Abnormal diastolic currents in ventricular myocytes from spontaneous hypertensive heart failure rats

Hypertension is a common cause of heart failure, and ventricular arrhythmias are a major cause of death in heart failure. The spontaneous hypertension heart failure (SHHF) rat model was used to study altered ventricular electrophysiology in hypertension and heart failure. We hypothesized that a reduction in the inward rectifier K(+) current (I(K1)) and expression of pacemaker current (I(f)) would favor abnormal automaticity in the SHHF ventricle. SHHF ventricular myocytes were isolated at 2 and 8 mo of age and during end-stage heart failure (>/=17 mo); myocytes from age-matched rats served as controls. Inward I(K1) was significantly reduced at both 8 and >/=17 mo in SHHF rats compared with controls. There was a reduction in inward I(K1) due to aging in the controls only at >/=17 mo. We found a significant increase in I(f) at all ages in the SHHF rats, compared with young controls. In controls, there was an age-dependent increase in I(f). Action potential recordings in the SHHF rats demonstrated abnormal automaticity, which was abolished by the addition of an I(f) blocker (10 muM zatebradine). Increased I(f) during hypertension alone or combined increases in I(f) with reduced I(K1) during the progression to hypertensive heart failure contribute to a substrate for arrhythmogenesis.     

A model of chronic heart failure in spontaneous hypertensive rats (SHR)

Common models of chronic heart failure (CHF) do not always result in parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure. The aim of this study was to establish and validate a new model of CHF in the rat. CHF was induced in Wistar Kyoto (WKY/NHsd) and spontaneously hypertensive (SHR/NHsd) rats by creating a permanent (8-week) occlusion of the left coronary artery 2 mm distal to the origin from the aorta by a modified technique. This resulted in a large infarction of the free left ventricular wall. The focus of attention was the validation of the geometric properties of the left ventricle and its contractility. The validation of the geometric properties of the left ventricle was done by a non-invasive magnetic resonance imaging (MRI) technique and by planimetry (stereology). Cardiodynamics (e.g. contractility) were evaluated in the isolated 'working heart' model. We were able to establish a new and predictive model of heart failure in the spontaneously hypertensive rat 8 weeks after coronary artery ligation. At this time point, the WKY rat did not show any symptoms of CHF. The model represents characteristic parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure (NYHA III-IV). Upon inspection, severe clinical symptoms of congestive heart failure were prominent, such as dyspnoea, subcutaneous oedema, pale-bluish limbs and impaired motion. Non-invasive sequential measurements by NMR techniques showed lung oedema, hydrothorax, large dilated left and right ventricular chambers and hypertrophy of the septum. The infarcted animals showed a reduced heart power, diminished contractility and enhanced heart work, much more so in the SHR/NHsd rat than in the WKY/NHsd rat. Furthermore the infarcted animals showed enhanced levels of hydroxyproline/proline ratios, again much more so in the SHR/NHsd rat than in the WKY/NHsd rat.     

Effect of docosahexaenoic acid (DHA) on arachidonic acid metabolism and platelet function

Studies from our laboratory have suggested a role for ferrous iron in the metabolism of arachidonic acid and demonstrated that inhibitors of prostaglandin synthesis exert their effect by complexing with the heme group of cyclooxygenase. Docosahexaenoic acid (DHA) is a potent competitive inhibitor of arachidonic acid metabolism by sheep vesicular gland prostaglandin synthetase. In this study we have evaluated the effect of exogenously added DHA on platelet function and arachidonic acid metabolism. DHA at 150 microM concentration inhibited aggregation of platelets to 450 microM arachidonic acid. At this concentration DHA also inhibited the second wave of the platelet response to the action of agonists such as epinephrine, adenosine diphosphate and thrombin. Inhibition induced by this fatty acid could be overcome by the agonists at higher concentrations. DHA inhibited the conversion of labeled arachidonic acid to thromboxane by intact, washed platelet suspensions. However, platelets in plasma incubated first with DHA then washed and stirred with labeled arachidonate generated as much thromboxane as control platelets. These results suggest that the polyenoic acids, if released in sufficient quantities in the vicinity of cyclooxygenase, could effectively compete for the heme site and inhibit the conversion of arachidonic acid.     

Low-intensity exercise training delays onset of decompensated heart failure in spontaneously hypertensive heart failure rats

Data regarding the effectiveness of chronic exercise training in improving survival in patients with congestive heart failure (CHF) are inconclusive. Therefore, we conducted a study to determine the effect of exercise training on survival in a well-defined animal model of heart failure (HF), using the lean male spontaneously hypertensive HF (SHHF) rat. In this model, animals typically present with decompensated, dilated HF between approximately 18 and 23 mo of age. SHHF rats were assigned to sedentary or exercise-trained groups at 9 and 16 mo of age. Exercise training consisted of 6 mo of low-intensity treadmill running. Exercise training delayed the onset of overt HF and improved survival (P < 0.01), independent of any effects on the hypertensive status of the rats. Training delayed the myosin heavy chain (MyHC) isoform shift from alpha- to beta-MyHC that was seen in sedentary animals that developed HF. Exercise was associated with a concurrent increase in cardiomyocyte length (approximately 6%), width, and area and prevented the increase in the length-to-width ratio seen in sedentary animals in HF. The increases in proteinuria, plasma atrial natriuretic peptide, and serum leptin levels observed in rats with HF were suppressed by low-intensity exercise training. No significant alterations in sarco(endo)plasmic reticulum Ca2+ ATPase, phospholamban, or Na+/Ca2+ exchanger protein expression were found in response to training. Our results indicate that 6 mo of low-intensity exercise training delays the onset of decompensated HF and improves survival in the male SHHF rat. Similarly, exercise intervention prevented or suppressed alterations in several key variables that normally occur with the development of overt CHF. These data support the idea that exercise may be a useful and inexpensive intervention in the treatment of HF.     

The antihypertensive action of arachidonic acid in the spontaneous hypertensive rat and its antagonism by anti-inflammatory agents

Changes in arterial blood pressure and heart rate were observed in the spontaneous hypertensive (SH) rat following the intravenous administration of arachidonic acid, the precursor of prostaglandin E₂ (PGE₂). The pronounced fall in blood pressure and the increase in heart rate induced by arachidonic acid were also observed in SH rats receiving either prostaglandin E₁ (PGE₁) or PGE₂. In SH rats receiving various anti-inflammatory agents the cardiovascular responses to arachidonic acid were inhibited, but the blood pressure responses to the E-type prostaglandins were not altered. The data are interpreted to suggest that cardiovascular changes induced by arachidonic acid are mediated via its conversion to PGE₂.     

Distribution of phosphatidylethanolamine arachidonic acid in platelet membranes

Arachidonic acid (20:4) and other fatty acids and aldehydes in phosphatidylethanolamine (PE) present on the platelet surface was determined. Surface-exposed PE was isolated by using 2,4,6-trinitrobenzenesulfonate, a nonpenetrating probe (Schick, P.K., Kurica, K.B. and Chacko, G.K. (1976) J. Clin. Invest. 57, 1221–1226). PE contains 50% total platelet arachidonic acid. Approx. 16% platelet PE is present on the platelet surface. The study showed that the fatty acid and aldehyde composition of PE on the platelet surface is virtually identical to that in PE present inside the platelet. Therefore, 8 nmol arachidonic acid are present in PE in the outer layer of the plasma membrane in 10⁹ platelets.     

The arachidonic acid effect on platelet nitric oxide level

Arachidonic acid can act as a second messenger regulating many cellular processes among which is nitric oxide (NO) formation. The aim of the present study was to investigate the molecular mechanisms involved in the arachidonic acid effect on platelet NO level. Thus NO, cGMP and superoxide anion level, the phosphorylation status of nitric oxide synthase, the protein kinase C (PKC), and NADPH oxidase activation were measured. Arachidonic acid dose-dependently reduced NO and cGMP level. The thromboxane A₂ mimetic U46619 behaved in a similar way. The arachidonic acid or U46619 effect on NO concentration was abolished by the inhibitor of the thromboxane A₂ receptor SQ29548 and partially reversed by the PKC inhibitor GF109203X or by the phospholipase C pathway inhibitor U73122. Moreover, it was shown that arachidonic acid activated PKC and decreased nitric oxide synthase (eNOS) activities. The phosphorylation of the inhibiting eNOSthr495 residue mediated by PKC was increased by arachidonic acid, while no changes at the activating ser1177 residue were shown. Finally, arachidonic acid induced NADPH oxidase activation and superoxide anion formation. These effects were greatly reduced by GF109203X, U73122, and apocynin. Likely arachidonic acid reducing NO bioavailability through all these mechanisms could potentiate its platelet aggregating power.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70-80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly 3H2O. The eicosanoid metabolites were fractionated into different polarity classes using reverse phase Sep-Pak C18 cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime-tert-butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotopic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

Essential fatty acid deficiency and platelet fatty acids of normotensive and genetically hypertensive rats

Termination of pregnancy in missed abortion and intra-uterine fetal death was accomplished using vaginal suppositories of 20 mg PGE₂ in 31 cases and the results were compared with oxytocin induction (with or without estrogen pre-treatment) in 17 cases at the doses routinely used in our hospital. The PG suppositories proved much more superior (96.7%) than oxytocin (47.7%), but induced a higher rate of side effects. The latter were not serious and were generally tolerated by the patients. There was a positive correlation between duration of fetal retention in utero and the induction expulsion time. The over all patient acceptance of the method was quite favourable and the approach appears to be a definite advance towards management of these cases.     

Effects of arachidonic acid and indomethacin on the in vitro release of prostaglandins by aortic strips of spontaneously hypertensive rats

Aortic strips removed from spontaneously hypertensive (SH) rats and preincubated with arachidonic acid (1.0 X 10(-5) g/ml) for 15 min produced two times more prostaglandin (PG) like material than aortae unexposed to the precursor of PG biosynthesis. The stimulating effect of arachidonic acid was largely inhibited by indomethacin (1.0 X 10(-5) g/ml). Also, the release of PG-like material by aortic strips derived from SH rats treated with an intravenous injection of indomethacin (10 mg/kg) was inhibited by 74% compared with the control tissues. These results raised the possibility that the in vivo conversion of arachidonic acid by large arteries of SH rats may contribute to the hypotensive effect of this PG precursor in SH rats.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70–80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and he represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly ³H₂O. The eicosanoid metabolites were fractioned into different polarity classes using reverse phase Sep-Pak C₁₈ cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime- -butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotropic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

Synthesis of all-trans arachidonic acid and its effect on rabbit platelet aggregation

A simple and high-yielding method to convert natural all-cis PUFA derivatives to the corresponding all-trans geometrical isomers is described. The method is based on the thiyl radical-catalyzed cis-trans isomerization. The all-trans isomer of arachidonic acid was found to cause rabbit platelet aggregation at concentrations higher than 0.1 mM and inhibition of PAF-induced platelet aggregation in a concentration dependent manner with an IC(50) in the micromolar range.     

Potential role of antioxidants during ethanol-induced changes in the fatty acid composition and arachidonic acid metabolites in male Wistar rats

Biochemical assessment of liver damage during ethanol-induced stress was done by measuring the activities of serum enzymes, viz., aspartate transaminase (AST) and alkaline phosphatase (ALP), which were significantly elevated in rats fed ethanol. Ethanol administration for a period of 60 days modifies the fatty acid composition, and the analysis of fatty acids showed that there was a significant increase in the concentrations of palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) in liver, kidney, and brain, whereas the concentrations of palmitoleic (16:1) and arachidonic acid (20:4) were significantly decreased. The breakdown products of arachidonic acids (20:4), prostaglandins, were elevated. The antioxidants curcumin and N-acetylcysteine (NAC) decreased the activities of serum AST and ALP. Curcumin and NAC decreased the concentrations of fatty acids, viz., palmitic, stearic, and oleic acid, whereas arachidonic acid and palmitoleic acid were elevated. The prostaglandin concentrations were also decreased after curcumin and N-acetylcysteine treatment. Thus the present investigation shows that curcumin and N-acetylcysteine prevent the fatty acid changes produced by ethanol and also reduce the inflammatory response of ethanol by reducing the level of prostaglandins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of platelet arachidonic acid 12-lipoxygenase by acetylenic acid compounds

Eighteen acetylenic fatty acids were tested as inhibitors of human platelet arachidonic acid 12-lipoxygenase. 4,7,10,13-Eicosatetraynoic (4,7,10,13-ETYA) acid emerged as the most potent compound. Additional experiments have shown that 4,7,10,13-ETYA selectively blocked the 12-lipoxygenase in washed human platelets with lesser activity against the cyclooxygenase. The ID50 value for lipoxygenase was 7.8 microM in comparison with an ID50 of 100 microM for the cyclooxygenase. The commonly used inhibitor 5,8,11,14-eicosatetraynoic acid inhibited both enzymes with equal potency. It appears that 4,7,10,13-ETYA may be a valuable lead for selective modulation of the 12-lipoxygenase pathway in platelet or other target tissues.