Toxic Effects Of Aconitine In Rats' Cardiomyocytes: A Study On Molecular Toxicology Mechanism

1 BackgroundToxic animals and plants poisoning are one of important componentelements of forensic toxicology with Chinese characteristic, and a part ofimportant content of forensic toxicology research in China. Aconite andAconitium plants, which are important Chinese medicine widely used inclinic, are typical representative for herb poisonous plants, and they arethe poisonous plant recorded earliest. Aconitium alkaloid is their chiefingredient; and Aconitine, chemical structure of which containsdiesterditerpene, is the most poisonous. The target organs of aconitinetoxic effect are mainly heart and nervous system. It has been confirmedthat toxic ingredient of aconitine, which plays roles of drug, have widespread pharmaceutical properties including cardio-tonic, analgesic,anti-inflammatory, tumor-suppressant, lowering blood pressure, reducinghemal wall and so on. As aconitine are of severe toxicity and therapy doseis so close to poisonous dose or lethal dose that use immodestly, such asimproper process, misuse and so on, would lead to poisoning, even death.Meanwhile, related homicide or veneficium suicide using Aconite andAconitium plants are matters of common occurrences, which indicate thatAconitine intoxication lies important position of toxic animals and plantspoisoning.In order to further improve the diagnosis and healing of aconitinepoisoning, specify the therapeutic action of Aconitium poisonous Chinesetraditional medicines for diseases and offer relevant theoretical foundationfor forensic evaluation of aconitine intoxication, the investigation of the mechanism of aconitine poisoning, especially the mechanism of toxiceffect on cardiomyocytes, turn into the important issue for research andapplication of Chinese traditional medicines, clinical emergency medicineand forensic toxicology study.At present, mechanism studies of toxic effect on aconitine poisoningare mainly focused on molecular action of cytoplasma membrane dynamicchange, most of which are limited to cardiomyocytes injury, detection ofion channel of single cardiac myocyte and so on. It is uncertain thatmolecular toxic mechanism of toxic effect on groups of ventricularmyocytes as target organ, especially influence on information transferbetween cardiomyocytes and mechanism of gene expression regulatory.2 Objectives●To systematic observe toxicological pathology change of cardiomyocytesafter incubation with different concentrations of aconitine,investigate the effect relationship between different poisonous dose,poisonous period and pathological change, optimize and specify cell ofaconitine target organ culture and basic method of moleculartoxicologic study.●To study the DNA damage of aconitine cultured cardiomyocytes andoffer theoretical principle for the relationship between DNA damageand aconitine intoxication mechanism from molecular toxicologicpoint of view.●To investigate the expression of protein kinase C (PKCα) andphosphorylation level in aconitine cultured cardiomyocytes and howPKCαphosphorylation affect Cx43 (Ser368) phosphorylation; affirmwhether PKCαand P-Cx43 (Ser368) phosphorylation be of cascadeeffect relation after aconitine incubation and play role in molecularmechanism of aconitine cardiomyocytes. ●To explore the influence of aconitine on calmodulin in cardiomyocytes.Observe calmodulinin involved in the way of toxicitymechanism of aconitine cardiomyocytes.●To analyze the character of aconitine poisoning and attentions inforensic identification.3 Methods3.1 Optimizing neonatal rats' cardomyocytes primary culturecondition.1-2 day-old Spague-Dawley rats, regardless of the gender, weredisinfected, and ventricles were minced and digested in solutioncontaining trypsinase to be myocardial cell suspension. After incubation at37℃in 5% CO₂, selective attachment of cardiomyocytes was induced forprimary culture cell to optimize primary cardomyocytes culture conditionand increase the purity, survival rates of ventricular myocytes and integrityof cardomyocytes group. Identify the activity of cultured cardiomyocytesby trypan blue staining method, and evaluate the purity of cardiomyocytesby anti-myocardium specific monoclonal antibody combining withindirect immunofluorescence. Specify and optimize in vitro neonatal rats'cardomyocytes primary culture condition and set up preferred basis forfollowing study.3.2 Setting up aconitine cultured model and studying toxic effect ofaconitine.On 6^th day of primary cardiomyocytes culture, after replenishmentwith serum-free medium for 16～18h incubation to wipe off interference ofserum, cardiomyocytes was incubated with different doses of aconitine tobe cultured models. Meanwhile, set up various intoxicated and controlgroups according to different objectives. Real-time monitoringmorphologic and functional change of aconitine cultured cardiomyocytes, and analyze the dose-effect manner of aconitine toxicity tocardiomyocytes injury.3.3 Studying the DNA damage of aconitine cultured cardiomyoeytes.Ventricular myocytes of 24 neonatal rats (randomly divided into 6groups) were incubated by differential adherent culture. On 6^th day ofcardiomyocytes primary culture, after replenishment with serum-freemedium for 16～18h incubation, unattached cells were adjusted to cellsuspension at a density of 2×10⁵ cell/ml. Add aconitine solution in cellsuspensions to aconitine mixture at different concentrations of 0.1μM/L,0.5μM/L, 1μM/L and 2μM/L for 30 min incubation. Set PBS to benegative control group. Detect DNA damage level of ventricular myocytesafter aconitine incubation by comet assay and acridine orange dye andanalyze the damage by CASP analysis software. All the steps repeated 6times.3.4 Investigating the expression of PKCαin aconitine culturedneonatal rats' cardiomyocytes.12 wells for primary cultured cardiomyocytes group were dividedinto 7groups, including normal group (Normal), aconitine incubated group(ACO), alkaline phosphatase treated group (A.P.), AAP treated group(AAP), aconitine incubated after AAP treated group (AAP+ACO), G(o|¨)6976 group (PKCαdepressant, G(o|¨) 6976) and AAP and G(o|¨) 6976 co-treatedgroup (AAP+G(o|¨) 6976). Extract total protein of cardiomyocytes ofexperimental groups and control group, and quantify the total protein byBCA standard protein regression curve. Quantify the change of expressionamounts of P-Cx43 (Ser-368), NP-Cx43 (Ser-368) and total PKCαandP-PKCα(Ser-657) in groups of cultured cardiomyocytes byWestern-bloting with anti-P-Cx43 (Ser-368) antibody, anti-NP-Cx43(Ser-368) antibody and anti-PKCαantibody and anti-P-PKCα(Ser-657)antibody. All the steps repeated 6 times. 3.5 Influence of the PKCαand P-PKCα(Ser-657) locusphosphorylation state in cardiomyocytes after aconitine incubation.Anti-rabbit-anti-rat PKCαpolyclonal antibody (dilution at 1:200),rabbit anti-mouse phosphorylation PKCα(Ser-657) functional antibody(dilution at 1:200), Fluorescein mark goat via rabbit IgG and Rhodaminemark goat via rabbit IgG (dilution at 1:100) were selected to detect thespecific phosphorylation state of P-PKCα(Ser-657) locus with laserscanning confocal microscope and cellular image fluorescent locationanalysis technique before and after aconitine incubation.3.6 Influence of the expression of calmodulin in aconitine culturedrats' cardiomyocytes.Multiplex fluorescence reaction system and amplification methodwere set up with Na⁺-Ca²⁺ crossover (NCX), phosphoric receptor sodiumalbumen (PLB), sarcoplasmic reticulum calcium pump Ca²⁺-ATP ase(SERCA₂), Ryanodine receptor (RyR₂) and housekeeping gene (β-actin)by fluorescence labeling technique and RT-PCR in 12 wells of primarycultured cardiomyocytes groups. Capillary electrophoresis was used by3100 DNA Sequencer to detect the difference and change of transcriptmRNA of NCX, SERCA₂, RyR₂ and PLB in aconitine cultured groups andcontrol group. All the steps repeated 6 times.3.7 Analyzing forensic postmortem examination documents of 10aconitine poisoning cases.Collect forensic postmortem examination documents of 10 aconitinepoisoning cases, including systematic forensic anatomical data andhistopathological outcomes, investigation documents, rescue record and soon for some of the cases. Sort the data by age, gender, poisoning cause,poisoning type, and pathological change, characteristic and so on.Summarize the toxicological pathologic change of aconitine poisoning and investigate attention of forensic identification for aconitine poisoning.3.8 Statistical analysis of experimental data.Data from the study analyzed by SSPS statistical software (version10.0, USA) were expressed as (?)±s.●Outcomes of comet assay were processed by CASP analysis software.Experimental data, such as head DNA (HDNA %), tail DNA(TDNA %), tail length (TL), tail moment (TM) and Olive tail moment(OTM) were measured and average value and standard deviation werecalculated and expressed by (?)±s. Each dosage group was comparedby one-way analysis of variance (ANOVA) with homogeneity ofvariance, and differences between groups were analyzed.●The differences between expressions of P-Cx43 (Ser-368) andNP-Cx43 (Ser-368), relative expression amounts of total PKCαandP-PKCα(Ser-657) from control group and each experimental groupwere analyzed with univariate two-way (ANOVA) by SSPS statisticalsoftware; and multiple comparisons were made using Games-Howellpost hoc test.●The difference and change of transcript mRNA of NCX, SERCA₂,RyR₂ and PLB in aconitine cultured groups and control group wereanalyzed by t-test with SPSS statistical software.4 Results4.1 Primary cardiomyocytes culture and condition optimization.When plated in culture well, the cardiomyocytes were bright pellets inshape and suspended in medium. After 12 hours, they began to crawl onthe bottom of the culture well and spontaneous beating could be found infew cells. After 24 hours, attached myocytes connected each other, looked like a net; and slow synchronous beating could be found in all cultures.After 48 hours, the cardiomyocytes stretched on the bottom of culturewells, and high-speed (80～120 times per minute) synchronous beatingmonolayers of syncytium were formed. On 6th day of culture, meanlivability of cardiomyocytes were 97.33 % using trypan blue stainingmethod. Applying immunofluorescent microscopy with specificmonoclonal anti-cTnI antibody, we estimated that the mean purity ofcultured cardiomyocytes were 97.1%.4.2 Setting-up models of cultured cardiomyocytes treated by aconitineand observing the toxic effect.Aconitine incubated models were set up and it was verified by H.E.staining that the morphological structure of cardiomyocytes were hardlychanged after incubation with different dosages of aconitine compared tothe control cultures. But cardiomyocytes showed asynchronic beat,slowing beat and even arrest. There were certain correlation amongtoxicological pathologic change, toxic dose and poisoning period.4.3 Observation of DNA damage of cultured rats' cardiomyocytesafter aconitine incubation.After different concentrations of aconitine incubation, tail DNA, taillength, tail moment ^TM and Olive tail moment of cultured neonatal rats'cardiomyocytes increased, and head DNA decreased while theconcentration of aconitine increased. And there were all extremelysignificant differences compared with the control group (P＜0.01). Theoutcomes indicated that higher the dose of aconitine, severer the DNAdamage of cardiomyocytes.4.4 The influence of expression of PKCαin cultured cardiomyocytes.4.4.1 Observation of expression of P-Cx43 (Ser-368) in cardimyocytes.Every experimental group was compared with the normal group. Andthe expressions of P-Cx43 (Ser-368) in cardiomyocytes with different treated patterns were not the same. The expression amounts of P-Cx43(Ser-368) in cardiomyocytes of ACO incubated group, A.R group and G(o|¨)6976 group decreased, and all increased in AAP group, AAP+ACO groupand AAP+G(o|¨) 6976 group. Games-Howell statistical method was selectedfor multiple comparisons of means between groups. Among ACOincubated group, A.P. group and G(o|¨) 6976 group, there was significantstatistical difference in ACO incubated group (P＜0.05), while there werehardly obvious difference among AAP group, AAP+ACO group andAAP+G(o|¨) 6976 group. The outcomes by Western Blotting presented thatafter ACO, A.R and G(o|¨) 6976 treated, the expression protein of Cx43(Ser-368) locus decreased, while after APP pre-treated increased obviously(P＜0.01).4.4.2 Observation of expression of NP-Cx43(Ser-368) in cardiomyocytes.The quantitative analytic outcomes of expression protein of NP-Cx43(Ser-368) locus phosphorylation state presented that the expression in A.P.group, ACO incubated group and G(o|¨) 6976 group reinforce (P＜0.01),especially A.P. group the most, while AAP group, AAP+ACO group andAAP+G(o|¨) 6976 group reduce (P＜0.01).4.4.3 Observation of expression of total PKCαin cardiomyocytes.The quantitative analytic result of total PKCαshowed that there wereno significant difference of expression of total PKCαin A.R group, ACOincubated group, G(o|¨) 6976 group, AAP group, AAP+ACO group andAAP+G(o|¨) 6976 group, compared with the control group (P＞0.05).4.4.4 Observation of expression of total P- PKCα(Ser-657) incardiomyocytes.Every experimental group was compared with the normal group. Andthe expressions of P-PKCα(Ser-657) in cardiomyocytes with differenttreated patterns were different. The P-PKCα(Ser-657) expressed incardiomyocytes of the normal group and ACO group, but none of A.Rgroup.Compared with the normal group, the expressions of P-PKCα (Ser-657) phosphorylation protein in cardiomyocytes of ACO group andG(o|¨) 6976 group decreased obviously (P＜0.01). And the expressions ofphosphorylation increased in AAP group, AAP+ACO group and AAP+G(o|¨)6976 group (P＜0.01).4.4.5 Observation of PKCαand PKCα(Ser-657) locus phosphorylationstate before and after aconitine incubationApplying laser scanning confocal microscope, the immumofluorescenedetection results presented:Total PKCαlabeled by Fluorescein, as green fluorescene signals, weredispersed distributed in cytoplasm of cardiomyocytes. There were nosignificant differences in ACO incubated group, G(o|¨) 6976 group, AAPgroup, AAP+ACO group and AAP+G(o|¨) 6976 co-treated group comparedwith the normal group.Total P-PKCα(Ser-657) labeled by Rhodamine red fluorescene signalslabeling, as red fluorescene signals, were dispersed distributed incytoplasm of cardiomyocytes. The red fluorescene signals weakenedobviously and there were extremely significant differences in ACOincubated group and G(o|¨) 6976 group compared with the normal group(P＜0.01). There were also extremely significant differences of redfluorescene signals in AAP group, AAP+ACO group and AAP+G6 6976co-treated group (P＜0.01).4.5 Influence of expression of calmodulin in aconitine cultured rats'cardiomyocytes.It was detected that mRNA of RyR₂ and NCX expressed more, andPLB and SERCA₂ less by RT-PCR combined with multiplex fluorescence.4.6 Analysis of forensic postmortem examination documents ofaconitine poisoning cases.Characteristic of pathological changes were mainly that:â‘ myocardium focal hemorrhage, transverse striation of cardiomyocytes wasunclear, sarcoplasm condensed and intercellular congestion;â‘¡focal or spotty necrosis of hepatocytes and fatty degeneration or hydropicdegeneration;â‘¢focal or globular hemorrhage of lung, focalpneumonedema;â‘£scattered globular hemorrhage in gastric mucosa ofsome cases;⑤globular hemorrhage or scattered necrosis in kidney of afew cases.5 Conclusions●5.1 Optimization and specify cardiomyocytes culture method,establishment of aconitine incubated in vitro neonatal rats' cell modeland reference for forensic toxicologic study.●5.2 DNA damage of aconitine incubated cardiomyocytes was ofobvious dose-effect relation, which would indicate that DNA damageplayed a role in toxic effect mechanism.●5.3 Aconitine incubation would influence phosphorylation state ofPKCαand phosphorylation expression of PKCα(Ser657) decreased,which further induced that phosphorylated Cx43 of cardiomyocytesweakened. It inferred that the change of phosphorylation state ofPKCαand Cx43 was one of the paths of the aconitine poisoning.●5.4 Aconitine influenced the expression of calmodulin incardiomyocytes, and mRNA of RyR₂ and NCX expressed more andPLB and SERCA₂ less. That calmodulin take part in toxic effectmechanism deserves further research.●5.5 Announcements of forensic identification for aconitine poisoningwere raised after collection of forensic postmortem examinationdocuments of 10 aconitine poisoning cases and summarize thecharacters of toxicological pathologic change.