Berberine, a benzodioxoloquinozine alkaloid, has been demonstrated experimentally to possess an antiarrhythmic action and a positive inotropic effect (1-10). Clinical studies also showed that this compound produces a beneficial effect in the treatment of cardiac arrhythmia and heart failure (11-13). It has been reported that berberine prolongs the action potential duration (APD) and effective refractory period (ERP), decreases the maximal velocity of depolarization (Vmax), and suppresses the development of delayed afterdepolarizations and triggered activity in cardiac myocytes. These are thought to be the important electrophysiologic mechanisms responsible for its antiarrhythmic action (6,9,10,14-16). Recently by using the patch clamp technique, we and other investigators showed that berberine blocks the cardiac adenosine triphosphate (ATP)-sensitive K+ channels, which might contribute to inhibiting the shortening of repolarization and subsequently preventing the development of cardiac arrhythmias during ischemia (17,18). Additionally, this alkaloid has been demonstrated to increase the open probability of single L-type Ca2+ channels in ventricular myocytes and to inhibit hyperpolarization-activated inward currents in sinoatrial node cells (19,20). However, the ionic basis for prolongation by berberine of cardiac repolarization remains elusive.
It is generally accepted that the effects of drugs on action-potential repolarization are an important factor for determining their antiarrhythmic and positive inotropic actions. Repolarization is controlled by several membrane currents including the delayed rectifier K+ current (IK), inward rectifier K+ current (IKl), L-type Ca2+ current (ICa), tetrodotoxin (TTX)-sensitive window Na current, and the Na+-Ca2+ exchange current (21). Therefore the prolongation of APD caused by drugs should be associated with a change in one or more of these currents.
The aim of this study was to examine the effects of berberine on membrane currents forming the repolarization phase of action potentials in single isolated guinea pig ventricular myocytes by using the patch-clamp technique. This should shed some light on the ionic mechanisms responsible for the effects of berberine.
Single myocyte preparations
The isolation procedure of single myocytes was described previously (18). In brief, male or female guinea pigs weighing 300-400 g were killed by cervical dislocation. Hearts were quickly mounted on a Langendorff apparatus for a retrograde coronary perfusion at a constant rate of 10 ml/min. After perfusion by Krebs-Henseleit solution (2 mM Ca2+) for 4 min, hearts continued to be perfused with nominally calcium-free Joklik solution for 4 min and then for 15 min with enzyme Joklik solution containing 0.4 mg/ml collagenase (Worthington, Freehold, NJ, U.S.A.), 0.5 mg/ml protease (Sigma), 0.2 mg/ml trypsin (Serva), 1 mg/ml albumin (Sigma), and 50 μM Ca2+. The solutions (pH 7.4) were gassed with 95% O2 and 5% CO2 and maintained at 37°C. After that, the single ventricular cells were obtained by the gentle agitation, and stored in the Joklik solution containing 1% albumin and 50 μM Ca2+ at room temperature for ≤6 h.
Single isolated cells were transferred to a 500-μl recording chamber placed on the stage of an inverted phase-contrast microscope. The chamber was continuously perfused at a rate of 4 ml/min with the solution (pH 7.4) prewarmed at 35 °C. Membrane currents were measured by the patch-clamp technique (22) by using a patch-clamp amplifier (L/M EPC7, List Medical Electronic, Lambrecht, FRG). Pipettes having 1.5-3 MΩ resistance were pulled from borosilicate glass capillaries. At the start of each experiment, the junction potential was adjusted to zero before the pipette was attached to the cell. After a gigaohm seal was obtained, the membrane patch was disrupted by a further slight suction to obtain the whole-cell configuration. Capacitance and series resistance were then compensated. For recording of transmembrane action potentials, the patch-clamp amplifier was switched to the current-clamp mode. Action potentials were induced by a depolarizing current pulse delivered through another microelectrode. Current and voltage signals were digitized at 3 KHz and stored on a disk. For the measurements of IK, both signals were digitized at 1 KHz. Data were reproduced and analyzed by an off-line computer.
The standard bath solution contained (in mM) 136.5 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 5.5 HEPES, and 5.5 glucose (pH adjusted to 7.4 with NaOH). The standard pipette solution consisted of (in mM) 80 K-aspartate, 50 KCl, 2 MgCl2, 5 NaCl, 11 HEPES, 0.1 EGTA, and 3 K2-ATP (pH = 7.4 with KOH). For the recording of the Na+-Ca2+ exchange current, the extracellular solution was (in mM) 136.5 NaCl, 4.0 CsCl, 1.0 BaCl2, 0.1 or 1.0 CaCl2, 2.0 MgCl2, 5.5 HEPES, and 5.5 glucose (pH adjusted to 7.4 with CsOH), including 20 μM ouabain and 2 μM verapamil to block the Na+-K+ ATPase and the L-type Ca2+ current. The pipette solution had (in mM): 80 Cs-aspartate, 50 CsCl, 20 NaCl, 5 HEPES, 42 EGTA, 21 CaCl2, 5 K2-creatine phosphate, 3 MgCl2, 10 Mg-ATP, and 20 tetraethylammonium-Cl (pH 7.4 with CsOH), which gave a final Ca2+ concentration of 69 nM according to Fabiato's computer programs (23). For measuring the L-type Ca2+ current, the composition of bath solution was (in mM) 136.5 NaCl, 5.4 CsCl, 1.8 CaCl2, 0.5 MgCl2, 5.5 HEPES, and 5.5 glucose (pH adjusted to 7.4 with NaOH). The pipette was filled with (in mM) 80 Cs-aspartate, 50 CsCl, 0.1 EGTA, 5 NaCl, 3 Mg-ATP, and 11 HEPES (pH 7.4 with CsOH).
Berberine was obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.), and dissolved in distilled water to make a stock solution with a concentration of 10 mM. Just before the experiments, the stock solution was added to the bath solution to achieve the final berberine concentration needed. Dofetilide was from Merck Research Laboratories (West Point, PA, U.S.A.). Both ouabain and verapamil were from Sigma Chemical Company.
All values are expressed as means ± SEM. Student's paired t test was used to determine the significance of differences between two observations within groups. Statistical analysis of the values between three observations was carried out by one-way analysis of variance (ANOVA) for repeated measurements. A p value of <0.05 was considered significant.
Effects on action potentials
In the control, APD at 90% repolarization (APD90) in isolated single ventricular myocytes paced at a frequency of 0.5 Hz was 421 ± 17 ms after 10-20 s of establishment of the whole-cell configuration. Action potential amplitude (APA) and resting potential (RP) were 128.9 ± 3 and 77.5 ± 2, respectively. These parameters were not significantly changed during 30 min of whole-cell recording. As can be seen in Fig. 1,[[AR FIG1]] application of 3 and 30 μM berberine caused a marked prolongation of APD90. This action became detectable within 2 min and reached a near steady-state at 10 min. Berberine at the concentration of 3 and 30 μM lengthened APD90 by 10.3 ± 1.1% (from 418 ± 21 to 461 ± 24 ms) and 24.7 ± 2.9% (from 411 ± 27 to 512 ± 22 ms), respectively (p < 0.01, n = 6). However, both APA and RP were not significantly altered after superfusion of berberine. These results bear a strong resemblance to those shown in multicellular preparations by using the standard microelectrode technique (6,10,14,16).
Effects on the delayed rectifier K+ current
To examine the possible interaction of berberine with IK, command voltage pulses of 5 s in duration from a holding potential of −40 mV to various membrane potentials between −30 and 80 mV in 10-mV increase steps were applied to ventricular cells at a frequency of 0.1 Hz. On repolarization to the potential of −30 mV, outward tail currents were induced. A measurement of its peak amplitude relative to the steady-state current after the tail-current decay provides an estimate of IK, because fast inward Na+ currents and T-type Ca2+ currents were inactivated by holding the cells at −40 mV, whereas L-type Ca2+ currents and the Na+ currents were totally blocked by 2 μM verapamil and 30 μM TTX in the bath solution. Under control conditions, the outward tail currents increased with depolarization and saturated at 60 mV.
Berberine (3 μM) significantly reduced the amplitude of the outward tail currents. After exposure to the higher concentration (30 μM), this compound produced a more potent inhibitory effect. Typical recordings before and after application of berberine (3 or 30 μM) obtained from the same cells depolarized from a holding potential of −40 mV to 60 mV are depicted in the top panels of Fig. 2. In the cells treated with 3 or 30 μM berberine, the outward tail currents were decreased from 155 and 162 to 116 and 104 pA, respectively. Quantitative data and statistical analysis of the effects of berberine are summarized in the middle panels of Fig. 2. It can be seen that little block developed at potentials more negative than 0 mV, but the peak outward tail currents were suppressed by berberine at voltages positive to 0 mV, and the suppression increased with the stronger depolarizations. When depolarized to 60 mV, the mean outward tail currents were decreased by 20.1 ± 1.8% (from 146 ± 11 to 117 ± 8 pA) with 3 μM, and by 37.5% ± 3.9% (from 142 ± 10 to 88 ± 9 pA) with 30 μM berberine (p < 0.01, n = 6). This suggests that the blocking effect by this agent might be dependent on the degree of channel activation.
To compare the half-maximal activation voltage (V50) and the slope factor (S) for the activation curve of the outward tail currents before and after application of berberine, the data were fit by the Boltzmann equation (1): where I is the recorded tail-current amplitude, Imax corresponds to the maximal current, and Vt is the test voltage. In the absence of the drug, V50 and S were 19.4 ± 1.7 and 18.3 ± 1.1 mV. Application of 3 and 30 μM berberine shifted V50 by 4.1 and 9.7 mV in the negative direction (from 19.2 ± 1.5 and 19.8 ± 1.8 mV to 15.1 ± 1.6 and 10.1 ± 2.2 mV, respectively; n = 6) without affecting the slope factor (Fig. 2).
Although analysis of the activation curves suggests that the block of the delayed rectifier K+ channels by berberine might be dependent on the degree of channel activation, whether the block is associated with intrinsic voltage dependence is unclear. To address this question, the fractional block (which was calculated as the amplitude of the outward tail currents at each potential after application of the drug normalized to that obtained before application of the drug, Idrug/Icontrol) was plotted against test potentials in the bottom panels of Fig. 2. With both concentrations, the degree of initial block increased steeply with stronger depolarizing potentials, but it reached a plateau level at potentials more positive than 60 mV, at which the channel activation was maximal in the absence of the drug. In other words, after the full activation of the channels was obtained, the extent of the block by berberine was not further increased with more positive potentials. This result indicates that berberine lacks intrinsic voltage-dependent block and is consistent with the view that the agent preferentially blocks the open-state channels.
It is well known that IK can be divided into two components, the rapidly activating IKr and the slowly activating IKs. Because berberine did not inhibit the IK tail currents at potential up to 0 mV, this alkaloid might block IKs but not IKr. Further to test this hypothesis, the effects of berberine on the isolated IKr and IKs were investigated. When cells are depolarized to −10 mV from a holding potential of −40 mV for 500 ms in Ca2+-free solution, only IKr is activated (24-26). Under these conditions, berberine (30 μM) had no effect on IKr. A typical example representive of five experiments and statistical data are shown in the top and bottom panels of Fig. 3A, respectively. By contrast, 0.2 μM dofetilide (the selective blocker of IKr; 26-28) almost completely inhibited the currents in the same cells (bottom panel of Fig. 3A). In the next inverse experiments, IKs was activated by stepping from a holding potential of −40 mV to 50 mV for 3 s under conditions in which IKr was blocked by 0.1 mM LaCl3 (24-26). Application of 30 μM berberine significantly inhibited IKs. Examples of current traces before and after application of this alkaloid are illustrated in the top panel of Fig. 3B. In a total of four cells tested, the mean amplitude of IKs was deceased by 34.1 ± 5.4% (from 140.6 ± 13.1 to 92.9 ± 8.2 pA, p < 0.05). However, exposure of myocytes to dofetilide (0.2 μM) did not affect the currents (bottom panel of Fig. 3B). These results further support the hypothesis that berberine blocks IKs but not IKr.
Effects on Na+-Ca2+ exchange currents
It is thought that the Na+-Ca2+ exchange current exerts a role in maintaining APD. Thus in this series of experiments, we tested the effects of berberine on this current with the same procedure reported by Kimura et al. (29). The current was induced by ramp pulses of ±90 mV/s (from −120 to 60 mV for 1 s) from a holding potential of −30 mV at a frequency of 0.1 Hz. The elevation of external Ca2+ concentration from 0.1 to 1.0 mM brought about a marked outward current, which was fully abolished by 5 mM NiCl (n = 6). Application of 3 μM berberine significantly augmented the outward Na+-Ca2+ exchange current. NiCl (5 mM) also completely blocked the increased outward current induced by berberine. With high concentration (30 μM), the alkaloid caused a greater Na+-Ca2+ exchange current. Sample current traces before and after berberine (3 and 30 μM) are shown in Fig. 4A. The mean values of the Na+-Ca2+ exchange current were increased from 0.76 ± 0.06 to 0.85 ± 0.07 nA with 3 μM berberine in the six cells tested and from 0.71 ± 0.05 to 0.88 ± 0.08 nA with 30 μM berberine in five other cells (p < 0.05; Fig. 4B).
Effects on L-type Ca2+ currents
To investigate the effects of berberine on ICa, a depolarizing pulse of 300-ms duration from a holding potential of −40 mV to 0 mV at a frequency of 0.5 Hz was applied to cardiomyocytes. In the absence of the drug, the peak ICa decayed with time because of the rundown phenomenon. The average value of ICa after 10 min from the establishment of the whole-cell recording was decreased by 7.5 ± 1.6% (from 1.44 ± 0.11 to 1.33 ± 0.08 nA) in a total of seven cells. In the presence of berberine, a substantial and concentration-dependent increase in the peak ICa was found. Examples of current traces are shown in Fig. 5A. Ten minutes after exposure to 3 and 30 μM berberine, the peak ICa was augmented by 17.5 ± 2.1% (p < 0.01, n = 6), and 33.7 ± 2.4 % (p < 0.01, n = 7), respectively. The time courses of its effects are summarized in Fig. 5B.
Figure 6 presents current-voltage relations for the peak ICa before and 10 min after the addition of berberine. The ventricular myocytes were depolarized for 300 ms from the holding potential of −40 mV to various potentials between −30 mV and 70 mV in increasing steps of 10 mV at a frequency of 0.2 Hz. In the presence of berberine, the peak ICa was significantly increased, but the threshold potential, the potential at which ICa was maximal, and the apparent reversal potential were not altered.
Effects on the inward rectifier current
To test the effects of berberine on IKl, the membrane potential was held at −40 mV, and hyperpolarizing voltage pulses of 500 ms in duration to various test potentials (from −50 mV to −100 mV) at a frequency of 0.5 Hz were applied to cells. In the control, the current-voltage relation showed inward rectification with its typical properties. With the increasing hyperpolarizing pulses, the steady-state current became less outward, crossed the zero current around −77.2 ± 4.4 mV, and then became increasingly inward. Application of 30 μM berberine did not produce a significant effect on IKl (Fig. 7). The lower concentration (3 μM) of this alkaloid also did not produce an effect (data not shown).
Effects on action potentials in the presence of tetrodotoxin
Similar to the results by Kiyosue and Arita (30), application of 10 μM TTX for 10 min significantly shortened APD90 from 405 ± 22 to 371 ± 16 ms (p < 0.05, n = 6) and did not alter APA and RP. As shown in Fig. 8, in the presence of 10 μM TTX, berberine (30 μM) retained the ability to lengthen APD. In a total of six cells, this alkaloid increased APD90 by 23.5 ± 1.7% (405 ± 18 vs. 501 ± 26 ms; p < 0.05), which was similar to that in the absence of 10 μM TTX (24.7 ± 2.9%). Because TTX does not inhibit its effects, berberine may also block the TTX-sensitive window Na+ current. If not, it would have been observed that TTX could partially antagonize the effects of berberine. In addition, no significant changes in both APA and RP were found in the presence of TTX plus berberine.
Further to test the hypothesis that berberine may block the TTX-sensitive window Na+ current, cells were first exposed to 30 μM berberine and then to 10 μM TTX. An example from this series of experiments is shown in the bottom panel of Fig. 8A. Application of TTX no longer caused the significant abbreviation of APD90 in cells pretreated with berberine. Similar findings were observed in three other cells (Fig. 8B). This result further indicates that berberine blocked the TTX-sensitive window Na+ current, which may limit its prolongation of APD.
Under the current-clamp configuration by using the whole-cell patch-clamp technique, berberine showed a remarkable concentration-dependent prolongation of APD in single isolated guinea pig ventricular cells. However, APA and RP were not altered. These results are consistent with those obtained from ventricular myocytes of isolated or in-vivo multicellular preparations by using the standard microelectrode technique (6,9,10,16).
It is widely accepted that IK plays a major role in cardiac repolarization. Many antiarrhythmic drugs such as quinidine, disopyramide, amiodarone, clofilium, and propafenone, which lengthen the APD, inhibit IK (31-37). In our study, we demonstrated that berberine exerts a concentration-dependent depression of IK. Thus this could be one of the mechanisms responsible for lengthening the APD. In addition, analysis of the activation curves for the IK tail currents indicates that this compound might preferentially block the delayed rectifier K+ channels while in the open state because (a) the degree of blockade by berberine tracks the voltage-dependent activation of the IK tail currents, (b) in the presence of the agent, the activation curve fitted by the Boltzmann equation was shifted in the hyperpolarized direction, and (c) no intrinsic voltage dependence is seen in its block of the delayed rectifier K+ channels. Evidence against this mode of action of berberine indicates that IK has been demonstrated to consist of two components, the rapidly activating IKr and the slowly activating IKs in guinea pig ventricular myocytes (38). However, based on the fact that IKr makes only a small contribution (<10%) to the total IK at long duration (38) and berberine does not inhibitKrI (see the following), this conclusion is tenable, although the magnitude of its action may be slightly underestimated.
IKr has a half-point activation voltage of −21.5 mV and is almost fully activated at 0 mV, whereas only ∼25% of IKs is activated at 0 mV, and its half-point activation voltage is 15.7 mV (38). Because berberine does not inhibit the IK tail currents at potentials up to 0 mV, this compound may block the slowly activating IKs without affecting the rapidly activating IKr. This hypothesis was further confirmed by experiments in which IKs and IKr were dissected out. As shown in Fig. 3, berberine inhibited IKs when activated by a 3-s depolarization from a holding potential of −40-50 mV in the presence of 1 mM La3+ to block IKr but had no detectable effect on IKr when activated by a 500-ms depolarization from −40 to −10 mV in Ca2+-free solution to eliminate IKs. Conversely, dofetilide, the selective blocker of IKr (26-28), greatly inhibited IKr but did not affect IKs Therefore the prolongation of APD by berberine is associated with the blockade of IKs.
There is increasing evidence that the Na+-Ca2+ exchange current may be involved in forming the cardiac action potential repolarization phase. This current can be inward or outward depending on the membrane potential and the actual Na+ and Ca2+ gradients. During the action potential plateau, as the membrane potential is positive to the reversal potential of Na+-Ca2+ exchange current, the current is outward and accompanied by the translocation of calcium into the cardiac cells. A calcium transient is elicited as the action potential is initiated, and it starts to decay to its original resting level after the plateau phase. This decrease of intracellular calcium concentration is associated to some extent with calcium extrusion through a Na+-Ca2+ exchange pathway. Therefore this current presumably flows in the outward direction somewhat longer than in the inward direction during an action potential (39). This study shows that berberine increased the Na+-Ca2+ exchange outward currents that were almost completely inhibited by 5 mM NiCl. It suggests that the prolongation of APD with the alkaloid is not be associated with the Na+-Ca2+ exchange current.
It has been demonstrated that this alkaloid possesses a positive inotropic effect on animal myocardium (1,3,4,6,14). Clinical trials also have shown that this compound is effective against heart failure. This effect is due, at least in part, to its increasing myocardial contractility (11,12). Because berberine increases the Na+-Ca2+ exchange current, calcium influx may be augmented by this pathway. This mechanism might be responsible in part for its positive inotropic effect.
For measuring the Na+-Ca2+ exchange outward currents, a high concentration of EGTA and CaCl2 was used (this study) by us and by other investigators (29). This would raise the intracellular osmolality. The increased osmolality might cause cell swelling, leading to a decrease in IKr, an increase in IKs, and activation of chloride currents (40-43). The possible implication of IKr and IKs could be excluded because K− was completely replaced by Cs− in the pipette and extracellular solution in this series of experiments, but further study is required to elucidate whether activation of cardiac chloride channels plays a role in the action of berberine.
Many antiarrhythmic drugs produce a negative inotropic action by direct or indirect inhibition of the cardiac L-type Ca2+ channels. Berberine has been shown to exert a beneficial effect on cardiac arrhythmias and heart failure in animals and humans (2,3,5,7-13). Our study indicates that berberine increases ICa in a concentration-dependent manner but did not affect its kinetics or current-voltage relation. By using the cell-attached configuration of the patch-clamp technique, it has been found that berberine to increase the open probability of single L-type Ca2+ channels in chick ventricular myocytes (19). These results imply the increase in ICa by berberine may contribute to lengthening the APD and augmenting the contractile force in myocardial cells. However, it should be kept in mind that the fact that berberine does not inhibit IK at potentials more negative than 0 mV means the basis for the prolongation of APD is solely the increased ICa, because the cell membrane is depolarized to potentials positive to 0 mV for several hundred milliseconds during an action potential at which IK is significantly blocked by berberine. The prolongation of APD by this compound is thus caused by both the inhibition of IKs and the increase of ICa, although a quantitative analysis for the contribution of each could not be made.
It is well known that cardiac function in many patients with cardiac arrhythmias is of low status. Cardiac arrhythmias may aggravate the existing abnormal hemodynamics or even induce cardiac decompensation. Thus the increased ICa and Na+-Ca2+ exchange currents and the resulting positive inotropic effects would be one of the advantages of this alkaloid in these cases. Riccioppo Neto (16) showed that berberine induces early afterdepolarizations in canine ventricular myocytes stimulated at a low frequency (0.1 Hz). The development of early after-depolarizations is related to an increase in cytosolic Ca2+ concentration (44). Therefore an increase in cytosolic Ca concentration caused by the increased Na+-Ca2+ exchange current and ICa induced by berberine might be a culprit for these early afterdeploarizations. Additionally, it should be noted that the blockade of the delayed rectifier K+ current by berberine may also contribute to its proarrhythmic effects because most drugs that block K+ channels have been shown to have a propensity to induce cardiac arrhythmias (45).
IKl is known to contribute to the final phase of repolarization and resting membrane potential. As depicted in Fig. 7, we did not find any detectable effects on this current at holding potential (−40 mV), at reversal potential, and during the potentials from −50 to −100 mV where IKl is predominant. In the current-clamped conditions, this alkaloid did not alter the resting potential (Figs. 1 and 7). These results, along with the fact that this alkaloid does not influence the resting potential in myocardial cells of multicellular preparations with the standard microelectrode technique (6,8-10,16) suggest that the prolongation of APD caused by berberine is not associated with effects on the inward rectifier K+ current.
A small but significant TTX-sensitive window Na+ current exists during cardiac repolarization. This current makes a contribution to maintaining the action potential plateau. TTX, lidocaine, and quinidine block this current and thus shorten the APD (30,46-49). Therefore it is expected that a drug that can increase the TTX-sensitive window Na+ current would be likely to lengthen the APD. TTX (10 μM) shortened APD90 but did not alter the prolongation of APD90 by berberine, 30 μM. Additionally, TTX (10 μM) lost the ability to shorten APD in the cells pretreated with berberine (30 μM). These results suggest that this alkaloid is able to inhibit the TTX-sensitive window Na+ current, which could tend to attenuate its prolongation of APD and may also explain why the extent of its prolongation is less than that seen with selective blockers of delayed rectifier K+ channels such as dofetilide (26-28) and E-4031 (38).
It is interesting that Sanchez-Chapula (50) recently showed that berberine is a selective IKr blocker and does not affect IKs and ICa in cat ventricular myocytes. These results are different from those observed in our study. Whether this major discrepancy results from the methods and animal species is not known, but evidence from a couple of studies supports the view that berberine is not a selective IKr blocker (17-20). By using the whole-cell or inside-out configurations of the patch-clamp technique, this alkaloid has been demonstrated to inhibit ATP-sensitive K+ channels in guinea pig myocardial cells (17,18), to increase the open probability of single L-type Ca2+ channels in chick ventricular myocytes (19), and to block the hyperpolarization-activated inward current (If) in rabbit sinoatrial node cells (20). Therefore further studies are required to reveal the discrepancy between Sanchez-Chapula's and others' results.
Acknowledgment: We thank Professor Joseph F. Spear (University of Pennsylvania, U.S.A.) for reading the manuscript and for the comments. This work was supported by National Natural Science Foundation of China (No 39200030 to Yong-Xiao Wang and 39400053 to Yun-Min Zheng).
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