这项研究得到了 DZHK (德国心血管研究中心)、EKFS (Kröner 费森尤斯基金会、卡尔亨利) 和 BMBF (德国教育部和研究部) 的支持, 以及波黑-Charité临床科学家项目资助由 Charité-Universitätsmedizin 柏林和柏林健康学院 (卡尔亨利)。

所有实验均经当地伦理委员会 (TVA T0060/15 和 T0003-15) 批准, 并与《关于护理和使用实验动物指南》 (美国国立卫生研究院) 达成一致。
注意: 过程的简化流程图如图 1所示。
1. Prearrangements
<ol><li>根据表1准备缓冲区。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>解决 方案</td>
			<td>铅</td>
			<td>Cb</td>
			<td>Db</td>
			<td>某人</td>
			<td>S1</td>
			<td>S2</td>
			<td>S3</td>
			<td>Nt</td>
		</tr>
<tr>
<td>试剂 (mM)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>Nacl</td>
			<td></td>
			<td>135</td>
			<td>135</td>
			<td>135</td>
			<td>135</td>
			<td>135</td>
			<td>135</td>
			<td>135</td>
			<td>135</td>
		</tr>
<tr>
<td>氯化钾</td>
			<td></td>
			<td>4。7</td>
			<td>4。7</td>
			<td>4。7</td>
			<td>4。7</td>
			<td>4。7</td>
			<td>4。7</td>
			<td>4。7</td>
			<td>4</td>
		</tr>
<tr>
<td>2PO4</td>
			<td></td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td></td>
		</tr>
<tr>
<td>Na2HPO4</td>
			<td></td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td>0。6</td>
			<td></td>
		</tr>
<tr>
<td>MgSO4</td>
			<td></td>
			<td>1。2</td>
			<td>1。2</td>
			<td>1。2</td>
			<td>1。2</td>
			<td>1。2</td>
			<td>1。2</td>
			<td>1。2</td>
			<td></td>
		</tr>
<tr>
<td>氯化镁</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>1</td>
		</tr>
<tr>
<td>HEPES</td>
			<td></td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
		</tr>
<tr>
<td>牛磺酸</td>
			<td></td>
			<td>30</td>
			<td>30</td>
			<td>30</td>
			<td>30</td>
			<td>30</td>
			<td>30</td>
			<td>30</td>
			<td></td>
		</tr>
<tr>
<td>葡萄糖</td>
			<td></td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td></td>
		</tr>
<tr>
<td>BDM</td>
			<td></td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td></td>
		</tr>
<tr>
<td>CaCl2</td>
			<td></td>
			<td></td>
			<td>1</td>
			<td>0.01</td>
			<td></td>
			<td>0.125</td>
			<td>0.25</td>
			<td>0。5</td>
			<td>1</td>
		</tr>
<tr>
<td>Bsa</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>150</td>
			<td>70</td>
			<td>70</td>
			<td>70</td>
			<td></td>
		</tr>
<tr>
<td>纯化酶共混 (中 Thermolysin)</td>
			<td></td>
			<td></td>
			<td></td>
			<td>0.195 Wünsch 单位/毫升</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>pH 值调整为</td>
			<td></td>
			<td>7。4</td>
			<td>7。4</td>
			<td>7。4</td>
			<td>7。4</td>
			<td>7。4</td>
			<td>7。4</td>
			<td>7。4</td>
			<td>7。4</td>
		</tr>
<tr>
<td>pH 值调整 at</td>
			<td></td>
			<td>37°c</td>
			<td>4°c</td>
			<td>37°c</td>
			<td>37°c</td>
			<td>37°c</td>
			<td>37°c</td>
			<td>37°c</td>
			<td>37°c</td>
		</tr>
<tr>
<td>pH 值调整与</td>
			<td></td>
			<td>氢氧化钠</td>
			<td>氢氧化钠</td>
			<td>氢氧化钠</td>
			<td>氢氧化钠</td>
			<td>氢氧化钠</td>
			<td>氢氧化钠</td>
			<td>氢氧化钠</td>
			<td>氢氧化钠</td>
		</tr>
</tbody></table>表 1: 缓冲区列表.PB: 灌注缓冲, 可储存3天在4°c (每动物300毫升)。CB: 插管缓冲器, 可以储存3天在4°c (每动物200毫升)。DB: 消化缓冲, 必须在白天使用 (每动物40毫升)。某人: 停止缓冲, 必须在白天使用 (每只动物2毫升)。S1: 步1缓冲, 必须在天之内使用 (每动物2毫升)。S2: 步2缓冲, 必须在天之内使用 (每动物2毫升)。S3: 步3缓冲, 必须在天之内使用 (每动物2毫升)。NT: 正常的 Tyrode, 必须在白天使用 (每只动物50毫升)。
<ol start="2"><li>
准备 Langendorff 设备 (图 2A)。
	<ol>
<li>冲洗系统与100毫升70% 乙醇, 其次是2冲洗100毫升蒸馏水。用200毫升的灌注缓冲 (PB) 填充系统。</li>
		<li>将蠕动泵的流量设置为3毫升/分钟。</li>
		<li>将 Langendorff 装置的铅温度校准到39摄氏度。<ol>
<li>将特制套管 [16 克, 长25毫米, 尖尖移除 (图 3A)] 放在 Langendorff 装置的顶部, 开始流动。打开加热模块, 调整加热模块的值, 以达到套管尖端的 PB 所需的温度。温度校准完成后, 将特制套管移至用于插管的注射器 (图 2B)。</li>
		</ol>
</li>
		<li>准备 Langendorff 系统旁边的压力控制装置 (图 3C)。将蝴蝶针安装在三脚架钳上, 准备3个阻塞节。 注: 胶原酶活性随温度变化。对左心房进行温度监测, 并对其进行动态调整, 在以后的过程中需要。</li>
	</ol>
</li>
	<li>根据图 2B设置器官切除和插管设备。用冰冷的插管缓冲器 (CB) 填满烧杯、注射器和培养皿, 准备插管结。</li>
	<li>
Heparinize 鼠的500国际单位肝素每100克的老鼠的体重如下。
	<ol>
<li>将2毫升100% 异氟醚放在适合啮齿动物的麻醉诱导室中。将21周大的 ZFS-1 肥胖鼠从笼子转移到感应室。允许大鼠进入深层麻醉, 由呼吸减速到其初始频率的一半表示。</li>
		<li>从感应腔中取出老鼠。用肝素注射器将500国际单位的肝素每100克注射到腹膜中。</li>
		<li>把老鼠送到笼子里, 让它醒过来。 注意: 在步骤1.4 中不需要保持不育。请稍候20分钟, 然后继续下一步。不完全抗凝可引起血液凝固, 导致微梗死, 对孤立心肌细胞的质量和产量有显著影响。</li>
	</ol>
</li>
</ol>2. 心脏准备
<ol><li>将2毫升100% 异氟醚放在适合啮齿动物的麻醉诱导室中。将21周大的 ZFS-1 肥胖鼠从笼子转移到感应室。允许大鼠进入深层麻醉, 由呼吸减速到其初始频率的一半表示。</li>
	<li>弄死动物通过斩首使用一个适合啮齿动物的断头台。把老鼠的四肢缠在聚苯乙烯泡沫表面上。用手术剪刀抬起并取出覆盖剑突过程的皮肤。在两侧的肋骨笼下面打开腹膜, 露出隔膜。</li>
	<li>用细剪刀沿前弧形切口切开隔膜。将两侧肋骨沿linea mediaclavicularis切开至锁骨骨, 使用手术剪刀, 在原位暴露纵隔。</li>
	<li>用细剪刀去除门区远端的肺部。切除胸腺以暴露主动脉弓。</li>
	<li>用镊子捏住心脏的底座, 轻轻地向下拉向动物的尾部。切过主动脉, 同时保持心脏的拉力, 留下5毫米长段的主动脉附着在心脏上。迅速转移心脏到50毫升冰冷 CB 在50毫升烧杯 (图 2B)。 注意: 在2.5–3.3 步骤中, 心脏在缺血性疾病中有效。避免超过3分钟以上的步骤, 以免损害心肌细胞。</li>
</ol>3. 插管
<ol><li>等待大约十年代心脏冷却下来并且占领收缩。将心脏转移到含有50毫升新鲜的冰冷 CB 的培养皿中。用镊子和剪刀小心地去除围绕主动脉的脂肪组织。</li>
	<li>插入自定义套管 (与步骤1.2.3 中使用的相同), 它连接到10毫升注射器, 充满冰冷的 CB, 3 毫米进入主动脉。将主动脉锁定在套管上, 将两个插管结 (图 3B) 中的一个放在套管尖端的压痕上。 注意: 小心不要通过导管穿透来损伤主动脉瓣。</li>
	<li>用5毫升冰冷的 CB 轻轻冲洗主动脉, 用注射器附着在导管上, 直到冠状动脉内没有更多的血液可见。用镊子轻轻按摩左心房, 注入剩余的5毫升 CB, 允许从腔内取出多余的血液进入培养皿。</li>
	<li>栓第二个插管结 (缝合: USP 3/0, 丝绸) 在凹痕远端到套管尖端。从注射器中卸下与心脏相连的套管。使用适当的适配器将连接心脏的套管装入 Langendorff。 注: 本协议在 Langendorff 器械灌注过程中使用心室腔内的高压。额外的插管结是需要通过避免任何顺缓冲泄漏通过主动脉, 以保持这种压力。</li>
</ol>4. 压力操纵和消化
<ol><li>启动 Langendorff 装置的蠕动泵, 用 PB 启动心脏组织的灌注。在心脏的底部, 不包括主动脉, 系上双头结 (缝合: USP 3/0, 丝)。重复这个步骤, 直到左心房和冠状动脉窦的通货膨胀被注意到。</li>
	<li>使用压力控制装置的蝶形针刺穿中庭 (图 3D), 并允许中庭收缩。通过调整蝶形软管的标高来操纵中庭的腔内压力。在整个程序的其余部分保持心房微微充气;监控并相应调整。 注意: 这一步需要迅速执行, 因为左心房的长期膨胀会导致心肌细胞死亡。</li>
	<li>通过定位左心房和左心室之间的温度探针来测量左心房的近似温度。相应地调整 Langendorff 加热模块的温度, 对准左心房37摄氏度的近似温度。</li>
	<li>灌注的心脏与 PB 共3分钟。</li>
	<li>将灌注转换为约14–18分钟的消化缓冲 (DB)。 注: 当左心房结构坍塌, 组织获得乳白色纹理时, 消化完成。</li>
	<li>用镊子捏住心房, 并进行轻微的拉力。用细剪刀取下左心房, 将中庭转移到含有2毫升停车缓冲 (SB) 的大称量船上, 充分淹没组织。</li>
	<li>按照实验室执行程序的指导方针, 处理余下的心脏组织。</li>
</ol>5. 细胞加工和钙的重新适应
<ol><li>用细剪刀将心房组织切成大约2毫米 x 2 毫米的小块。</li>
	<li>用转移吸管对组织块进行温和的吸入和弹射, 分散组织。继续这个过程大约5分钟, 直到组织的宏观离解能被观察。 注意: 在这个步骤中避免任何气泡, 因为将细胞暴露在空气中会导致心肌死亡。</li>
	<li>将细胞转移到15毫升锥形管上。允许组织块在三十年代解决. 将上清液转移到另一15毫升锥形管中。允许单元格结算15分钟。</li>
	<li>除去并丢弃上清。添加2毫升的步骤1缓冲区 (见表 1)。让心肌细胞安定10分钟。 注: 被丢弃的上清液还包括成纤维细胞和内皮干细胞。如果需要使用这些单元格进行实验14、23, 请参阅其他协议。该颗粒将主要包含心房心肌细胞, 可以通过光镜证实。步骤6.1 对心房心肌细胞的显微特征进行了讨论。</li>
	<li>除去并丢弃上清。添加2毫升的步骤2缓冲区。让心肌细胞安定10分钟。</li>
	<li>除去并丢弃上清。添加2毫升的步骤3缓冲区。让心肌细胞安定10分钟。</li>
	<li>除去并丢弃上清。添加250µL 的正常 Tyrode (NT), 其中包含1毫米 Ca2 +。</li>
	<li>将含有心房心肌细胞的正常 Tyrode 的50µL 转移到一个玻璃底盘上, 涂上25% 层层粘连蛋白 (并允许事先晾干)。让心肌细胞安定10分钟。</li>
	<li>填充一个玻璃底菜与500µL 的 NT 包含1毫米 CaCl2。</li>
</ol>6. 励磁收缩耦合功能评价
<ol><li>使用20X 放大倍数 (图 4A和4B) 评估光镜下的细胞形态和活力。随机选择大约100个细胞, 并将它们归类为可行或难以为继, 以估计细胞隔离过程的可行性。 注意: 可行细胞的特点是对称小节结构, 缺乏膜泡, 和一个棒形。</li>
	<li>
在完成步骤5.9 的20分钟内, 用 Ca2 +敏感荧光染料加载单元格。
	<ol>
<li>将10µM Fluo4-AM 添加到 NT 的500µL 中. 从玻璃底菜上取出上清液 (从步骤 5.9)。将包含 Fluo4-AM 的 NT 添加到玻璃底菜中。在室温下孵育混合物20分钟。除去并丢弃上清。使用 NT 500 µL 清洗样品2x。</li>
	</ol>
</li>
	<li>
用共焦显微镜可视化 Ca2 +励磁, 如下所示。
	<ol>
<li>将玻璃底部的盘子转移到共焦显微镜上, 用40X 放大倍数的显微镜 (激光强度为 5.8%, 激发 488 nm, 发射 515 nm) 可视化 Ca2 +励磁。</li>
		<li>使用适当的 superfusion 设备将 NT 加热到37摄氏度的单元格灌注。或者, 用显微镜安装的热孵化器保持细胞的温暖。</li>
		<li>刺激在一个电场的心肌细胞与商用, 显微镜安装刺激电极频率为1赫兹和电流 24 a. 等待1分钟, 允许细胞达到 Ca2 +处理的稳态状态。</li>
		<li>将扫描线平行于横向轴, 在原子核与随机选取的宏观收缩细胞的边缘之间进行半方向。通过重复扫描获取线扫描图像。</li>
		<li>使用自由可用的成像软件估计信号强度后立即电刺激 (F0) 整个细胞。在1刺激周期 (F) 的过程中, 在整个细胞中绘制信号强度。除以 (f) (f0) 获得各自的 Ca2 +瞬态。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57953/57953fig1.jpg"> 图 1: 隔离过程的简化流程图.该程序是高度敏感的时间, 直到酶消化的肌细胞完成。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57953/57953fig1large.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/57953/57953fig2.jpg"> 图 2: 在隔离之前准备设备.(A) 本小组展示自制的 Langendorff 装置: (1) 带铅的套式反应容器;(2) 带 CB 的套壳反应容器;(3) 3 路旋塞阀;(4) 注射器;(5) 蠕动泵;(6) 加热浸泡;(7) 夹套气泡陷阱;和 (8) 套管和心脏。(B) 本小组展示了为优化工作流程设置插管和器官切除的方法: (1) 100 毫升烧杯, 50 毫升冰冷 CB;(2) 10 毫升带有冰冷 CB 的注射器;(3) 带有冰冷 CB 的培养皿;(4) 光源;(5) 特制套管带插管结 (参见图 3A和3B);(6) 细、弯钳;(7) 组织钳;(8) 细钳, 角度 45°;(9) 腹部手术剪刀;(10) 精细手术剪刀;(11) 4 x 30 克针;(12) 15 克针。(C) 本小组展示了用于共焦成像的显微镜安装设备: (1) 电刺激电极;(2) superfusion 笔;(3) 飞机状态的玻璃底菜;(4) 浸入式铂电刺激电极。(D) 本小组展示了共焦成像的显微镜设置: (1) 共焦显微镜;(2) superfusion 笔;(3) superfusion 缓冲油藏;(4) superfusion 流量调节器;(5) superfusion 加热模块;(6) 计算机工作站;(7) 电刺激器。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57953/57953fig2large.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/57953/57953fig3.jpg"> 图 3: 定制设备.(A) 此面板显示操纵的15克套管与鲁尔锁。箭头表示插管节的两个凹痕。(B) 这些都是插管节, 两个双肩结互相放置在一起, 以快速收紧。箭头指示需要收紧结点的位置和顺序。(C) 此面板显示装配式压力控制装置。21克蝶形针被钩成三脚架钳。软管与针的高度保持一致。螺丝顶部打开。蝶形软管的标高可以根据箭头的指示改变, 以改变左心房的腔内压力。(D) 左心房被压控装置刺穿。这张照片显示了一个理想的充气左心房。椭圆标记了上手结的位置。(E) 左心房被压控装置刺穿。这张图片显示一个过度膨胀的左心房, 这将导致低产量的可行的飞机状态。椭圆标记了上手结的位置。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57953/57953fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<ol>
	<li>Alberti, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harmonizing+the+metabolic+syndrome:+a+joint+interim+statement+of+the+International+Diabetes+Federation+Task+Force+on+Epidemiology+and+Prevention;+National+Heart,+Lung,+and+Blood+Institute;+American+Heart+Association;+World+Heart+Federation;+International+Atherosclerosis+Society;+and+International+Association+for+the+Study+of+Obesity.">Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity.</a> Circulation. 120 (16), 1640-1645 (2009).</li><li>. IDF Consensus Worldwide Definition of the Metabolic Syndrome Available from: <a target="_blank" href="https://www.idf.org/e-library/consensus-statements/60-idfconsensus-worldwide-definitionof-the-metabolic-syndrome.html">https://www.idf.org/e-library/consensus-statements/60-idfconsensus-worldwide-definitionof-the-metabolic-syndrome.html</a> (2006)</li><li>Melenovsky, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+atrial+remodeling+and+function+in+advanced+heart+failure+with+preserved+or+reduced+ejection+fraction.">Left atrial remodeling and function in advanced heart failure with preserved or reduced ejection fraction.</a> Circulation: Heart Failure. 8 (2), 295-303 (2015).</li><li>Goette, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EHRA/HRS/APHRS/SOLAECE+expert+consensus+on+atrial+cardiomyopathies:+definition,+characterization,+and+clinical+implication.">EHRA/HRS/APHRS/SOLAECE expert consensus on atrial cardiomyopathies: definition, characterization, and clinical implication.</a> EP Europace. 18 (10), 1455-1490 (2016).</li><li>Schotten, U., Verheule, S., Kirchhof, P., Goette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+mechanisms+of+atrial+fibrillation:+a+translational+appraisal.">Pathophysiological mechanisms of atrial fibrillation: a translational appraisal.</a> Physiological Reviews. 91 (1), 265-325 (2011).</li><li>Hohendanner, F., DeSantiago, J., Heinzel, F. R., Blatter, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dyssynchronous+calcium+removal+in+heart+failure-induced+atrial+remodeling.">Dyssynchronous calcium removal in heart failure-induced atrial remodeling.</a> American Journal of Physiology-Heart and Circulatory Physiology. 311 (6), H1352-H1359 (2016).</li><li>Hohendanner, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol-1,4,5-trisphosphate+induced+Ca2++release+and+excitation-contraction+coupling+in+atrial+myocytes+from+normal+and+failing+hearts.">Inositol-1,4,5-trisphosphate induced Ca2+ release and excitation-contraction coupling in atrial myocytes from normal and failing hearts.</a> The Journal of Physiology. 593 (6), 1459-1477 (2015).</li><li>Tada, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+mineralocorticoid+receptor+on+experimental+cerebral+aneurysms+in+rats.">Role of mineralocorticoid receptor on experimental cerebral aneurysms in rats.</a> Hypertension. 54 (3), 552-557 (2009).</li><li>Iwasaki, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+promotion+with+long-term+repetitive+obstructive+sleep+apnea+in+a+rat+model.">Atrial fibrillation promotion with long-term repetitive obstructive sleep apnea in a rat model.</a> Journal of the American College of Cardiology. 64 (19), 2013-2023 (2014).</li><li>Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+natriuretic+factor-SV40+T+antigen+transgenes+produce+tumors+and+cardiac+arrhythmias+in+mice.">Atrial natriuretic factor-SV40 T antigen transgenes produce tumors and cardiac arrhythmias in mice.</a> Science. 239 (4843), 1029-1033 (1988).</li><li>Claycomb, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HL-1+cells:+a+cardiac+muscle+cell+line+that+contracts+and+retains+phenotypic+characteristics+of+the+adult+cardiomyocyte.">HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (6), 2979-2984 (1998).</li><li>Sartiani, L., Bochet, P., Cerbai, E., Mugelli, A., Fischmeister, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+the+hyperpolarization-activated,+non-selective+cation+current+I(f)+in+immortalized+HL-1+cardiomyocytes.">Functional expression of the hyperpolarization-activated, non-selective cation current I(f) in immortalized HL-1 cardiomyocytes.</a> The Journal of Physiology. 545 (Pt 1), 81-92 (2002).</li><li>Louch, W. E., Sheehan, K. A., Wolska, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+cardiomyocyte+isolation,+culture,+and+gene+transfer.">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> Journal of Molecular and Cellular Cardiology. 51 (3), 288-298 (2011).</li><li>Gunduz, D., Hamm, C. W., Aslam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Isolation+of+High+Quality+Cardiomyocytes,+Endothelial+Cells,+and+Fibroblasts+from+an+Adult+Rat+Heart.">Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart.</a> Journal of Visualized Experiments. (123), e55601 (2017).</li><li>Li, D., Wu, J., Bai, Y., Zhao, X., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+cardiomyocytes+for+cell+signaling+and+in+vitro+cardiac+hypertrophy.">Isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy.</a> Journal of Visualized Experiments. (87), e51357 (2014).</li><li>Graham, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+functional+characterization+of+adult+mouse+cardiomyoctyes.">Isolation, culture, and functional characterization of adult mouse cardiomyoctyes.</a> Journal of Visualized Experiments. (79), e50289 (2013).</li><li>Roth, G. M., Bader, D. M., Pfaltzgraff, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+physiological+analysis+of+mouse+cardiomyocytes.">Isolation and physiological analysis of mouse cardiomyocytes.</a> Journal of Visualized Experiments. (91), e51109 (2014).</li><li>Thum, T., Borlak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+Ca2++tolerant+cardiomyocytes+from+the+adult+rat:+improvements+and+applications.">Isolation and cultivation of Ca2+ tolerant cardiomyocytes from the adult rat: improvements and applications.</a> Xenobiotica. 30 (11), 1063-1077 (2000).</li><li>Egorova, M. V., Afanas'ev, S. A., Popov, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+isolation+of+cardiomyocytes+from+adult+rat+heart.">A simple method for isolation of cardiomyocytes from adult rat heart.</a> Bulletin of Experimental Biology and Medicine. 140 (3), 370-373 (2005).</li><li>Kohncke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Kv+channel+recordings+in+murine+atrial+and+ventricular+cardiomyocytes.">Isolation and Kv channel recordings in murine atrial and ventricular cardiomyocytes.</a> Journal of Visualized Experiments. (73), e50145 (2013).</li><li>Wagner, E., Brandenburg, S., Kohl, T., Lehnart, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+tubular+membrane+networks+in+cardiac+myocytes+from+atria+and+ventricles.">Analysis of tubular membrane networks in cardiac myocytes from atria and ventricles.</a> Journal of Visualized Experiments. (92), e51823 (2014).</li><li>Hohendanner, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+metabolic+syndrome-related+atrial+decompensation+in+a+rat+model+of+HFpEF.">Cellular mechanisms of metabolic syndrome-related atrial decompensation in a rat model of HFpEF.</a> Journal of Molecular and Cellular Cardiology. 115, 10-19 (2017).</li><li>Seluanov, A., Vaidya, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> Journal of Visualized Experiments. (44), e2033 (2010).</li><li>Bootman, M. D., Higazi, D. R., Coombes, S., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling+during+excitation-contraction+coupling+in+mammalian+atrial+myocytes.">Calcium signalling during excitation-contraction coupling in mammalian atrial myocytes.</a> Journal of Cell Science. 119 (Pt 19), 3915-3925 (2006).</li><li>Smyrnias, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+T-tubule+system+in+adult+rat+ventricular+and+atrial+myocytes,+and+its+role+in+excitation-contraction+coupling+and+inotropic+stimulation.">Comparison of the T-tubule system in adult rat ventricular and atrial myocytes, and its role in excitation-contraction coupling and inotropic stimulation.</a> Cell Calcium. 47 (3), 210-223 (2010).</li><li>Pritchett, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diastolic+dysfunction+and+left+atrial+volume:+a+population-based+study.">Diastolic dysfunction and left atrial volume: a population-based study.</a> Journal of the American College of Cardiology. 45 (1), 87-92 (2005).</li><li>Linz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cathepsin+A+mediates+susceptibility+to+atrial+tachyarrhythmia+and+impairment+of+atrial+emptying+function+in+Zucker+diabetic+fatty+rats.">Cathepsin A mediates susceptibility to atrial tachyarrhythmia and impairment of atrial emptying function in Zucker diabetic fatty rats.</a> Cardiovascular Research. 110 (3), 371-380 (2016).</li><li>Ackers-Johnson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simplified,+Langendorff-Free+Method+for+Concomitant+Isolation+of+Viable+Cardiac+Myocytes+and+Nonmyocytes+From+the+Adult+Mouse+Heart.">A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart.</a> Circulation Research. 119 (8), 909-920 (2016).</li><li>Ramanathan, T., Skinner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+blood+flow.">Coronary blood flow.</a> Continuing Education in Anaesthesia Critical Care &amp; Pain. 5 (2), 61-64 (2005).</li><li>Bond, M. D., Van Wart, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+individual+collagenases+from+Clostridium+histolyticum.">Characterization of the individual collagenases from Clostridium histolyticum.</a> Biochemistry. 23 (13), 3085-3091 (1984).</li><li>Deel, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+model+to+study+the+effects+of+matrix+stiffening+on+Ca(2+)+handling+and+myofilament+function+in+isolated+adult+rat+cardiomyocytes.">In vitro model to study the effects of matrix stiffening on Ca(2+) handling and myofilament function in isolated adult rat cardiomyocytes.</a> The Journal of Physiology. 595 (14), 4597-4610 (2017).</li><li>Wuensch, E., Heidrich, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[On+the+Quantitative+Determination+of+Collagenase].">[On the Quantitative Determination of Collagenase].</a> Hoppe-Seyler's Zeitschrift f&#252;r physiologische Chemie. 333, 149-151 (1963).</li><li>Conceicao, G., Heinonen, I., Lourenco, A. P., Duncker, D. J., Falcao-Pires, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+heart+failure+with+preserved+ejection+fraction.">Animal models of heart failure with preserved ejection fraction.</a> Netherlands Heart Journal. 24 (4), 275-286 (2016).</li><li>Horgan, S., Watson, C., Glezeva, N., Baugh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+diastolic+dysfunction+and+heart+failure+with+preserved+ejection+fraction.">Murine models of diastolic dysfunction and heart failure with preserved ejection fraction.</a> Journal of Cardiac Failure. 20 (12), 984-995 (2014).</li></ol>

作者没有什么可透露的。

在这里, 我们首先描述了一个协议, 从大鼠模型中分离出的单细胞飞机状态, 显示显着心房重塑22。这一过程具有独特的挑战性, 因为过量的脂肪组织可以使手术准备, 以及主动脉插管, 越来越困难。表 2中提供的故障排除指南解决了隔离过程中最常见的问题。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>问题</td>
			<td>...

大都会描述了糖尿病 type-2 和心血管疾病的一系列危险因素, 包括增加的动脉血压, 血脂异常 (升高甘油三酯和降低高密度脂蛋白胆固醇), 增加禁食葡萄糖和中央肥胖症1。据估计, 世界大都会的流行率为 25–30%, 持续上升2。HFpEF 是一种异质临床综合征, 常与大都会相关。HFpEF 和前阶段 (即高血压性心脏病) 的心脏重塑也伴随着心房3的重塑。左心房的收缩功能和结构改变与死亡率增加、心房颤动和新发心力衰竭4有关。心房重塑的特点是离子通道功能的变化, 钙2 +稳态, 心房结构, 成纤维细胞活化, 组织纤维化5。与 HFpEF 相关的左心房重塑及其潜在的病理机制尚不清楚, 需要进一步深入调查。动物模型已被证明是一个宝贵的工具, 并导致在心房心肌6,7,8,9领域的许多进展。
孤立单细胞心肌细胞的研究经常被用来证实和补充体内的发现。一个孤立和潜在的后续细胞培养, 允许调查信号通路, 离子通道电流, 和励磁收缩耦合。在生理条件下, 心肌细胞不会增殖。在转基因小鼠中, 心房钠素转录调控序列与猿猴病毒40大 T 抗原的融合导致了第一个永生化的飞机状态, 命名为 AT-110。AT-1 细胞的进一步发育引起了 HL-1 细胞的形成, 它不仅可以连续传代, 而且能自发收缩11。然而, 与新近分离的细胞相比, 它们表现出结构和功能上的差异, 如组织超微结构、发育肌原纤维11的高发生率和极化活化的内电流12。大鼠和小鼠心室心肌细胞 (VCM) 的分离是建立在13、14、15、16、17、18,19. 一般情况下, 切除心脏被安装到一个 Langendorff 设备和 retrogradely 灌注与一个 Ca2 +自由缓冲含有消化酶, 如胶原酶和蛋白酶。然后以逐步的方式将钙重新引入到生理条件中。然而, 即使专门用于隔离飞机状态的协议20、21, 由于纤维化和压力相关的差异, 它们在心房重塑的疾病模型中的效用是有限的。
在这篇文章中, 我们已经实施了一个协议, 以隔离的动物, 显示心房重塑 (特别是为 ZFS1大鼠模型 HFpEF)22的心房单细胞心肌细胞。现有的隔离协议得到了优化, 并辅以一个简单的, 定制的装置来控制和修改心脏腔腔内压力, 导致更高的形态和功能完整的心肌细胞的产量。下面的协议为研究员提供了一步一步的指南, 详细描述了定制的设备, 一个解决方案列表, 以及一个全面的故障排除指南。

<table><tbody><tr><td>ZSF-1 Obese rat</td><td>Charles River Laboratories, Inc.</td><td></td><td>21 weeks old</td></tr><tr><td>Fine Iris Scissors</td><td>Fine Science Tools GmbH</td><td>14094-11</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools GmbH</td><td>14001-18</td><td></td></tr><tr><td>Micro Dressing Forceps (curved, serrated)</td><td>Aesculap, Inc.</td><td>BD312R</td><td></td></tr><tr><td>Tissue Forceps (straight, 1 x 2 teeth)</td><td>Aesculap, Inc.</td><td>BD537R</td><td></td></tr><tr><td>Tying Forceps (angled)</td><td>Aesculap, Inc.</td><td>MA624R</td><td></td></tr><tr><td>Rodent and Small Animal Guillotine</td><td>Kent Scientific Corp.</td><td>DCAP</td><td></td></tr><tr><td>Low Cost Induction Chamber 3.0 L</td><td>Kent Scientific Corp.</td><td>SOMNO-0730&amp;nbsp;</td><td></td></tr><tr><td>Butterfly Winged Infusion Set 21 G</td><td>Hospira, Inc.</td><td>181106101</td><td></td></tr><tr><td>Abbocath 16 G</td><td>Hospira, Inc.</td><td>0G7149702</td><td></td></tr><tr><td>Microlance Hypodermic Needle</td><td>Becton Dickinson GmbH</td><td>301300</td><td>modify needle to make cannula</td></tr><tr><td>Braun Original Perfusor Syringe 50 ml</td><td>B. Braun Melsungen AG</td><td>8728810F</td><td></td></tr><tr><td>Braun Inject Solo Syringe 10 ml</td><td>B. Braun Melsungen AG</td><td>2057926</td><td></td></tr><tr><td>Beaker 50ml</td><td>Duran Group (DWK Life Sciences GmbH)</td><td>21 106 17</td><td></td></tr><tr><td>Duroplan petri dish (100 x 20 mm)</td><td>Duran Group (DWK Life Sciences GmbH)</td><td>21 755 48</td><td></td></tr><tr><td>Seraflex Suture USP 3/0</td><td>SERAG-WIESSNER GmbH &amp;amp; Co. KG</td><td>IC208000</td><td></td></tr><tr><td>VWR disposable Square Weighin Boats 100ml</td><td>VWR, Inc.</td><td>10803-148</td><td></td></tr><tr><td>Styrofoam surface</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich, Inc.</td><td>71380</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich, Inc.</td><td>P4504</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich, Inc.</td><td>P5379</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich, Inc.</td><td>S0876</td><td></td></tr><tr><td>Magensium sulfate heptahydrate</td><td>Sigma-Aldrich, Inc.</td><td>230391</td><td></td></tr><tr><td>Magensium chloride</td><td>Sigma-Aldrich, Inc.</td><td>M8266</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, Inc.</td><td>H3375</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich, Inc.</td><td>T0625</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich, Inc.</td><td>G7528</td><td></td></tr><tr><td>2,3-Butanedione monoxime</td><td>Sigma-Aldrich, Inc.</td><td>B0753</td><td></td></tr><tr><td>Calcium chloride solution (1 M)</td><td>Sigma-Aldrich, Inc.</td><td>21115</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich, Inc.</td><td>A9647</td><td></td></tr><tr><td>Liberase</td><td>Roche (Sigma-Aldrich, Inc.)</td><td>LIBTM-RO</td><td></td></tr><tr><td>Heparin</td><td>Rotexmedica GmbH</td><td>3862357</td><td></td></tr><tr><td>Forene (Isoflurane)</td><td>Abbvie Deutschland GmbH &amp;amp; Co. KG</td><td>10182054</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma-Aldrich, Inc.</td><td>L2020</td><td></td></tr><tr><td>WillCo glass-bottom dish 500&amp;micro;l 0.005mm</td><td>WillCo Wells B.V.</td><td>HBST-3522</td><td></td></tr><tr><td>Fluo4 AM</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>F14201</td><td>5&amp;micro;M for 20min at RT</td></tr><tr><td>Di-8-ANNEPS</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>D3167</td><td>10&amp;micro;M for 45 min at 37&amp;deg; C&amp;nbsp;</td></tr><tr><td>Mitotracker RED FM</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>M22425</td><td>20nM for 30 min at 37&amp;deg; C</td></tr><tr><td>Jacketed reaction vessel 500 ml</td><td>Gebr. Rettberg GmbH</td><td>107024414</td><td></td></tr><tr><td>Jacketed reaction vessel 1000 ml</td><td>Gebr. Rettberg GmbH</td><td>107025414</td><td></td></tr><tr><td>Jacketed bubble trap</td><td>Gebr. Rettberg GmbH</td><td>134720001</td><td></td></tr><tr><td>ED heating immersion circulator</td><td>Julabo GmbH</td><td>9116000</td><td></td></tr><tr><td>Reglo Digital MS-2/6 peristaltic pump</td><td>Ismatec (Cole-Parmer Gmbh)</td><td>ISM 831</td><td></td></tr><tr><td>Voltcraft Thermometer 302 K/J</td><td>Conrad Electronic SE</td><td>030300546</td><td></td></tr><tr><td>Tubing</td><td></td><td></td><td></td></tr><tr><td>LSM 700 microscope</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>ZEN 2.3 imaging software</td><td>Carl Zeiss, Inc.</td><td>410135-1011-240&amp;nbsp;</td><td></td></tr><tr><td>Single channel heater controller TC-324B</td><td>Warner Instruments, LLC</td><td>64-2400</td><td></td></tr><tr><td>8 channel perfusion system</td><td>Warner Instruments, LLC</td><td>64-0185</td><td></td></tr><tr><td>8 channel Multi-Line In-Line Solution Heaters</td><td>Warner Instruments, LLC</td><td>64-0105</td><td></td></tr></tbody></table>

isolation of atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction

在本文中, 我们描述了一个优化的, Langendorff 的程序, 以隔离的大鼠代谢综合征 (飞机状态) 的模型, 与保存的射血分数 (HFpEF) 的一个人心肌细胞。随着心房重塑是死亡率的独立预测因子, 与心房重塑和心房颤动相关的心房心肌与 HFpEF 的发病率呈上升趋势。孤立单细胞心肌细胞的研究经常被用来证实和补充体内的发现。循环血管 rarefication 和间质组织纤维化是一个潜在的限制因素, 成功的单细胞分离飞机状态从动物模型的这种疾病。
我们通过使用一种能够在隔离过程中手动调节心脏腔腔内压力的装置来解决这个问题, 大大提高形态学和功能完整的飞机状态的产量。所获得的细胞可用于各种不同的实验, 如细胞培养和功能性钙成像 (即励磁收缩耦合)。
我们为研究员提供一步一步的协议, 一个优化的解决方案列表, 详细的说明准备必要的设备, 和全面的故障排除指南。虽然最初的实施程序可能是相当困难的, 一个成功的适应将使读者能够执行最先进的隔离在大鼠模型中与 HFpEF 有关的广泛的实验。

在21周的年龄, 60–90% 的可行飞机状态 (估计如步骤6.1 所述), 在钙重新适应 (步骤 5.4–5.7), 可以从 ZSF-1 肥胖大鼠的这种方法 (图 4A)。在大鼠, 飞机状态的特点是一个不同的和更异质表型相比, VCMs24,25。图 4B显示了一个人与保存的膜和小节结构, 两个功能完整的细胞的强指标。
<p class="...

Watch this Scientific Journal Video about Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction at JoVE.com

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

代谢综合征心力衰竭模型大鼠心房心肌细胞的分离与保留射血分数的关系

在这里, 我们描述了一个优化的, Langendorff 的程序, 从一个大鼠模型的代谢综合征心力衰竭与保存的弹射分数分离的单细胞心房心肌细胞。对心脏腔腔内压进行人工调节, 以产生功能完整的心肌细胞, 适合于励磁收缩耦合研究。

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

isolation-atrial-cardiomyocytes-from-rat-model-metabolic-syndrome

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.1K 次观看.  Charité University Medicine. 在这里, 我们描述了一个优化的, Langendorff 的程序, 从一个大鼠模型的代谢综合征心力衰竭与保存的弹射分数分离的单细胞心房心肌细胞。对心脏腔腔内压进行人工调节, 以产生功能完整的心肌细胞, 适合于励磁收缩耦合研究。

视频: Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

科研

医学

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

生物学

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

朗根多夫方法的修改，用于从成年小鼠中同时分离心房和心室肌细胞

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

一种持续分离小鼠心肌细胞的简单有效的方法

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

从成年大鼠心脏同时分离高质量心肌细胞，内皮细胞和成纤维细胞

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

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 ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

来自成人鼠心脏的心外肌细胞、心室肌细胞和非肌细胞的同步隔离和文化

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

代谢综合征心力衰竭模型大鼠心房心肌细胞的分离与保留射血分数的关系

摘要

探索更多视频

此视频中的章节