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.

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).

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.

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 I_to 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.

The total I_to 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 I_to, pre-pulse (100 msec, −40 mV) was used to inactivate I_to prior to activation steps with allicin, and I_to was measured by subtracting the currents before and after that pre-pulse. By dividing the measured current amplitude by the membrane capacitance (pA/pF), I_to values were reported as current densities.

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

Using the current-voltage (I–V) association for I_to, the voltage-dependent of steady-state activation curve for I_to was fitted to the Boltzmann equation as follows: I/I_max = 1/[1+exp((V_T - V_1/2)/k)], where I_max is maximum current, V_T is the membrane potential, V_1/2 is the midpoint potential for activation and K is a slope factor (22).

The two-step voltage-clamp protocol was applied for steady-state inactivation of I_to, 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 I_to at each pulse potential was normalized to the maximal amplitude of this current (I/I_max). Data were fitted to the Boltzmann equation.

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.

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.

Allicin at 10, 30, 100 and 300 µmol/l was applied, respectively. I_to 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.

Fig. 2 displays the I–V association for I_to density prior to and following the application of 10, 30, 100 and 300 µmol/l allicin. The I_to 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.

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

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

The results revealed that a low dose of allicin had no significant effect on the voltage-dependence of the inactivation (I/I_max) of I_to (control, V_1/2 = −28.2±4.7 mV; 10 µmol/l allicin, V_1/2 = −32.2±3.8 mV; 30 µmol/l allicin, V_1/2 = −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 I_to toward the negative potential (100 µmol/l allicin, V_1/2 = −36.9±4.1 mV; 300 µmol/l allicin, V_1/2 = −55.3±5.0 mV; n=6; P<0.05 compared with the control).

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