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Characteristics of hERG and hNav1.5 channel blockade by sulcardine sulfate, a novel anti-arrhythmic compound
Author’s Accepted Manuscript Characteristics of hERG and hNav1.5 channel blockade by sulcardine sulfate, a novel antiarrhythmic compound Weihai Chen, Lu Gan, Yiping Wang www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(18)30701-5 https://doi.org/10.1016/j.ejphar.2018.12.009 EJP72114
To appear in: European Journal of Pharmacology Received date: 26 May 2018 Revised date: 29 November 2018 Accepted date: 5 December 2018 Cite this article as: Weihai Chen, Lu Gan and Yiping Wang, Characteristics of hERG and hNav1.5 channel blockade by sulcardine sulfate, a novel antiarrhythmic compound, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.12.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characteristics of hERG and hNav1.5 channel blockade by sulcardine sulfate, a novel anti-arrhythmic compound
Weihai Chen1,2*, Lu Gan1, Yiping Wang2
1
Key Laboratory of Cognition and Personality (Southwest University), Ministry of
Education; Faculty of Psychology, Southwest University, Chongqing, China; 2
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai, China
*
Corresponding author. Weihai Chen, PhD Faculty of Psychology, Southwest
University Tianshen Road 2, Beibei District Chongqing, China Tel.: 86-13594021891. [email protected]
Abstract Sulcardine sulfate (sulcardine) is a novel anti-arrhythmic compound, which blocks multiple channels and was shown to be safe and tolerated in clinical trials. The aim of the present study was to investigate the electrophysiological characteristics of sulcardine on the hERG and hNav1.5 channels. The hERG and hNav1.5 channels were heterologously stably expressed in human embryonic kidney 293 cells, and the effects of sulcardine on the hERG and hNav1.5 channels were recorded using the standard whole-cell patch-clamp technique. Sulcardine inhibited hERG channels in a
concentration-dependent and reversible manner (IC50 = 94.3 μM). In addition, sulcardine shifted the activation curve of hERG channels to more negative potentials. The relative block of sulcardine on hERG channels was close to zero at the time point corresponding to channel opening, which was achieved by applying a depolarizing voltage, and quickly increased afterward. Sulcardine inhibited hNav1.5 channels in a concentration-dependent and reversible manner (IC50 = 15.0 μM) and shifted the inactivation curve of hNav1.5 channels to more negative potentials. The blockade of sulcardine on hNav1.5 channels was use-dependent. In conclusion, sulcardine is a potent hNav1.5 channel blocker with a mild inhibitory effect on hERG channels and preferentially binds to both hERG and hNav1.5 channels in the open and inactivated states rather than in the resting state.
Keywords: sulcardine sulfate; human ether-a-go-go-related gene (hERG); the cardiac sodium channels (hNav1.5); arrhythmia; torsade de pointes arrhythmia
1. Introduction Most anti-arrhythmic medications exert their effects by blocking sodium, potassium, or calcium ion channels. However, the Cardiac Arrhythmia Suppression Trial (CAST) test has shown that sodium channel blockers increase the rate of mortality owing to arrhythmia (The CAST Investigators, 1989). Cardiac sodium channel blockade can slow intracardiac conduction, which is manifested as prolongation of the QRS complex on the ECG (Delk et al., 2007; Lu et al., 2010). Furthermore, it is suggested that ventricular tachycardia and other arrhythmias caused
by sodium channel blockers increase morbidity and mortality (Delk et al., 2007; Thanacoody and Thomas, 2005). More recently, research on anti-arrhythmic medications has focused on agents that can prolong the action potential and the refractory period, such as potassium channel blockers (i.e., Vaughan-Williams class III anti-arrhythmic drugs). Unfortunately, potassium channel blockers may excessively prolong the QT interval and raise the risk of torsade de pointes arrhythmia, which is a lethal ventricular arrhythmia (Vandenberg et al., 2001) (Waldo et al., 1996). Human ether-a-go-go-related gene potassium (hERG) channels conduct the rapid delayed rectifier potassium currents (IKr) (Sanguinetti and Tristani-Firouzi, 2006). Inhibition of hERG channels tends to prolong the action potential and the QT interval, and this prolongation can be used to treat arrhythmias but also causes acquired long QT syndrome and the life-threatening torsade de pointes arrhythmia (Hancox et al., 2008). In addition, the cardiac sodium channels (hNav1.5), encoded by the human cardiac sodium channel SCN5A gene, are responsible for voltage-dependent sodium currents and electrical excitation of atrial and ventricular myocytes (Erdemli et al., 2012; Gellens et al., 1992). Interestingly, the sodium channel blockade can markedly reduce the likelihood of torsade de pointes arrhythmia, which may be triggered by potassium channel blockers (Antzelevitch et al., 2004; Cairns et al., 1997; Dujardin et al., 2008; Julian et al., 1997; Khan and Gowda, 2004; Thomas and Behr, 2016). These data suggest that sodium channel blockade may increase the safety of potassium channel blockers and reduce the likelihood of torsade de pointes arrhythmia.
Sulcardine sulfate (sulcardine) {N-[4-hydroxy-3,5-bis(pyrrolidin-1-ylmethyl)benzyl]-4-methoxybenzenesulcardinefo namide sulfate} (Fig 1) is a novel anti-arrhythmic drug developed by the Shanghai Institute of Materia Medica, China. Sulcardine exhibits various anti-arrhythmic activities on experimental arrhythmia models in animals (Bai et al., 2012). Furthermore, sulcardine decreased the amplitude and the maximal upstroke velocity of action potentials in a concentration-dependent manner and also prolonged action potential duration and the refractory period in in vitro studies (Bai et al., 2012; Guo et al., 2011). The electrophysiological characteristics of sulcardine may be related to the blockade on multiple channels including sodium, potassium, and calcium channels (Bai et al., 2012; Guo et al., 2011). More importantly, sulcardine is a safe and well-tolerated drug in healthy Chinese subjects, and no serious adverse events have been reported (Chen et al., 2017), even in a repeated administration clinical study (twice daily for 5 days) (Wang et al., 2017). These data suggest that sulcardine is a promising anti-arrhythmic drug with less likelihood of pro-arrhythmia. As discussed above, the blockade of sodium channels can markedly reduce the likelihood of torsade de pointes arrhythmia, which may be triggered by blocking the hERG channel. To further explore the ionic mechanisms underlying the efficacy and safety of sulcardine, we used the standard whole-cell patch-clamp technique. In this way, we could study the electrophysiological characteristics of sulcardine on the hERG and hNav1.5 channels, which were stably expressed in human embryonic kidney 293 (HEK293) cells. The blocking characteristics of sulcardine on the hERG
and hNav1.5 channels may provide substantial evidence to explain the ionic mechanisms underlying its efficacy and safety.
2. Materials and methods 2.1 Cell culture We used a conventional protocol to construct the cell lines that stably expressed hERG and hNav1.5 channels respectively (Chen et al., 2010). Briefly, the hERG/pcDNA3 plasmid was generously donated by Dr. G. Robertson (University of Wisconsin), and the hNav1.5/pcDNA3 plasmid was generously donated by Dr. Robert S. Kass (Columbia University College of Physicians & Surgeons). Human embryonic kidney cells 293 (HEK293) were transfected with the plasmids using the lipofection transfection reagent (Tiangen Biotech CO., LTD, Beijing, China). The HEK293 cells stably expressing hERG and hNav1.5 channels were cultured respectively in Dulbecco’s modified eagle medium (DMEM, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA, USA) and 0.4 g/L geneticin (G418) in an atmosphere of 95% air and 5% CO2 (Chen et al., 2010). 2.2 Solutions and drug administration The external bath solution for recording hERG currents contained (in mM): NaCl 137, KCl 4, CaCl2 1.8, MgCl2 1, HEPES 10 and Glucose 10; the pH was adjusted to 7.4 with NaOH). The pipette solution contained (in mM): KCl 130, CaCl2 1, MgCl2 5, Na2-ATP 5, EGTA 5 and HEPES 10; the pH was adjusted to 7.4 using KOH.
For studying hNav1.5 currents, cells were superfused with a bath solution consisting of (in mM) choline chloride 100, NaCl 20, KCl 5.4, Na2HPO4 0.33, glucose 10, CdCl2 0.1, CaCl2 1 and HEPES 10; the pH was adjusted to 7.4 with NaOH. The composition of the internal solution (in mM) was CsCl 120, MgCl2 5, EGTA 11, HEPES 10, Na2ATP 5 and CaCl2 1; the pH was adjusted to 7.4 using CsOH. The chemicals used in the external and internal solutions were purchased from the Sigma-Aldrich Chemical Company (Shanghai, China). The sulcardine was kindly provided by the Department of Medicinal Chemistry of the Shanghai Institute of Materia Medica (Shanghai, China). Sulcardine was diluted in the external solution to the desired concentration (1—1000 μM) before use. 2.3 Electrophysiological recordings We used the whole-cell patch-clamp recording technique to record currents at room temperature (22–26°C) using an Axopatch 200A amplifier (Axon Instruments, Foster City, USA) with pClamp/Clampex software (Axon Instruments, Foster City, CA, USA). Electrodes were constructed from borosilicate glass using a micropipette puller (P-97, Sutter Instrument Co., Novato, CA, USA). The final resistance of the electrode was 2 to 3 MΩ when filled with the pipette solution. 2.4 Statistical analysis For the patch-clamp experiments, data are expressed as means ± standard deviation (S.D.), where n represents the number of cell experiments performed. Statistical significance was analyzed using a paired Student’s t-tests or one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests for multiple
comparisons. GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used for the statistical analysis of the data. Differences were considered statistically significant at a p-value < 0.05. Concentration-response curves were fitted with Hill equations of the form: E= Emax /[1+(IC50/C) nH]
(1)
where E is the effect at concentration C, Emax is the maximal effect, IC50 is the concentration for half-maximal block and nH is the Hill coefficient. The steady-state activation curve was analyzed by determining the conductance from the equation: Gm = I/(Em – Erev)
(2)
where I is the peak current, Em is the test voltage and Erev is the estimated reversal potential. This was plotted against the corresponding voltage. Plots were fitted with the Boltzmann equation: G/Gmax = 1/1 + exp [(V1/2 – Vm)/k]
(3)
where G is the conductance following the test potential Vm, Gmax is the maximal conductance, V1/2 and k represent the voltage of activation midpoint and slope factor, respectively. The steady-state inactivation curve was determined by fitting the values of the normalized current to the test potential with the Boltzmann equation of the following form: I/Imax = 1/1 + exp [(Vm – V1/2)/k]
(4)
where I is the current amplitude following the test potential Vm, Imax is the maximal current, V1/2 is the half-maximal inactivation potential and k is the slope factor of the
inactivation curve.
3. Results 3.1 Sulcardine inhibited hERG currents in a concentration-dependent manner Sulcardine inhibited hERG currents in a concentration-dependent manner (Fig. 2A). The currents were recorded after at least 2 min of exposure to each concentration of sulcardine. The cell membrane was depolarized to +20 mV for 2 s, and then repolarized to –40 mV for 1.6 s, which elicited a large, slowly decaying, outward tail current that is the feature of hERG currents (Robertson, 2000). The holding potential was –80 mV in all experiments performed in this study unless indicated otherwise. To test the concentration-dependent inhibition by sulcardine, peak tail currents in the presence of sulcardine were normalized to their respective control values and plotted as relative current amplitudes in Fig 2B (n = 5). One-way ANOVA and Bonferroni post hoc tests showed that sulcardine significantly inhibited hERG currents at 30—1000 μM (all P < 0.05). The mean data-points were fitted with Hill equations, and the half-maximal inhibitory concentration (IC50) was 94.3 μM with a Hill coefficient of –1.267. We used cisapride as the positive control compound, the IC50 of which was 24.7 nM (n = 5), similar to a previous report (IC50 = 26 nM) (Kirsch et al., 2004). As shown in Fig 2C, the blocking effects of sulcardine can be partially reversed after washout. After a control recording of 2 min, the hERG channels were exposed to sulcardine at 300 μM and reached steady-state conditions after exposure for 1 min. After 5 min washout, the blocking effect of sulcardine on hERG was partially reversed (Fig 2C, n = 5). In time-course control tests, the hERG tail currents,
after perfusing with the bath solution for 30 min, were 97.8 ± 3.9% of the initial value (n = 6).
3.2 Sulcardine inhibited hERG currents in a voltage-dependent manner Fig 3 shows the effects of sulcardine on the hERG current-voltage (I-V) relationship. hERG currents were elicited from a holding potential of –80 mV by 2-second depolarizing steps from –80 mV to +80 mV, applied in 10-mV increments at 0.1 Hz to produce activation currents, followed by a repolarizing step to –40 mV for 1.6 s to elicit outward tail currents. Representative currents were recorded in control conditions and 10 min after the perfusion with sulcardine at 100 μM in the same cell (Fig 3A). Fig 3B shows the I-V curve at the end of the activating currents. Under control conditions, the activation threshold of hERG currents was –40 mV and the maximum of the activating currents was +20 mV before an observed current reduction at higher test pulse potentials due to inward rectification. A 10-min application of sulcardine at 100 μM reduced the current maximum (at +20 mV) by 52.5 ± 11.5% (n = 5). As shown in Fig 3C, tail currents were saturated after a test pulse potential of +30 mV or above and sulcardine at 100 μM reduced the peak tail current amplitude (after a test pulse to +80 mV) by 59.2 ± 3.3% (n = 5). The inhibitory ratios at each test pulse potential for activating and tail currents were shown in Fig 3D and 3E. The inhibitory ratio at each test pulse potential was calculated as (Icontrol–Isulcardine)/Icontrol. As shown in Fig 3D, sulcardine inhibited activation currents by 19.0 ± 24.3% at 0 mV, and the maximal inhibitory ratio was achieved at +30 mV by 54.5 ± 8.4%, followed
by a progressively decreased inhibitory effect accompanying the increment of test pulse potentials. As shown in Fig 3E, sulcardine inhibited tail currents by 22.9 ± 22.3% at 0 mV and reached a maximal inhibitory ratio at +80 mV (n = 5). Normalized activating and tail currents in the absence and presence of sulcardine are shown in Fig 3F and 3G. The normalized activating and tail currents were fitted with a Boltzmann function and the mean half-maximal activation voltages (V1/2) of the activation curves of the hERG activating and tail currents were significantly shifted by 8.2 ± 2.7 mV and 9.0 ± 1.3 mV respectively towards more negative potentials (P < 0.01, n = 5, paired t-test).
3.3 Sulcardine inhibited hERG currents in a state-dependent manner We used three approaches to investigate the state-dependent block of hERG channels by sulcardine. In the first protocol, the membrane potential was increased from –80 mV to 0 mV for 7.5 s to produce a large activation current, which was first recorded in the absence of sulcardine. The cells were then perfused with sulcardine at 100 μM for 10 min without any intermittent stimulus, while the channels were kept in the resting state at –80 mV. Measurements in the presence of sulcardine were then conducted. As shown in Fig 4A, the blockade was close to zero at the time point of channel opening and quickly increased afterward. This blockade pattern was evidenced by the statistical analysis presented in Fig 4B, which illustrates the time-dependent increase of the inhibitory ratio, expressed as (Icontrol–Isulcardine)/Icontrol, at each time point after depolarization. As shown in Fig 4B, at 0.05 and 0.1 s after
depolarization, the inhibitory ratio was close to zero. At 1, 2 and 7.5 s after depolarization, the inhibitory ratio increased to 29.7 ± 38.0%, 40.3 ± 26.3% and 57.7 ± 10.1%, respectively (n = 5). This is consistent with the blockade of the hERG channels in the open state, but not in the resting state, which is a common pattern among hERG antagonists (Scholz et al., 2003; Thomas et al., 2002). The second protocol was used to test if sulcardine inhibited hERG channels in the inactivated state. A long-term depolarization potential to +80 mV (4000 ms) from a holding potential of –80 mV was used to inactivate the channels, and then a second test pulse to 0 mV (3500 ms) was applied to rapidly recover the channels from inactivation. Typical current traces before and after incubation with sulcardine for 10 min are shown in Fig 4C. Fig 4D represents the inhibitory ratio at each time point during the second test pulse (0 mV). Sulcardine inhibited hERG channels during the preceding inactivation (+80 mV pulse) and an additional time-dependent blockade of open channels was not observed during the 0 mV pulse. As shown in Fig 4D, at each time point from 0.05 to 3.5 s during repolarization at 0 mV, the inhibitory ratio was close to 40% (n = 6). These data indicate that sulcardine inhibited hERG channels to a maximal degree during the first inactivation test potential to +80 mV while the majority of channels stayed in the inactivated state, suggesting that sulcardine blocks hERG channels in the inactivated state, rather than in the resting state. The third protocol, an “envelope of tails” test, was conducted to test whether the hERG channel blockade by sulcardine was taking place during depolarization (Kamiya et al., 2001). Cells were firstly depolarized to +30 mV from a holding
potential of –80 mV for a variable duration ranging from 25 ms to 1600 ms, followed by a repolarizing step to –50 mV to induce hERG tail currents at 0.1 Hz. Tail currents at –50 mV were measured after each test pulse. We carried out the “envelope of tails” test in the same cell both before and after the application of sulcardine at 100 μM. Typical current traces before and after the application of sulcardine are shown in Fig 5A and 5B. hERG tail current amplitude increased with the depolarizing pulse duration over the first few pulses and then reached a steady maximal amplitude. The time constants of the hERG current activation were estimated by fitting the peak amplitude of the tail currents with a single exponential function (Fig 5C). The fitting yielded time constants of 283.1 ± 48.4 ms and 201.1 ± 53.4 ms before and after the application of sulcardine at 100 μM respectively (P < 0.01, n = 6, paired t-test, Fig 5D). The shortened time constants suggest that blockade by sulcardine was enhanced by the further activation of currents. That is, with the increment of activation duration, sulcardine showed more potent relative blockade on hERG channels, which indicates that sulcardine tends to inhibit inactivated and/or open hERG channels.
3.4 Sulcardine inhibited hNav1.5 currents in a dose-dependent manner Large fast inward hNav1.5 currents were induced by a depolarization pulse to –30 mV for 30 ms from a holding potential of –80 mV. hNav1.5 channels were exposed to each concentration of sulcardine for 2 min (1—300 μM). Typical current tracings before and after application of sulcardine are shown in Fig 6A. One-way ANOVA and Bonferroni post hoc test showed that sulcardine at 10 μM—300 μM significantly
inhibited hNav1.5 currents (all P < 0.05). The mean data-points were fitted using the Hill equation (Fig 6B). The calculated IC50 was 15.0 μM (Hill coefficient = –1.048, n = 7). Fig 6C shows that the inhibition of the hNav1.5 currents by sulcardine reached maximal conditions immediately. After washout, the blockade of sulcardine on hNav1.5 currents was partially reversed (n = 5). Tetrodotoxin was used as a positive control, the IC50 of tetrodotoxin being 6.3 μΜ (n = 6), which is similar to previous reports (IC50 = 5.7 μΜ) (Gellens et al., 1992). In time-course control tests, the hNav1.5 peak currents remained 98.7 ± 3.9 % of the initial value (n = 6) at the end of the test (20 min).
3.5 Sulcardine inhibited hNav1.5 currents in a state-dependent manner To clarify the electrophysiological mechanisms underlying the inhibitory effect of sulcardine on hNav1.5 currents, we investigated the effects of sulcardine on the activation and inactivation curves of hNav1.5 currents. The activation curves were obtained using depolarization test pulses from –70 mV to –10 mV from a holding potential of –80 mV with 10 mV increments at 0.2 Hz (Fig 7A). Paired student’s t-tests indicated that sulcardine did not influence the voltage dependence of activation (Fig 7B). Half-maximal current activation voltages (Vh) were –40.7 ± 1.4 mV for the controls and –41.5 ± 2.1 mV for the sulcardine-treated cells, respectively (P > 0.05, n = 6). Slope factors (k) were 3.1 ± 0.4 mV for controls and 3.8 ± 0.2 mV for the sulcardine-treated cells, respectively (P > 0.05, n = 6). Inactivation curves were obtained using a two-pulse protocol in which currents were first elicited by the
conditioning pulses from –150 to –50 mV from a holding potential of –80 mV for 1 second with 10 mV increments, followed by the depolarization to –30 mV at 0.2 Hz (McNulty and Hanck, 2004). As shown in Fig 7C, the smooth curves represent the best fits for the data using a Boltzmann function. Sulcardine at 30 μM shifted half-maximal current inactivation voltage (Vh) from –81.5 ± 3.3 mV to –89.7 ± 4.6 mV (P < 0.01, n = 6, paired t-test). However, sulcardine did not change the slope factor (k), which was 5.5 ± 0.9 mV and 6.2 ± 1.3 mV before and after the application of sulcardine at 30 μM, respectively (P > 0.05, n = 6, paired t-test). These results indicate that sulcardine preferentially blocks inactivated hNav1.5 channels. As shown in Fig 7C, sulcardine preferentially blocks inactivated hNav1.5 channels, it seems like that membrane depolarization may facilitate sodium channel blockade by sulcardine. To confirm this postulation, we compared the extent of blockade by sulcardine (1— 300 μM) at the resting state (–120 mV holding potential) to that at the inactivated state (–65 mV holding potential), using voltage steps of –30 mV to elicit hNav1.5 currents. At a holding potential of –65 mV, the calculated IC50 of sulcardine was 8.8 μM (Hill coefficient = –0.9578, n = 8), nevertheless, at the resting state (–120 mV holding potential), only 300 μM sulcardine slightly reduced hNav1.5 currents (8.2 ± 3.9%, n = 5), and an IC50 value could not be calculated in the tested concentration range (1—300 μM). These data suggest that sulcardine has a higher affinity for the inactivated state of the hNav1.5 channels (Fig 8A). Recovery from fast inactivation before and after the application of sulcardine at 30 μM is shown in Fig 8B. A double pulse protocol was used to elicit hNav1.5
currents by a 1000 ms prepulse (Ip) to –30 mV from a holding potential of –80 mV, followed by varying intervals at –80 mV, and a 30 ms test pulse (It) to –30 mV (Wang et al., 2004). Relative values of hNav1.5 (It/Ip) were plotted against the interpulse interval in the absence and presence of sulcardine at 30 μM (n = 6). Solid lines were obtained by fitting with a single exponential function. The pooled time constants of hNav1.5 currents recovery from fast inactivation in the absence and presence of sulcardine at 30 μM were 54.3 ± 6.2 ms and 111.9 ± 33.4 ms, respectively (P < 0.01, n = 6, paired t-test, Fig 8B). These data indicate that sulcardine preferentially binds to inactivated hNav1.5 channels and delays hNav1.5 channels recovery from the inactivation state to the resting state.
3.6 Sulcardine inhibited hNav1.5 currents in a use-dependent manner The earlier experiments suggest the blockade of sulcardine on hNav1.5 channels is state-dependent. That is, sulcardine preferentially binds to the inactivated channels rather than the resting ones. To further determine the state characteristics of the block of sulcardine, the tonic and use-dependent block of sulcardine on hNav1.5 channels was tested. The tonic and use-dependent block were evaluated by holding channels at a very negative potential (–130 mV) in which most channels were in the resting state, exposing channels to sulcardine at 30 μM for 5 min and then initiating a train of pulses at various frequencies. Fig 9A shows the superimposed records of hNav1.5 currents obtained with repetitive depolarizing pulses at rates of 0.5 and 20 Hz. At 0.5 Hz, the amplitude of hNav1.5 currents evoked by the first pulse in the pulse train was
4.5 ± 2.0 nA in the control and 4.2 ± 2.2 nA with sulcardine (P > 0.05, n = 7, paired t-test), and at 20 Hz, the corresponding values were 4.5 ± 2.0 nA and 4.0 ± 2.1 nA (P > 0.05, n = 7, paired t-test). These data suggest that at the holding potential of –130 mV, sulcardine does not inhibit hNav1.5 currents at the first pulse, which means no tonic block was observed. With the increase in the frequencies of stimuli, sulcardine showed a higher potential blocking effect (Fig 9B). The averaged final-pulse currents normalized to the first-pulse currents (I16th pulse / I1st pulse ) in the presence of sulcardine were 0.97 ± 0.04 at 0.5 Hz, 0.91 ± 0.04 at 5 Hz (compared with 0.5 Hz, P < 0.05), 0.82 ± 0.09 at 10 Hz (compared with 0.5 Hz, P < 0.01), and 0.69 ± 0.2 at 20 Hz (compared with 0.5 Hz, P < 0.01)(Fig 9B). The normalized currents of the hNav1.5 currents elicited by each pulse successively applied are plotted in Fig 9C. In the absence of sulcardine, 16 successive pulses to –30 mV for 5 ms did not induce a significant decrease in hNav1.5 currents, even at the highest frequency (20 Hz). In the presence of 30 μM sulcardine, the hNav1.5 currents’ amplitude tended to successively decrease and then reach a steady-state level at 10 Hz and 20 Hz (Fig 9C). These data showed that sulcardine preferentially binds to open hNav1.5 channels rather than resting ones.
4. Discussion The present study demonstrated that sulcardine blocks hERG and hNav1.5 currents. Thus, the blocking characteristics of sulcardine on hERG and hNav1.5 channels may account for its potential anti-arrhythmic activity and safety. Channel
kinetics studies showed that sulcardine preferentially binds to hERG and hNav1.5 channels in the open and inactivated states. In the present study, we investigated the effects of sulcardine on hERG channels stably expressed in HEK293 for the first time. Our findings demonstrated that sulcardine inhibited hERG channels in a concentration- and state-dependent manner (Fig 2 and 3). We found that sulcardine inhibited hERG channels in a voltage-dependent manner, increasing at positive potentials, reflecting in a hyperpolarizing shift of voltage-dependence of activation (Fig 3). Furthermore, the blockade by sulcardine was close to zero at the time of channel opening by the application of the depolarizing voltage and quickly increased afterward (Fig 4 A and 4B). This high velocity of the blockade observed in the onset of the depolarization indicates that the blockade preferably happens at the open state (Scholz et al., 2003). Of noted, because the inhibitory effect was very small at the start of depolarization (i.e., at 0.05 and 0.1 s) and the current traces were even reversed before and after sulcardine exposure in some cells, standard deviations were very high, but with the increase of depolarization, the inhibitory effect tended to a steady state and the standard deviations became small. Besides, the maximal block was achieved in the inactivated state with no further development of an open channel blockade (Fig 4C and D). As such the blockade by sulcardine is dependent on the open and inactivated states, but not on the resting state. The electrophysiological mechanism of the hERG blockade by sulcardine is consistent with that of other agents known to produce acquired long QT syndrome by inhibiting hERG channels (Gu et al., 2009; Kikuchi et
al., 2005; Su et al., 2004). According to the modulated receptor hypothesis, the actions of class I blockers on sodium channels differs when the channels are in resting, open or inactivated states (Hille, 1977; Hondeghem and Katzung, 1977). Sulcardine has a slight effect on the resting hNav1.5 channels (i.e., at a holding potential of –120 mV, Fig 8), but significantly inhibited sodium current with a greater potency at the holding potential of –65 mV (Fig 7 and 8), suggesting that sulcardine preferentially blocks the hNav1.5 channels in an inactivated state. The preferential blockade of the inactivated sodium channels suggests that sulcardine likely reduces the excitability of pathological heart tissue accompanied with membrane depolarization, as is the case for instance in acute myocardial ischemia (Shaw and Rudy, 1997). We also observed a use-dependent inhibition of the hNav1.5 channels in the cells treated with sulcardine at a holding potential of –130 mV but saw no evidence of tonic block (Fig 9). The phenomenon of the use-dependent block is generally interpreted as a drug molecule binding to channels in an open and inactivated state during membrane depolarization. Since the hNav1.5 channels spend more time in the open and inactivated state as the interpulse interval shortens, the decrease in hNav1.5 currents at high rates of stimulation reflects an accumulation of drug-associated channels. In the Phase I clinical study, the highest level of the plasma concentration approximately reached 2.64 ± 1.48 μM after multiple dose administration and no severe adverse reaction was reported at this plasma concentration (Wang et al., 2017).
The plasma concentration is near the least concentration (10 μM) that can inhibit hNav1.5 channels, but far away from the least concentration (30 μM) required to inhibit hERG channels. As Phase II clinical study is still in progress, we cannot determine the accurate range of effective blood concentration of sulcardine. As such, we may postulate that the major ionic mechanism underlying the anti-arrhythmic effect of sulcardine is the inhibitory effect on hNav1.5 channels. Furthermore, the IC50 value of the hERG channel blockade is approximately 36-fold more than the highest plasma concentration. Therefore, the low risk of torsade de pointes arrhythmia triggered by sulcardine may be due to the lesser effect it has on hERG channels. Multiple channel blockade may also contribute to the lesser likelihood of torsade de pointes arrhythmia triggered by sulcardine (Chen et al., 2017; Wang et al., 2017). For example, sodium channel blockade may decrease the likelihood of torsade de pointes arrhythmia which may be triggered by hERG channel blockers, because sodium channel blockers may shorten the excessively prolonged QT intervals and normalize the repolarization (Windle et al., 2001). Fortunately, sulcardine exhibits a potent blocking effect on hNav1.5 channels, which may contribute to the efficacy and safety of sulcardine and its lesser likelihood of torsade de pointes arrhythmia. Also, sulcardine exhibited a strong inhibition of late sodium currents in human myocytes (IC50 = 16.5 ± 1.4 μM) (Guo et al., 2011). Late sodium current blockade can shorten excessively prolonged action potentials and QT intervals induced by hERG channel blockers and prevent torsade de pointes arrhythmia (Johannesen et al., 2016). As such, blockade of peak and late sodium currents by sulcardine may not only contribute to
the anti-arrhythmic effect but also reduce the likelihood of torsade de pointes arrhythmia triggered by hERG channel blockade. In addition, sulcardine inhibited calcium channels in isolated guinea pig ventricular myocytes (IC50 = 69.2 μM) (Bai et al., 2012) and in human myocytes (IC50 = 32.2 ± 2.9 μM) (Guo et al., 2011). These data indicate that calcium channel blockade may be involved in the antiarrhythmic effect and reduce the likelihood of torsade de pointes arrhythmia triggered by hERG channel blockade because calcium blockade may attenuate the prolonged QT interval induced by hERG channel blockade (Huang et al., 2007; Johannesen et al., 2014). Therefore, multiple channel blockade may be the main reason the anti-arrhythmic effect and safety of sulcardine.
Conclusion We clarified the electrophysiological characteristics of sulcardine on hERG and hNav1.5 channels. Sulcardine is a potent hNav1.5 channel blocker with a mild inhibitory effect on hERG channels. Channel kinetics studies showed that sulcardine preferentially binds to hERG and hNav1.5 channels in the open and inactivated states rather than the resting state. It has been proven that the combination of sodium and potassium channel-blocking actions may be promising both regarding the anti-arrhythmic activity and side effect minimization profiles. Therefore, we proposed that sulcardine is a promising drug for the treatment of arrhythmias with less tendency of pro-arrhythmia.
Acknowledgment This work was supported by the grants from the National Basic Research Program of China (No. 2009CB930300) and the Key Program of the State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (No. SIMM 0907KF-02). Conflict of interests The authors declare no conflict of interests.
Author contributions Weihai Chen and Yiping Wang designed experiments; Weihai Chen carried out experiments; Weihai Chen and Lu Gan analyzed experimental results. Weihai Chen and Lu Gan wrote the paper. References Antzelevitch, C., Belardinelli, L., Zygmunt, A.C., Burashnikov, A., Di Diego, J.M., Fish, J.M., Cordeiro, J.M., Thomas, G., 2004. Electrophysiological effects of ranolazine, a novel antianginal agent with antiarrhythmic properties. Circulation 110, 904-910. Bai, D.L., Chen, W.Z., Bo, Y.X., Dong, Y.L., Kang, A.L., Sun, W.K., Wang, W., Hu, Z.L., Wang, Y.P., 2012. Discovery of N-(3,5-bis(1-pyrrolidylmethyl)-4-hydroxybenzyl)-4-methoxybenzenesulfamide (sulcardine) as a novel anti-arrhythmic agent. Acta Pharmacol Sin 33, 1176-1186. Cairns, J.A., Connolly, S.J., Roberts, R., Gent, M., 1997. Randomised trial of outcome after myocardial infarction in patients with frequent or repetitive ventricular premature depolarisations: CAMIAT. Canadian Amiodarone Myocardial Infarction Arrhythmia Trial Investigators. Lancet 349, 675-682. Chen, Q., Qian, H.J., Wang, W., Zhang, M.Q., Lu, D.Y., Lu, C., Jin, J.M., Hu, C.Y., Liu, G.Y., Jia, J.Y., Zheng, H.C., Li, X.N., Yu, C., Wang, Y.P., Liu, Y., 2017. Pharmacokinetics, safety, and tolerability of sulcardine sulfate: an open-label, single-dose, randomized study in healthy Chinese subjects. Fundam Clin Pharmacol 31, 120-125. Chen, W.H., Wang, W.Y., Zhang, J., Yang, D., Wang, Y.P., 2010. State-dependent blockade of human ether-a-go-go-related gene (hERG) K(+) channels by changrolin
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P., 1997. Randomised trial of effect of amiodarone on mortality in patients with left-ventricular dysfunction after recent myocardial infarction: EMIAT. European Myocardial Infarct Amiodarone Trial Investigators. Lancet 349, 667-674. Kamiya, K., Mitcheson, J.S., Yasui, K., Kodama, I., Sanguinetti, M.C., 2001. Open channel block of HERG K(+) channels by vesnarinone. Mol Pharmacol 60, 244-253. Khan, I.A., Gowda, R.M., 2004. Novel therapeutics for treatment of long-QT syndrome and torsade de pointes. Int J Cardiol 95, 1-6. Kikuchi, K., Nagatomo, T., Abe, H., Kawakami, K., Duff, H.J., Makielski, J.C., January, C.T., Nakashima, Y., 2005. Blockade of HERG cardiac K+ current by antifungal drug miconazole. Br J Pharmacol 144, 840-848. Kirsch, G.E., Trepakova, E.S., Brimecombe, J.C., Sidach, S.S., Erickson, H.D., Kochan, M.C., Shyjka, L.M., Lacerda, A.E., Brown, A.M., 2004. Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern. J Pharmacol Toxicol Methods 50, 93-101. Lu, H.R., Rohrbacher, J., Vlaminckx, E., Van Ammel, K., Yan, G.X., Gallacher, D.J., 2010. Predicting drug-induced slowing of conduction and pro-arrhythmia: identifying the 'bad' sodium current blockers. Br J Pharmacol 160, 60-76. McNulty, M.M., Hanck, D.A., 2004. State-dependent mibefradil block of Na+ channels. Mol Pharmacol 66, 1652-1661. Robertson, G.A., 2000. LQT2 : amplitude reduction and loss of selectivity in the tail that wags the HERG channel. Circ Res 86, 492-493. Sanguinetti, M.C., Tristani-Firouzi, M., 2006. hERG potassium channels and cardiac arrhythmia. Nature 440, 463-469. Scholz, E.P., Zitron, E., Kiesecker, C., Lueck, S., Kathofer, S., Thomas, D., Weretka, S., Peth, S., Kreye, V.A., Schoels, W., Katus, H.A., Kiehn, J., Karle, C.A., 2003. Drug binding to aromatic residues in the HERG channel pore cavity as possible explanation for acquired Long QT syndrome by antiparkinsonian drug budipine. Naunyn Schmiedebergs Arch Pharmacol 368, 404-414. Shaw, R.M., Rudy, Y., 1997. Electrophysiologic effects of acute myocardial ischemia: a theoretical study of altered cell excitability and action potential duration. Cardiovasc Res 35, 256-272. Su, Z., Martin, R., Cox, B.F., Gintant, G., 2004. Mesoridazine: an open-channel blocker of human ether-a-go-go-related gene K+ channel. J Mol Cell Cardiol 36, 151-160. Thanacoody, H.K., Thomas, S.H., 2005. Tricyclic antidepressant poisoning : cardiovascular toxicity. Toxicol Rev 24, 205-214. The Cardiac Arrhythmia Suppression Trial (CAST) Investigators, 1989. Preliminary report: effect of encainide and flecainide on mortality in a randomized trial of arrhythmia suppression after myocardial infarction. N Engl J Med 321, 406-412. Thomas, D., Gut, B., Wendt-Nordahl, G., Kiehn, J., 2002. The antidepressant drug fluoxetine is an inhibitor of human ether-a-go-go-related gene (HERG) potassium channels. J Pharmacol Exp Ther 300, 543-548. Thomas, S.H., Behr, E.R., 2016. Pharmacological treatment of acquired QT prolongation and torsades de pointes. Br J Clin Pharmacol 81, 420-427.
Vandenberg, J.I., Walker, B.D., Campbell, T.J., 2001. HERG K+ channels: friend and foe. Trends Pharmacol Sci 22, 240-246. Waldo, A.L., Camm, A.J., deRuyter, H., Friedman, P.L., MacNeil, D.J., Pauls, J.F., Pitt, B., Pratt, C.M., Schwartz, P.J., Veltri, E.P., 1996. Effect of d-sotalol on mortality in patients with left ventricular dysfunction after recent and remote myocardial infarction. The SWORD Investigators. Survival With Oral d-Sotalol. Lancet 348, 7-12. Wang, W., Qian, H.J., Xin, L., Zhang, M.Q., Lu, D.Y., Jin, J.M., Liu, G.Y., Jia, J.Y., Zheng, H.C., Yu, C., Wang, Y.P., Zhu, F., Liu, Y., 2017. Multiple Dose Pharmacokinetics and Safety of Sulcardine Sulfate in Healthy Chinese Male Subjects: An Open-Label Phase I Clinical Study. Eur J Drug Metab Pharmacokinet 42, 593-599. Wang, W., Wang, Y.P., Hu, G.Y., 2004. Tetrahydroacridine inhibits voltage-dependent Na(+) current in guinea-pig ventricular myocytes. Acta Pharmacol Sin 25, 1138-1144. Windle, J.R., Geletka, R.C., Moss, A.J., Zareba, W., Atkins, D.L., 2001. Normalization of ventricular repolarization with flecainide in long QT syndrome patients with SCN5A:DeltaKPQ mutation. Ann Noninvasive Electrocardiol 6, 153-158.
Fig 1 Sulcardine sulfate (sulcardine) {N-[4-hydroxy-3,5-bis(pyrrolidin-1-ylmethyl)benzyl]-4-methoxybenzenesulcardinefo namide sulfate}. Fig 2 Inhibition of hERG channels by sulcardine. (A) Representative current traces recorded from the same cell before and after superfusion with sulcardine (10 μM, 30 μM and 100 μM, 300 μM, 1 mM respectively). (B) Concentration-response relationship of the effects of sulcardine on hERG peak tail currents (n = 5). The IC50 was 94.3 μM (Hill coefficient = –1.267). (C) Time course of hERG tail current inhibition by 300 μM sulcardine (n = 5). Fig 3 Effects of sulcardine on the voltage dependence of hERG channel activation. (A) Examples of recordings of hERG currents before and after exposure to sulcardine at 100 μM. The arrowhead indicates zero current level. (B) The corresponding activating
current amplitude at the end of the preceding test pulse is shown as a function of the test pulse potentials. Following the application of sulcardine at 100 μM, the maximal peak current amplitude (at +20 mV) was reduced by 52.5 ± 11.5% (n = 5). (C) Tail current amplitude as a function of the preceding test pulse potentials. The application of 100 μM sulcardine reduced the peak tail current amplitude by 59.2 ± 3.3% (n = 5). (D) Sulcardine inhibited activation currents by 19.0 ± 24.3% at 0 mV, and the maximal inhibitory ratio was achieved at +30 mV, followed by a progressive decrease accompanying the increment of test pulse potentials. (E) Sulcardine inhibited tail currents by 22.9 ± 22.3% at 0 mV and reached a maximal inhibitory ratio at +80 mV (n = 5). (F and G) Normalized hERG activating and tail currents were shifted by 8.2 ± 2.7 mV and 9.0 ± 1.3 mV respectively towards more negative potentials (P < 0.01, n = 5, paired t-test). Fig 4 Sulcardine blocked hERG channels in the open and inactivated states. (A) Representative current traces recorded before and after incubation with sulcardine at 100 μM (for 10 min, without intermittent test pulses). (B) The time-dependent increase of the inhibitory ratio at each time point after depolarization. At 0.05 and 0.1 s after depolarization, the inhibitory ratio was close to zero. At 1, 2 and 7.5 s after depolarization, the inhibitory ratio increased to 29.7 ± 38.0%, 40.3 ± 26.3% and 57.7 ± 10.1%, respectively (n = 5). (C) Inhibition of inactivated channels by 100 μM sulcardine. (D) The corresponding normalized relative block during the test pulse to 0 mV. Maximum inhibition was achieved at the beginning of the second pulse and no further time-dependent blockade occurred upon channel opening during the second
voltage step. At each time point from 0.05 s to 3.5 s at 0 mV, the inhibitory ratio was close to 40% (n = 6). Fig 5 Development of hERG channel blockade during depolarization was assessed using the “envelope of tails” protocol (lower panel). (A and B) Representative original current traces of the hERG channel, elicited by the “envelope of tails” protocol in the absence and presence of sulcardine at 100 μM. (C) The peak tail current elicited by a repolarizing step to –50 mV was plotted as a function of the test pulse duration. Solid lines were obtained by fitting with a single exponential function. (D) Sulcardine at 100 μM shortened the time constant of hERG current activation. *P < 0.05 compared to the control, n = 6. Fig 6 Inhibition of hNav1.5 channels by sulcardine. (A) Representative current traces recorded from the same cell under control conditions and after superfusion with sulcardine (3 μM, 10 μM and 30 μM, 100 μM, 300 μM respectively). (B) Concentration-response relationship of the effects of sulcardine on hNav1.5 peak currents (n = 7). The IC50 was 15.0 μM with a Hill coefficient of –1.048. (C) Time course of hNav1.5 current inhibition by sulcardine at 100 μM (n = 6). Fig 7 The effect of sulcardine on voltage-dependent steady-state activation and inactivation of hNav1.5 channels. The solid curves represent the best fits with the Boltzmann equation (see text). (A) Examples of recordings of hNav1.5 currents before and after exposure to sulcardine at 30 μM. (B) Normalized hNav1.5 conductance (GNa/Gmax) plotted as a function of membrane potential. Sulcardine did not affect the voltage dependence of activation. (P > 0.05, n = 6). (C) The effect of
sulcardine on the voltage–dependence of steady-state inactivation of hNav1.5 channels. The steady-state inactivation curves for the hNav1.5 channel were obtained by normalizing the current amplitudes (I) to the maximal value (Imax) and plotted as a function of the conditioning potentials in each condition. Sulcardine shifted half-maximal current inactivation (Vh) from –81.5 ± 3.3 mV to –89.7 ± 4.6 mV (P < 0.01, n = 6, paired t-test). Fig 8 (A) Concentration-response data for sulcardine to block hNav1.5 channels at holding potentials of –120 and –65 mV. At a holding potential of –65 mV, the IC50 was 8.8 μM (Hill coefficient was –0.9578, n = 8), but at a holding potential of –120 mV, only sulcardine at 300 μM slightly reduced the current by 8.2% ± 3.9% and IC 50 could not be calculated (n = 5). (B) Recovery from fast inactivation in the absence and presence of sulcardine at 30 μM. The double pulse protocol is shown in the inset. Ip was the current amplitude induced by 1000 ms prepulse potential from a holding potential of –80 mV to –30 mV, and It was the current amplitude induced by the 30 ms test pulse to –30 mV. Relative values of hNav1.5 (It/Ip) were plotted against the interpulse interval in the absence and presence of sulcardine at 30 μM. The pooled time constants of hNav1.5 current recovery from fast inactivation in the absence and presence of 30 μM sulcardine were 54.3 ± 6.2 ms and 111.9 ± 33.4 ms, respectively (P < 0.01, n = 6). Fig 9 Use-dependent block of sulcardine on hNav1.5 channels. (A) Superimposed recordings obtained during a train of 16 successive depolarizing pulses applied at a frequency of either 0.5 or 20 Hz before and during exposure to sulcardine at 30 μM.
The amplitude of the first pulse both at 0.5 and 2 Hz was not inhibited by sulcardine. (B) With the increase in the frequencies of the stimuli, sulcardine showed a higher potential blocking effect. *P < 0.05, **P < 0.01 compared to when they were delivered at 0.5 Hz (n = 7). (C) A relationship between the normalized hNav1.5 currents (normalized to the 1st pulse) and the number of pulses applied at different rates in the absence and presence of sulcardine (n = 7). In the absence of sulcardine, the increase in frequencies did not reduce the normalized amplitude (left panel). In the presence of sulcardine, at 0.5 Hz, the normalized currents did not decrease with the increase of the pulses, but at 10 and 20 Hz, the normalized currents gradually decreased with the increase of pulses (right panel).
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PII: DOI: Reference:
S0014-2999(18)30701-5 https://doi.org/10.1016/j.ejphar.2018.12.009 EJP72114
To appear in: European Journal of Pharmacology Received date: 26 May 2018 Revised date: 29 November 2018 Accepted date: 5 December 2018 Cite this article as: Weihai Chen, Lu Gan and Yiping Wang, Characteristics of hERG and hNav1.5 channel blockade by sulcardine sulfate, a novel antiarrhythmic compound, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.12.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characteristics of hERG and hNav1.5 channel blockade by sulcardine sulfate, a novel anti-arrhythmic compound
Weihai Chen1,2*, Lu Gan1, Yiping Wang2
1
Key Laboratory of Cognition and Personality (Southwest University), Ministry of
Education; Faculty of Psychology, Southwest University, Chongqing, China; 2
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai, China
*
Corresponding author. Weihai Chen, PhD Faculty of Psychology, Southwest
University Tianshen Road 2, Beibei District Chongqing, China Tel.: 86-13594021891. [email protected]
Abstract Sulcardine sulfate (sulcardine) is a novel anti-arrhythmic compound, which blocks multiple channels and was shown to be safe and tolerated in clinical trials. The aim of the present study was to investigate the electrophysiological characteristics of sulcardine on the hERG and hNav1.5 channels. The hERG and hNav1.5 channels were heterologously stably expressed in human embryonic kidney 293 cells, and the effects of sulcardine on the hERG and hNav1.5 channels were recorded using the standard whole-cell patch-clamp technique. Sulcardine inhibited hERG channels in a
concentration-dependent and reversible manner (IC50 = 94.3 μM). In addition, sulcardine shifted the activation curve of hERG channels to more negative potentials. The relative block of sulcardine on hERG channels was close to zero at the time point corresponding to channel opening, which was achieved by applying a depolarizing voltage, and quickly increased afterward. Sulcardine inhibited hNav1.5 channels in a concentration-dependent and reversible manner (IC50 = 15.0 μM) and shifted the inactivation curve of hNav1.5 channels to more negative potentials. The blockade of sulcardine on hNav1.5 channels was use-dependent. In conclusion, sulcardine is a potent hNav1.5 channel blocker with a mild inhibitory effect on hERG channels and preferentially binds to both hERG and hNav1.5 channels in the open and inactivated states rather than in the resting state.
Keywords: sulcardine sulfate; human ether-a-go-go-related gene (hERG); the cardiac sodium channels (hNav1.5); arrhythmia; torsade de pointes arrhythmia
1. Introduction Most anti-arrhythmic medications exert their effects by blocking sodium, potassium, or calcium ion channels. However, the Cardiac Arrhythmia Suppression Trial (CAST) test has shown that sodium channel blockers increase the rate of mortality owing to arrhythmia (The CAST Investigators, 1989). Cardiac sodium channel blockade can slow intracardiac conduction, which is manifested as prolongation of the QRS complex on the ECG (Delk et al., 2007; Lu et al., 2010). Furthermore, it is suggested that ventricular tachycardia and other arrhythmias caused
by sodium channel blockers increase morbidity and mortality (Delk et al., 2007; Thanacoody and Thomas, 2005). More recently, research on anti-arrhythmic medications has focused on agents that can prolong the action potential and the refractory period, such as potassium channel blockers (i.e., Vaughan-Williams class III anti-arrhythmic drugs). Unfortunately, potassium channel blockers may excessively prolong the QT interval and raise the risk of torsade de pointes arrhythmia, which is a lethal ventricular arrhythmia (Vandenberg et al., 2001) (Waldo et al., 1996). Human ether-a-go-go-related gene potassium (hERG) channels conduct the rapid delayed rectifier potassium currents (IKr) (Sanguinetti and Tristani-Firouzi, 2006). Inhibition of hERG channels tends to prolong the action potential and the QT interval, and this prolongation can be used to treat arrhythmias but also causes acquired long QT syndrome and the life-threatening torsade de pointes arrhythmia (Hancox et al., 2008). In addition, the cardiac sodium channels (hNav1.5), encoded by the human cardiac sodium channel SCN5A gene, are responsible for voltage-dependent sodium currents and electrical excitation of atrial and ventricular myocytes (Erdemli et al., 2012; Gellens et al., 1992). Interestingly, the sodium channel blockade can markedly reduce the likelihood of torsade de pointes arrhythmia, which may be triggered by potassium channel blockers (Antzelevitch et al., 2004; Cairns et al., 1997; Dujardin et al., 2008; Julian et al., 1997; Khan and Gowda, 2004; Thomas and Behr, 2016). These data suggest that sodium channel blockade may increase the safety of potassium channel blockers and reduce the likelihood of torsade de pointes arrhythmia.
Sulcardine sulfate (sulcardine) {N-[4-hydroxy-3,5-bis(pyrrolidin-1-ylmethyl)benzyl]-4-methoxybenzenesulcardinefo namide sulfate} (Fig 1) is a novel anti-arrhythmic drug developed by the Shanghai Institute of Materia Medica, China. Sulcardine exhibits various anti-arrhythmic activities on experimental arrhythmia models in animals (Bai et al., 2012). Furthermore, sulcardine decreased the amplitude and the maximal upstroke velocity of action potentials in a concentration-dependent manner and also prolonged action potential duration and the refractory period in in vitro studies (Bai et al., 2012; Guo et al., 2011). The electrophysiological characteristics of sulcardine may be related to the blockade on multiple channels including sodium, potassium, and calcium channels (Bai et al., 2012; Guo et al., 2011). More importantly, sulcardine is a safe and well-tolerated drug in healthy Chinese subjects, and no serious adverse events have been reported (Chen et al., 2017), even in a repeated administration clinical study (twice daily for 5 days) (Wang et al., 2017). These data suggest that sulcardine is a promising anti-arrhythmic drug with less likelihood of pro-arrhythmia. As discussed above, the blockade of sodium channels can markedly reduce the likelihood of torsade de pointes arrhythmia, which may be triggered by blocking the hERG channel. To further explore the ionic mechanisms underlying the efficacy and safety of sulcardine, we used the standard whole-cell patch-clamp technique. In this way, we could study the electrophysiological characteristics of sulcardine on the hERG and hNav1.5 channels, which were stably expressed in human embryonic kidney 293 (HEK293) cells. The blocking characteristics of sulcardine on the hERG
and hNav1.5 channels may provide substantial evidence to explain the ionic mechanisms underlying its efficacy and safety.
2. Materials and methods 2.1 Cell culture We used a conventional protocol to construct the cell lines that stably expressed hERG and hNav1.5 channels respectively (Chen et al., 2010). Briefly, the hERG/pcDNA3 plasmid was generously donated by Dr. G. Robertson (University of Wisconsin), and the hNav1.5/pcDNA3 plasmid was generously donated by Dr. Robert S. Kass (Columbia University College of Physicians & Surgeons). Human embryonic kidney cells 293 (HEK293) were transfected with the plasmids using the lipofection transfection reagent (Tiangen Biotech CO., LTD, Beijing, China). The HEK293 cells stably expressing hERG and hNav1.5 channels were cultured respectively in Dulbecco’s modified eagle medium (DMEM, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA, USA) and 0.4 g/L geneticin (G418) in an atmosphere of 95% air and 5% CO2 (Chen et al., 2010). 2.2 Solutions and drug administration The external bath solution for recording hERG currents contained (in mM): NaCl 137, KCl 4, CaCl2 1.8, MgCl2 1, HEPES 10 and Glucose 10; the pH was adjusted to 7.4 with NaOH). The pipette solution contained (in mM): KCl 130, CaCl2 1, MgCl2 5, Na2-ATP 5, EGTA 5 and HEPES 10; the pH was adjusted to 7.4 using KOH.
For studying hNav1.5 currents, cells were superfused with a bath solution consisting of (in mM) choline chloride 100, NaCl 20, KCl 5.4, Na2HPO4 0.33, glucose 10, CdCl2 0.1, CaCl2 1 and HEPES 10; the pH was adjusted to 7.4 with NaOH. The composition of the internal solution (in mM) was CsCl 120, MgCl2 5, EGTA 11, HEPES 10, Na2ATP 5 and CaCl2 1; the pH was adjusted to 7.4 using CsOH. The chemicals used in the external and internal solutions were purchased from the Sigma-Aldrich Chemical Company (Shanghai, China). The sulcardine was kindly provided by the Department of Medicinal Chemistry of the Shanghai Institute of Materia Medica (Shanghai, China). Sulcardine was diluted in the external solution to the desired concentration (1—1000 μM) before use. 2.3 Electrophysiological recordings We used the whole-cell patch-clamp recording technique to record currents at room temperature (22–26°C) using an Axopatch 200A amplifier (Axon Instruments, Foster City, USA) with pClamp/Clampex software (Axon Instruments, Foster City, CA, USA). Electrodes were constructed from borosilicate glass using a micropipette puller (P-97, Sutter Instrument Co., Novato, CA, USA). The final resistance of the electrode was 2 to 3 MΩ when filled with the pipette solution. 2.4 Statistical analysis For the patch-clamp experiments, data are expressed as means ± standard deviation (S.D.), where n represents the number of cell experiments performed. Statistical significance was analyzed using a paired Student’s t-tests or one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests for multiple
comparisons. GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used for the statistical analysis of the data. Differences were considered statistically significant at a p-value < 0.05. Concentration-response curves were fitted with Hill equations of the form: E= Emax /[1+(IC50/C) nH]
(1)
where E is the effect at concentration C, Emax is the maximal effect, IC50 is the concentration for half-maximal block and nH is the Hill coefficient. The steady-state activation curve was analyzed by determining the conductance from the equation: Gm = I/(Em – Erev)
(2)
where I is the peak current, Em is the test voltage and Erev is the estimated reversal potential. This was plotted against the corresponding voltage. Plots were fitted with the Boltzmann equation: G/Gmax = 1/1 + exp [(V1/2 – Vm)/k]
(3)
where G is the conductance following the test potential Vm, Gmax is the maximal conductance, V1/2 and k represent the voltage of activation midpoint and slope factor, respectively. The steady-state inactivation curve was determined by fitting the values of the normalized current to the test potential with the Boltzmann equation of the following form: I/Imax = 1/1 + exp [(Vm – V1/2)/k]
(4)
where I is the current amplitude following the test potential Vm, Imax is the maximal current, V1/2 is the half-maximal inactivation potential and k is the slope factor of the
inactivation curve.
3. Results 3.1 Sulcardine inhibited hERG currents in a concentration-dependent manner Sulcardine inhibited hERG currents in a concentration-dependent manner (Fig. 2A). The currents were recorded after at least 2 min of exposure to each concentration of sulcardine. The cell membrane was depolarized to +20 mV for 2 s, and then repolarized to –40 mV for 1.6 s, which elicited a large, slowly decaying, outward tail current that is the feature of hERG currents (Robertson, 2000). The holding potential was –80 mV in all experiments performed in this study unless indicated otherwise. To test the concentration-dependent inhibition by sulcardine, peak tail currents in the presence of sulcardine were normalized to their respective control values and plotted as relative current amplitudes in Fig 2B (n = 5). One-way ANOVA and Bonferroni post hoc tests showed that sulcardine significantly inhibited hERG currents at 30—1000 μM (all P < 0.05). The mean data-points were fitted with Hill equations, and the half-maximal inhibitory concentration (IC50) was 94.3 μM with a Hill coefficient of –1.267. We used cisapride as the positive control compound, the IC50 of which was 24.7 nM (n = 5), similar to a previous report (IC50 = 26 nM) (Kirsch et al., 2004). As shown in Fig 2C, the blocking effects of sulcardine can be partially reversed after washout. After a control recording of 2 min, the hERG channels were exposed to sulcardine at 300 μM and reached steady-state conditions after exposure for 1 min. After 5 min washout, the blocking effect of sulcardine on hERG was partially reversed (Fig 2C, n = 5). In time-course control tests, the hERG tail currents,
after perfusing with the bath solution for 30 min, were 97.8 ± 3.9% of the initial value (n = 6).
3.2 Sulcardine inhibited hERG currents in a voltage-dependent manner Fig 3 shows the effects of sulcardine on the hERG current-voltage (I-V) relationship. hERG currents were elicited from a holding potential of –80 mV by 2-second depolarizing steps from –80 mV to +80 mV, applied in 10-mV increments at 0.1 Hz to produce activation currents, followed by a repolarizing step to –40 mV for 1.6 s to elicit outward tail currents. Representative currents were recorded in control conditions and 10 min after the perfusion with sulcardine at 100 μM in the same cell (Fig 3A). Fig 3B shows the I-V curve at the end of the activating currents. Under control conditions, the activation threshold of hERG currents was –40 mV and the maximum of the activating currents was +20 mV before an observed current reduction at higher test pulse potentials due to inward rectification. A 10-min application of sulcardine at 100 μM reduced the current maximum (at +20 mV) by 52.5 ± 11.5% (n = 5). As shown in Fig 3C, tail currents were saturated after a test pulse potential of +30 mV or above and sulcardine at 100 μM reduced the peak tail current amplitude (after a test pulse to +80 mV) by 59.2 ± 3.3% (n = 5). The inhibitory ratios at each test pulse potential for activating and tail currents were shown in Fig 3D and 3E. The inhibitory ratio at each test pulse potential was calculated as (Icontrol–Isulcardine)/Icontrol. As shown in Fig 3D, sulcardine inhibited activation currents by 19.0 ± 24.3% at 0 mV, and the maximal inhibitory ratio was achieved at +30 mV by 54.5 ± 8.4%, followed
by a progressively decreased inhibitory effect accompanying the increment of test pulse potentials. As shown in Fig 3E, sulcardine inhibited tail currents by 22.9 ± 22.3% at 0 mV and reached a maximal inhibitory ratio at +80 mV (n = 5). Normalized activating and tail currents in the absence and presence of sulcardine are shown in Fig 3F and 3G. The normalized activating and tail currents were fitted with a Boltzmann function and the mean half-maximal activation voltages (V1/2) of the activation curves of the hERG activating and tail currents were significantly shifted by 8.2 ± 2.7 mV and 9.0 ± 1.3 mV respectively towards more negative potentials (P < 0.01, n = 5, paired t-test).
3.3 Sulcardine inhibited hERG currents in a state-dependent manner We used three approaches to investigate the state-dependent block of hERG channels by sulcardine. In the first protocol, the membrane potential was increased from –80 mV to 0 mV for 7.5 s to produce a large activation current, which was first recorded in the absence of sulcardine. The cells were then perfused with sulcardine at 100 μM for 10 min without any intermittent stimulus, while the channels were kept in the resting state at –80 mV. Measurements in the presence of sulcardine were then conducted. As shown in Fig 4A, the blockade was close to zero at the time point of channel opening and quickly increased afterward. This blockade pattern was evidenced by the statistical analysis presented in Fig 4B, which illustrates the time-dependent increase of the inhibitory ratio, expressed as (Icontrol–Isulcardine)/Icontrol, at each time point after depolarization. As shown in Fig 4B, at 0.05 and 0.1 s after
depolarization, the inhibitory ratio was close to zero. At 1, 2 and 7.5 s after depolarization, the inhibitory ratio increased to 29.7 ± 38.0%, 40.3 ± 26.3% and 57.7 ± 10.1%, respectively (n = 5). This is consistent with the blockade of the hERG channels in the open state, but not in the resting state, which is a common pattern among hERG antagonists (Scholz et al., 2003; Thomas et al., 2002). The second protocol was used to test if sulcardine inhibited hERG channels in the inactivated state. A long-term depolarization potential to +80 mV (4000 ms) from a holding potential of –80 mV was used to inactivate the channels, and then a second test pulse to 0 mV (3500 ms) was applied to rapidly recover the channels from inactivation. Typical current traces before and after incubation with sulcardine for 10 min are shown in Fig 4C. Fig 4D represents the inhibitory ratio at each time point during the second test pulse (0 mV). Sulcardine inhibited hERG channels during the preceding inactivation (+80 mV pulse) and an additional time-dependent blockade of open channels was not observed during the 0 mV pulse. As shown in Fig 4D, at each time point from 0.05 to 3.5 s during repolarization at 0 mV, the inhibitory ratio was close to 40% (n = 6). These data indicate that sulcardine inhibited hERG channels to a maximal degree during the first inactivation test potential to +80 mV while the majority of channels stayed in the inactivated state, suggesting that sulcardine blocks hERG channels in the inactivated state, rather than in the resting state. The third protocol, an “envelope of tails” test, was conducted to test whether the hERG channel blockade by sulcardine was taking place during depolarization (Kamiya et al., 2001). Cells were firstly depolarized to +30 mV from a holding
potential of –80 mV for a variable duration ranging from 25 ms to 1600 ms, followed by a repolarizing step to –50 mV to induce hERG tail currents at 0.1 Hz. Tail currents at –50 mV were measured after each test pulse. We carried out the “envelope of tails” test in the same cell both before and after the application of sulcardine at 100 μM. Typical current traces before and after the application of sulcardine are shown in Fig 5A and 5B. hERG tail current amplitude increased with the depolarizing pulse duration over the first few pulses and then reached a steady maximal amplitude. The time constants of the hERG current activation were estimated by fitting the peak amplitude of the tail currents with a single exponential function (Fig 5C). The fitting yielded time constants of 283.1 ± 48.4 ms and 201.1 ± 53.4 ms before and after the application of sulcardine at 100 μM respectively (P < 0.01, n = 6, paired t-test, Fig 5D). The shortened time constants suggest that blockade by sulcardine was enhanced by the further activation of currents. That is, with the increment of activation duration, sulcardine showed more potent relative blockade on hERG channels, which indicates that sulcardine tends to inhibit inactivated and/or open hERG channels.
3.4 Sulcardine inhibited hNav1.5 currents in a dose-dependent manner Large fast inward hNav1.5 currents were induced by a depolarization pulse to –30 mV for 30 ms from a holding potential of –80 mV. hNav1.5 channels were exposed to each concentration of sulcardine for 2 min (1—300 μM). Typical current tracings before and after application of sulcardine are shown in Fig 6A. One-way ANOVA and Bonferroni post hoc test showed that sulcardine at 10 μM—300 μM significantly
inhibited hNav1.5 currents (all P < 0.05). The mean data-points were fitted using the Hill equation (Fig 6B). The calculated IC50 was 15.0 μM (Hill coefficient = –1.048, n = 7). Fig 6C shows that the inhibition of the hNav1.5 currents by sulcardine reached maximal conditions immediately. After washout, the blockade of sulcardine on hNav1.5 currents was partially reversed (n = 5). Tetrodotoxin was used as a positive control, the IC50 of tetrodotoxin being 6.3 μΜ (n = 6), which is similar to previous reports (IC50 = 5.7 μΜ) (Gellens et al., 1992). In time-course control tests, the hNav1.5 peak currents remained 98.7 ± 3.9 % of the initial value (n = 6) at the end of the test (20 min).
3.5 Sulcardine inhibited hNav1.5 currents in a state-dependent manner To clarify the electrophysiological mechanisms underlying the inhibitory effect of sulcardine on hNav1.5 currents, we investigated the effects of sulcardine on the activation and inactivation curves of hNav1.5 currents. The activation curves were obtained using depolarization test pulses from –70 mV to –10 mV from a holding potential of –80 mV with 10 mV increments at 0.2 Hz (Fig 7A). Paired student’s t-tests indicated that sulcardine did not influence the voltage dependence of activation (Fig 7B). Half-maximal current activation voltages (Vh) were –40.7 ± 1.4 mV for the controls and –41.5 ± 2.1 mV for the sulcardine-treated cells, respectively (P > 0.05, n = 6). Slope factors (k) were 3.1 ± 0.4 mV for controls and 3.8 ± 0.2 mV for the sulcardine-treated cells, respectively (P > 0.05, n = 6). Inactivation curves were obtained using a two-pulse protocol in which currents were first elicited by the
conditioning pulses from –150 to –50 mV from a holding potential of –80 mV for 1 second with 10 mV increments, followed by the depolarization to –30 mV at 0.2 Hz (McNulty and Hanck, 2004). As shown in Fig 7C, the smooth curves represent the best fits for the data using a Boltzmann function. Sulcardine at 30 μM shifted half-maximal current inactivation voltage (Vh) from –81.5 ± 3.3 mV to –89.7 ± 4.6 mV (P < 0.01, n = 6, paired t-test). However, sulcardine did not change the slope factor (k), which was 5.5 ± 0.9 mV and 6.2 ± 1.3 mV before and after the application of sulcardine at 30 μM, respectively (P > 0.05, n = 6, paired t-test). These results indicate that sulcardine preferentially blocks inactivated hNav1.5 channels. As shown in Fig 7C, sulcardine preferentially blocks inactivated hNav1.5 channels, it seems like that membrane depolarization may facilitate sodium channel blockade by sulcardine. To confirm this postulation, we compared the extent of blockade by sulcardine (1— 300 μM) at the resting state (–120 mV holding potential) to that at the inactivated state (–65 mV holding potential), using voltage steps of –30 mV to elicit hNav1.5 currents. At a holding potential of –65 mV, the calculated IC50 of sulcardine was 8.8 μM (Hill coefficient = –0.9578, n = 8), nevertheless, at the resting state (–120 mV holding potential), only 300 μM sulcardine slightly reduced hNav1.5 currents (8.2 ± 3.9%, n = 5), and an IC50 value could not be calculated in the tested concentration range (1—300 μM). These data suggest that sulcardine has a higher affinity for the inactivated state of the hNav1.5 channels (Fig 8A). Recovery from fast inactivation before and after the application of sulcardine at 30 μM is shown in Fig 8B. A double pulse protocol was used to elicit hNav1.5
currents by a 1000 ms prepulse (Ip) to –30 mV from a holding potential of –80 mV, followed by varying intervals at –80 mV, and a 30 ms test pulse (It) to –30 mV (Wang et al., 2004). Relative values of hNav1.5 (It/Ip) were plotted against the interpulse interval in the absence and presence of sulcardine at 30 μM (n = 6). Solid lines were obtained by fitting with a single exponential function. The pooled time constants of hNav1.5 currents recovery from fast inactivation in the absence and presence of sulcardine at 30 μM were 54.3 ± 6.2 ms and 111.9 ± 33.4 ms, respectively (P < 0.01, n = 6, paired t-test, Fig 8B). These data indicate that sulcardine preferentially binds to inactivated hNav1.5 channels and delays hNav1.5 channels recovery from the inactivation state to the resting state.
3.6 Sulcardine inhibited hNav1.5 currents in a use-dependent manner The earlier experiments suggest the blockade of sulcardine on hNav1.5 channels is state-dependent. That is, sulcardine preferentially binds to the inactivated channels rather than the resting ones. To further determine the state characteristics of the block of sulcardine, the tonic and use-dependent block of sulcardine on hNav1.5 channels was tested. The tonic and use-dependent block were evaluated by holding channels at a very negative potential (–130 mV) in which most channels were in the resting state, exposing channels to sulcardine at 30 μM for 5 min and then initiating a train of pulses at various frequencies. Fig 9A shows the superimposed records of hNav1.5 currents obtained with repetitive depolarizing pulses at rates of 0.5 and 20 Hz. At 0.5 Hz, the amplitude of hNav1.5 currents evoked by the first pulse in the pulse train was
4.5 ± 2.0 nA in the control and 4.2 ± 2.2 nA with sulcardine (P > 0.05, n = 7, paired t-test), and at 20 Hz, the corresponding values were 4.5 ± 2.0 nA and 4.0 ± 2.1 nA (P > 0.05, n = 7, paired t-test). These data suggest that at the holding potential of –130 mV, sulcardine does not inhibit hNav1.5 currents at the first pulse, which means no tonic block was observed. With the increase in the frequencies of stimuli, sulcardine showed a higher potential blocking effect (Fig 9B). The averaged final-pulse currents normalized to the first-pulse currents (I16th pulse / I1st pulse ) in the presence of sulcardine were 0.97 ± 0.04 at 0.5 Hz, 0.91 ± 0.04 at 5 Hz (compared with 0.5 Hz, P < 0.05), 0.82 ± 0.09 at 10 Hz (compared with 0.5 Hz, P < 0.01), and 0.69 ± 0.2 at 20 Hz (compared with 0.5 Hz, P < 0.01)(Fig 9B). The normalized currents of the hNav1.5 currents elicited by each pulse successively applied are plotted in Fig 9C. In the absence of sulcardine, 16 successive pulses to –30 mV for 5 ms did not induce a significant decrease in hNav1.5 currents, even at the highest frequency (20 Hz). In the presence of 30 μM sulcardine, the hNav1.5 currents’ amplitude tended to successively decrease and then reach a steady-state level at 10 Hz and 20 Hz (Fig 9C). These data showed that sulcardine preferentially binds to open hNav1.5 channels rather than resting ones.
4. Discussion The present study demonstrated that sulcardine blocks hERG and hNav1.5 currents. Thus, the blocking characteristics of sulcardine on hERG and hNav1.5 channels may account for its potential anti-arrhythmic activity and safety. Channel
kinetics studies showed that sulcardine preferentially binds to hERG and hNav1.5 channels in the open and inactivated states. In the present study, we investigated the effects of sulcardine on hERG channels stably expressed in HEK293 for the first time. Our findings demonstrated that sulcardine inhibited hERG channels in a concentration- and state-dependent manner (Fig 2 and 3). We found that sulcardine inhibited hERG channels in a voltage-dependent manner, increasing at positive potentials, reflecting in a hyperpolarizing shift of voltage-dependence of activation (Fig 3). Furthermore, the blockade by sulcardine was close to zero at the time of channel opening by the application of the depolarizing voltage and quickly increased afterward (Fig 4 A and 4B). This high velocity of the blockade observed in the onset of the depolarization indicates that the blockade preferably happens at the open state (Scholz et al., 2003). Of noted, because the inhibitory effect was very small at the start of depolarization (i.e., at 0.05 and 0.1 s) and the current traces were even reversed before and after sulcardine exposure in some cells, standard deviations were very high, but with the increase of depolarization, the inhibitory effect tended to a steady state and the standard deviations became small. Besides, the maximal block was achieved in the inactivated state with no further development of an open channel blockade (Fig 4C and D). As such the blockade by sulcardine is dependent on the open and inactivated states, but not on the resting state. The electrophysiological mechanism of the hERG blockade by sulcardine is consistent with that of other agents known to produce acquired long QT syndrome by inhibiting hERG channels (Gu et al., 2009; Kikuchi et
al., 2005; Su et al., 2004). According to the modulated receptor hypothesis, the actions of class I blockers on sodium channels differs when the channels are in resting, open or inactivated states (Hille, 1977; Hondeghem and Katzung, 1977). Sulcardine has a slight effect on the resting hNav1.5 channels (i.e., at a holding potential of –120 mV, Fig 8), but significantly inhibited sodium current with a greater potency at the holding potential of –65 mV (Fig 7 and 8), suggesting that sulcardine preferentially blocks the hNav1.5 channels in an inactivated state. The preferential blockade of the inactivated sodium channels suggests that sulcardine likely reduces the excitability of pathological heart tissue accompanied with membrane depolarization, as is the case for instance in acute myocardial ischemia (Shaw and Rudy, 1997). We also observed a use-dependent inhibition of the hNav1.5 channels in the cells treated with sulcardine at a holding potential of –130 mV but saw no evidence of tonic block (Fig 9). The phenomenon of the use-dependent block is generally interpreted as a drug molecule binding to channels in an open and inactivated state during membrane depolarization. Since the hNav1.5 channels spend more time in the open and inactivated state as the interpulse interval shortens, the decrease in hNav1.5 currents at high rates of stimulation reflects an accumulation of drug-associated channels. In the Phase I clinical study, the highest level of the plasma concentration approximately reached 2.64 ± 1.48 μM after multiple dose administration and no severe adverse reaction was reported at this plasma concentration (Wang et al., 2017).
The plasma concentration is near the least concentration (10 μM) that can inhibit hNav1.5 channels, but far away from the least concentration (30 μM) required to inhibit hERG channels. As Phase II clinical study is still in progress, we cannot determine the accurate range of effective blood concentration of sulcardine. As such, we may postulate that the major ionic mechanism underlying the anti-arrhythmic effect of sulcardine is the inhibitory effect on hNav1.5 channels. Furthermore, the IC50 value of the hERG channel blockade is approximately 36-fold more than the highest plasma concentration. Therefore, the low risk of torsade de pointes arrhythmia triggered by sulcardine may be due to the lesser effect it has on hERG channels. Multiple channel blockade may also contribute to the lesser likelihood of torsade de pointes arrhythmia triggered by sulcardine (Chen et al., 2017; Wang et al., 2017). For example, sodium channel blockade may decrease the likelihood of torsade de pointes arrhythmia which may be triggered by hERG channel blockers, because sodium channel blockers may shorten the excessively prolonged QT intervals and normalize the repolarization (Windle et al., 2001). Fortunately, sulcardine exhibits a potent blocking effect on hNav1.5 channels, which may contribute to the efficacy and safety of sulcardine and its lesser likelihood of torsade de pointes arrhythmia. Also, sulcardine exhibited a strong inhibition of late sodium currents in human myocytes (IC50 = 16.5 ± 1.4 μM) (Guo et al., 2011). Late sodium current blockade can shorten excessively prolonged action potentials and QT intervals induced by hERG channel blockers and prevent torsade de pointes arrhythmia (Johannesen et al., 2016). As such, blockade of peak and late sodium currents by sulcardine may not only contribute to
the anti-arrhythmic effect but also reduce the likelihood of torsade de pointes arrhythmia triggered by hERG channel blockade. In addition, sulcardine inhibited calcium channels in isolated guinea pig ventricular myocytes (IC50 = 69.2 μM) (Bai et al., 2012) and in human myocytes (IC50 = 32.2 ± 2.9 μM) (Guo et al., 2011). These data indicate that calcium channel blockade may be involved in the antiarrhythmic effect and reduce the likelihood of torsade de pointes arrhythmia triggered by hERG channel blockade because calcium blockade may attenuate the prolonged QT interval induced by hERG channel blockade (Huang et al., 2007; Johannesen et al., 2014). Therefore, multiple channel blockade may be the main reason the anti-arrhythmic effect and safety of sulcardine.
Conclusion We clarified the electrophysiological characteristics of sulcardine on hERG and hNav1.5 channels. Sulcardine is a potent hNav1.5 channel blocker with a mild inhibitory effect on hERG channels. Channel kinetics studies showed that sulcardine preferentially binds to hERG and hNav1.5 channels in the open and inactivated states rather than the resting state. It has been proven that the combination of sodium and potassium channel-blocking actions may be promising both regarding the anti-arrhythmic activity and side effect minimization profiles. Therefore, we proposed that sulcardine is a promising drug for the treatment of arrhythmias with less tendency of pro-arrhythmia.
Acknowledgment This work was supported by the grants from the National Basic Research Program of China (No. 2009CB930300) and the Key Program of the State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (No. SIMM 0907KF-02). Conflict of interests The authors declare no conflict of interests.
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Fig 1 Sulcardine sulfate (sulcardine) {N-[4-hydroxy-3,5-bis(pyrrolidin-1-ylmethyl)benzyl]-4-methoxybenzenesulcardinefo namide sulfate}. Fig 2 Inhibition of hERG channels by sulcardine. (A) Representative current traces recorded from the same cell before and after superfusion with sulcardine (10 μM, 30 μM and 100 μM, 300 μM, 1 mM respectively). (B) Concentration-response relationship of the effects of sulcardine on hERG peak tail currents (n = 5). The IC50 was 94.3 μM (Hill coefficient = –1.267). (C) Time course of hERG tail current inhibition by 300 μM sulcardine (n = 5). Fig 3 Effects of sulcardine on the voltage dependence of hERG channel activation. (A) Examples of recordings of hERG currents before and after exposure to sulcardine at 100 μM. The arrowhead indicates zero current level. (B) The corresponding activating
current amplitude at the end of the preceding test pulse is shown as a function of the test pulse potentials. Following the application of sulcardine at 100 μM, the maximal peak current amplitude (at +20 mV) was reduced by 52.5 ± 11.5% (n = 5). (C) Tail current amplitude as a function of the preceding test pulse potentials. The application of 100 μM sulcardine reduced the peak tail current amplitude by 59.2 ± 3.3% (n = 5). (D) Sulcardine inhibited activation currents by 19.0 ± 24.3% at 0 mV, and the maximal inhibitory ratio was achieved at +30 mV, followed by a progressive decrease accompanying the increment of test pulse potentials. (E) Sulcardine inhibited tail currents by 22.9 ± 22.3% at 0 mV and reached a maximal inhibitory ratio at +80 mV (n = 5). (F and G) Normalized hERG activating and tail currents were shifted by 8.2 ± 2.7 mV and 9.0 ± 1.3 mV respectively towards more negative potentials (P < 0.01, n = 5, paired t-test). Fig 4 Sulcardine blocked hERG channels in the open and inactivated states. (A) Representative current traces recorded before and after incubation with sulcardine at 100 μM (for 10 min, without intermittent test pulses). (B) The time-dependent increase of the inhibitory ratio at each time point after depolarization. At 0.05 and 0.1 s after depolarization, the inhibitory ratio was close to zero. At 1, 2 and 7.5 s after depolarization, the inhibitory ratio increased to 29.7 ± 38.0%, 40.3 ± 26.3% and 57.7 ± 10.1%, respectively (n = 5). (C) Inhibition of inactivated channels by 100 μM sulcardine. (D) The corresponding normalized relative block during the test pulse to 0 mV. Maximum inhibition was achieved at the beginning of the second pulse and no further time-dependent blockade occurred upon channel opening during the second
voltage step. At each time point from 0.05 s to 3.5 s at 0 mV, the inhibitory ratio was close to 40% (n = 6). Fig 5 Development of hERG channel blockade during depolarization was assessed using the “envelope of tails” protocol (lower panel). (A and B) Representative original current traces of the hERG channel, elicited by the “envelope of tails” protocol in the absence and presence of sulcardine at 100 μM. (C) The peak tail current elicited by a repolarizing step to –50 mV was plotted as a function of the test pulse duration. Solid lines were obtained by fitting with a single exponential function. (D) Sulcardine at 100 μM shortened the time constant of hERG current activation. *P < 0.05 compared to the control, n = 6. Fig 6 Inhibition of hNav1.5 channels by sulcardine. (A) Representative current traces recorded from the same cell under control conditions and after superfusion with sulcardine (3 μM, 10 μM and 30 μM, 100 μM, 300 μM respectively). (B) Concentration-response relationship of the effects of sulcardine on hNav1.5 peak currents (n = 7). The IC50 was 15.0 μM with a Hill coefficient of –1.048. (C) Time course of hNav1.5 current inhibition by sulcardine at 100 μM (n = 6). Fig 7 The effect of sulcardine on voltage-dependent steady-state activation and inactivation of hNav1.5 channels. The solid curves represent the best fits with the Boltzmann equation (see text). (A) Examples of recordings of hNav1.5 currents before and after exposure to sulcardine at 30 μM. (B) Normalized hNav1.5 conductance (GNa/Gmax) plotted as a function of membrane potential. Sulcardine did not affect the voltage dependence of activation. (P > 0.05, n = 6). (C) The effect of
sulcardine on the voltage–dependence of steady-state inactivation of hNav1.5 channels. The steady-state inactivation curves for the hNav1.5 channel were obtained by normalizing the current amplitudes (I) to the maximal value (Imax) and plotted as a function of the conditioning potentials in each condition. Sulcardine shifted half-maximal current inactivation (Vh) from –81.5 ± 3.3 mV to –89.7 ± 4.6 mV (P < 0.01, n = 6, paired t-test). Fig 8 (A) Concentration-response data for sulcardine to block hNav1.5 channels at holding potentials of –120 and –65 mV. At a holding potential of –65 mV, the IC50 was 8.8 μM (Hill coefficient was –0.9578, n = 8), but at a holding potential of –120 mV, only sulcardine at 300 μM slightly reduced the current by 8.2% ± 3.9% and IC 50 could not be calculated (n = 5). (B) Recovery from fast inactivation in the absence and presence of sulcardine at 30 μM. The double pulse protocol is shown in the inset. Ip was the current amplitude induced by 1000 ms prepulse potential from a holding potential of –80 mV to –30 mV, and It was the current amplitude induced by the 30 ms test pulse to –30 mV. Relative values of hNav1.5 (It/Ip) were plotted against the interpulse interval in the absence and presence of sulcardine at 30 μM. The pooled time constants of hNav1.5 current recovery from fast inactivation in the absence and presence of 30 μM sulcardine were 54.3 ± 6.2 ms and 111.9 ± 33.4 ms, respectively (P < 0.01, n = 6). Fig 9 Use-dependent block of sulcardine on hNav1.5 channels. (A) Superimposed recordings obtained during a train of 16 successive depolarizing pulses applied at a frequency of either 0.5 or 20 Hz before and during exposure to sulcardine at 30 μM.
The amplitude of the first pulse both at 0.5 and 2 Hz was not inhibited by sulcardine. (B) With the increase in the frequencies of the stimuli, sulcardine showed a higher potential blocking effect. *P < 0.05, **P < 0.01 compared to when they were delivered at 0.5 Hz (n = 7). (C) A relationship between the normalized hNav1.5 currents (normalized to the 1st pulse) and the number of pulses applied at different rates in the absence and presence of sulcardine (n = 7). In the absence of sulcardine, the increase in frequencies did not reduce the normalized amplitude (left panel). In the presence of sulcardine, at 0.5 Hz, the normalized currents did not decrease with the increase of the pulses, but at 10 and 20 Hz, the normalized currents gradually decreased with the increase of pulses (right panel).
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