Wistar大鼠乳鼠与成年鼠心室肌细胞的分离与性质鉴定

目的 探索和优化稳定的适于电生理实验研究的乳鼠及成年大鼠心室肌细胞分离方法。方法 切碎乳鼠心室肌,胰蛋白酶消化,差速贴壁2 h纯化心室肌细胞,台盼蓝染色判定心肌细胞活力,体外培养48 h后分别行倒置显微镜观察细胞形态,免疫组化鉴定,微电极阵列记录细胞搏动频率和场电位。采用Langendorff灌流成年大鼠心脏,主动脉逆行插管,胶原酶域反复灌流消化约30 min,无钙台氏液冲洗心脏5 min,剪下心室肌组织,台氏液中室温下剪碎,吹打,孵育5 min后,用200目筛网过滤,将细胞悬液用逐步复钙法复钙后,室温静置1 h,用于膜片钳记录。结果 经4 -6次消化后,乳鼠心室组织消化完全,细胞存活率大于80%。倒置显微镜下观察,细胞呈梭形、多角形。 12 h有少部分细胞搏动,48 h细胞交织成网,搏动呈同步性,搏动频率30 - 80次/分。 琢鄄辅肌动蛋白（琢鄄actin）经免疫组化检测,纯度达96%。 Langendorff灌流酶解法可获得形态呈杆状、横纹清晰、膜周边光滑完整、立体感强的单个成年鼠心肌细胞,存活率85%,复钙后存活率50%,可用于膜片钳记录。结论 采用本方法可以获得高产量与高质量的用于电生理检测的心室肌细胞。     

大鼠心肌细胞急性分离方法及影响因素探讨

目的探讨酶解法急性分离大鼠心肌细胞的相关影响因素。方法采用改良Langendorff主动脉逆行灌流法对大鼠全心工作细胞进行酶解分离,分析分离过程中的诸多条件,从中优化最佳的分离方案,为膜片钳实验提供合格细胞。结果分离所用灌流液pH值为7.35~7.40,分离细胞的存活率最高。不同复钙方法对后续细胞存活有明显影响。三步梯度复钙后的细胞存活率明显高于一步、二步复钙法。细胞分离时,灌流速度对细胞存活率有较大影响,3mL/min流速时细胞存活率最高,不同灌流流程对分离细胞的质和量较大影响。采用钙-无钙台氏液先后灌流后再行酶解分离方式可获得长杆状、棱角分明、立体感强的心肌细胞,该细胞复钙后存活率为(56.8±2.6)%;直接使用无钙台氏液灌流后再行酶解分离方法获得(57.2±3.3)%的心肌细胞,其复钙后存活率为(36.7±3.5)%,前述方法明显优于后者。结论灌流液的pH值、灌流速度、细胞分离后的复钙方法及不同的灌流流程等因素可对大鼠心肌细胞的急性分离结果产生显著影响。     

成年大鼠心室肌细胞的急性分离方法

目的介绍一种新型的成年大鼠心肌细胞的急性分离方法。方法麻醉大鼠后快速取心脏,为心脏先循环灌流无钙台氏液,后循环灌流酶液,酶解完成后取左心室,迅速置于含0.5 mg/ml BSA的KB液(恒温37℃)中拉碎,吹打数次后离心,去上清,温育在含0.5 mg/ml BSA的KB溶液中35 min,梯度复钙。结果首次分离细胞存活率在80%~90%,复钙完成仍有40%~50%的细胞维持杆状,横纹清晰且90%以上细胞处于静止状态。结论通过该方法可获得稳定、高比例的存活心肌细胞,能够为成年大鼠心肌细胞原代培养及电生理学研究提供高品质细胞。     

微电极阵芯片技术在心肌电生理研究中应用的可行性

目的探讨60极微电极阵芯片(MEA)技术在整体心脏、心肌组织片和培养心肌等电生理研究中的应用。方法①80只豚鼠,开胸取心脏并以2.5ml/min速度Langendorff灌流心脏20min,信号稳定后采样;②60只SD大鼠,开胸取心,剪取5mm×5mm大小的心脏组织片,置于MEA盘,台氏液间歇灌流5min,用5μV×1ms的刺激脉冲连续刺激,刺激间隔为1s,比较心房和心室组织片场电位(FPs)形态和传导时间;③20窝出生后3天以内的昆明小白鼠,消化分离心肌细胞,培养于MEA盘内,进行组织信号采样。结果①大鼠整体心脏灌流30～90min内能够记录到稳定的自主节律下的FPs信号,心室肌场电位时程(FPdur)210±78ms;心房肌FPdur164±58ms;房室传导时间320±150ms;并能记录到FPs激动顺序图。②心肌组织靶点取样,台氏液间歇灌流和电刺激下,组织的兴奋性可以稳定保持2h。心室肌组织片FPdur115.80±11.61ms,心房组织片FPdur83.71±6.48ms,刺激信号在心房组织片的传导时间66.46±6.73ms,心室组织片传导时间47.40±5.62ms;③小鼠乳鼠心脏原代培养心肌24h时后开始有散在、不同步的细胞克隆搏动和点状FPs信号,72h后细胞融合,开始记录到基本同步的群动和多位点FPs信号,培养心肌自发搏动频率150±100次/分。结论 MEA技术可以稳定记录小动物整体灌流心脏,心脏定点取样组织片,培养心肌细胞60极场电位和电传导特性。     

正常耐钙Spraque-Dawley大鼠心室肌细胞分离方法及体会

目的探讨稳定分离用于膜片钳实验的耐钙Spraque-Dawley大鼠心室肌细胞的方法。方法在自制心脏灌流装置上,采用三步法行逆行主动脉灌流获得单个耐钙心肌细胞,以膜片钳全细胞方式记录离子流。结果在分离过程中如整个心脏保持红润,则细胞数量在90%以上,耐钙细胞KB液中孵育后在70%左右;在分离过程中心脏局部保持红润部位的细胞数量在80%以上,耐钙细胞在60%左右,而苍白区细胞数量变异较大,但一般均在50%以下,且耐钙细胞较少;在分离过程中如整个心脏始终苍白,则细胞数量不仅低于30%,且几乎没有耐钙细胞。结论在心肌细胞分离过程中如严格按照本文的介绍,能获得大量具有正常电生理特性的耐钙心肌细胞。     

犬三层心室肌细胞的分离和鉴别

目的探讨有效分离和鉴别犬三层心室肌细胞的方法。方法用带有左室前降支的犬心肌组织块灌流分离心室肌细胞,得到的心肌细胞先根据解剖部位大致分成三层,再采用膜片钳技术,在电流钳模式下,随机选择各层15个细胞记录动作电位(AP),根据AP的形态、时程、频率依赖性进一步判断是否为相应层的心室肌细胞。结果经左室前降支插管灌流心肌组织块,可以得到数量多、状态好的心肌细胞。在15个心室肌细胞中,能准确判断其层次的有:外层7个、中层5个、内层8个。结论经冠状动脉插管灌流分离犬心室肌细胞是可行的,结合解剖部位和动作电位特点,能有效鉴别不同层的心室肌细胞。     

成年家兔心房肌细胞的分离和电生理记录

目的建立一种简单稳定的成年家兔心房肌细胞的分离、培养方法并进行电生理记录。方法麻醉后取出成年家兔心脏,采用Langendorff灌流装置及急性酶裂解法分离心房肌细胞,差速贴壁法进行纯化后培养于DMEM培养基。在倒置显微镜下观察细胞形态,利用透射电镜观察细胞超微结构,用免疫荧光染色法对心房肌细胞进行鉴定,利用全细胞膜片钳技术记录动作电位和内向钙电流和外向钾电流。结果本方法分离的心房肌细胞纯度和细胞存活率较高,并使用膜电钳技术成功记录了L-型钙电流和瞬时外向钾电流。结论该方法简便有效,细胞存活率高且为进一步进行各种电生理实验打下了基础。     

豚鼠心房肌细胞的分离及其离子流记录

为探讨豚鼠心房肌细胞的分离方法及其离子流记录,采用酶解技术分离豚鼠心房肌细胞,应用膜片钳全细胞记录技术,记录细胞膜L型钙流(L-I_(Ca))及延迟整流性钾流(I_K)。结果获得了具正常电生理活性的单个细胞及高阻封接的形成,成功记录了L-型钙流和延迟整流性钾流。认为本方法分离的单个心房肌细胞具正常的电生理活性,可用于研究心房肌细胞离子通道活性。     

豚鼠心房肌细胞的分离与其离子流记录

为探讨豚鼠心房肌细胞的分离方法及其离子离子流记录，采用酶解技术分离豚鼠心房肌细胞，应用膜片钳全细胞记录技术，记录细胞膜Ｌ型钙流（Ｌ－Ｉｃａ）及延迟整流性钾流（Ｉｋ）。结果获得了具正常电生理活性的单个细胞及高阻封接的形成，成功记录了Ｌ－型钙流和延迟整流钾流，认为本方法分离的单个心房肌细胞具正常的电生理活性，可用于研究心房肌细胞离子通道活性。     

改良成年小鼠心室肌细胞分离方法

目的 小鼠是优良的背景基因动物,分离成体原代心肌细胞可以真实地从细胞和分子水平反映单个心室肌细胞的功能。小鼠心室肌细胞的分离和大鼠相比较困难,分离获得的细胞存活率较低,为提高小鼠心室肌细胞的成活率和收获量,本研究团队通过艰苦的摸索,改进了小鼠成体心肌细胞的分离方法。方法 该方法采用心脏在体主动脉插管,行独创的前加压灌流法冲洗心脏后接入改进的langendorff装置,用胶原酶液II灌流、消化心脏后,将心脏剪碎并用吸管吹打,经200目滤网过滤后获得单个小鼠心室肌细胞。本研究检测并对比了在体和离体主动脉插管获得的心肌细胞的状态：梯度复钙后,在倒置显微镜下观察细胞的形态和杆状心室肌细胞的数目。结果 研究结果显示：改良的主动脉逆行灌流法在缩短手术时间的同时,提高了小鼠心室肌细胞收获量和存活率。本课题组利用医用三通管,独创倒置灌流排气法,可以迅速排空灌流装置中的气体;利用注射器和灌流针结合,独创前加压灌流法,可以快速彻底地冲洗心脏中的血液;并采用实验室常规眼科镊和血管钳巧妙组合成协助装置,可以单人独立完成插管,不需要助手协助,节约人力,节省经费开支。结论 这一方法的改进使整个实验流程简化,缩短了实验时间,操作简便易学;同时该分离方法稳定、可靠、有效,可以为同类型实验提供思路和借鉴。     

犬右房不同部位Kv4.3、KchIP2、Cav1.2 mRNA的表达

目的研究犬右房不同部位短暂外向钾电流、L型钙电流亚单位mRNA的表达情况,探讨其在致房性心律失常中的意义。方法应用逆转录-聚合酶链反应半定量分析犬界嵴、梳状肌、右心耳的短暂外向钾电流α亚单位(Kv4.3)、β亚单位(KchIP2)及L型钙电流的α亚单位(Cav1.2)mRNA的表达量(以β-actin为内参照)。结果界嵴和梳状肌Kv4.3、KchIP2 mRNA高于右心耳(P<0.05或0.01);界嵴Cav1.2 mRNA高于梳状肌和右心耳(P均<0.05),而梳状肌和右心耳之间没有差异。结论Kv4.3、KchIP2、Cav1.2 mRNA在右房空间表达上的差异与其相应离子流在右房空间上的差异一致,可能是其离子流差异的分子基础。     

Potential ionic mechanism for repolarization differences between canine right and left atrium

Experimental and clinical evidence suggests a critical role for the left atrium (LA) in atrial fibrillation (AF). In animal models, repolarization is faster in the LA than in the right atrium (RA), leading to more stable reentry circuits with a shorter intrinsic period in the LA. The ionic mechanisms underlying LA-RA repolarization differences are unknown. Therefore, we evaluated ionic currents and action potentials (APs) with the whole-cell patch clamp in isolated canine atrial myocytes. The density of the rapid delayed rectifier current (I(Kr)) was greater in the LA (eg, 1.83+/-0.10 pA/pF at +20 mV) than in the RA (1.15+/-0.07 pA/pF, P<0.01; n=16 cells per group). The slow and ultrarapid delayed rectifier, the inward rectifier, L-type Ca(2+), and transient outward K(+) currents were all comparable in the LA and RA. There were no differences in kinetic or voltage-dependent properties of currents in LA versus RA. Western blots of ether-a-go-go-related gene (ERG) protein in three RA and corresponding LA regions showed significantly greater ERG expression in LA. AP duration (APD) was shorter in the LA versus RA in both isolated cells and multicellular preparations, and the effective refractory period (ERP) was shorter in the LA compared with the RA in vivo. Dofetilide had significantly larger APD- and ERP-increasing effects in the LA compared with RA, and LA-RA repolarization differences were eliminated by exposure to dofetilide. We conclude that LA myocytes have larger I(Kr) than do RA myocytes, contributing importantly to the shorter APD and ERP in LA. The larger LA I(Kr) may participate in the ability of the LA to act as a "driver region" for AF, with potentially important implications for understanding AF mechanisms and antiarrhythmic therapy.     

Demonstration of calcium-activated transient outward chloride current and delayed rectifier potassium currents in Swine atrial myocytes

Cellular electrophysiology is not fully understood in the atrium of pig heart. The objective of the present study was to determine whether transient outward current (I(to)), ultra-rapid delayed rectifier potassium current (I(Kur)), and rapid and slow delayed rectifier K(+) currents (I(Kr) and I(Ks)) were present in pig atrium. The whole-cell patch technique was applied to record membrane currents and action potentials in myocytes isolated from pig atrium. It was found that an I(to) was activated upon depolarization voltage steps to between -10 and +60 mV from -50 mV in pig atrial cells, and the I(to) was sensitive to the inhibition by the blockade of L-type calcium (Ca(2+)) current, showed a "bell-shaped" I-V relationship, typical of I(to2) (i.e. I(Cl.Ca)). The I(to2) was inhibited by the chloride (Cl(-)) channel blocker anthracene-9-carboxylic acid (9-AC, 200 micromol/l) or 4,4'-diisothiocyanostilben-2,2'disulfonic acid (200 micromol/l), and by Cl(-) substitution in the superfusate. I(Kur) was found in pig atrial myocytes, and the current showed properties of weak inward rectification and use- and frequency-dependent reduction. I(Kur) was resistant to tetraethylammonium, but sensitive to inhibition by 4-aminopyridine (4-AP) (IC(50) = 71.7 +/- 3.5 micromol/l). In addition, E-4031-sensitive I(Kr) and chromanol 293B-sensitive I(Ks) were observed in pig atrial myocytes. Blockade of I(to2), I(Kur), I(Kr) or I(Ks) with corresponding blockers significantly prolonged atrial action potentials. These results indicate that Ca(2+)-activated I(to2), 4-AP-sensitive I(Kur), E-4031-sensitive I(Kr), and 293B-sensitive I(Ks) are present in pig atrial myocytes, and these currents play important roles in action potential repolarization of pig atria.     

Blockade of atrial-specific K+-currents increases atrial but not ventricular contractility by enhancing reverse mode Na+/Ca2+-exchange

BACKGROUND: AVE0118 (2'-{[2-(4-Methoxy-phenyl)-acetylamino]-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide) blocks atrial ultrarapid delayed rectifier currents (I(Kur)) and prolongs the atrial action potential (AP) plateau without affecting ventricular repolarisation. In patients with atrial contractile dysfunction due to atrial tachyarrhythmias, this response might increase atrial contractility without risk of ventricular proarrhythmia. This study was designed to evaluate the inotropic mechanisms of AVE0118. METHODS AND RESULTS: In isometrically contracting atrial trabeculae, AVE0118 increased contractile force by 55.4% in sinus rhythm patients (n = 9) and by 107.4% in patients with atrial fibrillation (n = 8). In freshly isolated canine atrial myocytes studied under perforated patch current clamp (37 degrees C), AVE0118 increased myocyte fractional shortening from 3.8+/-0.6 to 9.6+/-0.8% and prolonged action potential duration at 30% repolarisation from 9+/-2 to 102+/-11 ms. Clamping cells to an AP waveform recorded during exposure to AVE0118 produced the same inotropic response as the drug itself. In action potential clamp, peak Ca2+ inward current (I(CaL)) current declined from 5.5+/-1.3 pA/pF during control to 4.1+/-0.7 pA/pF when an AP recorded in the presence of AVE0118 was used as command waveform. However, I(CaL) was more sustained with AVE0118 and the time integral did not change (135+/-37 vs. 173+/-30 pA/pFms, p = ns). Importantly, blockade of reverse mode Na+/Ca2+-exchanger activity with 5 microM KBR7943 or using a Na+-free pipette solution abolished the positive inotropic effect of the AP recorded in the presence of AVE0118. In ventricular myocytes AVE0118 did not elicit a positive inotropic response. CONCLUSIONS: Block of I(Kur) by AVE0118 enhances atrial contractility both in patients with sinus rhythm and atrial fibrillation. The positive inotropic effect is atrial-specific and due to the changes of the action potential configuration which enhances Ca2+ entry via reverse mode Na+/Ca2+ exchange.     

心房颤动24h和48h对犬心房肌有效不应期及L型钙电流的影响

目的 研究犬心房颤动（房颤）持续24h和48 h,心房肌有效不应期和L型钙电流变化的一致性及其机制.方法 健康成年杂种犬18只,分为对照组、24 h房颤组、48 h房颤组,每组6只.600次/min起搏高右心房建立犬房颤模型,应用程序刺激的方法测定右心房有效不应期（ERP）,通过Langendorff左回旋支灌流分离心房肌细胞,并通过全细胞膜片钳技术记录L型钙电流（ICa-L）变化.应用免疫组织化学方法检测各组心房组织L型电压依赖性钙通道（LVDCC）α1c蛋白表达.用图像分析系统对组织化学抗原表达进行半定量分析.结果 所有实验动物均可诱发出房颤.心房ERP的变化在最初6h较对照组明显缩短,后直至48 h呈进行性缩短.快速起搏6h后ERP（129.50±8.64）ms较对照组（141.00±15.23）ms缩短（12.13±2.24）ms（P＜0.01）,24 h后心房ERP（123.00± 13.37） ms缩短（19.23±2.14）ms（P＜0.01）,48 h缩短（28.15±4.26）ms（P＜0.01）.同时在房颤持续过程中24h房颤可引起ICa-L电流密度（-4.83±0.30）pA/pF较对照组（-6.69±0.08）pA/pF减小,48 h（-3.70±0.50）pA/pF减少的程度更重.24 h房颤及48 h房颤犬的左、右心房组织LVDCC α1c蛋白表达均较对照组明显减少（P＜0.05）,48 h房颤组减少程度更重（P＜0.01）.结论 房颤持续24 h,心房肌ERP显著缩短、ICa-L密度降低.房颤持续48 h仍然维持L型钙通道改变特征.提示：钙通道阻断剂可用于持续24 h房颤,预防房颤复发.     

盐酸关附甲素对豚鼠离体心房肌电生理特性作用的研究

目的观察盐酸关附甲素（AHH）对豚鼠离体心房肌动作电位及有效不应期的影响,探讨其抗心律失常作用的机制。方法脱颈法处死豚鼠后,迅速开胸取出心脏,移至台氏液中。从左心耳剪取大约3mm×10mm大小的肌条,固定于浴槽中,用持续通以混合气体（95%O2＋5%CO2）的台式液灌流,流速为10ml/min,以频率为1Hz、脉宽2ms的阈上刺激平衡至少45min。利用玻璃微电极技术测定基础动作电位和有效不应期后,以25、75、250、500μmol/L的AHH灌流液灌流5min后,以同样条件测定药后动作电位和有效不应期。结果 500μmol/LAHH使零相最大除极速度（Vmax）下降12.4%,平均复极化斜率（ASRP）下降19.2%（p〈0.01）,对动作电位时程（APD）有延长趋势但未达到统计学差异;250μmol/L和500μmol/L两个浓度延长心房有效不应期的作用达到统计学差异,其作用呈频率依赖性。结论 1、高浓度盐酸关附甲素降低心房肌Vmax,有降低动作电位幅度（APA）趋势,具有延缓心房内传导的作用;2、盐酸关附甲素对心房动作电位时程无明显影响,但降低动作电位复极2相斜率,相对延缓心房复极;3、高浓度盐酸关附甲素频率依赖性延长心房有效不应期,可能是其抗心律失常的主要作用机制。     

Comparison of ion channel distribution and expression in cardiomyocytes of canine pulmonary veins versus left atrium

BACKGROUND: Cardiomyocytes in pulmonary vein (PV) sleeves are important in atrial fibrillation (AF), but underlying mechanisms are poorly understood. Pulmonary veins have different ionic current properties compared to left atrium, with pulmonary vein inward-rectifier currents being smaller and delayed-rectifier currents larger than in left atrium. METHODS: Expression and distribution of the inward-rectifier subunits Kir2.1 and Kir2.3, the rapid delayed-rectifier alpha-subunit ERG, the slow delayed-rectifier alpha-subunit KvLQT1, the beta-subunit minK, the L-type Ca(2+)-subunit Ca(v)1.2, and the Na(+),Ca(2+)-exchanger were quantified by Western blot on isolated cardiomyocytes and localized by immunohistochemistry in tissue sections obtained from canine hearts. RESULTS: Western blotting indicated significantly greater expression of ERG (by 28%, P<0.05) and KvLQT1 (by 34%, P<0.05) in pulmonary vein versus left atrial (LA) cardiomyocytes, but smaller Kir2.3 and similar Kir2.1, Ca(v)1.2 and Na(+),Ca(2+)-exchanger expression in PV. Kir2.1 exhibited weak transverse tubular distribution in both regions. Kir2.3 localized to intercalated disks in both regions, and to transverse tubules in left atrium but not pulmonary vein. ERG staining was more intense in pulmonary vein than left atrium, localizing to transverse tubules in both regions and intercalated disks in pulmonary veins. KvLQT1 was more intensely expressed in pulmonary veins, with a transverse tubular and intercalated disk localization, versus a more diffuse signal in left atrium. The Na(+),Ca(2+)-exchanger localized to transverse tubules, plasma membranes and intercalated disks with similar intensity in each region. CONCLUSIONS: Greater ERG and KvLQT1 abundance in pulmonary vein cardiomyocytes, lower abundance of Kir2.3 in pulmonary veins and differential pulmonary vein subcellular distribution of Kir2.3, ERG and KvLQT1 subunits may contribute to ionic current differences between pulmonary vein and left atrial cardiomyocytes.     

Postnatal development of atrial repolarization in the mouse

OBJECTIVES AND METHODS: This study examines postnatal development of action potential duration (APD) and voltage-dependent K(+) currents in mouse atrial myocytes and compares the expression levels of corresponding K(+) channels between adult and neonatal mouse atrial tissues. APD and K(+) currents were compared between atrial myocytes isolated from postnatal Day-1, Day-7, Day-20, and adult mice. RESULTS: All K(+) currents examined underwent significant up-regulation during postnatal life in mouse atrium, resulting in a dramatic shortening of the APD. The ultrarapid delayed rectifier (I(Kur)) was absent in the developing mouse heart and only contributed to repolarization in the adult mouse atrium, whereas the density of the other K(+) currents increased earlier during the developmental period. Indeed, the major changes in the expression of the inward rectifier current (I(K1)) occurred within the first week of life, the density of the Ca(2+)-independent transient outward K(+) current (I(to)) gradually increased while the development of the steady-state outward K(+) current (I(ss)) was completed within the first 3 weeks of life. Results of RNase protection assay and Western blot analysis confirmed that the postnatal development of the mouse atrial K(+) currents correlates with an increase in expression levels of underlying K(+) channel isoforms. CONCLUSION: These findings indicate that in mouse atrium, each K(+) current exhibits a specific postnatal development, suggesting that regulatory factors taking place during development are major determinants of the functional role of K(+) channels in cardiac repolarization. The mouse atrium is, therefore, a very interesting model to gain information on the mechanisms regulating K(+) channel activity.