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Am J Physiol Heart Circ Physiol 294: H2031-H2039, 2008. First published February 29, 2008; doi:10.1152/ajpheart.01357.2007
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An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes

Yejia Song,1 John C. Shryock,2 and Luiz Belardinelli2

1Division of Cardiovascular Medicine, University of Florida, Gainesville, Florida; and 2CV Therapeutics, Inc., Palo Alto, California

Submitted 21 November 2007 ; accepted in final form 28 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
This study determined the role of a slowly inactivating component of sodium current (INa), late INa, to induce delayed afterdepolarizations (DADs) and triggered activity. We hypothesized that an increase of late INa may induce not only early afterdepolarizations (EADs), but also intracellular calcium overload and DADs. Guinea pig atrial myocytes were studied using the whole cell patch-clamp technique. Anemone toxin II (ATX-II) (5–10 nmol/l) was used to enhance late INa. Ranolazine (10 µmol/l) and TTX (2 µmol/l) were applied to block ATX-II-induced late INa. ATX-II prolonged action potential duration and induced EADs. In the continuous presence of ATX-II, following the appearance of EADs, both DADs and sustained triggered activity occurred. Triggered activity was abolished and DADs were reduced by either ranolazine or TTX. Consistent with induction of DADs, ATX-II induced the transient inward current (ITI). The amplitude of ITI was significantly reduced by ranolazine. ATX-II induced only EADs, but no DADs, in the presence of the sodium-calcium exchange inhibitor KB-R7943 or the sarcoplasmic reticulum calcium release channel inhibitor ryanodine, or when the calcium chelator EGTA or BAPTA was included in the pipette solution. In conclusion, an increase of late INa, in addition to inducing EADs, can cause cellular calcium overload and induce DADs and sustained triggered activity in atrial myocytes. The data reveal that an increase of late INa is a novel mechanism for initiation of atrial arrhythmic activity.

action potential; calcium overload; transient inward current

CARDIAC AFTERDEPOLARIZATIONS are transient membrane depolarizations that may occur either before [early afterdepolarizations (EADs)] or after [delayed afterdepolarizations (DADs)] completion of action potential repolarization. When their amplitude is sufficiently large, and their timing appropriate, EADs and DADs may activate additional depolarizing current and cause rapid, repetitive action potentials, i.e., triggered activity. Experimental evidence strongly suggests that afterdepolarizations and triggered activity are one of the major cardiac arrhythmogenic mechanisms (12, 30, 46, 47).

The mechanisms of inducing DADs and EADs apparently differ. It is generally agreed that DADs are the consequence of intracellular Ca2+ overload (21). Excessive Ca2+ loading results in oscillatory Ca2+ releases from the sarcoplasmic reticulum (SR) during diastole, which, in turn, activate a transient inward current (ITI) (20, 23). ITI is the underlying ionic current responsible for DADs (23). In contrast, induction of EADs requires a prolongation of the action potential duration (APD) with reactivation of Na+ or Ca2+ channels (9, 19). An increased magnitude of inward Na+-Ca2+ exchange current (INCX) facilitates EAD formation (43), but EADs may occur without significant cellular Ca2+ loading (5, 19, 29, 38). Thus inhibition of Ca2+ release from the SR by ryanodine or intracellular Ca2+ buffering by BAPTA both diminish intracellular Ca2+ transients and suppress DADs, but not EADs (29).

The slowly inactivating component of sodium current (INa), late INa, is a depolarizing current that increases the duration of the ventricular action potential (4, 17, 22) and thereby facilitates induction of EADs in ventricular myocytes (11, 36). Late INa can be enhanced by anemone toxin II (ATX-II) (18) and is sensitive to inhibition by tetrodotoxin (TTX) (10, 27) and the anti-ischemic/antianginal drug ranolazine (1, 36). Due to its persistence throughout the plateau of the action potential, an increase of late INa results in a substantial Na+ loading of cells (26, 28, 32, 35). This, in turn, may lead to cellular Ca2+ overload (13) when intracellular Na+ is exchanged for extracellular Ca2+ via the sodium-calcium exchanger (NCX) (32). In addition, the late INa-induced prolongation of APD can increase Ca2+ entry by facilitation of reactivation of Ca2+ channels that have inactivated earlier during the long action potential plateau (34). In support of this hypothesis, blocking INa has been shown to reduce sodium-dependent calcium loading (16, 35).

Focal ectopic activity arising from triggered activity is considered as one of the major mechanisms underlying atrial tachyarrhythmias (30, 46). The above evidence suggests that Ca2+-dependent triggered activity may be caused by agents and pathological conditions that enhance late INa. Therefore, in this study, we examined the hypothesis that calcium overload due to an increase of late INa may activate ITI and induce DADs in atrial myocytes.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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Cell isolation. Experiments were performed on guinea pig isolated atrial myocytes. Hartley guinea pigs were ordered from Charles River Laboratories (Wilmington, MA), and myocytes were isolated as previously described (36). Briefly, hearts of adult guinea pigs of either sex were isolated and perfused via the aorta with warm (35°C) and oxygenated solutions as follows: 1) Tyrode solution containing (in mmol/l) 135 NaCl, 4.6 KCl, 1.8 CaCl2, 1.1 MgSO4, 10 glucose, and 10 HEPES, pH 7.4, for 5 min; 2) Ca2+-free solution containing (in mmol/l) 100 NaCl, 30 KCl, 2 MgSO4, 10 glucose, 10 HEPES, 15 taurine, and 5 pyruvate, pH 7.4, for 5 min; and 3) Ca2+-free solution containing collagenase (120 U/ml) and albumin (2 mg/ml) for 20 min. At the end of the perfusion, the atria were minced and gently shaken for 10 min in solution 3 to free single cells for study. Animal use was approved by the University of Florida Institutional Animal Care and Use Committee, and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Electrophysiological recording. Transmembrane voltages and currents were measured with an Axopatch-200 amplifier, a Digidata-1322A digitizer, and pCLAMP-9 software (Axon Instruments, Union City, CA). The whole cell patch-clamp technique was used in electrophysiological recording. All experiments were performed at 36°C. Action potentials were elicited by 3-ms depolarizing pulses. The APD was measured from the beginning of depolarization to the time when 30% (APD30) and 90% (APD90) of repolarization were completed. The amplitude of EADs was measured from the take-off potential to the peak of the largest EAD during an action potential. The values obtained from five consecutive action potentials were then averaged. The amplitude of DAD was determined from the resting membrane potential to the peak of the largest (usually the first) DAD following an action potential. To measure ITI, a 200-ms depolarizing pulse from –80 to +20 mV was applied. ITI was induced after the membrane potential repolarized to the holding potential of –80 mV. The amplitude of ITI was measured from the holding current at –80 mV to the peak inward current. Late INa was activated by 300-ms voltage-clamp pulses from –90 to –40 mV. The magnitude of late INa was calculated as integrated current during the last 50 ms of depolarizing pulse.

During measurements of action potential and ITI, myocytes were incubated in the above-mentioned Tyrode solution. The recording pipettes were filled with a solution containing (in mmol/l) 120 potassium-aspartate, 20 KCl, 1 MgSO4, 4 Na2ATP, 0.1 Na3GTP, and 10 HEPES, pH 7.3. When measuring late INa, K+ in both Tyrode and pipette solutions was replaced with Cs+ to minimize contamination with potassium currents, and nitrendipine (10 µmol/l) was added to the bath solution to block calcium channels. All electrical stimuli were applied at a frequency of 0.16 Hz. This relatively slow stimulation frequency was chosen to avoid overdrive-induced Ca2+ overload.

Chemicals. Ranolazine was obtained from CV Therapeutics (Palo Alto, CA), KB-R7943 was from Tocris (Ellisville, MO), and TTX, ATX-II, and ryanodine were from Sigma (St. Louis, MO). Ryanodine was dissolved in dimethyl sulfoxide to form a 10 mmol/l stock solution, which was then diluted to 1 µmol/l in bath solution. All other chemicals were directly dissolved in water. Ranolazine and TTX were applied at a low concentration to selectively block late INa, whereas ATX-II was used to enhance the current.

Statistics. Data are expressed as means ± SE. Values of n indicate the number of cells studied. Percentage of inhibition by TTX or adenosine of the effects of ATX-II was calculated using the formula [(ATX-II – TTX or adenosine)/(ATX-II – control)] x 100, where ATX-II, TTX or adenosine, and control indicate measurements obtained in the presence of ATX-II alone, ATX-II plus TTX or adenosine, and in the absence of drugs, respectively. The paired Student's t-test was used for statistical analysis of paired data, and the one-way repeated-measures ANOVA followed by Student-Newman-Keuls test was applied for multiple comparisons. A P value <0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Effects of ATX-II, ranolazine, and TTX on action potential and afterdepolarization. ATX-II (10 nmol/l) prolonged the APD and induced EADs (Fig. 1B) in all cells tested (n = 31). Following the appearance of EADs, first DADs (Fig. 1C) and then sustained triggered activity (Fig. 1, D and E) were observed when calcium chelators were not included in the pipette solution (n = 19). When the rapid triggered activity occurred, the amplitude of EADs was greatly reduced (Fig. 1E), probably due to a rate-dependent shortening of the APD.


Figure 1
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  		Fig. 1. Typical effect of anemone toxin II (ATX-II) to induce triggered activity of a single myocyte. A: an action potential recorded in the absence of drug (control). B–E: consecutive records of action potentials recorded in the continuous presence of 10 nmol/l ATX-II. B: 3 min after application of ATX-II. Early afterdepolarizations (EADs; thin arrow) appeared. C: 18 s after record B. Delayed afterdepolarizations (DADs; thick arrow) became apparent. D: DAD-triggered action potentials were observed and were followed by sustained triggered activity (E).

 
To determine whether ATX-II-induced sustained triggered activity could be attenuated by inhibition of late INa, ranolazine (10 µmol/l, n = 6) or TTX (2 µmol/l, n = 4) was added to the bath solution when the rapid triggered activity occurred. Figure 2 shows records obtained from an experiment, in which ranolazine and TTX were applied successively in the presence of ATX-II. As shown in the figure, the sustained triggered activity was completely abolished by either ranolazine (Fig. 2D) or TTX (Fig. 2G). In this example, ATX-II-induced DADs were also suppressed by ranolazine (Fig. 2D) or TTX (Fig. 2G). In some experiments DADs could be detected in the presence of ranolazine after termination of sustained triggered activity (not shown). However, the amplitude of DADs was significantly smaller in the presence of ATX-II plus ranolazine, compared with that in the presence of ATX-II alone. On average, the amplitude of DADs was reduced by ranolazine by 92 ± 7%, from 8.1 ± 1.0 to 0.9 ± 0.8 mV (n = 6, P < 0.05). In the presence of TTX, there were no detectable DADs. EADs were reproducibly induced and increased after washing out ranolazine (Fig. 2E) or TTX (Fig. 2H) in the presence of ATX-II, indicating that EADs were also inhibited by ranolazine and TTX.


Figure 2
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  		Fig. 2. Ranolazine (Ran) and TTX abolished ATX-II-induced sustained triggered activity. Action potential records were obtained from a single cell. A and B: sequential changes of membrane potentials during the course of experiment. A: in the absence of drugs; B–H: in the presence of 10 nmol/l ATX-II. B: EADs (thin arrow) and DADs (thick arrow) were induced after an initial treatment with ATX-II for 4 min. C: sustained triggered activity occurred after a further exposure of the cell to ATX-II. D: triggered activity was abolished by 10 µmol/l Ran. E: EADs and DADs appeared after washing out Ran for 1 min. F: sustained triggered activity resumed after washing out Ran for 2 min. G: triggered activity was terminated by 2 µmol/l TTX. H: EADs and DADs reappeared after washout of TTX.

 
In the absence of ATX-II, ranolazine alone (10 µmol/l, n = 18) shortened the APD30 from 40 ± 4 to 34 ± 3 ms (P < 0.05), but had no significant effect on the APD90 (ranolazine 95 ± 4 ms vs. control 92 ± 4 ms; P > 0.05). TTX (2 µmol/l) caused a slight but statistically insignificant shortening of APD. The values of APD30 were 38 ± 4 and 35 ± 3 ms, and values of APD90 were 87 ± 7 and 82 ± 8 ms, respectively, in the absence and presence of TTX alone (n = 3, P > 0.05).

Ranolazine and TTX reduce ATX-II-stimulated late INa. INa was activated by 300-ms-long depolarizing pulses. A small, sustained, inward current could be observed at the end of pulse, and this current was identified as late INa. ATX-II (5 nmol/l) increased late INa from –0.774 ± 0.172 to –3.396 ± 0.459 nC (integrated over 50 ms; n = 12; P < 0.05). Myocytes exposed to ATX-II were then treated with either ranolazine (10 µmol/l) or TTX (2 µmol/l) in the continuous presence of ATX-II. In ranolazine experiments (n = 7), late INa was increased by ATX-II from –0.996 ± 0.253 to –3.539 ± 0.666 nC and was decreased by ranolazine to 2.336 ± 0.474 nC (P < 0.05), a 48 ± 5% inhibition of ATX-II-induced late INa (Fig. 3). Similarly, in TTX experiments (n = 6), late INa was enhanced by ATX-II from –0.685 ± 0.071 to –3.098 ± 0.409 nC and was reduced to –1.586 ± 0.224 nC after addition of 2 µM TTX (P < 0.05) (Fig. 3), equivalent to an inhibition of 63 ± 3% of ATX-II-induced late INa.


Figure 3
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  		Fig. 3. Ran and TTX each reduced the magnitude of ATX-II-induced late sodium current (INa). A and B: membrane currents recorded from a single myocyte activated by 300-ms voltage-clamp pulses from –90 to –40 mV. Current traces were sequentially recorded in the absence of drug (control) (a) and in the presence of 5 nmol/l ATX-II (b), ATX-II + Ran (10 µmol/l) (c), ATX-II (washout of Ran) (d), and ATX-II + TTX (2 µmol/l) (e). C and D: effects of Ran and TTX (n = 6), respectively, on ATX-II-induced late INa. Late INa was calculated by integration of the current during the last 50 ms of the depolarizing pulse to –40 mV. Significant difference from *control and **ATX-II alone: P < 0.05.

 
Ranolazine attenuates ATX-II-induced ITI. The ITI is generated under conditions of intracellular Ca2+ overload and is the underlying ionic mechanism for DADs (20, 23). In the absence of drugs, ITI was not observed in the present study. Consistent with its effect to induce DADs, ATX-II (10 nmol/l) induced ITI in atrial myocytes (Fig. 4B). The amplitude of ITI was reversibly reduced by ranolazine (10 µmol/l; Fig. 4C) in the presence of 10 nmol/l ATX-II by 87 ± 6%, from 59 ± 12 to 12 ± 7 pA (n = 10, P < 0.05).


Figure 4
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  		Fig. 4. Ran attenuated ATX-II-induced transient inward current (ITI) in a single cell. Shown are membrane currents elicited by 200-ms voltage-clamp pulses from –80 to +20 mV. A: absence of drug. B: exposure to 10 nmol/l ATX-II-induced ITI (arrow). C: ATX-II failed to induce ITI in the presence of Ran. D: ITI reappeared after washout of Ran in the continued presence of ATX-II.

 
Interventions known to reduce Ca2+ overload or spontaneous Ca2+ release abolished ATX-II-induced DADs, but not EADs. ATX-II is reported to cause a Na+-dependent positive inotropic effect (17) that progresses to Ca2+ overload (13) and triggered activity (18). Cellular Ca2+ overload is known to give rise to spontaneous releases of Ca2+ from the SR, ITI, and DADs (20, 23). To establish the association of Ca2+ overload with the occurrence of ITI, DADs, and triggered activity induced by ATX-II, experiments were carried out using the NCX inhibitor KB-R7943, the SR calcium release inhibitor ryanodine, and the calcium chelators EGTA and BAPTA to reduce or prevent Ca2+ overload and spontaneous Ca2+ releases.

In the series of experiments using KB-R7943 (n = 5; Fig. 5), ATX-II (10 nmol/l) alone induced both EADs and DADs (amplitude = 13 ± 3 mV). When KB-R7943 (0.1 µmol/l) was present, ATX-II-induced only EADs, but no DADs. The ATX-II-induced EADs appeared not to be affected by KB-R7943. The amplitudes of EADs in the absence and presence of KB-R7943 were 17 ± 1 and 18 ± 1 mV, respectively (P > 0.05). Similarly, in ryanodine experiments (n = 5; Fig. 6), ATX-II (10 nmol/l)-induced DADs (amplitude = 10 ± 1 mV) were completely abolished by ryanodine (1 µmol/l), but the EADs were not significantly altered. The amplitudes of EADs in the absence and presence of ryanodine were 16 ± 3 and 16 ± 2 mV, respectively (P > 0.05). When EGTA (1 mmol/l, n = 6; Fig. 7A) or BAPTA (1 mmol/l, n = 6; Fig. 7B) was added to the pipette solution to buffer changes in the intracellular calcium concentration, only EADs, but no DADs, were induced by ATX-II (10 nmol/l). The amplitudes of ATX-II-induced EADs in the presence of EGTA and BAPTA were 18 ± 2 and 15 ± 1 mV, respectively, which were comparable to those observed in the presence of ATX-II alone.


Figure 5
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  		Fig. 5. KB-R7943 (KBR) prevented ATX-II-induced DADs. All action potential records were obtained from a single cell. Each panel consists of 5 consecutive and superimposed records. A: absence of drug. B: in the presence of 10 nmol/l ATX-II for 7 min, both EADs (thin arrow) and DADs (thick arrow) were induced. C: after washout of ATX-II for 4 min, EADs and DADs were not observed. D: in the presence of both 0.1 µmol/l KBR and 10 nmol/l ATX-II for 7 min, EADs but not DADs were observed. E: after washout of KBR in the continued presence of ATX-II for 3 min, both EADs and DADs were again observed.

 

Figure 6
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  		Fig. 6. Ryanodine abolished ATX-II-induced DADs and triggered activity. All action potential records were obtained from a single cell. Each panel consists of 5 consecutive and superimposed records. A: action potentials recorded in the absence of drug. B: ATX-II (10 nmol/l) induced both EADs (thin arrow) and DADs (thick arrow). C: in the continued presence of ATX-II, bursts of trigger action potentials were observed. D: ryanodine (1 µmol/l) abolished ATX-II-induced DADs and triggered action potentials, but EADs were still present.

 

Figure 7
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  		Fig. 7. ATX-II failed to induce DADs when either EGTA (1 mmol/; A) or BAPTA (1 mmol/l; B) was included in pipette solution. Action potentials were recorded from different single cells. Each panel shows 5 consecutive and superimposed records. In the presence of EGTA or BAPTA, ATX-II (10 nmol/l) induced EADs (arrows), but not DADs or triggered activity.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The major findings of the study were as follows: 1) an increase of late INa by ATX-II was followed by the sequential appearance of EADs, DADs, and sustained triggered activity; 2) the late INa enhancer ATX-II induced Ca2+-dependent ITI; 3) the effects of ATX-II were reduced or abolished by the late INa inhibitors ranolazine and TTX; 4) occurrences of DADs and sustained triggered activity in the presence of ATX-II were abolished by Ca2+ chelating agents (BAPTA and EGTA), the NCX blocker (KB-R7943), and the SR Ca2+ release inhibitor (ryanodine), but the induction by ATX-II of EADs was not prevented by these interventions. Although ATX-II is well known to prolong the duration of the ventricular action potential and induce EADs and triggered activity (17, 18, 36), this study is the first to show that ITI is induced by ATX-II. These data indicate that an increase of late INa directly prolongs duration of the action potential and facilitates EADs, and indirectly causes cellular Ca2+ overloading that leads to ITI and DADs. Because EADs are associated with bradycardia, whereas cellular Ca2+ loading and DADs are facilitated by tachycardia (6), an increase of late INa may contribute to arrhythmogenesis, facilitated by either brady- or tachycardias.

In guinea pig ventricular myocytes, an increase of late INa by ATX-II rarely leads to DADs, unless inward rectifier K+ current (IK1) is also reduced by barium (Y. Song, online supplement) (The online version of this article contains supplemental data.) The different responses of atrial and ventricular myocytes to ATX-II are likely to reflect differences in channel function and/or mechanisms of ion homeostasis in the two tissues. In the atrium, the magnitude of IK1 is less than in the ventricle (15, 45). In addition, the density of Na+ channels is greater (24) and the activity of Na+-K+-ATPase is lower (44) in atrial than in ventricular myocardium, and a strong NCX activity exists in the atrium (3, 25). Each of these factors may predispose the atrial myocyte to the development of Ca2+ overload-induced ITI and DADs. However, late INa-induced ITI and DADs have also been observed in mouse ventricular myocytes expressing the {Delta}KPQ mutant Na+ channel (14). Regardless, the effect of augmented late INa to cause DAD-related sustained triggered activity appears to be tissue (i.e., atria vs. ventricle), species, and condition dependent (e.g., IK1 may be reduced in heart failure and myocardial infarction, but enhanced in atrial fibrillation) (31).

ATX-II-induced EADs were not suppressed by the SR Ca2+ release inhibitors ryanodine and thapsigargin (5, 37) (Fig. 6), the Ca2+ chelators EGTA and BAPTA (Fig. 7), or the NCX inhibitor KB-R7943 (Fig. 5). Because an increase of intracellular Na+ concentration should increase outward INCX at plateau potentials, ATX-II-induced EADs are unlikely to depend on inward INCX following Ca2+ release from the SR. Rather, they are inhibited by TTX and ranolazine (5, 36, 37) and thus depend on Na+ influx during the action potential plateau.

The development of DADs and sustained triggered activity occurred after the appearance of EADs (Fig. 1), consistent with a gradual accumulation of intracellular Ca2+, secondary to an increase of late INa. In contrast to EADs, DADs were abolished by KB-R7943 (Fig. 5), ryanodine (Fig. 6), or intracellular application of EGTA or BAPTA (Fig. 7). ITI, DADs, and sustained triggered activity were also abolished by the late INa inhibitors ranolazine and TTX (Figs. 2–4). This finding is similar to the observation that ITI of myocytes expressing the LQT3 mutant Na+ channel {Delta}KPQ was EGTA, TTX, and flecainide sensitive (14). The results of both studies indicate that an increase of late INa can lead to ITI, DADs, and sustained triggered activity via a mechanism that likely involves cellular Ca2+ overload. This interpretation is supported by previous studies showing that an increased intracellular Na+ concentration is followed by signs of Ca2+ overload (16, 17, 35, 40, 42, 48). The effect of 0.1 µmol/l KB-R7943 to inhibit DADs in the presence of ATX-II was consistent with the hypothesis that ATX-II-induced Ca2+ overload is due to an increased Ca2+ entry via the NCX in response to a rise of intracellular Na+, although a direct inhibition by KB-R7943 of DADs cannot be excluded at present.

Neither TTX nor ranolazine, at the concentrations used, is known to significantly reduce peak L-type calcium channel current and INCX (1). Whereas 2 µmol/l TTX may reduce peak INa, ranolazine inhibits peak INa only at concentrations severalfold higher than that used in this study (40). On the other hand, both ranolazine and TTX significantly inhibit late INa at study concentrations (1, 40). Furthermore, DADs induced by either forskolin or ouabain are not reduced by ranolazine (Y. Song, unpublished data). Hence, the findings indicate that both ranolazine and TTX suppress ITI indirectly by reduction of late INa and, therefore, of late INa-induced Na+ and Ca2+ overload.

Implications. The magnitude of late INa is known to be increased by a wide variety of both inherited and acquired Na+ channelopathies (2, 4, 40, 41). Increases of late INa are commonly associated with bradyarrhythmia-triggered and pause-induced arrhythmic activity (8). In contrast, Ca2+ overload, ITI, and DADs are associated with an increase of pacing (heart) rate. The finding that prolonged action potentials, EADs, DADs, and ITI are induced by ATX-II, therefore, potentially implicates altered Na+ channel activity with increased late INa in the genesis of arrhythmias associated with both brady- and tachycardias. This hypothesis is consistent with the results of a recent clinical trial that the late INa inhibitor ranolazine significantly reduced incidence of cardiac arrhythmias in patients with acute coronary syndrome (33). The action of ranolazine to effectively inhibit late INa and triggered activity of atrial myocytes is also in agreement with a recent report that ranolazine is an atrium-selective Na+-channel blocker and suppresses atrial fibrillation in the dog (7).

Limitations. An obvious limitation of the present study is that measurements of intracellular concentrations of Na+ and Ca2+ and INCX were not done, and therefore assumptions of actions of drugs on these parameters need to be tempered by caution. Previous studies using fluorescent Na+ and Ca2+ indicators showed that ATX-II increased intracellular concentrations of Na+ in guinea pig and rat ventricular myocytes (17) and of Ca2+ in rat hearts (13). The ATX-II-induced increase of intracellular Ca2+ in rat hearts could be prevented by low concentrations (4 and 9 µmol/l) of ranolazine (13). In addition, hydrogen peroxide-induced intracellular Na2+ and Ca2+ overload, which was associated with an enhanced late INa, was attenuated by 10 µmol/l ranolazine (35). The arrhythmogenic effects of enhancing late INa by other mechanisms should also be investigated. Nonetheless, the principle that an increase of late INa can induce Ca2+ overload and arrhythmias has been validated by reports of the effects of "gain of function" Na+ channel mutations to cause cardiac electrical and mechanical dysfunction (4, 14, 39).

In conclusion, the results of this study suggest that enhanced late INa is a potential mechanism to cause both EADs and Ca2+ overloading that results in ITI, DADs, and triggered activity. Inhibitors of late INa may, therefore, be of therapeutic benefit to reduce the contributions of EADs, ITI, and DADs to arrhythmogenesis.


   GRANTS
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 ABSTRACT
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Y. Song is the recipient of a research grant from CV Therapeutics, Inc.


   DISCLOSURES
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J. Shryock and L. Belardinelli are employees of CV Therapeutics, Inc.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Song, Division of Cardiovascular Medicine, Univ. of Florida, 1600 SW Archer Rd., Rm. M-411, Gainesville, FL 32610–0277 (e-mail: songy{at}medicine.ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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