Characterisation of the ventilatory response to hypoxia in a model of transgenic anemic mice

doi:10.1016/j.resp.2005.03.011

Respiratory Physiology & Neurobiology

Volume 150, Issue 1, 25 January 2006, Pages 19-26

https://doi.org/10.1016/j.resp.2005.03.011 Get rights and content

Abstract

Both polycythemia and the increase in hypoxic ventilatory response (HVR) are considered as important factors of acclimatization to hypoxia. The objective of this study was to characterise the ventilation pattern at different inspired oxygen fraction in a model of chronic anemic mice. These mice have a targeted disruption in the 5′ untranslated region of the Epo gene that reduces Epo expression such that the homozygous animal is severely anemic. Ventilation in normoxia in Epo-TAg^h mice was significantly greater than in wild type, and the difference was mainly due to a higher tidal volume. HVR was higher in Epo-TAg^h mice at every FIO₂ suggesting a higher chemosensitivity. Resting oxygen consumption was maintained in anemic mice. Maximal oxygen consumption was 30% lower while hemoglobin was 60% lower in anemic mice compared to wild type. This small decrease in maximal oxygen consumption is probably due a greater cardiac output and/or a better tissue oxygen extraction and would allow these anemic mice to acclimatize to hypoxia in spite of low oxygen carrying capacity. In conclusion, Epo-TAg^h anemic mice showed increased ventilation and hypoxic ventilatory response. However, whether these adaptations will contribute to acclimatization in chronic hypoxia remains to be determined.

Introduction

The ventilatory response to hypoxia successively includes the acute ventilatory response to hypoxia, the relative decrease in ventilation (hypoxic ventilatory decline) after some minutes of exposure, and the time-dependent increase in ventilation that occurs with chronic hypoxic exposure of a few hours to several weeks. It is now well known that an increase in the acute hypoxic ventilatory response (HVR) contributes to the ventilatory acclimatization to hypoxia (Bisgard and Neubauer, 1995, Powell et al., 1998, Powell et al., 2000, Reeves et al., 1993).

In anemia, a majority of reports indicates an absence of hyperventilation in normoxia (Woodson et al., 1978), or a small increase in ventilation related to the hypotension due to peripheral vasodilatation in hypoxia (Saiki et al., 1994, Sardella and Ou, 1993). The oxygen carrying capacity of the blood is reduced, resulting in tissue hypoxia. Nevertheless at rest in normoxia, oxygen delivery is maintained by an increase in cardiac output and tissue O₂ extraction (Gonzalez et al., 1994, Ickx et al., 2000, Kurdak et al., 1995) to preserve oxygen consumption (Ickx et al., 2000, Vaslef et al., 2001). However, it has been also shown a fall in oxygen consumption secondary to acute anemia induced by hemodilution (Kurdak et al., 1995).

In addition to the constraint of a decrease arterial O₂ content (CaO₂), exposure to hypoxia adds the constraint of a decrease arterial O₂ pressure (PaO₂) that triggers integrated responses at the respiratory, cardiovascular and hematological levels.

This study focuses on the time course and effect of acute hypoxic exposure on the ventilatory response in the erythropoietin SV-40 T antigen mouse (Epo-TAg^h). This mouse has a targeted disruption in the 5′ untranslated region of the Epo gene that reduces the Epo expression such that the homozygous animal is severely anemic (Binley et al., 2002). The use of the Epo-TAg^h allows us to determine the effect of anemia on HVR, without the adverse effects of bleeding or hemodilution.

Polycythemia is considered as an important factor of acclimatization to hypoxia, together with the increase of ventilation. Because, the beneficial role of polycythemia has been questioned, it was of interest to characterise the ventilation pattern and response to hypoxia in a model of chronic anemic mice.

Section snippets

Animals

Animal studies were performed in accordance with guidelines established by the French Ministère de l’Agriculture.

Six anemic SV-40 T antigen (Epo-Tag^h) and six healthy wild type (C57B16/CBA) mice, 6- to 8-week-old, were investigated. Animals were kept in a room with free access to food and water. Temperature (21 ± 2 °C) and humidity (49 ± 3%) were checked daily. The animals were weighed before the experiments. At the end of the experiments a blood sample was taken from the tail vessel for hematocrit

Animals characteristics (Tables 1 and 2)

Body weights were similar in both groups. Hematocrit as well as hemoglobin concentration were significantly 60% lower in Epo-TAg^h mice demonstrating severe anemia. Resting oxygen consumption was similar in both groups in normoxia while ${\dot{V}}_{O_{2} max}$ w as 30% lower in Epo-TAg^h (Table 1, Table 2).

Anemic mice showed cardiac hypertrophy, as assessed by the higher right and left ventricular weight. No change was observed in right ventricular systolic pressure and resting heart rate in Epo-TAg^h mice

Discussion

This study is the first to characterise the hypoxic ventilatory response in a model of anemic mice. Our main results showed that: (1) anemic Epo-TAg^h mice hyperventilated in normoxia due to a greater V_T; (2) hypoxic ventilatory response was higher in Epo-TAg^h mice with a maximum at 12%, suggesting a higher chemosensitivity; (3) the maximal response of ventilation occurred after 5 min of exposure in both groups; (4) ventilation in Epo-TAg^h mice declined at FIO₂ < 12% and with time exposure; (5)

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Pharmacological, but not genetic, alteration of neural Epo modifies the CO<inf>2</inf>/H<sup>+</sup> central chemosensitivity in postnatal mice
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Research from our laboratories also contributed to this new concept of Epo. By using transgenic mice showing over- or deficient-expression of cerebral Epo, we reported altered modulation of basal ventilation and hypoxic hyperventilation, both at postnatal and adult ages (Ballot et al., 2015a; Caravagna et al., 2015; El Hasnaoui-Saadani et al., 2009; Khemiri et al., 2011; Macarlupu et al., 2006; Pichon et al., 2016b; Soliz et al., 2005; Voituron et al., 2014). Moreover, the basal ventilation and hypoxic ventilatory response were also drastically reduced (by about 50%) when the Epo/sEpoR ratio was altered by injecting soluble EpoR (sEpoR, the natural competitive antagonist of Epo) in the cisterna magna (intra-cisternal injection) of WT mice (Ballot et al., 2015b).
Cerebral erythropoietin (Epo) plays a crucial role for respiratory control in newborn rodents. We showed previously that soluble Epo receptor (sEpoR: an Epo antagonist) reduces basal ventilation and hypoxic hyperventilation at postnatal day 10 (P10) and in adult mice. However, at these ages (P10 and adulthood), Epo had no effect on central chemosensitivity. Nevertheless, it is known that the sensitivity to CO₂/H⁺ during the mammalian respiratory network maturation process is age-dependent. Accordingly, in this study we wanted to test the hypothesis that cerebral Epo is involved in the breathing stimulation induced by the activation of central CO₂/H⁺ chemoreceptors at earlier postnatal ages. To this end, en bloc brainstem-spinal cord preparations were obtained from P4 mice and the fictive breathing response to CO₂-induced acidosis or metabolic acidosis was analyzed. This age (P4) was chosen because previous research from our laboratory showed that Epo altered (in a dose- and time-dependent manner) the fictive ventilation elicited in brainstem-spinal cord preparations. Moreover, as it was observed that peripheral chemoreceptors determined the respiratory sensitivity of central chemoreceptors to CO₂, the use of this technique restricts our observations to central modulation. Our results did not show differences between preparations from control and transgenic animals (Tg21: overexpressing cerebral Epo; Epo-TAg^h: cerebral Epo deficient mice). However, when Tg21 brainstem preparations were incubated for 1 h with sEpoR, or with inhibitors of ERK/Akt (thus blocking the activation of the Epo molecular pathway), the fictive breathing response to CO₂-induced acidosis was blunted. Our data suggest that variation of the Epo/sEpoR ratio is central to breathing modulation during CO₂ challenges, and calls attention to clinical perspectives based on the use of Epo drugs at birth in hypoventilation cases.
Epo deficiency alters cardiac adaptation to chronic hypoxia
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As we found a decrease in LV hypertrophy and functional LV adaptation, a depressed HIF-1α/VEGF pathway as well as a reduced oxygen delivery, our results suggest that cardiac adaptive mechanisms that take place with chronic Epo deficiency and hypoxia may require extensive Epo effects (angiogenesis, cardioprotection) on the heart. On the other hand, our previous results in Epo-TAgh mice demonstrate that high levels of Epo are not necessary for survival in chronic moderate hypoxia (Macarlupu et al., 2006a,b). Similarly, the pika (Ochotona curzoniae) a lagomorph with low Hb (10 g/dL) is adapted to high altitude and does not develop RV hypertrophy (Ge et al., 1998).
The involvement of erythropoietin in cardiac adaptation to acute and chronic (CHx) hypoxia was investigated in erythropoietin deficient transgenic (Epo-TAg^h) and wild-type (WT) mice. Left (LV) and right ventricular functions were assessed by echocardiography and hemodynamics. HIF-1α, VEGF and Epo pathways were explored through RT-PCR, ELISA, Western blot and immunocytochemistry. Epo gene and protein were expressed in cardiomyocytes of WT mice in normoxia and hypoxia. Increase in blood hemoglobin, angiogenesis and functional cardiac adaptation occurred in CHx in WT mice, allowing a normal oxygen delivery (O₂T). Epo deficiency induced LV hypertrophy, increased cardiac output (CO) and angiogenesis, but O₂T remained lower than in WT mice. In CHx Epo-TAg^h mice, LV hypertrophy, CO and O₂T decreased. HIF-1α and Epo receptor pathways were depressed, suggesting that Epo-TAg^h mice could not adapt to CHx despite activation of cardioprotective pathways (increased P-STAT-5/STAT-5). HIF/Epo pathway is activated in the heart of WT mice in hypoxia. Chronic hypoxia induced cardiac adaptive responses that were altered with Epo deficiency, failing to maintain oxygen delivery to tissues.
Time course of ventilatory acclimatisation to hypoxia in a model of anemic transgenic mice
2006, Respiratory Physiology and Neurobiology
We questioned the assumption that polycythemia is essential for adaptation to chronic hypoxia. Thus, the objective of our study was to determine if anemic Epo-TAg^h mice could survive in hypoxia despite low oxygen carrying capacity. We explored the possibility that ventilatory acclimatisation is involved in the strategy used by anemic transgenic mice to adapt to chronic hypoxia. Epo-TAg^h and Wild Type mice were exposed during 2 weeks at a barometric pressure of 450 Torr. After 1, 5 and 14 days of exposure, ventilation at different inspired oxygen fraction was measured in both groups. Ventilation during acclimatisation to hypoxia was significantly greater in Epo-TAg^h than in Wild Type. The difference was mainly due to a higher tidal volume that could explain a higher arterial PO₂ in Epo-TAg^h mice. Epo-Tag^h mice did not develop right ventricle hypertrophy after 2 weeks of exposure to hypoxia while Wild Type did. Hemoglobin concentration was 60% lower in anemic mice versus Wild Type after acclimatisation. In conclusion, ventilatory acclimatisation contributed to the adaptation of Epo-Tag^h mice in chronic hypoxia despite low arterial oxygen carrying capacity.
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Red blood cell deformability is very slightly decreased in erythropoietin deficient mice
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Characterisation of the ventilatory response to hypoxia in a model of transgenic anemic mice

Abstract

Introduction

Section snippets

Animals

Animals characteristics (Tables 1 and 2)

Discussion

Respir. Physiol.

Blood

Respir. Physiol.

Respir. Physiol.

Respir. Physiol.

Peripheral and central effects of hypoxia

Mechanisms of improvement in pulmonary gas exchange during isovolemic hemodilution

J. Appl. Physiol.

Increasing P(50) does not improve DO(2CRIT) or systemic VO(2) in severe anemia

Am. J. Physiol.

Increasing maximal heart rate increases maximal O2 uptake in rats acclimatized to simulated altitude

J. Appl. Physiol.

Effect of hematocrit on systemic O2 transport in hypoxic and normoxic exercise in rats

J. Appl. Physiol.

Interactive effects of anemia and muscle oxidative capacity on exercise endurance

J. Appl. Physiol.

Effect of hemoglobin concentration on maximal O2 uptake in canine gastrocnemius muscle in situ

J. Appl. Physiol.

Increasing maximal heart rate increases maximal O₂ uptake in rats acclimatized to simulated altitude

Effect of hematocrit on systemic O₂ transport in hypoxic and normoxic exercise in rats

Effect of hemoglobin concentration on maximal O₂ uptake in canine gastrocnemius muscle in situ