Antioxidant enzyme gene expression in congestive heart failure following mycardial infarction

Increased oxidative stress and reduction in antioxidant enzymes have been suggested to be involved in the pathophysiology of congestive heart failure subsequent to myocardial infarction (MI). The objective of the present study was to characterize changes in the mRNA abundance and protein levels for the enzymatic antioxidants, superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase during the sequelae of congestive heart failure in rats. MI was produced by the ligation of the left coronary artery and hearts from controls and 1, 4 and 16 week PMI groups were analyzed. Losartan treatment (2 mg/ml in drinking water, daily) was started at 4 weeks and continued for 12 weeks. The mRNA levels for SOD were reduced by about 40% at 1-week PMI, were near to the control levels at 4-week PMI and at 16 weeks PMI, the levels were reduced by about 73% below the controls. GSHPx mRNA levels remained unchanged at all time points. The mRNA levels for catalase remained unchanged at 1 and 4 weeks PMI and were significantly reduced by about 44% at 16 weeks PMI as compared to the controls. The protein levels for MnSOD, CuZnSOD, GSHPx at 1 and 16 weeks remained unchanged in treated and untreated PMI groups. However, the protein levels for catalase was significantly increased in the control and PMI groups treated with Losartan. It is concluded that changes in the SOD and catalase activities during severe heart failure correlated with changes in mRNA for these enzymes. The precise mechanism/s for the improvement in antioxidant reserve and protein levels after Losartan treatment is/are unclear at this time.     

p38-MAPK and PKB/Akt,possible role players in red palm oil-induced protection of the isolated perfused rat heart?

It has been shown that dietary red palm oil (RPO) supplementation improves reperfusion function. However, no exact protective cellular mechanisms have been established. To determine a potential mechanism for functional improvement, we examined the regulation of both mitogen-activated protein kinases (MAPKs) and PKB/Akt in the presence and absence of dietary RPO supplementation in ischemia/reperfusion-induced injury. Wistar rats were fed a control diet or control diet plus 7 g RPO/kg diet for 6 weeks. Hearts were excised and mounted on an isolated working heart perfusion apparatus. Cardiac function was measured before and after hearts were subjected to 25 min of total global ischemia. Hearts subjected to the same conditions were freeze clamped and used to characterize the degree of phosphorylation of extracellular signal-regulated kinase, p38, c-Jun NH(2)-terminal protein kinase (JNK) and PKB/Akt. Dietary RPO supplementation significantly improved aortic output recovery (72.1 +/- 3.2% vs. 54.0 +/- 3.2%, P < .05). This improved aortic output recovery was associated with significant increases in p38 and PKB/Akt phosphorylation during reperfusion when compared with control hearts. Furthermore, a significant decrease in JNK phosphorylation and attenuation of poly(ADP-ribose) polymerase cleavage occurred in the RPO-supplemented group during reperfusion. Our results suggest that dietary RPO supplementation caused differential phosphorylation of the MAPKs and PKB/Akt during ischemia/reperfusion-induced injury. These changes in phosphorylation were associated with improved functional recovery and reduced cleavage of an apoptotic marker, arguing that dietary RPO supplementation may confer protection via the MAPK and PKB/Akt signaling pathways during ischemia/reperfusion-induced injury.     

Super,Red Palm and Palm Oleins Improve the Blood Pressure,Heart Size,Aortic Media Thickness and Lipid Profile in Spontaneously Hypertensive Rats

Background

Oleic acid has been shown to lower high blood pressure and provide cardiovascular protection. Curiosity arises as to whether super olein (SO), red palm olein (RPO) and palm olein (PO), which have high oleic acid content, are able to prevent the development of hypertension.

Methodology/Principal Findings

Four-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were fed 15% SO, RPO or PO supplemented diet for 15 weeks. After 15 weeks of treatment, the systolic blood pressure (SBP) of SHR treated with SO, RPO and PO were 158.4±5.0 mmHg (p<0.001), 178.9±2.7 mmHg (p<0.001) and 167.7±2.1 mmHg (p<0.001), respectively, compared with SHR controls (220.9±1.5 mmHg). Bradycardia was observed with SO and PO. In contrast, the SBP and heart rate of treated WKY rats were not different from those of WKY controls. The SO and PO significantly reduced the increased heart size and thoracic aortic media thickness observed in untreated SHR but RPO reduced only the latter. No such differences, however, were observed between the treated and untreated WKY rats. Oil Red O enface staining of thoracic-abdominal aorta did not show any lipid deposition in all treated rats. The SO and RPO significantly raised serum alkaline phosphatase levels in the SHR while body weight and renal biochemical indices were unaltered in both strains. Serum lipid profiles of treated SHR and WKY rats were unchanged, with the exception of a significant reduction in LDL-C level and total cholesterol/HDL ratio (atherogenic index) in SO and RPO treated SHR compared with untreated SHR.

Conclusion

The SO, RPO and PO attenuate the rise in blood pressure in SHR, accompanied by bradycardia and heart size reduction with SO and PO, and aortic media thickness reduction with SO, RPO and PO. The SO and RPO are antiatherogenic in nature by improving blood lipid profiles in SHR.     

Up-regulation of myocardial connexin-43 in spontaneously hypertensive rats fed red palm oil is most likely implicated in its anti-arrhythmic effects

The purpose of this study was to test our hypothesis that red palm oil (RPO) intake may affect abnormalities of myocardial connexin-43 (Cx43) and protein kinase Cε (PKCε) signaling, and consequently the propensity of the spontaneously hypertensive rat heart (SHR) heart to arrhythmias. SHR and Wistar-Kyoto (WKY) rats fed a standard rat chow plus red palm oil (200?μL/day) for 5?weeks were compared with untreated rats. Cytosolic but not particulate PKCε expression as well as Cx43-mRNA, total Cx43 proteins, and its phoshorylated forms were increased, and disordered localization of Cx43 was attenuated in the left ventricle of RPO-fed SHR compared with untreated rats. These alterations were associated with suppression of early post-ischemic-reperfusion-related ventricular tachycardia and electrically inducible ventricular fibrillation. However, the treatment dose of RPO caused down-regulation of myocardial Cx43, but did not alter its cell membrane distribution or overall PKCε expression in WKY rats. It was, however, associated with poor arrhythmia protection, suggesting overdosing. Results indicate that SHR benefit from RPO intake, particularly because of its apparent anti-arrhythmic effects. This protection can be, in part, attributed to the preservation of cell-to-cell communication via up-regulation of myocardial Cx43, but not with PKCε activation.     

Proposed mechanisms for red palm oil induced cardioprotection in a model of hyperlipidaemia in the rat

High-cholesterol diets alter myocardial and vascular NO-cGMP signaling and have been implicated in ischaemic/reperfusion injury. We investigated the effects of dietary red palm oil (RPO) containing fatty acids, carotonoids, tocopherols and tocotrienols on myocardial ischaemic tolerance and NO-cGMP pathway function in the rat. Wistar rats were fed a standard rat chow+/-RPO, or a standard rat chow+cholesterol+/-RPO diet. Myocardial mechanical function and NO-cGMP signaling pathway intermediates were determined before, during and after 25 min ischaemia. RPO-supplementation improved aortic output recovery and increased myocardial ischaemic cGMP concentrations. Simulated ischaemia (hypoxia) increased cardiomyocyte nitric oxide levels in the two RPO supplemented groups, but not in control non-supplemented groups. RPO supplementation also increased hypoxic nitric oxide levels in the control diet fed, but not the cholesterol fed rats. These data suggest that dietary RPO may improve myocardial ischaemic tolerance by increasing bioavailability of NO and improving NO-cGMP signaling in the heart.     

Reduced neuronal nitric oxide synthase expression contributes to cardiac oxidative stress and nitroso-redox imbalance in ob/ob mice.

Disruption of leptin signaling in the heart may contribute to obesity-related cardiac disease, as leptin deficient (oblob) mice display cardiac hypertrophy, increased cardiac apoptosis and reduced survival. Since leptin maintains a tonic level of neuronal nitric oxide synthase (NOS1) expression in the brain, we hypothesized that leptin deficiency would decrease NOS1 cardiac expression, in turn activating xanthine oxidoreductase (XOR) and creating nitroso-redox imbalance. We studied 2- to 6-month-old oblob (n=26) and C57Bl/6 controls (n=27). Cardiac NOS1 protein abundance (P<0.01) and mRNA expression (P=0.03) were reduced in oblob (n=10 and 6, respectively), while NOS3 protein abundance and mRNA expression were unaltered. Importantly, cardiac NOS1 protein abundance was restored towards normal in oblob mice after leptin treatment (n=3; P<0.05 vs leptin untreated oblob mice). NO metabolite (nitrite and nitrate) production within the myocardium was also reduced in oblob mice (n=5; P=0.02). Furthermore, oxidative stress was increased in oblob mice as GSH/GSSG ratio was decreased (n=4; P=0.02). Whereas XOR activity measured by Amplex Red fluorescence was increased (n=8; P=0.04), XOR and NADPH oxidase subunits protein abundance were not changed in oblob mice (n=6). Leptin deficiency did not disrupt NOS1 subcellular localization, as NOS1 co-localized with ryanodine receptor but not with caveolin-3. In conclusion, leptin deficiency is linked to decreased cardiac expression of NOS1 and NO production, with a concomitant increase in XOR activity and oxidative stress, resulting in nitroso-redox imbalance. These data offer novel insights into potential mechanisms of myocardial dysfunction in obesity.     

The effect of triiodothyronine on myocardial protein kinases

Rats treated with T3 (triiodothyronine) showed an increased heart weight after 3 days reaching 100% after 3 weeks of treatment compared to untreated controls. Cytosol protein kinases were not significantly different in the T3 treated rats compared to controls. The protein kinase activity of the NHP (nonhistone proteins) increased after 2 hours and doubled after 3 days for each substrate tested. After 1 week of T3 treatment the protein kinase activity returned to the control value and remained at the control level for the remainder of the 3 week experimental period. A study of the distribution of protein kinase activity in the NHP by disc gel electrophoresis showed that there was a difference in the distribution of some peaks in the T3 treated animals compared to the controls. T3 in concentrations from 10(-11) to 10(-3) M had no in vitro effect on the phosvitin kinase activity of NHP and of cytosol.     

TNF-α、IL-1β及LPS对血管平滑肌细胞一氧化氮合酶基因表达的影响及作用机制研究

通过ＲＮＡ印迹分析和亚硝酸盐含量测定检查ＴＮＦ－α、ＩＬ－１β和ＬＰＳ对大鼠血管平滑肌细胞（ＶＳＭＣ）诱导型一氧化氮合酶（ｉＮＯＳ）基因表达及ＮＯ生成的影响．结果表明,ＴＮＦ－α、ＩＬ－１β和ＬＰＳ均能显著诱导ＶＳＭＣｉＮＯＳ基因表达和促进ＮＯ生成,其作用强度与浓度和作用时间有关;双因素（ＴＮＦ－α＋ＬＰＳ,ＬＰＳ＋ＩＬ－１β）对诱导ｉＮＯＳ基因表达及ＮＯ生成产生协同作用．ＰｏｌｙｍｙｘｉｎＢ和地塞米松可部分抑制ＴＮＦ－α对ｉＮＯＳ基因表达的诱导作用及ＮＯ生成     

Nitric oxide synthase-2 modulates chemokine production by Trypanosoma cruzi-infected cardiac myocytes

An intense inflammatory process is associated with Trypanosoma cruzi infection. We investigated the mediators that trigger leukocyte activation and migration to the heart of infected mice. It is known that nitric oxide (NO) modulates the inflammatory response. During T. cruzi infection, increased concentrations of NO are produced by cardiac myocytes (CMs) in response to IFN-gamma and TNF. Here, we investigated whether NO, IFN-gamma and TNF regulate chemokine production by T. cruzi-infected CMs. In addition, we examined the effects of the NOS2 deficiency on chemokine expression both in cultured CMs and in hearts obtained from infected mice. After infection of cultured WT CMs with T. cruzi, the addition of IFN-gamma and TNF increased both mRNA and protein levels of the chemokines CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL5. Interestingly, T. cruzi-infected NOS2-deficient CMs produced significantly higher levels of CCL2, CCL4, CCL5 and CXL2 in the presence of IFN-gamma and TNF. Infection of NOS2-null mice resulted in a significant increase in the expression of both chemokine mRNA and protein levels in the heart of, compared with hearts obtained from, infected WT mice. Our data indicate that NOS2 is a potent modulator of chemokine expression which is critical to triggering the generation of the inflammatory infiltrate in the heart during T. cruzi infection.     

Administration of Chromium(III) and Manganese(II) as a Potential Protective Approach Against Daunorubicin-Induced Cardiotoxicity: in vitro and in vivo Experimental Evidence

Daunorubicin (DNR) is a widely used antitumor drug, but its application is limited because of its cardiotoxic side effects. The present study was designed to investigate the interaction between DNR and cardiac myosin (CM) in the presence of chromium(III) (Cr³⁺) and manganese(II) (Mn²⁺) using fluorescence spectrometry under simulative physiological conditions with the aim of exploring the influence of metal ion on DNR-CM complex and finding out an aggressive approach to abrogate of DNR-induced cardiotoxicity. In detail, the quenching and binding constant of ternary system, including metal ion, DNR, and CM, were measured and compared with the DNR-CM. The data from in vitro experiments indicate that the presence of Cr³⁺ or Mn²⁺ distinctly decreased the binding force between DNR and CM, and alleviated the cardiac toxicity caused by DNR. In addition, the variations in mice body weight and myocardial enzyme level were examined by in vivo experiments. Animals receiving Cr³⁺ or Mn²⁺ supplementation of DNR showed preservation of the normal pattern of the heart, especially 2.0 mg Cr³⁺/kg body wt or 50.0 mg Mn²⁺/kg body wt exhibited an obviously protective effect accompanied with body weight raise when compared with the mice treated with DNR alone, decreased the ratio of heart to body weight (BW) and the ratio of left ventricular mass to BW to the normal levels, and inhibited the leak of myocardial enzyme caused by DNR. As a result, this study suggests that pretreatment of lower dose of Cr³⁺ (2 mg/kg wt) and moderate dose of Mn²⁺ (50 mg/kg wt) might be useful and play an important role in ameliorating the cardiotoxicity of DNR treatment in cancer patients.     

Norepinephrine regulates the in vivo expression of the L-type calcium channel

The _1c subunit (DHP receptor) of the L-type Ca²⁺ channel is important for calcium homeostasis in cardiac muscle. The DHPr provides the primary mechanism for calcium influx during contraction. Published results demonstrate three in vitro signaling pathways that are important in the regulation of DHPr gene expression in neonatal cardiac myocytes, the protein kinase A (PKA), protein kinase C (PKC) pathways, and intracellular calcium. To determine whether these pathways are important in vivo, we treated adult rats with infusions of isoproterenol, or norepinephrine at 200 g/kg/h and assessed DHPr mRNA and protein levels. Following a 3-day infusion isoproterenol (ISO) and norepinephrine (NE) produced a small but insignificant reduction in DHPr mRNA levels. When the infusions were continued for 7 days isoproterenol increased DHPr mRNA accumulation to control levels while NE stimulated a 35% increase in DHPr mRNA levels and a 35% increase in protein abundance when compared to controls (p < 0.05). Furthermore, contractility and Ca²⁺ transient measurements of isolated cardiac myocytes from NE infused animals also display shortened duration of contraction/relaxation and increased intracellular free Ca²⁺ (DFFI) in response to electrical stimulation (p < 0.01). We conclude norepinephrine treatment alters DHPr mRNA and protein levels, and augments excitation-contraction coupling, and thus may be important for modulating cardiac calcium homeostasis in vivo.     

Alterations of the levels of primary antioxidant enzymes in different grades of human astrocytoma tissues

Malignant astrocytoma is the most commonly occurring brain tumour in humans. Oxidative stress is implicated in the development of cancers. Superoxide dismutase 2 (SOD2) was found to exert tumour suppressive effect in basic research, but increased SOD2 protein level was associated with higher aggressiveness of human astrocytomas. However, studies reporting alterations of antioxidant enzymes in human astrocytomas often employed less accurate methods or included different types of tumours. Here we analysed the mRNA levels, activities, and protein levels of primary antioxidant enzymes in control brain tissues and various grades of astrocytomas obtained from 40 patients. SOD1 expression, SOD1 activity, and SOD1 protein level were lower in Grade IV astrocytomas. SOD2 expression was lower in low-grade (Grades I and II) and Grade III astrocytomas than in controls, but SOD2 expression and SOD2 protein level were higher in Grade IV astrocytomas than in Grade III astrocytomas. Although there was no change in SOD2 activity and a lower activity of citrate synthase (CS), the MnSOD:CS ratio increased in Grade IV astrocytomas compared with controls and low-grade astrocytomas. Furthermore, SOD1 activity, CS activity, SOD1 expression, GPX4 expression, and GPX4 protein level were inversely correlated with the malignancy, whereas catalase activity, catalase protein, SOD2 protein level, and the SOD2:CS ratio were positively correlated with the degree of malignancy. Lower SOD2:CS ratio was associated with poor outcomes for Grade IV astrocytomas. This is the first study to quantify changes of various primary antioxidant enzymes in different grades of astrocytomas at different levels concurrently in human astrocytomas.     

Duodenal expression of iron transport molecules in patients with hereditary hemochromatosis or iron deficiency

Disturbances of iron metabolism are observed in chronic liver diseases. In the present study, we examined gene expression of duodenal iron transport molecules and hepcidin in patients with hereditary hemochromatosis (HHC) (treated and untreated), involving various genotypes (genotypes which represent risk for HHC were examined), and in patients with iron deficiency anaemia (IDA). Gene expressions of DMT1, ferroportin, Dcytb, hephaestin, HFE and TFR1 were measured in duodenal biopsies using real-time PCR and Western blot. Serum hepcidin levels were measured using ELISA. DMT1, ferroportin and TFR1 mRNA levels were significantly increased in post-phlebotomized hemochromatics relative to controls. mRNAs of all tested molecules were significantly increased in patients with IDA compared to controls. The protein expression of ferroportin was increased in both groups of patients but not significantly. Spearman rank correlations showed that DMT1 versus ferroportin, Dcytb versus hephaestin and DMT1 versus TFR1 mRNAs were positively correlated regardless of the underlying cause, similarly to protein levels of ferroportin versus Dcytb and ferroportin versus hephaestin. Serum ferritin was negatively correlated with DMT1 mRNA in investigated groups of patients, except for HHC group. A decrease of serum hepcidin was observed in IDA patients, but this was not statistically significant. Our data showed that although untreated HHC patients do not have increased mRNA levels of iron transport molecules when compared to normal subjects, the expression is relatively increased in relation to body iron stores. On the other hand, post-phlebotomized HHC patients had increased DMT1 and ferroportin mRNA levels possibly due to stimulated erythropoiesis after phlebotomy.