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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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>可能的原因</td>
			<td>解决 方案</td>
		</tr>
<tr>
<td>无流过蝶形软管</td>
			<td>主动脉插管结不正确闭合</td>
			<td>收紧前取出脂肪组织</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>添加第三插管结与各自的压痕</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>手拉结, 增加力</td>
		</tr>
<tr>
<td></td>
			<td>堵塞针</td>
			<td>调整位置到流明</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>用注射器轻轻拉解除阻止</td>
		</tr>
<tr>
<td>右心房/冠状动脉窦未充气</td>
			<td>心脏基底不正确闭合</td>
			<td>带附加节点的块流</td>
		</tr>
<tr>
<td></td>
			<td>右心房在准备过程中损坏</td>
			<td>闭合与夹子或结的泄漏</td>
		</tr>
<tr>
<td>差消化/心房不软化</td>
			<td>通过错误的温度降低酶活性</td>
			<td>调整温度为37°c</td>
		</tr>
<tr>
<td></td>
			<td>非活性/降解酶</td>
			<td>取代</td>
		</tr>
<tr>
<td></td>
			<td>老/过度纤维化心房</td>
			<td>增加消化时间 (步骤为 50%)</td>
		</tr>
<tr>
<td></td>
			<td>损伤的主动脉瓣</td>
			<td>插管时浅穿透</td>
		</tr>
<tr>
<td>细胞产量差</td>
			<td>心房过度消化</td>
			<td>减少消化时间 (在步骤 25%)</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>通过降低蝶形软管降低腔内压力</td>
		</tr>
<tr>
<td></td>
			<td>心房未消化</td>
			<td>增加消化时间 (步骤 25%)</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>提高蝶形软管的腔内压力</td>
		</tr>
<tr>
<td></td>
			<td>细胞弥散过于激进</td>
			<td>更温柔</td>
		</tr>
</tbody></table>表 2: 疑难解答指南.此表显示隔离过程及其各自解决方案的常见问题。
在最近的一项研究中, 我们已经表明, ZFS-1 肥胖大鼠模型显示广泛的心房重塑与增加心房大小22。左心房大小的增加已被确认为舒张功能障碍26的重要预后标志, 并导致微血管血管 rarefication 相对于组织。这导致在隔离过程中通过主动脉逆行灌注来减少消化缓冲的分布, 从而使单细胞隔离在这个模型中特别具有挑战性。其他标志性的特点, 心房重塑, 心房纤维化, 并增加纤维化已显示 ZDF 大鼠-一个大鼠模型的代谢性糖尿病和一个 ZFS1 大鼠的父母菌株27。胶原沉积损害消化酶的功效, 从细胞外基质中解放心肌细胞, 因此需要进一步调整细胞隔离程序28。
压力装置促进隔离效果的机制, 在心房重塑模型中的作用很可能与左心房冠状动脉血流的局部衰减有关。冠状动脉血流的一个主要成分是冠状动脉灌注压力, 它被定义为冠状动脉压与各腔29的终舒张压力之间的梯度。在被描述的过程中, 心脏基地的堵塞导致冠状动脉压力和心脏腔内压力的全球增加。随后穿刺左心房促进局部, 选择性下降的腔内压力在左心房腔。因此, 不仅建立了大的冠状动脉灌注压力梯度, 而且从充血的左心室到左心房的分流消化液也增加了灌注量。
此外, 消化酶的选择对其分离至关重要: 胶原酶 I 和 II 的纯化酶共混物已被证明优于低靶和不纯酶, 如胶原酶30。这种酶不仅允许更高的产量的形态学和功能完整的心肌细胞, 但也最大限度地减少任何聚集的单细胞后, 他们的隔离31。胶原酶的纯化酶与其他高酶或中 thermolysin 含量的混合, 最常用于大鼠心肌隔离。虽然 VCMs 是最好的隔离在更高的浓度, 最好的结果, 心房肌细胞获得 0.195 Wünsch 单位/毫升的浓度, 使用该协议32。
随着大中枢相关 HFpEF 的患病率上升2 , 心房心肌导致心房重塑和心房颤动的临床意义十分密切, 这一领域的研究具有重要的意义。许多新的动物模型为 HFpEF 正在出现33,34和心房重构符合增加的发病率的心房节律紊乱是疾病的标志性特征。该方法允许研究人员从大鼠模型中分离出可行的单心肌细胞, 并进行心房重塑, 以获得极高的产量和保存的机械和电功能。

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 border="0" cellpadding="0" cellspacing="0" width="935">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">ZSF-1 Obese rat</td>
			<td style="width:292px;">Charles River Laboratories, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:227px;">21 weeks old</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Fine Iris Scissors</td>
			<td style="width:292px;">Fine Science Tools GmbH</td>
			<td style="width:136px;">14094-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Surgical Scissors</td>
			<td style="width:292px;">Fine Science Tools GmbH</td>
			<td style="width:136px;">14001-18</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Micro Dressing Forceps (curved, serrated)</td>
			<td style="width:292px;">Aesculap, Inc.</td>
			<td style="width:136px;">BD312R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Tissue Forceps (straight, 1 x 2 teeth)</td>
			<td style="width:292px;">Aesculap, Inc.</td>
			<td style="width:136px;">BD537R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Tying Forceps (angled)</td>
			<td style="width:292px;">Aesculap, Inc.</td>
			<td style="width:136px;">MA624R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Rodent and Small Animal Guillotine</td>
			<td style="width:292px;">Kent Scientific Corp.</td>
			<td style="width:136px;">DCAP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Low Cost Induction Chamber 3.0 L</td>
			<td style="width:292px;">Kent Scientific Corp.</td>
			<td style="width:136px;">SOMNO-0730&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Butterfly Winged Infusion Set 21 G</td>
			<td style="width:292px;">Hospira, Inc.</td>
			<td style="width:136px;">181106101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Abbocath 16 G</td>
			<td style="width:292px;">Hospira, Inc.</td>
			<td>0G7149702</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Microlance Hypodermic Needle</td>
			<td style="width:292px;">Becton Dickinson GmbH</td>
			<td style="width:136px;">301300</td>
			<td>modify needle to make cannula</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Braun Original Perfusor Syringe 50 ml</td>
			<td style="width:292px;">B. Braun Melsungen AG</td>
			<td style="width:136px;">8728810F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Braun Inject Solo Syringe 10 ml</td>
			<td style="width:292px;">B. Braun Melsungen AG</td>
			<td style="width:136px;">2057926</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Beaker 50ml</td>
			<td style="width:292px;">Duran Group (DWK Life Sciences GmbH)</td>
			<td>21 106 17</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Duroplan petri dish (100 x 20 mm)</td>
			<td style="width:292px;">Duran Group (DWK Life Sciences GmbH)</td>
			<td style="width:136px;">21 755 48</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Seraflex Suture USP 3/0</td>
			<td style="width:292px;">SERAG-WIESSNER GmbH &#38; Co. KG</td>
			<td style="width:136px;">IC208000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">VWR disposable Square Weighin Boats 100ml</td>
			<td style="width:292px;">VWR, Inc.</td>
			<td style="width:136px;">10803-148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Styrofoam surface</td>
			<td style="width:292px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Sodium chloride</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">71380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Potassium chloride</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">P4504</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Potassium phosphate monobasic</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">P5379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Sodium phosphate dibasic</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">S0876</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Magensium sulfate heptahydrate</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">230391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Magensium chloride</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">M8266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">HEPES</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Taurine</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">T0625</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Glucose</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">G7528</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">2,3-Butanedione monoxime</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">B0753</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Calcium chloride solution (1 M)</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">21115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Bovine Serum Albumin</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">A9647</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Liberase</td>
			<td style="width:292px;">Roche (Sigma-Aldrich, Inc.)</td>
			<td style="width:136px;">LIBTM-RO</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Heparin</td>
			<td style="width:292px;">Rotexmedica GmbH</td>
			<td style="width:136px;">3862357</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Forene (Isoflurane)</td>
			<td style="width:292px;">Abbvie Deutschland GmbH &#38; Co. KG</td>
			<td style="width:136px;">10182054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">L2020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">WillCo glass-bottom dish 500&#181;l 0.005mm</td>
			<td style="width:292px;">WillCo Wells B.V.</td>
			<td style="width:136px;">HBST-3522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Fluo4 AM</td>
			<td style="width:292px;">Invitrogen (Thermo Fisher Scientific, Inc.)</td>
			<td>F14201</td>
			<td>5&#181;M for 20min at RT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Di-8-ANNEPS</td>
			<td style="width:292px;">Invitrogen (Thermo Fisher Scientific, Inc.)</td>
			<td>D3167</td>
			<td>10&#181;M for 45 min at 37&#176; C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Mitotracker RED FM</td>
			<td style="width:292px;">Invitrogen (Thermo Fisher Scientific, Inc.)</td>
			<td style="width:136px;">M22425</td>
			<td>20nM for 30 min at 37&#176; C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Jacketed reaction vessel 500 ml</td>
			<td style="width:292px;">Gebr. Rettberg GmbH</td>
			<td>107024414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Jacketed reaction vessel 1000 ml</td>
			<td style="width:292px;">Gebr. Rettberg GmbH</td>
			<td>107025414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Jacketed bubble trap</td>
			<td style="width:292px;">Gebr. Rettberg GmbH</td>
			<td>134720001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">ED heating immersion circulator</td>
			<td style="width:292px;">Julabo GmbH</td>
			<td>9116000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Reglo Digital MS-2/6 peristaltic pump</td>
			<td style="width:292px;">Ismatec (Cole-Parmer Gmbh)</td>
			<td style="width:136px;">ISM 831</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Voltcraft Thermometer 302 K/J</td>
			<td style="width:292px;">Conrad Electronic SE</td>
			<td style="width:136px;">030300546</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Tubing</td>
			<td style="width:292px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">LSM 700 microscope</td>
			<td style="width:292px;">Carl Zeiss, Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">ZEN 2.3 imaging software</td>
			<td style="width:292px;">Carl Zeiss, Inc.</td>
			<td style="width:136px;">410135-1011-240&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Single channel heater controller TC-324B</td>
			<td style="width:292px;">Warner Instruments, LLC</td>
			<td style="width:136px;">64-2400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">8 channel perfusion system</td>
			<td style="width:292px;">Warner Instruments, LLC</td>
			<td style="width:136px;">64-0185</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">8 channel Multi-Line In-Line Solution Heaters</td>
			<td style="width:292px;">Warner Instruments, LLC</td>
			<td style="width:136px;">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。
获得的飞机状态可以用多种方式进行处理。如图 5所示, 这些细胞可能会被用来研究形态学和/或功能的荧光染料所载。例如, di-8-ANEPPS 被用来描绘心房鼠细胞中的管状系统 (图 5A)。在另一组实验中, 采用线粒体染色的远红色荧光染料 (例如, mitotracker 红 FM) 检测心房重塑中的胞浆线粒体 (图 5B)。如图 5C所示, 与此协议隔离的飞机状态也适用于活细胞 Ca2 +成像, 并显示完整的励磁收缩耦合, 1 赫兹场刺激。所有的图像可用于进一步分析使用的各种算法, 可供研究员6,7 (图 5D)。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57953/57953fig4.jpg"> 图 4: 可行的单细胞 AM 的各自产量.(A) 本小组显示在 NT 重新适应飞机状态1毫米 Ca2 +后, 隔离的收益率. (B) 这个小组显示一个孤立的单细胞心房心肌细胞。小节结构、细胞膜和细胞核明显可见。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57953/57953fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57953/57953fig5.jpg"> 图 5: 荧光染料对大鼠飞机状态的染色.(A) 本小组展示了用荧光染料 Di8ANNEPS 染色的细胞膜和管状网络。(B) 本小组显示荧光染料 mitotracker-红 FM 染色线粒体. (C) 此面板显示了一个 Ca2 +-励磁的横向线扫描与荧光染料 Fluo4-AM 在一个单一的细胞。箭头指示电刺激, 进行1赫兹. (D) 此面板显示了从面板C获得的 longitudenal Ca2 +瞬变。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57953/57953fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

Research

JoVE Journal

Medicine

10.0K 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

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

缺血性心力衰竭在临床前模型的转基因

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

科研

医学

The WATCHMAN Left Atrial Appendage Closure Device for Atrial Fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting an estimated 6 million people in the United States 1. Since AF affects primarily elderly people, its prevalence increases parallel with age. As such, it is expected that 15.9 million Americans will be affected by the year 2050 2. Ischemic stroke occurs in 5% of non-anticoagulated AF patients each year. Current treatments for AF include rate control, rhythm control and prevention of stroke 3.
The American College of Cardiology, American Heart Association, and European Society of Cardiology currently recommended rate control as the first course of therapy for AF 3. Rate control is achieved by administration of pharmacological agents, such as &beta;-blockers, that lower the heart rate until it reaches a less symptomatic state 3. Rhythm control aims to return the heart to its normal sinus rhythm and is typically achieved through administration of antiarrhythmic drugs such as amiodarone, electrical cardioversion or ablation therapy. Rhythm control methods, however, have not been demonstrated to be superior to rate-control methods 4-6. In fact, certain antiarrhythmic drugs have been shown to be associated with higher hospitalization rates, serious adverse effects 3, or even increases in mortality in patients with structural heart defects 7. Thus, treatment with antiarrhythmics is more often used when rate-control drugs are ineffective or contraindicated. Rate-control and antiarrhythmic agents relieve the symptoms of AF, including palpitations, shortness of breath, and fatigue 8, but don't reliably prevent thromboembolic events 6.
Treatment with the anticoagulant drug warfarin significantly reduces the rate of stroke or embolism 9,10. However, because of problems associated with its use, fewer than 50% of patients are treated with it. The therapeutic dose is affected by drug, dietary, and metabolic interactions, and thus requires detailed monitoring. In addition, warfarin has the potential to cause severe, sometimes lethal, bleeding 2. As an alternative, aspirin is commonly prescribed. While aspirin is typically well tolerated, it is far less effective at preventing stroke 10. Other alternatives to warfarin, such as dabigatran 11 or rivaroxaban 12 demonstrate non-inferiority to warfarin with respect to thromboembolic events (in fact, dabigatran given as a high dose of 150 mg twice a day has shown superiority). While these drugs have the advantage of eliminating dietary concerns and eliminating the need for regular blood monitoring, major bleeding and associated complications, while somewhat less so than with warfarin, remain an issue 13-15.
Since 90% of AF-associated strokes result from emboli that arise from the left atrial appendage (LAA) 2, one alternative approach to warfarin therapy has been to exclude the LAA using an implanted device to trap blood clots before they exit. Here, we demonstrate a procedure for implanting the WATCHMAN Left Atrial Appendage Closure Device. A transseptal cannula is inserted through the femoral vein, and under fluoroscopic guidance, inter-atrial septum is crossed. Once access to the left atrium has been achieved, a guidewire is placed in the upper pulmonary vein and the WATCHMAN Access Sheath and dilator are advanced over the wire into the left atrium. The guidewire is removed, and the access sheath is carefully advanced into the distal portion of the LAA over a pigtail catheter. The WATCHMAN Delivery System is prepped, inserted into the access sheath, and slowly advanced. The WATCHMAN device is then deployed into the LAA. The device release criteria are confirmed via fluoroscopy and transesophageal echocardiography (TEE) and the device is released.

The accompanying video describes a procedure for percutaneous placement of the WATCHMAN Left Atrial Appendage (LAA) Device. The WATCHMAN is a nitinol device designed to be permanently implanted at, or slightly distal to, the opening of the left atrial appendage (LAA) to trap blood clots before they exit the LAA, preventing thromboembolic stroke.

守望的左心耳封闭装置心房颤动

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting an estimated 6 million people in the United States 1. Since AF affects primarily ...

A New Single Chamber Implantable Defibrillator with Atrial Sensing: A Practical Demonstration of Sensing and  Ease of Implantation

Implantable cardioverter-defibrillators (ICDs) terminate ventricular tachycardia (VT) and ventricular fibrillation (VF) with high efficacy and can protect patients from sudden cardiac death (SCD). However, inappropriate shocks may occur if tachycardias are misdiagnosed. Inappropriate shocks are harmful and impair patient quality of life. The risk of inappropriate therapy increases with lower detection rates programmed in the ICD. Single-chamber detection poses greater risks for misdiagnosis when compared with dual-chamber devices that have the benefit of additional atrial information. However, using a dual-chamber device merely for the sake of detection is generally not accepted, since the risks associated with the second electrode may outweigh the benefits of detection. Therefore, BIOTRONIK developed a ventricular lead called the LinoxSMART S DX, which allows for the detection of atrial signals from two electrodes positioned at the atrial part of the ventricular electrode. This device contains two ring electrodes; one that contacts the atrial wall at the junction of the superior vena cava (SVC) and one positioned at the free floating part of the electrode in the atrium. The excellent signal quality can only be achieved by a special filter setting in the ICD (Lumax 540 and 740 VR-T DX, BIOTRONIK). Here, the ease of implantation of the system will be demonstrated.

Dual-chamber implantable cardioverter-defibrillators (ICDs) may improve detection of atrial fibrillation as well as differentiation of tachycardias. However, this advantage is undermined by complications associated with the second electrode, which is required in conventional dual chamber devices. Therefore, BIOTRONIK has developed a new electrode called the LinoxSMART S DX that, when used in conjunction with the Lumax DX ICD, offers dual-chamber detection without the risks associated with the second electrode.

心房遥感新的单一商会植入去纤颤器:一个实用的遥感示范和易于植入

Implantable cardioverter-defibrillators (ICDs) terminate ventricular tachycardia (VT) and ventricular fibrillation (VF) with high efficacy and can protect ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型:教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

从孤立的大鼠心脏的间质渗出物收集的倒置心脏模型

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

成年大鼠心肌细胞的分离与培养

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

心动过速诱发的心肌病作为猪慢性心力衰竭模型的研究

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

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 ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

建立干细胞治疗心肌梗塞心力衰竭的猪模型

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation of signal transduction pathways, to counteract, in the short-term, for the cardiac myocyte loss and or the increase in wall stress. However, prolonged activation of these pathways becomes detrimental leading to the initiation and propagation of cardiac remodeling leading to changes in left ventricular geometry and increases in left ventricular volumes; a phenotype seen in patients with systolic heart failure (HF). Here, we describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction (MOD) by ascending aortic banding (AAB) via a vascular clip with an internal area of 2 mm2. The surgery is performed in 200 g Sprague-Dawley rats. The MOD HF phenotype develops at 8-12 weeks after AAB and is characterized noninvasively by means of echocardiography. Previous work suggests the activation of signal transduction pathways and altered gene expression and post-translational modification of proteins in the MOD HF phenotype that mimic those seen in human systolic HF; therefore, making the MOD HF phenotype a suitable model for translational research to identify and test potential therapeutic anti-remodeling targets in HF. The advantages of the MOD HF phenotype compared to the overt systolic HF phenotype is that it allows for the identification of molecular targets involved in the early remodeling process and the early application of therapeutic interventions. The limitation of the MOD HF phenotype is that it may not mimic the spectrum of diseases leading to systolic HF in human. Moreover, it is a challenging phenotype to create, as the AAB surgery is associated with high mortality and failure rates with only 20% of operated rats developing the desired HF phenotype.

We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

压力过载诱导中度重塑和收缩功能障碍的大鼠模型，而不是过度收缩性心力衰竭

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation ...