Cardioprotection by Peroxidase Activity of Prostaglandin H Synthases in Ischemia/Reperfusion Injury

HomeCirculationVol. 147, No. 19Cardioprotection by Peroxidase Activity of Prostaglandin H Synthases in Ischemia/Reperfusion Injury Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBCardioprotection by Peroxidase Activity of Prostaglandin H Synthases in Ischemia/Reperfusion Injury Guizhu Liu, Qian Liu, Naifu Wan, Xia Shen, Hui Cui, Cheng Dong, Xu Zhang, Huiyong Yin, Jian Wang, Colin D. Funk and Ying Yu Guizhu LiuGuizhu Liu Department of Pharmacology, Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, China (G.L., Q.L., X.Z., Y.Y.). Wuxi School of Medicine, Jiangnan University, Jiangsu, China (G.L.). State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China (G.L., J.W.). *G. Liu and Q. Liu contributed equally. Search for more papers by this author , Qian LiuQian Liu https://orcid.org/0000-0002-2788-6222 Department of Pharmacology, Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, China (G.L., Q.L., X.Z., Y.Y.). *G. Liu and Q. Liu contributed equally. Search for more papers by this author , Naifu WanNaifu Wan Search for more papers by this author , Xia ShenXia Shen School of Life Science and Technology, Shanghai Tech University, China (X.S., H.C., H.Y.). Search for more papers by this author , Hui CuiHui Cui School of Life Science and Technology, Shanghai Tech University, China (X.S., H.C., H.Y.). Search for more papers by this author , Cheng DongCheng Dong https://orcid.org/0000-0002-2891-8759 Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, China (C.D.). Search for more papers by this author , Xu ZhangXu Zhang https://orcid.org/0000-0002-8103-8486 Department of Pharmacology, Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, China (G.L., Q.L., X.Z., Y.Y.). Search for more papers by this author , Huiyong YinHuiyong Yin School of Life Science and Technology, Shanghai Tech University, China (X.S., H.C., H.Y.). Chinese Academy of Sciences Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, Innovation Center for Intervention of Chronic Disease and Promotion of Health, Chinese Academy of Sciences, China (H.Y.). Department of Biomedical Sciences, City University of Hong Kong, China (H.Y.). Search for more papers by this author , Jian WangJian Wang https://orcid.org/0000-0002-1278-256X Department of Vascular and Cardiology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, China (N.W.). Search for more papers by this author , Colin D. FunkColin D. Funk Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada (C.D.F.). Search for more papers by this author and Ying YuYing Yu Correspondence to: Ying Yu, MD, PhD, Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, 22 Qixiangtai Road, Tianjin 300070, China. Email E-mail Address: [email protected] https://orcid.org/0000-0002-6476-1752 Department of Pharmacology, Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, China (G.L., Q.L., X.Z., Y.Y.). Search for more papers by this author Originally published8 May 2023https://doi.org/10.1161/CIRCULATIONAHA.122.061145Circulation. 2023;147:1467–1470Prostaglandin H synthase (PGHS) is the key enzyme in the biosynthesis of an array of lipid mediators: prostaglandins and thromboxane. The 2 isoforms are referred to as PGHS1 and PGHS2. Both are heme-containing, glycosylated dimeric proteins that are anchored to 1 leaflet of the endoplasmic reticulum and nuclear membrane bilayer. Each possesses 2 distinct catalytic activities residing within the same enzyme: a cyclooxygenase activity that catalyzes the bis-dioxygenations of arachidonic acid to yield hydroperoxy-endoperoxide PGG2 and a peroxidase activity that reduces the hydroperoxide of PGG2 to PGH2.1 Nonsteroidal antiinflammatory drugs exert their actions by inhibiting the cyclooxygenase activity of PGHSs and reducing prostaglandin production. Crystal structures of the enzyme-substrate complex have demonstrated distinct cyclooxygenase and peroxidase active sites, the mode of binding of arachidonic acid, and its product (PGH2) in apo and heme forms.1 The peroxidase site, on the exterior of the protein, displays particular structural homology with other peroxidases even though there is very low sequence homology. Moreover, peroxidase of PGHS undergoes redox cycling to generate biologically reactive intermediates (such as oxyferryl heme-derived compounds). Many fundamental questions about the symbiotic relationship of the cyclooxygenase and peroxidase activities remain. For example, how did these 2 distinct enzyme activities coalesce to form a single enzyme, and are there independent metabolic activities and physiological functions in vivo?Distinct PGHS point mutations at certain residues such as cyclooxygenase catalytic residue tyrosine (Y) 385 or aspirin-mediated inactivation site (serine [S] 532 and S516 in murine PGHS1 and PHGS2, respectively) and the proximal heme ligand histidine (H; H390 and H374 in murine PGHS1 and PHGS2, respectively) can be induced to disrupt one activity while leaving the other largely intact.2 Therefore, cyclooxygenase-only (PGHS1POX−/−) and peroxidase-only PGHS1 (PGHS1COX−/−) mice (Figure [A–C]) and cyclooxygenase-only (PGHS2POX−/−) and peroxidase-only PGHS2 (PGHS2COX−/−) mice (Figure [D–F]) were created by traditional homologous recombination and CRISPR-Cas9 strategies. These point mutations did not overtly influence protein expression of PGHS isoforms (Figure [A and D]) but resulted in a marked reduction of PGHS1/2–derived prostaglandins and thromboxane production as measured by liquid chromatography–mass spectrometry analysis in tissues as PGHS1/2 deficiency, respectively.Download figureDownload PowerPointFigure. Ablation of peroxidase activity in PGHS isozymes exaggerates I/R-induced cardiac injury by impairing 20-HETE oxidation. A, Effect of prostaglandin H synthase (PGHS) 1 mutations on PGHS1 and PGHS2 proteins in peritoneal macrophages; n=6. B, Effect of PGHS1 mutations on cyclooxygenase (COX) activity of microsomes from peritoneal macrophages as measured by oxygen consumption. P<0.05 vs wild type (WT); n=6. C, Effect of PGHS1 mutations on peroxidase (POX) activity of microsomes from peritoneal macrophages. *P<0.05 vs WT (n=6). D, Effect of PGHS2 mutations on PGHS1 and PGHS2 protein expression from lipopolysaccharide (LPS)-stimulated (5 μg/mL) peritoneal macrophages; n=6. E, Effect of PGHS2 mutations on COX activity of microsomes from LPS-stimulated peritoneal macrophages. *P<0.05 vs WT; n=6. F, Effect of PGHS2 mutations on POX activity of microsomes from LPS-stimulated peritoneal macrophages. *P<0.05 vs WT; n=6. G and H, Representative photographs (G) and quantitative data for area at risk (AAR; red area) and infarct size (H, white area) in hearts from PGHS mutant mice followed by ischemia/reperfusion (I/R) injury (45 minutes of ischemia/24 hours of reperfusion). *P<0.05 vs WT; n=10 mice per group. Scale bar, 1 mm. I, Recovery of left ventricular developed pressure (LVDP) as percentage of baseline after I/R injury in PGHS mutant mice. *P<0.05 vs WT; n=10 mice per group. J, Representative photomicrographs (left) and quantitative data (right) for cardiomyocyte apoptosis in PGHS mutant mice followed by I/R injury as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. *P<0.05 vs WT; n=6 mice per group. K, Arachidonic acid–derived eicosanoid generation in LPS-stimulated macrophages from PGHS2 mutant mice; n=6 mice per group. L, Effect of PGHS2 mutations of production of 5-, 12-, 15-, 20-hydroxy-eicosatetraenoic (20-HETE) in LPS-stimulated macrophages. *P<0.05 vs WT; n=6. M, Effect of 5-, 12-, 15-, 20-HETE (100 nmol/L) on apoptosis in HL-1 cardiomyocytes. *P<0.05 vs control; n=6. N, Cardiac 20-HETE production in PGHS mutant mice after I/R injury. *P<0.05 vs WT; n=6. O, 20-Carboxy-arachidonic acid (20-COOH-AA) production in LPS-stimulated macrophages from PGHS2 mutant mice after 20-HETE treatment (30 μmol/L). *P<0.05 vs WT; n=6. P, 20-COOH-AA levels in heart tissues from PGHS mutant mice after I/R injury. *P<0.05 vs WT; n=6. Q, Effect of silencing GPR75 or peroxisome proliferator-activated receptor-γ (PPARγ) on 20-HETE–induced apoptosis in cardiomyocytes, assessed by flow cytometry. *P<0.05 vs vehicle; n=6. R, Effect of silencing GPR75 or PPARγ on the caspase-3 activity in 20-HETE–treated cardiomyocytes; n=6. S through U, Effect of adeno-associated virus–mediated GPR75 knockdown on area at risk (AAR; red area) and infarct size (white area, S and T) in hearts and LVDP recovery (U) in PGHS2POX−/− mice after I/R injury. *P<0.05 vs WT; n=8 mice per group. Scale bar, 1 mm. V, Potential model indicating POX activity of PGHSs mediating conversion of 20-HETE to 20-COOH-AA and controlling cardiomyocyte apoptosis. Data represent mean±SEM. Statistical significance was evaluated with Kruskal-Wallis tests followed by the Dunn test.Nonsteroidal antiinflammatory drugs are associated with a significantly increased risk of heart failure,3 and genetic deletion of genes encoding PGHS1 or PGHS2 exaggerates cardiac injury in mice after ischemia/reperfusion (I/R), which cannot be rescued by the addition of exogenous prostaglandins.4 These observations suggest that PGHS isozymes display a cardioprotective effect that is independent of their downstream prostaglandin products. To further delineate the potential roles of peroxidase and cyclooxygenase activities of PGHSs, cardiac function of these mutant mice was monitored after myocardial I/R injury (45 minutes of ischemia followed by 24 hours of reperfusion). As shown in the Figure (G through I), peroxidase activity ablation (PGHSPOX−/−) dramatically aggravated I/R-induced cardiac infarct size and compromised recovery of left ventricular developed pressure in mice, as in PGHS knockout (PGHS−/−) mice, whereas cyclooxygenase activity deficiency (PGHSCOX−/−) had no significant effects (Figure [G–I]). Pathologically, more apoptotic cardiomyocytes were observed in the heart tissues (PGHSPOX−/−) of mice after I/R injury (Figure [J]). Indomethacin treatment markedly attenuated cardiac function recovery after I/R injury in all the mutants, as previously reported.4Targeted metabolomics analysis on arachidonic acid metabolites (data are available in MetaboLights at https://www.ebi.ac.uk/metabolights/, reference no. MTBLS6527) confirmed reduced prostaglandin production in macrophages from cyclooxygenase-only, peroxidase-only, and global PGHS2−/− mice (Figure [K]). It is interesting that ablation of peroxidase, not cyclooxygenase activity, markedly boosted the generation of various hydroxy-eicosatetraenoic (HETE) acid isomers in macrophages (Figure [K and L]). Among them, 20-HETE is proapoptotic in cultured cardiomyocytes (Figure [M]). In addition, peroxidase deficiency led to augmented 20-HETE production in heart tissues from PGHSPOX−/− mice (Figure [N]). We also found that 20-HETE was further oxidized by peroxidase of PGHS to form 20-carboxy-arachidonic acid in macrophages (Figure [O]) and heart tissues from PGHS mutant mice followed by I/R injury (Figure [P]). 20-HETE exerts its biological functions through binding to peroxisome proliferator-activated receptor-γ or GPR75 receptor.5 Knockdown of GPR75 receptors, not peroxisome proliferator-activated receptor-γ, attenuated 20-HETE–induced apoptosis and caspase-3 activity in cardiomyocytes (Figure [Q and R]). Moreover, adeno-associated virus serotype 9–mediated cardiac GPR75 silencing (AAV9-CTnT-shRNA-GPR75) reversed I/R-induced cardiac injury in cyclooxygenase-only PGHS2 mice, as evidenced by reduced infarct size and increased left ventricular developed pressure recovery (Figure [S–U]). Therefore, peroxidase activity of PGHSs appears to confer cardioprotection against I/R injury through metabolic conversion of 20-HETE (Figure [V]).All animal protocols were approved by the Animal Care and Use Committee of Tianjin Medical University. All the data, materials, and methods supporting the findings of this study are available from the corresponding author on request.Article InformationSources of FundingThis work was supported by the National Natural Science Foundation of China (82030015, 82241016, 82261160656, 82270284), the National Key R&D Program of China (2021YFC2701104), the Tianjin Municipal Commission of Education (2020ZD12), the Haihe Laboratory of Cell Ecosystem Innovation Fund (22HHXBSS00048), and the Guangdong Department of Science and Technology (2019A1515010672). Dr Yu is a fellow at the Jiangsu Collaborative Innovation Center for Cardiovascular Disease Translational Medicine.Nonstandard Abbreviations and Acronyms20-COOH-AA20-carboxy-arachidonic acidHETEhydroxy-eicosatetraenoicI/Rischemia/reperfusionPGHSprostaglandin H synthaseDisclosures None.Footnotes*G. Liu and Q. Liu contributed equally.For Sources of Funding and Disclosures, see page 1469.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Ying Yu, MD, PhD, Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, 22 Qixiangtai Road, Tianjin 300070, China. Email yuying@tmu.edu.cnReferences1. Rouzer CA, Marnett LJ. Structural and chemical biology of the interaction of cyclooxygenase with substrates and non-steroidal anti-inflammatory drugs.Chem Rev. 2020; 120:7592–7641. doi: 10.1021/acs.chemrev.0c00215CrossrefMedlineGoogle Scholar2. Yu Y, Fan J, Chen XS, Wang D, Klein-Szanto AJ, Campbell RL, FitzGerald GA, Funk CD. Genetic model of selective COX2 inhibition reveals novel heterodimer signaling.Nat Med. 2006; 12:699–704. doi: 10.1038/nm1412CrossrefMedlineGoogle Scholar3. 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