Comparsion Of The Arrhythmogenic Effects Between ACO And MACO On Ventricular Myocytes Of Guinea-pig And Their Underlying Cellular Mechanisms

Aconite crude drugs belong to ranunculaceae plants, which has been usedin clinics for2000years in our country. Aconitumchinense is the main root,fuzi is the lateral root and tianxiong is the single root of ranunculaceae plants,all of them can be used as drugs. Fuzi has the effects of increment of heartfunction, anti-inflammatory, antinociceptive, antitumor and treat coronarydisease, rheumatic heart disease. Aconitum alkaloid is the major toxic element,which can cause the disorders bouth in central nerve system and incardiovascular system, especially in the latter, which severely limits theirclinical use. So the therapeutic effects and toxic effects co-exist in clinicalapplication. The cardio toxic of ACO mainly refers to the arrhythmogeniceffects, which include the induction of premature ventricular contractions(PVC), ventricular tachycardia (VT), torsades de pointes (Tdp), ventricularfibrillation (VF), and mortality with the increase of doses. ACO and MACOwere thought to be the important factors in leading arrhythmia. Because of thereliable and durable arrhythmogenic effects, ACO has also commonly beenused as a classic tool for inducing arrhythmias in experimental animals orinvestigation the effects of anti-arrhythmic drugs. However, the mechanismfor ACO induction of arrhythmia is still unclear. MACO, a structurally relatedanalog of ACO, shares a common C19-norditerpenoid skeleton and differsfrom ACO only by a methyl group instead of an ethyl group at the nitrogenatom. MACO is pharmacologically the most active component, and is reportedto have various pharmacological effects such as positive inotropic effects,analgesic and anti-inflammatory effects and cause contraction in deferens andileum, but the arrhythmogenic potential of MACO was still unknown.The occurrence of arrhythmia was due to the ion transportation imbalance between influx and efflux ions across the membrance. Cardiacaction potential was produced by the movement of various ion channels acrossthe cardiomyocyte membrane. It is generally believed that the arrhythmigeniceffects of ACO caused by open of TTX-sensitive Na+channels. The persistentactivation of Na+channels by ACO arises from the inhibition of channelinactivation, thus leading to Na+influx, which may be accompanied by Ca2+overload via electrogenic Na+-Ca2+exchange system and eventually inducesarrhythmia. In addition, ACO inhibited HERG, Ikurand L-type Ca2+currents indifferent degrees. The effects of ACO on above channels could prolong APDand induce DAD, TA. However, in the low concentration Ca2+of extracellularsolution, ACO shortened APD and also induced DAD and TA had beenreported. So the arrhythmogenic effect of ACO is related with the changes ofAPD. The effects of expressing prolongation or shorten of APD maybedependent on the composition of extracellular solution or animals used in theexperiments. By using the conventional glass electrode technique in recordingthe AP of papillary muscle in guinea-pig, we found ACO induced arrhythmiasvia shortening APD, it can not be interpreted only by activating sodiumchannel currents. The Na+/K+pump is a member of the P-type ATPase pumpsand has two major subunits: α and β. It can utilize the energy released by ATPhydrolysis to pump Na+out of the cell against concentration gradient inexchange for K+in an electrogenic process (3Na+:2K+), which is essential forthe steady state maintance of RMP and cell excitability. ACO inhibited Na+/K+pump in central nervous system significantly had been reported, but itwas uncertain whether it inhibited Na+/K+pump in cardiacmyocytes.Since the arrhythmogenic effects of ACO can not be interpretedcompletely by INaactivation, HERG and Ikurinhibition or the influence onL-type Ca2+currents, and the effects of MACO on heart is not clear. In thepresent study, we investigated and compared the arrhythmogenic effects ofACO and MACO in vivo, and analyzed their mechanisms by ventricularmyocytes in guinea-pigs. This investigation may provide some theoreticalbasis for avoiding or reducing the arrhythmogenic side-effect of ACO. Furthermore, it could provide some evidence for choosing appropriatearrhythmia models in pharmacology research.Part oneThe effects comparision between ACO and MACO on electrocardiogramin guinea-pigObjective: To investigate the effects of ACO and MACO onelectrocardiogram in guinea-pig, then analyzed and compared theircardiotoxicity.Methods: Experiments were performed in vivo on25male guinea-pigs(300~350g). The animals were divided into3groups randomly: solvent group(n=5), ACO group (25μg/Kg, n=10), and MACO group (25μg/Kg, n=10).After weighting, the guinea-pigs were anaesthetized by1.2%sodiumpentobarbital in a volume of0.3ml/100g (i.p). The experimental animalswere injected with ACO or MACO in a dosage of25μg/Kg via femoral vein,and those of control group were injected with normal saline at the same way.Then the standard lead Ⅱ ECGs were recorded by RM-6240CD systembefore and after giving ACO, MACO or normal saline for120minutes.Results: The ECG of control group was not changed obviously within120minutes. The ECG of ACO group was not changed obviously before andafter giving ACO within20minutes. However, different types of arrhythmiawere appeared which including VPB (22.79±7.90minutes,8/10), AVB(22.75±13.70minutes,5/10), VT (42.30±15.69minutes,5/10), VF(39.65±7.17minutes,3/10), then the animals died (56.32±11.40minutes,4/10).After giving MACO, the ECGs were changed rapidly and obviously, theyappeared VPB (1.46±0.66minutes,9/10), AVB (1.46±0.66minutes,8/10), VT(2.53±0.87minutes,9/10), VF (3.50±1.19minutes,9/10), mortality(23.01±14.92minutes,10/10) also appeared.Conclusions: The onset of arrhythmias of APB, AVB, VF and VT occurssignificantly earlier in MACO group compaired with than those in ACO group (P<0.01), and the ratio of VT and mortality were higher in MACOGROUP than those in ACO group (P<0.05).Part twoEffect comparision of ACO and MACO on AP in guinea-pig ventricularmyocytes and their underlying mechanismsObjective: To investigate the effects of ACO and MACO on APrespectively, and analyze their underlying mechanisms in guinea-pigventricular myocytes.Methods: The perforated patch-clamp technique in current-clamp modewas used as follows:(1) Investigating the effects of ACO or MACO on APrespectively in guinea-pig ventricular myocytes.(2) Recording the effects ofACO or MACO on AP respectively in guinea-pig ventricular myocytes inthe presence of SMV (a non-selective Na+/K+pump antagonist) or OUA (aselective Na+/K+pump antagonist).(3) Recording the effects of ACO orMACO on AP respectively in guinea-pig ventricular myocytes in the presenceof TTX (a selective Na+channel antagonist).Results:(1) Bouth ACO and MACO shortened APD30, APD90in aconcentration-dependent manner, at the higher concentration of ACO (3×10-6M), MACO (3×10-7M), the RMP and APA were decreased, the DAD and TAappeared in guinea-pig ventricular myocytes, EAD also appeared in thepresence of MACO.(2) SMV only counteracted the effects of lowerconcentration ACO (3×10-7M) on shortening APD30and APD90, and it alsoeliminated the effects of higher concentration of ACO (3×10-6M) on RMP andAPA, the DAD and TA also appeared compared with that using ACO alone.SMV can not counteracted the effects of MACO on shortening APD30, APD90,but it eliminated the effects of higher concentration of MACO (3×10-7M) onRMP and APA, but there were no effects on DAD, EAD and TA. OUA (10-7M)shortened APD30, APD90significantly, counteracted the effects of lowerconcentration of ACO (3×10-7M) on shortening APD30, APD90, and it also eliminated the effects of high concentration of ACO (3×10-6M) on RMP andAPA, the DAD and TA also appeared compared with that using ACO alone.OUA only counteracted the effects of high concentration of MACO (3×10-7M)on RMP, APA.(3) TTX (2×10-6M) decreased RMP level markedly. In thepresence of TTX, the effects of lower concentration ACO (3×10-7M) on APwere counteracted completely, the higher concentration of ACO (3×10-6M)just shortened APD30, APD90significantly, and there were no DAD and TAappeared. The effects of lower concentration MACO (10-7M) on AP werecounteracted completely, the higher concentration of MACO (3×10-7M) onlyexpressed the shorten APD30, APD90significantly, and EAD also appearedoccasionally.Conclusion:1. Bouth ACO and MACO induced DAD by APD shorten,MACO also induced EAD. In the same concentration, the effects of MACOwere stronger than that of ACO.2. The shorten effects of ACO on APD were caused by inhibiting INa/Kand activating INa, but that of MACO was caused mainly by activating INa.Part threeThe effects of ACO and MACO on Na+/K+pump current (INa/K) andsodium channel current (INa) in guinea-pig ventricular myocytes.Objective: To investigate the effects of ACO and MACO on INa/Kand INain guinea-pig ventricular myocytes.Methods: The whole-cell patch clamp technique in voltage-clamp modewas used as follows:(1) Indentifying the electrophysiology characteristic ofINa/K.(2) Recording the effects of ACO and MACO on INa/K, respectively.(3)Recording the effects of ACO and MACO on INa, respectively.Results:(1) The currents we recorded consist with theelectrophysiological characteristics of INa/Kin guinea-pig ventricular myocytes.(2) ACO inhibited bouth INa/Kand stimulated INain a concentration-dependentmanner,the inhibition ration for INa/Kwas22.8±4.46%, while the increasement ration for INawas25.5±9.2%in the presence of ACO3×10-7M; ACO3×10-6M inhibited INa/Kby48.6±14.6%and increased INaby37.2±9.5%respectively;MACO also inhibited bouth INa/Kand stimulated INain aconcentration-dependent manner, the inhibition ration for INa/Kwas28.0±7.5%,while the increase ration for INawas13.0±1.9%in the presence of MACO10-7M; the INa/Kinhibition ration was41.8±9.3%, and increasement ration was26.1±5.3%in the presence of MACO3×10-7M.Conclusions: Bouth ACO and MACO inhibited INa/Kand activated INaina concentration-dependent manner. The inhibition effects of MACO on INa/Kislarger than that of ACO in the same concentration while the activation ofMACO on INais similar with that of ACO.