We are supported by the University of Wisconsin-Madison Medical School start-up (A.V.G.).

所有实验均按照健康指南实验动物的护理和使用国家研究院（NIH酒吧。80-23号）进行。在这些研究中使用的所有方法和协议已通过威斯康星动物护理和使用协议委员会大学继美国国立卫生研究院公布了关怀和实验动物的使用指南（出版号85-23，1996年修订）。在这项研究中使用的所有动物人文关怀遵照指南实验动物的护理和使用。 1.心脏拆卸和灌注的Langendorff <ol><li>之前心脏隔离，温暖蒂罗德溶液至37℃，通过使用水浴和一个水套。 </li><li>麻醉用5％异氟醚/ 95％O 2鼠标。 </li><li>通过检查疼痛反射的损失确保麻醉适当的水平。 </li><li>执行开胸手术。通过采用弧形5.5"梅奥剪刀和5.5"凯利止血为打开胸腔牛肝菌使1厘米上切割胸廓的前面。 <ol><li>迅速从胸部（内30秒）中提取的心脏。用4个"弯钳光圈抢肺组织及使用4.3心包切出的肺，胸腺和心脏一起"IRIS剪刀。不要直接抢心脏。 </li><li>在充氧（95％O 2/5％CO 2）清洗，恒定温度（36.8±0.4℃）改性台氏液。使用下列溶液组成（以mM计）:128.2氯化钠，4.7氯化钾，1.19的NaH 2 PO 4，1.05 氯化镁 ，1.3氯化钙2，20.0 碳酸氢钠 ，和11.1葡萄糖（pH为7.35±0.05）。 </li></ol></li><li>而沐浴在同一溶液中，识别肺，胸腺，和脂肪组织。小心地从心脏解剖他们。用弯3"Vannas，蒂宾根剪刀和4.3"＃5镊子。 </li><li>然后确定主动脉导管插入它放到一个特制的21g的套管。使用两个弯曲的4.3"＃5B力PS。 </li><li>插管后，同时灌注（通过在〜5毫升/分钟的流速套管;这应该基于主动脉的压力进行调整，见下文）和superfuse（在浴中以〜50毫升/分钟的流速）与在整个实验温热并过滤台罗德氏溶液的心脏。通过在管线11微米尼龙过滤器通过将灌注溶液，以防止冠脉循环的堵塞。 </li><li>使用通过3路阀鲁尔锁连接到灌注线路连接的压力换能器（或压力计），监测动脉压和维护60和80毫米汞柱之间。如果需要的话，保持在此范围内的主动脉压力调节灌注速度。 </li></ol>从的Langendorff灌流整体心脏的SAN 2.光学标测<ol><li> 仪器仪表 <ol><li>水平放置的心脏和管脚使用0.1毫米直径销为p心室心尖到腔室中的硅涂覆底r事件流引起的在实验过程中的运动。12 </li><li>通过肺静脉中插入一个小（0.012"内径×0.005"），硅管进入左心室（LV），左心房（LA）和二尖瓣（MV）。用丝线缝合（4-0），以探测结缔组织修复管。 </li><li>的心脏，其后侧朝上（ 图1A， 左图 ）位置。 </li><li>针对RA附属物（RAA）使用0.1毫米直径minutien销腔的硅底部的边缘。调整它的电平，以使所述RA平坦的后表面，并允许它被定位在相机的焦平面上。这将使从心房的最大表面面积光学测量。 </li><li>绞索优越（SVC）和下（IVC）腔静脉通过丝线缝合（4-0），拉伸和缝合线的另一端的引脚到硅涂覆室（ 见图3A）的底部。确保缝线不会阻挡视光学领域。 </li><li>放置定做起搏电极上的RAA的边缘。为了使电极，利用涂硅0.25毫米直径银线，用0.5毫米的电极间距离和缺乏硅在起搏端一个1毫米的长度。然后将两个心电图12毫米针（29号）的左，右心室基地附近单极电极。放置心室的顶点附近的地面ECG电极。 </li></ol></li><li> 染色 <ol><li>因为这两种荧光染料和机电解偶联剂II型肌球蛋白是光敏感，执行在黑暗的房间下面描述的所有过程。首先准备电压敏感染料RH-237或二-4- ANNEPS作为原液，1.25毫克/毫升的二甲亚砜（2.5毫摩尔），等分试样是（每次30微升）和该等分试样储存在-20℃。 </li><li>稀释5-10微升染料储备溶液在1ml温热（37℃）的Tyrode溶液中，然后将其注入到冠状动脉灌注线在使用在线路厄注入口一段5-7分钟。 </li><li>制备II型肌球蛋白作为预先原液（2毫克/毫升的二甲亚砜，6.8毫摩尔），并将其存储在4℃。 </li><li>稳定20分钟后，在灌注液加入0.5毫升温热（37℃）II型肌球蛋白和稀0.1毫升II型肌球蛋白在1ml温热（37℃）的Tyrode溶液。然后注入到冠状动脉灌注线多使用一个直列鲁尔注射口一段5-7分钟。 </li></ol></li></ol>从隔离心房制备的SAN 3.光学测绘<ol><li> 仪器仪表 <ol><li>对于孤立的心房准备，隔离并在全心脏的准备步骤1.1-1.5上述导管插入心脏。 </li><li>解剖心室来自前侧的路程。参见图1A， 右图 ， 削减细节＃1。 </li><li>通过三尖瓣VA切开RALVE（电视）沿着所述电视-SVC轴。参见图1A， 右图 ， 切＃2的细节。 </li><li>切嵴的四肢内侧打开RAA。参见图1A， 右图， 切＃3。 </li><li>通过执行从先前切割＃3的中线切口的RAA的右下角的边缘打开前心房游离壁，压平及针心房游离壁到硅涂覆室的底部。见由图1A中的空心箭头所示的方向， 切＃4。保留心室组织的周缘为钉扎的制备以防止心房损伤。 </li><li>同样，开放LA通过的MV沿左心耳（左心耳）的MV-上角切割。 </li><li>要打开左心耳，从打开的左心耳中间切开，经过前心房游离壁，直到左心耳的中间篮下。 </li><li>打开心房前壁游离壁ALO纳克相同的方向，然后压平，并将其管脚到的Sylgard涂覆室的底部。 </li><li>部分除去房间隔组织。这将减少来自组织的光信号不在焦点的散射。因此，无论是对LA和RA以及SAN和房室结（AVJ）是在此制备（ 图1B）进行访问。 </li><li>抬起由从硅涂覆腔室的底部约0.5mm的最终制剂中，以便允许从两个心外膜和心内膜表面灌流。 </li><li> Superfuse用温热的制备（37℃）的Tyrode溶液在〜12毫升/分钟临近SVC聚焦流的恒定速率和〜要浴表面灌流为30 mL /分钟。 </li><li>放置在解剖RAA边缘的定做起搏的Ag / AgCl 2电极（直径0.25mm）的。然后将两个心电图12毫米针（29G）的电极（单极）的RAA和LAA附近，分别为。将地面心电图选乘坐附近的AVJ。 </li></ol></li><li> 染色 <ol><li>进行电压敏感染料（RH-237或二-4- ANNEPS）的直接应用。对于这一点，稀释1-2微升染料原液在温热（37℃）的Tyrode溶液为1毫米，并使用1毫米移液器缓慢释放制剂的表面上的稀释染料。 </li><li>可替代地，通过类似于上述用于全心脏制剂（步骤2.2.1-2.2.2）中描述的的Langendorff灌注心脏的冠状动脉灌注执行与电压敏感染料心房染色。 <ol><li>的冠状动脉灌注染色后，如所描​​述隔离心房制备。需要达到一个满意的荧光水平时执行额外的表面染色。连结心房隔离不漂白的染料，并且不影响染色的质量。 </li></ol></li><li>固定用机电解偶联剂，II型肌球蛋白的制备方法。稀释3-5微升BLEbbistatin在温热（37℃）的Tyrode溶液原液和缓慢释放制剂的表面和周围溶液的稀释II型肌球蛋白。由于II型肌球蛋白已被证明是怕光，13,14避免长时间暴露在光线下的准备和染色过程中。 </li><li>执行额外的II型肌球蛋白的应用程序多达需要抑制收缩。通常最多3个附加的应用程序（每个应用3-5微升）可用于充分地抑制以精确地重建SAN和心房激动的运动伪影。 </li><li>添加其他0.5毫升回暖II型肌球蛋白在灌注液。在此过程中，暂停灌流30秒，以允许染料/ II型肌球蛋白染色的心房组织。 </li></ol></li></ol> 4.光映射设置注:在其他地方提供的光学标测系统的详细描述12 <ol><li>使用相机的TEM2000帧/秒或更高，和一系列聚光透镜的poral分辨率达到100微米/像素或更高的空间分辨率。这是必需的重建的SAN活化和结内传播。 </li><li>以减少从振动溶液运动伪影，修复溶液在心脏/分离的心房制备的表面上的小玻璃盖。 </li><li>使用由恒流，低噪声卤素灯提供激发光（520±45纳米的波长）。通过使用柔性分叉导光引导过滤的光束到制剂。 </li><li>通过长通滤波器（&gt; 715纳米）过滤所发出的荧光。收集，数字化和保存在相机制造商提供的使用计算机软件获得的荧光信号。 </li></ol> 5.数据处理注意:光学测绘数据被收集并作为在不同时间点的一系列荧光强度矩阵的存储。每个像素represeNTS在特定的时间点从在组织上制备的特定位置收集的荧光强度的量度。背景荧光是每个像素自动移除，以允许通过膜电压的变化产生并反转为与由微电极系统测量动作电位（AP）的对应的荧光变化更好的可视化。在聚焦审查被提供在处理的光学成像数据，包括图像分割，空间滤波，时间滤波，和基线漂移除去，的不同步骤的信息。15 <ol><li>手动或通过使用特殊的算法（例如，阈值或边缘检测）15，以确定组织分界，创建的1（包括像素）和0（排除像素）的二进制掩模和掩模应用于序列中的每个帧。此过程将创建一个二进制图像，突出的进行进一步的处理感兴趣的领域。 </li><li>为了提高选择的信噪比的iCal数据，感兴趣的选定区域内过滤信号。对于这一点，使用空间（ 即由所需的卷积箱或内核中定义相邻的荧光象素的平均，例如3×3或，为噪声数据，5×5）和/或时间滤波（例如，巴特沃思，切比雪夫型1，切比雪夫型2，椭圆等 ）（ 图2）。请记住过滤诱发文物的可能性，解释数据的时候。有关详细信息，请参阅审查。15 </li><li>在需要的时候在光学记录除去基线漂移（通过使用高通滤波或多项式拟合到原始信号），然后从0（最小荧光）到1（最大荧光）正常化的每个像素的信号。 </li><li>选择，它包括一个单一的AP传播的所有像素的激活时间的时间窗口。其激活时间指定每个像素的最大上行程衍生物的时间（DF / dt 最大值 ，其中F是fluorescenCE强度）。使用所有像素的激活时间，重建等时激活图。因此，每个等时会显示在同一时间激活的像素。 </li><li>以重构的AP的持续时间（APD）分布图，对每一个像素计算的激活时间和时间之间的持续时间在复极的特定级别（例如，在复极化，APD 80的80％）。使用所有的像素值APD，重建APD分布等时的地图。因此，每个等时会显示具有相同的APD的像素。 </li><li>要计算AP传播传导速度，适合准备在5.4计算的激活时间数据的表面。对于这一点，使用多项式曲面拟合或本地内核表面平坦化，然后从嵌合面的梯度计算局部传导速度矢量。 </li><li>以创建复极图，其复极时间分配的每个像素，定义为最大二阶导数（D 2 F / dt的2）在OAP，或90％复极化的时间结束时，光信号的。 </li></ol>

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<li>Verheijck, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+features+of+the+mouse+sinoatrial+node+in+relation+to+connexin+distribution%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.</a> Cardiovasc Res. 52 (1), 40-50 (2001).</li>
<li>Krishnaswamy, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+parasympathetic+nervous+system+regulation+of+the+sinoatrial+node+in+Akita+diabetic+mice%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">Altered parasympathetic nervous system regulation of the sinoatrial node in Akita diabetic mice.</a> J Mol Cell Cardiol. 82, 125-135 (2015).</li>
<li>Nygren, A., Lomax, A. E., Giles, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneity+of+action+potential+durations+in+isolated+mouse+left+and+right+atria+recorded+using+voltage-sensitive+dye+mapping%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">Heterogeneity of action potential durations in isolated mouse left and right atria recorded using voltage-sensitive dye mapping.</a> Am J Physiol Heart Circ Physiol. 287 (6), H2634-H2643 (2004).</li>
<li>Efimov, I. R., Fedorov, V. V., Joung, B., Lin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+cardiac+pacemaker+circuits%3A+methodological+puzzles+of+the+sinoatrial+node+optical+mapping%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Mapping cardiac pacemaker circuits: methodological puzzles of the sinoatrial node optical mapping.</a> Circ Res. 106 (2), 255-271 (2010).</li>
<li>Aschar-Sobbi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+atrial+arrhythmia+susceptibility+induced+by+intense+endurance+exercise+in+mice+requires+TNFalpha%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Increased atrial arrhythmia susceptibility induced by intense endurance exercise in mice requires TNFalpha.</a> Nat Commun. 6, 6018(2015).</li>
<li>Hansen, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atrial+fibrillation+driven+by+micro-anatomic+intramural+re-entry+revealed+by+simultaneous+sub-epicardial+and+sub-endocardial+optical+mapping+in+explanted+human+hearts%5BTitle%5D)+AND+%22Eur+Heart+J%22%5BJournal%5D)">Atrial fibrillation driven by micro-anatomic intramural re-entry revealed by simultaneous sub-epicardial and sub-endocardial optical mapping in explanted human hearts.</a> Eur Heart J. 36 (35), 2390-2401 (2015).</li>
<li>Lang, D., Petrov, V., Lou, Q., Osipov, G., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+control+of+heart+rate+in+a+rabbit+heart%5BTitle%5D)+AND+%22J+Electrocardiol%22%5BJournal%5D)">Spatiotemporal control of heart rate in a rabbit heart.</a> J Electrocardiol. 44 (6), 626-634 (2011).</li>
<li>Kanlop, N., Sakai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+mapping+study+of+blebbistatin-induced+chaotic+electrical+activities+in+isolated+rat+atrium+preparations%5BTitle%5D)+AND+%22J+Physiol+Sci%22%5BJournal%5D)">Optical mapping study of blebbistatin-induced chaotic electrical activities in isolated rat atrium preparations.</a> J Physiol Sci. 60 (2), 109-117 (2010).</li>
<li>Glukhov, A. V., Flagg, T. P., Fedorov, V. V., Efimov, I. R., Nichols, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+K%28ATP%29+channel+pharmacology+in+intact+mouse+heart%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">Differential K(ATP) channel pharmacology in intact mouse heart.</a> J Mol Cell Cardiol. 48 (1), 152-160 (2010).</li>
<li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+sinoatrial+node+function+and+intra-atrial+conduction+in+murine+gain-of-function+Scn5a%2B%2FDeltaKPQ+hearts+suggest+an+overlap+syndrome%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/DeltaKPQ hearts suggest an overlap syndrome.</a> Am J Physiol Heart Circ Physiol. 302 (7), H1510-H1523 (2012).</li>
</ol>

No conflicts of interest are declared.

在这里，我们提出了两种类型的鼠标SAN准备工作:1）完整的SAN中的Langendorff灌注整个心脏，和2）SAN中分离出来的，开房准备。这两种类型的准备服务于不同的实验目的。在的Langendorff灌注整个心脏制备方法中，完整的心房结构被保留，这使得它能够在折返性心动过速研究复杂房性心律失常，例如心房纤颤，以及SAN和心房之间的相互作用。6相反，在分离的心房制备，心房被打开并展平为伪二...

Novel scientific breakthroughs that lead to leaps in the understanding of human physiology are often preceded by technological advances. Fluorescent optical mapping, for example, enables investigation of multiple physiological parameters in both cells and tissues.1,2 It significantly improved our understanding of how the anatomical structure is associated with electrophysiological functions and dysfunctions. In the heart, the natural pacemaker and conduction systems such as the sinoatrial node (SAN) and the atrioventricular junction consist of nodal myocytes that are insulated by the surrounding atrial myocytes.3-5 Such organization creates complex three-dimensional structures with specialized electrical properties. Both structural and functional remodeling of these pacemaker structures has been recognized to form significant electrophysiological heterogeneities.6,7 Understanding the mechanism underlying how such heterogeneities result in SAN dysfunctions and atrial arrhythmogenesis will dramatically benefit the clinical treatment of these diseases. It requires a technique to visualize the propagation of electrical signals at the tissue level, such as optical mapping. 
Recently, accumulating evidence has proven the advance of optical mapping in studies of atrial electrophysiology and pathology.2 However, novel and rigorous studies are dependent on the accurate interpretation of experimental data, whose validity and stability rely on careful experiment protocols. Genetically modified mice are extensively used for research as animal models of human diseases including sick sinus syndrome, pacemaker abnormalities and atrial arrhythmias.6,8-11 Thus, a combination of fluorescent optical mapping with transgenic mouse models provides a powerful tool to study cardiac electrical abnormalities associated with various pathologies. In this paper, we present a protocol for high-resolution optical mapping of the mouse SAN and atrium. Specifically, we discuss and compare different dye loading approaches, time effects on dye bleaching and heart rate stability during the experiment.

<table><tbody><tr><td>water jacket</td><td>Radnoti</td><td>1660 Series Tissue Bath for Large Organ or Single Cell Isolation Procedures</td><td></td></tr><tr><td>water bath / circulator</td><td>Fisher Scientific</td><td>1016S</td><td></td></tr><tr><td>pressure amplifier</td><td>AD Instruments</td><td>MLT0670</td><td></td></tr><tr><td>EMD Millipore Nylon Net Filters</td><td>Fisher Scientific</td><td>NY1102500</td><td></td></tr><tr><td>Pressure transducer</td><td>AD Instruments</td><td>MLT0670</td><td></td></tr><tr><td>Stainless Steel Minutien Pins - 0.1 mm Diameter</td><td>Fine Science Tools</td><td>26002-10&amp;nbsp;</td><td></td></tr><tr><td>Perfusion pump</td><td>World Precision Instruments</td><td>PERIPRO-4LS</td><td></td></tr><tr><td>Superfusion pump</td><td>World Precision Instruments</td><td>PERIPRO-4HS</td><td></td></tr><tr><td>Vannas Tubingen scissors&amp;nbsp;</td><td>World Precision Instruments</td><td>503379</td><td></td></tr><tr><td>Dumont forceps</td><td>World Precision Instruments</td><td>501201, 500085</td><td></td></tr><tr><td>Mayo scissors</td><td>World Precision Instruments</td><td>501750</td><td></td></tr><tr><td>Kelly hemostatic forceps</td><td>World Precision Instruments</td><td>501241</td><td></td></tr><tr><td>Iris forceps</td><td>World Precision Instruments</td><td>15917</td><td></td></tr><tr><td>Iris scissors</td><td>World Precision Instruments</td><td>501263</td><td></td></tr><tr><td>ECG 12 mm needle (29-gauge) electrodes (monopolar)&amp;nbsp;</td><td>AD Instruments</td><td>MLA1203</td><td></td></tr><tr><td>in-line Luer injection port</td><td>Ibidi</td><td>10820</td><td></td></tr><tr><td>Ultima-L CMOS camera&amp;nbsp;</td><td>SciMedia</td><td>MiCAM-05&amp;nbsp;</td><td></td></tr><tr><td>halogen lamp</td><td>Moritex USA Inc</td><td>MHAB-150W</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>S217-500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>S369-500</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233-3</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Blebbistatin</td><td>Tocris Bioscience</td><td>1760</td><td></td></tr><tr><td>RH237</td><td>ThermoFisher Scientific</td><td>S1109</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr></tbody></table>

high-resolution optical mapping of the mouse sino-atrial node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

从的Langendorff灌流心脏完整的SAN的光学标测 重构为自发窦性心律的RA激活等高线图的一个典型的例子示于图3为一个的Langendorff灌注小鼠心脏。早期活化点临近的SVC的intercaval区，其中在SAN是解剖学上定义内。在1.0和0.5毫秒的采样率获得3,16两个协活化等高线图显示在图3B中所示。 <p class="jove_content" fo:keep-together.within-page="1"...

Watch this Scientific Journal Video about High-resolution Optical Mapping of the Mouse Sino-atrial Node at JoVE.com

High-resolution Optical Mapping of the Mouse Sino-atrial Node

在这里，我们提出了从鼠标右心房，特别是窦房结电活动的光学标测的协议，在一个较高的空间和时间分辨率。

鼠标窦房结的高分辨率光学测绘

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

high-resolution-optical-mapping-of-the-mouse-sino-atrial-node

Research

JoVE Journal

Biology

15.6K 次观看.  University of Wisconsin-Madison School of Medicine and Public Health. 在这里，我们提出了从鼠标右心房，特别是窦房结电活动的光学标测的协议，在一个较高的空间和时间分辨率。 

视频: High-resolution Optical Mapping of the Mouse Sino-atrial Node

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

科研

生物工程

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. ...

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

高分辨率的心内膜和心外膜光学在拉伸诱发心房颤动的羊模型的映射

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

医学

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

光学测绘的Langendorff灌注大鼠心脏

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

生物学

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 ...

用于全面心脏成像的光片显微镜和组织透明化

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

作者聚焦：用于研究啮齿动物心脏心肌结构的定制光片成像

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

光片成像揭示啮齿动物心脏的心脏结构

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

小鼠窦房和房室结的全卡口免疫荧光染色、共聚焦成像和 3D 重建

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

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 ...

鼠标窦房结的高分辨率光学测绘

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