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Introduction

Allicin is the active compound in garlic, a well-researched remedy that is widely used as a spice and food (1,2). It has been reported that garlic may reduce cholesterol levels, lower blood pressure, inhibit platelet aggregation, activate fibrinolysis and prevent atherosclerosis, while it also has antioxidant and anticancer effects (3–12). Garlic has also been reported to have an antiarrhythmic effect (13,14), which has been observed in ventricular and supraventricular arrhythmias (13). The incidence of ischemia/reperfusion-induced ventricular fibrillation in isolated perfused rat hearts was found to be reduced by garlic powder (15). Garlic significantly decreases the upper limit of vulnerability of ventricular fibrillation and improves defibrillation efficacy in a dose-dependent pattern (16,17). Martín et al (18) revealed that allicin inhibited the myocardial contraction and slowed the sinus rhythm. In a further study, Martín et al (19) demonstrated that garlic dialysate was able to prolong the effective refractory period and the sinus node recovery time of isolated rat atria, in addition to suppressing premature ventricular contractions and ventricular tachycardia in ouabain-intoxicated canines.

A study by Deng et al (20) revealed that allicin was able to inhibit transient outward potassium currents (Ito) in human atrial myocytes. However, the direct effect of allicin on Ito in ventricular myocytes has yet to be elucidated. Therefore, in the present study, the effects of allicin on Ito in ventricular myocytes isolated from mice were investigated, using the whole-cell patch clamp recording technique to test the effect of allicin on Ito, as detected via Ito amplitude and kinetics, including Ito activation, inactivation and recovery.

Materials and methods

Ethical approval

All animal procedures were approved by the Institutional Animal Care and Use Committee at Renmin Hospital of Wuhan University (Wuhan, China). The animals used in the present study were male C57 mice, aged 8–10 weeks.

Drugs and solution

Tyrode's solution was composed of the following: 130 mmol/l NaCl, 5.4 mmol/l KCl, 1.8 mmol/l CaCl2, 1 mmol/l MgCl2, 0.3 mmol/l Na2HPO4, 10 mmol/l HEPES and 10 mmol/l glucose. The pH of the solution was adjusted to pH 7.4 using NaOH. In addition, Ca2+-free Tyrode's solution was used, without CaCl2. The collagenase solution was composed of Ca2+-free Tyrode's solution containing 0.6 mg/ml collagenase type II (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 0.1% bovine serum albumin, 20 mM taurine and 30 µM CaCl2. Kraft-Brühe (KB) solution included 10 mmol/l taurine, 70 mmol/l glutamic acid, 25 mmol/l KCl, 10 mmol/l KH2PO4, 22 mmol/l glucose and 0.5 mmol/l ethylene glycol tetraacetic acid (EGTA). The pH of the KB solution was adjusted to pH 7.2 using KOH. Tyrode's solution was supplemented with 10, 30, 100 and 300 µmol/l allicin during allicin treatment. Furthermore, the pipette solution used in the study consisted of 110 mmol/l K-aspartate, 20 mmol/l KCl, 8 mmol/l NaCl, 1 mmol/l MgC12, 1 mmol/l CaC12, 4 mmol/l MgATP, 0.1 mmol/l EGTA and 10 mmol/l HEPES, and was adjusted to pH 7.2 using KOH. Allicin was purchased from Xuzhou Ryen Pharm Co., Ltd (Xuzhou, China).

Isolation of ventricular cardiac myocytes

A total of 36 C57BL/6 mice, weighing 25.1±3.4 g, were heparinized [100 U; intraperitoneal injection (i.p.); Wangbang Co., Xuzhou, China] 15 min prior to sacrifice, anaesthetized by pentobarbital sodium (60 mg/kg; i.p.; Sigma-Aldrich, St. Louis, MO, USA) and sacrificed by cervical dislocation. Hearts were rapidly removed and retrogradely perfused at a temperature of 37°C with the following solutions, according to Langendorff technique (21): i) Tyrode's solution (5 min); ii) Ca2+-free Tyrode's solution (5 min); iii) collagenase solution (15 min); and iv) KB solution (5 min). Subsequent to the perfusion, the left ventricular free wall was dissected from the heart and placed in ice-cold KB solution. The tissue was then minced and titrated to free individual myocytes. Isolated cardiac myocytes were stored in KB solution at 4°C until required.

Electrophysiology recording

Whole-cell patch clamp was performed on the myocytes using an EPC-9 amplifier (Heka Elektronik, Lambrecht, Germany), as previously described (21), and data was recorded and analyzed with a Pulse/Pulsefit software interface (version 8.31; Heka Elektronik). During the experiments, 1.5 ml myocytes were placed in the experimental chamber and mounted on the stage of an inverted microscope (IX70; Olympus Corporation, Tokyo, Japan) and perfused with Tyrode solution supplemented with 10, 30, 100 and 300 µmol/l allicin for 5 min at a rate of 2–3 ml/min at room temperature. In order to elucidate the effect of allicin on Ito in mouse ventricular myocytes, 6 cells were observed per solution influx, in triplicate. Pipettes had resistances of 2.5–3.5 MΩ when filled with pipette solution. Series resistance (Rs) was between 4–8 MΩ and was compensated by 80–90% to reduce the Rs. Current signals were filtered at 3 kHz by an 8-pole Bessel filter, digitized at a sampling rate of 1 kHz and recorded on a computer running Pulse/Pulsefit software, which was additionally used for the generation of voltage pulses and data analysis.

Ito recording

The total Ito was determined by 500 msec depolarizing pulses varying from −50 to +60 mV in 10 mV increments from a holding potential of −80 mV. In order to examine Ito, pre-pulse (100 msec, −40 mV) was used to inactivate Ito prior to activation steps with allicin, and Ito was measured by subtracting the currents before and after that pre-pulse. By dividing the measured current amplitude by the membrane capacitance (pA/pF), Ito values were reported as current densities.

The IC50 of allicin on Ito was fitted with Hill function using OriginPro version 8.0 software as follows: E = Emax[1+(D⁄C)b], where E is the effect at concentration C, Emax is the maximum effect, D is the concentration for half-maximum action (IC50) and b is the Hill coefficient.

Steady-state activation curve of Ito

Using the current-voltage (I–V) association for Ito, the voltage-dependent of steady-state activation curve for Ito was fitted to the Boltzmann equation as follows: I/Imax = 1/[1+exp((VT - V1/2)/k)], where Imax is maximum current, VT is the membrane potential, V1/2 is the midpoint potential for activation and K is a slope factor (22).

Steady-state inactivation of Ito

The two-step voltage-clamp protocol was applied for steady-state inactivation of Ito, as previously described (21). The process involved an inactivating pre-pulse period that varied from −110 mV to +10 mV with a 1 sec pre-pulse, followed by a fixed 400 ms test pulse to +40 mV. The test current amplitude of Ito at each pulse potential was normalized to the maximal amplitude of this current (I/Imax). Data were fitted to the Boltzmann equation.

Recovery from inactivation of Ito

The time-dependence of reactivation was measured using an inactivating pulse (−40 mV, maintained for 500 msec). Following this, at variable time intervals (10–200 msec), a 500 msec test pulse at +40 mV was performed. The ratio of the current amplitude produced by the test pulse to the inactivating pulse (P2/P1) was plotted as a function of the time intervals. The time constant was calculated by data fitted to exponential functions.

Statistical analysis

All data are expressed as the mean ± standard deviation. Statistical analysis was performed using a Student's t test and analysis of variance, performed on SPSS version 17.0 software (SPSS, Inc. Chicago, IL, USA). Patch-clamp data were analyzed using Origin version 8.0 (OriginLab Corporation, Northampton, MA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of allicin on voltage-dependent Ito

Allicin at 10, 30, 100 and 300 µmol/l was applied, respectively. Ito was blocked by allicin in a concentration-dependent manner. Currents were gradually decreased with the increase of allicin concentration. The representative current blocked by allicin at 10, 30, 100 and 300 µmol/l is shown in Fig. 1.

Figure 1. Effects of allicin on transient outward potassium currents (Ito) in mouse ventricular myocytes. The data reveal the representative voltage-dependent Ito (A) under control conditions, and (B) in the presence of 10 µmol/l allicin, (C) 30 µmol/l allicin, (D) 100 µmol/l allicin and (E) 300 µmol/l allicin. Data are presented as mean ± standard deviation.

Fig. 2 displays the I–V association for Ito density prior to and following the application of 10, 30, 100 and 300 µmol/l allicin. The Ito was not significantly suppressed by allicin in the low dose (10 µmol/l; P>0.05); however, it was significantly suppressed by higher doses (30, 100 and 300 µmol/l; P<0.05; n=6) compared with the control.

Figure 2. Current-voltage association for Ito in mouse ventricular myocytes following treatment with various allicin concentrations. Allicin inhibited Ito in a concentration-dependent manner. Data are presented as the mean ± standard deviation. *P<0.05 vs. control, n=6. Ito, transient outward potassium current.

In addition, Fig. 3 shows the dose-response association for the inhibition of Ito by allicin. At a potential of +60 mv, treatment with 1, 10, 30, 100 and 300 µmol/l allicin decreased the peak Ito by 1.5, 17.8, 35.3, 63.2 and 76.9%, respectively. The IC50 of allicin on Ito was fitted with Hill function and calculated as 41.6 µmol/l (n=6 cells in each group) using OriginPro 8.0 software.

Figure 3. Dose-response association for inhibition of Ito by allicin. At a potential of +60 mV, treatment with 1, 10, 30, 100 and 300 µmol/l allicin decreased the peak Ito current by 1.5, 17.8, 35.3, 63.2 and 76.9%, respectively. The IC50 of allicin on Ito was fitted with Hill function and calculated to be 41.6 µmol/l, using OriginPro version 8.0 software (n=6). Ito, transient outward potassium current.

Effects of allicin on the steady-state activation curve of Ito

Allicin was not found to have a significant effect on the voltage-dependence of the steady-state activation curve of Ito (P>0.05; Fig. 4).

Figure 4. Voltage-dependent activation curve of Ito using the current-voltage association for Ito. Allicin had no significant effect on the voltage-dependence of the steady-state activation curve of Ito (P>0.05). Data are presented as the mean ± standard deviation. Ito, transient outward potassium current; Imax, maximum current.

Effects of allicin on the steady-state inactivation of Ito

The results revealed that a low dose of allicin had no significant effect on the voltage-dependence of the inactivation (I/Imax) of Ito (control, V1/2 = −28.2±4.7 mV; 10 µmol/l allicin, V1/2 = −32.2±3.8 mV; 30 µmol/l allicin, V1/2 = −30.1±3.6 mV; n=6; P>0.05, compared with the control). However, as shown in Fig. 5, high doses of allicin significantly shifted the voltage-dependence of the inactivation of Ito toward the negative potential (100 µmol/l allicin, V1/2 = −36.9±4.1 mV; 300 µmol/l allicin, V1/2 = −55.3±5.0 mV; n=6; P<0.05 compared with the control).

Figure 5. Voltage dependence of the inactivation (I/Imax) of Ito following treatment with various allicin concentrations. The voltage dependence of the inactivation of Ito was negatively shifted after treatment with high concentrations of allicin (100 and 300 µmol/l; *P<0.05 vs. control; n=6). Data are presented as the mean ± standard deviation. I/Imax, current/maximum current; Ito, transient outward potassium current.

Effects of allicin on the recovery from inactivation of Ito

Allicin was not found to have a significant effect on the recovery from the inactivation of Ito following allicin treatment (P>0.05; Fig. 6).

Figure 6. Effects of allicin on recovery from the inactivation of Ito. Allicin was not found to have a significant effect on the recovery from the inactivation of Ito (P>0.05 vs. control). Ito, transient outward potassium current; I/Imax, current/maximum current.

Discussion

In the present study, allicin significantly inhibited Ito in mouse ventricular myocytes in a concentration-dependent manner. High-dose allicin (≥100 µmol/l) was able to significantly shift the voltage-dependence of the steady-state inactivation curve of Ito towards an increasingly negative potential. However, allicin did not have a significant effect on steady-state activation, or recovery from the inactivation of Ito.

Traditional Chinese medicine has been used for thousands of years for the treatment of cardiovascular diseases (23,24). In recent decades, garlic has been found to possess antiarrhythmic effects (13,14). Several reports (25–27) have indicated that allicin is the predominant active component that is responsible for the majority of the biological activities of garlic, including attenuating ischemic injury, lowering blood pressure and antiarrhythmic effects (6,28,29). The chemical structure of allicin is CH2=CH-CH2-S(O)-S-CH2-CH=CH2, and it has been has been manufactured synthetically and produced worldwide (18). Although garlic has been discovered to be a significant antiarrhythmic agent, the exact mechanism has yet to be elucidated.

In the present study, allicin significantly inhibited Ito in mouse ventricular myocytes; however, it had no significant effect on steady-state activation, or recovery from inactivation of Ito, which is in agreement with previous findings (20). In the study by Deng et al (20), the research target was human atrial myocytes, and it was demonstrated that 30 µmol/l allicin was able to negatively shift the voltage-dependence of the steady-state inactivation curve of Ito. By contrast, in the present study, only high-dose allicin (≥100 µmol/l) was able to significantly shift the steady-state inactivation curve of Ito towards an increasingly negative potential. This may be due to allicin having different effects in different tissues and species. Allicin exerts its suppressive effect on Ito by changing the quantity and kinetic properties of Ito. In human atrial monocytes, Ito contributes to cardiac repolarization, whilst in the hearts of mice, Ito has a role in action potential repolarization (30,31). Notably, Ito is not uniformly distributed within the left ventricle in humans, mice and certain other mammals (32–35). In the left ventricular free wall, Ito is larger in epicardial compared with endocardial regions, which contributes to the regional variations of action potential (AP) profiles and results in a prominent AP notch in the epicardium, but not in the endocardium (36). It has been confirmed that a prominent Ito is important in physiological and pathophysiological process (37–41). The high incidence of phase 2 reentry and ventricular fibrillation during myocardial ischemia was partly due to the prominent Ito-mediated epicardial AP dome (42). In patients with coronary heart disease, the incidence of sudden mortality in men was significantly higher compared with that in women (43,44). This may be a result of a more prominent Ito in men compared with women (39). Thus, Ito block may be an effective therapy for arrhythmia (37).

In the present study, it was revealed that allicin was able to inhibit Ito, and may be the mechanism through which allicin exerts its antiarrhythmic effect. Antiarrhythmic therapeutics with low toxicity and low reverse use-dependence (RUD) effects are a focal point in antiarrhythmic drug research. Xing et al (23) confirmed that allicin has similar effects to amiodarone on the conduction system and cardiac electrophysiology. However, allicin possesses no RUD and this may contribute to multi-channel blockers. Furthermore, allicin appears to be safe for use in the majority of conditions (2) and is therefore likely to be a promising antiarrhythmic therapy.

In conclusion, the present study revealed that allicin inhibits Ito in mouse ventricular myocytes, which may be the mechanism through which allicin exerts its antiarrhythmic effect. Thus, allicin has demonstrated potential to be a promising antiarrhythmic therapy in the future; however, whether allicin exerts the same effect in other tissues or species requires further investigation.

Acknowledgements

The present study was financially supported by the Fundamental Research Funds for the Central Universities (grant no. 2012302020205).

References