【作者】 章诗伟；

【导师】 刘良；

【作者基本信息】 华中科技大学 ， 法医学， 2007， 博士

【摘要】 【研究背景】乌头属植物（Aconitium plants）是被子植物亚门毛茛科中一类重要的有毒植物,也是我国最早有记载的有毒植物之一;全世界已知的就有350种以上,主要生长在北半球温带地区;在国内,目前已被鉴定的约200余种,广泛分布于除海南以外的其他省区。乌头属植物及其制剂,在我国民间和传统中医治疗中已有二千多年的使用历史,疗效确切。现代药理学研究证实,乌头类药材大多具有抗炎消肿、麻醉止痛、强心降压、免疫抑制以及肿瘤抑制等广泛功效,有较高的药用价值。但该类药材,特别是其植物块根中,多含有剧毒的二萜类双酯型生物碱–乌头碱（aconitine）,其治疗量与中毒量或致死量接近,使用中因个体对乌头耐受性的差异、用法不当,以及误服或投毒等原因而发生中毒、甚至死亡的报道屡见不鲜。近年来在日本和欧美国家,利用乌头块根或乌头碱纯品服毒自杀或投毒他杀的案例也时有报道。乌头碱主要作用于神经系统和心脏。严重的心律失常,尤其是室速和室颤,是导致其中毒死亡的主要原因。以往运用离子通道结构分析和全细胞膜片钳技术等研究手段证实,乌头碱能与心肌细胞膜电压门控型Na⁺通道结合,抑制其失活,使Na⁺持续内流、延长细胞膜除极化时程而引发心律失常。但值得注意的是,近年来的实验和临床研究认为,心肌细胞间电耦联的失衡与心律失常的发生关系更为密切。因此,研究乌头碱对分离的单个心肌细胞离子通道的作用,不能全面反映其对作为功能性合胞体的心肌细胞群的整体结构和功能的影响。另一方面,有学者曾推测,既然乌头碱能作用于心肌细胞膜Na⁺通道,使Na⁺持续内流,那么在理论上,可能会通过Na⁺-Ca²⁺交换机制而导致胞内游离Ca²⁺浓度升高。因此有必要对乌头碱干预后,心肌细胞内游离Ca²⁺浓度变化做进一步研究。随着实验科学的不断发展,细胞培养技术因具有简便、可靠、可比性强和重复性好等优点,已被广泛应用于生物学和医学研究领域。体外培养的心肌细胞,不但保存了其结构和功能上的某些特点（如:自发性同步搏动）,还排除了机体神经、体液等各种复杂因素干扰,有利于从细胞和分子水平对心肌进行生理、病理、药理和毒理等多方面的研究。因此,体外心肌培养技术,可以用于在控制条件下（如:剂量、时间和染毒途径等）研究毒物对心肌细胞结构、功能和代谢等方面的影响,阐明毒物的毒性作用机制,在法医毒理病理学研究中值得应用和推广。【目的】1.建立体外培养条件下,不同剂量标准品乌头碱对新生大鼠心室肌细胞染毒的法医毒理学实验模型,从细胞学水平研究乌头碱对心肌细胞的毒性作用机制;2.测定不同剂量乌头碱染毒前后,培养的新生大鼠心室肌细胞缝隙连接蛋白Connexin43（Cx43）蛋白总量表达的情况,及磷酸化与非磷酸化Cx43亚型之间含量变化的规律;3.从蛋白磷酸化研究的角度,利用功能性抗体,在形态学水平,观察乌头碱染毒前后,心室肌细胞缝隙连接通道中,Cx43蛋白在其羧基端肽段特定氨基酸残基位点的磷酸化状态;4.运用荧光探针fluo-4 NW （no wash）游离Ca²⁺标记技术,实时动态检测乌头碱染毒前后,心室肌细胞内[Ca²⁺]振荡在幅度和频率上变化,对其变化机理进行分析;并进一步探讨乌头碱染毒后,心肌细胞内[Ca²⁺]振荡的变化与Cx43蛋白磷酸化状态改变之间的内在联系。【方法】1.体外培养条件下,新生大鼠心室肌细胞乌头碱染毒实验模型的建立选用SPF（Specific Pathogen Free）级,出生1～2 d的健康Sprague–Dawley大鼠（雌雄不拘）,每次取10只作心室肌细胞原代培养。于培养的第6 d更换无血清培养液维持12 h后,按相应的干预计划,对心肌细胞进行乌头碱染毒及后续检测,各批次实验均设立正常对照组。2.不同剂量乌头碱染毒前后,心肌细胞中Cx43总量及其磷酸化亚型含量的测定设立0.25、0.5、0.75、1.0、1.5和2.0μM/L共6个不同剂量的乌头碱染毒组,染毒1 h后,提取细胞膜蛋白,进行电泳分离,运用定量蛋白印迹分析技术,检测对照组及染毒后,心肌细胞Cx43蛋白总量及其磷酸化与非磷酸化亚型表达的变化。3.乌头碱染毒前后,心肌细胞Cx43蛋白羧基端肽段上特定位点磷酸化状态的观察选择抗磷酸化Cx43和抗非磷酸化Cx43两种功能性抗体,应用激光扫描共聚焦显微镜,结合细胞荧光图像定量分析技术,检测对照组和乌头碱染毒1 h后,心肌细胞Cx43蛋白在其羧基端第368位丝氨酸残基（Ser368）位点磷酸化状态的改变。4.乌头碱染毒前后,心肌细胞内Ca²⁺振荡变化的实时动态检测移除无血清培养液,向培养孔中加入空白或含一定浓度乌头碱的fluo-4 NW游离Ca²⁺荧光标记负载液,染毒1 h后,应用激光扫描共聚焦显微镜,选择二维平面扫描模式,在氩离子激光激发下,实时动态监测和记录染毒前后,心肌细胞浆和胞核内游离Ca²⁺振荡模式的变化。5.实验数据的统计学分析本实验采用SSPS统计软件（SSPS Inc., version 10.0, USA）,数据以X±S表示。在定量蛋白印迹分析实验中,选择单变量双因素方差分析（univariate two-way analysis of variance,ANOVA）,对正常对照组和各实验组Cx43蛋白总量及其各磷酸化亚型含量进行分析,组间均数的多重比较选用Games-Howell检验;各组之间,磷酸化与非磷酸化Cx43蛋白比值差异的比较,选用Kendall’s W协同系数检验。在免疫荧光检测实验中,选用嵌套设计的方差分析（nested ANOVA）,对正常对照组和各实验组P-Cx43 （Ser368）及NP-Cx43 （Ser368）阳性信号值之间的差异进行比较。认定P < 0.05时,组间均数有显著统计学差异。【结果】一、培养心肌细胞的形态、纯度,及其乌头碱染毒前后的细胞形态学比较1.倒置相差显微镜下观察:刚接种时的心肌细胞悬浮于培养液中,为均一、分散的圆形折光颗粒;12 h更换培养液时,细胞已开始贴壁生长,偶尔可见个别细胞自发搏动;培养24 h后,贴壁细胞互相连接呈稀疏网状,并出现缓慢的同步化搏动;48 h后,心肌细胞在培养孔底部进一步伸展,呈现快速同步化搏动的细胞单层,搏动频率约每分钟150 180次;2.培养第6 d,心肌细胞在培养孔底部充分伸展,形成稳定的同步化搏动细胞单层,频率约120次/min,选用抗心肌肌钙蛋白I （cTnI）特异性单克隆抗体,结合免疫荧光法检测培养物,证明绝大多数为心肌细胞;3.常规H.E.染色结果表明,与正常对照组比较,不同剂量乌头碱染毒后,心肌细胞形态结构未出现明显改变。二、不同剂量乌头碱对心肌细胞Cx43蛋白总量及其磷酸化亚型含量的影响1.兔抗总Cx43多克隆抗体可检测出Cx43蛋白条带的3种亚型,分子量大小为43、40和39 kDa,分别代表磷酸化P2带,磷酸化P1带和非磷酸化NP带。在正常对照组中,Cx43蛋白条带基本集中在43 kDa处（P2带）;碱性磷酸酶处理后,条带整体向前迁移到39 kDa处（NP带）;2.在染毒组中,0.25μM/L乌头碱干预后,Cx43条带开始向前弥散,出现P1带;乌头碱浓度提高到0.5μM/L时,NP带开始显现;当染毒浓度达到1.0μM/L后,NP带明显,但P2带和P1带显著变淡;1.5及2.0μM/L组的染毒效果和1.0μM/L染毒组相当;3. Cx43蛋白条带光密度定量分析结果显示:正常对照组、碱磷酶处理组或不同浓度乌头碱染毒组,心肌细胞中Cx43蛋白总量无显著差异;但乌头碱作用后,磷酸化的（P2 + P1） Cx43含量减少,而非磷酸化的NP Cx43含量增多。这提示,乌头碱虽不影响心肌细胞Cx43蛋白总量,但却能诱发其脱磷酸化,且呈一定的浓度依赖性。三、乌头碱对心肌细胞Cx43蛋白,羧基端肽段上特定位点磷酸化状态的影响1.本实验选择Cx43羧基端肽段,第368位丝氨酸残基（Ser368）的磷酸化状态作为研究对象。免疫荧光检测结果显示,在对照组中,标记P-Cx43 （Ser368）的绿色荧光信号分布于心肌细胞间交界处;而标记NP-Cx43 （Ser368）的红色荧光信号偶见于胞浆;2.染毒后,P-Cx43 （Ser368）信号明显减弱;而NP-Cx43 （Ser368）信号则显著增强,在心肌细胞间边界清晰可见。定量分析结果进一步说明,染毒后P-Cx43 （Ser368）信号减弱及NP-Cx43 （Ser368）信号增强,与对照组比较,均有显著性差异。实验证实,乌头碱能诱导Cx43蛋白羧基端第368位丝氨酸残基发生明显脱磷酸化。四、乌头碱对心肌细胞内[Ca²⁺]振荡的影响1.无论是否经乌头碱染毒,[Ca²⁺]在心肌细胞浆和细胞核中均表现为同步振荡;2.在对照组中,心肌细胞内[Ca²⁺]振荡不规则,但相对平稳,振荡幅度小,偶尔可见较明亮的Ca²⁺火花;3.乌头碱染毒后,Ca²⁺浓度在细胞浆和细胞核均出现剧烈振荡,典型的Ca²⁺火花闪烁现象消失,但细胞浆中Ca²⁺的平均浓度和对照组相比,并未显著升高。【结论】1.在实验选择的染毒浓度范围内（0.25 2.0μM/L）,不同剂量的乌头碱对培养心肌细胞的基本形态结构无明显的破坏作用;2.和正常对照组比较,不同浓度乌头碱染毒组,心肌细胞中Cx43蛋白总量未出现显著改变,说明乌头碱不影响Cx43蛋白总量的表达;3.随着乌头碱染毒浓度的提高,磷酸化的（P2 + P1） Cx43含量逐渐减少,而非磷酸化的NP Cx43含量逐渐增多,提示乌头碱可诱发心肌细胞中Cx43蛋白脱磷酸化,且呈一定的浓度依赖性;4.正常状态下,培养心肌细胞中Cx43蛋白羧基端肽段上,第368位丝氨酸残基（Ser368）处在磷酸化状态,乌头碱可诱导该位点发生明显脱磷酸化。由于Cx43蛋白的磷酸化状态对协调心肌细胞间电耦联关系密切,因此,诱导Cx43蛋白在特定位点脱磷酸化,是乌头碱对心肌细胞毒性作用的重要环节;5.乌头碱能作用于心肌细胞膜Na⁺通道,使Na⁺持续内流。理论上,胞内Na⁺浓度升高后,可通过Na⁺–Ca²⁺交换机制而导致胞内游离Ca²⁺浓度升高。但实验中我们发现,和对照组相比,乌头碱染毒后,胞浆中的平均Ca²⁺浓度并未显著升高,但细胞内Ca²⁺出现剧烈振荡。这提示,乌头碱染毒后,心肌细胞内并未发生Ca²⁺超载,但Ca²⁺浓度的剧烈振荡,很可能影响到心肌细胞内某些对[Ca²⁺]振荡频率敏感的信号转导过程。更多还原

【Abstract】 [Background]Aconitium plants are a group of important poisonous plants, belonging to the Ranunculaceae family of Angiosperm subphylum. They are one of the earliest recorded poisonous plants in China. More than 350 species of this plant have been identified in the world. They are widely distributed in the mountains or cooler regions of northern hemisphere. Up till now, over two hundred species of Aconitium plants are found in China except for Hainan Province.Aconitium plants and their preparations have been used in Chinese folk recipe and traditional medicine for more than two thousands years for their particularly curative effects. It has been confirmed by modern pharmacological studies that most of Aconitium medicinal materials have wide spread pharmaceutical properties including anti-inflammatory, detumescence, anesthetic, analgesic, cardio-tonic, hypotensive, immunosuppressant, tumor-suppressant effects and so on. Thus Aconitium plants possess considerable pharmaceutical value, however a set of various Aconitium alkaloids, belonging to the group of diesterditerpene alkaloids, are present in the tubers （root）, stalks, flowers, and leaves of these plant. The substance aconitine is the most toxic Aconitium alkaloids. Aconitine poisoning is a matter of common occurrence in China, lies in the insignificant difference in aconitine dosage among therapy, poisoning and death; the individual differences in pharmaceutical tolerance of aconitine; improper or accidental take in common people; and put in poison by homicide. In resent years, suicides and homicides cases relating to aconitine poisoning are reported occasionally in Japan and some Western countries.The toxicological effects of aconitine act mainly on the nervous system and the heart. Life-threatening arrhythmias, including ventricular tachycardia and ventricular fibrillation, are the major reasons for the aconitine induced death. By using of the structure analysis of ion channels and the whole cell patch clamp technique, it had been verified that aconitine can bind with high affinity to the open state of voltage-gated sodium channels, suppress their inactivation, and facilitate the excitation of the membrane, by increasing Na⁺ in-flow and prolonging depolarization, which bring about various types of ventricular tachyarrhythmia. It should be noticed that since the 1990’, accumulated result from experimental and clinical studies have shown that the electrical uncoupling among cardiomyocytes is closely related to the occurrence tachyarrhythmia. Therefore, study of the toxicology effects of aconitine on ion channels in single cardiomyocytes can not comprehensively reflect the structural and functional changes in aconitine treated cardiomyocytes which work as co-functional syncytium. On the other hand, some researchers hypothesized that theoretically, the aconitine-induced sustained influx of Na⁺ may eventually lead to intracellular Ca²⁺ overload via a Na⁺–Ca²⁺ exchange mechanism. Therefore, real-time measurement of intracellular [Ca²⁺] changes might be necessary to elucidate aconitine-induced Ca²⁺ disequilibrium in cardiomyocytes.Accompany with the development of experimental methods and possessing the advantages of simplicity, reliability, strong comparability and good reproduction, cell culture techniques have been widely apply in biological and medical realms. In vitro cultured cardiomyocytes not only maintain certain characteristics （e.g. spontaneous synchronously beating）, but also eliminate the affects of complicated organism factors from neuron and humor. It is suitable for scientific research in physiology, pathology, pharmacology and toxicology at cellular and molecular level. By using of cardiomyocyte culture, we could study the toxicological effects of poisons on the structure, function and metabolism of cardiomyocytes under controlled conditions （e.g. dose, time and ways of poisonous）. Therefore, the in vitro cell culture technique is worthy to be applied and popularized in forensic toxicological studies. [Objectives]1. To set up an experiment model of forensic toxicology under in vitro cell culture conditions, for incubation of the cultured neonatal rat ventricular myocytes with different dosages of standard aconitine, and investigating the aconitine induced toxicological effects on cultured cardiomyocytes;2. To exam the expressing of total amount of gap junctional protein– Connexin43 （Cx43） and the quantitative changes between phosphorylated Cx43 and non-phosphorylated Cx43 subtypes in the cultured neonatal rat ventricular myocytes before and after incubation of the cultures with aconitine;3. From the purpose of protein phosphorylation study, using functional antibodies to investigate the phosphorylation status at specific amino acid residues in the C-terminal domain of gap junctional Cx43 in cardiomyocytes at morphological level;4. Applying fluo-4 NW （no wash） fluorescent Ca²⁺ indicator, to real-timely measure the amplitude and the frequency changes of intracellular [Ca²⁺] oscillation in cardiomyocytes before and after incubated the cultures with aconitine. And then to further approach the internal relations between the changes of intracellular [Ca²⁺] oscillation pattern and the changes of Cx43 phosphorylation status in aconitine treated cultured cardiomyocytes.[Methods]1. Setting up toxicological model of neonatal rat ventricular myocytes by aconitine under in vitro culture conditionsSPF （Specific Pathogen Free） grade, 1-2 day old healthy Sprague–Dawley rats were selected, regardless of the gender. In each separate experimental batch, ten rats were used for in vitro ventricular myocytes culture. Toxicological experiments were initiated on day 6 of culture. After replenishing with serum free medium, the cultures were incubated for another 12 hours. Then the cultured cardiomyocytes were incubated with aconitine and were examined, according to the experiment design. Control groups were set each time.2. Examining the expression changes in total Cx43 protein and its phosphorylated or nonphosphorylated isoforms in cultured cardiomyocytes treated with different dosages of aconitine The cultured cardiomyocytes were incubated with aconitine at 6 different concentrations of 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0μM/L. after 1 hour of incubation, total cellular membrane protein of cultured cardiomyocytes were extracted and then separated by electrophoresis. Applying the quantitative Western blot analysis, we examined the changes in total Cx43 protein and its phosphorylated or nonphosphorylated subtypes in samples of control or aconitine treated cardiomyocytes.3. Observing the changes of Cx43 phosphorylation status in its C-terminal domain at specific residues in cultured cardiomyocytes before or after aconitine incubationTwo kinds of functional antibodies: rabbit anti-Ser368 phosphorylated Cx43 and mouse anti-Ser368 nonphosphorylated Cx43 were used in our experiment. We applied laser scanning confocal microscopy and quantitative analysis for cellular fluorescent images to detect the Cx43 phosphorylation status changes in the C-terminal domain at the 368th serine amino acid residue （Ser368） in cardiomyocytes from controls or cultures treated with 1 hour of aconitine.4. Real-time dynamic detection of the changes in [Ca²⁺] oscillation patterns in cultured cardiomyocytes before or after aconitine incubationAfter removing of the serum free medium, fluo-4 NW loading solutions （blank or containing 1.00μM/L aconitine） were added to the culture wells for fluorescent indication of free Ca²⁺ in cardiomyocytes. We applied laser scanning confocal microscopy, under the rectangle scanning mode, to monitor and record the changes of [Ca²⁺] oscillation patterns in cardiomyocytes from controls or cultures treated with 1 hour of aconitine.5. Statistical analysis of experiment dataData are expressed as mean±SD. In western blot studies, differences of Cx43 isoform levels between groups were analyzed with univariate two-way analysis of variance （ANOVA）; and multiple comparisons were made using Games-Howell post hoc test; differences of phosphorylated and nonphosphorylated Cx43 protein percentage between groups were determined by Kendall’s W nonparametric test. In immunofluorescence detection, proportional differences of Ser368 phosphorylated or nonphosphorylated Cx43 signal positive areas were analyzed by nested ANOVA. A value of P < 0.05 was considered statistically significant. [Results]Ⅰ: Morphology, degree of purity and morphological contrast of neonatal rat ventricular myocytes from control cultures or aconitine treated cultures1. When plated in culture well, the cardiomyocytes were bright pellets in shape and suspended in medium. After 12 hours, they began to crawl on the bottom of the culture well and spontaneous beating could be found in few 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 bottles, and high-speed （120 times per minute） synchronous beating monolayers of syncytium were formed.2. On day 6 of culture, we applied immunofluorescent microscopy and identified the cultures, using mouse monoclonal anti cTnI antibody. We estimated that > 95% of the cells were cardiac myocytes.3. It was verified by H.E. staining that compared to the control cultures, the morphological structure of cardiomyocytes were hardly changed after incubation of the cultures with different dosages of aconitine.Ⅱ: The effects of different dosages of aconitine on the amount of total Cx43 protein and its phosphorylated or nonphosphorylated subtypes1. Three Cx43 protein bands: 43 kDa, 40 kDa and 39 kDa in SDS-polyacrylamide gel could be detected by rabbit anti-total Cx43 antibody. These bands represent two phosphorylated isoforms （P2 and P1） and a non-phosphorylated isoform （NP） respectively. Under control conditions, most of Cx43 accumulate at 43 kDa （P2 band）; after treatment of the cultures with alkaline phosphatase, the overall band migrates forward to 39 kDa （NP band）;2. After 0.25μM/L aconitine treatment, the band of Cx43 migrated forward, and the P1 band could be detected; after 0.5μM/L aconitine treatment, the NP band emerged; when the concentration of aconitine was elevated to 1.0μM/L, the intensity of NP band increased significantly, concomitant with significant decreased intensity of band P2 and band P1; the effects of 1.5 or 2.0μM/L aconitine were about equal to that of 1.0μM/L aconitine;3. Quantitative analysis of Cx43 band intensity showed that there were no significant differences in total amount of Cx43 in cardiomyocytes among controls, alkaline phosphatase treated cultures or aconitine treated cultures; however, after aconitine treatment, the phosphorylated Cx43 （P2 + P1） decreased, concomitant with the increase in nonphosphorylated Cx43. The observations indicated that treatment of cardiomyocytes with aconitine did not affect the total amount of Cx43, whereas aconitine induced the dephosphorylation of Cx43 and present a concentration-dependent effect.Ⅲ: The effects of aconitine on the phosphorylation status in Cx43 C-terminal domain at specific residues1. In the present study, we examined the Cx43 phosphorylation status changes in the C-terminal domain at the 368th serine amino acid residue （Ser368）. Results from immunofluorescent staining revealed that under control conditions, P-Cx43 （Ser368） positive gap junction plaques （green fluorescence） were sparse as broken lines along the perimeters of cardiomyocytes; in contrast, only a few particles of NP-Cx43 （Ser368） positive signal （red fluorescence） were seen in the cytoplasm;2. Incubation of the cultures with aconitine led to a dramatic loss of P-Cx43 （Ser368）; whereas the NP-Cx43 （Ser368） particles were found abundantly accumulated at the points of intercellular borders. Quantitative immunofluorescent data indicate that compared to the controls, treatment of the cultures with aconitine resulted in a distinct decrease in P-Cx43 （Ser368） concomitant with a distinct increase in NP-Cx43 （Ser368）. So, it is clear that aconitine-induced dephosphorylation of Cx43 occurs at Ser368 in its C-terminal domain.Ⅳ: The effects of aconitine on the intracellular [Ca²⁺] oscillation patterns in cultured cardiomyocytes1. No matter whether treated with aconitine or not, the [Ca²⁺] oscillated synchronously in both nuclear and cytoplasmic regions of a same cardiomyocyte;2. Under control conditions, [Ca²⁺] oscillations were irregular but relatively stable accompanied by occasional small calcium sparks;3. After incubation of the cultures with aconitine, strenuous high frequency [Ca²⁺] oscillations emerged in both nuclear and cytoplasmic regions and typical calcium sparks disappeared; however, the average [Ca²⁺] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incubation, compared to the controls. [Conclusions]1. In the selected concentration range （0.25 2.0μM/L）, different dosages of aconitine do not disrupt the conformational structure of cultured cardiomyocytes;2. Compared to the controls, after treatment of the cardiomyocytes with different dosages of aconitine, the total amounts of Cx43 do not change, which indicate that aconitine do not interfere the expression of Cx43 protein;3. Accompanied by the elevation of aconitine concentration, the phosphorylated Cx43 （P2 + P1） decreased, concomitant with an increase in the nonphosphorylated Cx43. The observations indicated that in cultured cardiomyocytes, aconitine induced the dephosphorylation of Cx43 and present a concentration-dependent effect.4. Under the control conditions, the 368th serine amino acid residue （Ser368）, which is located in the C-terminal domain of Cx43, is phosphorylated. After treatment of the cultures with aconitine, the Ser368 residue undergoes dephosphorylation. Since the phosphorylation status of Cx43 is of very importance to the maintenance of the electric coupling among cardiomyocytes, inducing the dephosphorylation of Cx43 at specific residue underlies the mechanisms of aconitine intoxication in cardiomyocytes.5. Aconitine can bind with high affinity to the open state of voltage-gated sodium channels; suppress their inactivation and increase Na⁺ in-flow. Theoretically, the aconitine-induced sustained influx of Na⁺ may eventually lead to intracellular Ca²⁺ overload via a Na⁺–Ca²⁺ exchange mechanism. In the present study, we found that although oscillated strenuously at high frequency, the average [Ca²⁺] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incubation, compared to the controls. The observations indicate that aconitine induce the changes in [Ca²⁺] oscillation frequency other than the Ca²⁺ overload in cardiomyocytes, which may affects some [Ca²⁺] oscillation frequency sensitive process of signal transduction in cardiomyocytes.更多还原