Optimal hematocrit for maximal performance in acute and chronic -treated mice

Beat Schulera, Margarete Arrasb,c, Stephan Kellera,d, Andreas Rettichc, Carsten Lundbya, Johannes Vogela, and Max Gassmanna,1

aInstitute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, 8057 Zurich, Switzerland; bDepartment of Clinical Research, University Hospital Zurich, 8091 Zurich, Switzerland; cInstitute of Laboratory Animal Science, University of Zurich, 8057 Zurich, Switzerland; and dCardiovascular Research, Institute of Physiology, University of Zurich and Cardiology, Cardiovascular Center, University Hospital Zurich, 8091 Zurich, Switzerland

Communicated by Ewald R. Weibel, University of Bern, Bern, Switzerland, November 9, 2009 (received for review June 19, 2009) Erythropoietin (Epo) treatment increases hematocrit (Htc) and, The optimal Htc hypothesis is in disagreement with several consequently, arterial O content. This in turn improves exercise studies (17, 18). All of these authors provide evidence that O 2 _ 2 performance. However, because elevated blood viscosity associated delivery and thus exercise performance, including VO2max, re- with increasing Htc levels may limit cardiac performance, it was sug- mained relatively constant with chronic excessive erythrocytosis. gested that the highest attainable Htc may not necessarily be asso- These findings indicate that adaptive mechanisms to excessive ciated with the highest attainable exercise capacity. To test the erythrocytosis exist. proposed hypothesis that an optimal Htc in acute and chronic Epo- To explore the consequences of excessive erythrocytosis in treated mice exists—i.e., the Htc that facilitates the greatest O2 flux vivo we developed a transgenic mouse line (termed tg6) that during maximal exercise—Htc levels of wild-type mice were acutely reaches Htc values of 0.8–0.9 as a result of a constitutive over- elevated by administering novel erythropoiesis-stimulating protein expression of human Epo cDNA (19). Adaptive mechanisms to (NESP; wtNESP). Furthermore, in the transgenic mouse line tg6 that excessive erythrocytosis include increased plasma nitric oxide reaches Htc levels of up to 0.9 because of constitutive overexpres- levels and enhanced erythrocyte flexibility (19, 20). These data sion of human Epo, the Htc was gradually reduced by application of suggest that a shift of the optimal Htc for a maximal endurance the hemolysis-inducing compound phenylhydrazine (PHZ; tg6PHZ). performance to a higher Htc value occurs in tg6 mice. PHYSIOLOGY Maximal cardiovascular performance was measured by using tele- The present study tested the hypothesis that there is an optimal _ metry in all exercising mice. Highest maximal O2 uptake (VO2max) Htc value that allows for maximal systemic endurance perform- and maximal time to exhaustion at submaximal exercise intensities ance. The effect of chronically elevated Htc on exercise capacity were reached at Htc values of 0.58 and 0.57 for wtNESP, and 0.68 and may differ from that of acutely elevated Htc. We propose that mice 0.66 for tg6PHZ, respectively. Rate pressure product, and thus also with excessive erythrocytosis adapt better to varying Htc levels than maximal working capacity of the heart, increased with elevated Htc animals that experience an acute increase in Htc level. For this _ values. Blood viscosity correlated with VO2max. Apart from the con- purpose, wild-type mice were injected with novel erythropoiesis firmation of the Htc hypothesis, we conclude that tg6PHZ adapted stimulating protein (NESP; wtNESP) to increase Htc, tg6 mice better to varying Htc values than wtNESP because of the higher were treated with the hemolysis-inducing compound phenyl- optimal Htc of tg6PHZ compared to wtNESP. Of note, blood viscosity hydrazine (PHZ; tg6PHZ), and both wild-type and tg6 mice that plays a critical role in limiting exercise capacity. did not receive treatment served as controls. Metabolic and car- diovascular measurements were obtained at rest and during blood viscosity | doping | excessive erythrocytosis | exercise performance | endurance performance, whereas whole-blood analysis including hemolysis rheology was carried out at rest.

n a normal physiological hematocrit (Htc) range, eryth- Results Iropoietin (Epo) treatment or red blood cell retransfusion that Male wild-type and tg6 mice were approximately 8 and 9 weeks increases hemoglobin concentration ([Hb]) improves maximal old, respectively, when beginning their respective treatments. As O uptake (VO_ ) and enhances endurance performance expected, differences in Htc were initially observed (Table S1): 2 2max ± (1–6). Little, however, is known regarding the impact of [Hb] whereas wild-type males had an Htc of 0.46 0.03, the con- alteration over a wide-ranging Htc. Augmented [Hb] values are stitutively Epo-overexpressing transgenic tg6 males suffered ± associated with a rise in blood viscosity and, consequently, with a from excessive erythrocytosis expressed Htc values of 0.78 _ 0.06. No differences in age, resting mean arterial blood pressure, higher peripheral vascular resistance that may reduce VO2max _ because of the falling (7, 8). Accordingly, the heart rate, O2 uptake (VO2), or respiratory exchange ratio (RER) exercise capacity of polycythemic patients with chronic ob- were observed between wild-type and tg6 animals at the beginning of the incremental exercise test. structive pulmonary disease (COPD) is improved after hemodi- lution (9). Because of these counteracting effects, it was The Impact of Htc Manipulation. The results of wild-type/wtNESP suggested that the optimal Htc may be lower than expected be- and tg6/tg6PHZ after approximately 4 and 3 weeks of treatment, – cause of limitations induced by a higher blood viscosity (8, 10 respectively, are depicted in Fig. S1 and Fig. S2. Both figures 12). Although the role of Htc on exercise performance seems show [Hb], arterial O2 saturation (SaO2), plasma, and blood obvious, there is only one ex vivo study available addressing the above hypothesis in isolated higher vertebrate muscles (11). It

should be noted, however, that this may vary under different Author contributions: B.S. and M.G. designed research; B.S., M.A., S.K., and J.V. performed circumstances because of the blood's non-Newtonian behavior research; B.S. and M.G. analyzed data; and B.S., A.R., C.L., and M.G. wrote the paper. (13). Factors affecting this variation include the species, the or- The authors declare no conflict of interest. gans involved, and whether the organism is in resting or ex- Freely available online through the PNAS open access option. – ercising conditions (11, 12, 14 16). Thus, the above mentioned 1To whom correspondence should be addressed. E-mail: [email protected]. fl observations do not always re ect the general situation in ex- This article contains supporting information online at www.pnas.org/cgi/content/full/ ercising mammals and humans. 0912924107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0912924107 PNAS Early Edition | 1of5 Downloaded by guest on September 27, 2021 volume in relation to the Htc level of each individual mouse used were expressed as a function of blood volume or total hemoglobin in this study. As shown in Fig. S1 a, there was a linear increase in mass, again a polynomial second-degree relation was observed. [Hb] with greater Htc values in all wild-type and tg6 mice used. The graphs illustrate that approximately twice as much blood Whereas SaO (Fig. S1 b) and plasma volume (Fig. S2 a) did not volume or total hemoglobin mass is necessary to reach maximal 2 _ significantly change in any animal, irrespective of treatment and/ VO2max in tg6PHZ compared to the levels required in wtNESP. or genotype, changes in blood volumes paralleled those seen in Excessive erythrocytosis is known to impair blood flow because Htc levels (Fig. S2 b). As is defined via the (degree two) poly- of an Htc-dependent elevation of blood viscosity, and consequently nomial equation, alterations at lower Htc values had a lesser less O is transported to the tissue (10). In turn, this reduces ex- 2 _ impact on blood volume changes than those that were observed at ercise capacity. We observed a correlation between VO2max and higher Htc levels. Indeed, the increment of blood volume at Htc blood viscosity (Fig. 4). Compared to wtNESP, Epo-overexpressing _ levels from 0.4 to 0.5 was approximately 19 mL/kg for wtNESP tg6 mice reached maximal VO2max at a higher blood viscosity. and 10 mL/kg for tg6PHZ, whereas the increment between 0.6 The association between blood viscosity and Htc in wild-type and and 0.7 was approximately 37 and 50 mL/kg, respectively. tg6 mice was similar with our previous findings (20).

Optimal Htc for Maximal Endurance Performance. Endurance per- Increasing Mean Arterial Blood Pressure, Constant Heart Rate, and _ formance consists of the product of an individual's VO and Altered Stroke Volume with Rising Htc Levels at VO_ . Mean _ 2max 2max duration of exercise at a certain percentage of that VO2max until arterial blood pressure, heart rate, and stroke volume were all exhaustion (21). Thus, to investigate the impact of varying Htc quantified at VO_ . Mean arterial blood pressure rose with _ 2max levels on endurance performance, individual data of VO2max increasing Htc levels in wtNESP and tg6PHZ (Fig. S3 a). (Fig. 1) and time to exhaustion (Fig. 2) were plotted against Htc. Overall, wtNESP reached higher mean arterial blood pressure _ Time to exhaustion and VO2max behaved as a polynomial sec- values compared to those in tg6PHZ. Heart rate did not change ond-degree function in both wild-type and tg6 mice. As such, with increasing Htc levels and also was not different from group _ greater changes in time to exhaustion and VO2max were observed to group (Fig. S3 b). at lower and higher Htc levels. Furthermore, time to exhaustion Previous studies have identified a correlation between stroke was more sensitive to Htc alterations than VO_ . Calculations volume and O pulse during exercise in humans (23, 24). Fig. S3 c _ 2max 2 _ showed that greatest VO2max and time to exhaustion values were shows O2 pulse as a function of Htc at VO2max. The O2 pulse of obtained at Htc values of 0.58 and 0.57 for wtNESP mice, re- wtNESP and tg6PHZ rises slightly with increasing Htc levels, spectively, and 0.68 and 0.66 for tg6PHZ mice, respectively. plateaus at respective maximum Htc values of 0.58 for wild-type mice and 0.68 for tg6 mice, and then continues to decreases with Effect of Varying Blood Volume, Total Hemoglobin Mass, and higher values. Fitted curves display polynomial second-degree Viscosity on Endurance Performance. Besides Htc, blood volume characteristics. In comparison with wtNESP, tg6PHZ showed a and total hemoglobin mass play an important role in cardio- greater impact of varying Htc levels on O2 pulse. vascular performance (2, 3, 22). To determine the blood volume _ _ and total hemoglobin mass that are required to reach maximal Increasing Myocardial VO2 with Rising Htc Levels at VO2max. To study endurance performance, both parameters were correlated with the impact of myocardial VO_ on VO_ , the rate pressure _ _ 2 2max _ VO2max. As shown in Fig. 3, when VO2max of wtNESP and tg6PHZ product was also correlated with Htc levels. Myocardial VO2

· VO2max

160

Ⴄ □ ] □ 140 □ □ ႤႤ -1 □ □ □ □□ Ⴄ Ⴄ □ Ⴄ Ⴄ Ⴄ

·min ·min □

-1 120

□ wt [ml·kg [ml·kg 100 Ⴄ tg6

2max 2max ■ wtNESP ▲ tg6PHZ

· VO 80 Max. wt/wtNESP Max. tg6/tg6PHZ Max. tg6/tg6PHZ 60 0.3 0.4 0.5 0.6 0.7 0.8 0.9 160 Htc

] 140 -1 ·min ·min

-1 120

y = -252.78x 2 + 292.75x + 59.812 [ml·kg [ml·kg 100 R 2 = 0.7319; P < 0.0001

2max 2max y = -337.58x 2 + 461.8x - 14.515 80 2 · VO R = 0.7771; P < 0.0001 Max. tg6/tg6PHZ Max. tg6/tg6PHZ Max. wt/wtNESP 60 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Htc _ Fig. 1. Relationship between hematocrit (Htc) and maximal O2 uptake (VO2max) in wtNESP and tg6PHZ mice. Single prints represent individual values. —, regression plot _ of wild-type (wt)/wtNESP; —, regression plot of tg6/tg6PHZ. Also depicted are maximal VO2max values of wt/wtNESP (Max. wt/wtNESP) and tg6/tg6PHZ (Max. tg6/tg6PHZ).

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.0912924107 Schuler et al. Downloaded by guest on September 27, 2021 Time to exhaustion

120 wt tg6 □ wtNESP 90 tg6PHZ □ □ □□ □ □ □ 60 □ □

30 □ Time to exhaustionTime [min] Max. tg6/tg6PHZ Max. wt/wtNESP 0 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Htc 2 120 y = -1225.5x + 1389x - 298.72 R2 = 0.7691; P < 0.0001

y = -575.87x2 + 764.75x - 170.32 90 R2 = 0.7719; P < 0.0001

60

30 Time to exhaustionTime [min] Max. wt/wtNESP Max. tg6/tg6PHZ 0 0.3 0.9

0.4 0.5 0.6 0.7 0.8 PHYSIOLOGY Htc Fig. 2. Relationship between hematocrit (Htc) and time to exhaustion in wtNESP and tg6PHZ mice. Singles prints represent individual values. —, regression plot of wild-type (wt)/wtNESP; —, regression plot of tg6/tg6PHZ. Also depicted are maximal time to exhaustion values of wt/wtNESP (Max. wt/wtNESP) and tg6/tg6PHZ (Max. tg6/tg6PHZ).

increased with increasing Htc values in both groups (Fig. 5). each organ may present with its own individual optimal Htc (11, Both graphs showed a similar slope, but wtNESP mice had 16). Therefore, the systemic Htc value must be interpreted as an higher rate pressure product values at corresponding Htc levels average of all organ-specific optimal Htc values, thereby pro- than did tg6PHZ, indicating that the hearts of wtNESP mice had viding the whole organism with adequate O2 supply. During a higher requirement of myocardial O2 supply. exercise more O2 is required by the working skeletal muscles and thus a shift from the optimal physiological to the optimal Htc Discussion value for maximal endurance performance is observed. To cover The present study demonstrates that optimal Htc values max- their high O2 demand during strenuous exercise, some terrestrial imizing systemic endurance performance in mammals exist. In vertebrate species, such as horses and dogs, but not mice, release doing so we observed that NESP-treated wild-type animals stored erythrocytes into the circulation by splenic contraction maximized exercise performance at a lower optimal Htc than (26, 27). This mechanism elevates the blood O2-carrying capacity hemolysed tg6PHZ mice constitutively overexpressing Epo. during exercise within seconds. As a consequence, Htc increases Furthermore, the data demonstrated that (i) the optimal Htc from ≈0.4 at rest to ≈0.6 at exercise (28, 29) facilitating an im- levels for maximal systemic exercise performance and maximal provement in exercise performance. These hematopoietic pa- stroke volume were similar; and (ii) blood volumes were dra- rameters return quickly to resting values when the animals stop matically increased at higher Htc levels. exercising, preventing a constant overload of the cardiovascular _ VO2max as well as time to exhaustion as well as initially rose, system (28–30). In light of our data, it is tempting to speculate reached a maximum value, and then decreased with increasing that horses and dogs temporarily elevate their Htc levels close to _ Htc values. Maximal VO2max and time to exhaustion values were the optimal value Htc to reach maximal systemic endurance found at Htc levels of 0.58 and 0.57 for wtNESP, and 0.68 and performance. Indeed, splenic contraction has also been observed 0.66 for tg6PHZ, respectively. Our data are in agreement with in humans during performing a maximal exercise test, as evi- the classical optimal Htc hypothesis (8, 10–12, 25), where it was denced by a reduction in its volume and two-thirds decrease in speculated that the optimal systemic Htc of mammals would be splenic erythrocyte content, but Htc levels increased only a very much higher than the commonly observed 0.45. These calcu- small percentage from rest to maximal working levels (31). _ lations were based on the conditions present in the circulatory At first glance, the observed relationship between VO2max and system at rest, as exercise induces changes in vessel diameters, Htc in our study is in contradiction to others described in human blood flow, internal temperature, and blood distribution. How- studies. Several investigators have shown that there is a strong ever, before this study there was no experimental proof of this correlation between VO_ and total hemoglobin mass or blood 2max _ concept in living mammals. Gaethgens and coworkers (11) volume (2, 3, 22), but not between VO2max and Htc (32). It is of studied the effect of blood perfusion creating various Htc levels note, however, that most human studies are carried out with Htc in isolated dog muscle during rhythmic isotonic exercise. Max- levels between 0.4 and 0.5. Thus, the ergogenic effect of total _ imal VO2 and contractile power reached a plateau at Htc levels hemoglobin mass and blood volume as the primary limiting factors between 0.4 and 0.7. The authors concluded that these results of performance in endurance sports seems only to be valid in the may not be transferable to whole-body systemic exercise because physiologically occurring Htc range. At higher Htc levels, other

Schuler et al. PNAS Early Edition | 3of5 Downloaded by guest on September 27, 2021 A Blood volume as long as heart rate is not altered. However, after reaching a 160 maximum of 0.57 for wtNESP and 0.68 for tg6PHZ, the blood viscosity may have a negative contribution to cardiac perform- ]

1- 140 ance. High blood viscosity increases arterial blood pressure and nim· diminishes venous return because of the increased peripheral

1- 120 resistance (12, 34), a fact that may reduce stroke volume and gk·lm[ thus exercise performance. A converse observation is made in 100 patients suffering from polycythemic COPD after phlebotomy xam2 y = -0.0022x2 + 0.6703x + 93.356 wt R2 = 0.6891; P < 0.0001 tg6 (9). In these patients, an improvement in exercise tolerance OV · 80 y = -0.001x2 + 0.5474x + 76.706 wtNESP appears to be due to an improvement in cardiac function as R2 = 0.4472; P < 0.0001 tg6PHZ evidenced, primarily, by an increased stroke volume. The close 60 _ correlation between VO2max and blood viscosity, as well as be- 50 100 150 200 250 300 350 400 450 tween stroke volume, arterial blood pressure, and Htc, confirms -1 Blood volume [ml·kg ] this explanation. Moreover, the fact that rate pressure product and arterial blood pressures were increasing with incremental Total hemoglobin mass _ B elevation of Htc levels shows that VO2max was unaffected by the 160 maximal work capacity of the heart at optimal Htc. The different optimal Htc levels obtained by increasing the ]

1- 140

nim· normal physiological Htc of wild-type mice and reducing the

1- 120 elevated Htc of tg6 mice could be resolved by at least two mech- gk·lm[ anisms: (i) enhancement of endothelial nitric oxide synthase ac-

100 y = -40.341x2 + 61.123x + 122.03 tivity, which results in peripheral vasodilation despite concomitant xam2 wt 2 increased endothelial-1 levels (19, 35), and (ii) regulated elevation R = 0.635; P < 0.0001 tg6

OV fl · 80 y = -14.648x2 + 48.38x + 108.38 wtNESP of blood viscosity by increasing erythrocyte exibility in tg6 mice R2 = 0.4006; P < 0.0001 tg6PHZ compared to their controls (20). Both mechanisms would induce a 60 shift to a higher optimal Htc value in the tg6 mice. Thus, tg6PHZ 0 0.5 1 1.5 2 2.5 3 might be able to adapt better to varying Htc levels than wtNESP. Total hemoglobin mass [g] Physiological adaptations to excessive erythrocytosis are also ob- _ served in dogs and humans (17, 18). Moreover, one case report in Fig. 3. Relationship between maximal O2 uptake (VO2max) and blood vol- ume (a) and total hemoglobin mass (b) during terminal determination in sport medicine describes a successful Finnish cross-country skier wtNESP and tg6PHZ mice. Singles prints represent individual values. —,re- with an autosomal dominant mutation in Epo receptor that re- gression plot of wild-type (wt)/wtNESP; —, regression plot of tg6/tg6PHZ. sulted in increased sensitivity of erythroid progenitors to Epo that ultimately led to Htc levels of 0.68 (17). That endurance athlete won several Olympic gold medals. Based on our study, we con- factors, such as blood viscosity, may counteract the positive effects clude that his Htc may be very close to the optimal Htc for max- of enhanced arterial O2 content because the total hemoglobin mass, imal endurance performance. unlike to Htc or [Hb], cannot be masked by other factors. In In summary, the results of the present study confirm the optimal agreement with this hypothesis, we found that the closest relation- Htc hypothesis during systemic exercise in mice. The reason for this _ ship betweenVO2max and total hemoglobin mass in wtNESP ani- is that blood viscosity increases with rising Htc levels, limiting the R2 P < mals is within the Htc range of 0.4 and 0.55 ( = 0.513; 0.001). blood's O2 transport capacity. Furthermore, animals with chronic The augmented blood volume at higher Htc values observed in excessive erythrocytosis adapted better to different Htc levels than tg6 mice is caused by the dramatically increased number of did acutely NESP-injected animals. At normoxia, the heart can erythrocytes, whereas plasma volume remains unchanged (19). tolerate higher rate pressure product at higher Htc levels. Thus, the An elevation in blood volume enhances end-diastolic volume optimal Htc values for maximal endurance performance were not (preload) and results in increased stroke volume via the Starling caused by heart failure or attainment of the maximal working ca- _ _ mechanism (33), which will lead to the enhancement of VO2max pacity of the heart, VO2max is mainly limited by O2 delivery.

Blood viscosity wt 160 tg6 wtNESP tg6PHZ ]

1- 140 nim·

1- 120 gk·lm[ PSENtw/ ZHP6gt/6gt .xaM ZHP6gt/6gt

100 2 xam2 y = -1.2907x + 12.664x + 113.88 R2 = 0.4339; P < 0.0001 tw .x tw OV · 80 y = -1.7469x2 + 24.414x + 64.448

aM R2 = 0.5419; P < 0.0001 60 24681012 Blood viscosity [mPas] _ Fig. 4. Relationship between maximal O2 uptake (VO2max) and blood viscosity during terminal determination in wtNESP and tg6PHZ mice. Singles prints _ represent individual values. —, regression plot of wild-type (wt)/wtNESP; —, regression plot of tg6/tg6PHZ. Also depicted are maximal VO2max values of wt/ wtNESP (Max. wt/wtNESP) and tg6/tg6PHZ (Max. tg6/tg6PHZ).

4of5 | www.pnas.org/cgi/doi/10.1073/pnas.0912924107 Schuler et al. Downloaded by guest on September 27, 2021 Rate pressure product

170 wt ] 1- tg6 tcudorperusserp et tcudorperusserp nim· wtNESP

1- 130 tg6PHZ staeb·gHmm[·0001

2 90 y = 53811x + 39477x + 78548 R2 = 0.8038; P < 0.0001 a R y = 41876x2 + 46022x + 66746 R2 = 0.7488; P < 0.0001 50 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Htc _ Fig. 5. Relationship between hematocrit (Htc) and rate pressure product pulse at maximal O2 uptake (VO2max) in wtNESP and tg6PHZ mice. Singles prints represent individual values. —, regression plot of wild-type (wt)/wtNESP; —, regression plot of tg6/tg6PHZ.

Materials and Methods that were 4 weeks old. In the remaining animals, dummy transmitters were Experimental Animals and Set-up. The constitutively Epo-overexpressing tg6 implanted (wild type, n = 21; tg6, n = 21). Adjustments of the Htc levels were mouse line was generated as described in ref. 19. Compared to wild-type started in 8- and 9-week-old animals, respectively. At an age of 12 weeks, control, the tg6 mouse line had a 10- to 12-fold increase in plasma Epo- the main experiments were conducted, including incremental as well as levels, resulting in Htc levels of up to 0.9 (19, 20). Approximately half of the constant workload exercise tests (see below) followed by measurements of offspring were hemizygous for the transgene and were used for the he- the SaO2, [Hb], Htc, blood viscosity, plasma, and blood volume. To exclude molysis-inducing experiments, whereas the other half were used as wild type the impact of circadian rhythm, all measurements were performed at the

for the hemoconcentration experiments. Males were 12 weeks old during identical time of day. PHYSIOLOGY the first exercise test (Table S1). In total, 41 wild-type mice and 40 tg6 mice Other detailed methods are provided in SI Materials and Methods. were analyzed. No weight loss occurred during the study period. Mice were kept in standard rodent cages (T3) with food and water supplied ad libitum Statistics. All data were analyzed by using StatView software (Version 4.57; in a 12 h/12 h light-dark cycle. The experimental protocols were approved by Abacus Concepts). The relationship between the two parameters was ana- the Kantonales Veterinäramt Zürich and were performed in accordance with lyzed with linear or polynomial regression. Significances were performed by a the Swiss animal protection laws and institutional guidelines. one-way analysis of variance (ANOVA). Results are expressed as mean ± SD. The experimental design is shown in Fig. S4. At an age of 3 weeks, only tg6 Statistical difference was set at P < 0.05. mice were splenectomized to keep Htc levels low, because extramedullary erythropoiesis occurs in the spleen (20). One week later, telemetric blood ACKNOWLEDGMENTS. This work was supported by the Forschungskredit pressure transmitters were implanted in 20 wild-type mice and 19 tg6 mice (University of Zurich) and the Swiss National Science Foundation.

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