Name: murine short axis ventricular heart slices for electrophysiological studies
Uploaded: 2017-06-04T14:00:00.000+00:00
Duration: 7 min 51 s
Description: Watch this Scientific Journal Video about Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies at JoVE.com

我们承认研讨会和神经生理学研究所的动物设施提供的支持。这项工作得到了Walter und Marga Boll-Stiftung，KölnFortune和Dezscher Stfung Herzforschung的支持。

动物处理必须符合当地动物福利委员会的指导方针和欧盟议会的2010/63 / EU指令。 准备解决方案<ol><li>制备没有Ca 2+ （组成为mM）的Tyrode溶液:NaCl 136，KCl 5.4，NaH 2 PO 4 0.33，MgCl 2 1，葡萄糖10，HEPES 5,2,3-丁二酮单肟（BDM）30。将pH调节至7.4用NaOH在4℃。 </li><li>用Ca 2+ （组成为mM）制备Tyrode溶液:NaCl 136，KCl 5.4，NaH 2 PO 4 0.33，MgCl 2 ，葡萄糖10，HEPES 5，BDM 30，CaCl 2 0.9。在4℃用NaOH调节pH至7.4。 </li><li>准备4％低熔点琼脂糖:将0.6 g低熔点琼脂糖置于15 mL Tyrode溶液中，不含Ca 2+ 。将混合物在微波炉中以750W加热10次，直到琼脂糖溶解。将溶液保持在37℃的恒定温度，＃176; C并连续搅拌。 </li><li>在37℃下保持不含血清的Dulbecco&#39;s改良的Eagle培养基（DMEM），用碳霉素（5％CO 2，95％O 2 ）鼓泡。 </li></ol> 2.准备切片机<ol><li>打开切片机。 </li><li>用冰将外切片机室填满。 </li><li>用冰冷的Tyrode溶液填充内切片机室，不含Ca 2+ ，并持续用100％O 2使溶液充氧。 </li><li>将钢刀片放入切片机刀片架上。 </li></ol> 3.小鼠心脏隔离<ol><li>皮下注射2500 U肝素。等待15分钟</li><li>通过颈椎脱位牺牲动物。 </li><li>通过胸骨切开术打开胸部。 </li><li>用小剪刀和镊子＃5仔细分析心包。 </li><li>将插管插入升主动脉，并用冰冷的Tyrode&#39;s 原位灌注冠状动脉不含Ca 2+的溶液，直到除去剩余的血液。 </li><li>用镊子和剪刀轻轻切除心脏，将冰块转移到冰冷的Tyrode溶液中，不含Ca 2+ 。 </li><li>用手术刀或剪刀将心房与心室分开。 </li></ol> 4.将心室嵌入4％低熔点琼脂糖中<ol><li>将顶点面朝上放置在琼脂糖模具中（ 图1 ）。将引脚放在模具中间的左心室。 </li><li>在37℃下用4％低熔点琼脂糖填充模具，直到心脏完全被覆盖。 </li><li>将模具放在冰上，以更快地硬化琼脂糖，防止组织漂浮。 </li><li>用手术刀从模具中取出含有心室的琼脂糖块。 </li><li>将块颠倒倒置并充满心室和琼脂糖块背面的间隙，由蜕皮的针留下，用4％的低熔点琼脂糖，使用20 G针的注射器。 </li><li>用手术刀修剪琼脂糖块，实现块的平坦底部和心脏顶点的直立位置。 </li></ol> 5.切片心脏组织<ol><li>用一滴氰基丙烯酸酯胶将块固定在切片机的样品架上。面向心脏顶点向上。 </li><li>将样品架放入切片机的内部样品室中，该样品室装满冰冷的Tyrode，不含Ca 2+ 。用Tyrode的解决方案完全覆盖琼脂糖块。 </li><li>根据进一步的应用（锋利的电极记录150-200μm）准备厚度为150-400μm的短轴切片，将刀片的振动频率保持在60-70 Hz，并将刀片向前移动尽可能慢。 </li><li>用细毛刷小心地从切片中取出剩余的琼脂糖。 </li><li>轻轻地输r片用巴斯德吸管浸入含有100％O 2的 0.9mM Ca 2+的Tyrode溶液中，并在冰上储存至少30分钟，以从切片程序中回收。 </li><li>然后，在37℃的DMEM中保持切片30分钟，用碳水充气，以便进一步使用之前洗去BDM。 </li></ol> 6.准备夏普电极设置<ol><li>在预热之前，在录制开始前30分钟打开所有电器。 </li><li>将3厘米的细胞培养皿放在放置在倒置显微镜上的加热板上。 </li><li>将定制的环形电极（ 图2 ）放入盘中，并连接前置放大器和刺激电极的接地线。 </li><li>将灌注系统的柔性管连接到盘上。 </li><li>用DMEM填充灌注系统的储存器，用碳水充气。 </li><li>打开灌注泵并设置灌注量te至2-3 mL / min。 </li><li>通过调节流量加热器和加热板将盘中DMEM的温度调节至37°C。 </li></ol> 7.行动潜在记录<ol><li>将心室切片放入装有DMEM的盘中。 </li><li>用倒置显微镜检查组织的结构完整性和活力（基于收缩功能）。 </li><li>用3 M KCL填充记录玻璃电极。 </li><li>用DMEM填充刺激电极。 </li><li>将记录电极和刺激电极放在电极支架上。 </li><li>将刺激电极仔细放在切片上并打开电刺激器。以1-2赫兹的刺激频率开始。 </li><li>将记录电极用显微操纵器移动到预期的记录位置。 </li><li>缓慢降低记录电极直到尖端接触组织。 </li><li>应用短矩形电脉冲记录电极穿透细胞膜。 </li><li>仔细重新定位记录电极，直到确保稳定的信号。 </li><li>开始记录动作电位。 </li></ol>

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心室切片使电生理，药理和机械研究具有体内保留的组织结构，并可将测量技术直接进入心脏的所有区域。已经在胚胎，新生儿和成人切片9,10,11中证明了生理作用潜力。生物活性染色证实切片的活力除了切片程序直接损伤的表层外，其余的活性染色9 。使用尖锐玻璃电极的切片内的动作电位记录可用于研究如本文所述的移植心肌细胞的整合和成熟，或加入心脏活性?...

由于Yamamot和Mcllwain于1966年在体外证实了脑片的电活动，所以在基础科学中经常使用薄片切片1 。此后，从脑2 ，肝3 ，肺4和心肌组织5,6,7的切片进行了电生理和药理学研究。 1990年8月描述了来自新生大鼠心脏的心室切片中的第一次膜片钳记录，但是这种技术被遗忘了一段时间。十多年后，我们组织制定了一种新的方法来准备鼠胚胎9 ，新生儿10和成人11心脏切片。这些活组织切片可用于急性实验（成人切片）s可以培养数小时）或短期培养实验（胚胎和新生儿切片可以培养几天）。切片在体内显示如电生理特征和均匀的激发扩散，如通过尖锐电极动作电位和微电极阵列记录所评估的。由于其"二维"形态，它们允许记录电极直接进入心室的所有区域，这使得它们成为电生理调查的有趣工具，并且与Langendorff灌注的整个心脏相比提出了新的实验选择。离子通道阻滞剂如维拉帕米（L型Ca 2+通道阻断剂），利多卡因（Na +通道阻断剂），4-氨基吡啶（非选择性电压依赖性K +通道阻滞剂）和亚麻酸（KCNQ K +通道阻塞） 9，11 </sup&gt;对应于已解离的心肌细胞的已知作用。等轴力测量显示正的力频率关系，强烈建议完整的收缩功能10 。这些研究结果表明，鼠心室切片适合作为生理和药理学研究的体外组织模型。此外，受体心脏的心室切片与尖锐的电极记录结合已经被证明是一个非常有用的工具，用于表征移植胎儿12,13,14和干细胞衍生的15个心肌细胞的电和机械整合以及成熟。 总之，心室切片是一种有价值且成熟的多细胞组织模型，应被认为与分离的心肌细胞和Langendorff灌注心脏互补在心血管研究中，提供体内类似组织结构（与分离的细胞相反）的主要优点，以及直接访问诸如尖锐电极记录到心脏的所有区域的测量技术（与全心脏制剂相反）。

<table><tbody><tr><td>Leica VT 1000s</td><td>Leica Microsystems, Wetzlar, Germany</td><td></td><td>Microtome with vibrating blade.</td></tr><tr><td>Stainless Steel Blades</td><td>Campden Instruments, Loughborough, England</td><td>7550-1-SS</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Sigma-Aldrich, St. Louise, USA</td><td>Z627992</td><td></td></tr><tr><td>Fine brush, &lt;em&gt;e.g.&lt;/em&gt; size 6 (4/32&quot;)</td><td>VWR, International, Radnor, USA</td><td>149-2125</td><td></td></tr><tr><td>Preparation table</td><td></td><td></td><td>self made</td></tr><tr><td>Molt for embedding ventricles in agarose</td><td></td><td></td><td>self made</td></tr><tr><td>1 mL Syringe</td><td>Becton, Dickinson; Franklin Lakes, USA</td><td>300013</td><td></td></tr><tr><td>27 G x 3/4`` Needles</td><td>Braun, Melsungen, Germany</td><td>4657705</td><td></td></tr><tr><td>20 G 11/2`` Needles</td><td>4657519</td><td></td><td></td></tr><tr><td>Small scissor</td><td>WPI, Sarasota, USA</td><td>501263</td><td></td></tr><tr><td>Tweezers #5, 0.1 x 0.06 mm tip</td><td>WPI, Sarasota, USA</td><td>500342</td><td></td></tr><tr><td>Oxygen gas (medical grade O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Linde, Munich, Germany</td><td></td><td></td></tr><tr><td>Carbogen gas (95 % O&lt;sub&gt;2&lt;/sub&gt;, 5 % CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Linde, Munich, Germany</td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich, St. Louise, USA</td><td>7647-14-5</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich, St. Louise, USA</td><td>746436</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>746495</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>NIST200B</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, St. Louise, USA</td><td>51558</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>S5761</td><td></td></tr><tr><td>D(+)-Glucose</td><td>Sigma-Aldrich, St. Louise, USA</td><td>G8270</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>M7506</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich, St. Louise, USA</td><td>S8045</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Henkel, D&amp;uuml;sseldorf, Germany</td><td></td><td></td></tr><tr><td>Low-melt Agarose</td><td>Roth, Karlsruhe, Germany</td><td>6351.2</td><td></td></tr><tr><td>Heparin-sodium-25000 I.E./5 mL</td><td>Ratiopharm, Ulm, Germany</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX</td><td>ThermoScientific, Waltham, USA</td><td>10566016</td><td></td></tr><tr><td>SEC-10LX Amplifier</td><td>npi electronic GmbH, Tamm, Germany</td><td>SEC-10LX</td><td></td></tr><tr><td>EPC 9</td><td>HEKA Elektronik GmbH, Lambrecht, Germany</td><td></td><td></td></tr><tr><td>Zeiss Axiovert 200</td><td>Zeiss, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>Low magnification Micromanipulator</td><td>Narashige, Tokyo, Japan</td><td>Nm-3</td><td></td></tr><tr><td>High magnification, three-axis micromanipulator</td><td>Narashige, Tokyo, Japan</td><td>MHW-3</td><td></td></tr><tr><td>Peristaltic perfusion pump</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>PPS2</td><td></td></tr><tr><td>2-channel temperature controller</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>TCO02</td><td></td></tr><tr><td>Square pulse stimulator</td><td>Natus Europe GmbH, Planegg, Germany</td><td>Grass SD9</td><td></td></tr><tr><td>Glass capillaries</td><td>WPI, Sarasota, USA</td><td>1B150F-1</td><td></td></tr></tbody></table>

murine short axis ventricular heart slices for electrophysiological studies

鼠心肌细胞已广泛用于心脏生理学和新治疗策略的体外研究。然而，解离的心肌细胞的多细胞制剂并不代表心肌细胞，非肌细胞和细胞外基质的复合体内结构，其影响心脏的机械和电生理学性质。在这里，我们描述了一种技术，用于制备具有保留的体内类似组织结构的成年小鼠心脏的活动性心室切片，并且证明其适用于电生理记录。切除心脏后，将心室与心房分离，用含有2,3-丁二酮一肟的无Ca 2+溶液灌注，并包埋在4％ 低熔点琼脂糖块中。将块放置在具有振动叶片的切片机上，并且制备厚度为150-400μm的组织切片，保持振动频率60-70 Hz的刀片速度，并尽可能缓慢地向前移动刀片。切片的厚度取决于进一步的应用。切片储存在冰冷的Tyrode溶液中，用0.9mM Ca 2+和2,3-丁二酮一肟（BDM）处理30分钟。之后，将切片转移到37℃的DMEM中30分钟以洗出BDM。切片可用于具有尖锐电极或微电极阵列的电生理研究，用于力测量分析收缩功能或调查移植的干细胞衍生的心肌细胞和宿主组织的相互作用。对于尖锐的电极记录，将切片置于倒置显微镜的加热板上的3cm细胞培养皿中。用单极电极刺激切片，用锋利的玻璃电极记录切片内心肌细胞的细胞内动作电位。

心肌梗死导致心肌细胞几乎不可逆的损失。使用干细胞衍生的心肌细胞进行外源性心脏再生的细胞替代疗法是一种有希望的治疗方法。移植细胞的电气整合和成熟对细胞替代疗法的安全性和效率至关重要。 为了评估整合和成熟，我们将来自表达增强的绿色荧光蛋白（eGFP）的诱导多能干细胞（iPSCM; 2次注射0.5×10 6 iPSCM /1...

Watch this Scientific Journal Video about Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies at JoVE.com

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

在这里，我们描述了成年小鼠可行心室切片的制备及其对尖锐电极动作电位记录的应用。这些多细胞制剂提供了保留在体内的类似组织结构，这使得它们成为体外电生理和药理学研究的有价值的模型。

鼠短轴心脏心脏切片用于电生理研究

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular ...

murine-short-axis-ventricular-heart-slices-for-electrophysiological

Research

JoVE Journal

Medicine

7.3K 次观看.  University of Cologne. 在这里，我们描述了成年小鼠可行心室切片的制备及其对尖锐电极动作电位记录的应用。这些多细胞制剂提供了保留在体内的类似组织结构，这使得它们成为体外电生理和药理学研究的有价值的模型。 

视频: Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

科研

生物工程

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

分离的月毛心室肌细胞的光学成像

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

高分辨率光学制图对小鼠心房电生理的评价

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

医学

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

生物学

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

前维沃·兰根多夫注入心脏的心电生理学研究

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

微电极阵列记录中天节点发射率，以识别小鼠内在心脏起搏缺陷

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

人体心室心肌细胞与颤音-切切心肌切片分离

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Generation of Porcine Ventricular Slices: A Vibratome-based Technique to Prepare Ultra-thin Slices of Pig Heart Tissue

Source: Ou, Q. et. al., <a href="https://www.jove.com/v/60913">Slicing and Culturing Pig Hearts under Physiological Conditions.</a> J. Vis. Exp. (2020)
This video demonstrates a vibratome-based procedure to generate viable, ultra-thin sections of pig heart. The prepared tissue slices can be cultured and used as model systems for pharmacological testing.

猪脑室切片的生成:一种基于振动切片机的技术，用于制备猪心脏组织的超薄切片

Source: Ou, Q. et. al., Slicing and Culturing Pig Hearts under Physiological Conditions. J. Vis. Exp. (2020)
This video demonstrates a vibratome-based ...