Carotid body hyperplasia and enhanced ventilatory responses to hypoxia in mice with heterozygous deficiency of PHD2

Abstract

Oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor (HIF) by a set of closely related prolyl hydroxylase domain enzymes (PHD1, 2 and 3) regulates a range of transcriptional responses to hypoxia. This raises important questions about the role of these oxygen-sensing enzymes in integrative physiology. We investigated the effect of both genetic deficiency and pharmacological inhibition on the change in ventilation in response to acute hypoxic stimulation in mice. Mice exposed to chronic hypoxia for 7 days manifest an exaggerated hypoxic ventilatory response (HVR) (10.8 ± 0.3 versus 4.1 ± 0.7 ml min(-1) g(-1) in controls; P < 0.01). HVR was similarly exaggerated in PHD2(+/-) animals compared to littermate controls (8.4 ± 0.7 versus 5.0 ± 0.8 ml min(-1) g(-1); P < 0.01). Carotid body volume increased (0.0025 ± 0.00017 in PHD2(+/-) animals versus 0.0015 ± 0.00019 mm(3) in controls; P < 0.01). In contrast, HVR in PHD1(-/-) and PHD3(-/-) mice was similar to littermate controls. Acute exposure to a small molecule PHD inhibitor (PHI) (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid) did not mimic the ventilatory response to hypoxia. Further, 7 day administration of the PHI induced only modest increases in HVR and carotid body cell proliferation, despite marked stimulation of erythropoiesis. This was in contrast with chronic hypoxia, which elicited both exaggerated HVR and cellular proliferation. The findings demonstrate that PHD enzymes modulate ventilatory sensitivity to hypoxia and identify PHD2 as the most important enzyme in this response. They also reveal differences between genetic inactivation of PHDs, responses to hypoxia and responses to a pharmacological inhibitor, demonstrating the need for caution in predicting the effects of therapeutic modulation of the HIF hydroxylase system on different physiological responses.

Figure 1. Effect of pre-exposure to chronic hypoxia on ventilatory responses to acute hypoxia

Minute ventilation (left), tidal volume (middle) and respiratory rate (right) before, during and after an acute hypoxic stimulus, in mice pre-exposed to continuous hypoxia (10% oxygen) for 7 days or maintained in normoxia. Acute exposure to 10% oxygen was made without (A) and with (B) the addition of 3% carbon dioxide (filled and open bars, respectively). Mean ± SEM; n= 8 for each group.

Figure 2. Ventilatory responses to acute hypoxia in PHD2^+/− and littermate control wild-type mice

Minute ventilation (left), tidal volume (middle) and respiratory rate (right) after acute exposure to 10% oxygen without (A) and with (B) the addition of 3% carbon dioxide (filled and open bars, respectively). Mean ± SEM; n= 7 for each group.

Figure 3. Effect of pre-exposure to chronic hypoxia on ventilatory responses to acute hypoxia in PHD2^+/− mice

Minute ventilation (left), tidal volume (middle) and respiratory rate (right) before, during and after an acute hypoxic stimulus, in PHD2^+/− mice pre-exposed to continuous hypoxia (10% oxygen) for 7 days or maintained in normoxia. Acute exposure to 10% oxygen was made without (A) and with (B) the addition of 3% carbon dioxide (filled and open bars, respectively). Mean ± SEM; n= 5 for each group.

Figure 4. Arterial oxygen saturation in acute hypoxia

Conscious, unrestrained PHD2^+/− animals (filled bars) and littermate control wild-type animals (open bars); animals in normoxia, 10% oxygen or 10% oxygen with 3% carbon dioxide. Mean ± SEM; n= 5 mice for each group; ***P < 0.0001; **P < 0.01.

Figure 5. Histological analysis of carotid bodies

A, representative sections of wild-type and PHD2^+/− carotid bodies (CB) immunostained with tyrosine hydroxylase (TH) (brown). B, morphometric analysis of TH-positive cells, carotid body volume, TH-positive cell density and TH-positive cell volume in the CB. Mean ± SEM; n= 5 mice for each group; **P < 0.01.

Figure 6. Ventilatory responses of PHD1^−/− and PHD3^−/− mice

Changes in minute ventilation in response to 10% oxygen with 3% carbon dioxide in PHD1^−/− (A) and PHD3^−/− (B) and littermate control wild-type mice. Acute exposure to hypoxia: 10% oxygen with 3% carbon dioxide (open bars). Mean ± SEM; n= 7 for each group (A); n= 5 for each group (B).

Figure 7. Comparison of effects of IOX3 treatment versus continuous hypoxia

Mice were treated with IOX3 (30 mg kg⁻¹ twice daily) or vehicle (HBSS with 5% DMSO) for 7 days. Minute ventilation in response to 10% oxygen with 3% carbon dioxide (open bars) in IOX3-treated or vehicle-treated animals immediately following (A) or 7 days after (B) the first injection. C, chemical structure of IOX3. E, carotid body BrdU incorporation after 7 days of IOX3 treatment; mean ± SEM; n= 11 for each group; *P < 0.05. D, F and G, carotid body proliferation in mice exposed to continuous hypoxia (10% oxygen) or normoxia for 7 days. Morphometric analysis of BrdU staining in CBs from wild-type (F) and PHD2^+/− (G) mice; mean ± SEM; n= 8 (F) or 5 mice (G) for each group; ***P < 0.0001. D, representative images of BrdU staining (brown) in wild-type mice; CB: carotid body; SCG: supracervical ganglion.