Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improves myocardial function and coronary perfusion in senescent hearts.

Cardiovascular ageing is associated with an increase in cardiac susceptibility to ischaemia and reperfusion and production of reactive oxygen species has been suspected to be responsible for this age-associated particular vulnerability. To determine whether administration of antioxidant treatment could afford some protection against ischaemia and reperfusion during aging, isolated perfused hearts from adult and senescent rats were submitted to normoxia (180 min), prolonged low-flow ischaemia (15% of initial coronary flow;180 min) or low-flow ischaemia/reperfusion (45 min/30 min), without or with antioxidant enzymes (superoxide dismutase+catalase; 50IU/ml). Contractile function and coronary perfusion were measured and protein oxidation was quantitated in left ventricle after normoxia, ischaemia and ischaemia/reperfusion. Protein oxidation was higher in senescent than in adult hearts after ischaemia-reperfusion, in contrast to prolonged ischaemia. During prolonged ischaemia, antioxidant treatment prevented coronary vasoconstriction at both ages and delayed contractile dysfunction in senescent hearts but did not limit protein oxidation. During reperfusion, antioxidant treatment prevented coronary vasoconstriction and protein oxidation at both ages and considerably improved recovery of contractile function in senescent hearts. In conclusion, antioxidant treatment fully protects the senescent heart against ischaemia/reperfusion but not against prolonged ischaemia injury, indicating that oxidative stress plays a central role in the age-associated vulnerability to ischaemia-reperfusion.     

Myocardial ischemia-reperfusion damage after pacing-induced tachycardia in patients with cardiac syndrome X

The presence of myocardial ischemia in syndrome X (chest pain, "ischemia-like" electrocardiogram changes, and normal coronary angiograms) is uncertain possibly because, when focally distributed, it may not cause contractile dysfunction or lactate production. We measured lipid hydroperoxides (ROOHs) and conjugated dienes (CDs), two sensitive, independent markers of ischemia-reperfusion oxidative stress, in paired aortic and great cardiac vein blood samples before and after pacing-induced tachycardia in nine patients with syndrome X. Diagnostic ischemic S-T segment changes during pacing were followed by a consistent increase in ROOH and CD levels in the great cardiac vein (from 4.83 +/- 1.18 micromol/l at baseline to 7.88 +/- 1.12 micromol/l and from 0.038 +/- 0.002 to 0.051 +/- 0.003 arbitrary units, respectively, P < 0.01). In controls, ROOH and CD levels did not change after pacing. The large postpacing cardiac release of lipid peroxidation products, consistently observed in all patients and similar to that previously observed after ischemia caused by percutaneous transluminal coronary angioplasty, is consistent with an ischemic origin of syndrome X.     

Chemiluminescent response to PMA in isolated rat liver after in situ ischemia-reperfusion

The median and left lateral lobes of rat liver in situ were rendered ischemic for 30 min, then blood flow reinstituted. After 1, 3, 6, 24, or 48 h, livers were removed and set up for isolated perfused organ study. Luminol enhanced chemiluminescence (LEC) was recorded from the surface of the median and left lateral lobes before and for 90 min following phorbol myristate acetate (PMA, 1.6 × 10⁻⁸ M) perfusion. An increase in PMA induced LEC was evident at 1 h and continued to increase up to 6 h. By 24 h the magnitude of the PMA response had returned to within control values. This indicates that a large influx of inflammatory cells had occurred in the liver following the in vivo ischemia-reperfusion insult and that these cells were well fixed in the tissue and capable of mounting a very large and sustained burst of radical production on stimulation with PMA. This combined in vivo/in vitro technique is ideally suited for the assessment of interventions designed to ameliorate damage following oxidative stress.     

Mitochondrial dysfunction in cardiac ischemia-reperfusion injury: ROS from complex I, without inhibition

A key pathologic event in cardiac ischemia reperfusion (I-R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I-R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1alpha sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I-R injury are discussed.     

Identification of quercetin 3-O-beta-D-glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin

The potential beneficial effect of dietary quercetin (3,3',4',5,7-pentahydroxyflavone) has attracted much attention in relation to the prevention of cardiovascular disease. It is generally recognized that dietary quercetin is subject to metabolic conversion resulting in conjugated forms during absorption and circulation. However, no quercetin conjugates have yet been identified from biological fluids or tissues. In the present study, we isolated and characterized two quercetin conjugates from the plasma of quercetin-administered rats. The blood plasma was collected from 26 rats 30 min after oral administration of quercetin (250 mg/kg body weight), concentrated, dissolved in 2% acetic acid aqueous solution (pH 2.65), and extracted with ethyl acetate. Two compounds (P2, P3) were obtained from the extract by repeated reversed-phase HPLC. On the other hand, two quercetin glucuronides were synthesized chemically and identified as quercetin 3-O-beta-D-glucuronide (Q3GA) and quercetin 4'-O-beta-D-glucuronide (Q4'GA), as determined from FABMS, 1H- and 13C-NMR, and HMBC data. The retention times of P2 and P3 in the HPLC chromatogram corresponded to those of Q3GA and Q4'GA, respectively. FABMS data demonstrated that P2 and P3 are quercetin monoglucuronides. 1H-NMR data for P2 were completely in agreement with those for Q3GA. P2 was therefore identified as Q3GA. This is, to our knowledge, the first evidence that Q3GA accumulates in vivo after oral administration of quercetin. Q3GA is likely to act as an effective antioxidant in blood plasma low-density lipoprotein, because this conjugated metabolite was found to possess a substantial antioxidant effect on copper ion-induced oxidation of human plasma low-density lipoprotein as well as 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.     

Mitochondrial function as a determinant of recovery or death in cell response to injury

Many pathological conditions can be the cause or the consequence of mitochondrial dysfunction. For instance anoxia, which is initiated by a critical reduction of oxygen availability for mitochondrial oxidations, is followed by a wide variety of mitochondrial alterations. A crucial role in the evolution of cell injury is to be attributed to the direction of operation of the F₀F₁ ATPase, which may turn mitochondria into the major consumers of cellular ATP in the futile attempt to restore the proton electrochemical gradient. On the other hand, functional mitochondria can paradoxically accelerate or exacerbate cell damage. This concept is particularly relevant for the ischemic myocardium. Indeed, inhibition of the respiratory chain or addition of uncouplers of oxidative phosphorylation can both limit the extent of enzyme release in the intact heart and prevent the onset of irreversible morphological changes in isolated myocytes. From studies on different tissues in a variety of pathological conditions a general consensus emerges on the role of intracellular Ca²⁺ overload as a pivotal link between cellular alterations and mitochondrial dysfunction. Oxidative phosphorylation is reduced by a massive mitochondrial uptake of Ca²⁺, resulting in a vicious cycle whereby the reduced ATP availability is followed by a failure of the mechanisms which extrude Ca²⁺ from the sarcoplasm. In addition, the rise in [Ca²⁺]_i could promote opening of the cyclosporin-sensitive mitochondrial permeability transition pore, leading to a sudden _m dissipation. Here, we review the changes in intracellular and intramitochondrial ionic homeostasis occurring during ischemia and reperfusion. In particular, we evaluate the potential contribution of the permeability transition pore to cellular damage and discuss the mechanisms which can determine the cellular fate from a mitochondrial point of view.     

Partial reversal by rutin and quercetin of impaired cardiac function in streptozotocin-induced diabetic rats

The present investigation was carried out to evaluate the effects of the cyclodextrin complexes quercetin and rutin on left ventricle dysfunction in streptozotocin-induced diabetic rats. Diabetes was induced by streptozotocin (45 mg/kg body mass, i.v.) in Sprague-Dawley rats. Echocardiography and biochemical and histological studies were carried out under normal control, diabetic untreated, normal and diabetic vehicle (beta-cyclodextrin, p.o.), quercetin- (100 and 300 mg/kg, p.o.), and rutin- (100 and 300 mg/kg, p.o.) treated normal and diabetic animals at varying time intervals (1 and 12 weeks). The increase in the serum triglycerides and cholesterol levels was attenuated in the cyclo dextrin complexes of rutin-treated animals significantly more than in the quercetin-treated and diabetic vehicle-treated animals. Left ventricular diastolic dysfunction was observed in diabetic vehicle-treated animals after 12 weeks of the study as determined by a significant decrease in E-wave (45.91%), an increase in the A-wave (75.55%), and a decrease in the E/A ratio (70.14%). However, the percent decrease (after 12 weeks) in the E-wave, increase in the A-wave, and decrease in the E/A ratio were less in the cyclodextrin complexes of rutin-treated animals (100 and 300 mg/kg), which had the following values: E-wave, 12.22% and 13.80%; A-wave, 25.90% and 10.40%; and E/A ratio, 31.01% and 20.52%. In the quercetin-treated animals (100 and 300 mg/kg), which had the following values: E-wave, 40.44% and 36.44%; A-wave, 52.98% and 29.28%; and E/A ratio, 61.70% and 51.11%. Histopathological studies revealed that the degree of myocardial necrosis was less in rutin-treated animals compared with quercetin and diabetic vehicle-treated animals: rutin < quercetin < beta-cyclodextrin. Myocardial fructose levels were significantly increased in the diabetic vehicle-treated animals after 12 weeks of the study, suggesting an increment in the myocardial polyol pathway activity. However, myocardial fructose levels were significantly decreased in the rutin- and quercetin-treated animals compared with the vehicle-treated animals, possibly owing to their aldose reductase inhibitory activity. Quercetin and rutin treatment did not influence the echocardiographical and histo logical parameters in normal animals. Results from the present investigation demonstrated that rutin has a cardioprotective activity, and we conclude that the observed cardioprotection with rutin may be due to its aldose reductase inhibitory activity, as the enhanced aldose reductase pathway is implicated in the development of left ventricle dysfunction by several studies.     

The cardiac contractile function and hemodynamic control in rats after chronic adriamycin treatment

Cardiac contractile function and hemodynamic parameters of control and adriamycin-treated (2 mg/kg once a week for 10 weeks) rats were studied both in the anesthetized (hexenal, 20 mg/kg) and conscious state. Radiolabelled microspheres (diameter, 15 microns) were used to measure systemic and regional hemodynamics. No significant differences between the control and adriamycin-treated groups in cardiac contractile function, total peripheral resistance, and regional blood flow (except muscles) was found in anesthetized animals. In the conscious state, a significantly higher (+70%) total peripheral resistance combined with lower blood flow in the skin and spleen was observed in adriamycin-treated rats. The response of the heart rate to changes in the arterial pressure induced by nitroglycerin and phenylephrine injection was greatly diminished after adriamycin treatment. Isoprenaline (0.64 micrograms.kg-1.min-1) increased left ventricular contractile indices approximately twofold and heart rate by 30% in the control group, while in adriamycin-treated rats only minor changes in these parameters were observed. However, cardiac output rose by 36% and total peripheral resistance fell by 36% in these animals. Results show that prolonged adriamycin treatment leads to decreased inotropic response to beta-adrenoceptor stimulation and reduced baroreflex control. These changes occur in the stage preceding congestive heart failure.     

Mitochondrial displacements in response to nanomechanical forces

Mechanical stress affects and regulates many aspects of the cell, including morphology, growth, differentiation, gene expression and apoptosis. In this study we show how mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, we used a combined fluorescence-atomic force microscope (AFM) to produce well defined nanomechanical perturbations of 10 nN while simultaneously tracking the real-time motion of fluorescently labelled mitochondria in live cells. The spatial displacement of the organelles in response to applied loads demonstrates the highly dynamic mechanical response of mitochondria in fibroblast cells. The average displacement of all mitochondrial structures analysed showed an increase of approximately 40%, post-perturbation ( approximately 160 nm in comparison to basal displacements of approximately 110 nm). These results show that local forces can produce organelle displacements at locations far from the initial point of contact (up to approximately 40 microm). In order to examine the role of the cytoskeleton in force transmission and its effect on mitochondrial displacements, both the actin and microtubule cytoskeleton were disrupted using Cytochalasin D and Nocodazole, respectively. Our results show that there is no significant change in mitochondrial displacement following indentation after such treatments. These results demonstrate the role of the cytoskeleton in force transmission through the cell and on mitochondrial displacements. In addition, it is suggested that care must be taken when performing mechanical experiments on living cells with the AFM, as these local mechanical perturbations may have significant structural and even biochemical effects on the cell.     

Critical role of extracellular heat shock cognate protein 70 in the myocardial inflammatory response and cardiac dysfunction after global ischemia-reperfusion

Previous studies showed that Toll-like receptor 4 (TLR4) modulates the myocardial inflammatory response to ischemia-reperfusion injury, and we recently found that cytokines link TLR4 to postischemic cardiac dysfunction. Although TLR4 can be activated in cultured cells by endogenous agents including heat shock protein 70, how it is activated during myocardial ischemia-reperfusion is unknown. In the present study, we examined 1) whether heat shock cognate protein 70 (HSC70), which is constitutively expressed in the myocardium, is released during ischemia-reperfusion; 2) whether extracellular HSC70 induces the myocardial inflammatory response and modulates cardiac function; and 3) whether HSC70 exerts these effects via TLR4. We subjected isolated mouse hearts to global ischemia-reperfusion via the Langendorff technique. Immunoblotting and immunostaining detected the release of HSC70 from the myocardium during reperfusion. Treatment with an antibody specific to HSC70 suppressed myocardial cytokine expression and improved cardiac functional recovery after ischemia-reperfusion. Recombinant HSC70 induced NF-kappaB activation and cytokine expression and depressed myocardial contractility in a TLR4-dependent manner. These effects required the substrate-binding domain of HSC70. Fluorescence resonance energy transfer analysis of isolated macrophages demonstrated that extracellular HSC70 interacts with TLR4. Therefore, this study demonstrates for the first time that 1) the myocardium releases HSC70 during ischemia-reperfusion, 2) extracellular HSC70 contributes to the postischemic myocardial inflammatory response and to cardiac dysfunction, 3) HSC70 exerts these effects through a TLR4-dependent mechanism, and 4) the substrate-binding domain of HSC70 is required to induce these effects. Thus extracellular HSC70 plays a critical role in regulating the myocardial innate immune response and cardiac function after ischemia-reperfusion.     

The effect of continuous light exposure of rats on cardiac response to ischemia-reperfusion and NO-synthase activity

Factors modulating cardiac susceptibility to ischemia-reperfusion (I/R) are permanently attracting the attention of experimental cardiology research. We investigated, whether continuous 24 h/day light exposure of rats can modify cardiac response to I/R, NO-synthase (NOS) activity and the level of oxidative load represented by conjugated dienes (CD) concentration. Two groups of male adult Wistar rats were studied: controls exposed to normal light/dark cycle (12 h/day light, 12 h/day dark) and rats exposed to continuous light for 4 weeks. Perfused isolated hearts (Langendorff technique) were exposed to 25 min global ischemia and subsequent 30 min reperfusion. The recovery of functional parameters (coronary flow, left ventricular developed pressure, contractility and relaxation index) during reperfusion as well as the incidence, severity and duration of arrhythmias during first 10 min of reperfusion were determined. The hearts from rats exposed to continuous light showed more rapid recovery of functional parameters but higher incidence, duration and severity of reperfusion arrhythmias compared to controls. In the left ventricle, the NOS activity was attenuated, but the CD concentration was not significantly changed. We conclude that the exposure of rats to continuous light modified cardiac response to I/R. This effect could be at least partially mediated by attenuated NO production.     

Myocardial injury after ischemia-reperfusion in mice deficient in Akt2 is associated with increased cardiac macrophage density

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium. This change was time dependent because macrophage density was similar between WT and Akt2-KO myocardium at 3 days post-I/R, but by 7 and 14 days post-I/R, macrophage density was significantly increased in Akt2-KO myocardium. Concomitantly, infarct size was larger and cardiac function was reduced in Akt2-KO mice subjected to I/R. However, when cryo-infarction produced similar infarct sizes in the anterior wall in both WT and Akt2-KO mice, macrophage density remained higher in Akt2-KO mouse myocardium, suggesting Akt2 regulates myocardial macrophage density independent of infarct size. Consistently, bone marrow from Akt2-KO mice enhanced myocardial macrophage density in both C57/B6 WT and Akt2-KO recipient mice. Finally, reciprocal ex-vivo coculturing of macrophages and cardiac myocytes showed that activated Akt2-KO peritoneal macrophages had reduced mobility and adhesion when compared with WT littermate controls. Thus, although Akt-2 KO mice did not affect the initial inflammation response after injury and Akt2 deficiency has been shown to impair cell migration or motility in macrophages, our data suggested a novel mechanism in which increasing retention of Akt2-KO macrophages resulted in increasing cardiac Akt2-KO macrophage density in the myocardial space.     

Influence of peroxynitrite on energy metabolism and cardiac function in a rat ischemia-reperfusion model

Ischemia-reperfusion generates peroxynitrite (ONOO-), which interacts with many of the systems altered by ischemia-reperfusion. This study examines the influence of endogenously produced ONOO- on cardiac metabolism and function. Nitro-L-arginine (an inhibitor of ONOO- biosynthesis) and urate (a scavenger of ONOO-) were utilized to investigate potential pathophysiological roles for ONOO- in a rat Langendorff heart model perfused with glucose-containing saline at constant pressure and exposed to 30 min of ischemia followed by 60 min of reperfusion. In this model, ischemia-reperfusion decreased contractile function (e.g., left ventricular developed pressure), cardiac work (rate-pressure product), efficiency of O2 utilization, membrane-bound creatine kinase activity, and NMR-detectable ATP and creatine phosphate without significantly altering the recovery of coronary flow, heart rate, lactate release, and muscle pH. Treatment with urate and nitro-L-arginine produced a substantial recovery of left ventricular developed pressure, rate-pressure product, efficiency of O2 utilization, creatine kinase activity, and NMR-detectable creatine phosphate and a partial recovery of ATP. The pattern of effects observed in this study and in previously published work with similar models suggests that ONOO- may alter key steps in the efficiency of mitochondrial high-energy phosphate generation.     

Serological response in rabbits to Listeria monocytogenes after oral or intragastric inoculation

Abstract The serological response in rabbits against Listeria monocytogenes after oral or intragastric inoculation was investigated. Both the number of sero-positive and the average serum titres were hgher in animals inoculated by the oral route. This difference was especially marked in rabbits inoculated with the lower dose (1×10³ colony-forming units (cfu)), which developed a strong serological response (average serum titre of 1280 after 4 inoculations) in most of the inoculated animals (80%), without any clinical signs. The implication of these results in the epidemiology of listeriosis is discussed.     

Lack of effect of oral L-carnitine treatment on lipid metabolism and cardiac function in chronically diabetic rats

L-Carnitine is necessary for the transfer of long-chain fatty acids into the mitochondrial matrix where energy production occurs. In the absence of L-carnitine, the accumulation of free fatty acids and related intermediates could produce myocardial subcellular alterations and cardiac dysfunction. Diabetic hearts have a deficiency in the total carnitine pool and develop cardiac dysfunction. This suggested that carnitine therapy may ameliorate alteration in cardiac contractile performance seen during diabetes. In this study, heart function was studied in streptozotocin diabetic rats given L-carnitine orally. Oral L-carnitine treatment (50-250 mg.kg-1.day-1) of 1- and 3-week diabetic rats increased plasma free and total carnitine and decreased plasma acyl carnitine levels. In both groups, myocardial total carnitine levels were increased. However, L-carnitine (200 mg.kg-1.day-1) treatment of diabetic rats for 6 weeks had no effect on plasma carnitine levels. Similarly, plasma lipids remained elevated whereas cardiac function was still depressed. These studies suggest that in the chronically diabetic rat, the route of administration of L-carnitine is an important factor in determining an effect.     

JNK1 is required to preserve cardiac function in the early response to pressure overload

Cardiac stress consistently activates c-Jun NH(2)-terminal kinase (JNK) pathways, however the role of different members of the JNK family is unclear. In this study, we applied pressure overload (TAC) in mice with selective deletion of the three JNK genes (Jnk1(-/-), Jnk2(-/-), and Jnk3(-/-)). Following TAC, all three JNK knockout mouse lines developed cardiac hypertrophy similar to wild-type mice (WT), but only JNK1(-/-) mice displayed a significant reduction in fractional shortening after 3 and 7 days of pressure overload, associated with a significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining and marked inflammatory infiltrate. After the acute deterioration stage, JNK1(-/-) mice underwent a slow recovery followed by a steady progression of cardiac dysfunction, becoming indistinguishable from WT after 12 weeks of TAC. These data suggest that JNK1 plays a protective role in response to pressure overload, preventing the early deterioration in cardiac function following an acute increase in afterload.