Ginsenoside Rb1 exerts antiarrhythmic effects by inhibiting INa and ICaL in rabbit ventricular myocytes

Ginsenoside Rb1 exerts its pharmacological action by regulating sodium, potassium and calcium ion channels in the membranes of nerve cells. These ion channels are also present in cardiomyocytes, but no studies have been reported to date regarding the effects of Rb1 on cardiac sodium currents (INa), L-type calcium currents (ICaL) and action potentials (APs). Additionally, the antiarrhythmic potential of Rb1 has not been assessed. In this study, we used a whole-cell patch clamp technique to assess the effect of Rb1 on these ion channels. The results showed that Rb1 inhibited INa and ICaL, reduced the action potential amplitude (APA) and maximum upstroke velocity (Vmax), and shortened the action potential duration (APD) in a concentration-dependent manner but had no effect on the inward rectifier potassium current (IK1), delayed rectifier potassium current (IK) or resting membrane potential (RMP). We also designed a pathological model at the cellular and organ level to verify the role of Rb1. The results showed that Rb1 abolished high calcium-induced delayed afterdepolarizations (DADs), depressed the increase in intracellular calcium ([Ca2+]i), relieved calcium overload and protected cardiomyocytes. Rb1 can also reduce the occurrence of ventricular premature beats (VPBs) and ventricular tachycardia (VT) in ischemia-reperfusion (I-R) injury.

Panax ginseng Meyer, a traditional herbal medicine, exerts effects of strengthening the body and prolonging life. Modern medical research has shown that it confers protective effects in the nervous system and the cardiovascular system [1][2][3] and also exerts antitumoral effects 4 . The main active ingredient of ginseng is ginsenoside, which is divided into two types: protopanaxadiol and protopanaxatriol 5 . Rb1 contains the highest content of protopanaxadiol 6,7 and has a variety of biological properties, such as antiaging, antiamnestic, anti-inflammatory effects 8,9 . Rb1 has been reported to protect vascular endothelial cells and maintain their normal physiological functions, primarily due to an associated increase in endothelial nitric oxide synthase expression and nitric oxide production [10][11][12] . Rb1 also inhibits cardiomyocyte apoptosis and death, reducing the area of myocardial infarction caused by ischemia-reperfusion (I-R) injury [13][14][15][16] . The above studies indicate that Rb1 has cardiovascular protective function.
Arrhythmia is a severe cardiovascular disease, and ventricular arrhythmias such as ventricular premature beats (VPBs) and ventricular tachycardia (VT) can lead to sudden cardiac death 17,18 . The normal rhythm of the heart is derived from its regular electrical activity, i.e., the transmembrane ion channel currents of cardiomyocytes. The most important currents are I Na , I CaL and potassium current. The occurrence of arrhythmias is related to disturbances in cardiac electrical activity. Therefore, many antiarrhythmic drugs act on the above three ion channel currents. Related studies have shown that Rb1 exerts pharmacological effects by regulating sodium ion channels, potassium channels and calcium channels on nerve cell membranes [19][20][21][22] . However, whether Rb1 affects the sodium channels, L-type calcium channels and action potentials (APs) of cardiomyocytes has not yet been reported. This study investigated the effects of Rb1 on I Na , I CaL , potassium current and APs, and explored its potential pharmacological effects of Rb1 against arrhythmia and cardiac cell calcium overload.  Fig. 2a,b). The IC 50 of I CaL was 42 μmol/L (n = 6; Fig. 2c). Rb1 (80 μmol/L) shifted the steady-state inactivation curve of I CaL to the left (more negative membrane potential) and altered the V 1/2 from −30 ± 0.10 to −35 ± 0.12 mV (n = 10, repeated measures ANOVA, p = 1.4E-6 < 0.01); When the Rb1 was rinsed off, the half-inactivation voltage returns to −31 ± 0.10 mV (n = 10, repeated measures ANOVA, p = 3.4E-6 < 0.01). However, Rb1 did not affect the steady-state activation curve of I CaL (n = 12, paired-samples t-test, p = 0.93 > 0.05; Fig. 2d). Figure 2e shows the inactivation current recordings of I CaL under normal conditions with 80 μmol/L Rb1 added and washed out. The results presented in Fig. 2e-g indicate that Rb1 reversibly inhibited I CaL . After adding 40 μmol/L Rb1, I CaL was reduced to 55 ± 8.4% (n = 8, repeated measures ANOVA, p = 0 < 0.01 vs. Control; Fig. 2g). When Rb1 was washed out, I CaL returned to 85 ± 7.7% of the original current (n = 8, repeated measures ANOVA, p = 5.2E-4 < 0.01 vs. Control; p = 3.0E-7 < 0.01 vs. Rb1; Fig. 2g).

Discussion
In this study, we observed that Rb1 can inhibit I Na and I CaL in a concentration-dependent manner and that this effect is reversible. Rb1 had no effect on the activation of these two currents, but could shift the inactivation curve to the left (negative potential) and accelerate their deactivation. The APA, V max , APD 50 and APD 90 values decreased in the presence of Rb1, but the RMP, I K1 and I K were not affected by Rb1. In addition, Rb1 could alleviate the increase in diastolic [Ca 2+ ] i induced by H-R and inhibit the spontaneous contraction and death of cardiomyocytes due to calcium overload. Rb1 could also reduce the occurrence of VPBs and VT caused by I-R injury. www.nature.com/scientificreports www.nature.com/scientificreports/ Voltage-gated sodium channels play an indispensable role in the excitability and conduction of ventricular myocytes. Their opening is rapid and constitutes the rising phase of the AP of ventricular myocytes. Inhibition of I Na can increase the threshold for triggering an AP and reduce the occurrence of transient depolarization,  www.nature.com/scientificreports www.nature.com/scientificreports/ reducing the risk of arrhythmia 23 . Rb1 was shown to reduce I Na (Fig. 1a) and shift the steady-state inactivation curve to the left (Fig. 1d), which has a similar effect as class I antiarrhythmic drugs. Class I antiarrhythmic drugs can inhibit arrhythmia by increasing the threshold of action potential, reducing abnormal automaticity, slowing down abnormal conductivity and prolonging the effective refractory period. Due to its inhibition of sodium current, the excitatory conduction slows down and leads to arrhythmia. Therefore, Rb1 may also have the same arrhythmogenic effect as a class I antiarrhythmic drug.
The I CaL is an inward current that constitutes the plateau of ventricular myocytes. The maintenance of the plateau period depends on the balance between the outward current and the inward current. When the I CaL increases, the repolarization reserve decreases and the APD extends. The extended plateau provides conditions for the reactivation of the calcium channels, which produces early afterdepolarizations (EADs) that induces arrhythmia 24 . Rb1 was observed to inhibit I CaL (Fig. 2a) and accelerate the inactivation of calcium channels (Fig. 2d), reducing the flow of calcium ions into cells. Therefore, Rb1 reduces [Ca 2+ ] i overload caused by H-R and inhibits arrhythmias, as [Ca 2+ ] i overload is an important factor in inducing arrhythmia 25,26 .
The AP is a comprehensive representation of the ion current of cardiomyocytes, and changes in ion currents affect the shape of APs. Rb1 reduced the APA and V max values, which was the result of I Na inhibition. When V max is reduced, cell excitability and conductivity are reduced, which can decrease the occurrence of ectopic excitability. The plateau of the AP is primary formed by the inward I CaL and the outward I K. Interestingly, Rb1 suppressed I CaL but had no effect on I K . In general, the inward current was reduced, and the APD was shortened, which is beneficial for reducing the occurrence of EADs and inhibiting long QT syndrome-induced arrhythmias 27,28 . I K1 is involved in the formation of the RMP, and because Rb1 had no effect on I K1 (Fig. 3a), it did not alter the RMP (Table 1). Rb1 has been reported to increase the slow component of delayed rectifier potassium current (I Ks ) in guinea pig ventricular myocytes 29 , which contradicts the results of the present study, and this contradiction may be caused by differences in the studied species.
Ca 2+ is an extremely important second messenger in the cell, participating in many physiological activities, such as contraction, secretion, gene expression and so on 30 . Under normal conditions, [Ca 2+ ] i influx and outflow are equal. When the inflow increases or the outflow decreases, [Ca 2+ ] i increases, causing calcium overload. Calcium overload can overactivate a variety of enzymatic reactions, affecting the normal physiological functions of cells 31 . Calcium overload also causes the sarcoplasmic reticulum calcium content to increase. Beyond a specific certain limit, the sarcoplasmic reticulum spontaneously releases Ca 2+ , causing DADs and posterior contraction and eventually leading to arrhythmia 32 . In this study, we showed that Rb1 significantly inhibits DADs induced by high calcium concentrations in ventricular myocytes and exerts anti-arrythmia activity in cardiac cells. The entry and outflow of Ca 2+ is essential for maintaining the normal physiological activities of cells. With respect to hypoxia, ischemia and heart failure, [Ca 2+ ] i increases and calcium overload occurs 33 . After 15 min of hypoxia and 15 min of reoxygenation in the H-R group, diastolic [Ca 2+ ] i increased significantly and a contraction rhythm disorder appeared, which was the result of calcium overload. In the Rb1 group, diastolic [Ca 2+ ] i was also increased after hypoxia and reoxygenation with additional Rb1. However, the percentage of increase was lower than that observed in the H-R group, and a systolic rhythm disorder was not observed. This result indicates that Rb1 can inhibit the increase in diastolic [Ca 2+ ] i caused by H-R, thereby inhibiting calcium overload, maintaining normal contraction rhythm of cardiomyocytes and protecting cardiomyocytes from H-R. This result may be related to the inhibition of I CaL .
All of the above experiments only explored the role of Rb1 at the cellular level and used unilateral indicators as a reference. To assess whether Rb1 has an antiarrhythmic effect, we performed an organ-level experiment. I-R injury can lead to disturbances in various currents in cardiomyocytes, thereby inducing ventricular arrhythmias 18 . In these experiments, we observed that the incidence of VPBs and VT was significantly reduced and that the onset time was delayed when hearts were reperfused with Rb1-containing perfusate (Fig. 7), indicating that Rb1 indeed confers resistance to arrhythmias.
Ginsenoside Rb1 can simultaneously inhibit I CaL and I Na , which acts to lower the APA, reduce V max , and shorten the APD of APs. Due to its effect on I CaL , Rb1 inhibits high calcium-induced DADs, cellular calcium overload induced by H-R. Due to its effect on both I CaL and I Na , Rb1 reduces ventricular arrhythmias induced by I-R injury, which may be the reason for its antiarrhythmic effect.

Materials and Methods
Preparation of ventricular myocytes. The animals used in this experiment are in line with the "Guidelines for the Care and Use of Laboratory Animals" formulated by Hubei Province, China, and approved by the Institutional Animal Care and Use Committee of Wuhan University of Science and Technology.
New Zealand white rabbits weighing 1.5-2 kg were screened as the experimental subjects (male and female had no effect on the experimental results). The rabbits were heparinized (2000 U) and anesthetized with xylazine (7.5 mg/kg i.m.) and ketamine (30 mg/kg, i.v.). After the heart was removed, the aorta was cannulated. Next, we fixed the heart on a Langendoff apparatus and retrogradely perfused it with Ca 2+ -free Tyrode's solution for 5 min to discharge the heart congestion. The solution was then changed to enzyme-containing Ca 2+ -free Tyrode's solution (collagenase 1 g/L, bovine serum albumin, BSA 1 g/L). After 40 min of perfusion, we used Kraft-Brühe (KB) solution to irrigate the heart to discharge residual enzymes. All solutions were preoxygenated (95% O 2 and 5% CO 2 ) and maintained at 37 °C. After removing the heart, the left ventricle was cut and placed in a small beaker containing KB solution. The cells were filtered through nylon mesh and stored in KB solution at 4 °C for later use. www.nature.com/scientificreports www.nature.com/scientificreports/ were purchased from Sigma Aldrich (Saint Louis, MO, USA). Rb1 was dissolved in methanol, and tests were performed that excluded the role of solvent in the observed effects.
The Ca 2+ -free Tyrode's solution contained (in mmol/L) 135 NaCl, 5. Ion current and AP recording. Temperatures were maintained at 22-25 °C in all experiments. The glass electrodes were pulled with a two-stage patch pipette puller (PP-830, Narishige Group, Tokyo, Japan) and thermally polished. The electrode resistance was 1.5-2 MΩ when filled with pipette solution. Currents and APs were recorded by an EPC-10 patch clamp amplifier (HEKA Electronic, Lambrecht, Pfalz, Germany), filtered at 2 kHz and sampled at 10 kHz. I Na was elicited by a 0.2 Hz and 300 ms depolarizing pulse from a holding potential of −90 mV to −20 mV. To record the current-voltage relationship of I Na , the depolarizing pulse was changed from −70 mV to +40 mV in 5-mV increments at a frequency of 0.5 Hz. For the steady-state I Na inactivation curve, currents were evoked by a 100 ms conditioning prepulse from −100 mV to −50 mV in 5 mV increments at a holding potential of −90 mV followed by a 100 ms depolarizing test pulse to −30 mV. The frequency was 0.5 Hz. I CaL was elicited by a 0.2 Hz and 300 ms depolarizing pulse from a holding potential of −40 mV to 0 mV. To record the current-voltage relationship of I CaL , the depolarizing pulse was changed from −40 mV to +50 mV in 5 mV increments at a frequency of 0.5 Hz. For the steady-state I CaL inactivation curve, currents were evoked by a 2000 ms conditioning prepulse from −50 mV to 0 mV in 5-mV increments at a holding potential of −40 mV followed by a 300 ms depolarizing test pulse to 0 mV. The frequency was 0.5 Hz. I K1 was elicited by a 1 Hz and 400 ms depolarizing pulse from −120 mV to +50 mV in 10 mV increments at a holding potential of −40 mV. I K was elicited by a 0.1 Hz and 3000 ms depolarizing pulse from −40 mV to +50 mV in 10 mV increments followed by a 5000 ms repolarization pulse to −40 mV at a holding potential of −40 mV. APs were elicited by 5 ms duration and current pulses of 1.5 times the diastolic threshold at a frequency of 1 Hz using the patch clamp technique in current clamp mode. The RMP, APA, V max , APD 50 and APD 90 parameters were assessed. Electrocardiogram (ECG) recording. Hearts were obtained as described in the preparation of ventricular myocytes. We fixed the heart on a Langendoff apparatus and retrogradely perfused it with Ca 2+ -free Tyrode's solution. Three silver electrodes were placed on the heart to elicit the ECG. The ECG was recorded and measured using a multichannel physiological signal acquisition and processing system (RM6240C, Chengdu Instrument Factory, Sichuan, China). After ECG stabilization, we perfused the heart with normal Tyrode's solution for 10 min and then stopped perfusion. We reperfused the heart 30 min later with fresh Tyrode's solution or Tyrode's solution containing 40 μmol/L Rb1 for 60 min. We counted the number, VPB onset time and occurrence of VT during 60 min of reperfusion. Five consecutive VPBs are considered VT.