小鼠心室肌细胞分离方法的改良及钾电流的记录

目的报道一种改良的小鼠心室肌细胞分离方法,并观察小鼠心室肌细胞动作电位以及钾电流的电生理特性。方法采用双酶消化法分离单个心室肌细胞,应用全细胞膜片钳技术记录动作电位和钾电流。先记录外向钾电流(Ipeak),用低浓度4-氨基吡啶(100μmol/L)使延迟整流钾电流(IKur)失活后记录瞬时外向钾电流(Ito),用Ipeak减去Ito即可得到IKur,在完全失活IKur及Ito后可记录到稳态钾电流(Iss)。结果本法分离所得心室肌细胞横纹清晰,具有正常电生理活性,细胞池中加入层粘连蛋白后有助于细胞贴壁,从而易于形成高阻封接,并记录出小鼠心室肌细胞特征性的动作电位和钾电流。结论本实验所采用的分离方法简便,获得的小鼠心室肌细胞易于封接,且具有正常的电生理活性。     

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。     

家兔肺静脉的单个心肌细胞电生理特性——局灶性心房颤动起源的意义

肺静脉是诱发阵发性心房颤动的重要起源灶 ,然而其高度致心律失常性机制尚不明了。本试验的目的是从肺静脉中分离单个的心肌细胞 ,并评价其电生理特性及致心律失常的可能性。采用从主动脉经左室至左房的逆行灌流酶消化法 ,分离得到家兔肺静脉的心肌细胞。然后用全细胞膜片钳技术研究单个肺静脉心肌细胞的动作电位和离子流。分离肺静脉可得到单个的起搏细胞 (76 % )和具有快反应动作电位的非起搏细胞。内向钙电流和短暂外向钾电流的特征在这两类细胞中类似。然而与非起搏细胞相比 ,起搏细胞有更小的内向整流性钾电流 (1.5 0± 0 .2 2ｖｓ 4.2…     

兔心室肌细胞的急性分离及电生理记录

目的建立一种简单、稳定的兔心室肌细胞分离方法并进行多种电生理记录。方法采用Langendorff灌流装置及急性酶解分离技术获取单个兔心室肌细胞,并利用全细胞膜片钳技术记录动作电位及多种膜电流。结果耐钙心室肌细胞的存活率在50%～60%,其静息膜电位在-80～90mV,并成功地记录到典型的动作电位及钾、钠、钙等电流。结论该方法简便、稳定,细胞存活率高且易于进行各种电生理实验。     

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。     

在人小肠上皮细胞膜上检出钙激活性钾通道(英文)

目的 :研究人类小肠上皮细胞 （Intestine40 7cell lines）膜上钙激活性钾通道的分子生物学特征及其电生理学特性。方法 :用 RT- PCR方法检测培养的人类小肠上皮细胞膜上钙激活性钾通道的表达形式 ,用膜片钳的全细胞记录及单通道记录法探讨了该通道的电生理学特点。结果 :RT- PCR证实该细胞系有 interm ediate- conductance（IK）钙激活性钾通道表达 ,而没有 large- conductance（BK）和 small- conductance（SK）钙激活性钾通道的表达。电生理学研究表明 ,Ionom ycin引导的全细胞电流显示该钙激活性钾电流具有内向整流性并可被特异度 IK钙激活性钾通道阻断剂 clotrimazole所抑制 ,但特异度 SK钙激活性钾通道阻断剂 apam in对该电流无明显抑制作用。单通道记录法证实该通道的电导为 30± 2 p S,通道活性对细胞内 Ca2 +浓度有明显依赖性。结论 :分子生物学和电生理研究证实了人类小肠上皮细胞膜上有 IK钙激活性钾通道 ,其生理学意义有待进一步研究     

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。     

人骨髓间充质干细胞体外诱导分化为心肌样细胞的L型钙通道研究

目的:观察人骨髓间充质干细胞(hMSCs)诱导分化为心肌样细胞的电生理特征,L型钙离子通道的电流强度。方法:体外分离培养hMSCs,以5-氮胞苷诱导分化为心肌样细胞。分别以体外分离培养的原代搏动的SD乳鼠心肌细胞(CMs)和同代次的hMSCs为阳性对照和阴性对照,利用膜片钳技术观察诱导分化为心肌样细胞的电生理特征L型钙离子通道的电流强度。结果:在20个心肌样细胞中,有6个细胞可记录到对硝苯地平敏感的内向钙电流,其最大内向峰值电流(ICa-Peak)在0mV为(112.60±8.26)pA;而hMSCs和乳鼠的CMs的ICa-Peak分别为(96.67±13.50)pA和(263.86±39.01)pA(n=6),将诱导的心肌样细胞与未经诱导的hMSCs的ICa-Peak进行比较,P0.05具有统计学意义。结论:hMSCs经5-氮胞苷诱导后,其L型钙离子通道的钙电流增加,在电生理特性上具有向CMs分化的潜能。     

降钙素基因相关肽对正常及模拟缺血缺氧心肌钙通道的作用及心肌细胞数学模型的仿真研究

作者运用细胞培养、膜片钳和激光共聚焦技术 ,观察了降钙素基因相关肽 （CGRP）对正常及模拟缺血、缺氧心肌细胞钙通道的作用及其电生理离子机制 ,并利用计算机重建技术在心肌细胞通道动力学及电流重建等方面进行了探索性研究 ,得到了如下提示性结论 :1CGRP对心肌的保护作用有赖于适当剂量和浓度 ;2本课题建立的急性心肌细胞分离方法 ,能够得到具有正常电生理特性的耐钙心肌细胞 ,是膜片钳实验和激光扫描共聚焦技术测定胞内钙的基础 ;3CGRP对急性缺血、缺氧心肌细胞具有保护作用 ,在急性缺血、缺氧时 ,急性分离的心肌细胞胞内钙离子浓度降低 ,CGRP可引起胞内钙浓度稍微升高 ,且 CGRP可改善因急性缺血、缺氧引起的胞内钙浓度的降低 ,其机制有待于进一步探讨 ;4CGRP能促使正常及模拟缺血缺氧状态下豚鼠心室肌细胞 ICa内流的增加 ,是 CGRP正性作用的主要离子机制 ;CGRP对钙通道的作用机制还有待进一步研究 ;5本课题建立的对几种离子电流的计算机重建方法 ,提示可以被用来讨论离子电流机制 ,是一种新的途径     

Familial hypertrophic cardiomyopathy-linked mutant troponin T causes stress-induced ventricular tachycardia and Ca2+-dependent action potential remodeling

The cardiac troponin T (TnT) I79N mutation has been linked to familial hypertrophic cardiomyopathy and high incidence of sudden death, despite causing little or no cardiac hypertrophy in patients. Transgenic mice expressing mutant human TnT (I79N-Tg) have increased cardiac contractility, but no ventricular hypertrophy or fibrosis. Enhanced cardiac function has been associated with myofilament Ca2+ sensitization, suggesting altered cellular Ca2+ handling. In the present study, we compare cellular Ca2+ transients and electrophysiological parameters of 64 I79N-Tg and 106 control mice in isolated myocytes, isolated perfused hearts, and whole animals. Ventricular action potentials (APs) measured in isolated I79N-Tg hearts and myocytes were significantly shortened only at 70% repolarization. No significant differences were found either in L-type Ca2+ or transient outward K+ currents, but inward rectifier K+ current (IK1) was significantly decreased. More critically, Ca2+ transients of field-stimulated ventricular I79N-Tg myocytes were reduced and had slow decay kinetics, consistent with increased Ca2+ sensitivity of I79N mutant fibers. AP differences were abolished when myocytes were dialyzed with Ca2+ buffers or after the Na+-Ca2+ exchanger was blocked by Li+. At higher pacing rates or in presence of isoproterenol, diastolic Ca2+ became significantly elevated in I79N-Tg compared with control myocytes. Ventricular ectopy could be induced by isoproterenol-challenge in isolated I79N-Tg hearts and anesthetized I79N-Tg mice. Freely moving I79N-Tg mice had a higher incidence of nonsustained ventricular tachycardia (VT) during mental stress (warm air jets). We conclude that the TnT-I79N mutation causes stress-induced VT even in absence of hypertrophy and/or fibrosis, arising possibly from the combination of AP remodeling related to altered Ca2+ transients and suppression of IK1.     

Effects of gender difference on cardiac myocyte dysfunction in streptozotocin-induced diabetic rats

The main characteristics of type 1 diabetic cardiomyopathy include depressed contractility and altered electrophysiological properties in ventricular myocytes. The goal of the present study was to determine the potential influence of gender in the diabetes-induced pathogenesis of ventricular myocyte function. Diabetes in both male and female rats was induced by a single intravenous injection of streptozotocin (STZ). Diabetic rats exhibited hyperglycemia and reduced body weight gain in both male and female groups. Neither contractile profiles nor activity of three types of K+ channels of ventricular myocytes was significantly different between nondiabetic male and female rats. Ventricular myocytes isolated from diabetic rats exhibited significant depression in cell contraction and relaxation, which was associated with depression of intracellular Ca2+ ([Ca2+]i) transient. The degrees of contractile depression were comparable in ventricular myocytes obtained from both male and female diabetic rats. Similarly, diabetes depressed three types of outward K+ currents (Ito, Ik, and Iss) to the same extent in both gender myocytes. These data demonstrate that in this animal model of diabetes, gender difference in cardiac myocyte functions was eliminated.     

Transient-outward K+ channel inhibition facilitates L-type Ca2+ current in heart

BACKGROUND: Transient outward current (I(to)) and L-type calcium current (I(Ca)) are important repolarization currents in cardiac myocytes. These two currents often undergo disease-related remodeling while other currents are spared, suggesting a functional coupling between them. Here, we investigated the effects of I(to) channel blockers, 4-aminopyridine (4-AP) and heteropodatoxin-2 (HpTx2), on I(Ca) in cardiac ventricular myocytes. METHODS AND RESULTS: I(Ca) was recorded in enzymatically dissociated mouse and guinea pig ventricular myocytes using the whole-cell voltage clamp method. In mouse ventricular myocytes, 4-AP (2 mM) significantly facilitated I(Ca) by increasing current amplitude and slowing inactivation. These effects were not voltage-dependent. Similar facilitating effects were seen when equimolar Ba2+ was substituted for external Ca2+, indicating that Ca2+ influx is not required. Measurements of Ca2+/calmodulin-dependent protein kinase (CaMKII) activity revealed significant increases in cells treated with 4-AP. Pretreatment of cells with 10 microM KN93, a specific inhibitor of CaMKII, abolished the effects of 4-AP on I(Ca.) To test the requirement of I(to), we studied guinea pig ventricular myocytes, which do not express I(to) channels. In these cells, 2 mM 4-AP had no effect on I(Ca) amplitude or kinetics. In both cell types, Ca2+-induced I(Ca) facilitation, a CaMKII-dependent process, was observed. However, 4-AP abolished Ca2+-induced I(Ca) facilitation exclusively in mouse ventricular myocytes. CONCLUSION: 4-AP, an I(to) blocker, facilitates L-type Ca2+ current through a mechanism involving the I(to) channel and CaMKII activation. These data indicate a functional association of I(Ca) and I(to) in cardiac myocytes.     

Electrophysiological properties of mouse bone marrow c-kit+ cells co-cultured onto neonatal cardiac myocytes

OBJECTIVE: Controversy about hematopoietic stem cells reprogramming into cardiac myocytes is currently supported by positive and negative findings. In fact, some reports have shown the ability of stem cells from the bone marrow (BM) to differentiate into cardiac myocytes and to contribute to myocardium repair, while others have reported the opposite. METHODS: C-kit(+) cells from mouse bone marrow were co-cultured onto neonatal cardiac myocytes. Hematopoietic stem cell-derived cells were analyzed by investigating the expression of cardiac markers and ion channels and by single-cell electrophysiological recordings. RESULTS: Groups of undifferentiated c-kit(+) cells displayed only outward currents. Co-cultured c-kit(+) stem cells on neonatal cardiac myocytes expressed cardiac markers and Na(+) and Ca(2+) voltage-gated ion channels. However, Na(+) and Ca(2+) currents were not detected by electrophysiological patch-clamp recordings even if caffeine and cyclopiazonic acid treatment showed the presence of intracellular calcium stores. This suggests that these channels, although expressed, were not functional and thus do not allow the coupling between excitation and contraction that is typical of cardiac myocytes. Nevertheless, co-cultured cells had a more hyperpolarized resting membrane potential and, at least in a subset of cells, displayed voltage-gated inward rectifier currents and outward currents. Co-cultured c-kit(+)-derived cells were not connected to surrounding cardiac myocytes through gap junctions. To induce a more pronounced differentiation, co-cultured cells were treated with BMP-4 and TGF-beta, two factors that were shown to trigger a cardiac myocyte differentiation pathway in embryonic stem (ES) cells. Even under these conditions, c-kit(+) cells did not differentiate into functionally active cardiac myocytes. However, TGF-beta/BMP-4-treated cells were hyperpolarized and showed and increased inward rectifier current density. CONCLUSIONS: Our study shows that mouse BM hematopoietic stem cells exhibit a limited plasticity to transdifferentiate into cardiac myocytes in culture.     

Neurally-mediated increase in calcineurin activity regulates cardiac contractile function in absence of hypertrophy

OBJECTIVE: The calcineurin pathway has been involved in the development of cardiac hypertrophy, yet it remains unknown whether calcineurin activity can be regulated in myocardium independently from hypertrophy and cardiac load. METHODS: To test that hypothesis, we measured calcineurin activity in a rat model of infrarenal aortic constriction (IR), which affects neurohormonal pathways without increasing cardiac afterload. RESULTS: In this model, there was no change in arterial pressure over the 4-week experimental period, and the left ventricle/body weight ratio did not increase. At 2 weeks after IR, calcineurin activity was increased 1.8-fold (P<0.05) and remained elevated at 4 weeks (1.7-fold, P<0.05). Similarly, the cardiac activity of calcium calmodulin kinase II (CaMKII) was increased significantly after IR, which confirms a regulation of Ca(2+)-dependent enzymes in this model. In cardiac myocytes, the increased activity of calcineurin was accompanied by a significant decrease in L-type Ca(2+) channel activity (I(Ca)) and contraction velocity (-dL/dt). Cardiac denervation prevented the activation of calcineurin after IR, which demonstrates that a neurohormonal mechanism is responsible for the changes in enzymatic activity. In addition, cardiac denervation suppressed the effects of IR on I(Ca) and -dL/dt, which shows that calcineurin activation is related to altered contractility. However, action potential duration, the densities of inward rectifier K(+) currents (I(K1)), and outward K(+) currents (I(to) and I(K)) were not altered in IR myocytes. CONCLUSIONS: Calcineurin can be activated in the heart through a neural stimulus, which induces alterations in Ca(2+) currents and contractility. These effects occur in the absence of myocyte hypertrophy, electrophysiological changes in action potential, and K(+) channel currents.     

Ionic currents that generate the spontaneous diastolic depolarization in individual cardiac pacemaker cells.

An enzymatic dispersion procedure has been developed to obtain viable, spontaneously active single myocytes from cardiac pacemaker tissue: the bullfrog (Rana catesbeiana) sinus venosus. Recordings of time- and voltage-dependent Ca2+ and K+ currents have been made by using a single suction-microelectrode technique. The results show that two time- and voltage-dependent currents interact to modulate the slope of the pacemaker potential. These are: (i) the decay of a delayed rectifier K+ current and (ii) the activation of a Ca2+ current. In addition, the data strongly suggest that cardiac pacemaker tissue does not have an inwardly rectifying background K+ current.     

Enhanced Ca2+ channel currents in cardiac hypertrophy induced by activation of calcineurin-dependent pathway

Cardiac-specific expression of an activated calcineurin protein in the hearts of transgenic (CLN) mice produces a profound hypertrophy that rapidly progresses to heart failure. While calcineurin is regulated by Ca2+, the potential effects of calcineurin on cardiac myocyte Ca2+ handling has not been evaluated. To this end, we examined L-type Ca2+ currents (I(Ca)) in left ventricular myocytes. CLN myocytes had larger (approximately 80%) cell capacitance and enhanced I(Ca) density (approximately 20%) compared with non-transgenic (NTG) littermates, but no change in the current-voltage relationship, single-channel conductance or protein levels of alpha 1 or beta 2 subunit of L-type Ca2+ channels. Interestingly, the kinetics of I(Ca) inactivation was faster (approximately two-fold) in CLN myocytes compared with NTG myocytes. Ryanodine application slowed the rate of I(Ca) inactivation in both groups and abolished the kinetic difference, suggesting that Ca2+ dependent inactivation is increased in CLN myocytes due to altered SR Ca2+ release. Treatment of CLN mice with Cyclosporine A (CsA), a calcineurin inhibitor, prevented myocyte hypertrophy and changes in I(Ca) activity and inactivation kinetics. However, there was no direct effect of CsA on I(Ca) in either NTG or CLN myocytes, suggesting that endogenous calcineurin activity does not directly regulate Ca2+ channel activity. This interpretation is consistent with the observation that I(Ca) density, inactivation kinetics and regulation by isoproterenol were normal in cardiac-specific transgenic mice expressing calcineurin inhibitory protein domains from either Cain or AKAP79. Taken together these data suggest that chronic activation of calcineurin is associated with myocyte hypertrophy and a secondary enhancement of intracellular Ca2+ handling that is tied to the hypertrophy response itself.     

Glucocorticoid regulation of cardiac K+ currents and L-type Ca2+ current in neonatal mice.

Previous studies have reported that dexamethasone (Dex) prolongs cardiac action potential repolarization in mice and rats. However, the cellular mechanisms of this effect have not been addressed. Because action potential duration is influenced by a complex interplay of both inward and outward currents, this study evaluated the role of K+ currents and the L-type Ca2+ current in response to chronic in vivo Dex treatment. Accordingly, neonatal mice were randomly allocated to treatment with Dex (1 mg/kg per day) or placebo (saline) given subcutaneously for 5 days. At 14 to 15 days of age, the L-type Ca2+ current and K+ currents were recorded in ventricular myocytes using whole-cell patch-clamp techniques. The density of peak outward K+ currents was significantly decreased in the chronic Dex-treated group, but the current measured at the end of a 1-second depolarization pulse was similar in both groups. We further measured the magnitudes of the fast-inactivating (I(to)) and the slowly inactivating (I(slow)) currents that contribute to the peak outward K+ currents. I(to) was reduced from 17.5+/-3.0 pA/pF (control) to 10.6+/-2.5 pA/pF (Dex) at +50 mV (P<0.05), but I(slow) was not significantly different. These data suggest that downregulation of I(to) is responsible for the reduced peak outward current. Time courses of the onset and offset of in vivo Dex effects were also assessed. A period of 3 days of treatment was required to observe the Dex effect on peak outward K(+) currents, whereas a 7-day period after discontinuation of Dex was required to recover the baseline current density. Acute in vitro treatment with Dex (1 micromol/L) had no effect on K+ current densities. In addition, chronic Dex treatment significantly increased the density of the L-type Ca2+ current (I(Ca-L)) from -7.2+/-0.5 pA/pF of control to -8.9+/-0.6 pA/pF of Dex at +10 mV, P<0.05. In conclusion, chronic in vivo Dex treatment decreases I(to) and increases I(Ca-L) in neonatal mouse ventricular myocytes, both of which contribute to the prolongation of cardiac action potential repolarization induced by glucocorticoids.     

Increase in T-type calcium current in atrial myocytes from adult rats with growth hormone-secreting tumors.

Growth hormone (GH) has pronounced effects on protein synthesis and cell growth in cardiac muscle from adult animals, although the mechanism of its action is not understood. Because Ca2+ has been implicated as a regulator of mitogenic processes in a number of tissues, we investigated whether GH affects the transmembrane movement of Ca2+ through voltage-activated channels of cardiac myocytes. Atrial and ventricular myocytes were isolated from adult rats with GH-secreting tumors and studied electrophysiologically by using patch-clamp techniques. Tumor-bearing rats re-enter an active growth phase and double their body weight over age-matched controls 8 weeks after introduction of the tumor. Atrial myocytes from tumor-bearing animals showed a 3-fold increase in the density of T-type Ca2+ current compared with cells from control animals, although the voltage dependency of activation and inactivation of T-type current was not altered. The increase in T-current density of atrial myocytes preceded by at least a week any measurable change in heart weight, body weight, or myocyte size. L-type Ca2+ currents in atrial and ventricular cells were not affected. The results suggest that a tumor-derived growth factor, most likely GH, can cause a specific enhancement of T-type Ca2+ current in atrial myocytes.     

Gender-based differences in cardiac repolarization in mouse ventricle

The mouse heart has become a widely used model for genetic studies of heart diseases. Thus, understanding gender differences in mouse cardiac repolarization is crucial to the interpretation of such studies. The objective of this study was to evaluate whether there are gender differences in cardiac repolarization in mouse ventricle and to gain insights into the ionic and molecular mechanisms underlying these differences. Action potential durations (APDs) and K(+) currents in male and female ventricular myocytes were compared using a patch-clamp technique. APD(20), APD(50), and APD(90) were found to be significantly longer in females than males. Examination of the different K(+) currents revealed that a significantly lower current density exists in female ventricular myocytes compared with male myocytes for the ultrarapid delayed rectifier K(+) current, I(Kur) (at +30 mV, male, 33.2+/-2.9 pA/pF [n= 22]; female, 20.9+/-1.73 pA/pF [n= 19], P<0.001). Consistent with these findings were the results of the ribonuclease protection assay, Western blots, and confocal analysis that showed a significantly lower expression level of Kv1.5 (coding for I(Kur)) in female compared with male ventricle. The additional K(+) currents present in mouse ventricle exhibited no gender differences. In agreement with these electrophysiological data, no differences in the expression levels for the K(+) channels underlying these currents were detected between both sexes. This study demonstrates that adult mice exhibit gender differences in cardiac repolarization. The expression of Kv1.5 and of its corresponding K(+) current, I(Kur), is significantly lower in female mouse ventricle, and as a result, the APD is lengthened.