Studies On The Potential Proarrhythmia Of Combined Use Of Azithromycin And Shengmai Intravenous Injection And Its Underlying Mechanism

Objective:To explore the proarrhythmic effect and the underlying mechanism of combined use of azithromycin and shengmai intravenous injection.Methods:1.In vivo electrocardiogram(ECG)recording:For in vivo ECG recordings,ECG(Ⅱ lead)was recorded from anesthetized guinea pigs(20%urethane 5 mL/kg,intraperitoneal)by the conventional in vivo ECG technique in which the HR,P-R,QRS and QTc intervals were analyzed.Lyophilized azithromycin maleate,shengmai intravenous injection or combination of both were dissolved in saline and administrated intravenously.The left and right upper limbs and left lower limbs were inserted into the needle electrode to record the ECG.2.Ex vivo electrocardiogram(ECG)recording:For ex vivo ECG recordings,an isolated guinea pig heart perfusion system was used to record ECG.The hearts from anaesthetized guinea pigs were excised and cannulated through the aorta above the coronary ostia at constant pressure(70 mmHg)with perfusion solution(37℃)containing(mM):NaCl 117,CaCl2 2.5,KCl 5.7,NaHCO.4.4,MgCl2 1.7,HEPES 20,NaH2PO41.5,Glucose 11,gassed with 95%O2 plus 5%CO2,pH 7.4 with NaOH.Drugs were added in the perfusion solution and infused via retrograde perfusion of the coronary artery.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.3.Recording of action pote-ntials from guinea pig single venticular myocyte:Guinea pig ventricular myocytes were obtained by enzymatic dissociation as previously described.Whole-cell current clamp configuration was used to record action potential(AP)with an amplifier(Axopatch 200B,Axon Instruments).The composition of internal solution was(mM):KCl 135,EGTA 10,Glucose 5,HEPES 10,Na2-ATP 3,Na-GTP 0.5,pH 7.3 with KOH.The external solution contained(mM):NaCl 117,KCl 5.7,NaHC03 4.4,MgCl2 1,7,HEPES 20,Glucose 20,Taurine 20,CaCl2 1.8,pH 7.4 with NaOH.All experiments were performed at room temperature(20-22℃).APs were elicited by passing appropriate current pulses at 0.1 z through the recording electrode.Action potential durations at 50%and 90%foll repolarization levels(APD50 and APD90)were measured.4.Recording of L-type Ca2+ currents from guinea pig left ventricular myocytes:Whole-cell voltage clamp technique was used to record L-type Ca2+ currents from guinea pig left ventricular myocytes.The recording external solution contained(mM):NaCl 132,CsCl 5.4,CaCl2 1.8,MgCl2 1.8,NaH2PO4 0.6,4-AP 5,HEPES 10,Glucose 10,Na-pyruvate 5,pH 7.4 with NaOH.The internal solution contained(mM):CsCl 130,MgCh 2,EGTA 11,HEPES 20,Glucose 10,Na2-ATP 2,Na-GTP 0.1,pH 7.3 with CsOH.To record L-type Ca2+ current,the depolarizing pulses applied from a holding potential of-80 mV were stepped to-40 mV for 30 ms(to inactivate Na+ current)and then to 0 mV for 300 ms at 0.1 Hz.5.Recording of Na+ currents from guinea pig left ventricular myocytes:Whole-cell voltage clamp technique was used to record Na+ currents from guinea pig left ventricular myocytes.The external solution contained(mM):NaCl 70,Choline-Cl 70,KCl 5.4,MgCl2 1,CaCl2 0.1,HEPES 10,Glucose 10,NaH2PO4 0.33,pH 7.4 with NaOH.The internal solution contained(mM):CsCl 120,EGTA 11,HEPES 10,Na2-ATP 5,MgCl2 5,CaCl2 1,Glucose 11,pH 7.3 with CsOH.The stimulation pulse for recording Na4+ current was stepped-40 mV for 50 ms from a holding potential of-80 mV at 0.1 Hz.Results:1.Effects of azithromycin,Shengmai intravenous injection and their combination on ECG of guinea pigs in vivo:The results showed that the one time relevant clinical dose of azithromycin injection(38.2 mg/kg)or one time relevant clinical dose of Shengmai injection(4.6 mL/kg)had no significant effect on the electrocardiogram(ECG)of guinea pigs.When administration of azithromycin(one time revelant clinical dose plus Shengmai injection(one time relevant clinical dose),heart rate was reduced and PR and QRS were prolonged significant.2.Effects of azithromycin,Shengmai intravenous injection and their combination on ECG in isolated guinea pigs:Azithromycin or Shengmai injection significantly reduce the heart rate and prolonged the P-R,QRS and QTc interval significantly in a concentration dependent manner.However,only one time relevant clinical concentration Azithromycin combined with one time relevant clinical concentration Shengmai injection could reduce heart rate,prolong the P-R and QRS interval significantly.3.Effects of azithromycin,shengmai injection and their combination on action potential of single ventricular myocytes in guinea pigs:Azithromycin(one time relevant clinical concentration of 41.5 mg/L)failed to significantly affect APD50,APD90 and amplitude of action potential.However,one time relevant clinical concentration of azithromycin(41.5 mg/L)combined with Shengmai Injection(one time relevant clinical concentration of 5 mL/L)reduced the amplitude of action potentials and shortened APD50 and APD90 significantly.4.Effects of azithromycin,shengmai injection and their combination on L-type calcium current in left ventricular myocytes of guinea pigs:Azithromycin(one time relevant clinical concentration of 41.5 mg/L)failed to significantly affect L-type Ca2+ current.However,one time relevant clinical concentration of azithromycin(41.5 mg/L)combined with Shengmai Injection(one time relevant clinical concentration of 5 mL/L)significantly inhibited L-type Ca2+ current.5.Effects of Azithromycin,Shengmai Injection and their combination on Na+ currentin left ventricular myocytes of guinea pigs:Azithromycin(one time relevant clinical concentration of 41.5 mg/L)failed to significantly affect Na + current.However,one time relevant clinical concentration of azithromycin(41.5 mg/L)combined with Shengmai Injection(one time relevant clinical concentration of 5 mL/L)significant inhibition of Na + current.Conclusion:1.In vivo and in vitro ECG demonstrated that one time relevant dose concentration azithromycin combined with one time relevant clinical dose concentration shengmai injection could cause bradyarrhythmia in guinea pig heart.2.One time relevant concentration azithromycin combined with one time relevant clinical concentration shengmai injection significantly inhibited the amplitude of action potential,shortened APD50 and APD90.This result further consolidated the potential arrhythmia induced by azithromycin and azithromycin.3.Bradyarrhythmia induced azithromycin combined with Shengmai injection was mediated by inhibiting L-type Ca2+ and Na+ currents.