Life Sciences 94 (2014) 99–105
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Protective effect of piperine on electrophysiology abnormalities of left atrial myocytes induced by hydrogen peroxide in rabbits Yan Liu a,b,1, Yu Zhang a,c,1, Kun Lin a, De-xian Zhang a,c, Miao Tian d, Hong-yang Guo a, Yu-tang Wang e, Yang Li f,⁎, Zhao-liang Shan a,⁎⁎ a
Department of Cardiology, Chinese PLA General Hospital, Beijing 100853, China Department of Cardiology, Liao He Hospital, Liao Ning 111000, China Nankai University School of Medicine, Nankai University, Tianjin 300071, China d Department of Cardiology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing 100048, China e Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing 100853, China f Institute of Geriatric Cardiology, Chinese PLA General Hospital, Beijing 100853, China b c
a r t i c l e
i n f o
Article history: Received 6 June 2013 Accepted 18 October 2013 Keywords: Piperine Hydrogen peroxide Atrial myocytes Action potential
a b s t r a c t Aims: Piperine had protective effects on oxidative stress damage of ventricular myocytes by hydrogen peroxide (H2O2). In this study we aimed to explore the protective effect of piperine on abnormalities of the cardiac action potential (AP) and several ion currents induced by hydrogen peroxide (H2O2) in single rabbit left atrial myocyte. Main methods: Conventional microelectrodes were used to record action potential duration (APD), resting membrane potential (RMP) and some ion currents (ICa,L,Ito,IK1 and Ikur,ect.), before and after H2O2 administration with or without piperine. Key findings: The piperine (7 μmol/L) had no significant effect on APD, ICa,L,Ito,IK1 and Ikur and their channel dynamics. In the presence of 50 μmol/L H2O2, APD50 and APD90 shortened (P b 0.01), amplitude of RMP decreased (P b 0.05), the peak of ICa,L reduced significantly (P b 0.05). Piperine (7 μmol/L) significantly alleviated the inhibiting effect of H2O2 on APD and ICa,L (P b 0.01) and protected the changes of ICa,L dynamics induced by H2O2. The peak current of Ito was reduced significantly (P b 0.05); Piperine (7 μmol/L) significantly alleviated the inhibiting effect of H2O2 on Ito (P b 0.01). In addition, piperine protected the changes of Ito dynamics induced by H2O2. The peak current of IK1 and IKUr was significantly reduced (P b 0.05); Piperine (7 μmol/L) alleviated the inhibiting effect of H2O2 on IK1 and IKUr significantly (P b 0.01). In addition, piperine protected the changes of IKUr dynamics induced by H2O2. Significance: These results suggest that piperine effectively protects atrial myocytes from oxidative stress injury in atrial electrophysiology. © 2013 Elsevier Inc. All rights reserved.
Introduction Piperine (PIP) widely exists in pepper plants and is a common drug in Chinese Medicine, Mongolian Medicine and Tibetan Medicine. It has protective effects on sulfydryl, antioxidant molecules and antioxidase in cells via the inhibition of free radicals, reactive oxygen species (ROS) and lipid peroxidation (Srinivasan, 2007). The study indicated that PIP had protective effects on oxidative stress damage of ventricular myocytes by hydrogen peroxide (H2O2) (Hu et al., 2009). Piperine exhibited antioxidant action in experimental conditions, both in vivo as well as in vitro, through its radical quenching effect and by preventing GSH depletion (Mittal and Gupta, 2000). Additionally, piperine also possesses antiinflammatory (Kumar et al., 2007), antidepressant (Li et al., ⁎ Corresponding author. Tel./fax: +86 10 66 93 6762. ⁎⁎ Corresponding author. Tel.: +86 10 5549 9210; fax: +86 10 5549 9209. E-mail addresses: [email protected] (Y. Li), [email protected] (Z. Shan). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.10.024
2007), antiplatelet (Park et al., 2007), antipyretic and analgesic (Ahmad et al., 2002) characteristics. The present study was designed to investigate the effects of H2O2 on the cardiac action potential (AP); L-type calcium current (ICa,L), transient outward potassium current (Ito), inward rectifier potassium current (Ik1) and ultra rapid delayed rectifier potassium (Ikur) of rabbits' left atrial myocytes using a whole-cell configuration of the patchclamp technique. Also studied, was the protective effect of PIP. Material and methods Experimental animals Forty New Zealand rabbits (weighing 1–1.5 kg) were used for the study. The investigation conformed to the Guide for the Care and Use of Laboratory Animals issued by the National Committee of Science and Technology of China, and the study was approved by the Animal Investigation Committee of the Chinese PLA General Hospital.
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Drugs and solutions Tyrode's solution (in mmol/L): NaCl 136, KCl 5.4, MgCl2 1.0, NaH2PO4 0.33, CaCl2 1.8, hydroxyethyl PIPerazine ethanesulfonic acid (HEPES) 10, and glucose 10 (pH 7.4). Kreb's buffer solution (in mmol/L): KCl 40, KH2PO4 25, MgSO4 3.0, KOH 80, L-glutamine 50.3, taurine 20, HEPES 10, glucose 10, and EGTA 0.5 (pH 7.4). For AP recording, the internal pipette solution contained (in mmol/L) KCl 120, NaCl 10, CaCl2 1, MgATP 5, EGTA 11 and HEPES 10 (pH 7.4). For ICa-L recording, the internal pipette solution contained (in mmol/L) CsCl 140, Na2ATP 5.0, TEA-Cl 10, EGTA 10, MgCl2 3.0, and HEPES 10 (pH 7.4), while the bath solution contained (in mmol/L) choline chloride 137, CsCl 4.6, CaCl2 1.8, MgCl2 1.0, TEA-Cl 10, glucose 10, 4-AP 5.0, and HEPES 5.0 (pH 7.4). For potassium current recording, the internal PIPette solution contained (in mmol/L) KCl 45, K-aspartate 85, Na-pyruvate 5, MgATP 5.0, EGTA 10, HEPES 10, glucose 11 (pH 7.4), while the bath solution contained (in mmol/L) N-methylD-glucamine (NMG) 149,MgCl2 5,CaCl2 0.65,HEPES 5. For ICa,L recording, 4-aminopyridine (4-AP) (50 μmol/L), Dofetilide (5 nmol/L) and tetraodontoxin (TTX) (100 μmol/L) were added to the superfusion to block IKUr, IKr, and INa. For Ito and IKur recording, BaCl2 (200 mM) and CdCl2 (200 mM) were added to the superfusion to block IK1 and ICa. For IK1 recording, Dof (5 nmol/L), TTX (100 μmol/L) and CdCl2 (200 mM) were added to the superfusion to block IKr, INa and ICa. Single atrial myocyte preparation Left atrial myocytes were isolated enzymatically from the atrium of New Zealand rabbits (weighing 1.0–1.5 kg) as previously described.8–10 Briefly, the rabbit was anesthetized with an iv. injection of sodium pentobarbital (40 mg/kg), and then was heparinized (300 U/kg i.p.). The heart was excised immediately and mounted on a Langendorff apparatus. The heart was then perfused retrogradely via the aorta with oxygenated, calcium-free Tyrode's solution for 5 min, and with the Tyrode's solution containing 1.4 mg/mL of type II collagenase (Invitrogen, USA) and 0.24 mg/mL of trypsinase (Merck, Germany) for 15–20 min. Subsequently, the left atrial appendage was cut into small pieces in a dish containing Kreb's buffer solution and shaken gently to ensure the dispersion of dissociated cardiac myocytes. The cells were kept at 4 °C in Kreb's buffer solution. All the solutions were continuously gassed with 95% O2 and 5% CO2 and were maintained at 37 °C. Pharmacology Cells were divided into 4 groups: in the control group, cells were cultured without treatment for 1.5 h (n = 15); in the PIP group, cells were cultured with PIP (7 μmol/L) for 1.5 h (n = 15); in the H2O2 group, cells were cultured without treatment for 1 h, then with H2O2 for 0.5 h (n = 15); in the PIP + H2O2 group, cells were cultured with PIP (7 μmol/L) for 1 h and then cultured with H2O2 for 0.5 h (n = 15). Electrophysiological recording Membrane currents were measured using a whole-cell configuration of the patch-clamp technique with Axonpatch 700B amplifiers (Axon Instruments, USA). To stabilize the current, experiments were performed 5 min after entering the whole-cell configuration. All the measurements were obtained at a temperature of 22 °C. Parameters of the AP were measured with 2-Hz electrical stimuli before and after drug administration in the LA without spontaneous activity, which was measured after 10 min of superfusion. The AP amplitude (APA) was obtained from the resting membrane potential (RMP) or maximum diastolic potential to the peak of AP depolarization. The AP durations at a repolarization of 90%, and 50% of the APA were measured as APD90, and APD50, respectively. The maximum upstroke velocity was acquired
using the maximum positive value of the first derivative of the AP. Spontaneous activity was defined as the constant occurrence of spontaneous APs in the absence of any electrical stimuli. Burst firing was defined as the occurrence of accelerated spontaneous potential (faster than the basal rate) with sudden onset and termination. The resistance of Pipettes in the bath solution ranged from 2 M to 5 M. No leak subtraction was applied during recording. The current densities were calculated by dividing the current amplitudes by the cell capacitance. Voltage dependence of channel activation or inactivation was obtained by fitting normalized curves with the Boltzmann function. Time course of recovery from inactivation (τ) was quantified by fitting measured data with a 1-exponential function. Statistical analysis Statistical analysis was performed with SPSS version 13.0 (Chicago, IL, USA). Data are presented as mean ± standard deviation. ANOVA was used for multiple-group comparisons, followed by a Bonferronicorrected t-test. A 2-tailed value of P b 0.05 was taken as statistically significant. Results The effects of H2O2 and PIP on AP H2O2 (50 μmol/L) could shorten atrial myocytes' APD50 and APD90 and decrease the amplitude of the resting membrane potential (RMP). PIP mitigate the changes partly. The AP amplitude (APA) of 4 groups changed little (Fig. 1 and Table 1). The effects of H2O2 and PIP on ICa,L Fig. 2A, B shows H2O2 (50 μmol/L) reduced the peak of ICa,L density from (−18.3 ± 1.2) pA/pF to (−9.3 ± 0.9) pA/pF at the test potential of 0 mV (P b 0.01). PIP alleviated the changes (−11.9 ± 1.2 pA/pF). H2O2 (50 μmol/L) made the I–V curve shift upward, but PIP alleviated the changes (Fig. 2C). In the presence of 50 μmol/L H2O2, the steadystate activation curve shifted to a more positive potential, the half activation potentials (V1/2) at which 50% of channels activated were from (−34.64 ± 2.15) mV to (−20.34 ± 2.20) mV (P b 0.01), but PIP made the V1/2 change to (−28.81 ± 3.95) mV, and the slope factors (k) of 4 groups were not changed, these may reveal that H2O2 reduced the current via decreasing the channels activation (Fig. 2D). In the presence of 50 μmol/L H2O2, the steady-state inactivation curve was shifted to a more negative potential, the half inactivation potentials (V1/2) at which 50% of channels inactivated were from (− 19.12 ± 0.67) mV to (− 29.47 ± 1.82) mV (P b 0.01), but PIP made the V1/2 change to (− 25.94 ± 1.74) mV, and the slope factors (k) of 4 groups were not changed, these may reveal that H2O2 reduced the current via increasing the channels inactivation (Fig. 2E). However, as in the examples shown in Fig. 2F, the time course of recovery from inactivation did not change. PIP alone did not influence these parameters of ICa,L significantly (Fig. 2). The effects of H2O2 and PIP on Ito Fig. 3A, B shows H2O2 (50 μmol/L) reduced the peak of Ito density from (42.6.3 ± 3.0) pA/pF to (20.8 ± 2.0) pA/pF (P b 0.05, n = 15). PIP alleviated the changes (31.8 ± 2.7 pA/pF). H2O2 (50 μmol/L) made the I–V curve shifted downward, but PIP alleviated the changes (Fig. 3C). In the presence of 50 μmol/L H2O2, the steady-state activation curve was shifted to a more positive potential (Fig. 3D), the half activation potentials (V1/2) at which 50% of channels activated were from (− 31.51 ± 2.27) mV to (− 19.62 ± 1.53) mV (P b 0.01), but PIP made the V1/2 change to (− 26.86 ± 0.53) mV (Fig. 3E), and the slope factors (k) of 4 groups were not changed (Fig. 3F), these may
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Fig. 1. Effects of H2O2 and PIP on AP. H2O2 (50 μmol/L) could shorten atrial myocytes' APD50 and APD90 and decrease the amplitude of the resting membrane potential (RMP). PIP could partly put back the changes. The AP amplitude (APA) of 4 groups was changed little. (n = 15).
reveal that H2O2 reduced the current via decreasing the channels activation. As in the examples shown in Fig. 4A, in the presence of 50 μmol/L H2O2, the steady-state inactivation curve was shifted to a more negative potential, but it was not significant. The half inactivation potentials (V1/2), the slope factors (k) and the time course of recovery from inactivation of 4 groups were not changed (Fig. 4B, C, D). The closedstate inactivation of Ito was accelerated in the presence of 50 μmol/L H2O2, and PIP alleviated the changes (Fig. 4E). PIP alone has no significant influence on these parameters of ICa,L (Fig. 3). The effects of H2O2 and PIP on IK1 H2O2 (50 μmol/L) reduced the peak of IK1 density from (−148.2 ± 16.7) pA/pF to (−64.2 ± 9.8) pA/pF (P b 0.01, n = 15). PIP alleviated the changes (−113.8 ± 15.4 pA/pF) (Fig. 5A, B). H2O2 (50 μmol/L) made the I–V curve shifted upward, but PIP alleviated the changes (Fig. 5C). The effects of H2O2 and PIP on IKUr H2O2 (50 μmol/L) reduced the peak of IKUr density from (16.0 ± 2.1) pA/pF to (6.1 ± 1.4) pA/pF (P b 0.05, n = 15). PIP alleviated the changes (11.3 ± 1.8 pA/pF) (Fig. 6A, B). H2O2 (50 μmol/L) made the I–V curve shifted downward, but PIP alleviated the changes (Fig. 6C). In the presence of 50 μmol/L H2O2, the steady-state activation curve was shifted to a more positive potential (Fig. 6D), the half activation potentials (V1/2) at which 50% of channels activated were from (−11.55 ± 0.78) mV to (−1.07 ± 0.08) mV (P b 0.01), but PIP made the V1/2 change to (−3.67 ± 0.26) mV (Fig. 6E), and the slope factors (k) of 4 groups were not changed (Fig. 6F). In the presence of 50 μmol/L H2O2, the steady-state inactivation curve was shifted to a more negative potential (Fig. 7A). The half inactivation potentials (V1/2) decreased from (− 6.76 ± 0.94) mV to (− 15.87 ± 0.62) mV (P b 0.01), but PIP elevated the V1/2 change
Table 1 Comparison of parameters of AP influenced by H2O2 and PIP.
APD50(ms) APD90(ms) RMP(mV) APA(mV)
Control
H2O2
H2O2 + PIP
PIP
110.3 ± 9.3 251.7 ± 13.5 −79.1 ± 2.9 115.3 ± 6.9
77.6 ± 6.2⁎ 202.1 ± 11.6⁎ −67.6 ± 3.2⁎ 107.6 ± 5.2
100.7 ± 8.8⁎⁎ 247.5 ± 15.5⁎⁎ −80.4 ± 3.4⁎⁎ 113.2 ± 5.4
118.4 ± 7.8⁎⁎ 243.0 ± 10.1⁎⁎ −80.5 ± 3.0⁎⁎ 118.3 ± 4.8
AP, action potential; APD, action potential duration; RMP, resting member potential; APA, action potential amplitude. ⁎P b 0.05 vs control group; ⁎⁎P b 0.05 vs H2O2 group; ⁎⁎⁎P b 0.05 vs H2O2 + PIP group.
to (− 11.23 ± 1.17) mV (Fig. 7B), the slope factors (k) were from (9.83 ± 0.57) mV to (12.46 ± 0.42) mV (P b 0.01), but PIP made the slope factors (k) change to (11.27 ± 0.57) mV (Fig. 7C). The time course of recovery from inactivation was decelerated in the presence of 50 μmol/L H2O2, especially in 100 ms, but PIP regained the changes (Fig. 7D, E). PIP alone did not influence these parameters of ICa,L significantly (Fig. 7). Discussion Low concentration of H2O2 is an usual reagent to made a oxidative stress model (Lin et al., 2010), so we used H2O2 (50 μmol/L) in our experiments. PIP (7 μmol/L) has been showed to have protective effects on sulfydryl, antioxidant molecules and antioxidase in cells via the inhibition of free radicals, reactive oxygen species (ROS) and lipid peroxidation (Hu et al., 2009), so we used PIP (7 μmol/L) in this study. In the present study, we found that H2O2 decrease APD50, APD90, ICa,L,Ito,Ik1 and Ikur, but PIP could partly regain these changes. H2O2 decreased ICa,L and APD, so the mechanism of that maybe: first, in the presence of H2O2, membrane structure of rabbit atrial myocytes changes. These changes made the steady-state activation curve of ICa,L shift to positive potentials; channel activation slowed, the steady-state inactivation was shifted to negative potentials, and channel inactivation accelerated. So the number of ion channels decreased in the same voltage and the ICa,L decreased. Second, the previous studies show when cells were damaged by oxidative stress, the damaged of mitochondria by oxygen radical made an obstruction of ATP produced. The activity of Na+ pump and Ca2+pump decreased, which were dependent on ATP, so concentration of Na+ in cells increased, exchanged of Na+ and Ca2+ increased, inflow of Ca2+ increased (Korantzopoulos et al., 2003), and the concentration of Ca2+ increased which could inhibit ICa,L. ICa,L is the main current in the second plateau of the Aps (Nattel et al., 2008), and it was the inward current. The decreased current accelerated repolarization in atrial myocytes, then APD was shortened. Lin et al. (2010) revealed that H2O2 could shorten APD of the left atrial myocytes, which was in conformity with our study. A shortened APD may cause arrhythmias such as atrial fibrillation, especially in atrial myocytes. H2O2 decreased IK1 and the amplitude of RMP. IK1 stabilized the resting membrane potential and played a fundamental role in the terminal phase of the action potential's repolarization in the cardiac myocytes (Dhamoon and Jalife, 2005). In the present study, H2O2 could inhibit IK1. Decreases in IK1 caused the amplitude of RMP to decrease. So H2O2 caused RMP to decrease. There were not so many reports of IK1 influencing H2O2. Ward and Giles (1997) revealed that H2O2 had no effects on IK1. But they were in ventricular myocytes. In atrial myocytes, the study firstly revealed that H2O2 inhibited IK1 in atrial myocytes. These
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Fig. 2. (A) Effects of H2O2 and PIP on ICa,L. (B) Effects of H2O2 and PIP on peak of ICa,L. (C) Effects of H2O2 and PIP on the I–V curve of ICa,L. (D) Effects of H2O2 and PIP on the steady-state activation curve of ICa,L. (E) Effects of H2O2 and PIP on the steady-state inactivation curve of ICa,L. (F) Effects of H2O2 and PIP on the time course of recovery from inactivation of ICa,L. (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
changes could cause a decreased amplitude of RMP, which made the threshold stimulus to be decreased. And then excitability of atrial myocytes increased. Another, IK1 was the background current, so its reduction could make automaticity of atrial myocytes increase. Those two effects cause excitability and automaticity of atrial myocytes to be increased, which would easily cause arrhythmias such as atrial fibrillation, especially in atrial myocytes. H2O2 inhibited Ito and changed the I–V curve of Ito. In the presence of H2O2, the steady-state activation curve was shifted to more a positive potential, which may reveal that H2O2 reduced the current via decreasing
the channel activation, and the closed-state inactivation of Ito was accelerated, which may reveal that H2O2 reduced the current via decreasing the number of open channels at the same time. Those two effects made the current decline. Studies of Pike Pike et al. (1993) and Lu et al. (2008) were in conformity with our study. Ward and Giles (Ward and Giles, 1997) revealed that H2O2 had no effects on Ito, but Tanaka et al. (Tanaka et al., 2001) revealed that H2O2 had an increased effect on Ito. I think the cause of this difference may be the different methods of patch plant technique. Ward and Tanaka used the amphotericin B-perforated patch voltage-clamp, but Pike, Lu and we used the
Fig. 3. (A) Effects of H2O2 and PIP on Ito. (B) Effects of H2O2 and PIP on peak of Ito. (C) Effects of H2O2 and PIP on the I–V curve of Ito. (D) Effects of H2O2 and PIP on the steady-state activation curve of Ito. (E) Effects of H2O2 and PIP on the half activation potentials (V1/2) of Ito. (F) Effects of H2O2 and PIP on the slope factors (k) of Ito. (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
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Fig. 4. (A) Effects of H2O2 and PIP on the steady-state inactivation curve of Ito. (B) Effects of H2O2 and PIP on the half inactivation potentials (V1/2) of Ito. (C) Effects of H2O2 and PIP on the slope factors (k) of Ito. (D) Effects of H2O2 and PIP on the time course of recovery from inactivation of Ito. (E) Effects of H2O2 and PIP on the closed-state inactivation of Ito. (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
conventional whole cell patch plant technique. Ito played an important role in the phase 1 of AP, which decreased Ito in atrial myocytes and was an important characteristic in the atrial electric remodeling, though the changes had no specifically significance in the clinical results. Ikur was a current expressed in atrial myocytes, not ventricular myocytes (Wang et al., 1993). Ikur was an outward current in the phase 2 of AP and could promote the phase 3 of AP, which was one of the determining factors in APD and the effective refractory period (Van Wagoner, 2000). In the presence of H2O2, Ikur was reduced in atrial myocytes and the I–V curve of Ikur was shifted downward. There were few reports about Ikur being influenced by H2O2. Only Zhang et al.
(Zhang et al., 2008) revealed that Ikur was influenced by H2O2, which was concentration dependent: H2O2 was in 0.1–0.75 μmol/L, promoted, and in 0.75–10 μmol/L, was inhibited. In the present study, the concentration we used was 50 μmol/L. Results of the present study were in accordance with that. H2O2 inhibited Ikur in atrial myocytes. The mechanism maybe: first, the steady-state activation curve of Ikur was shifted to positive potentials, which made the number of active ion channels decrease in the same voltage channel, so the current was reduced. Second, the steady-state inactivation was shifted to negative potentials, which made the number of inactive ion channels increase in the same voltage channel, so the current was reduced. Third, the time of recovery
Fig. 5. (A) Effects of H2O2 and PIP on IK1. (B) Effects of H2O2 and PIP on peak of IK1. (C) Effects of H2O2 and PIP on the I–V curve of IK1. (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
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Fig. 6. (A) Effects of H2O2 and PIP on IKUr. (B) Effects of H2O2 and PIP on peak of IKUr. (C) Effects of H2O2 and PIP on the I–V curve of IKUr. (D) Effects of H2O2 and PIP on the steady-state activation curve of IKUr. (E) Effects of H2O2 and PIP on the half activation potentials (V1/2) of IKUr. (F) Effects of H2O2 and PIP on the slope factors (k) of IKUr. (Contrast to control group, a P b 0.05;contrast to H2O2 (50 μmol/L), cP b 0.05).
from inactivation slowed, which made prolonged the recovery time, so the current was reduced. Three changes in ion channel dynamic caused Ikur to be reduced. H2O2 was an usual reagent which made an oxidative stress model. Many studies indicated that oxidative stress and atrial fibrillation had
a very close relationship (Lin et al., 2010; Korantzopoulos et al., 2003; Nattel et al., 2008; Huang et al., 2009) So, the inhibited effects on APD, ICa,L, Ito, Ik1 and Ikur by H2O2 may be the mechanism of oxidative stress caused atrial myocytes electrical remodeling and then caused atrial fibrillation.
Fig. 7. (A) Effects of H2O2 and PIP on the steady-state inactivation curve of Ikur. (B) Effects of H2O2 and PIP on the half inactivation potentials (V1/2) of Ikur. (C) Effects of H2O2 and PIP on the slope factors (k) of ITO. (D) Effects of H2O2 and PIP on the time course of recovery from inactivation of ITO (800 ms). (E) Effects of H2O2 and PIP on the time course of recovery from inactivation of Ikur (100 ms). (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
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The present study indicated that PIP could alleviate the abnormalities of APD, ICa,L, Ito, Ik1 and Ikur induced by H2O2 in atrial myocytes. PIP could increase contents of antioxidant in cells, reduce the attack on polyunsaturated fatty acids by oxygen radicals, and decrease lipid peroxidation. Piperine had a free radical scavenging capacity (Mittal and Gupta, 2000). Piperine inhibited lipid peroxide formation and reduced the increase in acid phosphatase in rats injected with carrageenin, a compound known to stimulate lipid peroxide formation (Dhuley et al., 1993). PIP could elevate activities of SOD and CAT, the two enzymes that help to scavenge superoxide ions and hydroxyl ions, increase activities of GST, which can act either to detoxify activated oxygen species such as H2O2 or to reduce lipid peroxides themselves, and also elevate activities of GPx and GSH, which may help to control hydroxyl radicals indirectly or directly (Vijayakumar et al., 2004). So PIP could reduce the damage to cells by oxidative stress reaction (Hu et al., 2009). The present study indicates PIP may prevent or delay the development of atrial remodeling and atrial fibrillation via blocking or delaying the remodeling of currents in atrial myocytes. The underling mechanism may be that PIP could reduce the damage of atrial myocytes induced by oxidative stress. Moreover, oxidative stress and atrial fibrillation have close relationship. So we may have the conclusion that PIP could treat and prevent atrial fibrillation.
Limitation This study does not include the experiment whether PIP rescues the outcome if it is administrated after H2O2. The aim of this study was to explore the protective effect of piperine. We will discuss its reverse effect in further study. Moreover, this study was only involving cell and currents and needs more evidence on animals in further study. In addition, we did not test the effect of different concentration of PIP in this study. However, in a previous study, PIP (7 μmol/L) has been showed to have protective effects on sulfydryl, antioxidant molecules and antioxidase in cells via the inhibition of free radicals, reactive oxygen species (ROS) and lipid peroxidation, so we used PIP (7 μmol/L) in this study.
Conclusions In conclusion, oxidative stress is associated with electric remodeling in atrial myocytes. PIP could protect atrial myocytes from electric remodeling by reducing oxidative stress reaction.
Conflict of interest statement The authors declare that there are no conflicts of interest.
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Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 30772886 and No. 81270308 to Zhao-liang SHAN) and Beijing Natural Science Foundation (No. 7122173 to Zhao-liang SHAN). References Ahmad M, Rahman MW, Rahman MT, Hossain CF. Analgesic principle from the bark of Careya arborea. Pharmazie 2002;57:698–710. Dhamoon AS, Jalife J. The inward rectifier current (IK1) controls cardiac excitability and is involved in arrhythmogenesis. Heart Rhythm 2005;2:316–24. Dhuley JN, Raman PH, Mujumdar AM, Naik SR. Inhibition of lipid peroxidation by piperine during experimental inflammation in rats. Indian J Exp Biol 1993;31:443–5. Hu Y, Guo DH, Liu P, Rahman K, Wang DX, Wang B. Antioxidant effects of a rhodobryum roseum extract and its active components in isoproterenol-induced myocardial injury in rats and cardiac myocytes against oxidative stress-triggered damage. Pharmazie 2009;64:53–7. Huang CX, Liu Y, Xia WF, Tang YH, Huang H. Oxidative stress: a possible pathogenesis of atrial fibrillation. Med Hypotheses 2009;72:466–7. Korantzopoulos P, Kolettis T, Siogas K, Goudevenos J. Atrial fibrillation and electrical remodeling: the potential role of inflammation and oxidative stress. Med Sci Monit 2003;9:RA225–9. Kumar S, Singhal V, Roshan R, Sharma A, Rembhotkar GW, Ghosh B. Piperine inhibits TNF-alpha induced adhesion of neutrophils to endothelial monolayer through suppression of NF-kappaB and IkappaB kinase activation. Eur J Pharmacol 2007;575: 177–86. Li S, Wang C, Li W, Koike K, Nikaido T, Wang MW. Antidepressant-like effects of piperine and its derivative, antiepilepsirine. J Asian Nat Prod Res 2007;9:421–30. Lin YK, Lin FZ, Chen YC, Cheng CC, Lin CI, Chen YJ, et al. Oxidative stress on pulmonary vein and left atrium arrhythmogenesis. Circ J 2010;74:1547–56. Lu Z, Abe J, Taunton J, Lu Y, Shishido T, McClain C, et al. Reactive oxygen species-induced activation of p90 ribosomal S6 kinase prolongs cardiac repolarization through inhibiting outward K+ channel activity. Circ Res 2008 Aug 1;103(3):269–78. Mittal R, Gupta RL. In vitro antioxidants activity of piperine. Methods Find Exp Clin Pharmacol 2000;22:271–4. Nattel S, Burstein B, Dobrev D. Atrial remodeling and atrial fibrillation: mechanisms and implications. Circ Arrhythm Electrophysiol 2008;1:62–73. Park BS, Son DJ, Park YH, Kim TW, Lee SE. Antiplatelet effects of acidamides isolated from the fruits of Piper longum L. Phytomedicine 2007;14:853–5. Pike GK, Bretag AH, Roberts ML. Modification of the transient outward current of rat atrial myocytes by metabolic inhibition and oxidant stress. J Physiol 1993;470: 365–82. Srinivasan K. Black pepper and its pungent principle-piperine: a review of diverse physiological effects. Crit Rev Food Sci Nutr 2007;47:735–48. Tanaka H, Habuchi Y, Suto F, Morikawa J. Effects of hydrogen peroxide on the transient outward current in rabbit atrial myocytess. Clin Exp Pharmacol Physiol 2001;28: 743–7. Van Wagoner DR. Pharmacologic relevance of K channel remodeling in atrial fibrillation. Mol Cell Cardiol 2000;32:1763–6. Vijayakumar RS, Surya D, Nalini N. Antioxidant efficacy of black pepper (Piper nigrum L.) and piperine in rats with high fat diet induced oxidative stress. Redox Rep 2004;9:105–10. Wang Z, Fermini B, Nattel S. Sustained depolarization-induced outward current in human atrial myocytes. Evidence of a novel delayed rectifier K + current similar to Kv 1.5 cloned channel currents. Circ Res 1993;73:1061–76. Ward CA, Giles WR. Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. J Physiol 1997;500:631–42. Zhang GW, Gu TX, Wang C, Yu L, Wen T. Two-way concentration-dependent effect of H2O2 on I(Kur) and I(Ca, L) in human atrial myocytes. Sheng Li Xue Bao 2008;60: 695–703.
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Protective effect of piperine on electrophysiology abnormalities of left atrial myocytes induced by hydrogen peroxide in rabbits Yan Liu a,b,1, Yu Zhang a,c,1, Kun Lin a, De-xian Zhang a,c, Miao Tian d, Hong-yang Guo a, Yu-tang Wang e, Yang Li f,⁎, Zhao-liang Shan a,⁎⁎ a
Department of Cardiology, Chinese PLA General Hospital, Beijing 100853, China Department of Cardiology, Liao He Hospital, Liao Ning 111000, China Nankai University School of Medicine, Nankai University, Tianjin 300071, China d Department of Cardiology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing 100048, China e Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing 100853, China f Institute of Geriatric Cardiology, Chinese PLA General Hospital, Beijing 100853, China b c
a r t i c l e
i n f o
Article history: Received 6 June 2013 Accepted 18 October 2013 Keywords: Piperine Hydrogen peroxide Atrial myocytes Action potential
a b s t r a c t Aims: Piperine had protective effects on oxidative stress damage of ventricular myocytes by hydrogen peroxide (H2O2). In this study we aimed to explore the protective effect of piperine on abnormalities of the cardiac action potential (AP) and several ion currents induced by hydrogen peroxide (H2O2) in single rabbit left atrial myocyte. Main methods: Conventional microelectrodes were used to record action potential duration (APD), resting membrane potential (RMP) and some ion currents (ICa,L,Ito,IK1 and Ikur,ect.), before and after H2O2 administration with or without piperine. Key findings: The piperine (7 μmol/L) had no significant effect on APD, ICa,L,Ito,IK1 and Ikur and their channel dynamics. In the presence of 50 μmol/L H2O2, APD50 and APD90 shortened (P b 0.01), amplitude of RMP decreased (P b 0.05), the peak of ICa,L reduced significantly (P b 0.05). Piperine (7 μmol/L) significantly alleviated the inhibiting effect of H2O2 on APD and ICa,L (P b 0.01) and protected the changes of ICa,L dynamics induced by H2O2. The peak current of Ito was reduced significantly (P b 0.05); Piperine (7 μmol/L) significantly alleviated the inhibiting effect of H2O2 on Ito (P b 0.01). In addition, piperine protected the changes of Ito dynamics induced by H2O2. The peak current of IK1 and IKUr was significantly reduced (P b 0.05); Piperine (7 μmol/L) alleviated the inhibiting effect of H2O2 on IK1 and IKUr significantly (P b 0.01). In addition, piperine protected the changes of IKUr dynamics induced by H2O2. Significance: These results suggest that piperine effectively protects atrial myocytes from oxidative stress injury in atrial electrophysiology. © 2013 Elsevier Inc. All rights reserved.
Introduction Piperine (PIP) widely exists in pepper plants and is a common drug in Chinese Medicine, Mongolian Medicine and Tibetan Medicine. It has protective effects on sulfydryl, antioxidant molecules and antioxidase in cells via the inhibition of free radicals, reactive oxygen species (ROS) and lipid peroxidation (Srinivasan, 2007). The study indicated that PIP had protective effects on oxidative stress damage of ventricular myocytes by hydrogen peroxide (H2O2) (Hu et al., 2009). Piperine exhibited antioxidant action in experimental conditions, both in vivo as well as in vitro, through its radical quenching effect and by preventing GSH depletion (Mittal and Gupta, 2000). Additionally, piperine also possesses antiinflammatory (Kumar et al., 2007), antidepressant (Li et al., ⁎ Corresponding author. Tel./fax: +86 10 66 93 6762. ⁎⁎ Corresponding author. Tel.: +86 10 5549 9210; fax: +86 10 5549 9209. E-mail addresses: [email protected] (Y. Li), [email protected] (Z. Shan). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.10.024
2007), antiplatelet (Park et al., 2007), antipyretic and analgesic (Ahmad et al., 2002) characteristics. The present study was designed to investigate the effects of H2O2 on the cardiac action potential (AP); L-type calcium current (ICa,L), transient outward potassium current (Ito), inward rectifier potassium current (Ik1) and ultra rapid delayed rectifier potassium (Ikur) of rabbits' left atrial myocytes using a whole-cell configuration of the patchclamp technique. Also studied, was the protective effect of PIP. Material and methods Experimental animals Forty New Zealand rabbits (weighing 1–1.5 kg) were used for the study. The investigation conformed to the Guide for the Care and Use of Laboratory Animals issued by the National Committee of Science and Technology of China, and the study was approved by the Animal Investigation Committee of the Chinese PLA General Hospital.
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Drugs and solutions Tyrode's solution (in mmol/L): NaCl 136, KCl 5.4, MgCl2 1.0, NaH2PO4 0.33, CaCl2 1.8, hydroxyethyl PIPerazine ethanesulfonic acid (HEPES) 10, and glucose 10 (pH 7.4). Kreb's buffer solution (in mmol/L): KCl 40, KH2PO4 25, MgSO4 3.0, KOH 80, L-glutamine 50.3, taurine 20, HEPES 10, glucose 10, and EGTA 0.5 (pH 7.4). For AP recording, the internal pipette solution contained (in mmol/L) KCl 120, NaCl 10, CaCl2 1, MgATP 5, EGTA 11 and HEPES 10 (pH 7.4). For ICa-L recording, the internal pipette solution contained (in mmol/L) CsCl 140, Na2ATP 5.0, TEA-Cl 10, EGTA 10, MgCl2 3.0, and HEPES 10 (pH 7.4), while the bath solution contained (in mmol/L) choline chloride 137, CsCl 4.6, CaCl2 1.8, MgCl2 1.0, TEA-Cl 10, glucose 10, 4-AP 5.0, and HEPES 5.0 (pH 7.4). For potassium current recording, the internal PIPette solution contained (in mmol/L) KCl 45, K-aspartate 85, Na-pyruvate 5, MgATP 5.0, EGTA 10, HEPES 10, glucose 11 (pH 7.4), while the bath solution contained (in mmol/L) N-methylD-glucamine (NMG) 149,MgCl2 5,CaCl2 0.65,HEPES 5. For ICa,L recording, 4-aminopyridine (4-AP) (50 μmol/L), Dofetilide (5 nmol/L) and tetraodontoxin (TTX) (100 μmol/L) were added to the superfusion to block IKUr, IKr, and INa. For Ito and IKur recording, BaCl2 (200 mM) and CdCl2 (200 mM) were added to the superfusion to block IK1 and ICa. For IK1 recording, Dof (5 nmol/L), TTX (100 μmol/L) and CdCl2 (200 mM) were added to the superfusion to block IKr, INa and ICa. Single atrial myocyte preparation Left atrial myocytes were isolated enzymatically from the atrium of New Zealand rabbits (weighing 1.0–1.5 kg) as previously described.8–10 Briefly, the rabbit was anesthetized with an iv. injection of sodium pentobarbital (40 mg/kg), and then was heparinized (300 U/kg i.p.). The heart was excised immediately and mounted on a Langendorff apparatus. The heart was then perfused retrogradely via the aorta with oxygenated, calcium-free Tyrode's solution for 5 min, and with the Tyrode's solution containing 1.4 mg/mL of type II collagenase (Invitrogen, USA) and 0.24 mg/mL of trypsinase (Merck, Germany) for 15–20 min. Subsequently, the left atrial appendage was cut into small pieces in a dish containing Kreb's buffer solution and shaken gently to ensure the dispersion of dissociated cardiac myocytes. The cells were kept at 4 °C in Kreb's buffer solution. All the solutions were continuously gassed with 95% O2 and 5% CO2 and were maintained at 37 °C. Pharmacology Cells were divided into 4 groups: in the control group, cells were cultured without treatment for 1.5 h (n = 15); in the PIP group, cells were cultured with PIP (7 μmol/L) for 1.5 h (n = 15); in the H2O2 group, cells were cultured without treatment for 1 h, then with H2O2 for 0.5 h (n = 15); in the PIP + H2O2 group, cells were cultured with PIP (7 μmol/L) for 1 h and then cultured with H2O2 for 0.5 h (n = 15). Electrophysiological recording Membrane currents were measured using a whole-cell configuration of the patch-clamp technique with Axonpatch 700B amplifiers (Axon Instruments, USA). To stabilize the current, experiments were performed 5 min after entering the whole-cell configuration. All the measurements were obtained at a temperature of 22 °C. Parameters of the AP were measured with 2-Hz electrical stimuli before and after drug administration in the LA without spontaneous activity, which was measured after 10 min of superfusion. The AP amplitude (APA) was obtained from the resting membrane potential (RMP) or maximum diastolic potential to the peak of AP depolarization. The AP durations at a repolarization of 90%, and 50% of the APA were measured as APD90, and APD50, respectively. The maximum upstroke velocity was acquired
using the maximum positive value of the first derivative of the AP. Spontaneous activity was defined as the constant occurrence of spontaneous APs in the absence of any electrical stimuli. Burst firing was defined as the occurrence of accelerated spontaneous potential (faster than the basal rate) with sudden onset and termination. The resistance of Pipettes in the bath solution ranged from 2 M to 5 M. No leak subtraction was applied during recording. The current densities were calculated by dividing the current amplitudes by the cell capacitance. Voltage dependence of channel activation or inactivation was obtained by fitting normalized curves with the Boltzmann function. Time course of recovery from inactivation (τ) was quantified by fitting measured data with a 1-exponential function. Statistical analysis Statistical analysis was performed with SPSS version 13.0 (Chicago, IL, USA). Data are presented as mean ± standard deviation. ANOVA was used for multiple-group comparisons, followed by a Bonferronicorrected t-test. A 2-tailed value of P b 0.05 was taken as statistically significant. Results The effects of H2O2 and PIP on AP H2O2 (50 μmol/L) could shorten atrial myocytes' APD50 and APD90 and decrease the amplitude of the resting membrane potential (RMP). PIP mitigate the changes partly. The AP amplitude (APA) of 4 groups changed little (Fig. 1 and Table 1). The effects of H2O2 and PIP on ICa,L Fig. 2A, B shows H2O2 (50 μmol/L) reduced the peak of ICa,L density from (−18.3 ± 1.2) pA/pF to (−9.3 ± 0.9) pA/pF at the test potential of 0 mV (P b 0.01). PIP alleviated the changes (−11.9 ± 1.2 pA/pF). H2O2 (50 μmol/L) made the I–V curve shift upward, but PIP alleviated the changes (Fig. 2C). In the presence of 50 μmol/L H2O2, the steadystate activation curve shifted to a more positive potential, the half activation potentials (V1/2) at which 50% of channels activated were from (−34.64 ± 2.15) mV to (−20.34 ± 2.20) mV (P b 0.01), but PIP made the V1/2 change to (−28.81 ± 3.95) mV, and the slope factors (k) of 4 groups were not changed, these may reveal that H2O2 reduced the current via decreasing the channels activation (Fig. 2D). In the presence of 50 μmol/L H2O2, the steady-state inactivation curve was shifted to a more negative potential, the half inactivation potentials (V1/2) at which 50% of channels inactivated were from (− 19.12 ± 0.67) mV to (− 29.47 ± 1.82) mV (P b 0.01), but PIP made the V1/2 change to (− 25.94 ± 1.74) mV, and the slope factors (k) of 4 groups were not changed, these may reveal that H2O2 reduced the current via increasing the channels inactivation (Fig. 2E). However, as in the examples shown in Fig. 2F, the time course of recovery from inactivation did not change. PIP alone did not influence these parameters of ICa,L significantly (Fig. 2). The effects of H2O2 and PIP on Ito Fig. 3A, B shows H2O2 (50 μmol/L) reduced the peak of Ito density from (42.6.3 ± 3.0) pA/pF to (20.8 ± 2.0) pA/pF (P b 0.05, n = 15). PIP alleviated the changes (31.8 ± 2.7 pA/pF). H2O2 (50 μmol/L) made the I–V curve shifted downward, but PIP alleviated the changes (Fig. 3C). In the presence of 50 μmol/L H2O2, the steady-state activation curve was shifted to a more positive potential (Fig. 3D), the half activation potentials (V1/2) at which 50% of channels activated were from (− 31.51 ± 2.27) mV to (− 19.62 ± 1.53) mV (P b 0.01), but PIP made the V1/2 change to (− 26.86 ± 0.53) mV (Fig. 3E), and the slope factors (k) of 4 groups were not changed (Fig. 3F), these may
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Fig. 1. Effects of H2O2 and PIP on AP. H2O2 (50 μmol/L) could shorten atrial myocytes' APD50 and APD90 and decrease the amplitude of the resting membrane potential (RMP). PIP could partly put back the changes. The AP amplitude (APA) of 4 groups was changed little. (n = 15).
reveal that H2O2 reduced the current via decreasing the channels activation. As in the examples shown in Fig. 4A, in the presence of 50 μmol/L H2O2, the steady-state inactivation curve was shifted to a more negative potential, but it was not significant. The half inactivation potentials (V1/2), the slope factors (k) and the time course of recovery from inactivation of 4 groups were not changed (Fig. 4B, C, D). The closedstate inactivation of Ito was accelerated in the presence of 50 μmol/L H2O2, and PIP alleviated the changes (Fig. 4E). PIP alone has no significant influence on these parameters of ICa,L (Fig. 3). The effects of H2O2 and PIP on IK1 H2O2 (50 μmol/L) reduced the peak of IK1 density from (−148.2 ± 16.7) pA/pF to (−64.2 ± 9.8) pA/pF (P b 0.01, n = 15). PIP alleviated the changes (−113.8 ± 15.4 pA/pF) (Fig. 5A, B). H2O2 (50 μmol/L) made the I–V curve shifted upward, but PIP alleviated the changes (Fig. 5C). The effects of H2O2 and PIP on IKUr H2O2 (50 μmol/L) reduced the peak of IKUr density from (16.0 ± 2.1) pA/pF to (6.1 ± 1.4) pA/pF (P b 0.05, n = 15). PIP alleviated the changes (11.3 ± 1.8 pA/pF) (Fig. 6A, B). H2O2 (50 μmol/L) made the I–V curve shifted downward, but PIP alleviated the changes (Fig. 6C). In the presence of 50 μmol/L H2O2, the steady-state activation curve was shifted to a more positive potential (Fig. 6D), the half activation potentials (V1/2) at which 50% of channels activated were from (−11.55 ± 0.78) mV to (−1.07 ± 0.08) mV (P b 0.01), but PIP made the V1/2 change to (−3.67 ± 0.26) mV (Fig. 6E), and the slope factors (k) of 4 groups were not changed (Fig. 6F). In the presence of 50 μmol/L H2O2, the steady-state inactivation curve was shifted to a more negative potential (Fig. 7A). The half inactivation potentials (V1/2) decreased from (− 6.76 ± 0.94) mV to (− 15.87 ± 0.62) mV (P b 0.01), but PIP elevated the V1/2 change
Table 1 Comparison of parameters of AP influenced by H2O2 and PIP.
APD50(ms) APD90(ms) RMP(mV) APA(mV)
Control
H2O2
H2O2 + PIP
PIP
110.3 ± 9.3 251.7 ± 13.5 −79.1 ± 2.9 115.3 ± 6.9
77.6 ± 6.2⁎ 202.1 ± 11.6⁎ −67.6 ± 3.2⁎ 107.6 ± 5.2
100.7 ± 8.8⁎⁎ 247.5 ± 15.5⁎⁎ −80.4 ± 3.4⁎⁎ 113.2 ± 5.4
118.4 ± 7.8⁎⁎ 243.0 ± 10.1⁎⁎ −80.5 ± 3.0⁎⁎ 118.3 ± 4.8
AP, action potential; APD, action potential duration; RMP, resting member potential; APA, action potential amplitude. ⁎P b 0.05 vs control group; ⁎⁎P b 0.05 vs H2O2 group; ⁎⁎⁎P b 0.05 vs H2O2 + PIP group.
to (− 11.23 ± 1.17) mV (Fig. 7B), the slope factors (k) were from (9.83 ± 0.57) mV to (12.46 ± 0.42) mV (P b 0.01), but PIP made the slope factors (k) change to (11.27 ± 0.57) mV (Fig. 7C). The time course of recovery from inactivation was decelerated in the presence of 50 μmol/L H2O2, especially in 100 ms, but PIP regained the changes (Fig. 7D, E). PIP alone did not influence these parameters of ICa,L significantly (Fig. 7). Discussion Low concentration of H2O2 is an usual reagent to made a oxidative stress model (Lin et al., 2010), so we used H2O2 (50 μmol/L) in our experiments. PIP (7 μmol/L) has been showed to have protective effects on sulfydryl, antioxidant molecules and antioxidase in cells via the inhibition of free radicals, reactive oxygen species (ROS) and lipid peroxidation (Hu et al., 2009), so we used PIP (7 μmol/L) in this study. In the present study, we found that H2O2 decrease APD50, APD90, ICa,L,Ito,Ik1 and Ikur, but PIP could partly regain these changes. H2O2 decreased ICa,L and APD, so the mechanism of that maybe: first, in the presence of H2O2, membrane structure of rabbit atrial myocytes changes. These changes made the steady-state activation curve of ICa,L shift to positive potentials; channel activation slowed, the steady-state inactivation was shifted to negative potentials, and channel inactivation accelerated. So the number of ion channels decreased in the same voltage and the ICa,L decreased. Second, the previous studies show when cells were damaged by oxidative stress, the damaged of mitochondria by oxygen radical made an obstruction of ATP produced. The activity of Na+ pump and Ca2+pump decreased, which were dependent on ATP, so concentration of Na+ in cells increased, exchanged of Na+ and Ca2+ increased, inflow of Ca2+ increased (Korantzopoulos et al., 2003), and the concentration of Ca2+ increased which could inhibit ICa,L. ICa,L is the main current in the second plateau of the Aps (Nattel et al., 2008), and it was the inward current. The decreased current accelerated repolarization in atrial myocytes, then APD was shortened. Lin et al. (2010) revealed that H2O2 could shorten APD of the left atrial myocytes, which was in conformity with our study. A shortened APD may cause arrhythmias such as atrial fibrillation, especially in atrial myocytes. H2O2 decreased IK1 and the amplitude of RMP. IK1 stabilized the resting membrane potential and played a fundamental role in the terminal phase of the action potential's repolarization in the cardiac myocytes (Dhamoon and Jalife, 2005). In the present study, H2O2 could inhibit IK1. Decreases in IK1 caused the amplitude of RMP to decrease. So H2O2 caused RMP to decrease. There were not so many reports of IK1 influencing H2O2. Ward and Giles (1997) revealed that H2O2 had no effects on IK1. But they were in ventricular myocytes. In atrial myocytes, the study firstly revealed that H2O2 inhibited IK1 in atrial myocytes. These
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Fig. 2. (A) Effects of H2O2 and PIP on ICa,L. (B) Effects of H2O2 and PIP on peak of ICa,L. (C) Effects of H2O2 and PIP on the I–V curve of ICa,L. (D) Effects of H2O2 and PIP on the steady-state activation curve of ICa,L. (E) Effects of H2O2 and PIP on the steady-state inactivation curve of ICa,L. (F) Effects of H2O2 and PIP on the time course of recovery from inactivation of ICa,L. (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
changes could cause a decreased amplitude of RMP, which made the threshold stimulus to be decreased. And then excitability of atrial myocytes increased. Another, IK1 was the background current, so its reduction could make automaticity of atrial myocytes increase. Those two effects cause excitability and automaticity of atrial myocytes to be increased, which would easily cause arrhythmias such as atrial fibrillation, especially in atrial myocytes. H2O2 inhibited Ito and changed the I–V curve of Ito. In the presence of H2O2, the steady-state activation curve was shifted to more a positive potential, which may reveal that H2O2 reduced the current via decreasing
the channel activation, and the closed-state inactivation of Ito was accelerated, which may reveal that H2O2 reduced the current via decreasing the number of open channels at the same time. Those two effects made the current decline. Studies of Pike Pike et al. (1993) and Lu et al. (2008) were in conformity with our study. Ward and Giles (Ward and Giles, 1997) revealed that H2O2 had no effects on Ito, but Tanaka et al. (Tanaka et al., 2001) revealed that H2O2 had an increased effect on Ito. I think the cause of this difference may be the different methods of patch plant technique. Ward and Tanaka used the amphotericin B-perforated patch voltage-clamp, but Pike, Lu and we used the
Fig. 3. (A) Effects of H2O2 and PIP on Ito. (B) Effects of H2O2 and PIP on peak of Ito. (C) Effects of H2O2 and PIP on the I–V curve of Ito. (D) Effects of H2O2 and PIP on the steady-state activation curve of Ito. (E) Effects of H2O2 and PIP on the half activation potentials (V1/2) of Ito. (F) Effects of H2O2 and PIP on the slope factors (k) of Ito. (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
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Fig. 4. (A) Effects of H2O2 and PIP on the steady-state inactivation curve of Ito. (B) Effects of H2O2 and PIP on the half inactivation potentials (V1/2) of Ito. (C) Effects of H2O2 and PIP on the slope factors (k) of Ito. (D) Effects of H2O2 and PIP on the time course of recovery from inactivation of Ito. (E) Effects of H2O2 and PIP on the closed-state inactivation of Ito. (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
conventional whole cell patch plant technique. Ito played an important role in the phase 1 of AP, which decreased Ito in atrial myocytes and was an important characteristic in the atrial electric remodeling, though the changes had no specifically significance in the clinical results. Ikur was a current expressed in atrial myocytes, not ventricular myocytes (Wang et al., 1993). Ikur was an outward current in the phase 2 of AP and could promote the phase 3 of AP, which was one of the determining factors in APD and the effective refractory period (Van Wagoner, 2000). In the presence of H2O2, Ikur was reduced in atrial myocytes and the I–V curve of Ikur was shifted downward. There were few reports about Ikur being influenced by H2O2. Only Zhang et al.
(Zhang et al., 2008) revealed that Ikur was influenced by H2O2, which was concentration dependent: H2O2 was in 0.1–0.75 μmol/L, promoted, and in 0.75–10 μmol/L, was inhibited. In the present study, the concentration we used was 50 μmol/L. Results of the present study were in accordance with that. H2O2 inhibited Ikur in atrial myocytes. The mechanism maybe: first, the steady-state activation curve of Ikur was shifted to positive potentials, which made the number of active ion channels decrease in the same voltage channel, so the current was reduced. Second, the steady-state inactivation was shifted to negative potentials, which made the number of inactive ion channels increase in the same voltage channel, so the current was reduced. Third, the time of recovery
Fig. 5. (A) Effects of H2O2 and PIP on IK1. (B) Effects of H2O2 and PIP on peak of IK1. (C) Effects of H2O2 and PIP on the I–V curve of IK1. (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
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Fig. 6. (A) Effects of H2O2 and PIP on IKUr. (B) Effects of H2O2 and PIP on peak of IKUr. (C) Effects of H2O2 and PIP on the I–V curve of IKUr. (D) Effects of H2O2 and PIP on the steady-state activation curve of IKUr. (E) Effects of H2O2 and PIP on the half activation potentials (V1/2) of IKUr. (F) Effects of H2O2 and PIP on the slope factors (k) of IKUr. (Contrast to control group, a P b 0.05;contrast to H2O2 (50 μmol/L), cP b 0.05).
from inactivation slowed, which made prolonged the recovery time, so the current was reduced. Three changes in ion channel dynamic caused Ikur to be reduced. H2O2 was an usual reagent which made an oxidative stress model. Many studies indicated that oxidative stress and atrial fibrillation had
a very close relationship (Lin et al., 2010; Korantzopoulos et al., 2003; Nattel et al., 2008; Huang et al., 2009) So, the inhibited effects on APD, ICa,L, Ito, Ik1 and Ikur by H2O2 may be the mechanism of oxidative stress caused atrial myocytes electrical remodeling and then caused atrial fibrillation.
Fig. 7. (A) Effects of H2O2 and PIP on the steady-state inactivation curve of Ikur. (B) Effects of H2O2 and PIP on the half inactivation potentials (V1/2) of Ikur. (C) Effects of H2O2 and PIP on the slope factors (k) of ITO. (D) Effects of H2O2 and PIP on the time course of recovery from inactivation of ITO (800 ms). (E) Effects of H2O2 and PIP on the time course of recovery from inactivation of Ikur (100 ms). (Contrast to control group, aP b 0.05; contrast to H2O2 (50 μmol/L), cP b 0.05).
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The present study indicated that PIP could alleviate the abnormalities of APD, ICa,L, Ito, Ik1 and Ikur induced by H2O2 in atrial myocytes. PIP could increase contents of antioxidant in cells, reduce the attack on polyunsaturated fatty acids by oxygen radicals, and decrease lipid peroxidation. Piperine had a free radical scavenging capacity (Mittal and Gupta, 2000). Piperine inhibited lipid peroxide formation and reduced the increase in acid phosphatase in rats injected with carrageenin, a compound known to stimulate lipid peroxide formation (Dhuley et al., 1993). PIP could elevate activities of SOD and CAT, the two enzymes that help to scavenge superoxide ions and hydroxyl ions, increase activities of GST, which can act either to detoxify activated oxygen species such as H2O2 or to reduce lipid peroxides themselves, and also elevate activities of GPx and GSH, which may help to control hydroxyl radicals indirectly or directly (Vijayakumar et al., 2004). So PIP could reduce the damage to cells by oxidative stress reaction (Hu et al., 2009). The present study indicates PIP may prevent or delay the development of atrial remodeling and atrial fibrillation via blocking or delaying the remodeling of currents in atrial myocytes. The underling mechanism may be that PIP could reduce the damage of atrial myocytes induced by oxidative stress. Moreover, oxidative stress and atrial fibrillation have close relationship. So we may have the conclusion that PIP could treat and prevent atrial fibrillation.
Limitation This study does not include the experiment whether PIP rescues the outcome if it is administrated after H2O2. The aim of this study was to explore the protective effect of piperine. We will discuss its reverse effect in further study. Moreover, this study was only involving cell and currents and needs more evidence on animals in further study. In addition, we did not test the effect of different concentration of PIP in this study. However, in a previous study, PIP (7 μmol/L) has been showed to have protective effects on sulfydryl, antioxidant molecules and antioxidase in cells via the inhibition of free radicals, reactive oxygen species (ROS) and lipid peroxidation, so we used PIP (7 μmol/L) in this study.
Conclusions In conclusion, oxidative stress is associated with electric remodeling in atrial myocytes. PIP could protect atrial myocytes from electric remodeling by reducing oxidative stress reaction.
Conflict of interest statement The authors declare that there are no conflicts of interest.
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Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 30772886 and No. 81270308 to Zhao-liang SHAN) and Beijing Natural Science Foundation (No. 7122173 to Zhao-liang SHAN). References Ahmad M, Rahman MW, Rahman MT, Hossain CF. Analgesic principle from the bark of Careya arborea. Pharmazie 2002;57:698–710. Dhamoon AS, Jalife J. The inward rectifier current (IK1) controls cardiac excitability and is involved in arrhythmogenesis. Heart Rhythm 2005;2:316–24. Dhuley JN, Raman PH, Mujumdar AM, Naik SR. Inhibition of lipid peroxidation by piperine during experimental inflammation in rats. Indian J Exp Biol 1993;31:443–5. Hu Y, Guo DH, Liu P, Rahman K, Wang DX, Wang B. Antioxidant effects of a rhodobryum roseum extract and its active components in isoproterenol-induced myocardial injury in rats and cardiac myocytes against oxidative stress-triggered damage. Pharmazie 2009;64:53–7. Huang CX, Liu Y, Xia WF, Tang YH, Huang H. Oxidative stress: a possible pathogenesis of atrial fibrillation. Med Hypotheses 2009;72:466–7. Korantzopoulos P, Kolettis T, Siogas K, Goudevenos J. Atrial fibrillation and electrical remodeling: the potential role of inflammation and oxidative stress. Med Sci Monit 2003;9:RA225–9. Kumar S, Singhal V, Roshan R, Sharma A, Rembhotkar GW, Ghosh B. Piperine inhibits TNF-alpha induced adhesion of neutrophils to endothelial monolayer through suppression of NF-kappaB and IkappaB kinase activation. Eur J Pharmacol 2007;575: 177–86. Li S, Wang C, Li W, Koike K, Nikaido T, Wang MW. Antidepressant-like effects of piperine and its derivative, antiepilepsirine. J Asian Nat Prod Res 2007;9:421–30. Lin YK, Lin FZ, Chen YC, Cheng CC, Lin CI, Chen YJ, et al. Oxidative stress on pulmonary vein and left atrium arrhythmogenesis. Circ J 2010;74:1547–56. Lu Z, Abe J, Taunton J, Lu Y, Shishido T, McClain C, et al. Reactive oxygen species-induced activation of p90 ribosomal S6 kinase prolongs cardiac repolarization through inhibiting outward K+ channel activity. Circ Res 2008 Aug 1;103(3):269–78. Mittal R, Gupta RL. In vitro antioxidants activity of piperine. Methods Find Exp Clin Pharmacol 2000;22:271–4. Nattel S, Burstein B, Dobrev D. Atrial remodeling and atrial fibrillation: mechanisms and implications. Circ Arrhythm Electrophysiol 2008;1:62–73. Park BS, Son DJ, Park YH, Kim TW, Lee SE. Antiplatelet effects of acidamides isolated from the fruits of Piper longum L. Phytomedicine 2007;14:853–5. Pike GK, Bretag AH, Roberts ML. Modification of the transient outward current of rat atrial myocytes by metabolic inhibition and oxidant stress. J Physiol 1993;470: 365–82. Srinivasan K. Black pepper and its pungent principle-piperine: a review of diverse physiological effects. Crit Rev Food Sci Nutr 2007;47:735–48. Tanaka H, Habuchi Y, Suto F, Morikawa J. Effects of hydrogen peroxide on the transient outward current in rabbit atrial myocytess. Clin Exp Pharmacol Physiol 2001;28: 743–7. Van Wagoner DR. Pharmacologic relevance of K channel remodeling in atrial fibrillation. Mol Cell Cardiol 2000;32:1763–6. Vijayakumar RS, Surya D, Nalini N. Antioxidant efficacy of black pepper (Piper nigrum L.) and piperine in rats with high fat diet induced oxidative stress. Redox Rep 2004;9:105–10. Wang Z, Fermini B, Nattel S. Sustained depolarization-induced outward current in human atrial myocytes. Evidence of a novel delayed rectifier K + current similar to Kv 1.5 cloned channel currents. Circ Res 1993;73:1061–76. Ward CA, Giles WR. Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. J Physiol 1997;500:631–42. Zhang GW, Gu TX, Wang C, Yu L, Wen T. Two-way concentration-dependent effect of H2O2 on I(Kur) and I(Ca, L) in human atrial myocytes. Sheng Li Xue Bao 2008;60: 695–703.
