[Frontiers In Bioscience, Landmark, 23, 298-309, January 1, 2018]

L-NAME improves doxycycline and ML-7 cardioprotection from oxidative stress

Iwona Bil-Lula1, Anna Krzywonos –Zawadzka1, Jolanta Sawicka2, Dariusz Bialy3, Magdalena Wawrzynska4, Mieczyslaw Wozniak1,2, Grzegorz Sawicki1,2

1Department of Clinical Chemistry, Wroclaw Medical University, Wroclaw, Poland, 2Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Canada, 3Department and Clinic of Cardiology, Wroclaw Medical University, Wroclaw, Poland, 4Department of Emergency Medicine, Wroclaw Medical University, Wroclaw, Poland

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Isolated heart perfusion according to Langendorff
3.2. Protocol of global ischemia/reperfusion
3.3. Concentration of proteins
3.4. Determination of LDH activity
3.5. Immunoblott analysis of MLC1
3.6. Troponin I content in rat hearts and coronary effluents
3.7. Statistical analysis
4. Results
4.1. Determination of the protective threshold for cardiac mechanical function
4.2. Protective effect of co-administration of subthreshold concentration of Doxy, ML-7 and L- NAME against cardiac I/R injury
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

Matrix metalloproteinase-2 (MMP-2) mediated degradation of myosin light chain 1 (MLC1) and troponin I (TnI) contributes to myocardial ischemia/reperfusion (I/R) injury. Modifications of MLC1 triggered by oxidative stress are mediated by myosin light chain kinase (MLCK), nitric oxide synthase (NOS), and MMP-2. Previous studies have shown that inhibiting both MLCK and MMP-2 protects against I/R injury. Here, we hypothesized that the addition of NOS inhibitor (L-NAME) at subprotective concentration to the mixture of subprotective concentrations of ML-7 and doxycycline (Doxy), will increase a synergistic cardioprotection of Doxy and ML-7 during I/R. Isolated rat hearts were subjected to global ischemia without or with administration of the mixture of inhibitors. Markers of I/R injury were measured in hearts and coronary effluents. Addition of L-NAME to the mixture of Doxy and ML-7 led to full recovery of heart contractility in comparison to combination of Doxy and ML-7. Improved heart contractility was associated with reduced degradation of TnI and MLC1. The combined administration of NOS, MMP-2 and MLCK inhibitors provides a novel strategy to protect heart from I/R injury.

7. REFERENCES

1. P.Y. Cheung, G. Sawicki, M. Wozniak, W. Wang, M.W. Radomski, R. Schulz: Matrix metalloproteinase-2 contributes to ischemia-reperfusion injury in the heart. Circulation. 101(15), 1833–1899 (2000)
DOI:10.1161/01.CIR.101.15.1833

2. J. Fert-Bober, H. Leon, J. Sawicka, R.S. Basran, R.M. Devon, R. Schulz, G. Sawicki: Inhibiting matrix metalloproteinase-2 reduces protein release into coronary effluent from isolated rat hearts during ischemia-reperfusion. Basic Res Cardiol 103(5), 431–443 (2008)
DOI:10.1007/s00395-008-0727-y

3. D.K. Arrell, I. Neverova, H. Fraser, E. Marbán, J.E. Van Eyk: Proteomic analysis of pharmacologically preconditioned cardiomyocytes reveals novel phosphorylation of myosin light chain 1. Circ Res 89(6), 480–487 (2001)
DOI:10.1161/hh1801.097240

4. V.J. Cadete, J. Sawicka, J.S. Jaswal, G.D. Lopaschuk, R. Schulz, D. Szczesna-Cordary, G. Sawicki: Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase 2. FEBS J 279(13), 2444–2454 (2012)
DOI:10.1111/j.1742-4658.2012.08622.x

5. A. Doroszko, D. Polewicz, J. Sawicka, J.S. Richardson, P-Y. Cheung, G, Sawicki: Cardiac dysfunction in an animal model of neonatal asphyxia is associated with increased degradation of MLC1 by MMP-2. Basic Res Cardiol 104(6), 669–679 (2009)
DOI:10.1007/s00395-009-0035-1

6. D. Polewicz, V.J. Cadete, A. Doroszko, B.E. Hunter, J. Sawicka, D. Szczesna-Cordary, P.E. Light, G. Sawicki: Ischemia induced peroxynitrite dependent modifications of cardiomyocyte MLC1 increases its degradation by MMP-2 leading to contractile dysfunction. J Cell Mol Med 15(5), 1136–1147 (2011)
DOI:10.1111/j.1582-4934.2010.01094.x

7. W. Wang, C.J. Schulze, W.L. Suarez-Pinzon, J.R.B. Dyck, G. Sawicki, R. Schulz: Intracellular action of matrix metalloproteinase-2 accounts for acute myocardial ischemia and reperfusion injury. Circulation 106(12), 1543–1549 (2002)
DOI:10.1161/01.CIR.0000028818.33488.7B

8. B. Cauwe, G. Opdenakker: Intracellular substrate cleavage: a novel dimension in the biochemistry, biology and pathology of matrix metalloproteinases. Crit Rev Biochem Mol Biol 45(5), 351–423 (2010)
DOI:10.3109/10409238.2010.501783

9. M. Sariahmetoglu, M. Skrzypiec-Spring, N. Youssef, A.L. Jacob-Ferreira, J. Sawicka, C. Holmes, G. Sawicki, R. Schulz: Phosphorylation status of matrix metalloproteinase 2 in myocardial ischaemia-reperfusion injury. Heart Br Card Soc 98(8), 656–662 (2012)
DOI:10.1136/heartjnl-2011-301250

10. R. Schulz: Intracellular targets of matrix metalloproteinase-2 in cardiac disease: rationale and therapeutic approaches. Annu Rev Pharmacol Toxicol 47, 211–242 (2007)
DOI:10.1146/annurev.pharmtox.47.120505.105230

11. S. Viappiani, A.C. Nicolescu, A. Holt, G. Sawicki, B.D. Crawford, H. León, T. van Mulligen, R. Schulz: Activation and modulation of 72kDa matrix metalloproteinase-2 by peroxynitrite and glutathione. Biochem Pharmacol 77(5), 826–834 (2009)DOI:10.1016/j.bcp.2008.11.004

12. D.M. Yellon, D.J. Hausenloy: Myocardial reperfusion injury. N Engl J Med 357(11), 1121–1135 (2007)
DOI:10.1056/NEJMra071667

13. M.R. Bergman, J.R. Teerlink, R. Mahimkar, L. Li, B.Q. Zhu, A. Nguyen, S.Dahi, J.S. Karliner, D.H. Lovett:Cardiac matrix metalloproteinase-2 expression independently induces marked ventricular remodeling and systolic dysfunction. Am J Physiol Heart Circ Physiol 292(4), H1847–1860 (2007)
DOI:10.1152/ajpheart.00434.2006

14. G. Sawicki, H. Leon, J. Sawicka, M. Sariahmetoglu, C.J. Schulze, P.G. Scot, D. Szczesna-Cordary, R. Schulz R: Degradation of myosin light chain in isolated rat hearts subjected to ischemia-reperfusion injury: a new intracellular target for matrix metalloproteinase-2. Circulation 112(4), 544–552 (2005)
DOI:10.1161/CIRCULATIONAHA.104.531616

15. M.A. Ali, W.J. Cho, B. Hudson, Z. Kassiri, H. Granzier, R. Schulz: Titin is a target of matrix metalloproteinase-2: implications in myocardial ischemia/reperfusion injury. Circulation 122(20), 2039–2047 (2010)
DOI:10.1161/CIRCULATIONAHA.109.930222

16. H.B. Lin, V.J. Cadete, B. Sra, J. Sawicka, Z. Chen, L.K. Bekar, F. Cayabyab, G. Sawicki: Inhibition of MMP-2 expression with siRNA increases baseline cardiomyocyte contractility and protects against simulated ischemic reperfusion injury. BioMed Res Int 2014, 10371 (2014)
DOI:10.1155/2014/810371

17. J. Kanski, A. Behring, J. Pelling, C. Schöneich: Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging. Am J Physiol Heart Circ Physiol 288(1), H371–381 (2005)
DOI:10.1152/ajpheart.01030.2003

18. J. Kanski, S.J. Hong, C. Schöneich: Proteomic analysis of protein nitration in aging skeletal muscle and identification of nitrotyrosine-containing sequences in vivo by nanoelectrospray ionization tandem mass spectrometry. J Biol Chem 280(25), 24261–24266 (2005)
DOI:10.1074/jbc.M501773200

19. S.F. Steinberg: Oxidative stress and sarcomeric proteins. Circ Res 112(2), 393–405 (2013)>
DOI:10.1161/CIRCRESAHA.111.300496

20. S.B. Scruggs, R.J. Solaro: The significance of regulatory light chain phosphorylation in cardiac physiology. Arch Biochem Biophys 510(2), 129–134 (2015)
DOI:10.1016/j.abb.2011.02.013

21. O. Tsukamoto, M Kitakaze. Biochemical and physiological regulation of cardiac myocyte contraction by cardiac-specific myosin light chain kinase. Circ J Off J Jpn Circ Soc 77(9), 2218–2225 (2013)
DOI:10.1253/circj.cj-13-0627

22. T. Koga, T. Koga, M. Awai, J.Tsutsui, B.Y. Yue, H. Tanihara: Rho-associated protein kinase inhibitor, Y-27632, induces alterations in adhesion, contraction and motility in cultured human trabecular meshwork cells. Exp Eye Res 82(3), 362–370 (2006)
DOI:10.1016/j.exer.2005.07.006

23. B. Burýsková, K. Hilscherová, L. Bláha, B. Marsálek, I. Holoubek: Toxicity and modulations of biomarkers in Xenopus laevis embryos exposed to polycyclic aromatic hydrocarbons and their N-heterocyclic derivatives. Environ Toxicol 21(6):590–598 (2006)
DOI:10.1002/tox.20222

24. J. Xie, A. Nair, T.W. Hermiston: A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types. Toxicol Vitro Int J Publ Assoc BIBRA 22(1), 261–266 (2008)
DOI:10.1016/j.tiv.2007.08.019

25. A.H. Drummond, P. Beckett, P.D. Brown, E.A. Bone, A.H. Davidson, W.A. Galloway, P. Huxley, D. Laber, M. McCourt, M. Whittaker, L.M. Wood, A. Wright: Preclinical and clinical studies of MMP inhibitors in cancer. Ann N Y Acad Sci 878, 228–235 (1999)
DOI:10.1111/j.1749-6632.1999.tb07688.x

26. R.E. Castoldi, G. Pennella, G.S. Saturno, P. Grossi, M. Brughera, M. Venturi: Assessing and managing toxicities induced by kinase inhibitors. Curr Opin Drug Discov Devel 10(1), 53–57 (2007)

27. V.J. Cadete, J. Sawicka, L.K. Bekar, G. Sawicki: Combined subthreshold dose inhibition of myosin light chain phosphorylation and MMP-2 activity provides cardioprotection from ischaemic/reperfusion injury in isolated rat heart. Br J Pharmacol 170(2), 380–390 (2013)
DOI:10.1111/bph.12289

28. I. Bil-Lula, H.B. Lin, D. Biały, M. Wawrzyńska, L. Diebel, J. Sawicka, M. Woźniak, G. Sawicki: Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP-2/MLCK-mediated cardiomyocyte protection from hypoxic injury. J Cell Mol Med 20(6), 1086-1094 (2016)
DOI:10.1111/jcmm.12827

29. J.M. Brown, C.W. White, L.S. Terada, M.A. Grosso, P.F. Shanley, D.W. Mulvin, A. Banerjee, G.J. Whitman, A.H. Harken, J.E. Repine: Interleukin 1 pretreatment decreases ischemia/reperfusion injury. Proc Natl Acad Sci U S A 87(13), 5026–5030 (1990)
DOI:10.1073/pnas.87.13.5026

30. B. Campbell, C.M. Chuhran, D.J. Lefer, A.M. Lefer: Cardioprotective effects of abciximab (ReoPro) in an isolated perfused rat heart model of ischemia and reperfusion. Methods Find Exp Clin Pharmacol 21(8), 529–534 (1999)
DOI:10.1358/mf.1999.21.8.794834

31. R.C. Kukreja, K.E. Loesser, A.A. Kearns, S.A. Naseem, M.L. Hess: Protective effects of histidine during ischemia-reperfusion in isolated perfused rat hearts. Am J Physiol 264(5 Pt 2), H1370–1381 (1993)

32. T. Silambarasan, J. Manivannan, M.K. Priya, N. Suganya, S. Chatterjee, B. Raja: Sinapic acid protects heart against ischemia/reperfusion injury and H9c2 cardiomyoblast cells against oxidative stress. Biochem Biophys Res Commun 456(4), 853–859 (2015)
DOI:10.1016/j.bbrc.2014.12.022

33. L. Wang, X. Cheng, C. Huang, B. Huang, Q. Liang: Rapamycin protects cardiomyocytes against anoxia/reoxygenation injury by inducing autophagy through the PI3k/Akt pathway. J Huazhong Univ Sci Technolog Med Sci 35(1), 10–15 (2015)
DOI:10.1007/s11596-015-1381-x

34. M. Rottenstreich, M. Rottenstreich, S. Shapira: Doxycycline induced oesophageal ulcers in a navy ship crewmember. Int Marit Health 66(3), 181–183 (2015)
DOI:10.5603/IMH.2015.0035

35. I. Morano: Effects of different expression and posttranslational modifications of myosin light chains on contractility of skinned human cardiac fibers. Basic Res Cardiol 87 Suppl 1, 129–141 (1992)
DOI:10.1007/978-3-642-72474-9_11

36. D.H. Goh, A. Ferrante: In vitro inhibition of natural killer cell activity by doxycycline. Int J Immunopharmacol 6(1), 51–54 (1984)
DOI:10.1016/0192-0561(84)90034-1

37. M. Sourdeval, C. Lemaire, C. Brenner, E. Boisvieux-Ulrich, F. Marano: Mechanisms of doxycycline-induced cytotoxicity on human bronchial epithelial cells. Front Biosci J Virtual Libr 11, 3036–3048 (2006)
DOI:10.2741/2031

38. G. Sawicki: Intracellular regulation of matrix metalloproteinase-2 activity: new strategies in treatment and protection of heart subjected to oxidative stress. Scientifica 2013, 130451 (2013)
DOI:10.1155/2013/130451

39. G. Ren, O. Dewald, N.G. Frangogiannis: Inflammatory mechanisms in myocardial infarction. Curr Drug Targets Inflamm Allergy 2(3), 242–256 (2003)
DOI:10.2174/1568010033484098

40. E. Robin, R.D. Guzy, G. Loor, H. Iwase, G.B. Waypa, J.D. Marks, T.L. Hoek, P.T. Schumacker: Oxidant stress during simulated ischemia primes cardiomyocytes for cell death during reperfusion. J Biol Chem 282(26), 19133–19143 (2007)
DOI:10.1074/jbc.M701917200

41. Q. Hu, D. Zhou, X. Li, N. Yang, P. Guo, D. Xu X. Li: Renoprotective effects of propofol on the expression of iNOS protein in rats with ischemia reperfusion injury. Int J Clin Exp Med 8(1), 776–780 (2015)

42. O. Feron, C. Dessy, D.J. Opel, M.A. Arstall, R.A. Kelly, T. Michel: Modulation of the endothelial nitric-oxide synthase-caveolin interaction in cardiac myocytes. Implications for the autonomic regulation of heart rate. J Biol Chem 273(46), 30249–30254 (1998)
DOI:10.1074/jbc.273.46.30249

43. W. Wang, G. Sawicki, R. Schulz: Peroxynitrite-induced myocardial injury is mediated through matrix metalloproteinase-2. Cardiovasc Res 53(1), 165–174 (2002)
DOI:10.1016/S0008-6363(01)00445-X

44. A.L. Jacob-Ferreira, R. Schulz: Activation of intracellular matrix metalloproteinase-2 by reactive oxygen-nitrogen species: Consequences and therapeutic strategies in the heart. Arch Biochem Biophys 540(1-2), 82–93 (2013)
DOI:10.1016/j.abb.2013.09.019

45. H. Lin, V.J. Cadete, J. Sawicka, M. Wozniak, G. Sawicki: Effect of the myosin light chain kinase inhibitor ML-7 on the proteome of hearts subjected to ischemia-reperfusion injury. J Proteomics 75(17), 5386–5395 (2012)
DOI:10.1016/j.jprot.2012.06.016

46. M. Hori, K. Nishida: Oxidative stress and left ventricular remodelling after myocardial infarction. Cardiovasc Res 81(3), 457–464 (2009)
DOI:10.1093/cvr/cvn335

47. M.A. Ali, R. Schulz: Activation of MMP-2 as a key event in oxidative stress injury to the heart. Front Biosci Landmark Ed 14, 699–716 (2009)

48. Q. Chen, M. Jin, F. Yang, J. Zhu, Q. Xiao, L. Zhang: Matrix metalloproteinases: inflammatory regulators of cell behaviors in vascular formation and remodeling. Mediators Inflamm 2013, 928315 (2013)
DOI:10.1155/2013/928315

49. F. Perlikos, K.J. Harrington, K.N. Syrigos: Key molecular mechanisms in lung cancer invasion and metastasis: a comprehensive review. Crit Rev Oncol Hematol 87(1), 1–11 (2013)
DOI:10.1016/j.critrevonc.2012.12.007

50. R.R. Rigor, Q. Shen, C.D. Pivetti, M.H. Wu, S.Y. Yuan: Myosin light chain kinase signaling in endothelial barrier dysfunction. Med Res Rev 33(5), 911–933 (2013)
DOI:10.1002/med.21270

51. G. Ermak, V.J. Cancasci, K.J.A. Davies: Cytotoxic effect of doxycycline and its implications for tet-on gene expression systems. Anal Biochem 318(1), 152–154 (2003)
DOI:10.1016/S0003-2697(03)00166-0

52. D. Wu, A. Cederbaum: Nitric oxide donors prevent while the nitric oxide synthase inhibitor l-NAME increases arachidonic acid plus CYP2E1-dependent toxicity. Toxicol Appl Pharmacol 216(2), 282–292 (2006)
DOI:10.1016/j.taap.2006.05.019

53. M. Tisljar, Z. Grabarević, B. Artuković, P. Dzaja, S. Cenan, T.A. Zelenika, R.G. Cooper, M. Dinarina-Sablić: The impact of L-NAME and L-arginine chronic toxicity induced lesions on ascites--pulmonary hypertension syndrome development in broiler chickens. Coll Antropol 35(2), 547–556 (2011)

54. F. Fazal, L. Gu, I. Ihnatovych, Y. Han, W. Hu, N. Antic, F. Carreira, J.F. Blomquist, T.J. Hope, D.S. Ucker, P. de Lanerolle: Inhibiting myosin light chain kinase induces apoptosis in vitro and in vivo. Mol Cell Biol 25(14), 6259–6266 (2005)
DOI:10.1128/MCB.25.14.6259-6266.2005

55. V.J.J. Cadete, S.A. Arcand, B.M. Chaharyn, A. Doroszko, J. Sawicka, D.D. Mousseau, G. Sawicki: Matrix metalloproteinase-2 is activated during ischemia/reperfusion in a model of myocardial infarction. Can J Cardiol 29(11), 1495–1503 (2013)
DOI:10.1016/j.cjca.2013.03.014

56. A. Doroszko, D. Polewicz, V.J.J Cadete, J. Sawicka, M. Jones, D. Szczesna-Cordary, P.Y. Cheung, G. Sawicki: Neonatal asphyxia induces the nitration of cardiac myosin light chain 2 that is associated with cardiac systolic dysfunction. Shock 34(6), 592–600 (2010)
DOI:10.1097/SHK.0b013e3181e14f1d

57. M.A.J. Moser, S. Arcand, H.B. Lin, C. Wojnarowicz, J. Sawicka, T. Banerjee, Y. Luo, G.R. Beck, P.P. Luke, G. Sawicki: Protection of the transplant kidney from preservation injury by inhibition of matrix metalloproteinases. Plos One 11(6), e0157508 (2016)
DOI:10.1371/journal.pone.0157508

Abbreviations:CF: coronary flow; Doxy: doxycycline; HR: heart rate; H/R: hypoxia/reoxygenation; eNOS: endothelial nitric oxide synthase; iNOS: inducible NOS; I/R: ischemia/reperfusion; L-NAME: non-selective inhibitor of iNOS/eNOS; LVDP: left ventricular developed pressure; MLC- myosin light chain; MLC1: myosin light chain type 1; MLC2: myosin light chain type 2; MLCK: myosin light chain kinase; ML-7: inhibitor of MLCK; MMPs: matrix metalloproteinases; MMP-2: matrix metalloproteinase 2; MYL3: ventricular isoform of MLC1; NO: nitric oxide; NOS: nitric oxide synthase; ONOO-: peroxynitrate; PVDF: polyvinylidene fluoride; ROS: reactive oxygen species; RPP: the difference of systolic and diastolic ventricular pressures; TnI: troponin I;

Key Words: Ischemia, Reperfusion, Isolated rat heart, Doxycycline, ML-7, L-NAME

Send correspondence to: Grzegorz Sawicki, Department of Pharmacology, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5 Canada., Telephone: 306-9666997, Fax: 306-9664298, E-mail: greg.sawicki@usask.ca