Andrographolide inhibits arrhythmias and is cardioprotective in rabbits

Abstract

Andrographolide has a protective effect on the cardiovascular system. To study its cardic-electrophysiological effects, action potentials and voltage-gated Na⁺ (I_Na), Ca²⁺ (I_CaL), and K⁺ (I_K1, I_Kr, I_to and I_Kur) currents were recorded using whole-cell patch clamp and current clamp techniques. Additionally, the effects of andrographolide on aconitine-induced arrhythmias were assessed on electrocardiograms in vivo. We found that andrographolide shortened action potential duration and reduced maximum upstroke velocity in rabbit left ventricular and left atrial myocytes. Andrographolide attenuated rate-dependence of action potential duration, and reduced or abolished delayed afterdepolarizations and triggered activities induced by isoproterenol (1 μM) and high calcium ([Ca²⁺]_o=3.6 mM) in left ventricular myocytes. Andrographolide also concentration-dependently inhibited I_Na and I_CaL, but had no effect on I_to, I_Kur, I_K1, or I_Kr in rabbit left ventricular and left atrial myocytes. Andrographolide treatment increased the time and dosage thresholds of aconitine-induced arrhythmias, and reduced arrhythmia incidence and mortality in rabbits. Our results indicate that andrographolide inhibits cellular arrhythmias (delayed afterdepolarizations and triggered activities) and aconitine-induced arrhythmias in vivo, and these effects result from I_Na and I_CaL inhibition. Andrographolide may be useful as a class I and IV antiarrhythmic therapeutic.

Keywords: L-type calcium current; action potential; andrographolide; arrhythmia; sodium current.

Figure 1. Effects of andrographolide (Andro) on APs, DADs, and DAD-induced TAs

Typical photomicrographs of a single LVM (left) or LAM (right) cell under 40× light microscope (A). Effects of andrographolide (5 or 10 μM) on APs recorded from rabbit LVMs (left) and LAMs (right) (B). Effect of andrographolide (5 or 10 μM) on APs stimulated at 0.5, 1, or 2 Hz (C). APs from 30 consecutive sweeps were averaged. The data of APD₉₀ recorded at different stimulation frequencies are shown; andrographolide shortened APD₉₀ and attenuated its RD in a concentration-dependent manner in LVMs (D). Data are shown as means±SD (n=9, ^#p<0.01 vs control, ^$p<0.01 vs 5 μM andrographolide). Andrographolide abolished ISO (1 μM) and high calcium ([Ca²⁺]_o=3.6 mM)-induced DADs and TAs in LVMs (E). Arrows indicate depolarizing pulse.

Figure 2. Andrographolide inhibited I_Na in a concentration dependent manner in LVMs and LAMs

Representative whole-cell recordings of I_Na in LVMs (A) and LAMs (B) with or without 1, 5, 10, or 20 μM andrographolide. Current voltage relationships for I_Na in LVMs (C) and LAMs (D) Data are shown as means±SD (ventricle, n=10; atrium, n=8). ^#p<0.01 vs control, ^%p<0.01 vs 1 μM, ^$p<0.01 vs 5 μM, ^&p<0.01 vs 10 μM andrographolide. Representative I_Na recordings before (E) and after (F) andrographolide treatment using the inactivation protocol in the inset. Steady-state activation and inactivation curves for I_Na in LVMs (G) and LAMs (H) with or without 20 μM andrographolide. Lines represent data fit to a Boltzmann distribution function. Dose-reaction relationship between andrographolide and percent inhibition of I_Na (ventricle, n=10; atrium, n=10) (I)⁺p>0.05 vs ventricle.

Figure 3. Andrographolide inhibition of I_CaL is reversible

I_CaL time course after membrane rupture in LVMs (A). Histograms of I_CaL current densities for control, 10 μM andrographolide, and washout (n=8) (B). ^#p<0.01 vs control, ^&p<0.01 vs 10 μM andrographolide). Data are shown as means±SD. Effects of andrographolide on I_CaL are reversible, and nifedipine (10 μM) blocked I_CaL completely in LVMs (C).

Figure 4. Andrographolide inhibited I_CaL in a concentration-dependent manner in LVMs and LAMs

Representative whole-cell recordings of I_CaL in LVMs (A) and LAMs (B) with or without 1, 5, 10, or 20 μM andrographolide. Current voltage relationships for I_CaL in LVMs (C) and LAMs (D) Data are shown as means±SD (ventricle, n=14; atrium, n=15). ^#p<0.01 vs control, ^% p<0.01 vs 1 μM, ^$ p<0.01 vs 5 μM, ^&p<0.01 vs 10 μM andrographolide. Representative I_CaL recordings before (E) and after (F) andrographolide treatment using the inactivation protocol in the inset.Steady-state activation and inactivation curves for I_CaL in LVMs (G) and LAMs (H) with or without 20 μM andrographolide. Lines represent the data fit to a Boltzmann distribution function. Dose-reaction relationship between andrographolide and percent inhibition of I_CaL (ventricle, n=8; atrium, n=10) (I) ⁺p>0.05 vs ventricle.

Figure 5. Effects of andrographolide on I_K1, I_Kr, I_to, and I_Kur

I_K1 (A) and its I-V relationship (C) in LVMs beforeand after andrographolide treatment. Data are shown as means±SD (n=14). Typical I_Kr current traces from a single LVM cell (B) and its I-V relationship (D) before and after andrographolide treatment (n=15). Representative I_to current recording (E) and its I-V relationship (F) in LAMs before and after andrographolide treatment (n=15). I_Kur I-V relationship in LAMs before and after application of andrographolide (n=12) (G). *p>0.05 vs control, ^@p>0.05 vs 10 μM andrographolide, ^!p>0.05 vs 20 μM andrographolide.

Figure 6. Effects of andrographolide on aconitine-induced arrhythmias

Histogram of the aconitine-induced arrhythmia time threshold (NS: n=10; Andro: VPC, n=10; VT, n=7) (A). Histogram of the aconitine threshold (NS: n=10; Andro: VPC, n=10; VT, n=7) (B). VPC and VT appeared in all 10 NS group rabbits, and in 10 and 7 Andro group rabbits, respectively. Rabbit mortality before and after andrographolide treatment (NS: n=10, Andro: n=10) (C). Incidence of aconitine-induced multiple ventricular arrhythmias in the two groups (D). VPC was triggered successfully in all animals, but VT and VF incidences were reduced in the Andro group. Time-dosage threshold curve of various types of ventricular arrhythmias (E). VPC, VT and VF were observed in all 10 NS group rabbits, and in 10, 7, and 1 of 10 Andro group rabbits, respectively. Typical ECG tracings showing various types of ventricular arrhythmias before (Fa) and after aconitine treatment (2 μg/kg/min) (Fb–d) VPC, VT, and VF began to appear at 17, 23, and 47 min (F). Typical ECG tracings before (Ga) and after andrographolide (10 mg/kg) and aconitine (2 μg/kg/min) treatment (Gb–d). The times of the four ECG tracings in (G) were consistent with those in (F). After andrographolide treatment, VPC appeared at 47 min, and arrhythmia did not appear at either 17 or 23 min. *p<0.01 vs NS. NS: normal saline; VPC: ventricular premature contraction; VT: ventricular tachycardia; VF: ventricular fibrillation.