Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism

Abstract

HIF prolyl hydroxylases (PHD1–3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparα pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2α and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.

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Figure 1: Loss of Phd1 reduces oxygen consumption.
Figure 2: Loss of Phd1 reprograms muscle glucose metabolism.
Figure 3: Hypoxia tolerance in ischemic Phd1-deficient muscle.
Figure 4: Reduced oxidative stress and energy production in ischemic Phd1-deficient muscle.
Figure 5: Role of Pparα and Hif-2α, and hypoxia tolerance by knockdown of Phd1.
Figure 6: Loss of Phd1 induces hypoxia tolerance.

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Acknowledgements

J.A. is sponsored by the Centro Nacional de Investigaciones Cardiovasculares (CNIC; Spain) and Flanders Institute of Biotechnology (VIB; Belgium); M.S. by the Deutsche Forschungsgemeinschaft (Germany) and the Lymphatic Research Foundation; K.V.G. by a doctoral fellowship from the K.U. Leuven; P.F. by a postdoctoral fellowship from the K.U. Leuven; and H.L. by a postdoctoral fellowship from Fond Québécois de la nature et des technologies. This work is supported, in part, by grant GOA2006/11/KULeuven from the University of Leuven, Belgium, grant IUAP05/02 from the Federal Government Belgium, grants FWO G.0265 and FWO G.0387 from the Flanders Research Foundation, Belgium, grant 5RO1GM037704 from the US National Institutes of Health (to F.L.), grant 12RO1DK47844 from the US National Institutes of Health (to R.A.H.), grant 2.4532.03 from the Belgian Fonds National de la Recherche Scientifique (to F.S.), and a grant from the Molecular Small Animal Imaging Center, University of Leuven (to L.M. and P.V.H.) and by a Programme Grant from the British Heart Foundation (to P.H.M. and P.J.R.). The authors thank A. Bouche, A. Carton, P. Chevron, M. De Mol, E. Gils, B. Hermans, L. Kieckens, W.Y. Man, W. Martens, A. Manderveld, E. Meyhi, L. Notebaert, J. Souffreau, C. Vanhuylebroeck, B. Vanwetswinkel, P. Van Wesemael, Q. Swennen, B. Kamers and S. Wyns for their contributions.

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J.A.: analysis of all data; design and performance of NMR spectroscopy, assays of metabolic enzymes, metabolites, indirect calorimetry, RNA and protein expression, and respiration studies; writing of the paper. M.S.: analysis of all data; design and performance of running tests, hind limb vascularization, EPR, respiration studies, indirect calorimetry and histological analysis; writing of the paper. K.V.G.: analysis of all data; design and performance of RNA interference experiments, metabolic assays, indirect calorimetry and RNA and protein expression; writing of the paper. P.F.: analysis of all data; design and performance of metabolic assays and mitochondrial respiration studies; writing of the paper. T.D. and P.V.H.: NMR spectroscopy. M.M., P.L., S.K.H., A.G., S.Z. and T.B.: protein expression analysis, cell culture experiments, mitochondrial performance assays. N.H.J., F.G., R.A.H. and R.D.: metabolic enzyme and metabolite assays; oxidative stress assays. D.L. and M.D.: generation of PHD1-deficient mice; genetic analysis of background; statistical analysis. A.D.-J. and T.V.: design of constructs expressing interference short hairpin RNAs. P.V.N., K.D.B. and P.H.: muscle exercise studies. C.W. and C.P.: design and construction of PHD1 targeting vector. M.T. and L. Moons.: histological analysis. R.N. and F. Sluse.: mitochondrial respiration studies. C.D., B.W., J.N. and L. Mortelmans: glucose metabolism studies. B.J. and B.G.: EPR studies. R.S.-M. and F.L.: mitochondrial ultrastructure. P.S.: micro-CT analysis. J.B.: indirect calorimetry. H.L. and E.G.: analysis and performance of mitochondrial respiration studies in isolated myofibers. P.V.V., F. Schuit and M.B.: analysis and design of metabolic experiments and oxidative stress assays; writing of the paper. P.R., P.M and P.C.: scientific direction; generation of PHD knockout mice; conception of hypoxia tolerance studies; design of experimental approaches; analysis of data; writing of the paper.

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Correspondence to Peter Carmeliet.

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P.H.M. is a director, consultant and equity holder in ReOx Ltd. P.J.R., P.H.M and C.W.P. are founding scientists of ReOx Ltd.

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Aragonés, J., Schneider, M., Van Geyte, K. et al. Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism. Nat Genet 40, 170–180 (2008). https://doi.org/10.1038/ng.2007.62

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