| Part one Investigation of the methods for acute isolatingcalcium-tolerant ventricular myocytes andelectrophysiolological recordingsObjective:It is important to obtain viable cells for long period of electrophysiologicalrecording.In this study,we explore the influences of ingredients of extracellularsuperfusate on acute isolation of calcium-tolerant ventricular myocytes and recording ondelayed rectifier potassium (IK) and L-type calcium current (ICa-L).Methods:Single ventricular myocytes of rabbits were obtained enzymatically byretrograde Langendorff perfusing apparatus.Before Langendorff perfusing,theextracellular superfusate was Tyrode's solution in experiment group and calcium-freeTyrode's solution in control group.The effect of extracellular superfusate Ca2+ oncalcium-tolerant ventricular myocytes available for electrophysiological recordings wereinvestigated.Whole-cell patch clamp recording technique was used to record the changes ofIK and ICa-L in experimental groups following the administration of extracellular with BaCl2and N-methyl-D-glu-camine (NMDG) in experiment group and extracellular Tyrode'ssolution in control group.Results:The survival rates of ventricular myocytes and calcium-tolerant myocyteswere significantly higher in experimental groups than in control group (71.0%±9.7% vs54.5%±10.1%,52.0%±.7.9% vs 38.5%±10.8%,n=10,P＜0.05).In experimental group, peak ICa-L was elicited stably and larger than in control group (15.2±7.1 pA/pF vs 7.4±2.4pA/pF,n=10,P＜0.05).As compared 20 min with 5 min,the rate of change in peak ICa-L was36.1%±9.1% in control group (n=10,P＜0.05),and 3.0%±4.7% in experimental group(n=10,P＞0.05).The time-dependent outward current and tail current of IK were higher inexperimental group than in control group (1.39±0.05 pA/pF vs 0.53±0.14 pA/pF,0.74±0.09 pA/pF vs 0.39±0.13 pA/pF,n=10,P＜0.05).Conclusion:Extracellular superfusate Ca2+ is helpful for isolating singlecalcium-tolerant cardiomyocytes.With BaCl2 and NMDG in extracellular superfusate,theionic currents of ICa-L and IK were elicited typically and easily.Part two Effects of sodium ferulate and amiodarone on L-typecalcium channel current in isolated rabbit ventricular myocytesObjective:To study the effect of sodium ferulate on L-type calcium current (ICa-L) inisolated rabbit ventricular myocytes.Methods:Compared with amiodarone- a typical classⅢantiarrhythmic drug,singleventricular myocytes of rabbits were isolated enzymatically and whole-cell patch clamprecording technique was used to record the changes of ICa-L following the administration ofsodium ferulate at 3,10,30 and 100μmol/L.Results:Both sodium ferulate and amiodarone could block the ICa-L which hadconcentration dependence,however,sodium ferulate inferior to amiodarone (P＜0.05).PeakICa-L was reduced 21.1%±3.8%,32.6%±2.6%,52.6%±4.6% and 71.4%±7.0% (n=5,P＜0.05) respectively at amiodarone 1,3,10 and 30μmol/L.In contrast,Peak ICa-L wasreduced 11.1%±2.4%,26.9%±6.2%,40.5%±5.0% and 61.9%±5.5% (n=5,P＜0.05)respectively at sodium ferulate 3,10,30 and 100μmol/L.IC50 (the concentration for half-maximal block) was 32.6μmol/L and 9.5μmol/L.The current-voltage curve movedupward,stead-state activated curve moved right and stead-state deactivated curve movedleft,furthermore,they delayed the recovery time course of ICa-L from inactive state. Conclusion:Sodium ferulate block L-type calcium channel,slow it's activate,rapidit's inactivate and delay the recovery time.These may be the related mechanisms of theantiarrhythmic effects.Part three Effects of sodium ferulate and amiodarone on potassiumchannel currents in isolated rabbit ventricular myocytesObjective:To study the effect of sodium ferulate on rapidly and slowly activatingcomponent of delayed rectifier potassium current (IKr,IKs),inwardly rectified potassiumcurrent (IK1) and transient outward potassium current (Ito)in isolated rabbit ventricularmyocytes.Methods:Compared with amiodarone - a typical classⅢantiarrhythmic drug,singleventricular myocytes of rabbits were isolated enzymatically and whole-cell patch clamprecording technique was used to record the changes of IKr,IKs,IK1 and Ito following theadministration of sodium ferulate at 3,10,30 and 100μmol/L.Results Sodium ferulate was inferior to amiodarone,though both of them could blockthe time-dependent outward current and tail current of IKr and IKs which had concentrationdependence.Peak tail currents of IKr were reduced 12.1%±2.5%,24.1%±3.0%,47.0%±5.8%,58.5%±8.3% (n=5,P＜0.05) respectively,while,peak tail currents of IKs werereduced 15.6%±6.4%,27.1%±6.5%,45.6%±5.7%,51.8%±6.6% (n=5,P＜0.05)respectively at sodium ferulate 3,10,30 and 100μmol/L.IC50 (the concentration for half-maximal block) were higher than amiodarone respectively (43.6 vs 3.48μmol/L,44.8 vs5.11μmol/L).Compared with amiodarone (30μmol/L),sodium ferulate (100μmol/L)decreased Ik1 by 24.3%±8.8% vs 29.7%±8.0% at test potential of -100 mV (n=5,P＜0.05),At -20mV,IK1 decreased by 17.7%±3.3% vs 20.1%±10.6% (n=5,P＜0.05).Thecurrent-voltage curve of IK1 moved left by sodium ferulate (30,100μmol/L) andamiodarone (10,30μmol/L) acute superfusion.However,neither of them alter Ito and it'scurrent-voltage curve. Conclusion:Sodium ferulate block potassium channels,these may be the relatedmechanisms of the antiarrhythmic effects.Part four Effects of sodium ferulate and amiodarone on sodiumchannel currents in isolated rabbit ventricular myocytesObjective:To study the effect of sodium ferulate on sodium current (INa) in isolatedrabbit ventricular myocytes.Methods:Compared with amiodarone- a typical classⅢantiarrhythmic drug,singleventricular myocytes of rabbits were isolated enzymatically and whole-cell patch clamprecording technique was used to record the changes of INa.Results:The amplitude of peak INa was decreased by acute amiodarone (30μmol/L)superfusion (20.9±3.63 pA/pF vs 12.0±2.69 pA/pF,n=5,P＜0.05),the inhibition was42.6%±4.87%,and current-voltage curves of INa moved upward.However,sodiumferulate (100μmol/L) superfusion had no effect on INa (16.6±4.52 pA/pF vs 15.8±3.21pA/pF,n=5,P＜0.05 ),the inhibition was 6.12%±5.41%,neither did sodium ferulate altercurrent-voltage curves of INa ( n=5,P＞0.05 ).Conclusion:Sodium ferulate had no effect on sodium channel current in Langendorff-perfused rabbit heart.
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