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Sildenafil reduces L-NAME-induced severe hypertension and worsening of myocardial ischaemia-reperfusion damage in the rat.

ABSTRACT: BACKGROUND AND PURPOSE: Phosphodiesterase-5 inhibitors are beneficial in pulmonary hypertension and congestive heart failure, the two conditions associated with coronary heart disease and ischaemia. We investigated whether sildenafil counteracts the cardiovascular alterations induced by N -nitro-L-arginine methyl ester (L-NAME) in the rat. EXPERIMENTAL APPROACH: Sildenafil was given orally to rats at doses of 0.37, 0.75 or 1.5 mg kg-1day-1 for four weeks, either alone or with L-NAME (35-40 mg kg-1 day-1 in the drinking water). Systolic blood pressure and urinary parameters (6-keto-prostaglandin F1alpha, thromboxane B2, 8-isoprostane-prostaglandin F2 and nitrite/nitrate) were measured in conscious rats. Isolated hearts were subjected to low flow ischaemia-reperfusion, and myocardial levels of guanosine 3', 5'cyclic monophosphate (cGMP) were determined. Endothelial vascular dysfunction was examined in aortic rings. KEY RESULTS: Sildenafil dose-dependently prevented the rise in systolic blood pressure in L-NAME-treated rats. This activity was associated with a normalization of urinary 8-isoprostane-prostaglandin F2alpha and other biochemical parameters. In perfused hearts, the post-ischaemic ventricular dysfunction was worse in preparations from L-NAME-treated rats than in controls. Sildenafil dose-dependently reduced this effect, and creatine kinase and lactate dehydrogenase release were lower too. cGMP levels, which were low in myocardial tissue from L-NAME-treated rats, were restored by sildenafil. In noradrenaline-precontracted aortic rings from L-NAME-treated rats acetylcholine lost its vasorelaxant effect, and sildenafil restored it. CONCLUSION AND IMPLICATIONS: In a rat model of chronic nitric oxide deprivation, where hypertension and aggravation of post-ischaemic ventricular dysfunction are associated with loss of vascular endothelium-relaxant function, sildenafil provided significant cardiovascular protection, primarily by maintaining tissue cGMP levels.

SUBMITTER: Rossoni G

PROVIDER: S-EPMC2189760 | biostudies-other | 2007 Mar

REPOSITORIES: biostudies-other

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Project description:Aims:Myocardial ischaemia followed by reperfusion (IR) causes an oxidative burst resulting in cellular dysfunction. Little is known about the impact of oxidative stress on cardiomyocyte lipids and their role in cardiac cell death. Our goal was to identify oxidized phosphatidylcholine-containing phospholipids (OxPL) generated during IR, and to determine their impact on cell viability and myocardial infarct size. Methods and results:OxPL were quantitated in isolated rat cardiomyocytes using mass spectrophotometry following 24?h of IR. Cardiomyocyte cell death was quantitated following exogenously added OxPL and in the absence or presence of E06, a 'natural' murine monoclonal antibody that binds to the PC headgroup of OxPL. The impact of OxPL on mitochondria in cardiomyocytes was also determined using cell fractionation and Bnip expression. Transgenic Ldlr-/- mice, overexpressing a single-chain variable fragment of E06 (Ldlr-/--E06-scFv-Tg) were used to assess the effect of inactivating endogenously generated OxPL in vivo on myocardial infarct size. Following IR in vitro, isolated rat cardiomyocytes showed a significant increase in the specific OxPLs PONPC, POVPC, PAzPC, and PGPC (P?<?0.05 to P?<?0.001 for all). Exogenously added OxPLs resulted in significant death of rat cardiomyocytes, an effect inhibited by E06 (percent cell death with added POVPC was 22.6?±?4.14% and with PONPC was 25.3?±?3.4% compared to 8.0?±?1.6% and 6.4?±?1.0%, respectively, with the addition of E06, P?<?0.05 for both). IR increased mitochondrial content of OxPL in rat cardiomyocytes and also increased expression of Bcl-2 death protein 3 (Bnip3), which was inhibited in presence of E06. Notably cardiomyocytes with Bnip3 knock-down were protected against cytotoxic effects of OxPL. In mice exposed to myocardial IR in vivo, compared to Ldlr-/- mice, Ldlr-/--E06-scFv-Tg mice had significantly smaller myocardial infarct size normalized to area at risk (72.4?±?21.9% vs. 47.7?±?17.6%, P?=?0.023). Conclusions:OxPL are generated within cardiomyocytes during IR and have detrimental effects on cardiomyocyte viability. Inactivation of OxPL in vivo results in a reduction of infarct size.

Project description:Research focus recently shifted to mitochondrial dynamics and the role of fusion and fission in cardioprotection. The aim of this study was to evaluate (i) the function and dynamics of mitochondria isolated from hearts exposed to ischaemia/reperfusion (I/R) (ii) the effects of melatonin, a powerful cardioprotectant, on mitochondrial dynamics in I/R. Isolated perfused rat hearts were stabilized for 30 min, subjected to 20 min global ischaemia, followed by 30 min reperfusion. Tissue was collected, mitochondria isolated for measurement of mitochondrial oxidative function and lysates from mitochondrial and cytosolic fractions prepared for western blotting. Melatonin (0.3 or 50 ?M) was administered for 10 min immediately before the onset of ischaemia and for 10 min at the onset of reperfusion. Infarct size was assessed after 35 min regional ischaemia/60 min reperfusion using triphenyltetrazolium staining. The results show that reperfusion significantly reduced mitochondrial QO2 (states 3 and 4), with minor effects by melatonin. Cytosolic Beclin 1 and the LC3 II/I ratio were reduced by ischaemia and increased by reperfusion. Both ischaemia and reperfusion reduced mitochondrial PINK1 and Parkin levels, while reperfusion increased p62. An alternative mitophagy pathway mediated by Rab9 is activated during myocardial ischaemia/reperfusion. Ischaemia reduced and reperfusion increased cytosolic ULK1 expression, associated with redistribution of Rab9 and Drp1 between the cytosol and mitochondria. Melatonin significantly reduced mitochondrial p62 expression upon reperfusion. Throughout the protocol, melatonin significantly (i) increased cytosolic total (t) and phospho (p) ULK1, and Rab9 levels (ii) increased the cytosolic and reduced the mitochondrial pDrp1 levels and p/t Drp1 ratio, suggesting inhibition of mitochondrial fission. Fusion was affected to a lesser extent. Cardioprotection by melatonin is associated with substantial effects on mitophagy, the significance thereof remains to be established.

Project description:BACKGROUND:?Fibronectin splicing variant containing extra domain A (Fn-EDA), which is an endogenous ligand for Toll-like receptor 4 (TLR4), is present in negligible amounts in the plasma of healthy humans, but markedly elevated in patients with co-morbid conditions including diabetes and hyperlipidaemia, which are risk factors for myocardial infarction (MI). Very little is known about the role of Fn-EDA in the pathophysiology of acute MI under these co-morbid conditions. MATERIALS AND METHODS:?We determined the role of Fn-EDA in myocardial ischaemia/reperfusion (I/R) injury in the hyperlipidaemic apolipoprotein E-deficient (ApoE-/-) mice. Infarct size, plasma cardiac troponin I (cTnI) levels, intravascular thrombosis (CD41-positive), neutrophil infiltration (Ly6 B.2-positive), neutrophil extracellular traps (citrullinated H3-positive) and myocyte apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling-positive) were assessed in myocardial I/R injury model (1-hour ischaemia/23 hours of reperfusion). RESULTS:?Irrespective of gender, Fn-EDA-/-ApoE-/- mice exhibited smaller infarct size and decreased cTnI levels concomitant with reduced post-ischaemic intra-vascular thrombi, neutrophils influx, neutrophil extracellular traps and myocyte apoptosis (p?<?0.05 vs. ApoE-/- mice). Genetic deletion of TLR4 attenuated myocardial I/R injury in ApoE-/- mice (p?<?0.05 vs. ApoE-/- mice), but did not further reduce in Fn-EDA-/- ApoE-/- mice suggesting that Fn-EDA requires TLR4 to mediate myocardial I/R injury. Bone marrow transplantation experiments revealed that Fn-EDA exacerbates myocardial I/R injury through TLR4 expressed on the haematopoietic cells. Infusion of a specific inhibitor of Fn-EDA, 15 minutes post-reperfusion, into ApoE-/- mice attenuated myocardial I/R injury. CONCLUSION:?Fn-EDA exacerbates TLR4-dependent myocardial I/R injury by promoting post-ischaemic thrombo-inflammatory response. Targeting Fn-EDA may reduce cardiac damage following coronary artery re-canalization after acute MI.

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Sildenafil reduces L-NAME-induced severe hypertension and worsening of myocardial ischaemia-reperfusion damage in the rat.

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