Cardioprotective role of myocardial endothelial nitric oxide synthase and serum nitric oxide induced by hexarelin in rats with myocardial infarction-induced heart failure




Heart failure
Myocardial infarction
Nitric oxide


How to Cite

Agbo, E., Liu, D. H., Liao, J. W., SAAHENE, R. O., & Barnes, P. (2021). Cardioprotective role of myocardial endothelial nitric oxide synthase and serum nitric oxide induced by hexarelin in rats with myocardial infarction-induced heart failure. Iranian Red Crescent Medical Journal, 23(6). (Original work published June 21, 2021)


Background: Growth hormone-releasing peptides (GHRP) have been reported to possess cardioprotective properties; nonetheless, their mechanisms of action are still not very clear.

Objectives: Some studies have suggested that modulation of endothelial nitric oxide synthase (eNOS) and the upregulation of nitric oxide (NO) are cardioprotective. Therefore, the present study strived to test the hypothesis that a potent GHRP analog (hexarelin) could increase serum nitric oxide level and regulate myocardial eNOS to alleviate the development of heart failure.

Methods: Myocardial infarction-induced heart failure in rats was established by permanent coronary artery ligation. The sham group, control group, and heart failure group all received normal saline (100 µg/kg; SC BID; 30days), while the rats in the hexarelin treatment group were treated with hexarelin (100 µg/kg, SC BID, 30 days). The rats were tested for myocardial apoptosis, oxidative stress, left ventricular function, various molecular analyses, as well as pathological and structural myocardial changes.

Results: Hexarelin treatment improved contractile function and attenuated myocardial histopathological damages, oxidative stress, fibrosis, as well as apoptosis. All these were accompanied by the upregulation of myocardial eNOS and an increase in serum NO concentration.

Conclusion: As evidenced by the obtained results, the anti-cardiac failure capacity of hexarelinin in a rat model is mediated by an increase in serum nitric oxide level and the up-modulation of myocardial eNOS; therefore, they can be considered therapeutic targets against heart failure.


  1. Ghigo E, Arvat E, Muccioli G, Camanni F. Growth hormone-releasing peptides. Eur J Endocrinol. 1997;136(5):445-60. doi: 10.1530/eje.0.1360445. [PubMed: 9186261].
  2. Mao Y, Tokudome T, Kishimoto I, Otani K, Hosoda H, Nagai C, et al. Hexarelin treatment in male ghrelin knockout mice after myocardial infarction. Endocrinology. 2013;154(10):3847-54. doi: 10.1210/en.2013-1291. [PubMed: 23861368].
  3. Pang J, Xu Q, Xu X, Yin H, Xu R, Guo S, et al. Hexarelin suppresses high lipid diet and vitamin D3-induced atherosclerosis in the rat. Peptides. 2010;31(4):630-8. doi: 10.1016/j.peptides.2009.11.007. [PubMed: 19931584].
  4. Xu X, Pang J, Yin H, Li M, Hao W, Chen C, et al. Hexarelin suppresses cardiac fibroblast proliferation and collagen synthesis in rat. Am J Physiol Heart Circ Physiol. 2007;293(5):H2952-8. doi: 10.1152/ajpheart.00004.2007. [PubMed: 17766487].
  5. Agbo E, Liu D, Li M, Saahene RO, Chen L, Zhao L, et al. Modulation of PTEN by hexarelin attenuates coronary artery ligation-induced heart failure in rats. Turk J Med Sci. 2019;49(3):945-58. doi: 10.3906/sag-1812-49. [PubMed: 31091855].
  6. Filigheddu N, Fubini A, Baldanzi G, Cutrupi S, Ghè C, Catapano F, et al. Hexarelin protects H9c2 cardiomyocytes from doxorubicin-induced cell death. Endocrine. 2001;14(1):113-9. doi: 10.1385/ENDO:14:1:113. [PubMed: 11322493].
  7. Agbo E, Li M, Wang Y, Saahene R, Massaro J, Tian G. Hexarelin protects cardiac H9C2 cells from angiotensin II-induced hypertrophy via the regulation of autophagy. Pharmazie. 2019;74(8):485-91. doi: 10.1691/ph.2019.9324. [PubMed: 31526442].
  8. Broglio F, Guarracino F, Benso A, Gottero C, Prodam F, Granata R, et al. Effects of acute hexarelin administration on cardiac performance in patients with coronary artery disease during by-pass surgery. Eur J Pharmacol. 2002;448(2):193-200. doi: 10.1016/s0014-2999(02)01934-9. [PubMed: 12144941].
  9. Sun Q, Ma Y, Zhang L, Zhao YF, Zang WJ, Chen C. Effects of GH secretagogues on contractility and Ca2+ homeostasis of isolated adult rat ventricular myocytes. Endocrinology. 2010;151(9):4446-54. doi: 10.1210/en.2009-1432. [PubMed: 20610573].
  10. Tivesten As, Bollano E, Caidahl K, Kujacic V, Sun XY, Hedner T, et al. The growth hormone secretagogue hexarelin improves cardiac function in rats after experimental myocardial infarction. Endocrinology. 2000;141(1):60-6. doi: 10.1210/endo.141.1.7249. [PubMed: 10614623].
  11. Cahill TJ, Kharbanda RK. Heart failure after myocardial infarction in the era of primary percutaneous coronary intervention: mechanisms, incidence and identification of patients at risk. World J Cardiol. 2017;9(5):407-15. doi: 10.4330/wjc.v9.i5.407. [PubMed: 28603587].
  12. Wiemer G, Itter G, Malinski T, Linz W. Decreased nitric oxide availability in normotensive and hypertensive rats with failing hearts after myocardial infarction. Hypertension. 2001;38(6):1367-71. doi: 10.1161/hy1101.096115. [PubMed: 11751719].
  13. Drexler H. Endothelium as a therapeutic target in heart failure. Circulation. 1998;98(24):2652-5. doi: 10.1161/01.cir.98.24.2652. [PubMed: 9851948].
  14. Cabrales P, Tsai AG, Frangos JA, Intaglietta M. Role of endothelial nitric oxide in microvascular oxygen delivery and consumption. Free Radic Biol Med. 2005;39(9):1229-37. doi: 10.1016/j.freeradbiomed.2005.06.019. [PubMed: 16214038].
  15. Trochu JN, Bouhour JB, Kaley G, Hintze TH. Role of endothelium-derived nitric oxide in the regulation of cardiac oxygen metabolism: implications in health and disease. Circ Res. 2000;87(12):1108-17. doi: 10.1161/01.res.87.12.1108. [PubMed: 11110767].
  16. Duda DG, Fukumura D, Jain RK. Role of eNOS in neovascularization: NO for endothelial progenitor cells. Trends Mol Med. 2004;10(4):143-5. doi: 10.1016/j.molmed.2004.02.001. [PubMed: 15162796].
  17. Scherrer-Crosbie M, Ullrich R, Bloch KD, Nakajima H, Nasseri B, Aretz HT, et al. Endothelial nitric oxide synthase limits left ventricular remodeling after myocardial infarction in mice. Circulation. 2001;104(11):1286-91. doi: 10.1161/hc3601.094298. [PubMed: 11551881].  
  18. Lygate C. Surgical models of hypertrophy and heart failure: Myocardial infarction and transverse aortic constriction. Drug Discov Today Dis Models. 2006;3(3):283-90. doi: 10.1016/j.ddmod.2006.10.002.
  19. Xu X, Ding F, Pang J, Gao X, Xu RK, Hao W, et al. Chronic administration of hexarelin attenuates cardiac fibrosis in the spontaneously hypertensive rat. Am J Physiol Heart Circ Physiol. 2012;303(6):H703-11. doi: 10.1152/ajpheart.00257.2011. [PubMed: 22842067].
  20. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288(5789):373-6. doi: 10.1038/288373a0. [PubMed: 6253831].
  21. Kubes P, Suzuki M, Granger D. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A. 1991;88(11):4651-5. doi: 10.1073/pnas.88.11.4651. [PubMed: 1675786].
  22. Kelly RA, Balligand JL, Smith TW. Nitric oxide and cardiac function. Circ Res. 1996;79(3):363-80. doi: 10.1161/01.res.79.3.363. [PubMed: 8781470].
  23. Atochin DN, Huang PL. Endothelial nitric oxide synthase transgenic models of endothelial dysfunction. Pflug Arch Eur J Phy. 2010;460(6):965-74. doi: 10.1007/s00424-010-0867-4. [PubMed: 20697735].
  24. Bauersachs J, Fleming I, Fraccarollo D, Busse R, Ertl G. Prevention of endothelial dysfunction in heart failure by vitamin E: attenuation of vascular superoxide anion formation and increase in soluble guanylyl cyclase expression. Cardiovasc Res. 2001;51(2):344-50. doi: 10.1016/s0008-6363(01)00319-4. [PubMed: 11470474].
  25. Sartório CL, Fraccarollo D, Galuppo P, Leutke M, Ertl G, Stefanon I, et al. Mineralocorticoid receptor blockade improves vasomotor dysfunction and vascular oxidative stress early after myocardial infarction. Hypertension. 2007;50(5):919-25. doi: 10.1161/HYPERTENSIONAHA.107.093450. [PubMed: 17846350].
  26. Treasure CB, Vita JA, Cox DA, Fish RD, Gordon JB, Mudge GH, et al. Endothelium-dependent dilation of the coronary microvasculature is impaired in dilated cardiomyopathy. Circulation. 1990;81(3):772-9. doi: 10.1161/01.cir.81.3.772. [PubMed: 2306829].
  27. Hornig B, Maier V, Drexler H. Physical training improves endothelial function in patients with chronic heart failure. Circulation. 1996;93(2):210-4. doi: 10.1161/01.cir.93.2.210. [PubMed: 8548890].
  28. Mohri M, Egashira K, Tagawa T, Kuga T, Tagawa H, Harasawa Y, et al. Basal release of nitric oxide is decreased in the coronary circulation in patients with heart failure. Hypertension. 1997;30(1):50-6. doi: 10.1161/01.hyp.30.1.50. [PubMed: 9231820].
  29. Qi XL, Stewart DJ, Gosselin H, Azad A, Picard P, Andries L, et al. Improvement of endocardial and vascular endothelial function on myocardial performance by captopril treatment in postinfarct rat hearts. Circulation. 1999;100(12):1338-45. doi: 10.1161/01.cir.100.12.1338. [PubMed: 10491380].
  30. Bauersachs J, Bouloumié A, Fraccarollo D, Hu K, Busse R, Ertl G. Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production. Circulation. 1999;100(3):292-8. doi: 10.1161/01.cir.100.3.292. [PubMed: 10411855].
  31. Shiomi T, Tsutsui H, Matsusaka H, Murakami K, Hayashidani S, Ikeuchi M, et al. Overexpression of glutathione peroxidase prevents left ventricular remodeling and failure after myocardial infarction in mice. Circulation. 2004;109(4):544-9. doi: 10.1161/01.CIR.0000109701.77059.E9. [PubMed: 14744974].
  32. Gao L, Yin H, Smith RS, Chao L, Chao J. Role of kallistatin in prevention of cardiac remodeling after chronic myocardial infarction. Lab Invest. 2008;88(11):1157-66. doi: 10.1038/labinvest.2008.85. [PubMed: 18762777].
  33. Rakhit RD, Mojet MH, Marber MS, Duchen MR. Mitochondria as targets for nitric oxide–induced protection during simulated ischemia and reoxygenation in isolated neonatal cardiomyocytes. Circulation. 2001;103(21):2617-23. doi: 10.1161/01.cir.103.21.2617. [PubMed: 11382733].
  34. Rastaldo R, Cappello S, Folino A, Berta GN, Sprio AE, Losano G, et al. Apelin-13 limits infarct size and improves cardiac postischemic mechanical recovery only if given after ischemia. Am J Physiol Heart Circ Physiol. 2011;300(6):H2308-15. doi: 10.1152/ajpheart.01177.2010. [PubMed: 21378145].
  35. Azizi Y, Faghihi M, Imani A, Roghani M, Nazari A. Post-infarct treatment with [Pyr1]-apelin-13 reduces myocardial damage through reduction of oxidative injury and nitric oxide enhancement in the rat model of myocardial infarction. Peptides. 2013;46:76-82. doi: 10.1016/j.peptides.2013.05.006. [PubMed: 23727032].
  36. Balligand JL, Ungureanu D, Kelly RA, Kobzik L, Pimental D, Michel T, et al. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest. 1993;91(5):2314-9. doi: 10.1172/JCI116461. [PubMed: 8486792].
  37. Brady A, Poole-Wilson PA, Harding SE, Warren JB. Nitric oxide production within cardiac myocytes reduces their contractility in endotoxemia. Am J Physiol. 1992;263(6 Pt 2):H1963-6. doi: 10.1152/ajpheart.1992.263.6.H1963. [PubMed: 1481919].
  38. Kobayashi N, Yoshida K, Nakano S, Ohno T, Honda T, Tsubokou Y, et al. Cardioprotective mechanisms of eplerenone on cardiac performance and remodeling in failing rat hearts. Hypertension. 2006;47(4):671-9. doi: 10.1161/01.HYP.0000203148.42892.7a. [PubMed: 16505212].
  39. Haywood GA, Tsao PS, Von Der Leyen HE, Mann MJ, Keeling PJ, Trindade PT, et al. Expression of inducible nitric oxide synthase in human heart failure. Circulation. 1996;93(6):1087-94. doi: 10.1161/01.cir.93.6.1087. [PubMed: 8653828].
  40. Drexler H, Kästner S, Strobel A, Studer R, Brodde OE, Hasenfuss G. Expression, activity and functional significance of inducible nitric oxide synthase in the failing human heart. J Am Coll Cardiol. 1998;32(4):955-63. doi: 10.1016/s0735-1097(98)00336-2. [PubMed: 9768717].
  41. Vejlstrup NG, Bouloumie A, Boesgaard S, Andersen CB, Nielsen-Kudsk JE, Mortensen SA, et al. Inducible nitric oxide synthase (iNOS) in the human heart: expression and localization in congestive heart failure. J Mol Cell Cardiol. 1998;30(6):1215-23. doi: 10.1006/jmcc.1998.0686. [PubMed: 9689595].
  42. Ikeda U, Murakami Y, Kanbe T, Shimada K. α-Adrenergic stimulation enhances inducible nitric oxide synthase expression in rat cardiac myocytes. J Mol Cell Cardiol. 1996;28(7):1539-45. doi: 10.1006/jmcc.1996.0144. [PubMed: 8841941].
  43. Habib FM, Springall DR, Davies GJ, Oakley CM, Polak J, Yacoub M. Tumour necrosis factor and inducible nitric oxide synthase in dilated cardiomyopathy. Lancet. 1996;347(9009):1151-5. doi: 10.1016/s0140-6736(96)90610-8. [PubMed: 8609750].
  44. Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med. 1990;323(4):236-41. doi: 10.1056/NEJM199007263230405. [PubMed: 2195340].
  45. Boersma E, Mercado N, Poldermans D, Gardien M, Vos J, Simoons ML. Acute myocardial infarction. Lancet. 2003;361(9360):847-58. doi: 10.1016/S0140-6736(03)12712-2. [PubMed: 12642064].
  46. Jugdutt BI. Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough? Circulation. 2003;108(11):1395-403. doi: 10.1161/01.CIR.0000085658.98621.49. [PubMed: 12975244].
  47. Kumar D, Jugdutt BI. Apoptosis and oxidants in the heart. J Lab Clin Med. 2003;142(5):288-97. doi: 10.1016/S0022-2143(03)00148-3. [PubMed: 14647032].
  48. Melo LG, Pachori AS, Kong D, Gnecchi M, Wang K, Pratt RE, et al. Molecular and cell-based therapies for protection, rescue, and repair of ischemic myocardium: reasons for cautious optimism. Circulation. 2004;109(20):2386-93. doi: 10.1161/01.CIR.0000128597.37025.00. [PubMed: 15159329].
  49. Feng Q, Lu X, Jones DL, Shen J, Arnold JM. Increased inducible nitric oxide synthase expression contributes to myocardial dysfunction and higher mortality after myocardial infarction in mice. Circulation. 2001;104(6):700-4. doi: 10.1161/hc3201.092284. [PubMed: 11489778].
  50. Jones SP, Greer JJ, Van-Haperen R, Duncker DJ, de Crom R, Lefer DJ. Endothelial nitric oxide synthase overexpression attenuates congestive heart failure in mice. Proc Natl Acad Sci U S A. 2003;100(8):4891-6. doi: 10.1073/pnas.0837428100. [PubMed: 1267698].
  51. Kupatt C, Hinkel R, Vachenauer R, Horstkotte J, Raake P, Sandner T, et al. VEGF165 transfection decreases postischemic NF‐κB‐dependent myocardial reperfusion injury in vivo: role of eNOS phosphorylation. FASEB J. 2003;17(6):705-7. doi: 10.1096/fj.02-0673fje. [PubMed: 12586740].
  52. Gyurko R, Kuhlencordt P, Fishman MC, Huang PL. Modulation of mouse cardiac function in vivo by eNOS and ANP. Am J Physiol Heart Circ Physiol. 2000;278(3):H971-81. doi: 10.1152/ajpheart.2000.278.3.H971. [PubMed: 10710367].
  53. Hoshida S, Yamashita N, Igarashi J, Nishida M, Hori M, Kamada T, et al. Nitric oxide synthase protects the heart against ischemia-reperfusion injury in rabbits. J Pharmacol Exp Ther. 1995;274(1):413-8. [PubMed: 7542338].
  54. Gurjar MV, Sharma RV, Bhalla RC. eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity. Arterioscler Thromb Vasc Biol. 1999;19(12):2871-7. doi: 10.1161/01.atv.19.12.2871. [PubMed: 10591663].
  55. Wang D, Yu X, Brecher P. Nitric oxide and N-acetylcysteine inhibit the activation of mitogen-activated protein kinases by angiotensin II in rat cardiac fibroblasts. J Biol Chem. 1998;273(49):33027-34. doi: 10.1074/jbc.273.49.33027. [PubMed: 9830056].
  56. Locatelli V, Rossoni G, Schweiger F, Torsello A, De Gennaro Colonna V, Bernareggi M, et al. Growth hormone-independent cardioprotective effects of hexarelin in the rat. Endocrinology. 1999;140(9):4024-31. doi: 10.1210/endo.140.9.6948. [PubMed: 10465272].
  57. Weber KT. Extracellular matrix remodeling in heart failure: a role for de novo angiotensin II generation. Circulation. 1997;96(11):4065-82. doi: 10.1161/01.cir.96.11.4065. [PubMed: 9403633].
  58. Spinale FG, Coker ML, Heung LJ, Bond BR, Gunasinghe HR, Etoh T, et al. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation. 2000;102(16):1944-9. doi: 10.1161/01.cir.102.16.1944. [PubMed: 11034943].
  59. Ruiz-Ortega M, Rodríguez-Vita J, Sanchez-Lopez E, Carvajal G, Egido J. TGF-β signaling in vascular fibrosis. Cardiovasc Res. 2007;74(2):196-206. doi: 10.1016/j.cardiores.2007.02.008. [PubMed: 17376414].
  60. Luo J, Gao X, Peng L, Sun H, Dai G. Effects of hydrochlorothiazide on cardiac remodeling in a rat model of myocardial infarction-induced congestive heart failure. Eur J Pharmacol. 2011;667(1-3):314-21. doi: 10.1016/j.ejphar.2011.06.012. [PubMed: 21703256].
  61. Jneid H, Alam M, Virani SS, Bozkurt B. Redefining myocardial infarction: what is new in the ESC/ACCF/AHA/WHF Third Universal Definition of myocardial infarction? Methodist Debakey Cardiovasc J. 2013;9(3):169-72. doi: 10.14797/mdcj-9-3-169. [PubMed: 24066201].
  62. Soriano FG, Guido MC, Barbeiro HV, Caldini EG, Lorigados CB, Nogueira AC. Endotoxemic myocardial dysfunction: subendocardial collagen deposition related to coronary driving pressure. Shock. 2014;42(5):472-9. doi: 10.1097/SHK.0000000000000232. [PubMed: 25051283].
  63. Kim NN, Villegas S, Summerour SR, Villarreal FJ. Regulation of cardiac fibroblast extracellular matrix production by bradykinin and nitric oxide. J Mol Cell Cardiol. 1999;31(2):457-66. doi: 10.1006/jmcc.1998.0887. [PubMed: 10093057].
  64. Janssens S, Pokreisz P, Schoonjans L, Pellens M, Vermeersch P, Tjwa M, et al. Cardiomyocyte-specific overexpression of nitric oxide synthase 3 improves left ventricular performance and reduces compensatory hypertrophy after myocardial infarction. Circ Res. 2004;94(9):1256-62. doi: 10.1161/01.RES.0000126497.38281.23. [PubMed: 15044322].
  65. Smith RS Jr, Agata J, Xia CF, Chao L, Chao J. Human endothelial nitric oxide synthase gene delivery protects against cardiac remodeling and reduces oxidative stress after myocardial infarction. Life Sci. 2005;76(21):2457-71. doi: 10.1016/j.lfs.2004.11.028. [PubMed: 15763077].
  66. Prabhu SD. Nitric oxide protects against pathological ventricular remodeling: reconsideration of the role of NO in the failing heart. Circ Res. 2004;94(9):1155-7. doi: 10.1161/01.Res.0000129569.07667.89. [PubMed: 15142968].
  67. Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest. 1998;101(11):2567-78. doi: 10.1172/JCI1560. [PubMed: 9616228].
  68. Hu A, Jiao X, Gao E, Koch WJ, Sharifi-Azad S, Grunwald Z, et al. Chronic β-adrenergic receptor stimulation induces cardiac apoptosis and aggravates myocardial ischemia/reperfusion injury by provoking inducible nitric-oxide synthase-mediated nitrative stress. J Pharmacol Exp Ther. 2006;318(2):469-75. doi: 10.1124/jpet.106.102160. [PubMed: 16574780].
  69. Kim YM, Bombeck CA, Billiar TR. Nitric oxide as a bifunctional regulator of apoptosis. Circ Res. 1999;84(3):253-6. doi: 10.1161/01.res.84.3.253. [PubMed: 10024298].
  70. Kim YM, Kim TH, Seol DW, Talanian RV, Billiar TR. Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release. J Biol Chem. 1998;273(47):31437-41. doi: 10.1074/jbc.273.47.31437. [PubMed: 9813055].
  71. Pang JJ, Xu RK, Xu XB, Cao JM, Ni C, Zhu WL, et al. Hexarelin protects rat cardiomyocytes from angiotensin II-induced apoptosis in vitro. Am J Physiol Heart Circ Physiol. 2004;286(3):H1063-9. doi: 10.1152/ajpheart.00648.2003. [PubMed: 14615277].