Advertisement

Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium: a novel function beyond muscle contraction

  • Yi-Shuai Zhang
  • Bin Liu
  • Xiu-Ju Luo
  • Ting-Bo Li
  • Jie-Jie Zhang
  • Jing-Jie Peng
  • Xiao-Jie Zhang
  • Qi-Lin Ma
  • Chang-Ping Hu
  • Yuan-Jian Li
  • Jun Peng
  • Qingjie Li
Original Contribution

Abstract

Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia–reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H2O2 and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H2O2 levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.

Keywords

Cardiac myosin light chain 2 Ischemia Reperfusion NADPH oxidase 2 Transcription 

Notes

Acknowledgments

This work was supported by the Major Research Plan of the National Natural Science Foundation of China (No. 91439104 to Jun Peng), National Nature Science Foundation of China (No. 81373409 to Jun Peng; No.81370250 to Qi-Lin Ma), Hunan Provincial Natural Science Foundation of China (No.13JJ2008 to Jun Peng) and Doctoral Fund of Ministry of Education of China (No. 20120162110056 to Jun Peng).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

395_2015_494_MOESM1_ESM.doc (1.3 mb)
Supplementary material 1 (DOC 1326 kb)

References

  1. 1.
    Ago T, Kuroda J, Pain J, Fu C, Li H, Sadoshima J (2010) Upregulation of Nox4 by hypertrophic stimuli promotes apoptosis and mitochondrial dysfunction in cardiac myocytes. Circ Res 106:1253–1264. doi: 10.1161/CIRCRESAHA.109.213116 PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Banfi B, Malgrange B, Knisz J, Steger K, Dubois-Dauphin M, Krause KH (2004) NOX3, a superoxide-generating NADPH oxidase of the inner ear. J Biol Chem 279:46065–46072. doi: 10.1074/jbc.M403046200 PubMedCrossRefGoogle Scholar
  3. 3.
    Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245–313. doi: 10.1152/physrev.00044.2005 PubMedCrossRefGoogle Scholar
  4. 4.
    Borejdo J, Ushakov DS, Akopova I (2002) Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle. Biophys J 82:3150–3159. doi: 10.1016/S0006-3495(02)75657-9 PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Bou R, Codony R, Tres A, Decker EA, Guardiola F (2008) Determination of hydroperoxides in foods and biological samples by the ferrous oxidation-xylenol orange method: a review of the factors that influence the method’s performance. Anal Biochem 377:1–15. doi: 10.1016/j.ab.2008.02.029 PubMedCrossRefGoogle Scholar
  6. 6.
    Brandes RP, Weissmann N, Schroder K (2010) NADPH oxidases in cardiovascular disease. Free Radic Biol Med 49:687–706. doi: 10.1016/j.freeradbiomed.2010.04.030 PubMedCrossRefGoogle Scholar
  7. 7.
    Braunersreuther V, Montecucco F, Asrih M, Pelli G, Galan K, Frias M, Burger F, Quindere AL, Montessuit C, Krause KH, Mach F, Jaquet V (2013) Role of NADPH oxidase isoforms NOX1, NOX2 and NOX4 in myocardial ischemia/reperfusion injury. J Mol Cell Cardiol 64:99–107. doi: 10.1016/j.yjmcc.2013.09.007 PubMedCrossRefGoogle Scholar
  8. 8.
    Caremani M, Melli L, Dolfi M, Lombardi V, Linari M (2013) The working stroke of the myosin II motor in muscle is not tightly coupled to release of orthophosphate from its active site. J Physiol 591:5187–5205. doi: 10.1113/jphysiol.2013.257410 PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    de Lanerolle P (2012) Nuclear actin and myosins at a glance. J Cell Sci 125:4945–4949. doi: 10.1242/jcs.099754 PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Hafstad AD, Nabeebaccus AA, Shah AM (2013) Novel aspects of ROS signalling in heart failure. Basic Res Cardiol 108:359. doi: 10.1007/s00395-013-0359-8 PubMedCrossRefGoogle Scholar
  11. 11.
    Huynh K, Kiriazis H, Du XJ, Love JE, Gray SP, Jandeleit-Dahm KA, McMullen JR, Ritchie RH (2013) Targeting the upregulation of reactive oxygen species subsequent to hyperglycemia prevents type 1 diabetic cardiomyopathy in mice. Free Radic Biol Med 60:307–317. doi: 10.1016/j.freeradbiomed.2013.02.021 PubMedCrossRefGoogle Scholar
  12. 12.
    Josephson MP, Sikkink LA, Penheiter AR, Burghardt TP, Ajtai K (2011) Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain. Biochem Biophys Res Commun 416:367–371. doi: 10.1016/j.bbrc.2011.11.044 PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Kiani FA, Fischer S (2014) Catalytic strategy used by the myosin motor to hydrolyze ATP. Proc Natl Acad Sci USA. doi: 10.1073/pnas.1401862111 PubMedCentralPubMedGoogle Scholar
  14. 14.
    Kleikers PW, Wingler K, Hermans JJ, Diebold I, Altenhofer S, Radermacher KA, Janssen B, Gorlach A, Schmidt HH (2012) NADPH oxidases as a source of oxidative stress and molecular target in ischemia/reperfusion injury. J Mol Med (Berl) 90:1391–1406. doi: 10.1007/s00109-012-0963-3 CrossRefGoogle Scholar
  15. 15.
    Krijnen PA, Meischl C, Hack CE, Meijer CJ, Visser CA, Roos D, Niessen HW (2003) Increased Nox2 expression in human cardiomyocytes after acute myocardial infarction. J Clin Pathol 56:194–199. doi: 10.1136/jcp.56.3.194 PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Li Q, Sarna SK (2009) Nuclear myosin II regulates the assembly of preinitiation complex for ICAM-1 gene transcription. Gastroenterology 137:1051–1060. doi: 10.1053/j.gastro.2009.03.040 (1060 e1051–e1053) PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Lin HB, Cadete VJ, Sawicka J, Wozniak M, Sawicki G (2012) Effect of the myosin light chain kinase inhibitor ML-7 on the proteome of hearts subjected to ischemia-reperfusion injury. J Proteomics 75:5386–5395. doi: 10.1016/j.jprot.2012.06.016 PubMedCrossRefGoogle Scholar
  18. 18.
    Martindale JJ, Metzger JM (2014) Uncoupling of increased cellular oxidative stress and myocardial ischemia reperfusion injury by directed sarcolemma stabilization. J Mol Cell Cardiol 67:26–37. doi: 10.1016/j.yjmcc.2013.12.008 PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Matsushima S, Tsutsui H, Sadoshima J (2014) Physiological and pathological functions of NADPH oxidases during myocardial ischemia–reperfusion. Trends Cardiovasc Med. doi: 10.1016/j.tcm.2014.03.003 PubMedCentralPubMedGoogle Scholar
  20. 20.
    Moss RL, Fitzsimons DP (2006) Myosin light chain 2 into the mainstream of cardiac development and contractility. Circ Res 99:225–227. doi: 10.1161/01.RES.0000236793.88131.dc PubMedCrossRefGoogle Scholar
  21. 21.
    Murdoch CE, Alom-Ruiz SP, Wang M, Zhang M, Walker S, Yu B, Brewer A, Shah AM (2011) Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction. Basic Res Cardiol 106:527–538. doi: 10.1007/s00395-011-0179-7 PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Pei H, Yu Q, Xue Q, Guo Y, Sun L, Hong Z, Han H, Gao E, Qu Y, Tao L (2013) Notch1 cardioprotection in myocardial ischemia/reperfusion involves reduction of oxidative/nitrative stress. Basic Res Cardiol 108:373. doi: 10.1007/s00395-013-0373-x PubMedCrossRefGoogle Scholar
  23. 23.
    Philimonenko VV, Janacek J, Harata M, Hozak P (2010) Transcription-dependent rearrangements of actin and nuclear myosin I in the nucleolus. Histochem Cell Biol 134:243–249. doi: 10.1007/s00418-010-0732-8 PubMedCrossRefGoogle Scholar
  24. 24.
    Rodgers BD (2005) Insulin-like growth factor-I downregulates embryonic myosin heavy chain (eMyHC) in myoblast nuclei. Growth Horm IGF Res 15:377–383. doi: 10.1016/j.ghir.2005.08.001 PubMedCrossRefGoogle Scholar
  25. 25.
    Sarshad A, Sadeghifar F, Louvet E, Mori R, Bohm S, Al-Muzzaini B, Vintermist A, Fomproix N, Ostlund AK, Percipalle P (2013) Nuclear myosin 1c facilitates the chromatin modifications required to activate rRNA gene transcription and cell cycle progression. PLoS Genet 9:e1003397. doi: 10.1371/journal.pgen.1003397 PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Shi Y, Niculescu R, Wang D, Patel S, Davenpeck KL, Zalewski A (2001) Increased NAD(P)H oxidase and reactive oxygen species in coronary arteries after balloon injury. Arterioscler Thromb Vasc Biol 21:739–745. doi: 10.1161/01.ATV.21.5.739 PubMedCrossRefGoogle Scholar
  27. 27.
    Sirker A, Zhang M, Shah AM (2011) NADPH oxidases in cardiovascular disease: insights from in vivo models and clinical studies. Basic Res Cardiol 106:735–747. doi: 10.1007/s00395-011-0190-z PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Takano H, Zou Y, Hasegawa H, Akazawa H, Nagai T, Komuro I (2003) Oxidative stress-induced signal transduction pathways in cardiac myocytes: involvement of ROS in heart diseases. Antioxid Redox Signal 5:789–794. doi: 10.1089/152308603770380098 PubMedCrossRefGoogle Scholar
  29. 29.
    Taye A, Saad AH, Kumar AH, Morawietz H (2010) Effect of apocynin on NADPH oxidase-mediated oxidative stress-LOX-1-eNOS pathway in human endothelial cells exposed to high glucose. Eur J Pharmacol 627:42–48. doi: 10.1016/j.ejphar.2009.10.045 PubMedCrossRefGoogle Scholar
  30. 30.
    Tullio F, Angotti C, Perrelli MG, Penna C, Pagliaro P (2013) Redox balance and cardioprotection. Basic Res Cardiol 108:392. doi: 10.1007/s00395-013-0392-7 PubMedCrossRefGoogle Scholar
  31. 31.
    van der Vliet A (2008) NADPH oxidases in lung biology and pathology: host defense enzymes, and more. Free Radic Biol Med 44:938–955. doi: 10.1016/j.freeradbiomed.2007.11.016 PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Wang M, Zhang J, Walker SJ, Dworakowski R, Lakatta EG, Shah AM (2010) Involvement of NADPH oxidase in age-associated cardiac remodeling. J Mol Cell Cardiol 48:765–772. doi: 10.1016/j.yjmcc.2010.01.006 PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Warren SA, Briggs LE, Zeng H, Chuang J, Chang EI, Terada R, Li M, Swanson MS, Lecker SH, Willis MS, Spinale FG, Maupin-Furlowe J, McMullen JR, Moss RL, Kasahara H (2012) Myosin light chain phosphorylation is critical for adaptation to cardiac stress. Circulation 126:2575–2588. doi: 10.1161/CIRCULATIONAHA.112.116202 PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Zambelli F, Pesole G, Pavesi G (2009) Pscan: finding over-represented transcription factor binding site motifs in sequences from co-regulated or co-expressed genes. Nucleic Acids Res 37:W247–W252. doi: 10.1093/nar/gkp464 PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Zhang YS, He L, Liu B, Li NS, Luo XJ, Hu CP, Ma QL, Zhang GG, Li YJ, Peng J (2012) A novel pathway of NADPH oxidase/vascular peroxidase 1 in mediating oxidative injury following ischemia–reperfusion. Basic Res Cardiol 107:266. doi: 10.1007/s00395-012-0266-4 PubMedCrossRefGoogle Scholar
  36. 36.
    Zweier JL, Talukder MA (2006) The role of oxidants and free radicals in reperfusion injury. Cardiovasc Res 70:181–190. doi: 10.1016/j.cardiores.2006.02.025 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Yi-Shuai Zhang
    • 1
    • 2
  • Bin Liu
    • 1
  • Xiu-Ju Luo
    • 1
  • Ting-Bo Li
    • 1
    • 2
  • Jie-Jie Zhang
    • 1
    • 2
  • Jing-Jie Peng
    • 1
  • Xiao-Jie Zhang
    • 1
    • 2
  • Qi-Lin Ma
    • 3
  • Chang-Ping Hu
    • 1
    • 2
  • Yuan-Jian Li
    • 1
    • 2
  • Jun Peng
    • 1
    • 2
  • Qingjie Li
    • 4
  1. 1.Department of Pharmacology, School of Pharmaceutical SciencesCentral South UniversityChangshaChina
  2. 2.Hunan Provincial Key Laboratory of Cardiovascular Research, School of Pharmaceutical SciencesCentral South UniversityChangshaChina
  3. 3.Department of Cardiovascular Medicine, Xiangya HospitalCentral South UniversityChangshaChina
  4. 4.Department of Internal MedicineThe University of Texas Medical Branch at GalvestonGalvestonUSA

Personalised recommendations