The mKATP Channels and protein-kinase C Are Involved in the Cardioprotective Effects of Genistein on Estrogen-Deficient Rat Hearts Exposed to Ischemia/Reperfusion: Energetic Study.

Estrogenic deficiency is considered a risk of coronary disease in women. The phytoestrogen genistein could be a safe preventive strategy. The first aim of this work was to validate a model of cardiac stunning in which natural estrogenic deficiency rats, ie, adult young male (YM) and aged female (AgF), are compared with young female rats (YF). The second aim was to study whether the in vivo administration of genistein prevents the stunning in estrogenic deficiency rats. The third aim was to evaluate whether in our estrogenic deficiency model exists a synergy between genistein and estradiol. The fourth aim was to characterize the underlying mechanisms of genistein. Stunning was induced by ischemia/reperfusion (I/R) in isolated hearts inside a calorimeter. The left ventricular pressure (P) and total heat rate (Ht) were simultaneously measured, while diastolic contracture and muscle economy (P/Ht) were calculated. During R, P/Ht and P recovered less in AgF and YM than in YF rat hearts. Genistein through i.p. (GST-ip) improved P and P/Ht in AgF and YM, but not in YF. In YM, the cardioprotections of GST-ip and estradiol were synergistic. After ischemia, GST-ip increased SR Ca leak causing diastolic contracture. The GST-ip cardioprotection neither was affected by blockade of PI3K-Akt, NO synthases, or phosphatases, but it was sensitive to blockade of protein-kinase C and mKATP channels. Results suggest that (1) estrogenic deficiency worsens cardiac stunning, (2) GST-ip was more cardioprotective in estrogenic deficiency and synergistic with estradiol, and (3) cardioprotection of GST-ip depends on the protein-kinase C and mKATP channel pathway activation.

Milrinone and levosimendan administered after reperfusion improve myocardial stunning in swine.

OBJECTIVES: We assessed the effect of milrinone application timing after reperfusion against myocardial stunning as compared with levosimendan in swine. Furthermore, we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the milrinone-induced cardioprotection. DESIGN: All swine were subjected to 12-minutes ischemia followed by 90-minutes reperfusion to generate stunned myocardium. Milrinone or levosimendan was administered intravenously either for 20 minutes starting just after reperfusion or for 70 minutes starting 20 minutes after reperfusion. In another group, SB203580, a selective p38 MAPK inhibitor, was administered with and without milrinone. Regional myocardial contractility was assessed by percent segment shortening (%SS). RESULTS: Milrinone starting just after reperfusion, but not starting 20 minutes after reperfusion, improved %SS at 30, 60, and 90 minutes after reperfusion compared with that in the control group. SB203580 abolished the beneficial effect of milrinone. On the other hand, levosimendan starting 20 minutes after reperfusion, but not for 20 minutes starting just after reperfusion, improved %SS at 60 and 90 minutes after reperfusion. CONCLUSIONS: Milrinone should be administered just after reperfusion to protect myocardial stunning through p38 MAPK, whereas levosimendan improvement of contractile function could be mainly dependent on its positive inotropic effect.

Cardiomyocyte-specific overexpression of an active form of Rac predisposes the heart to increased myocardial stunning and ischemia-reperfusion injury.

The GTP-binding protein Rac regulates diverse cellular functions including activation of NADPH oxidase, a major source of superoxide production (O(2)(·-)). Rac1-mediated NADPH oxidase activation is increased after myocardial infarction (MI) and heart failure both in animals and humans; however, the impact of increased myocardial Rac on impending ischemia-reperfusion (I/R) is unknown. A novel transgenic mouse model with cardiac-specific overexpression of constitutively active mutant form of Zea maize Rac D (ZmRacD) gene has been reported with increased myocardial Rac-GTPase activity and O(2)(·-) generation. The goal of the present study was to determine signaling pathways related to increased myocardial ZmRacD and to what extent hearts with increased ZmRacD proteins are susceptible to I/R injury. The effect of myocardial I/R was examined in young adult wild-type (WT) and ZmRacD transgenic (TG) mice. In vitro reversible myocardial I/R for postischemic cardiac function and in vivo regional myocardial I/R for MI were performed. Following 20-min global ischemia and 45-min reperfusion, postischemic cardiac contractile function and heart rate were significantly reduced in TG hearts compared with WT hearts. Importantly, acute regional myocardial I/R (30-min ischemia and 24-h reperfusion) caused significantly larger MI in TG mice compared with WT mice. Western blot analysis of cardiac homogenates revealed that increased myocardial ZmRacD gene expression is associated with concomitant increased levels of NADPH oxidase subunit gp91(phox), O(2)(·-), and P(21)-activated kinase. Thus these findings provide direct evidence that increased levels of active myocardial Rac renders the heart susceptible to increased postischemic contractile dysfunction and MI following acute I/R.

Administration of the Rho-kinase inhibitor fasudil before ischemia or just after reperfusion, but not 30 min after reperfusion, protects the stunned myocardium in swine.

OBJECTIVES: We assessed the effect of administration time for fasudil treatment of the stunned myocardium in 40 anesthetized open chest swine. MATERIALS AND METHODS: All swine were subjected to 12 min ischemia followed by reperfusion to generate stunned myocardium. Group A (n = 11) received saline in place of fasudil both before ischemia and after reperfusion. Group B (n = 10) received 30 min intravenous fasudil at a rate of 13 mug/kg/min starting 45 min before ischemia and received saline after reperfusion. Groups C (n = 10) and D (n = 9) received saline before ischemia, and received fasudil at a rate of 13 microg kg(-1) min(-1) starting just before reperfusion in group C and 30 min after reperfusion in group D. In both groups, treatment lasted 30 min. Myocardial contractility was assessed by percent segment shortening (%SS). RESULTS AND DISCUSSION: Three swine in group A, 2 swine in each of groups B and C, and one swine in group D had ventricular fibrillation or tachycardia after reperfusion and were excluded from further analysis. The changes of %SS from baseline at 90 min after reperfusion in groups B and C were 68 +/- 8% and 75 +/- 8%, respectively, which were significantly higher than in group A or D (47 +/- 10% or 43 +/- 8%). CONCLUSION: We conclude that fasudil administered before ischemia or just after reperfusion, but not 30 min after reperfusion, protects the stunned myocardium.

Lack of the effect of superoxide dismutase and catalase on Na+,K+-ATPase activity in stunned rabbit hearts.

Reactive oxygen species (ROS) have been implicated in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. In this study, we examined protective effects of antioxidant enzymes, superoxide dismutase (SOD) and catalase, against ischemia/reperfusion-induced cardiac dysfunction and inhibition of Na+,K+-ATPase activity. Isolated Langendorff-perfused rabbit hearts were subjected to 15 min of global normothermic ischemia followed by 10 min reperfusion. The hearts treated with SOD plus catalase did not show significant recovery of left ventricular (LV) end-diastolic pressure compared with untreated ischemic reperfused hearts. Treatment with antioxidants had no protective effects on developed LV pressure or its maximal positive and negative first derivatives (+/-LVdP/dt). Myocardial stunning was accompanied by significant loss in sarcolemmal Na+,K+-ATPase activity and thiol group content. Inhibition of enzyme activity and oxidation of SH groups were not prevented by antioxidant enzymes. These results suggest that administration of SOD and catalase in perfusate do not protect significantly against cardiac dysfunction in stunned rabbit myocardium.

Endothelial nitric oxide synthase overexpression provides a functionally relevant angiogenic switch in hibernating pig myocardium.

OBJECTIVES: We investigated whether retroinfusion of liposomal endothelial nitric oxide synthase (eNOS) S1177D complementary deoxyribonucleic acid (cDNA) would affect neovascularization and function of the ischemic myocardium. BACKGROUND: Recently, we demonstrated the feasibility of liposomal eNOS cDNA transfection via retroinfusion in a model of acute myocardial ischemia/reperfusion. In the present study, we used this approach to target a phosphomimetic eNOS construct (eNOS S1177D) into chronic ischemic myocardium in a pig model of hibernation. METHODS: Pigs (n = 6/group) were subjected to percutaneous implantation of a reduction stent graft into the left anterior descending artery (LAD), inducing total occlusion within 28 days. At day 28, retroinfusion of saline solution containing liposomal green fluorescent protein or eNOS S1177D cDNA (1.5 mg/animal, 2 x 10 min) was performed. Furthermore, L-nitroarginine-methylester (L-NAME) was applied orally from day 28, where indicated. At day 28 and day 49, fluorescent microspheres were injected into the left atrium for perfusion analysis. Regional functional reserve (at atrial pacing 140/min) was assessed at day 49 by subendocardial segment shortening (SES) (sonomicrometry, percent of ramus circumflexus region). RESULTS: The eNOS S1177D overexpression increased endothelial cell proliferation as well as capillary and collateral growth at day 49. Concomitantly, eNOS S1177D overexpression enhanced regional myocardial perfusion from 62 +/- 4% (control) to 77 +/- 3% of circumflex coronary artery-perfused myocardium, unless L-NAME was co-applied (69 +/- 5%). Similarly, eNOS S1177D cDNA improved functional reserve of the LAD (33 +/- 5% vs. 7 +/- 3% of circumflex coronary artery-perfused myocardium), except for L-NAME coapplication (13 +/- 6%). CONCLUSIONS: Retroinfusion of eNOS S1177D cDNA induces neovascularization via endothelial cell proliferation and collateral growth. The resulting gain of perfusion enables an improved functional reserve of the hibernating myocardium.

[Stunned myocardium after acute ischemic stroke]. / Atontamiento miocárdico luego del accidente cerebrovascular isquémico.

The so-called stunned myocardium, defined as transitory myocardial contractile dysfunction, has been clearly demonstrated in diverse clinical situations. However, stunned myocardium related to ischemic stroke has been poorly identified. We describe two patients with diagnosis of acute ischemic stroke who developed eletrocardiographic changes, cardiac enzyme increasing levels and myocardial dysfunction secondary to abnormal cardiac wall motion. At the same time the patients developed acute lung injury with rapid resolution, perhaps as a consequence of neurocardiogenic components.

Atontamiento miocardico luego del accidente cerebrovascular isquemico / Stunned myocardium after acute ischemic stroke

El atontamiento miocárdico, definido como una disfunción contráctil transitoria del miocardio, ha sido demostrado claramente en distintas situaciones clínicas. Sin embargo, el atontamiento miocárdico asociado a un accidente cerebrovascular isquémico ha sido escasamente notificado. Describiremos dos pacientes con diagnóstico de accidente cerebrovascular isquémico agudo que presentaron cambios electrocardiográficos, elevación enzimática y trastornos en la motilidad cardíaca, compatibles con disfunciónmiocárdica. Simultáneamente desarrollaron injuria pulmonar aguda rápidamente reversible, originada probablemente por un doble componente neuro-cardiogénico.

The so-called stunned myocardium, defined as transitory myocardial contractile dysfunction, has been clearly demonstrated in diverse clinical situations. However, stunned myocardium related to ischemic stroke has been poorly identified. We describetwo patients with diagnosis of acute ischemic stroke who developed elctrocardiographic changes, cardiac enzyme increasing levels and myocardial dysfunction secondary to abnormal cardiac wall motion. At the same time the patients developed acute lung injury with rapid resolution, perhaps as a consequence of neurocardiogenic components.

Atontamiento miocardico luego del accidente cerebrovascular isquemico / Stunned myocardium after acute ischemic stroke

El atontamiento miocárdico, definido como una disfunción contráctil transitoria del miocardio, ha sido demostrado claramente en distintas situaciones clínicas. Sin embargo, el atontamiento miocárdico asociado a un accidente cerebrovascular isquémico ha sido escasamente notificado. Describiremos dos pacientes con diagnóstico de accidente cerebrovascular isquémico agudo que presentaron cambios electrocardiográficos, elevación enzimática y trastornos en la motilidad cardíaca, compatibles con disfunciónmiocárdica. Simultáneamente desarrollaron injuria pulmonar aguda rápidamente reversible, originada probablemente por un doble componente neuro-cardiogénico.(AU)

The so-called stunned myocardium, defined as transitory myocardial contractile dysfunction, has been clearly demonstrated in diverse clinical situations. However, stunned myocardium related to ischemic stroke has been poorly identified. We describetwo patients with diagnosis of acute ischemic stroke who developed elctrocardiographic changes, cardiac enzyme increasing levels and myocardial dysfunction secondary to abnormal cardiac wall motion. At the same time the patients developed acute lung injury with rapid resolution, perhaps as a consequence of neurocardiogenic components.(AU)

Atontamiento miocardico luego del accidente cerebrovascular isquemico / Stunned myocardium after acute ischemic stroke

El atontamiento miocárdico, definido como una disfunción contráctil transitoria del miocardio, ha sido demostrado claramente en distintas situaciones clínicas. Sin embargo, el atontamiento miocárdico asociado a un accidente cerebrovascular isquémico ha sido escasamente notificado. Describiremos dos pacientes con diagnóstico de accidente cerebrovascular isquémico agudo que presentaron cambios electrocardiográficos, elevación enzimática y trastornos en la motilidad cardíaca, compatibles con disfunciónmiocárdica. Simultáneamente desarrollaron injuria pulmonar aguda rápidamente reversible, originada probablemente por un doble componente neuro-cardiogénico.(AU)

The so-called stunned myocardium, defined as transitory myocardial contractile dysfunction, has been clearly demonstrated in diverse clinical situations. However, stunned myocardium related to ischemic stroke has been poorly identified. We describetwo patients with diagnosis of acute ischemic stroke who developed elctrocardiographic changes, cardiac enzyme increasing levels and myocardial dysfunction secondary to abnormal cardiac wall motion. At the same time the patients developed acute lung injury with rapid resolution, perhaps as a consequence of neurocardiogenic components.(AU)

Immunocytochemical evidence for inducible nitric oxide synthase and cyclooxygenase-2 expression with nitrotyrosine formation in human hibernating myocardium.

BACKGROUND: Myocardial hibernation may result from repetitive episodes of transient ischaemia leading to prolonged dysfunction. Inducible nitric oxide synthase (iNOS) expression has been demonstrated in animals following brief, non-lethal ischaemia-reperfusion injury. We therefore, hypothesised that in human hibernating myocardium: 1). iNOS would be present; 2). the reaction of nitric oxide and superoxide would form the strong oxidant peroxynitrite; 3) that this process would be accompanied by the expression of cyclooxygenase-2 (Cox-2) which interacts with NOS and whose products could further affect myocardial function. METHOD AND RESULTS: In sixteen patients with coronary artery disease (CAD), left ventricular biopsies were obtained from chronically dysfunctional segments subtended by a stenotic artery (> 75 %) and shown to be viable by (18)F-fluorodeoxyglucose positron emission tomography. Comparison was made with myocardial biopsies (n = 8) from normally contracting myocardium in patients undergoing coronary surgery, from unused transplant donors and at post-mortem. Regional wall motion score improved in all patients 6 months post-revascularisation (from 2.7 +/- 0.7 to 1.5 +/- 0.5; p < 0.001), confirming hibernation. Immunocytochemistry localized reactivity to iNOS, Cox-2 and nitrotyrosine (a marker of peroxynitrite formation) to cardiomyocytes from hibernating segments. No difference in reactivity to endothelial NOS was seen between hibernating and control cardiomyocytes. CONCLUSION: Cox-2 and iNOS are co-expressed in hibernating myocardium with nitrotyrosine suggesting nitric oxide production and peroxynitrite formation. We propose that this is secondary to ischaemia-reperfusion and that the products of these enzymes may have consequences for myocardial contractile function and survival.

Reversible myocardial dysfunction in critically ill, noncardiac patients: a review.

OBJECTIVE: To review reversible myocardial dysfunction affecting critically ill patients without cardiac pathology. DATA SOURCES: The bibliography for the study was compiled through a search of different databases for the period 1966-2001. References cited in the selected articles also were reviewed. STUDY SELECTION: The selection criteria included all articles published on reversible myocardial dysfunction in critically ill patients. CONCLUSIONS: Reversible myocardial dysfunction may develop in a situation of critical pathology, but the etiology of reversible myocardial dysfunction is not fully understood. This dysfunction may be accompanied by increases in enzyme concentrations and electrocardiographic changes. Reversible myocardial dysfunction probably is underdiagnosed, although its presence is associated with a worsening of the prognosis and with more specific therapeutic options. Further studies are necessary to define its true incidence and clinical implications.

Effect of myocardial stunning on thiol status, myofibrillar ATPase and troponin I proteolysis.

To investigate the mechanism underlying postischemic contractile dysfunction (myocardial stunning) we examined myocardial sulflhydryl group content, myofibrillar Ca2+-dependent Mg2+-ATPase activity and protein profile after global ischemia and reperfusion. The Langerdorff-perfused rabbit hearts were subjected to 15 min normothermic ischemia followed by 10 min reperfusion and myofibrils were isolated from homogenates of left ventricular tissues. Depressed contractile function during reperfusion was accompanied by a decrease in total sulfhydryl group content. However, myofibrillar protein profile was unchanged and Western immunoblotting analysis showed no significant differences in troponin I immunoreactive bands between control and stunned hearts. Likewise, myofibrillar Mg2+-ATPase activity was unaltered after ischemia and reperfusion. We conclude that myocardial stunning is not caused by altered myofibrillar function and protein degradation but may be partly due to the oxidative modification of as yet undefined proteins.

Role of cyclic guanosine monophosphate in late preconditioning in conscious rabbits.

BACKGROUND: Although NO has been shown to serve both as the trigger and the mediator of the late phase of ischemic preconditioning (PC), it is unknown whether NO acts via activation of soluble guanylate cyclase (sGC). The objective of this study was to investigate the role of sGC in late PC in conscious rabbits using the selective sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). METHODS AND RESULTS: A total of 172 conscious rabbits were used. When nonpreconditioned rabbits were subjected to a sequence of 4-minute coronary occlusion/4-minute reperfusion cycles, myocardial cyclic guanosine monophosphate (cGMP) levels increased significantly at the end of the third and sixth occlusions. In rabbits preconditioned 24 hours earlier (on day 1) with six occlusion/reperfusion cycles, myocardial cGMP levels on day 2 were significantly higher than in nonpreconditioned rabbits even before ischemia but did not increase further during a second sequence of 4-minute occlusion/reperfusion cycles. Administration of ODQ before the six occlusion/reperfusion cycles on day 1 did not prevent the development of late PC against either stunning or infarction on day 2. In contrast, administration of ODQ on day 2 completely ablated the late PC effect against both stunning and infarction. CONCLUSIONS: These results indicate that enhanced synthesis of cGMP by sGC is not necessary for ischemia to trigger a late PC effect but is required for the protection to become manifest 24 hours later. This implies that NO participates in late PC via two distinct mechanisms; ie, it triggers late PC on day 1 via a cGMP-independent mechanism and it mediates late PC on day 2 via a cGMP-dependent mechanism.

Xanthine oxidase contributes to preconditioning's preservation of left ventricular developed pressure in isolated rat heart: developed pressure may not be an appropriate end-point for studies of preconditioning.

Studies of preconditioning frequently use the isolated rat heart model in which recovery of post-ischemic function is the end-point. However, function following an episode of ischemia/reperfusion represents a composite of both stunning, which is related to free radical production and is not attenuated by preconditioning, and tissue salvage, the primary effect of preconditioning. Brief ischemia/reperfusion is also known to diminish adenosine release during subsequent ischemia by a mechanism independent of preconditioning's anti-infarct effect. Reduced purine release would diminish generation of free radicals by xanthine oxidase in rat heart and thus produce less stunning. In this paradigm preserved post-ischemic function in rat heart might look similar to salvage by preconditioning, but its mechanism would be quite different and not be relevant to the xanthine oxidase-deficient human heart. This hypothesis was tested in isolated rat hearts. Control or ischemically preconditioned hearts were subjected to 30 min of global ischemia and 60 min of reperfusion, either in the presence or absence of 25 micromol/l allopurinol, an inhibitor of xanthine oxidase. In non-preconditioned hearts allopurinol increased left ventricular developed pressure after 60 min of reperfusion from 26 +/- 5 mmHg in control hearts to 47 +/- 7 mmHg, whereas developed pressure in preconditioned hearts following reperfusion was 59 +/- 5 mmHg and was unaffected by allopurinol. Developed pressure in non-preconditioned hearts treated with allopurinol was midway between that for untreated control and preconditioned hearts suggesting that at least 50% of the recovery of developed pressure in preconditioned hearts may be related to free radical-induced stunning. In xanthine oxidase-deficient rabbit hearts, return of function was not different between non-preconditioned and preconditioned hearts. Therefore, post-ischemic developed pressure in the rat is significantly affected by purine-dependent stunning, and, hence, may be an unreliable marker of tissue salvage and also a poor index of what might be cardioprotective in man.

Hemin, inducer of heme-oxygenase 1, improves functional recovery from myocardial stunning in conscious dogs.

OBJECTIVE: To examine the effects of pretreatment with hemin, an inducer of the potential antioxidative enzyme heme-oxygenase 1 (HO-1) or heat-shock protein 32, on myocardial stunning. DESIGN: Randomized animal study. SETTING: Animal laboratory of a university hospital. PARTICIPANTS: Chronically instrumented mongrel dogs (n = 44). INTERVENTIONS: Dogs underwent chronic instrumentation for measurement of hemodynamics and myocardial wall thickening fraction (WTF). Experiments with 12 dogs were performed on separate days in a crossover fashion: (1) 10 minutes of left anterior descending (LAD) coronary artery occlusion after application of hemin (9 mg/kg/d) for 1 week and (2) 10 minutes of LAD coronary artery occlusion without hemin pretreatment. In control experiments (n = 32), the reversible induction of HO-1, using gel electrophoresis and Western blotting, was determined. MEASUREMENTS AND MAIN RESULTS: WTF was measured as a baseline value before hemin administration and at predetermined time points until complete recovery from stunning. LAD artery occlusion caused a significant reduction in the WTF in the LAD-perfused area with and without hemin, without significant hemodynamic changes. At all time points, after 1 minute of reperfusion, the WTF as percentage of baseline values was significantly higher after hemin pretreatment (p < 0.05). Baseline WTF values were reached after 24 hours with and after >48 hours without hemin pretreatment (p < 0.05). CONCLUSION: Hemin pretreatment attenuates myocardial stunning in conscious dogs.

Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits.

We examined the role of cyclooxygenase-2 (COX-2) in the late phase of ischemic preconditioning (PC). A total of 176 conscious rabbits were used. Ischemic PC (six cycles of 4-min coronary occlusions/4-min reperfusions) resulted in a rapid increase in myocardial COX-2 mRNA levels (+231 +/- 64% at 1 h; RNase protection assay) followed 24 h later by an increase in COX-2 protein expression (+216 +/- 79%; Western blotting) and in the myocardial content of prostaglandin (PG)E(2) and 6-keto-PGF(1alpha) (+250 +/- 85% and +259 +/- 107%, respectively; enzyme immunoassay). Administration of two unrelated COX-2 selective inhibitors (NS-398 and celecoxib) 24 h after ischemic PC abolished the ischemic PC-induced increase in tissue levels of PGE(2) and 6-keto-PGF(1alpha). The same doses of NS-398 and celecoxib, given 24 h after ischemic PC, completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that COX-2 activity is necessary for this phenomenon to occur. Neither NS-398 nor celecoxib lowered PGE(2) or 6-keto-PGF(1alpha) levels in the nonischemic region of preconditioned rabbits, indicating that constitutive COX-1 activity was unaffected. Taken together, these results demonstrate that, in conscious rabbits, up-regulation of COX-2 plays an essential role in the cardioprotection afforded by the late phase of ischemic PC. Therefore, this study identifies COX-2 as a cardioprotective protein. The analysis of arachidonic acid metabolites strongly points to PGE(2) and/or PGI(2) as the likely effectors of COX-2-dependent protection. The recognition that COX-2 mediates the antistunning and antiinfarct effects of late PC impels a reassessment of current views regarding this enzyme, which is generally regarded as detrimental.

Enhanced expression and localization of heme oxygenase-1 during recovery phase of porcine stunned myocardium.

Myocardial adaptation to ischemia involves up-regulated expression of a number of genes implicated in conferring cytoprotection. We have previously shown that myocardial ischemia followed by reperfusion leads to a co-ordinated expression of mRNAs encoding heme oxygenase-1 (HO-1) and ubiquitin in pigs. HO-1 participates in biological reaction leading to the formation of the antioxidant, bilirubin and the putative cellular messenger, carbon monoxide. In the present study, we examined the expression and cellular localization of HO-1 in the heart during myocardial stunning in anesthetized pigs. After thoracotomy, the LAD was occluded for 10 min and reperfused for 30 min (group I, n = 4), again occluded for 10 min and reperfused for 30 min (group II, n = 6), 90 min (group III, n = 4), 210 min (group IV, n = 5) and for 390 min (group V, n = 4). Myocardial tissue specimens were collected in 10% formalin as well as in liquid nitrogen and processed for immunohistochemistry and mRNA expression analysis, respectively. In the distribution territory of the LAD (experimental, E), systolic wall thickening was significantly decreased (39 +/- 6%) as compared to that of the area perfused by left circumflex coronary artery (LCx, control) in group I and remained depressed in all subsequent groups. Northern blot analysis revealed that the expression of a single mRNA species of 1.8 kb encoding HO-1 was significantly induced in E as compared to control in groups II and III with maximum mRNA levels in group II (1.9 +/- 0.4 fold vs. control). Immunoreactive HO-1 was localized in the cytoplasm of cardiomyocytes as well as in the perivascular regions in all groups. Semiquantitative analysis of HO-1 staining showed significantly enhanced levels of HO-1 in perivascular region in E as compared to respective controls derived from groups III and IV. These results suggest that myocardial adaptive response to ischemia involves up-regulation of HO-1 in cells of perivascular region indicating that this enzyme may participate in regulating vascular tone via CO and thereby, contributing in pathophysiologically important defense mechanism(s) in the heart.

Isoform-selective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning.

Although isoform-selective translocation of protein kinase C (PKC) epsilon appears to play an important role in the late phase of ischemic preconditioning (PC), the mechanism(s) responsible for such translocation remains unclear. Furthermore, the signaling pathway that leads to the development of late PC after exogenous administration of NO in the absence of ischemia (NO donor-induced late PC) is unknown. In the present study we tested the hypothesis that NO activates PKC and that this is the mechanism for the development of both ischemia-induced and NO donor-induced late PC. A total of 95 chronically instrumented, conscious rabbits were used. In rabbits subjected to ischemic PC (six 4-minute occlusion/4-minute reperfusion cycles), administration of the NO synthase inhibitor Nomega-nitro-L-arginine (group III), at doses previously shown to block the development of late PC, completely blocked the ischemic PC-induced translocation of PKCepsilon but not of PKCeta, indicating that increased formation of NO is an essential mechanism whereby brief ischemia activates the epsilon isoform of PKC. Conversely, a translocation of PKCepsilon and -eta quantitatively similar to that induced by ischemic PC could be reproduced pharmacologically with the administration of 2 structurally unrelated NO donors, diethylenetriamine/NO (DETA/NO) and S-nitroso-N-acetylpenicillamine (SNAP), at doses previously shown to elicit a late PC effect. The particulate fraction of PKCepsilon increased from 35+/-2% of total in the control group (group I) to 60+/-1% after ischemic PC (group II) (P<0.05), to 54+/-2% after SNAP (group IV) (P<0.05) and to 52+/-2% after DETA/NO (group V) (P<0.05). The particulate fraction of PKCeta rose from 66+/-5% in the control group to 86+/-3% after ischemic PC (P<0.05), to 88+/-2% after SNAP (P<0.05) and to 85+/-1% after DETA/NO (P<0.05). Neither ischemic PC nor NO donors had any appreciable effect on the subcellular distribution of PKCalpha, -beta1, -beta2, -gamma, -delta, - micro, or -iota/lambda; on total PKC activity; or on the subcellular distribution of total PKC activity. Thus, the effects of SNAP and DETA/NO on PKC closely resembled those of ischemic PC. The DETA/NO-induced translocation of PKCepsilon (but not that of PKCeta) was completely prevented by the administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg (group VI) (particulate fraction of PKCepsilon, 38+/-4% of total, P<0.05 versus group V; particulate fraction of PKCeta, 79+/-2% of total). The same dose of chelerythrine completely prevented the DETA/NO-induced late PC effect against both myocardial stunning (groups VII through X) and myocardial infarction (groups XI through XV), indicating that NO donors induce late PC by activating PKC and that among the 10 isozymes of PKC expressed in the rabbit heart, the epsilon isotype is specifically involved in the development of this form of pharmacological PC. In all groups examined (groups I through VI), the changes in the subcellular distribution of PKCepsilon protein were associated with parallel changes in PKCepsilon isoform-selective activity, whereas total PKC activity was not significantly altered. Taken together, the results provide direct evidence that isoform-selective activation of PKCepsilon is a critical step in the signaling pathway whereby NO initiates the development of a late PC effect both after an ischemic stimulus (endogenous NO) and after treatment with NO-releasing agents (exogenous NO). To our knowledge, this is also the first report that NO can activate PKC in the heart. The finding that NO can promote isoform-specific activation of PKC identifies a new biological function of this radical and a new mechanism in the signaling cascade of ischemic PC and may also have important implications for other pathophysiological conditions in which NO is involved and for nitrate therapy.

[Exogenous adenosine and postischemic dysfunction in the isolated rabbit heart]. / Adenosina exógena y disfuncion postisquémica en el corazon aislado de conejo.

It is recognized that adenosine lessens the systolic alterations of the postischemic ventricular dysfunction ("stunned myocardium"), but little is known about the drug's effects on the diastolic phase of the cardiac cycle. The aim of this work was to determine the effect of adenosine when it was administered: a) before ischemia and during reperfusion, and b) from the early reperfusion period to the end of the experiment on the systolic and diastolic function of the "stunned myocardium". An additional objective was to determine whether adenosine modifies the release of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH), in the "stunned myocardium". Rabbit isolated isovolumic hearts were perfused according to Langendorff's technique, and subjected to 15 minutes global ischemia and 30 minutes reperfusion. A small latex balloon was inserted into the left ventricle via the left atrium which allowed to measure the ventricular end-diastolic pressure (diastolic stiffness) and calculate the developed pressure, the maximal rate of pressure generation and maximal rate of pressure decay (+dP/dtmax and -dP/dtmax), the ratio between these two variables (+P/-P), and the time constant of isovolumic relaxation (tau, Tau). The adenosine administered both before the ischemia period, and at the beginning of reperfusion, attenuated the systolic and diastolic stiffness alterations without modifying the isovolumic relaxation. The administration of adenosine did not diminish the CPK and LDH release significantly when it was given before the ischemia period or the beginning of reperfusion.