Effect of Daurisoline on hERG Channel Electrophysiological Function

Sep 13, 2012 - prolonged action potential duration (APD) in a normal use-dependent manner. However, the electrophysiological mechanisms for 1-induced ...
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Effect of Daurisoline on hERG Channel Electrophysiological Function and Protein Expression Qiangni Liu, Xiaofang Mao, Fandian Zeng, Si Jin, and Xiaoyan Yang* Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, The Key Laboratory of Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan, Hubei, 430030, People’s Republic of China ABSTRACT: Daurisoline (1) is a bis-benzylisoquinoline alkaloid isolated from the rhizomes of Menispermum dauricum. The antiarrhythmic effect of 1 has been demonstrated in different experimental animals. In previous studies, daurisoline (1) prolonged action potential duration (APD) in a normal use-dependent manner. However, the electrophysiological mechanisms for 1-induced prolongation of APD have not been documented. In the present study, the direct effect of 1 was investigated on the hERG current and the expression of mRNA and protein in human embryonic kidney 293 (HEK293) cells stably expressing the hERG channel. It was shown that 1 inhibits hERG current in a concentration- and voltage-dependent manner. In the presence of 10 μM 1, steady-state inactivation of V1/2 was shifted negatively by 15.9 mV, and 1 accelerated the onset of inactivation. Blockade of hERG channels was dependent on channel opening. The expression and function of hERG were unchanged by 1 at 1 and 10 μM, while hERG expression and the hERG current were decreased significantly by 1 at 30 μM. These results indicate that 1, at concentrations below 30 μM, exerts a blocking effect on hERG, but does not affect the expression and function of the hERG channel. This may explain the relatively lower risk of long QT syndrome after long-term usage.

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aurisoline (1) is a bis-benzylisoquinoline alkaloid extracted from the rhizomes of Menispermum dauricum, which possesses a variety of pharmacological activities. In addition to antiplatelet aggregation, anti-inflammatory, and neuron-protective properties,1−5 1 has also been shown to have antiarrhythmic effects in various experimental animals, in which the effect seems to be more potent than that of dauricine, its structural analogue.6,7 Our previous studies have shown that 1 can suppress pro-arrhythmia early afterdepolarization, delayed afterdepolarization, and triggered activity.7−9 However, the underlying electrophysiological mechanism is not clear. Although 1 has not been used in patients, its analogue dauricine was evaluated in a clinical trail of 402 patients with atrial and ventricular arrhythmias. Dauricine was found to be efficacious in 91.5% of patients.10 Owing to the difficulty in extracting dauricine from the rhizomes of M. dauricum, it was postulated that the total alkaloids of M. dauricum may be of value as a treatment, and a phase I clinical trial is being performed to evaluate a total-alkaloid formulation.11 To understand the efficacy and safety of this total-alkaloid formulation of M. dauricum, it is necessary to further examine the underlying electrophysiological mechanism of 1, which is an important alkaloid constituent of the total-alkaloid formulation. Prolongation of the action potential duration (APD) is an important mechanism of an antiarrhythmic drug. It was shown that 1 prolonged APD in a normal use-dependent manner on papillary muscles of guinea pig.12 In vivo studies showed that 1 also decreased cardiac monophasic action potential amplitude (MAPA) and prolonged monophasic action potential duration at 50% and 90% (MAPD50 and MAPD90) and the effective refractory period (ERP) in a normal use-dependent manner in rabbits.13 In addition, daurisoline (1) inhibited the slow inward calcium currents generated by L-type calcium channels.8 © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Inhibition of hERG channels by daurisoline (1). (A) IhERG was elicited by the voltage protocol shown in the inset in the absence and presence of 30 μM 1. (B) Representative currents in the absence, presence (5 min), and following washout (5 min) of 30 μM 1.

However, the rapidly activating delayed rectifier potassium current (IKr) plays the most critical role in the repolarization of Received: March 27, 2012

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repolarization. However, the electrophysiological mechanisms for 1-induced prolongation of APD have not been documented. In previous studies, the effect of 1 on IKs was studied on single ventricular myocytes from guinea pigs or rabbits. In ventricular myocytes from guinea pigs, IKs‑tail was inhibited by 50 ± 16% in the presence of 100 μM 1, and IKr‑tail was inhibited by 39 ± 12% by 10 μM 1.17 In rabbit ventricular myocytes, 1 inhibited IKr‑tail with an IC50 of about 19.9 μM and blocked IKs‑tail with an IC50 of about 52.5 μM.18 Compared with IKs, inhibition of IKr by 1 was more apparent. Therefore, in the present study, the direct effects

cardiomyocytes. The human ether-a-go-go-related gene (hERG) encodes the α subunit of the IKr channel,14 which plays a central role in regulating cardiac excitability and maintaining normal cardiac rhythm. The hERG channel has been demonstrated to contribute to arrhythmia and sudden cardiac death, and suppression of hERG also seems to be associated with the pro-arrhythmic potential of antiarrhythmic drugs.15 There are two well-described mechanisms for loss of function of hERGblocking hERG or interrupting trafficking of the hERG protein to the cell membrane16both of which contribute to the prolongation of

Figure 2. Concentration-dependent and voltage-dependent inhibition of hERG channels by daurisoline (1). (A) Protocol and representative traces from a hERG cell before and after application of 10 μM 1. (B) Representative traces with different concentrations of 1 (0, 1, 3, 10, 30 μM) when the membrane potential was depolarized to +60 mV. (C) I/V plot of IhERG‑step (pA/pF) (n = 8, *p < 0.05, 1 vs control). (D) I/V plot of IhERG‑tail (pA/pF) (n = 8, *p < 0.05, 1 vs control). (E) Mean fractional block of IhERG‑step and IhERG‑tail with 1 (1, 3, 10, 30 μM). (F) Mean fractional block of IhERG‑step and IhERG‑tail with 10 μM 1 when the membrane potential was stepped from −20 mV to +60 mV. The inhibition was stronger at positive potentials between +10 and +60 mV than at potentials between −20 and 0 mV (n = 8, *p < 0.05). B

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Concentration- and voltage-dependent inhibitions of the hERG channel by 1 were also investigated. Figure 2A shows the protocol and the original current recordings from a cell stably expressing hERG, in the absence and presence of 10 μM 1. Currents were recorded in 1-free Tyrode’s solution, and then 10 μM 1 was perfused into the bath for 5 min. Figure 2B shows representative current traces in the presence of different concentrations of 1 (0, 1, 3, 10, 30 μM) when the membrane potential was depolarized to +40 mV. Figure 2C and D show the current amplitude at the end of depolarization (IhERG‑step) and IhERG‑tail density-to-voltage (I−V) relationship curves in the absence or presence of 1 (1, 3, 10, 30 μM). Daurisoline (1) showed a maximal inhibitory effect on IhERG‑step at +20 mV and on IhERG‑tail at +60 mV. Figure 2E summarizes the inhibition ratios of 1 on IhERG‑step at +20 mV and on IhERG‑tail at +60 mV. At concentrations of 1, 3, 10, and 30 μM, the inhibition ratios for IhERG‑step were 32.2 ± 4.2%, 41.6 ± 2.6%, 62.1 ± 5.9%, and 74.8 ± 6.8%, respectively; the IC50 was 9.1 μM. In turn, the inhibition ratios for IhERG‑tail were 16.7 ± 5.8%, 31.1 ± 4.5%, 55.1 ± 7.2%, and 81.2 ± 7.0%, respectively; the IC50 was 9.6 μM. Figure 2F shows the voltage-dependent inhibition of IhERG‑step and IhERG‑tail at a concentration of 10 μM. Compared to the proportion of blockade at potentials between −20 and 0 mV, the proportion of blockade increased at positive potentials between +10 and +60 mV (n = 8, p < 0.05). The kinetics of hERG activation, inactivation, deactivation, and reactivation were investigated in the presence of 1. To analyze

of 1 were investigated on the hERG current, mRNA, and protein expression in human embryonic kidney 293 (HEK293) cells stably expressing the hERG channel. It was shown that 1 inhibited IhERG in the open state and induced a marked shift of hERG inactivation curves toward more negative potentials. The hERG channel function was significantly decreased after incubation for 24 h with 30 μM 1.

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RESULTS AND DISCUSSION In HEK293 cells stably expressing the hERG channel, IhERG was evoked by a 1-s step depolarization from a holding potential of −80 mV to +60 mV, followed by a 3-s repolarization to −50 mV once every 10 s. The peak tail current (IhERG‑tail) was used to qualify the inhibition of IhERG. As shown in Figure 1A, a steadystate blockade was reached within 2 min (i.e., at the 12th pulse) in the presence of 30 μM 1. Figure 1B shows a recording of IhERG under baseline conditions, during a 5-min addition of 30 μM 1 and during a 5-min washout of the compound. The blocking effect was reversed gradually following washout.

Figure 3. Effect of daurisoline (1) on steady-state activation and inactivation of hERG channels. (A) Steady-state activation curve before and after application of 10 μM 1. (B) Protocol used to record steady-state inactivation curve and representative traces before and after 10 μM 1. (C) Steady-state inactivation curves before and after application of 10 μM 1. (D) Time constants of steady-state inactivation before and after 10 μM 1. Daurisoline (1) increased the time constants of steady-state inactivation from −120 mV to −60 mV (n = 7, *p < 0.05). C

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Figure 4. Effects of daurisoline (1) on the onset of inactivation and recovery from inactivation of hERG channels. (A) A three-pulse protocol was used to study the onset of inactivation of the hERG current; representative traces before and after 10 μM 1. (B) Onset-of-inactivation curves before and after 10 μM 1 (n = 7). (C) The fully activated I−V protocol was used to study the recovery from inactivation of the hERG channel and the representative traces before and after 10 μM 1. (D) Time constants of the onset of inactivation and recovery from inactivation before and after 10 μM 1 (n = 7, *p < 0.05, **p < 0.01, 1 vs control).

The effect of 1 on the onset of inactivation of IhERG was investigated using a three-pulse protocol as shown in Figure 4A. The channels were first inactivated by clamping the membrane at +40 mV for 2.5 s, followed by a prepulse to −100 mV for 10 ms. The prepulse was long enough to allow rapid recovery of channels from inactivation, but short enough to prevent significant channel deactivation. Following the recovery prepulse, a series of test pulses were delivered to potentials ranging from −120 to +30 mV, resulting in large, outward inactivating currents. The onset-of-inactivation curves in the absence and presence of 10 μM 1 are shown in Figure 4B. The onset-of-inactivation curve was shifted negatively by 10 μM 1 between −50 and +10 mV. As shown in Figure 4D, the time constants for the onset of inactivation were obtained by fitting an exponential function to the decaying current traces during the third pulse of the protocol. Daurisoline (1) at a concentration of 10 μM markedly shortened the time constants for the onset of inactivation between −20 and +10 mV (from 33.1 ± 2.4 ms to 22.8 ± 1.0 ms at −20 mV, p < 0.05, n = 8). To determine the time course of recovery from inactivation, the fully activated I−V protocol shown in Figure 4C was used: a 2.5-s depolarizing pulse to +40 mV to inactivate the hERG channels, followed by various repolarizing pulses to test potentials between −120 and +30 mV in increments of 10 mV. The time constant for recovery from inactivation was determined by fitting a monoexponential function to the initial increase in tail-current amplitude at potentials between −70 and −20 mV. As shown in Figure 4D, there was

the steady-state activation kinetics, the protocol shown in Figure 2A was used. As shown in Figure 3A, IhERG‑tail was normalized and plotted against the test pulse potential, giving the steady-state activation curve. The curve was fitted to a Boltzmann equation: 1/Imax = 1/[1+ exp(V1/2 − Vt)/k], where V1/2 is the half activation potential. There was no shift in the steady-state activation curve in the presence of 10 μM 1: (V1/2) control, 2.8 ± 3.3 mV; 10 μM 1, −2.6 ± 4.2 mV; n = 6, p > 0.05; (k) control, 10.5 ± 0.7; 10 μM 1, 13.2 ± 2.2; n = 6, p > 0.05. To analyze the steady-state inactivation kinetics, a special protocol was used (Figure 3B). The channels were inactivated at a holding potential of +40 mV and were recovered from inactivation in steps at various potentials from −120 to +20 mV (12 ms) in increments of 10 mV. The resulting peak outward currents were measured at +20 mV. The inactivating outward current amplitude (measured at +20 mV) was normalized and plotted against the test pulse potential, giving the steady-state inactivation curve shown in Figure 3C. This curve was fitted with a Boltzmann equation to yield inactivation V1/2 and k values. The V1/2 was shifted negatively by 15.9 mV, from −48.7 ± 7.6 mV (control) to −64.6 ± 5.2 mV (10 μM 1) (p < 0.05, n = 9). The corresponding k values were −21.9 ± 0.7 (control) and −22.5 ± 1.0 (10 μM 1) (p > 0.05, n = 9). The decay phase of IhERG was fitted to a monoexponential function to determine the inactivation time constants. Daurisoline (1) at a concentration of 10 μM increased the steady-state inactivation time constant from −120 mV to −60 mV (Figure 3D, p < 0.05, n = 7). D

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Figure 5. Development of hERG channel current blocking by daurisoline (1) using a long step-pulse protocol and an envelope-of-tails protocol. (A) Long step-pulse protocol and the representative recordings of hERG current before and after exposure to 10 μM 1. (B) Drug-sensitive current expressed as a proportion of the current in the absence and presence of 10 μM 1. (C) Envelope protocol and representative recordings of hERG current before and after exposure to 10 μM 1. (D) IhERG‑tail relative to control plotted against the pulse duration.

clamped at a holding potential of −80 mV and depolarized to +30 mV for a variable duration from 50 to 4800 ms in increments of 250 ms (Figure 5C, upper panel). The lower panel of Figure 5C shows a representative recording before and after exposure to 1. The IhERG‑tail relative to control was plotted against the pulse duration (Figure 5D), and the time-dependent decay curve was fitted using a monoexponential function. The time constant at +30 mV was 278 ± 62 ms (n = 5). The plot also shows that the extent of IhERG‑tail inhibition by 1 was significantly less after a brief depolarization step than after a long-duration pulse. The extent of blockade was only about 18.5% when the pulse duration was 50 ms, which suggests that 1 has a very low affinity with hERG in the inactivation or resting state. These data indicate the inhibitory effect increases significantly at membrane potentials more positive than +10 mV (Figure 2F), which elicit maximal hERG activation, and develop further in response to a longer voltage stimulation and envelope voltage protocol, which suggests channel opening is required for blockade of the hERG channel by 1. It has been reported that some drugs (e.g., cardiac glycosides, fluconazole, and ketoconazole) are able to cause the loss of hERG function by interrupting the trafficking of hERG protein to the cell membrane.22−24 To determine whether 1 has this action, hERG channel mRNA, protein expression, and IhERG‑tail were evaluated after incubation with 1 for 24 h. Figure 6A shows RTPCR (upper panel) and qRT-PCR (lower panel) analysis of hERG channel mRNA levels. The expression of hERG mRNA was markedly decreased by 30 μM 1. Figure 6B shows Western blot analysis for hERG protein expression. As previously described, hERG channels showed protein bands at ∼135 kDa (immature core−glycosylated channel protein located in the

no significant change in the time constant of recovery from inactivation after incubation with 1. In addition, 1 failed to shift the steady-state activation curve, suggesting that it does not affect the activation gating of the hERG channel. However, V1/2 for the steady-state inactivation curve was shifted negatively by 1, and the time constant for steady-state inactivation increased. Daurisoline (1) also shifted negatively the onset-of-inactivation curve between −50 and +10 mV, and the onset of inactivation was accelerated in the presence of 1 at test potentials between −20 and +10 mV. These results indicate that 1, like other hERG channel blockers (e.g., azimilide),19 affects hERG-channel inactivation. In order to study the time dependence for the onset of IhERG inhibition by 1, two experimental protocols were used. First, a long-duration (10 s) depolarization step from −80 mV to 0 mV was applied.20 The protocol was applied in the absence of 1, discontinued when the cells were equilibrated with 10 μM 1 for 5 min, and then reapplied in the presence of 10 μM 1. Representative traces in the absence and presence of 1 are shown in Figure 5A. Daurisoline (1) suppressed IhERG to a lesser extent at the beginning of activation than at the end of the depolarization step, which is consistent with the action of an open-channel blocker. Fractional block of IhERG ([Icontrol − IDS]/IDS) was calculated at different time points throughout the protocol, and the mean fractional block data were plotted against time (Figure 5B). The inhibitory effect increased with time of depolarization, but showed no significant change in blockade beyond ∼2000 ms. The result indicates that the blockade shows time dependence, with blockade developing over the first 700 to 800 ms (p < 0.001, n = 5). The time course for the development of inhibition by 1 was also assessed using an envelope-of-tail test.21 The cell was E

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affect hERG function after a 24-h incubation at 1 or 10 μM, but decreased hERG mRNA levels significantly, as well as protein expression, and IhERG at the 30 μM concentration. According to our previous studies, the mean peak concentration is about 1.7 μM when a 2.5 mg/kg bolus intravenous dose of 1 is given to rabbits or dogs,27,28 and this concentration is enough to inhibit arrhythmia. At this concentration, the inhibition ration of IhERG is about 20%. The degree of IhERG blockade is not a reliable predictor for risk of torsade de pointes (TdP). Some agents that inhibit IhERG/IKr or prolong the QT interval are not associated with TdP, such as verapamil and amidarone.29 Earlier, our group has reported that 1 inhibits L-type calcium current (ICa‑L) and suppresses dofetilide-induced early afterdepolaization.30,31 It has been suggested that blockade of ICa‑L may actually prevent the development of TdP, despite the ability of the compound to reduce IKr. In summary, it has been shown that daurisoline (1) directly blocks hERG channels in the open and inactivate state. In addition, it does not affect the expression of the hERG protein when the concentration is below 30 μM, which probably minimizes the risk of long QT syndrome after long-term usage. Inhibition of hERG appears to be the molecular mechanism underlying daurisoline-induced APD prolongation.

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EXPERIMENTAL SECTION

Cell Culture and Chemicals. The HEK293 cell line stably expressing hERG was generously provided by Dr. Guirong Li (University of Hong Kong). Cells were maintained in Dulbecco’s modified Eagle medium containing 500 mg/mL G418 and 10% fetal bovine serum. All reagents were obtained from Invitrogen Corporation. Tyrode’s solution contained (mmol) NaCl 137, KCl 4.0, MgCl2 1.0, CaCl2 1.8, HEPES 10.0, glucose 10; pH was adjusted to 7.4 with NaOH. The pipet solution contained (mmol) KCl 130, MgCl2 1.0, HEPES 10, EGTA 5.0, Mg2-ATP 5.0; pH was adjusted to 7.4 with KOH. Daurisoline (1) (purity >98%) was purchased from Shanghai Winherb Medical Technology Corporation (Shanghai, People’s Republic of China). This agent was dissolved in HCl (1 M) as a stock solution (1 mM, pH adjusted to 6.8 with NaOH) and then diluted to the final concentration before each experiment. Electrophysiological Recordings. Cells were harvested from the flask by treatment with 0.125% trypsin and 0.01% EDTA, centrifuged, and then stored in Tyrode’s solution containing 5% bovine serum albumin at 4 °C. Cells were studied within 8 h of harvesting. The whole cell patch-clamp technique was used as described previously.32 The cell capacitance and series resistance (Rs) were compensated for by about 50−70%, giving rise to Rs values of 2−5 MΩ. Command potential generation and data acquisition were performed with a patch-clamp amplifier (EPC-10, Germany). Quantitative Real-Time RT-PCR. hERG mRNA levels were measured by reverse transcription polymerase chain reaction (RTPCR). Total RNA was extracted with Trizol reagent (Invitrogen, Grand Island, NY, USA). cDNA was synthesized using First Strand kit (Invitrogen, Grand Island, NY, USA). Semiquantitative PCR was run to test the specificity of primers, using the following cycle conditions: 94 °C for 5 min; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s, 35 cycles, and 72 °C for 7 min. The reaction mixture (20 μL) for real-time PCR was as follows: 2× SYBR Green PCR master mix (Applied Biosystems, Carlsbad, CA, USA), 10 μL; 0.25 μM primers; and 6 μL of RT product. The real-time PCR reaction was performed under the following conditions: 95 °C for 10 min, followed by 75 cycles at 95 °C for 15 s and 60 °C for 1 min. The primers for hERG were as follows: (forward) 5′ ACC ACA CAT GCA CCG CCA GG 3′; (reverse) 5′ TTC TTC CCC AGG ATG GCC ACG A 3′. GAPDH was used as internal reference to normalize the relative mRNA level. The primers for GAPDH were as follows: (forward) 5′ AGG CTG GGG CTC ATT TGC AGG 3′; (reverse) 5′ TGG CCA GGG GTG CTA AGC AGT 3′.

Figure 6. Effect of daurisoline (1) on hERG channel protein and mRNA expression and IhERG after 24 h incubation. (A) RT-PCR (upper panel) and qRT-PCR (lower panel) of hERG mRNA level. (B) Western blot analysis of hERG proteins (n = 3,*p < 0.05, **p < 0.01, 1 vs control). (C) I/V plot of IhERG‑tail (pA/pF) (n = 7, *p < 0.05, **p < 0.01, 1 vs control).

endoplasmic reticulum) and at ∼155 kDa (mature fully glycosylated channel protein transported to the cell surface).25,26 The intensity of the 155- and 135-kDa bands remained unchanged after treatment of cells with 1 or 10 μM 1 (p > 0.05, n = 3). However, 30 μM 1 produced a significant decrease in both the 155- and 135-kDa hERG channel proteins (p < 0.05, n = 3). Figure 6C shows IhERG‑tail after incubation with 1 for 24 h, indicating a significant decrease in IhERG‑tail density in the presence of 30 μM 1. It was shown that 1 inhibits hERG channels in a concentrationand voltage-dependent manner. Daurisoline (1) can accelerate hERG-channel inactivation, and the open state is required for the inhibition of hERG by 1. In addition, hERG mRNA and protein expression were evaluated, as well as IhERG, in the absence and presence of 1 (1, 10, 30 μM) for 24 h. Daurisoline (1) did not F

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Western Blot Analysis. The expression of hERG protein was measured by Western blot. Cells were harvested with ice-cold phosphate-buffered saline, and the total protein was extracted. Protein was determined according to the Bradford method (Sigma, St Louis, MO, USA). Denatured protein was separated using 8% SDS-PAGE gels with 200 μg of protein per lane. Polyclonal rabbit anti-hERG antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and antiβ-actin antibody (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used to label hERG and β-actin protein, respectively. The protein bands were quantified by densitometry, and data were normalized to β-actin. Statistics. Values are presented as mean ± standard error of the mean. Single-factor analysis of variance or two-tailed Student’s t tests were used to conduct the statistics. Values of p < 0.05 were considered statistically significant.

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(18) Liu, Q. N. Cellular and Ionic Mechanisms of Dauricine and Daurisoline on Acquired Long QT Syndrome. Ph.D. thesis, University of Science and Technollogy, Wuhan, People’s Republic of China, 2010. (19) Yang, B. F.; Xu, D. H.; Xu, C. Q.; Li, Z.; Du, Z. M.; Wang, H. Z.; Dong, D. L. Acta Pharmacol. Sin. 2004, 25, 554−560. (20) Tang, Q.; Jin, M. W.; Xiang, J. Z.; Dong, M. Q.; Sun, H. Y.; Lau, C. P.; Li, G. R. Biochem. Pharmacol. 2007, 74, 1596−1607. (21) Kamiya, K.; Mitcheson, J. S.; Yasui, K.; Kodama, I.; Sanguinetti, M. C. Mol. Pharmacol. 2001, 60, 244−253. (22) Takemasa, H.; Nagatomo, T.; Abe, H.; Kawakami, K.; Igarashi, T.; Tsurugi, T.; Kabashima, N.; Tamura, M.; Okazaki, M.; Delisle, B. P.; January, C. T.; Otsuji, Y. Br. J. Pharmacol. 2008, 153, 439−447. (23) Han, S.; Zhang, Y.; Chen, Q.; Duan, Y.; Zheng, T.; Hu, X.; Zhang, Z.; Zhang, L. Eur. J. Pharmacol. 2011, 650, 138−144. (24) Wang, L.; Wible, B. A.; Wan, X.; Ficker, E. J. Pharmacol. Exp. Ther. 2007, 320, 525−534. (25) Ficker, E.; Dennis, A. T.; Wang, L.; Brown, A. M. Circ. Res. 2003, 92, e87−100. (26) Thomas, D.; Kiehn, J.; Katus, H. A.; Karle, C. A. Cardiovasc. Res. 2003, 60, 235−241. (27) Gu, S. F.; Shi, S. J.; Chen, H. Yaoxue Xuebao 2003, 38, 908−910. (28) Shi, S. J.; Chen, H.; Gu, S. F.; Zeng, F. D. Acta Pharmacol. Sin. 2003, 24, 1011−1015. (29) Roden, D. M. N. Engl. J. Med. 2004, 350, 1013−1022. (30) Belardinelli, L.; Antzelevitch, C.; Vos, M. A. Trends Pharmacol. Sci. 2003, 24, 619−625. (31) Belardinelli, L.; Shryock, J. C.; Wu, L.; Song, Y. Heart Rhythm 2005, 2, S16−22. (32) Tian, M.; Dong, M. Q.; Chiu, S. W.; Lau, C. P.; Li, G. R. Br. J. Pharmacol. 2006, 147, 289−297.

AUTHOR INFORMATION

Corresponding Author

*Tel and fax: +86 027 83691785. E-mail: [email protected]. edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project was supported by grants from the National Natural Science Foundation of China (30572346, 81000080, and 81072634) and grants from the Ministry of Education of China (NCET-10-0409, 2011TS069).

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