Objective:The purpose of the present study was to quantify the effect of chronic hypoxia on endothelial nitric oxide synthase (eNOS) gene and protein expression of fetal coronary artery segments and cardiac tissue of fetal guinea pig hearts.
Methods:Time-mated pregnant guinea pigs (term = 65 days) were housed in room air (NMX, n = 6) or in a hypoxic chamber containing 10.5% O2 for 14 days (HPX14, n = 6). At near term (60 days gestation), fetuses were excised from anesthetized animals via hysterotomy and hearts were removed and weighed. Both coronary artery segments and cardiac ventricle were excised from the same hearts, frozen, and stored at -80C until ready for study. eNOS mRNA was quantified using real-time polymerase chain reaction (PCR) based on SYBR Green I labeling (BioRad Laboratories, Hercules, CA) using eNOS primers obtained from GeneBank normalized to 18S. eNOS proteins were quantified by Western immunoblotting using eNOS antibody (1:200) and normalized to normoxic controls. eNOS cell-specific localization in the fetal guinea pig heart was performed by double immunofluorescence staining.
Results:Both coronary artery endothelial cells (EC) and cardiomyocytes (CM) but not vascular smooth muscle cells of normoxic hearts exhibited positive immunostaining of eNOS protein. Chronic hypoxia significantly (P <.05) increased both eNOS mRNA and proteins level of coronary artery segments (by 210.6% and 51.4%, respectively) but decreased (P <.05) mRNA and protein of cardiac tissue (by 50.0% and 40.6%, respectively) in the same hearts.
Conclusions:Chronic fetal hypoxia, after 14 days, induces sustained changes in eNOS gene and eNOS protein expression that differ between coronary and cardiac tissue in the fetal guinea pig heart. This study suggests that while the functional roles of altered eNOS expression in hypoxic fetal hearts remain unclear, the site at which eNOS expression is altered may be important in the adaptive response of the fetal heart to hypoxia.
1. Zhang L. Prenatal hypoxia and cardiac programming. J Soc Gynecol Invest2005;12:2-13.
2.
2. Giussani DA, Riquelme RA, Moraga FA, et al. Chemorelflex and endocrine components of cardiovascular responses to acute hypoxemia in the llama fetus. Am J Physiol1996;271:R73-R83.
3.
3. Hanson M. Role of chemoreceptors in effects of chronic hypoxia. Comp Biochem Physiol A Mol Integr Physiol1998;119:695-703.
4.
4. Moore LG. Fetal growth restriction and maternal oxygen transport during high altitude pregnancy. High Alt Med Biol2003;4:141-156.
5.
5. Barker DJP. Fetal origins of coronary heart disease. Br Med J1995;311:171-174.
6.
6. Davis L, Thornburg KL, Giraud GD. The effects of anaemia as a programming agent in theh fetal heart. J Physiol2005;565:35-41.
7.
7. Ostadal B, Ostadalova I, Dhalla NS. Development of cardiac sensitivity to oxygen deficiency: Comparative and ontogenetic aspects. Physiol Rev1999;79:635-659.
8.
8. Reller MD, Morton MJ, Giraud GD, Wu DE, Thornburg KL. Maximal myocardial blood flow is enhanced by chronic hypoxemia in late gestation fetal sheep. Am J Physiol1992;263:H1327-H1329.
9.
9. Gilbert RD, Pearce WJ, Longo LD. Fetal cardiac and cerebrovascular acclimatization responses to high altitude long-term hypoxia. High Alt Med Biol2003;4:203-213.
10.
10. Martin C, Yu AY, Jiang BH, et al. Cardiac hypertrophy in chronically anemia fetal shheep: Increased vascularization is associated with increased myocardial expression of vascular endothelial growth factor and hypoxia-inducible factor-1. Am J Obstet Gynecol1998;178:527-534.
15. Reller MD, Burson MA, Lohr J, Morton MJ, Thornburg KL. Nitric oxide is am important determinant of coronary flow at rest and during hypoxemic stress in fetal lambs. Am J Physiol1995;269:H2074-H2081.
16.
16. Wothe D, Hohimer A, Morton M, Thornburg K, Giraud G, Davis L. Increased coronary blood flow signals growth of coronary resistance vessels in near-term ovine fetusews. Am J Physiol Regul Integrative Comp Physiol2002;282:R295-R302.
18. Brown IP, Thompson CI, Belloni FL. Role of nitric oxide in hypoxic coronary vasodilation in isolated perfused guinea pig heart. Am J Physiol1993;264:H821-H882.
19.
19. Bredt DS. Nitric oxide signaling specificity—The heart of the problem. J Cell Sci2003;116:9-15.
20.
20. Balligand JL, Cannon PJ. Nitric oxide synthases and cardiac muscle. Autocrine and paracrine influences. Arterioscler Thromb Vasc Biol1997;17:1846-1858.
21.
21. Piech A, Dessy C, Havaux X, Feron O, Balligand JL. Differential regulation of nitric oxide synthase and their allosteric regulators in heart and vessles of hypertensive rats. Cardiovasc Res2003;57:456-467.
22.
22. Massion PB, Balligand JL. Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): Lessons from genetically modified mice. J Physiol2003;546:63-75.
23.
23. Shi Y, Pritchard KA, Holman P, et al. Chronic myocardial hypoxia increases nitric oxide synthase and decreases caveolin-3. Free Radic Biol Med2000;29:695-693.
24.
24. Simko F, Simko J. The potential role of nitric oxide in the hypertrophic growth of the left ventricle. Physiol Rev2000;49:37-46.
25.
25. Kojda G, Kottenberg K. Regulation of basal myocardial function by NO. Cardiovasc Res1994;41:514-523.
26.
26. Xu XP, Pollock JS, Tanner MA, Myers PR. Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells. Cardiovase Res1995;30:841-847.
27.
27. Chen J-X, Meyrick B. Hypoxia increases Hsp90 binding to eNOS via PI3K-Akt in porcine coronary artery endothelium. Lab Invest2004;84:182-190.
28.
28. Fitzpatrick CM, Shi Y, Hutchins WC, et al. Cardioprotection in chronically hypoxic rabbits persists upon exposure to normoxia: Role of nitric oxide synthase and KATP channels. Am J Physiol Heart Circ Physiol2005;288:H62-H68.
29.
29. Shi Y, Baker JE, Zhang C. Tweddell JS, Su J, Pritchhard KA. Chronic hypoxia increases endothelial nitric oxide synthase generation of nitric oxide by increasing heat shock protein 90 association and serine phosphorylation. Circ Res2002;91:300-306.
30.
30. Felaco M, Grilli A, Gorbunov N, et al. Endothelial NOS expression and ischemia-reperfusion in isolated working rat heart from hypoxic and hyperoxic conditions. Biochim Biophys Acta2000;1524:203-211.
31.
31. Gess B, Schricker K, Pfeifer M, Kurtz A. Acute hypoxia upregulates NOS gene expression in rats. Am J Physiol1997;273:R905-R910.
32.
32. Phelan MW, Faller DV. Hypoxia decreases constitutive nitric oxide synthase transcript and protein in cultural endothelial cells. J Cell Physiol1996;167:469-476.
33.
33. McQuillan LP, Leung GK, Marsden PA, Kostyk SK, Kourembanas S. Hypoxia inhibits expression of eNOS via transcriptional and postranscriptional mechanisms. Am J Physiol1994;267:H1921-H1927.
34.
34. Toporsian M, Govindaraju K, Nagi M, Eidelman D, Thibault G, Ward ME. Downregulation of endothelial nitric oxide synthase in rat aorta after prolonged hypoxia in vivo. Circ Res2000;86:671-675.
36. Thompson LP, Dong Y. Chronic hypoxia decreases endothelial nitric oxide synthase protein expression in fetal guinea pig hearts. J Soc Gynecol Investig2005;12:388-395.
37.
37. Nau PN, Van Natta T, Ralphe JC, et al. Metabolic adaptation of the fetal and postnatal ovine heart: Regulatory role of hypoxia-inducible factors and nuclear respiratory factor-1. Pediatr Res2002;52:269-278.
38.
38. Van Natta TL, Ralphe C, Mascio CE, Bedell KA, Scholz TD, Segar JL. Ontogeny of vascular growth factors in perinatal sheep myocardium. J Soc Gynecol Investig2004;11:503-510.
39.
39. Roghair RD, Lamb FS, Bedell KA, Smith OM, Scholz TD, Segar JL. Late-gestation etamethasone enhances coronary artery responsiveness to angiotensin II in fetal sheep. Am J Physiol Regul Integr Comp Physiol2004;286:R80-R88.
40.
40. Bubner B, Baldwin IT. Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep2004;23:263-271.
41.
41. Mayhew TM, Gregson C, Pharaoh A, Fagan DC. Numbers of nuclei in different tissue compartments of fetal ventricular myocardium from 16 to 35 weeks of gestation. Virchows Arch1998;433:167-172.
42.
42. Arnet UA McMillan A, Dinerman JL, Ballermann B, Lowenstein CJ. Regulation of endothelial nitric-oxide synthase during hypoxia. J Biol Chem1996;271:15069-15073.
43.
43. Semenza GL. O2-regulated gene expression: Transcriptional control of cardiorespiratory physiology by HIF-1. J Appl Physiol2004;96:1173-1177.
44.
44. Forstermann U, Boissel J-P, Kleinert H. Expressional control of thhe `constitutive' isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J1998;12:773-790.
45.
45. Zhang R, Min W, Sessa WC. Functional analysis of the human endothelial nitric oxide synthase promoter. J Biol Chem1995;270:15320-15326.
46.
46. Govers R, Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol2001;280:F193-F206.
47.
47. Nishida K, Harrison DG, Navas JP, et al. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest1992;90:2092-2096.
48.
48. Wang XQ, Vaziri ND. Erythropoietin depresses nitric oxide synthase expression by human endothelial cells. Hypertension1999;33:894-899.
49.
49. Prandota J. Possible pathomechanisms of sudden infant death syndrome: Key role of chronic hypoxia, infection/inflammation states, cytokine irregularities and metabolic trauma in genetically predisposed infants. Am J Ther2004;11:517-546.
50.
50. Mari D, DiBerardino F, Cugno M. Chronic heart failure and the immune system. Clin Rev Allergy Immunol2002;23:325-340.
51.
51. Paulus WJ. The role of nitric oxide in the failing heart. Heart Fail Rev2001;6:105-118.
52.
52. Neumann P, Gertzberg N, Johnson A. TNF-alpha induces a decrease in eNOS promoter activity. Am J Physiol Lung Cell Mol Physiol2004;286:L452-L459.
53.
53. Jung F, Palmer LA, Zhou N, Johns RA. Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes. Circ Res2000;86:319-325.
54.
54. Rouet-Benzineb P, Eddahibi S, Raffestin B, et al. Induction of cardiac nitric oxide synthase 2 in rats exposed to chronic hypoxia. J Mol Cell Cardiol1999;31:1697-1708.
55.
55. Williams JM, Hhull AD, Pearce WJ. Maturational modulation of endothelium-dependent vasodilation in ovine cerebral arteries. Am J Physiol Regul Integr Comp Physiol2005;288:R149-R157.
56.
56. White CR, Hamade MW, Siami K, et al. Maturation enhances fluid shear-induced activation of eNOS in perfused ovine carotids. Am J Physiol Heart Circ Physiol2005;289:H2220-H2227.
57.
57. Vaziri ND, Wang XQ. cGMP-mediated negative-feedback regulation of endothelial nitric oxide eynthase expression by nitric oxide. Hypertension1999;34:1237-1241.
58.
58. Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH. Chronic exercise in dogs increase coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res1994;74:349-353.
59.
59. Xiao Z, Zhang Z, Diamond SL. Shear stress induction of the endothelial nitric oxide synthase gene is calcium-dependent but not calcium-activated. J Cell Physiol1997;171:205-211.
60.
60. Boo YC, Jo H. Flow-dependent regulation of endothelial nitric oxide synthase: Role of protein kinases. Am J Physiol Cell Physiol2003;285:C499-C508.
