J Korean Med Sci.  2009 Jun;24(3):403-412. 10.3346/jkms.2009.24.3.403.

Effects of Mixed Herbal Extracts from Parched Puerariae Radix, Gingered Magnoliae Cortex, Glycyrrhizae Radix and Euphorbiae Radix (KIOM-79) on Cardiac Ion Channels and Action Potentials

• Park SJ
• Choi KS
• Shin DH
• Kim JS
• Jang DS
• Youm JB
• Choe H
• Earm YE
• Kim SJ

• KMID: 1779155
• DOI: http://doi.org/10.3346/jkms.2009.24.3.403

Abstract

KIOM-79, a mixture of ethanol extracts from four herbs (parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhizae radix and Euphorbiae radix), has been developed for the potential therapeutic application to diabetic symptoms. Because screening of unexpected cardiac arrhythmia is compulsory for the new drug development, we investigated the effects of KIOM-79 on the action potential (AP) and various ion channel currents in cardiac myocytes. KIOM-79 decreased the upstroke velocity (Vmax) and plateau potential while slightly increased the duration of action potential (APD). Consistent with the decreased Vmax and plateau potential, the peak amplitude of Na+ current (INa) and Ca2+ current (ICa,L) were decreased by KIOM-79. KIOM-79 showed dual effects on hERG K+ current; increase of depolarization phase current (Idepol) and decreased tail current at repolarization phase (Itail). The increase of APD was suspected due to the decreased Itail. In computer simulation, the change of cardiac action potential could be well simulated based on the effects of KIOM-79 on various membrane currents. As a whole, the influence of KIOM-79 on cardiac ion channels are minor at concentrations effective for the diabetic models (0.1-10 microg/mL). The results suggest safety in terms of the risk of cardiac arrhythmia. Also, our study demonstrates the usefulness of the cardiac computer simulation in screening drug-induced long-QT syndrome.

Keyword

Heart; Action Potentials; Long-QT Syndrome; Herbal Extract; Ion Channels; hERG

MeSH Terms

Action Potentials/*drug effects
Animals
Cell Line
Computer Simulation
Female
Ginger/chemistry
Humans
Ion Channels/*physiology
Long QT Syndrome/diagnosis
Male
Membrane Potentials/drug effects/physiology
Myocytes, Cardiac/*drug effects/physiology
Patch-Clamp Techniques
Plant Extracts/*pharmacology
Pueraria/chemistry
Purkinje Fibers/drug effects/physiology
Rabbits
Rats
Rats, Sprague-Dawley

Figure

• 

  Fig. 1 Effect of KIOM-79 on the APs in rabbit cardiac purkinje fibers. (A) Representative recording of triggered APs in control and in the presence of 10 µg/mL KIOM-79 (red trace). (B) Bar graphs showing shortened total amplitude (TA) and rate of maximum depolarization at phase 0 (Vmax) in the presence of 10 µg/mL KIOM-79 (upper panel). APD90 was increased by KIOM-79 (10 µg/mL) while APD50 was not affected (lower panels). *P value <0.05.

• 

  Fig. 2 Effect of KIOM-79 on voltage-gated Na+ current (INa) in rat ventricular myocytes. (A) A representative trace of membrane currents in rat ventricular myocytes obtained with Cs+ pipette solution. A transient INa was activated with a test depolarization (-35 mV) from holding potential of -80 mV. The INa was decrease by 10 µg/mL KIOM-79 (arrow). (B) Summary of the effects of KIOM-79 on the peak amplitude of INa. In each cell, the current amplitudes measured at the peak of current were normalized to the control amplitude and mean±SEM values were plotted (n=6). *P value <0.05.

• 

  Fig. 3 Effect of KIOM-79 on voltage-gated L-type Ca2+ current (ICaL) in rat ventricular myocytes. (A) The inward currents were recorded in rat ventricular myocytes with the Cs+ pipette solution. A representative current obtained by depolarizing pulse from -50 to 0 mV (200 msec) is shown. KIOM-79 (10 µg/mL) slightly decreased the inward current. (B) To obtain the I-V curve of ICaL, the membrane voltage was held at -50 mV and incremental step-like pulses were from -40 to 40 mV (10 mV intervals, 200 msec duration). The amplitudes of I-V curves were decreased by 10 µg/mL KIOM-79. Each symbol represents mean±SEM of current amplitudes normalized to the cell capacitance (pA/pF, n=5). *P value <0.05.

• 

  Fig. 4 Effects of KIOM-79 on hERG current. (A) Representative hERG current traces recorded from HEK293 cells before and after application of 10 µg/mL KIOM-79. The currents were recorded with a test depolarization from the holding potential of -80 to +20 mV (1 sec), with tail currents recorded upon repolarization to -50 mV for 1 sec. (B) Dose dependent relationship for the increase of Idepol (•) and the decrease of Itail (○) by KIOM-79 (µg/mL): 0.1 (n=3), 1 (n=8), 10 (n=10), 100 (n=5). Each symbol represents mean±SEM of current amplitudes normalized to control currents (%). The amplitudes of Idepol and Itail were measured at the timing indicated by downward arrows in the pulse protocol of panel (A). (C, D) I-V curves for Idepol and Itail before and after application of 10 µg/mL KIOM-79. Each symbol represents mean±SEM of current amplitudes normalized to the cell capacitance (pA/pF, n=6). *P value <0.05 vs. control.

• 

  Fig. 5 Effects of the three different fractions of KIOM-79 on hERG current. Left panels; representative current traces obtained by step like pulses same as Fig. 4A. Right panels; dose dependent relationships for Idepol (closed circle) and Itail (open circle). (A) No significant effect of the water extract (n=6). (B) Effects of butanol (BuOH) fraction demonstrating the increase of Idepol and the decrease of Itail (n=5). (C) Effects of ethylacetate (EtOAC) fraction demonstrating the increase of Idepol while no effect on Itail (n=6). (D) Effects of hexane fraction demonstrating the increase of Idepol and the decrease of Itail (n=6). Each symbol represents mean±SEM of current amplitudes normalized to control currents (%). *P value <0.05 vs. control.

• 

  Fig. 6 Computer simulation of the effects of KIOM-79 on the voltage-dependence of hERG channel activity. (A) A proposed change in voltage-dependence of steady-state inactivation of hERG to reproduce the effects of KIOM-79 on hERG current in Fig. 4. In order to reduce the slope of the voltage-dependence of steady-state inactivation, the opening and closing rate constants were modified as follows. αh=1.0/{1.6*exp([Vm-10]/17.0)+0.7*exp([Vm-10]/300.0)}; βh=1.0/{0.067*exp(-[Vm+30]/17.0)+0.63*exp(-[Vm+30]/150.0)}. The voltage-dependence of opening rate constant was shifted to the right by 10 mV, while that of closing rate constant was shifted to the left by 30 mV. Note that the voltage-dependence shows a deviation from typical Boltzman distribution since there are two exponential terms in the equations describing αh and βh. (B) Reconstructed hERG currents from the altered rate constants of inactivation. Altered inactivation increased the amplitude of membrane current activated by a depolarizing step from -80 mV to +20 mV, whereas it reduced the amplitude of membrane current activated by a repolarizing step from +20 to -50 mV.

• 

  Fig. 7 Computer simulation of the changes in AP shape by altered properties of hERG, INa and ICaL channels. (A) Changes in AP shape by applying the altered inactivation of hERG (Fig. 6) and the decrease in Na+-channel density (75% of control) to the computational model. By this modification, the APD90 of reconstructed action potential was prolonged from 208.7 to 230.7 msec, and the peak amplitude of phase 0 was decreased, similar with the results from Fig. 1. (B) When the conductance of ICaL was reduced (80% of control) in addition to the above changes in hERG and INa, the plateau potential was also affected reproducing the results from the cardiac Purkinje fiber of rabbits. The degree of prolongation in APD90 (ΔAPD90=15.5 ms) was rather small compared with the results (ΔAPD90=22.0 msec) in (A).

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