University of Zurich - UZH · 2010. 11. 29. · Acute and chronic exposure to hypoxia alters ventilatory pattern but not minute ventilation of mice overexpressing erythropoietin Jorge

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Acute and chronic exposure to hypoxia alters ventilatory patternbut not minute ventilation of mice overexpressing erythropoietin

Soliz, J; Soulage, C; Hermann, D M; Gassmann, M

Soliz, J; Soulage, C; Hermann, D M; Gassmann, M (2007). Acute and chronic exposure to hypoxia altersventilatory pattern but not minute ventilation of mice overexpressing erythropoietin. American Journal ofPhysiology. Regulatory, Integrative and Comparative Physiology, 293(4):R1702-R1710.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 2007, 293(4):R1702-R1710.

Soliz, J; Soulage, C; Hermann, D M; Gassmann, M (2007). Acute and chronic exposure to hypoxia altersventilatory pattern but not minute ventilation of mice overexpressing erythropoietin. American Journal ofPhysiology. Regulatory, Integrative and Comparative Physiology, 293(4):R1702-R1710.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 2007, 293(4):R1702-R1710.

Acute and chronic exposure to hypoxia alters ventilatory patternbut not minute ventilation of mice overexpressing erythropoietin

Abstract

Apart from enhancing red blood cell production, erythropoietin (Epo) has been shown to modulate theventilatory response to reduced oxygen supply. Both functions are crucial for the organism to cope withincreased oxygen demand. In the present work, we analyzed the impact of Epo and the resultingexcessive erythrocytosis in the neural control of normoxic and hypoxic ventilation. To this end, we usedour transgenic mouse line (Tg6) that shows high levels of human Epo in brain and plasma, the latterleading to a hematocrit of approximately 80%. Interestingly, while normoxic and hypoxic ventilation inTg6 mice was similar to WT mice, Tg6 mice showed an increased respiratory frequency but a decreasedtidal volume. Knowing that Epo modulates catecholaminergic activity, the altered catecholaminergicmetabolism measured in brain stem suggested that the increased respiratory frequency in Tg6 mice wasrelated to the overexpression of Epo in brain. In the periphery, higher response to hyperoxia (Dejourstest), as well as reduced tyrosine hydroxylase activity in carotid bodies, revealed a higherchemosensitivity to oxygen in transgenic mice. Moreover, in line with the decreased activity of therate-limiting enzyme for dopamine synthesis, the intraperitoneal injection of a highly specific peripheralventilatory stimulant, domperidone, did not stimulate hypoxic ventilatory response in Tg6 mice. Theseresults suggest that high Epo plasma levels modulate the carotid body's chemotransduction. All together,these findings are relevant for understanding the cross-talk between the ventilatory and erythropoieticsystems exposed to hypoxia.

Acute and chronic exposure to hypoxia alters ventilatory pattern but notminute ventilation of mice overexpressing erythropoietin

Jorge Soliz,1 Christophe Soulage,2 Dirk M. Hermann,3 and Max Gassmann1

1Institute of Veterinary Physiology, Vetsuisse Faculty, and Zurich Center for Integrative Human Physiology, University ofZurich, Zurich; 2Laboratoire de Physiologie Integrative Cellulaire et Moleculaire, Universite Claude Bernard, VilleurbanneCedex, France; 3Department of Neurology, University Hospital Zurich, Zurich, Switzerland

Submitted 18 May 2007; accepted in final form 19 July 2007

Soliz J, Soulage C, Hermann DM, Gassmann M. Acute andchronic exposure to hypoxia alters ventilatory pattern but not minuteventilation of mice overexpressing erythropoietin. Am J Physiol RegulIntegr Comp Physiol 293: R1702–R1710, 2007. First published July25, 2007; doi:10.1152/ajpregu.00350.2007.—Apart from enhancingred blood cell production, erythropoietin (Epo) has been shown tomodulate the ventilatory response to reduced oxygen supply. Bothfunctions are crucial for the organism to cope with increased oxygendemand. In the present work, we analyzed the impact of Epo and theresulting excessive erythrocytosis in the neural control of normoxicand hypoxic ventilation. To this end, we used our transgenic mouseline (Tg6) that shows high levels of human Epo in brain and plasma,the latter leading to a hematocrit of �80%. Interestingly, whilenormoxic and hypoxic ventilation in Tg6 mice was similar to WTmice, Tg6 mice showed an increased respiratory frequency but adecreased tidal volume. Knowing that Epo modulates catecholamin-ergic activity, the altered catecholaminergic metabolism measured inbrain stem suggested that the increased respiratory frequency in Tg6mice was related to the overexpression of Epo in brain. In theperiphery, higher response to hyperoxia (Dejours test), as well asreduced tyrosine hydroxylase activity in carotid bodies, revealed ahigher chemosensitivity to oxygen in transgenic mice. Moreover, inline with the decreased activity of the rate-limiting enzyme fordopamine synthesis, the intraperitoneal injection of a highly specificperipheral ventilatory stimulant, domperidone, did not stimulate hy-poxic ventilatory response in Tg6 mice. These results suggest thathigh Epo plasma levels modulate the carotid body’s chemotransduc-tion. All together, these findings are relevant for understanding thecross-talk between the ventilatory and erythropoietic systems exposedto hypoxia.

carotid body; brain stem; ventilatory control

UPON HYPOXIC EXPOSURE, ERYTHROPOIETIN (Epo) synthesis in kid-ney is accelerated, resulting in augmented Epo plasma levelsand leading to enhanced hemoglobin synthesis and increasedhematocrit levels (10, 40). Epo also has several nonerythropoi-etic functions (12). As such, Epo was recently identified as akey factor controlling the hypoxic ventilatory response (HVR)(39). We recently reported that Epo receptor (EpoR) is presentin peripheral chemoreceptors and central respiratory areascontrolling ventilation (VE), including glomus cells in carotidbodies (the primary sensory organ that monitors arterial oxy-gen), NK-1R substance preceptor immunopositive neurons inthe pre-Botzinger complex (proposed as the generator of re-spiratory rhythm), and the nucleus tractus solitarii in dorsalbrain stem (relays input from peripheral chemoreceptors to the

central respiratory areas) (39). Furthermore, it was demon-strated that cerebrally produced Epo per se influences thecentral respiratory activity, thereby increasing the respiratoryfrequency (fR) in response to hypoxia. Recently, we demon-strated that intracerebroventricular infusion of soluble EpoR, anegative regulator of Epos binding to the EpoR, abolishes theventilatory acclimatization to chronic hypoxia (38).

Apart from enhancing VE, erythrocytosis is also necessary toincrease oxygen-carrying capacity during exposure to chronichypoxia. As such, physiological erythrocytosis develops inindividuals living at a high altitude. Enhanced erythrocytosis isalso found in sports, the most prominent case being that of anoutstanding Olympic endurance athlete (19) harboring an au-tosomal dominant erythrocytosis (33) that resulted in hemato-crit levels up to 68%. Despite these beneficial effects, eryth-rocytosis may have a pathological impact. This occurs inlowlanders as a consequence of numerous respiratory disordersand in some highlanders when high altitude hypoxia patholog-ically stimulates dramatic increases in hemoglobin resulting inchronic mountain sickness (35). It is believed that CMS is theconsequence of a ventilatory deacclimatization to hypoxia,leading to central hypoventilation, severe hypoxemia, in-creased erythropoiesis, and excessive erythrocytosis (27, 35).In turn, excessive erythrocytosis leads to life-threatening cya-nosis, hyperemia, increased viscosity, thrombosis, and pulmo-nary hypertension in humans and mouse (13, 27, 35, 43).Because Epo and the adaptation to erythrocytosis are of generalinterest in sport medicine and are significant factors concerninglife at high altitudes, we studied the normoxic and HVR in atransgenic mice (Tg6) that due to constitutive overexpressionof human Epo in brain and lung (13) develop chronic eryth-rocytosis (36, 44). Despite Tg6 mice showing hematocritvalues up to 80–90%, we found no differences in minute VE

between Tg6 and control [wild-type (WT)] mice during acuteand chronic hypoxia. Nevertheless, Tg6 mice showed dramaticchanges in the ventilatory pattern with enlarged fR but de-creased tidal volume (VT), both under acute and chronichypoxic conditions. These findings suggest that elevated Epolevels in brain and blood are important factors regulatingoxygen homeostasis in conditions of restricted oxygen supply.

MATERIAL AND METHODS

Transgenic animals. The Epo-overexpressing transgenic mouseline was generated by microinjection of human Epo cDNA drivenby the human platelet-derived growth factor (PDGF) B-chain

Address for reprint requests and other correspondence: M. Gassmann,Institute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center forIntegrative Human Physiology, Winterthurerstrasse 260, CH-8057 Zurich,Switzerland (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Regul Integr Comp Physiol 293: R1702–R1710, 2007.First published July 25, 2007; doi:10.1152/ajpregu.00350.2007.

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promoter into the pronuclei of fertilized oocytes derived fromB6C3 hybrid mice (36). The resulting transgenic mouse lineTgN(PDGFBEPO)321ZbZ (Tg6) showed increased Epo levels inplasma (12-fold compared with WT) and brain (26-fold comparedwith WT), accompanied by a doubled hematocrit value (48). Thiserythrocytic line was backcrossed to C57BL/6 mice for more than 12generations by mating hemizygous males to WT C57BL/6 females.Half of the offspring were hemizygous for the transgene, while theother half were WT and thus were used as control animals. Allexperiments were performed in 3- to 4-mo-old male mice. Animalexperimental protocols were approved by the Veterinarant des KFZurich.

Ventilatory measurements by plethysmography. Respiration wasmonitored by the whole body flow through plethysmography tech-nique as previously described (39). Briefly, mice were placed in a600-ml chamber continuously supplied with airflow at 0.7–0.8 l/minusing flow restrictors. VE was calculated as the product of VT and fRand normalized to 100 g body wt (i.e., ml �min�1 �100 g�1). As soon asthe animal was familiarized with the plethysmographic chamber (�1 h),measurements of baseline VE (normoxia, 21% O2) and hypoxic VE

were performed. Acute hypoxia was achieved by flushing air balancedin N2 using a gas-mixing pump (Digamix type M302a-F; H.Wosthoff). The fraction of inspired O2 (FIO2) in the chamber wasgradually decreased from 21% to 10% O2 over 15 min. Respiratoryrecordings at 10% O2 were performed for 20 min. The oxygenconcentration in the chamber was then further reduced to 6% over thenext 15 min and recordings were performed for 20 min in the morehypoxic environment. At the end of each experiment, body weightwas routinely measured to express VT in milliliters per 100 g in bodytemperature and pressure (saturated) conditions. fR was defined asrespirations per minute. Hemoglobin was quantified using standardmethodology, and body temperature in normoxia and hypoxia wasmeasured using a rectal thermocouple (Fluke). The same measure-ments of VE and temperature were performed in animals that previ-ously were exposed to chronic hypoxia (see below).

Chronic exposure to hypoxia was performed as described byPrabhakar and colleagues (20, 24). Briefly, mice were housed in ahomemade hypoxic chamber connected to a gas-mixing pump (Diga-mix type M302 a-F; H. Wosthoff). The chamber oxygen concentrationwas gradually reduced from room air to 10% over 30 min andmaintained at 10% for 3 days. During this period, mice were allowedfree access to food and water. One hour after returning the mice toroom air, baseline VE and HVR at 10% and 6% O2 were recorded byplethysmography as described above.

To avoid any movement of the animals during the brief exposure to100% O2, the hyperoxic test [Dejours test (7)] was performed inanesthetized mice. Two minutes after injecting urethane solution (1.2g/kg) mice showed regular VE and normal fR. Baseline respirationwas recorded while animals breathed 21% O2 for 20 s. The plethys-mographic chamber was then quickly saturated with 100% O2, and thedecline of VE was recorded over 20 s. Respiratory variables wereanalyzed, and the magnitude of the transient VE decline was calcu-lated as the difference between baseline and hyperoxic respirationparameters.

Baseline VE in normoxia and VE response to hypoxia (10% and 6%O2) were evaluated 1–2 h after injection of domperidone (1 mg/kg ip;kindly provided by Janssen-Cilag; dissolved in 0.9% saline solutionwith 1 equivalent of tartaric acid). Note that according to the provider,domperidone is a highly specific peripheral D2-dopaminergic receptoragonist that does not cross the blood-brain barrier. Control animalswere injected with similar volumes of 0.9% NaCl.

An open-circuit system allowed measurement of O2 consumption(VO2, ml �min�1 �100 g�1; atmospheric temperature and pressure indry air) and CO2 production (VCO2, ml �min�1 �100 g�1) in normoxiaand hypoxia (10% and 6% O2). Mice were placed in a chamber wherea steady 0.2 l/min flow of air was maintained. The fractions of O2 and

CO2 at the inflow and the outflow of the chamber were measured byO2 and CO2 analyzers (Qubit Systems, Kingston, Ontario).

Quantitation of catecholamines in brain stem and carotid bodies.Catecholaminergic cell groups were obtained from successive trans-verse brain stem sections (60-�m thick), according to a mouse brainatlas (30) as described previously (47). In brief, A6 and A5 (in pons)and A1C1 and A2C1 (in caudal region) were dissected from the brainstem. Different animals were used to determine norepinephrine (NE)content (in brain stem slices) or tyrosine hydroxylase (TH) activity (inbrain stem slices and in carotid bodies), the latter analysis requiring aprevious injection of 3-hydroxybenzylhydrazine dihydrochloride(NSD 1015; 75 mg/kg ip in saline solution; Sigma, St. Louis, MO).Twenty minutes after injection, animals were decapitated, and theenzymatic activity of TH was indirectly evaluated by measuring theaccumulation of L-dihydroxyphenylalanine (L-DOPA) over 20 min,following the blockade of L-DOPA decarboxylase with NSD 1015.Both NE and L-DOPA were quantified by HPLC coupled withelectrochemical detection as described earlier (17). The mobile phaseconsisted of 0.1 M potassium phosphate buffer pH 3.0 containing 0.15mM disodic EDTA at a flow rate of 0.8 ml/min. L-DOPA wasmeasured at �0.65 V. The detection limit, calculated by doubling thenoise ratios and expressed in picomoles of injected amounts, was �0.03 pmol, and the intra-assay coefficient was 0.2%.

Statistical analysis. Analysis was performed using the StatViewsoftware (Abacus Concepts, Berkeley, CA). The reported values aremeans � SD. For simple measurements, data were analyzed byone-way ANOVA followed by a post hoc protected least significantdifference Fisher test. For hypoxic VE responses, data were analyzedby two-way ANOVA for repeated measurements. Differences wereconsidered significant at P � 0.05.

RESULTS

Basal VE and metabolic parameters showed no differencesin transgenic and WT mice. Minute VE, fR, and VT, as well asrectal temperature and metabolic variables, were evaluated intransgenic Tg6 and WT control mice under basal restingconditions (normoxia, 21% O2; Table 1). Neither the minuteVE nor the ventilatory pattern, i.e., VT and fR were found to bedifferent between the two strains in a normoxic environment.Additionally, no differences were noticed in metabolic param-eters, such as body temperature, oxygen consumption, orcarbon dioxide production (Table 1 and Fig. 1, F–H, 21% O2).

Tg6 mice show higher fR but lower VT upon exposure toacute hypoxia. The first attempt to regulate oxygen homeosta-sis during hypoxia is hyperventilation (31). We measured theVE response in unanesthetized WT and transgenic mice to twolevels of normobaric hypoxia (10% O2 and 6% O2). HVR wassimilar between WT and transgenic male mice (Fig. 1A).However, the VE pattern of Tg6 mice was substantially af-fected; while fR of moderate (10% O2) and severe hypoxia (6%O2) were significantly augmented (Fig. 1B; P � 0.0001), VTwas extensively decreased (Fig. 1C; P � 0.001). The compar-ison of the relative proportions of fR and VT in the HVRclearly shows that VT is the main contributor in the hypoxicresponse of WT mice during hypoxia (Fig. 1D). On thecontrary, the fR is the major provider for the HVR in trans-genic animals (Fig. 1E). Under our experimental conditions,body temperature, oxygen consumption, or carbon dioxideproduction (Fig. 1, F–H) were similar, thus the change in themetabolic drive cannot account for the observed changes inbreathing pattern.

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Transgenic mice show altered breathing patterns after ac-climatization to hypoxia. Long-term hypoxic exposure leads toa progressive increase in minute VE that is termed ventilatoryacclimatization to hypoxia (32). In mice this process is com-pleted after 3 days (24, 29). Accordingly, transgenic and WTcontrol mice were exposed to 10% O2 for 3 days, and thenreturned to normoxia to evaluate VE during normoxia and acutehypoxia. Both transgenic and WT mice showed similar in-creases in the level of VE during normoxia, indicating thatventilatory acclimatization to hypoxia occurred (Table 1 andFig. 2, F–H, 21% O2). During the hypoxic challenges, Tg6 andWT mice exhibited similar minute VE (Fig. 2A), but showdifferences in terms of hypoxic VE pattern. In agreement withthe measurements performed under acute hypoxic conditions,Tg6 mice showed higher fR (Fig. 2B) and lower VT (Fig. 2C)responses to hypoxia compared with WT control animals. Thecomparison of the relative proportions of fR and VT in theHVR show that after chronic hypoxia in both WT and Tg6mice, HVR is mainly supported by VT rather than fR; how-ever, the contribution of VT to WT ventilatory response ishigher in WT compared with transgenic mice (Fig. 2, D and E).As observed previously, alteration in body temperature ormetabolic rate (Fig. 2, F–H) did not change and thus cannotaccount for the observed alterations in the VE pattern.

Transgenic animals had altered catecholaminergic brainstem levels. We recently reported that the overexpression ofEpo in neuronal cells alter catecholamine activity in respiratory-related brain stem areas (39). In that work, we also showed thepresence of EpoRs on brain stem catecholamine neurons in WTmice. Because the Tg6 mouse line overexpresses 26-fold moreEpo in brain compared with WT, we hypothesized that theseanimals would exhibit an altered level of catecholamines in thebrain stem respiratory areas, which could contribute to theobserved changes in VE. Our previous work strongly suggestedthat Epo stimulation of fR during hypoxia is, at least in part,mediated by an effect on catecholaminergic synthesis in thebrain stem (39). Thus, we evaluated TH activity and the NEcontent in pontial A6 and A5 and medullar A1C1 and A2C2brain stem catecholaminergic groups. Compared with WTmice, we found that transgenic Tg6 animals showed higher THactivity and NE content in A5 cell group only (Fig. 3B). Incontrast, the catecholamine synthesis in A6, A1C1, and A2C2cell groups showed no differences in either mouse lines (Fig. 3,A, C, and D). Knowing that increased catecholamine levels inpontial groups contribute to augmentation of respiratory rateduring hypoxia (8, 14), our observations suggest that the

elevated fR observed in Tg6 mice during acute and afterchronic hypoxia is mediated by elevated cerebral Epo levelsthat in turn modulate the synthesis rate of NE in brain stemcatecholaminergic A5 cells. Since previous publicationsshowed that catecholamines modulate VE under normoxic aswell as hypoxic conditions (14, 15), the present data suggestthat the altered levels of catecholamines in brain stem respira-tory areas contribute to the observed changes in fR and VTduring acute and chronic hypoxic VE.

Tg6 mice exhibit higher peripheral chemosensitivity tooxygen. The basal contribution of peripheral chemoreceptors toresting minute VE and carotid body sensitivity is in agreementwith the magnitude of the transient VE decline in response to abrief period of hyperoxia (Dejours test). We have previouslydescribed the presence of EpoRs on carotid body cells in WTmice (39). Thus, in anesthetized WT and Tg6 mice, we re-corded the VE during normoxia (during 20 s) and after a suddenexposure to hyperoxia (100% O2) of a further 20-s period. Thehyperoxia-induced ventilatory decline was doubled in Tg6mice compared with corresponding WT mice (Fig. 4A). In Tg6animals, the ventilatory difference was mainly due to a higherdecline of fR rather than VT (Fig. 4, B and C). In parallel, andconsidering that Epo is a potent factor modulating release ofcatecholamines in cells with neural characteristics includingPC12 cells (22, 26, 41, 49), we also have evaluated TH activityin carotid bodies of WT and Tg6 mice. Tg6 mice showedsignificantly reduced TH activity compared with control WTanimals (Fig. 4D). As TH is the rate-limiting enzyme fordopamine synthesis in carotid bodies, this data suggests thatinhibitory dopaminergic influences are reduced in Tg6 mice.Altogether, these results imply that the Epo-mediated activa-tion of carotid bodies against changes in arterial oxygen pres-sure is higher in transgenic mice compared with WT.

Intraperitoneal injection of domperidone does not stimulateHVR in Tg6 mice. Domperidone is a highly specific peripheralD2-dopaminergic receptor-antagonist that causes a potent stim-ulation of VE and carotid sinus nerve discharge (16, 18). Whendomperidone was injected into mice, we found that normoxic(21% O2) and hypoxic (10 and 6% O2) VE were stimulated inWT animals (Fig. 5, A–C). However, consistent with thereduced TH activity, this drug had no effect on normoxic orhypoxic VE of transgenic animals (Fig. 5, D–F). This resultsuggests that the basic neurochemistry of the peripheral che-moreceptor is altered in Tg6 animals and that high Epo plasmalevels are implicated in this chemosensory rearrangement.

Table 1. Metabolic parameters

Normoxia Normoxia After Chronic Hypoxia

Type of mice WT Tg6 WT Tg6Number of mice used 10 9 10 8Body weight, g 27.9�1.71 26.3�2.03 26.2�0.68 25.7�0.97Body temperature, °C 37.7�0.67 36.7�0.23 37.4�0.95 36.3�0.71Hemoglobin, g/dl 13.8�0.80 24.7�5.15 19.6�0.8 24.2�9.02VE, ml �min�1 �100 g�1 86.5�4.69 93.5�7.09 117.2�6.35 120.8�9.92fR, resp/min 143.1�8.60 157.7�4.80 165.1�9.94 173.8�5.81VT, ml/100 g 0.61�0.02 0.59�0.4 0.72�0.03 0.69�0.05VO2, ml �min�1 �100 g�1 10.2�0.79 9.5�0.61 17.5�1.80 17.8�2.01VCO2, ml �min�1 �100 g�1 9.3�0.58 8.7�0.78 14.2�1.72 15.1�2.09

WT, wild-type mice; Tg6, transgenic mouse line TgN(PDGFBEPO)321ZbZ; VE, ventilation; VT, tidal volume; fR, respiratory frequency; VO2, O2

consumption; VCO2, CO2 production.

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DISCUSSION

In the present study, the impact of high Epo levels andexcessive erythrocytosis on respiratory responses and ventila-tory acclimatization to hypoxia were examined. We used atransgenic mouse line showing constitutive high levels ofhuman Epo in brain (26-fold/WT) and plasma (12-fold/WT).The hematocrit values of these mice was 80–90% (36, 43, 44).Despite these remarkable characteristics, our results showedthat normoxic and hypoxic VE in transgenic mice were similarto WT control. Tg6 mice however, showed marked alterationin the ventilatory pattern upon hypoxic exposure. fR in Tg6mice was significantly higher, while VT decreased. In line with

earlier work, our results suggest that the elevated cerebral Epolevels enhance fR in hypoxia and that Epo plasma levelsparticipate in the modulation of the carotid body chemotrans-duction.

Following our previously published methodology (39), ourexperimental protocol included ventilatory measurements dur-ing normoxia, moderate hypoxia (10% O2), and severe hypoxia(6% O2). These experiments were conducted in unanesthetizedand unrestrained mice. The advantage of this protocol is that italleviated the effects that anesthesia may have had on breathingand allowed us to monitor changes in body metabolism that areknown to occur during hypoxia (21, 24, 38). Changes in blood

Fig. 1. Ventilatory (VE) response to acute hypoxia in wild-type (Wt) and transgenic (Tg6) male mice. Hypoxia was achieved in 2 steps of 15-min gradualreduction of fraction of inspired oxygen (FIO2) (represented by a black triangle); first step from 21% to 10% O2, and second step from 10% to 6% O2. VE,respiratory frequency (fR), and tidal volume (VT) were evaluated in Wt control and Tg6 mice over 20 min at 10% and at 6% O2 (A–C). The relative meanproportions of fR and VT in the ventilatory response to hypoxia were calculated and are represented in the bars; since VT is the major component in Wtventilatory response, transgenic VE is mainly supported by fR (D and E). Metabolic parameters; body temperature (F), VO2 (G) and VCO2 (H) were alsodetermined in Wt and Tg6 mice. *P � 0.0001. Data are means � SD for n � 10 animals per group.



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pressure and arterial gases could not be monitored in unanes-thetized mice; nevertheless VO2 and VCO2 were evaluated. VO2

and VCO2 values, together with the VE data, gave a reliableindication of potential differences between the tested groups.Considering, for example, that arterial CO2 pressure is propor-tional to the metabolic rate and inversely proportional to VE,our results imply that arterial blood gases in normoxia andhypoxia were similar between both mouse groups.

Compared with WT mice, minute VE under normoxia, aswell as the HVR, during acute and after chronic hypoxia wasnot altered in Tg6 mice. What triggers the neural control of VE

in Tg6 mice–high Epo levels in brain and circulation or the

elevated hemoglobin concentration? A direct impact of hemat-ocrit on the neural control of VE has not been reported so far.As blood viscosity rose exponentially with the augmentation ofhematocrit (6), we expected an exponential increase of pulmo-nary vascular resistance (28). Interestingly, however, despitehematocrit values of 80%, blood pressure, heart rate, andcardiac output in Tg6 mice were not abnormal (36). As shownearlier, the adaptive mechanisms to excessive erythrocytosis inTg6 mice involve increased activity of endothelial nitric oxide(NO) synthase (eNOS). Thus, despite a concomitant increaseof the potent vasoconstrictor endothelin-1 (34), the eNOS-mediated enhanced NO synthesis in Tg6 mice results in gen-

Fig. 2. Basal VE and VE response to hypoxia measured in Wt and Tg6 mice acclimatized to chronic hypoxia (3 days at 10% O2). After acclimatization to hypoxia,animals were returned to room air and basal VE was evaluated. Hypoxia was achieved with a gradual reduction of FIO2 (black triangle); from 21% to 10% O2

(over 15 min) and from 10% to 6% O2 (over 15 min). Hypoxic ventilatory response (HVR) was evaluated during 20 min at 10% and at 6% O2 (A–C). The relativemean proportions of fR and VT were calculated and represented in bars (D and E). After chronic hypoxic VT is the major component of HVR both in Wt andTg6 mice; however, VT contribution is higher in Wt compared with transgenics. Metabolic parameters; body temperature (F), VO2 (G), and VCO2 (H) were alsodetermined in Wt and Tg6 mice. *P � 0.001. Data are means � SD for n � 10 animals per group.



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eralized peripheral vasodilatation (36). In parallel, one shouldkeep in mind that elevation of the NO metabolism in the lungwas also found in Tibetans and Bolivian Aymara as adaptivemechanism to hypoxia (2). Another adaptive mechanism toexcessive erythrocytosis that most probably contributes tonormal VE in Tg6 mice is the subtle increase of blood viscositythat is due to an increased flexibility of the transgenic eryth-rocytes (5, 43). We also speculate that increased flexibility ofthe red blood cells in Tg6 animals contributes to normaloxygen uptake and delivery. As such, we observed no differ-ences in VO2 and VCO2 between Tg6 and control WT mice.Taken together, these observations do not support the notion

that elevated hematocrit level influences the neuronal regula-tion of VE.

On the other hand, despite no differences in minute VE, theventilatory pattern was remarkably altered. While not signifi-cant, somewhat higher fR and lower VT are already observedunder normoxic conditions, and hypoxia potentiated thesedifferences. During hypoxic VE fR increased significantly,while VT decreased. We reported earlier that cerebral Epoenhanced fR under hypoxic conditions (39). We also showedrecently that bilateral transsection of carotid sinus nerve (che-modenervation) in WT mice leads to dramatic respiratorydepression during hypoxia, but high levels of brain Epo main-

Fig. 3. In vivo tyrosine hydroxylase (TH) activity and norepinephrine (NE) levels in brain stem catecholaminergic cell groups (A1C1 and A2C2 in the medullaoblongata; A5 and A6 in the pons) were determined by HPLC. TH activity is expressed as picomoles of L-dihydroxyphenylalanine (L-DOPA) formed in 20 minfollowing blockade of L-DOPA decarboxylase. Significant differences were found in the A5 cell group. *P � 0.05; n � 10–12 animals per group.

Fig. 4. Peripheral chemosensitivity to oxygen evaluated by a hyperoxic test and TH activity in carotid bodies. Ventilation (VE), VT, and fR declined (transitionfrom 21% to 100% O2) in response to hyperoxic testing (Dejours test). The experiment was performed in urethane-anesthetized Wt and Tg6 mice. Tg6 mice weremore sensitive to hyperoxia. Data are means � SD for n � 8 animals per group. *P � 0.001. TH activity is expressed as picomoles of L-DOPA accumulatedin 20 min following blockade of L-DOPA decarboxylase using 3-hydroxybenzylhydrazine dihydrochloride (NSD 1015). Noradrenaline levels were determinedusing HPLC coupled to electrochemical detection. *P � 0.05. Data are means � SD for n � 10–12 animals per group.



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tained fR despite the absence of signal information comingfrom the peripheral chemoreceptors (39). Because Tg6 micealso overexpress Epo in brain (26-fold/WT), it is tempting toattribute the higher hypoxic fR to local presence of Epo inbrain. In addition, our previous results strongly suggested thatEpo increased fR by altering the activity in brain stem cat-echolaminergic centers. High content of NE in A5 group cellsincreases the hypoxic fR (8, 14). In line with these observa-tions, we found that Tg6 mice showed significant increases ofTH activity and NE content in A5 cells compared with WTcontrol mice (Fig. 3B). In conclusion, the present results,together with previous data (39), support the hypothesis thatincreased cerebral Epo level modulates the catecholaminesynthesis in brain stem. Once exposed to hypoxia, this alter-ation affects the ventilatory response via increasing the fR.Despite the tight correlation between HVR and catecholamin-ergic metabolism in brain stem, we do not discard otherpossible mechanisms by which cerebral Epo modulates thehypoxic VE.

Apart from stimulating erythropoiesis (10, 40), plasma Epoaffects hypoxic VE most probably by interacting with carotidbody glomus cells. Carotid bodies are the main peripheralsensors responding to decreased oxygen content and mediate

the integrated cardiorespiratory responses to hypoxemia. Wedemonstrated earlier that intravenous injection of recombinanthuman Epo in WT mice induced a significant alteration inbreathing pattern in response to severe hypoxia (39). Anincreased fR and a profoundly decreased VT, compared withvehicle-injected mice, suggested that plasma Epo levels par-ticipated in the breathing control. The present results using Tg6mice support this hypothesis. Moreover, reduced activity of TH(the rate-limiting enzyme for dopamine synthesis in carotidbodies) in transgenic carotid bodies, suggest that the inhibitoryfunction of dopamine is reduced in transgenic animals. In linewith these results, we found that Tg6 mice exhibited a higherperipheral chemosensitivity as indicated by the response to thehyperoxic challenge (Dejours test). The greater ventilatorydecline observed during hyperoxia implies that transgeniccarotid bodies exert a stronger tonicity, and this effect is mostprobably due to direct stimulation by plasma Epo.

Despite good correlations between elevated central andplasma Epo levels and acute and chronic hypoxic breathingpatterns in Tg6 mice, we do not discard other factors that maycontribute to this process. Indeed, as mentioned earlier weshowed that as a consequence of excessive erythrocytosis, Tg6animals elevate expression of eNOS by threefold (36). The

Fig. 5. VE response in normoxia and acute hypoxia uponintraperitoneal injection of domperidone. Basal VE was evalu-ated after 1–2 h after injection of domperidone. Hypoxia wasachieved with a gradual reduction of FIO2 (black triangle); from21% to 10% O2 (over 15 min) and from 10% to 6% O2 (over 15min). HVR was evaluated during 20 min at 10% and at 6% O2.Control animals were injected with similar volume of 0.9%NaCl solution. *P � 0.001; n � 7–8 animals per group.



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generalized vasodilatation produced by eNOS activity preventsTg6 animals from cardiovascular dysfunction, hypertension,and thromboembolic complication (36). However, the eNOS-mediated production of NO also impacts on the peripheralrespiratory centers in hypoxia (42, 45, 46). As such, our data donot allow discrimination between the effect produced by NOand plasma Epo. However, as Epo is a potent factor leading tothe release of catecholamines in PC12 cells, a reliable model ofperipheral chemosensitive cells (22, 26, 41, 49), it is reasonableto suggest that Epo and not NO is the responsible factor of thecatecholamine alteration observed in carotid bodies. The com-bined impact of Epo and NO in the peripheral respiratorycenters is a matter of current investigation.

Very recently, the specificity of the commercially availableanti-EpoR-antibodies has been challenged (11). As regardingneuronal cells, however, we and others have described 1) thepresence of EpoR mRNA in the brain of several mammals,including humans, by RT-PCR (3, 4, 9, 23, 25, 37); 2) thepresence of specific Epo binding sites in the brain by usingradiolabeled Epo (9); 3) the impact of Epo on PC12 cells (areliable cell model of peripheral chemosensitive cells) (1, 26,41, 49); and 4) the inhibition of the ventilatory acclimatizationto chronic hypoxia in WT mice by intracerebroventicularinfusion of soluble EpoR (38). Thus, there is convincingevidence that EpoR is expressed in neural cells.

In conclusion, the ventilatory response to acute and chronichypoxia remains similar between Tg6 and WT animals. Incontrast, the ventilatory pattern was significantly altered–fRwas widely increased and VT was extensively decreased inTg6 compared with WT mice. In line with previous findings,our results imply that enhanced cerebral Epo levels stimulatefR in response to hypoxia. On the other hand, the modulationof the hypoxic ventilatory pattern in Tg6 carotid bodies mightbe a consequence of several factors, among them the high Epoplasma levels being a major player. These results support thehypothesis that enhanced levels of cerebral and plasma Epo areimportant factors in the regulation of oxygen homeostasis and,moreover, might have decisive implications in the ventilatoryacclimatization and deacclimatization process to high altitudehypoxia.

ACKNOWLEDGMENTS

The authors thank Stephan Keller for technical help, Martha Tissot VanPatot for proofreading the manuscript, and Vincent Joseph for fruitful discus-sions.

GRANTS

The present study was supported by grants from Roche Foundation forAnemia Research, the Swiss National Science Foundation, and the EU projectsEuroxy and PulmoTension (to J. Soliz and M. Gassmann).

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University of Zurich - UZH · 2010. 11. 29. · Acute and chronic exposure to hypoxia alters ventilatory pattern but not minute ventilation of mice overexpressing erythropoietin Jorge