Research ArticleHeart Failure

Accumulation of 5-oxoproline in myocardial dysfunction and the protective effects of OPLAH

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Science Translational Medicine  08 Nov 2017:
Vol. 9, Issue 415, eaam8574
DOI: 10.1126/scitranslmed.aam8574
  • Fig. 1. RNA-seq analysis on the reactivation of the cardiac fetal program.

    (A) Flow chart displaying the steps taken for the identification of novel cardiac fetal genes (n indicates the number of murine hearts/left ventricles used). qRT-PCR, quantitative real-time polymerase chain reaction. (B) Venn diagram of the number of differentially expressed genes identified in the different groups: up-regulated in development, down-regulated in development, down-regulated in HF, and up-regulated in HF. (C) Heat map depicting the expression profiles of the 68 identified cardiac fetal genes during murine development (E12, E18, PP2, 4 weeks old, and 20 weeks old) and cardiac injury (20 weeks old after IR).

  • Fig. 2. OPLAH in vitro characterization.

    (A) Representative immunoblotting analysis of OPLAH expression during hESC cardiomyocyte differentiation. (B) Representative immunoblotting analysis of OPLAH protein expression in NRVCs, rat fibroblasts, murine endothelioma cells, and rat smooth muscle cells. (C) Quantified OPLAH protein expression in NRVCs (n = 4), rat fibroblasts (n = 4), murine endothelioma cells (n = 4), and rat smooth muscle cells (n = 5). (D) qRT-PCR mRNA expression of OPLAH in NRVCs (n = 7), rat fibroblasts (n = 8), murine endothelioma cells (n = 8), and rat smooth muscle cells (n = 8). (E) Immunoblotting analysis of OPLAH expression in three animal models for HF. Top: OPLAH expression in Sprague Dawley rats (control; n = 4) versus renin overexpression TG rats (REN2; n = 5). Middle: OPLAH expression in sham (control; n = 3) versus IR (n = 5) C57BL/6 mice. Bottom: OPLAH expression in sham (control; n = 3) versus MI (n = 3) rats. (F) qRT-PCR mRNA expression of OPLAH in NRVM exposed to ISO (n = 4), PE (n = 4), stretch (n = 17), hypoxia (n = 31), H2O2 (n = 16), and no-treatment controls (n = 23). (G) Quantified OPLAH protein expression in NRVCs infected with the control, short hairpin OPLAH (shOPLAH), or human OPLAH overexpression (hOPLAH) constructs (n = 6, 11, and 5, respectively). (H) 5-Oxoproline concentrations in NRVCs infected with the control, shOPLAH, or hOPLAH adenoviral construct (n = 3). (I) CellROX analysis of adenoviral-infected NRVCs with control, shOPLAH, or hOPLAH vector exposed to 24 hours of hypoxia, 24 hours of H2O2 (500 μM), or 2 hours of 5-oxoproline (10 mM) culture conditions [for all conditions, n = 10 (control), 15 (shOPLAH), and 15 (hOPLAH)]. CellROX data are presented as fold change of relative fluorescence units (RFU) [arbitrary units (A.U.)] per microgram protein. Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ###P < 0.001; ####P < 0.0001, as calculated by Student’s t test or one-way analysis of variance (ANOVA). “*” indicates significant difference compared to control, whereas “#” denotes differences between groups other than control. n indicates the number of biological replicates of cell experiments or the number of animals.

  • Fig. 3. ERRα is involved in the regulation of OPLAH.

    (A) OPLAH expression in hESC-derived cardiomyocytes exposed to increasing concentrations of XCT-790 (vehicle, n = 6; 1 μM, n = 6; 3 μM, n = 6; 10 μM, n = 4). (B) OPLAH expression in hESC-derived cardiomyocytes exposed to increasing concentration of 4-hydroxytamoxifen (vehicle, n = 5; 1 μM, n = 6; 3 μM, n = 6; 10 μM, n = 5). (C) CellROX analysis of hESC-derived cardiomyocytes exposed to increasing concentrations of XCT-790 (vehicle, n = 48; 1 μM, n = 48; 3 μM, n = 64; 10 μM, n = 39; n indicates technical replicates). (D) ERRα mRNA expression in hESC-derived cardiomyocytes exposed to increasing concentrations of XCT-790 (vehicle, n = 3; 1 μM, n = 6; 3 μM, n = 4; 10 μM, n = 4). (E) Peroxisome proliferator–activated receptor γ coactivator 1α (PGC-1α) mRNA expression in hESC-derived cardiomyocytes exposed to increasing concentrations of XCT-790 (vehicle, n = 4; 1 μM, n = 6; 3 μM, n = 6; 10 μM, n = 3). (F) CellROX analysis of adenoviral-infected H9C2 cells with control or hOPLAH adenoviral vector exposed to increasing concentration of XCT-790 (vehicle, n = 24; 1 μM, n = 24; 3 μM, n = 24; 10 μM, n = 16; n indicates technical replicates). (G) qRT-PCR mRNA expression of ERRα and OPLAH at different stages of development and IR injury [E12, n = 8; E18, n = 11; PP2, n = 7; 4 weeks, n = 3; 20 weeks (sham), n = 3; 20 weeks (IR), n = 3]. CellROX data are presented as fold change of RFU (A.U.) per microgram protein. Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; #P < 0.05; ##P < 0.01; ###P < 0.001, as calculated by Student’s t test or one-way ANOVA. “*” indicates significant difference compared to control, whereas “#” denotes differences between groups other than control. n indicates the number of biological replicates of cell experiments.

  • Fig. 4. OPLAH-TG mice have improved cardiac function after IR injury.

    (A) Construct used to develop the OPLAH-TG (TG) mice. (B to D) OPLAH expression in LV and kidney tissue of TG and WT mice (n = 12 and 4, respectively). (B) Representative immunoblotting analysis of OPLAH protein. (C) Quantified OPLAH protein. (D) Quantified OPLAH mRNA. (E) Representative LV tissue sections with Masson’s trichrome staining of TG and WT after IR (scale bars, 2 mm) and quantification of infarct size (TG IR, n = 17; WT IR, n = 15). (F) Representative MRI images of WT and TG mouse hearts, and summary of LV ejection fraction and stroke volume (TG sham, n = 6; TG IR, n = 17; WT sham, n = 15; WT IR, n = 15). (G) Representative immunoblotting analysis of OPLAH and ERRα protein expression in the left ventricle of WT sham and IR mice. GAPDH, glyceraldehyde phosphate dehydrogenase. (H) 5-Oxoproline concentrations in LV tissue from TG versus WT mice 4 weeks after IR injury (TG sham, n = 9; TG IR, n = 8; WT sham, n = 11; WT IR, n = 7). (I) TAC of LV tissue from TG versus WT mice 4 weeks after IR injury (n = 3). (J) GSH/GSSG ratio present in LV tissue from TG versus WT mice 4 weeks after IR injury. Data are presented as fold change of microgram GSH/GSSG per microgram protein (TG sham, n = 3; TG IR, n = 4; WT sham, n = 4; WT IR n = 4). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001; #P < 0.05; ##P < 0.01, as calculated by Student’s t test or one-way ANOVA. “*” indicates significant difference compared to control, whereas “#” denotes differences between groups other than control. n indicates the number of animals.

  • Fig. 5. OPLAH-TG mice show improved cardiac function after MI.

    (A) Representative MRI images of sham and MI hearts of WT and TG mice, and summary of LV ejection fraction and stroke volume (TG sham, n = 6; TG MI, n = 13; WT sham, n = 6; WT MI, n = 15). (B) Representative LV tissue sections with Masson’s trichrome staining of TG and WT (scale bars, 3 mm), and summary infarct size (TG MI, n = 13; WT MI, n = 15). (C) 5-Oxoproline concentrations (in micromolars per microgram protein) in LV tissue from TG versus WT mice 4 weeks after MI (TG sham, n = 5; TG IR, n = 6; WT sham, n = 5; WT IR, n = 6). (D) TAC of LV tissue from TG versus WT mice 4 weeks after MI (n = 3). (E) GSH/GSSG ratio present in LV tissue from TG versus WT mice 4 weeks after MI. Data are presented as fold change of microgram GSH/GSSG per microgram protein (n = 3). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01, as calculated by Student’s t test or one-way ANOVA. “*” indicates significant difference compared to control, whereas “#” denotes differences between groups other than control. n indicates the number of animals.

  • Fig. 6. Circulating 5-oxoproline in murine and human HF.

    (A) 5-Oxoproline concentration in LV tissue of REN2 rats (n = 5) compared to control Sprague Dawley rats (n = 4). (B) 5-Oxoproline concentrations in the plasma from control Sprague Dawley rats (n = 4) versus renin overexpression TG Sprague Dawley rats (REN2; n = 5). (C) 5-Oxoproline concentration in the human plasma from healthy controls (control; n = 10) and acute HF patients (n = 10). (D) Kaplan-Meier plot of all-cause mortality and HF hospitalization at 18 months in COACH HF patients. Patient population is divided into tertiles of plasma 5-oxoproline concentrations (T1, 3.2 to 9.2 μM; T2, 9.3 to 13.2 μM; T3, 13.3 to 35.0 μM). 5-Oxoproline concentrations (in micromolars per microgram protein) are presented as fold change. Data are presented as means ± SEM **P < 0.01; ****P < 0.0001, as calculated by Student’s t test. “*” indicates significant difference compared to control. n indicates the number of animals or individual human samples.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/415/eaam8574/DC1

    Materials and Methods

    Fig. S1. Top 29 novel cardiac fetal genes mRNA expression profiles across a human organ panel.

    Fig. S2. OPLAH is localized in the cytosol of cardiomyocytes.

    Fig. S3. OPLAH protein expression in HF animal models.

    Fig. S4. Mechanical stretch results in oxidative stress and OPLAH depletion in NRVCs.

    Fig. S5. Hypoxia induces oxidative stress and OPLAH depletion in NRVCs.

    Fig. S6. H2O2 induces oxidative stress and OPLAH depletion in NRVCs.

    Fig. S7. OPLAH short hairpin and overexpression constructs in NRVCs.

    Fig. S8. ERRα is involved in the regulation of OPLAH in NRVCs.

    Fig. S9. ERRα expression in NRVCs exposed to stretch, hypoxia, or H2O2.

    Fig. S10. OPLAH-TG mice have reduced fibrosis compared to WT mice after IR injury.

    Fig. S11. OPLAH-TG mice show no difference in LV hypertrophy compared to WT mice after IR injury or after MI.

    Fig. S12. OPLAH-TG mice have reduced cleaved Caspase-3–positive cells in the left ventricle after MI.

    Fig. S13. Schematic of OPLAH regulation in the cardiomyocyte.

    Fig. S14. Calibration curves for LC-MS ISs 13C-5-oxoproline and l-glutamic acid.

    Table S1. Significantly up-regulated genes during murine cardiac development (provided as an Excel file).

    Table S2. Significantly down-regulated genes during murine cardiac development (provided as an Excel file).

    Table S3. Top cardiac fetal reprogramming genes (provided as an Excel file).

    Table S4. Characteristics of renin overexpression rats (REN2) and Sprague Dawley rats.

    Table S5. Characteristics of WT mice after IR injury.

    Table S6. Characteristics of Sprague Dawley rats after MI.

    Table S7. Characteristics of OPLAH overexpression (TG) and WT mice at baseline (sham) and after IR injury.

    Table S8. Characteristics of OPLAH overexpression (TG) and WT mice at baseline (sham) and after MI.

    Table S9. Baseline characteristics of all 535 patients compared to total COACH cohort (n = 1023).

    Table S10. Baseline characteristics of all 535 patients at discharge, divided into tertiles of 5-oxoproline (in micromolars).

    Table S11. Regression analyses of 5-oxoproline association with HF biomarkers.

    Table S12. Regression analyses of multivariable model corrected for univariable associations.

    Table S13. Survival analyses.

    Table S14. OPLAH-specific shRNA–targeting oligonucleotides and cloning primers for human OPLAH overexpression.

    Table S15. List of primers used in this study.

    Table S16. List of antibodies used in this study.

    Table S17. Accuracy and precision results for 5-oxoproline.

    Table S18. Accuracy and precision results for l-glutamic acid.

    Table S19. Individual subject level data for experiments with n < 20 (provided as an Excel file).

  • Supplementary Material for:

    Accumulation of 5-oxoproline in myocardial dysfunction and the protective effects of OPLAH

    Atze van der Pol, Andres Gil, Herman H. W. Silljé, Jasper Tromp, Ekaterina S. Ovchinnikova, Inge Vreeswijk-Baudoin, Martijn Hoes, Ibrahim J. Domian, Bart van de Sluis, Jan M. van Deursen, Adriaan A. Voors, Dirk J. van Veldhuisen, Wiek H. van Gilst, Eugene Berezikov, Pim van der Harst, Rudolf A. de Boer, Rainer Bischoff, Peter van der Meer*

    *Corresponding author. Email: p.van.der.meer{at}umcg.nl

    Published 8 November 2017, Sci. Transl. Med. 9, eaam8574 (2017)
    DOI: 10.1126/scitranslmed.aam8574

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Top 29 novel cardiac fetal genes mRNA expression profiles across a human organ panel.
    • Fig. S2. OPLAH is localized in the cytosol of cardiomyocytes.
    • Fig. S3. OPLAH protein expression in HF animal models.
    • Fig. S4. Mechanical stretch results in oxidative stress and OPLAH depletion in NRVCs.
    • Fig. S5. Hypoxia induces oxidative stress and OPLAH depletion in NRVCs.
    • Fig. S6. H2O2 induces oxidative stress and OPLAH depletion in NRVCs.
    • Fig. S7. OPLAH short hairpin and overexpression constructs in NRVCs.
    • Fig. S8. ERRα is involved in the regulation of OPLAH in NRVCs.
    • Fig. S9. ERRα expression in NRVCs exposed to stretch, hypoxia, or H2O2.
    • Fig. S10. OPLAH-TG mice have reduced fibrosis compared to WT mice after IR injury.
    • Fig. S11. OPLAH-TG mice show no difference in LV hypertrophy compared to WT mice after IR injury or after MI.
    • Fig. S12. OPLAH-TG mice have reduced cleaved Caspase-3–positive cells in the left ventricle after MI.
    • Fig. S13. Schematic of OPLAH regulation in the cardiomyocyte.
    • Fig. S14. Calibration curves for LC-MS ISs 13C-5-oxoproline and L-glutamic acid.
    • Table S4. Characteristics of renin overexpression rats (REN2) and Sprague Dawley rats.
    • Table S5. Characteristics of WT mice after IR injury.
    • Table S6. Characteristics of Sprague Dawley rats after MI.
    • Table S7. Characteristics of OPLAH overexpression (TG) and WT mice at baseline (sham) and after IR injury.
    • Table S8. Characteristics of OPLAH overexpression (TG) and WT mice at baseline (sham) and after MI.
    • Table S9. Baseline characteristics of all 535 patients compared to total COACH cohort (n = 1023).
    • Table S10. Baseline characteristics of all 535 patients at discharge, divided into tertiles of 5-oxoproline (in micromolars).
    • Table S11. Regression analyses of 5-oxoproline association with HF biomarkers.
    • Table S12. Regression analyses of multivariable model corrected for univariable associations.
    • Table S13. Survival analyses.
    • Table S14. OPLAH-specific shRNA–targeting oligonucleotides and cloning primers for human OPLAH overexpression.
    • Table S15. List of primers used in this study.
    • Table S16. List of antibodies used in this study.
    • Table S17. Accuracy and precision results for 5-oxoproline.
    • Table S18. Accuracy and precision results for L-glutamic acid.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Significantly up-regulated genes during murine cardiac development (provided as an Excel file).
    • Table S2. Significantly down-regulated genes during murine cardiac development (provided as an Excel file).
    • Table S3. Top cardiac fetal reprogramming genes (provided as an Excel file).
    • Table S19. Individual subject level data for experiments with n < 20 (provided as an Excel file).

    [Download Supplementary Tables]

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