【作者】 王勇；

【导师】 王嘉陵；

【作者基本信息】 华中科技大学 ， 药理学， 2013， 博士

【摘要】 心脏的功能活动是心肌动作电位变化和收缩力变化的统一体,心肌动作电位和收缩力受到心率的影响。心肌的兴奋收缩过程伴随着细胞内外离子浓度的变化,其中钙离子是参与心脏活动最关键的离子。经典瞬时受体电位通道(transient receptor potential canonical channel, TRPC)是TRP的一个亚家族,包括TRPC1-7共七个成员。TRPC广泛分布于心血管系统,是一类对钙通透的非选择性阳离子通道。TRPC的功能及表达异常可见于心肌肥厚、心律失常、心肌缺血等多种心血管疾病。心率的变化会造成细胞内离子的跨膜移动,进而影响心肌动作电位时程(action potential duration, APD)和收缩力,这一过程是否有TRPC参与,值得探讨。目的：通过电刺激控制心率,观察各种刺激频率对不同种属动物APD及收缩力的影响；探讨APD-频率依赖性与TRPC的关系,进一步分析TRPC在APD-频率依赖性中的作用；药物的延长APD作用是否有TRPC的参与。方法：1.各种刺激频率对不同种属动物心肌APD的影响采用心外膜接触电极记录不同频率刺激下豚鼠、家兔及大鼠在体心室单相动作电位(monophasic action potential, MAP),标准微电极技术记录大鼠心室乳头肌的动作电位,观察各种刺激频率对不同种属动物APD的影响。2.心肌APD-频率依赖性与TRPC表达的关系选用家兔为实验对象,采用心外膜接触电极记录家兔在体心室MAP,在4.5Hz、6Hz、7.5Hz不同频率下给予电刺激控制心率,10min后取心脏,做TRPC的mRNA及蛋白表达分析。3.甲基莲心碱对家兔心肌APD-频率依赖性及TRPC表达的影响(1)家兔静脉注射甲基莲心碱(2.5mg/kg)、胺碘酮(5mg/kg),记录并比较给药前后不同刺激频率下(4.5-7Hz)MAP的变化。(2)家兔静脉注射甲基莲心碱(2.5mg/kg)、胺碘酮(5mg/kg),给药后选取4.5Hz连续电刺激10min,迅速取心脏做TRPC的mRNA及蛋白表达分析。4.各种刺激频率对不同种属动物心肌收缩力的影响(1)对大鼠、家兔心室乳头肌标本进行离体组织灌流,依次给予不同频率刺激标本,记录不同刺激频率下心室乳头肌收缩力变化。(2)给予1Hz基础频率刺激3min,然后停止刺激,记录静息不同时间恢复刺激时心室乳头肌首次收缩力的变化。结果：1.各种刺激频率对不同种属动物心肌APD的影响(1)在体豚鼠心室肌MAP记录,在4.5Hz、5Hz、5.5Hz、6Hz、6.5Hz、7Hz刺激条件下,心肌APD90从对照水平(4.5Hz)的96.84±5.43ms,分别缩短至91.77±3.88ms (5Hz)、87.07±3.78ms (5.5Hz)、81.79±3.92ms (6Hz)、76.84±2.91ms (6.5Hz)、76.52±1.40ms (7Hz);与对照水平(4.5Hz)相比,缩短的百分比分别为5.2±0.9%、10.1±1.1%、15.6±1.5%、20.5±1.3%、20.4±3.0%。表明：在4.5-7Hz范围,随频率的增加APD逐渐缩短,呈现明显的APD-频率依赖性。(2)在体家兔心室肌MAP记录,在4.5Hz、5Hz、5.5Hz、6Hz、6.5Hz、7Hz、7.5Hz刺激条件下,心肌APD90从对照水平(4.5Hz)的141.53±3.85ms,分别缩短至138.44±3.45ms(5Hz)、131.65±3.23ms(5.5Hz)、125.94±3.11ms(6Hz)、119.77±3.31ms (6.5Hz)、112.64±1.83ms (7Hz)、104.69±1.58ms (7.5Hz);与对照水平(4.5Hz)相比,缩短的百分比分别为2.1±1.5%、6.9±1.3%、10.9±1.7%、15.3±1.8%、20.2±2.1%、25.8±2.0%。提示：在4.5-7.5Hz范围,随频率的增加APD逐渐缩短,呈现明显的APD-频率依赖性。(3)在体大鼠心室肌MAP记录,频率范围在5-8.5Hz；离体大鼠心室乳头肌跨膜动作电位记录,范围在1-8Hz,随频率的增加APD等无明显变化,未见显著的APD-频率依赖性。2.心肌APD-频率依赖性与TRPC的关系在4.5Hz、6Hz和7.5Hz三个刺激频率组中,与4.5Hz组(对照水平)相比,6Hz组、7.5Hz组家兔心肌TRPC3的mRNA表达水平均显著增加(P<0.05)。在同样的刺激条件下,心肌TRPC1～2、TRPC4-7的mRNA表达均无显著影响。与4.5Hz组(对照水平)相比,6Hz、7.5Hz组增加TRPC3的蛋白表达,后者显著增加TRPC3的蛋白表达水平(P<0.05)。同样条件下,心肌TRPC1和TRPC6的蛋白表达水平无显著影响。表明：随着刺激频率(4.5-7.5Hz)的增加,家兔心肌TRPC3的mRNA水平和蛋白表达都逐渐增高。3.甲基莲心碱对家兔心肌APD-频率依赖性及TRPC的影响(1)静脉注射5mg/kg胺碘酮(AM),刺激频率4.5Hz.5Hz、5.5Hz、6Hz、6.5Hz,与给药前水平相比,APD90延长的百分比分别为11.4±4.1%、10.6±3.4%、6.5±1.6%、7.7±1.8%、8.8±3.0%。静脉注射2.5mg/kg甲基莲心碱(Nef),刺激频率4.5Hz、5Hz、5.5Hz、6Hz.6.5Hz、7Hz,与给药前水平相比,APD90延长的百分比分别为5.8±1.6%、2.0±1.7%、2.4±0.4%、3.1±0.5%、2.5±0.3%、6.2±1.2%。提示：在所给刺激频率范围,胺碘酮(AM)和甲基莲心碱(Nef)均能延长家兔心室肌APD。两药作用性质相似,在各个刺激频率下,延长动作电位的比例(%)均无显著差异,呈非频率依赖性特点。(2)与对照组(4.5Hz)相比,Nef(2.5mg/kg)明显增加TRPC3的mRNA和蛋白表达水平(P<0.05),而AM(5mg/kg)对TRPC3的mRNA和蛋白表达无明显作用。同样条件下,Nef和AM给药组对TRPC1-2.TRPC4-7的mRNA表达无显著差异,对TRPC1和TRPC6的蛋白表达水平也无显著影响。4.各种刺激频率对不同种属动物心肌收缩力的影响(1)在0.4Hz、0.6Hz、0.8Hz、1.0Hz、1.5Hz.2Hz、3Hz刺激条件下,大鼠心室乳头肌收缩力分别从对照水平(以0.2Hz收缩力为100%)降至78.2±3.4%、66.3±4.9%、60.4±5.6%、56.0±6.0%、51.5±6.8%、49.3±6.9%、43.7±5.8%,表明随着刺激频率的增高(0.2-3Hz),收缩力逐渐减弱。同样刺激频率条件下,家兔心室乳头肌收缩力分别从对照水平(以0.2Hz收缩力为100%)升至109.6±3.5%、120.2±5.4%、130.6±7.5%、141.44±10.4%、142.7±12.4%、137.3±13.9%、125.5±14.4%,在0.2-1.5Hz范围,收缩力逐渐增强。(2)静息15s、30s、60s、120s、180s和240s,大鼠心室乳头肌收缩力分别从对照水平(以静息1s时收缩力为100%)增加至299.8±7.3%、386.7±14.9%、451.3±32.3%、455.7±35.8%、451.1±44.0%、445.9±46.6%,表明随静息时间的延长,收缩力呈逐渐增强。而在同样条件下,家兔心室乳头肌收缩力分别从对照水平(以静息1s时收缩力为100%)降至88.1±2.3%、82.7±1.9%、78.7±2.0%、77.8±2.6%、76.7±3.1%、76.5±3.5%,表明随静息时间的延长,收缩力逐渐减弱。结论：1.刺激频率对大鼠、豚鼠和家兔心脏APD的影响不同。在本实验所用频率范围,豚鼠和家兔心肌呈明显的APD-频率依赖性缩短,而对大鼠心肌APD无明显影响。大鼠和家兔心室乳头肌在收缩力-频率相关性方面也存在差别。2.家兔心肌APD-频率依赖性缩短的作用,可能与频率改变(增加)伴随的TRPC3增加有关。3.Nef能明显延长家兔心肌APD并呈现非频率依赖性特征(各频率下延长APD的作用无明显差异),提示Nef的这一作用与TRPC3增加有关。更多还原

【Abstract】 Functional activity of the heart is the integrity of action potential and myocardial contractility, which is affected by heart rate. Myocardial excitation-contraction process is accompanied by changes of intracellular ion concentration, and the calcium ion is the most important inorganic ion involved in cardiac activity. TRPC (transient receptor potential canonical) is one subfamily of TRP, including7isoforms (TRPC1-7). TRPC is widely distributed in the cardiovascular system, which is a non-selective cation channel with permeability for calcium. Abnormal function and expression of TRPC can be seen in various kinds of cardiovascular diseases, such as cardiac hypertrophy, arrhythmia, myocardial ischemia. Heart rate changes can affect the transmembrane movement of intracellular ions, as well as the myocardial action potential duration (APD) and contractile force. Whether TRPC participate in this process, it is worth being investigated further.Objective:Controlling heart rate through the electric stimulation, we observe the relationship between myocardial APD or contractile force and stimulation frequency in different kinds of animal, and investigate frequency dependence of APD associated with the expression of TRPC. We further analysis the roles of TRPC in this process, and whether TRPC is involved in the drug effects of APD prolonging.Methods:1. The relationship between myocardial APD and stimulation frequency in different species of animalTo observe the relationship between APD and stimulation frequency in different species of animal, monophasic action potentials (MAP) were recorded using contact electrode technique in different programmed stimulus frequencies on rats, guinea pigs, or rabbits ventricular epicardium in vivo. And transmembrane action potentials were recorded using conventional microelectrode techniques on ventricular papillary muscle of rats.2. Frequency dependence of action potential duration and its influences on TRPC expression of rabbit myocardiumPacing at the stimulus frequency of4.5Hz、6Hz、7.5Hz, we recorded MAP using contact electrode technique on rabbit ventricular epicardium in vivo. Ten minutes later, hearts were taken out and the expressions of TRPCs were detected using RT-PCR or Western blotting.3. Effects of neferine on TRPCs expression and frequency dependence of action potential duration of rabbit myocardium(1) Given intravenous injection of neferine (2.5mg/kg), amiodarone (5mg/kg) in rabbits, the changes of MAP were recorded and compared at different stimulus frequencies (4.5Hz～7Hz) before and after administration.(2) Given intravenous injection of neferine (2.5mg/kg), amiodarone (2.5mg/kg) in rabbits, a continuous electrical stimulation of10min was applied on rabbit myocardium after administration. Then the hearts were taken out rapidly and the expressions of TRPC were detected.4. Force-frequency relationship of ventricular papillary muscle in different species of animal(1) Ventricular papillary muscles were isolated from heart of rats or rabbits and immediately perfused with organ bath. Different frequency electrical stimulations were applied on the preparation in sequence and the contraction force was recorded. (2) In order to investigate the influences of post-rest twitch, different rest intervals between1and240s were prescribed from a basal stimulation rate of1Hz for3min, and the first contraction amplitude after resume was recorded.Results:1. The relationship between myocardial APD and stimulation frequency in different species of animal(1) At the frequency of4.5Hz,5Hz,5.5Hz,6Hz,6.5Hz,7Hz, myocardial APD gradually shortened with the increase of stimulation frequency in the MAP recording of guinea pigs. The APD90from the control level96.84±5.43ms (4.5Hz), respectively reduced to91.77±3.88ms (5Hz),87.07±3.78ms (5.5Hz),81.79±3.92ms (6Hz),76.84±2.91ms (6.5Hz),76.52±1.40ms (7Hz). Compared with the control (4.5Hz), the shorten percentages correspondingly were5.2±0.9%,10.1±1.1%,15.6±1.5%,20.5±1.3%,20.4±3.0%.(2) At the frequency of4.5Hz,5Hz,5.5Hz,6Hz,6.5Hz,7Hz,7.5Hz, a clear frequency dependence of APD was showed in the MAP recording of rabbit myocardium in vivo. Myocardial APD90from the control level (4.5Hz)141.53±3.85ms, respectively, reduced to138.44±3.45ms (5Hz),131.65±3.23ms (5.5Hz),125.94±3.11ms(6Hz),119.77±3.31ms (6.5Hz),112.64±1.83ms (7Hz),104.69±1.58ms (7.5Hz). Compared with the control (4.5Hz), the shorten percentages were2.1±1.5%,6.9±1.3%,10.9±1.7%,15.3±1.8%,20.2±2.1%,25.8±2.0%.(3) There was no obvious frequency dependence of action potential duration in rat MAP in vivo within the range of5-8.5Hz or isolated ventricular papillary muscle TAP (transmembrane action potentials) at the range of1～8Hz.2. Frequency dependence of action potential duration and its influences on TRPC expression of rabbit myocardiumIn the groups of4.5,6and7.5Hz, TRPC3mRNA and protein expression of rabbit myocardial was gradually raised with the increase of stimulus frequency. In contrast with control level (4.5Hz), the mRNA expression of TRPC3remarkably grew in the6Hz and7.5Hz group (P<0.05), while that of myocardial TRPC1-2, TRPC4-7showed no significant change. Compared with4.5Hz group, the protein expression of TRPC3obviously increased in the7.5Hz group (P<0.05). The expression of TRPC1and TRPC6had no significant difference in the three groups.3. Effects of neferine on frequency dependence of action potential duration and TRPCs expression of rabbit myocardium(1) At different stimulus frequencies, neferine and amiodarone could prolong APD in rabbit myocardial, and showed characters of rate independence. Compared with that before administration at the frequency of4.5Hz,5Hz,5.5Hz,6Hz,6.5Hz, the prolonging percentage were respectively11.4±4.1%,10.6±3.4%,6.5±1.6%,7.7±1.8%,8.8±3.0%after intravenous injection of amiodarone (5mg/kg) in rabbits. At the frequency of4.5Hz,5Hz,5.5Hz,6Hz,6.5Hz,7Hz, the prolonging percentage of neferine (2.5mg/kg) injection correspondingly were5.8±1.6%,2.0±1.7%,2.4±0.4%,3.1±0.5%,2.5±0.3%,6.2±1.2%compared with that before administration.(2) Neferine (2.5mg/kg) could significantly increase the expression of TRPC3mRNA and protein in the stimulation of4.5Hz, while amiodarone (5mg/kg) had no such effects. Under the same stimulus conditions, the medication administration groups had no significant difference on the mRNA expression of TRPC1-2and TRPC4-7, and on the protein expression of TRPC1and TRPC6compared with4.5Hz control group.4. Force-frequency relationship of ventricular papillary muscle in different species of animal(1)With the increase of frequency (0.2-3Hz), contraction force gradually weakened in papillary muscles of rats, while enhanced in that of rabbits within0.2-1.5Hz range. At the frequency of0.4Hz,0.6Hz,0.8Hz,1.0Hz,1.5Hz,2Hz and3Hz, contraction force percentage of rat papillary muscles from the control level (0.2Hz)100%, respectively reduced to78.2±3.4%,66.3±4.9%,60.4±5.6%,56.0±6.0%,51.5±6.8%,49.3±6.9%and 43.7±5.8%. Under the same stimulus conditions, that of rabbit papillary muscles from the control level (0.2Hz)100%, correspondingly raised to109.6±3.5%,120.2±5.4%,130.6±7.5%,141.4±10.4%,142.7±12.4%,137.3±13.9%and125.5±14.4%.(2) There was clear rest decay of twitch amplitude in rabbit papillary muscles, while rat ventricular muscles exhibited rest potentiation of twitch force after different rest intervals between1and240s. After rest15s,30s,60s,120s,180s and240s, contraction force percentage of rat papillary muscles from the control level (rest Is)100%, respectively raised to299.8±7.3%,386.7±14.9%,451.3±32.3%,455.7±35.8%,451.1±44.0%and445.9±46.6%. Under the same rest conditions, that of rabbit papillary muscles from the control level (rest Is)100%, correspondingly reduced to88.1±2.3%,82.7±1.9%,78.7±2.0%,77.8±2.6%,76.7±3.1%and76.5±3.5%.Conclusions:1. There are species differences in the relationship between myocardial APD and stimulation frequency. An obviously APD shorten with the increase of frequency is showed in guinea pigs and rabbits, while this phenomenon is not observed in rat at the frequency ranges in our experiments. There are also distinctions between rat and rabbit papillary muscles in the force-frequency relationship.2. Frequency dependent shorten of APD is associated with the increase of TRPC3expression in rabbit myocardium.3. Neferine could significantly increase the expression of TRPC3rabbit myocardial, and the prolongation of APD showed characters of rate independence.更多还原