Objective: Comprised of the alcohol-water extracts of three herbs: lonicera (shuanghua), scute (huangqin), and forsythia (liangqiao), Shuanghuanglian (SHL) is a modem formula meant to be taken by mouth or orally, that was devised in the 1960s for the treatment of a variety of infections.SHL was later developed in an injection form, and adopted for the treatment of respiratory tract infections.Due to severe adverse reactions and deaths, injectable SHL was withdrawn from the market on February 12, 2009 by the Ministry of Public Health and State Food and Drug Administration (SFDA) of China.Since the mechanism for injectable SHLs severe adverse reaction is still poorly understood, the purpose of this study is to investage the potential role of SHL for cardiotoxicty and for proarrhythmia, which may have contributed to the clinical risks associated with injectable SHL.Methods: The experimentals are carried out using techniques set by ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use).1.In vivo electrocardiogram (ECG) recording Ⅱ lead ECG was recorded by conventional in vivo ECG technique.The P-R interval and the rate-corrected QT intervals (QTc) intervals were analyzed.2.Ex vivo electrocardiogram (ECG) recording: Langendoff isolated heart perfusion system was used to record ECG.The positive electrode was placed at the cardiac apex, the negative electrode at right atrium and the reference electrode at the root of the aorta.The P-R interval and the rate-corrected QT intervals (QTc) were analyzed.3.Recording of action potentials from guinea pig heart papillary muscle and canine purkinje fiber: The hearts were taken from anesthetized animals (guinea pig and canine) and placed in ice cold water.The guinea pig heart papillary muscle and canine purkinje fiber were separated and placed in a recording perfusion solution chamber at 35.0 ± 0.5℃ gased with 95% O2 plus 5% CO2 (pH 7.4 with NaOH).The action potential during at 50 % depolarization level (APD50) and at 90 % depolarization level (APD90) were analyzed from papillary muscle and purkinje fiber using conventional intracellular recording technique.4.Recording of L-type Ca2+ current from guinea pig left ventricular myocyte.Patch clamp technique was used to record the L-type Ca2+ current from guinea pig left ventricular myocyte.The recording external solution (37 ℃) contained (in mM): NaCl 138, KCl 9, CaCl2 1 ,MgCl2 1, TEA-C1 10, HEPES 10, pH 7.4 with NaOH.The internal solution contained (inmM): CsCl 130, MgCl22, EGTA 11, Glucose 10, HEPES 20, Na2ATP2, GTP0.1, pH 7.2 with CsOH.To record L-type Ca2+ current, depolarizing pulses applied from a holding potential of-80 mV were stepped to-40 mV for 20 ms (to inactivate Na+ current) and then to 0 mV for 300 ms.5.Recording ofhNavl.5 current from HEK cell line cloned with hNav1.5 cDNA: The sequence of the hNav1.5 cDNA (NM_000335.4) was synthesized and cloned into the expression vector pcDNA3.1 with the G418 resistance.The plasmid was transfected into the HEK cell line by lipofectamine 2000.During the screening process, G418 at 500 μg/ml was used.The cell lines, which showed good and stable current by manual patch clamp, were used for the further experiments.The recording external solution (37 ℃) contained (in raM): NaCl 25, TEACl 122, MgCl2 1, Glucose 10, HEPES 10, CaCl2 1.8, pH 7.4 with NaOH.The internal solution contained (in mM): CsF 129, MgCl2 2, EGTA 11, HEPES 10, Na2ATP 3, pH 7.2 with CsOH.To record hNav1.5 current, depolarizing pulses applied from a holding potential of-80 mV were stepped to-20 mV for 20 ms.6.Recording ofhERG current from HEK cell line cloned with hERG cDNA: The sequence of the bERG cDNA (NM_000238.2) was synthesized and cloned into the expression vector pcDNA3.1 with the G418 resistance.The plasmid was transfected into the HEK cell line by lipofectamine 2000.During the screening process, G418 at 500 μg/ml was used.The cell lines,which showed good and stable current by manual patch clamp, were used for the further experiments.The recording external solution (37 ℃) contained (in mM): NaCl 137, MgCl2 1.2, KCl 5.4, Glucose 10, HEPES 10, CaCl2 2, pH 7.4 with NaOH.The internal solution contained (in mM): KCl 140, MgCl2 1, EGTA 5, Na2ATP 5, HEPES 10, pH 7.2 with KOH.The tail currents were evoked in room temperature once every 30 s by a 5000 ms-50 mV repolarizing pulse following a 4800 ms +50 mV depolarizing pulse with a hold voltage of-80 inV.A 50 ms depolarized pulse to-50 mV at the beginning of the voltage protocol served as a baseline for calculating the amplitude of the peak tail current.Results: 1.SHL significantly prolonged the in vivo ECG P-R interval: SHL 325.5 mg/kg (one time clinical dose) and 1627.5 mg/kg (five time clinical dose) significantly prolonged P-R intervals from control (59 ± 5) ms to (74 ± 10) ms and (88 ± 20) ms, respectively (P<0.05, n=15) and did not significantly change QTc (P>0.05, n=15).SHL 3255.0 mg/kg (ten time clinical dose) significantly prolonged P-R and QTc intervals from control (58 ± 5) ms and (247 ± 16) ms to (133 ± 29) ms and (301 ± 65 ms), respectively (p<0.05, n=15) with significant intra-ventricular block.2.SHL significantly prolonged in vitro ECG P-R interval: SHL 3 g/L (ten time clinical dose) and 9 g/L (30 time clinical dose) significantly prolonged the in vitro ECG P-R intervals from control (84 ± 17) ms to (113 ± 39) ms and (130 ± 23) ms, respectively (p<0.05, n=7) and did not significantly change QTc intervals.Both above doses of SHL caused significant intra-ventricular block.3.SHL abolished action potentials recorded from left ventricle papillary muscles of guinea pig and canine right purkinje fibers: SHL 9 g/L and 6 g/L abolished action potentials recorded from left ventricle papillary muscles of guinea pig and canine right purkinje fibers respectively and did not affect the durings of action potentials from both tissues.Washout of SHL could recover the action potentials from both tissues.4.SHL suppressed the L-type Ca2+ current from left ventricular myocyte of guinea pig: SHL suppressed the L-type Ca2+ current from left ventricular myocyte of guinea pig in a dose-dependent manner.IC50 of SHL for L-type Ca2+ current was 1.76 ± 0.5 g/L which is approximately six time clinical dose (one clinical dose is 0.3 g/L).5.SHL suppressed the hNav1.5 current: SHL suppressed the hNav1.5 current in a dose-dependent manner.IC50 of SHL for the hNav1.5 current was 0.93 ± 0.09 g/L which is approximately three time clinical dose (one clinical dose is 0.3 g/L).6.SHL suppressed the hERG current: SHL suppressed the bERG current in a dose-dependent manner.IC50 of SHL for the hERG current was 2.8 ± 0.3 g/L which is approximately nine time clinical dose (one clinical dose is 0.3 g/L).Conclusion: 1.One time clinical dose of SHL significantly prolonged guinea pig P-R interval of in vivo ECG and did not affect QTc.Ten time clinical SHL caused the intra-ventricular block of in vivo guinea pig.2.20 and 30 time clinical dose of SHL abolished action potentials from canine right ventricular purkinje fiber and guinea pig left ventricular papillary muscles respectively.The IC50S of SHL for blocking hNav1.5 current, L-type Ca2+ current and bERG current were three, six and nine time clinical doses respectively.3.SHL, from low to high concentrations, blocked hNav1.5 current, L-type Ca2+ current and hERG current respectively.SHL caused bradyarrhythmias, not tachyarrhythmia mainly by inhibiting Na+ current.