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The rapid generation of net inward sodium current (peak INa) via voltage-gated sodium channel (VGSC) activation is responsible for the fast initiation of the cardiac action potential. VGSCs normally inactivate within 10 ms and thus sodium entry is usually limited to the depolarization phase of the action potential. However, under several circumstances, the inactivation process is dysfunctional, resulting in the development of a persistent current (late INa). This is a key feature of several pathologies, including long QT syndrome 3 (LQT3), in which congenital mutations in the SCN5A gene lead to impaired inactivation of cardiac VGSCs, resulting in a proarrhythmic prolonged action potential (Wang et al., 1995; Janse, 1999). The increased influx of sodium during the action potential plateau phase may promote the formation of after-depolarizations, leading to severe types of ventricular tachycardia such as torsade de pointes (Janse, 1999). Furthermore, late INa has been implicated in heart failure and during ischaemia-reperfusion (I/R) injury (Maltsev et al., 1998; Xiao and Allen, 1999). The accumulation of cytosolic sodium through persistent VGSC activity may also lead to calcium overload via increased reverse-mode sodium–calcium exchanger current (INa/Ca) (Ravens and Himmel, 1999). This may cause the formation of premature after-depolarizations and triggered activity, leading to ventricular arrhythmias as well as irreversible cell injury via calcium loading and subsequent hypercontracture (Janse, 1999).
VGSC inhibitors represent an important class of antiarrhythmic agents that include lidocaine and mexiletine (Liu et al., 2003). Although some of these compounds possess desirable features, such as frequency-dependence of block, they may inhibit peak INa to the same extent as late INa. Identification of novel VGSC inhibitors that selectively inhibit late INa may provide useful, pathology-specific, pharmacological tools. For example, the antianginal compound ranolazine is proposed to act via inhibition of late INa and provide cardioprotection by a subsequent reduction of calcium overload without altering normal conduction (Antzelevitch et al., 2004).
Many bioactive compounds contain one or more phenol rings in their structure and several of them have demonstrated cardioprotective efficacy. In particular, red grape products such as red wine are important sources of several dietary polyphenols with concentrations estimated to range from 0.5 to 200 μg ml−1 for catechin and epicatechin, 0.25–50 μg ml−1 for quercetin and 0.05–8.5 μg ml−1 for resveratrol (Bertelli et al., 1998; Ray et al., 1999; Leonard et al., 2003; Gambuti et al., 2004; Manach et al., 2004). These polyphenolic constituents are thought to be responsible for the well-documented cardiovascular benefits of red wine and for the ‘French Paradox’ of low mortality from cardiovascular disease (Renaud and de Lorgeril, 1992).
Resveratrol is a well-characterized grape polyphenol that has exhibited a multitude of cardioprotective properties in vitro and in vivo (Frankel et al., 1993; Bertelli et al., 1995; Pace-Asciak et al., 1995; Rotondo et al., 1998; Ray et al., 1999; Hung et al., 2000; Cao and Li, 2004), including antiplatelet (Bertelli et al., 1995; Pace-Asciak et al., 1995), antioxidative (Frankel et al., 1993; Cao and Li, 2004), anti-ischaemic (Ray et al., 1999; Hung et al., 2000) and antiarrhythmic (Hung et al., 2000) effects. In models of I/R injury, resveratrol improves functional recovery (Ray et al., 1999) and reduces both infarct size (Ray et al., 1999; Hung et al., 2000) and the severity of resultant ventricular arrhythmias (Hung et al., 2000). The reductions in I/R-induced cell damage may result from increased nitric oxide synthesis and other antioxidant effects of resveratrol but a separate, as yet uncharacterized mechanism may be involved, related to a reduction of I/R-induced arrhythmias (Hung et al., 2004), suggestive of an effect on cardiac ion channel function. For example, resveratrol has recently shown inhibition of L-type calcium channels and reductions in action potential duration (Liew et al, 2005).
The red grape polyphenols catechin, quercetin and resveratrol share the common structural feature of one or more phenolic rings with VGSC-blocking drugs such as lidocaine (Figure 1c). As the phenol group found in lidocaine is thought to impart late VGSC block (Zamponi and French, 1993; Haeseler et al., 2002), we hypothesized that red grape polyphenolic compounds may also inhibit peak and/or late INa, contributing to the documented beneficial effects of these grape polyphenols. Supporting this hypothesis, it has recently been shown that resveratrol inhibits neuronal VGSCs (Kim et al., 2005).
Figure 1. (a) Representative traces of recombinant INa through VGSCs expressed in tsA201 cells in the presence of 0 or 15 μg ml−1 grape extract. (b) Grape extract (15 μg ml−1, n=4, P<0.05) inhibited peak recombinant INa but did not wash out. (c) Structural formulae of resveratrol, catechin and quercetin, antioxidant polyphenols with substituted aromatic rings. Lidocaine is a VGSC blocker containing a similar aromatic group. N-acetylcysteine is an antioxidant without structural similarity to these compounds.
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Grape products contain flavonoids and stilbenes, including catechins, quercetin and resveratrol, which have demonstrated benefits in vitro and in vivo. Consumption of red wine (20–30 g alcohol day−1) has been associated with a 40% reduction in risk of coronary heart disease (CHD) and a diet rich in flavonoids (30 mg day−1) has been linked to a 50% decrease in CHD mortality (Renaud and de Lorgeril, 1992). While polyphenols have cardioprotective effects related to their antioxidant properties (Frankel et al., 1993; Hung et al., 2000; Brito et al., 2002), it has emerged in recent years that they may also interact with intracellular and cell-surface proteins, including ion channels, independently of these actions (Li et al., 2000; Dobrydneva et al., 2002; Wallerath et al., 2002; Cao and Li, 2004; Orsini et al., 2004; Kim et al., 2005). Examples of these include potassium channels (Orsini et al., 2004), calcium channels (Dobrydneva et al., 2002) and neuronal sodium channels (Kim et al., 2005).
In addition, we now show that several grape polyphenols directly inhibit cardiac VGSCs and that this mechanism may contribute to the cardioprotective effects of these polyphenols. Specifically, our data demonstrate that application of red grape extract at a concentration relevant to the diet (15 μg ml−1; Manach et al., 2004), significantly inhibits wild-type VGSCs (Figure 1a and b). Three of the most common polyphenols in grape extract are quercetin, catechin and resveratrol (Figure 1c), and these polyphenols, in that order of potency, dose-dependently inhibit INa (Figure 2e).
We also provide direct evidence that the polyphenol resveratrol may exert some of its effects via inhibition of late INa. Two models of late INa were used, differing in their mechanisms: the LQT3 mutant R1623Q introduces an intrinsic change in the structure of the VGSC, uncoupling activation and inactivation (Kambouris et al., 1998), and the toxin ATXII, by destabilizing the inactivated state (Chahine et al., 1996), allows the VGSC to more readily return to the open state. The latter model may be used to pharmacologically manipulate late INa in the recombinant system and use these results as the basis for experiments on native myocytes. Resveratrol inhibits late INa to a ∼2-fold greater extent than peak INa in both models of late INa (Figures 4d and 5b). In comparison, quercetin also displayed some selectivity in inhibiting late INa vs peak INa block, although not as great as resveratrol and only in the ATXII model of late INa (Figure 4d vs Figure 5b and d). These differences may be attributable to the structural differences between resveratrol and quercetin discussed below.
The ability of resveratrol to preferentially inhibit late INa in the heart has direct relevance to pathophysiological states such as LQT3, I/R injury and arrhythmias. In LQT3, prolongation of the APD can be attenuated by reduced late INa, leading to a lower likelihood of after-depolarization formation and subsequent torsade de pointes arrhythmias. The antianginal agent ranolazine exhibits selective inhibition of this current, protecting the heart by preventing APD prolongation (Antzelevitch et al., 2004). In the case of I/R injury, increases in intracellular sodium via late INa and sodium–hydrogen exchanger activity (Lazdunski et al., 1985; Van Emous et al., 1997; Karmazyn et al., 1999) are thought to facilitate calcium overload via reverse-mode INa/Ca, thus a reduction in sodium load via late INa block by polyphenols may reduce or prevent increases in intracellular calcium, reducing cellular damage in the form of irreversible cellular hypercontracture and the generation of arrhythmias. In either of these situations, frequency-independent inhibition of peak INa may be ineffective or even detrimental. Our results show that while resveratrol both prevents and reverses increases in diastolic calcium induced by ATXII, a VGSC-specific toxin (Figure 6), produces a three-fold delay in ATXII-induced contractile dysfunction (Figure 7c) and reduces the incidence of abnormal contractions, defined as premature peaks (Figure 7d), it has no effects in the absence of pathophysiological conditions (results not shown) and does not produce a cessation of synchronously stimulated calcium transients or myocyte contractility, indicating that it is acting primarily through late and not peak VGSC inhibition. The observed lack of direct effect of resveratrol on reverse-mode INa/Ca (Figure 8) further suggests that the beneficial effects of resveratrol on dysfunctional calcium handling and contractility induced by ATXII involve a specific inhibition of persistent VGSC, leading indirectly to reduced reverse-mode INa/Ca. This does not exclude an additional role for L-type calcium channel inhibition (Liew et al., 2005), which could explain discrepancies between the effect of resveratrol on INa and on calcium handling and contractility. It is plausible that other ion channels may also be modulated by these polyphenols, which should be further characterized in future studies.
VGSC blockers, like polyphenols, have a substituted aromatic ring as a common structural feature (Figure 1c). It is thought to be the moiety responsible for binding to their target site, a cluster of hydrophobic residues at the interface of four segments lining the VGSC pore (Ragsdale et al., 1994; Haeseler et al., 2001; Yarov-Yarovoy et al., 2002). In studies in which lidocaine is chemically ‘dissected’ into its hydrophilic and hydrophobic components (Zamponi and French, 1993; Haeseler and Leuwer, 2002; Haeseler et al., 2002), the polar, amine portion exhibits fast, use-dependent open-state block involving changes in channel conductance (Zamponi and French, 1993), while the phenolic part exhibits less frequency-dependence in its inhibition of INa, in which there are changes in open probability and rapid recovery from inactivated-state block (Zamponi and French, 1993; Haeseler et al., 2002). Our finding that resveratrol does not incur use-dependent block of VGSCs further confirms the importance of the charged amine moiety in use-dependent block by lidocaine. It has been theorized that lack of use-dependence of resveratrol could be related to its rapid dissociation from the VGSC (Kim et al., 2005); however, our observations of an absence of washout indicate this to be unlikely. Furthermore, our data indicate that, as with simple phenolics, voltage-dependence of activation and steady-state availability are not affected by resveratrol or quercetin (Figure 2f and g).
A key finding from this study is the ∼2-fold selectivity of resveratrol for late over peak INa compared to the absence of selectivity in the case of quercetin. An analysis of their structures (Figure 1c) suggests that quercetin's bulkier structure, richer in electron-donating groups, may sterically hinder interaction with residues deeper in the VGSC's pore responsible for allowing necessary conformational changes for a late current to result (Yarov-Yarovoy et al., 2002; Leuwer et al., 2004). Differences in the sodium channel residues involved in coupling of activation to inactivation and in inactivation state stabilization may be responsible for discrepancies between results for resveratrol and quercetin in the two models of late INa. In addition, our observation that quercetin is a much more effective VGSC inhibitor than catechin (IC50 values of 19.4 and 76.8 μM, respectively) despite almost identical structures suggests that the presence of the conjugated carbonyl on quercetin's second ring structure may contribute to the observed increase in VGSC inhibitory efficacy.
Additional benefits of resveratrol and other red grape polyphenols may be contributed by the well-documented antioxidant properties of the polyphenols in reducing reperfusion-induced free-radical damage (Hung et al., 2002; Valdez et al., 2002). Changes in redox potential or surface charge may also account for some ionic current block (Bhatnagar et al., 1990), therefore it is possible that the antioxidant properties of polyphenols may contribute to the observed VGSC inhibition. However, parallel experiments with N-acetylcysteine, a structurally unrelated antioxidant lacking a substituted phenolic group (Figure 1c), demonstrated no effect on INa (Figure 2d and e), making this an unlikely mechanism in this case.
Polyphenol concentrations effective for free-radical scavenging are in the 5–20 μM range in vitro (Alvarez et al., 2002; Leonard et al., 2003) and in perfused rat hearts showing ischaemic recovery (Ray et al., 1999), indicating potentially clinically relevant doses. While concentrations of individual polyphenols in the 50 μM range, as used in this study, are unlikely to be reached in human plasma after moderate red wine consumption (Vitaglione et al., 2005), a polyphenol-rich diet may include several dietary sources, raising the total polyphenol concentration above that for each compound alone, possibly imparting additive effects on inhibition of INa.
Our observations that red grape extract and resveratrol are resistant to washout (Figures 1b and 3d) indicate that membrane partitioning and subsequent binding to VGSCs may contribute to the efficacy of these polyphenols even at lower apparent plasma concentrations. This is supported by evidence suggesting that dietary polyphenols may accumulate in tissues, resulting in a higher local concentration of these compounds. For example, resveratrol concentrations were 2.4-fold higher in mouse liver and heart than the concentrations reached in plasma (Sale et al., 2004). Moreover, many polyphenols exist in both their aglycone and their glycoside forms in plasma (Vitrac et al., 2003; Manach et al., 2004); the latter are cleaved by endogenous β-glucosidases to increase polyphenol bioavailability.
In summary, we have demonstrated that several common red grape polyphenols are effective inhibitors of peak and/or late INa. This mechanism may contribute to the observed protective effects of red grape/wine ingestion on cardiac function during I/R injury. This novel protective mechanism involves improved myocyte calcium handling and contractility that is downstream of inhibition of late INa. Given the important role that ion channels play in a variety of disease states, further studies on the modulatory effects of grape polyphenols on other types of ion channels are warranted.