Wayne R. Leifert

Cardioprotective actions of grape polyphenols.

The aim of this review is to discuss the accumulating evidence that suggests that grape extracts and purified grape polyphenols possess a diverse array of biological actions and may be beneficial in the prevention of some inflammatory-mediated diseases including cardiovascular disease. The active components from grape extracts, which include the grape seed, grape skin, and grape juice, that have been identified thus far include polyphenols such as resveratrol, phenolic acids, anthocyanins, and flavonoids. All possess potent antioxidant properties and have been shown to decrease low-density lipoprotein-cholesterol oxidation and platelet aggregation. These compounds also possess a range of additional cardioprotective and vasoprotective properties including antiatherosclerotic, antiarrhythmic, and vasorelaxation actions. Although not exclusive, antioxidant properties of grape polyphenols are likely to be central to their mechanism(s) of action, which also include cellular signaling mechanisms and interactions at the genomic level. This review discusses some of the evidence favoring the consumption of grape extracts rich in polyphenols in the prevention of cardiovascular disease. Consumption of grape and grape extracts and/or grape products such as red wine may be beneficial in preventing the development of chronic degenerative diseases such as cardiovascular disease.

Inhibition of cardiac sodium currents in adult rat myocytes by n-3 polyunsaturated fatty acids.

1 The acute effects of n‐3 polyunsaturated fatty acids were determined on whole‐cell sodium currents recorded in isolated adult rat ventricular myocytes using patch clamp techniques. 2 The n‐3 polyunsaturated fatty acids docosahexaenoic acid (22:6, n‐3), eicosapentaenoic acid (20:5, n‐3) and α‐linolenic acid (18:3, n‐3) dose‐dependently blocked the whole‐cell sodium currents evoked by a voltage step to −30 mV from a holding potential of −90 mV with EC50 values of 6.0 ± 1.2, 16.2 ± 1.3 and 26.6 ± 1.3 μM, respectively. 3 Docosahexaenoic acid, eicosapentaenoic acid and α‐linolenic acid at 25 μM shifted the voltage dependence of activation of the sodium current to more positive potentials by 9.2 ± 2.0, 10.1 ± 1.1 and 8.3 ± 0.9 mV, respectively, and shifted the voltage dependence of inactivation to more negative potentials by 22.3 ± 0.9, 17.1 ± 3.7 and 20.5 ± 1.0 mV, respectively. In addition, the membrane fluidising agent benzyl alcohol (10 mM) shifted the voltage dependence of activation to more positive potentials by 7.8 ± 2.5 mV and shifted the voltage dependence of inactivation to more negative potentials (by −24.6 ± 3.6 mV). 4 Linoleic acid (18:2, n‐6), oleic acid (18:1, n‐9) and stearic acid (18:0) were either ineffective or much less potent at blocking the sodium current or changing the voltage dependence of the sodium current compared with the n‐3 fatty acids tested. 5 Docosahexaenoic acid, eicosapentaenoic acid, α‐linolenic acid and benzyl alcohol significantly increased sarcolemmal membrane fluidity as measured by fluorescence anisotropy (steady‐state, rss, values of 0.199 ± 0.004, 0.204 ± 0.006, 0.213 ± 0.005 and 0.214 ± 0.009, respectively, compared with 0.239 ± 0.002 for control), whereas stearic, oleic and linoleic acids did not alter fluidity (the rss was not significantly different from control). 6 The potency of the n‐3 fatty acids docosahexaenoic acid, eicosapentaenoic acid and α‐linolenic acid to block cardiac sodium currents is correlated with their ability to produce an increase in membrane fluidity.

Cardiovascular Biology of Interleukin-6

Interleukin-6 (IL-6) is a multifunctional pro-inflammatory cytokine that is tightly regulated and expressed at low levels in healthy individuals. Increased IL-6 expression has been associated with a variety of diseases, including inflammatory conditions such as atherosclerosis and cardiovascular disease (obesity, myocardial infarction and type II diabetes). Cytokines including IL-6 and tumour necrosis factor alpha as well as acute phase proteins such as C-reactive protein (CRP) and fibrinogen are key biochemical risk factors for the development of these disease conditions. IL-6 is the key cytokine responsible for the stimulus of synthesis and secretion of CRP. IL-6 activates cell surface signalling via the assembly of IL-6, the IL-6 receptor (IL-6R) and the signalling receptor gp130. Assembly of the (hexameric) signalling complex of IL-6, IL-6R and gp130 occurs in a sequential manner and therefore this signalling complex lends itself to several potential sites for drug targeting. This review discusses some of the mechanisms of IL-6 signalling on various aspects of cardiovascular biology as well as some recent developments in drug targeting of this complex.

Termination of asynchronous contractile activity in rat atrial myocytes by n-3 polyunsaturated fatty acids

A protective effect of the n-3 polyunsaturated fatty acids (PUFAs) in preventing ventricular fibrillation in experimental animals and cultured cardiomyocytes has been demonstrated in a number of studies. In this study, a possible role for the n-3 PUFAs in the treatment of atrial fibrillation (AF) was investigated at the cellular level using atrial myocytes isolated from young adult rats as the experimental model. Electrically-stimulated, synchronously-contracting myocytes were induced to contract asynchronously by the addition of 10 μM isoproterenol. Asynchronous contractile activity was reduced following acute addition of the n-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) at 10 μM, compared with no fatty acid addition (from 99.0 ±: 1.0% to 30.7 ± 5.2% (p < 0.05) for DHA and 23.8 ± 2.8% (p < 0.01) for EPA), while the saturated fatty acid, docosanoic acid (DA) and the methyl ester of DHA (DHA m.e.) did not exert a significant effect on asynchronous contractile activity. Asynchronous contractile activity was also reduced to 1.7 ± 1.7% in the presence of the membrane fluidising agent, benzyl alcohol (p < 0.001 vs no fatty acid addition). Cell membrane fluidity was determined by steady state fluorescence anisotropy using the fluorescent probe, TMAP-DPH. Addition of DHA, EPA or benzyl alcohol significantly increased sarcolemmal membrane fluidity (decreased anisotropy, rss) of atrial myocytes compared with no addition of fatty acid (control) (from rss = 0.203 ±0.004 to 0.159 ± 0.004 (p < 0.01) for DHA, 0.166 ± 0.001 (p < 0.01) for EPA and 0.186 ±0.003 (p < 0.05) for benzyl alcohol, while DA and DHA m.e. were without effect. It is concluded that the n-3 PUFAs exert anti-asynchronous effects in rat atrial myocytes by a mechanism which may involve changes in membrane fluidity.

Grape seed and red wine polyphenol extracts inhibit cellular cholesterol uptake, cell proliferation, and 5-lipoxygenase activity.

Accumulating evidence suggests that grape seed and wine polyphenol extracts possess a diverse array of actions and may be beneficial in the prevention of inflammatory-mediated disease such as cardiovascular disease and cancer. This study aimed to determine whether the reported pleiotropic effects of several polyphenolic extracts from grape seed products or red wine would also include inhibition of cholesterol uptake and cell proliferation, and inhibit a known specific target of the inflammatory process, that is, 5-lipoxygenase (5-LOX). Incubation of HT29, Caco2, HepG2, or HuTu80 cells in a medium containing [(3)H]cholesterol in the presence of a grape seed extract (GSE) or red wine polyphenolic compounds (RWPCs) inhibited [(3)H]cholesterol uptake by up to 66% (which appeared maximal). The estimated IC(50) values were 60 and 83 microg/mL for RWPC and GSE, respectively. Similar cholesterol uptake inhibitory effects were observed using the fluorescent cholesterol analogue NBD cholesterol. The inhibition of cholesterol uptake was independent of the samples (GSE and RWPC) potent antioxidative capacity. Red wine polyphenolic compound and GSE dose dependently inhibited HT29 colon adenocarcinoma cell proliferation, which was accompanied by an increase in apoptosis. In addition, RWPC and GSE inhibited 5-LOX activity with the IC(50) values being 35 and 13 microg/mL, respectively. Two of 3 other GSEs tested also significantly inhibited 5-LOX activity. Inhibition of cholesterol uptake and proinflammatory 5-LOX activity may be beneficial in preventing the development of chronic degenerative diseases such as cardiovascular disease and cancer.

G-Protein-Coupled Receptors in Drug Discovery: Nanosizing Using Cell-Free Technologies and Molecular Biology Approaches

Signal transduction by G-protein-coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to activation of a genericmolecular switch involving heterotrimeric G-proteins and guanine nucleotides. Signal transduction has been studied extensively with both cell-based systems and assays comprising isolated signaling components. Interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, highthroughput screening, biosensors, and so on will focus greater attention on assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell-based assays are adequate for many GPCRs, it is likely that these formatswill limit the development of higher density GPCRassay platforms mandatory for other applications. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR assay platforms adaptable for such applications as microarrays. The authors review current cell-free GPCR assay technologies and molecular biological approaches for construction of novel, functional GPCR assays.

Dietary fish oil prevents asynchronous contractility and alters Ca2+ handling in adult rat cardiomyocytes

This study examined the effects of dietary incorporation of n-3 polyunsaturated fatty acids (PUFAs) into cardiac membrane phospholipids on Ca(2+) handling (using Fura-2) and arrhythmic contractility in electrically-stimulated, adult rat ventricular cardiomyocytes. Dietary lipid supplementation with fish oil (FO) for 3 weeks significantly increased the proportion of total n-3 polyunsaturated fatty acids (in particular, docosahexaenoic acid) in ventricular membrane phospholipids compared with a saturated fat (SF) supplemented diet (26.2 +/- 0.9% vs 6.9 +/- 0.9%, respectively, P < 0.001). Cardiomyocytes isolated from the FO group were significantly (P < 0.001) less susceptible to isoproterenol-induced arrhythmic contractile activity compared with the SF group over a range of isoproterenol concentrations. Isoproterenol (0.5 &mgr;M) stimulation increased end-diastolic and systolic [Ca(2+)](i) to a similar extent in both groups. The time constant of Ca(2+) transient decay was significantly increased in the FO group compared with the SF group (98.4 +/- 2.8 ms, n = 8 and 86.9 +/- 2.1 ms, n = 8, P < 0.01, respectively). The effect of dietary n-3 PUFA incorporation into membrane phospholipids was not associated with changes in sarcoplasmic reticulum Ca(2+) content (measured by rapid application of caffeine) or membrane fluidity. The increase in the time constant of decay of Ca(2+) transients following dietary supplementation with FO may indicate altered functioning of the sarcolemmal Na(+)-Ca(2+) exchanger by n-3 PUFA incorporation into membrane phospholipids.

Effects of dietary n-3 fatty acids on contractility, Na+ and K+ currents in a rat cardiomyocyte model of arrhythmia

The n-3 polyunsaturated fatty acids (PUFAs) have been reported to prevent ventricular fibrillation in human clinical studies and in studies involving experimental animals and isolated cardiomyocytes. This study aimed to determine whether dietary n-3 PUFAs could prevent isoproterenol and free radical-induced arrhythmic (asynchronous) contractile activity in adult rat cardiomyocytes and whether whole-cell Na(+) and K(+) currents measured by patch-clamp techniques were affected. Dietary supplementation with fish oil for 3 weeks significantly increased the proportion of total n-3 PUFAs in ventricular membrane phospholipids compared with saturated fat supplementation (18.8 +/- 0.6% vs. 8.1 +/- 1.0%, respectively). Cardiomyocytes from the fish oil group were less susceptible to isoproterenol-induced asynchronous contractile activity than were those from the saturated fat group [EC(50) values: 892 +/- 130 nM, n = 6 and 347 +/- 91 nM, n = 6 (P < 0.05), respectively]. Fish oil supplementation also prolonged the time taken to develop asynchronous contractile activity induced by superoxide and hydrogen peroxide. The voltage dependence of inactivation of Na(+) currents were significantly altered (-73.5 +/- 1.2 mV, n = 5 vs. -76.7 +/- 0.7 mV, n = 5, P < 0.05, for saturated fat and fish oil treated groups, respectively). The voltage dependence of activation of Na(+) and K(+) currents was not significantly affected by the dietary fish oil treatment. These results demonstrate the antiarrhythmic effects of dietary fish oil in a cardiomyocyte model of arrhythmia.

G‐protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H1‐histamine or M2‐muscarinic receptors

This paper describes a novel strategy to create a microarray of G‐protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H1‐histamine receptor and the M2‐muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol‐modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM‐D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self‐sorting liposome array of GPCRs which would underpin a variety of future novel applications.

Membrane fluidity changes are associated with the antiarrhythmic effects of docosahexaenoic acid in adult rat cardiomyocytes

Previous studies using neonatal rat cardiomyocytes have reported antiarrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs). In this study, we examined the effects of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) on the spontaneous contractile activity and membrane fluidity of adult rat ventricular myocytes. Cardiomyocytes were induced to contract spontaneously by continuous superfusion of a solution containing the arrhythmogenic agents isoproterenol (a beta-adrenergic receptor agonist) or lysophosphatidylcholine. The percentage of cardiomyocytes displaying spontaneous contractions induced by isoproterenol when pretreated with the saturated fatty acid docosanoic acid was 48.1 +/- 7.7%; the percentage for cardiomyocytes pretreated with DHA was 7.1 +/- 2.4% (P < 0.01). DHA significantly prevented lysophosphatidylcholine-induced spontaneous contractions (17.7 +/- 6.5%) compared with treatment with the saturated fatty acid stearic acid (78.0 +/- 7.3%, P < 0.01). The membrane fluidizing agent benzyl alcohol also significantly prevented spontaneous contractions in cardiomyocytes. Membrane fluidity was determined by steady-state fluorescence anisotropy (r(ss)) using the fluorescent probe N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl) trimethyl-ammonium p-toluene-sulfonate (TMAP-DPH). DHA and benzyl alcohol dose-dependently decreased the r(ss); however, saturated fatty acids were without effect. These results suggest that the antiarrhythmic mechanisms of the n-3 PUFAs such as DHA may involve changes in membrane fluidity.