Effects of Astragaloside IV on Action Potentials and Ionic Currents in Guinea-Pig Ventricular Myocytes

Abstract

Astragaloside IV (AS-IV) is one of the main active constituents of Astragalus membranaceus, which has various actions on the cardiovascular system. However, its electrophysiological mechanisms are not clear. In the present study, we investigated the effects of AS-IV on action potentials and membrane currents using the whole-cell patch clamp technique in isolated guinea-pig ventricular myocytes. AS-IV prolonged the action potential duration (APD) at all three tested concentrations. The peak effect was achieved with 1×10⁻⁶ m, at which concentration AS-IV significantly prolonged the APD at 95% repolarization from 313.1±38.9 to 785.3±83.7 ms. AS-IV at 1×10⁻⁶ m also enhanced the inward rectifier K⁺ currents (I_K1) and inhibited the delayed rectifier K⁺ currents (I_K). AS-IV (1×10⁻⁶ m) strongly depressed the peak of voltage-dependent Ca²⁺ channel current (I_CaL) from −607.3±37.5 to −321.1±38.3 pA. However, AS-IV was not found to affect the Na⁺ currents. Taken together, AS-IV prolonged APD of guinea-pig ventricular myocytes, which might be explained by its inhibition of I_K. AS-IV also influences Ca²⁺ signaling through suppressing I_CaL.

Astragaloside IV (AS-IV) is one of the main active ingredients of Astragalus membranaceus (HuangQi). Chemically, it is a cycloartane triterpene saponin (Fig. 1). AS-IV has multiple pharmacological functions including anti-inflammation,^1,2) antioxidation,^3–5) antivirus,^6,7) anti-apoptosis,^8,9) anti-hyperglycemic,^10,11) neuroprotection,¹³⁾ gastroprotection,¹⁴⁾ anti-chronic-asthma,¹⁵⁾ and anti-lung-cancer.¹⁶⁾ One of AS-IV’s targets is the cardiovascular system, and especially the heart. There are emerging evidences that AS-IV has cardio-protective effects, for example, AS-IV treatment inhibited compensatory hypertrophy of myocardial cells and lowered the number of apoptotic myocytes in long-term heart failure in rats.¹⁷⁾ These beneficial effects of AS-IV may involve the prevention of the depression of the activity of Ca²⁺-ATPase on sarcoplasmic reticulum.¹⁸⁾

Fig. 1. Chemical Structure of AS-IV

However, to our knowledge, there have been no evaluations of the effects of AS-IV upon the electrophysiological characteristics of cardiac ventricular myocytes. In the present study, we have investigated the effects of AS-IV on action potentials and ionic currents from freshly isolated ventricular myocytes of guinea-pig heart using the whole-cell patch clamp technique. We have found that AS-IV prolonged the action potential duration (APD) and inhibited the delayed rectifier K⁺ currents (I_K) and voltage-gated Ca²⁺ currents (I_CaL).

Materials and Methods

Animals and Cell Preparation

Healthy, 350±50 g, adult guinea-pigs were purchased from China Medical University Animal Center. Single ventricular myocytes of guinea-pig heart were obtained with an enzymatic dissociation protocol.^19,20) Briefly, animals were anaesthetized intraperitoneally with 100 mg/kg sodium pentoparbital. The heart was dissected out quickly and was mounted on a Langendorff apparatus for digestion. After perfusion with Tyrode solution for 3 min, Ca²⁺-free Tyrode solution for 6 min, and then Ca²⁺-free Tyrode solution containing 0.08% collagenase (Yakult, Japan) for 15 min, the heart was washed thoroughly with 60 mL Kraftbrühe-modified (KB) solution. All solutions were maintained at 37°C. Then the digested left ventricle was cut away, shredded and filtered through a 105 µm stainless-steel mesh. The filtrate was collected and centrifuged with KB solution. The dispersed cells were maintained in the storage solution at 4°C until use.

Solutions and Drugs

For the isolation ventricular myocytes of guinea-pig heart, Tyrode solution contained (in mm): NaCl 135, KCl 5.4, NaH₂PO₄ 0.33, MgCl₂ 1.0, glucose 5.5, CaCl₂ 1.8, and N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) 10 (pH 7.4 adjusted by NaOH). KB solution contained (in mm): KOH 70, Glutamic acid 50, KCl 40, Taurine 20, KH₂PO₄ 20, Glucose 10, ethylene glycol bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) 0.5, MgCl₂ 3 and HEPES 10 (pH 7.4 by KOH). The whole-cell patch clamp technique was employed to record the action potential and membrane channel currents. The basic external perfusion solution was the Tyrode solution, except indicated especially. AS-IV (C₄₁H₆₈O₁₄) was isolated from Astragalus membranaceus var. mongholicus by chromatographic techniques and identified through spectral analysis. The purity (>99%) of AS-IV was determined with the high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method. AS-IV was dissolved in dimethyl sulphoxide (DMSO), then diluted in the perfusion solution (final concentration of DMSO <0.1%). Three concentrations of AS-IV (1×10⁻⁷, 1×10⁻⁶, 1×10⁻⁵ m) were prepared in Tyrode solution.

Experimental Groups

Four groups of cells were used in electrophysiological experiments. The control group was bathed in normal Tyrode solution. The three experimental groups were bathed in Tyrode solution which contained 1×10⁻⁷ m, 1×10⁻⁶ m, and 1×10⁻⁵ m AS-IV, respectively. Experiments were conducted in cells immersed in these solutions for 30 min to 2 h.

Action Potential Recording

Action potentials were recorded from isolated myocytes using the patch clamp technique in current-clamp mode.²¹⁾ Cells were bathed in solution in a recording chamber placed on an Olympus inverted microscope. Experiments were conducted at room temperature. A high-resistance seal (giga-seal) was established on a cell with Tyrode solution or drug solutions perfusion. The resistance of the pipette filled with the internal solution was 2–5 MΩ. Data were recorded with a patch-clamp amplifier (Axon 200B, Axon, America) filtered at 1 kHz, and fed to a computer at a sampling rate of 5 kHz. Action potentials were evoked with a 3 ms, 4 nA rectangular pulse. The external perfusion solution was the Tyrode solution. The patch pipettes were filled with (in mm): K-aspartate 110, KCl 30, MgCl₂ 5, K₂ATP 5, Na₂CrP 5, HEPES 5, EGTA 10, and CaCl₂ 1.43 (pH 7.2 by KOH).

Whole-Cell Membrane Current Recording

Membrane currents were recorded in voltage-clamp mode. After obtaining a high-resistance seal (giga-seal) to cell membrane, the whole-cell configuration was formed by rupturing the membrane with negative pressure. Na⁺, K⁺ and Ca²⁺ membrane currents were recorded with appropriate stimulus protocols.

The external perfusion solution for recording voltage-gated Na⁺ currents (I_Na) was (in mm): Choline chloride 100, NaCl 50, MgCl₂ 2, HEPES 10, Glucose 10, CsCl 20, CdCl₂ 0.3 (pH 7.3 by NaOH); and the pipette solution contained (in mm): CsCl 130, MgCl₂ 2, NaCl 10, HEPES 10 (pH 7.3 by CsOH). To record inward rectified K⁺ currents (I_K1), BaCl₂ (0.5 mm) were added to the external solution at the end of experiments, and the Ba²⁺-subtracted currents were considered as I_K1 component. To record the delayed rectified K⁺ currents (I_K), nifedipine (3 µm) and BaCl₂ (0.5 mm) were added into the Tyrode solution as the external solution to block I_CaL and I_K1 currents, respectively; and the pipette solution was the same as applied in action potential recording. The tail current of I_K (I_K tail) which evoked on returning to a holding potential represented I_K and has been analyzed. For recording voltage-gated L-type Ca²⁺ channel currents (I_CaL), the internal and external solutions were the same as administrated in action potential recording.

Statistical Analysis

The results were expressed as mean±standard error (S.E.). Statistical significance was determined by Student’s t-test or one-way ANOVA. p-values of less than 0.05 were considered significant.

Results

Effects of AS-IV on Action Potentials

All three concentrations of AS-IV significantly prolonged the APD (Fig. 2). The peak effect was achieved with AS-IV at 1×10⁻⁶ m. At that concentration, AS-IV increased APD₂₅, APD₅₀ and APD₉₅ to 8.5, 3.0 and 2.5 times the control values (p=0.0055, p=0.0005, p=0.0001). AS-IV at 1×10⁻⁷ m and 1×10⁻⁵ m showed similar but lesser effects on APD. We also found that neither the resting membrane potential (RMP) nor the action potential amplitude (APA) was significantly changed by AS-IV at all three concentrations tested (Table 1).

Fig. 2. The Effects of AS-IV upon Action Potentials in Isolated Ventricular Myocytes of Guinea-Pig Heart

Action potentials were recorded in current-clamp mode. The supra-threshold current pulses were applied every 3 s. (A) Representative action potential recordings obtained from different groups of ventricular myocytes in normal Tyrode solution (Control) and in the presence of the indicated concentrations of AS-IV. (B) The mean values of action potential duration at 25%, 50% and 95% repolarization (APD₂₅, APD₅₀, APD₉₅) in the presence of AS-IV at 1×10⁻⁷ m, 1×10⁻⁶ m, and 1×10⁻⁵ m. n=4–8. * p<0.05, ** p<0.01, and *** p<0.001 versus control.

Table 1. Effects of AS-IV on Action Potential Parameters

	RMP (mV)	APA (mV)	APD25 (ms)	APD50 (ms)	APD95 (ms)
Control	−75.5±1.7	158±4.6	13.1±4.1	189.2±40.3	313.1±38.9
10−7 m AS-IV	−74.5±2.2	150.3±5.1	32.4±8.9	423.6±72.0*	499.6±65.5*
10−6 m AS-IV	−77.6±4.5	153.3±7.4	111.5±18.9**	574.3±77.4***	785.3±83.7***
10−5 m AS-IV	−75.9±6.1	161.2±23.4	73.4±16.6**	276.9±91	503.7±42.1*

RMP: resting membrane potential; APA: action potential amplitude; APD₂₅, APD₅₀ and APD₉₅: action potential duration at 25%, 50% and 95% of repolarization. n=4–8. * p<0.05, ** p<0.01, and *** p<0.001, compared with control group.

Effects of AS-IV on Na⁺ Currents

The voltage-dependent Na⁺ currents (I_Na) are responsible for the rising phase of the action potential in cardiac myocytes, we thus examined the effects of AS-IV on I_Na. Neither the I–V curves (Fig. 3A) nor the inward peak amplitude of I_Na (Fig. 3B) was affected by AS-IV. These suggest that AS-IV does not affect I_Na, which is consistent with the result that AS-IV is failed to affect APA. Representative traces of I_Na are shown in Fig. 3C.

Fig. 3. A Lack of Effects of AS-IV on I_Na

(A) Current–voltage (I–V) curves for I_Na. Open circles (○) represent control data, filled circles (●), filled triangles (▲) and filled squares (■) represent the effects of 1×10⁻⁷ m, 1×10⁻⁶ m, and 1×10⁻⁵ m AS-IV, respectively. The amplitudes of I_Na were normalized by cell capacitance. n=9. (B) The peak amplitude of I_Na in control and in the presence of different concentrations of AS-IV (1×10⁻⁷, 1×10⁻⁶, 1×10⁻⁵ m). There was no significant difference among the four groups. (C) Representative I_Na recorded from different myocytes with the indicated concentrations of AS-IV. The voltage-clamp protocol is shown in the inset. I_Na was elicited by 50 ms voltage steps from the holding potential of −120 mV to between −80 and +20 mV in 10 mV increments.

Effects of AS-IV on K⁺ Currents

We then proceeded to investigate the effects of AS-IV on K⁺ currents which are known to contribute to the repolarization of the action potential. Voltage-clamp protocols used to elicit both inward rectifier K⁺ currents (I_K1) and delayed rectifier K⁺ currents (I_K) were shown in the inlets of Figs. 4 and 5. Ba²⁺-subtracted current was taken as I_K1 (see Materials and Methods), AS-IV was likely to increase I_K1 (Fig. 4), but decrease I_K at all three concentrations (Fig. 5). However, there were no significant differences among three different concentrations of AS-IV. Especially, the middle dose (1×10⁻⁶ m) of AS-IV induced the smallest increase in I_K1 (Fig. 4B); and this was also the only concentration of AS-IV which showed significant suppression of I_K (Fig. 5B).

Fig. 4. Effects of AS-IV on I_K1

(A) Analysis of I_K1 in a ventricular muocyte of guinea-pig heart. The dash dot line with diamonds (◆) shows the current due to a voltage-clamp step stimulation applied to a ventricular cell in nifedipine (3 µm) containing Tyrode solution. Following the superfusion of 0.5 mm BaCl₂ in the Tyrode solution, a second stimulation was applied. Membrane current under this condition is shown as the dash dot line with stars (★). The value of pure I_K1 is the difference current between without and with BaCl₂ shown as the solid line with open circles (○). (B) Current–voltage (I–V) curves for I_K1 currents recorded with the inset voltage-clamp protocol. From a holding potential of −40 mV, 3 s steps were applied from −130 to +20 mV with 10 mV steps. Open circles (○) in each graph represent control data, filled circles (●), filled triangles (▲) and filled squares (■) represent results obtained in the presence of 1×10⁻⁷ m, 1×10⁻⁶ m and 1×10⁻⁵ m AS-IV, respectively. n=6. * p<0.05 versus control.

Fig. 5. Effects of AS-IV on I_K

(A) Representative tail current traces of I_K in control, 1×10⁻⁷ m, 1×10⁻⁶ m, and 1×10⁻⁵ m AS-IV. (B) I–V curves for I_K tail currents were recorded during 3-s depolarizing test pulses from −30 to +40 mV, followed by 0.2 s repolarization to −40 mV. Open circles (○) in each graph represent control data, filled circles (●), filled triangles (▲) and filled squares (■) represent results obtained in the presence of 1×10⁻⁷ m, 1×10⁻⁶ m and 1×10⁻⁵ m AS-IV, respectively. n=6. * p<0.05, ** p<0.01, *** p<0.001 versus control. The I_K tail currents were recorded in Tyrode solution with 3 µm nifedipine and 0.5 mm BaCl₂ to avoid I_CaL and I_K1 contamination.

Effects of AS-IV on Ca²⁺ Currents

The effect of AS-IV in prolonging the APD suggested that membrane voltage-gated L-type Ca²⁺ currents (I_CaL) might be affected by AS-IV. Thus we examined the effects of AS-IV on I_CaL. Free Ca²⁺ concentration in the patch pipette solution was 80 nm, which is the physiological intracellular Ca²⁺ concentration at rest condition. Following 60-ms voltage steps from the holding potential of −80 to −40 mV to inactivate the I_Na, I_CaL was then evoked by subsequent 200-ms depolarizations from −40 to +40 mV in 5 mV increments. AS-IV decreased the amplitude of I_CaL at all tested potentials (Fig. 6A). All three doses of AS-IV significantly reduced the inward peak amplitude of I_CaL to 61.2±7.2% (p<0.05), 52.8±6.3% (p<0.01), and 60.9±5.8% (p<0.05) of the control group value, respectively (Fig. 6B). Interestingly, as we may see from the reduction percentage, the middle dose (1×10⁻⁶ m) of AS-IV reduced the I_CaL the most effectively. It also induced a significant negative shift of the I_CaL inactivation curve [V_1/2 (10⁻⁶ m)=−24.4±1.4 mV versus V_1/2 (control)=−19.4±0.7 mV, p=0.005], while 1×10⁻⁷ m and 1×10⁻⁵ m AS-IV had no effect [V_1/2 (10⁻⁷ m)=−19.9±1.0 mV, P=0.673; V_1/2 (10⁻⁵ m)=−20.1±1.4 mV, p=0.675] (Fig. 6C). In no case did the slope factor of inactivation change, k (10⁻⁷ m)=6.2±0.6, k (10⁻⁶ m)=7.4±0.7, k (10⁻⁵ m)=6.1±0.3, versus k (control)=6.7±0.4.

Fig. 6. Effect of AS-IV on I_CaL

(A) Current–voltage (I–V) curves for I_CaL. The voltage-clamp protocol was a 200 ms voltage step from −40 to +40 mV with a 5 mV increment from a holding potential of −80 mV. Open circles (○) represent control data, filled circles (●), filled triangles (▲) and filled squares (■) represent 1×10⁻⁷ m, 1×10⁻⁶ m and 1×10⁻⁵ m AS-IV, respectively. n=7. (B) a) Mean maximum values of I_CaL in the different groups of myocytes. b) Representative current traces in control (○), 1×10⁻⁷ m (●), 1×10⁻⁶ m (▲), and 1×10⁻⁵ m (■) AS-IV. ** p<0.01, *** p<0.001 versus control. The broken lines indicated the zero current level. (C) Inactivation curves for voltage-gated Ca²⁺ channels. I: normalized channel availability. Data points were fitted with the Boltzmann equation: y=A₂+(A₁−A₂)/(1+exp((V−V_1/2)/k)), where A₁, A₂, V_1/2, and k are the initial value, final value, mid-point voltage, and slope factor, respectively. Insert: voltage clamp protocol with the pre-steps ranging from −40 to +10 mV, followed by the 100 ms test step to 0 mV.

Discussion

HuangQi, as a tradition Chinese medicine, has been widely used for heart disease treatment in clinical conditions. AS-IV, as one of its active elements, has been revealed its pharmacological mechanisms to some degree. Recent researches have suggested that AS-IV might protect from cardiomyopathy via diverse effects upon a variety of intracellular signaling pathways, including the activation of Src/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway,²²⁾ inhibiting tumor growth factor (TGF)-β1²³⁾ and ERK1/2 signaling pathways,²⁴⁾ upregulating superoxide dismutase-1 levels,^3,25) antioxidative and nitric oxide-inducing pathway,²⁶⁾ and improving intracellular calcium handling.^27,28) The aim of our study is to clarify the basic electrophysiological effects of AS-IV on cardiac myocytes, and we have found that AS-IV prolonged APD, and decreased I_K and I_CaL in guinea-pig cardiac myocytes.

The action potential is formed by multiple ion currents gated by different ion channels. Na⁺ channels are responsible for the initial membrane depolarization (phase 0) during the action potential. AS-IV had no effect on I_Na, and correspondingly, APA (Fig. 3). Current clamp results (Table 1) also showed that AS-IV had no effect on RMP. I_K1 is the major determinant of RMP, but AS-IV, which was shown to increase I_K1, had no effect on this parameter. We noticed that although AS-IV increased I_K1 at −130 mV, −120 mV and −110 mV (Fig. 4), indeed, it had no measurable effect on I_K1 at the membrane potentials around RMP, for example at −70 mV. Perhaps this could explain why it has no influence upon RMP.

The action potential plateau and repolarization result from complex affections between Ca²⁺ and K⁺ channel currents. Delayed repolarization and thus APD elongation may result from decreased outward currents or enhanced inward currents during the action potential plateau and the repolarization phase. I_K is the major outward current involved in respolarization of ventricular myocytes. It consists of two major components with different biophysical properties and different drug sensitivities: one is rapid activation component (I_Kr), which rectifies inwardly at depolarized membrane potential due to C-type inactivation, the other is slow activation component (I_Ks), which has almost linear current voltage-relationship.^29,30) Reductions in I_Kr prolong the APD, which consequently activate more I_Ks to prevent excess repolarization delay. In most mammalian species, I_Kr including the human ether-à-go-go-related gene (hERG) currents, play a pivotal role in the repolarization of cardiac action potentail. Actually I_Kr/hERG represents an important target for pharmacological management of arrhythmias.³¹⁾ Blockers of I_Kr prolong the cardiac APD, exhibiting actions of Class III antiarrhythmic drugs. For example, teisamil, a novel antiarrythmic compound, inhibited the K⁺ currents, thus contributed to the potent antiarrythmic action, underlying predominant Class III antiarrhythmic characteristics.³²⁾ In the present study, although I_Kr was not observed alone, we found that the total I_K currents were inhibited by AS-IV (Fig. 5). It has been reported that AS-IV remarkably decreased the incidence of ventricular arrhythmia on the arrhythmia mice model caused by toad venom.³³⁾ We speculate that the inhibition of I_K by AS-IV may cause APD prolongation, which could be a mechanism for its terminating effects on tachyarrhythmia. On the other hand, excessive prolongation of APD with drugs which inhibit I_K may lead to torsades de pointes (TdP) arrhythmias associated with acquired long QT syndrome,³⁴⁾ especially the drugs which prolong the APD through the inhibition of hERG channels. To predict the potential cardiac toxicity of compounds, the effects of compounds on hERG channels should be observed in the early stage of drug development. Interestingly, our data showed that AS-IV at the higher concentration (1×10⁻⁵ m) didn’t exhibit potent inhibition on I_K and prolongation of APD, whereas the middle dose (1×10⁻⁶ m) of AS-IV reduced the I_K current the most. This kind of dose-dependency was different from most western medicines, which may benefit for the clinical application of AS-IV safely.

An important inward current that contributes to the plateau phase is I_CaL. We found that AS-IV reduced I_CaL (Fig. 6). Inhibition of I_CaL would normally shorten APD, but given the observation that AS-IV prolonged APD, its inhibitory effect on I_CaL must have been compensated by some other effects, for example the reduction of I_K. Therefore, AS-IV could be regard as a multi-channel blocker. Rather than pure channel blockers, compounds with multiple channel targets have come to be preferred as antiarrhythmics.^35,36) Dronedarone, a new Class III antiarrhythmic, with fewer adverse effects, had multi-channel blocking effect, inhibiting I_Na, I_K and I_CaL, and was highly effective in treating many arrhythmias.³⁷⁾ Verapamil, a Ca²⁺ channel blocker, classified as a Class IV antiarrhythmic drug, has been further reported not only to block the Ca²⁺ channel, but also to exert prolonging APD by blocking K⁺ channels. It is believed that this multi-electrophysiology actions probably explained why verapamil was free from QT prolongation in human.³⁸⁾ It seems that AS-IV not only inhibited I_K to prolong APD, but also suppressed I_CaL to prevent APD over-prolongation which can further cause arrhythmias. This multi-target effect could be taken into account when considering the possible clinical application of AS-IV as an antiarrhythmic drug.

We found that the dose-dependent effect of AS-IV upon the parameters measured in the present study was not typical “S” shaped, especially the effects on I_CaL. Actually, this kind of phenomenon has also been reported in other systems. For example, as Han et al. observed, the effects of calmodulin (CaM) on Ca²⁺ channel activity in inside-out patch was bell-shaped, and they believed that the dual effects of CaM were due to two CaM-binding sites, which had opposite effects in the regulation of Ca²⁺ channel activity.¹⁹⁾ Waston et al. suggested another complex response pattern with multiple peaks, in which the actions of estrogens and xenoestrogens via non-genomic signaling mechanisms, including their oscillating time courses and non-monotonic concentration-responses, were observed. They suggested that the multi-peak patterns could be probably dictated by the influences from the complement of estrogen receptors engaged and the availability of signaling partners in given tissues and cell types.³⁹⁾ A possible explanation for the none-‘S’-shaped dose-dependency of AS-IV’s effects on the electrophysiological properties of cardiac myocytes is that it is also a complex involving multiple factors, not only including the membrane ionic channels but also other intracellular signaling pathways. In our study, we also found that AS-IV at 1×10⁻⁵ m and 1×10⁻⁷ m significantly prolonged APD, but only slightly inhibited I_K, which is thought the major current affecting APD. This phenomenon also suggests that AS-IV may have effects on other targets, such as intracellular Ca²⁺ handling. Therefore, further studies are required for searching the other targets of AS-IV, especially the intracellular pathways that may affect channel activities and APD.

In summary, the present study provided some explanations for potential mechanism of AS-IV’s effects on the heart by observing the alterations of electrophysiological parameters of cardiac ventricular myocytes. These effects might be expected to protect from cardiac disorders, for instance tachyarrhythmia, mainly via prolonging the APD and/or blocking the Ca²⁺ and the K⁺ channels.

Acknowledgment

The study was supported by Research Grants form Nation Nature Science Foundation of China Grants to L. Y. Hao (30870907, 31071004) and X. S. Huang (30600783).

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