This document summarizes a presentation given by Prof. Max Gassmann on the impacts of erythropoietin (Epo) on circulation and exercise performance. Some key points: - Epo increases red blood cell production and hematocrit levels, improving oxygen delivery. However, too high hematocrit levels can reduce performance due to increased blood viscosity. - Transgenic mice overexpressing Epo had hematocrits over 80% and showed reduced exercise capacity, as well as organ damage over time. - The optimal hematocrit for maximal endurance performance in mice is around 57-68%. Above this, increased blood viscosity limits performance. - Long-term complications can occur in humans and animals

1. San Francisco
Prof. Max Gassmann
Erythropoietin’s impact on
and exercise performance
Chairman of the Institute of Veterinary Physiology,
Vetsuisse Faculty, and of the
Zurich Center for Integrative Human Physiology
(ZIHP), University of Zurich
Gaming the System:
Doping in Sports
swissnex San Francisco
August 20, 2012
Chart 1

2. circulation
reduced
oxygen
Epo
Epo
anti-apoptotic effect
bone on erythroid
oxygen marrow precursor cells
sensing:
HIF-2 + neg.Feedback
-
Epo-synthesis
Hb-O2 erythrocytes
O2 oxygen
uptake +
hematocrit +
Institute of Veterinary Physiology Chart 2

3. rhEpo has been developed to treat anemic
patients and not to dope cheating athletes
Institute of Veterinary Physiology Chart 3

4. Mouse models to study the impact
of acute and chronically elevated
Epo levels
• 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
Ruschitzka et al., PNAS 2000
Wiessner et al., JCBF&M 2001
Institute of Veterinary Physiology Chart 4

5. Epo-overexpressing transgenic
mouse lines tg21 and tg6:
hematological parameters
wt+tg21 tg6
plasma Epo U/l 22 260
red blood cells 1012 / l 6.2 13.8
hemoglobin g/l 140 235
hematocrit % 40 79 ( 90)
reticulocyte counts % 1.7 3.4
blood volume ml 2.2 5.5
arterial pO2 mmHg 125 133
arterial oxygen content ml / dl 17 31
Institute of Veterinary Physiology Chart 5

6. The higher the hematocrit the higher the performance?

7. Reduced exercise performance in
Epo-overexpressing tg6 mice with a
hematocrit of exercise performance in
Reduced ~80 %
EPO- overexpressing mice with a hematocrit of ~80 %
swimming running
0.22 100
exercise time (min)
swim speed (m/s)
0.20
wt 75
0.18
50
0.16
25
0.14 tg6
0.12 0
20 40 60 80 100 120 wt tg6
seconds
Institute of Veterinary Physiology Chart 7

8. An endurance athlete copes with a
chronically elevated hematocrit
Eero Mäntyranta (born 1937)
hematocrit: 68%
won 6 gold medals between 1960-1966

9. An optimal hematocrit for maximal
performance?
wt tg6
Hematocrit: 0.4 Hematocrit: 0.8-0.9


Hematocrit Hematocrit
NESP (“Epo”) Phenylhydrazine (PHZ)
leads to hemolysis
acute Treadmill chronic
Institute of Veterinary Physiology Chart 9

10. Maximal performance is reached at
hematocrit levels 0.57-0.68
·
VO2max
Time to exhaustion
.
Schuler et al., PNAS 2010

11. What is the limiting factor? Viscosity!
∙
VO2max vs. blood viscosity

12. Conclusions
The present study confirms our hypothesis that
optimal hematocrit during systemic exercise in
mice exists.
Maximal working capacity of the heart was not
reached at the optimal hematocrit for maximal
endurance performance.
Blood viscosity is the most likely candidate to limit
exercise performance.

13. 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

14. Complications in mice suffering from
Epo-induced excessive erythrocytosis
Institute of Veterinary Physiology Chart 14

15. Degenerative process in the liver of 5
month-old tg6 mice
wt tg6
tg6
->: hemosiderin 50µm
 : inflammation
Institute of Veterinary Physiology Chart 15

16. Degenerative processes in the sciatic
nerve of 5 month-old tg6 mice
20µm 2µm
A: Axoplasma SC: Schwam cells  axonal swelling
ML: Myelin lamellae Co: Collagen fibres  deformed fibres
Institute of Veterinary Physiology Chart 16

17. Hindlimb paralysis and neuromuscular
degeneration in tg6 mice
Motor disorders characterized by bi- or unilateral spastic
contraction and paralysis of the hindlimbs
Institute of Veterinary Physiology Chart 17

18. Degenerated skeletal muscle fibres
in tg6 mice
wt tg6
= muscle capillaries
Institute of Veterinary Physiology Chart 18

19. circulation
reduced
oxygen
Epo
Epo
anti-apoptotic effect
bone on erythroid
oxygen marrow precursor cells
sensing:
HIF-2 + neg.Feedback
-
Epo-synthesis
Hb-O2 erythrocytes
O2 oxygen
uptake +
hematocrit +
Institute of Veterinary Physiology Chart 19

20. Repoxygen® was intended for gene therapy
Recombinant
epo
oxygen HIF
gene therapy
HRE hEpo DNA
- HIF is present in all cells of the human body
- 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
Institute of Veterinary Physiology Chart 20

21. Repoxygen®: no further development
HIF
- Is not in clinical trials HRE hEpo DNA
- Economics: Repoxygen vs. classical project is stopped
rhEpo
- 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
Institute of Veterinary Physiology Chart 21

22. oxygen
low normal
blood loss (normoxia)
anemia
altitude
Hypoxia-Inducible Factor Hypoxia-Inducible Factor
(HIF): stabilized (HIF): degraded by PHD‘s
HIF HIF
PHD„s
+ O2
induce
HRE HIF target genes DNA
Hypoxia - Erythropoietin (Epo)
Response - Vascular Endothelial Growth Factor
Element - etc.
Institute of Veterinary Physiology Chart 22

23. Acknowledgement
s
B. Schuler E. Vannoni
J. Vogel D. Wolfer
A. Bogdanova C. Lundby
L. Ogunshola F. Ruschitzka
J. Soliz Th. Lüscher
B. Grenacher M. Maggiorini
M. Alvarez
R. Jacobs
V. Diaz
Institute of Veterinary Physiology Chart 23

24. Institute of Veterinary Physiology Chart 24

25. Institute of Veterinary Physiology Chart 26

26. Epo-overexpressing tg6 mouse:
resting hemodynamic parameters
wt tg6
heart rate beats/min 569 ± 18 605 ± 9
systolic blood pressure mmHg 119 ± 5 120 ± 5
cardiac output ml/min 6.4 ± 1.7 6.7 ± 1.7
Ruschitzka et al., PNAS 2000
Institute of Veterinary Physiology Chart 27

27. How do tg6 mice adapt to excessive
erythrocytosis?
by NO-mediated vasodilation
by regulating blood viscosity
Institute of Veterinary Physiology Chart 28

28. Elevated eNOS synthesis in Epo-
overexpressing tg6 mice
control wt tg6
eNOS (140 kDa)
control wt tg6 tg6
Institute of Veterinary Physiology Chart 29

29. Regulated elevation of blood
viscosity in tg6 mice
Hct blood
0.43 wt
80 0.89 wt, hemoconc.
0.88 tg
.
apparent blood viscocity (mPa.s)
60
40 Note:
tg plasma
viscosity is not
altered
20
0 Vogel et al. Blood 2001
11 23 90
shear rate s-1

30. Enhanced elongation of tg6 erythrocytes
(ektacytometry)
tg6
elongation of erythrocytes (%)
wt
10
*
* *
5 *
*
*
0
12 25 90
shear rate (s-1)
**=p<0.01
Vogel et al., Blood 2003
Institute of Veterinary Physiology Chart 31

31. 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.