Activation of the Prolyl Hydroxylase Oxygen-sensor Results in Induction of GLUT1, Heme Oxygenase-1, and Nitric-oxide Synthase Proteins and Confers Protection from Metabolic Inhibition to Cardiomyocytes

Recently an oxygen-sensing/transducing mechanism has been identified as a family of O2-dependent prolyl hydroxylase domain-containing enzymes (PHD). In normoxia, PHD hydroxylates a specific proline residue that directs the degradation of constitutively synthesized hypoxia-inducible factor-1α. During hypoxia, the cessation of hydroxylation of this proline results in less degradation and thus increases hypoxia-inducible factor-1α protein levels. In this study we have examined the consequences of activating the PHD oxygen-sensing pathway in cultured neonatal myocytes using ethyl-3,4 dihydroxybenzoate and dimethyloxalylglycine, inhibitors that, similar to hypoxia, inhibit this family of O2-dependent PHD enzymes. Increased glucose uptake and enhanced glycolytic metabolism are classical cellular responses to hypoxia. Ethyl-3,4 dihydroxybenzoate treatment of cardiomyocyte cultures for 24 h increased 3Hdeoxy-4-glucose uptake concurrent with an induction of GLUT1 protein. In addition, ethyl-3,4 dihydroxybenzoate, dimethyloxalylglycine, and hypoxia treatments were found to induce protein levels of nitricoxide synthase-2 and heme oxygenase-1, two important cardioregulatory proteins whose expression in response to hypoxic conditions is poorly understood. In conjunction with these changes in gene expression, activation of the PHD oxygen-sensing mechanism was found to preserve myocyte viability in the face of metabolic inhibition with cyanide and 2-deoxyglucose. These results point to a key role for the PHD pathway in the phenotypic changes that are observed in a hypoxic myocyte and may suggest a strategy to pharmacologically induce protection in heart. Recently an oxygen-sensing/transducing mechanism has been identified as a family of O2-dependent prolyl hydroxylase domain-containing enzymes (PHD). In normoxia, PHD hydroxylates a specific proline residue that directs the degradation of constitutively synthesized hypoxia-inducible factor-1α. During hypoxia, the cessation of hydroxylation of this proline results in less degradation and thus increases hypoxia-inducible factor-1α protein levels. In this study we have examined the consequences of activating the PHD oxygen-sensing pathway in cultured neonatal myocytes using ethyl-3,4 dihydroxybenzoate and dimethyloxalylglycine, inhibitors that, similar to hypoxia, inhibit this family of O2-dependent PHD enzymes. Increased glucose uptake and enhanced glycolytic metabolism are classical cellular responses to hypoxia. Ethyl-3,4 dihydroxybenzoate treatment of cardiomyocyte cultures for 24 h increased 3Hdeoxy-4-glucose uptake concurrent with an induction of GLUT1 protein. In addition, ethyl-3,4 dihydroxybenzoate, dimethyloxalylglycine, and hypoxia treatments were found to induce protein levels of nitricoxide synthase-2 and heme oxygenase-1, two important cardioregulatory proteins whose expression in response to hypoxic conditions is poorly understood. In conjunction with these changes in gene expression, activation of the PHD oxygen-sensing mechanism was found to preserve myocyte viability in the face of metabolic inhibition with cyanide and 2-deoxyglucose. These results point to a key role for the PHD pathway in the phenotypic changes that are observed in a hypoxic myocyte and may suggest a strategy to pharmacologically induce protection in heart. The cardiovascular system displays exquisite sensitivity to oxygen levels with a constellation of both acute and chronic responses to changes in oxygen tension. It is of interest to explore the specific changes in gene expression and the signaling pathways that mediate the response of the heart cell to hypoxia. Recently a family of O2-dependent prolyl hydroxylase domain-containing enzymes (PHD) 1The abbreviations used are: PHD, prolyl hydroxylase domain-containing enzymes; HIF-1α, hypoxia-inducible factor-1α; HO-1, heme oxygenase-1; NOS-2, inducible nitric-oxide synthase; DMEM, Dulbecco's modified Eagle's medium; HBSS, Hank's balanced salt solution; ERK, extracellular signal-regulated kinase; EDHB, ethyl 3,4-dihydroxybenzoate; DMOG, dimethyloxalylglycine. have been identified as an oxygen-sensing/transducing pathway (1Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2130) Google Scholar, 2Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar). Hydroxylation of a specific proline in the degradation domain of hypoxia-inducible factor-1α (HIF-1α) by the PHD enzyme directs its constitutive degradation (3Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3917) Google Scholar, 4Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4489) Google Scholar). In hypoxia, failure of the oxygen-dependent PHD to hydroxylate this proline interrupts the targeting of HIF for proteasomal degradation. In addition, in low oxygen conditions, the failure to hydroxylate an asparagine residue in the transactivating domain of HIF results in an increased transactivating potential (5Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Science. 2002; 295: 858-861Crossref PubMed Scopus (1280) Google Scholar). Three mammalian members of this oxygen-dependent prolyl hydroxylase enzyme family (PHD 1, 2, and 3) have been identified to date (1Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2130) Google Scholar, 2Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar). Other possible cellular targets of these prolyl and asparaginyl hydroxylase enzymes, beside HIF, remain unidentified. The expression of many genes is induced or modulated in response to hypoxia. A well characterized example of a hypoxia-inducible gene is Glut1. In the case of GLUT1, its induction by hypoxia occurs mostly at the transcriptional level through an HIF-dependent mechanism (6Behrooz A. Ismail-Beigi F. J. Biol. Chem. 1997; 272: 5555-5562Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 7Behrooz A. Ismail-Beigi F. News Physiol. Sci. 1999; 14: 105-110PubMed Google Scholar). Like GLUT1, heme oxygenase-1 (HO-1) has been characterized as a hypoxia-inducible gene in cardiomyocyte cells (8Borger D.R. Essig D.A. Am. J. Physiol. 1998; 274: H965-H973PubMed Google Scholar). HO-1 is a stress-responsive protein that has recently been shown to ameliorate the damage sustained by the heart from an ischemia-reperfusion insult (9Hangaishi M. Ishizaka N. Aizawa T. Kurihara Y. Taguchi J. Nagai R. Kimura S. Ohno M. Biochem. Biophys. Res. Commun. 2000; 279: 582-588Crossref PubMed Scopus (112) Google Scholar, 10Clark J.E. Foresti R. Sarathchandra P. Kaur H. Green C.J. Motterlini R. Am. J. Physiol. 2000; 278: H643-H651Crossref PubMed Google Scholar). Inducible nitric-oxide synthase (NOS-2) is another important cardioregulatory protein whose expression is thought to be regulated by oxygen tension. The induction of NOS-2 expression after an ischemic insult has been postulated to underlie the phenomenon of delayed ischemic preconditioning (11Bolli R. Manchikalapudi S. Tang X.L. Takano H. Qiu Y. Guo Y. Zhang Q. Jadoon A.K. Circ. Res. 1997; 81: 1094-1107Crossref PubMed Scopus (279) Google Scholar). NOS-2 induction by cytokines is modulated by oxygen tension (12Kacimi R. Long C.S. Karliner J.S. Circulation. 1997; 96: 1937-1943Crossref PubMed Scopus (64) Google Scholar, 13Jung F. Palmer L.A. Zhou N. Johns R.A. Circ. Res. 2000; 86: 319-325Crossref PubMed Scopus (272) Google Scholar). However the ability of hypoxia, per se, to induce NOS-2 protein expression is unclear. In this study we have employed specific inhibitors of the prolyl hydroxylase class of enzymes to activate the PHD oxygen-sensing pathway in cardiomyocytes to explore which responses in the ischemic heart might be attributable to this oxygen-sensing mechanism. Because the expression of GLUT1, HO-1, and NOS-2 has been suggested to be modulated by hypoxia and/or HIF, we examined whether the pharmacological activation of the PHD enzyme oxygen-sensing pathway would effect the expression of these genes. We found that prolyl hydroxylase inhibitors lead to the induction of GLUT1, HO-1, and NOS-2 proteins in heart cells. Further experimentation revealed that a phenotype resistant to metabolic insult accompanied these changes in gene expression. These results point to a central role for the PHD oxygen-sensing mechanism in the response of the heart to hypoxia. Myocyte Cultures—Primary neonatal cardiomyocytes were isolated from 1- to 2-day-old CD-1 mouse pups using a previously described (14Wright G. Singh I.S. Hasday J.D. Farrance I.K. Hall G. Cross A.S. Rogers T.B. Am. J. Physiol. 2002; 282: H872-H879PubMed Google Scholar) method that produces cultures that exhibit spontaneous beating and >95% myocyte purity. Cells were plated on gelatin-coated plates at a density of 2 × 106 cells per 60 mm diameter culture dish, in DMEM/F12 medium supplemented with 10% fetal bovine serum. After 16 h myocytes were washed and placed in maintenance media consisting of DMEM/F12 supplemented with insulin/transferrin/selenium solution (ITS+2) (Sigma) and 1% fetal calf serum. All experiments were conducted on days 3–5 after isolation of the myocytes. To achieve hypoxia, cultures were placed in an airtight Plexiglas chamber that was vigorously purged (10 min) and then continuously perfused with 95/5% argon/CO2 gas mixture (∼400 cm3 per min). Glucose Uptake Assay—Glucose uptake was assayed by placing cultures, treated as indicated, in Hank's balanced salt solution (HBSS) containing 2 μCi 3H2-deoxyglucose and 10 μm cold 2-deoxyglucose, ± cytochalasin B (50 μm) for 10 min at room temperature. Uptake that was not inhibited by cytochalasin did not exceed 10% of total and was subtracted from values as nonspecific background. Cultures were washed with phosphate-buffered saline, and cells were lysed directly on plates with 0.05 m NaOH. Extracts were counted via scintillation. Parallel non-radiolabeled cultures treated as above were harvested for protein determinations. Western Blots—Western blot analysis was performed by standard procedures using SDS-PAGE Tris/glycine pre-cast gels in the NOVEX electrophoresis system. Western blot analyses were performed and detected using ECL chemiluminescence detection reagents (Amersham Biosciences) and protocols. Antibodies directed against GLUT1 (Alpha Diagnostic, San Antonio, TX), NOS-2 (Santa Cruz), HO-1 (Santa Cruz), phospho-ERK1 275: 469-476Crossref PubMed Scopus (36) Google Scholar, 16Friedman L. Higgin J.J. Moulder G. Barstead R. Raines R.T. Kimble J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4736-4741Crossref PubMed Scopus (61) Google Scholar). Prolyl Hydroxylase Inhibition Increases Glucose Uptake and GLUT1 Protein—An increase in glucose uptake and utilization is a classic cellular response to hypoxia or cultures were treated with the prolyl hydroxylase inhibitor to the potential of glucose uptake by PHD pathway has been characterized as a specific inhibitor that to both the and of the prolyl hydroxylase domain T. K. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. Gunzler V. Hanauske-Abel H.M. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of for the inhibition of prolyl hydroxylase in cell was as μm T. K. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). 3H2-deoxyglucose uptake by neonatal myocytes cultured in medium and treated with or for 16 h was increased and is to increase glucose uptake by the transcriptional activation of the GLUT1 gene (6Behrooz A. Ismail-Beigi F. J. Biol. Chem. 1997; 272: 5555-5562Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). is that increased expression of GLUT1 protein the of glucose uptake by cultures were found to have increased GLUT1 protein levels as by Western blot the induction of the GLUT1 gene in to of the Prolyl Hydroxylase HO-1 is a cardioregulatory protein that has previously been shown to be induced by hypoxia in neonatal cardiomyocytes (8Borger D.R. Essig D.A. Am. J. Physiol. 1998; 274: H965-H973PubMed Google Scholar). It was of interest to explore the that the induction of HO-1 by hypoxia be to the activation of the PHD oxygen treatment of cardiomyocyte cultures was found to induce HO-1 protein levels using previously to be at prolyl hydroxylase enzyme in cell T. K. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). potential and were found to be by treatment that HO-1 induction be to a The induction of HO-1 by prolyl hydroxylase inhibition was confirmed using the PHD inhibitor As with EDHB, induced HO-1 protein levels at with to inhibit PHD enzymes in cell (15Tschank G. Brocks D.G. Engelbart K. Mohr J. Baader E. Gunzler V. Hanauske-Abel H.M. Biochem. J. 1991; 275: 469-476Crossref PubMed Scopus (36) Google with HO-1 induction at μm mRNA and in to of the Prolyl Hydroxylase is in heart after an ischemic insult Y. Guo Y. Zhang J. W. R. J. Cell. 2002; Full Text PDF PubMed Scopus Google Scholar) and hypoxia P. S. M. S. S. J. Cell. 1999; Full Text PDF PubMed Scopus Google whether hypoxia, per se, is for NOS-2 protein expression. shown that PHD inhibitors induced two hypoxia-inducible GLUT1 and HO-1, was of interest to on NOS-2 expression. activation of the NOS-2 gene in cardiomyocyte cultures was observed with μm) as in the and increase in NOS-2 mRNA levels with real-time PCR The induction of NOS-2 mRNA was at the level of with a of NOS-2 protein detected was found to induce NOS-2 protein levels at the found to induce HO-1 and with the to inhibit prolyl hydroxylase inhibitors increase NOS-2 protein levels. Western blot analysis of NOS-2 in from cardiomyocytes treated with the of and for HO-1 and NOS-2 in shown that the pharmacological activation of the PHD oxygen-sensing pathway was to induce expression of HO-1 and NOS-2, was important to the effect of hypoxia on expression levels. To that HO-1 and NOS-2 are induced by hypoxia, cardiomyocyte cultures were placed in hypoxic culture conditions for h and Western analysis of HO-1 and NOS-2 protein levels was HO-1 and NOS-2 proteins levels were found to be induced by hypoxic culture conditions of the a on the that the changes in gene expression induced by the hypoxic a whose is to preserve myocyte viability in conditions of low oxygen low the viability of myocytes metabolic was in cultures ± PHD inhibitor of the pathway for 16 h to 260 min of metabolic inhibition cell After metabolic the of cells with EDHB, or that for trypan blue was ± ± and ± The effect of PHD inhibitor treatment was confirmed in cultures using propidium and study has examined the consequences of pharmacological activation of the PHD oxygen-sensing pathway in of the oxygen with inhibitors of the PHD enzymes was found to induce GLUT1, HO-1, and NOS-2 In the case of NOS-2 protein these to the first to that hypoxia and the activation of the PHD oxygen are to NOS-2 protein these results suggest a role for the PHD oxygen in the and responses of the heart a family of oxygen-dependent prolyl enzymes has been identified as a cellular oxygen-sensing and mechanism. It was found that a specific hydroxylation of proline of its with a protein that targets proteins for degradation by the In low oxygen conditions, PHD is proline is not thus the of HIF and proteins and to the of degradation (3Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3917) Google Scholar, 4Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4489) Google Scholar). In this prolyl hydroxylation is by the protein a prolyl that is a of the family of enzymes to via proline hydroxylation A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar, Raines R.T. 2002; PubMed Scopus Google Scholar). Three mammalian prolyl proteins (PHD 1, 2, and 3) have been identified and characterized as oxygen-sensing enzymes A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar). family of PHD enzymes the and for R. T. J. PubMed Scopus Google Scholar). and are to inhibit prolyl hydroxylation in L. Higgin J.J. Moulder G. Barstead R. Raines R.T. Kimble J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4736-4741Crossref PubMed Scopus (61) Google Scholar). In to prolyl hydroxylation as a has recently been shown that an oxygen-dependent asparaginyl hydroxylase the transactivating domain of HIF (5Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Science. 2002; 295: 858-861Crossref PubMed Scopus (1280) Google Scholar, D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. R.K. 2002; PubMed Scopus Google Scholar). of hydroxylation of by hypoxia to an increase in its for the and in this increased HIF, targets of this prolyl and asparaginyl hydroxylase family have not been In this study we have employed pharmacological that hypoxia in that inhibit the of members of this family of prolyl hydroxylase enzymes and activate this The pharmacological activation of this oxygen-sensing pathway has to to explore the whose induction may be important to the response of the cardiomyocyte to and hypoxia. The well characterized cellular response to oxygen is to increase glycolytic Glucose GLUT1, as well as glycolytic enzymes are hypoxia-inducible genes 1998; PubMed Scopus Google Scholar). In the case of GLUT1, its induction by hypoxia occurs mostly at the transcriptional level through an HIF-dependent mechanism (6Behrooz A. Ismail-Beigi F. J. Biol. Chem. 1997; 272: 5555-5562Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The of GLUT1 levels in response to conditions an important mechanism that for increased glycolytic metabolism to These that, with the activation of the cellular oxygen-sensing PHD inhibitors induce expression of GLUT1 and glucose a study R. M. L. J. F. S. Tian R. Circulation. 2002; PubMed Scopus Google Scholar) has found that GLUT1 the heart from oxygenase-1 as is a cardioregulatory protein previously shown (8Borger D.R. Essig D.A. Am. J. Physiol. 1998; 274: H965-H973PubMed Google Scholar) to be induced by hypoxia in HO-1 the reaction in heme and as is an of the and that HO-1 the of these D. Am. J. Cell Biol. 2002; PubMed Scopus Google Scholar). the HO-1 gene an P.J. J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google the of HIF in the induction of HO-1 in response to hypoxia has been by J. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. PubMed Scopus Google Scholar, P. D. M. V. J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). have that hypoxia HO-1 in cardiomyocytes and have the PHD oxygen as the of this NOS-2 is in heart in chronic hypoxia and after an ischemic It has been a central role in the delayed phase of ischemic preconditioning (11Bolli R. Manchikalapudi S. Tang X.L. Takano H. Qiu Y. Guo Y. Zhang Q. Jadoon A.K. Circ. Res. 1997; 81: 1094-1107Crossref PubMed Scopus (279) Google Scholar). Recently nitric-oxide has been to phenotype in ischemic heart cells G. D. N. D.L. Circulation. 2002; PubMed Scopus (64) Google Scholar, G. H. M. R. Circ. Res. 2000; PubMed Scopus Google Scholar). to a role in NOS-2 induction in heart. The NOS-2 has for that are regulated by hypoxia and oxygen R. J. PubMed Scopus Google Scholar, G. T. A. L. J. PubMed Scopus Google Scholar). These and the NOS-2 is by hypoxia in and cardiomyocytes F. Palmer L.A. Zhou N. Johns R.A. Circ. Res. 2000; 86: 319-325Crossref PubMed Scopus (272) Google Scholar, G. T. A. L. J. PubMed Scopus Google Scholar). However whether hypoxia, per se, is for the induction of NOS-2 protein levels (12Kacimi R. Long C.S. Karliner J.S. Circulation. 1997; 96: 1937-1943Crossref PubMed Scopus (64) Google Scholar, 13Jung F. Palmer L.A. Zhou N. Johns R.A. Circ. Res. 2000; 86: 319-325Crossref PubMed Scopus (272) Google Scholar). that hypoxia, and activation of the PHD oxygen are for the induction of NOS-2 protein. the of this signaling mechanism for the increased expression of NOS-2 a of conditions is to be These may suggest a potential role for the PHD mechanism in the responses of heart to an oxygen delayed The changes in gene expression that are by PHD pathway activation are to a response of the cardiomyocyte to ischemic In of this we have found that the pharmacological activation of this PHD oxygen-sensing mechanism of cellular viability heart cells are treated with metabolic In this the changes that we have to in response to PHD inhibitors induction of NOS-2, HO-1, and may a of the heart cells to a The pharmacological activation of the PHD oxygen-sensing pathway results in the induction of GLUT1, HO-1, and Because of these proteins have been to have these may suggest a strategy to pharmacologically a phenotype to heart the of an ischemic These results that inhibition of PHD is for the induction of NOS-2 and HO-1