Effects of different fractions of a red wine non-alcoholic extract on ischemia-reperfusion injury

We have recently demonstrated the cardioprotective effects of a non-alcoholic extract of Argentinian red wine (RWE) on ischemia-reperfusion injury. The aim of the present study was to assess the relative contribution of four phenolic fractions separated from RWE by liquid/liquid extraction with solvents of decreasing hydrophobicity, to the myocardial protection achieved by the original extract. Isovolumic perfused rat hearts treated with each fraction 10 min before ischemia and the first 10 min of reperfusion were submitted to a 20-min global ischemic period followed by 30 min of reperfusion. The treatment with the fraction rich in polymeric proanthocyanidins (fraction IV = aqueous residue) significantly improved the postischemic recovery of left ventricular developed pressure (LVDP) and +dP/dt (max) (111 +/- 5% and 117 +/- 6% vs 61 +/- 4%, 62 +/- 5% , respectively, detected in control hearts) and abolished the increase of left ventricular end diastolic pressure (LVEDP) (8 +/- 2 mmHg vs 42 +/- 4 mmHg in untreated hearts). However, the fraction rich in anthocyanins (III: butanol) elicited a cardioprotective action weaker than the original extract. On the other hand, the representative of either resveratrol or flavan-3-ols and flavonols (fractions I and II) failed to induce this type of response. LDH release and TBARS concentration were significantly lowered after treatment with fraction IV alone. These data show that the fraction rich in polymeric proanthocyanidins exerts a protective effect against myocardial alterations derived from ischemia and reperfusion comparable to the original RWE. This beneficial effect can be correlated to the ability of that fraction to attenuate the degree of lipid peroxidation.     

Myocardial triglyceride turnover during reperfusion of isolated rat hearts subjected to a transient period of global ischemia.

Triglyceride turnover in reperfused/ischemic rat hearts was investigated. Hearts were initially perfused under aerobic conditions for a 1-h "pulse" perfusion with 1.2 mM [1-14C]palmitate to label the endogenous lipid pools, followed by a 30-min period of no-flow ischemia or a 10-min period of retrograde perfusion (control). Hearts were then reperfused under aerobic conditions with buffer containing 1.2 mM [9,10-3H]palmitate. All buffers contained 11 mM glucose and 500 microunits/ml insulin. Rates of endogenous triglyceride lipolysis and synthesis were measured during reperfusion, whereas rates of exogenous palmitate oxidation were measured both prior to ischemia and during reperfusion following ischemia. During reperfusion of ischemic hearts, a 20% increase in exogenous fatty acid oxidation rates was seen compared with pre-ischemic rates. Despite an initial burst of endogenous fatty acid oxidation, no acceleration of steady state endogenous triglyceride lipolysis was seen compared with their nonischemic hearts. In contrast, a significant increase in triglyceride synthesis was observed. Triglyceride turnover was also measured in a series of hearts reperfused following ischemia in the absence of exogenous fatty acids. A significant enhancement of functional recovery was seen compared with hearts reperfused with 1.2 mM palmitate. In addition, a significant increase in fatty acid oxidation from endogenous triglyceride lipolysis was observed. We conclude that the heart quickly recovers its ability to oxidize exogenous fatty acids during reperfusion and that although triglyceride lipolysis is not accelerated during reperfusion of ischemic hearts in the presence of 1.2 mM palmitate, a significant increase in triglyceride synthesis does occur.     

Effects of long-term treatment with eicosapentaenoic acid on the heart subjected to ischemia/reperfusion and hypoxia/reoxygenation in rats

The effects of eicosapentaenoic acid (EPA) and long-term treatment with EPA-ethylester (EPA-E) were examined in perfused rat hearts subjected to ischemia/reperfusion and adult rat cardiomyocytes subjected to hypoxia/reoxygenation. EPA (0.1 M) improved postischmic contractile dysfunction of the ischemic/reperfused heart. EPA (10 M) attenuated hypoxia/reoxygenation-induced morphological deterioration of cardiomyocytes. The results suggest the presence of direct cardioprotective effects of EPA. Rats were orally treated for 4 weeks with 1 g/kg/day of EPA-E to elucidate ex vivo effects of EPA, and the fatty acid composition of cardiac phospholipids was determined. The percent ratio of EPA in total fatty acids of cardiac phospholipids increased whereas that of arachidonic acid decreased. The percent ratio of n-3/n-6 fatty acid did not increase. Treatment with EPA-E did not improve the post-ischemic contractile function, but attenuated the ischemia/reperfusion-induced release of prostaglandins during reperfusion. Treatment with EPA-E preserved a better morphological appearance of the cardiomyocytes subjected to hypoxia/reoxygenation. The results suggest that the mechanisms responsible for cytoprotective effects of hypoxic/reoxygeanted cardiomyocytes or inhibition of metabolic alterations of the ischemic/reperfused heart by long-term EPA-E treatment did not contribute substantially to recovery of post-ischemic contractile dysfunction. The direct in vitro effects of EPA may play a role in the protection of the heart from ischemia/reperfusion or hypoxia/reoxygenation injury.     

Effect of different whole body hyperthermic sessions on the heat shock response in mice liver and brain

The apoptotic death of cardiomyocytes due to ischemia/reperfusion is one of the major complications of heart disease. Ischemia/reperfusion has been shown to lead to the activation of the stress-activated protein (SAP) kinases and the p38/reactivating kinase (p38/RK). In this study, the direct effect of an aqueous Flos carthami (FC) extract on SAP kinases was investigated. When isolated rat hearts were perfused by Langendorff mode with media containing FC extract prior to the induction of global ischemia and the subsequent reperfusion, SAP kinase activity was inhibited 95%. Untreated ischemic/reperfused hearts showed a 57% elevation in the activity of SAP kinase. The in vitro effect of these FC extracts on SAP kinase was also tested. At a concentration of 10 g/ml, the aqueous FC extract resulted in 50% inhibition of SAP kinase activity in ischemic heart tissue. Our results showed that FC affected both the interaction of SAP kinase with c-jun as well as the phosphotransferase reaction. These results clearly demonstrate that extracts from Flos carthami exerted inhibitory effects on SAP kinase. The administration of the FC extract may lead to a modulation of the apoptotic effect of SAP kinase activation induced during ischemia/reperfusion.     

Role of ERK1/2 in the anti-apoptotic and cardioprotective effects of nitric oxide after myocardial ischemia and reperfusion

Objective: Experimental results from cultured cells suggest that there is cross-talk between nitric oxide (NO) and extracellular signal-regulated kinase (ERK) in their anti-apoptotic effect. However, the cross-talk between these two molecules in either direction has not been confirmed in the whole organ or whole animal level. The aim of the present study was to determine whether ERK may play a role in the anti-apoptotic and cardioprotective effects of NO in myocardial ischemia/reperfusion (MI/R). Methods: Isolated perfused mouse hearts were subjected to 20 min of global ischemia and 120 min of reperfusion and treated with vehicle or an NO donor (SNAP, 10 μM) during reperfusion. To determine the role of ERK1/2 in the anti-apoptotic and cardioprotective effects of NO, hearts were pre-treated (10 min before ischemia) with U0126, a selective MEK1/2 inhibitor (1 μM). Results: Treatment with SNAP exerted significant cardioprotective effects as evidenced by reduced cardiac apoptosis (TUNEL and caspase 3 activity, p < 0.01), and improved cardiac functional recovery (p < 0.01). In addition, treatment with SNAP resulted in a 2.5-fold increase in ERK activation when compared with heart receiving vehicle. Pre-treatment with U0126 slightly increased post-ischemic myocardial apoptosis but had no significant effect on cardiac functional recovery in this isolated perfused heart model. However, treatment with U0126 completely blocked SNAP-induced ERK activation and markedly, although not completely, inhibited the cardioprotection exerted by SNAP. Conclusion: These results demonstrate that nitric oxide exerts its anti-apoptotic and cardioprotective effects, at least in part, by activation of ERK in ischemic/reperfused heart. The first two authors contribute equally to this study.     

Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion

Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60, 90 or 120 min of reperfusion. At the end of each experiment, the heart was processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG® in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 90 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were completely abolished by subjecting the myocardium to repeated short-term ischemia and reperfusion which also reduced the ischemic reperfusion injury as evidenced by better recovery of left ventricular performance in the preconditioned myocardium. The results of this study indicate that reperfusion of ischemic heart, but not ischemia, induces apoptotic cell death and DNA fragmentation which can be inhibited by myocardial adaptation to ischemia.     

Effects of L-carnitine and its derivatives on postischemic cardiac function,ventricular fibrillation and necrotic and apoptotic cardiomyocyte death in isolated rat hearts

The study aimed to examine whether L-carnitine and its derivatives, acetyl-L-carnitine and propionyl-L-carnitine, were equally effective and able to improve postischemic cardiac function, reduce the incidence of reperfusion-induced ventricular fibrillation, infarct size, and apoptotic cell death in ischemic/reperfused isolated rat hearts. There are several studies indicating that L-carnitine, a naturally occurring amino acid and an essential cofactor, can improve mechanical function and substrate metabolism not only in hypertrophied or failing myocardium but also in ischemic/reperfused hearts. The effects of L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine, on the recovery of heart function, incidence of reperfusion-induced ventricular fibrillation (VF), infarct size, and apoptotic cell death after 30 min ischemia followed by 120 min reperfusion were studied in isolated working rat hearts. Hearts were perfused with various concentrations of L-carnitine (0.5 and 5 mM), acetyl-L-carnitine (0.5 and 5 mM), and propionyl-L-carnitine (0.05, 0.5, and 5 mM), respectively, for 10 min before the induction of ischemia. Postischemic recovery of CF, AF, and LVDP was significantly improved in all groups perfused with 5 mM of L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine. Significant postischemic ventricular recovery was noticed in the hearts perfused with 0.5 mM of propionyl-L-carnitine, but not with the same concentration of L-carnitine or L-acetyl carnitine. The incidence of reperfusion VF was reduced from its control value of 90 to 10% (p < 0.05) in hearts perfused with 5 mM of propionyl-L-carnitine only. Other doses of various carnitines failed to reduce the incidence of VF. The protection in CF, AF, LVDP, and VF reflected in a reduction in infarct size and apoptotic cell death in hearts treated with various concentrations of carnitine derivatives. The difference between effectiveness of various carnitines on the recovery of postischemic myocardium may be explained by different membrane permeability properties of carnitine and its derivatives.     

O2 delivery and redox state are determinants of compartment-specific reactive O2 species in myocardial reperfusion

Reperfusion of the ischemic myocardium leads to a burst of reactive O(2) species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early O(2) delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 min and then reperfused with either 95% or 20% O(2)-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial O(2) delivery during the first 5 min of reperfusion was 84.7 +/- 4.2 ml O(2)/min with 20% O(2)-saturated buffer and 354.4 +/- 22.8 ml O(2)/min with 95% O(2) (n = 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O(2) compared with 20% O(2). However, the resorufin signal (extracellular ROS) was significantly increased with 20% O(2) compared with 95% O(2) during reperfusion. Perfusion of hearts with 20 mM glucose reduced the (.)NADH during ischemia (P < 0.001) and the (.)ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial O(2) delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O(2) delivery promotes intracellular ROS and low O(2) delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor for reperfusion ROS production.     

The relative contribution of glucose and fatty acids to ATP production in hearts reperfused following ischemia

High levels of fatty acids decrease the extent of mechanical recovery of hearts reperfused following a transient period of severe ischemia. Glucose oxidation rates during reperfusion are low under these conditions, which can result in a decreased recovery of mechanical function. Stimulation of glucose oxidation with the carnitine palmitoyl transferase I inhibitor, Etomoxir, or by directly stimulating pyruvate dehydrogenase activity with dichloroacetate (DCA) results in an improvement in mechanical function during reperfusion of previously ischemic hearts. Addition of DCA (1 mM) to hearts perfused with 11 mM glucose and 1.2 mM palmitate results in an increase in contribution of glucose oxidation to overall ATP production from 6 to 23%, with a parallel decrease in that of fatty acid oxidation from 90 to 69%. In aerobic hearts, endogenous myocardial triglycerides are an important source of fatty acids for -oxidation. Using hearts in which the myocardial triglycerides were pre-labeled, the contribution of both endogenous and exogenous fatty acid oxidation to myocardial ATP production was determined in hearts perfused with 11 mM glucose, 1.2 mM palmitate and 500 µU/ml insulin. In hearts reperfused following a 30 min period of global no flow ischemia, 91.9% of ATP production was derived from endogenous and exogenous fatty acid oxidation, compared to 87.7% in aerobic hearts. This demonstrates that fatty acid oxidation quickly recovers following a transient period of severe ischemia. Furthermore, therapy aimed at overcoming fatty acid inhibition of glucose oxidation during reperfusion of ischemic hearts appears to be beneficial to recovery of mechanical function.     

Postischemic Deactivation of Cardiac Aldose Reductase: ROLE OF GLUTATHIONE S-TRANSFERASE P AND GLUTAREDOXIN IN REGENERATION OF REDUCED THIOLS FROM SULFENIC ACIDS*

Aldose reductase (AR) is a multifunctional enzyme that catalyzes the reduction of glucose and lipid peroxidation-derived aldehydes. During myocardial ischemia, the activity of AR is increased due to the oxidation of its cysteine residues to sulfenic acids. It is not known, however, whether the activated, sulfenic form of the protein (AR-SOH) is converted back to its reduced, unactivated state (AR-SH). We report here that in perfused mouse hearts activation of AR during 15 min of global ischemia is completely reversed by 30 min of reperfusion. During reperfusion, AR-SOH was converted to a mixed disulfide (AR-SSG). Deactivation of AR and the appearance of AR-SSG during reperfusion were delayed in hearts of mice lacking glutathione S-transferase P (GSTP). In vitro, GSTP accelerated glutathiolation and inactivation of AR-SOH. Reduction of AR-SSG to AR-SH was facilitated by glutaredoxin (GRX). Ischemic activation of AR was increased in GRX-null hearts but was attenuated in the hearts of cardiospecific GRX transgenic mice. Incubation of AR-SSG with GRX led to the regeneration of the reduced form of the enzyme. In ischemic cardiospecific AR transgenic hearts, AR was co-immunoprecipitated with GSTP, whereas in reperfused hearts, the association of AR with GRX was increased. These findings suggest that upon reperfusion of the ischemic heart AR-SOH is converted to AR-SSG via GSTP-assisted glutathiolation. AR-SSG is then reduced by GRX to AR-SH. Sequential catalysis by GSTP and GRX may be a general redox switching mechanism that regulates the reduction of protein sulfenic acids to cysteines.     

Beneficial effects of a novel IH636 grape seed proanthocyanidin extract and a niacin-bound chromium in a hamster atherosclerosis model

Atherosclerosis is a disease of the arteries in which fatty plaques develop on the inner arterial wall, which eventually obstructs blood flow. Identified risk factors for atherosclerosis include genetics, diet, lifestyle, smoking, circulating lipid and cholesterol levels, and molecular and circulating signals of chronic vascular inflammation. The link between flavonoids and atherosclerosis is based partly on the evidence that some flavonoids possess antioxidant properties and have been shown to be potent inhibitors of LDL oxidation in vitro. Hypercholesterolemia, a significant cardiovascular risk factor is prevalent in the American population. Grape seed proanthocyanidin extracts are known to exhibit a broad spectrum of chemopreventive and cardioprotective properties against oxidative stress. A recent study has shown that a combination of IH636 grape seed proanthocyanidin extract (GSPE) and a niacin-bound chromium (NBC) can decrease total cholesterol, LDL and oxidized LDL levels in hypercholesterolemic human subjects. In this study, we assessed the efficacy of GSPE supplementation in hamsters, singly and in combination with NBC, since these animals have a similar lipid profile to hypercholesterolemic humans when fed a hypercholesterolemic diet of 0.2% cholesterol and 10% coconut oil (HCD). After 10 weeks of feeding HCD, these animals developed foam cells, which is a biomarker of early stages of atherosclerosis. Atherosclerosis (% of aorta covered with foam cells) was reduced by approximately 50% and 63% following supplementation of these animals with 50 mg/kg and 100 mg/kg of GSPE, respectively, in conjunction with a HCD, while approximately 32% reduction was observed following supplementation of GSPE plus NBC. A minimum of 7–9 animals were used in each study group. GSPE alone and in combination with NBC exerted a pronounced effect on the cholesterol, and triglyceride levels, as well as oxidative lipid damage as demonstrated by the formation of thiobarbituric acid reactive substances (TBARS). This data demonstrates that GSPE and NBC may provide significant health benefits by dramatically ameliorating the incidence of atherosclerosis as demonstrated by reducing the formation of foam cells.     

Effect of ischemia and reperfusion on antioxidant enzymes and mitochondrial inner membrane proteins in perfused rat heart

Experiments were performed to investigate the effects of 60 min severe global ischemia followed by 30 min reperfusion on the antioxidant enzymatic system in the isolated perfused rat heart. Ischemia induced a significant increase of cytoplasmic and mitochondrial selenium-dependent glutathione peroxidase (EC 1.11.1.9) activity. In reperfused hearts, only the mitochondrial form showed a further significant increase. Glutathione reductase (EC 1.6.4.2) was increased in ischemic hearts, whilst the reperfused hearts showed a decrease towards the level found in aerobic hearts. Mitochondrial superoxide dismutase (EC 1.15.1.1) activity was depressed in ischemic as well as in reperfused hearts, though the cytoplasmic form was unmodified. Catalase (EC 1.11.1.6), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glutathione transferase (EC 2.5.1.18) activities were unchanged throughout the experiment. Ischemia and reperfusion induced a significant fall in tissue-reduced glutathione content concomitant with an increase of its oxidized form. We have also studied the mitochondrial inner membrane proteins for both molecular weight, with Coomassie blue, and thiol status, with monobromobimane stain, using a sodium dodecyl sulfate polyacrylamide gel electrophoresis technique. Neither ischemia nor reperfusion effected any relevant modification of the molecular weight of the mitochondrial inner-membrane proteins either in the presence or absence of a reducing agent. However, two of these proteins with an apparent molecular weight of 52 0000 and 12 000 showed a decrease in the monobromobimane stain, probably due to the oxidation of their thiol groups.     

Effect of ischemia and reperfusion of the myocardium on in vitro beta-oxidation of fatty acids

The in vivo oxidation of perfused [14C]-labeled fatty acids has been shown to decrease dramatically in hypoxic hearts. This study addresses the influence of ischemia and reperfusion on the enzymic activities of beta-oxidation of fatty acids in mitochondria and of peroxisomal origin. The rate of beta-oxidation of fatty acids in the isolated mitochondria from myocardium of swine fed control diet declined about 20% by the ischemic insult induced by hypothermic cardioplegic arrest. Upon reperfusion, the rate of mitochondrial beta-oxidation returned to a normal level. In clofibrate-fed animals, the rate of mitochondrial beta-oxidation did not vary significantly between control, ischemic, and perfused tissues. Furthermore, neither in control nor in clofibrate-fed animals did the rates of peroxisomal beta-oxidation of fatty acids vary significantly in the ischemic or reperfused tissues as compared to that of preischemic controls. These results suggest that ischemia does not contribute to any loss of enzymic activity in beta-oxidation of fatty acid cycles either in mitochondria or peroxisomes. Furthermore, the feeding of 0.5% (w/w) clofibrate to pigs increased the rate of mitochondrial beta-oxidation of fatty acids only by 50% while that of peroxisomes increased threefold. A similar threefold increase in catalase activity was also produced by clofibrate feeding. These results suggest that the heart plays a role in the hypolipidemic action of clofibrate.     

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.     

Dendroaspis natriuretic peptide protects the post-ischemic myocardial injury

The present study used isolated rat hearts to investigate whether (1) Dendroaspis natriuretic peptide (DNP) is protective against post-ischemic myocardial dysfunction, and (2) whether the cardioprotective effects of DNP is related to alteration of Bcl-2 family protein levels. The excised hearts of Sprague-Dawley rats were perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. Left ventricular end-diastolic pressure (LVEDP, mmHg), left ventricular developed pressure (LVDP, mmHg) and coronary flow (CF, ml/min) were continuously monitored. In the presence of 50 nM DNP, all hearts were perfused for a total of 100 min consisting of a 20 min pre-ischemic period followed by a 30 min global ischemia and 50 min reperfusion. Lactate dehydrogenase (LDH) activity in the effluent was measured during reperfusion. Treatment with DNP alone improved the pre-ischemic LVEDP and post-ischemic LVEDP significantly comparing with the untreated control hearts during reperfusion. However, DNP did not affect the LVDP, heart rate (HR, beats/min), and CF. Bcl-2, an anti-apoptotic protein expressed in ischemic myocardium of DNP+ischemia/reperfusion (I/R) group, was higher than that in I/R alone group. Bax, a pro-apoptotic protein expressed in ischemic myocardium of DNP+I/R group, has no significant difference compared with I/R alone group. These results suggest that the protective effects of DNP against I/R injury would be mediated, at least in part, through the increased ratio of Bcl-2 to Bax protein after ischemia-reperfusion.     

Urotensin II protects ischemic reperfusion injury of hearts through ROS and antioxidant pathway

Urotensin II (UII) is a vasoactive peptide which is bound to a G protein-coupled receptor. UII and its receptor are upregulated in ischemic and chronic hypoxic myocardium, but the effect of UII on ischemic reperfusion (I/R) injury is still controversial. The aim of the present study was to investigate whether UII protects heart function against I/R injury. Global ischemia was performed using isolated perfused Langendorff hearts of Sprague-Dawley rats. Hearts were perfused with Krebs-Henseleit buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Pretreatment with UII (10nM) for 10min increased recovery percentage of the post-ischemic left ventricular developed pressure and ±dp/dt, and decreased post-ischemic left ventricular end-diastolic pressure as compared with I/R group. UII decreased infarct size and an increased lactate dehydrogenase level during reperfusion. Cardioprotective effects of UII were attenuated by pretreatment with UII receptor antagonist. The hydrogen peroxide activity was increased in UII-treated heart before ischemia. The Mn-SOD, catalase, heme oxygenase-1 and Bcl-2 levels were increased, and the Bax and caspase-9 levels were decreased in UII-treated hearts. These results suggest that UII has cardioprotective effects against I/R injury partly through activating antioxidant enzymes and reactive oxygen species.     

The co-administration of proanthocyanidins and an obesogenic diet prevents the increase in intestinal permeability and metabolic endotoxemia derived to the diet

The consumption of Westernized diets leads to hyperphagia and obesity, as well as intestinal alterations. In the present study, we evaluated the effect of the administration of a grape seed proanthocyanidin extract (GSPE) at different time points on the modulation of intestinal barrier function (intestinal permeability and metabolic endotoxemia), in rats with high-fat/high-carbohydrate diet-induced obesity. Animals were fed a cafeteria diet (CAF) supplemented with a preventive (PRE-CAF) or simultaneously intermittent (SIT-CAF) GSPE treatment (500 mg/kg bw). Changes in the plasma levels of an orally administered marker of intestinal permeability (ovalbumin, OVA), lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) were analyzed after animals were fed the obesogenic diet for 8, 12 and 17 weeks. In addition, ex vivo variations in transepithelial electrical resistance (TEER), the expression of tight junction (TJ) genes and the activity of myeloperoxidase (MPO) in the small and large intestines were monitored at the end of the experiment. The CAF diet increased OVA, LPS, MPO and TNF-α levels, accompanied by decreased TEER values in the small and large intestines. Interestingly, both GSPE treatments prevented these detrimental effects of the CAF diet, being the SIT-CAF group the most effective after 17 weeks of diet intervention. For the first time, this study provides evidence of the ameliorative effect of a proanthocyanidin extract, administered before or together with an obesogenic diet, on barrier dysfunction, as measured by intestinal permeability and metabolic endotoxemia.     

Ischemic preconditioning and superoxide dismutase protect against endothelial dysfunction and endothelium glycocalyx disruption in the postischemic guinea-pig hearts

The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. In conclusion: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.     

Oxygen reperfusion is limited in the postischemic hypertrophic myocardium

Studies have shown that hypertrophied hearts are unusually vulnerable to ischemia. Compromised O2 supply has been postulated as a possible explanation for this phenomenon on the basis of elongated O2 diffusion distance and altered coronary vasculature found in hypertrophied myocardium. To examine the postulate, perfused heart experiments followed the metabolic and functional responses of hypertrophic myocardium to ischemia. 1H/31P NMR was used to measure cellular oxygenation and energy level during ischemia-reperfusion. The left ventricles from spontaneously hypertensive rats (SHR) were enlarged by 48%. With this moderate degree of hypertrophy, cellular O2 and energy levels were normal during baseline perfusion. After an ischemic episode, however, cellular O2 was severely deprived in the SHR hearts compared with the normal hearts. Depressed postischemic O2 reperfusion correlated well with depressed energetic and functional recovery. The results from the current study thus demonstrate a critical relationship between reperfused O2 level and functional recovery in hypertrophic myocardium. The role of reperfused O2, however, is time dependent. During early reperfusion, factor(s) other than O2 appear to limit functional recovery. It is when the mechanical function of the heart approaches a new steady state that O2 becomes a dominant factor. Meanwhile, the finding of a normal O2 level in preischemic SHR hearts defies the notion of preexisting hypoxia as a primer of ischemic damage.     

Myocardial Antioxidant Defense Mechanisms: Time Related Changes After Reperfusion of the Ischemic Rat Heart

It is well known that reperfusion damage of ischemic myocardium may be attributed to alterations in the antioxidant defense system against free radical aggression. In addition, the degree of myocardial damage may depend on the duration and severity of ischemia that precedes reperfusion. We carried out serial ischemic experiments (10, 30, 60 and 120 min) in ex-vivo rat hearts followed by 30 min reperfusion and we assayed the glutathione-dependent enzymatic activities (selenium-dependent glutathione-peroxidase: GSH-Px; selenium-independent glutathione peroxidase: GST-Px; glutathione-transferase: GST and glutathione-reductase: GS-SG-Red), Catalase activity (CAT) and non-proteic thiol compounds (NP-SH) at the end of reperfusion. We found a significant reduction of NP-SH, GSH-Px and CAT in ischemic/ reperfused hearts from 30 min on, while GST activity was increased. In addition, we observed the appearance of a selenium-independent glutathione peroxidase activity (GST-Px) belonging to the GST system. In conclusion, we found the longer the duration of ischemia the greater the inbalance between the myocardial antioxidant system especially the GST activation, suggesting in particular for GST-Px, a role in the control of the damage against oxygen toxicity during ischemia/reperfusion.