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A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents
Journal of Pharmacological Sciences xxx (xxxx) xxx
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A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents Huiyuan Hu a, 1, Shi Zhou a, 1, Xiaodong Sun a, Yingchun Xue a, Ling Yan a, Xuanxuan Sun a, Ming Lei b, Jinming Li a, Xiaorong Zeng b, **, Liying Hao a, * a
Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang, 110122, PR China Key Laboratory of Medical Electrophysiology of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, Luzhou, Sichuan, 646000, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 August 2019 Received in revised form 14 December 2019 Accepted 20 December 2019 Available online xxx
N-methyl berbamine (N-MB) is a berberine derivative. Its analogue berbamine has been reported to have remarkable antiarrhythmic and ischemic protective effects. However, the pharmacological effects of NMB are ill-defined. In this study, molecular docking was used to evaluate the binding of N-MB to CaV1.2 Ca2þ and KV11.1 Kþ channels, and the effects of N-MB on action potential and ionic currents were observed in the ventricular myocytes of rabbits, HEK293 cells stably transfected with the hCaV1.2 gene and CHO cells stably transfected with hERG (human ether-a-go-go related gene). The results showed that N-MB was able to bind to both CaV1.2 and KV11.1 channels. Following a perfusion with N-MB, the durations of action potentials (APD20, APD50 and APD90) were extended, and the outward tail current, Itail, as well as the hERG current, IhERG, were inhibited, while the amplitude of action potential (APA) was only slightly reduced. N-MB also decreased the peak amplitude of the L-type Ca2þ channel current, ICaL, as well as the CaV1.2 current, ICaV1.2; this may limit the prolongation of APD. In conclusion, N-MB is a potent and natural antiarrhythmic multitarget drug that may elicit its antiarrhythmic effect through blocking both Ca2þ and Kþ channel currents. © 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: N-methyl berbamine Action potential Ca2þ current Kþ current Multitarget
Introduction N-methyl berbamine (N-MB) is a kind of bisbenzylisoquinoline alkaloid extracted from the roots of plants in the Berberis genus.1 Berbamine is a well-known bisbenzylisoquinoline alkaloid isolated from the roots, bark and stems of plants from Berberis L., such as Berberis poiretii Schneid, a traditional Chinese herbal medicine.2,3 It has been reported that berbamine hydrochloride has remarkable
* Corresponding author. Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, No. 77, Puhe Road, Shenyang North New Aera, Shenyang, 110122, PR China. Fax: þ86 24 3193 9448. ** Corresponding author. Key Laboratory of Medical Electrophysiology of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, 319 Zhongshan Road, Jiangyang District Luzhou, Sichuan, 646000, PR China. Fax: þ86 830 3160619. E-mail addresses: [email protected] (X. Zeng), [email protected] (L. Hao). Peer review under responsibility of Japanese Pharmacological Society. 1 Co-first author.
antiarrhythmic4 and ischemic protective effects.3,5 As an N-methyl analogue of berbamine, N-MB should have similar pharmacological effects. However, the cardiovascular effects of N-MB are ill-defined. The occurrence of arrhythmia is closely related to the abnormality of myocardial electrical activity. The main mechanisms are autonomic abnormalities of cardiomyocytes, reentry caused by impulsive conduction disorder, and triggering activities caused by afterdepolarization.6 The existing antiarrhythmic drugs achieve their effects through decreasing autonomy, reducing afterdepolarization, and inhibiting conduction or prolonging the effective refractory period (ERP) to eliminate reentry. Antiarrhythmic Kþ channel blockers can reduce abnormal autonomy by blocking Kþ efflux and prolonging the action potential duration (APD). Ca2þ channel blockers can inhibit intracellular Ca2þ overload and reduce delayed afterdepolarization (DAD). Naþ channel blockers and Kþ channel blockers can prolong the ERP of fast response cells, while Ca2þ channel blockers and Kþ channel blockers can prolong the ERP of slow response cells and eliminate reentry activation.7 In our recent study, a potent antiarrhythmic effect of N-MB was achieved
https://doi.org/10.1016/j.jphs.2019.12.008 1347-8613/© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Fig. 1. Binding of N-MB to 3D structure models of CaV1.2 and KV11.1 channels. A: Chemical structure of N-MB; BeD: Schematic diagrams of N-MB (B), verapami (C) and diltiazem (D) interacting with specific amino acids of CaV1.2 channel. EeG: Schematic diagrams of N-MB (E), E-4031 (F) and dofetilide (G) interacting with specific amino acids of KV11.1 channel.
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Table 1 Compositions of the solutions used in the electrophysiological analysis (mM). APD and ICaL recording
Itail recording
IhERG recording on CHO
ICaV1.2 recording on HEK293
External (pH 7.40 by NaOH)
Pipette (pH 7.40 by KOH)
External (pH 7.40 by NaOH)
Pipette (pH 7.40 by KOH)
External (pH 7.40 by NaOH)
Pipette (pH 7.20 by KOH)
External (pH 7.40 by NaOH)
Pipette (pH 7.20 by CsOH)
NaCl KCl MgCl2 CaCl2 NaH2PO4 Hepes Glucose
K-Aspartate KCl MgCl2 K2ATP Na2CrP Hepes EGTA CaCl2
NaCl KCl CaCl2 MgCl2 NaH2PO4 Hepes NaOH Glucose
KCl K2ATP Creatine phosphate Hepes
NaCl KCl CaCl2 MgCl2 Hepes Glucose
KCl MgCl2 CaCl2 EGTA Hepes
NaCl BaCl2 Hepes TEA-Cl Glucose
CsCl MgATP EGTA Hepes TEA-Cl
135 5.4 1 1.8 0.33 10 5.5
110 30 1 5 5 5 10 1.43
143 5.40 1.8 0.5 0.3 5 2.3 5
130 5 5 5
140 5 1 1.25 10 10
140 1 1 10 10
120 10 10 20 10
130 5 10 10 10
Note: EGTA, ethylene glycol tetraacetic acid.
in dog Purkinje fibers through extending the APD and ERP. However, the underlying mechanisms and their effects on membrane ionic currents have not been clarified. Although medicinal plants have been known since ancient times to have therapeutic effects on human cardiovascular diseases, their mechanisms of action have not been completely understood until now.8 It is widely known that the process for drug discovery and development is time consuming, expensive and challenging. Acting as a virtual shortcut, computer-aided drug discovery (CADD) tools can assist in shortening this time, as well as potentially reducing the cost of research and development of drugs.9 CADD methods have played a major role in the development of therapeutically important small molecules for over three decades.10 Among these, the popular structure-based approach, molecular docking, has been widely used to model the interaction between a small molecule and a protein, which allows researchers to characterize the behavior of small molecules in the binding site of target proteins, in addition to elucidating fundamental biochemical processes.11
Table 2 Predicted binding sites of the chemicals and ion channels. Ion channels
Chemicals
London G scores
Combination sites (A/B chain)
Combination types
CaV1.2
N-MB
14.88 14.88 14.71 14.71 14.71 14.01 13.74 13.74 13.58 11.70 11.42 11.42 11.28 11.28 11.25 11.50 11.07 10.97 10.97 9.02 10.79 10.79 10.79 10.79 10.62 10.76 10.51 10.51 10.51 10.51
ASN 1040 (A) LYS 1046 (A) ASP 737 (A) SER 740 (A) GLU 340 (A) ARG 1372 (A) ASN 1314 (A) LYS 1299 (A) ARG 1372 (A) ASN 1282 (A) GLU 1252 (A) LYS 1299 (A) GLU 1252 (A) LYS 1299 (A) GLU 1252 (A) VAL 476 (A) ASN 470 (A) ASP 540 (A) TYR 475 (A) CYS 729 (A) HIS 402 (A) ARG 541 (A) LYS 407 (A) HIS 492 (A) TYR 403 (A) TYR 403 (A) ARG 541(A) ARG 537(A) HIS 492(A) TYR 403 (A)
H-donor H-acceptor H-donor pi-H pi-H H-acceptor H-acceptor H-acceptor H-acceptor H-acceptor H-donor H-acceptor H-donor H-acceptor H-donor pi-H H-donor H-donor pi-H H-donor H-donor H-acceptor H-acceptor H-acceptor pi-H pi-H H-acceptor H-acceptor H-acceptor pi-H
Verapamil
Diltiazem
KV11.1
N-MB
E-4031
Dofetilide
In this study, the effects of N-MB on the action potential, L-type Ca2þ current and outward rectifying Kþ current in the cardiac ventricular myocytes of rabbits, CHO cells transfected with hERG (human ether-a-go-go related gene), and HEK293 cells transfected with the hCaV1.2 gene were investigated by using the patch clamp technique; the molecular binding site of N-MB to these ion channels was predicted with Molecular Operating Environment (MOE) software. Our results showed that N-MB could extend the action potential of the cardiomyocytes through inhibiting both the L-type Ca2þ current and the outward rectifying Kþ current, which reveals the possible anti-arrhythmic mechanisms of N-MB and could promote the clinical application of this chemical to traditional Chinese medicine.
Materials and methods Drugs and solutions N-MB (purity > 98.2%) was purchased from the Institute of Applied Ecology, Chinese Academy of Sciences (Shenyang, China) and its structure is shown in Fig. 1A. The Tyrode solution contained (mM): NaCl 135, KCl 5.4, MgCl2 1.0, CaCl2 1.8, NaH2PO4 0.33, Hepes 10, glucose 5.5 (pH 7.4). Kraftbrühe-modified (KB) solution consisted of (mM): L-glutamic acid 50, KCl 40, taurine 20, KH2PO4 20, Hepes 10, glucose 10, MgCl2 3, EGTA 0.5 (pH 7.4). The compositions of external and pipette solutions used for APD, ICaL, Itail, IhERG, and ICaV1.2 recording are shown in Table 1.
Molecular docking The SWISS-MODEL homology modeling server (http:// swissmodel.expasy.org) was used to model the rabbit cardiomyocyte CaV1.2 channel a1 subunit (NP_00112994.1) and KV11.1 channel a subunit HERG (NP_001075853.1).12 The target sequences of CaV1.2 channel or KV11.1 channel were input into the website server, and the appropriate templates (PDB: 5gjv.1.A for CaV1.2 and 5va1.1.A for KV11.1) were searched and selected, which has the highest homology with the target sequences.13 Automated mode was used during modeling.14,15 The binding of N-MB or Ca2þ channel blockers, i.e. verapamil and diltiazem, to the CaV1.2 channel and the binding of N-MB or specific KV11.1 channel blockers, i.e. E-4031 and dofetilide, to the KV11.1 channel were simulated by using MOE molecular docking software, respectively. The binding sites with the highest binding affinity were chosen based on the lowest binding free energy of the protein-molecule complex calculated with London dG method. The specific Naþ channel blocker tetrodotoxin (TTX) was used as the negative control drug.
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Fig. 2. N-MB extended action potentials in the ventricular myocytes of rabbits. Representative traces of action potential in ventricular myocytes of control (A), 1 mM N-MB (B) and washout (C) groups; D: Measurements of APD20, APD50 and APD90 in ventricular myocytes of control, l mM N-MB and washout groups. ***P < 0.001, ****P < 0.0001, vs. Control; ns, not significant. n ¼ 5 in each group.
Preparation of single ventricular myocytes of rabbits The rabbits of either sex, weighing 1.0e1.5 kg, were provided by the Experimental Animal Center of the China Medical University (SCXK Liao, No. 2008e0005). All animals received humane care in compliance with the guidelines of National Institutes of Health. The rabbits were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and the hearts were excised and mounted in a Langendorff perfusion system quickly. The hearts were first perfused with normal Tyrode solution for 10e20 min at 37 C, which was followed by perfusion with Ca2þ-free Tyrode solution for 10 min and finally perfusion with a collagenase-containing solution (collagenase type I 0.6 g/L and bovine serum albumin 1 g/L) for about 20 min. After digestion, the enzyme was washed out with KB solution. The hearts were removed and left ventricles were minced, and the ventricular myocytes were isolated followed by filtration with a strainer.16 The myocytes were washed twice by centrifugation (1000 rpm for 3 min) and stored in KB solution at 4 C till use. Cell culture CHO cells stably transfected with hERG gene were cultured in a 37 C, 5% CO2 incubator, and passaged at a ratio of 1:5 every 48 h. Culture medium formula included 90% F12 (Invitrogen, CA, USA), 10% fetal bovine serum (Gibco, NY, USA), 100 mg/mL G418 (Invitrogen, CA, USA) and 100 mg/mL hygromycin B (Invitrogen, CA, USA). HEK293 cells stably transfected with hCaV1.2 gene were cultured in a 37 C, 5% CO2 incubator, and passaged at a ratio of 1:4 every 48 h. Culture medium formula included 90% DMEM (Invitrogen, CA, USA), 10% fetal bovine serum (Gibco, NY, USA), 2 mM Lglutamine, 100 mg/mL G418 (Invitrogen, CA, USA), 100 mg/mL hygromycin B (Invitrogen, CA, USA) and 40 mg/mL Zeocin
(Invitrogen, CA, USA). On the day of the experiment, the cells were digested with 0.25% Trypsin-EDTA (Gibco, NY, USA) solution and then transferred to a test dish for electrophysiological recording. Electrophysiology In current clamping mode, a current pulse from Axopatch-1D amplifier was applied through the probe to the single cell and the action potential was elicited from the resting membrane potential level. Then, it was rapidly changed to voltage clamping mode in which the membrane potential was depolarized to þ50 mV from a holding potentia1 of 40 mV in 10 mV steps. Under this condition, a slow inward currents and an outward tail current were elicited at the beginning and the end of a 300 ms clamping pulse. The currents and action potential tracings were recorded before and after the NMB treatment. The whole cell patch-clamp technique was used to record IhERG current in CHO cells stably transfected with hERG gene. The resistance of the pipette filled with solution was 2e5 MU. The clamping voltage was controlled by using the pClamp 10.0 software with a sampling frequency of 10 kHz and a filter frequency of 2 kHz. When obtaining the whole cell recording, the CHO cells were clamped at 80 mV and IhERG current was induced by a depolarization of 2 s with the step voltage from 80 mV to þ20 mV, followed by a repolarization to 50 mV for 1 s before returning to 80 mV. The CaV1.2 current in HEK293 cells stably transfected with the hCaV1.2 gene was also recorded by using the whole cell patch-clamp technique. After obtaining the whole cell recording, the CaV1.2 current was elicited using a series of depolarization pulses ranging from 80 to 0 mV for 100 ms, from a holding potential of 80 mV. The inhibition rate of IhERG (peak IhERG was induced at 50 mV) and CaV1.2 current (peak CaV1.2 current was induced at 0 mV) was
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Fig. 3. Effects of N-MB on ionic currents in the ventricular myocytes of rabbits. A: Representative traces of membrane currents (top) and voltage protocol (bottom) in ventricular myocytes of control (left), 1 mM N-MB (middle) and washout (right) groups; B: Currentevoltage relationship curves of ICaL under control conditions (open circles) and after the application of l mM N-MB (filled circles). Cells were held at 40 mV and depolarized from 40 to þ50 mV in l0 mV steps; C: The voltage dependence of steady-state activation of ICaL. Data were represented as normalized conductance and fitted to a curve based on a Boltzmann equation; DeF: Quantitative comparison of the peak amplitudes of ICaL (D), halfmaximal activation potential (V1/2, E) and slope factor (K, F) under control conditions and after the application of l mM N-MB; G: Representative current traces of Itail in ventricular myocytes of control (top), 1 mM N-MB (middle) and washout (bottom) groups; H: Currentevoltage relationship curve for Itail recorded in ventricular myocytes of control (open circles), 1 mM N-MB (filled circles) and washout (filled triangles) groups; I: Quantitative comparison of Itail current in ventricular myocytes of control, 1 mM N-MB and washout groups. **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. Control; ns, not significant. n ¼ 3e6 in each group.
calculated by the following formula: Inhibition % ¼ [1 e (I/ Io)] 100%, where I and Io represent the magnitude of IhERG or CaV1.2 current before and after N-MB treatment, respectively. All the experiments were performed at room temperature (20e22 C). The steady-state activation kinetics were evaluated by fitting the conductance (G)/peak conductance (Gmax)-voltage relationship to a Boltzmann equation as follows: G/Gmax ¼ 1/{1 þ exp [(V1/2-Vm)/K]}, where Vm is the test potential, V1/2 is the membrane potential at which half of the channels are activated, and K is the slope factor.
Table 3 Inhibition of N-MB on IhERG and ICaV1.2. Inhibition of N-MB on IhERG
Inhibition of N-MB on ICaV1.2
Drugs
Inhibition rate (%)
Drugs
Inhibition rate (%)
0.1 mM N-MB 1 mM N-MB 10 mM N-MB 300 nM cisapride
4.43 34.5 96.0 98.3
1.23 mM N-MB 3.70 mM N-MB 11.1 mM N-MB 33.3 mM N-MB 100 mM N-MB
7.72 ± 3.21 (n ¼ 4) 3.65 ± 9.51 (n ¼ 4) 22.0 ± 11.7 (n ¼ 4) 60.6 ± 7.55 (n ¼ 4) 88.1 ± 3.12 (n ¼ 5)
± ± ± ±
0.95 3.37 0.58 0.33
(n (n (n (n
¼ ¼ ¼ ¼
5) 7) 6) 4)
Statistical analysis All data were expressed as mean ± standard error (S.E.) and analyzed with the pClamp10.0 and Origin 8.0 software. The Student's t-test was used to compare the mean values between the two groups and P < 0.05 was considered statistically significant. Results N-MB was predicted to bind to CaV1.2 and KV11.1 channels The binding of N-MB to the ion channels was analyzed with MOE through the London dG scoring function. According to the optimized molecular placement method, the top five constellation results were displayed. As a result, N-MB was shown to bind to CaV1.2 channels (Table 2 and Fig. 1B). As positive controls, the binding of Ca2þ channel blockers verapamil and diltiazem was also simulated; this showed that the binding sites of verapamil and diltiazem to the CaV1.2 channel are mostly located in the fourth transmembrane structure domain (Fig. 1C and D), as previously reported.17 The specific Naþ channel blocker TTX was used as a
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Fig. 4. N-MB dose-dependently inhibited CaV1.2 current in HEK293 cells. A: Representative traces of CaV1.2 currents recorded in HEK293 cells stably transfected with hCaV1.2 gene in the absence (control) or presence of 11.1 mM, 33.3 mM and 100 mM N-MB, respectively; B: Doseeresponse curve of N-MB on CaV1.2 current; C: Currentevoltage relationship curves of CaV1.2 current recorded in the absence (control) or presence of 30 mM N-MB; D: The voltage dependence of steady-state activation of CaV1.2 current recorded in the absence (control) or presence of 30 mM N-MB. Data were represented as normalized conductance and fitted to a curve based on a Boltzmann equation. n ¼ 4e5 in each group.
negative control, which did not bind to the CaV1.2 channel model. The results show that N-MB could bind to the CaV1.2 channel with a similar binding score as those for verapamil and diltiazem (N-MB, ASN 1040, -14.88; verapamil, ARG 1372, -14.01; diltiazem, GLU 1252, -11.42), indicating that N-MB might form a stable conformation with Ca2þ channels. N-MB could also bind to the KV11.1 channel. Both the London dG score and the binding site of N-MB to KV11.1 were similar to those of the positive control drugs (N-MB, VAL 476, -11.50; E-4031, HIS 402, -10.79; Dofetilide, TYR 403, -10.76), indicating that N-MB might bind to Kþ channels with strong binding affinity (Table 2; Fig. 1EeG). Therefore, compared with the corresponding specific blockers, N-MB showed similar binding properties to CaV1.2 and KV11.1 channels.
N-MB prolonged the APD in rabbit cardiac ventricular myocytes Based on the molecular docking findings that N-MB could bind to both Ca2þ and Kþ channels, the electrophysiological effects of NMB on the APD and the ion channels were then investigated. A set of typical waveforms of the action potential and the transmembrane ionic currents of a ventricular myocyte under control conditions is shown in Figs. 2A and 3A. In the current-clamping mode, the resting potential of single ventricular myocytes was 80 ± 2 mV (n ¼ 5) with no current injection (I ¼ 0). The action potential was then elicited by applying a current pulse of 2 nA for 10 ms. Following a 5 min perfusion with l mM of NMB, the durations of action potentials (APD20, APD50 and APD90) were dramatically extended from 170.36 ± 14.93 ms, 277.06 ± 15.41 ms and 309.98 ± 14.59 ms to 393.06 ± 20.91 ms, 603.04 ± 16.14 ms and 661.02 ± 19.55 ms, respectively (Fig. 2D,
n ¼ 5), and the amplitude of action potential (APA) was partially reduced from 124.46 ± 1.15 mV to 111.92 ± 1.39 mV (Fig. 2B, n ¼ 5). These changes in action potentials were reversible after 5e10 min washout with normal Tyrode solution, as shown in Fig. 2C.
N-MB dose-dependently inhibited ICaL After the action potential of rabbit ventricular cardiomyocytes was elicited, the clamping circuit was rapidly changed to the voltage clamp mode and the membrane potential was kept at 40 mV to inactivate fast inward Naþ currents and purposefully elicit the slow inward Ca2þ current, ICaL. Meanwhile, a time-dependent outward tail current, Itail, was recorded at the end of the command potential. However, inconsistent with the changes in APD (s), the peak values of ICaL at different membrane potentials were significantly reduced rather than enhanced (the amplitude of ICaL on 10 mV: in control, 1484.67 ± 95.40 pA, n ¼ 6; in 1 mM NMB, 1004.86 ± 75.42 pA, n ¼ 5, Fig. 3A and D). The currentevoltage (IeV) relationship curve showed that N-MB did not change the potential (approximately 10 mV) at which the maximal value of peak ICaL occurred. Additionally, N-MB neither changed the potential (approximately 30 mV) at which Ca2þ channels were activated nor altered the reversal potential (approximately þ60 mV, Fig. 3B). Neither the half-maximal activation potential, Vl/2 (in control, 17.24 ± 1.84 mV, n ¼ 6; in 1 mM NMB, 17.87 ± 1.30 mV, n ¼ 5) nor the slope factor, K (in control, 5.00 ± 1.68 mV, n ¼ 6; in 1 mM N-MB, 5.05 ± 1.24 mV, n ¼ 5) were altered by 1 mM N-MB (Fig. 3C, E, 3F). The effect of 1 mM N-MB could be partially washed out (data not shown).
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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To verify the above effect of N-MB on ICaL, we further investigated the effects of N-MB on CaV1.2 currents by using HEK293 cells stably transfected with the hCaV1.2 gene. The results showed that N-MB could inhibit CaV1.2 currents in a concentration-dependent manner (Table 3, Fig. 4A) with an IC50 of 23.5 mM (Fig. 4B). Similar to the response of native ICaL, N-MB did not change the shape of the currentevoltage (IeV) relationship curve (taken at 30 mM N-MB, Fig. 4C). The slope factor K (in control, 16.13 ± 2.54 mV, n ¼ 5; in 30 mM N-MB, 17.07 ± 3.22 mV, n ¼ 4) did not change either (Fig. 4D), but Vl/2 was altered from 2.44 ± 3.06 mV of the control (n ¼ 5) to 13.77 ± 3.03 mV in the presence of 30 mM N-MB (n ¼ 4).
The tail currents of the delayed rectifying currents contain IKr and IKs (slow delayed rectifier current). Since IKr is the main component of Itail, the effect of N-MB on IKr, represented as IhERG, was then determined in CHO cells stably transfected with the hERG gene. N-MB inhibited IhERG in a dose-dependent manner (Table 3, Fig. 5A); the estimated IC50 was 2.45 mM. Despite the significant decrease in the amplitude of peak IhERG by l mM N-MB (in control, 636.57 ± 85.95 pA, n ¼ 7; in 1 mM N-MB, 363.59 ± 43.45 pA, n ¼ 5, Fig. 5B and E), the potential (approximately 40 mV) at which Kþ channels were activated did not change (Fig. 5C). The Vl/2 was altered from 17.74 ± 0.79 mV (n ¼ 7) to 29.30 ± 1.17 mV (n ¼ 5) by 1 mM N-MB, and the slope factor K was changed from 8.53 ± 0.75 mV (n ¼ 7) to 6.99 ± 1.15 mV (n ¼ 5, Fig. 5DeG).
N-MB inhibited the rapid component of the delayed rectifier Kþ current (IKr)
Discussion
However, consistent with the changes in APD, N-MB decreased the amplitude of the tail currents of the delayed rectifying currents (Itail) of rabbit cardiac ventricular myocytes. Under the control condition, Itail had a distinct outward rectifying characteristic (Fig. 3G). Overall, 1 mM N-MB dramatically reduced the amplitude of Itail to the zero level at different membrane potentials, from 20 mV to þ50 mV; this was reversible after 5e10 min washing with normal Tyrode solution (the amplitude of Itail on þ50 mV: in control, 95.97 ± 1.63 pA, n ¼ 3; in 1 mM N-MB, 0.10 ± 0.44 pA, n ¼ 3; in washout, 100.07 ± 1.36 pA, n ¼ 3, Fig. 3GeI).
Arrhythmia is a common cardiovascular disease, and its primary treatment is drug therapy. According to their mechanism of action, antiarrhythmic drugs are mainly divided into four categories.18 Class III antiarrhythmic drugs achieve their effects through inhibiting IK and slowing down the repolarization process. However, with the application of these drugs, the APD may be extremely prolonged and the plateau period may be extended, which may induce an increase in Ca2þ influx and early depolarization; this can lead to arrhythmia and thus limiting their usage.19 Therefore, new antiarrhythmic drugs with multitargets on ion channels in cardiomyocytes are more promising.20 In the present study, using the
Fig. 5. N-MB dose-dependently inhibited IhERG in CHO cells. A: Representative traces of IhERG recorded in the absence (control) or presence of 0.1, 1, 10 mM N-MB, respectively; B: Voltage protocol and current traces of IhERG. Cells were held at 80 mV and depolarized from 60 mV to þ60 mV in 10 mV steps, followed by a repolarization to 50 mV for 1 s before returning to 80 mV; C: Currentevoltage relationship curves of KV11.1 channels in the absence (open circles) or presence (filled circles) of l mM N-MB; D: The voltage dependence of steady-state activation of IhERG; EeG: Quantitative comparison of peak amplitudes of IhERG (E), half maximal activation potential (V1/2, F) and slope factor (K, G) in the absence or presence of 1 mM N-MB. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. Control. n ¼ 5e7 in each group.
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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molecular docking and patch clamp technique, we found that N-MB could extend the action potential of cardiomyocytes through inhibiting the outward-rectifying Kþ current. Additionally, the Ltype Ca2þ current was blocked as well, which might limit the prolongation of APD and prevent arrhythmogenic risk. In a previous study, we found that N-MB inhibited Ca2þ currents and ATP-sensitive Kþ currents, IK-ATP, in guinea pig ventricular myocytes.21,22 N-MB could also decrease the IK in isolated rat hepatocytes.1 In the present study, molecular docking was applied to explore the binding of N-MB to CaV1.2 and Kþ channels. We found that N-MB could form a stable conformation with CaV1.2 and KV11.1 Kþ channels with similar binding affinity as their corresponding positive control drugs. Since selective KV11.1 channel blockers have negative frequency-dependent and arrhythmogenic properties, their clinical use is limited. Our molecular docking results suggested that N-MB could bind to both CaV1.2 and KV11.1 channels. Therefore, the cardiovascular effects of N-MB might differ from those of Kþ channel blockers. The APD of cardiomyocytes mainly depends on the length of the plateau, and the formation of the plateau period, especially its amplitude and duration, is the result of a balance between the inward and outward currents. If the inward slow current increases and/or the outward current decreases, the plateau duration increases, and vice versa.23 There are three main inward ion currents in the plateau: ICaL, Naþ-Ca2þ exchange current (INa/Ca) and slow Naþ channel current (INa); among these, the most important is ICaL, which deactivates slowly and persists throughout the plateau. There are also three outward currents involved in the plateau: inward rectifier Kþ channel current (IK1), IKr and platform Kþ channel current (IKP); of these, IKr is the main outward current of the plateau.24 In the present study, we found that N-MB could extend the APD, but that it had an inhibitory effect on ICaL in cardiomyocytes. Therefore, the effect of N-MB on the delayed outward rectifying Kþ current was investigated. As expected, N-MB significantly suppressed the Itail (IK) current, especially the IKr current, with increasing the half-maximal activation potential and decreasing the slope factor. The hERG gene encodes the pore-forming subunit of the channel that conducts IKr in the heart.12,25 IKr is a crucial current for cardiac repolarization and participates in APD determination.26,27 Loss of function in hERG channels due to genetic mutations or medications results in long QT syndrome (LQTS), which tends to affect individuals with fatal cardiac arrhythmias.27e29 It is also well-known that hERG is an important target for the treatment of arrhythmias.30 Amiodarone is a commonly used class III antiarrhythmic agent in clinical practice that can inhibit both IKr and IKs.31 However, it also shows the potential for causing QT interval prolongation, and thus has an inherent risk of LQTS.32 In this study, N-MB exerted its advantage by inhibiting IKr while blocking the ICaL current; this might prevent extensive APD prolonging and subsequently reduce the risk of induced LQTS. Moreover, the reduction of APA by N-MB indicated that N-MB might inhibit the INa in phase 0 of the action potential33; however, this remains to be confirmed by further studies. These data suggest that N-MB might be a potent multitarget drug acting on all three of the main ion channels, i.e., Naþ, Kþ and Ca2þ channels. N-MB also showed inhibitory effects on the ICaL current. N-MB dose-dependently inhibited the CaV1.2 current. In addition, the finding that N-MB treatment resulted in a shift to the left of the activation curve of ICaL in HEK293 cells that stably expressed the CaV1.2 calcium channel suggested that this drug not only affected the absolute conductance of the Ca2þ channel but also reduced the amount by affecting the opening of the Ca2þ channel. Further study is needed for kinetic analysis of the inactivation of the Ca2þ channel by N-MB to fully explain the effect of N-MB on Ca2þ currents.
In conclusion, N-MB extended the action potential duration through inhibiting the delayed outward rectifying Kþ current. Meanwhile, N-MB also partially decreased the slow inward current, indicating that N-MB may perform its antiarrhythmic effect through blocking both Ca2þ and Kþ channels. Our study provides important data of a candidate multitarget drug for future clinical use in cardiac arrhythmia. Declaration of Competing Interest None. Acknowledgement This work was supported by grants from the National Natural Science Foundation of China [31471091, 81100108], the Key Laboratory of Medical Electrophysiology (Southwest Medical University), Ministry of Education of China [No. 201605 and No. 201601] and Scientific Research Project of Department of Education of Liaoning Province [JC2019035]. References 1. Li JM, Cui GY, Liu DJ, et al. Effects of N-methyl berbamine on delayed outward potassium current in isolated rat hepatocytes. Zhongguo Yaoli Xuebao. 1998;19(1):24e26. https://www.ncbi.nlm.nih.gov/pubmed/10375752. 2. Xie JW, Ma T, Gu Y, et al. Berbamine derivatives: a novel class of compounds for anti-leukemia activity. Eur J Med Chem. 2009;44(8):3293e3298. https://doi.org/ 10.1016/j.ejmech.2009.02.018. 3. Zheng YJ, Gu SS, Li XX, et al. Berbamine postconditioning protects the heart from ischemia/reperfusion injury through modulation of autophagy. Cell Death Dis. 2017;8:1e13. https://doi.org/10.1038/cddis.2017.7. 4. Guo ZB, Fu JG. Progress of cardiovascular pharmacologic study on berbamine. Zhongguo Zhong Xi Yi Jie He Za Zhi. 2005;25:765e768. https://www.ncbi.nlm. nih.gov/pubmed/16152843. 5. Zhang CM, Gao L, Zheng YJ, et al. Berbamine protects the heart from ischemia/ reperfusion injury by maintaining cytosolic Ca2þ homeostasis and preventing calpain activation. Circ J. 2012;76(8):1993e2002. https://doi.org/10.1253/ circj.cj-11-1431. 6. Fu DG. Cardiac arrhythmias: diagnosis, symptoms, and treatments. Cell Biochem Biophys. 2015;73:291e296. https://doi.org/10.1007/s12013-015-0626-4. 7. Singh SN, Patrick J. Antiarrhythmic drugs. Curr Treat Options Cardiovasc Med. 2004;6(5):357e364. https://doi.org/10.1007/s11936-004-0019-2. 8. Jauhari N, Gupta S, Saxena S, et al. Computational studies of synthetic and plant-derived compounds against cardiovascular disease targets. Int J Pharm Sci Drug Res. 2016;8:144e148. https://doi.org/10.25004/IJPSDR.2016.080303. 9. Leelananda SP, Steffen L. Computational methods in drug discovery. Beilstein J Org Chem. 2016;12:2694e2718. https://doi.org/10.3762/bjoc.12.267. 10. Gregory S, Sandeepkumar K, Jens M, et al. Computational methods in drug discovery. Pharmacol Rev. 2014;66:334e395. https://doi.org/10.1124/ pr.112.007336. 11. Mahalakshmi M, Arjunan S, Vadivel B. Molecular docking studies on apigenin as a target with MAPK p38 for cardiovascular diseases. Int J Res Dev Pharm Life Sci. 2016;5:2241e2244. https://www.omicsonline.org/open-access/moleculardocking-studies-on-apigenin-as-a-target-with-mapk-p38-forcardiovasculardiseases-.pdf. 12. Sanguinetti MC, Jiang C, Curran ME, et al. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell. 1995;8:299e307. https://doi.org/10.1016/0092-8674(95) 90340-2. 13. Wang W, MacKinnon R. Cryo-EM structure of the open human ether- a-go-gorelated Kþ channel HERG. Cell. 2017;169(3):422e430. https://doi.org/10.1016/ j.cell.2017.03.048. 14. Xiang Z. Advances in homology protein structure modeling. Curr Protein Pept Sci. 2006;7(3):217e227. https://www.ncbi.nlm.nih.gov/pubmed/16787261. 15. Vyas VK, Ukawala RD, Ghate M, et al. Homology modeling a fast tool for drug discovery: current perspectives. Indian J Pharm Sci. 2012;74(1):1e17. https:// doi.org/10.4103/0250-474X.102537. 16. Isenberg G, Klockner U. Calcium tolerant ventricular myocytes prepared by preincubation in a "KB medium". Pflüg Arch. 1982;395:6e18. https://doi.org/ 10.1007/bf00584963. 17. Tang L, El-Din TG, Swanson TM, et al. Structural basis for inhibition of a voltage-gated Ca2þ channel by Ca2þ antagonist drugs. Nature. 2016;537: 117e121. https://doi.org/10.1038/nature19102. 18. Lei M, Wu L, Terrar DA, et al. Modernized classification of cardiac antiarrhythmic drugs. Circulation. 2018;138:1879e1896. https://doi.org/10.1161/ CIRCULATIONAHA.118.035455.
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H. Hu et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx 19. Shah SA, Kluger J, White CM. Monotherapy versus combination therapy with class III antiarrhythmic agents to attenuate transmural dispersion of repolarization: a potential risk factor for torsade de pointes. Pharmacotherapy. 2007;27(9):1297e1305. https://doi.org/10.1592/phco.27.9.1297. 20. Savelieva I, Camm J. Anti-arrhythmic drug therapy for atrial fibrillation: current anti-arrhythmic drugs, investigational agents, and innovative approaches. Europace. 2008;10(6):647e665. https://doi.org/10.1093/europace/eun130. 21. Li JM, Sun XD, Chang TH, et al. Effect of N-methyl berbamine on slow inward current in single cardiac cell of Guinea pigs. Chin J Pharmacol Toxicol. 1992;6: 307e308. http://www.cnki.com.cn/Article/CJFDTotal-YLBS199204020.htm. 22. Li JM, Zhang SL, Yang G, et al. Effect of N-methyl berbamine on ATP-sensitive Kþ current in single ventricular myocytes of Guinea pig. Chin Pharmacol Bull. 1999;15:542e544. http://www.cnki.com.cn/Article/CJFDTOTALYAOL199906019.htm. 23. Tamargo J, Ricardo C, Carmen V, et al. Pharmacology of cardiac potassium channels. Cardiovasc Res. 2004;62(1):9e33. https://doi.org/10.1016/ j.cardiores.2003.12.026. 24. Li GR, Dong MQ. Pharmacology of cardiac potassium channels. Adv Pharmacol. 2010;59(1):93e134. https://doi.org/10.1016/S1054-3589(10)59004-5. 25. Trudeau MC, Warmke JW, Ganetzky B, et al. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science. 1995;269:92e95. https:// doi.org/10.1126/science.7604285. 26. Curran ME, Splawski I, Timothy KW, et al. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell. 1995;80:795e803. https://doi.org/10.1016/0092-8674(95)90358-5.
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27. Keating MT, Sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell. 2001;104:569e580. https://doi.org/10.1016/s0092-8674(01) 00243-4. 28. Vandenberg JI, Perry MD, Perrin MJ, et al. hERG Kþ channels: structure, function, and clinical significance. Physiol Rev. 2012;92(3):1393e1478. https:// doi.org/10.1152/physrev.00036.2011. 29. Grandi E, Sanguinetti MC, Bartos DC, et al. Potassium channels in the heart: structure, function and regulation. J Physiol. 2017;595(7):2209e2228. https:// doi.org/10.1113/JP272864. 30. Zhao X, Yao H, Yin HL, et al. Ginkgo biloba extract and ginkgolide antiarrhythmic potential by targeting hERG and ICa-L channel. J Pharmacol Sci. 2013;123(4):318e327. http://europepmc.org/abstract/med/24225402. 31. Hiroko IN, Mihoko HN, Naito AT, et al. Application of human induced pluripotent stem cell-derived cardiomyocytes sheets with microelectrode array system to estimate antiarrhythmic properties of multi-ion channel blockers. J Pharmacol Sci. 2018;137(4):372e378. https://www.sciencedirect.com/ science/article/pii/S1347861318301312. 32. Lehtonen A, Fodstad H, Laitinen-Forsblom P, et al. Further evidence of inherited long QT syndrome gene mutations in antiarrhythmic drug-associated torsades de pointes. Heart Rhythm. 2007;4(5):603e607. https://www.sciencedirect. com/science/article/abs/pii/S1547527107000501?via%3Dihub. 33. Jiang W, Zeng M, Cao Z, et al. Icariin, a novel blocker of sodium and calcium channels, eliminates early and delayed afterdepolarizations, as well as triggered activity, in rabbit cardiomyocytes. Front Physiol. 2017;8:342. https:// doi.org/10.3389/fphys.2017.00342.
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A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents Huiyuan Hu a, 1, Shi Zhou a, 1, Xiaodong Sun a, Yingchun Xue a, Ling Yan a, Xuanxuan Sun a, Ming Lei b, Jinming Li a, Xiaorong Zeng b, **, Liying Hao a, * a
Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang, 110122, PR China Key Laboratory of Medical Electrophysiology of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, Luzhou, Sichuan, 646000, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 August 2019 Received in revised form 14 December 2019 Accepted 20 December 2019 Available online xxx
N-methyl berbamine (N-MB) is a berberine derivative. Its analogue berbamine has been reported to have remarkable antiarrhythmic and ischemic protective effects. However, the pharmacological effects of NMB are ill-defined. In this study, molecular docking was used to evaluate the binding of N-MB to CaV1.2 Ca2þ and KV11.1 Kþ channels, and the effects of N-MB on action potential and ionic currents were observed in the ventricular myocytes of rabbits, HEK293 cells stably transfected with the hCaV1.2 gene and CHO cells stably transfected with hERG (human ether-a-go-go related gene). The results showed that N-MB was able to bind to both CaV1.2 and KV11.1 channels. Following a perfusion with N-MB, the durations of action potentials (APD20, APD50 and APD90) were extended, and the outward tail current, Itail, as well as the hERG current, IhERG, were inhibited, while the amplitude of action potential (APA) was only slightly reduced. N-MB also decreased the peak amplitude of the L-type Ca2þ channel current, ICaL, as well as the CaV1.2 current, ICaV1.2; this may limit the prolongation of APD. In conclusion, N-MB is a potent and natural antiarrhythmic multitarget drug that may elicit its antiarrhythmic effect through blocking both Ca2þ and Kþ channel currents. © 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: N-methyl berbamine Action potential Ca2þ current Kþ current Multitarget
Introduction N-methyl berbamine (N-MB) is a kind of bisbenzylisoquinoline alkaloid extracted from the roots of plants in the Berberis genus.1 Berbamine is a well-known bisbenzylisoquinoline alkaloid isolated from the roots, bark and stems of plants from Berberis L., such as Berberis poiretii Schneid, a traditional Chinese herbal medicine.2,3 It has been reported that berbamine hydrochloride has remarkable
* Corresponding author. Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, No. 77, Puhe Road, Shenyang North New Aera, Shenyang, 110122, PR China. Fax: þ86 24 3193 9448. ** Corresponding author. Key Laboratory of Medical Electrophysiology of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, 319 Zhongshan Road, Jiangyang District Luzhou, Sichuan, 646000, PR China. Fax: þ86 830 3160619. E-mail addresses: [email protected] (X. Zeng), [email protected] (L. Hao). Peer review under responsibility of Japanese Pharmacological Society. 1 Co-first author.
antiarrhythmic4 and ischemic protective effects.3,5 As an N-methyl analogue of berbamine, N-MB should have similar pharmacological effects. However, the cardiovascular effects of N-MB are ill-defined. The occurrence of arrhythmia is closely related to the abnormality of myocardial electrical activity. The main mechanisms are autonomic abnormalities of cardiomyocytes, reentry caused by impulsive conduction disorder, and triggering activities caused by afterdepolarization.6 The existing antiarrhythmic drugs achieve their effects through decreasing autonomy, reducing afterdepolarization, and inhibiting conduction or prolonging the effective refractory period (ERP) to eliminate reentry. Antiarrhythmic Kþ channel blockers can reduce abnormal autonomy by blocking Kþ efflux and prolonging the action potential duration (APD). Ca2þ channel blockers can inhibit intracellular Ca2þ overload and reduce delayed afterdepolarization (DAD). Naþ channel blockers and Kþ channel blockers can prolong the ERP of fast response cells, while Ca2þ channel blockers and Kþ channel blockers can prolong the ERP of slow response cells and eliminate reentry activation.7 In our recent study, a potent antiarrhythmic effect of N-MB was achieved
https://doi.org/10.1016/j.jphs.2019.12.008 1347-8613/© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Fig. 1. Binding of N-MB to 3D structure models of CaV1.2 and KV11.1 channels. A: Chemical structure of N-MB; BeD: Schematic diagrams of N-MB (B), verapami (C) and diltiazem (D) interacting with specific amino acids of CaV1.2 channel. EeG: Schematic diagrams of N-MB (E), E-4031 (F) and dofetilide (G) interacting with specific amino acids of KV11.1 channel.
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Table 1 Compositions of the solutions used in the electrophysiological analysis (mM). APD and ICaL recording
Itail recording
IhERG recording on CHO
ICaV1.2 recording on HEK293
External (pH 7.40 by NaOH)
Pipette (pH 7.40 by KOH)
External (pH 7.40 by NaOH)
Pipette (pH 7.40 by KOH)
External (pH 7.40 by NaOH)
Pipette (pH 7.20 by KOH)
External (pH 7.40 by NaOH)
Pipette (pH 7.20 by CsOH)
NaCl KCl MgCl2 CaCl2 NaH2PO4 Hepes Glucose
K-Aspartate KCl MgCl2 K2ATP Na2CrP Hepes EGTA CaCl2
NaCl KCl CaCl2 MgCl2 NaH2PO4 Hepes NaOH Glucose
KCl K2ATP Creatine phosphate Hepes
NaCl KCl CaCl2 MgCl2 Hepes Glucose
KCl MgCl2 CaCl2 EGTA Hepes
NaCl BaCl2 Hepes TEA-Cl Glucose
CsCl MgATP EGTA Hepes TEA-Cl
135 5.4 1 1.8 0.33 10 5.5
110 30 1 5 5 5 10 1.43
143 5.40 1.8 0.5 0.3 5 2.3 5
130 5 5 5
140 5 1 1.25 10 10
140 1 1 10 10
120 10 10 20 10
130 5 10 10 10
Note: EGTA, ethylene glycol tetraacetic acid.
in dog Purkinje fibers through extending the APD and ERP. However, the underlying mechanisms and their effects on membrane ionic currents have not been clarified. Although medicinal plants have been known since ancient times to have therapeutic effects on human cardiovascular diseases, their mechanisms of action have not been completely understood until now.8 It is widely known that the process for drug discovery and development is time consuming, expensive and challenging. Acting as a virtual shortcut, computer-aided drug discovery (CADD) tools can assist in shortening this time, as well as potentially reducing the cost of research and development of drugs.9 CADD methods have played a major role in the development of therapeutically important small molecules for over three decades.10 Among these, the popular structure-based approach, molecular docking, has been widely used to model the interaction between a small molecule and a protein, which allows researchers to characterize the behavior of small molecules in the binding site of target proteins, in addition to elucidating fundamental biochemical processes.11
Table 2 Predicted binding sites of the chemicals and ion channels. Ion channels
Chemicals
London G scores
Combination sites (A/B chain)
Combination types
CaV1.2
N-MB
14.88 14.88 14.71 14.71 14.71 14.01 13.74 13.74 13.58 11.70 11.42 11.42 11.28 11.28 11.25 11.50 11.07 10.97 10.97 9.02 10.79 10.79 10.79 10.79 10.62 10.76 10.51 10.51 10.51 10.51
ASN 1040 (A) LYS 1046 (A) ASP 737 (A) SER 740 (A) GLU 340 (A) ARG 1372 (A) ASN 1314 (A) LYS 1299 (A) ARG 1372 (A) ASN 1282 (A) GLU 1252 (A) LYS 1299 (A) GLU 1252 (A) LYS 1299 (A) GLU 1252 (A) VAL 476 (A) ASN 470 (A) ASP 540 (A) TYR 475 (A) CYS 729 (A) HIS 402 (A) ARG 541 (A) LYS 407 (A) HIS 492 (A) TYR 403 (A) TYR 403 (A) ARG 541(A) ARG 537(A) HIS 492(A) TYR 403 (A)
H-donor H-acceptor H-donor pi-H pi-H H-acceptor H-acceptor H-acceptor H-acceptor H-acceptor H-donor H-acceptor H-donor H-acceptor H-donor pi-H H-donor H-donor pi-H H-donor H-donor H-acceptor H-acceptor H-acceptor pi-H pi-H H-acceptor H-acceptor H-acceptor pi-H
Verapamil
Diltiazem
KV11.1
N-MB
E-4031
Dofetilide
In this study, the effects of N-MB on the action potential, L-type Ca2þ current and outward rectifying Kþ current in the cardiac ventricular myocytes of rabbits, CHO cells transfected with hERG (human ether-a-go-go related gene), and HEK293 cells transfected with the hCaV1.2 gene were investigated by using the patch clamp technique; the molecular binding site of N-MB to these ion channels was predicted with Molecular Operating Environment (MOE) software. Our results showed that N-MB could extend the action potential of the cardiomyocytes through inhibiting both the L-type Ca2þ current and the outward rectifying Kþ current, which reveals the possible anti-arrhythmic mechanisms of N-MB and could promote the clinical application of this chemical to traditional Chinese medicine.
Materials and methods Drugs and solutions N-MB (purity > 98.2%) was purchased from the Institute of Applied Ecology, Chinese Academy of Sciences (Shenyang, China) and its structure is shown in Fig. 1A. The Tyrode solution contained (mM): NaCl 135, KCl 5.4, MgCl2 1.0, CaCl2 1.8, NaH2PO4 0.33, Hepes 10, glucose 5.5 (pH 7.4). Kraftbrühe-modified (KB) solution consisted of (mM): L-glutamic acid 50, KCl 40, taurine 20, KH2PO4 20, Hepes 10, glucose 10, MgCl2 3, EGTA 0.5 (pH 7.4). The compositions of external and pipette solutions used for APD, ICaL, Itail, IhERG, and ICaV1.2 recording are shown in Table 1.
Molecular docking The SWISS-MODEL homology modeling server (http:// swissmodel.expasy.org) was used to model the rabbit cardiomyocyte CaV1.2 channel a1 subunit (NP_00112994.1) and KV11.1 channel a subunit HERG (NP_001075853.1).12 The target sequences of CaV1.2 channel or KV11.1 channel were input into the website server, and the appropriate templates (PDB: 5gjv.1.A for CaV1.2 and 5va1.1.A for KV11.1) were searched and selected, which has the highest homology with the target sequences.13 Automated mode was used during modeling.14,15 The binding of N-MB or Ca2þ channel blockers, i.e. verapamil and diltiazem, to the CaV1.2 channel and the binding of N-MB or specific KV11.1 channel blockers, i.e. E-4031 and dofetilide, to the KV11.1 channel were simulated by using MOE molecular docking software, respectively. The binding sites with the highest binding affinity were chosen based on the lowest binding free energy of the protein-molecule complex calculated with London dG method. The specific Naþ channel blocker tetrodotoxin (TTX) was used as the negative control drug.
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Fig. 2. N-MB extended action potentials in the ventricular myocytes of rabbits. Representative traces of action potential in ventricular myocytes of control (A), 1 mM N-MB (B) and washout (C) groups; D: Measurements of APD20, APD50 and APD90 in ventricular myocytes of control, l mM N-MB and washout groups. ***P < 0.001, ****P < 0.0001, vs. Control; ns, not significant. n ¼ 5 in each group.
Preparation of single ventricular myocytes of rabbits The rabbits of either sex, weighing 1.0e1.5 kg, were provided by the Experimental Animal Center of the China Medical University (SCXK Liao, No. 2008e0005). All animals received humane care in compliance with the guidelines of National Institutes of Health. The rabbits were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and the hearts were excised and mounted in a Langendorff perfusion system quickly. The hearts were first perfused with normal Tyrode solution for 10e20 min at 37 C, which was followed by perfusion with Ca2þ-free Tyrode solution for 10 min and finally perfusion with a collagenase-containing solution (collagenase type I 0.6 g/L and bovine serum albumin 1 g/L) for about 20 min. After digestion, the enzyme was washed out with KB solution. The hearts were removed and left ventricles were minced, and the ventricular myocytes were isolated followed by filtration with a strainer.16 The myocytes were washed twice by centrifugation (1000 rpm for 3 min) and stored in KB solution at 4 C till use. Cell culture CHO cells stably transfected with hERG gene were cultured in a 37 C, 5% CO2 incubator, and passaged at a ratio of 1:5 every 48 h. Culture medium formula included 90% F12 (Invitrogen, CA, USA), 10% fetal bovine serum (Gibco, NY, USA), 100 mg/mL G418 (Invitrogen, CA, USA) and 100 mg/mL hygromycin B (Invitrogen, CA, USA). HEK293 cells stably transfected with hCaV1.2 gene were cultured in a 37 C, 5% CO2 incubator, and passaged at a ratio of 1:4 every 48 h. Culture medium formula included 90% DMEM (Invitrogen, CA, USA), 10% fetal bovine serum (Gibco, NY, USA), 2 mM Lglutamine, 100 mg/mL G418 (Invitrogen, CA, USA), 100 mg/mL hygromycin B (Invitrogen, CA, USA) and 40 mg/mL Zeocin
(Invitrogen, CA, USA). On the day of the experiment, the cells were digested with 0.25% Trypsin-EDTA (Gibco, NY, USA) solution and then transferred to a test dish for electrophysiological recording. Electrophysiology In current clamping mode, a current pulse from Axopatch-1D amplifier was applied through the probe to the single cell and the action potential was elicited from the resting membrane potential level. Then, it was rapidly changed to voltage clamping mode in which the membrane potential was depolarized to þ50 mV from a holding potentia1 of 40 mV in 10 mV steps. Under this condition, a slow inward currents and an outward tail current were elicited at the beginning and the end of a 300 ms clamping pulse. The currents and action potential tracings were recorded before and after the NMB treatment. The whole cell patch-clamp technique was used to record IhERG current in CHO cells stably transfected with hERG gene. The resistance of the pipette filled with solution was 2e5 MU. The clamping voltage was controlled by using the pClamp 10.0 software with a sampling frequency of 10 kHz and a filter frequency of 2 kHz. When obtaining the whole cell recording, the CHO cells were clamped at 80 mV and IhERG current was induced by a depolarization of 2 s with the step voltage from 80 mV to þ20 mV, followed by a repolarization to 50 mV for 1 s before returning to 80 mV. The CaV1.2 current in HEK293 cells stably transfected with the hCaV1.2 gene was also recorded by using the whole cell patch-clamp technique. After obtaining the whole cell recording, the CaV1.2 current was elicited using a series of depolarization pulses ranging from 80 to 0 mV for 100 ms, from a holding potential of 80 mV. The inhibition rate of IhERG (peak IhERG was induced at 50 mV) and CaV1.2 current (peak CaV1.2 current was induced at 0 mV) was
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Fig. 3. Effects of N-MB on ionic currents in the ventricular myocytes of rabbits. A: Representative traces of membrane currents (top) and voltage protocol (bottom) in ventricular myocytes of control (left), 1 mM N-MB (middle) and washout (right) groups; B: Currentevoltage relationship curves of ICaL under control conditions (open circles) and after the application of l mM N-MB (filled circles). Cells were held at 40 mV and depolarized from 40 to þ50 mV in l0 mV steps; C: The voltage dependence of steady-state activation of ICaL. Data were represented as normalized conductance and fitted to a curve based on a Boltzmann equation; DeF: Quantitative comparison of the peak amplitudes of ICaL (D), halfmaximal activation potential (V1/2, E) and slope factor (K, F) under control conditions and after the application of l mM N-MB; G: Representative current traces of Itail in ventricular myocytes of control (top), 1 mM N-MB (middle) and washout (bottom) groups; H: Currentevoltage relationship curve for Itail recorded in ventricular myocytes of control (open circles), 1 mM N-MB (filled circles) and washout (filled triangles) groups; I: Quantitative comparison of Itail current in ventricular myocytes of control, 1 mM N-MB and washout groups. **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. Control; ns, not significant. n ¼ 3e6 in each group.
calculated by the following formula: Inhibition % ¼ [1 e (I/ Io)] 100%, where I and Io represent the magnitude of IhERG or CaV1.2 current before and after N-MB treatment, respectively. All the experiments were performed at room temperature (20e22 C). The steady-state activation kinetics were evaluated by fitting the conductance (G)/peak conductance (Gmax)-voltage relationship to a Boltzmann equation as follows: G/Gmax ¼ 1/{1 þ exp [(V1/2-Vm)/K]}, where Vm is the test potential, V1/2 is the membrane potential at which half of the channels are activated, and K is the slope factor.
Table 3 Inhibition of N-MB on IhERG and ICaV1.2. Inhibition of N-MB on IhERG
Inhibition of N-MB on ICaV1.2
Drugs
Inhibition rate (%)
Drugs
Inhibition rate (%)
0.1 mM N-MB 1 mM N-MB 10 mM N-MB 300 nM cisapride
4.43 34.5 96.0 98.3
1.23 mM N-MB 3.70 mM N-MB 11.1 mM N-MB 33.3 mM N-MB 100 mM N-MB
7.72 ± 3.21 (n ¼ 4) 3.65 ± 9.51 (n ¼ 4) 22.0 ± 11.7 (n ¼ 4) 60.6 ± 7.55 (n ¼ 4) 88.1 ± 3.12 (n ¼ 5)
± ± ± ±
0.95 3.37 0.58 0.33
(n (n (n (n
¼ ¼ ¼ ¼
5) 7) 6) 4)
Statistical analysis All data were expressed as mean ± standard error (S.E.) and analyzed with the pClamp10.0 and Origin 8.0 software. The Student's t-test was used to compare the mean values between the two groups and P < 0.05 was considered statistically significant. Results N-MB was predicted to bind to CaV1.2 and KV11.1 channels The binding of N-MB to the ion channels was analyzed with MOE through the London dG scoring function. According to the optimized molecular placement method, the top five constellation results were displayed. As a result, N-MB was shown to bind to CaV1.2 channels (Table 2 and Fig. 1B). As positive controls, the binding of Ca2þ channel blockers verapamil and diltiazem was also simulated; this showed that the binding sites of verapamil and diltiazem to the CaV1.2 channel are mostly located in the fourth transmembrane structure domain (Fig. 1C and D), as previously reported.17 The specific Naþ channel blocker TTX was used as a
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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Fig. 4. N-MB dose-dependently inhibited CaV1.2 current in HEK293 cells. A: Representative traces of CaV1.2 currents recorded in HEK293 cells stably transfected with hCaV1.2 gene in the absence (control) or presence of 11.1 mM, 33.3 mM and 100 mM N-MB, respectively; B: Doseeresponse curve of N-MB on CaV1.2 current; C: Currentevoltage relationship curves of CaV1.2 current recorded in the absence (control) or presence of 30 mM N-MB; D: The voltage dependence of steady-state activation of CaV1.2 current recorded in the absence (control) or presence of 30 mM N-MB. Data were represented as normalized conductance and fitted to a curve based on a Boltzmann equation. n ¼ 4e5 in each group.
negative control, which did not bind to the CaV1.2 channel model. The results show that N-MB could bind to the CaV1.2 channel with a similar binding score as those for verapamil and diltiazem (N-MB, ASN 1040, -14.88; verapamil, ARG 1372, -14.01; diltiazem, GLU 1252, -11.42), indicating that N-MB might form a stable conformation with Ca2þ channels. N-MB could also bind to the KV11.1 channel. Both the London dG score and the binding site of N-MB to KV11.1 were similar to those of the positive control drugs (N-MB, VAL 476, -11.50; E-4031, HIS 402, -10.79; Dofetilide, TYR 403, -10.76), indicating that N-MB might bind to Kþ channels with strong binding affinity (Table 2; Fig. 1EeG). Therefore, compared with the corresponding specific blockers, N-MB showed similar binding properties to CaV1.2 and KV11.1 channels.
N-MB prolonged the APD in rabbit cardiac ventricular myocytes Based on the molecular docking findings that N-MB could bind to both Ca2þ and Kþ channels, the electrophysiological effects of NMB on the APD and the ion channels were then investigated. A set of typical waveforms of the action potential and the transmembrane ionic currents of a ventricular myocyte under control conditions is shown in Figs. 2A and 3A. In the current-clamping mode, the resting potential of single ventricular myocytes was 80 ± 2 mV (n ¼ 5) with no current injection (I ¼ 0). The action potential was then elicited by applying a current pulse of 2 nA for 10 ms. Following a 5 min perfusion with l mM of NMB, the durations of action potentials (APD20, APD50 and APD90) were dramatically extended from 170.36 ± 14.93 ms, 277.06 ± 15.41 ms and 309.98 ± 14.59 ms to 393.06 ± 20.91 ms, 603.04 ± 16.14 ms and 661.02 ± 19.55 ms, respectively (Fig. 2D,
n ¼ 5), and the amplitude of action potential (APA) was partially reduced from 124.46 ± 1.15 mV to 111.92 ± 1.39 mV (Fig. 2B, n ¼ 5). These changes in action potentials were reversible after 5e10 min washout with normal Tyrode solution, as shown in Fig. 2C.
N-MB dose-dependently inhibited ICaL After the action potential of rabbit ventricular cardiomyocytes was elicited, the clamping circuit was rapidly changed to the voltage clamp mode and the membrane potential was kept at 40 mV to inactivate fast inward Naþ currents and purposefully elicit the slow inward Ca2þ current, ICaL. Meanwhile, a time-dependent outward tail current, Itail, was recorded at the end of the command potential. However, inconsistent with the changes in APD (s), the peak values of ICaL at different membrane potentials were significantly reduced rather than enhanced (the amplitude of ICaL on 10 mV: in control, 1484.67 ± 95.40 pA, n ¼ 6; in 1 mM NMB, 1004.86 ± 75.42 pA, n ¼ 5, Fig. 3A and D). The currentevoltage (IeV) relationship curve showed that N-MB did not change the potential (approximately 10 mV) at which the maximal value of peak ICaL occurred. Additionally, N-MB neither changed the potential (approximately 30 mV) at which Ca2þ channels were activated nor altered the reversal potential (approximately þ60 mV, Fig. 3B). Neither the half-maximal activation potential, Vl/2 (in control, 17.24 ± 1.84 mV, n ¼ 6; in 1 mM NMB, 17.87 ± 1.30 mV, n ¼ 5) nor the slope factor, K (in control, 5.00 ± 1.68 mV, n ¼ 6; in 1 mM N-MB, 5.05 ± 1.24 mV, n ¼ 5) were altered by 1 mM N-MB (Fig. 3C, E, 3F). The effect of 1 mM N-MB could be partially washed out (data not shown).
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To verify the above effect of N-MB on ICaL, we further investigated the effects of N-MB on CaV1.2 currents by using HEK293 cells stably transfected with the hCaV1.2 gene. The results showed that N-MB could inhibit CaV1.2 currents in a concentration-dependent manner (Table 3, Fig. 4A) with an IC50 of 23.5 mM (Fig. 4B). Similar to the response of native ICaL, N-MB did not change the shape of the currentevoltage (IeV) relationship curve (taken at 30 mM N-MB, Fig. 4C). The slope factor K (in control, 16.13 ± 2.54 mV, n ¼ 5; in 30 mM N-MB, 17.07 ± 3.22 mV, n ¼ 4) did not change either (Fig. 4D), but Vl/2 was altered from 2.44 ± 3.06 mV of the control (n ¼ 5) to 13.77 ± 3.03 mV in the presence of 30 mM N-MB (n ¼ 4).
The tail currents of the delayed rectifying currents contain IKr and IKs (slow delayed rectifier current). Since IKr is the main component of Itail, the effect of N-MB on IKr, represented as IhERG, was then determined in CHO cells stably transfected with the hERG gene. N-MB inhibited IhERG in a dose-dependent manner (Table 3, Fig. 5A); the estimated IC50 was 2.45 mM. Despite the significant decrease in the amplitude of peak IhERG by l mM N-MB (in control, 636.57 ± 85.95 pA, n ¼ 7; in 1 mM N-MB, 363.59 ± 43.45 pA, n ¼ 5, Fig. 5B and E), the potential (approximately 40 mV) at which Kþ channels were activated did not change (Fig. 5C). The Vl/2 was altered from 17.74 ± 0.79 mV (n ¼ 7) to 29.30 ± 1.17 mV (n ¼ 5) by 1 mM N-MB, and the slope factor K was changed from 8.53 ± 0.75 mV (n ¼ 7) to 6.99 ± 1.15 mV (n ¼ 5, Fig. 5DeG).
N-MB inhibited the rapid component of the delayed rectifier Kþ current (IKr)
Discussion
However, consistent with the changes in APD, N-MB decreased the amplitude of the tail currents of the delayed rectifying currents (Itail) of rabbit cardiac ventricular myocytes. Under the control condition, Itail had a distinct outward rectifying characteristic (Fig. 3G). Overall, 1 mM N-MB dramatically reduced the amplitude of Itail to the zero level at different membrane potentials, from 20 mV to þ50 mV; this was reversible after 5e10 min washing with normal Tyrode solution (the amplitude of Itail on þ50 mV: in control, 95.97 ± 1.63 pA, n ¼ 3; in 1 mM N-MB, 0.10 ± 0.44 pA, n ¼ 3; in washout, 100.07 ± 1.36 pA, n ¼ 3, Fig. 3GeI).
Arrhythmia is a common cardiovascular disease, and its primary treatment is drug therapy. According to their mechanism of action, antiarrhythmic drugs are mainly divided into four categories.18 Class III antiarrhythmic drugs achieve their effects through inhibiting IK and slowing down the repolarization process. However, with the application of these drugs, the APD may be extremely prolonged and the plateau period may be extended, which may induce an increase in Ca2þ influx and early depolarization; this can lead to arrhythmia and thus limiting their usage.19 Therefore, new antiarrhythmic drugs with multitargets on ion channels in cardiomyocytes are more promising.20 In the present study, using the
Fig. 5. N-MB dose-dependently inhibited IhERG in CHO cells. A: Representative traces of IhERG recorded in the absence (control) or presence of 0.1, 1, 10 mM N-MB, respectively; B: Voltage protocol and current traces of IhERG. Cells were held at 80 mV and depolarized from 60 mV to þ60 mV in 10 mV steps, followed by a repolarization to 50 mV for 1 s before returning to 80 mV; C: Currentevoltage relationship curves of KV11.1 channels in the absence (open circles) or presence (filled circles) of l mM N-MB; D: The voltage dependence of steady-state activation of IhERG; EeG: Quantitative comparison of peak amplitudes of IhERG (E), half maximal activation potential (V1/2, F) and slope factor (K, G) in the absence or presence of 1 mM N-MB. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. Control. n ¼ 5e7 in each group.
Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008
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molecular docking and patch clamp technique, we found that N-MB could extend the action potential of cardiomyocytes through inhibiting the outward-rectifying Kþ current. Additionally, the Ltype Ca2þ current was blocked as well, which might limit the prolongation of APD and prevent arrhythmogenic risk. In a previous study, we found that N-MB inhibited Ca2þ currents and ATP-sensitive Kþ currents, IK-ATP, in guinea pig ventricular myocytes.21,22 N-MB could also decrease the IK in isolated rat hepatocytes.1 In the present study, molecular docking was applied to explore the binding of N-MB to CaV1.2 and Kþ channels. We found that N-MB could form a stable conformation with CaV1.2 and KV11.1 Kþ channels with similar binding affinity as their corresponding positive control drugs. Since selective KV11.1 channel blockers have negative frequency-dependent and arrhythmogenic properties, their clinical use is limited. Our molecular docking results suggested that N-MB could bind to both CaV1.2 and KV11.1 channels. Therefore, the cardiovascular effects of N-MB might differ from those of Kþ channel blockers. The APD of cardiomyocytes mainly depends on the length of the plateau, and the formation of the plateau period, especially its amplitude and duration, is the result of a balance between the inward and outward currents. If the inward slow current increases and/or the outward current decreases, the plateau duration increases, and vice versa.23 There are three main inward ion currents in the plateau: ICaL, Naþ-Ca2þ exchange current (INa/Ca) and slow Naþ channel current (INa); among these, the most important is ICaL, which deactivates slowly and persists throughout the plateau. There are also three outward currents involved in the plateau: inward rectifier Kþ channel current (IK1), IKr and platform Kþ channel current (IKP); of these, IKr is the main outward current of the plateau.24 In the present study, we found that N-MB could extend the APD, but that it had an inhibitory effect on ICaL in cardiomyocytes. Therefore, the effect of N-MB on the delayed outward rectifying Kþ current was investigated. As expected, N-MB significantly suppressed the Itail (IK) current, especially the IKr current, with increasing the half-maximal activation potential and decreasing the slope factor. The hERG gene encodes the pore-forming subunit of the channel that conducts IKr in the heart.12,25 IKr is a crucial current for cardiac repolarization and participates in APD determination.26,27 Loss of function in hERG channels due to genetic mutations or medications results in long QT syndrome (LQTS), which tends to affect individuals with fatal cardiac arrhythmias.27e29 It is also well-known that hERG is an important target for the treatment of arrhythmias.30 Amiodarone is a commonly used class III antiarrhythmic agent in clinical practice that can inhibit both IKr and IKs.31 However, it also shows the potential for causing QT interval prolongation, and thus has an inherent risk of LQTS.32 In this study, N-MB exerted its advantage by inhibiting IKr while blocking the ICaL current; this might prevent extensive APD prolonging and subsequently reduce the risk of induced LQTS. Moreover, the reduction of APA by N-MB indicated that N-MB might inhibit the INa in phase 0 of the action potential33; however, this remains to be confirmed by further studies. These data suggest that N-MB might be a potent multitarget drug acting on all three of the main ion channels, i.e., Naþ, Kþ and Ca2þ channels. N-MB also showed inhibitory effects on the ICaL current. N-MB dose-dependently inhibited the CaV1.2 current. In addition, the finding that N-MB treatment resulted in a shift to the left of the activation curve of ICaL in HEK293 cells that stably expressed the CaV1.2 calcium channel suggested that this drug not only affected the absolute conductance of the Ca2þ channel but also reduced the amount by affecting the opening of the Ca2þ channel. Further study is needed for kinetic analysis of the inactivation of the Ca2þ channel by N-MB to fully explain the effect of N-MB on Ca2þ currents.
In conclusion, N-MB extended the action potential duration through inhibiting the delayed outward rectifying Kþ current. Meanwhile, N-MB also partially decreased the slow inward current, indicating that N-MB may perform its antiarrhythmic effect through blocking both Ca2þ and Kþ channels. Our study provides important data of a candidate multitarget drug for future clinical use in cardiac arrhythmia. Declaration of Competing Interest None. Acknowledgement This work was supported by grants from the National Natural Science Foundation of China [31471091, 81100108], the Key Laboratory of Medical Electrophysiology (Southwest Medical University), Ministry of Education of China [No. 201605 and No. 201601] and Scientific Research Project of Department of Education of Liaoning Province [JC2019035]. References 1. Li JM, Cui GY, Liu DJ, et al. Effects of N-methyl berbamine on delayed outward potassium current in isolated rat hepatocytes. Zhongguo Yaoli Xuebao. 1998;19(1):24e26. https://www.ncbi.nlm.nih.gov/pubmed/10375752. 2. Xie JW, Ma T, Gu Y, et al. Berbamine derivatives: a novel class of compounds for anti-leukemia activity. Eur J Med Chem. 2009;44(8):3293e3298. https://doi.org/ 10.1016/j.ejmech.2009.02.018. 3. Zheng YJ, Gu SS, Li XX, et al. Berbamine postconditioning protects the heart from ischemia/reperfusion injury through modulation of autophagy. Cell Death Dis. 2017;8:1e13. https://doi.org/10.1038/cddis.2017.7. 4. Guo ZB, Fu JG. Progress of cardiovascular pharmacologic study on berbamine. Zhongguo Zhong Xi Yi Jie He Za Zhi. 2005;25:765e768. https://www.ncbi.nlm. nih.gov/pubmed/16152843. 5. Zhang CM, Gao L, Zheng YJ, et al. Berbamine protects the heart from ischemia/ reperfusion injury by maintaining cytosolic Ca2þ homeostasis and preventing calpain activation. Circ J. 2012;76(8):1993e2002. https://doi.org/10.1253/ circj.cj-11-1431. 6. Fu DG. Cardiac arrhythmias: diagnosis, symptoms, and treatments. Cell Biochem Biophys. 2015;73:291e296. https://doi.org/10.1007/s12013-015-0626-4. 7. Singh SN, Patrick J. Antiarrhythmic drugs. Curr Treat Options Cardiovasc Med. 2004;6(5):357e364. https://doi.org/10.1007/s11936-004-0019-2. 8. Jauhari N, Gupta S, Saxena S, et al. Computational studies of synthetic and plant-derived compounds against cardiovascular disease targets. Int J Pharm Sci Drug Res. 2016;8:144e148. https://doi.org/10.25004/IJPSDR.2016.080303. 9. Leelananda SP, Steffen L. Computational methods in drug discovery. Beilstein J Org Chem. 2016;12:2694e2718. https://doi.org/10.3762/bjoc.12.267. 10. Gregory S, Sandeepkumar K, Jens M, et al. Computational methods in drug discovery. Pharmacol Rev. 2014;66:334e395. https://doi.org/10.1124/ pr.112.007336. 11. Mahalakshmi M, Arjunan S, Vadivel B. Molecular docking studies on apigenin as a target with MAPK p38 for cardiovascular diseases. Int J Res Dev Pharm Life Sci. 2016;5:2241e2244. https://www.omicsonline.org/open-access/moleculardocking-studies-on-apigenin-as-a-target-with-mapk-p38-forcardiovasculardiseases-.pdf. 12. Sanguinetti MC, Jiang C, Curran ME, et al. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell. 1995;8:299e307. https://doi.org/10.1016/0092-8674(95) 90340-2. 13. Wang W, MacKinnon R. Cryo-EM structure of the open human ether- a-go-gorelated Kþ channel HERG. Cell. 2017;169(3):422e430. https://doi.org/10.1016/ j.cell.2017.03.048. 14. Xiang Z. Advances in homology protein structure modeling. Curr Protein Pept Sci. 2006;7(3):217e227. https://www.ncbi.nlm.nih.gov/pubmed/16787261. 15. Vyas VK, Ukawala RD, Ghate M, et al. Homology modeling a fast tool for drug discovery: current perspectives. Indian J Pharm Sci. 2012;74(1):1e17. https:// doi.org/10.4103/0250-474X.102537. 16. Isenberg G, Klockner U. Calcium tolerant ventricular myocytes prepared by preincubation in a "KB medium". Pflüg Arch. 1982;395:6e18. https://doi.org/ 10.1007/bf00584963. 17. Tang L, El-Din TG, Swanson TM, et al. Structural basis for inhibition of a voltage-gated Ca2þ channel by Ca2þ antagonist drugs. Nature. 2016;537: 117e121. https://doi.org/10.1038/nature19102. 18. Lei M, Wu L, Terrar DA, et al. Modernized classification of cardiac antiarrhythmic drugs. Circulation. 2018;138:1879e1896. https://doi.org/10.1161/ CIRCULATIONAHA.118.035455.
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Please cite this article as: Hu H et al., A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.12.008