems likely that endurance athletes from other sporting disciplines may respond simi-

larly. Based on the present study and previous work, it has become evident that there is an

altitude threshold around 3300-3500 m below which an increase in [Hb] has a beneficial


effect on V& O2max, whereas this advantage disappears at higher altitudes. One reason that V& O2max does not increase with the elevation of [Hb] at higher altitudes is probably that

there is a reduction in peak leg blood flow in chronic hypoxia (Calbet et al., 2003b;

Lundby et al., 2007). This implies that the extra O2 carrying capacity gained with the ac-

climatization-elicited elevation in [Hb] is not made fully made available to the exercising

muscles, but deviated to secure oxygenation of other vascular beds. This hypothesis, how-

ever, needs to be tested experimentally.


7. General discussion and conclusions This thesis investigated the impact of erythropoietin (Epo) on endurance performance un-

der normoxic condition and upon acclimatization to moderate altitude in mice and humans.

To be able to study metabolic and cardiovascular parameters in exercising mice, telemetry

and indirect calorimetry during exercise were combined and improved in first study. In a

newly developed injury regime, the transmitter body was subcutaneously placed in the

midline of mice’s back. This is more comfortable for the animals during submaximal as

well as maximal exercise and improves blood pressure signal. Furthermore, the success of

the implantation depends on the choice of the postoperative regime. Note that this might

also be relevant for other injuries. This new method was used in the second study to inves-

tigate the effect of varying hematocrit (Htc) levels on exercise performance and the cardio-

vascular system. To this end, Htc levels of wild type (wt) were acutely elevated by novel

erythropoiesis stimulating protein (NESP) administration. On the other hand, Htc levels of

our transgenic mice line termed tg6 that reach Htc levels of up to 0.9 due to the overex-

pression of Epo were reduced by the hemolysis-inducing compound phenylhydrazine

(PHZ). Maximal oxygen (O2) uptake (V& O2max) and time to exhaustion increased with in-

creasing Htc levels to a peak at 0.58 and 0.57 for wtNESP mice, and 0.68 and 0.66 for

tg6PHZ, respectively. Increasing blood viscosity seems to be a reasonable candidate to

limit exercise performance

The third study investigated the effect of altitude acclimatization on exercise performance

and hemoglobin concentration ([Hb]). To this end, elite endurance athletes were exposed

during a period of 21 days to 2360 m altitude. V& O2max and time to exhaustion increased

over the experimental period, and the main increment occurred within the first 14 days of

altitude acclimatization. The altitude-induced elevation in [Hb] seems to be responsible for

the improvement in exercise performance.

Overall, the findings of the present work suggest that the main effect of Epo on exercise

performance is aimed at improving O2 carrying capacity by increasing [Hb]. Paradoxically,

the associated increased blood viscosity that accompanies elevated Htc levels impairs O2-

delivery and thus, exercise performance. Interestingly, the effects were more pronounced at

submaximal intensities than at V& O2max. This is in line with the observation by Thomsen

and co-workers (2007) showing that recombinant human Epo (rhEpo) treatment in healthy

humans over 14 days increases V& O2max by approximately 12% and prolongs submaximal

exercise by approximately 54%. This implies that other factors may also play a role. A


number of data have been gathered on the impact of Epo under pathological condition in

order to study its non-hematopoietic functions. Systemically administered recombinant

human erythropoietin (rhEpo) crosses the blood-brain barrier and has neuroprotective ef-

fects on the central nervous system (Brines et al., 2000). Furthermore, Epo and Epo recep-

tors are also expressed in the brain (Jelkmann, 2005). Elevated Epo-levels in plasma and

brain enhance the ventilatory response to severe acute hypoxia as well as the acclimatiza-

tion to chronic hypoxic exposure (Soliz et al., 2005). Moreover, it was shown in healthy

humans that rhEpo administration positively affects the self reported mood, physical condi-

tioning and strength scores (Miskowiak et al., 2008; Ninot et al., 2006). All of them could

have an impact on exercise performance of our subjects.

The question arises as to whether the optimal Htc for best aerobic exercise performance

alters upon exposure to altitude. It is likely that Htc level is optimized because of its influ-

ences on blood O2 transport capacity. During altitude acclimatization, [Hb] increases and

may lead to an arterial O2 content (CaO2) that is higher than that at sea level. However, the

acclimatization process is multifactorial. Thus, it is difficult to define as whether the opti-

mal Htc for best aerobic exercise performance is altered at altitude. For instance, more than

controversial is the effect of hypoxia on the degree of capillarisation (Lundy et al., 2004;

Hoppeler et al., 2008). Increased muscle capillarisation might reduce pheripheral resistance

in the exercising skeletal muscle resulting in a shift of the optimal Htc to a higher Htc

value. Therefore, more studies are required to unravel this issue.

Elevated blood viscosity may not only limit exercise performance, but also is potentially

dangerous for the health of the athletes. The main risk occurs with Htc above 0.55 (Jelk-

mann, 2003). Risks include heart failure, myocardial infarction, peripheral thromboem-

bolic events and pulmonary embolism. During competition, athletes are at special risk,

because blood viscosity may further increase due to sweating and the loss of body fluid.

Thus, sport associations have introduced Htc thresholds, above which athletes are not al-

lowed to compete to stop them from damaging their health. These limiting values are dif-

ferent depending on the association and are not scientifically proven. More investigation to

study the morphological and functional consequences of varying Htc levels would support

scientific Htc limitation to protect the athlete’s lives. Moreover, scientific evidence would

demonstrate the life-threatening consequences of long-term rhEpo-abuse. This is necessary

to sensitize athletes, trainers, administrators and also, importantly, spectators. It is becom-

ing even more important, as anti-doping agencies will be confronted with a new technol-

ogy – gene doping. Genetically manipulated athletes will be difficult to identify. Thus,


more scientific proofs about the health risks of long-term Epo abuse will probably hinder

many athletes from abusing their bodies in this way.

On the other hand, rhEpo and its derivate are used for the treatment of anemia such as

chronic renal failure, HIV-infection or cancer. Thereby, the treatment is depending on se-

verity of the illness and the ability for regeneration of the injured organs. Thus, novel sci-

entific knowledges of Epo’s positive and negative side effects to human health are also

important for those patients suffering from anemic diseases.

Finally, Epo has a future in clinical medicine for a number of therapies in areas including

neuroscience and cardiovascular diseases because it is a safe drug. Unfortunately, it is also

misused by scrupulous athletes. Indeed, many publications and reviews are available on

this topic. In spite of the efforts in the last years, there is still much work to do in order to

understand the multiple functions of Epo.


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9. List of abbreviations CaO2 arterial oxygen content

Epo erythropoietin

Hif hypoxia-inducible factor

Hif-α α subunit of hypoxia-inducible factor

Hif-1 hypoxia inducible factor-1

Hif-1α α subunit of hypoxia-inducible factor-1

Hif-1β β subunit of hypoxia-inducible factor-1



[Hb] hemoglobin concentration

Htc hematocrit

O2 oxygen

NESP novel erythropoiesis stimulating protein

PHZ phenylhydrazine

pO2 oxygen partial pressure

RER respiratory exchange ratio

rhEpo recombinant human erythropoietin

SaO2 arterial oxygen saturation

SD standard deviation

tg6 transgenic mouse line overexpressing human erythropoietin cDNA

tg6PHZ transgenic mouse line overexpressing human erythropoietin cDNA, treated with phenylhydrazine

wt wild type mouse

wtNESP wild type mouse treated with novel erythropoiesis stimulating protein

VEGF vascular endothelial growth factor

V& CO2 carbon dioxide production

V& O2 oxygen uptake

V& O2max maximal oxygen uptake


10. Curriculum Vitae

Name: Beat Schuler

Date of birth: 16 June 1972

Citizen of: Schübelbach, Schwyz

Education:

1979-1986 Primary school, Siebnen

1986-1989 Secondary school, Siebnen

1989-1992 Apprenticeship as a landscaper, Lachen

1990-1992 Vocational training college, Wetzikon

1992-1996 Academic High school (Interstaatliche Maturitätsschule für Erwachsene) in Sargans, Matura type C (Mathematics-Natural Science)

1996-1998 Undergraduate studies in Physics at the Swiss Federal Institute of Technol-ogy Zurich (ETHZ), Zurich

1998-2003 Graduate degree in Biology at the Swiss Federal Institute of Technology Zurich (ETHZ), Zurich (dipl. Natw. ETH)

2002-2003 Diploma thesis at the Institute for Movement Sciences, Swiss Federal Insti-tute of Technology Zurich (ETHZ), Zurich

2003 - Education of the didactics (ETH), Zurich

2005 Research period, Prof. Dr. JP Richalet, Université Paris 13, Bobigny Cedex, France

2006 LTK Modul 2: Training for People Responsible for Directing Animal Ex-periments, Zurich

2004-2009 PhD student at the Swiss Federal Institute of Technology Zurich (ETHZ), conducted at the Institute of Veterinary Physiology, University of Zurich, Zurich, in the group of Prof. Dr. M. Gassmann


11. Publications

Scientific Publications

Föller M, Feil S, Ghoreschi K, Koka S, Gerling A, Thunemann M, Hofmann F, Schuler B, Vogel J, Pichler B, Kasinathan RS, Nicolay JP, Huber SM, Lang F, Feil R. Anemia and splenomegaly in cGKI-deficient mice. Proc Natl Acad Sci U S A 105(18): 6771-6776, 2008.

Föller M, Kasinathan RS, Koka S, Huber SM, Schuler B, Vogel J, Gassmann M, Lang F. Enhanced susceptibility to suicidal death of erythrocytes from transgenic mice overexpressing erythropoietin. Am J Physiol Regul Integr Comp Physiol 293(3): R1127-R1134, 2007.

Schuler B, Gassmann M. Optimale Ankunftszeit vor einem Wettkampf auf moderater Höhe. Schweiz Z Sportmed 55: 107-108, 2007.

Schuler B, Thomsen JJ, Gassmann M, Lundby C. Timing the arrival at 2340 m altitude for aerobic performance. Scand J Med Sci Sports 17(5): 588-94, 2007.

Schuler B, Lundby C, Gassmann M. HIF-1 and the adaptation of man to high altitude. Schweiz Z Sportmed 52(2): 82-87, 2005.

Proceedings and Abstracts

Kuhn G, Schneider P, Schuler B, Meier M, Vogel J, Müller R. Excessive Erythropoiesis affects Bone Architecture. ASBMR 30th Annual Meeting. 12-16 September 2008, Montreal, Canada.

Schuler B, Thomsen JJ, Gassmann M, Lundby C. Time course of improvement in en-durance performance during altitude acclimatization. 11th annual Congress of Euro-pean College of Sport Science, 5-8 July 2006, Lausanne, Switzerland.

Arras M, Rettich A, Käsermann HP, Schuler B, Cinelli P, Bürki K. Real-time monitor-ing of post-operative recovery in mice by telemetry, 2th ZIHP Symposia, 22 Septem-ber 2006, Zurich Switzerland.

Schuler B, Thomsen JJ, Gassmann M, Lundby C. Optimal arrival for competing at moderate altitude in elite endurance athletes, 2th ZIHP Symposia, 22 September 2006, Zurich Switzerland.

Other publications

- Gedopte Mäuse ohne Ausdauer, CAS-Wissenschaftsjournalismus, 4 November 2008.

- Der Dopingverdacht rennt mit, 10vor10, Swiss Television SF, 15 August 2008.

- 175 Years Anniversary of the University of Zurich, University of Zurich, 17-18 April 2008.

- Zaubersaft Blut, ARTE (Association à la Télévision Européenne), 1 August, 2008.

- Doping- ein Hintergrundbericht, Sportpanorama, Swiss Television SF, 16 December 2007.


- Leistungstests mit Epo-Mäusen, Unipublic, 17 June 2007.

Oral presentation - Optimale Ankunftszeit vor Wettkämpfen auf moderater Höhe, 3. Internationales Sym-

posium „Höhenphysiologie und Praxis”, Reiteralpe, Germany, 16-19 October 2007.

Grants - Doping and Gendoping: The impact of Epo and NESP on exercise performance and

health, Forschungskredit, University of Zurich, Zurich, Switzerland, 2008, CHF: 94576.


12. Acknowledgements I wish to thank everyone who was involved in this research project and who contributed to

its completion. The following people are worthy of special mention:

Prof. Dr. Max Gassmann, my direct supervisor and mentor, who gave me the opportunity

to conduct my thesis in his institute. Many thanks for his generous scientific support from

the time of the development of the project outline until the publication. His confidence,

friendship and personal advice encouraged me to believe in my work and to continue my

future career in science.

Prof. Dr. Urs Boutellier who consented to officiate as a referee at the ETH Zurich and in-

spired my scientific interest during the diploma thesis within his group “exercise physiol-

ogy”. Since that time I have profited from his enormous experience and knowledge. I also

thank him for his open and fruitful discussions.

Prof. Dr. Jean-Claude Perriard from the Institute of Cell Biology, ETH Zurich, who kindly

consented to be my co-referee. His engaged and motivating feedback contributed deci-

sively to the success of this project.

Dr. Margarete Arras and Prof. Dr. Kurt Bürki from the Institute of Laboratory Animal Sci-

ence. Without their willingness to collaborate, this project would not have been able to

have been carried out. I am looking to further fruitful and successful collaboration. Both of

them always had time to help me to solve all kinds of mouse problems.

Prof. Dr. Johannes Vogel and Stephan Keller who always came on time to the University

Hospital. I enjoyed very much working together with such reliable and cooperative people.

In addition, I thank Stephan for introducing me to the world of wines and whiskies.

Dr. Béatrice Bürgi and Andreas Rettich for the critical reading of the manuscript and tech-

nical support.

Many thanks to my colleagues at the Institute of Veterinary Physiology and the Institute of

Laboratory Animal Science.

My partner, my friends and my mother who stood by me and had a lot of understanding

when I was working every night at the University Hospital.

Zurich Center for Integrative Human Physiology (ZIHP) to allocate techniques of the core

facility.

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