ИШЕМИИ ПРЕРЫВИСТОЙ ФЕНОМЕНИШЕМИЧЕСКАЯ БОЛЕЗНЬ СЕРДЦАСЕРДЦА БОЛЕЗНИЦИТОТОКСИНЫ /ВРЕД ВОЗДАЗОТА ОКСИДЫЭНДОТЕЛИЙ /ПОВРЕЖДМИОКАРДА РЕПЕРФУЗИОННЫЕ ПОВРЕЖДЕНИЯРЕПЕРФУЗИОННОЕ ПОВРЕЖДЕНИ

Eugenia, B. Manukhina

H., Fred Downey

Jie, Sun

Myoung-gwi, Ryou

Robert, T. Mallet

Electronic arhive of Vitebsk State Medical University Library  (Репозиторий библиотеки Витебского государственного медицинского университета)

РАЗДЕЛ I. ДИСФУНКЦИЯ ЭНДОТЕЛИЯ КРОВЕНОСНЫХ СОСУДОВ: ОБЩИЕ МЕДИКО-БИОЛОГИЧЕСКИЕ ВОПРОСЫHYPOXIC CONDITIONING SUPPRESSES CYTOTOXICNITRIC OXIDE PRODUCTION BY ENDOTHELIUM UPON REPERFUSION FOLLOWING ACUTE MYOCARDIAL ISCHEMIAH. Fred Downey1, Myoung-gwi Ryou1, Jie Sun1, Eugenia B.Manukhina2, Robert T. Mallet1Integrative Physiology, University o f North Texas Health Science Center at Fort Worth', USA;Regulatory Mechanisms o f Adaptation, Institute of General Pathology and Pathophysiology2, Russian FederationThere is increasing evidence that hypoxic conditioning, in particular intermittent and repetitive exposure to moderate hypoxia, induces adaptive responses that protect the heart from ischemic insults.1 A number of mechanisms have been proposed to account for the cardioprotective re­sponses to hypoxia stress. These mechanisms include 1) activation of A TP-sensitive K+ channels in cellular2,3 and mitochondrial4'6 membranes, 2) production of cardioprotective proteins including glycolytic enzymes7,8 and heat-shock proteins,912 3) release of erythropoietin,13 4) increased pro­duction of NO by enhanced inducible nitric oxide synthase,14"16 5) angio- genesis,17 6) attenuation of apoptosis,18 and enhancement of antioxidative defenses.19’21 Although there is evidence that hypoxia-induced increase in NO is cardioprotective,22,23 there is also evidence that excessive NO might exacerbate myocardial damage.24,25In the first study of hypoxia induced cardioprotection in a large ani­mal, we found remarkable attenuation of myocardial infarct size and absence of lethal arrhythmias in hypoxia-conditioned dogs.26 As shown in Figure 1, dogs that completed a program of intermittent, moderate hypoxia (9.5 -10.0% FI02, 5-10 min/cycle, 5-8 cycles/d with intervening 4 min normoxia for 20 d) had negligible myocardial infarction following 1 hr occlusion and 5 hr reperfusion of the left anterior descending coronary arteiy (LAD). Hearts of untreated con­trol dogs had large infarctions, and the majority of these untreated animals de­veloped ventricular tachycardia or ventricular fibrillation. Collateral blood flow to the ischemic territory did not differ among the groups, so cardioprotection by intermittent hypoxia was not due to collateral angiogenesis.3IS /AAR AAR/LVFigure 1. Intermittent, normobaric hypoxia conditioning (IHC) prevents myocar­dial infarction. Masses o f  myocardial infarct (infarct size; IS), ischemic area at risk (AAR), and left ventricular free wall + septum (LV) were measured after 1 hour occlu­sion o f  the left anterior descending coronary artery and 5 h reperfusion. Values are means ± SEM from 7 acute non-conditioned dogs (open bars), 12 dogs hypoxia- conditioned for 20 consecutive days (IHC; cross-hatched bars), and 10 dogs sham- conditioned with normoxic air for 20 consecutive days (hatched bars). *P <  0.001 vs. both non-hypoxic groups.Whereas increased myocardial NO may be a beneficial result of hy­poxia conditioning, excessive release of NO by endothelium during reper­fusion may be deleterious. Thus, we hypothesized that hypoxia condition­ing suppresses myocardial formation of excessive NO by the endothelial NO synthase isoform (eNOS) upon coronary artery reperfusion. In the ab­sence of hypoxic conditioning, excessive NO would react with superoxide to produce cytotoxic peroxynitrite and lead to myocardial injury.27Materials and MethodsMongrel dogs were conditioned by a 20 d program of intermittent normobaric hypoxia as described above,26 and compared with non- conditioned control dogs. One day after the conditioning program was completed, myocardium was sampled to measure left ventricular NO syn­thase (NOS) activity (colorimetric assay) and eNOS content (Western blot). In other anesthetized dogs, myocardial nitrite release, an index of NO for­mation 28,29 was measured in coronary venous blood during reperfusion fol­lowing 1 h occlusion of the left anterior descending coronary artery (LAD).ResultsFigure 2 shows that hypoxic conditioning lowered left ventricular to­tal NOS activity 45%, from 92 ± 8 to 43 ± 6 mU/g protein (P<0.01).4са »о1200)с£ 100 ■«»Юф0 м ££ 601> 40•РОяй 28 оLCX LAD RVFigure 2. Myocardial nitric oxide synthase (NOS) activity. Total NOS activity was measured in left ventricular myocardium from the left circumflex (LCX) and left anterior descending (LAD) coronary artery perfusion territories, and in right ventricular myocardium (RV) Values are means + SEM from 7 non-conditioned (control) and 6 hypoxia-conditioned (IHC) dogs. *P < 0.05 vs. control.Consistent with these data is the finding that nitrite release into coro­nary venous blood upon reperfusion was sharply reduced in hypoxic condi­tioned dogs.Figure 3 illustrates that peak nitrite release immediately after reperfu­sion was greatly reduced in the hypoxia conditioned dogs,and Figure 4A shows that significantly less total nitrite was released during the first 5 min of reperfusion in these dogs. If coronary flow were reduced during reperfusion in the hypoxia-conditioned dogs, reduced endo­thelial shear stress might lessen NO production and decrease nitrite release. Figure 4B shows that peak flow in the reperfused LAD was similar in con­trol and hypoxia conditioned dogs, suggesting that NO does not play an es­sential role in early reperfusion hyperemia; moreover, lower nitrite release in the hypoxia-conditioned dogs was not due to decreased flow-dependent shear stress. It is interesting that between 5 and 10 min of reperfusion, both flow and nitrite release were greater in the control hearts.Figure 5 shows a Western blot for analysis of eNOS in samples of LAD perfused myocardium from three hypoxia-conditioned and four con­trol dogs. It is apparent that eNOS content was reduced in the hypoxia- conditioned myocardium.5кcf------- t> —  IHCReperfusion (min)Figure 3. Left ventricular myocardial nitrite (NCV) release. NO2' was measured in plasma sampled simultaneously from the aorta and interventricular coronary vein during pre-ischemic baseline (BL) and reperfusion following 60 min LAD occlusion (OCCL). Means ± SEM from 5 experiments per group.Figure 4, Cumulative myocardial N 0 2' release (panel A) and LAD flow (panel B) during the first 5 min o f  LAD reperfusion. Means + SEM from 5 experiments per group. *P < 0.05 vs. control.6Con- IHC ControlCon- IHC ControlFigure 5. Western blot o f eNOS in left ventricular myocardium. Total protein (50 ng) from homogenates o f  left ventricular myocardium was electrophoretically sepa­rated, transferred to nylon membrane, and probed with eNOS-specific antibody. Actin (Coomassie blue-stained gel) served as loading control.DiscussionHypoxia conditioning protects myocardium from ischemic injury. It is likely that multiple mechanisms contribute to this protection. Blocking NO synthesis can exacerbate myocardial ischemic damage,22 so NO is an important cardioprotective agent. However, moderate reductions in NO synthesis are cardioprotective,30’31 suggesting that beyond an optimal con­centration, excessive NO may contribute to ischemic damage. This is es­pecially likely in the setting of coronary artery reperfusion, where endothe­lial release of NO would be accelerated to the extent that cytotoxic perox­ide and other NO derivates might be formed in the presence of elevated su­peroxide.27The present findings demonstrate that hypoxic conditioning of dogs reduces myocardial NOS activity and release of nitrite into coronary ve­nous blood at reperfusion. Since hypoxia is expected to elevate iNOS,14-16 the present finding of reduced total NOS activity could be explained if eNOS were concurrently reduced. Our Western blot results demonstrate that this is the case. Thus, we conclude that abruptly reperfused coronary endothelium releases excessive NO, so cytotoxic peroxynitrite and other degradation products of NO are formed. Hypoxia conditioning attenuates eNOS activity, prevents excessive NO upon reperfusion and, thus, protects myocardium from ischemia-reperfusion injury.7AcknowledgementsThis work was supported by the U.S. National Institutes o f Health (grants HL064785 and HL071684), the Netherlands Organization for Sci­entific Research (NWO grant 047-014-016), and the Russian Foundation fo r Basic Research (grant 03-04-49065). Arthur G. Williams, Jr. provided excellent technical support.References1. Manukhina EB, Downey HF, Mallet RT. Role o f  nitric oxide in cardiovas­cular adaptation to intermittent hypoxia. Exp Biol Med, in press.2. Shigematsu S, Arita M. Anoxia-induced activation o f ATP-sensitive K+ channels in guinea pig ventricular cells and its modulation by glycolysis. Cardiovasc Res 1997; 35:273-2823. Liu H, Chen H, Yang X, Cheng J. ATP sensitive K+ channel may be in­volved in the protective effects o f  preconditioning in isolated guinea pig cardiomyo- cytes. Chin Med J 2001; 114178-1824. Asemu G, Papousek F, 05t&dal B, Kolar F. Adaptation to high altitude hy­poxia protects the rat heart against ischemia-induced arrhythmias. Involvement o f  mi­tochondrial Katp channel. J Mol Cell Cardiol 1999; 31:1821-18315. Neckar J, Szarszoi O, Koten L, Papousek F, Ostadal B, Grover GJ, Kolar F. Effects o f  mitochondrial Кдтр modulators on cardioprotection induced by chronic high altitude hypoxia in rats. Cardiovasc Res 2002; 55:567-5756. Zhu H-F, Dong J-W, Zhu W-Z, Ding H-L, Zhou Z-N. ATP-dependent po­tassium channels involved in the cardiac protection induced by intermittent hypoxia against ischemia/reperfusion injury. Life Sci 2003; 73:1275-12877. Bass A, Ostadal B, Prochazka J, Pelouch V, Samanek M, Stejskalova M. In­termittent high altitude-induced changes in energy metabolism in the rat myocardium and their reversibility. Physiol Bohemoslov 1989; 38:155-1618. Daneshrad Z, Garcia Riera MP, Verdys M, Rossi A. Differential responses to chronic hypoxia and dietary restriction o f  aerobic capacity and enzyme levels in the rat myocardium. Mol Cell Biochem 2000; 210: 159-1669. Deindl E, Kolar F, Neubauer E, Vogel S, Schaper W, Ostadal B. Effect o f  intermittent high altitude hypoxia on gene expression in rat heart and lung. Physiol Res 2003; 52:147-15710. Kacimi R, Chentoufi J, Honbo N, Long CS, Karliner JS. Hypoxia differen­tially regulates stress proteins in cultured myocytes: role o f  the p38 stress-activated kinase signaling cascade, and relation to cytoprotection. Cardiovasc Res 2000; 46:139- 15011. Meerson FZ, Malyshev IYu, Zamotrinsky AV. Differences in adaptive stabilization o f structures in response to stress and hypoxia relate to the accumulation of  hsp70 isofoims. Mol Cell Biochem 1992; 111:87-9512. Zhong N, Zhang Y, Fang QZ, Zhou ZN. Intermittent hypoxia exposure- induced heat-shock protein 70 expression increases resistance o f  rat heart to ischemic injury. 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Myocardial capillary angiogenesis and coronary flow in ischemia tolerance rat by adaptation to in­termittent high altitude hypoxia. Acta Pharmacol Sin 2002; 23:305-31018. Dong J-W, Zhu H-F, Zhu W-Z, Ding H-L, Ma T-M, Zhou Z-N. Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression. Cell Res 2003; 13:385-39119. Irlbeck M, Iwai T, Lemer T, Zimmer H-G. Effects o f  angiotensin II receptor blockade on hypoxia-induced right ventricular hypertrophy in rats. J Mol Cell Cardiol 1997;29:2931-293920. Nakanishi K, Tajima F, Nakamura A, Yagura S, Ookawara T, Yamashita H, Suzuki K, Taniguchi N, Ohno H. Effects o f  hypobaric hypoxia on antioxidant enzymes in rats. J Physiol 1995; 489.3:869-87621. Zhu H-F, Dong J-W, Zhu W-Z, Dmg H-L, Zhou Z-N. ATP-dependent po­tassium channels involved in the cardiac protection induced by intermittent hypoxia against ischemia/reperfusion injury. Life Sci 2003; 73:1275-128722. Xi L, Tekin D, Gursoy E, Salloum F, Levasseur JE, Kukreja RC. Evidence that NOS2 acts as a trigger and mediator o f  late preconditioning induced by acute sys­temic hypoxia. Am J Physiol Heart Circ Physiol 2002; 283:H5-H1223. Baker JE, Holman P, Kalyanaraman B, Pritchard KA Jr. Adaptation of  hearts to chronic hypoxia increases tolerance to subsequent ischemia by increased nitric oxide production. Adv Exp Med Biol 1998;454:203-21724. Zhang Y, Davies LR, Martin SM, Coddington WJ, Miller FJ Jr, Buettner GR, Kerber RE. The nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) in­creases free radical generation and degrades left ventricular function after myocardial ischemia-reperfusion. Resuscitation 2003; 59:345-35225. Ungvari Z, Gupte SA, Recchia FA, Batkai S, Pacher P. Role o f  oxidative- nitrosative stress and downstream pathways in various forms o f  cardiomyopathy and heart failure. Curr Vase Pharmacol 2005; 3:221-22926. Zong P, Setty S, Sun W, Martinez R, Tune JD Ehrenbourg IV, Tkatchouk EN, Mallet RT, Downey HF. Intermittent hypoxic training protects canine myocardium from infarction. Exp Biol Med 2004; 229:806-81227. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol Cell Physiol 1996; 271:C1424-C143728. Lauer T, Preik M, Rassaf T, Strauer BE, Deussen A, Feelisch M, Kelm M. Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activ­ity but lacks intrinsic vasodilator action. Proc Nat Acad Sci USA 2001; 98:12814- 1281929. Kelinbongard P, Dejam A, Lauer T, Rassaf T, Schindler A, Picker O,9Scheeren T, Godecke A, Schrader J, Schulz R, Heusch G, Schaub G, Bryan N, Feelisch M, Kelm M. Plasma nitrite reflects constitutive nitric oxide synthase activity in mam­mals. Free Radic Biol Med 2003; 35:790-79630. Kobara M, Tatsumi T, Takeda M, Мало A, Yamanaka S, Shiraishi J, Keira N, Matoba S, Asayama J, Nakagawa M. The dual effects o f  nitric oxide synthase in­hibitors on ischemia-reperfusion injury in rat hearts. Basic Res Cardiol 2003; 98:319- 32831. Yasmin W, Strynadka KD, Schulz R. Generation o f  peroxynitrite contrib­utes to ischemia-reperfusion injury in isolated rat hearts. Cardiovasc Res 1997; 33:422- 432NO-ЗАВИСИМЫЕ МЕХАНИЗМЫ ФОРМИРОВАНИЯ КИСЛОРОДСВЯЗУЮЩИХ СВОЙСТВ ГЕМОГЛОБИНАЗинчук В.В.У О «Гродненский государственный медицинский университет»,БеларусьВ большинстве исследований, термин "дисфункция эндотелия" используется как синоним нарушений эндотелиального метаболизма Ь-аргинин-NO системы, так как монооксид азота (NO) является сиг­нальным агентом, участвующим в опосредовании его функций и ос­новное его количество, образуемого в организме, осуществляется в эндотелии. Снижение синтеза NO может быть вызвано: отсутствием исходного субстрата - L-аргинина, отсутствием кофакторов, снижен­ной экспрессией фермента и повышением уровня эндогенных инги­биторов фермента (например, асимметрично диметилированных про­изводных аргинина). Кроме того, снижение антиоксидантной способ­ности организма также может вести к зависимой от N 0  дисфункции эндотелия, так как снижение его эффектов может вызываться усилен­ной его инактивацией в результате повышенной выработки кислород­ных радикалов.В последние годы проблема изучения физиологических эф­фектов N 0 приобрела новый аспект, а именно его взаимодействие с различными компонентами крови, и в частности, с гемоглобином. Предложена теория сосудистого «сохранения» и транспорта N 0  гемо­глобином. В литературе широко обсуждались различные стороны это­го вопроса, что получило отражение в ряде публикаций с весьма ин­тригующими заголовками: «Гемоглобин: транспортер NO, инактива­тор NO или ни то ни другое?» [Hobbs A.J. et al., 2002]; «Реакции азота с гемоглобином: взгляд через шторм SNO» [Gladwin М.Т. et al., 2003];10

Hypoxic conditioning suppresses cytotoxic nitricoxide production by endothelium upon reperfusion following acute myocardial ischemia

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Hypoxic conditioning suppresses cytotoxic nitricoxide production by endothelium upon reperfusion following acute myocardial ischemia

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