Adora2b-elicited Per2 stabilization promotes a HIF-dependent metabolic switch crucial for myocardial adaptation to ischemia

Journal name:
Nature Medicine
Volume:
18,
Pages:
774–782
Year published:
DOI:
doi:10.1038/nm.2728
Received
Accepted
Published online

Abstract

Adenosine signaling has been implicated in cardiac adaptation to limited oxygen availability. In a wide search for adenosine receptor A2b (Adora2b)-elicited cardioadaptive responses, we identified the circadian rhythm protein period 2 (Per2) as an Adora2b target. Adora2b signaling led to Per2 stabilization during myocardial ischemia, and in this setting, Per2−/− mice had larger infarct sizes compared to wild-type mice and loss of the cardioprotection conferred by ischemic preconditioning. Metabolic studies uncovered a limited ability of ischemic hearts in Per2−/− mice to use carbohydrates for oxygen-efficient glycolysis. This impairment was caused by a failure to stabilize hypoxia-inducible factor-1α (Hif-1α). Moreover, stabilization of Per2 in the heart by exposing mice to intense light resulted in the transcriptional induction of glycolytic enzymes and Per2-dependent cardioprotection from ischemia. Together, these studies identify adenosine-elicited stabilization of Per2 in the control of HIF-dependent cardiac metabolism and ischemia tolerance and implicate Per2 stabilization as a potential new strategy for treating myocardial ischemia.

At a glance

Figures

  1. Consequences of adenosine signaling on Per2 induction.
    Figure 1: Consequences of adenosine signaling on Per2 induction.

    (a) Transcript concentrations of individual adenosine receptors (ADORA1 ADORA2A, ADORA2B and ADORA3) in cardiac tissue from patients with severe ischemic heart disease (IHD) or controls (C). n = 10 patients per condition. Statistical significance was determined by Student's t test. Data are mean ± s.d. (b) Canonical pathway analysis. A canonical pathway analysis was ued to identify the pathways from the IPA (Ingenuity Pathway Analysis) library of canonical (typical) pathways that were most significant to the dataset. (cf) Hearts from Adora2b−/− and WT littermate controls were analyzed. (c) Per2 mRNA levels in hearts over a period of 24 hours (with ZT0 set as 6 am). n = 6 mice per group. *P < 0.05 by one-way analysis of variance (ANOVA) with Dunnett's post-hoc test. Data are mean ± s.d. NS, not significant. (d) Per2 mRNA concentrations in hearts subjected to in situ ischemic preconditioning (IP) followed by reperfusion for the indicated time periods. n = 6 mice per group. Statistical significance was calculated by one-way ANOVA followed by Dunnett's post-hoc test compared to controls (C). Data are mean ± s.d. (e) Per2 protein expression determined by western blot following ischemic preconditioning without reperfusion. WT, wild type; KO, Adora2b−/−. One representative blot of three is shown. Actb, β-actin. (f) Comparison of the immunoreactivity for Per2 in preconditioned cardiac tissue (IP-WT and IP-Adora2b−/−) or sham-operated control tissue (C-WT and C-Adora2b−/−). One representative image of three for each condition is shown. Scale bar, 100 μm. (g) Adora2b and Per2 mRNA concentrations in isolated adult mouse cardiomyocytes from wild-type or Adora2b−/− mice after in vitro exposure to HPC. n = 4 hearts per group. Statistical significance was calculated by Student's t test. Data are mean ± s.d. (h) PER2 mRNA (left) or protein (right) concentrations in cardiac tissues from patients with severe ischemic heart disease (IHD) or controls (C). A representative immunoblot for PER2 protein from the indicated subjects is shown below (see Supplementary Fig. 9). ACTB (β-actin) was used a loading control. n = 10 patients per condition. Statistical significance was calculated by Student's t test. Data are mean ± s.d. (i) Chromatin immunopreciptitation analysis to detect CREB protein binding to the PER2 promoter using HMEC-1 cells treated with BAY 60-6583 for 20 min. Real-time RT-PCR for human PER2 promoter or satellite DNA (negative control) (top). Products obtained by PCR for the PER2 promoter analyzed on a 1% agarose gel (bottom). Data are mean ± s.d. n = 3. *P < 0.05 by Student's t test. Ab, antibody; IgG, immunoglobulin G. (j) Luciferase activity measured in transfected HMEC-1 cells with a full-length PER2 promoter construct (FLPER2) or the indicated truncated versions, subcloned into the pGL4 luciferase reporter vector. The pGL4 vector used alone served as a negative control. As a positive control, a pMetLuc reporter vector containing a specific promoter for CRE was used (CREB). The cells were treated with vehicle or BAY 60-6583 or were cotransfected with a dominant negative CREB (Dom neg CREB) construct, as indicated. Schematic diagrams of the truncated versions of the PER2 promoter, as well as the location of putative CREB binding sites, are shown in Supplementary Figure 11. *P < 0.05 by one-way ANOVA with Dunnett's post-hoc test for luciferase activity over baseline. Data are mean ± s.d. n = 6.

  2. Influence of transcriptional, translational and post-translational mechanisms on Per2 protein expression.
    Figure 2: Influence of transcriptional, translational and post-translational mechanisms on Per2 protein expression.

    (a) Immunoblot analysis of PER2 protein using synchronized HMEC-1 cells treated with vehicle or BAY 60-6583 for the indicated time periods. One of three representative experiments is shown and is quantified below; boxed protein signals indicate the largest difference in PER2 signal strength between controls and cells treated with BAY 60-6583. *P < 0.05 by Student's t-test. Data are mean ± s.d. n = 3. (b,c) Immunoblot analysis of PER2 protein in synchronized HMEC-1 cells treated with vehicle, BAY 60-6583, or forskolin with or without actinomycin (ACT) (b) or cycloheximide (CXM) (c). Protein signals were quantified using densitometry, and the fold change over control is given below the blots. The effectiveness of ACT and CXM is shown in Supplementary Figure 12a,b. (d) Proposed model of adenosine-dependent alteration on post-translational PER2 protein stability (left). Immunoblot analysis of PER2 protein in synchronized HMEC-1 cells with or without addition of the proteasome inhibitor AM114 (right). One of three representative blots is shown. (e) Protein lysates were obtained from synchronized HMEC-1 cells treated with vehicle or BAY 60-6583 and were immunoprecipitated with PER2 antibody. The presence of ubiquitin (UBC) in the immunoprecipitate was determined by immunoblot analysis (left). Co-IP, coimmunoprecipitation. One representative blot of three is shown. Changes in protein concentration of PER2 (middle) and coimmunoprecipitated UBC (right) after BAY 60-6583 treatment, as determined by densitometry. *P < 0.05 by Student's t test. n = 3. Data are mean ± s.d. (f) Immunoblot analysis for total CUL1 or neddylated CUL1 (CUL1NEDD8) in synchronized HMEC-1 cells treated with vehicle or BAY 60-6583. One of three representative blots is shown. (g) Immunoblot analysis for CULNEDD8 in synchronized HMEC-1 cells treated with BAY 60-6583 alone or after pretreatment with the ADORA2B antagonist PSB 1115. One of three representative experiments is shown. (h,i) Immunoblot analysis for CULNEDD8 (h) and PER2 (i) in synchronized HMEC-1 cells after siRNA knockdown of CSN5 (siCSN5) or treatment with nonspecific control siRNA (csiR). The cells were treated with vehicle or BAY 60-6583 as indicated. One of three representative experiments is shown. (j) Immunoblot analysis for CULNEDD8 and Per2 in cardiac myocytes isolated from wild-type or Adora2b−/− mice exposed to HPC or control conditions. One representative blot of three independent experiments is shown. n = 1 mouse per experiment.

  3. Functional role of Per2 during myocardial ischemia and ischemic preconditioning.
    Figure 3: Functional role of Per2 during myocardial ischemia and ischemic preconditioning.

    (a) Infarct sizes and troponin I concentrations in plasma samples from Per2−/− mice and WT littermate controls that were exposed to in situ myocardial ischemia followed by reperfusion or received ischemic preconditioning or treatment with BAY 60-6583 before myocardial ischemia. Infarct sizes are expressed as the percent of the area at risk that was exposed to myocardial ischemia. Data are mean ± s.d. n = 6 mice per group. Statistical significance was calculated by two-factor ANOVA with Bonferroni's post-hoc test. −IP, ischemia and reperfusion only; +IP, ischemic preconditioning. (b) Representative infarct staining in hearts from Per2−/− mice exposed to ischemia and reperfusion only (top) or to ischemic preconditioning (middle) or BAY 60-6583 (bottom) before myocardial ischemia (blue, retrograde Evan's blue staining; red and white, area at risk; white, infarcted tissue). Scale bar, 50 μm. (c) Infarct sizes and troponin I concentrations in plasma samples from Per2−/− mice or WT littermate controls treated with either vehicle or BAY 60-6583. Data are mean ± s.d. n = 6 mice per group. Statistical significance was calculated by two-factor ANOVA with Bonferroni's post-hoc test. (d) Electron microscopy of wild-type and Per2−/− heart tissue. At baseline, wild-type and Per2−/− mice showed normal sarcolemmal structures, however, in some areas, the Per2−/− tissue showed enhanced glycogen content (white arrow), swollen mitochondria (white asterisk) and lipid accumulation within mitochondria (black asterisk). Scale bar, 500 nm. (e) Long-chain fatty acid concentrations (top) and immunoblot analysis of Cpt1 (bottom) in heart tissue from Per2−/− mice and WT littermate controls subjected to in situ ischemic preconditioning. Data are mean ± s.d. n = 3 mice per group. Statistical significance was calculated by Student's t test compared to control mice without ischemic preconditioning (C). One representative blot of three is shown. (fh) Per2−/− mice and WT littermate controls were exposed to in situ myocardial ischemia with or without ischemic preconditioning. (f) Creatine phosphate (CrP) concentrations, as determined by NMR. (g,h) Lactate concentrations and the corresponding NMR spectra. n = 3 mice per group. *P < 0.05 by Student's t test compared to baseline. Data are mean ± s.d.

  4. Consequences of Per2 deficiency on cardiac metabolism during myocardial ischemia and reperfusion.
    Figure 4: Consequences of Per2 deficiency on cardiac metabolism during myocardial ischemia and reperfusion.

    (ag) Metabolic parameters in Per2−/− mice and WT littermate controls exposed to in situ myocardial ischemia alone or to ischemia preceded by ischemic preconditioning. Both conditions were analyzed with and without reperfusion. For conditions without reperfusion, 13C-glucose was administered before ischemia, and with reperfusion, 13C-glucose was given at the onset of reperfusion. C, control; I, ischemia; IR, ischemia and reperfusion; IP, ischemic preconditioning. Data are mean ± s.d., n = 3 mice per group. P values were calculated by two-factor analysis of variance with Bonferroni's post-hoc test. Shown are 13C6-glucose concentrations (a), 13C6-fructose-1,6-bisphosphate concentrations (b), 13C3-pyruvate concentrations (c), 13C6-lactate concentrations (d),TCA cycle flux rates determined by the ratio of 13C3-glutamate to total creatine (e,f), and glycogen concentrations (g). Data are mean ± s.d., n = 3 mice per group. Statistical significance was calculated by two-factor ANOVA with Bonferroni's post-hoc test. (h) Summary of key metabolites of all experimental conditions in Per2−/− mice and WT littermate controls. Arrows indicate changes in experimental groups compared to their untreated control group. ↔, black, no changes; ↑ or ↓, black, slight increase or decrease compared to control; ↑↑ or ↓↓, black, strong increase or decrease compared to control; ↑↑↑ or ↓↓↓, black, very strong increase or decrease compared to control. Red arrows indicate differences between Per2−/− mice with the indicated treatments and equivalently-treated wild-type mice. WT, wildtype, I, ischemia alone; IP, ischemic preconditioning; IR, ischemia followed by reperfusion; Gluc, 13C6- glucose; Lac, 13C6-lactate; F1,6p, 13C6-fructose-1,6-bisphosphate, Pyr, 13C3-pyruvate; TCA, tricarboxylic acid cycle flux.

  5. Hif-1[alpha] functions as a link between adenosine-mediated Per2 signaling and metabolism.
    Figure 5: Hif-1α functions as a link between adenosine-mediated Per2 signaling and metabolism.

    (a) Luciferase activity measured in the area at risk of hearts over a 24-h period (with ZT0 set as 6 am) from Hif-1α reporter mice, Per2 reporter mice, and Per2−/− mice containing the HIF-1α reporter (Hif-1α in Per2−/−). *P < 0.05 by one-way ANOVA with Dunnett's post-hoc test compared to baseline. Data are mean ± s.d. n = 3 mice per group. (b,c) Immunoblot analysis for Hif-1α or Per2 in Per2−/− mice or mice with cardiac-specific knockout of Hif1a (cardiac Hif1a−/−) subjected to ischemic preconditioning compared to untreated controls (C). Cardiac tissue in the area at risk was used. One representative blot of three is shown. (d) Immunoblot analysis for Hif-1α in isolated adult cardiomyocytes from wild-type or Per2−/− mice exposed to ambient hypoxia (Hx) for the indicated periods of time. One representative blot of three is shown. (e) Luciferase activity (as a readout of Hif-1α expression) in the area at risk of mice of the indicated genotypes exposed to myocardial ischemia-reperfusion with or without ischemic preconditioning. Data are mean ± s.d. n = 4 mice per group. Statistical significance was calculated by two-factor ANOVA with Bonferroni's post-hoc test. (f) Transcriptional regulation of glycolytic enzymes in control HMEC-1 cells or HMEC-1 cells overexpressing oxygen-stable HIF-1α (HIF-1α OE) without or with siRNA-mediated PER2 knockdown (HIF-1α OE + PER2 KD). Cells were also treated with vehicle or BAY 60-6583 as indicated. Shown are the transcript concentrations of phosphofructokinase-m (PFKM), pyruvate kinase (PK), pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase a (LDHA). Data are mean ± s.d., n = 3. Statistical significance was calculated by one-way ANOVA with Dunnett's post-hoc test. (g) Coimmunoprecipitation of Per2 and HIF-1α. Protein lysates from the hearts of wild-type mice subjected to ischemic preconditioning were immunoprecipitated with either Hif-1α or Per2 antibody, and the presence of the other protein in the immunoprecipitates was detected by immunoblotting. One representative blot of three is shown. (h,i) Immunoblotting for Hif-1α in hearts or isolated myocytes from wild-type or Adora2b−/− mice exposed to ischemic preconditioning or ambient hypoxia. One representative blot of three is shown. (j) Transcript concentrations of Pfkm, Pk, Pdk1 and Ldha in the hearts from wild-type and Adora2b−/− mice with or without ischemic preconditioning. Data are mean ± s.d. n = 3 mice per group. Statistical significance was calculated by one-way ANOVA with Dunnett's post-hoc test.

  6. Light-induced stabilization of cardiac Per2 expression provides potent protection from myocardial ischemia.
    Figure 6: Light-induced stabilization of cardiac Per2 expression provides potent protection from myocardial ischemia.

    (a) Experimental model for studying light-induced stabilization of cardiac Per2. (b) Immunoblotting for Per2 in the hearts of wild-type mice after exposure to 12 h of darkness and then to indicated time periods of intense light (13,000 lx; daylight) compared to control mice maintained at room light (top). Quantitation of the results by densitometry (below). n = 4 mice per group. *P < 0.05 by Student's t test. Data are mean ± s.d. (c) Transcript concentrations of Pfkm, Pgk1, Pk and Pdk1 in the hearts of Per2−/− or WT littermate control mice after 4 h of daylight exposure. n = 4 mice per condition. Statistical significance was calculated by one-way ANOVA with Dunnett's post-hoc test. Data are mean ± s.d. (d,e) Myocardial injury, as assessed by infarct size (d) or measurement of troponin I plasma concentrations (e), in Per2−/− or WT littermate control mice exposed to light over the indicated time periods followed by exposure to in situ myocardial ischemia-reperfusion. n = 6 mice per group. Statistical significance was calculated by two-factor ANOVA with Bonferroni's post-hoc test. Data are mean ± s.d. (f) Troponin I in wild-type mice subjected to in situ myocardial ischemia reperfusion and Per2 expression as measured in hearts of untreated Per2 reporter mice at various times of day (with ZT0 set as 6 am) over a 24-h time period (left). Representative images of infarcts in mice subjected to myocardial ischemia-reperfusion at the indicated times of day (blue, retrograde Evan's blue staining; red and white, area at risk; white, infarcted tissue (right; scale bar, 50 μm). *P < 0.05. n = 8 mice per group. Statistical significance was calculated by one-way ANOVA with Dunnett's post-hoc test. Data are mean ± s.d. (g) Schematic model of Adora2b-dependent Per2 stabilization and its role in regulating glycolysis and cardiac metabolism during myocardial ischemia. pCREB, phosphorylated CREB.

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Author information

Affiliations

  1. Mucosal Inflammation Program, Department of Anesthesiology, University of Colorado Denver, Aurora, Colorado, USA.

    • Tobias Eckle,
    • Katherine Hartmann,
    • Stephanie Bonney,
    • Susan Reithel,
    • Douglas J Kominsky &
    • Holger K Eltzschig
  2. Institute of Neurology (Edinger Institute), University of Frankfurt, Frankfurt, Germany.

    • Michel Mittelbronn
  3. Division of Cardiology, Department of Medicine, University of Colorado Denver, Aurora, Colorado, USA.

    • Lori A Walker,
    • Brian D Lowes &
    • Peter M Buttrick
  4. University of Victoria–Genome BC Proteomics Centre, Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.

    • Jun Han &
    • Christoph H Borchers
  5. Mucosal Inflammation Program, Department of Medicine, University of Colorado Denver, Aurora, Colorado, USA.

    • Sean P Colgan

Contributions

T.E. designed and supervised the study, wrote the manuscript and did mouse surgery. K.H. did western blots, RT-PCRs and siRNA knockdown studies. S.B. did western blots, coimmunoprecipitation, promoter studies, ELISAs and animal experiments. S.R. did western blots, RT-PCRs, ELISAs and mouse experiments. M.M. did immunohistochemistry and electron microscopy. L.A.W. isolated mouse myocytes and supervised the study. B.D.L. provided human heart samples. J.H., C.H.B. and D.J.K. did metabolic analysis. P.M.B., S.P.C. and H.K.E. supervised the study and wrote the manuscript.

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The authors declare no competing financial interests.

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