Gaming the System: Doping in Sports - Max gassmann

Erythropoietin’s impact on therapyand exercise performance Chairman of the Institute of Veterinary

Physiology, Vetsuisse Faculty, and of the Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich

Prof. Max Gassmann

Chart 1

Gaming the System:Doping in Sportsswissnex San FranciscoAugust 20, 2012

San Francisco

Institute of Veterinary Physiology Chart 2

reduced

oxygen

Epo

circulation

Epo

bone

marrow

anti-apoptotic effect

on erythroid

precursor cells

erythrocytes

hematocrit

oxygen

uptake

O2

Hb-O2

neg.Feedback

Epo-synthesis

oxygen

sensing:

HIF-2 +

-

+

+


rhEpo has been developed to treat anemic patients and not to dope cheating

athletes


• rhEpo injection (i.p.) into wild type mouse

• transgenic mouse constitutively overexpressing human Epo in brain only (tg21)

• transgenic mouse constitutively overexpressing human Epo (26 fold) in brain and

reaching a 12 fold increase in plasma Epo levels (tg6) excessive erythrocytosis

Mouse models to study the impact of acute and chronically

elevated Epo levels

Ruschitzka et al., PNAS 2000

Wiessner et al., JCBF&M 2001


Epo-overexpressing transgenic mouse lines tg21 and tg6:hematological parameters

plasma Epo

red blood cells

hemoglobin

hematocrit

reticulocyte counts

blood volume

arterial pO2

arterial oxygen content

U / l

1012 / l

g / l

%

%

ml

mmHg

ml / dl

22

6.2

140

40

1.7

2.2

125

17

260

13.8

235

79 (90)

3.4

5.5

133

31

wt+tg21 tg6

The higher the hematocrit the higher the performance?


Reduced exercise performance inEpo-overexpressing tg6 mice with a hematocrit of ~80 %

An endurance athlete copes with a chronically elevated hematocrit

Eero Mäntyranta (born 1937)

hematocrit: 68%

won 6 gold medals between 1960-1966


Treadmill

wt

Hematocrit: 0.4

NESP (“Epo”)

Hematocrit

An optimal hematocrit for maximal performance?

acute

Phenylhydrazine (PHZ)

leads to hemolysis

tg6

Hematocrit: 0.8-0.9

Hematocrit

chronic

Time to exhaustion

VO2max

Maximal performance is reached at hematocrit levels 0.57-0.68

·

Schuler et al., PNAS 2010

.

What is the limiting factor? Viscosity!

VO2max

vs. blood viscosity ∙

Conclusions

Maximal working capacity of the heart was not reached at the optimal hematocrit for

maximal endurance performance.

The present study confirms our hypothesis that optimal hematocrit during systemic

exercise in mice exists.

Blood viscosity is the most likely candidate to limit exercise performance.

Mäntyranta expresses a truncated form of the Epo receptor

Truncated Epo receptor causes dominantly inherited benign human erythrocytosis by

de la Chapelle et al., PNAS 1993


Complications in mice suffering from

Epo-induced excessive erythrocytosis


Degenerative process in the liver of 5 month-old tg6 mice

50µm

wt tg6

tg6

->: hemosiderin

: inflammation


Degenerative processes in the sciatic nerve of 5 month-old tg6 mice

A: Axoplasma SC: Schwam cells axonal swelling

ML: Myelin lamellae Co: Collagen fibres deformed fibres

20µm 2µm


Motor disorders characterized by bi- or unilateral spastic contraction and paralysis of the hindlimbs

Hindlimb paralysis and neuromuscular degeneration in tg6 mice


Degenerated skeletal muscle fibresin tg6 mice

= muscle capillaries

tg6wt


reduced

oxygen

Epo

circulation

Epo

bone

marrow

anti-apoptotic effect

on erythroid

precursor cells

erythrocytes

hematocrit

oxygen

uptake

O2

Hb-O2

neg.Feedback

Epo-synthesis

oxygen

sensing:

HIF-2 +

-

+

+


Repoxygen® was intended for gene therapy

Recombinant

epo

oxygen

gene therapy

- Thus oxygen-regulated Epo synthesis should be possible in muscle, too

- Introduction into muscle by: - viral vectors

- direct muscular injection

- hEpo expression is expected to be oxygen-regulated

- HIF is present in all cells of the human body

DNAhEpo

HIF

HRE


Repoxygen®: no further development

- Additional problems:

- normal Epo expression: kidney (fetal liver) but not muscle

- HRE is variable

- oxygen-regulated Repoxygen expression in humans: unknown

- Introduction into human body:- viral vectors

- direct muscular injection

- CAVE: uncontrolled Epo expression polycythemia

serious risks including death

- Economics: Repoxygen vs. classical

rhEpo

project is stopped

- Is not in clinical trialsDNA

hEpo

HIF

HRE


oxygen

low normal

(normoxia)blood loss

anemia

altitude

PHD‘s

+ O2

DNA

Hypoxia-Inducible Factor

(HIF): stabilized

HIF

HRE HIF target genes

- Erythropoietin (Epo)

- Vascular Endothelial Growth Factor

- etc.

Hypoxia

Response

Element

induce

Hypoxia-Inducible Factor

(HIF): degraded by PHD‘s

HIF


B. Schuler

J. Vogel

A. Bogdanova

L. Ogunshola

J. Soliz

B. Grenacher

M. Alvarez

R. Jacobs

V. Diaz

Acknowledgements

E. Vannoni

D. Wolfer

C. Lundby

F. Ruschitzka

Th. Lüscher

M. Maggiorini




Epo-overexpressing tg6 mouse: resting hemodynamic parameters

heart rate

systolic blood pressure

cardiac output

beats/min

mmHg

ml/min

569 ± 18

119 ± 5

6.4 ± 1.7

605 ± 9

120 ± 5

6.7 ± 1.7

wt tg6

Ruschitzka et al., PNAS 2000


How do tg6 mice adapt to excessive erythrocytosis?

by NO-mediated vasodilation

by regulating blood viscosity


Elevated eNOS synthesis in Epo-overexpressing tg6 mice

control wt tg6

eNOS (140 kDa)

wt tg6 tg6control

Regulated elevation of blood viscosity in tg6 mice

.

11 23 90

0

20

40

60

80

Hct

0.43

0.89

0.88

blood

wt

wt, hemoconc.

tg

shear rate s-1

ap

pa

ren

t b

loo

d v

isc

oc

ity

(m

Pa.s

)

Note:

tg plasma viscosity is not

altered

Vogel et al. Blood 2001


Enhanced elongation of tg6 erythrocytes (ektacytometry)

**

**

**

tg6

wt

elo

ng

atio

n o

f er

yth

rocy

tes

(%)

0

5

10

12 25 90

**=p<0.01shear rate (s-1)

Vogel et al., Blood 2003

Erythrocytes of camillidae are elliptical

100x

The erythrocytes of llamas, alpacas and camels are

small, flat and oval-shaped.

The lifespan of the llama‘s erythrocyte is 60 days.