Gerald A. Cordis

The red wine antioxidant resveratrol protects isolated rat hearts from ischemia reperfusion injury.

The consumption of red wine has been reported to impart a greater benefit in the prevention of coronary heart disease than the consumption of other alcoholic beverages. This beneficial effect is increasingly being attributed to certain antioxidants comprising the polyphenol fraction of red wine such as transresveratrol. In the present study, we investigated the potential cardioprotective effects of resveratrol in the face of ischemia reperfusion (I/R) injury. Isolated perfused working rat hearts after stabilization were perfused with Krebs-Henseleit Bicarbonate buffer (KHB) either in the presence or absence of transresveratrol (RVT) at a concentration of 10 microM for 15 min prior to subjecting them to 30 min of global ischemia followed by 2 h of reperfusion. Left ventricular functions were monitored at various timepoints throughout the reperfusion period to assess the extent of postischemic recovery in comparison with baseline values. Coronary perfusate samples were also collected to determine malonaldehyde (MDA) levels. The results demonstrated that RVT exhibited significant myocardial protection. This was evidenced by improved recovery of post-ischemic ventricular function including developed pressure and aortic flow as compared to the control group (KHB). Values for developed pressure in the RVT-treated group were significantly higher than those in the control group throughout the reperfusion period (71.09+/-4.88 mm Hg vs. 58.47+/-3.88 mm Hg, 68.87+/-5.07 mm Hg vs. 49.74+/-2.65 mm Hg and 51.67+/-3.95 mm Hg vs. 30.50+/-4.80 mm Hg at reperfusion timepoints R-15, R-60, and R-120, respectively). From R-30 onwards, aortic flow was markedly higher in the RVT treated group as compared with the control group, the differences being most significant at R-90 (32.45+/-2.19 ml/min vs. 19.83+/-1.62 ml/min) and R-120 (27.15+/-2.27 ml/min vs. 14.10+/-1.69 ml/min). In contrast to the KHB treated group, the RVT-treated group displayed significant reduction in MDA formation especially in the immediate early reperfusion period (63.71+/-8.19 pM/ml vs. 130.86+/-4.76 pM/ml, 63.84+/-15.62 pM/ml vs. 156.99+/-18.93 pM/ml, 71.29+/-2.80 pM/ml vs. 129.5+/-10.30 pM/ml and 56.25+/-5.79 pM/ml vs. 127.99+/-3.50 pM/ml at timepoints R-1, R-3, R-5, and R-7, respectively) indicating a reduction in I/R injury related oxidative stress. Infarct size was markedly reduced in the RVT group when compared with the control group (10.57+/-0.35% vs. 36.27+/-5.28%). In vitro studies revealed RVT to be a potent scavenger of peroxyl radicals suggestive of a probable mechanism involved in the protective ability of RVT. The results of this study indicate that resveratrol possesses cardioprotective effects which may be attributed to its peroxyl radical scavenging activity.

Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts

While much is known about the beneficial effects of myocardial stress adaptation, relatively less information is available about the adaptive mechanisms. To explore the signaling pathways of stress adaptation, isolated working rat hearts were divided into three groups. Group I was adapted to stress by conventional technique of repeated ischemia and reperfusion consisting of 5 min of ischemia followed by 10 min of reperfusion, repeated four times. Group II was treated with 100 μM of genistein, a tyrosine kinase inhibitor, followed by preconditioning as described for group I. The third group, perfused with buffer only for 60 min, served as control. All hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. The results of our study demonstrated better postischemic myocardial functions in the preconditioned hearts as evidenced by increased aortic flow, coronary flow, developed pressure and lesser amount of tissue injury as evidenced by the decreased creatine kinase release. The preconditioning effects were associated with enhancement of phospholipase D activity in the heart. The preconditioning effect was almost abolished by the genistein treatment which also prevented the enhancement of phospholipase D activities. Additionally, preconditioning of the rat hearts stimulated protein kinase C, MAP kinase, and MAPKAP kinase 2 activities which were inhibited by genistein. The results identifies for the first time tyrosine kinase‐phospholipase D as potential signaling pathway for ischemic preconditioning, and implicates the involvement of multiple protein kinases in myocardial adaptation to ischemia.

Anti-apoptotic protein survivin plays a significant role in tubular morphogenesis of human coronary arteriolar endothelial cells by hypoxic preconditioning

Brief exposure of endothelial cells to oxidative stress induced by hypoxia followed by reoxygenation enhances tube formation. Our study provides evidence that hypoxic preconditioning accelerates tubular morphogenesis along with the activation of reactive oxygen species‐inducible nuclear transcription factor‐κB (NF‐κB), phosphatidylinositol 3‐kinase (PI3‐kinase) and broad‐spectrum anti‐apoptotic protein survivin in human coronary arteriolar endothelial cells (HCAEC). The formation of tubular morphogenesis was inhibited by using the PI3‐kinase and NF‐κB antagonists LY294002 and SN50 respectively. The activation of survivin by hypoxic preconditioning was also inhibited by LY294002 and SN50 along with increased apoptosis in HCAEC. These data demonstrate a crucial role of PI3‐kinase/Akt/NF‐κB/survivin signaling in tubular morphogenesis of HCAEC triggered by hypoxic preconditioning.

Nitric oxide signaling in ischemic heart

OBJECTIVE Several recent studies have implicated a role of endogenous nitric oxide (NO) in the pathophysiology of myocardial ischemic/reperfusion injury. However, the mechanism by which NO exerts its beneficial/detrimental effects remains unknown. This study examined the intracellular signaling of NO by studying the role of the NO-cGMP signaling pathway on the phospho-diesteratic breakdown and turnover of phosphoinositides during myocardial ischemia and reperfusion. METHODS Isolated working rat hearts were made ischemic for 30 min followed by 30 min of reperfusion. A separate group of hearts were pre-perfused with 3 mM L-arginine for 10 min prior to ischemia. The release of NO was monitored using an on-line amperometric sensor. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [3H]myoinositol and [14C]arachidonic acid. Hearts were then perfused for 10 min in the presence or absence of L-arginine via the Langendorff mode. Ischemia was induced for 30 min followed by 30 min of reperfusion. Experiments were terminated before L-arginine and after L-arginine treatment, after ischemia, and during reperfusion. Biopsies were processed to determine the isotopic incorporation into various phosphoinositols as well as phosphatidic acid and diacylglycerol. cGMP was assayed by radioimmunoassay and SOD content was determined by enzymatic analysis. RESULTS The release of NO was diminished following ischemia and reperfusion and was augmented by L-arginine. L-Arginine reduced ischemic/reperfusion injury as evidenced by the enhanced myocardial functional recovery. cGMP, which remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The cGMP level persisted up to 10 min of reperfusion and then dropped slightly. Reperfusion of ischemic myocardium resulted in significant accumulation of radiolabeled inositol phosphate, inositol bisphosphate, and inositol triphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly during reperfusion. Reperfusion of the ischemic heart prelabeled with [14C]-arachidonic acid resulted in modest increases in [14C]diacylglycerol and [14C]phosphatidic acid. Pretreatment of the heart with L-arginine significantly reversed this enhanced phosphodiesteratic breakdown during ischemia and early reperfusion. However, at the end of the reperfusion the inhibitory effect of L-arginine on the phosphodiesterases seems to be reduced. In L-arginine-treated hearts, SOD activity was progressively decreased with the duration of reperfusion time. CONCLUSIONS The results suggest for the first time that NO plays a significant role in transmembrane signaling in the ischemic myocardium. The signaling seems to be transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. This signaling effect appears to be reduced as reperfusion progresses. These results, when viewed in the light of free radical chemistry of NO, suggest that such on- and off-signaling of NO may be linked to its interaction with the superoxide radical generated during the reperfusion of ischemic myocardium.

Phospholipase D Plays a Role in Ischemic Preconditioning in Rabbit Heart

BACKGROUND Activation of protein kinase C (PKC) is thought to be a critical step in ischemic preconditioning. Many receptor agonists activate PKC via stimulation of phospholipase C (PLC), which degrades membrane phospholipids to diacylglycerol (DAG), an important PKC cofactor. However, adenosine receptors, critical components of the prototypical preconditioning pathway, are not thought to couple to PLC in the cardiomyocyte. We therefore tested whether ischemic preconditioning or adenosine might instead activate phospholipase D (PLD) to produce DAG. METHODS AND RESULTS PLD activity was measured in isolated rabbit hearts. Ischemic injury was evaluated in either isolated rabbit hearts or dispersed myocytes. PLD activity doubled from a control level of 74.8 +/- 10.0 to 140.0 +/- 11.5 mumol.min-1.g-1 (P < .025) after two 5-minute periods of global ischemia separated by 5 minutes of reperfusion. A similar increase was noted after the heart had been exposed to (R)-N6-(2-phenylisopropyl)-adenosine [(R)-PIA] for 20 minutes. When sodium oleate, which activates PLD, was administered to isolated hearts before a 30-minute coronary occlusion, infarct size (15.6 +/- 2.0% of the risk zone) was significantly smaller than in untreated hearts (30.4 +/- 2.2%; P < .01). Exposure to sodium oleate significantly prolonged the rate of isolated myocyte survival during simulated ischemia. Propranolol 100 mumol/L, which blocks DAG production from metabolites produced by PLD catalysis, completely abolished the protective effects of both metabolic preconditioning and (R)-PIA exposure in myocytes. CONCLUSIONS We conclude that PLD stimulation is involved in the protection of ischemic preconditioning in the rabbit heart.

L-arginine reduces endothelial inflammation and myocardial stunning during ischemia/reperfusion.

BACKGROUND This study evaluated whether the nitric oxide precursor L-arginine could reduce ischemia/reperfusion injury by preventing leukocyte-endothelial interactions. METHODS Normothermic regional ischemia was induced in the open-chest working pig heart for 30 minutes followed by 90 minutes of reperfusion. A preischemic 10-minute intravenous infusion of 4 mg.kg-1.min-1 of L-arginine (n = 12) was compared with 12 control pigs. Nitric oxide release was measured from the coronary sinus using an amperometric probe. Left ventricular function, malonaldehyde, creatine kinase, myocardial oxygen extraction, and the soluble adhesion molecules (intracellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1) were measured. RESULTS Nitric oxide release was significantly reduced from baseline throughout ischemia/reperfusion only in the control group. Systolic and diastolic function, and myocardial oxygen extraction were also significantly decreased during early reperfusion in the control compared with the L-arginine group. Peak creatine kinase release was not significantly different between groups. The incidence of ventricular fibrillation, malonaldehyde release, and soluble intracellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1 were each significantly decreased during reperfusion in the L-arginine group. CONCLUSIONS L-Arginine reduced lipid peroxidation, plasma levels of soluble adhesion molecules, myocardial stunning, and arrhythmias. These results support an excessive endothelial injury/inflammatory response after regional ischemia/reperfusion that can be ameliorated through augmented nitric oxide.

Effects of preconditioning on reperfusion arrhythmias, myocardial functions, formation of free radicals, and ion shifts in isolated ischemic/reperfused rat hearts.

The effects of preconditioning on development of reperfusion-induced ventricular fibrillation (VF), ventricular tachycardia (VT), free radical formation, and ion shifts, particularly those of Na, K, Ca, and Mg, were studied in isolated rat heart. Hearts were randomly divided into four groups: group I, aerobically perfused time-matched controls with no preconditioning or ischemia; group II, hearts subjected to 30-min global ischemia followed by 30-min reperfusion; group III, hearts subjected to one cycle of preconditioning, consisting of 5-min global ischemia plus 10-min reperfusion, followed by 30-min global ischemia plus 30-min reperfusion; and group IV, hearts subjected to four cycles of preconditioning (5-min ischemia plus 10-min reperfusion) followed by 30-min ischemia plus 30-min reperfusion. The incidences of VF and VT were reduced from their nonpreconditioned ischemic values of 100 and 100% in group II to 83 and 92% in group III and to 33% (p < 0.05) and 41% (p < 0.05) in group IV, respectively. Maximum malondialdehyde formation, as an indirect marker of free radicals, was observed after 30-min ischemia followed by 10-min reperfusion (0.72 +/- 0.1 nmol/ml) in the nonpreconditioned ischemic group (protocol II). One and four cycles of preconditioning reduced formation of malondialdehyde from the nonpreconditioned ischemic value of 0.72 +/- 0.1 to 0.35 +/- 0.02 and 0.26 +/- 0.02 nmol/ml (p < 0.05), respectively. The same trend was observed when free radical formation was directly detected by salicylic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of oxidative stress in heart by estimating the dinitrophenylhydrazine derivative of malonaldehyde

Accurate estimation of the oxidative stress in heart is necessary because the pathogenesis of many heart diseases are believed to be mediated at least in part from the development of oxidative stress resulting from the generation of oxygen free radicals and reduced antioxidant defense system. The most widely used method for this purpose has been the estimation of malonaldehyde (MDA), a lipid peroxidation product, by the thiobarbituric acid (TBA) reaction method. However, because of the nonspecificity of this method, the results are often erroneous. The present report describes a method using high-performance liquid chromatography (HPLC) to estimate MDA. To develop the oxidative stress, two different models were used: ischaemic-reperfused heart and perfusing the heart with a hydroxyl radical (OH+) generating system. The coronary effluents obtained from the isolated rat heart before ischaemia and during the reperfusion of ischaemic heart, as well as during the perfusion of the heart with the OH+ generating system were collected, derivatized with 2,4-dinitrophenylhydrazine (DNPH) and extracted with pentane. Aliquots of 25 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column. The products were eluted isocratically with a mobile phase containing acetonitrile-water-acetic acid (40:60:0.1, v/v/v), measured at 307 nm using a Waters M-490 multichannel UV detector and collected for gas chromatography-mass spectrometry (GC-MS). The peaks were identified by co-chromatography with DNPH derivatives of authentic standards, peak addition, and by GC-MS. The retention time for MDA-DNPH was 5.3 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparisons of ESR and HPLC methods for the detection of OH. radicals in ischemic/reperfused hearts. A relationship between the genesis of free radicals and reperfusion arrhythmias

In this study we compared two methods, electron spin resonance (ESR) spectroscopy and high performance liquid chromatography (HPLC), which are currently used to detect directly hydroxyl radical (OH.) formation in the ischemic and reperfused heart. Isolated buffer-perfused rat hearts were subjected to 30 min of normothermic global ischemia followed by 30 min of reperfusion. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) was used as a spin-trap agent to detect OH. radicals by ESR and HPLC. In additional HPLC studies, salicylic acid was infused into the heart for the detection of OH. radicals. In all studies, the effects of superoxide dismutase (SOD) and catalase (CAT) on the OH. generation were examined. The results of our studies indicate that, irrespective of the method, OH. was always detected when an ischemic heart was reperfused and showed ventricular fibrillation. The OH. concentration increased dramatically between 60 and 90 sec of reperfusion, peaked between 180 and 210 sec, and then progressively decreased. In all cases, both SOD and CAT were able to reduce the formation of OH. radicals, with SOD being relatively more effective. Our results indicate that OH. was produced only in the fibrillating hearts that peaked between 180 and 210 sec (1.64 +/- 0.09 nmol/mL measured by ESR), but not in the non-fibrillating hearts. Although SOD or CAT reduced the OH. formation, they had no effects on the incidence of reperfusion-induced ventricular fibrillation (VF) and ventricular tachycardia (VT). However, when SOD (5 x 10(4) IU/L) was coadministered with CAT (5 x 10(4) IU +/- L), the incidence of reperfusion-induced VF (total) and VT was reduced from their control value of 92 and 100 to 33 (P < 0.05) and 50% (P < 0.05), respectively. The results of this study indicate that the HPLC method, as well as ESR, can be used to detect OH. formation in ischemic/reperfused hearts. Because of the convenience, reproducibility and greater sensitivity, the HPLC technique may be more suitable for OH. detection. Our results further suggest the potential therapeutic value of the combination therapy of SOD and CAT for the reduction of reperfusion-induced VF and VT.

High-performance liquid chromatographic peak identification of 2,4-dinitrophenylhydrazine derivatives of lipid peroxidation aldehydes by photodiode array detection.

Malonaldehyde (MDA), a product of lipid peroxidation, is a presumptive marker for the development of oxidative stress in tissues and plasmas. In this study we report the photodiode array detection of the 2,4-dinitrophenylhydrazine (DNPH) derivatives of MDA using HPLC. Oxidative stress was produced by injecting (i.p.) bacterial lipopolysaccharide (LPS) into rats at a dose of 100 micrograms/kg, or i.v. into rabbits (1 microgram/kg), or added to freshly drawn human blood (200 ng/ml). Blood was collected at several time points up to 5 h, centrifuged, and equal volumes of 20% TCA were used to precipitate proteins from the plasma. The supernatants were derivatized with DNPH, and the aldehyde-DNPHs were extracted with pentane. After evaporation, aliquots of 10 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column, chromatographed with an acetonitrile-water-acetic acid gradient mobile phase and scanned using Waters 996 photodiode array detector. Peak identification and homogeneity was determined by comparing the experimental peaks and UV scans with those of authentic standards. A significant increase in the DNPH derivative of malonaldehyde (MDA-DNPH), but not of the other aldehyde-DNPH derivatives of formaldehyde (FDA), acetaldehyde (ADA), acetone and propionaldehyde (PDA) was seen over the first hour after LPS administration in anesthetized rats, while in conscious rabbits this trend lasted up to 3 h. The retention times as well as the UV scans of the derivatized aldehydes matched the authentic standards. Thus, photodiode array detection has proved valuable in establishing this HPLC method for estimating oxidative stress. This technique could accurately measure pmol amounts of MDA-DNPH indicating the usefulness of photodiode array detection method for estimating small changes in the oxidative stress.