小鼠胚胎心肌细胞的分离及电生理特性

本文旨在探索小鼠胚胎心肌细胞的分离方法并观察其电生理特性。应用胶原酶B消化法获得不同时期单个小鼠胚胎心肌细胞;利用全细胞膜片钳技术,记录胚胎心肌细胞的超极化激活的非选择性内向阳离子电流(If)和L-钙电流(ICa-L),并用电流钳记录其自发性动作电位。胚胎心肌细胞通过相差显微镜依据其形态和自发性收缩进行鉴定。本法分离所获得的胚胎心肌细胞容易进行全细胞膜片钳记录,可用于记录If,ICa-L．电流和自发性动作电位,己证实胚胎心肌细胞If和Ica-L的电生理特性与成年起搏细胞或心肌细胞相似。本实验建立的分离方法简单、稳定、有效、可靠,最早可获得8．5d的胚胎心肌细胞。胚胎心肌细胞的电生理记录为探索胚胎心肌细胞的电生理特性提供了一个可用的模型,并可能为某些心脏疾病产生的机制提供实验依据。     

24小时快速起搏右心房对兔心房单向动作电位的影响

目的应用心内电生理技术研究心房快速起搏(RAP)对兔心房单向动作电位(MAP)的影响。方法成年新西兰兔20只随机分为二组:假手术组、模型组各10只。经颈内静脉将电极置入右心房。以600次/分行RAP,同时分析在0、4、8、12和24h的单向动作电位时程(MAPD)。结果假手术组在实验的时间段内右房游离壁MAP复极90%时程(MAPD90)无明显差别。RAP8h,起搏组右房游离壁MAPD90较P0有明显缩短,从起搏前(112.50±9.57)ms至起搏8h分别缩短到(51.25±4.79)ms,分别缩短了61.25ms。结论房颤(AF)时心房MAPD90缩短。MAP技术可安全地用于研究AF时的电重构(ER),能提供准确的电生理改变的信息。     

利用膜片钳技术标记脑片神经元的方法

目的: 介绍一种利用膜片钳技术标记脑片神经元形态的方法。方法: 利用振动切片机切好实验目标部位的脑片,用含有Neurobiotin^TM Tracer的电极内液灌注玻璃微电极,并进行全细胞膜片钳记录;实验结束后将脑片先用4％多聚甲醛固定、漂洗,再用含有Streptavidin-Texas Red和Triton X-100的PB染色,2 h后即可用荧光显微镜观察着色的神经元。结果: 将细胞膜电压钳制在-70 mV,阶跃刺激后神经元表现为逐渐增大的膜电流。电流钳模式记录时,阶跃刺激使神经元去极化,达到阈电位后爆发动作电位。荧光显微镜下可看到胞体和主要突起清晰完整的神经元形态。结论: 本方法适用于在膜片钳实验后观察所记录的神经元的形态特征,操作方便,图像直观清晰。     

N-乙酰半胱氨酸对犬心房快速起搏电重构的影响

目的:探讨N-乙酰半胱氨酸(NAC)对犬心房快速起搏电重构的影响。方法:取16只犬,随机分为对照组和NAC干预组。NAC组按照15mg/kg/d剂量给予NAC口服6周时间。在犬右房置入电极,快速起搏右心房,诱发房颤并维持2小时。在起搏前后分别测定有效不应期(AERP)。结果:房颤后对照组AERP显著缩短,AERP频率适应性下降(P<0.05);而NAC组房颤前后AERP和AERP频率适应性均无明显变化。结论:在心房快速起搏致房颤2h的模型中,NAC对心房电重构具有明显的保护作用。     

山羊房颤模型中左心房与肺静脉外膜碎裂电位的动态比较

目的: 比较在持续性房颤发生、发展过程中,房颤模型山羊左心房与肺静脉外膜碎裂电位(CFAEs)的变化,以期探讨肺静脉外膜碎裂电位(CFAEs)在持续性房颤中的作用。方法: 选取10只雌性山羊,使用左心房快速刺激,发送输出电压为6 V、周长为20 ms的脉冲1 s,间隔2 s后重复发放,以此方法建立持续性房颤模型(房颤持续＞24 h),平均刺激时间为(9.5±2.3)d。在整个房颤发生、发展过程中,记录并比较刺激前后左心房及肺静脉外膜CFAEs的动态变化。结果: 随着刺激时间延长,心房不应期(AERP)逐渐缩短,房颤持续时间逐渐延长,左心房及肺静脉外膜的CFAEs逐渐增多,肺静脉外膜CFAEs的比例始终多于左心房。当房颤持续24 h,肺静脉外膜CFAEs几乎持续存在(房颤初发与房颤持续24 h相比,2.7%±3.6% vs 92.6%±6.4%,P＜0.05),肺静脉外膜CFAEs比例明显高于左心房(P＜0.05)。结论: 心外膜CFAEs具有位置特异性,与电重构相关联。房颤发生发展中心外膜CFAEs逐渐增多,可能在房颤的维持中起重要作用。     

乙醇对大鼠心肌动作电位及人Kv1.5通道的影响

为了研究乙醇对心肌动作电位的作用及其机制,本实验采用标准玻璃微电极细胞内记录技术记录离体大鼠心肌细胞的动作电位(action potential,AP),采用全细胞膜片钳技术记录HEK293细胞上表达的人Kv1.5(human Kv1.5,hKv1.5)通道电流,观察6.25、12.5、25.0、50.0、100.0及...     

脉冲磁场对神经元动作电位发放的影响

磁场作用于生物体产生的生物效应被广泛研究。本文将脉冲磁场作用于神经元细胞,试图观察Na+通道的变化及其引起的动作电位的变化。选择频率15 Hz、强度1 mT的脉冲磁场刺激昆明小鼠大脑皮质神经元,随后进行全细胞膜片钳实验。结果显示,脉冲磁场延缓了Na+通道电流的激活,促进Na+通道电流的失活。基于经典的细胞三层介电模型,模拟了在脉冲磁场下细胞膜电位分布的极化图,结果显示诱发的膜电位大小与脉冲磁场的频率和强度有关。基于Hodgkin-Huxley(H-H)模型,仿真了脉冲磁场感应的电流作用于离子通道所产生的动作电位曲线,与不加刺激时候的曲线相比较,当外加电流在-1.32~0μA时,动作电位的产生频率减小,幅值降低;当外加电流大于0μA时,动作电位的产生频率增大,幅值变化不明显;当外加电流小于-1.32μA时,动作电位的上升时间快速变短,峰值剧烈降低,无法形成完整的动作电位,即无动作电位。以上结果提示,磁场刺激可通过调节Na+通道影响神经元动作电位的发放频率和幅值。     

味觉感受细胞离子通道和动作电位的建模与仿真

结合膜片钳测量的味觉感受细胞离子通道实验数据,提出了一个哺乳动物味觉感受细胞动作电位的数学模型.首先,建立了味觉感受细胞的电压门控Na＋通道和外向延迟整流K＋通道的模型,在此基础上建立了味觉感受细胞的单细胞计算模型.其次,仿真研究了味觉感受细胞在电刺激和酸味刺激下产生的动作电位,以及离子通道动力学特性对其的影响.该模型对于研究味觉感受细胞在味觉物质刺激下产生的动作电位及其离子通道的工作机制,以及味觉信息在外周神经的传递和信息编码具有指导意义。     

N-乙酰半胱氨酸对犬心房快速起搏电重构的影响

目的：探讨N-乙酰半胱氨酸（NAC）对犬心房快速起搏电重构的影响。方法：取16只犬,随机分为对照组和NAC干预组。NAC组按照15mg／kg／d剂量给予NAC口服6周时间。在犬右房置入电极,快速起搏右心房,诱发房颤并维持2小时。在起搏前后分别测定有效不应期（AERP）。结果：房颤后对照组AERP显著缩短,AERP频率适应性下降（P〈0．05）;而NAC组房颤前后AERP和AERP频率适应性均无明显变化。结论：在心房快速起搏致房颤2h的模型中,NAC对心房电重构具有明显的保护作用。     

心室不同部位起搏对正常犬和扩张型心肌病心力衰竭犬模型在体心肌跨室壁复极离散的影响

实验以正常犬和扩张型心肌病心力衰竭犬(dilated cardiomyopathy congestive heart failure,DCM-CHF)模型为对象、以心肌跨室壁复极离散的相关参数为指标,研究左心室心外膜起搏、双心室起搏(模拟临床上心室再同步治疗的方法)后的心肌电生理特性变化。实验以快速右心室起搏的方法制备DCM-CHF犬模型;正常犬和DCM-CHF犬均经射频消融希氏束制备三度房室传导阻滞模型;采用同步记录犬体表心电图和内膜下、中层、外膜下三层心肌单相动作电位(monophasic action potentials,MAP)的方法,测定不同部位起搏时的QT间期、Tpeak-Tend(Tp-Te)间期和三层心肌的单相动作电位时程(MAP duration,MAPD)、跨室壁复极离散度(transmural dispersion of repolaization,TDR)。结果显示:在正常犬,左室心外膜与双心室起搏后三层心肌的MAPD均延长,同时TDR增大(左室心外膜起搏47.16 ms、双心室起搏37.54 ms、右室心内膜起搏26.75 ms,P<0.001),体表心电图Tp-Te间期的变化与之平行;在DCM-CHF犬较正常犬已表现出中层心肌MAPD延长(276.30 ms vs 257.35 ms,P<0.0001)和TDR(33.8 ms vs 27.58 ms,P=0.002)增大的基础上,左室心外膜参与起搏后仍进一步使三层心肌的MAPD延长和TDR增大。研究结果提示,左室心外膜起搏和双心室起搏后使内膜下、中层     

Crosstalk between L-type calcium channels and ZnT-1, a new player in rate-dependent cardiac electrical remodeling

Crosstalk between two membrane transport systems is an established mechanism underlying regulation. In this study, we investigated the interaction between ZnT-1, a putative plasma membrane zinc transporter, and L-type voltage-dependent calcium channels (LTCC). In the atrium of the myocardium decreased activity of the LTCC is a dominant feature of patients with atrial fibrillation. The trigger for this inhibition has been attributed to the rapid firing rates and consequent calcium overload in the atrial cardiomyocytes. However, the underlying mechanism of LTCC inhibition is still to be elucidated. Here, we showed that the expression of ZnT-1 inhibits the activity of L-type channels during electrical remodeling induced by rapid pacing. (i) Direct manipulations of ZnT-1 expression in cultured cardiomyocytes either by ZnT-1 overexpression or by ZnT-1 silencing with siRNA, decreased or enhanced, respectively, the barium influx through the LTCC. (ii) Co-expression of ZnT-1 with LTCC in Xenopus oocytes decreased whole cell barium current through LTCC. (iii) Rapid pacing of cultured cardiomyocytes (4 h, 100 ms cycle) increased ZnT-1 protein expression and inhibited the voltage-dependent divalent cation influx through the LTCC. Moreover, silencing ZnT-1 with siRNA prevented the rapid pacing induced inhibition of the LTCC (iv) Atrial pacing of anesthetized adult rats (4 h, 50 ms cycle) led to a significant increase in atrial ZnT-1 protein expression in parallel with the typical decrease of the refractory period in the atria. Taken together, these findings demonstrate that crosstalk between ZnT-1 and the L-type calcium channels may underlie atrial response to rapid pacing, suggesting that ZnT-1 is a significant participant in rate-dependent cardiac electrical remodeling.     

Effects of electrical and structural remodeling on atrial fibrillation maintenance: a simulation study

Atrial fibrillation, a common cardiac arrhythmia, often progresses unfavourably: in patients with long-term atrial fibrillation, fibrillatory episodes are typically of increased duration and frequency of occurrence relative to healthy controls. This is due to electrical, structural, and contractile remodeling processes. We investigated mechanisms of how electrical and structural remodeling contribute to perpetuation of simulated atrial fibrillation, using a mathematical model of the human atrial action potential incorporated into an anatomically realistic three-dimensional structural model of the human atria. Electrical and structural remodeling both shortened the atrial wavelength--electrical remodeling primarily through a decrease in action potential duration, while structural remodeling primarily slowed conduction. The decrease in wavelength correlates with an increase in the average duration of atrial fibrillation/flutter episodes. The dependence of reentry duration on wavelength was the same for electrical vs. structural remodeling. However, the dynamics during atrial reentry varied between electrical, structural, and combined electrical and structural remodeling in several ways, including: (i) with structural remodeling there were more occurrences of fragmented wavefronts and hence more filaments than during electrical remodeling; (ii) dominant waves anchored around different anatomical obstacles in electrical vs. structural remodeling; (iii) dominant waves were often not anchored in combined electrical and structural remodeling. We conclude that, in simulated atrial fibrillation, the wavelength dependence of reentry duration is similar for electrical and structural remodeling, despite major differences in overall dynamics, including maximal number of filaments, wave fragmentation, restitution properties, and whether dominant waves are anchored to anatomical obstacles or spiralling freely.     

Rapid pacing of embryoid bodies impairs mitochondrial ATP synthesis by a calcium-dependent mechanism--a model of in vitro differentiated cardiomyocytes to study molecular effects of tachycardia

Tachycardia may cause substantial molecular and ultrastructural alterations in cardiac tissue. The underlying pathophysiology has not been fully explored. The purpose of this study was (I) to validate a three-dimensional in vitro pacing model, (II) to examine the effect of rapid pacing on mitochondrial function in intact cells, and (III) to evaluate the involvement of L-type-channel-mediated calcium influx in alterations of mitochondria in cardiomyocytes during rapid pacing. In vitro differentiated cardiomyocytes from P19 cells that formed embryoid bodies were paced for 24 h with 0.6 and 2.0 Hz. Pacing at 2.0 Hz increased mRNA expression and phosphorylation of ERK1/2 and caused cellular hypertrophy, indicated by increased protein/DNA ratio, and oxidative stress measured as loss of cellular thiols. Rapid pacing additionally provoked structural alterations of mitochondria. All these changes are known to occur in vivo during atrial fibrillation. The structural alterations of mitochondria were accompanied by limitation of ATP production as evidenced by decreased endogenous respiration in combination with decreased ATP levels in intact cells. Inhibition of calcium inward current with verapamil protected against hypertrophic response and oxidative stress. Verapamil ameliorated morphological changes and dysfunction of mitochondria. In conclusion, rapid pacing-dependent changes in calcium inward current via L-type channels mediate both oxidative stress and mitochondrial dysfunction. The in vitro pacing model presented here reflects changes occurring during tachycardia and, thus, allows functional analyses of the signaling pathways involved.     

Rapid pacing of embryoid bodies impairs mitochondrial ATP synthesis by a calcium-dependent mechanism—A model of in vitro differentiated cardiomyocytes to study molecular effects of tachycardia

Tachycardia may cause substantial molecular and ultrastructural alterations in cardiac tissue. The underlying pathophysiology has not been fully explored. The purpose of this study was (I) to validate a three-dimensional in vitro pacing model, (II) to examine the effect of rapid pacing on mitochondrial function in intact cells, and (III) to evaluate the involvement of L-type-channel-mediated calcium influx in alterations of mitochondria in cardiomyocytes during rapid pacing. In vitro differentiated cardiomyocytes from P19 cells that formed embryoid bodies were paced for 24 h with 0.6 and 2.0 Hz. Pacing at 2.0 Hz increased mRNA expression and phosphorylation of ERK1/2 and caused cellular hypertrophy, indicated by increased protein/DNA ratio, and oxidative stress measured as loss of cellular thiols. Rapid pacing additionally provoked structural alterations of mitochondria. All these changes are known to occur in vivo during atrial fibrillation. The structural alterations of mitochondria were accompanied by limitation of ATP production as evidenced by decreased endogenous respiration in combination with decreased ATP levels in intact cells. Inhibition of calcium inward current with verapamil protected against hypertrophic response and oxidative stress. Verapamil ameliorated morphological changes and dysfunction of mitochondria. In conclusion, rapid pacing-dependent changes in calcium inward current via L-type channels mediate both oxidative stress and mitochondrial dysfunction. The in vitro pacing model presented here reflects changes occurring during tachycardia and, thus, allows functional analyses of the signaling pathways involved.     

Chronic atrial fibrillation does not further decrease outward currents. It increases them

Rapid atrial pacing causes electrical remodeling that leads to atrial fibrillation (AF). AF can further remodel atrial electrophysiology to maintain AF. Our previous studies showed that there was a marked difference in the duration of AF in dogs that have been atrial paced at 400 beats/min for 6 wk. We hypothesized that this difference is based on the changes in the degree of electrical remodeling caused by rapid atrial pacing versus that by AF. Right atrial cells were isolated from control dogs (Con, N = 28), from dogs with chronic AF (cAF dogs, N = 13, episodes lasting at least 6 days), or from dogs with nonsustained or brief episodes of AF (nAF dogs, N = 10, episodes lasting minutes to hours). Both transient outward (Ito) and sustained outward K+ current (Isus) densities/functions were determined using whole cell voltage-clamp techniques. In nAF cells, Ito density was reduced by 69% at +40 mV: from 7.1 +/- 0.5 pA/pF (Con, n = 59) to 2.2 +/- 0.2 pA/pF (nAF, n = 24) (P < 0.05). The voltage dependence of inactivation of Ito was shifted positively and decay kinetics were changed; however, recovery from inactivation was not altered in nAF cells. In contrast, Ito density in cAF cells was both significantly different from Con cells and larger than that in nAF cells [at +40 mV, 3.5 +/- 0.3 pA/pF (cAF, n = 29), P < 0.05]. In cAF cells, recovery from inactivation and decay of Ito were both slow; yet, voltage dependence inactivation of Ito approached that of Con cells. Furthermore, "recovered" Ito of cAF cells was more sensitive to tetraethylammonium than currents of Con and nAF cells. Isus densities of nAF and cAF cells did not differ. Both nAF and cAF cells have reduced Ito versus Con cells, but Ito remodeling of nAF cells differed from that of cAF cells. Ito in cAF dogs was likely remodeled by AF per se, whereas that in nAF dogs was likely the consequence of the rapid rate in the absence of sustained AF.     

Bioengineering approaches to mature induced pluripotent stem cell-derived atrial cardiomyocytes to model atrial fibrillation

Induced pluripotent stem cells (iPSCs) serve as a robust platform to model several human arrhythmia syndromes including atrial fibrillation (AF). However, the structural, molecular, functional, and electrophysiological parameters of patient-specific iPSC-derived atrial cardiomyocytes (iPSC-aCMs) do not fully recapitulate the mature phenotype of their human adult counterparts. The use of physiologically inspired microenvironmental cues, such as postnatal factors, metabolic conditioning, extracellular matrix (ECM) modulation, electrical and mechanical stimulation, co-culture with non-parenchymal cells, and 3D culture techniques can help mimic natural atrial development and induce a more mature adult phenotype in iPSC-aCMs. Such advances will not only elucidate the underlying pathophysiological mechanisms of AF, but also identify and assess novel mechanism-based therapies towards supporting a more ‘personalized’ (i.e. patient-specific) approach to pharmacologic therapy of AF.