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.

INTRODUCTION

The traditional medical herb, Andrographis paniculata (Burm.f.) Wall. ex Nees, has been used for centuries to treat a variety of diseases in China. Andrographolide, its major bioactive component, has many pharmacological effects, including antitumor [1, 2], anti-inflammatory [1–4], antiviral [2], anti-hypertension [5], antioxidant [3, 6, 7], antihyperglycaemic [2], hepatoprotective [2, 7], and cardioprotective properties [5–11]. Recent studies found that andrographolide protected rat cardiomyocytes against hypoxia/reoxygenation injury by upregulating cellular-reduced glutathione levels and antioxidant enzyme activities [6]. Andrographolide protected mouse hearts from LPS-induced cardiac malfunctions, and thus may prevent myocardial malfunction during sepsis [8]. Because platelet activation is closely linked to coronary heart disease, andrographolide could be used to treat patients with thromboembolic disorders due to its antiplatelet activity [9–11].

Andrographolide has been used clinically for many years, and its cardioprotective properties are known. However, its effects on sodium (I_Na), calcium (I_CaL), and potassium channels (I_K1, I_Kr, I_to, I_Kur), and action potentials (APs) in cardiomyocytes have not been reported. This study explored the effects of andrographolide on the aforementioned ion channels and action potentials (APs) to determine its antiarrhythmic mechanism of action.

RESULTS

Effects of andrographolide on APs in rabbit left ventricular and left atrial myocytes

In this study, left ventricular myocytes (LVMs) and left atrial myocytes (LAMs) were isolated at the same time and were tested alternately, so comparisons between LVM and LAM electrical activity parameters were reasonable. LVMs (Figure 1A, left) were larger and wider in size than LAMs (right), and had more intensive transverse striations.

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.

APs were elicited by depolarizing pulses delivered in a width of 4 ms, 1.5-fold above the threshold at a rate of 1 Hz using the current-clamp technique. Figure 1B shows typical LVM (left) and LAM (right) morphology, and the effect of andrographolide on APs in these cells. Andrographolide shortened action potential duration at 50% repolarization (APD₅₀) and 90% repolarization (APD₉₀), and decreased maximum upstroke velocity (V_max) in a concentration-dependent manner without changing resting membrane potential (RMP) or action potential amplitude (APA) in LVMs or LAMs. The effects of andrographolide on AP parameters were the same for LVMs and LAMs (Table 1).

Table 1: Effects of andrographolide on LVM and LAM AP parameters

Parameter	Andrographolide
	Control		5 μM		10 μM
	Ventricle	Atrium	Ventricle	Atrium	Ventricle	Atrium
RMP	-88 ± 2	-77 ± 4	-88 ± 2	-77 ± 5+	-88 ± 2	-77 ± 6+
APA	129 ± 4	129 ± 11	129 ± 5	128 ± 11+	128 ± 4	126 ± 11+
APD50	172 ± 7	91 ± 4	147 ± 8##	77 ± 6##+	131 ± 6##$$	66 ± 4##$$+
APD90	195 ± 8	137 ± 8	164 ± 7##	119 ± 6#+	142 ± 3##$$	111 ± 6##$*
Vmax	175 ± 7	249 ± 10	156 ± 8# #	226 ± 5##+	143 ± 7##	213 ± 7##$+

Data are presented as means±SD (ventricle, n=15; atrium, n=8).

^##p<0.01 vs control, ^#p<0.05 vs control, ^$$p<0.01 vs 5 μM andrographolide, ^$p<0.05 vs 5 μM andrographolide, ⁺p>0.05 vs ventricle, ^*p<0.05 vs ventricle.

RMP: resting membrane potential (mV); APA: action potential amplitude (mV); APD₅₀: action potential duration at 50% repolarization (ms); APD₉₀: action potential duration at 90% repolarization (ms); V_max: maximum upstroke velocity (mV/ms).

Effects of andrographolide on rate-dependent repolarization of APs in LVMs

APs were recorded using depolarizing pulses delivered in a width of 4 ms, 1.5-fold above the threshold at a rate of 1 Hz using the current-clamp technique. APs were recorded at stimulation frequencies of 0.5, 1, and 2 Hz with or without andrographolide after the AP stabilized at 1 Hz. Andrographolide shortened APD₉₀ in a concentration-dependent manner and attenuated rate-dependence (RD) of APD (Figure 1C–1D).

Effects of andrographolide on isoproterenol- and high calcium-induced delayed afterdepolarizations and triggered activities

APs were elicited by string stimulation containing 10 pulses at a basic cycle length (BCL) of 300 ms and string stimulation frequency of 0.125 Hz. APs were recorded after stabilization. Isoproterenol (ISO, 1 μM) was added to the external bath solution and the calcium concentration ([Ca²⁺]_o) was increased to 3.6 mM at the same time. Under control circumstances, there were no delayed afterdepolarizations (DADs) or triggered activities (TAs). In contrast, ISO (1 μM) and high calcium ([Ca²⁺]_o=3.6 mM) induced DADs and TAs in 7/7 LVMs following 10 consecutive pulses at a BCL of 300 ms (Figure 1E). The addition of 20 μM andrographolide abolished TAs and markedly reduced or abolished DADs.

Effects of andrographolide on sodium current (I_Na) in LVMs and LAMs

I_Na was recorded using 300 ms depolarizing voltage steps between -70 mV and +40 mV in 5 mV increments from a holding potential of -90 mV at a rate of 0.5 Hz with the cells perfused with normal bath solution and recorded the current as control group. After currents stabilized, cells were perfused with solution containing andrographolide (1, 5, 10, or 20 μM). When sealing resistance was stable, currents were recorded under different concentrations and different voltages. Results in a single cell (Figure 2A–2B) and in a group cell (Figure 2C–2D) with or without andrographolide in LVMs and LAMs showed that andrographolide decreased I_Na in a concentration-dependent manner without shifting the voltage at which I_Na amplitude was maximal.

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.

Steady-state inactivation curves for sodium currents in the absence or presence of 20 μM andrographolide were evaluated from a holding potential of -90 mV. A 100 ms conditioning pre-pulse was applied from -100 mV to +40 mV in 5 mV increments followed by 100 ms depolarizing test pulses to -30 mV at a rate of 0.5 Hz to evoke I_Na. Figure 2E–2F show typical examples of one single cell from LVMs and LAMs, respectively. Figure 2G shows steady-state activation and inactivation curves fitted with a Boltzmann equation for I_Na under control conditions and after 20 μM andrographolide treatment in LVMs. For the steady-state activation of I_Na, with and without 20 μM andrographolide, V_1/2 values were -46.28±0.31mV and -45.97±0.30 mV (n=8, p>0.05), with k=4.13±0.28 mV and 4.21±0.27 mV (n=8, p>0.05), respectively. For the steady-state inactivation of I_Na, with and without 20 μM andrographolide, V_1/2 values were -76.63±0.38 mV and -88.52±0.84 mV (n=8, p<0.01), with k=8.60±0.45 mV and 8.25±0.53 mV (n=8, p>0.05), respectively.

Figure 2H shows the steady-state activation and inactivation curves fitted with a Boltzmann equation for I_Na with or without 20 μM andrographolide in LAMs. For the steady-state activation of I_Na, with or without 20 μM andrographolide, V_1/2 values were -47.81±0.33 mV and -48.03±0.21 mV (n=8, p>0.05), with k=4.93±0.31 mV and 4.83±0.19 mV (n=8, p>0.05), respectively. For the steady-state inactivation of I_Na, with or without 20 μM andrographolide, V_1/2 values were -79.58±0.39 mV and -88.64±0.04 mV (n=8, p<0.01), with k=9.97±0.45 mV and 9.17±0.65 mV (n=8, p>0.05), respectively. While andrographolide did not affect activation, but the voltage dependence of I_Na inactivation curve was shifted toward negative membrane potential, and andrographolide accelerated the inactivation process without changing its slope factor (Figure 2G–2H). Andrographolide inhibited I_Na in LVMs and LAMs with an IC₅₀ of 10.41±3.18 μM and 8.28±1.26 μM, respectively (Figure 2I). The effects of andrographolide on I_Na were similar between LVMs and LAMs (ventricle, n=10; atrium, n=10; p>0.05 vs ventricle).

Effects of andrographolide on L-type calcium current (I_CaL) in LVMs and LAMs

I_CaL was recorded using a 300 ms depolarizing pulse from -40 mV to +50 mV in 5 mV increments at a rate of 0.5 Hz with a holding potential of -40 mV. One of the difficulties of studying I_CaL is the run-down phenomenon in which the membrane ruptures. Figure 3A shows I_CaL current density changes over time after membrane rupture without addition of any drug in LVMs. I_CaL tends to stabilize at 7–25 min after membrane rupture. To show that andrographolide inhibition of I_CaL was independent of the run-down phenomenon, the drug was added after I_CaL peak amplitude stabilized (about 7 min after membrane rupture). The experiment was accomplished within 25 min after membrane rupture to avoid interference by the run-down phenomena.

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 3C shows typical I_CaL current traces from a single LVM cell under control conditions or with 10 μM andrographolide, washout, and 10 μM nifedipine. 10 μM andrographolide inhibited I_CaL, and I_CaL amplitude recovered progressively after withdrawal of andrographolide (washout). Then, to identify I_CaL, current was recorded after addition of 10 μM nifedipine. I_CaL was almost completely blocked by 10 μM nifedipine, indicating that the current recorded with the above pulse stimulation was I_CaL. These results demonstrated that andrographolide inhibition of I_CaL is significant and reversible (Figure 3B–3C).

Figure 4A–4B shows typical I_CaL current traces from single LVM or LAM cells before (control) and after andrographolide treatment. I_CaL current amplitude tended to stabilize within 1 min after andrographolide addition. Results in a typical single LVM or LAM cell (Figure 4A–4B) and in a group cell (Figure 4C–4D) with or without andrographolide showed that andrographolide inhibited I_CaL in a concentration-dependent manner. Figure 4C–4D shows the I_CaL I–V relationship after andrographolide application, which indicated that andrographolide inhibited I_CaL without shifting the voltage at which I_CaL amplitude was maximal.

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.

The steady-state I_CaL inactivation curves with or without 20 μM andrographolide were evaluated from a holding potential of -40 mV. A 2000 ms conditioning pre-pulse was applied from -50 mV to 0 mV in 5 mV increments followed by 300 ms depolarizing test pulses to 0 mV at a rate of 0.5 Hz to evoke I_CaL. Figure 4E–4F show typical example for one single cell of LVMs and LAMs, respectively. Figure 4G shows the steady-state activation and inactivation curves fitted with a Boltzmann equation for I_CaL under control conditions and after 20 μM andrographolide application in LVMs. For the steady-state activation of I_CaL, with or without 20 μM andrographolide, V_1/2 values were -9.26±0.44 mV and -8.53±0.53 mV (n=14, p>0.05), with k=6.69±0.41 mV and 6.51±0.49 mV (n=14, p>0.05), respectively. For the steady-state inactivation of I_CaL, with or without 20 μM andrographolide, V_1/2 values were -28.44±0.23 mV and -32.87±0.35 mV (n=7, p<0.01), with k=4.55±0.21 mV and 6.09±0.31 mV (n=7, p<0.01), respectively. Figure 4H shows the steady-state activation and inactivation curves fitted with a Boltzmann equation for I_CaL with or without 20 μM andrographolide in LAMs. For the steady-state activation of I_CaL, with or without 20 μM andrographolide, V_1/2 values were -10.99±0.31 mV and -10.66±0.33 mV (n=13, p>0.05), with k=8.32±0.33 mV and 8.89±0.37 mV (n=13, p>0.05), respectively. For the steady-state inactivation of I_CaL, with or without 20 μM andrographolide, V_1/2 values were -30.54±0.29 mV and -34.68±0.54 mV (n=8, p<0.01), with k=4.93±0.27 mV and 6.02±0.45 mV (n=8, p<0.01), respectively. These results suggest that andrographolide did not affect I_CaL activation, but shifted the voltage dependence of I_CaL inactivation curve toward negative membrane potential. Andrographolide also accelerated the inactivation process and increased its slope factor (Figure 4G–4H). Andrographolide inhibited I_CaL in LVMs and LAMs with an IC₅₀ of 11.91±1.55 μM and 10.08±1.15 μM, respectively (Figure 4I). Andrographolide had similar effects on I_CaL between LVMs and LAMs (ventricle, n=8; atrium, n=10. p>0.05 vs ventricle).

Effect of andrographolide on inward rectifying potassium current (I_k1) in LVMs

I_K1 was elicited by 400 ms depolarization voltage from -120 mV to +50 mV in 5 mV increments at a rate of 1 Hz with a holding potential of -40 mV. Figure 5A shows typical I_K1 current traces from a single cell before and after 40 μM andrographolide treatment. Neither 20 μM nor 40 μM andrographolide affected I_K1 (Figure 5C).

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.

Effect of andrographolide on rapid delayed rectifier potassium current (I_Kr) in LVMs

I_Kr was elicited by 3000 ms depolarization voltage from -40 mV to +50 mV in 10 mV increments followed by a 5000 ms repolarization pulse to -40 mV at a rate of 0.1 Hz with a holding potential of -40 mV. Figure 5B shows typical single cell I_Kr current traces before and after 40 μM andrographolide application. Neither 10 μM nor 40 μM andrographolide affected I_Kr (Figure 5D).

Effect of andrographolide on transient outward potassium current (I_to) in LAMs

I_to was recorded using a 400 ms depolarization voltage from -40 mV to +50 mV in 10 mV increments at a rate of 0.1 Hz with a holding potential of -80 mV. I_Na was eliminated by 100 ms, with -40 mV depolarizing pre-pluses. Figure 5E shows typical I_to current traces from a single cell before and after 40 μM andrographolide application. Neither 20 μM nor 40 μM andrographolide affected I_to (Figure 5F).

Effect of andrographolide on ultra-rapid delayed rectifier potassium current (I_Kur) in LAMs

I_Kur was recorded with an 80 ms pre-pulse to +30 mV to inactivate I_to, then repolarized to -50 mV, lasting 50 ms, followed by a 150 ms test pulse to +60 mV from -40 mV in 10 mV increments, at a rate of 0.5 Hz with a holding potential of -50 mV. Neither 20 μM nor 40 μM andrographolide affected I_Kur (Figure 5G).

Effect of andrographolide on aconitine-induced ventricular arrhythmias in rabbits

Ventricular premature contraction (VPC), ventricular tachycardia (VT), and ventricular fibrillation (VF) appeared in all 10 NS group rabbits, and in 10, 7, and 1 of 10 Andro group rabbits, respectively. Andrographolide treatment prior to aconitine increased the time threshold (Figure 6A) and aconitine cumulative dosage (Figure 6B) required to induce VPC, VT, and VF compared with the NS group. Andrographolide treatment also reduced rabbit mortality (Figure 6C) and aconitine-induced ventricular arrhythmia incidence (Figure 6D–6E).

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.

DISCUSSION

This study found that andrographolide inhibited I_Na and I_CaL in LVMs and LAMs in a concentration-dependent manner. Andrographolide (5 and 10 μM) decreased APD₅₀, APD₉₀, and V_max without affecting RMP and APA in these cells. Treatment also shortened APD and attenuated RD of APD in LVMs in a concentration-dependent manner. We found that 20 μM andrographolide attenuated TAs and DADs induced by ISO (1 μM) and high calcium (3.6 mM) in LVMs. Finally, andrographolide had no influence on I_K1 and I_Kr in LVMs, or I_to and I_Kur in LAMs. Our results suggest that andrographolide had cardioprotective effects in rabbits with aconitine-induced arrhythmias.

Andrographolide shifted the I_Na inactivation curve toward negative membrane potential and accelerated the inactivation process without changing its slope factor. Decreased I_Na amplitude and accelerated I_Na inactivation indicated that the number of Na⁺ flowing into the cell was reduced. Since the transient sodium current initiates the action potential [12], increasing the quantity of current passed through the membrane would enhance cell excitability. Thus, andrographolide decreased cell excitability, which suggests its potential use in treatment of arrhythmias caused by enhanced excitability. In cardiomyocytes, intracellular Na⁺ concentration ([Na⁺]_i) is an important Ca²⁺ homeostasis and cell contractility modulator [13]. [Na⁺]_i is increased under certain pathological conditions, such as myocardial ischemia [14]. In this study, andrographolide inhibited I_Na. Therefore, andrographolide reversed increased [Na⁺]_i levels under pathological conditions by inhibiting I_Na, which could help alleviate the Na⁺-dependent calcium overload in cardiomyocytes under some pathological conditions [15]. These results indicate that andrographolide might serve as a class I antiarrhythmic, cardioprotective therapeutic.

Normal I_CaL regulation is key to intracellular Ca²⁺ ([Ca²⁺]_i) homeostasis. The inorganic calcium ion (Ca²⁺), an important messenger in living cells, is responsible for signal transduction and regulation of vital functions [16]. Disruption of [Ca²⁺]_i homeostasis (calcium overload) is closely related to multiple pathological conditions, including arrhythmia [17], myocardial ischemia/reperfusion [18], heart failure [19], digitalis intoxication [20], and hypercalcinemia [21]. Andrographolide inhibited I_CaL concentration-dependently, and shifted the I_CaL inactivation curve toward negative membrane potential by amplifying its V_1/2 and k values. When k was increased, the inactivation process accelerated. I_CaL inactivation acceleration and decreased I_CaL amplitude indicated that the number of Ca²⁺ flowing into cell was reduced. Since andrographolide decreased [Ca²⁺]_i, it might alleviate Ca²⁺ overloading in cardiomyocytes. Andrographolide could inhibit increased [Ca²⁺]_i and restore intracellular Ca²⁺ homeostasis, suggesting its potential use as a class IV anti-arrhythmic drug.

Andrographolide did not influence rabbit cardiomyocyte RMP, which was consistent with our finding that it had no effect on LVM I_K1. Andrographolide (5 and 10 μM) had no effect on APA, but reduced V_max. A V_max reduction can decrease cardiomyocyte excitability and slow down the excitement conduction velocity, which is helpful for inhibiting reentrant tachycardia. Since andrographolide had no influence on I_K1, I_Kr, I_to, or I_Kur, andrographolide mainly shortens APD₅₀ and APD₉₀ by inhibiting I_CaL. In this study, andrographolide not only shortened AP duration, but also decreased the RD of APD concentration-dependently. When RD decreased, the transmural dispersion of repolarization also diminished. Therefore, andrographolide may reduce the transmural repolarization heterogeneity of APD. Enlargement of APD transmural repolarization heterogeneity leads to the occurrence of arrhythmias [22]. Andrographolide reduced RD, indicating that it may reduce the transmural repolarization heterogeneity of APD, thus also inhibiting reentrant arrhythmias.

ISO and high calcium causes Ca²⁺-overload by increasing [Ca²⁺]_i, thereby resulting in DADs and TAs [23–25]. DAD-mediated TA can increase abnormal autorhythmicity, causing the majority of sudden cardiac deaths during nonischemic heart failure [24, 25]. In this study, 20 μM andrographolide inhibited DADs and TAs evoked by ISO and high calcium. The potential mechanism may be related to andrographolide inhibition of I_CaL and I_Na to alleviate intracellular Ca²⁺- and Na⁺-overloading.

This study also found that andrographolide has no effect on I_K1, I_Kr, I_to, and I_Kur. Enhanced reverse rate-dependence (RRD) of APD caused by drugs that inhibit I_Kr is an undesirable response, as it reduces drug efficacy during tachycardia and may promote proarrhythmic activity after tachycardia termination [26, 27]. Andrographolide as a potential antiarrhythmia therapeutic does not induce such side effects.

Finally, in vivo results indicated that andrographolide had cardioprotective effects in rabbits with aconitine-induced arrhythmias. Aconitine binds to voltage-dependent cardiac Na⁺ channels [28, 29] and prolongs their open state, favoring entry of a large quantity of Na⁺ into cytosol. This may be accompanied by Ca²⁺ overload via an electrogenic Na⁺-Ca²⁺ exchange (NCX) system, and eventually induces triggered activity [30, 31]. Furthermore, aconitinine can increase I_CaL by accelerating the activation process and delaying inactivation process [32, 33]. We found that andrographolide increased the time threshold and cumulative aconitine dosage required to induce VPC, VT, and VF. It also reduced incidences of aconitine-induced VT and VF, as well as mortality in rabbits. These results indicated that andrographolide had the protective effects on aconitine-induced arrhythmias. And this antiarrhythmic effect of andrographolide was related to its inhibition of I_Na and I_CaL.

To the best of our knowledge, this is the first demonstration about the effects of andrographolide on cardiac myocyte electrophysiologyical properties, and its possible antiarrhythmic mechanisms is related to its inhibitory effects on I_Na and I_CaL. Our results of this study indicate that andrographolide may be a potential class I and IV antiarrhythmia agent.

MATERIALS AND METHODS

Ethics statement

Use of rabbits in this investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1996) and the Guide for the Care and Use of Laboratory Animals (Hubei Province, China), and was approved by the Institutional Animal Care and Use Committee of Wuhan University of Science and Technology.

Cardiomyocyte isolation

Single cardiac ventricular and atrial myocytes were enzymatically isolated from adult New Zealand white rabbits (1.7–2 kg weight, 24–32 weeks of age, of either sex), as described preciously [34]. Rabbits were provided by the Experimental Animal Center of Wuhan University of Science and Technology. Rabbits were heparinized (2000 U) and anesthetized with ketamine (30 mg/kg, intravenously) and xylazine (7.5 mg/kg, intramuscularly). After the corneal reflex disappeared, rabbits were fixed on their backs to the test stands. Hearts were quickly removed after thoracotomy and put in a petri dish filled with 37°C, oxygen (95% O₂ and 5% CO₂) saturated, Ca²⁺- free Tyrode’s solution containing (mM): NaCl 135, NaH₂PO₄ 0.33, KCl 5.4, MgCl₂ 1.0, HEPES 10, glucose 10 (pH 7.4, adjusted with NaOH) to stop the heartbeat. Fat and connective tissues were removed from the heart. To eliminate residual blood in the heart cavity, the aorta was cannulated and perfused retrogradely using a modified Langendorff apparatus (perfusion pressure 70 cm H₂O₂), then perfused for 5 more min with oxygen saturated, Ca²⁺-free Tyrode’s solution (at 37°C). Hearts were then digested for 30 min with the same solution containing enzyme (collagenase type I, 1 g/L) and bovine serum albumin (BSA, 1 g/L) at 37°C. The enzyme-containing solution was pumped at a constant rate using a peristaltic pump, and gas (95% O₂ and 5% CO₂) was allowed to pass through this solution. The perfusate was then switched to preheated and oxygen saturated Kraftbrühe (KB) solution (mM): KOH 70, KCl 40, KH₂PO₄ 20, glutamic acid 50, taurine 20, EGTA 0.5, glucose 10, HEPES 10, MgSO₄ 3 (pH 7.4, adjusted with KOH) for 5 min to replace the Ca²⁺-free Tyrode’s solution. All solutions were bubbled with 95% O₂ and 5% CO₂ and maintained at 37°C. The left ventricle and left atrium were then cut into small chucks separately and gently agitated in KB solution (BSA, 1 g/L). Cells were filtered using nylon mesh and stored in KB solution (BSA, 1 g/L) until use.

Aconitine-induced arrhythmias in rabbits

Adult New Zealand white rabbits were randomly divided into two groups (n=10, each): i) Normal saline (NS) group; ii) andrographolide (Andro) group. Rabbits were then anesthetized and fixed on their backs to test stands. Polyethylene tubing was inserted into the ear marginal vein to administer aconitine, NS, and andrographolide. Standard limb lead II electrocardiogram (ECG) was measured using the BL-420F data acquisition and analysis system (Chengdu TaiMeng, Sichuan, China) following subcutaneous penetration of electrodes into four limbs. After 10 min of stabilization, NS (10 ml) was injected into NS group rabbits in 5 min through the ear marginal vein. Five min later, aconitine (2 μg/kg/min, flow rate: 80 μl/min) was injected using an infusion pump to induce arrhythmias. Andro group rabbits were treated with 10 ml andrographolide (10 mg/kg) in 5 min prior to aconitine (2 μg/kg/min, flow rate: 80 μl/min). ECG recordings were conducted in both groups for 120 min following aconitine administration. The cumulative dosage of aconitine required to induce VPC, VT and VF was calculated. VPC, VT, and VF time thresholds were recorded.

Drugs and solutions

Collagenase type I and CsCl were purchased from Gibco (Invitrogen Co., Paisley, UK). Bovine serum albumin (BSA) and HEPES were obtained from Roche (Basel, Switzerland). L-glutamic acid and taurine were purchased from Wuhan Zhongnan Chemical Reagent Co. (Wuhan, China). E-4031 and chromanol 293B were obtained from Tocris Bioscience (Minneapolis, MN, USA). All other drugs and reagents were purchased from Sigma Aldrich (Saint Louis, MO, USA). Andrographolide was dissolved in methyl alcohol, chromanol 293B in DMSO, and nifedipine in ethyl alcohol. All drugs were dissolved and diluted into the external recording solution immediately before use. Final methyl alcohol, DMSO, and ethyl alcohol concentrations in the test solutions were ≤0.1% to assure no effects on recorded currents. Stock solution of other drugs were prepared in dH₂O. Drug concentrations refer to that in the external recording solution.

The AP recording bath solution contained (mM): NaCl 140, KCl 4.0, CaCl₂ 1.8, MgCl₂ 1.0, HEPES 5.0, Glucose 10.0 (pH 7.4, adjusted with NaOH) and the pipette solution contained (mM): K-aspartate 110, KCl 30, HEPES 10, EGTA 0.1, MgATP 5, Creatine phosphate 5, CAMP 0.05 (pH 7.2, adjusted with KOH).

The I_Na recording bath solution contained (mM): NaCl 30, CaCl₂ 1.0, CsCl 105, MgCl₂ 1.0, HEPES 5.0, Glucose 5 (pH 7.4, adjusted with CsOH) and the intracellular pipette solution contained (mM): CsCl 120, Na₂ATP 5, MgCl₂ 5, CaCl₂ 1.0, TEA-Cl 10, EGTA 10, HEPES 10 (pH 7.3, adjusted with CsOH). Nifedipine (0.01 mM) was added to the bath solution to block L-type Ca²⁺ channels.

The I_CaL recording bath solution contained (mM): NaCl 135, CsCl 5.4, CaCl₂ 1.8, MgCl₂ 1.0, BaCl₂ 0.3, NaH₂PO₄ 0.33, Glucose 10, EGTA 10 (pH 7.4, adjusted with NaOH), and the pipette solution contained (mM): CsCl 120, Na₂ATP 5, MgCl₂ 5, CaCl₂ 1.0, TEA-Cl 10, EGTA 10, HEPES 10 (pH 7.3, adjusted with CsOH).

Extracellular solution for I_K1 recordings contained (mM): NaCl 137, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.0, NaH₂PO₄ 0.33, HEPES 10, Glucose 10, CdCl₂ 0.3 (to block L-type Ca²⁺ channels) (pH 7.4, adjusted with NaOH). Intracellular solution for I_K1 recordings contained (mM): KCl 140, MgCl₂ 1.0, K₂ATP 5, EGTA 10, HEPES 5.0 (pH 7.3, adjusted with KOH).

The I_to and I_Kur recording bath solution contained (mM): NaCl 140, KCl 5.4, MgCl₂ 1.0, CaCl₂ 1.8, NaH₂PO₄ 0.33, HEPES 5, Glucose 10 (pH 7.4, adjusted with NaOH) and the intracellular pipette solution contained (mM): KCl 20, K-aspartate 110, MgCl₂ 1.0, HEPES 10, EGTA 5, GTP 0.1, Na₂-phosphocreatine 5, Mg₂ATP 5 (pH 7.2, adjusted with KOH). For recording I_to or I_Kur, 200 μM BaCl₂ and 200 μM CdCl₂ were added to the bath solution to block I_K1 and I_CaL, respectively. Atropine (1.0 μM) was used to block I_K,Ach. To inhibit sodium current, an inactivating pre-pulse (-40 mV, lasting 100 ms) was used when recording I_to. While recording I_Kur, sodium current was inhibited by a holding potential of 50 mV and equimolar N-methyl-D-glucamine replacement of Na⁺ in the bath solution.

For recording I_Kr, the external solution contained (mM): NaCl 135, KCl 5.4, CaCl₂ 1.0, CdCl₂ 0.2, NaH₂PO₄ 0.33, MgCl₂ 1.0, HEPES 5, Glucose 5 (pH 7.4, adjusted with NaOH). The intracellular pipette solution contained (mM): KCl 140, MgCl₂ 1.0, HEPES 5, EGTA 10, Na₂ATP 2 (pH 7.25, adjusted with KOH).

Current recordings

Transmembrane action potentials and ion currents were recorded from isolated myocytes using a patch clamp amplifier (EPC-10, Heka Electronic, Lambrecht, Pfalz, Germany) with a current or voltage clamp technique, respectively. All experiments were performed at 37±0.5°C and bath solutions were bubbled with 95% O₂ and 5% CO₂. Before patch clamp recording, myocytes were transferred into a recording chamber mounted on an inverted microscope stage (Nikon TE2000-S) and allowed to adhere to the glass bottom of the chamber for 5 min. A motorized micromanipulator (MP285, Sutter, USA) propelled the recording electrode to the chosen cells. Membranes were then sealed and ruptured with negative pressure. Sealing resistance was maintained >1GΩ. A 80% compensation of series resistance was achieved without ringing. Capacitance and series resistances were adjusted to minimize the contribution of the capacitive transients. Patch electrodes were pulled with a two-stage patch pipette puller (PP-830, Narishige Group, Tokyo, Japan) and then fire-polished. Patch electrodes had a resistance of 1.8–2.5 MΩ when filled with pipette solution. Current signals were conducted by the Ag/AgCl electrode and amplified by an EPC-10 amplifier, then filtered at 1.5 kHz, digitized at 10 kHz, and stored on a computer hard disk for analysis.

Data analysis

Applicate FitMaster (v2x32; HEKA) was used for data analysis and SPSS 18.0 for statistical analysis. Figures were plotted using Origin software (V7.0, OriginLab Co., MA, USA). Summary data were presented as means±SD. Current density was obtained by dividing current amplitude by cell capacitance. Data from steady-state I_Na activation and inactivation were fitted to the Boltzmann equation: Y=1/[1+exp(Vm -V_1/2)/k], where Vm is the membrane potential, V_1/2 the half-activation or half-inactivation potential, and k the slope factor. For steady-state activation curves, Y represents relative conductance. Chord conductance was calculated using the ratio of the current to electromotive force for potential in individual current-voltage relationships. Conductances were normalized to their individual maximal conductance. For steady-state inactivation curves, Y represents relative current (I_Na/I_Namax; I_CaL/I_CaLmax). Differences between groups were determined using one-way analysis of variance (ANOVA) followed by the Tukey multiple comparison test or Student’s paired t-test. P<0.05 was considered statistically significant.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

GRANT SUPPORT

This work was supported by the National Natural Science Foundation of China (No 81670302).