Basal late sodium current is a significant contributor to the duration of action potential of guinea pig ventricular myocytes

Abstract In cardiac myocytes, an enhancement of late sodium current (IN aL) under pathological conditions is known to cause prolongation of action potential duration (APD). This study investigated the contribution of IN aL under basal, physiological conditions to the APD. Whole‐cell IN aL and the APD of ventricular myocytes isolated from healthy adult guinea pigs were measured at 36°C. The IN aL inhibitor GS967 or TTX was applied to block IN aL. The amplitude of basal IN aL and the APD at 50% repolarization in myocytes stimulated at a frequency of 0.17 Hz were ‐0.24 ± 0.02 pA/pF and 229 ± 6 msec, respectively. GS967 (0.01–1 μmol/L) concentration dependently reduced the basal I NaL by 18 ± 3–82 ± 4%. At the same concentrations, GS967 shortened the APD by 9 ± 2 to 25 ± 1%. Similarly, TTX at 0.1–10 μmol/L decreased the basal I NaL by 13 ± 1–94 ± 1% and APD by 8 ± 1–31 ± 2%. There was a close correlation (R 2 = 0.958) between the percentage inhibition of IN aL and the percentage shortening of APD caused by either GS967 or TTX. MTSEA (methanethiosulfonate ethylammonium, 2 mmol/L), a NaV1.5 channel blocker, reduced the I NaL by 90 ± 5%, suggesting that the NaV1.5 channel isoform is the major contributor to the basal I NaL. KN‐93 (10 μmol/L) and AIP (2 μmol/L), blockers of CaMKII, moderately reduced the basal I NaL. Thus, this study provides strong evidence that basal endogenous I NaL is a significant contributor to the APD of cardiac myocytes. In addition, the basal I NaL of guinea pig ventricular myocytes is mainly generated from NaV1.5 channel isoform and is regulated by CaMKII.


Introduction
The late Na + current (I NaL ) is a component of the fast inward I Na , which remains activated during the plateau and the repolarization of a cardiac action potential (Noble and Noble 2006;Antzelevitch et al. 2014). I NaL is increased in congenital and acquired pathological conditions, such as long QT syndrome type 3, cardiac hypertrophy, heart failure, and myocardial ischemia (Belardinelli et al. 2015;Makielski 2016). An enhancement of I NaL under these pathological conditions may cause a prolongation of the action potential duration (APD) and is considered potentially arrhythmogenic (Belardinelli et al. 2015;Makielski 2016). Inhibition of I NaL by I NaL blockers, such as ranolazine, has shown promising antiarrhythmic value (Belardinelli et al. 2015;Makielski 2016). However, because the amplitude of I NaL under physiological conditions is relatively small, its role (i.e., the I NaL in the absence of drug or pathological modification) in cardiac repolarization has not been fully recognized.
Several lines of evidence suggest that the inward I NaL may play a significant role in maintaining cardiac depolarization under physiological conditions. (1) I NaL can remain activated throughout the action potential plateau, where the membrane resistance is high (Weidmann 1951). Therefore, even a small net inward current may cause a significant lengthening of the plateau and thus the APD.
(2) The APD is shortened in the presence of TTX, an inhibitor of I NaL (Coraboeuf et al. 1979;Kiyosue and Arita 1989). (3) In canine ventricular myocytes, the density of I NaL is greater in the mid-myocardium, compared with that in the epi-and endomyocardium (Zygmunt et al. 2001). In keeping with that, TTX-induced APD shortening is greater in the mid-myocardium than in the epi-and endomyocardium (Zygmunt et al. 2001). (4) In failing hearts of both human and canine models, inactivation of I NaL of ventricular myocytes is further slowed, compared with that in myocytes of normal hearts (Maltsev et al. 2007). The enhanced I NaL contributes to the prolongation of APD (Maltsev et al. 2007), and conversely, inhibition of I NaL by ranolazine shortens the APD of ventricular myocytes isolated from a canine heart failure model (Undrovinas et al. 2006).
The goal of this study was to determine the contribution of basal I NaL to the APD of ventricular myocytes of healthy guinea pigs. In the past, a precise evaluation of the contribution of basal I NaL to the APD has been hindered by the small amplitude of the current and the lack of a selective inhibitor. Most studies of I NaL have been conducted in the presence of I NaL enhancers, such as anemone toxin II (ATX-II) (Isenberg and Ravens 1984;Song et al. 2004). In this study, the selective I NaL inhibitor GS967 (Belardinelli et al. 2013) and low concentrations of TTX were applied to selectively block I NaL . The amplitude of I NaL in this study was not preenhanced by drugs or special experimental conditions, except for one series of experiments in which the I NaL enhancer ATX-II was applied to verify the specificity of the action of GS967. Thus, the subject of this study was cardiac I NaL under basal conditions. The role of basal I NaL in maintaining the depolarization of the ventricular action potential was assessed by comparing the percentage inhibition of I NaL with the percentage shortening of APD. In addition, we examined whether the basal I NaL is generated from Na V 1.5 channels, and whether the basal I NaL is regulated by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), respectively, by applying the selective Na V 1.5 channel blocker MTSEA (methanethiosulfonate ethylammonium) (Haufe et al. 2005;O'Reilly and Shockett 2012) and the CaMKII inhibitors KN-93 and AIP (autocamtide-2-related inhibitory peptide).

Materials and Methods
Animal use was approved by the Institutional Animal Care and Use Committee, and conformed to the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Hearts of guinea pigs of either sex were isolated and perfused via the aorta with warm (35°C) and oxygenated solutions in the following order: (1) Tyrode solution containing (in mmol/L) 135 NaCl, 4.6 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 10 glucose, and 10 HEPES, pH 7.4, for 5 min; (2) Ca 2+ -free solution containing (in mmol/L) 100 NaCl, 30 KCl, 2 MgCl 2 , 10 glucose, 10 HEPES, 15 taurine, and 5 pyruvate, pH 7.4, for 5 min; and (3) Ca 2+ -free solution containing collagenase (120 units/mL) and albumin (2 mg/mL), for 20 min. At the end of the perfusion, the ventricles were minced and gently shaken for 10 min in the collagenase solution to release single cells. Only the quiescent myocytes with clear striations were used for this study.
Transmembrane voltages and currents were recorded using the whole-cell patch-clamp technique. Data were acquired and analyzed with an Axopatch-200 amplifier, a Digidata-1440A digitizer, and pCLAMP-10 software. All experiments were performed at 36°C.
For measurements of action potentials, cells were incubated in the Tyrode solution (bath solution). The recording pipettes were filled with a solution containing (in mmol/L) 120 K-aspartate, 20 KCl, 1 MgSO 4 , 4 Na 2 ATP, 0.1 Na 3 GTP, and 10 HEPES, pH 7.3. A depolarizing pulse was applied every 6 sec to elicit action potentials. The APD was determined from the beginning of depolarization to the time when 30% (APD 30 ), 50% (APD 50 ), and 90% (APD 90 ) of repolarization were completed.
For measurements of I NaL , myocytes were superfused with a bath solution containing (in mmol/L) 135 NaCl, 1.8 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES, 4.6 CsCl, 0.05 NiCl 2 , and 0.01 nitrendipine, pH 7.4. The recording pipettes were filled with a solution containing (in mmol/L) 120 Cs-aspartate, 20 CsCl, 1 MgSO 4 , 4 Na 2 ATP, 0.1 Na 3 GTP, and 10 HEPES, pH 7.2. Sodium current was activated by 200-250 msec long voltage-clamp pulses applied every 10 sec, from a holding potential of À90 mV to a test potential of À30 or À50 mV. The amplitude of I NaL was calculated as the average amplitude of current during the last 100 msec of a depolarizing pulse.
GS967 was synthesized by Gilead Sciences. MTSEA was purchased from Toronto Research Chemicals, KN-93 and KN-92 from Calbiochem, AIP from Tocris, and ATX-II from Sigma. KN-93, KN-92, and AIP were applied through the recording pipette solution; other drugs were added to the bath solutions. The duration of each drug treatment was 3 min before recording.
Data are expressed as mean AE SEM. Sample size (n) is shown as number of cells/from number of hearts. Statistical analyses were conducted using SigmaPlot software. Concentration-response relationship and EC 50 for GS967 inhibition of I NaL were calculated from a standard four-parameter logistic curve fitted with the following equation: Þ ÀHillslope Coefficient of determination (R 2 ) was calculated from a standard linear regression curve fitted with the following model: The t-test or one-way ANOVA followed by Holm-Sidak method was applied for statistical analysis. A P < 0.05 was considered statistically significant.

Contribution of basal I NaL to APD
To verify the action of GS967 as an I NaL blocker, the effect GS967 on I NaL induced by the I NaL enhancer ATX-II was examined. In this series of experiments, I NaL was activated by voltage-clamp pulses from À90 to À50 mV. ATX-II (5 nmol/L) increased the amplitude of I NaL at À50 mV from À0.12 AE 0.01 to À0.47 AE 0.03 pA/pF (n = 24/9, P < 0.001). GS967 reversibly and concentration dependently inhibited the I NaL in the presence of ATX-II. GS967 at concentrations of 0.1, and 0.3 lmol/L significantly (P < 0.001, n = 12/5) reduced the amplitude of ATX-II-stimulated I NaL by 41 AE 2% and 93 AE 5%, respectively (Fig. 1). In another group of myocytes (n = 12/4), the ATX-II-stimulated I NaL was inhibited by 0.03 and 1 lmol/L GS967 by 24 AE 3% and 100%, respectively (P < 0.001, not shown).
There was a close correlation (R 2 = 0.958) between the percentage inhibition of basal I NaL and the percentage shortening of APD caused by either GS967 or TTX (Fig. 4), indicating that basal I NaL significantly contributes to the APD.
Inhibition of basal I NaL by Na V 1.5 channel blocker MTSEA is a selective blocker of Na V 1.5 channels (Haufe et al. 2005;O'Reilly and Shockett 2012). In this study, MTSEA (2 mmol/L) was added to the bath solution to Figure 1. Concentration-dependent inhibition by GS967 of ATX-II (5 nmol/L)-induced I NaL . Inward currents were activated by depolarizing pulses from À90 to À50 mV. Panel A, superimposed currents recorded in the order of a-e from a single myocyte before (control) and after drug treatments. Panel B, summary of the average amplitude of I NaL recorded before (A) and after (B-E) drug treatments, as shown in panel A (n = 12/5). *P < 0.001 versus control; † P < 0.001 versus ATX-II alone.  determine whether the basal I NaL of myocytes was generated from the Na V 1.5 channels. I NaL was activated by depolarizing pulses from À90 to À30 mV. In this series of experiments, MTSEA decreased the amplitude of I NaL by 90 AE 5%, from À0.20 AE 0.03 to 0.03 AE 0.01 pA/pF (n = 12/6, P < 0.001; Fig. 5). The result suggests that under the experimental conditions, the Na V 1.5 channel is the major contributor to the I NaL of guinea pig ventricular myocytes.

Decrease in basal I NaL by CaMKII inhibitors
Activation of CaMKII was reported to slow sodium channel inactivation. We used the CaMKII inhibitors KN-93 and AIP, and an inactive analog of KN-93 and KN-92, as a negative control, to determine whether CaMKII plays a significant role in maintaining basal I NaL . The three drugs were applied through the recording pipette solution to three separate groups of myocytes, respectively. I NaL was activated by voltage-clamp pulses from À90 to À30 mV. The amplitude of I NaL measured in the absence of drugs was À0.24 AE 0.02 pA/pF. KN-93 (10 lmol/L) and AIP (2 lmol/L) reduced the I NaL to À0.17 AE 0.02 pA/pF (n = 11/3, P < 0.05) and À0.17 AE 0.02 pA/pF (n = 11/5, P < 0.05), respectively (Fig. 6), whereas KN-92 (10 lmol/L) had no effect on I NaL (À0.24 AE 0.02 pA/pF, n = 12/4; Fig. 6).

Discussion
This study revealed that the basal I NaL is of sufficient magnitude to affect the duration of the action potential of ventricular myocytes isolated from healthy guinea pigs. In the presence of the I NaL blocker GS967 or low concentrations of TTX, the reduction in I NaL was closely correlated with the shortening of APD (Figs. 2-4). Furthermore, the study showed that the basal I NaL of guinea pig ventricular myocytes was mainly generated from the Na V 1.5 channels (Fig. 5) and was regulated by CaMKII (Fig. 6). Thus, the results of the present study suggest that the basal, CaMKII-mediated Na V 1.5 I NaL is a significant and physiological contributor to the action potential duration of guinea pig ventricular myocytes.
The action potentials of cardiac ventricular myocytes are characterized by a prominent plateau phase (phase 2) (Draper and Weidmann 1951). Repolarization is delayed during the plateau phase, and thus the duration of a myocardial action potential is largely determined by the length of the plateau phase. The action potential plateau is caused by a  balance between depolarizing currents, including the inward L-type (I CaL ) and T-type (I CaT ) Ca 2+ current, Na + -Ca 2+ exchange current (I NCX ) and I NaL , and repolarizing currents, such as the outward delayed rectifier K + current (I K ) (Ten Eick et al. 1992). The amplitude of I NaL under physiological conditions is relatively small. In this study, the average amplitude of I NaL was À0.24 AE 0.02 pA/pF. However, because I NaL remains activated throughout the plateau phase and the membrane resistance is known to be high at the plateau (Weidmann 1951), even a small inward current such as I NaL could play a significant role in maintaining cardiac depolarization, and thereby the duration of action potential. Myocytes used in this study were isolated from the whole ventricles; therefore, the present results represented the average effect of basal I NaL on the duration of ventricular action potentials. As it has been reported that the density of I NaL is greater in the mid-myocardium than that in the epi-and endomyocardium (Zygmunt et al. 2001), it will be interesting to know if there is a regional difference in the contribution of I NaL to the APD among different parts of ventricular myocardium. Assessment of the contribution of basal I NaL to APD requires the use of an inhibitor that, at least at certain concentrations, selectively and concentration dependently reduces I NaL , and has no effect on other ion currents that can modulate the APD. We used the I NaL inhibitor GS967 at concentrations and conditions in which its inhibition was selective for the I NaL (Belardinelli et al. 2013). For comparison, the Na + channel blocker TTX applied at low concentrations was used to confirm that the inward current recorded was a Na + -channel current. The selectivity of GS967 to inhibit I NaL has been studied using rabbit ventricular myocytes (Belardinelli et al. 2013). The results of that study showed that, at a holding potential of À120 mV and a stimulation frequency of 0.1-3 Hz, GS967 (0.1-5 lmol/L) concentration dependently blocked ATX-II-stimulated I NaL without reducing the peak I Na . In addition, GS967 (1-3 lmol/L) had no significant effect on I CaL , I CaT , and ATP-sensitive K + current, although GS967 at a high concentration of 10 lmol/L caused a small (17%) inhibition of the rapid component of I K . In this study of guinea pig ventricular myocytes, GS967 concentration dependently inhibited ATX-II-induced I NaL (Fig. 1), further confirming that this compound is a suitable pharmacological tool to investigate the role of I NaL in cardiac repolarization. GS967 at 1 lmol/L blocked the ATX-II stimulated and the basal I NaL by 100% and 82 AE 4%, respectively. Thus, it appears that the potency of GS967 to inhibit I NaL is greater in the presence, than in the absence, of ATX-II. This could be due to a sensitization by ATX-II of sodium channels to the inhibitory action of GS967, as it has been found that sodium channel site-3 toxins (such as ATX-II) can enhance the binding and action of site-1 toxin (such as TTX) and local anesthetics on this channel (Nishio et al. 1991).
A contribution of basal I NaL to the APD was suggested by a previous study (Kiyosue and Arita 1989). In that study, TTX at a concentration of 60 lmol/L caused a decrease in APD of ventricular myocytes isolated from healthy guinea pigs. However, TTX at such a high concentration could block not only the peak I Na , but also the L-and T-type Ca 2+ channels (Sun et al. 2008;Hegyi et al. 2012), which would also lead to a shortening of the APD. To verify the role of I NaL in modulation of APD, we used the selective I NaL blocker GS967 and low concentrations of TTX to determine the effect of an inhibition of basal I NaL on the APD. Our results showed that GS967 and TTX at a concentration as low as 0.01 lmol/L and 0.1 lmol/L, respectively, could cause a significant shortening of the APD (Fig. 3). Furthermore, a quantitative The Physiological Society and the American Physiological Society analysis indicated that the inhibition of basal I NaL and the shortening of APD caused by GS967 and TTX were closely correlated (Fig. 4). Na V 1.5 channel has been recognized as the dominant sodium channel of ventricular myocytes (Gellens et al. 1992;Maltsev et al. 2008;Veerman et al. 2015). In addition to the Na V 1.5 channel, other sodium channel isoforms may also contribute to the cardiac sodium current. One study reported that A-803467, a Na V 1.8 channel blocker, blocked I NaL of mouse and rabbit ventricular myocytes, suggesting that Na V 1.8 channel contributes to cardiac I NaL ( (Yang et al. 2012). In contrast, another study found that A-803467 had no effect on sodium current of mouse ventricular myocytes (Verkerk et al. 2012). In this study, the selective Na V 1.5 channel blocker MTSEA (Haufe et al. 2005; O'Reilly and Shockett 2012) decreased the amplitude of basal I NaL by 90 AE 5%, indicating that under the conditions of our experiments, the Na V 1.5 channel isoform is a major contributor to basal I NaL of guinea pig ventricular myocytes.
Cardiac myocytes overexpressing CaMKII showed an enhanced I NaL (Wagner et al. 2006). In this study, we investigated the role of CaMKII in regulating basal I NaL by comparing the amplitude of I NaL in the absence and presence of the CaMKII inhibitor KN-93 or AIP. Because KN-93 and its inactive analog KN-92 may have CaMKII-independent effects on ion channels if applied extracellularly (Rezazadeh et al. 2006), these drugs and AIP were applied intracellularly through the pipette solution. Our results showed that the amplitude of basal I NaL was decreased by either KN-93 or AIP, but not by KN-92, suggesting a significant role of CaMKII in regulating cardiac I NaL under basal conditions (Fig. 6). However, CaMKII phosphorylation may not be the only mechanism to maintain basal I NaL .
Other mechanisms, such as protein kinase C (Ma et al. 2012), may also be involved in the regulation of basal I NaL .
In summary, in this study we investigated the role of basal I NaL in modulating the cardiac APD, by quantitatively determining the relationship between the amplitude of I NaL and the duration of action potential. The results showed a close correlation between a decrease in I NaL and a shortening of the APD, and thus provide strong evidence that basal endogenous I NaL is a significant contributor to the APD of cardiac myocytes. The present results also demonstrated that the basal I NaL of guinea pig ventricular myocytes is mainly generated from Na V 1.5 channel isoform and is regulated by CaMKII.