Resveratrol Attenuates the Na+-Dependent Intracellular Ca2+ Overload by Inhibiting H2O2-Induced Increase in Late Sodium Current in Ventricular Myocytes

Background/Aims Resveratrol has been demonstrated to be protective in the cardiovascular system. The aim of this study was to assess the effects of resveratrol on hydrogen peroxide (H2O2)-induced increase in late sodium current (I Na.L) which augmented the reverse Na+-Ca2+ exchanger current (I NCX), and the diastolic intracellular Ca2+ concentration in ventricular myocytes. Methods I Na.L, I NCX, L-type Ca2+ current (I Ca.L) and intracellular Ca2+ properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes. Results Resveratrol (10, 20, 40 and 80 µM) decreased I Na.L in myocytes both in the absence and presence of H2O2 (300 µM) in a concentration dependent manner. Ranolazine (3–9 µM) and tetrodotoxin (TTX, 4 µM), I Na.L inhibitors, decreased I Na.L in cardiomyocytes in the presence of 300 µM H2O2. H2O2 (300 µM) increased the reverse I NCX and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H2O2 of the diastolic intracellular Ca2+ fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca2+ transient rise and decay. As expected, 2 µM TTX had no effect on I Ca.L. Conclusion Resveratrol protects the cardiomyocytes by inhibiting the H2O2-induced augmentation of I Na.L.and may contribute to the reduction of ischemia-induced lethal arrhythmias.


Introduction
Despite intensive research has been conducted in recent years, cardiac arrhythmias remain a serious problem. Late sodium current (I Na.L ) has been recognized as an important factor contributing to the abnormal repolarization in ischemic and failured hearts [1]. I Na.L plays an important role in determining the action potential duration (APD) [2] and the alteration of the intracellular Na + concentration ([Na + ] i ) [3,4]. It has also been reported that hypoxia increased I Na.L in rat ventricular myocytes [4], and the increase in Na + inflow during hypoxia increased [Na + ] i which in turn rose the intracellular Ca 2+ concentration ([Ca 2+ ] i ) via the Na + -Ca 2+ exchanger (NCX) resulting in a Na +dependent intracellular Ca 2+ overload induced by I Na.L [5,6,7]. An increase in [Ca 2+ ] i caused cardiac arrhythmias and irreversible cell damage [8]. Furthermore, increased I Na.L caused arrhythmic activity and contractile dysfunction [9,10]. Therefore, inhibition of I Na.L is considered to be a new potential target for therapeutic intervention in patients with myocardial ischaemia and heart failure [10][11][12][13][14].
Resveratrol (trans-3, 49, 5-trihydroxystilbene), a polyphenol in various vegetables and fruits, is abundant in grapes. The root extracts of Polygonum cuspidatum, a constituent of Chinese and Japanese folk medicine, is also a good source of resveratrol [15]. Sufficient clinical and epidemiological evidence showed that the consumption of red wine reduced the incidence of mortality and morbidity in patients with coronary heart disease [16]. Among all the evidence, the well-known one is now popularly termed as the ''French paradox'' [16,17]. Resveratrol has been considered to be responsible for the cardiovascular benefits after moderate wine consumption [18]. It is speculated that resveratrol may act as an antioxidant, which modulates the vascular cell functions [19], inhibits platelet aggregation [20], and reduces lipoprotein oxidation [21], to serve as a cardioprotective agent. H 2 O 2 , a reactive oxygen species, is a by-product of oxidative metabolism in which energy activation and electron reduction are involved, and was enhanced during ischemia-reperfusion of the heart [22]. Excessive amount of H 2 O 2 augmented I Na.L in ventricular myocytes [10,23], but the reducing agents, e.g., dithiothreitol (DTT) and glutathione (GSH), reversed these changes induced by either H 2 O 2 or hypoxia [24,25]. Since resveratrol acts as an antioxidant [26], we presumed that it might inhibit the increase in I Na.L induced by H 2 O 2 .
To further clarify the pharmacological mechanisms and the scope of application of the agent, it is critical to determine the effect of resveratrol on I Na.L . Previous investigation showed that 50 mM of resveratrol reduced I Na.L in a recombinant expression system with the R1623Q LQT3 mutation [27]. To our knowledge, the effect of resveratrol on I Na.L in ventricular myocytes with increased H 2 O 2 has not been reported. Therefore, this study was designed to address the impact of resveratrol on the Na +dependent Ca 2+ overload induced by H 2 O 2 -induced increase in I Na.L in ventricular myocytes, with the intention to shed some light on its potential clinical application in the future.

Isolation of Ventricular Myocytes
Adult New Zealand white rabbits (body weight 1.7-2 kg) of either sex were heparinized (2000 U) and anesthetized with ketamine (30 mg kg 21 i.v.) and xylazine (7.5 mg kg 21 i.m.). Hearts were excised rapidly and perfused retrogradely on a Langendorff apparatus for 5 min with a Ca 2+ -free Tyrode's solution containing (in mM): NaCl 135, KCl 5.4, MgCl 2 1, NaH 2 PO 4 0.33, HEPES 10 and glucose 10 (pH 7.4, adjusted with NaOH), and then a Tyrode's solution containing enzyme (collagenase type I, 0.1 g/l) and bovine serum albumin (BSA, 0.5 g/l) for 40-50 min. The perfusate was finally switched to KB solution containing (in mM): KOH 70, taurine 20, glutamic acid 50, KCl 40, KH 2 PO 4 20, MgCl 2 3, EGTA 0.5, HEPES 10, and glucose 10 (pH 7.4), for 5 min. All perfusates were bubbled with 100% O 2 and maintained at 37uC. The left ventricles were then cut into small chunks and gently agitated in KB solution. The cells were filtered through nylon mesh and stored in KB solution at 25uC. The use of animals in this investigation was approved by the Institutional Animal Care and Use Committee of Wuhan University of Science and Technology and conformed to the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication no. 85-23, revised 1996) and the Guide for the Care and Use of Laboratory Animals of Hubei Province, China.

Protocol of Experiments
Isolated cells were perfused with Tyrode's solution saturated oxygenated with 100% O 2 (control) and were then exposed to Tyrode's solution containing 300 mM H 2 O 2 for 7 min. Next, isolated cells were perfused with Tyrode's solution containing both 300 mM H 2 O 2 and one of the following, resveratrol (10, 20, 40 or

Electrical Recordings
Experiments were performed at room temperature (22-24uC). Rabbit ventricular myocytes were placed into a recording chamber that was bathed with normal extracellular solution, in the absence and presence of drug (s), at a rate of 2 ml min 21 . I Na.L , I NCX and I Ca.L were recorded in voltage clamp mode by using whole-cell patch-clamp techniques in rabbit ventricular myocytes. Patch electrodes were pulled with a two-stage puller (PP-830, Narishige Group, Tokyo, Japan). Their resistances were in the range of 1-1.5 MV. Capacitance and series resistances were adjusted to obtain minimal contribution of the capacitive transients. A 60% to 80% compensation of the series resistance was usually achieved without ringing. Currents were obtained with an EPC 9 amplifier (Heka Electronic, Lambrecht, Pfalz, Germany) and a Multiclamp 700B amplifier (Axon Instruments, Inc. USA), filtered at 2 kHz, digitized at 10 kHz, and stored on a computer hard disk for further analysis.

Intracellular Ca 2+ Fluorescence Measurement
Myocytes were loaded with fura-2-AM (0.5 mM) for 10 min at 25uC, and fluorescence measurements were recorded with a dual excitation fluorescence photomultiplier system (Ionoptix). Myocytes were imaged through an Olympus IX-70 Fluor 40 6 oil objective. The cells were field stimulated with a suprathreshold (150%) voltage and at a frequency of 0.5 Hz, 3-ms duration, using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator (Brunswick, NE, USA). The polarity of the stimulatory electrodes was reversed frequently to avoid possible build up of electrolyte by-products. Cells were exposed to light emitted by a 75-W lamp and passed through either a 340-or 380-nm filter (bandwidths were 615 nm) while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the cells at 340 nm for 0.5 s then at 380 nm for the duration of the recording protocol (333 Hz sampling rate). The 360 excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular Ca 2+ level were inferred from the ratio of the fura-fluorescence intensity (FFI) at both wavelengths. Intracellular Ca 2+ fluorescence measurements were assessed using the following indices: diastolic intracellular Ca 2+ level (diastolic FFI) (340/380 ratio), electrically stimulated rise in intracellular Ca 2+ (gFFI) (340/380 ratio), maximal velocity of Ca 2+ rise and Ca 2+ decay (340/380 ratio).

Data Analysis
Whole-cell recordings were analyzed using clampfit 9.0 (Axon Instruments, Inc.USA) and PulseFit (V8.74, HEKA). Figures were plotted by Origin (V7.0, OriginLab Co., MA, USA). All amplitudes of I Na.L were tested at 200 ms in depolarization testing pulse to eliminate the influence of transient sodium current (I Na.T ). Statistical significance between two groups and multiple groups were evaluated by Student's t-test and one-way analysis of variance (ANOVA), respectively. All values were expressed as mean 6 SD, and the number of cells (n) in each group was given. P,0.05 was considered to be statistically significant.

Effects of Resveratrol and TTX on I Na.L Under Normal Condition
To identify I Na.L , the current was recorded first in the absence and then in the presence of 4 mM TTX with 300 ms voltage steps from a holding potential (HP) of 2120 to 220 mV. The values of current recorded before (control condition) and after application of TTX were 20.40060.050 and 20.15460.038 pA pF 21 (n = 6, P,0.05 versus control), respectively, indicating that this TTXsensitive current recorded was I Na.L . When I Na.L was recorded under normal condition using depolarizing pulses with a duration of 300 ms applied at 0.25 Hz from a HP of 2120 mV in 10 mV increments between 270 and 220 mV, administration of 10, 20, 40 and 80 mM resveratrol resulted in decreased amplitudes of I Na.L in a concentration dependent manner ( Figure 1A, 1B). Figure 1B showed the I-V relationship of I Na.L after the administration of 10, 20, 40 and 80 mM resveratrol, without a shift of the voltage at which the I Na.L amplitude was maximal ( Figure 1B). Figure 1C shows the inhibition amounts of 10, 20, 40 and 80 mM resveratrol on the I Na.L with an IC 50  Currents were recorded using depolarizing pulses with a duration of 300 ms at a rate of 0.25 Hz from a HP of 2120 mV, in 10 mV increments between 270 and 220 mV. Administration of resveratrol at concentrations of 10, 20, 40 and 80 mM resulted in a decrease in the amplitudes of I Na.L in a concentration dependent manner in myocytes exposed to H 2 O 2 ( Figure 2). H 2 O 2 (300 mM) increased the amplitudes of I Na.L but 10, 20, 40 and 80 mM resveratrol decreased the amplitudes of I Na.L in the continued presence of H 2 O 2 (Figure 2A). Shown in figure 2B are the I-V relationships of I Na.L after the sequential application of 300 mM H 2 O 2 , 10, 20, 40 and 80 mM resveratrol respectively, without a shift of the voltage at which the I Na.L amplitude was maximal. Figure 2C shows the inhibition amounts of 10, 20, 40 and 80 mM  figure 3B are the I-V relationships of I Na.L after the sequential application of 300 mM H 2 O 2 in the absence and presence of 3, 6 and 9 mM ranolazine respectively, without a shift of the voltage at which the I Na.L amplitude was maximal. Figure 3C shows the inhibition amounts of 3, 6 and 9 mM ranolazine on the gI Na.L induced by 300 mM H 2 O 2 with 300 ms voltage steps from a HP of 2120 to 220 mV with an IC 50

Effects of Resveratrol, Ranolazine and TTX on Increased Electrogenic I NCX by H 2 O 2
Electrogenic I NCX was measured to determine whether the reverse NCX was activated by the increase of I Na.L ,. Membrane currents were elicited using ramp voltage-clamp pulses from a HP of 240 mV to +60 mV for 100 ms and then ramped to 2120 mV over a period of 2 seconds (i.e. at 90 mV s 21 ) before returning to 240 mV. The current-time relationship was constructed from the declining slope of the ramp pulse ( Figure 4A, 4B, 4D, 4E, 4G, 4H). Figure 4B, 4E, 4H shows the Ni 2+ -sensitive (NCX) current obtained by subtracting the data in the trace d, h or l from the data in the trace a, e, i, b, f, j, c, g or k in the panel 4A, 4D or 4G. Figure 4C, 4F, 4I are I NCX measured at voltage levels of +50 mV and 2100 mV, respectively, as the Ni 2+ -sensitive current by subtracting the current recorded in the presence from that in the absence of 5 mM NiCl 2 .

Effects of TTX on I Ca.L
Recent evidence suggests a potential for TTX to inhibit L-type Ca 2+ channels [28]. The results in this study showed that 2 mM TTX inhibited H 2 O 2 -induced augmentations in diastolic Ca 2+ concentration and amplitude of calcium transients. To identify the effect of 2 mM TTX on intracellular Ca 2+ was from its blocking of I Na.L but not the inhibition of L-type Ca 2+ channels, I Ca.L was measured. The results indicated that at a low concentration (2 mM) TTX is relatively a selective I Na.L blocker. Using depolarizing pulses with a duration of 300 ms applied at 0.5 Hz from a HP of 280 mV, in 10 mV increments between 240 and +40 mV, I Ca.L was recorded in the absence ( Figure 6A) and presence ( Figure 6B) of 2 mM TTX. Figure 6C showed the effect of TTX (2 mM) application on the current-voltage relationship of I Ca.L . TTX at concentration of 2 mM had no effect on I Ca.L , accounting for that the effects of 2 mM TTX to inhibit H 2 O 2induced augmentations in diastolic Ca 2+ concentration and amplitude of calcium transients were from its inhibition on I Na.L and subsequently the reverse I NCX .

Discussion
The mechanisms underlying the genesis of ischemia-and reperfusion-induced arrhythmias are notoriously complex and controversial. There has been an interest in the concept that oxygen free radicals play a role in the pathogenesis of myocardial ischemia and infarction. It has been reported that a burst of H 2 O 2 , an important reactive oxygen species, is generated in the myocardium during ischemia and reperfusion [29][30][31][32][33] and causes Ca 2+ overload through many ways [34,35,36,37]. For example, the activation of ryanodine receptors with H 2 O 2 could also account for the increased cytosolic Ca 2+ levels found with ROS production, which could account for the Ca 2+ overload in cells [34]. Furthermore, the excessive amount of H 2 O 2 could increase I Na.L in cardiomyocytes, subsequently leading to intracellular Ca 2+ overload through reverse NCX (the Na + -dependent Ca 2+ overload induced by I Na.L ) and ultimately causing cell damage [10,23,38,39]. Reducing agent, e.g., dithiothreitol (DTT) and reduced glutathione (GSH), could reverse the increased I Na.L by H 2 O 2 and hypoxia [5,24,25]. Resveratrol, a natural antioxidant, has beneficial effects against coronary heart disease. Previous studies have shown that resveratrol effectively suppressed ischemia/reperfusion-induced arrhythmia [40,41] and reduced both peak I Na and I Na.L in the R1623Q LQT3 mutation in a recombinant expression system [27]. But the effect of resveratrol on the increased I Na.L and reverse I NCX under proarrhythmic conditions (H 2 O 2 ) in rabbit ventricular myocytes has not been investigated yet. The data from this study addressed the impact of resveratrol on the Na + -dependent Ca 2+ overload.
In this study, I Na.L was increased by H 2 O 2 (Figure 2, 3). Ranolazine attenuated the increased I Na.L by H 2 O 2 in a concentration dependent manner ( Figure 3) and 4 mM TTX attenuated the increased I Na.L increased by H 2 O 2 as well. These data are consistent with other reports and our previous studies that the I Na.L inhibitors ranolazine and TTX significantly inhibited late I Na.L at clinical relevant concentrations [42,43]. H 2 O 2 -induced intracellular Na + and Ca 2+ overload was associated with an enhanced I Na.L and therefore was attenuated by the I Na.L inhibitors ranolazine and TTX [10]. The I Na.L blocking agents may be effective in preventing arrhythmias by reducing [Na + ] i load and subsequently the [Ca 2+ ] i load. However, ranolazine has been suggested to inhibit the cardiac ryanodine receptor (IC 50 = 10 mM) [44], which could also modulate intracellular Ca 2+ levels. Ranolazine is currently approved as an antianginal agent that reduces the Na + -dependent Ca 2+ overload via inhibition of the I Na.L and thus improves diastolic tone and oxygen handling during myocardial ischemia [7]. I Na.L is an important contributing factor to intracellular Ca 2+ overload in the pathogenesis of myocardial ischemia and infarction. In rabbit ventricular myocytes, low concentrations of TTX (1.5-4.0 mM) did not alter the L-type Ca 2+ current ( Figure 6) and I Na.T [6,25,45], but obviously inhibited the I Na.L . Accordingly TTX was used to confirm the process of Na + -dependent Ca 2+ overload induced by H 2 O 2 . The effect of resveratrol on I Na.L is similar to ranolazine and TTX. Resveratrol inhibited I Na.L in both normal and H 2 O 2treated cells in a concentration dependent manner (Figure 1, 2). This result is consistent with our previous studies that DTT and reduced glutathione could reverse the increase in I Na.L induced by either H 2 O 2 or hypoxia [24,25], indicating resveratrol may act as an antioxidant to eliminate the detrimental effects of H 2 O 2 on I Na.L . Changes in redox potential or surface charge may account for some ionic current block [46], therefore it is possible that the antioxidant properties of resveratrol may contribute to the I Na.L inhibition observed in this study.
Recently, it has been reported that reverse I NCX was increased along with the increased I Na.L during hypoxia and was decreased along with the I Na.L inhibition by TTX in hypoxic ventricular myocytes, suggesting that the increased I Na.L contributed to the increase in the reverse I NCX [6]. In this study, 300 mM H 2 O 2 increased the reverse I NCX while the inward I NCX was not affected obviously, whereas ranolazine or TTX attenuated the increase in the reverse I NCX significantly (Figure 4). Different from I Na.T , I Na.L can be blocked by a low concentration of ranolazine and TTX, and the consequent reduction of Na + loading via the decrease of the I Na.L can prevent the increase in the reverse I NCX -induced intracellular Ca 2+ accumulation [47]. Ranolazine (4 mM) and TTX (4 mM) decreased the reverse I NCX through the inhibition of I Na.L. Similarly, resveratrol (20 mM) attenuated the increase in the reverse I NCX by H 2 O 2 . Thus, we concluded that the effect of resveratrol to inhibit the increased reverse I NCX caused by H 2 O 2 was from its inhibition of I Na.L .
In this study, 150 mM H 2 O 2 significantly increased the amplitude of calcium transients and diastolic calcium concentration in the ventricular cell which could be reversed by TTX (2 mM). The intracellular Ca 2+ overload caused by ROS was due to an increase in [Na + ] i followed with an increase in Ca 2+ influx via the reverse mode of the NCX [48]. Then the large entry of Ca 2+ into the cell will cause intracellular Ca 2+ overload [49,50]. TTX also inhibited L-type Ca 2+ channel with an IC 50 value of 5562 mM [28]. In this study in rabbit ventricular myocytes, 2 mM TTX inhibited I Na.L and restrained Ca 2+ overload induced by H 2 O 2 but not affected L-type Ca 2+ channels ( Figure 6), supporting that I Na.L played an important role in the genesis of Ca 2+ overload induced by H 2 O 2 . TTX also reversed the increase in calcium transients amplitude and diastolic calcium concentration through inhibiting the increased I Na.L by H 2 O 2 . Resveratrol (10 mM) also restrained the increased calcium transients amplitude and the diastolic calcium concentration induced by H 2 O 2 (150 mM). Therefore the effects of resveratrol on the Na + -dependent Ca 2+ overload induced by enhanced I Na.L were similar to 2 mM TTX, suggesting that the reduction of Ca 2+ overload by resveratrol may have similar mechanism to TTX, i.e., inhibition of I Na.L . Indeed, resveratrol has also been suggested to inhibit the ryanodine receptor-induced intracellular Ca 2+ increase [51] which may account for the reduction of [Ca 2+ ] i . The results in this study indicated that resveratrol reduced both I Na.L and reverse I NCX which was responsible for the reversal of intracellular Ca 2+ overload in the presence of H 2 O 2 . Resveratrol may inhibit both the ryanodine receptor-induced intracellular Ca 2+ overload and I Na.L -induced increase in reverse I NCX to attenuate the intracellular Ca 2+ overload. Further research will be needed to clarify the contribution of the two pathways by resveratrol in the absence and presence of H 2 O 2 .
Conclusions I Na.L is an important target for resveratrol to prevent or treat ventricular arrhythmias. I Na.L increased by H 2 O 2 induces intracellular Ca 2+ overload (the increased diastolic calcium concentration) through the increase in the reverse I NCX . The inhibitive effect of resveratrol on H 2 O 2 -induced I Na.L may reduce the concentration of [Na + ] i , lower [Ca 2+ ] i by attenuating reverse NCX to eliminate Ca + overload, and ultimately inhibit the electrical abnormalities.