Zhang GQ et al / Acta Pharmacol Sin 2003 Dec; 24 (12): 1212-1216

Puerarin blocks Na+ current in rat ventricular myocytes1

ZHANG Guang-Qin, HAO Xue-Mei2, DAI De-Zai3, FU Yu2, ZHOU Pei-Ai2, WU Cai-Hong2

Research Division of Pharmacology, China Pharmaceutical University, Nanjing 210009, China; 2National Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China

1 Project supported by the National Natural Science Foundation of China (No 39970850).

3 Correspondence to Prof DAI De-Zai. Phn 86-25-327-1270. Fax 86-25-330-2827. E-mail njpdai@jlonline.com

Received 2002-11-15 Accepted 2003-05-30

KEY WORDS puerarin; myocardium; patch-clamp techniques; sodium channels

ABSTRACT

AIM: To study the effect of puerarin (Pue) on Na+ channel in rat ventricular myocytes. METHODS: Whole-cell patch-clamp technique was applied on isolated cardiomyocytes from rats. RESULTS: Pue inhibited cardiac INa in a positive rate-dependent and dose-dependent manner, with an IC50 of 349 µmol/L. The kinetics of blockage of cardiac sodium channel by Pue resembled the ClassIa/Ic of antiarrhythmic agents. Pue 300 µmol/L did not alter the shape of the I-V curve of INa, but markedly shifted the steady-state inactivation curve of INa towards more negative potential by 15.9 mV, and postponed the recovery of INa inactivation state from (21.9±1.6) ms to (54.4±3.4) ms (P<0.01). It demonstrated that the steady state of inactivation was affected by Pue significantly. CONCLUSION: Pue protected ventricular myocytes against cardiac damage and arrhythmias by inhibiting recovery from inactivation of cardiac Na+ channels.

INTRODUCTION

Puerarin (Pue) prevents the heart from arrhythmias and improves myocardial reperfusion injury[1,2] , and has been applied in the treatment of cardiovascular diseases in clinical settings. Recent findings showed that Pue blocked L-type calcium channel and K+ channel in isolated guinea pig ventricular myocytes[3-5], inhibited the transient outward, and delayed rectified K+ current in mouse hippocampal CA1 neurons[6].

The sodium channel in cardiomyocytes regulates the influx of INa which causes depolarization of the membrane, therefore, to affect voltage gated K+ chan-nels. Myocardial ischemia is likely to induce changes in the INa amplitude which affects the velocity of car diac impulse conduction and the refractory period of cardiac excitability, leading to arrhythmogenesis. So suppression of the cardiac Na+ channels is always regarded as an important indication for development of agents to potentially eliminate cardiac arrhythmias under clinical conditions. The suppressive effect of Pue on Na+ channels was reported in neurons[7]. However, it is well known that the gene encoding and molecular construction of the Na+ channels are different in the neural and cardiac tissue[8,9]. So it is interesting to observe that the mode of Pue on the cardiac Na+ channel in order to understand mechanisms of Pue action on the heart.

MATERIALS AND METHODS

Isolation of cardiac myocytes Single ventricular myocytes were isolated from the hearts of adult Sprague-Dawley rats (200-250 g, , Grade II, Certificate No 0003, the Experimental Animal Center of Beijing) as previously described[7]. Briefly, rats were anesthetized with pentobarbital sodium (30 mg/kg, ip). Hearts were rapidly excised and retrogradely perfused on a Langendorff apparatus, with a Ca2+-free Tyrode's solution of the following composition (mmol/L): NaCl 137, KCl 5.4, NaH2PO4 1.2, MgSO4 1.2, Hepes 10, glucose 10, pH 7.4 for 5 min, then the perfusate was switched to Ca2+-free Tyrode's solution containing collagenase B 0.5 g/L (Boehringer Mannheim Corp), protease XIV 0.2 g/L (Sigma Chemical Co), and bovine serum albumin (BSA) 1 g/L. The perfusate was oxygenated (95 % O2+5 % CO2) and maintained at 37 ºC. The ventricles were removed, cut into small chunks, and gently shaken in enzymatic solution. The cells were filtered through nylon mesh and washed twice with KB solution by centrifugation. Finally, the cells were stored in KB solution containing (mmol/L): KOH 70, KCl 40, KH2PO4 20, glutamic acid 50, MgCl2 3, taurine 20, egtazic acid 0.5, Hepes 10, glucose 10, pH 7.4.

Electrical recordings Dissected cells were transferred to a 0.5-mL chamber mounted on the plate of an inverted microscope (Nikon 810185, Japan), and perfused with normal Tyrode's solution at 2 mL/min. The whole cell patch-clamp technique was used to record membrane currents. Patch pipettes with a resistance of 1-3 M, were pulled by a two-step puller (Narishige PP-93, Japan), and filled with an internal solution containing (mmol/L): CsF 125, CsCl 20, NaCl2 10, egtazic acid 5, Hepes 5, pH 7.2. Membrane currents were filtered by the amplifier at 3 kHz, sampled at 5 kHz and stored in a PC 486 computer using the Labmaster TL-1 interface (Axon Ins, USA). Current and voltage protocol generation and data acquisition and analysis were performed by the pClamp software (Ver 6.01, Axon Ins, USA).

To measure whole cell INa, myocytes were perfused with an external solution consisting of (mmol/L) choline chloride 100, NaCl2 20, CsCl 5, 4-AP 3, MgCl2 1.2, CaCl2 1.8, glucose 5.6, Hepes 5, CoCl2 3, TEA 20, pH 7.4. All experiments were carried out at room temperature (23 ºC to 25 ºC). INa amplitude was measured as the peak inward current with reference to the current at the end of the test pulse.

Chemicals Pue, a gift from Department of Phyto-chemistry, China Pharmaceutical University, was dissolved in extracellular solutions. The 4-AP (4-aminopyridine), CsCl, CsF, TEA, CoCl2, egtazic acid, and BSA were purchased from Sigma Chemical Co.

Data analysis All data were analyzed by pCLAMP 6.0 procedures (Axon Ins, USA) and Sigmaplot (Jandel Scientific) software. All values were expressed as mean±SD. Statistical significance was analyzed by paired or unpaired t-test.

RESULTS

Concentration-dependent inhibition of cardiac INa by Pue INa was elicited by a 30-ms pulse to -40 mV from the holding potential at -90 mV. TTX 30 µmol/L completely blocked the current to confirm that the current was cardiac INa (n=5, not shown). Effect of Pue reached a steady state around 8-10 min. So INa was recorded after application of Pue for 10 min. The suppressive effect of Pue on the cardiac INa currents was concentration-dependent (Fig 1) in the range of 10-1000 µmol/L with the IC50 value of 349 µmol/L and the maximal suppression was up to 68.4 %±15.8 % by Pue 1000 µmol/L (n=7). After washout of Pue, the cardiac INa recovered substantially and the effect of Pue was reversible in drug-free solution (n=7 , Fig 2A).

Fig 1. Concentration-dependent effect of Pue on INa in rat ventricular myocytes. n=7 cells. Mean±SD.

Effects of Pue on current-voltage relationship (I-V) of cardiac INa The I-V relationship of INa was shown in Fig 2B. Current traces were elicited by 30-ms depolarizing pulses to potentials ranging from -80 mV to +40 mV in 10-mV increments at 0.5 Hz, when the holding potential was -90 mV. Before the application of drug, the current began to be activated at -60 mV, reaching maximum amplitude near -40 mV. The reversal potential was (36.0±0.7) mV. Pue 300 µmol/L suppressed the cardiac INa, without modifying the maximum activation potential and the reversal potential [(37.1±1.3) mV]. The peak of INa was decreased by 45 %±5 % at -40 mV and the shape of I-V curve was not altered by Pue (P>0.05, n=9).

Fig 2. Effect of Pue 300 µmol/L on I-V relationship of INa in rat ventricular myocytes. A) Current traces obtained in the absence and presence of the drug. B) I-V relationship of INa. n=9 cells. Mean±SD. bP<0.05, cP<0.01 vs control.

Effects of Pue on activation and inactivation kinetics of cardiac INa On the basis of data obtained from I-V relationship, the activation curves of cardiac INa were determined before and after application of Pue 300 µmol/L. The activation curve was fitted by the Boltzmann equation G/Gmax=1/[1+exp(Vh -V)/k] , where G is the membrane conductance at potential V, Vh is the half activation voltage, k is a slope factor. The Vh measured before and after the application of Pue was (-52.2±2.6) mV and (-57±3) mV (P>0.05), respectively, with k value as (4.8±0.7) mV and (5.1±0.9) mV, separately (P>0.05, n=9). Pue slightly shifted the steady-state activation curve of cardiac INa towards more positive potential by 4.9 mV, but did not obviously affect the activation characteristics of INa.

The steady-state inactivation curves of INa were obtained by use of a double-pulse protocol, a 3-s condition prepulse of various potentials (from -120 mV to +30 mV, holding potential at -90 mV) was followed by a 30-ms test pulse to -40 mV. The inactivation curve was also fitted by the Boltzmann equation I/Imax=1/[1+exp(V-V h)/k], where Vh is the half inactivation voltage, k is a slope factor. The Vh and k of the cardiac INa were (-67±3) mV and (8.3±1.2) mV in control; and (-82±4) mV (P<0.01) and (13.7±1.5) mV (P<0.01), in the presence of Pue 300 µmol/L (n=9), respectively. The steady-state inactivation curve of INa was obviously shifted towards more negative potential by 15.9 mV in the presence of Pue 300 µmol/L (Fig 3).

Fig 3. Effect of Pue 300 µmol/L on activation and inactivation kinetics of INa in rat ventricular myocytes. n=9 cells.

Effects of Pue on the recovery kinetics of cardiac INa The time course of recovery of INa from steady state inactivation was studied using a conventional double-pulse protocol. A 3-s pre-pulse to -40 mV from holding potential of -90 mV was followed by various recovery durations and then by a test pulse to -40 mV for 50 ms. The recovery process of cardiac INa could be fitted by a single exponent and the time constant of recovery from the steady state inactivation was (21.9± 1.6) ms and (54.4±3.4) ms in control and Pue 300 µmol/L group (P<0.01, n=9), respectively. It was confirmed that Pue delayed the recovery time constant of cardiac INa from inactivation state (Fig 4).

Fig 4. Effect of Pue 300 µmol/L on the recovery curve of INa from inactivation in rat ventricular myocytes. n=9 cells. Mean±SD.

Positive rate-dependent effect of Pue on cardiac INa The rate-dependent effect of Pue 300 µmol/L was studied by a series of 30 depolarizing pulses (30 ms duration) from a holding potential of -90 mV to -40 mV at different stimulation frequencies (1, 2, and 4 Hz) (n=8). The suppression of the sodium channel was enhanced along with an increase in the frequency of stimuli showing a positive rate-dependence. The kinetic of positive rate-dependence was rapidly onset but it was very slow approaching to a plateau (Fig 5).

Fig 5. Positive frequency-dependent effect of Pue 300 µmol/L on the INa in rat ventricular myocytes. n=8 cells.

DISCUSSION

Pue inhibited cardiac INa in a concentration-dependent and positive rate-dependent manner . The positive rate-dependence developed rapidly but reached the plateau slowly in a manner resembling the characteristics of Class Ia/Ic antiarrhythmic agents[11].

The blocking effect of Pue seems to be more sensitive in cardiac myocytes than in the neuron[7]. The potency of the inhibition of sodium channels by Pue is 3 fold more potent in cardiac myocytes than that in neuron, IC50 349 vs 934 µmol/L[7]. The difference of sodium channels in neuronal and cardiac tissue has been reported[8,9], concentrations of TTX to suppress the neuronal sodium channels are in the range of nmol/L but as high as 1-10 µmol/L to suppress the cardiac INa[9]. The difference in the pharmacological action stemmed from differentiated coding by genes. Subunits of sodium channels, one subunit and two (1 & 2) subunits, are instituted in a large family which consists of some isoforms in the brain, heart, and skeletal muscle. The cardiac subunit in possession of 4 domains of channels is encoded by gene SCN5A, which is located at chromosome 3 p14, totally different from the gene that encodes neuronal sodium channels (Tab 1). There are three isoforms of neuronal Na+ channels, the SCN1A, SCN2A, and SCN3A[12]. Different genes contribute to different structure of the cardiac -subunit of which there is systeine at position 373 in the amino sequence against phenylalanine/tyrosine in the neurons resulting in the divorced responses to TTX (Tab 1).

Tab 1. Comparison of the a subunit of sodium channel in the brain and heart.

The inactivation curve of the cardiac INa was shifted by Pue toward more negative potential but it was not affected in the neurons. Furthermore the time constant of recovery from inactivation was markedly increased in the cardiac sodium channels but not in the neurons[7].

Cardiac injury caused by myocardial ischemia or hypoxia induces the accumulation of cytosol Na+ which leads to an overload of Ca2+ by activation of Na+-Ca2+ exchanger[12]. During hypoxia/ischemia, intracellular acidosis is commonly observed due to activation of Na+-H+ exchange via a rise in cytosolic Na+ concentration[13]. Na+-H+ exchange and Na+ channels are considered as the two main sources for developing intracellular Na+ accumulation under pathological processes[12,13]. The effectiveness of Na+-channel blockers in cardio-protection, such as limiting post-ischemic arrhythmias and improving mechanical recovery, is due to inhibition of Na+-H+ exchange, Na+ channel, or Na+/Ca2+ exchange[14-16].

The blocking effect of Pue on the sodium channels is not selective. Because Pue also blocked Ca2+ and K+ channels[4-6]. This phenomena of non-selective effect on ion channels could also be seen in other compounds isolated from plant origin: berberine, palmatine, and the derivative CPU 86017[17,18]. In clinical practice Pue has been applied to treat myocardial ischemia. These findings suggest that Pue can block the inactivation state of Na+ channel and delay recovery of Na+ channel from inactivation state to inhibit the cardiac arrhythmias.

REFERENCES