TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia.

Bleeding and thrombosis are known as common complications of polycythemia for a long time. However, the role of coagulation system in erythropoiesis is unclear. Here, we discover that an anticoagulant protein tissue factor pathway inhibitor (TFPI) plays an essential role in erythropoiesis via the control of heme biosynthesis in central macrophages. TFPI levels are elevated in erythroblasts of human erythroblastic islands with JAK2V617F mutation and hypoxia condition. Erythroid lineage-specific knockout TFPI results in impaired erythropoiesis through decreasing ferrochelatase expression and heme biosynthesis in central macrophages. Mechanistically, the TFPI interacts with thrombomodulin to promote the downstream ERK1/2-GATA1 signaling pathway to induce heme biosynthesis in central macrophages. Furthermore, TFPI blockade impairs human erythropoiesis in vitro, and normalizes the erythroid compartment in mice with polycythemia. These results show that erythroblast-derived TFPI plays an important role in the regulation of erythropoiesis and reveal an interplay between erythroblasts and central macrophages.

The ribosomal chaperone NACA recruits PHD2 to cotranslationally modify HIF-α.

Prolyl hydroxylase domain protein 2 (PHD2)-catalyzed modification of hypoxia-inducible factor (HIF)-α is a key event in oxygen sensing. We previously showed that the zinc finger of PHD2 binds to a Pro-Xaa-Leu-Glu (PXLE) motif. Here, we show that the zinc finger binds to this motif in the ribosomal chaperone nascent polypeptide complex-α (NACA). This recruits PHD2 to the translation machinery to cotranslationally modify HIF-α. Importantly, this cotranslational modification is enhanced by a translational pause sequence in HIF-α. Mice with a knock-in Naca gene mutation that abolishes the PXLE motif display erythrocytosis, a reflection of HIF pathway dysregulation. In addition, human erythrocytosis-associated mutations in the zinc finger of PHD2 ablate interaction with NACA. Tibetans, who have adapted to the hypoxia of high altitude, harbor a PHD2 variant that we previously showed displays a defect in zinc finger binding to p23, a PXLE-containing HSP90 cochaperone. We show here that Tibetan PHD2 maintains interaction with NACA, thereby showing differential interactions with PXLE-containing proteins and providing an explanation for why Tibetans are not predisposed to erythrocytosis.

Epo-IGF1R cross talk expands stress-specific progenitors in regenerative erythropoiesis and myeloproliferative neoplasm.

We found that in regenerative erythropoiesis, the erythroid progenitor landscape is reshaped, and a previously undescribed progenitor population with colony-forming unit-erythroid (CFU-E) activity (stress CFU-E [sCFU-E]) is expanded markedly to restore the erythron. sCFU-E cells are targets of erythropoietin (Epo), and sCFU-E expansion requires signaling from the Epo receptor (EpoR) cytoplasmic tyrosines. Molecularly, Epo promotes sCFU-E expansion via JAK2- and STAT5-dependent expression of IRS2, thus engaging the progrowth signaling from the IGF1 receptor (IGF1R). Inhibition of IGF1R and IRS2 signaling impairs sCFU-E cell growth, whereas exogenous IRS2 expression rescues cell growth in sCFU-E expressing truncated EpoR-lacking cytoplasmic tyrosines. This sCFU-E pathway is the major pathway involved in erythrocytosis driven by the oncogenic JAK2 mutant JAK2(V617F) in myeloproliferative neoplasm. Inability to expand sCFU-E cells by truncated EpoR protects against JAK2(V617F)-driven erythrocytosis. In samples from patients with myeloproliferative neoplasm, the number of sCFU-E-like cells increases, and inhibition of IGR1R and IRS2 signaling blocks Epo-hypersensitive erythroid cell colony formation. In summary, we identified a new stress-specific erythroid progenitor cell population that links regenerative erythropoiesis to pathogenic erythrocytosis.

Breakthrough science: hypoxia-inducible factors, oxygen sensing, and disorders of hematopoiesis.

Hypoxia-inducible factors (HIFs) were discovered as activators of erythropoietin gene transcription in response to reduced oxygen (O2) availability. O2-dependent hydroxylation of HIFs on proline and asparagine residues regulates protein stability and transcriptional activity, respectively. Mutations in genes encoding components of the O2-sensing pathway cause familial erythrocytosis. Several small-molecule inhibitors of HIF prolyl hydroxylases are currently in clinical trials as erythropoiesis-stimulating agents. HIFs are overexpressed in bone marrow neoplasms, and the development of HIF inhibitors may improve outcomes in these disorders.

Heritable disorders of oxygen sensing.

Hypoxia-inducible factors (HIFs) activate gene transcription in response to reduced O2 availability and play critical roles in development, physiology, and disease pathogenesis. Mutations that dysregulate HIF activity are the genetic basis for tumor predisposition in the von Hippel-Lindau syndrome and excess red blood cell production in hereditary erythrocytosis.

HIF2α regulates the synthesis and release of epinephrine in the adrenal medulla.

The adrenal gland and its hormones regulate numerous fundamental biological processes; however, the impact of hypoxia signaling on adrenal function remains poorly understood. Here, we reveal that deficiency of HIF (hypoxia inducible factors) prolyl hydroxylase domain protein-2 (PHD2) in the adrenal medulla of mice results in HIF2α-mediated reduction in phenylethanolamine N-methyltransferase (PNMT) expression, and consequent reduction in epinephrine synthesis. Simultaneous loss of PHD2 in renal erythropoietin (EPO)-producing cells (REPCs) stimulated HIF2α-driven EPO overproduction, excessive RBC formation (erythrocytosis), and systemic hypoglycemia, which is necessary and sufficient to enhance exocytosis of epinephrine from the adrenal medulla. Based on these results, we propose that the PHD2-HIF2α axis in the adrenal medulla regulates the synthesis of epinephrine, whereas in REPCs, it indirectly induces the release of this hormone. Our findings are also highly relevant to the testing of small molecule PHD inhibitors in phase III clinical trials for patients with renal anemia. KEY MESSAGES: HIF2α and not HIF1α modulates PNMT during epinephrine synthesis in chromaffin cells. The PHD2-HIF2α-EPO axis induces erythrocytosis and hypoglycemia. Reduced systemic glucose facilitates exocytosis of epinephrine from adrenal gland.

Heritable disorders of oxygen sensing.

Hypoxia-inducible factors (HIFs) activate gene transcription in response to reduced O2 availability and play critical roles in development, physiology, and disease pathogenesis. Mutations that dysregulate HIF activity are the genetic basis for tumor predisposition in the von Hippel-Lindau syndrome and excess red blood cell production in hereditary erythrocytosis.

Protective role of estrogen against excessive erythrocytosis in Monge's disease.

Monge's disease (chronic mountain sickness (CMS)) is a maladaptive condition caused by chronic (years) exposure to high-altitude hypoxia. One of the defining features of CMS is excessive erythrocytosis with extremely high hematocrit levels. In the Andean population, CMS prevalence is vastly different between males and females, being rare in females. Furthermore, there is a sharp increase in CMS incidence in females after menopause. In this study, we assessed the role of sex hormones (testosterone, progesterone, and estrogen) in CMS and non-CMS cells using a well-characterized in vitro erythroid platform. While we found that there was a mild (nonsignificant) increase in RBC production with testosterone, we observed that estrogen, in physiologic concentrations, reduced sharply CD235a+ cells (glycophorin A; a marker of RBC), from 56% in the untreated CMS cells to 10% in the treated CMS cells, in a stage-specific and dose-responsive manner. At the molecular level, we determined that estrogen has a direct effect on GATA1, remarkably decreasing the messenger RNA (mRNA) and protein levels of GATA1 (p < 0.01) and its target genes (Alas2, BclxL, and Epor, p < 0.001). These changes result in a significant increase in apoptosis of erythroid cells. We also demonstrate that estrogen regulates erythropoiesis in CMS patients through estrogen beta signaling and that its inhibition can diminish the effects of estrogen by significantly increasing HIF1, VEGF, and GATA1 mRNA levels. Taken altogether, our results indicate that estrogen has a major impact on the regulation of erythropoiesis, particularly under chronic hypoxic conditions, and has the potential to treat blood diseases, such as high altitude severe erythrocytosis.

Therapeutic inhibition of HIF-2α reverses polycythemia and pulmonary hypertension in murine models of human diseases.

Polycythemia and pulmonary hypertension are 2 human diseases for which better therapies are needed. Upregulation of hypoxia-inducible factor-2α (HIF-2α) and its target genes, erythropoietin (EPO) and endothelin-1, causes polycythemia and pulmonary hypertension in patients with Chuvash polycythemia who are homozygous for the R200W mutation in the von Hippel Lindau (VHL) gene and in a murine mouse model of Chuvash polycythemia that bears the same homozygous VhlR200W mutation. Moreover, the aged VhlR200W mice developed pulmonary fibrosis, most likely due to the increased expression of Cxcl-12, another Hif-2α target. Patients with mutations in iron regulatory protein 1 (IRP1) also develop polycythemia, and Irp1-knockout (Irp1-KO) mice exhibit polycythemia, pulmonary hypertension, and cardiac fibrosis attributable to translational derepression of Hif-2α, and the resultant high expression of the Hif-2α targets EPO, endothelin-1, and Cxcl-12. In this study, we inactivated Hif-2α with the second-generation allosteric HIF-2α inhibitor MK-6482 in VhlR200W, Irp1-KO, and double-mutant VhlR200W;Irp1-KO mice. MK-6482 treatment decreased EPO production and reversed polycythemia in all 3 mouse models. Drug treatment also decreased right ventricular pressure and mitigated pulmonary hypertension in VhlR200W, Irp1-KO, and VhlR200W;Irp1-KO mice to near normal wild-type levels and normalized the movement of the cardiac interventricular septum in VhlR200Wmice. MK-6482 treatment reduced the increased expression of Cxcl-12, which, in association with CXCR4, mediates fibrocyte influx into the lungs, potentially causing pulmonary fibrosis. Our results suggest that oral intake of MK-6482 could represent a new approach to treatment of patients with polycythemia, pulmonary hypertension, pulmonary fibrosis, and complications caused by elevated expression of HIF-2α.

VHL gene methylation contributes to excessive erythrocytosis in chronic mountain sickness rat model by upregulating the HIF-2α/EPO pathway.

AIMS: Hypoxia-inducible factors (HIFs) play important roles in the pathogenesis of erythrocytosis in chronic mountain sickness (CMS). von Hippel-Lindau (VHL) is a key regulator of hypoxia that can direct the poly-ubiquitylation and degradation of HIFs. Epigenetic mechanisms are believed to contribute toward adaption to chronic hypoxia. Here, we investigated the contribution and mechanism of VHL methylation in rats with erythrocytosis in CMS. MAIN METHODS: The methylation status of VHL was measured via bisulfite sequencing PCR, while VHL, DNMT1, DNMT3α, and DNMT3ß expression were assessed using real-time reverse transcription PCR and western blotting. HIF-2α and EPO expression levels in bone marrow were determined via immunohistochemical staining, and erythroid hyperplasia in bone marrow sections were observed with hematoxylin and eosin staining. KEY FINDINGS: We found that chronic hypoxia triggered erythroid hyperplasia in the bone marrow and increased the quantity of peripheral red blood cells in CMS rats. Chronic hypoxia significantly induced methylation at the CpG site in the VHL promoter, decreased VHL expression, and increased HIF-2α and EPO expression. Chronic hypoxia increased DNMT3α and DNMT3ß expression, consistent with the decrease in VHL expression. The DNA methyltransferase inhibitor 5-azacytidine reduced chronic hypoxia-induced erythroid proliferation in the bone marrow of rats with CMS by suppressing VHL methylation and DNMTs expression. SIGNIFICANCE: Our study suggests that VHL methylation contributes toward excessive erythrocytosis in CMS by upregulating the HIF-2α/EPO pathway in the bone marrow of rats. We demonstrated that the DNMT inhibitor 5-azacytidine can attenuate erythroid hyperplasia in the bone marrow by demethylating the VHL promoter.

Increased Pulmonary GM-CSF Causes Alveolar Macrophage Accumulation. Mechanistic Implications for Desquamative Interstitial Pneumonitis.

Desquamative interstitial pneumonia (DIP) is a rare, smoking-related, diffuse parenchymal lung disease characterized by marked accumulation of alveolar macrophages (AMs) and emphysema, without extensive fibrosis or neutrophilic inflammation. Because smoking increases expression of pulmonary GM-CSF (granulocyte/macrophage-colony stimulating factor) and GM-CSF stimulates proliferation and activation of AMs, we hypothesized that chronic exposure of mice to increased pulmonary GM-CSF may recapitulate DIP. Wild-type (WT) mice were subjected to inhaled cigarette smoke exposure for 16 months, and AM numbers and pulmonary GM-CSF mRNA levels were measured. After demonstrating that smoke inhalation increased pulmonary GM-CSF in WT mice, transgenic mice overexpressing pulmonary GM-CSF (SPC-GM-CSF+/+) were used to determine the effects of chronic exposure to increased pulmonary GM-CSF (without smoke inhalation) on accumulation and activation of AMs, pulmonary matrix metalloproteinase (MMP) expression and activity, lung histopathology, development of polycythemia, and survival. In WT mice, smoke exposure markedly increased pulmonary GM-CSF and AM accumulation. In unexposed SPC-GM-CSF+/+ mice, AMs were spontaneously activated as shown by phosphorylation of STAT5 (signal inducer and activator of transcription 5) and accumulated progressively with involvement of 84% (interquartile range, 55-90%) of the lung parenchyma by 10 months of age. Histopathologic features also included scattered multinucleated giant cells, alveolar epithelial cell hyperplasia, and mild alveolar wall thickening. SPC-GM-CSF+/+ mice had increased pulmonary MMP-9 and MMP-12 levels, spontaneously developed emphysema and secondary polycythemia, and had increased mortality compared with WT mice. Results show cigarette smoke increased pulmonary GM-CSF and AM proliferation, and chronically increased pulmonary GM-CSF recapitulated the cardinal features of DIP, including AM accumulation, emphysema, secondary polycythemia, and increased mortality in mice. These observations suggest pulmonary GM-CSF may be involved in the pathogenesis of DIP.

Update on mutations in the HIF: EPO pathway and their role in erythrocytosis.

Identification of the underlying defects in congenital erythrocytosis has provided mechanistic insights into the regulation of erythropoiesis and oxygen homeostasis. The Hypoxia Inducible Factor (HIF) pathway plays a key role in this regard. In this pathway, an enzyme, Prolyl Hydroxylase Domain protein 2 (PHD2), constitutively prolyl hydroxylates HIF-2α, thereby targeting HIF-2α for degradation by the von Hippel Lindau (VHL) tumor suppressor protein. Under hypoxia, this modification is attenuated, resulting in the stabilization of HIF-2α and transcriptional activation of the erythropoietin (EPO) gene. Circulating EPO then binds to the EPO receptor (EPOR) on red cell progenitors in the bone marrow, leading to expansion of red cell mass. Loss of function mutations in PHD2 and VHL, as well as gain of function mutations in HIF-2α and EPOR, are well established causes of erythrocytosis. Here, we highlight recent developments that show that the study of this condition is still evolving. Specifically, novel mutations have been identified that either change amino acids in the zinc finger domain of PHD2 or alter splicing of the VHL gene. In addition, continued study of HIF-2α mutations has revealed a distinctive genotype-phenotype correlation. Finally, novel mutations have recently been identified in the EPO gene itself. Thus, the cascade of genes that at a molecular level leads to EPO action, namely PHD2 - > HIF2A - > VHL - > EPO - > EPOR, are all mutational targets in congenital erythrocytosis.

Normal and pathological erythropoiesis in adults: from gene regulation to targeted treatment concepts.

Pathological erythropoiesis with consequent anemia is a leading cause of symptomatic morbidity in internal medicine. The etiologies of anemia are complex and include reactive as well as neoplastic conditions. Clonal expansion of erythroid cells in the bone marrow may result in peripheral erythrocytosis and polycythemia but can also result in anemia when clonal cells are dysplastic and have a maturation arrest that leads to apoptosis and hinders migration, a constellation typically seen in the myelodysplastic syndromes. Rarely, clonal expansion of immature erythroid blasts results in a clinical picture resembling erythroid leukemia. Although several mechanisms underlying normal and abnormal erythropoiesis and the pathogenesis of related disorders have been deciphered in recent years, little is known about specific markers and targets through which prognosis and therapy could be improved in anemic or polycythemic patients. In order to discuss new markers, targets and novel therapeutic approaches in erythroid disorders and the related pathologies, a workshop was organized in Vienna in April 2017. The outcomes of this workshop are summarized in this review, which includes a discussion of new diagnostic and prognostic markers, the updated WHO classification, and an overview of new drugs used to stimulate or to interfere with erythropoiesis in various neoplastic and reactive conditions. The use and usefulness of established and novel erythropoiesis-stimulating agents for various indications, including myelodysplastic syndromes and other neoplasms, are also discussed.

HIF-2α-pVHL complex reveals broad genotype-phenotype correlations in HIF-2α-driven disease.

It is definitively established that mutations in transcription factor HIF-2α are causative of both neuroendocrine tumors (class 1 disease) and polycythemia (class 2 disease). However, the molecular mechanism that underlies this emergent genotype-phenotype relationship has remained unclear. Here, we report the structure of HIF-2α peptide bound to pVHL-elongin B-elongin C (VBC) heterotrimeric complex, which shows topographical demarcation of class 1 and 2 mutations affecting residues predicted, and demonstrated via biophysical analyses, to differentially impact HIF-2α-pVHL interaction interface stability. Concordantly, biochemical experiments showed that class 1 mutations disrupt pVHL affinity to HIF-2α more adversely than class 2 mutations directly or indirectly via impeding PHD2-mediated hydroxylation. These findings suggest that neuroendocrine tumor pathogenesis requires a higher HIF-2α dose than polycythemia, which requires only a mild increase in HIF-2α activity. These biophysical data reveal a structural basis that underlies, and can be used to predict de novo, broad genotype-phenotype correlations in HIF-2α-driven disease.

Expansion of EPOR-negative macrophages besides erythroblasts by elevated EPOR signaling in erythrocytosis mouse models.

Activated erythropoietin (EPO) receptor (EPOR) signaling causes erythrocytosis. The important role of macrophages for the erythroid expansion and differentiation process has been reported, both in baseline and stress erythropoiesis. However, the significance of EPOR signaling for regulation of macrophages contributing to erythropoiesis has not been fully understood. Here we show that EPOR signaling activation quickly expands both erythrocytes and macrophages in vivo in mouse models of primary and secondary erythrocytosis. To mimic the chimeric condition and expansion of the disease clone in the polycythemia vera patients, we combined Cre-inducible Jak2V617F/+ allele with LysM-Cre allele which expresses in mature myeloid cells and some of the HSC/Ps (LysM-Cre;Jak2V617F/+ mice). We also generated inducible EPO-mediated secondary erythrocytosis models using Alb-Cre, Rosa26-loxP-stop-loxP-rtTA, and doxycycline inducible EPAS1-double point mutant (DPM) alleles (Alb-Cre;DPM mice). Both models developed a similar degree of erythrocytosis. Macrophages were also increased in both models without increase of major inflammatory cytokines and chemokines. EPO administration also quickly induced these macrophages in wild-type mice before observable erythrocytosis. These findings suggest that EPOR signaling activation could induce not only erythroid cell expansion, but also macrophages. Surprisingly, an in vivo genetic approach indicated that most of those macrophages do not express EPOR, but erythroid cells and macrophages contacted tightly with each other. Given the importance of the central macrophages as a niche for erythropoiesis, further elucidation of the EPOR signaling mediated-regulatory mechanisms underlying macrophage induction might reveal a potential therapeutic target for erythrocytosis.

Notch Downregulation and Extramedullary Erythrocytosis in Hypoxia-Inducible Factor Prolyl 4-Hydroxylase 2-Deficient Mice.

Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2gt/gt) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

Senp1 drives hypoxia-induced polycythemia via GATA1 and Bcl-xL in subjects with Monge's disease.

In this study, because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge's disease) but not others living at the same altitude in the Andes, we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology, genomics, and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all, respectively, CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding, we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology.

[Influence of HIF- 2α on the expression of GATA- 1 in bone marrow CD71(+) cell of high altitude polycythemia rat model].

OBJECTIVE: To explore the influence of hypoxia-inducible factor-2 αlpha (HIF-2α) on the expression of erythroid-specific transcription factor GATA-1 in bone marrow CD71(+) cells of rat model with high altitude polycythemia (HAPC). METHODS: A total of 48 male SD rats were selected and randomly divided into normal control group and HAPC group. HAPC model was established at an altitude of 4 300 meters in the natural environment and verified by bone marrow cell classification and counting, hematologic parameters and serum EPO detection. Bone marrow CD71 (+) cells were separated by a combination of methods with density gradient centrifugation and magnetic activated cell sorting. The changes of expression level of HIF-2α, GATA-1 mRNA and proteins were detected by Q-PCR and Western blot. CD71 (+) cells were cultured under hypoxia condition and transfected with selected optimal HIF- 2α shRNAi3 for 96 h. And the expression level of HIF-2α and GATA-1 mRNA and proteins were detected by Q- PCR and Western blot. RESULTS: The results of bone marrow cell counts, the hematologic parameters and the serum EPO content showed that the HAPC rat model was successfully established. The expression of HIF-2α and GATA-1 mRNA and protein in bone marrow CD71(+) cells of HAPC group was higher than that in control group (P<0.05). And HIF-2α and GATA-1 of HAPC group were positively correlated at the expression levels of mRNA and protein, respectively (r=0.923, P<0.01; r=0.838, P<0.01). However, the expression of HIF-2α and GATA-1 mRNA and protein in HAPC group was significantly lower than that in control groups after interfered by HIF-2α shRNAi3 for 96 h (P<0.05). CONCLUSION: The effect of HIF-2α on GATA-1 expression may be correlated with the pathogenesis of HAPC.

The von Hippel-Lindau Chuvash mutation in mice alters cardiac substrate and high-energy phosphate metabolism.

Hypoxia-inducible factor (HIF) appears to function as a global master regulator of cellular and systemic responses to hypoxia. HIF pathway manipulation is of therapeutic interest; however, global systemic upregulation of HIF may have as yet unknown effects on multiple processes. We used a mouse model of Chuvash polycythemia (CP), a rare genetic disorder that modestly increases expression of HIF target genes in normoxia, to understand what these effects might be within the heart. An integrated in and ex vivo approach was employed. Compared with wild-type controls, CP mice had evidence (using in vivo magnetic resonance imaging) of pulmonary hypertension, right ventricular hypertrophy, and increased left ventricular ejection fraction. Glycolytic flux (measured using [(3)H]glucose) in the isolated contracting perfused CP heart was 1.8-fold higher. Net lactate efflux was 1.5-fold higher. Furthermore, in vivo (13)C-magnetic resonance spectroscopy (MRS) of hyperpolarized [(13)C1]pyruvate revealed a twofold increase in real-time flux through lactate dehydrogenase in the CP hearts and a 1.6-fold increase through pyruvate dehydrogenase. (31)P-MRS of perfused CP hearts under increased workload (isoproterenol infusion) demonstrated increased depletion of phosphocreatine relative to ATP. Intriguingly, no changes in cardiac gene expression were detected. In summary, a modest systemic dysregulation of the HIF pathway resulted in clear alterations in cardiac metabolism and energetics. However, in contrast to studies generating high HIF levels within the heart, the CP mice showed neither the predicted changes in gene expression nor any degree of LV impairment. We conclude that the effects of manipulating HIF on the heart are dose dependent.