Barbaloin inhibits ventricular arrhythmias in rabbits by modulating voltage-gated ion channels

Abstract

Barbaloin (10-β-D-glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)-anthracenone) is extracted from the aloe plant and has been reported to have anti-inflammatory, antitumor, antibacterial, and other biological activities. Here, we investigated the effects of barbaloin on cardiac electrophysiology, which has not been reported thus far. Cardiac action potentials (APs) and ionic currents were recorded in isolated rabbit ventricular myocytes using whole-cell patch-clamp technique. Additionally, the antiarrhythmic effect of barbaloin was examined in Langendorff-perfused rabbit hearts. In current-clamp recording, application of barbaloin (100 and 200 μmol/L) dose-dependently reduced the action potential duration (APD) and the maximum depolarization velocity (Vmax), and attenuated APD reverse-rate dependence (RRD) in ventricular myocytes. Furthermore, barbaloin (100 and 200 μmol/L) effectively eliminated ATX II-induced early afterdepolarizations (EADs) and Ca²⁺-induced delayed afterdepolarizations (DADs) in ventricular myocytes. In voltage-clamp recording, barbaloin (10-200 μmol/L) dose-dependently inhibited L-type calcium current (I_Ca.L) and peak sodium current (I_Na.P) with IC₅₀ values of 137.06 and 559.80 μmol/L, respectively. Application of barbaloin (100, 200 μmol/L) decreased ATX II-enhanced late sodium current (I_Na.L) by 36.6%±3.3% and 71.8%±6.5%, respectively. However, barbaloin up to 800 μmol/L did not affect the inward rectifier potassium current (I_K1) or the rapidly activated delayed rectifier potassium current (I_Kr) in ventricular myocytes. In Langendorff-perfused rabbit hearts, barbaloin (200 μmol/L) significantly inhibited aconitine-induced ventricular arrhythmias. These results demonstrate that barbaloin has potential as an antiarrhythmic drug.

Figure 1

Effects of barbaloin on action potentials (APs) in rabbit ventricular myocytes. (A) Representative traces showing that barbaloin exerted reversible inhibitory effects on APs. (B) Representative traces of APs recorded continuously at a frequency of 1 Hz before (control) and after the administration of 100 and 200 μmol/L barbaloin. The gray traces are the changes in the APs after the administration of barbaloin. (C) Bar graphs showing the mean APD percentage values for the control, 100 μmol/L barbaloin-treated and wash-out groups (n=10. ^**P<0.01 vs control. ^##P<0.01 vs 100 μmol/L barbaloin). (D) The data for the APD₉₀, which were averaged from 30 sweeps recorded at different cycle lengths (CLs) (n=8. ^**P<0.01 vs control). (E) Representative single traces of APs recorded in an alternating manner at frequencies of 0.5, 1 and 2 Hz before (control) and after the administration of 100 and 200 μmol/L barbaloin.

Figure 2

Barbaloin suppressed ATX II-induced early afterdepolarizations (EADs) and action potential duration (APD) prolongation in rabbit ventricular myocytes. (A) Representative single APs recorded at a stimulation frequency of 0.25 Hz. (B, C, D, E and F) show representative traces from the groups subjected to control, 10 nmol/L ATX II, 10 nmol/L ATX II and 100 μmol/L barbaloin, 10 nmol/L ATX II and 200 μmol/L barbaloin and wash-out treatment, respectively. These 3 consecutive APs were recorded at a stimulation frequency of 0.25 Hz with a CL of 12.5 s.

Figure 3

Barbaloin suppressed delayed afterdepolarizations (DADs) induced by the extracellular addition of CaCl₂ in rabbit ventricular myocytes. Ten consecutive APs were recorded at a frequency of 5 Hz with a CL of 7 s. (A, B, C, D and E) show representative traces from the groups subjected to control, 3.6 mmol/L CaCl₂, 3.6 mmol/L CaCl₂ and 100 μmol/L barbaloin, 3.6 mmol/L CaCl₂ and 200 μmol/L barbaloin and wash-out treatment, respectively. F. Incidence of DADs in the control group, in the presence of 3.6 mmol/L CaCl₂ alone, after the addition of 100 μmol/L barbaloin and 200 μmol/L barbaloin and in the wash-out group (n=9. ^**P<0.01 vs control. ^##P<0.01 vs 3.6 mmol/L CaCl₂. ^&&P<0.01 vs 100 μmol/L barbaloin. ^$$P<0.01 vs 200 μmol/L barbaloin).

Figure 4

Effects of barbaloin on L-type calcium current (I_Ca.L) in rabbit ventricular myocytes. (A) Representative traces showing that barbaloin exerted reversible inhibitory effects on I_Ca.L. (B) The bar graphs show the mean I_Ca.L percentage values for the control, 100 μmol/L barbaloin-treated and wash-out groups (n=8. ^**P<0.01 vs control. ^##P<0.01 vs 100 μmol/L barbaloin). (C) Representative I_Ca.L traces from the control and 50, 100 and 200 μmol/L barbaloin-treated groups. (D) I_Ca.L current-voltage (I–V) relationships in the control and 10, 50, 100 and 200 μmol/L barbaloin-treated groups (n=8. ^*P<0.05, ^**P<0.01 vs control. ^#P<0.05, ^##P<0.01 vs 10 μmol/L barbaloin. ^&P<0.05, ^&&P<0.01 vs 50 μmol/L barbaloin. ^$P<0.05, ^$$P<0.01 vs 100 μmol/L barbaloin). (E) The concentration-response relationship curve for the effects of barbaloin on I_Ca.L, fitted to the Hill equation. (F) Representative traces for I_Ca.L steady-state inactivation in the control and 200 μmol/L-treated barbaloin groups. (G) Curves for I_Ca.L steady-state activation and inactivation before (control) and after the administration of 200 μmol/L barbaloin, fitted to the Boltzmann equation.

Figure 5

Effects of barbaloin on ATX II-induced late sodium current (I_Na.L) enhancements and under normal conditions in rabbit ventricular myocytes. (A) Representative single traces of the effects of 4 μmol/L TTX on ATX II-induced I_Na.L enhancement. (B) Representative single traces showing that barbaloin exerted reversible inhibitory effects on ATX II-induced I_Na.L enhancement. (C) Representative single traces of the effects of 200 μmol/L barbaloin on I_Na.L under normal conditions. (D) Single traces of the effects of barbaloin on ATX II-induced I_Na.L enhancements at a membrane potential of −20 mV. (E) Representative traces of the effects of barbaloin on ATX II-induced I_Na.L enhancements at membrane potentials of −80, −60, −50, −40 and −20 mV. (F) Bar graphs showing the mean ATX II-increased I_Na.L percentage values for the control, 100 μmol/L barbaloin-treated and wash-out groups (n=10. ^**P<0.01 vs control. ^##P<0.01 vs 100 μmol/L barbaloin). (G) The I-V relationship for the effects of barbaloin on ATX II-induced I_Na.L enhancement (n=8. ^*P<0.05, ^**P<0.01 vs control. ^#P<0.05, ^##P<0.01 vs 10 nmol/L ATX II. ^&P<0.05, ^&&P<0.01 vs 100 μmol/L barbaloin).

Figure 6

Effects of barbaloin on peak sodium current (I_Na.P) in rabbit ventricular myocytes. (A) Representative traces showing that barbaloin exerted reversible inhibitory effects on I_Na.P. (B) Bar graphs showing the mean I_Na.P percentage values for the control, 400 μmol/L barbaloin-treated and wash-out groups (n=10. ^*P<0.05, ^**P<0.01 vs control. ^##P<0.01 vs 400 μmol/L barbaloin). (C) Representative I_Na.P traces from the control and 100, 400 and 800 μmol/L barbaloin-treated groups. (D) I-V relationships for I_Na.P in the control and 10, 100, 400 and 800 μmol/L barbaloin-treated groups (n=8. ^*P<0.05, ^**P<0.01 vs control. ^#P<0.05, ^##P<0.01 vs 10 μmol/L barbaloin. ^&P<0.05, ^&&P<0.01 vs 100 μmol/L barbaloin. ^$P<0.05, ^$$P<0.01 vs 400 μmol/L barbaloin). (E) The concentration-response relationship curve for the effects of barbaloin on I_Na.P, fitted to the Hill equation. (F) Representative traces of I_Na.P steady-state inactivation in the control and 200 μmol/L barbaloin-treated groups. (G) Curves for I_Na.P steady-state activation and inactivation before (control) and after the administration of 200 μmol/L barbaloin, fitted to the Boltzmann equation.

Figure 7

Barbaloin had similar effects on inward rectifier potassium current (I_K1) and rapidly activated delayed rectifier potassium current (I_Kr) in rabbit ventricular myocytes. (A) Representative I_Kr traces from the control and 100 and 800 μmol/L barbaloin-treated groups. (B) I–V relationships for I_Kr-tail in the control and 100 and 800 μmol/L barbaloin-treated groups (n=8, P>0.05 for both 100 and 800 μmol/L barbaloin vs control). (C) Representative I_K1 traces from the control and 100 and 800 μmol/L barbaloin-treated groups. (D) I–V relationships for I_K1 in the control and 100 and 800 μmol/L barbaloin-treated groups (n=8, P>0.05 for both 100 and 800 μmol/L barbaloin vs control).

Figure 8

Effects of barbaloin on aconitine-induced ventricular arrhythmias in Langendorff-perfused rabbit hearts. (A) Representative ECG traces at onset time in the aconitine-treated group. (B) Representative ECG traces at the same time as A in the barbaloin and aconitine co-treatment group. (C) The heart rates of the Langendorff-perfused rabbit hearts for the first 30 min of recording time in four different groups. The arrows indicate the time when barbaloin or aconitine was added to the perfusate. (D) The onset time of aconitine-induced ventricular arrhythmias in the aconitine-treated and barbaloin and aconitine co-treatment groups (n=8. ^**P<0.01 vs aconitine group). (E) The incidence of aconitine-induced ventricular arrhythmias in the aconitine-treated and barbaloin and aconitine co-treatment groups (n=8, ^**P<0.01 vs aconitine group).