Torsade de pointes (TdP) arrhythmia is a potentially fatal form of ventricular arrhythmia that occurs under conditions in which cardiac repolarization is delayed (as indicated by prolonged QT intervals in electrocardiograph recordings).1-3) A likely mechanism for QT interval prolongation and TdP arrhythmias is blockade of the rapid component of the cardiac delayed rectifier K ϩ current (I Kr ), which is encoded by human ether-à-go-go-related gene (HERG). 1,4,5) More than 100 non-cardiovascular drugs can induce TdP arrhythmias or QT interval prolongations in electrocardiograms. [6][7][8][9] Regulatory guidelines [CPMP/986/96 (1997) and ICH S7B (2005)] recommend the use of preclinical in vivo and in vitro (in which a broad range of concentrations should be used) studies to detect the QT-prolonging effects and arrhythmogenic potential of new chemical entities. [10][11][12] The HERG assay (which tests I Kr ) is one of the recommended in vitro models for the detection of potential drug-induced long QT.
11)Sophocarpine (SC) is a dehydroderivative of the bis-quinolizidine alkaloid matrine. 13) In China, it has entered clinical development as an injection solution with a superior inhibitory effect on Coxsackie B virus that should result in improved efficacy in the treatment of viral myocarditis. [14][15][16][17] The inhibitory actions of several matrine-type alkaloids on I Kr have recently been reported. 18,19) We investigated the effects of SC on HERG-encoded K ϩ channels to assess the risk of this drug causing cardiac arrhythmia associated with QT interval prolongation.We set out to establish: (i) the effects of SC on the amplitude, concentration dependence and kinetics of currents conducted by HERG channels stably expressed in human embryonic kidney (HEK293) cells. This was done using a wholecell patch clamp method; and (ii) the effects of SC on HERG protein levels in HEK293 cells. This was achieved using Western blot analysis and immunofluorescence methods.
MATERIALS AND METHODS
HERG-Expressing Cell LinesExperiments were performed on HEK293 cells that stably expressed the wild-type HERG gene (provided by Professor Zhi-Guo Wang, Montreal Heart Institute, Montreal, Canada). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Hyclone) supplemented with 10% (v/v) fetal calf serum (FCS, Gibco) and 200-mM geneticin (G-418, Gibco) at 37°C in a humidified atmosphere of 5% CO 2 .Sophocarpine SC (Dushun Ltd., Ningxia, China) was dissolved in deionized water to obtain a 10-mM stock solution, which was stored at Ϫ20°C.Electrophysiological Recordings HERG currents were measured using the whole-cell configuration of the patch clamp technique. 8,9,20) Heat-polished patch pipettes had final resistances of 2-4 MW when filled with a pipette solution containing (in mM): 130 KCl, 1 MgCl 2 · 6H 2 O, 10 HEPES, 5 Mg-ATP, 5 EGTA and 0.1 guanosine triphosphate (GTP) (pH 7.3 with KOH). The extracellular solution contained (in mM): 136 NaCl, 5.4 KCl, 5 HEPES, 1 MgCl 2 · 6H 2 O, 1 CaCl 2 and 10 glucose (pH 7.4 with NaOH). The f...
