Molecular and Cellular Biochemistry

, Volume 436, Issue 1–2, pp 167–178 | Cite as

Activation of adrenergic receptor in H9c2 cardiac myoblasts co-stimulates Nox2 and the derived ROS mediate the downstream responses

Article

Abstract

In recent years, NADPH oxidases (Noxes) have emerged as an important player in cardiovascular pathophysiology. Despite the growing evidences on the role of specific Nox isoforms, mechanisms of their activation, targets of reactive oxygen species (ROS) generated, and their downstream effects are poorly understood as yet. In this study, we treated H9c2 cardiac myoblasts with norepinephrine (NE, 2 µM), inducing ROS generation that was inhibited by Nox2-specific peptide inhibitor gp91ds-tat. Organelle-specific hydrogen peroxide-sensitive probe HyPer showed that the site of ROS generation is primarily in the cytosol, to some extent in the endoplasmic reticulum (ER) but not the mitochondria. Modulation of mRNAs of marker genes of cardiac hypertrophy i.e. induction in ANP and β-MHC, and reduction in α-MHC by NE treatment was prevented by specific inhibition of Nox2 by gp91ds-tat. Induction of ANP and β-MHC at the protein level were also attenuated by the inhibition of Nox2. Induction of c-Jun and FosB, the two members of the transcription factor family AP-1, were also blocked by the inhibition of Nox2 by gp91ds-tat. Induction of promoter-reporter constructs harboring multiple AP-1 elements and the upstream of FosB and ANP genes by NE were also blocked by the inhibition of Nox2 by gp91ds-tat and a dominant negative mutant of p22phox, a constituent of Nox2 that prevents its activation. This study for the first time establishes the significant role of Nox2 in mediating the NE-induced pathological adrenergic signaling in cardiac myoblasts.

Keywords

Redox signaling Reactive oxygen species NADPH oxidase Cardiac hypertrophy Norepinephrine gp91ds-tat 

Notes

Acknowledgements

This work was supported by The Department of Biotechnology, Government of India, under Grant (BT/PR4268/BRB/10/1016/2011), awarded to SKG. NS is a recipient of a JRF/SRF from the Indian Council of Medical Research, Government of India.

Compiance with ethical standards

Ethical approval

This article does not contain any studies performed with animals.

Supplementary material

11010_2017_3088_MOESM1_ESM.tif (131 kb)
Supplemental Fig.1s Schematic representation of construction of HyPer-ER ER signal sequence and retrieval sequence were inserted in-frame at N-terminal and C-terminal of pHyPer-Cyto ORF (open reading frame) to generate pHyPer-ER (details given in materials and methods). Then pHyper-Cyto and pHyPer-ER constructs were transiently transfected in H9c2 cells. After 36 hr of transfection, images were captured at Ex max of 488nm by Nikon Eclipse Ti-E fluorescence microscope. Supplementary material 1 (TIFF 131 kb)
11010_2017_3088_MOESM2_ESM.docx (7 kb)
Supplementary material 2 (DOCX 7 kb)

References

  1. 1.
    Ho E, Karimi Galougahi K, Liu C-C et al (2013) Biological markers of oxidative stress: applications to cardiovascular research and practice. Redox Biol 1:483–491. doi: 10.1016/j.redox.2013.07.006 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Murray TVA, Ahmad A, Brewer AC (2014) Reactive oxygen at the heart of metabolism. Trends Cardiovasc Med 24:113–120. doi: 10.1016/j.tcm.2013.09.003 CrossRefPubMedGoogle Scholar
  3. 3.
    Zhang Y, Tocchetti CG, Krieg T, Moens AL (2012) Oxidative and nitrosative stress in the maintenance of myocardial function. Free Radic Biol Med 53:1531–1540. doi: 10.1016/j.freeradbiomed.2012.07.010 CrossRefPubMedGoogle Scholar
  4. 4.
    Sawyer DB (2011) Oxidative stress in heart failure: what are we missing? Am J Med Sci 342:120–124. doi: 10.1097/MAJ.0b013e3182249fcd CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Wadley AJ, Aldred S, Coles SJ (2016) An unexplored role for Peroxiredoxin in exercise-induced redox signalling? Redox Biol 8:51–58. doi: 10.1016/j.redox.2015.10.003 CrossRefPubMedGoogle Scholar
  6. 6.
    Forman HJ, Ursini F, Maiorino M (2014) An overview of mechanisms of redox signaling. J Mol Cell Cardiol 73:2–9. doi: 10.1016/j.yjmcc.2014.01.018 CrossRefPubMedGoogle Scholar
  7. 7.
    Latimer HR, Veal EA (2016) Peroxiredoxins in regulation of MAPK signalling pathways; sensors and barriers to signal transduction. Mol Cells 39:40–45. doi: 10.14348/molcells.2016.2327 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lee J-G, Baek K, Soetandyo N, Ye Y (2013) Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells. Nat Commun 4:1568. doi: 10.1038/ncomms2532 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Stangherlin A, Reddy AB (2013) Regulation of circadian clocks by redox homeostasis. J Biol Chem 288:26505–26511. doi: 10.1074/jbc.R113.457564 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Groitl B, Jakob U (2014) Thiol-based redox switches. Biochim Biophys Acta 1844:1335–1343. doi: 10.1016/j.bbapap.2014.03.007 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Madamanchi NR, Runge MS (2013) Redox signaling in cardiovascular health and disease. Free Radic Biol Med 61:473–501. doi: 10.1016/j.freeradbiomed.2013.04.001 CrossRefPubMedGoogle Scholar
  12. 12.
    Ciccarelli M, Santulli G, Pascale V et al (2013) Adrenergic receptors and metabolism: role in development of cardiovascular disease. Front Physiol 4:265. doi: 10.3389/fphys.2013.00265 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ferrara N, Komici K, Corbi G et al (2014) β-adrenergic receptor responsiveness in aging heart and clinical implications. Front Physiol 4:396. doi: 10.3389/fphys.2013.00396 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Clerk A (2003) The radical balance between life and death. J Mol Cell Cardiol 35:599–602CrossRefPubMedGoogle Scholar
  15. 15.
    Fu Y-C, Chi C-S, Yin S-C et al (2004) Norepinephrine induces apoptosis in neonatal rat cardiomyocytes through a reactive oxygen species-TNF alpha-caspase signaling pathway. Cardiovasc Res 62:558–567. doi: 10.1016/j.cardiores.2004.01.039 CrossRefPubMedGoogle Scholar
  16. 16.
    Gupta MK, Neelakantan TV, Sanghamitra M et al (2006) An assessment of the role of reactive oxygen species and redox signaling in norepinephrine-induced apoptosis and hypertrophy of H9c2 cardiac myoblasts. Antioxid Redox Signal 8:1081–1093. doi: 10.1089/ars.2006.8.1081 CrossRefPubMedGoogle Scholar
  17. 17.
    Thakur A, Alam MJ, Ajayakumar MR et al (2015) Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation. Redox Biol 5:243–252. doi: 10.1016/j.redox.2015.05.005 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Sirokmány G, Donkó Á, Geiszt M (2016) Nox/Duox family of NADPH oxidases: lessons from knockout mouse models. Trends Pharmacol Sci 37:318–327. doi: 10.1016/j.tips.2016.01.006 CrossRefPubMedGoogle Scholar
  19. 19.
    von Löhneysen K, Noack D, Wood MR et al (2010) Structural insights into Nox4 and Nox2: motifs involved in function and cellular localization. Mol Cell Biol 30:961–975. doi: 10.1128/MCB.01393-09 CrossRefGoogle Scholar
  20. 20.
    Heppner DE, van der Vliet A (2016) Redox-dependent regulation of epidermal growth factor receptor signaling. Redox Biol 8:24–27. doi: 10.1016/j.redox.2015.12.002 CrossRefPubMedGoogle Scholar
  21. 21.
    Spencer NY, Engelhardt JF (2014) The basic biology of redoxosomes in cytokine-mediated signal transduction and implications for disease-specific therapies. Biochemistry (Mosc) 53:1551–1564. doi: 10.1021/bi401719r CrossRefGoogle Scholar
  22. 22.
    Brandes RP, Weissmann N, Schröder K (2014) Nox family NADPH oxidases: molecular mechanisms of activation. Free Radic Biol Med 76:208–226. doi: 10.1016/j.freeradbiomed.2014.07.046 CrossRefPubMedGoogle Scholar
  23. 23.
    Granger DN, Kvietys PR (2015) Reperfusion injury and reactive oxygen species: the evolution of a concept. Redox Biol 6:524–551. doi: 10.1016/j.redox.2015.08.020 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Lassègue B, San Martín A, Griendling KK (2012) Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system. Circ Res 110:1364–1390. doi: 10.1161/CIRCRESAHA.111.243972 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Fisher AB (2009) Redox signaling across cell membranes. Antioxid Redox Signal 11:1349–1356. doi: 10.1089/ars.2008.2378 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Theccanat T, Philip JL, Razzaque AM et al (2016) Regulation of cellular oxidative stress and apoptosis by G protein-coupled receptor kinase-2; The role of NADPH oxidase 4. Cell Signal 28:190–203. doi: 10.1016/j.cellsig.2015.11.013 CrossRefPubMedGoogle Scholar
  27. 27.
    Kawahara T, Ritsick D, Cheng G, Lambeth JD (2005) Point mutations in the proline-rich region of p22phox are dominant inhibitors of Nox1- and Nox2-dependent reactive oxygen generation. J Biol Chem 280:31859–31869. doi: 10.1074/jbc.M501882200 CrossRefPubMedGoogle Scholar
  28. 28.
    Malinouski M, Zhou Y, Belousov VV et al (2011) Hydrogen peroxide probes directed to different cellular compartments. PLoS One 6:e14564. doi: 10.1371/journal.pone.0014564 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Burch PM, Yuan Z, Loonen A, Heintz NH (2004) An extracellular signal-regulated kinase 1- and 2-dependent program of chromatin trafficking of c-Fos and Fra-1 is required for cyclin D1 expression during cell cycle reentry. Mol Cell Biol 24:4696–4709. doi: 10.1128/MCB.24.11.4696-4709.2004 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Jindal E, Goswami SK (2011) In cardiac myoblasts, cellular redox regulates FosB and Fra-1 through multiple cis-regulatory modules. Free Radic Biol Med 51:1512–1521. doi: 10.1016/j.freeradbiomed.2011.07.008 CrossRefPubMedGoogle Scholar
  31. 31.
    Belousov VV, Fradkov AF, Lukyanov KA et al (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3:281–286. doi: 10.1038/nmeth866 CrossRefPubMedGoogle Scholar
  32. 32.
    Banerjee P, Bandyopadhyay A (2014) Cytosolic dynamics of annexin A6 trigger feedback regulation of hypertrophy via atrial natriuretic peptide in cardiomyocytes. J Biol Chem 289:5371–5385. doi: 10.1074/jbc.M113.514810 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Yariswamy M, Yoshida T, Valente AJ et al (2016) Cardiac-restricted overexpression of TRAF3 interacting protein 2 (TRAF3IP2) results in spontaneous development of myocardial hypertrophy, fibrosis, and dysfunction. J Biol Chem 291:19425–19436. doi: 10.1074/jbc.M116.724138 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Yan L, Zhang JD, Wang B et al (2013) Quercetin inhibits left ventricular hypertrophy in spontaneously hypertensive rats and inhibits angiotensin II-induced H9C2 cells hypertrophy by enhancing PPAR-γ expression and suppressing AP-1 activity. PLoS One 8:e72548. doi: 10.1371/journal.pone.0072548 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Windak R, Müller J, Felley A et al (2013) The AP-1 transcription factor c-Jun prevents stress-imposed maladaptive remodeling of the heart. PLoS One 8:e73294. doi: 10.1371/journal.pone.0073294 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Meng Q, Xia Y (2011) c-Jun, at the crossroad of the signaling network. Protein Cell 2:889–898. doi: 10.1007/s13238-011-1113-3 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Wang J, Paradis P, Aries A et al (2005) Convergence of protein kinase C and JAK-STAT signaling on transcription factor GATA-4. Mol Cell Biol 25:9829–9844. doi: 10.1128/MCB.25.22.9829-9844.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Knowlton KU, Baracchini E, Ross RS et al (1991) Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during alpha-adrenergic stimulation of neonatal rat ventricular cells. Identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression. J Biol Chem 266:7759–7768PubMedGoogle Scholar
  39. 39.
    Dey S, Sidor A, O’Rourke B (2016) Compartment-specific control of reactive oxygen species scavenging by antioxidant pathway enzymes. J Biol Chem 291:11185–11197. doi: 10.1074/jbc.M116.726968 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Schaar CE, Dues DJ, Spielbauer KK et al (2015) Mitochondrial and cytoplasmic ROS have opposing effects on lifespan. PLoS Genet 11:e1004972. doi: 10.1371/journal.pgen.1004972 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Maejima Y, Kuroda J, Matsushima S et al (2011) Regulation of myocardial growth and death by NADPH oxidase. J Mol Cell Cardiol 50:408–416. doi: 10.1016/j.yjmcc.2010.12.018 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Bell RM, Cave AC, Johar S et al (2005) Pivotal role of NOX-2-containing NADPH oxidase in early ischemic preconditioning. FASEB J Off Publ Fed Am Soc Exp Biol 19:2037–2039. doi: 10.1096/fj.04-2774fje Google Scholar
  43. 43.
    Montezano AC, Touyz RM (2014) Reactive oxygen species, vascular Noxs, and hypertension: focus on translational and clinical research. Antioxid Redox Signal 20:164–182. doi: 10.1089/ars.2013.5302 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Jang S, Lewis TS, Powers C et al (2016) Elucidating mitochondrial electron transport chain supercomplexes in the heart during ischemia-reperfusion. Antioxid Redox Signal. doi: 10.1089/ars.2016.6635 Google Scholar
  45. 45.
    Qin F, Siwik DA, Pimentel DR et al (2014) Cytosolic H2O2 mediates hypertrophy, apoptosis, and decreased SERCA activity in mice with chronic hemodynamic overload. Am J Physiol Heart Circ Physiol 306:H1453–H1463. doi: 10.1152/ajpheart.00084.2014 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Matsushima S, Kuroda J, Zhai P et al (2016) Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling. J Clin Investig 126:3403–3416. doi: 10.1172/JCI85624 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Burgoyne JR, Rudyk O, Cho H et al (2015) Deficient angiogenesis in redox-dead Cys17Ser PKARIα knock-in mice. Nat Commun 6:7920. doi: 10.1038/ncomms8920 CrossRefPubMedGoogle Scholar
  48. 48.
    Derochette S, Serteyn D, Mouithys-Mickalad A et al (2015) EquiNox2: a new method to measure NADPH oxidase activity and to study effect of inhibitors and their interactions with the enzyme. Talanta 144:1252–1259. doi: 10.1016/j.talanta.2015.08.007 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia

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