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KCNE1 (IKs) potassium channels
European Journal of Pharmacology 590 (2008) 317–321
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Tanshinone IIA: A new activator of human cardiac KCNQ1/KCNE1 (IKs) potassium channels Dong-Dong Sun a,1, Hai-Chang Wang a,1, Xiao-Bin Wang b, Ying Luo b, Zhen-Xiao Jin c, Zhi-Chao Li b, Gui-Rong Li d, Ming-Qing Dong b,⁎ a
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China Department of Pathology and Pathophysiology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China Department of Cardiovascular Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China d Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China b c
A R T I C L E
I N F O
Article history: Received 21 December 2007 Received in revised form 21 May 2008 Accepted 2 June 2008 Available online 7 June 2008 Keywords: Tanshinone IIA hKCNQ1/hKCNE1 IKs Kv1.5 hERG IK1 HEK 293 cell Potassium channel Ion channel Patch clamp Human
A B S T R A C T Tanshinone IIA, one of the main active components from Chinese herb Danshen, is widely used to treat cardiovascular diseases including arrhythmia in Asian countries especially in China. However, the mechanisms underlying its anti-arrythmia effects are not clear. In this study we investigate the effects of tanshinone IIA on human KCNQ1/KCNE1 potassium channels (IKs), human ether-a-go-go-related gene potassium channels (hERG), Kv1.5 potassium channels, inward rectifier potassium channels (IK1) expressed in HEK 293 cells using patch clamp technique. Tanshinone IIA potently and reversibly enhanced the amplitude of IKs in a concentration dependent manner with an EC50 of 64.5 μM, accelerated the activation rate of IKs channels, decelerated their deactivation and shifted the voltage dependence of IKs activation to negative direction. Isoproteronol, a stimulator of β-adrenergic receptor, at 1 μM and sodium nitroprusside (SNP), a NO donor, at 1 mM, had no significant effects on the enhancement of IKs by 30 μM tanshinone IIA. N-[2-(pbromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), a selective protein kinase A inhibitor, at 0.1 μM and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), a selective nitric oxide-sensitive guanylyl cyclase inhibitor, at 10 μM, also had no significant effects on the enhancement of IKs by 30 μM tanshinone IIA. Tanshinone IIA did not affect expressed hERG channels, Kv1.5 channels and IK1 channels. These results indicate that tanshinone IIA directly and specifically activate human cardiac KCNQ1/KCNE1 potassium channels (IKs) in HEK 293 cell through affecting the channels' kinetics. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The slowly activating delayed rectifier potassium current IKs plays an important role in the repolarization of human cardiac action potential. It has been demonstrated that IKs is a heteromultimeric complex composed of pore-forming α subunit and accessory β subunit encoded, respectively, by the KCNQ1 and KCNE1 genes (Barhanin et al., 1996; Sanguinetti et al., 1996). Loss or reduction of IKs channels function, due to genetic mutations in the KCNQ1 and KCNE1 genes or electrical remodeling in diseased heart (Barhanin et al., 1996; Li et al., 2004; Nabauer and Kaab, 1998; Xu et al., 2001), induces a decrease in net repolarizing current. The reduced repolarization reserve results in delayed repolarization, lengthened action
⁎ Corresponding author. Department of Pathology and Pathophysiology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China. Tel.: +86 29 84773105; fax: +86 29 84774548. E-mail address: [email protected] (M.-Q. Dong). 1 Contribute equally to the paper. 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.06.005
potential duration, early after depolarization and prolongation of QT interval in the electrocardiogram. All these alterations increase the risk of life-threatening ventricular arrhythmia for the affected persons. Activators of IKs are promising candidates to regain IKs channel function and treat the IKs dysfunction related arrhythmia, thus it is potentially important in the clinical setting if a drug is found to be able to specifically enhance IKs. However, only a few compounds are found to have activating effects on IKs (Salata et al., 1998; Seebohm et al., 2003; Bai et al., 2004; Zhang et al., 2006). Danshen, a dried root of Salvia miltiorrhiza, is a traditional Chinese medicinal herb. It has been widely used to treat many kinds of diseases for thousands of years in China. Tanshinone IIA is one of the main active components from Danshen, exhibiting a variety of cardiovascular effects including vasorelaxation, protection against ischemia– reperfusion injury and antiarrhythmic effects (Gao et al., 2008; Hu et al., 1981; Zhou et al., 2005). It is widely used to treat angina pectoris, hyperlipidemia, acute ischemic stroke and arrhythmia in Asian countries especially in China. Although its success in clinics for treating cardiovascular diseases, the exact mechanism underlying its
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therapeutic basis especially for arrhythmia is poorly understood. Recently, tanshinone IIA was found to shorten action potential duration in rat heart, inhibit L-type Ca2+ current in guinea pig ventricular myocytes, inhibit IK1 currents in rat ventricular myocytes (Xu et al., 1997; Zhu et al., 2005), which is regarded as the partial mechanism of its anti-arrhythmia effects. However, there are at present no published reports in which effects of tanshinone IIA on human cardiomyocyte potassium currents have been studied. In the present study, therefore, we determine whether tanshinone IIA regulates recombinant human cardiac IKs (hKCNQ1/hKCNE1), hERG (human ether-a-go-go-related gene), IK1 (Kir2.1) and Kv1.5 channels expressed in HEK 293 cells. The results demonstrate that tanshinone IIA potently and specifically enhances IKs through affecting the channels' kinetics and its effect is independent of protein kinase A (PKA) activation, protein kinase G (PKG) activation and channel nitrosylation. 2. Materials and methods 2.1. Cell culture HEK 293 cell lines stably expressing human cardiac KCNQ1/KCNE1 channel (IKs), human cardiac hERG channel (hERG), human cardiac Kv1.5 channel (Kv1.5), human cardiac Kir2.1 channel (IK1) respectively were cultured at 37 °C, 5% CO2 and 95% air in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum and 200 μg/ml hygromycin (for IKs) or 1 mg/ml G418 (for hERG, Kv1.5 and IK1), and subcultured every 3–4 days. For patch clamp experiments, the cells were subcultured on a coverslip and incubated overnight then transferred to an open chamber for currents recording. 2.2. Solutions and drugs The standard Tyrode's solution contained (in mM): NaCl 140, KCl 5.0, MgCl2 1.0, CaCl2 1.0, NaH2PO4 0.33, HEPES 5.0, glucose 10, pH adjusted to 7.4 with NaOH. The pipette solution contained (in mM): KCl 20, K-aspartate 110, MgCl2 1.0, HEPES 10, EGTA 5.0, and GTP 0.1, Na2-phosphocreatine 5.0, Mg2-ATP 5.0, pH adjusted to 7.2 with KOH. In perforated patch recording mode, 200 μg/ml amphotericin B (Sigma-Aldrich) was added into the pipette solution. Tanshinone IIA (sulfonate, purity is 99%) was purchased from Xi'an Honson Biotechnology Co. Ltd (Xi`an, China). All other chemicals are from Sigma. Tanshinone IIA, isoproterenol and sodium nitroprusside (SNP) were freshly prepared and kept in the dark at 4 °C. N-[2-(pbromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) were prepared as 5 and 20 mM stock solution in dimethylsulfoxide (DMSO) respectively.
200B amplifier and Clampex 8.0 software (Axon Instruments). Current signals were low-pass filtered at 2 kHz and stored on the hard disk of an IBM computer. Values are presented as mean ± S.E.M. Nonlinear curve fitting was performed using Clampfit 9.0 (Axon) and Sigmaplot (SPSS Science, Chicago, IL, U.S.A.). Paired Student's t-tests were used to evaluate the statistical significance of differences between two group means. ANOVA was used for multiple groups. Values of P ≤ 0.05 were considered statistically significant. 3. Results 3.1. Concentration-dependent enhancement of IKs by tanshinone IIA The effects of tanshinone IIA on IKs stably expressed in HEK 293 were shown in Fig. 1. IKs currents were elicited by a 3-s depolarization pulse from holding potential of −80 mV to various test potentials between −60 mV and +60 mV in a 20 mV step, then back to −40 mV for 3-s at 0.05 Hz. IKs were measured from zero level to the current at the end of 3-s depolarization step voltage. Fig. 1A shows the original traces of IKs recorded in one representative cell in the absence and in the presence of tanshinone IIA at 10 μM, 100 μM and after washout of the drug respectively. Tanshinone IIA substantially enhanced IKs and the effect was partially washed out. Fig. 1B shows the time course of 30 μM tanshinone IIA on IKs in a representative cell. IKs was elicited by continuously applying 3-s depolarization pulses from holding potential of −80 mV to test potential of +40 mV in a 20-s interval. IKs gradually increased after drug application, reached to its maximal effect in 2 min and recovered upon the drug washout for about 3 min. The original traces at corresponding time points are shown in the right inset of the panel. Fig. 2A displays the I–V relationships of mean values of IKs step current in the absence and presence of 10 μM and 100 μM tanshinone IIA and washout for 5 min. Tanshinone IIA significantly increased IKs at
2.3. Data acquisition and analysis The experiments were conducted at room temperature (25 °C). The whole-cell and perforated patch-clamp techniques were used as described previously. Briefly, the borosilicate glass electrodes (1.5 mm OD) were pulled with Brown–Flaming puller (model P-97, Sutter Instrument Co., Novato, CA, U.S.A.) and had tip resistances of 1–2 MΩ when filled with pipette solution. 3 M KCl–agar salt bridge was used as reference electrode. Liquid junction potentials were compensated before the pipette touched the cell. After giga seal was obtained, the cell membrane was ruptured by gentle suction to establish the wholecell configuration to record hERG, Kv1.5 and IK1. Perforated patch configuration was used to record IKs to minimize rundown of the current. The series resistance was electrically compensated to minimize voltage errors. The membrane currents were converted to digital signals with Digidata 1200 (Axon Instruments, Forster City, CA, U.S.A) at a sampling rate of 10 kHz and recorded with an Axonpatch
Fig. 1. Effects of tanshinone IIA on I Ks stably expressed in HEK 293 cells. (A) Representative original IKs traces recorded in one typical cell under control condition, in the presence of 10 μM, 100 μM tanshinone IIA and after washout of the drug respectively. IKs was induced with 3 - s voltage step from −60 mV to +60 mV then back to −40 mV as shown in the inset. (B) Time course of normalized IKs step current recorded at + 40 mV after a 3-s voltage step to + 40 from −80 mV as shown in right inset. Original superimposed IKs current traces at corresponding time points indicated by lower-case italic alphabets are shown at the right of the panel. In this and the following figures, TAN represents tanshinone IIA.
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to +60 mV (all n = 7, P b 0.01 or P b 0.05); however, the deactivation time constant was increased by tanshinone IIA at potentials of −90 mV to −40 mV (all n = 7, P b 0.01 or P b 0.05). 3.4. Effects of β-adrenergic receptor stimulator, NO donor, PKA inhibitor and guanylate cyclase inhibitor on tanshinone IIA induced I Ks enhancement
Fig. 2. Concentration dependent effects of tanshinone IIA on IKs. (A) I–V relationships of IKs step currents under control condition, in the presence of 10 μM, 100 μM tanshinone IIA, and washout. Tanshinone IIA significantly enhanced IKs with increasing concentration and the enhancement was almost reversed upon washout. (B) Concentration– response curve of IKs step current enhanced by tanshinone IIA at +40 mV. Symbols are the mean values of enhancement. Solid lines are the best fit Hill equation: E = Emax / [1 + (EC50 / C)b], where E is the effect at concentration C, Emax is the maximal effect, EC50 is the concentration for half-maximal effect, and b is the Hill coefficient. IC50 was 64.5 μM, b was 0.66, and Emax was 151.8%.
test potentials of −20 mV to +60 mV (all n = 6, P b 0.01 vs control). The mean value of IKs magnitude at +40 mV in the absence and presence of 10 μM and 100 μM tanshinone IIA was 473.4 ± 14.4 pA, 630.5 ± 24.8 pA and 938.8 ± 47.9 pA respectively. Fig. 2B shows the concentration– response relationship curve for the enhancement of IKs by tanshinone IIA, which was fitted to Hill equation: E = Emax / [1 + (EC50 / C)b], where E is the effect at concentration C, Emax is the maximal effect, EC50 is the concentration for half-maximal effect, and b is the Hill coefficient. The EC50 at 40 mV is 64.5 μM with a b value of 0.66 (Emax= 151.8%).
It is known that IKs is enhanced by various signals, including PKA activation (Lo and Numann, 1998; Marx et al., 2002; Sanguinetti et al., 1991; Volders et al., 2003), protein kinase C (PKC) activation (Lo and Numann, 1998) and the direct S-nitrosylation of channel protein (Bai et al., 2004). Previous experiments have shown that endogenous βadrenergic receptor–cAMP–PKA pathway and NO–cGMP–PKG pathway exists in HEK 293 cells (Browning et al., 2001;Browning et al., 2000; Fukao et al., 1999;Ming-Qing Dong et al., 2006;Yamamoto et al., 2001). So we examined whether enhancement of tanshinone IIA on IKs is mediated through these signal pathways. IKs was elicited with the same protocol as in Fig. 1. Firstly, we studied the effects of PKA activation and NO application on the tanshinone IIA induced enhancement of IKs. The cells were pretreated for 10 min with 1 μM isoproterenol, a β-adrenergic receptor stimulator, or 1 mM SNP, a NO donor, then additional 30 μM tanshinone IIA was applied into the bath solution still containing 1 μM isoproterenol or 1 mM SNP. As shown in Fig. 4, pretreatment with 1 μM isoproterenol (Fig. 4A) or 1 mM SNP (Fig. 4B) had no effects
3.2. Effects of tanshinone IIA on voltage-dependence of IKs activation The effects of tanshinone IIA at 30 μM on the voltage dependence of IKs activation (Fig. 3A) determined by constructing the activation curve of normalized IKs step currents. IKs step currents were normalized to the maximum amplitude measured at +60 mV from the end of the 3-s depolarization pulse, plotted against the test potentials, and was fitted to the Boltzman function: f = 1 / [1+ EXP ((V0.5 −Vt) /S)], where Vt is the test potential, V0.5 is the membrane voltage at which half-activation occurs, and S is the slope factor. Tanshinone IIA at 30 μM produced a significant leftward shift in the voltage-dependence of IKs activation without effect on slope factor. The V0.5 was 24.1 ± 1.9 mV and 16.1 ± 1.2 mV (P b 0.05, n = 7), while the S was 17.6 ± 2.4 and 19.1 ± 2.3 (P = NS, n = 7), in the absence and in the presence of 30 μM tanshinone IIA respectively. 3.3. Effects of tanshinone IIA on activation and deactivation kinetics of IKs Further, the effects of tanshinone IIA on activation and deactivation kinetics of IKs were examined. The data points of activation of IKs, which were recorded from the start to the end of the 3-s test voltage step, were fitted to monoexponential function; while the data points of deactivation of IKs, which were recorded from the start to the end of the 3-s tail test voltage step, were fitted to monoexponential function. Fig. 3B shows superimposed IKs traces recorded in one representative cell before and after 30 μM tanshinone IIA application. The currents were induced with 3-s test voltage step to +40 mV then back to −40 mV from a holding potential of −80 mV as shown in the inset. Fig. 3C displays the activation time constants of IKs elicited with the same protocol as in Fig. 1, at different depolarization test voltages in the absence and presence of 30 μM tanshinone IIA. Fig. 3D displays the deactivation time constants of IKs at tail test voltages of −90 to −40 mV after a 3-s depolarization voltage step to +40 mV (shown in the inset) in the absence and presence of 30 μM tanshinone IIA. The activation time constant of IKs was reduced by 30 μM tanshinone IIA at −20 mV
Fig. 3. Effects of tanshinone IIA on the voltage dependence of activation and kinetics of IKs. (A) Effects of tanshinone IIA on the voltage dependence of IKs activation determined by constructing the activation curve of normalized IKs step currents, and fitted to the Boltzman function: f = 1 / [1 + EXP ((V0.5 − Vt) / S)], where Vt is the test potential, V0.5 is the membrane voltage at which half-activation occurs, and S is the slope factor. IKs were induced with the same protocol as in Fig. 1A, measured at the end of 3-s depolarization voltage, normalized to the maximum amplitude at +60 mV, and plotted against the test potential. Tanshinone IIA at 30 μM produced a significant leftward shift in the voltagedependence of IKs activation without effect on slope factor. The V0.5 was 24.1 ± 1.9 mV and 16.1 ± 1.2 mV (P b 0.05, n = 7), while the S was 17.6 ± 2.4 and 19.1 ± 2.3 (P = NS, n = 7), in the absence (circle) and in the presence (square) of 30 μM tanshinone IIA respectively. (B) Superimposed IKs traces recorded in one representative cell in the absence and presence of 30 μM tanshinone IIA. The currents were induced with 3-s voltage step to +40 mV then back to −40 mV from a holding potential of −80 mV as shown in the inset. (C) Activation time constants of IKs at different depolarization voltages in the absence (circle) and presence (square) of 30 μM tanshinone IIA, determined with the same protocol as in (A). (D) Deactivation time constants of IKs at test potential of −90 to −40 mV after a 3-s depolarization voltage step to +40 mV (shown in the inset) in the absence (circle) and presence (square) of 30 μM tanshinone IIA. The activation time constants were reduced by 30 μM tanshinone IIA at −20 mV to +60 mV (n = 7, P b 0.01 or P b 0.05); whereas, the deactivation time constant were increased by 30 μM tanshinone IIA at potentials of − 90 mV to −40 mV (n = 7, P b 0.01 or P b 0.05).
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on the enhancement of IKs by tanshinone IIA. The enhancement of IKs at step voltage of +40 mV was 143.8 ± 6.0%, 145.2 ± 5.3%, and 148.3 ± 4.4% in the presence of 30 μM tanshinone IIA, 30 μM tanshinone IIA plus 1 μM isoproterenol and 30 μM tanshinone IIA plus 1 mM SNP respectively (all P = NS, n = 6) (Fig. 4C). Then we studied the effects of PKA blockade and cGMP reduction on the tanshinone IIA induced increase of IKs. Similarly, the cells were pretreated for 10 min with 0.1 μM H89, a selective potent PKA inhibitor, or 10 μM ODQ, a selective NO-sensitive guanylate cyclase inhibitor, then additional 30 μM tanshinone IIA was applied into the bath solution still containing 0.1 μM H89 or 10 μM ODQ. Pretreatment with 0.1 μM H89 or 10 μM ODQ had no significant effects on the tanshinone IIA induced increase of IKs. The increase of IKs at step of +40 mV was 143.8 ± 6.0%, 141.8 ± 6.9% and 149.0 ± 5.4% in the presence of 30 μM tanshinone IIA, 30 μM tanshinone IIA plus 0.1 μM H89, and 30 μM tanshinone IIA plus 10 μM ODQ respectively (all P = NS, n = 6) (Fig. 4C). 3.5. Effects of tanshinone IIA on recombinant Kv1.5, IK1 and hERG channels The effects of tanshinone IIA on human cardiac Kv1.5, IK1 and hERG expressed in HEK 293 cells were also examined. Kv1.5 currents were elicited by 0.5-s test pulses between −60 mV and +60 mV in 20 mV step from a holding potential of −80 mV, then back to −40 mV at 0.1 Hz. IK1 currents were elicited by a 3-s test pulse between −120 mV and +10 mV
Fig. 5. Effects of tanshinone IIA on expressed Kv1.5, IK1and hERG. (A–C) Recorded representative traces of expressed Kv1.5 (A), IK1 (B) and hERG (C) in HEK 293 cells, before and after application of 100 μM tanshinone IIA respectively. Kv1.5 currents were elicited by 0.5-s test pulses between −60 mV and +60 mV in a 20 mV step from a holding potential of − 80 mV, then back to −40 mV at 0.1 Hz. IK1 currents were elicited by a 3-s test pulse between − 120 mV and +10 mV in a 10 mV step from a holding potential of −80 mV, then back to −40 mV at 0.1 Hz. hERG currents were elicited by a 1-s test pulses between −60 mV and +60 mV in a 20 mV step from a holding potential of −80 mV, then back to −40 mV for 3-s at 0.05 Hz.
in a 10 mV step from a holding potential of −80 mV, then back to −40 mV at 0.1 Hz. hERG currents were elicited by a 1-s test pulses between −60 mV and +60 mV in a 20 mV step from a holding potential of −80 mV, then back to −40 mV for 3-s at 0.05 Hz. Fig. 5A–C shows the representative traces of Kv1.5, IK1 and hERG currents recorded in one cell respectively, in the absence and in the presence of 100 μM tanshinone IIA. Tanshinone IIA at 100 μM had no significant effects on the expressed Kv1.5, IK1 and hERG currents at all test potentials (all P=NS, n=6). The mean absolute values for current amplitudes before and after 100 μM tanshinone IIA addition is 720.2 ± 50.1 pA and 708.8 ± 40.4 pA for Kv1.5 at+ 40 mV, 3.2 ± 0.2 nA and 3.1 ± 0.2 nA for IK at −120 mV, 635.4 ± 40.5 pA and 620.8 ± 38.4 pA for hERG at +40 mV respectively. The currents were measured at the end of the test pulse (for Kv1.5 and IK1) or measured as the maximal tail current for hERG. 4. Discussion
Fig. 4. Effects of isoproterenol, SNP, H89 and ODQ on tanshinone IIA induced IKs enhancement (A–B) Time course of IKs in two typical experiment recorded with a 3-s step to +40 from − 80 mV (right inset) under the conditions of the pretreatment with 1 μM isoproterenol (A) or 1 mM SNP (B). Current traces at corresponding time points indicated by lower-case italic alphabets are shown at the right of the panel. (C) Histogram summarizes the percentage values of IKs under the conditions of application of 30 μM tanshinone IIA, 30 μM tanshinone IIA plus 1 μM isoproterenol (ISO), 30 μM tanshinone IIA plus 1 mM SNP, 30 μM tanshinone IIA plus 0.1 μM H89 and 30 μM tanshinone IIA plus 10 μM ODQ (all n = 6, P = NS).
The present study firstly demonstrates that tanshinone IIA potently enhances recombinant human cardiac IKs current in HEK 293 cells in a concentration dependent way, shifts the activation curve to negative potential, accelerates the activation and decelerates the deactivation of IKs. We also examined the effects of tanshinone IIA on recombinant human cardiac Kv1.5, IK1, hERG and found that tanshinone IIA did not significantly affect these currents. These results suggest that tanshinone IIA is a specific activator of IKs and may be used to prevent and treat long QT syndrome and arrhythmia induced by cardiomyocyte action potential repolarization delay and reduction of IKs channels function, especially in the patho-physiological process of the electrical remodeling in diseased heart. IKs is reported to be regulated by a number of endogenous or intracellular molecules and pathways, including ATP, cAMP, angiotensin II, testosterone, protein kinase A, tyrosine kinases etc. (Bai et al., 2005; Lo and Numann, 1998; Marx et al., 2002; Matsumoto et al., 1999; Missan et al., 2006; Zankov et al., 2006). Among of them, PKA activation is the most important for its underlying the sympathetic
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nervous system regulation of cardiac action potential duration and heart rate. However, only several compounds are found to enhance IKs (Salata et al., 1998; Bai et al., 2004; Zhang et al., 2006). R-L3, a novel benzodiazepine, activates IKs by slowing the rate of IKs deactivation and shifted the activation curve of IKs to negative potential (Salata et al., 1998). Ginsenoside Re, a major ingredient of Panax ginseng, enhances IKs through direct S-nitrosylation of the channel protein (Bai et al., 2004). Resveratrol, a natural ingredient of grape skin, selectively enhances IKs without effects on IKr (Zhang et al., 2006). The present study demonstrated that tanshinone IIA enhanced IKs through accelerating the activation and decelerating the deactivation of IKs channel, which is partially similar to that of benzodiazepine R-L3. In the present study, we pretreated the cells with 1 μM isoproterenol, which is enough to activate PKA through stimulating β-adrenergic receptor, and 0.1 μM H89, a selective potent PKA inhibitor, to explore whether the increase of IKs by tanshinone IIA is related to PKA activation. Pretreatment with isoproterenol or H89 did not affect the effects of tanshinone IIA on IKs, showing that the effects of tanshinone IIA are independent of PKA activation. Similarly, pretreatment with 1 mM SNP, a NO donor or 10 μM ODQ, a selective nitric oxide-sensitive guanylyl cyclase, also did not affect the effects of tanshinone IIA. Because SNP can produce NO which can activate cGMP then PKG or directly nitrosylate channel protein, these results suggest that PKG activation and channel nitrosylation are not involved in the increase of tanshinone IIA on IKs. The effects of tanshinone IIA on IK1 in our study are different from that in rat (Yu et al., 2002), in which tanshinone IIA inhibited IK1. The different experiment conditions may be the major reason. Several lines of evidences show that at least three members of Kir2.x subfamily (Kir2.1, Kir2.2, and Kir 2.3) contribute to native cardiac IK1 through assembling as homo- and/or heterotetrameric channels although Kir2.1 may be the predominant determinant of IK1 (Dhamoon et al., 2004; Lopatin and Nichols, 2001; Plaster et al., 2001; Wang et al., 1998). In the present study Kir2.1 gene was transfected into HEK 293 cells and the Kir2.1 channel was used as a model for cardiac IK1. So besides the species differences between human and rat, lacking ancillary subunits and/or lacking heteromultimer formation in HEK 293 cells is possibly the major reason for the discrepant results between our study and YU's. One limitation of the present study is that the effects of tanshinone IIA on IKs were measured in HEK 293 cells. Although the characterizations of recombinant IKs stably expressed in HEK 293 cells are similar to that of native IKs in cardiac myocytes (Ming-Qing Dong et al., 2006), it should be taken care to extend the effects to native human cardiac IKs. Future work is necessary to confirm the enhancement of tanshinone IIA on native IKs in human cardiac myocytes and determine its possible binding sites on the channels. In summary, the present study provides the novel information that tanshinone IIA directly and specifically enhances recombinant human cardiac IKs independent of PKA activation, PKG activation and channel nitrosylation in HEK 293 cells. Tanshinone is a new activator of human cardiac IKs. Acknowledgement This work was supported by National Natural Science Foundation of China (No 30400155 and No 30700265). References Bai, C.X., Takahashi, K., Masumiya, H., Sawanobori, T., Furukawa, T., 2004. Nitric oxidedependent modulation of the delayed rectifier K+ current and the L-type Ca2+ current by ginsenoside Re, an ingredient of Panax ginseng, in guinea-pig cardiomyocytes. Br. J. Pharmacol. 142, 567–575. Bai, C.X., Kurokawa, J., Tamagawa, M., Nakaya, H., Furukawa, T., 2005. Nontranscriptional regulation of cardiac repolarization currents by testosterone. Circulation 112, 1701–1710. Barhanin, J., Lesage, F., Guillemare, E., Fink, M., Lazdunski, M., Romey, G., 1996. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature 384, 78–80.
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Tanshinone IIA: A new activator of human cardiac KCNQ1/KCNE1 (IKs) potassium channels Dong-Dong Sun a,1, Hai-Chang Wang a,1, Xiao-Bin Wang b, Ying Luo b, Zhen-Xiao Jin c, Zhi-Chao Li b, Gui-Rong Li d, Ming-Qing Dong b,⁎ a
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China Department of Pathology and Pathophysiology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China Department of Cardiovascular Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China d Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China b c
A R T I C L E
I N F O
Article history: Received 21 December 2007 Received in revised form 21 May 2008 Accepted 2 June 2008 Available online 7 June 2008 Keywords: Tanshinone IIA hKCNQ1/hKCNE1 IKs Kv1.5 hERG IK1 HEK 293 cell Potassium channel Ion channel Patch clamp Human
A B S T R A C T Tanshinone IIA, one of the main active components from Chinese herb Danshen, is widely used to treat cardiovascular diseases including arrhythmia in Asian countries especially in China. However, the mechanisms underlying its anti-arrythmia effects are not clear. In this study we investigate the effects of tanshinone IIA on human KCNQ1/KCNE1 potassium channels (IKs), human ether-a-go-go-related gene potassium channels (hERG), Kv1.5 potassium channels, inward rectifier potassium channels (IK1) expressed in HEK 293 cells using patch clamp technique. Tanshinone IIA potently and reversibly enhanced the amplitude of IKs in a concentration dependent manner with an EC50 of 64.5 μM, accelerated the activation rate of IKs channels, decelerated their deactivation and shifted the voltage dependence of IKs activation to negative direction. Isoproteronol, a stimulator of β-adrenergic receptor, at 1 μM and sodium nitroprusside (SNP), a NO donor, at 1 mM, had no significant effects on the enhancement of IKs by 30 μM tanshinone IIA. N-[2-(pbromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), a selective protein kinase A inhibitor, at 0.1 μM and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), a selective nitric oxide-sensitive guanylyl cyclase inhibitor, at 10 μM, also had no significant effects on the enhancement of IKs by 30 μM tanshinone IIA. Tanshinone IIA did not affect expressed hERG channels, Kv1.5 channels and IK1 channels. These results indicate that tanshinone IIA directly and specifically activate human cardiac KCNQ1/KCNE1 potassium channels (IKs) in HEK 293 cell through affecting the channels' kinetics. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The slowly activating delayed rectifier potassium current IKs plays an important role in the repolarization of human cardiac action potential. It has been demonstrated that IKs is a heteromultimeric complex composed of pore-forming α subunit and accessory β subunit encoded, respectively, by the KCNQ1 and KCNE1 genes (Barhanin et al., 1996; Sanguinetti et al., 1996). Loss or reduction of IKs channels function, due to genetic mutations in the KCNQ1 and KCNE1 genes or electrical remodeling in diseased heart (Barhanin et al., 1996; Li et al., 2004; Nabauer and Kaab, 1998; Xu et al., 2001), induces a decrease in net repolarizing current. The reduced repolarization reserve results in delayed repolarization, lengthened action
⁎ Corresponding author. Department of Pathology and Pathophysiology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China. Tel.: +86 29 84773105; fax: +86 29 84774548. E-mail address: [email protected] (M.-Q. Dong). 1 Contribute equally to the paper. 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.06.005
potential duration, early after depolarization and prolongation of QT interval in the electrocardiogram. All these alterations increase the risk of life-threatening ventricular arrhythmia for the affected persons. Activators of IKs are promising candidates to regain IKs channel function and treat the IKs dysfunction related arrhythmia, thus it is potentially important in the clinical setting if a drug is found to be able to specifically enhance IKs. However, only a few compounds are found to have activating effects on IKs (Salata et al., 1998; Seebohm et al., 2003; Bai et al., 2004; Zhang et al., 2006). Danshen, a dried root of Salvia miltiorrhiza, is a traditional Chinese medicinal herb. It has been widely used to treat many kinds of diseases for thousands of years in China. Tanshinone IIA is one of the main active components from Danshen, exhibiting a variety of cardiovascular effects including vasorelaxation, protection against ischemia– reperfusion injury and antiarrhythmic effects (Gao et al., 2008; Hu et al., 1981; Zhou et al., 2005). It is widely used to treat angina pectoris, hyperlipidemia, acute ischemic stroke and arrhythmia in Asian countries especially in China. Although its success in clinics for treating cardiovascular diseases, the exact mechanism underlying its
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therapeutic basis especially for arrhythmia is poorly understood. Recently, tanshinone IIA was found to shorten action potential duration in rat heart, inhibit L-type Ca2+ current in guinea pig ventricular myocytes, inhibit IK1 currents in rat ventricular myocytes (Xu et al., 1997; Zhu et al., 2005), which is regarded as the partial mechanism of its anti-arrhythmia effects. However, there are at present no published reports in which effects of tanshinone IIA on human cardiomyocyte potassium currents have been studied. In the present study, therefore, we determine whether tanshinone IIA regulates recombinant human cardiac IKs (hKCNQ1/hKCNE1), hERG (human ether-a-go-go-related gene), IK1 (Kir2.1) and Kv1.5 channels expressed in HEK 293 cells. The results demonstrate that tanshinone IIA potently and specifically enhances IKs through affecting the channels' kinetics and its effect is independent of protein kinase A (PKA) activation, protein kinase G (PKG) activation and channel nitrosylation. 2. Materials and methods 2.1. Cell culture HEK 293 cell lines stably expressing human cardiac KCNQ1/KCNE1 channel (IKs), human cardiac hERG channel (hERG), human cardiac Kv1.5 channel (Kv1.5), human cardiac Kir2.1 channel (IK1) respectively were cultured at 37 °C, 5% CO2 and 95% air in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum and 200 μg/ml hygromycin (for IKs) or 1 mg/ml G418 (for hERG, Kv1.5 and IK1), and subcultured every 3–4 days. For patch clamp experiments, the cells were subcultured on a coverslip and incubated overnight then transferred to an open chamber for currents recording. 2.2. Solutions and drugs The standard Tyrode's solution contained (in mM): NaCl 140, KCl 5.0, MgCl2 1.0, CaCl2 1.0, NaH2PO4 0.33, HEPES 5.0, glucose 10, pH adjusted to 7.4 with NaOH. The pipette solution contained (in mM): KCl 20, K-aspartate 110, MgCl2 1.0, HEPES 10, EGTA 5.0, and GTP 0.1, Na2-phosphocreatine 5.0, Mg2-ATP 5.0, pH adjusted to 7.2 with KOH. In perforated patch recording mode, 200 μg/ml amphotericin B (Sigma-Aldrich) was added into the pipette solution. Tanshinone IIA (sulfonate, purity is 99%) was purchased from Xi'an Honson Biotechnology Co. Ltd (Xi`an, China). All other chemicals are from Sigma. Tanshinone IIA, isoproterenol and sodium nitroprusside (SNP) were freshly prepared and kept in the dark at 4 °C. N-[2-(pbromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) were prepared as 5 and 20 mM stock solution in dimethylsulfoxide (DMSO) respectively.
200B amplifier and Clampex 8.0 software (Axon Instruments). Current signals were low-pass filtered at 2 kHz and stored on the hard disk of an IBM computer. Values are presented as mean ± S.E.M. Nonlinear curve fitting was performed using Clampfit 9.0 (Axon) and Sigmaplot (SPSS Science, Chicago, IL, U.S.A.). Paired Student's t-tests were used to evaluate the statistical significance of differences between two group means. ANOVA was used for multiple groups. Values of P ≤ 0.05 were considered statistically significant. 3. Results 3.1. Concentration-dependent enhancement of IKs by tanshinone IIA The effects of tanshinone IIA on IKs stably expressed in HEK 293 were shown in Fig. 1. IKs currents were elicited by a 3-s depolarization pulse from holding potential of −80 mV to various test potentials between −60 mV and +60 mV in a 20 mV step, then back to −40 mV for 3-s at 0.05 Hz. IKs were measured from zero level to the current at the end of 3-s depolarization step voltage. Fig. 1A shows the original traces of IKs recorded in one representative cell in the absence and in the presence of tanshinone IIA at 10 μM, 100 μM and after washout of the drug respectively. Tanshinone IIA substantially enhanced IKs and the effect was partially washed out. Fig. 1B shows the time course of 30 μM tanshinone IIA on IKs in a representative cell. IKs was elicited by continuously applying 3-s depolarization pulses from holding potential of −80 mV to test potential of +40 mV in a 20-s interval. IKs gradually increased after drug application, reached to its maximal effect in 2 min and recovered upon the drug washout for about 3 min. The original traces at corresponding time points are shown in the right inset of the panel. Fig. 2A displays the I–V relationships of mean values of IKs step current in the absence and presence of 10 μM and 100 μM tanshinone IIA and washout for 5 min. Tanshinone IIA significantly increased IKs at
2.3. Data acquisition and analysis The experiments were conducted at room temperature (25 °C). The whole-cell and perforated patch-clamp techniques were used as described previously. Briefly, the borosilicate glass electrodes (1.5 mm OD) were pulled with Brown–Flaming puller (model P-97, Sutter Instrument Co., Novato, CA, U.S.A.) and had tip resistances of 1–2 MΩ when filled with pipette solution. 3 M KCl–agar salt bridge was used as reference electrode. Liquid junction potentials were compensated before the pipette touched the cell. After giga seal was obtained, the cell membrane was ruptured by gentle suction to establish the wholecell configuration to record hERG, Kv1.5 and IK1. Perforated patch configuration was used to record IKs to minimize rundown of the current. The series resistance was electrically compensated to minimize voltage errors. The membrane currents were converted to digital signals with Digidata 1200 (Axon Instruments, Forster City, CA, U.S.A) at a sampling rate of 10 kHz and recorded with an Axonpatch
Fig. 1. Effects of tanshinone IIA on I Ks stably expressed in HEK 293 cells. (A) Representative original IKs traces recorded in one typical cell under control condition, in the presence of 10 μM, 100 μM tanshinone IIA and after washout of the drug respectively. IKs was induced with 3 - s voltage step from −60 mV to +60 mV then back to −40 mV as shown in the inset. (B) Time course of normalized IKs step current recorded at + 40 mV after a 3-s voltage step to + 40 from −80 mV as shown in right inset. Original superimposed IKs current traces at corresponding time points indicated by lower-case italic alphabets are shown at the right of the panel. In this and the following figures, TAN represents tanshinone IIA.
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to +60 mV (all n = 7, P b 0.01 or P b 0.05); however, the deactivation time constant was increased by tanshinone IIA at potentials of −90 mV to −40 mV (all n = 7, P b 0.01 or P b 0.05). 3.4. Effects of β-adrenergic receptor stimulator, NO donor, PKA inhibitor and guanylate cyclase inhibitor on tanshinone IIA induced I Ks enhancement
Fig. 2. Concentration dependent effects of tanshinone IIA on IKs. (A) I–V relationships of IKs step currents under control condition, in the presence of 10 μM, 100 μM tanshinone IIA, and washout. Tanshinone IIA significantly enhanced IKs with increasing concentration and the enhancement was almost reversed upon washout. (B) Concentration– response curve of IKs step current enhanced by tanshinone IIA at +40 mV. Symbols are the mean values of enhancement. Solid lines are the best fit Hill equation: E = Emax / [1 + (EC50 / C)b], where E is the effect at concentration C, Emax is the maximal effect, EC50 is the concentration for half-maximal effect, and b is the Hill coefficient. IC50 was 64.5 μM, b was 0.66, and Emax was 151.8%.
test potentials of −20 mV to +60 mV (all n = 6, P b 0.01 vs control). The mean value of IKs magnitude at +40 mV in the absence and presence of 10 μM and 100 μM tanshinone IIA was 473.4 ± 14.4 pA, 630.5 ± 24.8 pA and 938.8 ± 47.9 pA respectively. Fig. 2B shows the concentration– response relationship curve for the enhancement of IKs by tanshinone IIA, which was fitted to Hill equation: E = Emax / [1 + (EC50 / C)b], where E is the effect at concentration C, Emax is the maximal effect, EC50 is the concentration for half-maximal effect, and b is the Hill coefficient. The EC50 at 40 mV is 64.5 μM with a b value of 0.66 (Emax= 151.8%).
It is known that IKs is enhanced by various signals, including PKA activation (Lo and Numann, 1998; Marx et al., 2002; Sanguinetti et al., 1991; Volders et al., 2003), protein kinase C (PKC) activation (Lo and Numann, 1998) and the direct S-nitrosylation of channel protein (Bai et al., 2004). Previous experiments have shown that endogenous βadrenergic receptor–cAMP–PKA pathway and NO–cGMP–PKG pathway exists in HEK 293 cells (Browning et al., 2001;Browning et al., 2000; Fukao et al., 1999;Ming-Qing Dong et al., 2006;Yamamoto et al., 2001). So we examined whether enhancement of tanshinone IIA on IKs is mediated through these signal pathways. IKs was elicited with the same protocol as in Fig. 1. Firstly, we studied the effects of PKA activation and NO application on the tanshinone IIA induced enhancement of IKs. The cells were pretreated for 10 min with 1 μM isoproterenol, a β-adrenergic receptor stimulator, or 1 mM SNP, a NO donor, then additional 30 μM tanshinone IIA was applied into the bath solution still containing 1 μM isoproterenol or 1 mM SNP. As shown in Fig. 4, pretreatment with 1 μM isoproterenol (Fig. 4A) or 1 mM SNP (Fig. 4B) had no effects
3.2. Effects of tanshinone IIA on voltage-dependence of IKs activation The effects of tanshinone IIA at 30 μM on the voltage dependence of IKs activation (Fig. 3A) determined by constructing the activation curve of normalized IKs step currents. IKs step currents were normalized to the maximum amplitude measured at +60 mV from the end of the 3-s depolarization pulse, plotted against the test potentials, and was fitted to the Boltzman function: f = 1 / [1+ EXP ((V0.5 −Vt) /S)], where Vt is the test potential, V0.5 is the membrane voltage at which half-activation occurs, and S is the slope factor. Tanshinone IIA at 30 μM produced a significant leftward shift in the voltage-dependence of IKs activation without effect on slope factor. The V0.5 was 24.1 ± 1.9 mV and 16.1 ± 1.2 mV (P b 0.05, n = 7), while the S was 17.6 ± 2.4 and 19.1 ± 2.3 (P = NS, n = 7), in the absence and in the presence of 30 μM tanshinone IIA respectively. 3.3. Effects of tanshinone IIA on activation and deactivation kinetics of IKs Further, the effects of tanshinone IIA on activation and deactivation kinetics of IKs were examined. The data points of activation of IKs, which were recorded from the start to the end of the 3-s test voltage step, were fitted to monoexponential function; while the data points of deactivation of IKs, which were recorded from the start to the end of the 3-s tail test voltage step, were fitted to monoexponential function. Fig. 3B shows superimposed IKs traces recorded in one representative cell before and after 30 μM tanshinone IIA application. The currents were induced with 3-s test voltage step to +40 mV then back to −40 mV from a holding potential of −80 mV as shown in the inset. Fig. 3C displays the activation time constants of IKs elicited with the same protocol as in Fig. 1, at different depolarization test voltages in the absence and presence of 30 μM tanshinone IIA. Fig. 3D displays the deactivation time constants of IKs at tail test voltages of −90 to −40 mV after a 3-s depolarization voltage step to +40 mV (shown in the inset) in the absence and presence of 30 μM tanshinone IIA. The activation time constant of IKs was reduced by 30 μM tanshinone IIA at −20 mV
Fig. 3. Effects of tanshinone IIA on the voltage dependence of activation and kinetics of IKs. (A) Effects of tanshinone IIA on the voltage dependence of IKs activation determined by constructing the activation curve of normalized IKs step currents, and fitted to the Boltzman function: f = 1 / [1 + EXP ((V0.5 − Vt) / S)], where Vt is the test potential, V0.5 is the membrane voltage at which half-activation occurs, and S is the slope factor. IKs were induced with the same protocol as in Fig. 1A, measured at the end of 3-s depolarization voltage, normalized to the maximum amplitude at +60 mV, and plotted against the test potential. Tanshinone IIA at 30 μM produced a significant leftward shift in the voltagedependence of IKs activation without effect on slope factor. The V0.5 was 24.1 ± 1.9 mV and 16.1 ± 1.2 mV (P b 0.05, n = 7), while the S was 17.6 ± 2.4 and 19.1 ± 2.3 (P = NS, n = 7), in the absence (circle) and in the presence (square) of 30 μM tanshinone IIA respectively. (B) Superimposed IKs traces recorded in one representative cell in the absence and presence of 30 μM tanshinone IIA. The currents were induced with 3-s voltage step to +40 mV then back to −40 mV from a holding potential of −80 mV as shown in the inset. (C) Activation time constants of IKs at different depolarization voltages in the absence (circle) and presence (square) of 30 μM tanshinone IIA, determined with the same protocol as in (A). (D) Deactivation time constants of IKs at test potential of −90 to −40 mV after a 3-s depolarization voltage step to +40 mV (shown in the inset) in the absence (circle) and presence (square) of 30 μM tanshinone IIA. The activation time constants were reduced by 30 μM tanshinone IIA at −20 mV to +60 mV (n = 7, P b 0.01 or P b 0.05); whereas, the deactivation time constant were increased by 30 μM tanshinone IIA at potentials of − 90 mV to −40 mV (n = 7, P b 0.01 or P b 0.05).
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on the enhancement of IKs by tanshinone IIA. The enhancement of IKs at step voltage of +40 mV was 143.8 ± 6.0%, 145.2 ± 5.3%, and 148.3 ± 4.4% in the presence of 30 μM tanshinone IIA, 30 μM tanshinone IIA plus 1 μM isoproterenol and 30 μM tanshinone IIA plus 1 mM SNP respectively (all P = NS, n = 6) (Fig. 4C). Then we studied the effects of PKA blockade and cGMP reduction on the tanshinone IIA induced increase of IKs. Similarly, the cells were pretreated for 10 min with 0.1 μM H89, a selective potent PKA inhibitor, or 10 μM ODQ, a selective NO-sensitive guanylate cyclase inhibitor, then additional 30 μM tanshinone IIA was applied into the bath solution still containing 0.1 μM H89 or 10 μM ODQ. Pretreatment with 0.1 μM H89 or 10 μM ODQ had no significant effects on the tanshinone IIA induced increase of IKs. The increase of IKs at step of +40 mV was 143.8 ± 6.0%, 141.8 ± 6.9% and 149.0 ± 5.4% in the presence of 30 μM tanshinone IIA, 30 μM tanshinone IIA plus 0.1 μM H89, and 30 μM tanshinone IIA plus 10 μM ODQ respectively (all P = NS, n = 6) (Fig. 4C). 3.5. Effects of tanshinone IIA on recombinant Kv1.5, IK1 and hERG channels The effects of tanshinone IIA on human cardiac Kv1.5, IK1 and hERG expressed in HEK 293 cells were also examined. Kv1.5 currents were elicited by 0.5-s test pulses between −60 mV and +60 mV in 20 mV step from a holding potential of −80 mV, then back to −40 mV at 0.1 Hz. IK1 currents were elicited by a 3-s test pulse between −120 mV and +10 mV
Fig. 5. Effects of tanshinone IIA on expressed Kv1.5, IK1and hERG. (A–C) Recorded representative traces of expressed Kv1.5 (A), IK1 (B) and hERG (C) in HEK 293 cells, before and after application of 100 μM tanshinone IIA respectively. Kv1.5 currents were elicited by 0.5-s test pulses between −60 mV and +60 mV in a 20 mV step from a holding potential of − 80 mV, then back to −40 mV at 0.1 Hz. IK1 currents were elicited by a 3-s test pulse between − 120 mV and +10 mV in a 10 mV step from a holding potential of −80 mV, then back to −40 mV at 0.1 Hz. hERG currents were elicited by a 1-s test pulses between −60 mV and +60 mV in a 20 mV step from a holding potential of −80 mV, then back to −40 mV for 3-s at 0.05 Hz.
in a 10 mV step from a holding potential of −80 mV, then back to −40 mV at 0.1 Hz. hERG currents were elicited by a 1-s test pulses between −60 mV and +60 mV in a 20 mV step from a holding potential of −80 mV, then back to −40 mV for 3-s at 0.05 Hz. Fig. 5A–C shows the representative traces of Kv1.5, IK1 and hERG currents recorded in one cell respectively, in the absence and in the presence of 100 μM tanshinone IIA. Tanshinone IIA at 100 μM had no significant effects on the expressed Kv1.5, IK1 and hERG currents at all test potentials (all P=NS, n=6). The mean absolute values for current amplitudes before and after 100 μM tanshinone IIA addition is 720.2 ± 50.1 pA and 708.8 ± 40.4 pA for Kv1.5 at+ 40 mV, 3.2 ± 0.2 nA and 3.1 ± 0.2 nA for IK at −120 mV, 635.4 ± 40.5 pA and 620.8 ± 38.4 pA for hERG at +40 mV respectively. The currents were measured at the end of the test pulse (for Kv1.5 and IK1) or measured as the maximal tail current for hERG. 4. Discussion
Fig. 4. Effects of isoproterenol, SNP, H89 and ODQ on tanshinone IIA induced IKs enhancement (A–B) Time course of IKs in two typical experiment recorded with a 3-s step to +40 from − 80 mV (right inset) under the conditions of the pretreatment with 1 μM isoproterenol (A) or 1 mM SNP (B). Current traces at corresponding time points indicated by lower-case italic alphabets are shown at the right of the panel. (C) Histogram summarizes the percentage values of IKs under the conditions of application of 30 μM tanshinone IIA, 30 μM tanshinone IIA plus 1 μM isoproterenol (ISO), 30 μM tanshinone IIA plus 1 mM SNP, 30 μM tanshinone IIA plus 0.1 μM H89 and 30 μM tanshinone IIA plus 10 μM ODQ (all n = 6, P = NS).
The present study firstly demonstrates that tanshinone IIA potently enhances recombinant human cardiac IKs current in HEK 293 cells in a concentration dependent way, shifts the activation curve to negative potential, accelerates the activation and decelerates the deactivation of IKs. We also examined the effects of tanshinone IIA on recombinant human cardiac Kv1.5, IK1, hERG and found that tanshinone IIA did not significantly affect these currents. These results suggest that tanshinone IIA is a specific activator of IKs and may be used to prevent and treat long QT syndrome and arrhythmia induced by cardiomyocyte action potential repolarization delay and reduction of IKs channels function, especially in the patho-physiological process of the electrical remodeling in diseased heart. IKs is reported to be regulated by a number of endogenous or intracellular molecules and pathways, including ATP, cAMP, angiotensin II, testosterone, protein kinase A, tyrosine kinases etc. (Bai et al., 2005; Lo and Numann, 1998; Marx et al., 2002; Matsumoto et al., 1999; Missan et al., 2006; Zankov et al., 2006). Among of them, PKA activation is the most important for its underlying the sympathetic
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nervous system regulation of cardiac action potential duration and heart rate. However, only several compounds are found to enhance IKs (Salata et al., 1998; Bai et al., 2004; Zhang et al., 2006). R-L3, a novel benzodiazepine, activates IKs by slowing the rate of IKs deactivation and shifted the activation curve of IKs to negative potential (Salata et al., 1998). Ginsenoside Re, a major ingredient of Panax ginseng, enhances IKs through direct S-nitrosylation of the channel protein (Bai et al., 2004). Resveratrol, a natural ingredient of grape skin, selectively enhances IKs without effects on IKr (Zhang et al., 2006). The present study demonstrated that tanshinone IIA enhanced IKs through accelerating the activation and decelerating the deactivation of IKs channel, which is partially similar to that of benzodiazepine R-L3. In the present study, we pretreated the cells with 1 μM isoproterenol, which is enough to activate PKA through stimulating β-adrenergic receptor, and 0.1 μM H89, a selective potent PKA inhibitor, to explore whether the increase of IKs by tanshinone IIA is related to PKA activation. Pretreatment with isoproterenol or H89 did not affect the effects of tanshinone IIA on IKs, showing that the effects of tanshinone IIA are independent of PKA activation. Similarly, pretreatment with 1 mM SNP, a NO donor or 10 μM ODQ, a selective nitric oxide-sensitive guanylyl cyclase, also did not affect the effects of tanshinone IIA. Because SNP can produce NO which can activate cGMP then PKG or directly nitrosylate channel protein, these results suggest that PKG activation and channel nitrosylation are not involved in the increase of tanshinone IIA on IKs. The effects of tanshinone IIA on IK1 in our study are different from that in rat (Yu et al., 2002), in which tanshinone IIA inhibited IK1. The different experiment conditions may be the major reason. Several lines of evidences show that at least three members of Kir2.x subfamily (Kir2.1, Kir2.2, and Kir 2.3) contribute to native cardiac IK1 through assembling as homo- and/or heterotetrameric channels although Kir2.1 may be the predominant determinant of IK1 (Dhamoon et al., 2004; Lopatin and Nichols, 2001; Plaster et al., 2001; Wang et al., 1998). In the present study Kir2.1 gene was transfected into HEK 293 cells and the Kir2.1 channel was used as a model for cardiac IK1. So besides the species differences between human and rat, lacking ancillary subunits and/or lacking heteromultimer formation in HEK 293 cells is possibly the major reason for the discrepant results between our study and YU's. One limitation of the present study is that the effects of tanshinone IIA on IKs were measured in HEK 293 cells. Although the characterizations of recombinant IKs stably expressed in HEK 293 cells are similar to that of native IKs in cardiac myocytes (Ming-Qing Dong et al., 2006), it should be taken care to extend the effects to native human cardiac IKs. Future work is necessary to confirm the enhancement of tanshinone IIA on native IKs in human cardiac myocytes and determine its possible binding sites on the channels. In summary, the present study provides the novel information that tanshinone IIA directly and specifically enhances recombinant human cardiac IKs independent of PKA activation, PKG activation and channel nitrosylation in HEK 293 cells. Tanshinone is a new activator of human cardiac IKs. Acknowledgement This work was supported by National Natural Science Foundation of China (No 30400155 and No 30700265). References Bai, C.X., Takahashi, K., Masumiya, H., Sawanobori, T., Furukawa, T., 2004. Nitric oxidedependent modulation of the delayed rectifier K+ current and the L-type Ca2+ current by ginsenoside Re, an ingredient of Panax ginseng, in guinea-pig cardiomyocytes. Br. J. Pharmacol. 142, 567–575. Bai, C.X., Kurokawa, J., Tamagawa, M., Nakaya, H., Furukawa, T., 2005. Nontranscriptional regulation of cardiac repolarization currents by testosterone. Circulation 112, 1701–1710. Barhanin, J., Lesage, F., Guillemare, E., Fink, M., Lazdunski, M., Romey, G., 1996. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature 384, 78–80.
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