Name: electrophysiological assessment of murine atria with high-resolution optical mapping
Uploaded: 2018-02-22T12:30:00.000+00:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping at JoVE.com

这项工作得到了改善研究环境的项目的支持, 从促进科学技术的特别协调基金 (16K09494), 为科学研究提供援助 (no, 26293052 号,到 T.F.)来自日本教育、文化、体育、科学技术部 (下个)。我们感谢 Brainvision 和 Tsubokura 先生的技术援助, 我们也感谢约翰. 马丁先生提供的语言帮助。

这项动物实验是根据东京医科牙科大学机构动物保育和使用委员会的规定批准和执行的。
1. 准备
<ol><li>库存解决方案<ol>
<li>将电压敏感染料 (di-4-ANEPPS 和 RH237) 和 Ca2 +指示器 (Rhod-2 AM) 与100% 二甲基亚砜 (亚砜) 溶解, 分别进行6毫米、10毫米和10毫米浓度的库存解决方案。</li>
		<li>溶解的励磁收缩 (E C) uncoupler, blebbistatin, 与90% 亚砜, 使50毫米的股票解决方案。</li>
		<li>整除在一个黑暗的房间里, 在一个0.2 毫升的 PCR 管中储存溶液, 用氮气代替储存管中的空气, 以避免氧化。</li>
		<li>在铝箔上单独包装铝管以保护光线, 并将其储存在-20 ˚C。</li>
	</ol>
</li>
	<li>工作解决方案<ol>
<li>准备 1 L 磷酸缓冲盐水 (PBS), 不含Ca2 + (137 毫米氯化钠, 2.7 毫米氯化钾, 8.0 毫米 Na2HPO 4, 1.5 毫米.</li>
		<li>准备1升的 Tyrode 溶液 (135 毫米氯化钠, 5.4 毫米氯化钾, 1.8 毫米 CaCl2, 0.53 毫米氯化镁2, 0.33 毫米 NaH2PO4, 5.5 毫米 d-葡萄糖, 5.0 毫米 HEPES, pH 7.40 调整由氢氧化钠)。</li>
		<li>过滤 Tyrode 的解决方案与一个0.22 µm 瓶顶过滤器, 并通气它良好的气泡曝气使用连接到 O2气瓶的空气石。</li>
	</ol>
</li>
</ol>2. Langendorff 灌注心脏的光学制图
<ol><li>使用两个互补金属氧化物半导体 (CMOS) 摄像头组装光学映射系统 (图 1a& b)<ol>
<li>将 CMOS 照相机、光束分配器、物镜、透镜左轮手枪、光源、分色镜、滤镜和其他部件装配到光学映射系统中, 如图 1a& b所示。</li>
		<li>通过改变炮塔中的物镜来选择放大倍数, 配备不同的放大 (1.6X 和 5X)。 注: CMOS 传感器的尺寸为10毫米 x 10 毫米, 有 100 x 100 分辨率, 这意味着5X 物镜, 该系统提供了20µm 空间分辨率。</li>
		<li>使用发光二极管 (LED) 灯设置励磁灯, 其中心波长为 530 nm, 通过带通滤波器 (520/35 nm), 并用分色镜 (560 nm) 反射。</li>
		<li>记录发射信号除以分光器 (665 nm), 其中设置相机1与一个长的通行证过滤器 (697/75 毫微米) 检测荧光使用膜电压敏感染料 (di 4-ANEPPS 或 RH237), 相机2与带通滤波器 (572/28 nm) 检测到Ca2 +指示器 (Rhod2-AM) 的信号。</li>
	</ol></li>
	<li>装配 Langendorff 灌注电路<ol>
<li>将聚氯乙烯 (PVC) 管连接到蠕动泵。</li>
		<li>将 PVC 管的进气侧放入 Tyrode 的溶液中, 如步骤1.2.3 中所述, 加气 100% O2 。</li>
		<li>将 PVC 管的放电端连接到手工制造的空气阱, 然后将5µm 过滤器、三路 stopcocks、压力传感器、加热玻璃线圈和21口径钝针 (针尺寸根据主动脉大小变化) 依次排列。, 使用其他 PVC 管。</li>
		<li>用 Tyrode 的溶液填充灌注电路, 避免电路中出现气泡, 并停止流直到心脏连接到电路。</li>
	</ol></li>
	<li>肝素化和麻醉<ol>
<li>注射普通肝素 (200 IU, 无论体重) 腹腔成一只老鼠使用25口径针和1毫升注射器。</li>
		<li>麻醉的腹腔注射戊巴比妥 (65 毫克/千克) 10 分钟后, 肝素注射液。</li>
	</ol></li>
	<li>插管和灌注<ol>
<li>将鼠标放在仰卧位置。确认动物因脚趾夹缺乏反应而被麻醉。使用剪刀打开剑突过程水平以下的腹壁。横切口在隔膜, 切开两侧的肋骨在内侧腋窝线没有任何损害心脏, 并翻转前胸壁向上。在心脏后向前弯曲钳, 并保持降主动脉, 食管和下腔静脉。然后, 用剪刀将心脏与邻近的血管和组织 (如主动脉、肺、气管、食道、脂肪组织和胸腺) 快速地放在一起。</li>
		<li>在培养皿中用10毫升的冰冷 PBS 冲洗心脏, 并取出相邻的组织。</li>
		<li>将21尺钝针连接到提升主动脉的尖端, 并将其用线固定在显微镜下。</li>
		<li>开始灌注的心脏10分钟, Tyrode 的解决方案曝气与 100% O2。</li>
		<li>与放大器和录音机相连的压力传感器连续监测灌注压力。</li>
		<li>在以下所有步骤中保持 80-100 mmHg (通常对应于2-5 毫升/分钟的流速) 的灌注压力。</li>
		<li>在初始灌注过程中 (步骤 2.4.4), 将薄聚乙烯 (PE) 管 (外径: 0.8 毫米) 插入 SVC, 并用螺纹固定。然后, 将心脏放入加热的玻璃腔室 (图 1b& c)。</li>
		<li>将24口径留置针 (外径: 0.7 毫米) 插入左心室 (LV) 腔内, 通过心室先端, 以避免对心房造成任何损伤, 并取出左外套管的内针离开 LV。</li>
		<li>介绍一个 1-法国大小定制电极导管通过 PE 管在 SVC, 以执行电刺激在 RA。如有需要, 将导管插入右心室 (RV) 进行心室起搏。</li>
		<li>插入一个针电极到心室先端, 连续记录在上升主动脉针电极和插管针之间的双极心电图 (心电图) (图 1c)。</li>
		<li>在整个研究中继续监测心电图和灌注压力。</li>
		<li>关掉灯, 在暗室里做下面的实验。</li>
		<li>转到步骤2.5。用于单个记录的膜电压或2.6。对于膜电压和 Ca2 +瞬态的双记录。</li>
	</ol></li>
	<li>膜电压单记录染色<ol>
<li>在这个染色协议中保持37˚C 加热的灌注溶液, 用于单层膜电压记录。</li>
		<li>稀释8.3 µL 的 di-4-ANEPPS 溶液 (6 毫米), 10 毫升 Tyrode 溶液 (最终浓度为5µM)。</li>
		<li>将稀释后的 di-4-ANEPPS 溶液的总容积 (10 毫升) 注入心脏, 通过灌注途径2-5 分钟, 然后用 Tyrode 的溶液冲洗5分钟。</li>
		<li>稀释5µL 的 blebbistatin 溶液 (50 毫米) 与1mL 的 Tyrode 溶液 (最终浓度为250µM)。</li>
		<li>通过灌注途径管理稀释 blebbistatin 溶液的总用量。</li>
		<li>跳过步骤 2.6, 然后继续步骤2.7 以进行冲洗。</li>
	</ol></li>
	<li>膜电压和 Ca2 +瞬态的双重记录的染色<ol>
<li>在室温下保持 Tyrode 的溶液。 注: 这种初始温度的设定与对膜电压单记录的染色不同 (步骤 2.5)。</li>
		<li>以与步骤2.5.4 和2.5.5 相同的方式管理 blebbistatin。</li>
		<li>混合3µL 的 Rhod2AM 溶液 (10 毫米) 和30µL 聚氧乙烯-丙烯嵌段共聚物 F-127 (20% 在亚砜), 然后稀释混合物与10毫升 Tyrode 的解决方案。</li>
		<li>通过与 blebbistatin 相同的路径, 将 Rhod2AM 解决方案 (3 µM) 的总金额加载到2-5 分钟以上。</li>
		<li>稀释7µL RH237 溶液 (10 毫米), 10 毫升 Tyrode 溶液。</li>
		<li>管理稀释 RH237 溶液的总用量 (7 µM) 超过2-5 分钟。</li>
		<li>完成上述程序后, 开始加热 Tyrode 的解决方案, 在37˚C。</li>
	</ol></li>
	<li>冲洗<ol>
<li>在以下实验中保持 Tyrode 溶液的温度为37˚C。</li>
		<li>灌注的心脏与 Tyrode 的解决方案, 至少5分钟洗出过量的染料。 注意: 在这个洗出步骤中没有必要增加流量 (请参阅步骤 2.4.6)。</li>
		<li>确认由于宫缩导致的任何运动伪影的消失, 以及灌注心脏的均匀染色。</li>
	</ol></li>
	<li>抽样和数据分析<ol>
<li>小心地将盖子玻璃 (25 毫米 x 60 毫米) 放在灌注的心脏上, 使心房表面平整, 不受太多机械应力的影响, 并防止运动工件从振动中得到解决。确认中庭适当附着在盖玻璃上。</li>
		<li>用 CMOS 相机记录荧光, 采样率高达0.1 毫秒, 用于 RH237 和 Rhod2AM 染色, 为 di 4 ANEPPS 和 1 ms。</li>
		<li>根据制造商对软件实际操作的说明, 使用分析软件对所获得的录音进行分析。</li>
		<li>在提取感兴趣的区域、漂移删除、时间筛选和 3 X 3 binning5之后, 创建激活映射和影片。</li>
	</ol></li>
</ol>3. 电生理研究
<ol><li>设置起搏电极和刺激器<ol>
<li>确认 RA 中的自定义起搏电极尖端与组织的接触 (请参阅步骤 2.4.9)。</li>
		<li>提供所有的起搏刺激从连接到可编程刺激器的起搏导管。</li>
	</ol>
</li>
	<li>起搏阈值的确定<ol>
<li>设置100和150毫秒之间的起搏间隔 (脉宽为0.4 毫秒), 避免任何内在节律超过起搏速率。</li>
		<li>提供恒定的起搏刺激, 至少20节拍, 以获得稳定的心房起搏。</li>
		<li>设置 5 mV 的起搏输出, 然后逐渐减少它, 直到交付的起搏无法去极化中庭。</li>
		<li>确认心房激发的存在 P 波在心电图, 和/或心房励磁信号由光学记录。</li>
		<li>确定能够将中庭去极化为起搏阈值的最小起搏输出。</li>
		<li>将以下研究的起搏输出设置为两次起搏阈值。</li>
	</ol>
</li>
	<li>恒定和爆裂起搏<ol>
<li>提供恒定的起搏99节拍, 从150毫秒开始的起搏间隔, 或最长的间隔, 避免超过内在的节奏。</li>
		<li>以5毫秒的步骤逐步减少起搏间隔, 降至40毫秒, 或直到达到无法获得1:1 个心房的间隔。</li>
	</ol>
</li>
	<li>单 extrastimulus 起搏<ol>
<li>将基本驱动器 (S1) 的起搏周期长度设置为120毫秒、100毫秒和80毫秒, 除非内在节律超过基本驱动器或起搏未能获得1:1 心房激发。</li>
		<li>将起搏刺激的次数设置为基本驱动列车的10节拍。</li>
		<li>将第一个 extrastimulus (S2) 设置为-10 毫秒, 以确定基本周期长度。</li>
		<li>在基本驱动器的最后一次起搏刺激后交付 S2。</li>
		<li>将 S2 的耦合间隔逐步缩短为5毫秒, 直到 S2 无法去极化中庭。</li>
		<li>确定有效的难治期 (ERP) 为去极化心房的最长 S2 间隔。</li>
		<li>评估 erp 至少3个不同的基本周期长度, 以评估 erp 的速率适应。</li>
	</ol>
</li>
	<li>双 extrastimuli 起搏诱导心房失常<ol>
<li>如果不出现心律失常, 将 S2 间隔重置为 S2 ERP 外的点20毫秒。</li>
		<li>添加第二个 extrastimulus (S3), 以与 S2 相同的间隔开始。</li>
		<li>S2 和 S3 之间的耦合间隔逐渐减少5毫秒, 直到 S3 无法去极化心房。</li>
		<li>将 S2 的间隔重置为 ERP 外部的10毫秒, 然后重复步骤3.5.3。</li>
		<li>将 S2 和 S3 间隔重置为每个 ERP 外部的点20毫秒。</li>
		<li>添加第三个 extrastimulus (S4), 以与 S3 相同的间隔开始。</li>
		<li>S3 和 S4 之间的耦合间隔逐渐减少5毫秒, 直到 S4 无法去极化心房。</li>
		<li>将 S3 间隔重置为 ERP 外部的点10毫秒, 然后重复步骤3.5.7。</li>
		<li>将 S2 和 S3 间隔重置为 ERP 之外的点10毫秒, 然后重复步骤3.5.7。</li>
		<li>用相似的程序性刺激定义心房失常的 inducibility。</li>
	</ol>
</li>
	<li>心房失常 inducibility 的评价<ol>
<li>在所有起搏协议中连续记录心电图 (步骤 3.3-3.5)。</li>
		<li>在每次刺激过程中和之后执行光学录音, 记录心房心动过速 (AT) 或重复性心房反应 (RAR) 的诱导。</li>
		<li>在 AF 或 RAR 诱发的情况下, 应用相同的起搏协议来确认重现性。</li>
	</ol>
</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock-induced+depolarization+of+refractory+myocardium+prevents+wave-front+propagation+in+defibrillation.">Shock-induced depolarization of refractory myocardium prevents wave-front propagation in defibrillation.</a> Circ. Res. 79, 957-973 (1996).</li><li>Lang, D., Sulkin, M., Lou, Q., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+action+potentials+and+calcium+transients+in+the+mouse+heart.">Optical mapping of action potentials and calcium transients in the mouse heart.</a> J. Vis. Exp. (55), e3275 (2011).</li><li>Eijsbouts, S. C. M., Majidi, M., Zandvoort, M. v., Allessie, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+atrial+dilation+on+heterogeneity+in+conduction+in+the+isolated+rabbit+heart.">Effects of acute atrial dilation on heterogeneity in conduction in the isolated rabbit heart.</a> J. Cardiovasc. Electr. 14, 269-278 (2003).</li><li>Ravelli, F., Allessie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Atrial+dilatation+on+refractory+period+and+vulnerability+to+atrial+fibrillation+in+the+isolated+Langendorff-perfused+rabbit+heart.">Effects of Atrial dilatation on refractory period and vulnerability to atrial fibrillation in the isolated Langendorff-perfused rabbit heart.</a> Circulation. 96, 1686-1695 (1997).</li><li>Sasano, T., McDonald, A. D., Kikuchi, K., Donahue, J. 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作者没有什么可透露的。

光学制图是研究心脏电生理学的一个很好的方法7, 是一个非常有用的工具, 不仅评估室性心律失常8,9, 但也心房一个10,11.同时映射跨膜电位和 Ca2 +瞬变有助于了解心律失常的基本机制与心力衰竭和其他心脏病的关系12,13。当比较其他的电生理评估方法, 如使用单个细胞或细胞片, 在灌注心脏的光学映射的绝对优势之一是评估在完整的心房传导模式和心室, 不仅在窦节律, 而且在诱发心律失常的14。试图利用小鼠的心脏, 特别是中庭, 作为人类的替代品遇到了困难, 主要是由于其体积较小, 然而, 老鼠是一个有吸引力的实验模型, 在评估的基因工程动物模型, 这个问题必须克服。我们的方法为解决这一目标提供了一个方向。
虽然我们的光学测绘仪器基本上与常规的小鼠心脏系统相似15, 但我们的方法具有对小鼠心房进行一些修改的优势。首先, 我们追求获得高达0.1 毫秒/帧和20µm/像素的高空间和时间分辨率, 这种高分辨率的映射有助于更精确地测量小鼠心房的传导速度和传播模式。第二, 为了避免任何不必要的机械损伤或心房伸展, 这可能改变电生理特性16,17, 留置针直接插入 LV 以减少腔内压力,而不是在前面的研究15中执行时将其插入到 LA 中。此外, 起搏刺激是通过一个定制的 1-法国大小电极导管放置在 RA, 但不是由针电极, 这可能会损害心房。在修复心房附属物时, 避免使用任何引脚, 在过去的研究中采用了15。第三, 在对心律失常的基本机制进行评估时, 诱导心房失常的程序性刺激协议至关重要18,19。我们执行编程刺激相同的临床电生理研究, 包括爆裂起搏和高达三重 extrastimuli 起搏, 改变了起搏间隔的鼠标心脏。因此, 除了基线测量参数外, 该协议还可以评估 AT 的 inducibility。在需要时, inducibility 的使用是用异丙肾上腺素或其他药物进行评估的。在我们的经验, 野生型小鼠几乎没有显示任何 ATs, 即使在一个完整的刺激协议。因此, inducibility 应该是评估多种病理条件的贡献的重要信息, 如基因突变、手术程序和药物管理11。这些修改可以优化完整的小鼠心房的精确电生理评估。
该方法也有一定的局限性。首先, 使用具有5X 物镜的最大空间分辨率, 视场 (FOV) 仅限于中庭的一部分 (i. e. 只有左心房附属物, 如图 2a所示)。为了获得中庭较大的 FOV, 1.6X 物镜有时更可取 (图 2b)。其次, 不用针固定心房, 有时很难正确测量心房传导特性, 因为心房表面弯曲。所以, 我们把盖子玻璃放在它的表面, 以使其平整, 而不是用别针固定。这种方法也有利于防止运动工件的振动的解决方案。第三, 用我们的方法, 很难获得它的整个 FOV, 因此, 在我们的方法中使用前和后视图比在图 2中所示的其他方法更重要。前视图的优点是在病理条件下, 特别是在附属物 (图 4) 中对再入的清楚观察。另一方面, 后视的优点是获得良好的心房后壁的看法, 可能是一个详细的记录触发活动的心肌袖。当难以获得合适的视图, 并以我们的方法使其曲面平坦时, 中庭可以通过引脚的最小张力固定。
用我们的方法, 有3可能的问题, 染色失败, 起搏, 心律失常诱导。对于染色失败, 如果没有或轻微荧光被观察, 你应该检查是否正确组装光学测绘仪器, 以及试剂是否适当储存和使用。灌注液的条件也是至关重要的, 这也会影响心脏本身的电生理特性, 因此, 溶液中的 pH 值、温度以及是否有足够的曝气条件必须严格监测。避免心脏的空气栓塞也很重要。对于起搏失败, 如果起搏刺激不能激发心房, 研究人员应检查线路是否正确使用电路测试仪。当起搏刺激正确输出时, 问题是电极与组织的接触。电极的重新定位可以解决这个问题, 我们使用起搏导管的方法很容易。对于心律失常诱导的困难, RV 起搏可用于在一些有限的情况下的诱导。使用 quadripolar 电极导管, 其中远端两电极和近端电极可以位于 rv 和 RA, 分别是很容易改变的起搏地点从 RA 到 rv。当同时心室活化信号遮盖心房激发信号时, 该导管也可用于转移心室兴奋。
这一方法将有助于评估新发现的 AF 相关基因, 如 GWAS, 特别是对于调查未能通过其他方法显示的基因表型之间的相互作用。随着设备和技术的进步, 肺静脉袖的电生理特性是 AF20的重要来源, 可以用这种方法对完整的心脏进行评估。

心脏包括4个室在哺乳动物。上两室为心房, 下部为心室。心室作为泵排出血液到全身或肺循环。心房接受血液从全身或肺静脉返回, 并协助将血液输送到心室以获得有效的心脏泵功能。从电生理方面来说, 心房的重要功能是调节心率。电信号来源于位于上腔静脉 (SVC) 与右心房 (ra) 交界处的窦节点, 然后传播到 RA 和左心房 (LA), 通过房室结和他的浦肯野进行心室传导。传导系统。
心律失常是心脏节律紊乱, 根据其来源分为心房和心室。心房颤动 (AF) 是最常见的心律失常的持续形式, 其特点是随机和快速兴奋的心房。最近的基因分析和全基因组联合研究 (GWAS) 显示了 AF 与基因突变或 monopolymorphisms1、2、3、4之间的关联。这些发现表明 AF 至少部分与遗传原因有关。因此, 用基因工程动物模型评价心房的基因型与表位相互作用是至关重要的。人们普遍认为, 老鼠是最成熟的基因改良哺乳动物。
为评价心脏组织的兴奋性, 研制了光学制图技术。然而, 光测图观察小鼠心房的大小相对较小。我们试图对小鼠心房进行详细的评估, 具有较高的时间和空间分辨率。

<table><tbody><tr><td>(-)-Blebbistatin</td><td>SIGMA</td><td>B0560-1MG</td><td>E-C decoupler to eliminate motion artifact during optical mapping</td></tr><tr><td>RH237</td><td>Biotium</td><td>61018</td><td>Voltage-sensitive dye</td></tr><tr><td>Rhod2AM</td><td>Biotium</td><td>50024</td><td>Ca indicator</td></tr><tr><td>Pluronic F-127 20% solution in DMSO</td><td>Biotium</td><td>#59000</td><td>To enhance the staining with Rhod2AM</td></tr><tr><td>Di-4-ANEPPS</td><td>Wako</td><td>041-29111</td><td>Voltage-sensitive dye</td></tr><tr><td>Dimethyl sulfoxide</td><td>Wako</td><td>046-21981</td><td>Solvent for reagents</td></tr><tr><td>Bottle top filter</td><td>Corning</td><td>430513</td><td>For filtering Tyrode&#039;s solution</td></tr><tr><td>Haparin Sodium</td><td>Mochida Pharmaceutical Co., Ltd</td><td>N/A</td><td>To avoid blood clots in the coronary artery</td></tr><tr><td>Air stone (&amp;phi;8 mm x 10 mm)</td><td>Tokyo Koshin Rikagaku Seisakusho</td><td>N/A</td><td>for aeration</td></tr><tr><td>Pentobarbital</td><td>Kyoritsu Seiyaku Corporation</td><td>N/A</td><td>For an anesthesia</td></tr><tr><td>Programmable stimulator</td><td>Fukuda Denshi</td><td>BC-05</td><td>Fukuda Denshi kindly rented us.</td></tr><tr><td>Power Lab</td><td>AD Instruments</td><td>Powerlab 26/8SP</td><td>To record blood pressure and electrocardiogram</td></tr><tr><td>Bio Amp</td><td>AD Instruments</td><td>ML132</td><td>Amprifier for electrocardiogram</td></tr><tr><td>BP Amp</td><td>AD Instruments</td><td>FE117</td><td>Amprifier for blood pressure</td></tr><tr><td>LabChart</td><td>AD Instruments</td><td>Version 7</td><td>Software to record and analyze blood pressure and electrocardiogram</td></tr><tr><td>Disposable BP transducer</td><td>AD Instruments</td><td>MLT0670</td><td>pressure transducer</td></tr><tr><td>1-Fr custom made electrode catheter</td><td>Unique Medical</td><td>N/A</td><td>To pace right atrium</td></tr><tr><td>Polyethylene tube (OD: 0.8 mm, ID: 0.5 mm)</td><td>Natume Seisakujo</td><td>SP31</td><td>Put into superior vena cava to introduce electrode catheter</td></tr><tr><td>Millex-SV 5.00 &amp;mu;m</td><td>Merk Millipore</td><td>SLSV025LS</td><td>To filter the circulating Tyrode</td></tr><tr><td>24-gauge indwelling needle</td><td>TERUMO</td><td>SR-FS2419</td><td>Introduced into left ventricle to reduce the pressure in chamber</td></tr><tr><td>21-gauge needle</td><td>TERUMO</td><td>SN-2170</td><td>We cut the tip of needle and blunted it by filing</td></tr><tr><td>25-guage needle</td><td>TERUMO</td><td>NN-2525R</td><td></td></tr><tr><td>1-ml syringe</td><td>TERUMO</td><td>SS-01T</td><td></td></tr><tr><td>PVC tube</td><td>TERUMO</td><td>SF-ET0525</td><td>for Langendorff&#039;s perfusion circuit</td></tr><tr><td>Three-way stopcock</td><td>TERUMO</td><td>TS-TL2K</td><td>for Langendorff&#039;s perfusion circuit</td></tr><tr><td>Petri dish</td><td>As one</td><td>3-1491-01</td><td></td></tr><tr><td>Custum made heating glass coil</td><td>Motohashi Rika</td><td>N/A</td><td>to keep temperature of perfusion solution</td></tr><tr><td>Custum made warming glass chamber</td><td>Motohashi Rika</td><td>N/A</td><td>to keep temperature of perfusion solution</td></tr><tr><td>Constant temperature circulating device</td><td>Lauda</td><td>E100</td><td>connected to heating coil and warming chamber</td></tr><tr><td>Cover glass (25 mm &amp;times; 60 mm)</td><td>Matsunami</td><td>C025601</td><td>Put on the atria to flatten the recording area</td></tr><tr><td>Perista pump</td><td>ATTO</td><td>SJ-1211</td><td>peristaltic pump</td></tr><tr><td>Stemi DV4</td><td>Carl Zeiss</td><td>N/A</td><td>Stereomicroscope</td></tr><tr><td>MiCAM ULTIMA-L2</td><td>Brainvision Inc.</td><td>UL-L2</td><td>Optical mapping System</td></tr><tr><td>BV_Ana Software</td><td>Brainvision Inc.</td><td>BV_Ana</td><td>Data Analysis Software</td></tr><tr><td>THT Macroscope</td><td>Brainvision Inc.</td><td>THT-ZS</td><td>Epi-Illumination Unit</td></tr><tr><td>LED Light Source</td><td>Brainvision Inc.</td><td>LEX2-G</td><td></td></tr><tr><td>Dichroic Mirror 560nm</td><td>Brainvision Inc.</td><td>DM560</td><td>Epi-Illuminatinon</td></tr><tr><td>Excitation Filter 520/35nm</td><td>Semrock, Inc.</td><td>FF01-520/35-25</td><td></td></tr><tr><td>Projection lens Plan S 1.0X</td><td>Carl Zeiss</td><td>435200-0000-000</td><td></td></tr><tr><td>Focus Drive</td><td>Carl Zeiss</td><td>435400-0000-000</td><td></td></tr><tr><td>Objective lens Revolver</td><td>Carl Zeiss</td><td>435302-0000-000</td><td></td></tr><tr><td>Manual Focus Column</td><td>Carl Zeiss</td><td>435400-0000-000</td><td></td></tr><tr><td>Macroscope Base</td><td>Carl Zeiss</td><td>435430-9901-000</td><td></td></tr><tr><td>Straight Light Guide</td><td>MORITEX Corporation</td><td>MSG10-2200S</td><td>Epi-Illuminatinon</td></tr><tr><td>Condenser Lens</td><td>MORITEX Corporation</td><td>ML-50</td><td></td></tr><tr><td>PLANAPO 5.0X</td><td>Leica Microsystems</td><td>10447243</td><td>Objective Lens</td></tr><tr><td>PLANAPO 1.0X</td><td>Leica Microsystems</td><td>10447157</td><td>Objective Lens</td></tr><tr><td>PLANAPO 1.6X</td><td>Leica Microsystems</td><td>10447050</td><td>Objective Lens</td></tr><tr><td>Beam-Splitter</td><td>Brainvision Inc.</td><td>FLSP-2</td><td></td></tr><tr><td>Dichroic Mirror 665nm</td><td>Brainvision Inc.</td><td>DM665</td><td>Beam-Splitter</td></tr><tr><td>Emission Filter 572/28nm</td><td>Edmund Optics</td><td>#84-100</td><td>Rhod2-AM</td></tr><tr><td>Emission Filter 697/75nm</td><td>Semrock, Inc.</td><td>FF01-697/75-25</td><td>RH237 and Di-4-ANEPPS</td></tr><tr><td>0.2 mL PCR tube</td><td>Greiner Bio-One</td><td>671201</td><td></td></tr><tr><td>aluminum foil</td><td>Toyo alumi</td><td>0020</td><td></td></tr></tbody></table>

electrophysiological assessment of murine atria with high-resolution optical mapping

最近针对心房颤动 (AF) 的全基因组联合研究表明, 心房的基因型和电生理表型之间有很强的联系。这鼓励我们利用基因工程的老鼠模型来阐明 AF 的机制。然而, 由于小鼠心房的体积较小, 很难评价其电生理特性。该协议描述了在 Langendorff 灌注小鼠心脏的高时间和空间分辨率的光学映射系统心房的电生理评价。光学映射系统采用双高速互补金属氧化物半导体相机和高放大物镜组合, 检测电压敏感染料和 Ca2 +指示器的荧光。专注于对小鼠心房的评估, 光学映射的面积为2毫米 x 2 毫米或10毫米 x 10 毫米, 有 100 x 100 分辨率 (20 µm/像素或100µm/像素), 最大值为10赫 (0.1 毫秒) 的采样速率。1-法国大小 quadripolar 电极起搏导管放置在右心房通过上腔静脉避免任何机械损伤心房, 和起搏刺激通过导管传递。采用程控刺激进行电生理研究, 包括恒定起搏、爆裂起搏和高达三重 extrastimuli 起搏。在自发或起搏的节奏下, 光学映射在右、左心房分别记录动作电位持续时间、活化图、传导速度和 Ca2 +瞬态。此外, 程序化刺激也决定了心房失常的 inducibility。在诱导心房快速心律失常过程中, 进行精确的活化映射, 以确定心房的兴奋传播。具有专门设置的光学映射可以对小鼠病理模型中心房进行彻底的电生理评估。

光学映射实验是使用这些设备执行的, 如图 1a所示。光学系统的架构在图 1b中说明。该系统提供了高分辨率分析的膜电位和 Ca2 +瞬态在中庭。
如图 1c和d所示, 隔离的心脏位于温度控制的腔室中。1-法国大小电极导管放置在 RA 通过 PE 管插入到 SVC, 另一个留置 pe 管插入到 lv 腔从 lv 先端, 以避免过度压力的心脏室。对于同时心电图记录, 阴极 (心电图引线 [-]) 连接到插管针, 阳极 (心电图铅 [+]) 连接到插入心室顶点的针电极。该总成的特点是避免任何机械损伤的心房。
对于膜电压的评估, 使用 di-4-ANEPPS 的荧光记录高达1万帧/秒采样速率 (图 2)。活化图是在恒定的起搏期间提供从 RA。精细图谱可以分析小鼠心房的详细传导模式。
图 3显示了在 RH237 和 Rhod2AM 期间, 从 RA 的恒定起搏过程中, 膜电压和 Ca2 +瞬态的代表性痕迹。我们将映射的采样率降低到1000帧/秒, 以便在当前设置下获得 Rhod2AM 信号的足够信号/噪声比。但是, 系统为分析动作电位与 Ca2 +瞬态之间的关系提供了足够的质量。
然后, 我们进行诱导的心房快速心律失常的编程刺激使用小鼠在病理条件下。图 4展示了在 di-4-ANEPPS 染色的小鼠心房中诱导的光学记录。在实验6前10天, 鼠标经过横向主动脉收缩程序, 将压力超负荷应用于中庭。我们通过三重 extrastimuli 起搏 (120/100/100/80) 诱导, 并观察到重入电路的传播。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56478/56478fig1.jpg"> 图1。光学制图系统.a.电生理学/光学制图系统的大会。b.光学映射系统组成示意图: 蓝色箭头表示光源产生的激发光。物镜可以与炮塔交换。从染色的心脏发出的光被分割在照相机1和照相机2之间。摄像头1检测到薄膜电压的长波长信号, 而2的摄像机记录波长, 其带通滤波器为 Ca2 +瞬态的 580 @ 20 nm。在手术过程中, 心电图和灌注压力不断记录, 心电图信号也同时导入到光学地图记录软件中。c.分离心脏的制备: 心脏是空心的, 通过上升主动脉的钝针灌注。同时心电图记录, 阴极连接到插管针, 阳极与针电极插入心室顶点。d.插管管和电极: 心脏是空心的以21尺钝针为灌注 (插管针)。1-法国大小刺激电极 (电极) 被放置在右心房通过聚乙烯管插入到上腔静脉。玻璃室通过加热的水在房间的腔内循环37˚C 加热。<a href="//cloudflare.jove.com/files/ftp_upload/56478/56478fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56478/56478fig2.jpg"> 图2。在中庭激活地图.a.具有5X 物镜的前视图中的激活映射, b.在后视图与1.6X 物镜。(左面板)隔离心脏和心房的示意图。(右面板)1万帧/秒 di-4-ANEPPS 染色的左心房活化图。录音是在恒定的起搏下进行的, 刺激电极放置在右心房。<a href="//cloudflare.jove.com/files/ftp_upload/56478/56478fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56478/56478fig3.jpg"> 图3。双记录动作电位和 Ca2 +瞬态在左心房。a.隔离心脏示意图。b.在左心房中使用 RH237 为 Ca2 +瞬态的膜电压 (Vm) 和 Rhod2AM 提供荧光强度的代表图像。录音是用5X 物镜在1000帧/秒进行的. c.同时记录心电图 (顶部), RH237 信号 (中间) 和 Rhod2AM 信号 (底部), 在恒定的起搏与刺激电极在右心房。黑色箭头表示刺激峰, 黄色箭头的心室激发。在 RH237 信号和 Rhod2AM 信号中也可以观察到心室的光散射效应 (黄色箭头)。d.合并的 RH237 和 Rhod2AM 跟踪。操作电位和瞬态 Ca2 +的更改同时被记录。<a href="//cloudflare.jove.com/files/ftp_upload/56478/56478fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56478/56478fig4.jpg"> 图4。心房心动过速期间的激活映射.(左面板)孤立的心脏示意图。(中间面板)心房心动过速 (AT) 的激活图。在由右心房提供的三重 extrastimuli 起搏诱发。(右面板)激活图在窦节律期间在同一鼠标心脏。<a href="//cloudflare.jove.com/files/ftp_upload/56478/56478fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
辅助影片 1.在中庭使用 di-4-ANEPPS 染色的膜电位的代表性电影。<a href="http://cloudflare.jove.com/files/ftp_upload/56478/Supplementary_movie_1.zip">请单击此处下载此文件.</a>
辅助影片 2.在心房心动过速和窦节律期间激活映射的代表性电影。<a href="http://cloudflare.jove.com/files/ftp_upload/56478/Supplementary_movie_2.zip">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping at JoVE.com

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

高分辨率光学制图对小鼠心房电生理的评价

该协议描述了利用具有高时间和空间分辨率的光学映射系统对小鼠心房的电生理评价, 包括膜电压的双记录和 Ca2 +瞬态的编程通过专门的电极导管进行刺激。

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

electrophysiological-assessment-murine-atria-with-high-resolution

Research

JoVE Journal

Medicine

9.7K 次观看.  Tokyo Medical and Dental University. 该协议描述了利用具有高时间和空间分辨率的光学映射系统对小鼠心房的电生理评价, 包括膜电压的双记录和 Ca2 +瞬态的编程通过专门的电极导管进行刺激。

视频: Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

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

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 ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

使用电子地图的光映射数据的高通量分析

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

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 ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

培养HL-1心房肌细胞单层的电压和钙双通道光映射

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

的内肌浆网钙光学标测<sup&gt; 2+</sup&gt;和的Langendorff灌注兔心脏跨膜电位

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

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.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

高分辨率光学制图对小鼠心房电生理的评价

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