Effects of erythropoietin on systemic hematocrit and oxygen transport in the splenectomized horse

Accepted Manuscript Title: EFFECTS OF ERYTHROPOIETIN ON SYSTEMIC HEMATOCRIT AND OXYGEN TRANSPORT IN THE SPLENECTOMIZED HORSE Author: Kenneth H. McKeever Beth A. McNally Kenneth W. Hinchcliff Robert A. Lehnhard David C. Poole PII: DOI: Reference:

S1569-9048(16)30009-X http://dx.doi.org/doi:10.1016/j.resp.2016.02.001 RESPNB 2612

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Respiratory Physiology & Neurobiology

Received date: Revised date: Accepted date:

5-9-2015 30-1-2016 2-2-2016

Please cite this article as: McKeever, Kenneth H., McNally, Beth A., Hinchcliff, Kenneth W., Lehnhard, Robert A., Poole, David C., EFFECTS OF ERYTHROPOIETIN ON SYSTEMIC HEMATOCRIT AND OXYGEN TRANSPORT IN THE SPLENECTOMIZED HORSE.Respiratory Physiology and Neurobiology http://dx.doi.org/10.1016/j.resp.2016.02.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

EFFECTS OF ERYTHROPOIETIN ON SYSTEMIC HEMATOCRIT AND OXYGEN TRANSPORT IN THE SPLENECTOMIZED HORSE Kenneth H. McKeever1, Beth A. McNally2, Kenneth W. Hinchcliff3 Robert A. Lehnhard4, and David C. Poole5

1Department

of Animal Science, Rutgers the State University of New Jersey, New Brunswick, NJ 08903; of Health, Physical Education and Recreation, The Ohio State University, Columbus, OH, 43210, 3Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Melbourne, Australia, 4Department of Kinesiology, University of Maine, Orono, Maine, and 5Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University. 2School

Running Head: Erythropoietin and Exercise

Address for Correspondence: Kenneth H. McKeever, Ph.D., FACSM Department of Animal Science Rutgers the State University of New Jersey New Brunswick, NJ 08901 Telephone:

(848) 932-9390

Fax:

(732) 932-6996

Email:

[email protected]

Highlights  Three weeks of erythropoietin treatment (EPO) in the splenectomized horse significantly increases systemic arterial hematocrit and [hemoglobin] concomitant with reduced plasma volume.

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

During maximal exercise, cardiac output is unchanged and V O2max (and exercise performance) is enhanced 19% by substantially augmented perfusive (increased 20%) and diffusive (increased 30%) O2 transport: the latter suggesting that the increased systemic hematocrit raises capillary [hemoglobin] and facilitates blood-myocyte O2 flux.



Whereas this polycythemia increases blood viscosity in situ, vascular pressures at V O2max are unchanged suggesting either that vascular changes increase hemodynamic conductance or, alternatively, equine blood in vivo during maximal exercise does not increase viscosity as seen in situ.

Abstract To test the hypotheses that erythropoietin (rhuEPO) treatment increases systemic hematocrit, maximal O2 uptake ( V O2max, by elevated perfusive and diffusive O2 conductances) and performance five female horses (4-13 y) received 15 IU/kg rhuEPO (erythropoietin) three times per week for three weeks. These horses had been splenectomized over 1 year previously to avoid confounding effects from the mobilization of splenic red blood cell reserves. Each horse performed three maximal exercise tests (one per month) on an inclined (4o) treadmill to the limit of tolerance; two control trials and one following EPO treatment. Measurements of hemoglobin concentration ([Hb] and hematocrit), plasma and blood volume, V O2, cardiac output as well as arterial and mixed venous blood gases were made at rest and during maximal exercise. EPO increased resting [Hb] by 18% from 13.3+0.6 to 15.7+0.8 g/dL (mean +SD) corresponding to an increased hematocrit from 36+2 to 46+2% concurrent with 23 and 10% reductions in plasma and blood volume, respectively (all P<0.05). EPO elevated V O2max by 20% from 25.7+1.7 to 30.9+3.4 L/min (P<0.05) via a 17% increase in arterial O2 content and 18% greater arteriovenous O2 difference in the face of an unchanged cardiac output. To achieve the greater V O2max after EPO, diffusive O2 conductance increased ~30% (from 580+76 to 752+ 166 ml O2/mmHg/min, P<0.05) which was substantially greater than the elevation of perfusive O2 conductance. These effects of EPO were associated with an increased exercise performance (total running time: control, 216 + 72; EPO, 264 + 48 s, P<0.05). We conclude that EPO substantially increases V O2max and performance in the splenectomized horse via improved perfusive and diffusive O2 transport. Key words: maximal aerobic capacity; perfusive and diffusive O2 transport; cardiac output; arteriovenous O2 difference

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1.

INTRODUCTION The maximal O2 uptake ( V O2max) defines the upper limit for O2 transport from the

atmosphere to its site of reduction in the mitochondrial electron transport chain. Across a broad range of species V O2max is determined by O2 supply as distinct from the mitochondrial oxidative capacity (rev. Wagner et al. 1997; Poole and Erickson, 2011; but see also Weibel and Hoppeler, 2005). Supporting the highly integrated functioning of the O2 transport system, for the whole body (Roca et al. 1989; 1992) or exercising muscle(s) (Hogan et al. 1988,1989,1990,1991), perfusive (blood flow x arterial [O2]) and diffusive (transmembrane O2 transport) conductances conflate to yield a given V O2max (rev. Wagner et al. 1997). It is generally, but not always (Gonzalez et al. 1994; Prommer et al. 2007), recognized that total hemoglobin (and therefore red blood cell) mass, an index of O2 delivery potential, correlates closely with V O2max in humans (Buick et al. 1980; Convertino, 1991; Ekblom et al. 1975; Gledhill, 1985; Saltin and Strange, 1992; Schaffartzik et al. 1993; Woodson et al. 1978; rev. Schmidt and Prommer, 2010), horses (Wagner et al. 1995) rats (Gonzalez et al. 1994) and dogs (Hsia et al. 2007). In marked contrast, relatively scant attention has been afforded the contribution of the whole body or muscle(s) O2 diffusing capacity and how it might be impacted by altered [hemoglobin] and what research there is on the topic paints a controversial picture. Currently accepted models (Federspiel and Popel, 1986; Groebe and Thews, 1990) and experimental evidence in frog skin (Malvin and Wood, 1992) support that O2 diffusing capacity is determined by the number of red blood cells in the capillaries immediately adjacent to the muscle fibers or tissue. Thus, if microvascular hematocrit were to change in concert with systemic hematocrit, alterations in systemic [hemoglobin] induced by blood transfusion or the action of erythropoietin, for example, should translate directly to proportional changes in O2 diffusing capacity during maximal exercise. However, direct measurements in the microcirculation suggest that capillary hematocrit (and changes thereof) may be dissociated from systemic (Sarelius, 1989). It is not surprising, therefore, that manipulations of systemic hematocrit may (dog, Hsai et al. 2007; control rat, Gonzalez et al. 1994; horse (splenectomy), 3

Wagner et al. 1995) or may not (humans, Lundby et al. 2008; altitude acclimatized, Gonzalez et al. 1994; horse (splenectomy+transfusion), Wagner et al. 1995) impact O2 diffusing capacity during maximal exercise. The horse (Wagner et al. 1995; Poole and Erickson, 2011) and other highly aerobic vertebrates such as the rainbow trout (Gallaugher et al. 1995), diving mammals such as the Weddell seal (Hurford et al. 1996) and dogs (Hsia et al. 1992) exhibit a powerful splenic contraction in response to exercise as initially considered by Barcroft and colleagues (Barcroft and Poole, 1927; Barcroft and Stephens, 1927). For instance, the horse, by releasing splenic red cell reserves (at 80-90% hematocrit) into the systemic circulation may raise systemic hematocrit from ~35% at rest to 60-70% during maximal exercise thereby almost doubling perfusive O2 transport capacity (rev. Poole and Erickson, 2011). Splenectomy prevents this hemoconcentration and reduces perfusive (i.e., cardiac output x arterial [O2]) and diffusive O2 conductance or capacity (Wagner et al. 1995). Interestingly, in a small group (n=3) of splenectomized horses transfused with nearly 12 L RBCs, both perfusive and diffusive O2 transport were higher, as expected, but surprisingly the magnitude of increase was far greater for perfusive (38%) than diffusive (14%) O2 transport (Wagner et al. 1995). Altitude acclimatized rats (Gonzalez et al. 1994) and erythropoietin-treated humans (Lundby et al. 2008) also have been reported not to increase O2 diffusing capacity. It is argued that, because the intact horse naturally achieves high systemic hematocrits, the splenectomized horse constitutes a highly physiological and ecologically-relevant model in which to address the effects of erythropoietin-increased systemic hematocrit on the relationships among perfusive and diffusive O2 capacity, V O2max and performance. Accordingly, given the scientific uncertainties regarding the relation between systemic hematocrit and O2 diffusing capacity as well as the strong association between V O2max and performance in horses and the potential for erythropoietin use and abuse in the racing industry, we tested the following original hypotheses in the splenectomized horse: 1. Erythropoietin will increase systemic hematocrit by elevating total red cell mass and decreasing plasma volume. 2. Irrespective of increased systemic hematocrit associated changes in blood

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viscosity, cardiac output at maximal exercise will not be impaired. 3. Increased systemic hematocrit will elevate V O2max by means of improvements in both perfusive and diffusive O2 conductances. 4. Elevations in V O2max will translate into improved exercise performance.

2.

MATERIALS AND METHODS

2.1

Subjects Splenectomized horses (n=5, mares) were selected for investigation to eliminate acute

and chronic confounding effects of the splenic red cell reserve on blood and red cell volume. Ages ranged from 4 to 13 y. The mares were not specifically exercise trained, yet acclimated to performing maximum incremental exercise tests on a high speed treadmill (Sato Treadmill, Uppsala, Sweden). Uncontrolled exercise among trials was prevented by individually housing mares in box stalls for the duration of the study. Each mare had its left carotid artery surgically relocated to a subcutaneous position to facilitate arterial catheterization and blood sampling. Splenectomy and arterial relocation surgeries were performed over one year prior to initiation of the experiments. This investigation was performed in accordance with the Guiding Principles in Care and Use of Animals of the American Physiological Society and all procedures were approved by the Institutional Laboratory Animal Care and Use Committees of The Ohio State University and Rutgers University.

2.2

Experimental Design Each horse participated in three incremental maximal exercise tests, i.e., two control

trials (1 and 2) and one experimental trial (erythropoietin). The two control trials, were separated by 4 wk, and were conducted prior to erythropoietin treatment. Before the erythropoietin trial, rhuEPO (15 IU/kg bw) was administered intravenously three times a week for 3 wk. Prior to each injection, 2 mL of venous blood were drawn into heparinized vacuum (Vacutainer, Becton Dickenson, Parsippany, NJ) tubes for the measurement of hematocrit, total solids, and [Hb]. Administration of rhuEPO (Epogen, Amgen, Inc., Thousand Oaks, CA) occurred

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between 1000 and 1400 h on days 1, 3, and 5 of each week. Each horse received oral iron supplementation (four 100 mg iron tablets) daily throughout the rhuEPO treatment phase. One of the major side effects of rhuEPO in humans is the development of polycythemia (McKeever, 1996; Spriet, 1991). Because limited dosage studies have been conducted on horses (Jaussaud et al. 1994; McKeever et al. 1993; Souillard et al. 1996), resting hematocrit was monitored throughout rhuEPO treatment to ensure that excessive increases in hematocrit were not present. The experimental test (erythropoietin) was conducted 4 wk after control trial 2 (1 wk after the last dose of rhuEPO).

2.3

Trial & Exercise Test Protocol Each horse was weighed immediately preceding the exercise trials. All of the tests were

conducted between 1000-1400 h in a climate controlled room at 20-21oC. Trials began with a 1 h catheterization and instrumentation period followed by a 30 min equilibration period while the horse stood quietly on the treadmill. Baseline data were collected during the last 5 min of this period. The horses then underwent an incremental maximal exercise test as described below. The exercise test was performed on a high speed treadmill (Sato I, Uppsala, Sweden) to measure V O2max and the hemodynamic responses to running. During the test horses ran up a fixed grade of 4o. The test started at an initial treadmill speed of 3 m/s. Speed was then increased 1 m/s every 1.5 min until the horse reached the limit of tolerance (Evans and Rose, 1988; Hinchcliff et al. 1996). That limit was determined by the inability of the horses to maintain the required pace with further increases in speed despite humane encouragement. V O2 was measured at 10 s intervals using an open circuit calorimeter (Oxymax-XL, Columbus Instruments, Columbus, OH). The calorimeter was calibrated prior to each trial using known concentrations of gas and a previously-reported nitrogen dilution technique (Fedak et al. 1981; Hinchcliff et al. 1996). Hemodynamic data were measured continuously and were recorded at 6 s intervals starting with the 5 min baseline data collection period until the end of the recovery period (McKeever et al. 1993ab). Data collected during the last 30 s of the treadmill test were averaged and used for statistical analysis. Mixed venous (pulmonary artery) and arterial blood

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samples were collected simultaneously at rest and during the last 15 s of the incremental exercise test.

2.4

Instrumentation & Hemodynamic Measurements. One hour before each experiment the mares were catheterized for blood pressure

measurements and sample collections. All catheterizations were performed using sterile techniques under local anesthesia with 2% lidocaine. The right atrial (RAP) and right ventricular (RVP) pressures were measured with a dual-sensor micromanometer catheter (Millar Instruments, Houston TX) inserted into the heart through an introducer catheter (7 French, Argon Medical Corp, Athens, TX) placed into the left jugular vein. A #240 polyethylene catheter and a thermistor (Physitemp Instruments, Clifton, NJ) were inserted into the right jugular vein and positioned in the pulmonary artery (via two introducer catheters) for sampling and temperature-correcting mixed venous blood. Carotid artery pressure was measured with a fluid-filled catheter (Intracath, 18 gauge, 12 in, Deseret, Sandy, UT) attached to a Gould P23 pressure transducer. A carotid catheter was inserted percutaneously into the surgicallyelevated left carotid artery. Cardiovascular measurements made using the pressure sensing catheters included RAP,RVP, heart rate (HR), and carotid artery pressure. Calculated variables included cardiac output ( V O2/(a-v)O2), stroke volume (SV = cardiac output/HR), mean arterial pressure [MAP = 1/3(systolic - diastolic blood pressure)+ diastolic blood pressure], and total peripheral resistance (TPR = MAP/cardiac output). The analog data from the pressure sensing catheters were collected continuously via a hemodynamic monitoring/recording system (Model VR-12, Pleasantville, NY). The output of the VR-12 was channeled through an analog-to-digital conversion system (Buxco Datalogger, Buxco Systems, Sharron, CT) for digitization, and stored both on paper and on computer disk for later analysis. The position of all catheters was verified before and after each trial using characteristic wave forms seen on the oscilloscope of the physiological recorder (Model VR-12, PPG Biomedical, Pittsburg, PA). Calibration and linearity of the Millar micromanometer and Gould P23 pressure transducers were checked before each experiment by comparing their output with that of a mercury manometer. The fluid filled transducers were zeroed at the level of the heart. 7

2.5

Blood Samples A total of 20 mL of blood was collected anaerobically at rest and maximal exercise.

Arterial and mixed venous samples (3 mL each) were drawn simultaneously into heparinized syringes and placed immediately on ice. Two 7 mL mixed venous samples were collected and placed into tubes containing ethylenediaminetetracetate (EDTA). Arterial blood samples were obtained from the carotid artery via the fluid-filled catheter used to measure MAP. Patency of the catheters was maintained by flushing with heparinized saline. Packed cell volume (PCV) and total solids (TS) were determined immediately following each trial using one of the 7 mL samples of mixed venous blood from each stage of the incremental exercise test. The PCV (or hematocrit) was measured in triplicate using well mixed samples and the microhematocrit technique. Total solids, used to estimate plasma protein concentration, were also measured in triplicate by refractometry (Refractometer, Cambridge Instruments Inc., Buffalo, NY). Intraassay coefficients of variation for PCV and TS were 1.6% (n=24) and 0.7% (n=24), respectively. The remaining portion of the 7 mL sample used for PCV and TS determinations was centrifuged at 1500 g for 15 min at 4oC. The supernatant was transferred into disposable sterile cryogenic vials (Corning) and frozen at -80oC for the measurement of plasma lactate concentration, which was determined in duplicate using a lactate analyzer (YSI Model L23, Yellow Springs, OH). Plasma volume determinations were made using a modified Evans Blue dye method (McKeever et al. 1988) during the instrumentation period, while the horses stood in stocks. Two 7 mL samples of blood were drawn and placed into tubes containing the anticoagulant EDTA. One of these samples was obtained prior to and one 15 min following injection of Evans blue dye. These samples were used for the determination of plasma volume (McKeever et al. 1988). Intra-assay coefficients of variation were 1.7% (n=20) for lactate concentration and 0.8% (n=6) for plasma volume determination. Hemoglobin concentrations and oxygen contents (CaO2, CvO2) of arterial and venous blood samples were measured using a hemoglobin-oximeter (Co-Oximeter Model 0M-3, Radiometer Inc., Copenhagen). Each sample was assayed in triplicate, and all samples were analyzed within 6 h following each trial. The intra-assay coefficients of variation for coefficients of [hemoglobin] and O2 content were 0.1% 8

and 0.8%, respectively, and were determined with nine sample replicates. PO2 was calculated from O2 content via %Hb saturation-PO2 curves obtained for equine blood (Fenger et al. 2000) using appropriate corrections. Body O2 diffusing capacity was estimated by a forward integration procedure as described initially by Bohr (1909) and used previously in humans (pulmonary, Wagner and West, 1972; pulmonary, Roca et al. 1989; leg, Roca et al. 1992) assuming that all residual O2 in the mixed venous blood resulted from diffusive O2 transport and that QO2-to- V O2 mismatch, shunt and admixture from tissues other than skeletal muscle were negligible and also that mitochondrial PO2 is close to zero as demonstrated during heavy/severe exercise in canine and human muscle (rev. Roca et al. 1992; Richardson et al. 1995). Specifically, the lumped diffusion parameter reflects the tissue diffusing capacity required to explain the measured mixed venous O2 at V O2max.

2.5

Blood Viscosity The second 7 mL EDTA blood sample collected from each stage of the exercise test was

utilized for blood viscosity determinations. Blood treated with EDTA is similar in viscosity to heparinized blood, and reliability of viscosity measurements is better using EDTA-treated than heparinized samples when the time of determination exceeds 2 h (Ernst et al. 1984; Rand et al. 1964). Blood viscosity was measured using a digital cone-plate viscometer (Model DVII, Brookfield Engineering Laboratories Inc., Stoughton, MA) with a CP-42 spindle. The temperatures of the machine and blood were kept constant at 37o C. The viscometer was standardized prior to blood viscosity determinations using a Newtonian silicone standard of 7.8 centipoise (Brookfield Engineering Laboratories Inc., Stoughton, MA). Blood samples of 1 mL were measured in triplicate at three spindle speeds, i.e., 60, 6, and 1.5 rpm. These spindle speeds are representative of the respective shear rates of 230.0, 23.0, and 5.75 s-1. The range of spindle speeds was chosen to determine the rheological properties of blood of splenectomized horses. Data determined at 60 rpm were used for comparison with other species. All tests were completed within 9 h of the trial on previously refrigerated blood. Preliminary studies showed that the viscosity of blood stored for 24 h changes less than 3%. Coefficients of variation for six replicate samples were 2.8% at 60 rpm, 8.5% at 6 rpm, and 8.5% at 1.5 rpm. 9

2.6

Statistical Analysis The data were checked for normal distribution and then analyzed using t-tests for

paired comparisons of the means (Glantz, 1987). t-tests were performed for comparison of Control trial #1 with Control trial #2 and coefficients of variation (CV) were calculated. The CV’s were used as a measure of the test-retest variability for the two control (i.e., pre-EPO) studies. It was calculated as the SD (of the difference between tests)/grand mean and expressed as a percentage (Colton, 1978). Where no differences were found between trials A and B, the results were averaged and that average was used to test the hypotheses for the effects of EPO. Values are expressed as mean + SD unless noted otherwise (Curran-Everatt, 2007; Cumming et al., 2007). The null hypothesis was rejected at P<0.05.

3. RESULTS No significant differences were found between the control trials, 1 and 2, for any of the variables measured at rest or at V O2max. Accordingly, statistical comparisons used to determine the effects of erythropoietin administration were made against averaged data from trails 1+2. The body mass of the horses was unchanged throughout the investigation (Table 1).

3.1

Vascular Volume, Hematocrit, and V O2max The pre-exercise baseline for hematocrit was 36 + 2 which was within normal limits.

Erythropoietin increased resting red cell volume by 13% (P<0.05), decreased plasma volume by 23% (P<0.05), and reduced total blood volume by 10% (P<0.05) (Figure 1). The concentration of plasma total solids was unchanged by erythropoietin (control: 9.8 + 0.5; EPO: 10.0 + 0.2 g/dL; p>0.05). All variables related to red cell mass and oxygen carrying capacity of the blood increased in EPO (Figures 1-3). Specifically, in the resting condition, erythropoietin increased [hemoglobin] 18% (P<0.05), venous hematocrit 25% (P<0.05) and arterial O2 content 17% (P<0.05). Erythropoietin elevated V O2max 20% from 25.7 + 1.7 to 30.9 + 3.4 L/min (i.e., control, 61 + 14: EPO 72 + 15 ml/kg/min, P<0.05) (Figures 2-4). Arteriovenous oxygen difference at V O2max increased 17% (P<0.05), which was driven by the increased arterial [O2] 10

(control, 18.1 + 1.0, erythropoietin, 21.1 + 1.4 ml/ 100 ml, P<0.05) and a modest non-significant decrease in mixed venous [O2] after erythropoietin.

3.2

Hemodynamics There were minimal effects of erythropoietin on the central and peripheral circulation at

rest or at V O2max (Table 1 and Figure 2). Neither resting cardiac output nor that at V O2max (control, 148 + 33; erythropoietin, 151 + 33, P>0.05) changed after erythropoietin treatment Table 2. Whereas resting and maximal exercise stroke volume increased numerically after erythropoietin, these responses were highly variable and hence not significant. HR at rest and maximal exercise was unchanged after erythropoietin. Similarly, no changes in RAP, total peripheral resistance or right ventricular dP/dtmax were seen with the elevated red cell mass after erythropoietin at rest or V O2max.

3.3

Perfusive and Diffusive O2 Fluxes The increase of arterial O2 delivery (i.e., perfusive O2 conductance) at V O2max in

erythropoietin (control, 26.9 + 1.4, erythropoietin, 32.0 + 2.0 L/min, P<0.05) was driven completely by the increased arterial [O2] (Figure 3) in the face of unchanged cardiac output (Figure 2). As seen from the increased slope in Figure 4, erythropoietin elevated O2 diffusing capacity to a significantly greater extent (30%) than seen for perfusive O2 delivery (19%).

3.4

Viscosity The elevated total red cell volume was associated with higher whole blood viscosity

(Figure 5) which also increased as a function of decreasing shear rates. Erythropoietin increased viscosity in blood sampled at rest at each viscometer speed as follows: 60 rpm, 22%; 6 rpm, 47%; 1.5 rpm, 67% (all P<0.05). Blood viscosity at V O2max was increased in similar fashion: 60 rpm, 17%; 6 rpm, 43%; 1.5 rpm, 54% (all P<0.05).

3.5

Performance

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Erythropoietin resulted in a 22% increase (P<0.05) in the total running time during the exercise test (Figure 6).

4.

DISCUSSION The principal original findings of this investigation include the demonstration that

erythropoietin treatment in the splenectomized horse increases hematocrit at rest and during maximal exercise which elevates arterial [O2] and, in the face of unchanged maximal exercising cardiac output despite elevated whole blood viscosity, significantly raises whole body O2 delivery. Thus, V O2max (and also exercise performance) is increased substantially by raising both perfusive (~19%) and diffusive (~30%) O2 conductances. Collectively, this evidence supports that these horses were O2 delivery-limited (at least in the control condition) and also that hemodynamic events in the muscle capillary bed, as they relate to the determinants of muscle and, ultimately, whole-body, O2 diffusing capacity are of crucial importance in facilitating blood-myocyte O2 flux during maximal exercise.

4.1

Agreement with existing literature The compelling weight of evidence indicates that horses, humans and dogs have a

sufficiently high mitochondrial oxidative capacity that V O2max is limited by O2 delivery rather than utilization per se. Thus, paradigms designed to increase whole body and muscle(s) O2 delivery by means of either elevated arterial [O2] or cardiac output/muscle(s) blood flow invariably elevate V O2max (rev. Poole and Erickson, 2011). The classic studies of Persson (Persson, 1967, 1973) documented the relationship between equine RBC volume and V O2max by determining the effects of splenectomy and exercise training. The present investigation demonstrates that this is true also for pharmacologically-induced increases of red cell volume in this species as well as for human end-stage renal disease patients (Lundin et al. 1991).

That stroke volume, heart rate, and cardiac output at maximal exercise were maintained at pre- erythropoietin treatment values is consistent with blood doping studies where the elevated perfusive O2 delivery has translated to an increased arterial-to-venous O2 difference 12

and thus V O2max (Ekblom et al. 1976; Robertson et al. 1982). However, the most pronounced contrast between the results obtained in the present investigation (and those of Abdulhadi et al. 1990 and Lundby et al. 2007) with erythropoietin versus blood doping is the 10% decrease in blood volume that occurred concomitant with a 13% increase of red cell mass. Generally, following acute changes in blood volume, increased red cell mass is compensated to a certain degree by an opposite change in plasma volume (Sjostrand, 1962). In this fashion, limitation or prevention of vascular expansion is facilitated by a diuresis triggered by atrial stretch receptors that respond to increased venous pressure (Convertino, 1991) in combination with increased capillary fluid filtration consequent to elevated capillary pressures (Guyton, 1981). Consequently, previous blood-doping studies involving single red cell infusions have resulted in little or no change in blood volumes. In contrast, in the present investigation, the increase in red cell volume was progressive rather than acute and such changes may lead to an increased rather than reduced total blood volume and vascular space (Hillman and Finch, 1992). Therefore we would not have predicted that the more gradual increase in red cell volume following 3 wk of erythropoietin treatment would reduce plasma and total blood volume. That body mass of the horses did not change significantly throughout the course of the study suggests that a shift of fluid from the vascular to the extravascular space occurred without a loss of total body water.

The present findings are in agreement with Wagner et al.’s demonstration that infusing 12 L RBCs into the splenectomized horse elevates V O2max by means of perfusive and diffusive O2 transport. However, the appreciably greater increase of O2 diffusing capacity with erythropoietin herein may indicate that the (presumably) younger red cell population generated with erythropoietin (compared with extra red cells transfused from donor horses, Wagner et al. 1995) have hemorheological properties within the microcirculation that more effectively raise blood-myocyte flux and hence O2 diffusing capacity. This may present a significant advantage of erythropoietin treatment over blood transfusion. As regards the disagreement with the human study of Lundby and colleagues (Lundby et al. 2008b), it is noteworthy that erythropoietin treatment in humans served to raise effluent muscle(s) venous 13

blood [O2] which increased further with subsequent hemodilution. In contrast, the horses in the present investigation demonstrated a modest (but not significant) decrease in mixed venous [O2]. The finding of increased O2 diffusing capacity with erythropoietin-induced elevation of hematocrit herein agrees with Gonzalez and colleagues (Gonzalez et al., 1994) findings in acutely red cell infused ~sea level rats but not in their altitude-acclimatized polycythemic counterparts. One significant difference between the two polycythemic rat groups was that altitude acclimatization increased total peripheral resistance which the authors speculated might have resulted from vascular remodeling and/or increased vasoconstrictor tone likely related to the chronic altitude/hypoxia. These findings suggest that, especially in animal models (rat, horse), with respect to muscle O2 transport it may be of crucial importance not just that there is polycythemia but how that polycythemia was achieved: erythropoietin being superior to chronic hypobaric hypoxia, for example.

4.2

Additional considerations The low V O2max values and general performance values (including low maximal

[lactate] and HR<220 observed in the present study reflect the effects of splenectomy on the exercise capacity of the horse. Similar observations have been observed in the literature (Persson, 1967; Persson. 1973; McKeever et al. 1993; Wagner et al. 1995). The increases in red cell volume and maximal aerobic capacity and decreases in resting plasma volume observed in the splenectomized horses of the present study are similar to those reported by McKeever et al. (2006) for intact horses administered a similar course of EPO. In the present study, the decrease in plasma volume was larger than the calculated increase in red cell volume, resulting in a decrease in calculated blood volume. This differs from the McKeever et al. (2006) where there was a statistically non-significant 5% increase in total blood volume. However, that finding should not be over-interpreted because their calculations relied on the hematocrit measured after mobilization of the splenic reserve during exercise (Persson, 1967; McKeever et al., 2006). One should also note that, the hematocrit collected during exercise is influenced both by the effect of splenic reserve mobilization and by exercise-induced shifts of fluid out of 14

the vascular compartment. The presence of resting hematocrits in the splenectomized horses herein avoids such confounds and negates critique concerning the validity of the calculation of blood and red cell volumes. Another concern with the present investigation regards the use of a Co-oximeter to measure the arterial and venous oxygen content without the simultaneous measurement of PO2 using a blood gas analyzer. This necessitated an estimation of PO2 via O2 content and % saturation. The authors used the appropriate O2 dissociation curves to derive PO2 with minimal error and specifically avoided calculating arterial PO2’s (and alveolar-arterial PO2 gradients) towards the plateau of the O2 dissociation curve where small differences in measured O2 content would potentially translate to substantial PO2 variations.

In the lung red blood cell transit time (i.e., that time available for gas exchange in the pulmonary capillary) decreases during maximal exercise as the ratio of cardiac output to pulmonary capillary blood volume decreases. As presented by Zavorsky et al. (2003), greater elevation of pulmonary capillary blood volume will serve to constrain the fall of red cell transit time and help preserve arterial oxygenation closer to that at rest (see also Wilkins et al. 2001 for the horse). In performance horses maximal cardiac outputs well over 400 L/min induce profound arterial hypoxemia despite extraordinarily high pulmonary vascular pressures elevating capillary volumes (rev. Poole and Erickson, 2011). The splenectomized horses herein reached cardiac outputs considerably lower than their performance horse counterparts (i.e., ~150 L/min) which would serve to limit any tendency for arterial hypoxemia. We did not measure pulmonary vascular pressures and therefore we can only surmise that the 10% reduction of total blood volume combined with the elevated hematocrit may have evoked changes in pulmonary capillary volume and thus red cell transit times. However, the outcome of these effects on arterial oxygenation is likely to have been trivial if anything at all.

4.3

Cardiac and hemodynamic effects Low dose EPO treatment did not produce any substantial changes in arterial blood

pressure either at rest or during maximal exercise. This is consistent with findings from studies on both normotensive human end-stage renal disease patients (Abdulhadi et al. 1990; Kamata 15

et al. 1991; Levin, 1991) and healthy men (Berglund and Ekblom, 1991) and raises the question as to why the elevated hematocrit and associated increases in viscosity, measured in situ (Figure 5), did not elevate arterial pressures particularly at cardiac outputs ~150 L/min. A partial explanation may be that hematocrits in the range observed (i.e., 43-50%) represent near-optimal values for viscosity in horses and humans (Buick et al. 1980; Richardson and Guyton, 1959; Robertson et al. 1982; Spriet et al. 1986) and also that, as noted by Fedde and Wood (1993), that shear-dependent qualities of equine blood may facilitate a “homeostasis of viscosity” during running such that in vivo viscosity does not raise vascular resistance. Of course, it is possible that if erythropoietin was to be given to non-splenectomized horses hematocrit may become so high that vascular pressures, and potentially cardiovascular function, are compromised. Although it has been noted that erythropoietin treatment is associated with increased cardiac power such that cardiac output might be maintained even at increased blood viscosities (rev. Boning et al. 2010). With respect to effective blood viscosity within the microcirculation it is pertinent that, in addition to the influence of hematocrit and plasma viscosity on whole blood viscosity, the shape and distensibility of red cells are crucial. Rheologically, one of the most distinguishing differential features between horse and human blood relates to the small size and greater flexibility of horse red cells (Amin and Sirs, 1985). Erythrocyte flexibility measured as mean packing rate is approximately 30% per min for horses compared to about 7% per min for humans (Amin et al. 1986). The increased flexibility in horse erythrocytes is conferred by an, as yet, unidentified factor that can increase human red cell flexibility substantially when human red cells are suspended in horse plasma (Amin and Sirs, 1982). This enhanced flexibility of equine red cells facilitates shape change (Amin et al. 1986) and, as such, is expected to ease their entry into and passage along muscle capillaries (Amin et al. 1986). This is likely an essential property of equine blood necessary for the elite performance horse to achieve extraordinary cardiac outputs (exceeding 450 L/min, Poole and Erickson, 2011) without raising systemic arterial pressures into the pathological range.

4.5

Mechanisms for increased perfusive and diffusive conductances at V O2max 16

As mentioned above, the greater perfusive O2 transport at V O2max was the product of an increased arterial [O2] concomitant with unchanged cardiac output. To achieve the elevated V

O2max arterial-to-venous O2 difference was widened by 18% which required a 30% increase

of O2 diffusing capacity as demonstrated in Figure 4. Empirical evidence in humans (Richardson et al. 1995) and dogs (Honig et al. 1988, 1989, 1990,1991; Hepple et al. 2000) suggests that the bulk of the diffusional resistance to blood-mitochondrial O2 flux occurs in close proximity to the red cell rather than within the myocyte. This supports the elegant and contemporary models developed by Federspiel and Popel (1986) and Groebe and Thews (1990) and the data of Malvin and Wood (1992) where O2 diffusing capacity is determined primarily by the number of red cells in the capillaries adjacent to the myocytes/tissue at any given time. Whereas it has been pointed out that alterations in systemic hematocrit may not be faithfully reflected by parallel changes at the capillary (Sarelius, 1989) the present findings suggest otherwise. Specifically, as most capillaries sustain red cell flux in muscle at rest there are not many more capillaries to be ‘recruited’ in contracting muscle (rev. Poole et al. 2011,2013). Hence, the recruitment of additional capillary surface area, and thus of O2 diffusing capacity during maximal exercise by erythropoietin likely occurs within capillaries that were already flowing at rest by at least two mechanisms: 1. The elevated systemic hematocrit is reflected in the capillaries thereby increasing RBC number adjacent to the muscle fibers. 2. Increased arterial [O 2] and widening the arterial-to-venous O2 difference may facilitate increasing the length of each capillary over which blood-myocyte O2 flux occurs i.e., a longitudinal recruitment of capillary surface area. It is also possibly that there may be an improvement in the matching of O2 delivery within muscle to local O2 demands (rev. Koga et al. 2014): However, given that horses may extract 90% or more O2 from the blood it is doubtful that there exists any substantial mismatch initially. In addition to these effects increased kinetics of O2 release resulting from elevated capillary [hemoglobin] (Roughton and Forster, 1957) would also be expected to increase O2 diffusing capacity. The increase O2 diffusing capacity demonstrated herein and its greater magnitude relative to that of perfusive O2 transport is similar to the effect of endurance-type exercise training in humans (Roca et al. 1992): though we argue that the mechanisms are very different. 17

Exercise training also increases perfusive O2 transport as seen herein but via increased cardiac output rather than arterial [O2] and that increased cardiac output is distributed among more capillaries generated via the angiogenic consequences of exercise training. Thus, the proliferation of capillaries and increased muscle capillary volume will offset the tendency for the training-induced cardiac output elevation to reduce mean capillary red cell transit time. Whether or not erythropoietin caused angiogenesis in the present investigation is unknown, though there is the suggestion that erythropoietin may stimulate capillary neogenesis possibly by enhancing tissue vascular endothelial growth factor levels (Alvarez et al. 1998) neither fiber type shift nor angiogenesis were found in human skeletal muscle after erythropoietin treatment (Lundby et al. 2008a). However, even in the absence of such an effect, without cardiac output (and presumably muscle(s) blood flow) changing, the increased O2 diffusing capacity found herein is best explained by transduction of the elevated systemic hematocrit into the capillary bed and a proportional increase in red blood cell number per unit capillary length.

5.0

SUMMARY Three weeks of erythropoietin treatment in the splenectomized horse significantly

increases systemic arterial hematocrit and [hemoglobin] concomitant with reduced plasma volume. During maximal exercise cardiac output is unchanged and V O2max (and exercise performance) is enhanced 19% by substantially augmented perfusive (increased 19%) and diffusive (increased 30%) O2 transport: the latter suggesting that the increased systemic hematocrit raises capillary [hemoglobin] and facilitates blood-myocyte O2 flux. Whereas this polycythemia increases blood viscosity in situ vascular pressures at V O2max are unchanged suggesting either that vascular changes increase hemodynamic conductance or, alternatively, equine blood in vivo during maximal exercise does not increase viscosity as seen in situ.

6.

ACKNOWLEDGEMENTS The authors thank Judith Dutson and Dr. James Farris for their help in conducting this

study and Sunny Geiser for her help in preparing the manuscript. Support for this project was 18

provided by the Ohio Thoroughbred and Standardbred Research Funds and New Jersey Agricultural Experimentation Station Project #99466, the Earle Mack Foundation, the NY Thoroughbred Owner and Breeders Association, and the Association of Racing Commissioners International.

7.

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24

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25

8.0

FIGURE LEGENDS

Figure 1

Effects of erythropoietin rhuEPO) administration on mean (+ SE) resting hemoglobin concentration, plasma volume and total blood volume. * p<0.05 erythropoietin (EPO) versus Control.

Figure 2

Effects of erythropoietin (rhuEPO) administration on mean (+ SE) maximal aerobic capacity ( V O2max) and cardiac output in splenectomized horses. * p<0.05 erythropoietin (EPO) versus Control, n.s., not significant.

Figure 3

Effects of erythropoietin (rhuEPO) administration on the relationship between maximal aerobic capacity ( V O2max) and oxygen delivery (mean + SE). * p<0.05 erythropoietin (EPO) versus Control.

Figure 4

Effects of erythropoietin (rhuEPO) administration on the relationship between maximal aerobic capacity ( V O2max) and estimated mean capillary O2 partial pressure (PO2) (mean + SE). The slope of the lines from the origin defines O2 diffusing capacity (ḊO2) according to Fick’s law and is expressed in ml O2/mmHg/min. * p<0.05 erythropoietin (EPO) versus Control.

Figure 5

Effects of erythropoietin (rhuEPO) administration on blood viscosity expressed in centipoise (cps) in horses at rest (top) and at V O2max (mean + SE) at spindle speeds of 60, 6 and 1.5 rpm which equate to shear rates of 230.0, 23.0, and 5.75 s-1, respectively (cps). * p<0.05 erythropoietin (EPO) versus Control.

26

Figure 6

Effects of erythropoietin (rhuEPO) administration on maximal running time at the point of exhaustion. *p<0.05 erythropoietin (EPO) versus Control.

27

Figure 1

18

16 15 14 13

18

16

*

14

Blood Volume (liters)

*

Plasma Volume (liters)

32

17

Hemoglobin (g/dL)

34

20

30 28

*

26 24 22 20

12

12 Control EPO

18 Control

EPO

Control

EPO

28

Figure 2

29

Figure 3

30

Figure 4

31

Resting Blood Viscosity (cps)

Figure 5

20

15

** **

10

**

5

0

60 rpm

Blood Viscosity at VO2max (cps)

Control EPO

6 rpm

1.5 rpm

20

Control EPO

** 15

** 10

* 5

0

60 rpm

6 rpm

1.5 rpm 32

Figure 6

400

Running Time (s)

* 300

200

100

0 Control

EPO

33

Table 1: Body Mass, Blood Parameters, and Vascular Volumes

GXT #1

Control GXT #2

429 + 23

423 + 23

Average Control Control GXTs 1 & C.V. 2 426 + 20 1.9%

13.8 + 0.6

12.9 + 0.4

13.3 + 0.6

5.3%

15.7 + 0.8*

36 + 2

37 + 2

36 + 2

1.3%

46 + 2*

9.6 + 0.6

10.0 + 0.4

9.8 + 0.5

2.3%

10.0 + 0.2

18.4 + 1.6

18.3 + 1.4

18.3 + 1.5

2.6%

14.1 + 1.2*

10.4 + 1.4

10.6 + 1.2

10.5 + 1.1

2.7%

12.0 + 1.5*

28.8 + 3.0

28.9 + 2.5

28.8 + 2.7

2.3%

26.1 + 2.0*

1Control

Body Mass (kg) Hemoglobin (g/dL) Hematocrit (%) Plasma Total Solids (g/dL) Plasma Volume (L) Red Cell Volume (L) Blood Volume (L)

Post-EPO GXT 411 + 21

1Values

represent means + SD for each variable measured during graded exercise tests to fatigue (GXT). Control GXT 1 and Control GXT 2 were performed 4 weeks apart and prior to administration of EPO. The “control” coefficient of variation (CV) is presented to demonstrate the variability between measurements made in Control GXT #1 versus Control GXT #2. Means with and asterisk (*) are different (P<0.05).

34

Table 2: Oxygen Uptake and Cardiovascular Data

1Control

Rest Oxygen Uptake (mL/Kg/min)

V

O2max

Rest

GXT #1

Control GXT #2

3.8 + 0.4

3.8 + 0.7

60.6 + 14.7

Average Control GXTs 1 & 2 3.8 + 0.2

60.4 + 12.8 60.5 + 13.8

Control C.V.

Post-EPO GXT

23.5%

4.4 + 0.6

5.7%

72.0 + 15.3*

34 + 7

32 + 10

33 + 9

32.0%

37 + 9

150 + 32

149 + 28

148 + 33

8.5%

151 +33

Rest

43 + 14

46 + 10

44 + 12

20.2%

44 + 11

V

201 + 3

200 + 3

201 + 3

1.7%

198 + 4

Rest

124 + 10

118 + 13

121 + 12

8.1%

120 + 12

V

O2max

132 + 21

135 + 15

133 + 18

6.8%

136 + 19

Total Peripheral Rest Resistance V O2max (mm Hg/L/min)

4.0 + 0.9

4.0 + 1.3

4.0 + 1.1

25.5%

3.0 + 1.0

0.9 + 0.1

0.9 + 0.1

0.9 + 0.1

12.8%

0.9 + 0.1

Cardiac Output (Liters/min) Heart Rate (beats/min) Mean Arterial Pressure (mm Hg)

V

O2max

O2max

Right Atrial Pressure (mm Hg)

Rest

5.0 + 0.9

0.9 + 1.6

2.3 + 2.4

99.7%

2.0 + 2.0

V

2.4 + 3.2

0.9 + 3.4

1.4 + 3.2

263.4%

-5.0 + 4.0

Right Ventricular Pressure (mm Hg)

Rest

54 + 8

53 + 5

53 + 5

4.8%

55 + 7

125 + 12

118 + 16

121 + 11

4.7%

122 + 15

V

O2max

O2max

1Values

represent means + SD for each variable measured during incremental exercise tests to fatigue (GXT). Control GXT 1 and Control GXT 2 were performed 4 weeks apart and prior to administration of EPO. The “control” coefficient of variation (CV) is presented to demonstrate the variability between measurements made in Control GXT #1 versus Control GXT #2. Means with and asterisk (*) are different (P<0.05).

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