Key points Sustained hypoxic exposure increases ventilatory sensitivity to hypoxia as part of physiological acclimatisation.Oxygen‐sensitive signals are transduced in animal cells by post‐translational hydroxylation of transcription factors termed hypoxia‐inducible factors (HIFs).Mice heterozygous for the principal ‘oxygen‐sensing’ HIF hydroxylase PHD2 (prolyl hydroxylase domain 2) show enhanced ventilatory sensitivity to hypoxia.To analyse the underlying mechanisms, functional (hypoxic ventilatory responses, HVRs) and anatomical (cellular proliferation within carotid bodies) responses were studied in genetic models of inducible and constitutive inactivation of PHD2 and its principal hydroxylation substrates, HIF‐1α and HIF‐2α.Inducible PHD2 inactivation enhanced HVR, similar to constitutive inactivation; both responses were almost entirely compensated for by specific inactivation of HIF‐2α.Inducible inactivation of HIF‐2α, but not HIF‐1α, strikingly reduced ventilatory acclimatisation to hypoxia and associated carotid body cell proliferation.These findings demonstrate a key role for PHD2 and HIF‐2α in ventilatory control and carotid body biology. AbstractVentilatory sensitivity to hypoxia increases in response to continued hypoxic exposure as part of acute acclimatisation. Although this process is incompletely understood, insights have been gained through studies of the hypoxia‐inducible factor (HIF) hydroxylase system. Genetic studies implicate these pathways widely in the integrated physiology of hypoxia, through effects on developmental or adaptive processes. In keeping with this, mice that are heterozygous for the principal HIF prolyl hydroxylase, PHD2, show enhanced ventilatory sensitivity to hypoxia and carotid body hyperplasia. Here we have sought to understand this process better through comparative analysis of inducible and constitutive inactivation of PHD2 and its principal targets HIF‐1α and HIF‐2α. We demonstrate that general inducible inactivation of PHD2 in tamoxifen‐treated Phd2f/f;Rosa26+/CreERT2 mice, like constitutive, heterozygous PHD2 deficiency, enhances hypoxic ventilatory responses (HVRs: 7.2 ± 0.6 vs. 4.4 ± 0.4 ml min−1 g−1 in controls, P < 0.01). The ventilatory phenotypes associated with both inducible and constitutive inactivation of PHD2 were strongly compensated for by concomitant inactivation of HIF‐2α, but not HIF‐1α. Furthermore, inducible inactivation of HIF‐2α strikingly impaired ventilatory acclimatisation to chronic hypoxia (HVRs: 4.1 ± 0.5 vs. 8.6 ± 0.5 ml min−1 g−1 in controls, P < 0.0001), as well as carotid body cell proliferation (400 ± 81 vs. 2630 ± 390 bromodeoxyuridine‐positive cells mm−2 in controls, P < 0.0001). The findings demonstrate the importance of the PHD2/HIF‐2α enzyme–substrate couple in modulating ventilatory sensitivity to hypoxia.
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 mm3 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.
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